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Sample records for gene delivery mediated

  1. Recent Trends of Polymer Mediated Liposomal Gene Delivery System

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    Shyamal Kumar Kundu

    2014-01-01

    Full Text Available Advancement in the gene delivery system have resulted in clinical successes in gene therapy for patients with several genetic diseases, such as immunodeficiency diseases, X-linked adrenoleukodystrophy (X-ALD blindness, thalassemia, and many more. Among various delivery systems, liposomal mediated gene delivery route is offering great promises for gene therapy. This review is an attempt to depict a portrait about the polymer based liposomal gene delivery systems and their future applications. Herein, we have discussed in detail the characteristics of liposome, importance of polymer for liposome formulation, gene delivery, and future direction of liposome based gene delivery as a whole.

  2. Polyelectrolyte nanoparticles mediate vascular gene delivery.

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    Zaitsev, Sergey; Cartier, Régis; Vyborov, Oleg; Sukhorukov, Gleb; Paulke, Bernd-Reiner; Haberland, Annekathrin; Parfyonova, Yelena; Tkachuk, Vsevolod; Böttger, Michael

    2004-09-01

    The purpose is to develop a non-viral gene delivery system that meets the requirements of colloidal stability of DNA complexes expressed in terms of no particle aggregation under physiologic conditions. The system should be used to transfect cardiovascular tissues. We used a strategy based on the formation of polyelectrolyte nanoparticles by deposition of alternatively charged polyelectrolytes onto a DNA core. Polyelectrolytes were transfer RNA as well as the synthetic polyanion, polyvinyl sulfate (PVS), and the polycation polyethylenimine (PEI). The PEI/DNA complex formed the DNA core. We observed that the DNA is condensed by polycations and further packaged by association with a polyanion. These nanoparticles exhibited negative surface charge and low aggregation tendency. In vivo rat carotid artery experiments revealed high transfection efficiency, not only with the reporter gene but also with the gene encoding human urokinase plasminogen activator (Hu-uPA). Hu-uPA is one of the proteins involved in the recovery of the blood vessels after balloon catheter injury and therefore clinically relevant. A strategy for in vivo gene transfer is proposed that uses the incorporation of polyanions as RNA or PVS into PEI/DNA complexes in order to overcome colloidal instability and to generate a negative surface charge. The particles proved to be transfectionally active in vascular gene transfer.

  3. Ultrasound-Mediated Local Drug and Gene Delivery Using Nanocarriers

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    Zhou, Qiu-Lan; Chen, Zhi-Yi; Yang, Feng

    2014-01-01

    With the development of nanotechnology, nanocarriers have been increasingly used for curative drug/gene delivery. Various nanocarriers are being introduced and assessed, such as polymer nanoparticles, liposomes, and micelles. As a novel theranostic system, nanocarriers hold great promise for ultrasound molecular imaging, targeted drug/gene delivery, and therapy. Nanocarriers, with the properties of smaller particle size, and long circulation time, would be advantageous in diagnostic and therapeutic applications. Nanocarriers can pass through blood capillary walls and cell membrane walls to deliver drugs. The mechanisms of interaction between ultrasound and nanocarriers are not clearly understood, which may be related to cavitation, mechanical effects, thermal effects, and so forth. These effects may induce transient membrane permeabilization (sonoporation) on a single cell level, cell death, and disruption of tissue structure, ensuring noninvasive, targeted, and efficient drug/gene delivery and therapy. The system has been used in various tissues and organs (in vitro or in vivo), including tumor tissues, kidney, cardiac, skeletal muscle, and vascular smooth muscle. In this review, we explore the research progress and application of ultrasound-mediated local drug/gene delivery with nanocarriers. PMID:25202710

  4. Ultrasound-Mediated Local Drug and Gene Delivery Using Nanocarriers

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    Qiu-Lan Zhou

    2014-01-01

    Full Text Available With the development of nanotechnology, nanocarriers have been increasingly used for curative drug/gene delivery. Various nanocarriers are being introduced and assessed, such as polymer nanoparticles, liposomes, and micelles. As a novel theranostic system, nanocarriers hold great promise for ultrasound molecular imaging, targeted drug/gene delivery, and therapy. Nanocarriers, with the properties of smaller particle size, and long circulation time, would be advantageous in diagnostic and therapeutic applications. Nanocarriers can pass through blood capillary walls and cell membrane walls to deliver drugs. The mechanisms of interaction between ultrasound and nanocarriers are not clearly understood, which may be related to cavitation, mechanical effects, thermal effects, and so forth. These effects may induce transient membrane permeabilization (sonoporation on a single cell level, cell death, and disruption of tissue structure, ensuring noninvasive, targeted, and efficient drug/gene delivery and therapy. The system has been used in various tissues and organs (in vitro or in vivo, including tumor tissues, kidney, cardiac, skeletal muscle, and vascular smooth muscle. In this review, we explore the research progress and application of ultrasound-mediated local drug/gene delivery with nanocarriers.

  5. Polymeric Nanoparticle-Mediated Gene Delivery for Lung Cancer Treatment.

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    Amreddy, Narsireddy; Babu, Anish; Muralidharan, Ranganayaki; Munshi, Anupama; Ramesh, Rajagopal

    2017-04-01

    In recent years, researchers have focused on targeted gene therapy for lung cancer, using nanoparticle carriers to overcome the limitations of conventional treatment methods. The main goal of targeted gene therapy is to develop more efficient therapeutic strategies by improving the bioavailability, stability, and target specificity of gene therapeutics and to reduce off-target effects. Polymer-based nanoparticles, an alternative to lipid and inorganic nanoparticles, efficiently carry nucleic acid therapeutics and are stable in vivo. Receptor-targeted delivery is a promising approach that can limit non-specific gene delivery and can be achieved by modifying the polymer nanoparticle surface with specific receptor ligands or antibodies. This review highlights the recent developments in gene delivery using synthetic and natural polymer-based nucleic acid carriers for lung cancer treatment. Various nanoparticle systems based on polymers and polymer combinations are discussed. Further, examples of targeting ligands or moieties used in targeted, polymer-based gene delivery to lung cancer are reviewed.

  6. Nanoparticle-mediated delivery of suicide genes in cancer therapy.

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    Vago, Riccardo; Collico, Veronica; Zuppone, Stefania; Prosperi, Davide; Colombo, Miriam

    2016-09-01

    Conventional chemotherapeutics have been employed in cancer treatment for decades due to their efficacy in killing the malignant cells, but the other side of the coin showed off-target effects, onset of drug resistance and recurrences. To overcome these limitations, different approaches have been investigated and suicide gene therapy has emerged as a promising alternative. This approach consists in the introduction of genetic materials into cancerous cells or the surrounding tissue to cause cell death or retard the growth of the tumor mass. Despite promising results obtained both in vitro and in vivo, this innovative approach has been limited, for long time, to the treatment of localized tumors, due to the suboptimal efficiency in introducing suicide genes into cancer cells. Nanoparticles represent a valuable non-viral delivery system to protect drugs in the bloodstream, to improve biodistribution, and to limit side effects by achieving target selectivity through surface ligands. In this scenario, the real potential of suicide genes can be translated into clinically viable treatments for patients. In the present review, we summarize the recent advances of inorganic nanoparticles as non-viral vectors in terms of therapeutic efficacy, targeting capacity and safety issues. We describe the main suicide genes currently used in therapy, with particular emphasis on toxin-encoding genes of bacterial and plant origin. In addition, we discuss the relevance of molecular targeting and tumor-restricted expression to improve treatment specificity to cancer tissue. Finally, we analyze the main clinical applications, limitations and future perspectives of suicide gene therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. PLGA Nanoparticles for Ultrasound-Mediated Gene Delivery to Solid Tumors

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    Marxa Figueiredo

    2012-01-01

    Full Text Available This paper focuses on novel approaches in the field of nanotechnology-based carriers utilizing ultrasound stimuli as a means to spatially target gene delivery in vivo, using nanoparticles made with either poly(lactic-co-glycolic acid (PLGA or other polymers. We specifically discuss the potential for gene delivery by particles that are echogenic (amenable to destruction by ultrasound composed either of polymers (PLGA, polystyrene or other contrast agent materials (Optison, SonoVue microbubbles. The use of ultrasound is an efficient tool to further enhance gene delivery by PLGA or other echogenic particles in vivo. Echogenic PLGA nanoparticles are an attractive strategy for ultrasound-mediated gene delivery since this polymer is currently approved by the US Food and Drug Administration for drug delivery and diagnostics in cancer, cardiovascular disease, and also other applications such as vaccines and tissue engineering. This paper will review recent successes and the potential of applying PLGA nanoparticles for gene delivery, which include (a echogenic PLGA used with ultrasound to enhance local gene delivery in tumors or muscle and (b PLGA nanoparticles currently under development, which could benefit in the future from ultrasound-enhanced tumor targeted gene delivery.

  8. Polyethylenimine-mediated gene delivery to the lung and therapeutic applications

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    Sante Di Gioia

    2008-09-01

    Full Text Available Sante Di Gioia, Massimo ConeseDepartment of Biomedical Sciences, University of Foggia, Foggia, ItalyAbstract: Nonviral gene delivery is now considered a promising alternative to viral vectors. Among nonviral gene delivery agents, polyethylenimine (PEI has emerged as a potent candidate for gene delivery to the lung. PEI has some advantages over other polycations in that it combines strong DNA compaction capacity with an intrinsic endosomolytic activity. However, intracellular (mainly the nuclear membrane and extracellular obstacles still hamper its efficiency in vitro and in vivo, depending on the route of administration and the type of PEI. Nuclear delivery has been increased by adding nuclear localization signals. To overcome nonspecific interactions with biological fluids, extracellular matrix components and nontarget cells, strategies have been developed to protect polyplexes from these interactions and to increase target specificity and gene expression. When gene delivery into airway epithelial cells of the conducting airways is necessary, aerosolization of complexes seems to be better suited to guarantee higher transgene expression in the airway epithelial cells with lower toxicity than observed with either intratracheal or intravenous administration. Aerosolization, indeed, is useful to target the alveolar epithelium and pulmonary endothelium. Proof-of-principle that PEI-mediated gene delivery has therapeutic application to some genetic and acquired lung disease is presented, using as genetic material either plasmidic DNA or small-interfering RNA, although optimization of formulation and delivery protocols and limitation of toxicity need further studies.Keywords: gene transfer, gene therapy, polyethylenimine, airway epithelial cells, lung, RNA interference

  9. Electric pulse-mediated gene delivery to various animal tissues

    DEFF Research Database (Denmark)

    Mir, Lluis M; Moller, Pernille H; André, Franck

    2005-01-01

    therapy, termed electrogenetherapy (EGT as well). By transfecting cells with a long lifetime, such as muscle fibers, a very long-term expression of genes can be obtained. A great variety of tissues have been transfected successfully, from muscle as the most extensively used, to both soft (e.g., spleen...

  10. PLGA-Chitosan nanoparticle-mediated gene delivery for oral cancer treatment: A brief review

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    Bakar, L. M.; Abdullah, M. Z.; Doolaanea, A. A.; Ichwan, S. J. A.

    2017-08-01

    Cancer becomes a serious issue on society with increasing of their growth and proliferation, either in well economic developed countries or not. Recent years, oral cancer is one of the most threatening diseases impairing the quality of life of the patient. Scientists have emphasised on application of gene therapy for oral cancer by using nanoparticle as transportation vectors as a new alternative platform in order to overcome the limitations of conventional approaches. In modern medicine, nanotechnologies’ application, such as nanoparticles-mediated gene delivery, is one of promising tool for therapeutic devices. The objective of this article is to present a brief review summarizes on the current progress of nanotechnology-based gene delivery treatment system targeted for oral cancer.

  11. Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs

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    Atsushi Miyanohara

    2016-01-01

    Full Text Available Effective in vivo use of adeno-associated virus (AAV-based vectors to achieve gene-specific silencing or upregulation in the central nervous system has been limited by the inability to provide more than limited deep parenchymal expression in adult animals using delivery routes with the most clinical relevance (intravenous or intrathecal. Here, we demonstrate that the spinal pia membrane represents the primary barrier limiting effective AAV9 penetration into the spinal parenchyma after intrathecal AAV9 delivery. We develop a novel subpial AAV9 delivery technique and AAV9-dextran formulation. We use these in adult rats and pigs to show (i potent spinal parenchymal transgene expression in white and gray matter including neurons, glial and endothelial cells after single bolus subpial AAV9 delivery; (ii delivery to almost all apparent descending motor axons throughout the length of the spinal cord after cervical or thoracic subpial AAV9 injection; (iii potent retrograde transgene expression in brain motor centers (motor cortex and brain stem; and (iv the relative safety of this approach by defining normal neurological function for up to 6 months after AAV9 delivery. Thus, subpial delivery of AAV9 enables gene-based therapies with a wide range of potential experimental and clinical utilizations in adult animals and human patients.

  12. Increased 1-aminocyclopropane-1-carboxylate deaminase activity enhances Agrobacterium tumefaciens-mediated gene delivery into plant cells.

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    Someya, Tatsuhiko; Nonaka, Satoko; Nakamura, Kouji; Ezura, Hiroshi

    2013-10-01

    Agrobacterium-mediated transformation is a useful tool for the genetic modification in plants, although its efficiency is low for several plant species. Agrobacterium-mediated transformation has three major steps in laboratory-controlled experiments: the delivery of T-DNA into plant cells, the selection of transformed plant cells, and the regeneration of whole plants from the selected cells. Each of these steps must be optimized to improve the efficiency of Agrobacterium-mediated plant transformation. It has been reported that increasing the number of cells transformed by T-DNA delivery can improve the frequency of stable transformation. Previously, we demonstrated that a reduction in ethylene production by plant cells during cocultivation with A. tumefaciens-expressing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase resulted in increased T-DNA delivery into the plant cells. In this study, to further improve T-DNA delivery by A. tumefaciens, we modified the expression cassette of the ACC deaminase gene using vir gene promoter sequences. The ACC deaminase gene driven by the virD1 promoter was expressed at a higher level, resulting in a higher ACC deaminase activity in this A. tumefaciens strain than in the strain with the lac promoter used in a previous study. The newly developed A. tumefaciens strain improves the delivery of T-DNA into Solanum lycopersicum (tomato) and Erianthus ravennae plants and thus may be a powerful tool for the Agrobacterium-mediated genetic engineering of plants. © 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  13. In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor cells in mini-pigs

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    Bez, Maxim; Sheyn, Dmitriy; Tawackoli, Wafa; Avalos, Pablo; Shapiro, Galina; Giaconi, Joseph C.; Da, Xiaoyu; Ben David, Shiran; Gavrity, Jayne; Awad, Hani A.; Bae, Hyun W.; Ley, Eric J.; Kremen, Thomas J.; Gazit, Zulma; Ferrara, Katherine W.; Pelled, Gadi; Gazit, Dan

    2017-01-01

    More than 2 million bone-grafting procedures are performed each year using autografts or allografts. However, both options carry disadvantages, and there remains a clear medical need for the development of new therapies for massive bone loss and fracture nonunions. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would induce efficient bone regeneration and fracture repair. To test this hypothesis, we surgically created a critical-sized bone fracture in the tibiae of Yucatán mini-pigs, a clinically relevant large animal model. A collagen scaffold was implanted in the fracture to facilitate recruitment of endogenous mesenchymal stem/progenitor cells (MSCs) into the fracture site. Two weeks later, transcutaneous ultrasound-mediated reporter gene delivery successfully transfected 40% of cells at the fracture site, and flow cytometry showed that 80% of the transfected cells expressed MSC markers. Human bone morphogenetic protein-6 (BMP-6) plasmid DNA was delivered using ultrasound in the same animal model, leading to transient expression and secretion of BMP-6 localized to the fracture area. Micro–computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to complete radiographic and functional fracture healing in all animals 6 weeks after treatment, whereas nonunion was evident in control animals. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchy-mal progenitor cells can effectively treat nonhealing bone fractures in large animals, thereby addressing a major orthopedic unmet need and offering new possibilities for clinical translation. PMID:28515335

  14. In vivo targeted gene delivery to peripheral neurons mediated by neurotropic poly(ethylene imine)-based nanoparticles

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    Lopes, Cátia DF; Oliveira, Hugo; Estevão, Inês; Pires, Liliana Raquel; Pêgo, Ana Paula

    2016-01-01

    A major challenge in neuronal gene therapy is to achieve safe, efficient, and minimally invasive transgene delivery to neurons. In this study, we report the use of a nonviral neurotropic poly(ethylene imine)-based nanoparticle that is capable of mediating neuron-specific transfection upon a subcutaneous injection. Nanoparticles were targeted to peripheral neurons by using the nontoxic carboxylic fragment of tetanus toxin (HC), which, besides being neurotropic, is capable of being retrogradely transported from neuron terminals to the cell bodies. Nontargeted particles and naked plasmid DNA were used as control. Five days after treatment by subcutaneous injection in the footpad of Wistar rats, it was observed that 56% and 64% of L4 and L5 dorsal root ganglia neurons, respectively, were expressing the reporter protein. The delivery mediated by HC-functionalized nanoparticles spatially limited the transgene expression, in comparison with the controls. Histological examination revealed no significant adverse effects in the use of the proposed delivery system. These findings demonstrate the feasibility and safety of the developed neurotropic nanoparticles for the minimally invasive delivery of genes to the peripheral nervous system, opening new avenues for the application of gene therapy strategies in the treatment of peripheral neuropathies. PMID:27354797

  15. Protein antigen delivery by gene gun-mediated epidermal antigen incorporation (EAI).

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    Scheiblhofer, Sandra; Ritter, Uwe; Thalhamer, Josef; Weiss, Richard

    2013-01-01

    The gene gun technology can not only be employed for efficient transfer of gene vaccines into upper layers of the skin, but also for application of protein antigens. As a tissue rich in professional antigen presenting cells, the skin represents an attractive target for immunizations. In this chapter we present a method for delivery of the model antigen ovalbumin into the skin of mice termed epidermal antigen incorporation and describe in detail how antigen-specific proliferation in draining lymph nodes can be followed by flow cytometry.

  16. Efficacy of In Vivo Electroporation-Mediated IL-10 Gene Delivery on Survival of Skin Flaps.

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    Seyed Jafari, S Morteza; Shafighi, Maziar; Beltraminelli, Helmut; Weber, Benedikt; Schmid, Ralph A; Geiser, Thomas; Gazdhar, Amiq; Hunger, Robert E

    2018-04-01

    Despite advances in understanding the underlying mechanisms of flap necrosis and improvement in surgical techniques, skin flap necrosis after reconstructive surgery remains a crucial issue. We investigated the efficacy of electroporation-mediated IL-10 gene transfer to random skin flap with an aim to accelerate wound healing and improve skin flap survival. Nine male Wistar rats (300-330 g) were divided in two groups (a) control group (n = 5), only surgery no gene transfer, and (b) experimental group, received electroporation-mediated IL-10 gene transfer 24 h before the surgery as prophylaxis (n = 4). Random skin flap (McFarlane) was performed in both groups. Planimetry, Laser Doppler imaging, and immunohistochemistry were used to evaluate the effect of IL-10 gene transfer between study groups at day 7. Electroporation-mediated IL-10 gene transfer decreased percentage of flap necrosis (p value = 0.0159) and increased cutaneous perfusion compared to the control group (p value = 0.0159). In addition, Spearman's rank correlation showed a significant negative correlation between percentage of flap necrosis and Laser Index (p value = 0.0083, r -0.83, respectively). Furthermore, significantly higher mean CD31 + vessel density was detected in the experimental group compared to the control group (p value = 0.0159). Additionally, semi-quantitative image analysis showed lower inflammatory cell count in experimental group compared to control group (p value = 0.0317). In vivo electroporation-mediated IL-10 gene transfer reduced necrosis, enhanced survival and vascularity in the ischemic skin flap.

  17. Substrate-mediated delivery of gene complex nanoparticles via polydopamine coating for enhancing competitiveness of endothelial cells.

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    Li, Bo-Chao; Chang, Hao; Ren, Ke-Feng; Ji, Jian

    2016-11-01

    Substrate-mediated delivery of functional plasmid DNA (pDNA) has been proven to be a promising strategy to promote competitiveness of endothelial cells (ECs) over smooth muscle cells (SMCs), which is beneficial to inducing fast endothelialization of implanted vascular devices. Thus, it is of great importance to develop universal approaches with simplicity and easiness to immobilize DNA complex nanoparticles on substrates. In this study, the bioinspired polydopamine (PDA) coating was employed in immobilization of DNA complex nanoparticles, which were composed of protamine (PrS) and plasmid DNA encoding with hepatocyte growth factor (HGF-pDNA) gene. We demonstrated that the DNA complex nanoparticles can be successfully immobilized onto the PDA surface. Consequently, the HGF expression of both ECs and SMCs were significantly improved when they cultured on the DNA complex nanoparticles-immobilized substrates. Furthermore, EC proliferation was specifically promoted due to bioactivity of HGF, leading to an enhancement of EC competitiveness over SMCs. Our findings demonstrated the substrate-mediated functional gene nanoparticle delivery through PDA coating as a simple and efficient approach. It may hold great potential in the field of interventional cardiovascular implants. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Carbon nanotube-mediated siRNA delivery for gene silencing in cancer cells

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    Hong, Tu; Guo, Honglian; Xu, Yaqiong

    2011-10-01

    Small interfering RNA (siRNA) is potentially a promising tool in influencing gene expression with a high degree of target specificity. However, its poor intracellular uptake, instability in vivo, and non-specific immune stimulations impeded its effect in clinical applications. In this study, carbon nanotubes (CNTs) functionalized with two types of phospholipid-polyethylene glycol (PEG) have shown capabilities to stabilize siRNA in cell culture medium during the transfection and efficiently deliver siRNA into neuroblastoma and breast cancer cells. Moreover, the intrinsic optical properties of CNTs have been investigated through absorption and fluorescence measurements. We have found that the directly-functionalized groups play an important role on the fluorescence imaging of functionalized CNTs. The unique fluorescence imaging and high delivery efficiency make CNTs a promising material to deliver drugs and evaluate the treatment effect simultaneously.

  19. Significant inhibition of Tembusu virus envelope and NS5 gene using an adenovirus-mediated short hairpin RNA delivery system.

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    Wang, Hongzhi; Feng, Qiang; Wei, Lei; Zhuo, Liling; Chen, Hao; Diao, Youxiang; Tang, Yi

    2017-10-01

    Tembusu virus (TMUV) is a mosquito-borne flavivirus, which was first isolated in the tropics during the 1970s. Recently, a disease characterized by ovarian haemorrhage and neurological symptoms was observed in ducks in China, which threatens poultry production. However, there is no suitable vaccination strategy or effective antiviral drugs to combat TMUV infections. Consequently, there is an urgent need to develop a new anti-TMUV therapy. In this study, we report an efficient short hairpin RNA (shRNA) delivery strategy for the inhibition of TMUV production using an adenovirus vector system. Using specifically designed shRNAs based on the E and NS5 protein genes of TMUV, the vector-expressed viral genes, TMUV RNA replication and infectious virus production were downregulated at different levels in Vero cells, where the shRNA (NS52) was highly effective in inhibiting TMUV. Using the human adenovirus type 5 shRNA delivery system, the recombinant adenovirus (rAd-NS52) inhibited TMUV multiplication with high efficiency. Furthermore, the significant dose-dependent inhibition of viral RNA copies induced by rAd-NS52 was found in TMUV-infected cells, which could last for at least 96h post infection. Our results indicated that the adenovirus-mediated delivery of shRNAs could play an active role in future TMUV antiviral therapeutics. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Adenovirus-mediated gene delivery to cells of the magnocellular hypothalamo-neurohypophyseal system

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    Vasquez, E. C.; Beltz, T. G.; Haskell, R. E.; Johnson, R. F.; Meyrelles, S. S.; Davidson, B. L.; Johnson, A. K.

    2001-01-01

    The objective of the present study was to define the optimum conditions for using replication-defective adenovirus (Ad) to transfer the gene for the green fluorescent protein (GFP) to the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei and cells of the neurohypophysis (NH). As indicated by characterizing cell survival over 15 days in culture and in electrophysiological whole cell patch-clamp studies, viral concentrations up to 2 x 10(7) pfu/coverslip did not affect viability of transfected PVN and NH cultured cells from preweanling rats. At 2 x 10(7) pfu, GFP gene expression was higher (40% of GFP-positive cells) and more sustained (up to 15 days). Using a stereotaxic approach in adult rats, we were able to directly transduce the PVN, SON, and NH and visualize gene expression in coronal brain slices and in the pituitary 4 days after injection of Ad. In animals receiving NH injections of Ad, the virus was retrogradely transported to PVN and SON neurons as indicated by the appearance of GFP-positive neurons in cultures of dissociated cells from those brain nuclei and by polymerase chain reaction and Western blot analyses of PVN and SON tissues. Adenoviral concentrations of up to 8 x 10(6) pfu injected into the NH did not affect cell viability and did not cause inflammatory responses. Adenoviral injection into the pituitary enabled the selective delivery of genes to the soma of magnocellular neurons. The experimental approaches described here provide potentially useful strategies for the treatment of disordered expression of the hormones vasopressin or oxytocin. Copyright 2000 Academic Press.

  1. Gene therapy-mediated delivery of targeted cytotoxins for glioma therapeutics.

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    Candolfi, Marianela; Xiong, Weidong; Yagiz, Kader; Liu, Chunyan; Muhammad, A K M G; Puntel, Mariana; Foulad, David; Zadmehr, Ali; Ahlzadeh, Gabrielle E; Kroeger, Kurt M; Tesarfreund, Matthew; Lee, Sharon; Debinski, Waldemar; Sareen, Dhruv; Svendsen, Clive N; Rodriguez, Ron; Lowenstein, Pedro R; Castro, Maria G

    2010-11-16

    Restricting the cytotoxicity of anticancer agents by targeting receptors exclusively expressed on tumor cells is critical when treating infiltrative brain tumors such as glioblastoma multiforme (GBM). GBMs express an IL-13 receptor (IL13Rα2) that differs from the physiological IL4R/IL13R receptor. We developed a regulatable adenoviral vector (Ad.mhIL-4.TRE.mhIL-13-PE) encoding a mutated human IL-13 fused to Pseudomonas exotoxin (mhIL-13-PE) that specifically binds to IL13Rα2 to provide sustained expression, effective anti-GBM cytotoxicity, and minimal neurotoxicity. The therapeutic Ad also encodes mutated human IL-4 that binds to the physiological IL4R/IL13R without interacting with IL13Rα2, thus inhibiting potential binding of mhIL-13-PE to normal brain cells. Using intracranial GBM xenografts and syngeneic mouse models, we tested the Ad.mhIL-4.TRE.mhIL-13-PE and two protein formulations, hIL-13-PE used in clinical trials (Cintredekin Besudotox) and a second-generation mhIL-13-PE. Cintredekin Besudotox doubled median survival without eliciting long-term survival and caused severe neurotoxicity; mhIL-13-PE led to ∼40% long-term survival, eliciting severe neurological toxicity at the high dose tested. In contrast, Ad-mediated delivery of mhIL-13-PE led to tumor regression and long-term survival in over 70% of the animals, without causing apparent neurotoxicity. Although Cintredekin Besudotox was originally developed to target GBM, when tested in a phase III trial it failed to achieve clinical endpoints and revealed neurotoxicity. Limitations of Cintredekin Besudotox include its short half-life, which demanded frequent or continued administration, and binding to IL4R/IL13R, present in normal brain cells. These shortcomings were overcome by our therapeutic Ad, thus representing a significant advance in the development of targeted therapeutics for GBM.

  2. AAV Vector-Mediated Gene Delivery to Substantia Nigra Dopamine Neurons: Implications for Gene Therapy and Disease Models

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    Katrina Albert

    2017-02-01

    Full Text Available Gene delivery using adeno-associated virus (AAV vectors is a widely used method to transduce neurons in the brain, especially due to its safety, efficacy, and long-lasting expression. In addition, by varying AAV serotype, promotor, and titer, it is possible to affect the cell specificity of expression or the expression levels of the protein of interest. Dopamine neurons in the substantia nigra projecting to the striatum, comprising the nigrostriatal pathway, are involved in movement control and degenerate in Parkinson′s disease. AAV-based gene targeting to the projection area of these neurons in the striatum has been studied extensively to induce the production of neurotrophic factors for disease-modifying therapies for Parkinson′s disease. Much less emphasis has been put on AAV-based gene therapy targeting dopamine neurons in substantia nigra. We will review the literature related to targeting striatum and/or substantia nigra dopamine neurons using AAVs in order to express neuroprotective and neurorestorative molecules, as well as produce animal disease models of Parkinson′s disease. We discuss difficulties in targeting substantia nigra dopamine neurons and their vulnerability to stress in general. Therefore, choosing a proper control for experimental work is not trivial. Since the axons along the nigrostriatal tract are the first to degenerate in Parkinson′s disease, the location to deliver the therapy must be carefully considered. We also review studies using AAV-a-synuclein (a-syn to target substantia nigra dopamine neurons to produce an α-syn overexpression disease model in rats. Though these studies are able to produce mild dopamine system degeneration in the striatum and substantia nigra and some behavioural effects, there are studies pointing to the toxicity of AAV-carrying green fluorescent protein (GFP, which is often used as a control. Therefore, we discuss the potential difficulties in overexpressing proteins in general in

  3. Increased 1-aminocyclopropane-1-carboxylate deaminase activity enhances Agrobacterium tumefaciens-mediated gene delivery into plant cells

    OpenAIRE

    Someya, Tatsuhiko; Nonaka, Satoko; Nakamura, Kouji; Ezura, Hiroshi

    2013-01-01

    Agrobacterium-mediated transformation is a useful tool for the genetic modification in plants, although its efficiency is low for several plant species. Agrobacterium-mediated transformation has three major steps in laboratory-controlled experiments: the delivery of T-DNA into plant cells, the selection of transformed plant cells, and the regeneration of whole plants from the selected cells. Each of these steps must be optimized to improve the efficiency of Agrobacterium-mediated plant transf...

  4. Adenovirus-mediated gene delivery to hypothalamic magnocellular neurons in mice

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    Vasquez, E. C.; Beltz, T. G.; Meyrelles, S. S.; Johnson, A. K.

    1999-01-01

    Vasopressin is synthesized by magnocellular neurons in supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei and released by their axon terminals in the neurohypophysis (NH). With its actions as an antidiuretic hormone and vasoactive agent, vasopressin plays a pivotal role in the control of body fluids and cardiovascular homeostasis. Because of its well-defined neurobiology and functional importance, the SON/PVN-NH system is ideal to establish methods for gene transfer of genetic material into specific pathways in the mouse central nervous system. In these studies, we compared the efficiency of transferring the gene lacZ, encoding for beta-galactosidase (beta-gal), versus a gene encoding for green fluorescent protein by using replication-deficient adenovirus (Ad) vectors in adult mice. Transfection with viral concentrations up to 2 x 10(7) plaque-forming units per coverslip of NH, PVN, and SON in dissociated, cultured cells caused efficient transfection without cytotoxicity. However, over an extended period of time, higher levels (50% to 75% of the cells) of beta-gal expression were detected in comparison with green fluorescent protein (5% to 50% of the cells). With the use of a stereotaxic approach, the pituitary glands of mice were injected with Ad (4 x 10(6) plaque-forming units). In material from these animals, we were able to visualize the expression of the beta-gal gene in the NH and in magnocellular neurons of both the PVN and SON. The results of these experiments indicate that Ad-Rous sarcoma virus promoter-beta-gal is taken up by nerve terminals at the injection site (NH) and retrogradely transported to the soma of the neurons projecting to the NH. We conclude that the application of these experimental approaches will provide powerful tools for physiological studies and potential approaches to deliver therapeutic genes to treat diseases.

  5. Surface-mediated functional gene delivery: an effective strategy for enhancing competitiveness of endothelial cells over smooth muscle cells.

    Science.gov (United States)

    Chang, Hao; Ren, Ke-feng; Wang, Jin-Lei; Zhang, He; Wang, Bai-liang; Zheng, Shan-mei; Zhou, Yuan-yuan; Ji, Jian

    2013-04-01

    The non-biorecognition of general biomaterials and inherent biospecificity of biological systems pose key challenges to the optimal functions of medical devices. In this study, we constructed the surface-mediated functional gene delivery through layer-by-layer self-assembly of protamine sulfate (PrS) and plasmid DNA encoding hepatocyte growth factor (HGF), aiming at specific enhancing endothelial cells (EC) compeititiveness over smooth muscle cells (SMC). Characterizations of the (PrS/HGF-pDNA) multilayered films present the linear buildup with homogeneous and flat topographical feature. The amount of DNA can be easily controlled. By using these multilayered films, both human umbilical vein endothelial cells (HUVEC) and human umbilical artery smooth muscle cells (HUASMC) can be directly transfected when they contact with the multilayered films. On transfection, increasing secretion of HGF has been detected in both HUVEC and HUASMC culture, which leads to selective promotion of HUVEC proliferation. In the co-culture experiment, we also exhibit the promoted and hindered growth of HUVEC and HUASMC, respectively, which could be attributed to the inverse influence of HUVEC on HUASMC. These results collectively demonstrate that our system can be served as a powerful tool for enhancing competitiveness of EC over SMC, which opens perspectives for the regulation of intercellular competitiveness in the field of interventional therapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Reversing Bacterial Resistance to Antibiotics by Phage-Mediated Delivery of Dominant Sensitive Genes

    Science.gov (United States)

    Edgar, Rotem; Friedman, Nir; Molshanski-Mor, Shahar

    2012-01-01

    Pathogen resistance to antibiotics is a rapidly growing problem, leading to an urgent need for novel antimicrobial agents. Unfortunately, development of new antibiotics faces numerous obstacles, and a method that resensitizes pathogens to approved antibiotics therefore holds key advantages. We present a proof of principle for a system that restores antibiotic efficiency by reversing pathogen resistance. This system uses temperate phages to introduce, by lysogenization, the genes rpsL and gyrA conferring sensitivity in a dominant fashion to two antibiotics, streptomycin and nalidixic acid, respectively. Unique selective pressure is generated to enrich for bacteria that harbor the phages carrying the sensitizing constructs. This selection pressure is based on a toxic compound, tellurite, and therefore does not forfeit any antibiotic for the sensitization procedure. We further demonstrate a possible way of reducing undesirable recombination events by synthesizing dominant sensitive genes with major barriers to homologous recombination. Such synthesis does not significantly reduce the gene's sensitization ability. Unlike conventional bacteriophage therapy, the system does not rely on the phage's ability to kill pathogens in the infected host, but instead, on its ability to deliver genetic constructs into the bacteria and thus render them sensitive to antibiotics prior to host infection. We believe that transfer of the sensitizing cassette by the constructed phage will significantly enrich for antibiotic-treatable pathogens on hospital surfaces. Broad usage of the proposed system, in contrast to antibiotics and phage therapy, will potentially change the nature of nosocomial infections toward being more susceptible to antibiotics rather than more resistant. PMID:22113912

  7. Optimization of transfection parameters for ultrasound/SonoVue microbubble-mediated hAng-1 gene delivery in vitro.

    Science.gov (United States)

    Zhou, Qing; Chen, Jin-Ling; Chen, Qian; Wang, Xiao; Deng, Qing; Hu, Bo; Guo, Rui-Qiang

    2012-12-01

    This study aimed to explore the effects of microbubble concentration, gene dosage, cell-microbubble mixing mode and fetal bovine serum (FBS) on gene delivery. 293T cells were transfected with Sonovue microbubbles carrying the hAng-1 gene via ultrasound irradiation. Various ultrasound exposure parameters and microbubble and DNA concentrations were investigated. In addition, FBS and the cell suspension or adherent mode was explored. Transfection efficiency and cell viability were used to determine the optimal transfection parameters. hAng-1 gene transfection efficiency gradually increased with elongation of ultrasound exposure and increasing microbubble concentration. However, if ultrasound irradiation exceeded 1.5 W/cm² and 30 sec or the microbubble concentration was over 20%, hAng-1 gene expression was significantly decreased, coupled with extensive cell death. Gene transfection levels were low under DNA concentrations less than 15 µg/ml. Furthermore, the gene transfer rate was significantly increased under cell suspension mode; FBS had no effect on hAng-1 gene transfection. The integrity of hAng-1 DNA was not affected by ultrasonic irradiation under optimal conditions. The optimal transfection parameters for the hAng-1 gene and Sonovue microbubble were ultrasound exposure of 1.5 W/cm² and 30 sec, 20% microbubbles, 15 µg/ml of DNA and under cell suspension mode.

  8. Ab-externo AAV-mediated gene delivery to the suprachoroidal space using a 250 micron flexible microcatheter.

    Directory of Open Access Journals (Sweden)

    Marc C Peden

    2011-02-01

    Full Text Available The current method of delivering gene replacement to the posterior segment of the eye involves a three-port pars plana vitrectomy followed by injection of the agent through a 37-gauge cannula, which is potentially wrought with retinal complications. In this paper we investigate the safety and efficacy of delivering adeno-associated viral (AAV vector to the suprachoroidal space using an ab externo approach that utilizes an illuminated microcatheter.6 New Zealand White rabbits and 2 Dutch Belted rabbits were used to evaluate the ab externo delivery method. sc-AAV5-smCBA-hGFP vector was delivered into the suprachoroidal space using an illuminated iTrackTM 250A microcatheter. Six weeks after surgery, the rabbits were sacrificed and their eyes evaluated for AAV transfection using immunofluorescent antibody staining of GFP.Immunostaining of sectioned and whole-mounted eyes demonstrated robust transfection in all treated eyes, with no fluorescence in untreated control eyes. Transfection occurred diffusely and involved both the choroid and the retina. No apparent adverse effects caused by either the viral vector or the procedure itself could be seen either clinically or histologically.The ab externo method of delivery using a microcatheter was successful in safely and effectively delivering a gene therapy agent to the suprachoroidal space. This method presents a less invasive alternative to the current method of virally vectored gene delivery.

  9. Ultrasound mediated transdermal drug delivery.

    Science.gov (United States)

    Azagury, Aharon; Khoury, Luai; Enden, Giora; Kost, Joseph

    2014-06-01

    Transdermal drug delivery offers an attractive alternative to the conventional drug delivery methods of oral administration and injections. However, the stratum corneum serves as a barrier that limits the penetration of substances to the skin. Application of ultrasound (US) irradiation to the skin increases its permeability (sonophoresis) and enables the delivery of various substances into and through the skin. This review presents the main findings in the field of sonophoresis in transdermal drug delivery as well as transdermal monitoring and the mathematical models associated with this field. Particular attention is paid to the proposed enhancement mechanisms and future trends in the fields of cutaneous vaccination and gene therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Transcription activator-like effector-mediated regulation of gene expression based on the inducible packaging and delivery via designed extracellular vesicles.

    Science.gov (United States)

    Lainšček, Duško; Lebar, Tina; Jerala, Roman

    2017-02-26

    Transcription activator-like effector (TALE) proteins present a powerful tool for genome editing and engineering, enabling introduction of site-specific mutations, gene knockouts or regulation of the transcription levels of selected genes. TALE nucleases or TALE-based transcription regulators are introduced into mammalian cells mainly via delivery of the coding genes. Here we report an extracellular vesicle-mediated delivery of TALE transcription regulators and their ability to upregulate the reporter gene in target cells. Designed transcriptional activator TALE-VP16 fused to the appropriate dimerization domain was enriched as a cargo protein within extracellular vesicles produced by mammalian HEK293 cells stimulated by Ca-ionophore and using blue light- or rapamycin-inducible dimerization systems. Blue light illumination or rapamycin increased the amount of the TALE-VP16 activator in extracellular vesicles and their addition to the target cells resulted in an increased expression of the reporter gene upon addition of extracellular vesicles to the target cells. This technology therefore represents an efficient delivery for the TALE-based transcriptional regulators. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. DNA vaccination for cervical cancer: Strategic optimisation of RALA mediated gene delivery from a biodegradable microneedle system.

    Science.gov (United States)

    Cole, Grace; Ali, Ahlam A; McCrudden, Cian M; McBride, John W; McCaffrey, Joanne; Robson, Tracy; Kett, Vicky L; Dunne, Nicholas J; Donnelly, Ryan F; McCarthy, Helen O

    2018-03-03

    Dissolvable microneedles can be employed to deliver DNA to antigen presenting cells within the skin. However, this technology faces two main challenges: the poor transfection efficacy of pDNA following release from the microneedle matrix, and the limited loading capacity of the micron-scale devices. Two-tier delivery systems combining microneedle platforms and DNA delivery vectors have increased efficacy but the challenge of increasing the loading capacity remains. This study utilised lyophilisation to increase the loading of RALA/pDNA nanoparticles within dissolvable PVA microneedles. As a result, delivery was significantly enhanced in vivo into an appropriate range for DNA vaccination (∼50 μg per array). Furthermore, modifying the manufacturing process was not detrimental to the microneedle mechanical properties or cargo functionality. It was demonstrated that arrays retained mechanical and functional stability over short term storage, and were able to elicit gene expression in vitro and in vivo. Finally, treatment with this novel formulation significantly retarded the growth of established tumours, and proved superior to standard intramuscular injection in a preclinical model of cervical cancer. Copyright © 2018. Published by Elsevier B.V.

  12. Synthetic sustained gene delivery systems.

    Science.gov (United States)

    Agarwal, Ankit; Mallapragada, Surya K

    2008-01-01

    Gene therapy today is hampered by the need of a safe and efficient gene delivery system that can provide a sustained therapeutic effect without cytotoxicity or unwanted immune responses. Bolus gene delivery in solution results in the loss of delivered factors via lymphatic system and may cause undesired effects by the escape of bioactive molecules to distant sites. Controlled gene delivery systems, acting as localized depot of genes, provide an extended sustained release of genes, giving prolonged maintenance of the therapeutic level of encoded proteins. They also limit the DNA degradation in the nuclease rich extra-cellular environment. While attempts have been made to adapt existing controlled drug delivery technologies, more novel approaches are being investigated for controlled gene delivery. DNA encapsulated in nano/micro spheres of polymers have been administered systemically/orally to be taken up by the targeted tissues and provide sustained release once internalized. Alternatively, DNA entrapped in hydrogels or scaffolds have been injected/implanted in tissues/cavities as platforms for gene delivery. The present review examines these different modalities for sustained delivery of viral and non-viral gene-delivery vectors. Design parameters and release mechanisms of different systems made with synthetic or natural polymers are presented along with their prospective applications and opportunities for continuous development.

  13. Adenovirus-mediated gene delivery: Potential applications for gene and cell-based therapies in the new era of personalized medicine

    Directory of Open Access Journals (Sweden)

    Cody S. Lee

    2017-06-01

    Full Text Available With rapid advances in understanding molecular pathogenesis of human diseases in the era of genome sciences and systems biology, it is anticipated that increasing numbers of therapeutic genes or targets will become available for targeted therapies. Despite numerous setbacks, efficacious gene and/or cell-based therapies still hold the great promise to revolutionize the clinical management of human diseases. It is wildly recognized that poor gene delivery is the limiting factor for most in vivo gene therapies. There has been a long-lasting interest in using viral vectors, especially adenoviral vectors, to deliver therapeutic genes for the past two decades. Among all currently available viral vectors, adenovirus is the most efficient gene delivery system in a broad range of cell and tissue types. The applications of adenoviral vectors in gene delivery have greatly increased in number and efficiency since their initial development. In fact, among over 2000 gene therapy clinical trials approved worldwide since 1989, a significant portion of the trials have utilized adenoviral vectors. This review aims to provide a comprehensive overview on the characteristics of adenoviral vectors, including adenoviral biology, approaches to engineering adenoviral vectors, and their applications in clinical and preclinical studies with an emphasis in the areas of cancer treatment, vaccination and regenerative medicine. Current challenges and future directions regarding the use of adenoviral vectors are also discussed. It is expected that the continued improvements in adenoviral vectors should provide great opportunities for cell and gene therapies to live up to its enormous potential in personalized medicine.

  14. Recombinant fusion proteins TAT-Mu, Mu and Mu-Mu mediate efficient non-viral gene delivery.

    Science.gov (United States)

    Rajagopalan, Rukkumani; Xavier, Jennifer; Rangaraj, Nandini; Rao, Nalam Madhusudhana; Gopal, Vijaya

    2007-04-01

    The inherent ability of certain peptides or proteins of viral, prokaryotic and eukaryotic origin to bind DNA was used to generate novel peptide-based DNA delivery protocols. We have developed a recombinant approach to make fusion proteins with motifs for DNA-binding ability, Mu and membrane transduction domains, TAT, and tested them for their DNA-binding, uptake and transfection efficiencies. In one of the constructs, the recombinant plasmid was designed to encode the Mu moiety of sequence MRRAHHRRRRASHRRMRGG in-frame with TAT of sequence YGRKKRRQRRR to generate TAT-Mu, while the other two constructs, Mu and Mu-Mu, harbor a single copy or two copies of the Mu moiety. Recombinant his-tag fusion proteins TAT-Mu, Mu and Mu-Mu were purified by overexpression of plasmid constructs using cobalt-based affinity resins. The peptides were characterized for their size and interaction with DNA, complexed with plasmid pCMVbeta-gal, and shown to transfect MCF-7, COS and CHOK-1 cells efficiently. Recombinant fusion proteins TAT-Mu, Mu and Mu-Mu were cloned and overexpressed in BL21(DE3)pLysS with greater than 95% purity. The molecular weight of TAT-Mu was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to be 11.34 kDa while those of Mu and Mu-Mu were 7.78 and 9.83 kDa, respectively. Live uptake analysis of TAT-Mu, Mu and Mu-Mu as DP (DNA+peptide) or DPL (DNA+peptide+lipid) complexes into MCF-7 cells, followed by immunostaining and laser scanning confocal microscopy, demonstrated that the complexes are internalized very efficiently and localized in the nucleus. DNA:peptide complexes (DP) transfect MCF-7, COS and CHOK-1 cells. The addition of cationic liposomes enhances the uptake of the ternary complexes (DPL) further and also brings about 3-7-fold enhancement in reporter gene expression compared to DP alone. Recombinant proteins that are heterologous fusions, having DNA-binding domains and nuclear localization epitopes

  15. Adeno-associated virus vector-mediated IL-10 gene delivery prevents type 1 diabetes in NOD mice

    Science.gov (United States)

    Goudy, Kevin; Song, Sihong; Wasserfall, Clive; Zhang, Y. Clare; Kapturczak, Matthias; Muir, Andrew; Powers, Matthew; Scott-Jorgensen, Marda; Campbell-Thompson, Martha; Crawford, James M.; Ellis, Tamir M.; Flotte, Terence R.; Atkinson, Mark A.

    2001-01-01

    The development of spontaneous autoimmune diabetes in nonobese diabetic (NOD) mice provides for their use as a model of human type 1 diabetes. To test the feasibility of muscle-directed gene therapy to prevent type 1 diabetes, we developed recombinant adeno-associated virus (rAAV) vectors containing murine cDNAs for immunomodulatory cytokines IL-4 or IL-10. Skeletal muscle transduction of female NOD mice with IL-10, but not IL-4, completely abrogated diabetes. rAAV-IL-10 transduction attenuated the production of insulin autoantibodies, quantitatively reduced pancreatic insulitis, maintained islet insulin content, and altered splenocyte cytokine responses to mitogenic stimulation. The beneficial effects were host specific, as adoptive transfer of splenocytes from rAAV IL-10-treated animals rapidly imparted diabetes in naive hosts, and the cells contained no protective immunomodulatory capacity, as defined through adoptive cotransfer analyses. These results indicate the utility for rAAV, a vector with advantages for therapeutic gene delivery, to transfer immunoregulatory cytokines capable of preventing type 1 diabetes. In addition, these studies provide foundational support for the concept of using immunoregulatory agents delivered by rAAV to modulate a variety of disorders associated with deleterious immune responses, including allergic reactions, transplantation rejection, immunodeficiencies, and autoimmune disorders. PMID:11717448

  16. Nanoparticle mediated PPARγ gene delivery on dental implants improves osseointegration via mitochondrial biogenesis in diabetes mellitus rat model.

    Science.gov (United States)

    Lee, Young-Hee; Kim, Jeong-Seok; Kim, Ji-Eun; Lee, Min-Ho; Jeon, Jae-Gyu; Park, Il-Song; Yi, Ho-Keun

    2017-07-01

    Diabetes mellitus (DM) has a detrimental effect on osseointegration, stability and longevity of implants due to osteoporosis. In this study, PPARγ-loaded dental implants were investigated for the improvement of osseointegration and peri-implantitis. Chitosan gold nanoparticles conjugated with PPARγ cDNA were introduced on titanium mini-implant surfaces for PPARγ release to rat mandibular. DM-induced rat mandible showed structural changes such as decreased bone mass and increased inflammatory molecules, and diminution of PPARγ expression and bone formation molecules compared to normal rats. PPARγ induced bone formation via reduction of inflammatory molecules even under glucose oxidative stress. Furthermore, PPARγ strongly activated mitochondrial biogenesis and cell viability via p-AMK and Wnt/β-catenin signaling. Consequently, PPARγ gene delivery on regional dental implants contributed osseointegration, new bone formation and mineralization in DM-induced rats. This study demonstrates that PPARγ can be used as a therapeutic gene with dental implantation in diabetic patients since regional PPARγ expression enhances osseointegration and implant longevity. Copyright © 2017. Published by Elsevier Inc.

  17. DNA vaccination for cervical cancer; a novel technology platform of RALA mediated gene delivery via polymeric microneedles.

    Science.gov (United States)

    Ali, Ahlam A; McCrudden, Cian M; McCaffrey, Joanne; McBride, John W; Cole, Grace; Dunne, Nicholas J; Robson, Tracy; Kissenpfennig, Adrien; Donnelly, Ryan F; McCarthy, Helen O

    2017-04-01

    HPV subtypes (16, 18) are associated with the development of cervical cancer, with oncoproteins E6 and E7 responsible for pathogenesis. The goal of this study was to evaluate our 'smart system' technology platform for DNA vaccination against cervical cancer. The vaccination platform brings together two main components; a peptide RALA which condenses DNA into cationic nanoparticles (NPs), and a polymeric polyvinylpyrrolidone (PVP) microneedle (MN) patch for cutaneous delivery of the loaded NPs. RALA condensed E6/E7 DNA into NPs not exceeding 100nm in diameter, and afforded the DNA protection from degradation in PVP. Sera from mice vaccinated with MN/RALA-E6/E7 were richer in E6/E7-specific IgGs, displayed a greater T-cell-mediated TC-1 cytotoxicity and contained more IFN-γ than sera from mice that received NPs intramuscularly. More importantly, MN/RALA-E6/E7 delayed TC-1 tumor initiation in a prophylactic model, and slowed tumor growth in a therapeutic model of vaccination, and was more potent than intramuscular vaccination. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Functionalized graphene oxide mediated adriamycin delivery and miR-21 gene silencing to overcome tumor multidrug resistance in vitro.

    Directory of Open Access Journals (Sweden)

    Feng Zhi

    Full Text Available Multidrug resistance (MDR is a major impediment to successful cancer chemotherapy. Co-delivery of novel MDR-reversing agents and anticancer drugs to cancer cells holds great promise for cancer treatment. MicroRNA-21 (miR-21 overexpression is associated with the development and progression of MDR in breast cancer, and it is emerging as a novel and promising MDR-reversing target. In this study, a multifunctional nanocomplex, composed of polyethylenimine (PEI/poly(sodium 4-styrenesulfonates (PSS/graphene oxide (GO and termed PPG, was prepared using the layer-by-layer assembly method to evaluate the reversal effects of PPG as a carrier for adriamycin (ADR along with miR-21 targeted siRNA (anti-miR-21 in cancer drug resistance. ADR was firstly loaded onto the PPG surface (PPGADR by physical mixing and anti-miR-21 was sequentially loaded onto PPGADR through electric absorption to form (anti-miR-21PPGADR. Cell experiments showed that PPG significantly enhanced the accumulation of ADR in MCF-7/ADR cells (an ADR resistant breast cancer cell line and exhibited much higher cytotoxicity than free ADR, suggesting that PPG could effectively reverse ADR resistance of MCF-7/ADR. Furthermore, the enhanced therapeutic efficacy of PPG could be correlated with effective silencing of miR-21 and with increased accumulation of ADR in drug-resistant tumor cells. The endocytosis study confirmed that PPG could effectively carry drug molecules into cells via the caveolae and clathrin-mediated endocytosis pathways. These results suggest that this PPG could be a potential and efficient non-viral vector for reversing MDR, and the strategy of combining anticancer drugs with miRNA therapy to overcome MDR could be an attractive approach in cancer treatment.

  19. Electroporation-mediated in vivo gene delivery of the Na+/K+-ATPase pump reduced lung injury in a mouse model of lung contusion.

    Science.gov (United States)

    Machado-Aranda, David A; Suresh, M V; Yu, Bi; Raghavendran, Krishnan

    2012-01-01

    Lung contusion (LC) is an independent risk factor for acute respiratory distress syndrome. The final common pathway in ARDS involves accumulation of fluid in the alveoli. In this study, we demonstrate the application of a potential gene therapy approach by delivering the Na+/K+-ATPase pump subunits in a murine model of LC. We hypothesized that restoring the activity of the pump will result in removal of excess alveolar fluid and additionally reduce inflammation. Under anesthesia, C57/BL6 mice were struck along the right posterior axillary line 1 cm above the costal margin with a cortical contusion impactor. Immediately afterward, 100 μg of plasmid DNA coding for the α,β of the Na+/K+-ATPase pump were instilled into the lungs (LC-electroporation-pump group). Contusion only (LC-only) and a sham saline instillation group after contusion were used as controls (LC-electroporation-sham). By using a BTX 830 electroporator, eight electrical pulses of 200 V/cm field strength were applied transthoracically. Mice were killed at 24 hours, 48 hours, and 72 hours after delivery. Bronchial alveolar lavage was recollected to measure albumin and cytokines by enzyme-linked immunosorbent assay. Pulmonary compliance was measured, and lungs were subject to histopathologic analysis. After the electroporation and delivery of genes coding for the α,β subunits of the Na+/K+-ATPase pump, there was a significant mitigation of acute lung injury as evidenced by reduction in bronchial alveolar lavage levels of albumin, improved pressure volume curves, and reduced inflammation seen on histology. Electroporation-mediated gene transfer of the subunits of the Na+/K+-ATPase pump enhanced recovery from acute inflammatory lung injury after LC.

  20. Freeze-dried allograft-mediated gene or protein delivery of growth and differentiation factor 5 reduces reconstructed murine flexor tendon adhesions

    DEFF Research Database (Denmark)

    Svensson, Sys Hasslund; Dadali, Tulin; Ulrich-Vinther, Michael

    2014-01-01

    Advances in allograft processing have opened new horizons for clinical adaptation of flexor tendon allografts as delivery scaffolds for antifibrotic therapeutics. Recombinant adeno-associated-virus (rAAV) gene delivery of the growth and differentiation factor 5 (GDF-5) has been previously...

  1. Ultrasound mediated nanoparticle drug delivery

    Science.gov (United States)

    Mullin, Lee B.

    Ultrasound is not only a powerful diagnostic tool, but also a promising therapeutic technology that can be used to improve localized drug delivery. Microbubble contrast agents are micron sized encapsulated gas filled bubbles that are administered intravenously. Originally developed to enhance ultrasound images, microbubbles are highly echogenic due to the gas core that provides a detectable impedance difference from the surrounding medium. The core also allows for controlled response of the microbubbles to ultrasound pulses. Microbubbles can be pushed using acoustic radiation force and ruptured using high pressures. Destruction of microbubbles can increase permeability at the cellular and vascular level, which can be advantageous for drug delivery. Advances in drug delivery methods have been seen with the introduction of nanoparticles, nanometer sized objects often carrying a drug payload. In chemotherapy, nanoparticles can deliver drugs to tumors while limiting systemic exposure due to abnormalities in tumor vasculature such large gaps between endothelial cells that allow nanoparticles to enter into the interstitial space; this is referred to as the enhanced permeability and retention (EPR) effect. However, this effect may be overestimated in many tumors. Additionally, only a small percentage of the injected dose accumulates in the tumor, which most the nanoparticles accumulating in the liver and spleen. It is hypothesized that combining the acoustic activity of an ultrasound contrast agent with the high payload and extravasation ability of a nanoparticle, localized delivery to the tumor with reduced systemic toxicity can be achieved. This method can be accomplished by either loading nanoparticles onto the shell of the microbubble or through a coadministration method of both nanoparticles and microbubbles. The work presented in this dissertation utilizes novel and commercial nanoparticle formulations, combined with microbubbles and a variety of ultrasound systems

  2. Novel Polymeric Nanoparticles for Pulmonary Gene Delivery

    Science.gov (United States)

    Fields, Rachel Jennifer

    NPs to deliver difficult to gene therapy reagents, such as PNAs, to cells within the lung, I loaded NPs with PNA and DNA and administered them via the intranasal route. Triplex forming peptide nucleic acids (PNAs) are gene therapy reagents that can mediate site-specific homologous recombination with genomic DNA when successfully delivered to the nucleus of cells in combination with donor DNA oligos. Delivery of NPs resulted in EGFP expression in transgenic mice with an aberrant EGFP gene that could be corrected and effectively expressed with nuclear delivery of a PNA/DNA. This work represents the first successful use of PNA/DNA mediated homologous recombination to target cells of the lung.

  3. Mechanistic evaluation of the transfection barriers involved in lipid-mediated gene delivery: Interplay between nanostructure and composition

    Science.gov (United States)

    Pozzi, D.; Marchini, C.; Cardarelli, F.; Salomone, F.; Coppola, S.; Montani, M.; Zabaleta, M. Elexpuru; Digman, M.A.; Gratton, E.; Colapicchioni, V.; Caracciolo, G.

    2014-01-01

    Here we present a quantitative mechanism-based investigation aimed at comparing the cell uptake, intracellular trafficking, endosomal escape and final fate of lipoplexes and lipid–protamine/deoxyribonucleic acid (DNA) (LPD) nanoparticles (NPs) in living Chinese hamster ovary (CHO) cells. As a model, two lipid formulations were used for comparison. The first formulation is made of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic lipid dioleoylphosphocholine (DOPC), while the second mixture is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine (DOPE). Our findings indicate that lipoplexes are efficiently taken up through fluid-phase macropinocytosis, while a less efficient uptake of LPD NPs occurs through a combination of both macropinocytosis and clathrin-dependent pathways. Inside the cell, both lipoplexes and LPD NPs are actively transported towards the cell nucleus, as quantitatively addressed by spatio-temporal image correlation spectroscopy (STICS). For each lipid formulation, LPD NPs escape from endosomes more efficiently than lipoplexes. When cells were treated with DOTAP–DOPC-containing systems the majority of the DNA was trapped in the lysosome compartment, suggesting that extensive lysosomal degradation was the rate-limiting factors in DOTAP–DOPC-mediated transfection. On the other side, escape from endosomes is large for DC-Chol–DOPE-containing systems most likely due to DOPE and cholesterol-like molecules, which are able to destabilize the endosomal membrane. The lipid-dependent and structure-dependent enhancement of transfection activity suggests that DNA is delivered to the nucleus synergistically: the process requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability. PMID:24296066

  4. Long-Term PEDF Release in Rat Iris and Retinal Epithelial Cells after Sleeping Beauty Transposon-Mediated Gene Delivery

    Directory of Open Access Journals (Sweden)

    Laura Garcia-Garcia

    2017-12-01

    Full Text Available Pigment epithelium derived factor (PEDF is a potent antiangiogenic, neurotrophic, and neuroprotective molecule that is the endogenous inhibitor of vascular endothelial growth factor (VEGF in the retina. An ex vivo gene therapy approach based on transgenic overexpression of PEDF in the eye is assumed to rebalance the angiogenic-antiangiogenic milieu of the retina, resulting in growth regression of choroidal blood vessels, the hallmark of neovascular age-related macular degeneration. Here, we show that rat pigment epithelial cells can be efficiently transfected with the PEDF-expressing non-viral hyperactive Sleeping Beauty transposon system delivered in a form free of antibiotic resistance marker miniplasmids. The engineered retinal and iris pigment epithelium cells secrete high (141 ± 13 and 222 ± 14 ng PEDF levels in 72 hr in vitro. In vivo studies showed cell survival and insert expression during at least 4 months. Transplantation of the engineered cells to the subretinal space of a rat model of choroidal neovascularization reduces almost 50% of the development of new vessels.

  5. [Adenovirus-mediated delivery of nm23-H1 gene inhibits growth of colorectal carcinoma cell line Lovo].

    Science.gov (United States)

    Wang, Qi; He, Xueling; Liu, Yan; Yin, Hailin

    2010-12-01

    This experimental study sought to find out the inhibitory effects of Ad-GFP-nm23-H1 on proliferation and metastasis of human colorectal carcinoma cell line Lovo, and, further, to gain an insight into some theoretical and methodical basis for instituting nm23-H1 gene therapy of cancers. MTT assay and Transwell chamber were used to detect the rates of proliferation and invasion as well as the adhesion of Lovo cells in vitro. The results demonstrated that the proliferation inhibition rates of Lovo cells treated with Ad-GFP-nm23-H1 of 10(10) PFU/ml, 10(9) PFU/ml and 10(8) PFU/ml were 84.9% +/- 1.51%, 48.5% +/- 7.23% and 22.5% +/- 5.47%, that the adherence inhibition rates of Lovo cells treated with Ad-GFP-nm23-H1 of 10(10) PFU/ml, 10(9) PFU/ml and 10(8) PFU/ml were 70.3% +/- 2.40%, 60.1% +/- 5.68% and 18.5% +/- 3.61%, and that the invasiveness inhibition rates of Lovo cells treated with Ad-GFP-nm23-H1 of 10(10) PFU/ml, 10(9) PFU/ml and 10(8) PFU/ml were 83.2% +/- 5.71%, 52.2% +/- 6.94% and 28.1% +/- 8.21%. These data suggested that Ad-GFP-nm23-H1 exerted significant inhibitory effects on the proliferation and metastasis of human colorectal carcinoma cell line Lovo in a dose-dependent way.

  6. Cationic Bolaamphiphiles for Gene Delivery

    Science.gov (United States)

    Tan, Amelia Li Min; Lim, Alisa Xue Ling; Zhu, Yiting; Yang, Yi Yan; Khan, Majad

    2014-05-01

    Advances in medical research have shed light on the genetic cause of many human diseases. Gene therapy is a promising approach which can be used to deliver therapeutic genes to treat genetic diseases at its most fundamental level. In general, nonviral vectors are preferred due to reduced risk of immune response, but they are also commonly associated with low transfection efficiency and high cytotoxicity. In contrast to viral vectors, nonviral vectors do not have a natural mechanism to overcome extra- and intracellular barriers when delivering the therapeutic gene into cell. Hence, its design has been increasingly complex to meet challenges faced in targeting of, penetration of and expression in a specific host cell in achieving more satisfactory transfection efficiency. Flexibility in design of the vector is desirable, to enable a careful and controlled manipulation of its properties and functions. This can be met by the use of bolaamphiphile, a special class of lipid. Unlike conventional lipids, bolaamphiphiles can form asymmetric complexes with the therapeutic gene. The advantage of having an asymmetric complex lies in the different purposes served by the interior and exterior of the complex. More effective gene encapsulation within the interior of the complex can be achieved without triggering greater aggregation of serum proteins with the exterior, potentially overcoming one of the great hurdles faced by conventional single-head cationic lipids. In this review, we will look into the physiochemical considerations as well as the biological aspects of a bolaamphiphile-based gene delivery system.

  7. Efficient Gene Delivery Mediated by a Helical Polypeptide: Controlling the Membrane Activity via Multivalency and Light-Assisted Photochemical Internalization (PCI).

    Science.gov (United States)

    Xu, Xin; Li, Yongjuan; Liang, Qiujun; Song, Ziyuan; Li, Fangfang; He, Hua; Wang, Jinhui; Zhu, Lipeng; Lin, Zhifeng; Yin, Lichen

    2018-01-10

    The development of robust and nontoxic membrane-penetrating materials is highly demanded for nonviral gene delivery. Herein, a photosensitizer (PS)-embedded, star-shaped helical polypeptide was developed, which combines the advantages of multivalency-enhanced intracellular DNA uptake and light-strengthened endosomal escape to enable highly efficient gene delivery with low toxicity. 5,10,15,20-Tetrakis-(4-aminophenyl) porphyrin as a selected PS initiated ring-opening polymerization of N-carboxyanhydride and yielded a star-shaped helical polypeptide after side-chain functionalization with guanidine groups. The star polypeptide afforded a notably higher transfection efficiency and lower cytotoxicity than those of its linear analogue. Light irradiation caused almost complete (∼90%) endosomal release of the DNA cargo via the photochemical internalization (PCI) mechanism and further led to a 6-8-fold increment of the transfection efficiency in HeLa, B16F10, and RAW 264.7 cells, outperforming commercial reagent 25k PEI by up to 3 orders of magnitude. Because the PS and DNA cargoes were compartmentalized distantly in the core and polypeptide layers, respectively, the generated reactive oxygen species caused minimal damage to DNA molecules to preserve their transfection potency. Such multivalency- and PCI-potentiated gene delivery efficiency was also demonstrated in vivo in melanoma-bearing mice. This study thus provides a promising strategy to overcome the multiple membrane barriers against nonviral gene delivery.

  8. Genetic nanomedicine: gene delivery by targeted lipoplexes.

    Science.gov (United States)

    Düzgüneş, Nejat; de Ilarduya, Conchita Tros

    2012-01-01

    Cationic liposome-DNA complexes (lipoplexes) are used for the delivery of plasmid DNA to cultured cells and various tissues in vivo. In this chapter, we describe the preparation and evaluation of plain and targeted lipoplexes, using targeting ligands, including epidermal growth factor and transferrin. Ligand-associated lipoplexes may be used to target DNA or other nucleic acid drugs to specific cells, particularly cancer cells that overexpress the receptors for the ligands. We provide examples of the enhancement of gene expression mediated by epidermal growth factor in murine and human oral squamous cell carcinoma cells, and human hepatoblastoma and rat colon adenocarcinoma cells. We also summarize the studies on the use of transferrin-lipoplexes for enhancing gene delivery to cervical carcinoma, murine colon carcinoma, and African green monkey kidney cells. We outline two animal models in which transferrin-lipoplexes have been used for antitumor therapy by delivering either the gene encoding interleukin-12 or a suicide gene: a CT26 murine colon carcinoma, and a syngeneic, orthotopic murine oral squamous cell carcinoma. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. Production of transgenic cattle highly expressing human serum albumin in milk by phiC31 integrase-mediated gene delivery.

    Science.gov (United States)

    Luo, Yan; Wang, Yongsheng; Liu, Jun; Lan, Hui; Shao, Minghao; Yu, Yuan; Quan, Fusheng; Zhang, Yong

    2015-10-01

    Transgenic cattle expressing high levels of recombinant human serum albumin (HSA) in their milk may as an alternative source for commercial production. Our objective was to produce transgenic cattle highly expressing HSA in milk by using phiC31 integrase system and somatic cell nuclear transfer (SCNT). The mammary-specific expression plasmid pIACH(-), containing the attB recognition site for phiC31 integrase, were co-transfected with integrase expression plasmid pCMVInt into bovine fetal fibroblast cells (BFFs). PhiC31 integrase-mediated integrations in genome of BFFs were screened by nested inverse PCR. After analysis of sequence of the PCR products, 46.0% (23/50) of the both attB-genome junction sites (attL and attR) were confirmed, and four pseudo attP sites were identified. The integration rates in BF3, BF11, BF19 and BF4 sites were 4.0% (2/50), 6.0% (3/50), 16.0% (8/50) and 20.0% (10/50), respectively. BF3 is located in the bovine chromosome 3 collagen alpha-3 (VI) chain isomer 2 gene, while the other three sites are located in the non-coding region. The transgenic cell lines from BF11, BF19 and BF4 sites were used as donors for SCNT. Two calves from transgenic cells BF19 were born, one died within a few hours after birth, and another calf survived healthy. PCR and Southern blot analysis revealed integration of the transgene in the genome of cloned calves. The nested reverse PCR confirmed that the integration site in cloned calves was identical to the donor cells. The western blotting assessment indicated that recombinant HSA was expressed in the milk of transgenic cattle and the expression level was about 4-8 mg/mL. The present study demonstrated that phiC31 integrase system was an efficient and safety gene delivery tool for producing HSA transgenic cattle. The production of recombinant HSA in the milk of cattle may provide a large-scale and cost-effective resource.

  10. A new electrospray method for targeted gene delivery.

    Science.gov (United States)

    Boehringer, Stephan; Ruzgys, Paulius; Tamò, Luca; Šatkauskas, Saulius; Geiser, Thomas; Gazdhar, Amiq; Hradetzky, David

    2018-03-05

    A challenge for gene therapy is absence of safe and efficient local delivery of therapeutic genetic material. An efficient and reproducible physical method of electrospray for localized and targeted gene delivery is presented. Electrospray works on the principle of coulombs repulsion, under influence of electric field the liquid carrying genetic material is dispersed into micro droplets and is accelerated towards the targeted tissue, acting as a counter electrode. The accelerated droplets penetrate the targeted cells thus facilitating the transfer of genetic material into the cell. The work described here presents the principle of electrospray for gene delivery, the basic instrument design, and the various optimized parameters to enhance gene transfer in vitro. We estimate a transfection efficiency of up to 60% was achieved. We describe an efficient gene transfer method and a potential electrospray-mediated gene transfer mechanism.

  11. Postnatal Cardiac Gene Editing Using CRISPR/Cas9 With AAV9-Mediated Delivery of Short Guide RNAs Results in Mosaic Gene Disruption.

    Science.gov (United States)

    Johansen, Anne Katrine; Molenaar, Bas; Versteeg, Danielle; Leitoguinho, Ana Rita; Demkes, Charlotte; Spanjaard, Bastiaan; de Ruiter, Hesther; Akbari Moqadam, Farhad; Kooijman, Lieneke; Zentilin, Lorena; Giacca, Mauro; van Rooij, Eva

    2017-10-27

    CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9)-based DNA editing has rapidly evolved as an attractive tool to modify the genome. Although CRISPR/Cas9 has been extensively used to manipulate the germline in zygotes, its application in postnatal gene editing remains incompletely characterized. To evaluate the feasibility of CRISPR/Cas9-based cardiac genome editing in vivo in postnatal mice. We generated cardiomyocyte-specific Cas9 mice and demonstrated that Cas9 expression does not affect cardiac function or gene expression. As a proof-of-concept, we delivered short guide RNAs targeting 3 genes critical for cardiac physiology, Myh6 , Sav1 , and Tbx20 , using a cardiotropic adeno-associated viral vector 9. Despite a similar degree of DNA disruption and subsequent mRNA downregulation, only disruption of Myh6 was sufficient to induce a cardiac phenotype, irrespective of short guide RNA exposure or the level of Cas9 expression. DNA sequencing analysis revealed target-dependent mutations that were highly reproducible across mice resulting in differential rates of in- and out-of-frame mutations. Finally, we applied a dual short guide RNA approach to effectively delete an important coding region of Sav1 , which increased the editing efficiency. Our results indicate that the effect of postnatal CRISPR/Cas9-based cardiac gene editing using adeno-associated virus serotype 9 to deliver a single short guide RNA is target dependent. We demonstrate a mosaic pattern of gene disruption, which hinders the application of the technology to study gene function. Further studies are required to expand the versatility of CRISPR/Cas9 as a robust tool to study novel cardiac gene functions in vivo. © 2017 American Heart Association, Inc.

  12. Newborn Analgesia Mediated by Oxytocin during Delivery

    Science.gov (United States)

    Mazzuca, Michel; Minlebaev, Marat; Shakirzyanova, Anastasia; Tyzio, Roman; Taccola, Giuliano; Janackova, Sona; Gataullina, Svetlana; Ben-Ari, Yehezkel; Giniatullin, Rashid; Khazipov, Rustem

    2011-01-01

    The mechanisms controlling pain in newborns during delivery are poorly understood. We explored the hypothesis that oxytocin, an essential hormone for labor and a powerful neuromodulator, exerts analgesic actions on newborns during delivery. Using a thermal tail-flick assay, we report that pain sensitivity is two-fold lower in rat pups immediately after birth than 2 days later. Oxytocin receptor antagonists strongly enhanced pain sensitivity in newborn, but not in 2-day-old rats, whereas oxytocin reduced pain at both ages suggesting an endogenous analgesia by oxytocin during delivery. Similar analgesic effects of oxytocin, measured as attenuation of pain-vocalization induced by electrical whisker pad stimulation, were also observed in decerebrated newborns. Oxytocin reduced GABA-evoked calcium responses and depolarizing GABA driving force in isolated neonatal trigeminal neurons suggesting that oxytocin effects are mediated by alterations of intracellular chloride. Unlike GABA signaling, oxytocin did not affect responses mediated by P2X3 and TRPV1 receptors. In keeping with a GABAergic mechanism, reduction of intracellular chloride by the diuretic NKCC1 chloride co-transporter antagonist bumetanide mimicked the analgesic actions of oxytocin and its effects on GABA responses in nociceptive neurons. Therefore, endogenous oxytocin exerts an analgesic action in newborn pups that involves a reduction of the depolarizing action of GABA on nociceptive neurons. Therefore, the same hormone that triggers delivery also acts as a natural pain killer revealing a novel facet of the protective actions of oxytocin in the fetus at birth. PMID:21519396

  13. Albumin-mediated delivery of siRNA

    DEFF Research Database (Denmark)

    Bienk, Konrad

    2015-01-01

    in autoimmune disorders, neurodegenerative disorders and cancer by specific gene silencing. The effective delivery of the therapeutic to target cells and tissues is currently a great challenge for successful siRNA therapy. In the bloodstream, free siRNA is rapidly cleared by renal excretion or degraded by serum...... been used to create a range of albumin variants with FcRn affinity both lower and higher than that of wild-type albumin. The aim of this project is to develop a siRNA carrier system utilizing albumin as a natural delivery vehicle. Cholesteryl functionalized siRNA was designed to facilitate binding...... formulation with albumin. Furthermore, the experiments revealed a preferential accumulation of the 2- cholesteryl siRNA in the liver after 6 hours. The finding of accumulation in the liver was utilized in vivo to illustrate liver specific Factor VII silencing of ~30%, mediated only by albumin as a delivery...

  14. Sodium Iodide Symporter (NIS)-Mediated Radionuclide (131I, 188Re) Therapy of Liver Cancer After Transcriptionally Targeted Intratumoral in Vivo NIS Gene Delivery

    Science.gov (United States)

    Klutz, Kathrin; Willhauck, Michael J.; Wunderlich, Nathalie; Zach, Christian; Anton, Martina; Senekowitsch-Schmidtke, Reingard; Göke, Burkhard

    2011-01-01

    Abstract We reported the therapeutic efficacy of 131I in hepatocellular carcinoma (HCC) cells stably expressing the sodium iodide symporter (NIS) under the control of the tumor-specific α-fetoprotein (AFP) promoter. In the current study we investigated the efficacy of adenovirus-mediated in vivo NIS gene transfer followed by 131I and 188Re administration for the treatment of HCC xenografts. We used a replication-deficient adenovirus carrying the human NIS gene linked to the mouse AFP promoter (Ad5-AFP-NIS) for in vitro and in vivo NIS gene transfer. Functional NIS expression was confirmed by in vivo γ-camera imaging, followed by analysis of NIS protein and mRNA expression. Human HCC (HepG2) cells infected with Ad5-AFP-NIS concentrated 50% of the applied activity of 125I, which was sufficiently high for a therapeutic effect in an in vitro clonogenic assay. Four days after intratumoral injection of Ad5-AFP-NIS (3×109 plaque-forming units) HepG2 xenografts accumulated 14.5% injected dose (ID)/g 123I with an effective half-life of 13 hr (tumor-absorbed dose, 318 mGy/MBq 131I). In comparison, 9.2% ID/g 188Re was accumulated in tumors with an effective half-life of 12.8 hr (tumor-absorbed dose, 545 mGy/MBq). After adenovirus-mediated NIS gene transfer in HepG2 xenografts administration of a therapeutic dose of 131I or 188Re (55.5 MBq) resulted in a significant delay in tumor growth and improved survival without a significant difference between 188Re and 131I. In conclusion, a therapeutic effect of 131I and 188Re was demonstrated in HepG2 xenografts after tumor-specific adenovirus-mediated in vivo NIS gene transfer. PMID:21488714

  15. Ultrasound-mediated structural changes in cells revealed by FTIR spectroscopy: A contribution to the optimization of gene and drug delivery

    Science.gov (United States)

    Grimaldi, Paola; Di Giambattista, Lucia; Giordani, Serena; Udroiu, Ion; Pozzi, Deleana; Gaudenzi, Silvia; Bedini, Angelico; Giliberti, Claudia; Palomba, Raffaele; Congiu Castellano, Agostina

    2011-12-01

    Ultrasound effects on biological samples are gaining a growing interest concerning in particular, the intracellular delivery of drugs and genes in a safe and in a efficient way. Future progress in this field will require a better understanding of how ultrasound and acoustic cavitation affect the biological system properties. The morphological changes of cells due to ultrasound (US) exposure have been extensively studied, while little attention has been given to the cells structural changes. We have exposed two different cell lines to 1 MHz frequency ultrasound currently used in therapy, Jurkat T-lymphocytes and NIH-3T3 fibroblasts, both employed as models respectively in the apoptosis and in the gene therapy studies. The Fourier Transform Infrared (FTIR) Spectroscopy was used as probe to reveal the structural changes in particular molecular groups belonging to the main biological systems. The genotoxic damage of cells exposed to ultrasound was ascertained by the Cytokinesis-Block Micronucleus (CBMN) assay. The FTIR spectroscopy results, combined with multivariate statistical analysis, regarding all cellular components (lipids, proteins, nucleic acids) of the two cell lines, show that Jurkat cells are more sensitive to therapeutic ultrasound in the lipid and protein regions, whereas the NIH-3T3 cells are more sensitive in the nucleic acids region; a meaningful genotoxic effect is present in both cell lines only for long sonication times while in the Jurkat cells also a significant cytotoxic effect is revealed for long times of exposure to ultrasound.

  16. In vivo gene knockdown in rat dorsal root ganglia mediated by self-complementary adeno-associated virus serotype 5 following intrathecal delivery.

    Directory of Open Access Journals (Sweden)

    Qinghao Xu

    Full Text Available We report here in adult rat viral vector mediate-gene knockdown in the primary sensory neurons and the associated cellular and behavior consequences. Self-complementary adeno-associated virus serotype 5 (AAV5 was constructed to express green fluorescent protein (GFP and a small interfering RNA (siRNA targeting mammalian target of rapamycin (mTOR. The AAV vectors were injected via an intrathecal catheter. We observed profound GFP expression in lumbar DRG neurons beginning at 2-week post-injection. Of those neurons, over 85% were large to medium-diameter and co-labeled with NF200, a marker for myelinated fibers. Western blotting of mTOR revealed an 80% reduction in the lumbar DRGs (L4-L6 of rats treated with the active siRNA vectors compared to the control siRNA vector. Gene knockdown became apparent as early as 7-day post-injection and lasted for at least 5 weeks. Importantly, mTOR knockdown occurred in large (NF200 and small-diameter neurons (nociceptors. The viral administration induced an increase of Iba1 immunoreactivity in the DRGs, which was likely attributed to the expression of GFP but not siRNA. Rats with mTOR knockdown in DRG neurons showed normal general behavior and unaltered responses to noxious stimuli. In conclusion, intrathecal AAV5 is a highly efficient vehicle to deliver siRNA and generate gene knockdown in DRG neurons. This will be valuable for both basic research and clinic intervention of diseases involving primary sensory neurons.

  17. Gene therapy prospects--intranasal delivery of therapeutic genes.

    Science.gov (United States)

    Podolska, Karolina; Stachurska, Anna; Hajdukiewicz, Karolina; Małecki, Maciej

    2012-01-01

    Gene therapy is recognized to be a novel method for the treatment of various disorders. Gene therapy strategies involve gene manipulation on broad biological processes responsible for the spreading of diseases. Cancer, monogenic diseases, vascular and infectious diseases are the main targets of gene therapy. In order to obtain valuable experimental and clinical results, sufficient gene transfer methods are required. Therapeutic genes can be administered into target tissues via gene carriers commonly defined as vectors. The retroviral, adenoviral and adeno-associated virus based vectors are most frequently used in the clinic. So far, gene preparations may be administered directly into target organs or by intravenous, intramuscular, intratumor or intranasal injections. It is common knowledge that the number of gene therapy clinical trials has rapidly increased. However, some limitations such as transfection efficiency and stable and long-term gene expression are still not resolved. Consequently, great effort is focused on the evaluation of new strategies of gene delivery. There are many expectations associated with intranasal delivery of gene preparations for the treatment of diseases. Intranasal delivery of therapeutic genes is regarded as one of the most promising forms of pulmonary gene therapy research. Gene therapy based on inhalation of gene preparations offers an alternative way for the treatment of patients suffering from such lung diseases as cystic fibrosis, alpha-1-antitrypsin defect, or cancer. Experimental and first clinical trials based on plasmid vectors or recombinant viruses have revealed that gene preparations can effectively deliver therapeutic or marker genes to the cells of the respiratory tract. The noninvasive intranasal delivery of gene preparations or conventional drugs seems to be very encouraging, although basic scientific research still has to continue.

  18. Recombinant adeno-associated virus-mediated gene delivery of long chain acyl coenzyme A dehydrogenase (LCAD) into LCAD-deficient mice.

    Science.gov (United States)

    Beattie, Stuart G; Goetzman, Eric; Tang, Qiuishi; Conlon, Thomas; Campbell-Thompson, Martha; Matern, Dietrich; Vockley, Jerry; Flotte, Terence R

    2008-10-01

    Very long chain acyl coenzyme A (CoA) dehydrogenase (VLCAD) deficiency is a relatively common mitochondrial beta-oxidation disorder. The most severe form of VLCAD deficiency presents with neonatal cardiomyopathy and hepatic failure and is generally fatal within the first year of life. Mice deficient for long chain acyl CoA dehydrogenase (LCAD) closely resemble the clinical syndrome observed in VLCAD-deficient humans. Recombinant adeno-associated viral (rAAV) vectors with pseudotype capsids were investigated for their potential towards correcting the phenotype observed in mice heterozygous (+/-) for LCAD (i.e. liver and muscle steatosis). rAAV containing the mouse LCAD cDNA (mLCAD) under the transcriptional control of the CMV/chicken beta-actin hybrid promoter were injected intramuscularly into the tibialis anterior (TA) muscle of LCAD(+/-) mice or injected into the portal vein to transduce hepatocytes. Ten weeks post-injection of rAAV1-mLCAD into the TA muscle, significantly increased levels of mLCAD within mitochondria were demonstrated by immunostaining of TA sections, immunoblotting of mitochondrial isolates and by the electron transfer flavoprotein (ETF) fluorescence reduction enzyme activity assay. Magnetic resonance spectroscopy of vector-injected TA muscle demonstrated a reduction in the lipid content compared to phosphate-buffered saline-injected mice, whereas a systemic effect was observed as a reduction in liver macrosteatosis. Eight weeks after portal vein injection of rAAV8-mLCAD into LCAD(+/-) mice, increased levels of mLCAD within hepatocyte mitochondria were demonstrated by immunostaining and also by the ETF assay. Scoring of the hepatosteatosis observed in partially deficient LCAD mice indicated a reduction in the lipid content within livers of vector-treated mice. These studies show that rAAV-mediated delivery of mLCAD was efficient and led to an amelioration of local and systemic pathologies observed in partially deficient LCAD mice. Copyright (c

  19. Bioreducible polymers for gene silencing and delivery.

    Science.gov (United States)

    Son, Sejin; Namgung, Ran; Kim, Jihoon; Singha, Kaushik; Kim, Won Jong

    2012-07-17

    Polymeric gene delivery vectors show great potential for the construction of the ideal gene delivery system. These systems harness their ability to incorporate versatile functional traits to overcome most impediments encountered in gene delivery: from the initial complexation to their target-specific release of the therapeutic nucleic acids at the cytosol. Among the numerous multifunctional polymers that have been designed and evaluated as gene delivery vectors, polymers with redox-sensitive (or bioreducible) functional domains have gained great attention in terms of their structural and functional traits. The redox environment plays a pivotal role in sustaining cellular homeostasis and natural redox potential gradients exist between extra- and intracellular space and between the exterior and interior of subcellular organelles. In some cases, researchers have designed the polymeric delivery vectors to exploit these gradients. For example, researchers have taken advantage of the high redox potential gradient between oxidizing extracellular space and the reducing environment of cytosolic compartments by integrating disulfide bonds into the polymer structure. Such polymers retain their cargo in the extracellular space but selectively release the therapeutic nucleic acids in the reducing space within the cytosol. Furthermore, bioreducible polymers form stable complex with nucleic acids, and researchers can fabricate these structures to impart several important features such as site-, timing-, and duration period-specific gene expression. Additionally, the introduction of disulfide bonds within these polymers promotes their biodegradability and limits their cytotoxicity. Many approaches have demonstrated the versatility of bioreducible gene delivery, but the underlying biological rationale of these systems remains poorly understood. The process of disulfide reduction depends on multiple variables in the cellular redox environment. Therefore, the quest to unravel various

  20. Modified montmorillonite as vector for gene delivery.

    Science.gov (United States)

    Lin, Feng-Huei; Chen, Chia-Hao; Cheng, Winston T K; Kuo, Tzang-Fu

    2006-06-01

    Currently, gene delivery systems can be divided into two parts: viral or non-viral vectors. In general, viral vectors have a higher efficiency on gene delivery. However, they may sometimes provoke mutagenesis and carcinogenesis once re-activating in human body. Lots of non-viral vectors have been developed that tried to solve the problems happened on viral vectors. Unfortunately, most of non-viral vectors showed relatively lower transfection rate. The aim of this study is to develop a non-viral vector for gene delivery system. Montmorillonite (MMT) is one of clay minerals that consist of hydrated aluminum with Si-O tetrahedrons on the bottom of the layer and Al-O(OH)2 octahedrons on the top. The inter-layer space is about 12 A. The room is not enough to accommodate DNA for gene delivery. In the study, the cationic hexadecyltrimethylammonium (HDTMA) will be intercalated into the interlayer of MMT as a layer expander to expand the layer space for DNA accommodation. The optimal condition for the preparation of DNA-HDTMA-MMT is as follows: 1 mg of 1.5CEC HDTMA-MMT was prepared under pH value of 10.7 and with soaking time for 2 h. The DNA molecules can be protected from nuclease degradation, which can be proven by the electrophoresis analysis. DNA was successfully transfected into the nucleus of human dermal fibroblast and expressed enhanced green fluorescent protein (EGFP) gene with green fluorescence emission. The HDTMA-MMT has a great potential as a vector for gene delivery in the future.

  1. Synthetic virology: engineering viruses for gene delivery.

    Science.gov (United States)

    Guenther, Caitlin M; Kuypers, Brianna E; Lam, Michael T; Robinson, Tawana M; Zhao, Julia; Suh, Junghae

    2014-01-01

    The success of gene therapy relies heavily on the performance of vectors that can effectively deliver transgenes to desired cell populations. As viruses have evolved to deliver genetic material into cells, a prolific area of research has emerged over the last several decades to leverage the innate properties of viruses as well as to engineer new features into them. Specifically, the field of synthetic virology aims to capitalize on knowledge accrued from fundamental virology research in order to design functionally enhanced gene delivery vectors. The enhanced viral vectors, or 'bionic' viruses, feature engineered components, or 'parts', that are natural (intrinsic to viruses or from other organisms) and synthetic (such as man-made polymers or inorganic nanoparticles). Various design strategies--rational, combinatorial, and pseudo-rational--have been pursued to create the hybrid viruses. The gene delivery vectors of the future will likely criss-cross the boundaries between natural and synthetic domains to harness the unique strengths afforded by the various functional parts that can be grafted onto virus capsids. Such research endeavors will further expand and enable enhanced control over the functional capacity of these nanoscale devices for biomedicine. © 2014 Wiley Periodicals, Inc.

  2. Immunoliposome-mediated delivery of neomycin phosphotransferase for the lineage-specific selection of differentiated/committed stem cell progenies: potential advantages over transfection with marker genes, fluorescence-activated and magnetic affinity cell-sorting.

    Science.gov (United States)

    Heng, Boon Chin; Cao, Tong

    2005-01-01

    A major challenge in the therapeutic application of stem cells in regenerative medicine is the lineage-specific selection of their committed/differentiated progenies for transplantation. This is necessary to avoid engraftment of undesired lineages at the transplantation site, i.e. fibroblastic scar tissue, as well as to enhance the efficacy of transplantation therapy. Commonly used techniques for lineage-specific selection of committed/differentiated stem cell progenies include marker gene transfection, fluorescence-activated (FACS) and magnetic-affinity (MACS) cell-sorting. Nevertheless, these have their disadvantages for therapeutic applications. Marker gene transfection invariably leads to permanent genetic modification of stem cells, which in turn limits their use in human clinical therapy due to overwhelming ethical and safety concerns. FACS requires expensive instrumentation and highly-skilled personnel, and is unsuited for handling bulk quantities of cells that would almost certainly be required for transplantation therapy. MACS is a cheaper alternative, but the level of purity attained is also reduced. A possible novel approach that has yet to be investigated is immunoliposome-mediated delivery of neomycin phosphotranferase (NPT) for lineage-specific selection of stem cell progenies. This would avoid permanent genetic modification to the cell, unlike recombinant NPT expression linked to activation of specific promoter sequences. Moreover, it could potentially provide a much more practical and cost-effective alternative for handling bulk quantities of cells that would be required for transplantation therapy, as compared to FACS or MACS. As such, this alternative approach needs to be rigorously investigated, in view of its potentially useful applications in stem cell therapeutics.

  3. Liposomes for Use in Gene Delivery

    Directory of Open Access Journals (Sweden)

    Daniel A. Balazs

    2011-01-01

    Full Text Available Liposomes have a wide array of uses that have been continuously expanded and improved upon since first being observed to self-assemble into vesicular structures. These arrangements can be found in many shapes and sizes depending on lipid composition. Liposomes are often used to deliver a molecular cargo such as DNA for therapeutic benefit. The lipids used to form such lipoplexes can be cationic, anionic, neutral, or a mixture thereof. Herein physical packing parameters and specific lipids used for gene delivery will be discussed, with lipids classified according to overall charge.

  4. Nonviral Delivery Systems For Cancer Gene Therapy: Strategies And Challenges.

    Science.gov (United States)

    Shim, Gayong; Kim, Dongyoon; Le, Quoc-Viet; Park, Gyu Thae; Kwon, Taekhyun; Oh, Yu-Kyoung

    2018-01-19

    Gene therapy has been receiving widespread attention due to its unique advantage in regulating the expression of specific target genes. In the field of cancer gene therapy, modulation of gene expression has been shown to decrease oncogenic factors in cancer cells or increase immune responses against cancer. Due to the macromolecular size and highly negative physicochemical features of plasmid DNA, efficient delivery systems are an essential ingredient for successful gene therapy. To date, a variety of nanostructures and materials have been studied as nonviral gene delivery systems. In this review, we will cover nonviral delivery strategies for cancer gene therapy, with a focus on target cancer genes and delivery materials. Moreover, we will address current challenges and perspectives for nonviral delivery-based cancer gene therapeutics. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  5. Discovery of Cationic Polymers for Non-viral Gene Delivery using Combinatorial Approaches

    Science.gov (United States)

    Barua, Sutapa; Ramos, James; Potta, Thrimoorthy; Taylor, David; Huang, Huang-Chiao; Montanez, Gabriela; Rege, Kaushal

    2015-01-01

    Gene therapy is an attractive treatment option for diseases of genetic origin, including several cancers and cardiovascular diseases. While viruses are effective vectors for delivering exogenous genes to cells, concerns related to insertional mutagenesis, immunogenicity, lack of tropism, decay and high production costs necessitate the discovery of non-viral methods. Significant efforts have been focused on cationic polymers as non-viral alternatives for gene delivery. Recent studies have employed combinatorial syntheses and parallel screening methods for enhancing the efficacy of gene delivery, biocompatibility of the delivery vehicle, and overcoming cellular level barriers as they relate to polymer-mediated transgene uptake, transport, transcription, and expression. This review summarizes and discusses recent advances in combinatorial syntheses and parallel screening of cationic polymer libraries for the discovery of efficient and safe gene delivery systems. PMID:21843141

  6. Enhancement of adenovirus-mediated gene delivery to rheumatoid arthritis synoviocytes and synovium by fiber modifications: role of arginine-glycine-aspartic acid (RGD)- and non-RGD-binding integrins.

    NARCIS (Netherlands)

    Toh, M.L.; Hong, S.S.; Loo, F.A.J. van de; Franqueville, L.; Lindholm, L.; Berg, W.B. van den; Boulanger, P.; Miossec, P.

    2005-01-01

    Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) do not express the coxsackie-adenovirus (Ad) receptor and are poorly permissive to Ad serotype 5 (Ad5). Genetically modified, coxsackie-Ad receptor-independent Ad5 vectors were studied for gene delivery in human RA FLS and synovium

  7. A novel needleless liquid jet injection methodology for improving direct cardiac gene delivery: An optimization of parameters, AAV mediated therapy and investigation of host responses in ischemic heart failure

    Science.gov (United States)

    Fargnoli, Anthony Samuel

    Heart disease remains the leading cause of mortality and morbidity worldwide, with 22 million new patients diagnosed annually. Essentially, all present therapies have significant cost burden to the healthcare system, yet fail to increase survival rates. One key employed strategy is the genetic reprogramming of cells to increase contractility via gene therapy, which has advanced to Phase IIb Clinical Trials for advanced heart failure patients. It has been argued that the most significant barrier preventing FDA approval are resolving problems with safe, efficient myocardial delivery, whereby direct injection in the infarct and remote tissue areas is not clinically feasible. Here, we aim to: (1) Improve direct cardiac gene delivery through the development of a novel liquid jet device approach (2) Compare the new method against traditional IM injection with two different vector constructions and evaluate outcome (3) Evaluate the host response resulting from both modes of direct cardiac injection, then advance a drug/gene combination with controlled release nanoparticle formulations.

  8. Tatp-mediated intracellular delivery of pharmaceutical nanocarriers.

    Science.gov (United States)

    Torchilin, V P

    2007-08-01

    CPPs (cell-penetrating peptides), including Tatp (transactivator of transcription peptide), have been successfully used for intracellular delivery of a wide variety of cargoes including various nanoparticulate pharmaceutical carriers such as liposomes, micelles and nanoparticles. Here, we will consider the major results obtained in this area with emphasis on Tatp-mediated delivery of liposomes and various transfection vectors. We will also address the development of 'smart' stimuli-sensitive nanocarriers, where the cell-penetrating function can only be activated when the nanocarrier is inside the biological target, thus minimizing the interaction with non-target cells.

  9. Self-Assembling Multifunctional Peptide Dimers for Gene Delivery Systems

    Directory of Open Access Journals (Sweden)

    Kitae Ryu

    2015-01-01

    Full Text Available Self-assembling multifunctional peptide was designed for gene delivery systems. The multifunctional peptide (MP consists of cellular penetrating peptide moiety (R8, matrix metalloproteinase-2 (MMP-2 specific sequence (GPLGV, pH-responsive moiety (H5, and hydrophobic moiety (palmitic acid (CR8GPLGVH5-Pal. MP was oxidized to form multifunctional peptide dimer (MPD by DMSO oxidation of thiols in terminal cysteine residues. MPD could condense pDNA successfully at a weight ratio of 5. MPD itself could self-assemble into submicron micelle particles via hydrophobic interaction, of which critical micelle concentration is about 0.01 mM. MPD showed concentration-dependent but low cytotoxicity in comparison with PEI25k. MPD polyplexes showed low transfection efficiency in HEK293 cells expressing low level of MMP-2 but high transfection efficiency in A549 and C2C12 cells expressing high level of MMP-2, meaning the enhanced transfection efficiency probably due to MMP-induced structural change of polyplexes. Bafilomycin A1-treated transfection results suggest that the transfection of MPD is mediated via endosomal escape by endosome buffering ability. These results show the potential of MPD for MMP-2 targeted gene delivery systems due to its multifunctionality.

  10. Microneedle-mediated delivery of viral vectored vaccines.

    Science.gov (United States)

    Zaric, Marija; Ibarzo Yus, Bárbara; Kalcheva, Petya Petrova; Klavinskis, Linda Sylvia

    2017-10-01

    Microneedle array platforms are a promising technology for vaccine delivery, due to their ease of administration with no sharp waste generated, small size, possibility of targeted delivery to the specified skin depth and efficacious delivery of different vaccine formulations, including viral vectors. Areas covered: Attributes and challenges of the most promising viral vector candidates that have advanced to the clinic and that have been leveraged for skin delivery by microneedles; The importance of understanding the immunobiology of antigen-presenting cells in the skin, in particular dendritic cells, in order to generate further improved skin vaccination strategies; recent studies where viral vectors expressing various antigens have been coupled with microneedle technology to examine their potential for improved vaccination. Expert opinion: Simple, economic and efficacious vaccine delivery methods are needed to improve health outcomes and manage possible outbreaks of new emerging viruses. Understanding what innate/inflammatory signals are required to induce both immediate and long-term responses remains a major hurdle in the development of the effective vaccines. One approach to meet these needs is microneedle-mediated viral vector vaccination. In order for this technology to fulfil this potential the industry must invest significantly to further develop its design, production, biosafety, delivery and large-scale manufacturing.

  11. Molecular Imaging of Biological Gene Delivery Vehicles for Targeted Cancer Therapy: Beyond Viral Vectors

    Energy Technology Data Exchange (ETDEWEB)

    Min, Jung Joon; Nguyen, Vu H. [Chonnam National University Medical School, Gwangju (Korea, Republic of); Gambhir, Sanjiv S. [Stanford University, California(United States)

    2010-04-15

    Cancer persists as one of the most devastating diseases in the world. Problems including metastasis and tumor resistance to chemotherapy and radiotherapy have seriously limited the therapeutic effects of present clinical treatments. To overcome these limitations, cancer gene therapy has been developed over the last two decades for a broad spectrum of applications, from gene replacement and knockdown to vaccination, each with different requirements for gene delivery. So far, a number of genes and delivery vectors have been investigated, and significant progress has been made with several gene therapy modalities in clinical trials. Viral vectors and synthetic liposomes have emerged as the vehicles of choice for many applications. However, both have limitations and risks that restrict gene therapy applications, including the complexity of production, limited packaging capacity, and unfavorable immunological features. While continuing to improve these vectors, it is important to investigate other options, particularly nonarrival biological agents such as bacteria, bacteriophages, and bacteria-like particles. Recently, many molecular imaging techniques for safe, repeated, and high-resolution in vivo imaging of gene expression have been employed to assess vector-mediated gene expression in living subjects. In this review, molecular imaging techniques for monitoring biological gene delivery vehicles are described, and the specific use of these methods at different steps is illustrated. Linking molecular imaging to gene therapy will eventually help to develop novel gene delivery vehicles for preclinical study and support the development of future human applications.

  12. Gene delivery in tissue engineering and regenerative medicine.

    Science.gov (United States)

    Fang, Y L; Chen, X G; W T, Godbey

    2015-11-01

    As a promising strategy to aid or replace tissue/organ transplantation, gene delivery has been used for regenerative medicine applications to create or restore normal function at the cell and tissue levels. Gene delivery has been successfully performed ex vivo and in vivo in these applications. Excellent proliferation capabilities and differentiation potentials render certain cells as excellent candidates for ex vivo gene delivery for regenerative medicine applications, which is why multipotent and pluripotent cells have been intensely studied in this vein. In this review, gene delivery is discussed in detail, along with its applications to tissue engineering and regenerative medicine. A definition of a stem cell is compared to a definition of a stem property, and both provide the foundation for an in-depth look at gene delivery investigations from a germ lineage angle. © 2014 Wiley Periodicals, Inc.

  13. Effective Nanoparticle-based Gene Delivery by a Protease Triggered Charge Switch

    DEFF Research Database (Denmark)

    Gjetting, Torben; Jølck, Rasmus Irming; Andresen, Thomas Lars

    2014-01-01

    (ethylene glycol) (PEG) chain is investigated. Utilizing ethanol-mediated nucleic acid encapsulation to prepare lipo-nanoparticles (LNPs), LNPs that are stable in serum are obtained. The LNPs constitute a highly effective gene delivery systems in vitro and possess the right features for further...

  14. Systemic gene delivery to the central nervous system using Adeno-associated virus

    Directory of Open Access Journals (Sweden)

    Mathieu eBOURDENX

    2014-06-01

    Full Text Available Adeno-associated virus (AAV-mediated gene delivery has emerged as an effective and safe tool for both preclinical and clinical studies of neurological disorders. The recent discovery that several serotypes are able to cross the blood-brain-barrier when administered systemically has been a real breakthrough in the field of neurodegenerative diseases. Widespread transgene expression after systemic injection could spark interest as a therapeutic approach. Such strategy will avoid invasive brain surgery and allow non-focal gene therapy promising for CNS diseases affecting large portion of the brain. Here, we will review the recent results achieved through different systemic routes of injection generated in the last decade using systemic AAV-mediated delivery and propose a brief assessment of their values. In particular, we emphasize how the methods used for virus engineering could improve brain transduction after peripheral delivery.

  15. Will Nanotechnology Bring New Hope for Gene Delivery?

    Science.gov (United States)

    Wong, Joanna K L; Mohseni, Rashin; Hamidieh, Amir Ali; MacLaren, Robert E; Habib, Nagy; Seifalian, Alexander M

    2017-05-01

    The high mortality rates of cancer patients receiving standard treatments emphasize the crucial need for alternative treatments. One promising strategy is to use organic nanoparticles (NPs) for gene delivery. Although multifunctional NPs theoretically have many desirable properties as gene vectors, there are several practical barriers to successful gene delivery. In this review we discuss the properties of NPs and overview in vitro and preclinical studies of organic NPs over the past 5 years. The results of recent clinical trials suggest that the NP field holds significant promise. The annual market value for successful gene delivery could exceed US$30 billion, and this will encourage researchers to study the application of NPs to therapeutic gene delivery. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Biomaterials mediated microRNA delivery for bone tissue engineering.

    Science.gov (United States)

    Sriram, M; Sainitya, R; Kalyanaraman, V; Dhivya, S; Selvamurugan, N

    2015-03-01

    Bone tissue engineering is an alternative strategy to overcome the problems associated with traditional treatments for bone defects. A number of bioactive materials along with new techniques like porous scaffold implantation, gene delivery, 3D organ printing are now-a-days emerging for traditional bone grafts and metal implants. Studying the molecular mechanisms through which these biomaterials induce osteogenesis is an equally hot field. Biomaterials could determine the fate of a cell via microRNAs (miRNAs). miRNAs are short non-coding RNAs that act as post-transcriptional regulators of gene expression and play an essential role for regulation of cell specific lineages including osteogenesis. Thus, this review focuses the recent trends on establishing a link of biomaterials with miRNAs and their delivery for bone tissue engineering applications. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. In vivo gene delivery by cationic tetraamino fullerene.

    Science.gov (United States)

    Maeda-Mamiya, Rui; Noiri, Eisei; Isobe, Hiroyuki; Nakanishi, Waka; Okamoto, Koji; Doi, Kent; Sugaya, Takeshi; Izumi, Tetsuro; Homma, Tatsuya; Nakamura, Eiichi

    2010-03-23

    Application of nanotechnology to medical biology has brought remarkable success. Water-soluble fullerenes are molecules with great potential for biological use because they can endow unique characteristics of amphipathic property and form a self-assembled structure by chemical modification. Effective gene delivery in vitro with tetra(piperazino)fullerene epoxide (TPFE) and its superiority to Lipofectin have been described in a previous report. For this study, we evaluated the efficacy of in vivo gene delivery by TPFE. Delivery of enhanced green fluorescent protein gene (EGFP) by TPFE on pregnant female ICR mice showed distinct organ selectivity compared with Lipofectin; moreover, higher gene expression by TPFE was found in liver and spleen, but not in the lung. No acute toxicity of TPFE was found for the liver and kidney, although Lipofectin significantly increased liver enzymes and blood urea nitrogen. In fetal tissues, neither TPFE nor Lipofectin induced EGFP gene expression. Delivery of insulin 2 gene to female C57/BL6 mice increased plasma insulin levels and reduced blood glucose concentrations, indicating the potential of TPFE-based gene delivery for clinical application. In conclusion, this study demonstrated effective gene delivery in vivo for the first time using a water-soluble fullerene.

  18. Preclinical evaluation of gene delivery methods for the treatment of loco-regional disease in breast cancer.

    LENUS (Irish Health Repository)

    Rajendran, Simon

    2012-01-31

    Preclinical results with various gene therapy strategies indicate significant potential for new cancer treatments. However, many therapeutics fail at clinical trial, often due to differences in tissue physiology between animal models and humans, and tumor phenotype variation. Clinical data relevant to treatment strategies may be generated prior to clinical trial through experimentation using intact patient tissue ex vivo. We developed a novel tumor slice model culture system that is universally applicable to gene delivery methods, using a realtime luminescence detection method to assess gene delivery. Methods investigated include viruses (adenovirus [Ad] and adeno-associated virus), lipofection, ultrasound (US), electroporation and naked DNA. Viability and tumor populations within the slices were well maintained for seven days, and gene delivery was qualitatively and quantitatively examinable for all vectors. Ad was the most efficient gene delivery vector with transduction efficiency >50%. US proved the optimal non-viral gene delivery method in human tumor slices. The nature of the ex vivo culture system permitted examination of specific elements. Parameters shown to diminish Ad gene delivery included blood, regions of low viability and secondary disease. US gene delivery was significantly reduced by blood and skin, while tissue hyperthermia improved gene delivery. US achieved improved efficacy for secondary disease. The ex vivo model was also suitable for examination of tissue-specific effects on vector expression, with Ad expression mediated by the CXCR4 promoter shown to provide a tumor selective advantage over the ubiquitously active cytomegalovirus promoter. In conclusion, this is the first study incorporating patient tissue models in comparing gene delivery from various vectors, providing knowledge on cell-type specificity and examining the crucial biological factors determining successful gene delivery. The results highlight the importance of in

  19. Preclinical evaluation of gene delivery methods for the treatment of loco-regional disease in breast cancer.

    LENUS (Irish Health Repository)

    Rajendran, Simon

    2011-04-01

    Preclinical results with various gene therapy strategies indicate significant potential for new cancer treatments. However, many therapeutics fail at clinical trial, often due to differences in tissue physiology between animal models and humans, and tumor phenotype variation. Clinical data relevant to treatment strategies may be generated prior to clinical trial through experimentation using intact patient tissue ex vivo. We developed a novel tumor slice model culture system that is universally applicable to gene delivery methods, using a realtime luminescence detection method to assess gene delivery. Methods investigated include viruses (adenovirus [Ad] and adeno-associated virus), lipofection, ultrasound (US), electroporation and naked DNA. Viability and tumor populations within the slices were well maintained for seven days, and gene delivery was qualitatively and quantitatively examinable for all vectors. Ad was the most efficient gene delivery vector with transduction efficiency >50%. US proved the optimal non-viral gene delivery method in human tumor slices. The nature of the ex vivo culture system permitted examination of specific elements. Parameters shown to diminish Ad gene delivery included blood, regions of low viability and secondary disease. US gene delivery was significantly reduced by blood and skin, while tissue hyperthermia improved gene delivery. US achieved improved efficacy for secondary disease. The ex vivo model was also suitable for examination of tissue-specific effects on vector expression, with Ad expression mediated by the CXCR4 promoter shown to provide a tumor selective advantage over the ubiquitously active cytomegalovirus promoter. In conclusion, this is the first study incorporating patient tissue models in comparing gene delivery from various vectors, providing knowledge on cell-type specificity and examining the crucial biological factors determining successful gene delivery. The results highlight the importance of in

  20. Functionalized nanoparticles for AMF-induced gene and drug delivery

    Science.gov (United States)

    Biswas, Souvik

    The properties and broad applications of nano-magnetic colloids have generated much interest in recent years. Specially, Fe3O4 nanoparticles have attracted a great deal of attention since their magnetic properties can be used for hyperthermia treatment or drug targeting. For example, enhanced levels of intracellular gene delivery can be achieved using Fe3O4 nano-vectors in the presence of an external magnetic field, a process known as 'magnetofection'. The low cytotoxicity, tunable particle size, ease of surface functionalization, and ability to generate thermal energy using an external alternating magnetic field (AMF) are properties have propelled Fe3O4 research to the forefront of nanoparticle research. The strategy of nanoparticle-mediated, AMF-induced heat generation has been used to effect intracellular hyperthermia. One application of this 'magnetic hyperthermia' is heat activated local delivery of a therapeutic effector (e.g.; drug or polynucleotide). This thesis describes the development of a magnetic nano-vector for AMF-induced, heat-activated pDNA and small molecule delivery. The use of heat-inducible vectors, such as heat shock protein ( hsp) genes, is a promising mode of gene therapy that would restrict gene expression to a local region by focusing a heat stimulus only at a target region. We thus aimed to design an Fe3O4 nanoparticle-mediated gene transfer vehicle for AMF-induced localized gene expression. We opted to use 'click' oximation techniques to assemble the magnetic gene transfer vector. Chapter 2 describes the synthesis, characterization, and transfection studies of the oxime ether lipid-based nano-magnetic vectors MLP and dMLP. The synthesis and characterization of a novel series of quaternary ammonium aminooxy reagents (2.1--2.4) is described. These cationic aminooxy compounds were loaded onto nanoparticles for ligation with carbonyl groups and also to impart a net positive charge on the nanoparticle surface. Our studies indicated that the

  1. Bacteriophages as vehicles for gene delivery into mammalian cells: prospects and problems.

    Science.gov (United States)

    Bakhshinejad, Babak; Sadeghizadeh, Majid

    2014-10-01

    The identification of more efficient gene delivery vehicles (GDVs) is essential to fulfill the expectations of clinical gene therapy. Bacteriophages, due to their excellent safety profile, extreme stability under a variety of harsh environmental conditions and the capability for being genetically manipulated, have drawn a flurry of interest to be applied as a newly arisen category of gene delivery platforms. The incessant evolutionary interaction of bacteriophages with human cells has turned them into a part of our body's natural ecosystem. However, these carriers represent several barriers to gene transduction of mammalian cells. The lack of evolvement of specialized machinery for targeted cellular internalization, endosomal, lysosomal and proteasomal escape, cytoplasmic entry, nuclear localization and intranuclear transcription poses major challenges to the expression of the phage-carried gene. In this review, we describe pros and cons of bacteriophages as GDVs, provide an insight into numerous barriers that bacteriophages face for entry into and subsequent trafficking inside mammalian cells and elaborate on the strategies used to bypass these barriers. Tremendous genetic flexibility of bacteriophages to undergo numerous surface modifications through phage display technology has proven to be a turning point in the uncompromising efforts to surmount the limitations of phage-mediated gene expression. The revelatory outcomes of the studies undertaken within the recent years have been promising for phage-mediated gene delivery to move from concept to reality.

  2. Kinetics of lipid-nanoparticle-mediated intracellular mRNA delivery and function

    Science.gov (United States)

    Zhdanov, Vladimir P.

    2017-10-01

    mRNA delivery into cells forms the basis for one of the new and promising ways to treat various diseases. Among suitable carriers, lipid nanoparticles (LNPs) with a size of about 100 nm are now often employed. Despite high current interest in this area, the understanding of the basic details of LNP-mediated mRNA delivery and function is limited. To clarify the kinetics of mRNA release from LNPs, the author uses three generic models implying (i) exponential, (ii) diffusion-controlled, and (iii) detachment-controlled kinetic regimes, respectively. Despite the distinct differences in these kinetics, the associated transient kinetics of mRNA translation to the corresponding protein and its degradation are shown to be not too sensitive to the details of the mRNA delivery by LNPs (or other nanocarriers). In addition, the author illustrates how this protein may temporarily influence the expression of one gene or a few equivalent genes. The analysis includes positive or negative regulation of the gene transcription via the attachment of the protein without or with positive or negative feedback in the gene expression. Stable, bistable, and oscillatory schemes have been scrutinized in this context.

  3. Rapid endosomal escape of prickly nanodiamonds: implications for gene delivery

    Science.gov (United States)

    Chu, Zhiqin; Miu, Kaikei; Lung, Pingsai; Zhang, Silu; Zhao, Saisai; Chang, Huan-Cheng; Lin, Ge; Li, Quan

    2015-06-01

    The prickly nanodiamonds easily entered cells via endocytosis followed by unique intracellular translocation characteristics—quick endosomal escape followed by stable residence in cytoplasm. Endosomal membrane rupturing is identified as the major route of nanodiamonds’ escaping the vesicle confinement and to the cytoplasm. Little cytotoxicity is observed to associate with the nanodiamonds’ cytosolic release. Such features enable its application for gene delivery, which requires both effective cellular uptake and cytosolic release of the gene. Taking green fluorescent protein gene as an example, we demonstrate the successful cytosolic delivery and expression of such a gene using the prickly nanodiamonds as carrier.

  4. Rapid endosomal escape of prickly nanodiamonds: implications for gene delivery.

    Science.gov (United States)

    Chu, Zhiqin; Miu, Kaikei; Lung, Pingsai; Zhang, Silu; Zhao, Saisai; Chang, Huan-Cheng; Lin, Ge; Li, Quan

    2015-06-30

    The prickly nanodiamonds easily entered cells via endocytosis followed by unique intracellular translocation characteristics—quick endosomal escape followed by stable residence in cytoplasm. Endosomal membrane rupturing is identified as the major route of nanodiamonds' escaping the vesicle confinement and to the cytoplasm. Little cytotoxicity is observed to associate with the nanodiamonds' cytosolic release. Such features enable its application for gene delivery, which requires both effective cellular uptake and cytosolic release of the gene. Taking green fluorescent protein gene as an example, we demonstrate the successful cytosolic delivery and expression of such a gene using the prickly nanodiamonds as carrier.

  5. Engineering PTEN-L for Cell-Mediated Delivery.

    Science.gov (United States)

    Lavictoire, Sylvie J; Gont, Alexander; Julian, Lisa M; Stanford, William L; Vlasschaert, Caitlyn; Gray, Douglas A; Jomaa, Danny; Lorimer, Ian A J

    2018-06-15

    The tumor suppressor PTEN is frequently inactivated in glioblastoma. PTEN-L is a long form of PTEN produced by translation from an alternate upstream start codon. Unlike PTEN, PTEN-L has a signal sequence and a tract of six arginine residues that allow PTEN-L to be secreted from cells and be taken up by neighboring cells. This suggests that PTEN-L could be used as a therapeutic to restore PTEN activity. However, effective delivery of therapeutic proteins to treat CNS cancers such as glioblastoma is challenging. One method under evaluation is cell-mediated therapy, where cells with tumor-homing abilities such as neural stem cells are genetically modified to express a therapeutic protein. Here, we have developed a version of PTEN-L that is engineered for enhanced cell-mediated delivery. This was accomplished by replacement of the native leader sequence of PTEN-L with a leader sequence from human light-chain immunoglobulin G (IgG). This version of PTEN-L showed increased secretion and an increased ability to transfer to neighboring cells. Neural stem cells derived from human fibroblasts could be modified to express this version of PTEN-L and were able to deliver catalytically active light-chain leader PTEN-L (lclPTEN-L) to neighboring glioblastoma cells.

  6. Self-assembled pentablock copolymers for selective and sustained gene delivery

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Bingqi [Iowa State Univ., Ames, IA (United States)

    2011-05-15

    The poly(diethylaminoethyl methacrylate) (PDEAEM) - Pluronic F127 - PDEAEM pentablock copolymer (PB) gene delivery vector system has been found to possess an inherent selectivity in transfecting cancer cells over non-cancer cells in vitro, without attaching any targeting ligands. In order to understand the mechanism of this selective transfection, three possible intracellular barriers to transfection were investigated in both cancer and non-cancer cells. We concluded that escape from the endocytic pathway served as the primary intracellular barrier for PB-mediated transfection. Most likely, PB vectors were entrapped and rendered non-functional in acidic lysosomes of non-cancer cells, but survived in less acidic lysosomes of cancer cells. The work highlights the importance of identifying intracellular barriers for different gene delivery systems and provides a new paradigm for designing targeting vectors based on intracellular differences between cell types, rather than through the use of targeting ligands. The PB vector was further developed to simultaneously deliver anticancer drugs and genes, which showed a synergistic effect demonstrated by significantly enhanced gene expression in vitro. Due to the thermosensitive gelation behavior, the PB vector packaging both drug and gene was also investigated for its in vitro sustained release properties by using polyethylene glycol diacrylate as a barrier gel to mimic the tumor matrix in vivo. Overall, this work resulted in the development of a gene delivery vector for sustained and selective gene delivery to tumor cells for cancer therapy.

  7. Gold nanoparticle mediated laser transfection for efficient siRNA mediated gene knock down.

    Directory of Open Access Journals (Sweden)

    Dag Heinemann

    Full Text Available Laser based transfection methods have proven to be an efficient and gentle alternative to established molecule delivery methods like lipofection or electroporation. Among the laser based methods, gold nanoparticle mediated laser transfection bears the major advantage of high throughput and easy usability. This approach uses plasmon resonances on gold nanoparticles unspecifically attached to the cell membrane to evoke transient and spatially defined cell membrane permeabilization. In this study, we explore the parameter regime for gold nanoparticle mediated laser transfection for the delivery of molecules into cell lines and prove its suitability for siRNA mediated gene knock down. The developed setup allows easy usage and safe laser operation in a normal lab environment. We applied a 532 nm Nd:YAG microchip laser emitting 850 ps pulses at a repetition rate of 20.25 kHz. Scanning velocities of the laser spot over the sample of up to 200 mm/s were tested without a decline in perforation efficiency. This velocity leads to a process speed of ∼8 s per well of a 96 well plate. The optimal particle density was determined to be ∼6 particles per cell using environmental scanning electron microscopy. Applying the optimized parameters transfection efficiencies of 88% were achieved in canine pleomorphic adenoma ZMTH3 cells using a fluorescent labeled siRNA while maintaining a high cell viability of >90%. Gene knock down of d2-EGFP was demonstrated and validated by fluorescence repression and western blot analysis. On basis of our findings and established mathematical models we suppose a mixed transfection mechanism consisting of thermal and multiphoton near field effects. Our findings emphasize that gold nanoparticle mediated laser transfection provides an excellent tool for molecular delivery for both, high throughput purposes and the transfection of sensitive cells types.

  8. Gold Nanoparticle Mediated Laser Transfection for Efficient siRNA Mediated Gene Knock Down

    Science.gov (United States)

    Heinemann, Dag; Schomaker, Markus; Kalies, Stefan; Schieck, Maximilian; Carlson, Regina; Escobar, Hugo Murua; Ripken, Tammo; Meyer, Heiko; Heisterkamp, Alexander

    2013-01-01

    Laser based transfection methods have proven to be an efficient and gentle alternative to established molecule delivery methods like lipofection or electroporation. Among the laser based methods, gold nanoparticle mediated laser transfection bears the major advantage of high throughput and easy usability. This approach uses plasmon resonances on gold nanoparticles unspecifically attached to the cell membrane to evoke transient and spatially defined cell membrane permeabilization. In this study, we explore the parameter regime for gold nanoparticle mediated laser transfection for the delivery of molecules into cell lines and prove its suitability for siRNA mediated gene knock down. The developed setup allows easy usage and safe laser operation in a normal lab environment. We applied a 532 nm Nd:YAG microchip laser emitting 850 ps pulses at a repetition rate of 20.25 kHz. Scanning velocities of the laser spot over the sample of up to 200 mm/s were tested without a decline in perforation efficiency. This velocity leads to a process speed of ∼8 s per well of a 96 well plate. The optimal particle density was determined to be ∼6 particles per cell using environmental scanning electron microscopy. Applying the optimized parameters transfection efficiencies of 88% were achieved in canine pleomorphic adenoma ZMTH3 cells using a fluorescent labeled siRNA while maintaining a high cell viability of >90%. Gene knock down of d2-EGFP was demonstrated and validated by fluorescence repression and western blot analysis. On basis of our findings and established mathematical models we suppose a mixed transfection mechanism consisting of thermal and multiphoton near field effects. Our findings emphasize that gold nanoparticle mediated laser transfection provides an excellent tool for molecular delivery for both, high throughput purposes and the transfection of sensitive cells types. PMID:23536802

  9. Evaluation and mechanism studies of PEGylated dendrigraft poly-L-lysines as novel gene delivery vectors

    Science.gov (United States)

    Huang, Rongqin; Liu, Shuhuan; Shao, Kun; Han, Liang; Ke, Weilun; Liu, Yang; Li, Jianfeng; Huang, Shixian; Jiang, Chen

    2010-07-01

    Dendrimers have attracted great interest in the field of gene delivery due to their synthetic controllability and excellent gene transfection efficiency. In this work, dendrigraft poly-L-lysines (DGLs) were evaluated as a novel gene vector for the first time. Derivatives of DGLs (generation 2 and 3) with different extents of PEGylation were successfully synthesized and used to compact pDNA as complexes. The result of gel retardation assay showed that pDNA could be effectively packed by all the vectors at a DGLs to pDNA weight ratio greater than 2. An increase in the PEGylation extent of vectors resulted in a decrease in the incorporation efficiency and cytotoxicity of complexes in 293 cells, which also decreased the zeta potential a little but did not affect the mean diameter of complexes. Higher generation of DGLs could mediate higher gene transfection in vitro. Confocal microscopy and cellular uptake inhibition studies demonstrated that caveolae-mediated process and macropinocytosis were involved in the cellular uptake of DGLs-based complexes. Also the results indicate that proper PEGylated DGLs could mediate efficient gene transfection, showing their potential as an alternate biodegradable vector in the field of nonviral gene delivery.

  10. Evaluation and mechanism studies of PEGylated dendrigraft poly-L-lysines as novel gene delivery vectors

    Energy Technology Data Exchange (ETDEWEB)

    Huang Rongqin; Liu Shuhuan; Shao Kun; Han Liang; Ke Weilun; Liu Yang; Li Jianfeng; Huang Shixian; Jiang Chen, E-mail: jiangchen@shmu.edu.cn [Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai 201203 (China)

    2010-07-02

    Dendrimers have attracted great interest in the field of gene delivery due to their synthetic controllability and excellent gene transfection efficiency. In this work, dendrigraft poly-L-lysines (DGLs) were evaluated as a novel gene vector for the first time. Derivatives of DGLs (generation 2 and 3) with different extents of PEGylation were successfully synthesized and used to compact pDNA as complexes. The result of gel retardation assay showed that pDNA could be effectively packed by all the vectors at a DGLs to pDNA weight ratio greater than 2. An increase in the PEGylation extent of vectors resulted in a decrease in the incorporation efficiency and cytotoxicity of complexes in 293 cells, which also decreased the zeta potential a little but did not affect the mean diameter of complexes. Higher generation of DGLs could mediate higher gene transfection in vitro. Confocal microscopy and cellular uptake inhibition studies demonstrated that caveolae-mediated process and macropinocytosis were involved in the cellular uptake of DGLs-based complexes. Also the results indicate that proper PEGylated DGLs could mediate efficient gene transfection, showing their potential as an alternate biodegradable vector in the field of nonviral gene delivery.

  11. Adenovirus-mediated delivery and expression of a cAMP-dependent protein kinase inhibitor gene to BEAS-2B epithelial cells abolishes the anti-inflammatory effects of rolipram, salbutamol, and prostaglandin E2: a comparison with H-89.

    Science.gov (United States)

    Meja, Koremu K; Catley, Matthew C; Cambridge, Lisa M; Barnes, Peter J; Lum, Hazel; Newton, Robert; Giembycz, Mark A

    2004-05-01

    cAMP-elevating drugs are thought to mediate their biological effects by activating the cAMP/cAMP-dependent protein kinase (PKA) cascade. However, this hypothesis is difficult to confirm due to a lack of selective inhibitors. Here, we have probed the role of PKA in mediating inhibitory effects of several cAMP-elevating drugs in BEAS-2B epithelial cells using an adenovirus vector encoding a PKA inhibitor protein (PKIalpha) and have compared it to H-89, a commonly used small molecule PKA inhibitor. Initial studies established efficient gene transfer and confirmed functionality of PKIalpha 48 h after virus infection. All cAMP-elevating drugs tested promoted the phosphorylation of cAMP response element-binding protein (CREB), activated a cAMP response element (CRE)-driven luciferase reporter gene, and suppressed both granulocyte/macrophage colony-stimulating factor (GM-CSF) generation and [(3)H]arachidonic acid (AA) release in response to interleukin-1beta and monocyte chemotactic protein (MCP)-1, respectively. These effects were abolished by PKIalpha. In contrast, H-89 behaved unpredictably under the same conditions. Thus, although CREB phosphorylation evoked by a range of cAMP-elevating drugs was abolished by H-89, neither activation of the CRE-dependent luciferase reporter gene construct nor the inhibition of GM-CSF generation was inhibited. Paradoxically, H-89 antagonized MCP-1-induced [(3)H]AA release and enhanced the inhibitory effect of submaximal concentrations of rolipram and 8-bromo-cAMP. We suggest that expression of PKIalpha in susceptible cells provides a simple and unambiguous way to assess the role of PKA in cAMP signaling and to probe the mechanism of action of other drugs and cAMP-dependent responses where the participation of PKA is equivocal. Furthermore, these data suggest that H-89 is not a selective inhibitor of PKA and should be avoided.

  12. Artificial Virus as Trump-card to Resolve Exigencies in Targeted Gene Delivery.

    Science.gov (United States)

    Ajithkumar, K C; Pramod, Kannissery

    2018-01-01

    Viruses are potent pathogens that can effectively deliver the genetic material to susceptible host cells. This capability is beneficially utilized to successfully deliver the genetic material. However, the use of virus mediated gene delivery is considered divisive, because the potentially replicable genomes recombine or integrate with the cell DNA resulting in immunogenicity, ranging from inflammation to death. Thus, the need for potentially effective non-viral gene delivery vehicles arises. Non-viral vectors, protein only particles and virus like particles (VLP) can be constructed which contain all the necessary functional moieties. These resemble viruses and are called artificial or synthetic virus. The artificial virus eliminates the disadvantages of viral vectors but retain the beneficial effects of the viruses. Need for further functionalization can be avoided by this approach because incorporation of requisite agents such as cell ligands, membrane active peptides, etc. into proteins is possible. The protein- DNA complexes resemble bacterial inclusion bodies. Nucleic acids influence conformation of protein units which subsequently result in cell uptake and finally to the cell nucleus. Such tunable systems mimic the activities of infected viruses and are used for the safe and effective delivery of drugs and genetic material in gene therapy. The versatility, stability and biocompatible nature of artificial virus along with high transfection efficacy have made it favorite for gene delivery purposes, in addition to being useful for various biomedical and drug delivery applications. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  13. Gene delivery with viral vectors for cerebrovascular diseases

    Science.gov (United States)

    Gan, Yu; Jing, Zheng; Stetler, R. Anne; Cao, Guodong

    2017-01-01

    Recent achievements in the understanding of molecular events involved in the pathogenesis of central nervous system (CNS) injury have made gene transfer a promising approach for various neurological disorders, including cerebrovascular diseases. However, special obstacles, including the post-mitotic nature of neurons and the blood-brain barrier (BBB), constitute key challenges for gene delivery to the CNS. Despite the various limitations in current gene delivery systems, a spectrum of viral vectors has been successfully used to deliver genes to the CNS. Furthermore, recent advancements in vector engineering have improved the safety and delivery of viral vectors. Numerous viral vector-based clinical trials for neurological disorders have been initiated. This review will summarize the current implementation of viral gene delivery in the context of cerebrovascular diseases including ischemic stroke, hemorrhagic stroke and subarachnoid hemorrhage (SAH). In particular, we will discuss the potentially feasible ways in which viral vectors can be manipulated and exploited for use in neural delivery and therapy. PMID:23276981

  14. Receptor-Mediated Endocytosis and Brain Delivery of Therapeutic Biologics

    Directory of Open Access Journals (Sweden)

    Guangqing Xiao

    2013-01-01

    Full Text Available Transport of macromolecules across the blood-brain-barrier (BBB requires both specific and nonspecific interactions between macromolecules and proteins/receptors expressed on the luminal and/or the abluminal surfaces of the brain capillary endothelial cells. Endocytosis and transcytosis play important roles in the distribution of macromolecules. Due to the tight junction of BBB, brain delivery of traditional therapeutic proteins with large molecular weight is generally not possible. There are multiple pathways through which macromolecules can be taken up into cells through both specific and nonspecific interactions with proteins/receptors on the cell surface. This review is focused on the current knowledge of receptor-mediated endocytosis/transcytosis and brain delivery using the Angiopep-2-conjugated system and the molecular Trojan horses. In addition, the role of neonatal Fc receptor (FcRn in regulating the efflux of Immunoglobulin G (IgG from brain to blood, and approaches to improve the pharmacokinetics of therapeutic biologics by generating Fc fusion proteins, and increasing the pH dependent binding affinity between Fc and FcRn, are discussed.

  15. Gene Delivery for Metastatic Prostate Cancer Cells

    National Research Council Canada - National Science Library

    Pang, Shen

    2001-01-01

    .... Enhanced by the bystander effect, the specific expression of the DTA gene causes significant cell death in prostate cancer cell cultures, with very low background cell eradication in control cell lines...

  16. Engineered nonviral nanocarriers for intracellular gene delivery applications

    International Nuclear Information System (INIS)

    Ojea-Jiménez, Isaac; Puntes, Victor F; Tort, Olivia; Lorenzo, Julia

    2012-01-01

    The efficient delivery of nucleic acids into mammalian cells is a central aspect of cell biology and of medical applications, including cancer therapy and tissue engineering. Non-viral chemical methods have been received with great interest for transfecting cells. However, further development of nanocarriers that are biocompatible, efficient and suitable for clinical applications is still required. In this paper, the different material platforms for gene delivery are comparatively addressed, and the mechanisms of interaction with biological systems are discussed carefully. (paper)

  17. Selenium nanoparticles: potential in cancer gene and drug delivery.

    Science.gov (United States)

    Maiyo, Fiona; Singh, Moganavelli

    2017-05-01

    In recent decades, colloidal selenium nanoparticles have emerged as exceptional selenium species with reported chemopreventative and therapeutic properties. This has sparked widespread interest in their use as a carrier of therapeutic agents with results displaying synergistic effects of selenium with its therapeutic cargo and improved anticancer activity. Functionalization remains a critical step in selenium nanoparticles' development for application in gene or drug delivery. In this review, we highlight recent developments in the synthesis and functionalization strategies of selenium nanoparticles used in cancer drug and gene delivery systems. We also provide an update of recent preclinical studies utilizing selenium nanoparticles in cancer therapeutics.

  18. VEGF targeting in mesotheliomas using an interleukin-6 signal inhibitor based on adenovirus gene delivery.

    Science.gov (United States)

    Adachi, Yasuo; Yoshio-Hoshino, Naoko; Aoki, Chieko; Nishimoto, Norihiro

    2010-06-01

    Malignant mesotheliomas reportedly secrete interleukin-6 (IL-6) which augments production of vascular endothelial growth factor (VEGF) from mesothelioma cells. We previously reported the development of a new receptor inhibitor of IL-6 (NRI) by genetically engineering tocilizumab, a humanized anti-IL-6 receptor monoclonal antibody. Since NRI is encoded on a single gene, it is easily applicable to a gene delivery system using virus vehicles. In this study, we report VEGF targeting through NRI expression based on adenovirus-mediated gene delivery in mesothelioma cells. We constructed an NRI expression vector in the context of a tropism-modified adenovirus vector that had enhanced infectivity in mesothelioma cells. This virus effectively induced NRI secretion from mesothelioma cells. This virus infection also reduced the VEGF production in mesothelioma cells. These results indicate that NRI shows potential as an agent in the treatment of mesotheliomas.

  19. Ultrasound-mediated oncolytic virus delivery and uptake for increased therapeutic efficacy: state of art

    Directory of Open Access Journals (Sweden)

    Nande R

    2015-11-01

    Full Text Available Rounak Nande,1 Candace M Howard,2 Pier Paolo Claudio,3,4 1Department of Biochemistry and Microbiology, Marshall University School of Medicine, Huntington, WV, 2Department of Radiology, University of Mississippi Medical Center, Jackson, MS, 3Department of BioMolecular Sciences and National Center for Natural Products Research, School of Pharmacy, University of Mississippi, MS, 4Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS, USA Abstract: The field of ultrasound (US has changed significantly from medical imaging and diagnosis to treatment strategies. US contrast agents or microbubbles (MB are currently being used as potential carriers for chemodrugs, small molecules, nucleic acids, small interfering ribonucleic acid, proteins, adenoviruses, and oncolytic viruses. Oncolytic viruses can selectively replicate within and destroy a cancer cell, thus making them a powerful therapeutic in treating late-stage or metastatic cancer. These viruses have been shown to have robust activity in clinical trials when injected directly into tumor nodules. However limitations in oncolytic virus’ effectiveness and its delivery approach have warranted exploration of ultrasound-mediated delivery. Gene therapy bearing adenoviruses or oncolytic viruses can be coupled with MBs and injected intravenously. Following application of US energy to the target region, the MBs cavitate, and the resulting shock wave enhances drug, gene, or adenovirus uptake. Though the underlying mechanism is yet to be fully understood, there is evidence to suggest that mechanical pore formation of cellular membranes allows for the temporary uptake of drugs. This delivery method circumvents the limitations due to stimulation of the immune system that prevented intravenous administration of viruses. This review provides insight into this intriguing new frontier on the delivery of oncolytic viruses to tumor sites.Keywords: microbubbles, ultrasound

  20. Ex vivo culture of patient tissue & examination of gene delivery.

    LENUS (Irish Health Repository)

    Rajendran, Simon

    2012-01-31

    This video describes the use of patient tissue as an ex vivo model for the study of gene delivery. Fresh patient tissue obtained at the time of surgery is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial.

  1. Pancreatic Cancer Gene Therapy: From Molecular Targets to Delivery Systems

    Energy Technology Data Exchange (ETDEWEB)

    Fillat, Cristina, E-mail: cristina.fillat@crg.es; Jose, Anabel; Ros, Xavier Bofill-De; Mato-Berciano, Ana; Maliandi, Maria Victoria; Sobrevals, Luciano [Programa Gens i Malaltia, Centre de Regulació Genòmica-CRG, UPF, Parc de Recerca Biomedica de Barcelona-PRBB and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Barcelona (Spain)

    2011-01-18

    The continuous identification of molecular changes deregulating critical pathways in pancreatic tumor cells provides us with a large number of novel candidates to engineer gene-targeted approaches for pancreatic cancer treatment. Targets—both protein coding and non-coding—are being exploited in gene therapy to influence the deregulated pathways to facilitate cytotoxicity, enhance the immune response or sensitize to current treatments. Delivery vehicles based on viral or non-viral systems as well as cellular vectors with tumor homing characteristics are a critical part of the design of gene therapy strategies. The different behavior of tumoral versus non-tumoral cells inspires vector engineering with the generation of tumor selective products that can prevent potential toxic-associated effects. In the current review, a detailed analysis of the different targets, the delivery vectors, the preclinical approaches and a descriptive update on the conducted clinical trials are presented. Moreover, future possibilities in pancreatic cancer treatment by gene therapy strategies are discussed.

  2. Physical non-viral gene delivery methods for tissue engineering.

    Science.gov (United States)

    Mellott, Adam J; Forrest, M Laird; Detamore, Michael S

    2013-03-01

    The integration of gene therapy into tissue engineering to control differentiation and direct tissue formation is not a new concept; however, successful delivery of nucleic acids into primary cells, progenitor cells, and stem cells has proven exceptionally challenging. Viral vectors are generally highly effective at delivering nucleic acids to a variety of cell populations, both dividing and non-dividing, yet these viral vectors are marred by significant safety concerns. Non-viral vectors are preferred for gene therapy, despite lower transfection efficiencies, and possess many customizable attributes that are desirable for tissue engineering applications. However, there is no single non-viral gene delivery strategy that "fits-all" cell types and tissues. Thus, there is a compelling opportunity to examine different non-viral vectors, especially physical vectors, and compare their relative degrees of success. This review examines the advantages and disadvantages of physical non-viral methods (i.e., microinjection, ballistic gene delivery, electroporation, sonoporation, laser irradiation, magnetofection, and electric field-induced molecular vibration), with particular attention given to electroporation because of its versatility, with further special emphasis on Nucleofection™. In addition, attributes of cellular character that can be used to improve differentiation strategies are examined for tissue engineering applications. Ultimately, electroporation exhibits a high transfection efficiency in many cell types, which is highly desirable for tissue engineering applications, but electroporation and other physical non-viral gene delivery methods are still limited by poor cell viability. Overcoming the challenge of poor cell viability in highly efficient physical non-viral techniques is the key to using gene delivery to enhance tissue engineering applications.

  3. Calcium gluconate in phosphate buffered saline increases gene delivery with adenovirus type 5.

    Directory of Open Access Journals (Sweden)

    Marko T Ahonen

    Full Text Available BACKGROUND: Adenoviruses are attractive vectors for gene therapy because of their stability in vivo and the possibility of production at high titers. Despite exciting preclinical data with various approaches, there are only a few examples of clear efficacy in clinical trials. Effective gene delivery to target cells remains the key variable determining efficacy and thus enhanced transduction methods are important. METHODS/RESULTS: We found that heated serum could enhance adenovirus 5 mediated gene delivery up to twentyfold. A new protein-level interaction was found between fiber knob and serum transthyretin, but this was not responsible for the observed effect. Instead, we found that heating caused the calcium and phosphate present in the serum mix to precipitate, and this was responsible for enhanced gene delivery. This finding could have relevance for designing preclinical experiments with adenoviruses, since calcium and phosphate are present in many solutions. To translate this into an approach potentially testable in patients, we used calcium gluconate in phosphate buffered saline, both of which are clinically approved, to increase adenoviral gene transfer up to 300-fold in vitro. Gene transfer was increased with or without heating and in a manner independent from the coxsackie-adenovirus receptor. In vivo, in mouse studies, gene delivery was increased 2-, 110-, 12- and 13-fold to tumors, lungs, heart and liver and did not result in increased pro-inflammatory cytokine induction. Antitumor efficacy of a replication competent virus was also increased significantly. CONCLUSION: In summary, adenoviral gene transfer and antitumor efficacy can be enhanced by calcium gluconate in phosphate buffered saline.

  4. Delivery of gene-expressing fragments using quantum dot

    Science.gov (United States)

    Hoshino, Akiyoshi; Manabe, Noriyoshi; Hanada, Sanshiro; Fujioka, Kouki; Yasuhara, Masato; Kondo, Akihiko; Yamamoto, Kenji

    2009-02-01

    Gene therapy is an attractive approach to supplement a deficient gene function. Although there has been some success with specific gene delivery using various methods including viral vectors and liposomes, most of these methods have a limited efficiency or also carry a risk for oncogenesis. Fluorescent nanoparticles, such as nanocrystal quantum dots (QDs), have potential to be applied to molecular biology and bioimaging, since some nanocrystals emit higher and longer lasting fluorescence than conventional organic probes do. We herein report that quantum dots (QDs) conjugated with nuclear localizing signal peptides (NLSP) successfully introduced the gene-fragments with promoter elements, which promoted the expression of the enhanced green fluorescent protein (eGFP) gene in mammalian cells. The expression of eGFP protein was observed when the QD/geneconstruct was added to the culture media. The gene-expression efficiency varied depending on multiple factors around QDs, such as 1) the reading direction of gene fragments, 2) the quantity of gene fragments attached on the surface of QD-constructs, 3) the surface electronic charges varied according to the structure of QD/gene-constructs, and 4) the particle size of QD/gene complex varied according to the structure and amounts of gene fragments. Using this QD/geneconstruct system, eGFP protein could be detected 28 days after the gene-introduction whereas the fluorescence of QDs was disappeared. This system therefore provides another method for the intracellular delivery of gene-fragments without using either viral vectors or specific liposomes. These results suggest that inappropriate treatment and disposal of QDs may still have risks to the environmental pollution including human health under certain conditions. Here we propose the further research for the immune and physiological responses in not only immune cells but also other cells, in order to clear the effect of all other nanoscale products as well as nanocrystal

  5. Gene therapy for meningioma: improved gene delivery with targeted adenoviruses

    NARCIS (Netherlands)

    Dirven, Clemens M. F.; Grill, Jacques; Lamfers, Martine L. M.; van der Valk, Paul; Leonhart, Angelique M.; van Beusechem, Victor W.; Haisma, Hidde J.; Pinedo, Herbert M.; Curiel, David T.; Vandertop, W. Peter; Gerritsen, Winald R.

    2002-01-01

    OBJECT: Due to their surgical inaccessibility or aggressive behavior, some meningiomas cannot be cured with current treatment strategies. Gene therapy is an emerging strategy for the treatment of brain tumors, which the authors investigated to determine whether adenoviruses could be used for gene

  6. Gene therapy for meningioma : improved gene delivery with targeted adenoviruses

    NARCIS (Netherlands)

    Dirven, CMF; Grill, J; Lamfers, MLM; Van der Valk, P; Leonhart, AM; Van Beusechem, VW; Haisma, HJ; Pinedo, HM; Curiel, DT; Vandertop, WP; Gerritsen, WR

    Object. Due to their surgical inaccessibility or aggressive behavior, some meningiomas cannot be cured with current treatment strategies. Gene therapy is an emerging strategy for the treatment of brain tumors, which the authors investigated to determine whether adenoviruses could be used for gene

  7. Synergistic effects between natural histone mixtures and polyethylenimine in non-viral gene delivery in vitro.

    Science.gov (United States)

    Schneeweiss, Anne; Buyens, Kevin; Giese, Matthias; Sanders, Niek; Ulbert, Sebastian

    2010-11-15

    Nanoparticles made of plasmid DNA (pDNA) and cationic polymers are promising strategies for non-viral gene delivery. However, many cationic polymers are toxic to cells when used in higher concentrations. Positively charged proteins, such as histones, are biodegradable and a good alternative, especially for potential in vivo applications. It has previously been shown that histones are able to complex DNA and mediate transfection of cells. To investigate possible synergistic effects between the different histone types and to avoid the use of recombinant proteins, we analysed whether natural histone mixtures would be functional as gene carriers. Core and linker histones from calf thymus and from chicken erythrocytes were used to transfect different cell lines. The protein mixtures efficiently complexed the pDNA, and the resulting particles entered the cells. However, only marginal expression of the gene encoded by the pDNA was observed. Transfection rates increased drastically when minimal amounts of the basic polymer polyethylenimine (PEI) were added to the particles. Neither PEI nor histones alone mediated any transfection under the conditions where a combination of both worked efficiently, and the combined particles were well tolerated by the cells. These results demonstrate that histone mixtures from natural sources in combination with minimal amounts of PEI can be used as gene carriers. This might have consequences for the development of novel gene delivery strategies, such as DNA vaccines, with minimal side-effects. Copyright © 2010 Elsevier B.V. All rights reserved.

  8. Application of Ferriferous Oxide Modified by Chitosan in Gene Delivery

    Directory of Open Access Journals (Sweden)

    Yu Kuang

    2012-01-01

    Full Text Available New approaches to improve the traditional gene carriers are still required. Here we explore Fe3O4 modified with degradable polymers that enhances gene delivery and target delivery using permanent magnetic field. Two magnetic Fe3O4 nanoparticles coated with chitosan (CTS and polyethylene glycol (PEG were synthesized by means of controlled chemical coprecipitation. Plasmid pEGFP was encapsulated as a reported gene. The ferriferous oxide complexes were approximately spherical; surface charge of CTS-Fe3O4 and PEG-Fe3O4 was about 20 mv and 0 mv, respectively. The controlled release of DNA from the CTS-Fe3O4 nanoparticles was observed. Concurrently, a desired Fe3O4 concentration of less than 2 mM was verified as safe by means of a cytotoxicity test in vitro. Presence of the permanent magnetic field significantly increased the transfection efficiency. Furthermore, the passive target property and safety of magnetic nanoparticles were also demonstrated in an in vivo test. The novel gene delivery system was proved to be an effective tool required for future target expression and gene therapy in vivo.

  9. Preeclampsia mediates the association between shorter height and increased risk of preterm delivery.

    Science.gov (United States)

    Morisaki, Naho; Ogawa, Kohei; Urayama, Kevin Y; Sago, Haruhiko; Sato, Shoji; Saito, Shigeru

    2017-10-01

    Maternal short stature has been observed to increase the risk of preterm birth; however, the aetiology behind this phenomenon is unknown. We investigated whether preeclampsia, an obstetric complication that often leads to preterm delivery and is reported to have an inverse association with women's height, mediates this association. We studied 218 412 women with no underlying diseases before pregnancy, who delivered singletons from 2005 to 2011 and were included in the Japan Society of Obstetrics and Gynecology perinatal database, which is a national multi-centre-based delivery database among tertiary hospitals. We assessed the risk of preterm delivery in relation to height using multivariate analysis, and how the association was mediated by risk of preeclampsia using mediation analysis. Each 5-cm decrement in height was associated with significantly higher risk of preterm delivery [relative risk 1.20; 95% confidence interval (CI): 1.13, 1.27] and shorter gestational age (-0.30; 95% CI: -0.44, -0.16 weeks). Mediation analysis showed that the effect of shorter height on increased risk of preterm delivery, due to an indirect effect mediated through increased risk of preeclampsia, was substantial for shorter gestational age (48%), as well as risk of preterm delivery (28%). When examining the three subtypes of preterm delivery separately, mediated effect was largest for provider-initiated preterm delivery without premature rupture of membranes (PROM) (34%), compared with spontaneous preterm delivery without PROM (17%) or preterm delivery with PROM (0%). Preeclampsia partially mediates the association between maternal short stature and preterm delivery.

  10. Local Gene Delivery System by Bubble Liposomes and Ultrasound Exposure into Joint Synovium

    Directory of Open Access Journals (Sweden)

    Yoichi Negishi

    2011-01-01

    Full Text Available Recently, we have developed novel polyethylene glycol modified liposomes (bubble liposomes; BL entrapping an ultrasound (US imaging gas, which can work as a gene delivery tool with US exposure. In this study, we investigated the usefulness of US-mediated gene transfer systems with BL into synoviocytes in vitro and joint synovium in vivo. Highly efficient gene transfer could be achieved in the cultured primary synoviocytes transfected with the combination of BL and US exposure, compared to treatment with plasmid DNA (pDNA alone, pDNA plus BL, or pDNA plus US. When BL was injected into the knee joints of mice, and US exposure was applied transcutaneously to the injection site, highly efficient gene expression could be observed in the knee joint transfected with the combination of BL and US exposure, compared to treatment with pDNA alone, pDNA plus BL, or pDNA plus US. The localized and prolonged gene expression was also shown by an in vivo luciferase imaging system. Thus, this local gene delivery system into joint synovium using the combination of BL and US exposure may be an effective means for gene therapy in joint disorders.

  11. Acidity-responsive gene delivery for "superfast" nuclear translocation and transfection with high efficiency.

    Science.gov (United States)

    Zhu, Jing-Yi; Zeng, Xuan; Qin, Si-Yong; Wan, Shuang-Shuang; Jia, Hui-Zhen; Zhuo, Ren-Xi; Feng, Jun; Zhang, Xian-Zheng

    2016-03-01

    In principle, not only efficient but rapid transfection is required since it can maximize the bioavailability of vector-carried gene prior to the cellular excretion. However, the "rapid" goal has been paid few attentions so far in the research field of vector-aided transfection. As a pioneering attempt, the present study designed a lysosome-targeting acidity-responsive nanoassembly as gene vectors, which proved the amazing potency to mediate the "Superfast" transnuclear gene transport and gene transfection with high efficiency in vitro and in vivo. The nanoassembly was constructed on the pH-reversible covalent boronic acid-diol coupling between 1,3-diol-rich oligoethylenimine (OEI-EHDO) and phenylboronic acid modified cholesterol (Chol-PBA). The rapid and efficient nuclei-tropic delivery and transfection was demonstrated to highly rely on the lysosome-acidity induced assembly destruction followed by the easy liberation of gene payloads inside cells. The nanoassembly-mediated transfection at 8 h can afford the outcome even comparable to that achieved at 48 h by the golden standard of PEI25k, and the transfection efficiency can still remain at a high level during 48 h. In contrast, time-dependent efficiency enhancement was identified for the transfections using PEI25k and OEI-EHDO as delivery vectors. Moreover, owing to the hydroxyl-rich surface, this delivery nanosystem presented strong tolerance to the serum-induced transfection inhibition that frequently occurred for the polycationic gene vectors such as PEI25k. The in vitro and in vivo results manifested the low toxicity of this bio-decomposable nanoassembly. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Protein kinase A inhibition modulates the intracellular routing of gene delivery vehicles in HeLa cells, leading to productive transfection

    NARCIS (Netherlands)

    Rehman, Zia Ur; Hoekstra, Dick; Zuhorn, Inge S.

    2011-01-01

    Cellular entry of nanoparticles for drug- and gene delivery relies on various endocytic pathways, including clathrin-and caveolae-mediated endocytosis. To improve delivery, i.e., the therapeutic and/or cell biological impact, current efforts are aimed at avoiding processing of the carriers along the

  13. Theory and in vivo application of electroporative gene delivery.

    Science.gov (United States)

    Somiari, S; Glasspool-Malone, J; Drabick, J J; Gilbert, R A; Heller, R; Jaroszeski, M J; Malone, R W

    2000-09-01

    Efficient and safe methods for delivering exogenous genetic material into tissues must be developed before the clinical potential of gene therapy will be realized. Recently, in vivo electroporation has emerged as a leading technology for developing nonviral gene therapies and nucleic acid vaccines (NAV). Electroporation (EP) involves the application of pulsed electric fields to cells to enhance cell permeability, resulting in exogenous polynucleotide transit across the cytoplasmic membrane. Similar pulsed electrical field treatments are employed in a wide range of biotechnological processes including in vitro EP, hybridoma production, development of transgenic animals, and clinical electrochemotherapy. Electroporative gene delivery studies benefit from well-developed literature that may be used to guide experimental design and interpretation. Both theory and experimental analysis predict that the critical parameters governing EP efficacy include cell size and field strength, duration, frequency, and total number of applied pulses. These parameters must be optimized for each tissue in order to maximize gene delivery while minimizing irreversible cell damage. By providing an overview of the theory and practice of electroporative gene transfer, this review intends to aid researchers that wish to employ the method for preclinical and translational gene therapy, NAV, and functional genomic research.

  14. Co-factors necessary for PPAR mediated transactivation of endogenous target genes

    DEFF Research Database (Denmark)

    Grøntved, Lars; Nielsen, Ronni; Stunnenberg, Henk

    of endogenous target gene in different cell types are elusive. To mutually compare the ability of the PPAR subtypes to activate endogenous target genes in a given cell, PPARa, PPARb/d and PPARg2 were HA tagged and rapidly, equally and synchronously expressed using adenoviral delivery. Within a few hours after...... subtype specific activation of target genes. Accumulating evidence suggests that transcriptional co-factors can function as master regulators for nuclear receptors and impose promoter selectivity. To study co-factor necessity for PPAR mediated transactivation of endogenous target genes, specific co...

  15. A novel Listeria monocytogenes-based DNA delivery system for cancer gene therapy.

    LENUS (Irish Health Repository)

    van Pijkeren, Jan Peter

    2012-01-31

    Bacteria-mediated transfer of plasmid DNA to mammalian cells (bactofection) has been shown to have significant potential as an approach to express heterologous proteins in various cell types. This is achieved through entry of the entire bacterium into cells, followed by release of plasmid DNA. In a murine model, we show that Listeria monocytogenes can invade and spread in tumors, and establish the use of Listeria to deliver genes to tumors in vivo. A novel approach to vector lysis and release of plasmid DNA through antibiotic administration was developed. Ampicillin administration facilitated both plasmid transfer and safety control of vector. To further improve on the gene delivery system, we selected a Listeria monocytogenes derivative that is more sensitive to ampicillin, and less pathogenic than the wild-type strain. Incorporation of a eukaryotic-transcribed lysin cassette in the plasmid further increased bacterial lysis. Successful gene delivery of firefly luciferase to growing tumors in murine models and to patient breast tumor samples ex vivo was achieved. The model described encompasses a three-phase treatment regimen, involving (1) intratumoral administration of vector followed by a period of vector spread, (2) systemic ampicillin administration to induce vector lysis and plasmid transfer, and (3) systemic administration of combined moxifloxacin and ampicillin to eliminate systemic vector. For the first time, our results reveal the potential of Listeria monocytogenes for in vivo gene delivery.

  16. Delivery of human NKG2D-IL-15 fusion gene by chitosan nanoparticles to enhance antitumor immunity

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Chen; Jie, Leng; Yongqi, Wang [Department of Immunology, School of Medicine, Yangzhou University, Yangzhou, 225009 (China); Weiming, Xiao [Department of Gastroenterology, The Second Clinical Medical College, Yangzhou University, Yangzhou, 225009 (China); Juqun, Xi [Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Senile Diseases, Yangzhou, 225009 (China); Yanbing, Ding [Department of Gastroenterology, The Second Clinical Medical College, Yangzhou University, Yangzhou, 225009 (China); Li, Qian [Department of Immunology, School of Medicine, Yangzhou University, Yangzhou, 225009 (China); Xingyuan, Pan [Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, 225009 (China); Mingchun, Ji [Department of Immunology, School of Medicine, Yangzhou University, Yangzhou, 225009 (China); Weijuan, Gong, E-mail: wjgong@yzu.edu.cn [Department of Immunology, School of Medicine, Yangzhou University, Yangzhou, 225009 (China); Department of Gastroenterology, The Second Clinical Medical College, Yangzhou University, Yangzhou, 225009 (China); Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Senile Diseases, Yangzhou, 225009 (China); Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, 225009 (China); Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009 (China)

    2015-07-31

    Nanoparticles are becoming promising carriers for gene delivery because of their high capacity in gene loading and low cell cytotoxicity. In this study, a chitosan-based nanoparticle encapsulated within a recombinant pcDNA3.1-dsNKG2D-IL-15 plasmid was generated. The fused dsNKG2D-IL-15 gene fragment consisted of double extracellular domains of NKG2D with IL-15 gene at downstream. The average diameter of the gene nanoparticles ranged from 200 nm to 400 nm, with mean zeta potential value of 53.8 ± 6.56 mV. The nanoparticles which were loaded with the dsNKG2D-IL-15 gene were uptaken by tumor cells with low cytotoxicity. Tumor cells pre-transfected by gene nanopartilces stimulated NK and T cells in vitro. Intramuscular injection of gene nanoparticles suppressed tumor growth and prolonged survival of tumor-bearing mice through activation of NK and CD8{sup +} T cells. Thus, chitosan-based nanoparticle delivery of dsNKG2D-IL-15 gene vaccine can be potentially used for tumor therapy. - Highlights: • Generation of a nanoparticle for delivery of dsNKG2D-IL-15 gene. • Characterization of the gene nanoparticle. • Antitumor activity mediated by the gene nanoparticle.

  17. Layered double hydroxide nanoparticles in gene and drug delivery.

    Science.gov (United States)

    Ladewig, Katharina; Xu, Zhi Ping; Lu, Gao Qing Max

    2009-09-01

    Layered double hydroxides (LDHs) have been known for many decades as catalyst and ceramic precursors, traps for anionic pollutants, catalysts and additives for polymers, but their successful synthesis on the nanometer scale a few years ago opened up a whole new field for their application in nanomedicine. The delivery of drugs and other therapeutic/bioactive molecules (e.g., peptides, proteins, nucleic acids) to mammalian cells is an area of research that is of tremendous importance to medicine and provides manifold applications for any new developments in the area of nanotechnology. Among the many different nanoparticles that have been shown to facilitate gene and/or drug delivery, LDH nanoparticles have attracted particular attention owing to their many desirable properties. This review aims to report recent progress in gene and drug delivery using LDH nanoparticles. It summarizes the advantages and disadvantages of using LDH nanoparticles as carriers for nucleic acids and drugs against the general background of bottlenecks that are encountered by cellular delivery systems. It describes further the models that have been proposed for the internalization of LDH nanoparticles into cells so far and discusses the intracellular fate of the particles and their cargo. The authors offer some remarks on how this field of research will progress in the near future and which challenges need to be overcome before LDH nanoparticles can be used in a clinical setting.

  18. A variable gene delivery carrier-biotinylated chitosan/polyethyleneimine

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Yi-Chen; Young, Tai-Horng [Institute of Polymer Science and Engineering, College of Engineering, National Taiwan University, Taipei 106, Taiwan (China); Chang, Fu-Hsiung [Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei 100, Taiwan (China); Wei, Ming-Feng, E-mail: thyoung@ntu.edu.t [Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei 100, Taiwan (China)

    2010-12-15

    A variable gene delivery system has been developed based on conjugating chitosan to biotin through a functionalized poly(ethylene glycol) (PEG) spacer, which can be used to further bind different molecules on the outer layer of a polymer/DNA complex by streptavidin (SA)-biotin linkage. In this study, TAT-conjugated SA was used as the model molecule to prove the conjugation function of the prepared complex. In addition, low-molecular-weight poly(ethyleneimine) (PEI) was added into the polymer/DNA complex to increase the transfection efficiency. The results of the luciferase assay show that the transfection efficiency of the prepared complex was significantly correlated with the amount of PEI and was further enhanced when TAT was conjugated to the complex by SA-biotin linkage. Considered to have negligible cytotoxic effects, the variable gene delivery complex prepared in this study would be of considerable potential as carriers for in vitro applications.

  19. Prodrug converting enzyme gene delivery by L. monocytogenes

    Directory of Open Access Journals (Sweden)

    Pilgrim Sabine

    2008-04-01

    Full Text Available Abstract Background Listeria monocytogenes is a highly versatile bacterial carrier system for introducing protein, DNA and RNA into mammalian cells. The delivery of tumor antigens with the help of this carrier into tumor-bearing animals has been successfully carried out previously and it was recently reported that L. monocytogenes is able to colonize and replicate within solid tumors after local or even systemic injection. Methods Here we report on the delivery of two prodrug converting enzymes, purine-deoxynucleoside phosphorylase (PNP and a fusion protein consisting of yeast cytosine deaminase and uracil phosphoribosyl transferase (FCU1 into cancer cells in culture by L. monocytogenes. Transfer of the prodrug converting enzymes was achieved by bacterium mediated transfer of eukaryotic expression plasmids or by secretion of the proteins directly into the host cell cytosol by the infecting bacteria. Results The results indicate that conversion of appropriate prodrugs to toxic drugs in the cancer cells occured after both procedures although L. monocytogenes-mediated bactofection proved to be more efficient than enzyme secretion 4T1, B16 and COS-1 tumor cells. Exchanging the constitutively PCMV-promoter with the melanoma specific P4xTETP-promoter resulted in melanoma cell-specific expression of the prodrug converting enzymes but reduced the efficiencies. Conclusion These experiments open the way for bacterium mediated tumor specific activation of prodrugs in live animals with tumors.

  20. Optimization of ultrasound parameters for microbubble-nanoliposome complex-mediated delivery.

    Science.gov (United States)

    Yoon, Young Il; Yoon, Tae-Jong; Lee, Hak Jong

    2015-10-01

    The aim of this study was to identify the optimal ultrasound (US) parameters for gene and drug delivery. In order to target SkBr3, which is a breast cancer cell overexpressing the Her2 receptor, trastuzumab (Herceptin) was used. Micobubble-nanoliposome complex (MLC) was mixed with trastuzumab and stored overnight. Finally, MLC was combined with Her2Ab. A US device equipped with a 1-MHz probe was used for delivery to the cell. Several parameters, including intensity (w/cm(2)), time (minutes), and duty cycle (%), were varied within a range from 1 w/cm(2), 1 minute, and 20% to 2 w/cm(2), 2 minutes, and 60%, respectively. A confocal laser scanning microscope (CLSM) was used to confirm the delivery of MLC to the cells after US treatment. MLC with fluorescent dyes and trastuzumab was synthesized successfully. By delivering MLC with Her2Ab to cells, the targeting effect of trastuzumab with MLC was confirmed by CLSM. The cell membranes showed green (fluorescein isothiocyanate) and red (Texas red) fluorescence but treatments with MLC without Her2Ab did not show any fluorescence. Optimal conditions for US-mediated delivery were 1 or 2 w/cm(2), 2 minutes, and 60% (uptake ratio, 95.9% for 1 w/cm(2) and 95.7% for 2 w/cm(2)) for hydrophobic materials and 2 w/cm(2), 2 minutes, and 60% (uptake ratio, 95.0%) for hydrophilic materials. The greater the strength, duty cycle, and period of US application within the tested range, the more efficiently the fluorescent contents were conveyed.

  1. Optimization of ultrasound parameters for microbubble-nanoliposome complex-mediated delivery

    Directory of Open Access Journals (Sweden)

    Young Il Yoon

    2015-10-01

    Full Text Available Purpose: The aim of this study was to identify the optimal ultrasound (US parameters for gene and drug delivery. Methods: In order to target SkBr3, which is a breast cancer cell overexpressing the Her2 receptor, trastuzumab (Herceptin was used. Micobubble-nanoliposome complex (MLC was mixed with trastuzumab and stored overnight. Finally, MLC was combined with Her2Ab. A US device equipped with a 1-MHz probe was used for delivery to the cell. Several parameters, including intensity (w/cm2, time (minutes, and duty cycle (%, were varied within a range from 1 w/cm2, 1 minute, and 20% to 2 w/cm2, 2 minutes, and 60%, respectively. A confocal laser scanning microscope (CLSM was used to confirm the delivery of MLC to the cells after US treatment. Results: MLC with fluorescent dyes and trastuzumab was synthesized successfully. By delivering MLC with Her2Ab to cells, the targeting effect of trastuzumab with MLC was confirmed by CLSM. The cell membranes showed green (fluorescein isothiocyanate and red (Texas red fluorescence but treatments with MLC without Her2Ab did not show any fluorescence. Optimal conditions for US-mediated delivery were 1 or 2 w/cm2, 2 minutes, and 60% (uptake ratio, 95.9% for 1 w/cm2 and 95.7% for 2 w/cm2 for hydrophobic materials and 2 w/cm2, 2 minutes, and 60% (uptake ratio, 95.0% for hydrophilic materials. Conclusion: The greater the strength, duty cycle, and period of US application within the tested range, the more efficiently the fluorescent contents were conveyed.

  2. Membrane-Mimic Nanoparticles for Drug and Gene Delivery

    KAUST Repository

    Alamoudi, Kholod

    2017-12-01

    Nanoscale organic particles have gained a prominent role in drug and gene delivery field. As the nature of the nanoparticle’s (NPs) surface plays a major role in their targeting efficiency, bioavailability, and cytotoxicity, membrane-mimic nanoparticles are considered highly attractive materials for in vivo and in vitro applications. Synthetic membrane vesicles (liposomes) and nanoconstructs built with native cancer cellular membrane are excellent scaffolds to improve cellular delivery. Liposomes have been extensively used due to their high loading capacity, biocompatibility and biodegradability. However, modifications with stimuli responsive materials are highly needed to improve their stability and turn them active participants in controlled delivery. Towards a nature inspired approach, reconstructed bilayers from cell membrane are a good candidate to enhance NP’s targeting ability and biocompatibility. The primary focus of this research is to develop smart responsive (lipid) membrane coated NPs with surface modifications for controlled and targeted drug and/or gene delivery for application in cancer therapy. Three approaches have been developed, namely i) liposomes as thermoresponsive nanocarriers for the delivery of genetic material; ii) magnetically photosensitive liposome hybrids and iii) biomimetic periodic mesoporous organo silica engineered for better a biocompatibility and targeting capabilities. In the first project synthetic liposomes were loaded with ammonium bicarbonate salt (ABC) and siRNA. The combination of lipids chosen and the relative ratios allowed the rapid release of the genetic material to the multi drug resistant cancer cells studied, upon external heat trigger. This design has improved the gene silencing efficiency via successful endosomal escape. In the second project, SPIO@Au nanoparticles were imbedded in the lipid bilayer to produce a photo/thermal responsive carrier that could be also used in cell imaging besides gene transfection

  3. Follistatin allows efficient retroviral-mediated gene transfer into rat liver

    International Nuclear Information System (INIS)

    Borgnon, Josephine; Djamouri, Fatima; Lorand, Isabelle; Rico, Virginie Di; Loux, Nathalie; Pages, Jean-Christophe; Franco, Dominique; Capron, Frederique; Weber, Anne

    2005-01-01

    Retroviral vectors are widely used tools for gene therapy. However, in vivo gene transfer is only effective in dividing cells, which, in liver, requires a regenerative stimulus. Follistatin is effective in promoting liver regeneration after 90% and 70% hepatectomy in rats. We studied its efficacy on liver regeneration and retroviral-mediated gene delivery in 50% hepatectomized rats. When human recombinant follistatin was infused into the portal vein immediately after 50% hepatectomy, hepatocyte proliferation was significantly higher than in control 50% hepatectomized rats. A single injection of virus particles administered 23 h after follistatin infusion resulted in more than 20% gene transduction efficiency in hepatocytes compared to 3% in control rats. It is concluded that a single injection of follistatin induces onset of proliferation in 50% hepatectomized rats and allows efficient retroviral-mediated gene transfer to the liver

  4. Pancreatic Cancer Gene Therapy: From Molecular Targets to Delivery Systems

    Directory of Open Access Journals (Sweden)

    Maria Victoria Maliandi

    2011-01-01

    Full Text Available The continuous identification of molecular changes deregulating critical pathways in pancreatic tumor cells provides us with a large number of novel candidates to engineer gene-targeted approaches for pancreatic cancer treatment. Targets—both protein coding and non-coding—are being exploited in gene therapy to influence the deregulated pathways to facilitate cytotoxicity, enhance the immune response or sensitize to current treatments. Delivery vehicles based on viral or non-viral systems as well as cellular vectors with tumor homing characteristics are a critical part of the design of gene therapy strategies. The different behavior of tumoral versus non-tumoral cells inspires vector engineering with the generation of tumor selective products that can prevent potential toxic-associated effects. In the current review, a detailed analysis of the different targets, the delivery vectors, the preclinical approaches and a descriptive update on the conducted clinical trials are presented. Moreover, future possibilities in pancreatic cancer treatment by gene therapy strategies are discussed.

  5. Mannosylated Chitosan Nanoparticles Based Macrophage-Targeting Gene Delivery System Enhanced Cellular Uptake and Improved Transfection Efficiency.

    Science.gov (United States)

    Peng, Yixing; Yao, Wenjun; Wang, Bo; Zong, Li

    2015-04-01

    Gene transfer mediated by mannosylated chitosan (MCS) is a safe and promising approach for gene and vaccine delivery. MCS nanoparticles based gene delivery system showed high in vivo delivery efficiency and elicited strong immune responses in mice. However, little knowledge about the cell binding, transfection efficiency and intracellular trafficking of MCS nanoparticles had been acquired. In this study, using gastrin-releasing peptide as a model plasmid (pGRP), the binding of MCS/pGRP nanoparticles to macrophages and the intracellular trafficking of MCS/pGRP nanoparticles in macrophages were investigated. MCS-mediated transfection efficiency in macrophages was also evaluated using pGL-3 as a reporter gene. The results showed that the binding and transfection efficiency of MCS nanoparticles in macrophages was higher than that of CS, which was attributed to the interaction between mannose ligands in MCS and mannose receptors on the surface of macrophages. Observation with a confocal laser scanning microscope indicated the cellular uptake of MCS/pGRP nanoparticles were more than that of CS/pGRP nanoparticles in macrophages. MCS/pGRP nanoparticles were taken up by macrophages and most of them were entrapped in endosomal/lysosomal compartments. After the nanoparticles escaping from endosomal/lysosomal compartments, naked pGRP entered the nucleus, and a few MCS might enter the nucleus in terms of nanoparticles. Overall, MCS has the potential to be an excellent macrophage-targeting gene delivery carrier.

  6. Evaluation of carrier-mediated siRNA delivery

    DEFF Research Database (Denmark)

    Colombo, Stefano; Nielsen, Hanne Mørck; Foged, Camilla

    2013-01-01

    Harnessing the RNA interference (RNAi) process with chemically synthesized small interfering RNA (siRNA) is dependent on the development of efficient delivery vehicles that can help overcome the numerous barriers existing for siRNA delivery. However, quantifying the intracellular amount of si......RNA delivery. An in vitro cell culture model system expressing enhanced green fluorescent protein (EGFP) was used to develop the assay, which was based on the intracellular quantification of a full-length double-stranded Dicer substrate siRNA by stem-loop RT qPCR. The result is a well-documented protocol...... for accurate and sensitive determination of the effective intracellular siRNA concentration upon transfection with different reagents. Specific guidelines for the customization of the protocol are provided and reported together with an example of its application for studying a specific siRNA delivery case...

  7. Mannan-Modified PLGA Nanoparticles for Targeted Gene Delivery

    Directory of Open Access Journals (Sweden)

    Fansheng Kong

    2012-01-01

    Full Text Available The studies of targeted gene delivery nanocarriers have gained increasing attention during the past decades. In this study, mannan modified DNA loaded bioadhesive PLGA nanoparticles (MAN-DNA-NPs were investigated for targeted gene delivery to the Kupffer cells (KCs. Bioadhesive PLGA nanoparticles were prepared and subsequently bound with pEGFP. Following the coupling of the mannan-based PE-grafted ligands (MAN-PE with the DNA-NPs, the MAN-DNA-NPs were delivered intravenously to rats. The transfection efficiency was determined from the isolated KCs and flow cytometry was applied for the quantitation of gene expression after 48 h post transfection. The size of the MAN-DNA-NPs was found to be around 190 nm and the Zeta potential was determined to be −15.46mV. The pEGFP binding capacity of MAN-DNA-NPs was (88.9±5.8% and the in vitro release profiles of the MAN-DNA-NPs follow the Higuchi model. When compared with non-modified DNA-NPs and Lipofectamine 2000-DNA, MAN-DNA-NPs produced the highest gene expressions, especially in vivo. The in vivo data from flow cytometry analysis showed that MAN-DNA-NPs displayed a remarkably higher transfection efficiency (39% than non-modified DNA-NPs (25% and Lipofectamine 2000-DNA (23% in KCs. The results illustrate that MAN-DNA-NPs have the ability to target liver KCs and could function as promising active targeting drug delivery vectors.

  8. Chitosan-mediated vaccine delivery through the intestinal epithelium

    OpenAIRE

    Cole, Hannah

    2016-01-01

    Chitosan is one of the most widely studied materials in the field of drug delivery. Chitosan nanoparticles produced by ionic gelation can encapsulate macromolecular drug cargo. This work explores the potential of a specific chitosan molecule, ultrapure chitosan chloride, for oral vaccine delivery in vitro using polarised Caco-2 epithelial cells and in vivo using Balb/C mice. The chosen antigen used in the chitosan nanoparticles was ovalbumin (OVA). Work revealed an optimum concentration ...

  9. BMP2 gene delivery to bone mesenchymal stem cell by chitosan-g-PEI nonviral vector

    Science.gov (United States)

    Yue, Jianhui; Wu, Jun; Liu, Di; Zhao, Xiaoli; Lu, William W.

    2015-04-01

    Nanotechnology has made a significant impact on the development of nanomedicine. Nonviral vectors have been attracting more attention for the advantage of biosafety in gene delivery. Polyethylenimine (PEI)-conjugated chitosan (chitosan-g-PEI) emerged as a promising nonviral vector and has been demonstrated in many tumor cells. However, there is a lack of study focused on the behavior of this vector in stem cells which hold great potential in regenerative medicine. Therefore, in this study, in vitro gene delivering effect of chitosan-g-PEI was investigated in bone marrow stem cells. pIRES2-ZsGreen1-hBMP2 dual expression plasmid containing both the ZsGreen1 GFP reporter gene and the BMP2 functional gene was constructed for monitoring the transgene expression level. Chitosan-g-PEI-mediated gene transfer showed 17.2% of transfection efficiency and more than 80% of cell viability in stem cells. These values were higher than that of PEI. The expression of the delivered BMP2 gene in stem cells enhanced the osteogenic differentiation. These results demonstrated that chitosan-g-PEI is capable of applying in delivering gene to stem cells and providing potential applications in stem cell-based gene therapy.

  10. Microbubble-mediated ultrasound drug-delivery and therapeutic monitoring.

    Science.gov (United States)

    Sennoga, Charles A; Kanbar, Emma; Auboire, Laurent; Dujardin, Paul-Armand; Fouan, Damien; Escoffre, Jean-Michel; Bouakaz, Ayache

    2017-09-01

    Recent developments in ultrasound imaging and ultrasound contrast agents (UCAs) improved diagnostic confidence in echography and set into motion their combined use as a tool for drug delivery and therapeutic monitoring. Non-invasive, precise and targeted delivery of drug molecules to pathological tissues by employing different mechanisms of drug release is becoming feasible. Areas covered: We sought to describe: the nature and features of UCAs; outline current contrast-specific imaging modes; before describing a variety of strategies for using ultrasound and microbubbles as a drug delivery system. Our expert opinion focusses on results and prospects of using ultrasound and microbubbles as a dual modality for drug delivery and therapeutic monitoring. Expert opinion: Today, ultrasound and microbubbles present a realistic prospect as drug delivery tools that have been demonstrated in a variety of animal models and clinical indications. Besides delivering drugs, ultrasound and microbubbles have demonstrated added value through therapeutic monitoring and assessment. Successful evaluation of the sonoporation mechanism(s), ultrasound parameters, drug type and dose will need to be addressed before translating this technology for clinic use. Ultimately, the development of a strategy for monitoring targeted delivery and its implementation in clinical practice would advance therapeutic treatment to a new qualitative level.

  11. Hydrogels: a journey from diapers to gene delivery.

    Science.gov (United States)

    Chawla, Pooja; Srivastava, Alok Ranjan; Pandey, Priyanka; Chawla, Viney

    2014-02-01

    Hydrogels are the biomaterials comprising network of natural or synthetic polymers capable of absorbing large amount of water. Hydrogels are "Smart Gels" or "Intelligent Gels" which can be made to respond to the various environmental conditions like temperature, pH, magnetic/electric field, ionic strength, inflammation, external stress etc. There are numerous potential applications of hydrogels in modern day life ranging from a diaper to gene delivery. This review succinctly describes the classification, properties and preparation methods along with numerous diverse applications of hydrogels like agricultural hydrogels, hydrogel for drug delivery, sensing, dental adhesives, wound healing and tissue regeneration, diet aid and gastric retention and in tissue engineering etc. Hydrogels can be regarded as highly valuable biomaterials for human-beings.

  12. Advances in ultrasound-targeted microbubble-mediated gene therapy for liver fibrosis

    Directory of Open Access Journals (Sweden)

    Cuiyuan Huang

    2017-07-01

    Full Text Available Hepatic fibrosis develops as a wound-healing scar in response to acute and chronic liver inflammation and can lead to cirrhosis in patients with chronic hepatitis B and C. The condition arises due to increased synthesis and reduced degradation of extracellular matrix (ECM and is a common pathological sequela of chronic liver disease. Excessive deposition of ECM in the liver causes liver dysfunction, ascites, and eventually upper gastrointestinal bleeding as well as a series of complications. However, fibrosis can be reversed before developing into cirrhosis and has thus been the subject of extensive researches particularly at the gene level. Currently, therapeutic genes are imported into the damaged liver to delay or prevent the development of liver fibrosis by regulating the expression of exogenous genes. One technique of gene delivery uses ultrasound targeting of microbubbles combined with therapeutic genes where the time and intensity of the ultrasound can control the release process. Ultrasound irradiation of microbubbles in the vicinity of cells changes the permeability of the cell membrane by its cavitation effect and enhances gene transfection. In this paper, recent progress in the field is reviewed with emphasis on the following aspects: the types of ultrasound microbubbles, the construction of an ultrasound-mediated gene delivery system, the mechanism of ultrasound microbubble–mediated gene transfer and the application of ultrasound microbubbles in the treatment of liver fibrosis.

  13. Long circulating polymeric nanoparticles for gene/drug delivery.

    Science.gov (United States)

    Hu, Jiaming; Sheng, Yan; Shi, Junfeng; Yu, Bohao; Yu, Zhiqiang; Liao, Guochao

    2017-12-07

    The major limitation in the improving polymeric nanoparticles into an efficient gene/drug delivery carrier is the rapid opsonization, phagocytic uptake by mononuclear phagocyte system and subsequent clearance from the bloodstream. The prolonged circulation time of nanoparticles in the blood is a prerequisite to realizing a controlled and targeted (passive or active targeting) release of the encapsulated gene/drug at the desired site of action. In this review, the factors such as biological barriers and physical barriers including particle size, shape, zeta potential, and hydrophilicity will be discussed, which can cause effects on blood clearance and organ accumulation. Some natural and synthetic polymers utilized in long-circulating nanoparticles will also be discussed. The most popular method to mask or camouflage nanoparticles is the adsorbed, grafted or conjugated of poly (ethylene glycol) (PEG) or other hydrophilic polymers (e.g. polysaccharides) to the particle surface. Surface modification of nanoparticles with these polymers results in an increased blood circulation time by several orders of magnitude in comparison to the bare nanoparticles. However, the circulation half-life of nanoparticles still cannot satisfy the need for clinical use. At present, identification of novel potential coating materials is an emerging field of interest in the design of long-circulating polymer-based nanoparticulate gene/drug delivery. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  14. Biolistic DNA delivery to mice with the low pressure gene gun.

    Science.gov (United States)

    Yen, Meng-Chi; Lai, Ming-Derg

    2013-01-01

    Biolistic DNA delivery is an approach to deliver plasmid to culture cells, plants, or animals. Plasmid DNA is usually transferred through bombardment of DNA-coated particles by highly pressurized gas in various kinds of delivery vehicles. The low pressure gene gun can deliver plasmid at lower pressure. Here, we describe methods of biolistic DNA delivery to mice using the low pressure gene gun.

  15. Efficient gene delivery using chitosan-polyethylenimine hybrid systems

    International Nuclear Information System (INIS)

    Jiang, Hu-Lin; Kim, Tae-Hee; Kim, You-Kyoung; Park, In-Young; Cho, Chong-Su; Cho, Myung-Haing

    2008-01-01

    Chitosan and chitosan derivatives have been investigated as non-viral vectors because they have several advantages, such as biocompatibility, biodegradability, low cytotoxicity and low immunogenicity. However, low transfection efficiency and low cell specificity must be solved for their use in clinical trials. In this paper, chitosan-polyethylenimine (PEI) hybrid systems such as chitosan/PEI blend and chitosan-graft-PEI are described for efficient gene delivery because the PEI has high transfection efficiency owing to a proton sponge effect and chitosan has biocompatibility. Also, hepatocyte specificity of the galactosylated chitosan is explained after combination with PEI

  16. Dual delivery systems based on polyamine analog BENSpm as prodrug and gene delivery vectors

    Science.gov (United States)

    Zhu, Yu

    Combination drug and gene therapy shows promise in cancer treatment. However, the success of such strategy requires careful selection of the therapeutic agents, as well as development of efficient delivery vectors. BENSpm (N 1, N11-bisethylnorspermine), a polyamine analogue targeting the intracellular polyamine pathway, draws our special attention because of the following reasons: (1) polyamine pathway is frequently dysregulated in cancer; (2) BENSpm exhibits multiple functions to interfere with the polyamine pathway, such as to up-regulate polyamine metabolism enzymes and down-regulate polyamine biosynthesis enzymes. Therefore BENSpm depletes all natural polyamines and leads to apoptosis and cell growth inhibition in a wide range of cancers; (3) preclinical studies proved that BENSpm can act synergistically with various chemotherapy agents, making it a promising candidate in combination therapy; (4) multiple positive charges in BENSpm enable it as a suitable building block for cationic polymers, which can be further applied to gene delivery. In this dissertation, our goal was to design dual-function delivery vector based on BENSpm that can function as a gene delivery vector and, after intracellular degradation, as an active anticancer agent targeting dysregulated polyamine metabolism. We first demonstrated strong synergism between BENSpm and a potential therapeutic gene product TRAIL. Strong synergism was obtained in both estrogen-dependent MCF-7 breast cancer cells and triple-negative MDA-MB-231 breast cancer cells. Significant dose reduction of TRAIL in combination with BENSpm in MDA-MB-231 cells, together with the fact that BENSpm rendered MCF-7 cells more sensitive to TRAIL treatment verified our rationale of designing BENSpm-based delivery platform. This was expected to be beneficial for overcoming drug resistance in chemotherapy, as well as boosting the therapeutic effect of therapeutic genes. We first designed a lipid-based BENSpm dual vector (Lipo

  17. Human gene therapy: novel approaches to improve the current gene delivery systems.

    Science.gov (United States)

    Cucchiarini, Magali

    2016-06-01

    Even though gene therapy made its way through the clinics to treat a number of human pathologies since the early years of experimental research and despite the recent approval of the first gene-based product (Glybera) in Europe, the safe and effective use of gene transfer vectors remains a challenge in human gene therapy due to the existence of barriers in the host organism. While work is under active investigation to improve the gene transfer systems themselves, the use of controlled release approaches may offer alternative, convenient tools of vector delivery to achieve a performant gene transfer in vivo while overcoming the various physiological barriers that preclude its wide use in patients. This article provides an overview of the most significant contributions showing how the principles of controlled release strategies may be adapted for human gene therapy.

  18. Virosome-mediated delivery of protein antigens to dendritic cells

    NARCIS (Netherlands)

    Bungener, L; Serre, K; Bijl, L; Leserman, L; Wilschut, J; Daemen, T; Machy, P

    2002-01-01

    Virosomes are reconstituted viral membranes in which protein can be encapsulated. Fusion-active virosomes, fusion-inactive virosomes and liposomes were used to study the conditions needed for delivery of encapsulated protein antigen ovalbumin (OVA) to dendritic cells (DCs) for MHC class I and 11

  19. Nanoparticle (MPG)-mediated delivery of small RNAs into human ...

    African Journals Online (AJOL)

    Aghomotsegin

    Divita and co-workers reported that MPG delivers active macromolecules permitting the control of the release of the cargo in the appropriate target subcellular compart- ment. Therefore, by tampering with the NLS sequence of. MPG, delivery between the nucleus and the cytoplasm can be discriminated and MPG containing ...

  20. Optical Molecular Imaging of Ultrasound-mediated Drug Delivery

    NARCIS (Netherlands)

    Derieppe, M.P.P.

    2015-01-01

    The goal of this PhD project was to develop optical molecular imaging methods to study drug delivery facilitated by ultrasound waves (US) and hyperthermia. Fibered confocal fluorescence microscopy (FCFM), together with dedicated image analysis, was used in vitro on a cell monolayer, and in vivo at

  1. PAIN RELIEF MEDIATED BY IMPLANTABLE DRUG-DELIVERY DEVICES

    NARCIS (Netherlands)

    HOEKSTRA, A

    Various totally implantable drug delivery systems from single access ports to micropumps are now available for administration of repeated boluses, and continuous or programmable infusions. In this respect, emphasis is given to a relatively cheap, totally implantable system for self-administering

  2. Dual peptide-mediated targeted delivery of bioactive siRNAs to oral cancer cells in vivo.

    Science.gov (United States)

    Alexander-Bryant, Angela A; Zhang, Haiwen; Attaway, Christopher C; Pugh, William; Eggart, Laurence; Sansevere, Robert M; Andino, Lourdes M; Dinh, Lu; Cantini, Liliana P; Jakymiw, Andrew

    2017-09-01

    Despite significant advances in cancer treatment, the prognosis for oral cancer remains poor in comparison to other cancer types, including breast, skin, and prostate. As a result, more effective therapeutic modalities are needed for the treatment of oral cancer. Consequently, in the present study, we examined the feasibility of using a dual peptide carrier approach, combining an epidermal growth factor receptor (EGFR)-targeting peptide with an endosome-disruptive peptide, to mediate targeted delivery of small interfering RNAs (siRNAs) into EGFR-overexpressing oral cancer cells and induce silencing of the targeted oncogene, cancerous inhibitor of protein phosphatase 2A (CIP2A). Fluorescence microscopy, real-time PCR, Western blot analysis, and in vivo bioimaging of mice containing orthotopic xenograft tumors were used to examine the ability of the dual peptide carrier to mediate specific delivery of bioactive siRNAs into EGFR-overexpressing oral cancer cells/tissues. Co-complexation of the EGFR-targeting peptide, GE11R9, with the endosome-disruptive 599 peptide facilitated the specific uptake of siRNAs into oral cancer cells overexpressing EGFR in vitro with optimal gene silencing observed at a 60:30:1 (GE11R9:599:siRNA) molar ratio. Furthermore, when administered systemically to mice bearing xenograft oral tumors, this dual peptide complex mediated increased targeted delivery of siRNAs into tumor tissues in comparison to the 599 peptide alone and significantly enhanced CIP2A silencing. Herein we provide the first report demonstrating the clinical potential of a dual peptide strategy for siRNA-based therapeutics by synergistically mediating the effective targeting and delivery of bioactive siRNAs into EGFR-overexpressing oral cancer cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Parallel synthesis and screening of polymers for nonviral gene delivery.

    Science.gov (United States)

    Barua, Sutapa; Joshi, Amit; Banerjee, Akhilesh; Matthews, Dana; Sharfstein, Susan T; Cramer, Steven M; Kane, Ravi S; Rege, Kaushal

    2009-01-01

    We describe the parallel synthesis and in vitro evaluation of a cationic polymer library for the discovery of nonviral gene delivery vectors. The library was synthesized based on the ring-opening polymerization reaction between epoxide groups of diglycidyl ethers and the amines of (poly)amines. Parallel screening of soluble library constituents led to the identification of lead polymers with high DNA-binding efficacies. Transfection efficacies of lead polymers were evaluated using PC3-PSMA human prostate cancer cells and murine osteoblasts in the absence and presence of serum. In vitro experiments resulted in the identification of a candidate polymer that demonstrated significantly higher transfection efficacies and lower cytotoxicities than poly(ethyleneimine) (pEI), the current standard for polymeric transfection agents. In addition, polymers that demonstrated moderately higher and comparable transfection efficacies with respect to pEI were also identified. Our results demonstrate that high-throughput synthesis and screening of polymers is a powerful approach for the identification of novel nonviral gene delivery agents.

  4. Cross-linked polyethylenimine–tripolyphosphate nanoparticles for gene delivery

    Science.gov (United States)

    Huang, Xianzhang; Shen, Sujing; Zhang, Zhanfeng; Zhuang, Junhua

    2014-01-01

    The high transfection efficiency of polyethylenimine (PEI) makes it an attractive potential nonviral genetic vector for gene delivery and therapy. However, the highly positive charge of PEI leads to cytotoxicity and limits its application. To reduce the cytotoxicity of PEI, we prepared anion-enriched nanoparticles that combined PEI with tripolyphosphate (TPP). We then characterized the PEI-TPP nanoparticles in terms of size, zeta potential, and Fourier-transform infrared (FTIR) spectra, and assessed their transfection efficiency, cytotoxicity, and ability to resist deoxyribonuclease (DNase) I digestion. The cellular uptake of PEI-TPP with phosphorylated internal ribosome entry site–enhanced green fluorescent protein C1 or FAM (fluorouracil, Adriamycin [doxorubicin] and mitomycin)-labeled small interfering ribonucleic acids (siRNAs) was monitored by fluorescence microscopy and confocal laser microscopy. The efficiency of transfected delivery of plasmid deoxyribonucleic acid (DNA) and siRNA in vitro was 1.11- to 4.20-fold higher with the PEI-TPP particles (7.6% cross-linked) than with the PEI, at all N:P ratios (nitrogen in PEI to phosphorus in DNA) tested. The cell viability of different cell lines was more than 90% at the chosen N:P ratios of PEI-TPP/DNA complexes. Moreover, PEI-TPP nanoparticles resisted digestion by DNase I for more than 2 hours. The time-dependent absorption experiment showed that 7.6% of cross-linked PEI-TPP particles were internalized by 293T cells within 1 hour. In summary, PEI-TPP nanoparticles effectively transfected cells while conferring little or no toxicity, and thus have potential application in gene delivery. PMID:25342902

  5. Non-Viral, Lipid-Mediated DNA and mRNA Gene Therapy of the Central Nervous System (CNS): Chemical-Based Transfection.

    Science.gov (United States)

    Hecker, James G

    2016-01-01

    Appropriate gene delivery systems are essential for successful gene therapy in clinical medicine. Cationic lipid-mediated delivery is an alternative to viral vector-mediated gene delivery. Lipid-mediated delivery of DNA or mRNA is usually more rapid than viral-mediated delivery, offers a larger payload, and has a nearly zero risk of incorporation. Lipid-mediated delivery of DNA or RNA is therefore preferable to viral DNA delivery in those clinical applications that do not require long-term expression for chronic conditions. Delivery of RNA may be preferable to non-viral DNA delivery in some clinical applications, because transit across the nuclear membrane is not necessary and onset of expression with RNA is therefore even faster than with DNA, although both are faster than most viral vectors. Here, we describe techniques for cationic lipid-mediated delivery of nucleic acids encoding reporter genes in a variety of cell lines. We describe optimized formulations and transfection procedures that we previously assessed by bioluminescence and flow cytometry. RNA transfection demonstrates increased efficiency relative to DNA transfection in non-dividing cells. Delivery of mRNA results in onset of expression within 1 h after transfection and a peak in expression 5-7 h after transfection. Duration of expression in eukaryotic cells after mRNA transcript delivery depends on multiple factors, including transcript stability, protein turnover, and cell type. Delivery of DNA results in onset of expression within 5 h after transfection, a peak in expression 24-48 h after transfection, and a return to baseline that can be as long as several weeks after transfection. In vitro results are consistent with our in vivo delivery results, techniques for which are described as well. RNA delivery is suitable for short-term transient gene expression due to its rapid onset, short duration of expression and greater efficiency, particularly in non-dividing cells, while the longer duration and

  6. Intracellular delivery of potential therapeutic genes: prospects in cancer gene therapy.

    Science.gov (United States)

    Bakhtiar, Athirah; Sayyad, Mustak; Rosli, Rozita; Maruyama, Atsushi; Chowdhury, Ezharul H

    2014-01-01

    Conventional therapies for malignant cancer such as chemotherapy and radiotherapy are associated with poor survival rates owing to the development of cellular resistance to cancer drugs and the lack of targetability, resulting in unwanted adverse effects on healthy cells and necessitating the lowering of therapeutic dose with consequential lower efficacy of the treatment. Gene therapy employing different types of viral and non-viral carriers to transport gene(s) of interest and facilitating production of the desirable therapeutic protein(s) has tremendous prospects in cancer treatments due to the high-level of specificity in therapeutic action of the expressed protein(s) with diminished off-target effects, although cancer cell-specific delivery of transgene(s) still poses some challenges to be addressed. Depending on the potential therapeutic target genes, cancer gene therapy could be categorized into tumor suppressor gene replacement therapy, immune gene therapy and enzyme- or prodrug-based therapy. This review would shed light on the current progress of delivery of potentially therapeutic genes into various cancer cells in vitro and animal models utilizing a variety of viral and non-viral vectors.

  7. Design and application of cationic amphiphilic β-cyclodextrin derivatives as gene delivery vectors

    Science.gov (United States)

    Wan, Ning; Huan, Meng-Lei; Ma, Xi-Xi; Jing, Zi-Wei; Zhang, Ya-Xuan; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

    2017-11-01

    The nano self-assembly profiles of amphiphilic gene delivery vectors could improve the density of local cationic head groups to promote their DNA condensation capability and enhance the interaction between cell membrane and hydrophobic tails, thus increasing cellular uptake and gene transfection. In this paper, two series of cationic amphiphilic β-cyclodextrin (β-CD) derivatives were designed and synthesized by using 6-mono-OTs-β-CD (1) as the precursor to construct amphiphilic gene vectors with different building blocks in a selective and controlled manner. The effect of different type and degree of cationic head groups on transfection and the endocytic mechanism of β-CD derivatives/DNA nanocomplexes were also investigated. The results demonstrated that the designed β-cyclodextrin derivatives were able to compact DNA to form stable nanocomplexes and exhibited low cytotoxicity. Among them, PEI-1 with PEI head group showed enhanced transfection activity, significantly higher than commercially available agent PEI25000 especially in the presence of serum, showing potential application prospects in clinical trials. Moreover, the endocytic uptake mechanism involved in the gene transfection of PEI-1 was mainly through caveolae-mediated endocytosis, which could avoid the lysosomal degradation of loaded gene, and had great importance for improving gene transfection activity.

  8. Low frequency sonophoresis mediated transdermal and intradermal delivery of ketoprofen.

    Science.gov (United States)

    Herwadkar, Anushree; Sachdeva, Vishal; Taylor, Leslie F; Silver, Herb; Banga, Ajay K

    2012-02-28

    The objective of this study was to test low frequency sonophoresis at 20 kHz for delivery of ketoprofen into and across the skin. Permeation studies were carried out in vitro on excised hairless rat skin over a period of 24h using Franz diffusion cells after which, skin samples were subjected to skin extraction to quantify the amount of drug present in skin. Parameters like ultrasound application time, duty cycle coupling medium and distance of ultrasound horn from skin were optimized. Transepidermal water loss (TEWL) was measured to indicate the extent of barrier disruption following sonophoresis. Confocal microscopy was used to visualize dye penetration through sonophoresis treated skin. Application of ultrasound significantly enhanced permeation of ketoprofen from 74.87 ± 5.27 μg/cm(2) for passive delivery to 491.37 ± 48.78 μg/cm(2) for sonophoresis. Drug levels in skin layers increased from 34.69 ± 7.25 μg following passive permeation to 212.62 ± 45.69 μg following sonophoresis. TEWL increased from 31.6 ± 0.02 (passive) to 69.5 ± 12.60 (sonophoresis) indicating disruption of barrier properties. Confocal microscopy images depicted enhanced dye penetration through sonophoresis treated skin confirming barrier disruption. Low frequency sonophoresis with optimized ultrasound parameters can be effectively used to actively enhance transdermal and topical delivery of ketoprofen. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. Nanoparticle-Mediated Pulmonary Drug Delivery: A Review

    Directory of Open Access Journals (Sweden)

    Mukta Paranjpe

    2014-04-01

    Full Text Available Colloidal drug delivery systems have been extensively investigated as drug carriers for the application of different drugs via different routes of administration. Systems, such as solid lipid nanoparticles, polymeric nanoparticles and liposomes, have been investigated for a long time for the treatment of various lung diseases. The pulmonary route, owing to a noninvasive method of drug administration, for both local and systemic delivery of an active pharmaceutical ingredient (API forms an ideal environment for APIs acting on pulmonary diseases and disorders. Additionally, this route offers many advantages, such as a high surface area with rapid absorption due to high vascularization and circumvention of the first pass effect. Aerosolization or inhalation of colloidal systems is currently being extensively studied and has huge potential for targeted drug delivery in the treatment of various diseases. Furthermore, the surfactant-associated proteins present at the interface enhance the effect of these formulations by decreasing the surface tension and allowing the maximum effect. The most challenging part of developing a colloidal system for nebulization is to maintain the critical physicochemical parameters for successful inhalation. The following review focuses on the current status of different colloidal systems available for the treatment of various lung disorders along with their characterization. Additionally, different in vitro, ex vivo and in vivo cell models developed for the testing of these systems with studies involving cell culture analysis are also discussed.

  10. PiggyBac transposon-mediated gene delivery efficiently generates stable transfectants derived from cultured primary human deciduous tooth dental pulp cells (HDDPCs) and HDDPC-derived iPS cells.

    Science.gov (United States)

    Inada, Emi; Saitoh, Issei; Watanabe, Satoshi; Aoki, Reiji; Miura, Hiromi; Ohtsuka, Masato; Murakami, Tomoya; Sawami, Tadashi; Yamasaki, Youichi; Sato, Masahiro

    2015-09-14

    The ability of human deciduous tooth dental pulp cells (HDDPCs) to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medicine. Realization of this potential depends on efficient and optimized protocols for the genetic manipulation of HDDPCs. In this study, we demonstrate the use of a PiggyBac (PB)-based gene transfer system as a method for introducing nonviral transposon DNA into HDDPCs and HDDPC-derived inducible pluripotent stem cells. The transfection efficiency of the PB-based system was significantly greater than previously reported for electroporation-based transfection of plasmid DNA. Using the neomycin resistance gene as a selection marker, HDDPCs were stably transfected at a rate nearly 40-fold higher than that achieved using conventional methods. Using this system, it was also possible to introduce two constructs simultaneously into a single cell. The resulting stable transfectants, expressing tdTomato and enhanced green fluorescent protein, exhibited both red and green fluorescence. The established cell line did not lose the acquired phenotype over three months of culture. Based on our results, we concluded that PB is superior to currently available methods for introducing plasmid DNA into HDDPCs. There may be significant challenges in the direct clinical application of this method for human dental tissue engineering due to safety risks and ethical concerns. However, the high level of transfection achieved with PB may have significant advantages in basic scientific research for dental tissue engineering applications, such as functional studies of genes and proteins. Furthermore, it is a useful tool for the isolation of genetically engineered HDDPC-derived stem cells for studies in tooth regenerative medicine.

  11. Systemic gene delivery transduces the enteric nervous system of guinea pigs and cynomolgus macaques.

    Science.gov (United States)

    Gombash, S E; Cowley, C J; Fitzgerald, J A; Lepak, C A; Neides, M G; Hook, K; Todd, L J; Wang, G-D; Mueller, C; Kaspar, B K; Bielefeld, E C; Fischer, A J; Wood, J D; Foust, K D

    2017-10-01

    Characterization of adeno-associated viral vector (AAV) mediated gene delivery to the enteric nervous system (ENS) was recently described in mice and rats. In these proof-of-concept experiments, we show that intravenous injections of clinically relevant AAVs can transduce the ENS in guinea pigs and non-human primates. Neonatal guinea pigs were given intravenous injections of either AAV8 or AAV9 vectors that contained a green fluorescent protein (GFP) expression cassette or phosphate-buffered saline. Piglets were euthanized three weeks post injection and tissues were harvested for immunofluorescent analysis. GFP expression was detected in myenteric and submucosal neurons along the length of the gastrointestinal tract in AAV8 injected guinea pigs. GFP-positive neurons were found in dorsal motor nucleus of the vagus and dorsal root ganglia. Less transduction occurred in AAV9-treated tissues. Gastrointestinal tissues were analyzed from young cynomolgus macaques that received systemic injection of AAV9 GFP. GFP expression was detected in myenteric neurons of the stomach, small and large intestine. These data demonstrate that ENS gene delivery translates to larger species. This work develops tools for the field of neurogastroenterology to explore gut physiology and anatomy using emerging technologies such as optogenetics and gene editing. It also provides a basis to develop novel therapies for chronic gut disorders.

  12. Experimental research on treating hepatic carcinoma by arterial injection of liposome mediated p53 genes

    International Nuclear Information System (INIS)

    Zhu Guangyu; Lu Qin; Teng Gaojun; Guo Jinhe; Yu Hui; Deng Gang; He Shicheng; Fang Wen; Li Guozhao; Wei Xiaoying

    2007-01-01

    Objective: To investigate the transfection and expression of p53 genes mediated by liposome and its feasibility in treatment of liver cancer by transcatheter arterial injection on rabbit VX2 hepatocarcinoma model. Methods: pCMV-myc-p53 plasmids, LipofectAMINE and p53-LipofectAMINE complex were infused into tumor's feeding artery of rabbit VX2 hepatocarcinoma model, respectively, and then protein of cancer tissue was extracted, followed by measuring gene transfection and expression by western blot and immunohistochemistry, p53-LipofectAMlNE complex in different doses were infused into tumor's feeding artery of rabbit VX2 hepatocarcinoma model with the gene transfection and expression detected by the same way. Results: Liposome-mediated p53 gene injected through catheter could be successfully transfected and expressed in the cancer tissue of rabbit VX2 hepatocarcinoma model, with transfection efficiency higher than the gene delivery alone. The efficiency and the gene dose has dose-effect relationship. Conclusions: Treatment of liver cancer by transcatheter arterial injection of p53 genes mediated by liposome is a feasible and effective method, with wide prospect of application. (authors)

  13. Gene delivery to the lungs: pulmonary gene therapy for cystic fibrosis.

    Science.gov (United States)

    Villate-Beitia, Ilia; Zarate, Jon; Puras, Gustavo; Pedraz, José Luis

    2017-07-01

    Cystic fibrosis (CF) is a monogenic autosomal recessive disorder where the defective gene, the cystic fibrosis transmembrane conductance regulator (CFTR), is well identified. Moreover, the respiratory tract can be targeted through noninvasive aerosolized formulations for inhalation. Therefore, gene therapy is considered a plausible strategy to address this disease. Conventional gene therapy strategies rely on the addition of a correct copy of the CFTR gene into affected cells in order to restore the channel activity. In recent years, genome correction strategies have emerged, such as zinc-finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats associated to Cas9 nucleases. These gene editing tools aim to repair the mutated gene at its original genomic locus with high specificity. Besides, the success of gene therapy critically depends on the nucleic acids carriers. To date, several clinical studies have been carried out to add corrected copies of the CFTR gene into target cells using viral and non-viral vectors, some of them with encouraging results. Regarding genome editing systems, preliminary in vitro studies have been performed in order to repair the CFTR gene. In this review, after briefly introducing the basis of CF, we discuss the up-to-date gene therapy strategies to address the disease. The review focuses on the main factors to take into consideration when developing gene delivery strategies, such as the design of vectors and plasmid DNA, in vitro/in vivo tests, translation to human use, administration methods, manufacturing conditions and regulatory issues.

  14. Nanoparticle-Mediated p53 Gene Therapy for Breast Cancer

    National Research Council Canada - National Science Library

    Prabha, Swayam

    2003-01-01

    The effect of different formulation parameters on nanoparticle-mediated gene transfection in vitro was studied Nanoparticles encapsulating plasmid DNA encoding fire fly luciferase were formulated using poly lactide (PLA...

  15. Preparation and characterization of magnetic gene vectors for targeting gene delivery

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, S.W.; Liu, G. [College of Chemistry, Chemical Engineering and Materials Science and Key Laboratory of Organic Synthesis of Jiangsu Province, Soochow University, SIP, Suzhou 215123 (China); Hong, R.Y., E-mail: rhong@suda.edu.cn [College of Chemistry, Chemical Engineering and Materials Science and Key Laboratory of Organic Synthesis of Jiangsu Province, Soochow University, SIP, Suzhou 215123 (China); State Key Laboratory of Multi-phase Complex Systems, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080 (China); Li, H.Z. [State Key Laboratory of Multi-phase Complex Systems, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080 (China); Li, Y.G., E-mail: ilguoliang@sohu.com [Department of radiology, the First Affiliated Hospital of Soochow University, Suzhou 215007 (China); Wei, D.G., E-mail: dougwei@deas.harvard.edu [Center for Nanoscale Systems, School of Engineering and Applied Science, Harvard University, 11 Oxford Street, Cambridge, MA 02139 (United States)

    2012-10-15

    Highlights: Black-Right-Pointing-Pointer PEI is ideal candidate polymer for the design of gene delivery systems. Black-Right-Pointing-Pointer PEI-CMD-MNPs exhibited a typical superparamagnetic behavior. Black-Right-Pointing-Pointer PEI-CMD-MNPs were well stable over the entire range of pH and NaCl concentration. Black-Right-Pointing-Pointer DNA-PEI-CMD-MNPs transfected cells by a magnet have higher transfection efficiency and gene expression efficiency. - Abstract: The PEI-CMD-MNPs were successfully prepared by the surface modification of magnetic Fe{sub 3}O{sub 4} nanoparticles with carboxymethyl dextran (CMD) and polyethyleneimine (PEI). The PEI-CMD-MNPs polyplexes exhibited a typical superparamagnetic behavior and were well stable over the entire range of pH and NaCl concentration. These PEI-CMD-MNPs were used as magnetic gene vectors for targeting gene delivery. The prepared MNPs at different surface modification stages were characterized using Fourier transform infrared (FT-IR), thermogravimetric analysis (TGA), field emissions canning electron microscopy (FE-SEM), powder X-ray diffraction (XRD) and dynamic laser light scattering (DLS) analysis. The magnetic properties were studied by vibrating sample magnetometer (VSM). To evaluate the performance of the magnetic nanoparticles as gene transfer vector, the PEI-CMD-MNPs were used to delivery green fluorescent protein (GFP) gene into BHK21 cells. The expression of GFP gene was detected by fluorescence microscope. DNA-PEI-CMD-MNPs polyplexes absorbed by the cells were also monitored by Magnetic resonance imaging (MRI). The transfection efficiency and gene expression efficiency of that transfected with a magnet were much higher than that of standard transfection.

  16. Starburst low-molecular weight polyethylenimine for efficient gene delivery.

    Science.gov (United States)

    Zhao, Yanjun; Yang, Rulei; Liu, Dong; Sun, Mingjing; Zhou, Lijun; Wang, Zheng; Wan, Ying

    2012-01-01

    Low-molecular weight polyethylenimine (LMW PEI) shows the advantage of low-cytotoxicity, but has been inefficient in gene delivery as a consequence of the low-charge density. A number of previous studies employed the approach of crosslinking to solve this problem. In this study, a starburst LMW PEI gene vector has been developed. It has a polyamidoamine (PAMAM) core conjugated with a shell composed of LWM PEI and polyethylene glycol (PEG), that is PAMAM-PEI-PEG. Plasmid DNA (pEGFP-N1) and human cervix epithelial carcinoma (HeLa) cells were used in the study. The results showed that the starburst LMW PEI could effectively condense DNA at N/P above 5. The polyplexes had a size of about 500 nm and a nearly neutral surface because of the PEG shielding effect. This novel gene vector is able to maintain the low-cytotoxicity of LMW PEI, whereas its transfection efficiency was significantly improved. Copyright © 2011 Wiley Periodicals, Inc.

  17. Perinatal systemic gene delivery using adeno-associated viral vectors

    Directory of Open Access Journals (Sweden)

    Rajvinder eKarda

    2014-11-01

    Full Text Available Neurodegenerative monogenic diseases can also affect a broad range of tissues and organs throughout the body. An effective treatment would require a systemic approach. The intravenous administration of novel therapies is ideal but is hampered by the inability of such drugs to cross the blood-brain barrier and precludes efficacy in the central nervous system. A number of these early lethal intractable diseases also present devastating irreversible pathology at birth or soon after. Therefore, any therapy would ideally be administered during the perinatal period to prevent, stop or ameliorate disease progression. The concept of perinatal gene therapy has moved a step further towards being a feasible approach to treating such disorders. This has primarily been driven by the recent discoveries that particular serotypes of adeno-associated virus (AAV gene delivery vectors have the ability to cross the blood-brain barrier following intravenous administration. Furthermore, this has been safely demonstrated in perinatal mice and non-human primates. This review focuses on the progress made in using AAV to achieve systemic transduction and what this means for developing perinatal gene therapy for early lethal neurodegenerative diseases.

  18. Microneedle-Mediated Delivery of Copper Peptide Through Skin.

    Science.gov (United States)

    Li, Hairui; Low, Yong Sheng Jason; Chong, Hui Ping; Zin, Melvin T; Lee, Chi-Ying; Li, Bo; Leolukman, Melvina; Kang, Lifeng

    2015-08-01

    Copper peptide (GHK-Cu) plays an important role in skin regeneration and wound healing. However, its skin absorption remains challenging due to its hydrophilicity. Here we use polymeric microneedle array to pre-treat skin to enhance GHK-Cu skin penetration. Two in vitro skin models were used to assess the capability of microneedles in facilitating skin delivery of GHK-Cu. Histological assay and confocal laser scanning microscopy were performed to characterize and quantify the microconduits created by the microneedles inside skin. Cellular and porcine models were used to evaluate the safety of microneedle-assisted copper peptide delivery. The depth and percentage of microneedle penetration were correlated with application forces, which in turn influenced the extent of enhancement in the skin permeability of GHK-Cu. In 9 h, 134 ± 12 nanomoles of peptide and 705 ± 84 nanomoles of copper permeated though the microneedle treated human skin, while almost no peptide or copper permeated through intact human skin. No obvious signs of skin irritation were observed with the use of GHK-Cu after microneedle pretreatment. It is effective and safe to enhance the skin permeation of GHK-Cu by using microneedles. This approach may be useful to deliver similar peptides or minerals through skin.

  19. Iontophoretic and Microneedle Mediated Transdermal Delivery of Glycopyrrolate

    Directory of Open Access Journals (Sweden)

    Meera Gujjar

    2014-12-01

    Full Text Available Purpose: The objective of this study was to investigate the use of iontophoresis, soluble microneedles and their combination for the transdermal delivery of glycopyrrolate. Methods: In vitro permeation was tested using full thickness porcine ear skin mounted onto Franz diffusion cells. Iontophoresis (0.5 mA/cm2 was done for 4 h using Ag/AgCl electrodes. For microneedles, three line array (27 needles/line of maltose microneedles were used to microporate the skin prior to mounting. Pore uniformity was determined by taking fluorescent images of distribution of calcein into pores and processing the images using an image analysis tool, which measured the fluorescent intensity in and around each pore to provide a pore permeability index (PPI. The donor chamber contained 500 µL of a 1 mg/mL solution of glycopyrrolate, and the receptor chamber contained 5 mL of 50 mM NaCl in deionized water. Samples were collected at predetermined time points over a period of 24 h and analyzed by HPLC. Skin irritation testing was performed with a 3D cell culture kit of human skin. MTT assay determined cell viability; viability less than 50% was considered irritant. Results: A control experiment which investigated passive permeation of glycopyrrolate delivered an average cumulative amount of 24.92 ± 1.77 µg/cm2 at 24 h, while microneedle pretreatment increased permeability to 46.54 ± 6.9 µg/cm2. Both iontophoresis (158.53 ± 17.50 µg/cm2 and a combination of iontophoresis and microneedles (182.43 ± 20.06 µg/ cm2 significantly increased delivery compared to passive and microneedles alone. Glycopyrrolate solution was found to be nonirritant with cell viability of 70.4% ± 5.03%. Conclusion: Iontophoresis and a combination of iontophoresis with microneedle pretreatment can be effectively used to enhance the transdermal delivery of glycopyrrolate. Glycopyrrolate was found to be non-irritant to skin.

  20. Optimization of conditions for gene delivery system based on PEI

    Directory of Open Access Journals (Sweden)

    Roya Cheraghi

    2017-01-01

    Full Text Available Objective(s: PEI based nanoparticle (NP due to dual capabilities of proton sponge and DNA binding is known as powerful tool for nucleic acid delivery to cells. However, serious cytotoxicity and complicated conditions, which govern NPs properties and its interactions with cells practically, hindered achievement to high transfection efficiency. Here, we have tried to optimize the properties of PEI/ firefly luciferase plasmid complexes and cellular condition to improve transfection efficiency. Materials and Methods: For this purpose, firefly luciferase, as a robust gene reporter, was complexed with PEI to prepare NPs with different size and charge. The physicochemical properties of nanoparticles were evaluated using agarose gel retardation and dynamic light scattering.  MCF7 and BT474 cells at different confluency were also transfected with prepared nanoparticles at various concentrations for short and long times. Results: The branched PEI can instantaneously bind to DNA and form cationic NPs. The results demonstrated the production of nanoparticles with size about 100-500 nm dependent on N/P ratio. Moreover, increase of nanoparticles concentration on the cell surface drastically improved the transfection rate, so at a concentration of 30 ng/ìl, the highest transfection efficiency was achieved. On the other side, at confluency between 40-60%, the maximum efficiency was obtained. The result demonstrated that N/P ratio of 12 could establish an optimized ratio between transfection efficiency and cytotoxicity of PEI/plasmid nanoparticles. The increase of NPs N/P ratio led to significant cytotoxicity. Conclusion: Obtained results verified the optimum conditions for PEI based gene delivery in different cell lines.

  1. Design of novel polysaccharidic nanostructures for gene delivery

    Energy Technology Data Exchange (ETDEWEB)

    Fuente, M de la; Seijo, B; Alonso, M J [Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Santiago de Compostela, Campus sur s/n, E-15782 Santiago de Compostela (Spain)], E-mail: ffmjalon@usc.es

    2008-02-20

    The goal of the present work was to develop a new synthetic nanosystem for gene delivery. For this purpose, we chose two polysaccharides, hyaluronic acid (HA) and chitosan (CS), as the main components of the nanocarrier. Nanoparticles with different hyaluronate:chitosan (HA:CS) mass ratios (0.5:1 and 1:1) and different polymer molecular weights (hyaluronate 170 (HA) or <10 kDa (HAO) and chitosan 125 (CS) or 10-12 (CSO) kDa) could be obtained using an ionic crosslinking method. These nanoparticles were loaded with pDNA and characterized for their size, zeta potential and pDNA association efficiency. Moreover, their toxicity and ability to transfect the model plasmid pEGFP-C1 were evaluated in the cell line HEK 293, as well as their intracellular fate. The results showed that HA:CS nanoparticles have a small size in the range of 110-230 nm, a positive zeta potential of +10 to +32 mV and a very high pDNA association efficiency of 87-99% (w/w). On the other hand, nanoparticles exhibited low cell toxicity and transfection levels up to 25% GFP expressing HEK 293 cells, lasting for the whole observation period of 10 days. We also provide basic information about the role of both polymers, HA and CS, and the effect of their molecular weight on the effectiveness of the resulting DNA nanocarrier, being the highest transfection levels observed with HAO:CSO 1:1 nanoparticles. In conclusion, HA:CS nanoparticles are promising carriers for gene delivery.

  2. Preparation and characterization of magnetic gene vectors for targeting gene delivery

    Science.gov (United States)

    Zheng, S. W.; Liu, G.; Hong, R. Y.; Li, H. Z.; Li, Y. G.; Wei, D. G.

    2012-10-01

    The PEI-CMD-MNPs were successfully prepared by the surface modification of magnetic Fe3O4 nanoparticles with carboxymethyl dextran (CMD) and polyethyleneimine (PEI). The PEI-CMD-MNPs polyplexes exhibited a typical superparamagnetic behavior and were well stable over the entire range of pH and NaCl concentration. These PEI-CMD-MNPs were used as magnetic gene vectors for targeting gene delivery. The prepared MNPs at different surface modification stages were characterized using Fourier transform infrared (FT-IR), thermogravimetric analysis (TGA), field emissions canning electron microscopy (FE-SEM), powder X-ray diffraction (XRD) and dynamic laser light scattering (DLS) analysis. The magnetic properties were studied by vibrating sample magnetometer (VSM). To evaluate the performance of the magnetic nanoparticles as gene transfer vector, the PEI-CMD-MNPs were used to delivery green fluorescent protein (GFP) gene into BHK21 cells. The expression of GFP gene was detected by fluorescence microscope. DNA-PEI-CMD-MNPs polyplexes absorbed by the cells were also monitored by Magnetic resonance imaging (MRI). The transfection efficiency and gene expression efficiency of that transfected with a magnet were much higher than that of standard transfection.

  3. PEGylated carboxymethyl chitosan/calcium phosphate hybrid anionic nanoparticles mediated hTERT siRNA delivery for anticancer therapy.

    Science.gov (United States)

    Xie, Ying; Qiao, Hongzhi; Su, Zhigui; Chen, Minglei; Ping, Qineng; Sun, Minjie

    2014-09-01

    Lack of safe and effective delivery vehicle is the main obstacle for siRNA mediated cancer therapy. In this study, we synthesized a pH-sensitive polymer of PEG grafted carboxymethyl chitosan (PEG-CMCS) and developed anionic-charged hybrid nanoparticles of PEG-CMCS and calcium phosphate (CaP) for siRNA delivery through a single-step self-assembly method in aqueous condition. The formed nanoparticles with charge of around -8.25 mv and average diameter of 102.1 nm exhibited efficient siRNA encapsulation and enhanced colloidal and serum stability. The test in vitro indicated that the nanoparticles entered into HepG2 cells by endocytosis, and achieved endosomal escape of siRNA effectively due to the pH-responsive disassembly of nanoparticles and dissolution of CaP in the endosome. Reporter gene silencing assay showed that luciferase siRNA delivered by the anionic nanoparticles could achieve gene silencing efficacy comparable to that of conventional Lipofectamine 2000. Additionally, dramatic hTERT knockdown mediated by the anionic nanoparticles transfection induced significant apoptosis of HepG2 cells in vitro. After intravenous injection in tumor-bearing BALB/c nude mice, the nanoparticles specifically accumulated into tumor regions by EPR effect, leading to efficient and specific gene silencing sequentially. Most importantly, the nanoparticles carrying hTERT siRNA inhibited tumor growth significantly via silencing hTERT expression and inducing cells apoptosis in HepG2 tumor xenograft. Moreover, comprehensive safety studies of the nanoparticles confirmed their superior safety both in vitro and in vivo. We concluded that the PEG-CMCS/CaP hybrid anionic nanoparticles possessed potential as a safe and effective siRNA delivery system for anticancer therapy. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Nebulisation of receptor-targeted nanocomplexes for gene delivery to the airway epithelium.

    Directory of Open Access Journals (Sweden)

    Maria D I Manunta

    Full Text Available Gene therapy mediated by synthetic vectors may provide opportunities for new treatments for cystic fibrosis (CF via aerosolisation. Vectors for CF must transfect the airway epithelium efficiently and not cause inflammation so they are suitable for repeated dosing. The inhaled aerosol should be deposited in the airways since the cystic fibrosis transmembrane conductance regulator gene (CFTR is expressed predominantly in the epithelium of the submucosal glands and in the surface airway epithelium. The aim of this project was to develop an optimised aerosol delivery approach applicable to treatment of CF lung disease by gene therapy.The vector suspension investigated in this study comprises receptor-targeting peptides, cationic liposomes and plasmid DNA that self-assemble by electrostatic interactions to form a receptor-targeted nanocomplex (RTN of approximately 150 nm with a cationic surface charge of +50 mV. The aerodynamic properties of aerosolised nanocomplexes produced with three different nebulisers were compared by determining aerosol deposition in the different stages of a Next Generation Pharmaceutical Impactor (NGI. We also investigated the yield of intact plasmid DNA by agarose gel electrophoresis and densitometry, and transfection efficacies in vitro and in vivo.RTNs nebulised with the AeroEclipse II BAN were the most effective, compared to other nebulisers tested, for gene delivery both in vitro and in vivo. The biophysical properties of the nanocomplexes were unchanged after nebulisation while the deposition of RTNs suggested a range of aerosol aerodynamic sizes between 5.5 µm-1.4 µm cut off (NGI stages 3-6 compatible with deposition in the central and lower airways.RTNs showed their ability at delivering genes via nebulisation, thus suggesting their potential applications for therapeutic interventions of cystic fibrosis and other respiratory disorders.

  5. Cross-linked polyethylenimine–tripolyphosphate nanoparticles for gene delivery

    Directory of Open Access Journals (Sweden)

    Huang XZ

    2014-10-01

    Full Text Available Xianzhang Huang,1 Sujing Shen,2 Zhanfeng Zhang,1 Junhua Zhuang1 1Department of Laboratory Science, Second Affiliated Hospital of Guangzhou University of Chinese Medicine, 2Department of Laboratory Science, Guangdong Second Provincial Traditional Chinese Medicine Hospital, Guangzhou, People’s Republic of China Abstract: The high transfection efficiency of polyethylenimine (PEI makes it an attractive potential nonviral genetic vector for gene delivery and therapy. However, the highly positive charge of PEI leads to cytotoxicity and limits its application. To reduce the cytotoxicity of PEI, we prepared anion-enriched nanoparticles that combined PEI with tripolyphosphate (TPP. We then characterized the PEI-TPP nanoparticles in terms of size, zeta potential, and Fourier-transform infrared (FTIR spectra, and assessed their transfection efficiency, cytotoxicity, and ability to resist deoxyribonuclease (DNase I digestion. The cellular uptake of PEI-TPP with phosphorylated internal ribosome entry site–enhanced green fluorescent protein C1 or FAM (fluorouracil, Adriamycin [doxorubicin] and mitomycin-labeled small interfering ribonucleic acids (siRNAs was monitored by fluorescence microscopy and confocal laser microscopy. The efficiency of transfected delivery of plasmid deoxyribonucleic acid (DNA and siRNA in vitro was 1.11- to 4.20-fold higher with the PEI-TPP particles (7.6% cross-linked than with the PEI, at all N:P ratios (nitrogen in PEI to phosphorus in DNA tested. The cell viability of different cell lines was more than 90% at the chosen N:P ratios of PEI-TPP/DNA complexes. Moreover, PEI-TPP nanoparticles resisted digestion by DNase I for more than 2 hours. The time-dependent absorption experiment showed that 7.6% of cross-linked PEI-TPP particles were internalized by 293T cells within 1 hour. In summary, PEI-TPP nanoparticles effectively transfected cells while conferring little or no toxicity, and thus have potential application in gene

  6. Host genes involved in Agrobacterium-mediated transformation

    NARCIS (Netherlands)

    Soltani, Jalal

    2009-01-01

    Agrobacterium is the nature’s genetic engineer that can transfer genes across the kingdom barriers to both prokaryotic and eukaryotic host cells. The host genes which are involved in Agrobacterium-mediated transformatiom (AMT) are not well known. Here, I studied in a systematic way to identify the

  7. Transgene transmission in chickens by sperm-mediated gene ...

    Indian Academy of Sciences (India)

    Transgenic animals have been successfully produced by mass gene transfer techniques such as sperm-mediated gene transfer (SMGT). The aim of this work was to demonstrate transgene transmission by SMGT in chickens using dimethylsulfoxide (DMSO) or ,-dimethylacetamide (DMAc) as transfectants after seminal ...

  8. Aptamer-Mediated Polymeric Vehicles for Enhanced Cell-Targeted Drug Delivery.

    Science.gov (United States)

    Tan, Kei X; Danquah, Michael K; Sidhu, Amandeep; Yon, Lau Sie; Ongkudon, Clarence M

    2018-02-08

    The search for smart delivery systems for enhanced pre-clinical and clinical pharmaceutical delivery and cell targeting continues to be a major biomedical research endeavor owing to differences in the physicochemical characteristics and physiological effects of drug molecules, and this affects the delivery mechanisms to elicit maximum therapeutic effects. Targeted drug delivery is a smart evolution essential to address major challenges associated with conventional drug delivery systems. These challenges mostly result in poor pharmacokinetics due to the inability of the active pharmaceutical ingredients to specifically act on malignant cells thus, causing poor therapeutic index and toxicity to surrounding normal cells. Aptamers are oligonucleotides with engineered affinities to bind specifically to their cognate targets. Aptamers have gained significant interests as effective targeting elements for enhanced therapeutic delivery as they can be generated to specifically bind to wide range of targets including proteins, peptides, ions, cells and tissues. Notwithstanding, effective delivery of aptamers as therapeutic vehicles is challenged by cell membrane electrostatic repulsion, endonuclease degradation, low pH cleavage, and binding conformation stability. The application of molecularly engineered biodegradable and biocompatible polymeric particles with tunable features such as surface area and chemistry, particulate size distribution and toxicity creates opportunities to develop smart aptamer-mediated delivery systems for controlled drug release. This article discusses opportunities for particulate aptamer-drug formulations to advance current drug delivery modalities by navigating active ingredients through cellular and biomolecular traffic to target sites for sustained and controlled release at effective therapeutic dosages while minimizing systemic cytotoxic effects. A proposal for a novel drug-polymer-aptamer-polymer (DPAP) design of aptamer-drug formulation with

  9. Ultrasound-mediated drug delivery by gas bubbles generated from a chemical reaction.

    Science.gov (United States)

    Lee, Sungmun; Al-Kaabi, Leena; Mawart, Aurélie; Khandoker, Ahsan; Alsafar, Habiba; Jelinek, Herbert F; Khalaf, Kinda; Park, Ji-Ho; Kim, Yeu-Chun

    2018-02-01

    Highly echogenic and ultrasound-responsive microbubbles such as nitrogen and perfluorocarbons have been exploited as ultrasound-mediated drug carriers. Here, we propose an innovative method for drug delivery using microbubbles generated from a chemical reaction. In a novel drug delivery system, luminol encapsulated in folate-conjugated bovine serum albumin nanoparticles (Fol-BSAN) can generate nitrogen gas (N 2 ) by chemical reaction when it reacts with hydrogen peroxide (H 2 O 2 ), one of reactive oxygen species (ROS). ROS plays an important role in the initiation and progression of cancer and elevated ROS have been observed in cancer cells both in vitro and in vivo. High-intensity focussed ultrasound (HIFU) is used to burst the N 2 microbubbles, causing site-specific delivery of anticancer drugs such as methotrexate. In this research, the drug delivery system was optimised by using water-soluble luminol and Mobil Composition of Matter-41 (MCM-41), a mesoporous material, so that the delivery system was sensitive to micromolar concentrations of H 2 O 2 . HIFU increased the drug release from Fol-BSAN by 52.9 ± 2.9% in 10 minutes. The cytotoxicity of methotrexate was enhanced when methotrexate is delivered to MDA-MB-231, a metastatic human breast cancer cell line, using Fol-BSAN with HIFU. We anticipate numerous applications of chemically generated microbubbles for ultrasound-mediated drug delivery.

  10. A Simple Zn2+ Complex-Based Composite System for Efficient Gene Delivery.

    Directory of Open Access Journals (Sweden)

    Zhe Zhang

    Full Text Available Metal complexes might become a new type of promising gene delivery systems because of their low cytotoxicity, structural diversity, controllable aqua- and lipo-solubility, and appropriate density and distribution of positive charges. In this study, Zn2+ complexes (1-10 formed with a series of ligands contained benzimidazole(bzimwere prepared and characterized. They were observed to have different affinities for DNA, dependent on their numbers of positive charges, bzim groups, and coordination structures around Zn2+. The binding induced DNA to condensate into spherical nanoparticles with ~ 50 nm in diameter. The cell transfection efficiency of the DNA nanoparticles was poor, although they were low toxic. The sequential addition of the cell-penetrating peptide (CPP TAT(48-60 and polyethylene glycol (PEG resulted in the large DNA condensates (~ 100 nm in diameter and the increased cellular uptake. The clathrin-mediated endocytosis was found to be a key cellular uptake pathway of the nanoparticles formed with or without TAT(48-60 or/and PEG. The DNA nanoparticles with TAT(48-60 and PEG was found to have the cell transfection efficiency up to 20% of the commercial carrier Lipofect. These results indicated that a simple Zn2+-bzim complex-based composite system can be developed for efficient and low toxic gene delivery through the combination with PEG and CPPs such as TAT.

  11. Gas-Stabilizing Gold Nanocones for Acoustically Mediated Drug Delivery.

    Science.gov (United States)

    Mannaris, Christophoros; Teo, Boon M; Seth, Anjali; Bau, Luca; Coussios, Constantin; Stride, Eleanor

    2018-04-25

    The efficient penetration of drugs into tumors is a major challenge that remains unmet. Reported herein is a strategy to promote extravasation and enhanced penetration using inertial cavitation initiated by focused ultrasound and cone-shaped gold nanoparticles that entrap gas nanobubbles. The cones are capable of initiating inertial cavitation under pressures and frequencies achievable with existing clinical ultrasound systems and of promoting extravasation and delivery of a model large therapeutic molecule in an in vitro tissue mimicking flow phantom, achieving penetration depths in excess of 2 mm. Ease of functionalization and intrinsic imaging capabilities provide gold with significant advantages as a material for biomedical applications. The cones show neither cytotoxicity in Michigan Cancer Foundation (MCF)-7 cells nor hemolytic activity in human blood at clinically relevant concentrations and are found to be colloidally stable for at least 5 d at 37 °C and several months at 4 °C. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Modulating polyplex-mediated gene transfection by small-molecule regulators of autophagy.

    Science.gov (United States)

    Zhong, Xiao; Panus, David; Ji, Weihang; Wang, Chun

    2015-03-02

    Nonviral gene transfection mediated by cationic polymer/DNA polyplexes often imposes stress and toxicity to cells. To better understand the relationship between cellular stress responses and polyplex-mediated transfection, polyplex-induced early autophagy in mouse fibroblasts was characterized and the impact of autophagy modulation on transgene expression evaluated. Transmission electron microscopy revealed the formation of double-membraned autophagosome in the cytoplasm of polyplex-transfected cells. Immunofluorescence staining and microscopy revealed intracellular LC3 punctation that was characteristic of early autophagy activation. Elevated expression of autophagosome-associated LC3 II protein was also detected by Western blot. When cells were treated with small-molecule modulators of autophagy, polyplex-mediated gene transfection efficiency was significantly affected. 3-Methyladenine (3-MA), an early autophagy inhibitor, reduced transfection efficiency, whereas rapamycin, an autophagy inducer, enhanced transgene expression. Importantly, the observed functional impact on gene transfection by autophagy modulation was decoupled from that of other modes of cellular stress response (apoptosis/necrosis). Treatment of cells by 3-MA or rapamycin did not affect the level of intracellular reactive oxygen species (ROS) but did decrease or increase, respectively, nuclear localization of polyplex-delivered plasmid DNA. These findings suggest new possibilities of enhancing polyplex-mediated gene delivery by codelivery of small-molecule regulators of autophagy.

  13. Direct Cytosolic Delivery of CRISPR/Cas9-Ribonucleoprotein for Efficient Gene Editing.

    Science.gov (United States)

    Mout, Rubul; Ray, Moumita; Yesilbag Tonga, Gulen; Lee, Yi-Wei; Tay, Tristan; Sasaki, Kanae; Rotello, Vincent M

    2017-03-28

    Genome editing through the delivery of CRISPR/Cas9-ribonucleoprotein (Cas9-RNP) reduces unwanted gene targeting and avoids integrational mutagenesis that can occur through gene delivery strategies. Direct and efficient delivery of Cas9-RNP into the cytosol followed by translocation to the nucleus remains a challenge. Here, we report a remarkably highly efficient (∼90%) direct cytoplasmic/nuclear delivery of Cas9 protein complexed with a guide RNA (sgRNA) through the coengineering of Cas9 protein and carrier nanoparticles. This construct provides effective (∼30%) gene editing efficiency and opens up opportunities in studying genome dynamics.

  14. Positive-negative-selection-mediated gene targeting in rice

    Directory of Open Access Journals (Sweden)

    Zenpei eShimatani

    2015-01-01

    Full Text Available Gene targeting (GT refers to the designed modification of genomic sequence(s through homologous recombination (HR. GT is a powerful tool both for the study of gene function and for molecular breeding. However, in transformation of higher plants, non-homologous end joining (NHEJ occurs overwhelmingly in somatic cells, masking HR-mediated GT. Positive-negative selection (PNS is an approach for finding HR-mediated GT events because it can eliminate NHEJ effectively by expression of a negative-selection marker gene. In rice—a major crop worldwide—reproducible PNS-mediated GT of endogenous genes has now been successfully achieved. The procedure is based on strong PNS using diphtheria toxin A-fragment as a negative marker, and has succeeded in the directed modification of several endogenous rice genes in various ways. In addition to gene knock-outs and knock-ins, a nucleotide substitution in a target gene was also achieved recently. This review presents a summary of the development of the rice PNS system, highlighting its advantages. Different types of gene modification and gene editing aimed at developing new plant breeding technology (NPBT based on PNS are discussed.

  15. Recruitment of endocytosis in sonopermeabilization-mediated drug delivery: a real-time study

    Science.gov (United States)

    Derieppe, Marc; Rojek, Katarzyna; Escoffre, Jean-Michel; de Senneville, Baudouin Denis; Moonen, Chrit; Bos, Clemens

    2015-07-01

    Microbubbles (MBs) in combination with ultrasound (US) can enhance cell membrane permeability, and have the potential to facilitate the cellular uptake of hydrophilic molecules. However, the exact mechanism behind US- and MB-mediated intracellular delivery still remains to be fully understood. Among the proposed mechanisms are formation of transient pores and endocytosis stimulation. In our study, we investigated whether endocytosis is involved in US- and MB-mediated delivery of small molecules. Dynamic fluorescence microscopy was used to investigate the effects of endocytosis inhibitors on the pharmacokinetic parameters of US- and MB-mediated uptake of SYTOX Green, a 600 Da hydrophilic model drug. C6 rat glioma cells, together with SonoVue® MBs, were exposed to 1.4 MHz US waves at 0.2 MPa peak-negative pressure. Collection of the signal intensity in each individual nucleus was monitored during and after US exposure by a fibered confocal fluorescence microscope designed for real-time imaging. Exposed to US waves, C6 cells pretreated with chlorpromazine, an inhibitor of clathrin-mediated endocytosis, showed up to a 2.5-fold significant increase of the uptake time constant, and a 1.1-fold increase with genistein, an inhibitor of caveolae-mediated endocytosis. Both inhibitors slowed down the US-mediated uptake of SYTOX Green. With C6 cells and our experimental settings, these quantitative data indicate that endocytosis plays a role in sonopermeabilization-mediated delivery of small molecules with a more predominant contribution of clathrin-mediated endocytosis.

  16. Genetic transformation of wheat via Agrobacterium-mediated DNA delivery.

    Science.gov (United States)

    Sparks, Caroline A; Doherty, Angela; Jones, Huw D

    2014-01-01

    The method described involves an initial incubation of wheat immature embryos in a liquid culture of Agrobacterium tumefaciens. The Agrobacterium strain is engineered to contain a binary vector with a gene of interest and a selectable marker gene placed between the T-DNA borders; the T-DNA is the region transferred to the plant cells, thus harnessing the bacterium's natural ability to deliver specific DNA into host cells. Following the initial inoculation with the Agrobacterium, the embryos are co-cultivated for several days after which the Agrobacterium is selectively destroyed using an antibiotic. Tissue culture of the embryos on plant media with a correct balance of hormones allows embryogenic callus formation followed by regeneration of plantlets, and in the later stages of tissue culture a selectable marker (herbicide) is included to minimize the incidence of non-transformed plants. This protocol has been used successfully to generate transformed plants of a wide range of wheat varieties, both spring and winter bread wheats (T. aestivum L.) and durum wheats (T. turgidum L.).

  17. Bioreducible poly(amido amine)s for non-viral gene delivery

    NARCIS (Netherlands)

    Lin, C.

    2008-01-01

    This thesis describes the design and development of bioreducible poly(amido amine)s as non-viral vectors for gene delivery in vitro and in vivo. The structural influences of these polymers on their physico-chemical properties and gene delivery properties, transfection capability and cytotoxicity in

  18. A sight on the current nanoparticle-based gene delivery vectors

    Science.gov (United States)

    Dizaj, Solmaz Maleki; Jafari, Samira; Khosroushahi, Ahmad Yari

    2014-05-01

    Nowadays, gene delivery for therapeutic objects is considered one of the most promising strategies to cure both the genetic and acquired diseases of human. The design of efficient gene delivery vectors possessing the high transfection efficiencies and low cytotoxicity is considered the major challenge for delivering a target gene to specific tissues or cells. On this base, the investigations on non-viral gene vectors with the ability to overcome physiological barriers are increasing. Among the non-viral vectors, nanoparticles showed remarkable properties regarding gene delivery such as the ability to target the specific tissue or cells, protect target gene against nuclease degradation, improve DNA stability, and increase the transformation efficiency or safety. This review attempts to represent a current nanoparticle based on its lipid, polymer, hybrid, and inorganic properties. Among them, hybrids, as efficient vectors, are utilized in gene delivery in terms of materials (synthetic or natural), design, and in vitro/ in vivo transformation efficiency.

  19. Delivery of proteins to mammalian cells via gold nanoparticle mediated laser transfection

    International Nuclear Information System (INIS)

    Heinemann, D; Kalies, S; Schomaker, M; Ertmer, W; Meyer, H; Ripken, T; Murua Escobar, H

    2014-01-01

    Nanoparticle laser interactions are in widespread use in cell manipulation. In particular, molecular medicine needs techniques for the directed delivery of molecules into mammalian cells. Proteins are the final mediator of most cellular cascades. However, despite several methodical approaches, the efficient delivery of proteins to cells remains challenging. This paper presents a new protein transfection technique via laser scanning of cells previously incubated with gold nanoparticles. The laser-induced plasmonic effects on the gold nanoparticles cause a transient permeabilization of the cellular membrane, allowing proteins to enter the cell. Applying this technique, it was possible to deliver green fluorescent protein into mammalian cells with an efficiency of 43%, maintaining a high level of cell viability. Furthermore, a functional delivery of Caspase 3, an apoptosis mediating protein, was demonstrated and evaluated in several cellular assays. Compared to conventional protein transfection techniques such as microinjection, the methodical approach presented here enables high-throughput transfection of about 10 000 cells per second. Moreover, a well-defined point in time of delivery is guaranteed by gold nanoparticle mediated laser transfection, allowing the detailed temporal analysis of cellular pathways and protein trafficking. (papers)

  20. Developing a Novel Gene-Delivery Vector System Using the Recombinant Fusion Protein of Pseudomonas Exotoxin A and Hyperthermophilic Archaeal Histone HPhA.

    Science.gov (United States)

    Deng, Xin; Zhang, Guoli; Zhang, Ling; Feng, Yan; Li, Zehong; Wu, GuangMou; Yue, Yuhuan; Li, Gensong; Cao, Yu; Zhu, Ping

    2015-01-01

    Non-viral gene delivery system with many advantages has a great potential for the future of gene therapy. One inherent obstacle of such approach is the uptake by endocytosis into vesicular compartments. Receptor-mediated gene delivery method holds promise to overcome this obstacle. In this study, we developed a receptor-mediated gene delivery system based on a combination of the Pseudomonas exotoxin A (PE), which has a receptor binding and membrane translocation domain, and the hyperthermophilic archaeal histone (HPhA), which has the DNA binding ability. First, we constructed and expressed the rPE-HPhA fusion protein. We then examined the cytotoxicity and the DNA binding ability of rPE-HPhA. We further assessed the efficiency of transfection of the pEGF-C1 plasmid DNA to CHO cells by the rPE-HPhA system, in comparison to the cationic liposome method. The results showed that the transfection efficiency of rPE-HPhA was higher than that of cationic liposomes. In addition, the rPE-HPhA gene delivery system is non-specific to DNA sequence, topology or targeted cell type. Thus, the rPE-HPhA system can be used for delivering genes of interest into mammalian cells and has great potential to be applied for gene therapy.

  1. Combinational Spinal GAD65 Gene Delivery and Systemic GABA-Mimetic Treatment for Modulation of Spasticity

    Science.gov (United States)

    Kakinohana, Osamu; Hefferan, Michael P.; Miyanohara, Atsushi; Nejime, Tetsuya; Marsala, Silvia; Juhas, Stefan; Juhasova, Jana; Motlik, Jan; Kucharova, Karolina; Strnadel, Jan; Platoshyn, Oleksandr; Lazar, Peter; Galik, Jan; Vinay, Laurent; Marsala, Martin

    2012-01-01

    Background Loss of GABA-mediated pre-synaptic inhibition after spinal injury plays a key role in the progressive increase in spinal reflexes and the appearance of spasticity. Clinical studies show that the use of baclofen (GABAB receptor agonist), while effective in modulating spasticity is associated with major side effects such as general sedation and progressive tolerance development. The goal of the present study was to assess if a combined therapy composed of spinal segment-specific upregulation of GAD65 (glutamate decarboxylase) gene once combined with systemic treatment with tiagabine (GABA uptake inhibitor) will lead to an antispasticity effect and whether such an effect will only be present in GAD65 gene over-expressing spinal segments. Methods/Principal Findings Adult Sprague-Dawley (SD) rats were exposed to transient spinal ischemia (10 min) to induce muscle spasticity. Animals then received lumbar injection of HIV1-CMV-GAD65 lentivirus (LVs) targeting ventral α-motoneuronal pools. At 2–3 weeks after lentivirus delivery animals were treated systemically with tiagabine (4, 10, 20 or 40 mg/kg or vehicle) and the degree of spasticity response measured. In a separate experiment the expression of GAD65 gene after spinal parenchymal delivery of GAD65-lentivirus in naive minipigs was studied. Spastic SD rats receiving spinal injections of the GAD65 gene and treated with systemic tiagabine showed potent and tiagabine-dose-dependent alleviation of spasticity. Neither treatment alone (i.e., GAD65-LVs injection only or tiagabine treatment only) had any significant antispasticity effect nor had any detectable side effect. Measured antispasticity effect correlated with increase in spinal parenchymal GABA synthesis and was restricted to spinal segments overexpressing GAD65 gene. Conclusions/Significance These data show that treatment with orally bioavailable GABA-mimetic drugs if combined with spinal-segment-specific GAD65 gene overexpression can represent a novel

  2. Polyamidoamine (PAMAM Dendrimers Modified with Cathepsin-B Cleavable Oligopeptides for Enhanced Gene Delivery

    Directory of Open Access Journals (Sweden)

    Seulgi Lee

    2017-06-01

    Full Text Available Because of the complex mechanisms mediating cancer onset, prognosis, and metastatic behavior, different therapeutic approaches targeting these mechanisms have been investigated. Recent advancements in nanocarrier-based drug and gene delivery methods have encouraged scientific groups to investigate various novel therapeutic techniques. In this study, a poly(amidoamine (PAMAM polymer-based gene carrier containing the cathepsin B-enzyme sensitive sequence (glycine-phenylalanine-leucine-glycine, GFLG was evaluated to determine transfection efficiency. Following the GFLG sequence, the surface of PAMAM generation 4 (G4 was conjugated with histidine (H and arginine (R for improved endosomal escape and cellular uptake, respectively. The successful synthesis of G4-GLFG-H-R was confirmed by 1H-nuclear magnetic resonance spectroscopy. The polyplex composed of G4-GLFG-H-R and pDNA was simulated by the enzyme cathepsin B and induced endosomal escape of pDNA, which was confirmed by gel electrophoresis. Compared with the G4 control, enzyme-sensitive G4-GLFG-H-R showed higher transfection efficiency and lower cytotoxicity in HeLa cells. These results demonstrated that G4-GLFG-H-R may be a highly potent and efficient carrier for gene therapy applications.

  3. Hyaluronic acid based self-assembling nanosystems for CD44 target mediated siRNA delivery to solid tumors

    Science.gov (United States)

    Ganesh, Shanthi; Iyer, Arun K.; Morrissey, David V.; Amiji, Mansoor M.

    2013-01-01

    Anticancer therapeutics employing RNA interference mechanism holds promising potentials for sequence-specific silencing of target genes. However targeted delivery of siRNAs to tumor tissues and cells and more importantly, their intracellular release at sites of interest still remains a major challenge that needs to be addressed before this technique could become a clinically viable option. In the current study, we have engineered and screened a series of CD44 targeting hyaluronic acid (HA) based self-assembling nanosystems for targeted siRNA delivery. The HA polymer was functionalized with lipids of varying carbon chain lengths/nitrogen content, as well as polyamines for assessing siRNA encapsulation. From the screens, several HA-derivatives were identified that could stably encapsulate/complex siRNAs and form self-assembled nanosystems, as determined by gel retardation assays and dynamic light scattering. Many HA derivatives could transfect siRNAs into cancer cells overexpressing CD44 receptors. Interestingly, blocking the CD44 receptors on the cells using free excess soluble HA prior to incubation of cy3-labeled-siRNA loaded HA nano-assemblies resulted in >90% inhibition of the receptor mediated uptake, confirming target specificity. In addition, SSB/PLK1 siRNA encapsulated in HA-PEI/PEG nanosystems demonstrated dose dependent and target specific gene knockdown in both sensitive and resistant A549 lung cancer cells overexpressing CD44 receptors. More importantly, these siRNA encapsulated nanosystems demonstrated tumor selective uptake and target specific gene knock down in vivo in solid tumors as well as in metastatic tumors. The HA based nanosystems thus portend to be promising siRNA delivery vectors for systemic targeting of CD44 overexpressing cancers including tumor initiating (stem-) cells and metastatic lesions. PMID:23410679

  4. Pokemon siRNA Delivery Mediated by RGD-Modified HBV Core Protein Suppressed the Growth of Hepatocellular Carcinoma.

    Science.gov (United States)

    Kong, Jing; Liu, Xiaoping; Jia, Jianbo; Wu, Jinsheng; Wu, Ning; Chen, Jun; Fang, Fang

    2015-10-01

    Hepatocellular carcinoma (HCC) is a deadly human malignant tumor that is among the most common cancers in the world, especially in Asia. Hepatitis B virus (HBV) infection has been well established as a high risk factor for hepatic malignance. Studies have shown that Pokemon is a master oncogene for HCC growth, suggesting it as an ideal therapeutic target. However, efficient delivery system is still lacking for Pokemon targeting treatment. In this study, we used core proteins of HBV, which is modified with RGD peptides, to construct a biomimetic vector for the delivery of Pokemon siRNAs (namely, RGD-HBc-Pokemon siRNA). Quantitative PCR and Western blot assays revealed that RGD-HBc-Pokemon siRNA possessed the highest efficiency of Pokemon suppression in HCC cells. In vitro experiments further indicated that RGD-HBc-Pokemon-siRNA exerted a higher tumor suppressor activity on HCC cell lines, evidenced by reduced proliferation and attenuated invasiveness, than Pokemon-siRNA or RGD-HBc alone. Finally, animal studies demonstrated that RGD-HBc-Pokemon siRNA suppressed the growth of HCC xenografts in mice by a greater extent than Pokemon-siRNA or RGD-HBc alone. Based on the above results, Pokemon siRNA delivery mediated by RGD-modified HBV core protein was shown to be an effective strategy of HCC gene therapy.

  5. Gold Nanoparticle Approach to the Selective Delivery of Gene Silencing in Cancer—The Case for Combined Delivery?

    Directory of Open Access Journals (Sweden)

    Rita Mendes

    2017-03-01

    Full Text Available Gene therapy arises as a great promise for cancer therapeutics due to its potential to silence genes involved in tumor development. In fact, there are some pivotal gene drivers that suffer critical alterations leading to cell transformation and ultimately to tumor growth. In this vein, gene silencing has been proposed as an active tool to selectively silence these molecular triggers of cancer, thus improving treatment. However, naked nucleic acid (DNA/RNA sequences are reported to have a short lifetime in the body, promptly degraded by circulating enzymes, which in turn speed up elimination and decrease the therapeutic potential of these drugs. The use of nanoparticles for the effective delivery of these silencers to the specific target locations has allowed researchers to overcome this issue. Particularly, gold nanoparticles (AuNPs have been used as attractive vehicles for the target-specific delivery of gene-silencing moieties, alone or in combination with other drugs. We shall discuss current trends in AuNP-based delivery of gene-silencing tools, considering the promising road ahead without overlooking existing concerns for their translation to clinics.

  6. Widespread gene transfer in the central nervous system of cynomolgus macaques following delivery of AAV9 into the cisterna magna

    Directory of Open Access Journals (Sweden)

    Christian Hinderer

    2014-01-01

    Full Text Available Adeno-associated virus serotype 9 (AAV9 vectors have recently been shown to transduce cells throughout the central nervous system of nonhuman primates when injected into the cerebrospinal fluid (CSF, a finding which could lead to a minimally invasive approach to treat genetic and acquired diseases affecting the entire CNS. We characterized the transduction efficiency of two routes of vector administration into the CSF of cynomolgus macaques—lumbar puncture, which is typically used in clinical practice, and suboccipital puncture, which is more commonly used in veterinary medicine. We found that delivery of vector into the cisterna magna via suboccipital puncture is up to 100-fold more efficient for achieving gene transfer to the brain. In addition, we evaluated the inflammatory response to AAV9-mediated GFP expression in the nonhuman primate CNS. We found that while CSF lymphocyte counts increased following gene transfer, there were no clinical or histological signs of immune toxicity. Together these data indicate that delivery of AAV9 into the cisterna magna is an effective method for achieving gene transfer in the CNS, and suggest that adapting this uncommon injection method for human trials could vastly increase the efficiency of gene delivery.

  7. Effect of ultrasound irradiation on bacterial internalization and bacteria-mediated gene transfer to cancer cells.

    Science.gov (United States)

    Ninomiya, Kazuaki; Yamada, Ryuji; Meisaku, Hitomi; Shimizu, Nobuaki

    2014-05-01

    The present study demonstrates that ultrasound irradiation can facilitate bacteria-mediated gene delivery (bactofection). Escherichia coli modified with avidin were employed as a vehicle for delivery of the green fluorescent protein (GFP) gene, a model heterologous gene, into the breast cancer cell line MCF-7. Avidin-mediated binding of E. coli to MCF-7 cells enhanced the internalization of E. coli by approximately 17%, irrespective of the use of ultrasound irradiation. Furthermore, the use of ultrasound irradiation increased the internalization by approximately 5%, irrespective of the presence of avidin on the E. coli cell surface. The percentages of GFP-expressing MCF-7 cells at 24h after bactofection were below 0.5% and 2% for the case with only avidin-modification of E. coli cell surface and only ultrasound irradiation, respectively. However, combining avidin modification with the ultrasound treatment increased this value to 8%. Thus, the use of avidin-modified bacteria in conjunction with ultrasound irradiation has potential as an effective strategy for tumor-targeted bactofection. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Image-Guided Hydrodynamic Gene Delivery: Current Status and Future Directions

    Directory of Open Access Journals (Sweden)

    Kenya Kamimura

    2015-08-01

    Full Text Available Hydrodynamics-based delivery has been used as an experimental tool to express transgene in small animals. This in vivo gene transfer method is useful for functional analysis of genetic elements, therapeutic effect of oligonucleotides, and cancer cells to establish the metastatic cancer animal model for experimental research. Recent progress in the development of image-guided procedure for hydrodynamics-based gene delivery in large animals directly supports the clinical applicability of this technique. This review summarizes the current status and recent progress in the development of hydrodynamics-based gene delivery and discusses the future directions for its clinical application.

  9. Complexation of oppositely charged polyelectrolytes in gene delivery and biology

    Science.gov (United States)

    Shklovskii, Boris

    2009-03-01

    Charge inversion of a DNA double helix by a positively charged flexible polymer (polyelectrolyte) is widely used to facilitate DNA contact with negative cell membranes for gene delivery. Motivated by this application in the first part of the talk I study the phase diagram a solution of long polyanions (PA) with a shorter polycations (PC) as a function the ratio of total charges of PC and PA in the solution, x, and the concentration of monovalent salt. Each PA attracts many PCs to form a complex. When x= 1, the complexes are neutral and condense in a macroscopic drop. When x is far away from 1, complexes are strongly charged and stable. PA are overcharged by PC at x > 1 and undercharged by PC at x < 1. As x approaches 1, PCs attached to PA disproportionate between complexes. Some complexes become neutral and condensed in a macroscopic drop while others become even stronger charged and stay free. The second part of the talk deals with biological example of PA -PC complexes namely self-assembly of vegetable viruses from long ss-RNA molecule paying role of scaffold and identical capsid proteins with long positive tails. I show that optimization Coulomb energy of the virus leads to the charge of RNA twice larger than the total charge of the capsid, in agreement with the experimental data. Then I discuss kinetics of the Coulomb complexation driven virus self-assembly. Capsid proteins stick to unassembled chain of ss RNA (which we call ``antenna'') and slide on it towards the assembly site. I show that at excess of capsid proteins such one-dimensional diffusion accelerates self-assembly more than ten times. On the other hand at excess of ss-RNA, antenna slows self-assembly down. Several experiments are proposed to verify the role of ss-RNA antenna in self-assembly.

  10. Genes and mediators of inflammation and development in osteoarthritis

    NARCIS (Netherlands)

    Bos, Steffan Daniël

    2010-01-01

    Osteoarthritis (OA) mainly affects the articular cartilage covering the bones. In this thesis we investigated the relation between levels of inflammatory mediators, genes involved in their regulation and the disease status of OA. We investigated the role of genetic variation at the interleukin(IL)-1

  11. Neuroprotection by biodegradable PAMAM ester (e-PAM-R)-mediated HMGB1 siRNA delivery in primary cortical cultures and in the postischemic brain.

    Science.gov (United States)

    Kim, Il-Doo; Lim, Chae-Moon; Kim, Jung-Bin; Nam, Hye Yeong; Nam, Kihoon; Kim, Seung-Woo; Park, Jong-Sang; Lee, Ja-Kyeong

    2010-03-19

    Although RNA interference (RNAi)-mediated gene silencing provides a powerful strategy for modulating specific gene functions, difficulties associated with siRNA delivery have impeded the development of efficient therapeutic applications. In particular, the efficacy of siRNA delivery into neurons has been limited by extremely low transfection efficiencies. e-PAM-R is a biodegradable arginine ester of PAMAM dendrimer, which is readily degradable under physiological conditions (pH 7.4, 37 degrees C). In the present study, we investigated the efficiency of siRNA delivery by e-PAM-R in primary cortical cultures and in rat brain. e-PAM-R/siRNA complexes showed high transfection efficiencies and low cytotoxicities in primary cortical cultures. Localization of fluorescence-tagged siRNA revealed that siRNA was delivered not only into the nucleus and cytoplasm, but also along the processes of the neuron. e-PAM-R/siRNA complex-mediated target gene reduction was observed in over 40% of cells and it was persistent for over 48 h. The potential use of e-PAM-R was demonstrated by gene knockdown after transfecting High mobility group box-1 (HMGB1, a novel cytokine-like molecule) siRNA into H(2)O(2)- or NMDA-treated primary cortical cultures. In these cells, HMGB1 siRNA delivery successfully reduced both basal and H(2)O(2)- or NMDA-induced HMGB1 levels, and as a result of that, neuronal cell death was significantly suppressed in both cases. Furthermore, we showed that e-PAM-R successfully delivered HMGB1 siRNA into the rat brain, wherein HMGB1 expression was depleted in over 40% of neurons and astrocytes of the normal brain. Moreover, e-PAM-R-mediated HMGB1 siRNA delivery notably reduced infarct volume in the postischemic rat brain, which is generated by occluding the middle cerebral artery for 60 min. These results indicate that e-PAM-R, a novel biodegradable nonviral gene carrier, offers an efficient means of transfecting siRNA into primary neuronal cells and in the brain and of

  12. Reducible poly(amido ethylenimine)s designed for triggered intracellular gene delivery

    NARCIS (Netherlands)

    Christensen, Lane V.; Christensen, L.; Chang, Chien-Wen; Kim, Won Jong; Kim, Sung Wan; Zhong, Zhiyuan; Lin, C.; Engbersen, Johannes F.J.; Feijen, Jan

    2006-01-01

    Poly(amido ethylenimine) polymers, a new type of peptidomimetic polymer, containing multiple disulfide bonds (SS-PAEIs) designed to degrade after delivery of plasmid DNA (pDNA) into the cell were synthesized and investigated as new carriers for triggered intracellular gene delivery. More

  13. Non-electrostatic complexes with DNA: towards novel synthetic gene delivery systems.

    Science.gov (United States)

    Soto, J; Bessodes, M; Pitard, B; Mailhe, P; Scherman, D; Byk, G

    2000-05-01

    We have developed new DNA complexing amphiphile based on Hoechst 33258 interaction with DNA grooves. The synthesis and physicochemical characterisation of lipid/DNA complexes, as compared to that of classical lipopolyamine for gene delivery, are described and discussed.

  14. Perfluorochemical Liquid-Adenovirus Suspensions Enhance Gene Delivery to the Distal Lung

    Directory of Open Access Journals (Sweden)

    Jeffrey A. Kazzaz

    2011-01-01

    Full Text Available We compared lung delivery methods of recombinant adenovirus (rAd: (1 rAd suspended in saline, (2 rAd suspended in saline followed by a pulse-chase of a perfluorochemical (PFC liquid mixture, and (3 a PFC-rAd suspension. Cell uptake, distribution, and temporal expression of rAd were examined using A549 cells, a murine model using luciferase bioluminescence, and histological analyses. Relative to saline, a 4X increase in transduction efficiency was observed in A549 cells exposed to PFC-rAd for 2–4 h. rAd transgene expression was improved in alveolar epithelial cells, and the level and distribution of luciferase expression when delivered in PFC-rAd suspensions consistently peaked at 24 h. These results demonstrate that PFC-rAd suspensions improve distribution and enhance rAd-mediated gene expression which has important implications in improving lung function by gene therapy.

  15. Adenoassociated virus serotype 9-mediated gene therapy for x-linked adrenoleukodystrophy.

    Science.gov (United States)

    Gong, Yi; Mu, Dakai; Prabhakar, Shilpa; Moser, Ann; Musolino, Patricia; Ren, JiaQian; Breakefield, Xandra O; Maguire, Casey A; Eichler, Florian S

    2015-05-01

    X-linked adrenoleukodystrophy (X-ALD) is a devastating neurological disorder caused by mutations in the ABCD1 gene that encodes a peroxisomal ATP-binding cassette transporter (ABCD1) responsible for transport of CoA-activated very long-chain fatty acids (VLCFA) into the peroxisome for degradation. We used recombinant adenoassociated virus serotype 9 (rAAV9) vector for delivery of the human ABCD1 gene (ABCD1) to mouse central nervous system (CNS). In vitro, efficient delivery of ABCD1 gene was achieved in primary mixed brain glial cells from Abcd1-/- mice as well as X-ALD patient fibroblasts. Importantly, human ABCD1 localized to the peroxisome, and AAV-ABCD1 transduction showed a dose-dependent effect in reducing VLCFA. In vivo, AAV9-ABCD1 was delivered to Abcd1-/- mouse CNS by either stereotactic intracerebroventricular (ICV) or intravenous (IV) injections. Astrocytes, microglia and neurons were the major target cell types following ICV injection, while IV injection also delivered to microvascular endothelial cells and oligodendrocytes. IV injection also yielded high transduction of the adrenal gland. Importantly, IV injection of AAV9-ABCD1 reduced VLCFA in mouse brain and spinal cord. We conclude that AAV9-mediated ABCD1 gene transfer is able to reach target cells in the nervous system and adrenal gland as well as reduce VLCFA in culture and a mouse model of X-ALD.

  16. Nanoparticle mediated P-glycoprotein silencing for improved drug delivery across the blood-brain barrier: a siRNA-chitosan approach.

    Directory of Open Access Journals (Sweden)

    Jostein Malmo

    Full Text Available The blood-brain barrier (BBB, composed of tightly organized endothelial cells, limits the availability of drugs to therapeutic targets in the central nervous system. The barrier is maintained by membrane bound efflux pumps efficiently transporting specific xenobiotics back into the blood. The efflux pump P-glycoprotein (P-gp, expressed at high levels in brain endothelial cells, has several drug substrates. Consequently, siRNA mediated silencing of the P-gp gene is one possible strategy how to improve the delivery of drugs to the brain. Herein, we investigated the potential of siRNA-chitosan nanoparticles in silencing P-gp in a BBB model. We show that the transfection of rat brain endothelial cells mediated effective knockdown of P-gp with subsequent decrease in P-gp substrate efflux. This resulted in increased cellular delivery and efficacy of the model drug doxorubicin.

  17. Nano-scale gene delivery systems; current technology, obstacles, and future directions.

    Science.gov (United States)

    Garcia-Guerra, Antonio; Dunwell, Thomas L; Trigueros, Sonia

    2018-01-07

    Within the different applications of nanomedicine currently being developed, nano-gene delivery is appearing as an exciting new technique with the possibility to overcome recognised hurdles and fulfill several biological and medical needs. The central component of all delivery systems is the requirement for the delivery of genetic material into cells, and for them to eventually reside in the nucleus where their desired function will be exposed. However, genetic material does not passively enter cells; thus, a delivery system is necessary. The emerging field of nano-gene delivery exploits the use of new materials and the properties that arise at the nanometre-scale to produce delivery vectors that can effectively deliver genetic material into a variety of different types of cells. The novel physicochemical properties of the new delivery vectors can be used to address the current challenges existing in nucleic acid delivery in vitro and in vivo. While there is a growing interest in nanostructure-based gene delivery, the field is still in its infancy, and there is yet much to discover about nanostructures and their physicochemical properties in a biological context. We carry out an organized and focused search of bibliographic databases. Our results suggest that despite new breakthroughs in nanostructure synthesis and advanced characterization techniques, we still face many barriers in producing highly efficient and non-toxic delivery systems. In this review, we overview the types of systems currently used for clinical and biomedical research applications along with their advantages and disadvantages, as well as discussing barriers that arise from nano-scale interactions with biological material. In conclusion, we hope that by bringing the far reaching multidisciplinary nature of nano-gene delivery to light, new targeted nanotechnology-bases strategies are developed to overcome the major challenges covered in this review. Copyright© Bentham Science Publishers; For

  18. Macrophage mediated PCI enhanced gene-directed enzyme prodrug therapy

    Science.gov (United States)

    Christie, Catherine E.; Zamora, Genesis; Kwon, Young J.; Berg, Kristian; Madsen, Steen J.; Hirschberg, Henry

    2015-03-01

    Photochemical internalization (PCI) is a photodynamic therapy-based approach for improving the delivery of macromolecules and genes into the cell cytosol. Prodrug activating gene therapy (suicide gene therapy) employing the transduction of the E. coli cytosine deaminase (CD) gene into tumor cells, is a promising method. Expression of this gene within the target cell produces an enzyme that converts the nontoxic prodrug, 5-FC, to the toxic metabolite, 5-fluorouracil (5-FU). 5-FC may be particularly suitable for brain tumors, because it can readily cross the bloodbrain barrier (BBB). In addition the bystander effect, where activated drug is exported from the transfected cancer cells into the tumor microenvironment, plays an important role by inhibiting growth of adjacent tumor cells. Tumor-associated macrophages (TAMs) are frequently found in and around glioblastomas. Monocytes or macrophages (Ma) loaded with drugs, nanoparticles or photosensitizers could therefore be used to target tumors by local synthesis of chemo attractive factors. The basic concept is to combine PCI, to enhance the ex vivo transfection of a suicide gene into Ma, employing specially designed core/shell NP as gene carrier.

  19. Transdermal baicalin delivery using diethylene glycol monoethyl ether-mediated cubic phase gel.

    Science.gov (United States)

    Zhang, Yongtai; Zhang, Kai; Guo, Teng; Li, Yuan; Zhu, Chunyun; Feng, Nianping

    2015-02-01

    This study investigated the transdermal permeability of baicalin, a hydrophobic and readily hydrolyzed drug, delivered by glyceryl monooleate (GMO)-based cubic phase gel (CPG) mediated with Transcotol(®) P (TP, diethylene glycol monoethyl ether). A range of CPGs was produced by varying GMO, water, and TP levels. Examination of their physicochemical properties revealed that the optically isotropic CPG showed higher viscosity than lamellar phase gels (LPG), and the baicalin cargo increased CPG viscosity. The GMO:TP ratio and water content also altered viscosity. CPG-mediated delivery increased baicalin's skin permeation, with 76.65- to 200.24-fold higher (p<0.05) transdermal flux than that of a Carbopol(®)-based hydrogel (HDG), and 6.72- to 17.55-fold (p<0.05) higher than that of LPG, with the same water content. Rat in vivo microdialysis showed that CPG produced sustained baicalin release, with superior pharmacokinetic parameters to those of HDG. Furthermore, cutaneous drug absorption was more efficient on rat abdominal skin, compared to that in the chest or scapular region. Effective fusion between the CPG lipid matrix and the stratum corneum may explain this enhancement of transdermal permeation. CPG containing TP therefore, achieved excellent transdermal drug delivery and good baicalin stability, indicating that this system represents a promising transdermal delivery vehicle. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Polymer-Mediated Delivery of siRNAs to Hepatocellular Carcinoma: Variables Affecting Specificity and Effectiveness

    Directory of Open Access Journals (Sweden)

    Rossella Farra

    2018-03-01

    Full Text Available Despite the advances in anticancer therapies, their effectiveness for many human tumors is still far from being optimal. Significant improvements in treatment efficacy can come from the enhancement of drug specificity. This goal may be achieved by combining the use of therapeutic molecules with tumor specific effects and delivery carriers with tumor targeting ability. In this regard, nucleic acid-based drug (NABD and particularly small interfering RNAs (siRNAs, are attractive molecules due to the possibility to be engineered to target specific tumor genes. On the other hand, polymeric-based delivery systems are emerging as versatile carriers to generate tumor-targeted delivery systems. Here we will focus on the most recent findings in the selection of siRNA/polymeric targeted delivery systems for hepatocellular carcinoma (HCC, a human tumor for which currently available therapeutic approaches are poorly effective. In addition, we will discuss the most attracting and, in our opinion, promising siRNA-polymer combinations for HCC in relation to the biological features of HCC tissue. Attention will be also put on the mathematical description of the mechanisms ruling siRNA-carrier delivery, this being an important aspect to improve effectiveness reducing the experimental work.

  1. Data on macrophage mediated muscle transfection upon delivery of naked plasmid DNA with block copolymers

    Directory of Open Access Journals (Sweden)

    Vivek Mahajan

    2016-06-01

    Full Text Available The data contains 14 figures supporting the research article “Horizontal gene transfer from macrophages to ischemic muscles upon delivery of naked DNA with Pluronic block copolymers” [1]. The data explains the surgical procedure and histological characterization of Murine Hind Limb Ischemia. The data also shows the kinetics of luciferase gene expression, spread of GFP expression through muscle and the colocalization of GFP with cellular markers in ischemic muscles injected with pDNA alone or pDNA/Pluronic. Finally the data shows the effect of Pluronic Block Copolymer to enhance total gene expression (cmv-promoter driven luciferase gene in coculture of DNA transfected MØs with muscle cells.

  2. Nose-to-brain delivery of macromolecules mediated by cell-penetrating peptides

    Directory of Open Access Journals (Sweden)

    Tingting Lin

    2016-07-01

    Full Text Available Brain delivery of macromolecular therapeutics (e.g., proteins remains an unsolved problem because of the formidable blood–brain barrier (BBB. Although a direct pathway of nose-to-brain transfer provides an answer to circumventing the BBB and has already been intensively investigated for brain delivery of small drugs, new challenges arise for intranasal delivery of proteins because of their larger size and hydrophilicity. In order to overcome the barriers and take advantage of available pathways (e.g., epithelial tight junctions, uptake by olfactory neurons, transport into brain tissues, and intra-brain diffusion, a low molecular weight protamine (LMWP cell-penetrating peptide was utilized to facilitate nose-to-brain transport. Cell-penetrating peptides (CPP have been widely used to mediate macromolecular delivery through many kinds of biobarriers. Our results show that conjugates of LMWP–proteins are able to effectively penetrate into the brain after intranasal administration. The CPP-based intranasal method highlights a promising solution for protein therapy of brain diseases.

  3. Profit status and delivery of hospice care for infants: the mediating role of pediatric knowledge.

    Science.gov (United States)

    Lindley, Lisa C; Mixer, Sandy; Mack, Jennifer W

    2012-12-01

    Infant mortality is a persistent problem in the United States, and yet, hospice care is rarely provided for infants at the end of life. To examine the influence of profit status on delivery of hospice care for infants, and to explore the mediating role of pediatric knowledge (pediatric program and pediatric knowledge) on the relationship between profit status and the delivery of infant care. Our study was a cross-sectional secondary analysis, using data from the 2008 California State Utilization Data File of Home Health Agencies and Hospice Facilities survey. Of the hospices in the sample, 13% delivered care for infants. Nonprofit hospices were more likely to delivery care for infants (B=0.67, phospices with a pediatric program were no more likely to deliver care to infants than hospices without a pediatric program (B=0.82, p>0.05). Experience with greater numbers of any pediatric patients facilitated the delivery of hospice infant care in nonprofits (B=0.86, pHospices may need to explore ways to direct resources toward assisting hospice staff members in gaining pediatric experience.

  4. Mathematical Modeling: A Tool for Optimization of Lipid Nanoparticle-Mediated Delivery of siRNA.

    Science.gov (United States)

    Mihaila, Radu; Ruhela, Dipali; Keough, Edward; Cherkaev, Elena; Chang, Silvia; Galinski, Beverly; Bartz, René; Brown, Duncan; Howell, Bonnie; Cunningham, James J

    2017-06-16

    Lipid nanoparticles (LNPs) have been used to successfully deliver small interfering RNAs (siRNAs) to target cells in both preclinical and clinical studies and currently are the leading systems for in vivo delivery. Here, we propose the use of an ordinary differential equation (ODE)-based model as a tool for optimizing LNP-mediated delivery of siRNAs. As a first step, we have used a combination of experimental and computational approaches to develop and validate a mathematical model that captures the critical features for efficient siRNA-LNP delivery in vitro. This model accurately predicts mRNA knockdown resulting from novel combinations of siRNAs and LNPs in vitro. As demonstrated, this model can be effectively used as a screening tool to select the most efficacious LNPs, which can then further be evaluated in vivo. The model serves as a starting point for the future development of next generation models capable of capturing the additional complexity of in vivo delivery. Copyright © 2017 Elena Cherkaev, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ USA. Published by Elsevier Inc. All rights reserved.

  5. Nanodiamonds-mediated doxorubicin nuclear delivery to inhibit lung metastasis of breast cancer.

    Science.gov (United States)

    Xiao, Jisheng; Duan, Xiaopin; Yin, Qi; Zhang, Zhiwen; Yu, Haijun; Li, Yaping

    2013-12-01

    Lung metastasis is one of the greatest challenges for breast cancer treatment. Here, a nanodiamonds (NDs)-mediated doxorubicin (DOX) delivery system was first designed to inhibit the lung metastasis of breast cancer effectively. DOX was non-covalently bound to NDs via physical adsorption in an aqueous solution, then DSPE-PEG 2K was coated to the NDs-DOX complex (NDX) to increase the dispersibility and prolong the circulation time. DSPE-PEG 2K coating NDX (DNX) displayed high drug loading and excellent ability to deliver DOX to the nucleus, thereby significantly enhancing cytotoxicity and inducing cell apoptosis. Furthermore, DNX showed good histocompatibility and could improve drug accumulation in lung, as a result, markedly inhibited the lung metastasis of breast cancer. The high anti-metastasis efficacy with the decreased systemic toxicity suggested that DNX could be a promising drug delivery system for the therapy of lung metastasis of breast cancer. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. AAV2-mediated in vivo immune gene therapy of solid tumours

    LENUS (Irish Health Repository)

    Collins, Sara A

    2010-12-20

    Abstract Background Many strategies have been adopted to unleash the potential of gene therapy for cancer, involving a wide range of therapeutic genes delivered by various methods. Immune therapy has become one of the major strategies adopted for cancer gene therapy and seeks to stimulate the immune system to target tumour antigens. In this study, the feasibility of AAV2 mediated immunotherapy of growing tumours was examined, in isolation and combined with anti-angiogenic therapy. Methods Immune-competent Balb\\/C or C57 mice bearing subcutaneous JBS fibrosarcoma or Lewis Lung Carcinoma (LLC) tumour xenografts respectively were treated by intra-tumoural administration of AAV2 vector encoding the immune up-regulating cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) and the co-stimulatory molecule B7-1 to subcutaneous tumours, either alone or in combination with intra-muscular (IM) delivery of AAV2 vector encoding Nk4 14 days prior to tumour induction. Tumour growth and survival was monitored for all animals. Cured animals were re-challenged with tumourigenic doses of the original tumour type. In vivo cytotoxicity assays were used to investigate establishment of cell-mediated responses in treated animals. Results AAV2-mediated GM-CSF, B7-1 treatment resulted in a significant reduction in tumour growth and an increase in survival in both tumour models. Cured animals were resistant to re-challenge, and induction of T cell mediated anti-tumour responses were demonstrated. Adoptive transfer of splenocytes to naïve animals prevented tumour establishment. Systemic production of Nk4 induced by intra-muscular (IM) delivery of Nk4 significantly reduced subcutaneous tumour growth. However, combination of Nk4 treatment with GM-CSF, B7-1 therapy reduced the efficacy of the immune therapy. Conclusions Overall, this study demonstrates the potential for in vivo AAV2 mediated immune gene therapy, and provides data on the inter-relationship between tumour

  7. TEAD mediates YAP-dependent gene induction and growth control.

    Science.gov (United States)

    Zhao, Bin; Ye, Xin; Yu, Jindan; Li, Li; Li, Weiquan; Li, Siming; Yu, Jianjun; Lin, Jiandie D; Wang, Cun-Yu; Chinnaiyan, Arul M; Lai, Zhi-Chun; Guan, Kun-Liang

    2008-07-15

    The YAP transcription coactivator has been implicated as an oncogene and is amplified in human cancers. Recent studies have established that YAP is phosphorylated and inhibited by the Hippo tumor suppressor pathway. Here we demonstrate that the TEAD family transcription factors are essential in mediating YAP-dependent gene expression. TEAD is also required for YAP-induced cell growth, oncogenic transformation, and epithelial-mesenchymal transition. CTGF is identified as a direct YAP target gene important for cell growth. Moreover, the functional relationship between YAP and TEAD is conserved in Drosophila Yki (the YAP homolog) and Scalloped (the TEAD homolog). Our study reveals TEAD as a new component in the Hippo pathway playing essential roles in mediating biological functions of YAP.

  8. Agrobacterium-mediated gene transfer in plants and biosafety considerations.

    Science.gov (United States)

    Mehrotra, Shweta; Goyal, Vinod

    2012-12-01

    Agrobacterium, the natures' genetic engineer, has been used as a vector to create transgenic plants. Agrobacterium-mediated gene transfer in plants is a highly efficient transformation process which is governed by various factors including genotype of the host plant, explant, vector, plasmid, bacterial strain, composition of culture medium, tissue damage, and temperature of co-cultivation. Agrobacterium has been successfully used to transform various economically and horticulturally important monocot and dicot species by standard tissue culture and in planta transformation techniques like floral or seedling infilteration, apical meristem transformation, and the pistil drip methods. Monocots have been comparatively difficult to transform by Agrobacterium. However, successful transformations have been reported in the last few years based on the adjustment of the parameters that govern the responses of monocots to Agrobacterium. A novel Agrobacterium transferred DNA-derived nanocomplex method has been developed which will be highly valuable for plant biology and biotechnology. Agrobacterium-mediated genetic transformation is known to be the preferred method of creating transgenic plants from a commercial and biosafety perspective. Agrobacterium-mediated gene transfer predominantly results in the integration of foreign genes at a single locus in the host plant, without associated vector backbone and is also known to produce marker free plants, which are the prerequisites for commercialization of transgenic crops. Research in Agrobacterium-mediated transformation can provide new and novel insights into the understanding of the regulatory process controlling molecular, cellular, biochemical, physiological, and developmental processes occurring during Agrobacterium-mediated transformation and also into a wide range of aspects on biological safety of transgenic crops to improve crop production to meet the demands of ever-growing world's population.

  9. GFP expression by intracellular gene delivery of GFP-coding fragments using nanocrystal quantum dots

    Science.gov (United States)

    Hoshino, Akiyoshi; Manabe, Noriyoshi; Fujioka, Kouki; Hanada, Sanshiro; Yasuhara, Masato; Kondo, Akihiko; Yamamoto, Kenji

    2008-12-01

    Gene therapy is an attractive approach to supplement a deficient gene function. Although there has been some success with specific gene delivery using various methods including viral vectors and liposomes, most of these methods have a limited efficiency or also carry a risk for oncogenesis. We herein report that quantum dots (QDs) conjugated with nuclear localizing signal peptides (NLSP) successfully introduced gene-fragments with promoter elements, which promoted the expression of the enhanced green fluorescent protein (eGFP) gene in mammalian cells. The expression of eGFP protein was observed when the QD/gene-construct was added to the culture media. The gene-expression efficiency varied depending on multiple factors around QDs, such as (1) the reading direction of the gene-fragments, (2) the quantity of gene-fragments attached on the surface of the QD-constructs, (3) the surface electronic charges varied according to the structure of the QD/gene-constructs, and (4) the particle size of QD/gene complex varied according to the structure and amounts of gene-fragments. Using this QD/gene-construct system, eGFP protein could be detected 28 days after the gene-introduction whereas the fluorescence of QDs had disappeared. This system therefore provides another method for the intracellular delivery of gene-fragments without using either viral vectors or specific liposomes.

  10. GFP expression by intracellular gene delivery of GFP-coding fragments using nanocrystal quantum dots

    International Nuclear Information System (INIS)

    Hoshino, Akiyoshi; Manabe, Noriyoshi; Fujioka, Kouki; Hanada, Sanshiro; Yamamoto, Kenji; Yasuhara, Masato; Kondo, Akihiko

    2008-01-01

    Gene therapy is an attractive approach to supplement a deficient gene function. Although there has been some success with specific gene delivery using various methods including viral vectors and liposomes, most of these methods have a limited efficiency or also carry a risk for oncogenesis. We herein report that quantum dots (QDs) conjugated with nuclear localizing signal peptides (NLSP) successfully introduced gene-fragments with promoter elements, which promoted the expression of the enhanced green fluorescent protein (eGFP) gene in mammalian cells. The expression of eGFP protein was observed when the QD/gene-construct was added to the culture media. The gene-expression efficiency varied depending on multiple factors around QDs, such as (1) the reading direction of the gene-fragments, (2) the quantity of gene-fragments attached on the surface of the QD-constructs, (3) the surface electronic charges varied according to the structure of the QD/gene-constructs, and (4) the particle size of QD/gene complex varied according to the structure and amounts of gene-fragments. Using this QD/gene-construct system, eGFP protein could be detected 28 days after the gene-introduction whereas the fluorescence of QDs had disappeared. This system therefore provides another method for the intracellular delivery of gene-fragments without using either viral vectors or specific liposomes.

  11. Magnetic nanoparticles for targeted therapeutic gene delivery and magnetic-inducing heating on hepatoma

    International Nuclear Information System (INIS)

    Yuan, Chenyan; Zhang, Jia; Li, Hongbo; Zhang, Hao; Wang, Ling; Zhang, Dongsheng; An, Yanli

    2014-01-01

    Gene therapy holds great promise for treating cancers, but their clinical applications are being hampered due to uncontrolled gene delivery and expression. To develop a targeted, safe and efficient tumor therapy system, we constructed a tissue-specific suicide gene delivery system by using magnetic nanoparticles (MNPs) as carriers for the combination of gene therapy and hyperthermia on hepatoma. The suicide gene was hepatoma-targeted and hypoxia-enhanced, and the MNPs possessed the ability to elevate temperature to the effective range for tumor hyperthermia as imposed on an alternating magnetic field (AMF). The tumoricidal effects of targeted gene therapy associated with hyperthermia were evaluated in vitro and in vivo. The experiment demonstrated that hyperthermia combined with a targeted gene therapy system proffer an effective tool for tumor therapy with high selectivity and the synergistic effect of hepatoma suppression. (paper)

  12. [Research progress in sperm mediated gene transfer technology].

    Science.gov (United States)

    Hao, Xiaoxiong; Zhu, Zheng; Cao, Mianfu; Li, Chengren; Lin, Yunlai

    2013-04-01

    With the rapid development of biotechnology, we can change the trait of organism using transgenetic technology. In recent years, there are growing interests in the establishment of sperm mediated gene transfer (SMGT) technology as an effective and convenient method to produce transgenic animals. SMGT technology is a transgenetic method, which is easy in operation and does little harm to the cell compared with the other transgenetic methods. In this review, we expound the background, development, mechanism, operation and application of SMGT.

  13. A geminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing.

    Science.gov (United States)

    Yin, Kangquan; Han, Ting; Liu, Guang; Chen, Tianyuan; Wang, Ying; Yu, Alice Yunzi L; Liu, Yule

    2015-10-09

    CRISPR/Cas has emerged as potent genome editing technology and has successfully been applied in many organisms, including several plant species. However, delivery of genome editing reagents remains a challenge in plants. Here, we report a virus-based guide RNA (gRNA) delivery system for CRISPR/Cas9 mediated plant genome editing (VIGE) that can be used to precisely target genome locations and cause mutations. VIGE is performed by using a modified Cabbage Leaf Curl virus (CaLCuV) vector to express gRNAs in stable transgenic plants expressing Cas9. DNA sequencing confirmed VIGE of endogenous NbPDS3 and NbIspH genes in non-inoculated leaves because CaLCuV can infect plants systemically. Moreover, VIGE of NbPDS3 and NbIspH in newly developed leaves caused photo-bleached phenotype. These results demonstrate that geminivirus-based VIGE could be a powerful tool in plant genome editing.

  14. α, ω-Cholesterol-Functionalized Low Molecular Weight Polyethylene Glycol as a Novel Modifier of Cationic Liposomes for Gene Delivery

    Directory of Open Access Journals (Sweden)

    Cui-Cui Ma

    2014-11-01

    Full Text Available Here, three novel cholesterol (Ch/low molecular weight polyethylene glycol (PEG conjugates, termed α, ω-cholesterol-functionalized PEG (Ch2-PEGn, were successfully synthesized using three kinds of PEG with different average molecular weight (PEG600, PEG1000 and PEG2000. The purpose of the study was to investigate the potential application of novel cationic liposomes (Ch2-PEGn-CLs containing Ch2-PEGn in gene delivery. The introduction of Ch2-PEGn affected both the particle size and zeta potential of cationic liposomes. Ch2-PEG2000 effectively compressed liposomal particles and Ch2-PEG2000-CLs were of the smallest size. Ch2-PEG1000 and Ch2-PEG2000 significantly decreased zeta potentials of Ch2-PEGn-CLs, while Ch2-PEG600 did not alter the zeta potential due to the short PEG chain. Moreover, the in vitro gene transfection efficiencies mediated by different Ch2-PEGn-CLs also differed, in which Ch2-PEG600-CLs achieved the strongest GFP expression than Ch2-PEG1000-CLs and Ch2-PEG2000-CLs in SKOV-3 cells. The gene delivery efficacy of Ch2-PEGn-CLs was further examined by addition of a targeting moiety (folate ligand in both folate-receptor (FR overexpressing SKOV-3 cells and A549 cells with low expression of FR. For Ch2-PEG1000-CLs and Ch2-PEG2000-CLs, higher molar ratios of folate ligand resulted in enhanced transfection efficacies, but Ch2-PEG600-CLs had no similar in contrast. Additionally, MTT assay proved the reduced cytotoxicities of cationic liposomes after modification by Ch2-PEGn. These findings provide important insights into the effects of Ch2-PEGn on cationic liposomes for delivering genes, which would be beneficial for the development of Ch2-PEGn-CLs-based gene delivery system.

  15. Ultrasound-guided drug delivery in cancer

    Energy Technology Data Exchange (ETDEWEB)

    Chowdhury, Sayan Mullick; Lee, Tae Hwa; Willmann, Jugen K. [Dept. of Radiology, Stanford University School of Medicine, Stanford (United States)

    2017-07-15

    Recent advancements in ultrasound and microbubble (USMB) mediated drug delivery technology has shown that this approach can improve spatially confined delivery of drugs and genes to target tissues while reducing systemic dose and toxicity. The mechanism behind enhanced delivery of therapeutics is sonoporation, the formation of openings in the vasculature, induced by ultrasound-triggered oscillations and destruction of microbubbles. In this review, progress and challenges of USMB mediated drug delivery are summarized, with special focus on cancer therapy.

  16. Ultrasound-guided drug delivery in cancer

    International Nuclear Information System (INIS)

    Chowdhury, Sayan Mullick; Lee, Tae Hwa; Willmann, Jugen K.

    2017-01-01

    Recent advancements in ultrasound and microbubble (USMB) mediated drug delivery technology has shown that this approach can improve spatially confined delivery of drugs and genes to target tissues while reducing systemic dose and toxicity. The mechanism behind enhanced delivery of therapeutics is sonoporation, the formation of openings in the vasculature, induced by ultrasound-triggered oscillations and destruction of microbubbles. In this review, progress and challenges of USMB mediated drug delivery are summarized, with special focus on cancer therapy

  17. Junk DNA enhances pEI-based non-viral gene delivery

    NARCIS (Netherlands)

    Gaal, E.V.B. van; Oosting, R.S.; Hennink, W.E.; Crommelin, D.J.A.; Mastrobattista, E.

    Gene therapy aims at delivering exogenous DNA into the nuclei of target cells to establish expression of a therapeutic protein. Non-viral gene delivery is examined as a safer alternative to viral approaches, but is presently characterized by a low efficiency. In the past years several non-viral

  18. Cell based-gene delivery approaches for the treatment of spinal cord injury and neurodegenerative disorders.

    Science.gov (United States)

    Taha, Masoumeh Fakhr

    2010-03-01

    Cell based-gene delivery has provided an important therapeutic strategy for different disorders in the recent years. This strategy is based on the transplantation of genetically modified cells to express specific genes and to target the delivery of therapeutic factors, especially for the treatment of cancers and neurological, immunological, cardiovascular and heamatopoietic disorders. Although, preliminary reports are encouraging, and experimental studies indicate functionally and structurally improvements in the animal models of different disorders, universal application of this strategy for human diseases requires more evidence. There are a number of parameters that need to be evaluated, including the optimal cell source, the most effective gene/genes to be delivered, the optimal vector and method of gene delivery into the cells and the most efficient route for the delivery of genetically modified cells into the patient. Also, some obstacles have to be overcome, including the safety and usefulness of the approaches and the stability of the improvements. Here, recent studies concerning with the cell-based gene delivery for spinal cord injury and some neurodegenerative disorders such as amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease are briefly reviewed, and their exciting consequences are discussed.

  19. Homologous recombination mediates functional recovery of dysferlin deficiency following AAV5 gene transfer.

    Directory of Open Access Journals (Sweden)

    William E Grose

    Full Text Available The dysferlinopathies comprise a group of untreatable muscle disorders including limb girdle muscular dystrophy type 2B, Miyoshi myopathy, distal anterior compartment syndrome, and rigid spine syndrome. As with other forms of muscular dystrophy, adeno-associated virus (AAV gene transfer is a particularly auspicious treatment strategy, however the size of the DYSF cDNA (6.5 kb negates packaging into traditional AAV serotypes known to express well in muscle (i.e. rAAV1, 2, 6, 8, 9. Potential advantages of a full cDNA versus a mini-gene include: maintaining structural-functional protein domains, evading protein misfolding, and avoiding novel epitopes that could be immunogenic. AAV5 has demonstrated unique plasticity with regards to packaging capacity and recombination of virions containing homologous regions of cDNA inserts has been implicated in the generation of full-length transcripts. Herein we show for the first time in vivo that homologous recombination following AAV5.DYSF gene transfer leads to the production of full length transcript and protein. Moreover, gene transfer of full-length dysferlin protein in dysferlin deficient mice resulted in expression levels sufficient to correct functional deficits in the diaphragm and importantly in skeletal muscle membrane repair. Intravascular regional gene transfer through the femoral artery produced high levels of transduction and enabled targeting of specific muscle groups affected by the dysferlinopathies setting the stage for potential translation to clinical trials. We provide proof of principle that AAV5 mediated delivery of dysferlin is a highly promising strategy for treatment of dysferlinopathies and has far-reaching implications for the therapeutic delivery of other large genes.

  20. Gene therapy of transplant arteriopathy by liposome-mediated transfection of endothelial nitric oxide synthase.

    Science.gov (United States)

    Iwata, A; Sai, S; Moore, M; Nyhuis, J; de Fries-Hallstrand, R; Quetingco, G C; Allen, M D

    2000-11-01

    Transplant arteriopathy is the major factor limiting long-term survival after cardiac transplantation. We have previously demonstrated that liposome-mediated gene delivery of endothelial nitric oxide synthase (eNOS) to donor hearts reduces ischemia-reperfusion injury by blocking NFkappaB activation, adhesion molecule expression, and leukocyte infiltration. In this study, we used gene transfer of eNOS in a rabbit carotid transplant model to see whether these same effects would similarly ameliorate transplant arteriopathy. Liposomes complexed to the gene encoding eNOS were injected into donor carotid arterial segments that were transplanted orthotopically into recipient carotid arteries (n = 10). Controls included transplanted carotids transfected with liposomes complexed to empty plasmids (no functional gene) (n = 4) and transplanted carotids treated with saline (n = 6). Transplanted arteries were harvested for processing at 21 days. Intima/media (I/M) area ratios were calculated by computerized image analysis. Infiltrating T-lymphocytes and macrophages, and expression of VCAM-1 and ICAM-1 were quantified on immunocytochemistry. The I/M ratio was significantly reduced in eNOS-transfected arteries compared with arteries transfected with empty plasmids and saline-treated controls. Compared to transplanted control arteries, eNOS-transfected arteries demonstrated significantly reduced T-cell infiltration into the intima and significantly reduced macrophage infiltration into the media. Cell surface expression of VCAM-1 and ICAM-1 were both reduced in eNOS-transfected arteries. ENOS gene delivery can suppress neointimal lesion formation and T-lymphocyte and macrophage infiltration in transplanted arteries, associated with a reduction in relevant adhesion molecule expression. Thus, gene therapy with eNOS may not only reduce ischemia-reperfusion injury but may also ameliorate transplant arteriopathy in transplanted hearts.

  1. Multifunctional pH-Responsive Folate Receptor Mediated Polymer Nanoparticles for Drug Delivery.

    Science.gov (United States)

    Cai, Xiaoqing; Yang, Xiaoye; Wang, Fang; Zhang, Chen; Sun, Deqing; Zhai, Guangxi

    2016-07-01

    Multifunctional pH-responsive folate receptor mediated targeted polymer nanoparticles (TPNps) were developed for docetaxel (DTX) delivery based on poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)poly (β-amino ester) (P123-PAE) and poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)-folate (P123-FA) copolymers. The DTX was loaded into the TPNps with a decent drug loading content of 15.02 ± 0.14 wt%. In vitro drug release results showed that the DTX was released from the TPNps at a pH-dependent manner. Tetrazolium dye (MTT) assay revealed that the bland polymer nanoparticles displayed almost nontoxicity at 200 μg/mL concentration. However, the DTX-loaded TPNps showed high anti-tumor activity at low IC50 (0.72 μg/mL) for MCF-7 cells following 48 h incubation. Cellular uptake experiments revealed that the TPNps had higher degree of cellular uptake than nontargeted polymer nanoparticles, indicating that the nanoparticles were internalized into the cells via FA receptor-mediated endocytosis. Moreover, the cellular uptake pathways for the FA grafted polymer were involved in energy-dependent, clathrin-mediated and caveolae-mediated endocytosis. The cell killing effect and cellular uptake of the DTX-TPNps by the MCF-7 cells were all enhanced by about two folds at pH 5.5 when compared with pH 7.4. The TPNps also significantly prolonged the in vivo retention time for the DTX. These results suggest that the biocompatible pH responsive folate-modified polymer nanoparticles present a promising safe nanosystem for intracellular targeted delivery of DTX.

  2. Self-focusing therapeutic gene delivery with intelligent gene vector swarms: intra-swarm signalling through receptor transgene expression in targeted cells.

    Science.gov (United States)

    Tolmachov, Oleg E

    2015-01-01

    are then attracted to and internalized into the intended target cells via the expressed cognate strongly binding extra-cellular receptor, causing escalation of gene transfer into these cells and increasing the copy number of the therapeutic gene expression modules. Such self-focusing swarms of gene vectors can be either homogeneous, with 'scout' and 'therapeutic' members of the swarm being structurally identical, or, alternatively, heterogeneous (split), with 'scout' and 'therapeutic' members of the swarm being structurally specialized. It is hoped that the proposed self-focusing cell-targeted gene vector swarms with receptor-mediated intra-swarm signalling could be particularly effective in 'top-up' gene delivery scenarios, achieving high-level and sustained expression of therapeutic transgenes that are prone to shut-down through degradation and silencing. Crucially, in contrast to low-precision 'general location' vector guidance by diffusible chemo-attractants, ear-marking non-diffusible receptors can provide high-accuracy targeting of therapeutic vector particles to the specific cell, which has undergone a 'successful cell-specific hit' by a 'scout' vector particle. Opportunities for cell targeting could be expanded, since in the proposed model of self-focusing it could be possible to probe a broad selection of intra-cellular determinants of cell-specificity and not just to rely exclusively on extra-cellular markers of cell-specificity. By employing such self-focusing gene vectors for the improvement of cell-targeted delivery of therapeutic genes, e.g., in cancer therapy or gene addition therapy of recessive genetic diseases, it could be possible to broaden a leeway for the reduction of the vector load and, consequently, to minimize undesired vector cytotoxicity, immune reactions, and the risk of inadvertent genetic modification of germline cells in genetic treatment in vivo. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Novel non-viral vectors for gene delivery: synthesis of a second-generation library of mono-functionalized poly-(guanidinium)amines and their introduction into cationic lipids.

    Science.gov (United States)

    Byk, G; Soto, J; Mattler, C; Frederic, M; Scherman, D

    1998-01-01

    The development of new gene delivery technologies is a prerequisite towards gene therapy clinical trials. Because gene delivery mediated by viral vectors remains of limited scope due to immunological and propagation risks, the development of new non-viral gene delivery systems is of crucial importance. We have synthesized a secondary library of mono-functionalized poly-(guanidinium)amines generated from a library of mono-functionalized polyamines applying the concept of "libraries from libraries." The method allows a quick and easy access to mono-functionalized geometrically varied poly-(guanidinium)amines. The new building blocks were introduced into cationic lipids to obtain novel poly-(guanidinium)amine lipids, which are potential DNA vectors for gene delivery. Copyright 1998 John Wiley & Sons, Inc.

  4. Chromosomal position effects on AAV-mediated gene targeting.

    Science.gov (United States)

    Cornea, Anda M; Russell, David W

    2010-06-01

    The effects of chromosomal position and neighboring genomic elements on gene targeting in human cells remain largely unexplored. To study these, we used a shuttle vector system in which murine leukemia virus (MLV)-based proviral targets present at different chromosomal locations and containing mutations in the neomycin phosphotransferase (neo) gene were corrected by adeno-associated virus (AAV)-mediated gene targeting. Sixteen identical target loci present in HT-1080 human sarcoma cells were all successfully corrected by gene targeting. The gene targeting frequencies varied by as much as 10-fold, and there was a clear bias for correction of one of the targets in clones containing two target sites. The targeting frequency at each site was correlated to the proximity and density of various genomic elements, and we found a significant association of higher targeting frequencies at loci near a subset of dinucleotide microsatellite repeats (r = -0.55, P targeting frequencies at the target loci (r = 0.52, P targeting frequencies. Our results indicate that certain chromosomal positions are preferred sites for gene targeting in human cells.

  5. Reprogramming of Human Fibroblasts to Induced Pluripotent Stem Cells with Sleeping Beauty Transposon-Based Stable Gene Delivery.

    Science.gov (United States)

    Sebe, Attila; Ivics, Zoltán

    2016-01-01

    Human induced pluripotent stem (iPS) cells are a source of patient-specific pluripotent stem cells and resemble human embryonic stem (ES) cells in gene expression profiles, morphology, pluripotency, and in vitro differentiation potential. iPS cells are applied in disease modeling, drug screenings, toxicology screenings, and autologous cell therapy. In this protocol, we describe how to derive human iPS cells from fibroblasts by Sleeping Beauty (SB) transposon-mediated gene transfer of reprogramming factors. First, the components of the non-viral Sleeping Beauty transposon system, namely a transposon vector encoding reprogramming transcription factors and a helper plasmid expressing the SB transposase, are electroporated into human fibroblasts. The reprogramming cassette undergoes transposition from the transfected plasmids into the fibroblast genome, thereby resulting in stable delivery of the reprogramming factors. Reprogramming by using this protocol takes ~4 weeks, after which the iPS cells are isolated and clonally propagated.

  6. Safety assessment of liver-targeted hydrodynamic gene delivery in dogs.

    Directory of Open Access Journals (Sweden)

    Kenya Kamimura

    Full Text Available Evidence in support of safety of a gene delivery procedure is essential toward gene therapy. Previous studies using the hydrodynamics-based procedure primarily focus on gene delivery efficiency or gene function analysis in mice. The current study focuses on an assessment of the safety of computer-controlled and liver-targeted hydrodynamic gene delivery in dogs as the first step toward hydrodynamic gene therapy in clinic. We demonstrate that the impacts of the hydrodynamic procedure were limited in the injected region and the influences were transient. Histological examination and the hepatic microcirculation measurement using reflectance spectrophotometry reveal that the liver-specific impact of the procedure involves a transient expansion of the liver sinusoids. No systemic damage or toxicity was observed. Physiological parameters, including electrocardiogram, heart rate, blood pressure, oxygen saturation, and body temperature, remained in normal ranges during and after hydrodynamic injection. Body weight was also examined to assess the long-term effects of the procedure in animals who underwent 3 hydrodynamic injections in 6 weeks with 2-week time interval in between. Serum biochemistry analysis showed a transient increase in liver enzymes and a few cytokines upon injection. These results demonstrate that image-guided, liver-specific hydrodynamic gene delivery is safe.

  7. Effects of Extracorporeal Shock Wave-Mediated Transdermal Local Anesthetic Drug Delivery on Rat Caudal Nerves.

    Science.gov (United States)

    Luh, Jer-Junn; Huang, Wan-Ting; Lin, Kwan-Hwa; Huang, Yi-You; Kuo, Po-Ling; Chen, Wen-Shiang

    2018-01-01

    Cavitation plays a substantial role in the clinical effects of extracorporeal shock wave therapy (ESWT). It is also generally accepted as a major mechanism in sonophoresis. To identify the enhancing effect of extracorporeal shock wave-mediated transdermal drug delivery, 24 Wistar rats were randomly assigned to four groups: (i) topical application of a eutectic mixture of local anesthetics (EMLA); (ii) 1-MHz ultrasound; (iii) ESWT pre-treatment combined with EMLA application; (iv) ESWT concurrent with EMLA application on rat tails. The degree of anesthesia was assessed using the amplitude and latency of sensory nerve action potentials within 5 min after a 60-min EMLA application. The results indicated that ESWT pre-treatment and concurrent ESWT accelerated the anesthetic effects of the EMLA cream on the tail nerve (p < 0.05). This finding might indicate that shock wave-mediated transdermal drug delivery is possible during the ESWT period. Copyright © 2018 World Federation for Ultrasound in Medicine and Biology. Published by Elsevier Inc. All rights reserved.

  8. Intracellular localisation of proteins to specific cellular areas by nanocapsule mediated delivery.

    Science.gov (United States)

    Wang, Huabin; Chen, Ligang; Sun, Xianchao; Fu, Ailing

    2017-09-01

    Nanocapsules are promising carriers with great potential for intracellular protein transport. Although many studies have intended to improve cell uptake efficacy, there is an increasing interest in understanding of subcellular distribution of cargoes inside cells, which is essential for purposeful delivery of biomolecules into specific sites within cells. Herein, we interrogate the intracellular localisation of exogenous proteins, including fluorescein isothiocyanate (FITC)-labelled bovine serum albumin (BSA) and green fluorescent protein (GFP), mediated by specially designed nanocapsules. The results show that the designed nanocapsules can deliver the two types of fluorescent proteins into different cellular destinations (cytosol, nucleus or the whole cell), depending on the composition of nanocapsules. Meanwhile, several impact factors that influence the distribution of proteins in cells have also been investigated, and the results suggest that the localisation of capsule-mediated proteins in cells is strongly affected by the surface properties of nanocapsules, the types of stabilisers and proteins, and environmental temperatures. The rational control of intracellular localised delivery of exogenous proteins as we demonstrated in this study might open new avenues to obtain desired magnitude of drug effects for modulating cell activity.

  9. An albumin-mediated cholesterol design-based strategy for tuning siRNA pharmacokinetics and gene silencing

    DEFF Research Database (Denmark)

    Bienk, Konrad; Hvam, Michael Lykke; Pakula, Malgorzata Maria

    2016-01-01

    Major challenges for the clinical translation of small interfering RNA (siRNA) include overcoming the poor plasma half-life, site-specific delivery and modulation of gene silencing. In this work, we exploit the intrinsic transport properties of human serum albumin to tune the blood circulatory ha...... of 28% (rHSA/siRNA) compared to 4% (naked siRNA) 6 days post-injection. This work presents a novel albumin-mediated cholesteryl design-based strategy for tuning pharmacokinetics and systemic gene silencing....

  10. Exosome Mediated Delivery of miR-124 Promotes Neurogenesis after Ischemia

    Directory of Open Access Journals (Sweden)

    Jialei Yang

    2017-06-01

    Full Text Available The intrinsic ability of neurogenesis after stroke has been proven weak, which results in insufficient repair of injury in the nerve system. Recent studies suggest multiple microRNAs (miRNAs are involved in the neuroremodeling process. Targeted miRNAs delivery for amplification of neurogenesis is promising in promoting the prognosis after ischemia. Here, we showed that modified exosomes, with rabies virus glycoprotein (RVG fused to exosomal protein lysosome-associated membrane glycoprotein 2b (Lamp2b, could efficiently deliver miR-124 to the infarct site. Systemic administration of RVG-exosomes loaded with miR-124 promoted cortical neural progenitors to obtain neuronal identity and protect against ischemic injury by robust cortical neurogenesis. Our study suggests that RVG-exosomes can be utilized therapeutically for the targeted delivery of gene drugs to the brain, thus having great potential for clinical applications.

  11. Imaging in solution of (Lys)(16)-containing bifunctional synthetic peptide/DNA nanoparticles for gene delivery.

    Science.gov (United States)

    Collins, Louise; Kaszuba, Michael; Fabre, John W

    2004-04-07

    The physical properties of non-viral vector/DNA nanoparticles in physiological aqueous solution are poorly understood. A Fluid Particle Image Analyser (FPIA), normally used for analysis of industrial and environmental fluids, was used to visualise individual (Lys)(16)-containing peptide/DNA particles. Eight (Lys)(16)-containing synthetic peptides were used to generate peptide/DNA particles at a constant + to - charge ratio of 2.8:1 with 10 microg/ml of plasmid DNA in phosphate buffered saline. Dynamic Light Scattering (DLS) and gene delivery studies were also performed. We present the first images of non-viral vector/DNA nanoparticles in physiological aqueous solution, together with precise measurements of individual particle size and shape in solution and, for the first time, an accurate measure of particle number. Particle size and shape, particle number, and efficiency for gene delivery varied markedly with different peptides. Under standard conditions for in vitro gene delivery, we estimate approximately 60 peptide/DNA nanoparticles per target cell, each containing approximately 70,000 plasmids. This novel capacity to image individual vector/DNA nanoparticles in solution and to count them accurately will enable a more precise assessment of non-viral gene delivery systems, and a more quantitative interpretation of gene delivery experiments.

  12. Dendrimer-coupled sonophoresis-mediated transdermal drug-delivery system for diclofenac.

    Science.gov (United States)

    Huang, Bin; Dong, Wei-Jiang; Yang, Gao-Yi; Wang, Wei; Ji, Cong-Hua; Zhou, Fei-Ni

    2015-01-01

    The purpose of the present study was to develop a novel transdermal drug-delivery system comprising a polyamidoamine dendrimer coupled with sonophoresis to enhance the permeation of diclofenac (DF) through the skin. The novel transdermal drug-delivery system was developed by using a statistical Plackett-Burman design. Hairless male Wistar rat skin was used for the DF-permeation study. Coupling media concentration, ultrasound-application time, duty cycle, distance from probe to skin, and a third-generation polyamidoamine-dendrimer concentration were selected as independent variables, while in vitro drug release was selected as a dependent variable. Independent variables were found to be statistically significant (Psonophoresis treatment (run 14) showed 257.3 µg/cm(2) cumulative drug permeated through the skin after 24 hours. However, when the same gel was applied to sonophoresis-treated skin, drastic permeation enhancement was observed. In the case of run 3, the cumulative drug that permeated through the skin was 935.21 µg/cm(2). It was concluded that dendrimer-coupled sonophoresis-mediated transdermal drug delivery system has the potential to enhance the permeation of DF through the skin.

  13. Neuroprotection from NMDA excitotoxic lesion by Cu/Zn superoxide dismutase gene delivery to the postnatal rat brain by a modular protein vector

    OpenAIRE

    Peluffo, Hugo; Acarin, Laia; Arís, Anna; González, Pau; Villaverde, Antoni; Castellano, Bernardo; González, Berta

    2006-01-01

    Abstract Background Superoxide mediated oxidative stress is a key neuropathologic mechanism in acute central nervous system injuries. We have analyzed the neuroprotective efficacy of the transient overexpression of antioxidant enzyme Cu/Zn Superoxide dismutase (SOD) after excitotoxic injury to the immature rat brain by using a recently constructed modular protein vector for non-viral gene delivery termed NLSCt. For this purpose, animals were injected with the NLSCt vector carrying the Cu/Zn S...

  14. Antibody-IL2 Fusion Protein Delivery by Gene Transfer

    National Research Council Canada - National Science Library

    Nicolet, Charles

    1997-01-01

    The purpose of the work described is to assess the feasibility of a gene therapy approach to deliver a specific antibody cytokine fusion protein called CC49-1L2 to a tumor expressing antigen reactive with the antibody...

  15. Functional gene silencing mediated by chitosan/siRNA nanocomplexes

    Energy Technology Data Exchange (ETDEWEB)

    Ji, A M; Su, D; Che, O; Li, W S; Sun, L; Zhang, Z Y; Xu, F [Department of Pharmaceutical Science, Zhujiang Hospital, Southern Medical University, Guangzhou 510282 (China); Yang, B, E-mail: andrewfxu1998@gmail.co [Department of Chemistry, Indiana University-Bloomington, Bloomington, IN 47405 (United States)

    2009-10-07

    Chitosan/siRNA nanoparticles to knock down FHL2 gene expression were reported in this work. The physicochemical properties such as particle size, surface charge, morphology and complex stability of chitosan nanoparticle-incorporated siRNA were evaluated. Nanoparticles which were formulated with chitosan/siRNA exhibited irregular, lamellar and dendritic structures with a hydrodynamic radius size of about 148 nm and net positive charges with zeta-potential value of 58.5 mV. The knockdown effect of the chitosan/siRNA nanoparticles on gene expression in FHL2 over-expressed human colorectal cancer Lovo cells was investigated. The result showed that FHL2 siRNA formulated within chitosan nanoparticles could knock down about 69.6% FHL2 gene expression, which is very similar to the 68.8% reduced gene expression when siRNA was transfected with liposome Lipofectamine. Western analysis further showed significant FHL-2 protein expression reduced by the chitosan/siRNA nanoparticles. The results also showed that blocking FHL2 expression by siRNA could also inhibit the growth and proliferation of human colorectal cancer Lovo cells. The current results demonstrated that chitosan-based siRNA nanoparticles were a very efficient delivery system for siRNA in vivo as previously reported.

  16. Functional gene silencing mediated by chitosan/siRNA nanocomplexes

    Science.gov (United States)

    Ji, A. M.; Su, D.; Che, O.; Li, W. S.; Sun, L.; Zhang, Z. Y.; Yang, B.; Xu, F.

    2009-10-01

    Chitosan/siRNA nanoparticles to knock down FHL2 gene expression were reported in this work. The physicochemical properties such as particle size, surface charge, morphology and complex stability of chitosan nanoparticle-incorporated siRNA were evaluated. Nanoparticles which were formulated with chitosan/siRNA exhibited irregular, lamellar and dendritic structures with a hydrodynamic radius size of about 148 nm and net positive charges with zeta-potential value of 58.5 mV. The knockdown effect of the chitosan/siRNA nanoparticles on gene expression in FHL2 over-expressed human colorectal cancer Lovo cells was investigated. The result showed that FHL2 siRNA formulated within chitosan nanoparticles could knock down about 69.6% FHL2 gene expression, which is very similar to the 68.8% reduced gene expression when siRNA was transfected with liposome Lipofectamine. Western analysis further showed significant FHL-2 protein expression reduced by the chitosan/siRNA nanoparticles. The results also showed that blocking FHL2 expression by siRNA could also inhibit the growth and proliferation of human colorectal cancer Lovo cells. The current results demonstrated that chitosan-based siRNA nanoparticles were a very efficient delivery system for siRNA in vivo as previously reported.

  17. Functional gene silencing mediated by chitosan/siRNA nanocomplexes

    International Nuclear Information System (INIS)

    Ji, A M; Su, D; Che, O; Li, W S; Sun, L; Zhang, Z Y; Xu, F; Yang, B

    2009-01-01

    Chitosan/siRNA nanoparticles to knock down FHL2 gene expression were reported in this work. The physicochemical properties such as particle size, surface charge, morphology and complex stability of chitosan nanoparticle-incorporated siRNA were evaluated. Nanoparticles which were formulated with chitosan/siRNA exhibited irregular, lamellar and dendritic structures with a hydrodynamic radius size of about 148 nm and net positive charges with zeta-potential value of 58.5 mV. The knockdown effect of the chitosan/siRNA nanoparticles on gene expression in FHL2 over-expressed human colorectal cancer Lovo cells was investigated. The result showed that FHL2 siRNA formulated within chitosan nanoparticles could knock down about 69.6% FHL2 gene expression, which is very similar to the 68.8% reduced gene expression when siRNA was transfected with liposome Lipofectamine. Western analysis further showed significant FHL-2 protein expression reduced by the chitosan/siRNA nanoparticles. The results also showed that blocking FHL2 expression by siRNA could also inhibit the growth and proliferation of human colorectal cancer Lovo cells. The current results demonstrated that chitosan-based siRNA nanoparticles were a very efficient delivery system for siRNA in vivo as previously reported.

  18. DNA Nanotechnology for Precise Control over Drug Delivery and Gene Therapy.

    Science.gov (United States)

    Angell, Chava; Xie, Sibai; Zhang, Liangfang; Chen, Yi

    2016-03-02

    Nanomedicine has been growing exponentially due to its enhanced drug targeting and reduced drug toxicity. It uses the interactions where nanotechnological components and biological systems communicate with each other to facilitate the delivery performance. At this scale, the physiochemical properties of delivery systems strongly affect their capacities. Among current delivery systems, DNA nanotechnology shows many advantages because of its unprecedented engineering abilities. Through molecular recognition, DNA nanotechnology can be used to construct a variety of nanostructures with precisely controllable size, shape, and surface chemistry, which can be appreciated in the delivery process. In this review, different approaches that are currently used for the construction of DNA nanostructures are reported. Further, the utilization of these DNA nanostructures with the well-defined parameters for the precise control in drug delivery and gene therapy is discussed. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Translational Advancement of Somatostatin Gene Delivery for Disease Modification and Cognitive Sparing in Intractable Epilepsy

    Science.gov (United States)

    2015-09-01

    efficacy against seizures. 15. SUBJECT TERMS Epilepsy; seizure; kindling; somatatostatin; traumatic brain injury; gene delivery; adeno­ associated  viral ...vector; neurogenesis; inflammation; neurodegeneration; hippocampus, memory 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF...delivery using adeno-associated viral vectors for SST or inactive control protein will be tested for ability to suppress seizure susceptibility and

  20. Self-assembling polycations for gene delivery: Effects of polymer structure and environmental pH

    OpenAIRE

    Samsonova, Olga

    2012-01-01

    SUMMARY In this thesis, novel polycationic vectors for therapeutic gene delivery were introduced with the focus on structure-function relationship in both physicochemical and biological aspects. First the transfection related differences of classical vectors with high and low branching grade and charge density: PEI and PLL, were examined under pH-alternating tumor tissue conditions. Further structural design for siRNA delivery was...

  1. Investigating Gene Function in Cereal Rust Fungi by Plant-Mediated Virus-Induced Gene Silencing.

    Science.gov (United States)

    Panwar, Vinay; Bakkeren, Guus

    2017-01-01

    Cereal rust fungi are destructive pathogens, threatening grain production worldwide. Targeted breeding for resistance utilizing host resistance genes has been effective. However, breakdown of resistance occurs frequently and continued efforts are needed to understand how these fungi overcome resistance and to expand the range of available resistance genes. Whole genome sequencing, transcriptomic and proteomic studies followed by genome-wide computational and comparative analyses have identified large repertoire of genes in rust fungi among which are candidates predicted to code for pathogenicity and virulence factors. Some of these genes represent defence triggering avirulence effectors. However, functions of most genes still needs to be assessed to understand the biology of these obligate biotrophic pathogens. Since genetic manipulations such as gene deletion and genetic transformation are not yet feasible in rust fungi, performing functional gene studies is challenging. Recently, Host-induced gene silencing (HIGS) has emerged as a useful tool to characterize gene function in rust fungi while infecting and growing in host plants. We utilized Barley stripe mosaic virus-mediated virus induced gene silencing (BSMV-VIGS) to induce HIGS of candidate rust fungal genes in the wheat host to determine their role in plant-fungal interactions. Here, we describe the methods for using BSMV-VIGS in wheat for functional genomics study in cereal rust fungi.

  2. An efficient nonviral gene-delivery vector based on hyperbranched cationic glycogen derivatives

    Directory of Open Access Journals (Sweden)

    Liang X

    2014-01-01

    Full Text Available Xuan Liang,1,* Xianyue Ren,2,* Zhenzhen Liu,1 Yingliang Liu,1 Jue Wang,2 Jingnan Wang,2 Li-Ming Zhang,1 David YB Deng,2 Daping Quan,1 Liqun Yang1 1Institute of Polymer Science, School of Chemistry and Chemical Engineering, Key Laboratory of Designed Synthesis and Application of Polymer Material, Key Laboratory for Polymeric Composite and Functional Materials of Ministry of Education, Sun Yat-Sen University, Guangzhou, People's Republic of China; 2Research Center of Translational Medicine, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, People's Republic of China *Both these authors contributed equally to this work Background: The purpose of this study was to synthesize and evaluate hyperbranched cationic glycogen derivatives as an efficient nonviral gene-delivery vector. Methods: A series of hyperbranched cationic glycogen derivatives conjugated with 3-(dimethylamino-1-propylamine (DMAPA-Glyp and 1-(2-aminoethyl piperazine (AEPZ-Glyp residues were synthesized and characterized by Fourier-transform infrared and hydrogen-1 nuclear magnetic resonance spectroscopy. Their buffer capacity was assessed by acid–base titration in aqueous NaCl solution. Plasmid deoxyribonucleic acid (pDNA condensation ability and protection against DNase I degradation of the glycogen derivatives were assessed using agarose gel electrophoresis. The zeta potentials and particle sizes of the glycogen derivative/pDNA complexes were measured, and the images of the complexes were observed using atomic force microscopy. Blood compatibility and cytotoxicity were evaluated by hemolysis assay and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, respectively. pDNA transfection efficiency mediated by the cationic glycogen derivatives was evaluated by flow cytometry and fluorescence microscopy in the 293T (human embryonic kidney and the CNE2 (human nasopharyngeal carcinoma cell lines. In vivo delivery of pDNA in model animals (Sprague Dawley

  3. Dendrimer-coupled sonophoresis-mediated transdermal drug-delivery system for diclofenac

    Directory of Open Access Journals (Sweden)

    Huang B

    2015-07-01

    Full Text Available Bin Huang,1 Wei-Jiang Dong,2 Gao-Yi Yang,3 Wei Wang,1 Cong-Hua Ji,1 Fei-Ni Zhou4 1Department of Ultrasound, First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, 2Department of Ultrasonography, Tongxiang Chinese Medicine Hospital, Jiaxing, 3Department of Ultrasound, Hangzhou Red Cross Hospital, 4Department of Medical Records and Statistics, First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China Abstract: The purpose of the present study was to develop a novel transdermal drug-delivery system comprising a polyamidoamine dendrimer coupled with sonophoresis to enhance the permeation of diclofenac (DF through the skin. The novel transdermal drug-delivery system was developed by using a statistical Plackett–Burman design. Hairless male Wistar rat skin was used for the DF-permeation study. Coupling media concentration, ultrasound-application time, duty cycle, distance from probe to skin, and a third-generation polyamidoamine-dendrimer concentration were selected as independent variables, while in vitro drug release was selected as a dependent variable. Independent variables were found to be statistically significant (P<0.05. DF gel without dendrimer and ultrasound treatment to skin (passive delivery, run 13 showed 56.69 µg/cm2 cumulative drug permeated through the skin, while the DF-dendrimer gel without sonophoresis treatment (run 14 showed 257.3 µg/cm2 cumulative drug permeated through the skin after 24 hours. However, when the same gel was applied to sonophoresis-treated skin, drastic permeation enhancement was observed. In the case of run 3, the cumulative drug that permeated through the skin was 935.21 µg/cm2. It was concluded that dendrimer-coupled sonophoresis-mediated transdermal drug delivery system has the potential to enhance the permeation of DF through the skin. Keywords: sonophoresis, ultrasound, polyamidoamine, permeation enhancers, stratum corneum

  4. Ligand-Modified Human Serum Albumin Nanoparticles for Enhanced Gene Delivery.

    Science.gov (United States)

    Look, Jennifer; Wilhelm, Nadine; von Briesen, Hagen; Noske, Nadja; Günther, Christine; Langer, Klaus; Gorjup, Erwin

    2015-09-08

    The development of nonviral gene delivery systems is a great challenge to enable safe gene therapy. In this study, ligand-modified nanoparticles based on human serum albumin (HSA) were developed and optimized for an efficient gene therapy. Different glutaraldehyde cross-linking degrees were investigated to optimize the HSA nanoparticles for gene delivery. The peptide sequence arginine-glycine-aspartate (RGD) and the HIV-1 transactivator of transduction sequence (Tat) are well-known as promising targeting ligands. Plasmid DNA loaded HSA nanoparticles were covalently modified on their surface with these different ligands. The transfection potential of the obtained plasmid DNA loaded RGD- and Tat-modified nanoparticles was investigated in vitro, and optimal incubation conditions for these preparations were studied. It turned out that Tat-modified HSA nanoparticles with the lowest cross-linking degree of 20% showed the highest transfection potential. Taken together, ligand-functionalized HSA nanoparticles represent promising tools for efficient and safe gene therapy.

  5. Delivery methods for site-specific nucleases: Achieving the full potential of therapeutic gene editing.

    Science.gov (United States)

    Liu, Jia; Shui, Sai-Lan

    2016-12-28

    The advent of site-specific nucleases, particularly CRISPR/Cas9, provides researchers with the unprecedented ability to manipulate genomic sequences. These nucleases are used to create model cell lines, engineer metabolic pathways, produce transgenic animals and plants, perform genome-wide functional screen and, most importantly, treat human diseases that are difficult to tackle by traditional medications. Considerable efforts have been devoted to improving the efficiency and specificity of nucleases for clinical applications. However, safe and efficient delivery methods remain the major obstacle for therapeutic gene editing. In this review, we summarize the recent progress on nuclease delivery methods, highlight their impact on the outcomes of gene editing and discuss the potential of different delivery approaches for therapeutic gene editing. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Gene therapy for C-26 colon cancer using heparin-polyethyleneimine nanoparticle-mediated survivin T34A

    Science.gov (United States)

    Zhang, Ling; Gao, Xiang; Men, Ke; Wang, BiLan; Zhang, Shuang; Qiu, Jinfeng; Huang, Meijuan; Gou, MaLing; Huang, Ning; Qian, ZhiYong; Zhao, Xia; Wei, YuQuan

    2011-01-01

    Background Gene therapy provides a novel method for the prevention and treatment of cancer, but the clinical application of gene therapy is restricted, mainly because of the absence of an efficient and safe gene delivery system. Recently, we developed a novel nonviral gene carrier, ie, heparin-polyethyleneimine (HPEI) nanoparticles for this purpose. Methods and results HPEI nanoparticles were used to deliver plasmid-expressing mouse survivin-T34A (ms-T34A) to treat C-26 carcinoma in vitro and in vivo. According to the in vitro studies, HPEI nanoparticles could efficiently transfect the pGFP report gene into C-26 cells, with a transfection efficiency of 30.5% ± 2%. Moreover, HPEI nanoparticle-mediated ms-T34A could efficiently inhibit the proliferation of C-26 cells by induction of apoptosis in vitro. Based on the in vivo studies, HPEI nanoparticles could transfect the Lac-Z report gene into C-26 cells in vivo. Intratumoral injection of HPEI nanoparticle-mediated ms-T34A significantly inhibited growth of subcutaneous C-26 carcinoma in vivo by induction of apoptosis and inhibition of angiogenesis. Conclusion This research suggests that HPEI nanoparticle-mediated ms-T34A may have a promising role in C-26 colon carcinoma therapy. PMID:22072877

  7. Effective gene delivery to Trypanosoma cruzi epimastigotes through nucleofection.

    Science.gov (United States)

    Pacheco-Lugo, Lisandro; Díaz-Olmos, Yirys; Sáenz-García, José; Probst, Christian Macagnan; DaRocha, Wanderson Duarte

    2017-06-01

    New opportunities have raised to study the gene function approaches of Trypanosoma cruzi after its genome sequencing in 2005. Functional genomic approaches in Trypanosoma cruzi are challenging due to the reduced tools available for genetic manipulation, as well as to the reduced efficiency of the transient transfection conducted through conventional methods. The Amaxa nucleofector device was systematically tested in the present study in order to improve the electroporation conditions in the epimastigote forms of T. cruzi. The transfection efficiency was quantified using the green fluorescent protein (GFP) as reporter gene followed by cell survival assessment. The herein used nucleofection parameters have increased the survival rates (>90%) and the transfection efficiency by approximately 35%. The small amount of epimastigotes and DNA required for the nucleofection can turn the method adopted here into an attractive tool for high throughput screening (HTS) applications, and for gene editing in parasites where genetic manipulation tools remain relatively scarce. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Non-viral gene delivery strategies for cancer therapy, tissue engineering and regenerative medicine

    Science.gov (United States)

    Bhise, Nupura S.

    Gene therapy involves the delivery of deoxyribonucleic acid (DNA) into cells to override or replace a malfunctioning gene for treating debilitating genetic diseases, including cancer and neurodegenerative diseases. In addition to its use as a therapeutic, it can also serve as a technology to enable regenerative medicine strategies. The central challenge of the gene therapy research arena is developing a safe and effective delivery agent. Since viral vectors have critical immunogenic and tumorogenic safety issues that limit their clinical use, recent efforts have focused on developing non-viral biomaterial based delivery vectors. Cationic polymers are an attractive class of gene delivery vectors due to their structural versatility, ease of synthesis, biodegradability, ability to self-complex into nanoparticles with negatively charged DNA, capacity to carry large cargo, cellular uptake and endosomal escape capacity. In this thesis, we hypothesized that developing a biomaterial library of poly(betaamino esters) (PBAE), a newer class of cationic polymers consisting of biodegradable ester groups, would allow investigating vector design parameters and formulating effective non-viral gene delivery strategies for cancer drug delivery, tissue engineering and stem cell engineering. Consequently, a high-throughput transfection assay was developed to screen the PBAE-based nanoparticles in hard to transfect fibroblast cell lines. To gain mechanistic insights into the nanoparticle formulation process, biophysical properties of the vectors were characterized in terms of molecular weight (MW), nanoparticle size, zeta potential and plasmid per particle count. We report a novel assay developed for quantifying the plasmid per nanoparticle count and studying its implications for co-delivery of multiple genes. The MW of the polymers ranged from 10 kDa to 100 kDa, nanoparticle size was about 150 run, zeta potential was about 30 mV in sodium acetate buffer (25 mM, pH 5) and 30 to 100

  9. Cell mediated therapeutics for cancer treatment: Tumor homing cells as therapeutic delivery vehicles

    Science.gov (United States)

    Balivada, Sivasai

    Many cell types were known to have migratory properties towards tumors and different research groups have shown reliable results regarding cells as delivery vehicles of therapeutics for targeted cancer treatment. Present report discusses proof of concept for 1. Cell mediated delivery of Magnetic nanoparticles (MNPs) and targeted Magnetic hyperthermia (MHT) as a cancer treatment by using in vivo mouse cancer models, 2. Cells surface engineering with chimeric proteins for targeted cancer treatment by using in vitro models. 1. Tumor homing cells can carry MNPs specifically to the tumor site and tumor burden will decrease after alternating magnetic field (AMF) exposure. To test this hypothesis, first we loaded Fe/Fe3O4 bi-magnetic NPs into neural progenitor cells (NPCs), which were previously shown to migrate towards melanoma tumors. We observed that NPCs loaded with MNPs travel to subcutaneous melanoma tumors. After alternating magnetic field (AMF) exposure, the targeted delivery of MNPs by the NPCs resulted in a mild decrease in tumor size (Chapter-2). Monocytes/macrophages (Mo/Ma) are known to infiltrate tumor sites, and also have phagocytic activity which can increase their uptake of MNPs. To test Mo/Ma-mediated MHT we transplanted Mo/Ma loaded with MNPs into a mouse model of pancreatic peritoneal carcinomatosis. We observed that MNP-loaded Mo/Ma infiltrated pancreatic tumors and, after AMF treatment, significantly prolonged the lives of mice bearing disseminated intraperitoneal pancreatic tumors (Chapter-3). 2. Targeted cancer treatment could be achieved by engineering tumor homing cell surfaces with tumor proteases cleavable, cancer cell specific recombinant therapeutic proteins. To test this, Urokinase and Calpain (tumor specific proteases) cleavable; prostate cancer cell (CaP) specific (CaP1 targeting peptide); apoptosis inducible (Caspase3 V266ED3)- rCasp3V266ED3 chimeric protein was designed in silico. Hypothesized membrane anchored chimeric protein (rCasp3V

  10. DNA-transporting nanoparticles : design and in vitro evaluation of DNA and formulation for non-viral gene delivery

    NARCIS (Netherlands)

    van Gaal, E.V.B.

    2010-01-01

    The aim of gene therapy is to treat, cure or prevent a disease by replacing defective genes, introducing new genes or changing the expression of a person’s genes. Success of gene therapy is dependent on successful delivery of DNA from the site of administration into cell nuclei. Naturally occurring

  11. Magnetotactic Bacterial Cages as Safe and Smart Gene Delivery Vehicles

    KAUST Repository

    Alsaiari, Shahad K.

    2016-07-27

    In spite of the huge advances in the area of synthetic carriers, their efficiency still poorly compares to natural vectors. Herein, we report the use of unmodified magnetotactic bacteria as a guidable delivery vehicle for DNA functionalized gold nanoparticles (AuNPs). High cargo loading is established under anaerobic conditions (bacteria is alive) through endocytosis where AuNPs are employed as transmembrane proteins mimics (facilitate endocytosis) as well as imaging agents to verify and quantify loading and release. The naturally bio-mineralized magnetosomes, within the bacteria, induce heat generation inside bacteria through magnetic hyperthermia. Most importantly after exposing the system to air (bacteria is dead) the cell wall stays intact providing an efficient bacterial vessel. Upon incubation with THP-1 cells, the magnetotactic bacterial cages (MBCs) adhere to the cell wall and are directly engulfed through the phagocytic activity of these cells. Applying magnetic hyperthermia leads to the dissociation of the bacterial microcarrier and eventual release of cargo.

  12. Vascular gene transfer and drug delivery in vitro using low-frequency ultrasound and microbubbles

    Science.gov (United States)

    Yang, Hong; Liu, Zhong-hua; Liu, Yi-yao; Lou, Chang-chun; Ren, Zheng-long; Miyoshi, Hirokazu

    2010-01-01

    Aim: To determine the effects of ultrasound exposure in combination with a microbubble contrast agent (SonoVue) on the cellular uptake and delivery of drugs/genes into human umbilical vein endothelial cells (HUVECs) as well as their biological effects on migration. Methods: HUVECs in suspension were exposed to pulsed ultrasound with a 10% duty cycle in combination with various concentrations of a microbubble contrast agent (SonoVue) using a digital sonifier at a frequency of 20 kHz and an intensity of 3.77 W/cm2 on the surface of a horn tip. Cell culture inserts were used to determine the cell migration ability. Results: Exposure to pulsed ultrasound resulted in enhanced green fluorescent protein (EGFP) gene transfection efficiencies ranging from 0.2% to 2%. The transfection efficiency of HUVECs was approximately 3-fold higher in the presence of SonoVue than in its absence at the effective exposure time of 6 s. For drug delivery to HUVECs using ultrasound, the delivery efficiencies of a low-molecular-weight model drug (TO-PRO®−1, MW 645.38) were significantly higher when compared to drug delivery without ultrasound, with a maximum efficiency of approximately 34%. However, the delivery efficiencies of a high-molecular-weight model drug (Dextran-Rhodamine B, MW 70 000) were low, with a maximum delivery efficiency of nearly 0.5%, and gene transfection results were similarly poor. The migration ability of HUVECs exposed to ultrasound was also lower than that of the control (no exposure). Conclusion: The use of low-frequency and low-energy ultrasound in combination with microbubbles could be a potent physical method of increasing drug/gene delivery efficiency. This technique is a promising nonviral approach that can be used in cardiovascular disease therapy. PMID:20348943

  13. Effect of adrenomedullin gene delivery on insulin resistance in type 2 diabetic rats

    Directory of Open Access Journals (Sweden)

    Hoda Y. Henein

    2011-01-01

    Full Text Available Type 2 diabetes mellitus is one of the common metabolic disorders that ultimately afflicts large number of individuals. Adrenomedullin (AM is a potent vasodilator peptide; previous studies reported development of insulin resistance in aged AM deficient mice. In this study, we employed a gene delivery approach to explore its potential role in insulin resistance. Four groups were included: control, diabetic, non-diabetic injected with the AM gene and diabetic injected with the AM gene. One week following gene delivery, serum glucose, insulin, triglycerides, leptin, adiponectin and corticosterone were measured as well as the insulin resistance index (HOMA-IR. Soleus muscle glucose uptake and RT-PCR of both AM and glucose transporter-4 (GLUT 4 gene expressions were assessed. A single tail vein injection of adrenomedullin gene in type 2 diabetic rats improved skeletal muscle insulin responsiveness with significant improvement of soleus muscle glucose uptake, HOMA-IR, serum glucose, insulin and triglycerides and significant increase in muscle GLUT 4 gene expression (P < 0.05 compared with the non-injected diabetic rats. The beneficial effects of AM gene delivery were accompanied by a significant increase in the serum level of adiponectin (2.95 ± 0.09 versus 2.33 ± 0.17 μg/ml in the non-injected diabetic group as well as a significant decrease in leptin and corticosterone levels (7.51 ± 0.51 and 262.88 ± 10.34 versus 10.63 ± 1.4 and 275.86 ± 11.19 ng/ml respectively in the non-injected diabetic group. The conclusion of the study is that AM gene delivery can improve insulin resistance and may have significant therapeutic applications in type 2 diabetes mellitus.

  14. Non-viral gene delivery systems: hurdles for bench-to-bedside transformation.

    Science.gov (United States)

    Helal, N A; Osami, A; Helmy, A; McDonald, T; Shaaban, L A; Nounou, M I

    2017-11-01

    Biologist and Nobel Prize winner James Watson's quote, "We used to think that our fate was in our stars, but now we know that, in large measure, our fate is in our genes", represents the initial food for thought that revolutionized the way medications and active pharmaceutical ingredients are defined (Rocholl 1996). This fate engraved in the genetic material, as mentioned in Watson's quote, fueled a tremendous revolution wave in gene therapy. Gene therapy is a promising technology for treating genetic and acquired diseases by modulating the expression of a specific gene in the pathological cells. This is achieved by introducing a DNA sequence or other nucleic acid material or oligonucleotides to the target cell (Kay, 2011). Moreover, gene therapy contributes to correction of genetic defects, expression of therapeutic proteins, and inhibition of the synthesis of malignant proteins. In this review article, different non-viral gene delivery systems and their applications are discussed in detail. We reviewed and tabulated over 90 papers and 50 patents from 2006 to date discussing non-viral gene delivery technologies, innovation, and bench-to-bedside transformation. Furthermore, we are going to shed light on the lack of standardization in the design and characterization of non-viral gene delivery systems worldwide, which is a major concern in this research's field. This review would aid in getting an eagle eye view through non-viral gene delivery technologies during the past 20 years. Such a view, capturing the advances, the hurdles, and experimental details, would aid expert researchers in tuning their experimentation strategies and help newcomers better initially design their studies to generate solid and comprehensive results that can be reliable and reproducible.

  15. Strategies for improving chemotherapeutic delivery to solid tumors mediated by vascular permeability modulation

    Science.gov (United States)

    Roy Chaudhuri, Tista

    An essential mode of distribution of blood-borne chemotherapeutic agents within a solid tumor is via the micro-circulation. Poor tumor perfusion, because of a lack of functional vasculature or a lack of microvessels, as well as low tumor vascular permeability, can prevent adequate deposition of even low molecular-weight agents into the tumor. The modulation of tumor vascular function and density can provides numerous strategies for improving intratumor deposition of chemotherapeutic agents. Here we investigated strategies to improve drug delivery to two tumor types that share in common poor drug delivery, but differ in the underlying cause. First, in an angiogenesis-driven brain tumor model of Glioblastoma, the vascular permeability barrier, along with poorly-functional vasculature, hinders drug delivery. A strategy of nanoparticle-based tumor 'priming' to attack the vascular permeability barrier, employing sterically stabilized liposomal doxorubicin (SSL-DXR), was investigated. Functional and histological evaluation of tumor vasculature revealed that after an initial period of depressed vascular permeability and vascular pruning 3--4 days after SSL-DXR administration, vascular permeability and perfusion were restored and then elevated after 5--7 days. As a result of tumor priming, deposition of subsequently-administered nanoparticles was enhanced, and the efficacy of temozolomide (TMZ), if administered during the window of elevated permeability, was increased. The sequenced regimen resulted in a persistent reduction of the tumor proliferative index and a 40% suppression of tumor volume, compared to animals that received both agents simultaneously. Second, in a hypovascular, pancreatic ductal adenocarcinoma model, disruption of tumor-stromal communication via sonic hedgehog (sHH) signaling pathway inhibition mediated an indirect vascular proliferation and a more than 2-fold increase in intratumor nanoparticle deposition. Enhanced delivery of SSL-DXR in tumors pre

  16. Predicting DNA-mediated drug delivery in interior carcinoma using electromagnetically excited nanoparticles.

    Science.gov (United States)

    Ghosh, Soham; Das, Tamal; Chakraborty, Suman; Das, Sarit K

    2011-09-01

    magnetizing field strength, nanoparticle size, diffusion coefficients, porous media parameters and different oligonucleotide sequences on temperature rise and site-specific drug release. The proposed model, thus, provides a generic framework for the betterment of nanoparticle mediated drug delivery, which is expected to impart significant impact on cancer therapy. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Attenuation of endoplasmic reticulum stress-related myocardial apoptosis by SERCA2a gene delivery in ischemic heart disease.

    Science.gov (United States)

    Xin, Wei; Lu, Xiaochun; Li, Xiaoying; Niu, Kun; Cai, Jimei

    2011-01-01

    Previous studies suggested that endoplasmic reticulum (ER) stress-associated apoptosis plays an important role in the pathogenesis of ischemic heart disease. Gene transfer of sarco/endoplasmic reticulum Ca(2+) ATPase 2a (SERCA2a) attenuates myocardial apoptosis in a variety of heart failure models. This study is to investigate the effects of SERCA2a gene delivery on the myocardial apoptosis and ER stress pathway in a porcine ischemic heart disease model. Eighteen pigs were either subjected to ameroid implantation in the coronary artery or sham operation. Eight wks after gene delivery, the protein level and activity of SERCA2a were measured. Myocardial apoptosis was determined using terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling assay. Regional myocardial perfusion and function were evaluated by (99m)Tc-sestamibi ((99m)Tc-MIBI) single photon emission computed tomography and echocardiography. The ER stress signaling was assessed by Western blot. SERCA2a protein level and activity were significantly decreased in the ischemic myocardium and restored to normal after SERCA2a gene transfer. Restoration of SERCA2a expression significantly improved the cardiac function, although no improvement of regional myocardial perfusion was detected. Restoration of SERCA2a significantly attenuated myocardial apoptosis and reversed the activation of unfolded protein response (UPR) pathway and the ER stress-associated apoptosis pathways. These findings demonstrate a robust role of SERCA2a in attenuation of ischemic myocardial apoptosis, correlating with reverse activation of the ER stress-associated apoptosis pathways, suggesting that the beneficial effects of SERCA2a gene transfer may involve the attenuation of ER stress-associated myocardial apoptosis.

  18. siRNA delivery using polyelectrolyte-gold nanoassemblies in neuronal cells for BACE1 gene silencing.

    Science.gov (United States)

    Chaudhary, Aparna; Garg, Sanjeev

    2017-11-01

    Small interfering RNA (siRNA) mediated RNA interference is a versatile therapeutic tool for many intractable genetic disorders. Various nanoassemblies specifically designed to deliver the siRNAs could be utilized for efficient siRNA delivery which is one of the major concern for the success of this therapeutic. Thus, in the present study, polyelectrolyte-gold nanoassemblies (PE-Gold NAs) were selected for siRNA delivery of an in vitro verified siRNA. Three different polyelectrolytes (polyethyleneimine, citraconic anhydride modified poly (allylamine) hydrochloride and poly l-arginine) were used to formulate the PE-Gold NAs using the layer-by-layer technique. Successful physico-chemical characterizations of these PE-Gold NAs were performed using UV-Visible, FTIR, 1 H-NMR spectroscopies, XRD, TEM, DLS and Zeta potential measurements. In vitro studies for the cytotoxicity, the uptake of these nanoassemblies and the gene silencing were carried out using these PE-Gold NAs in N2a and NB4 1A3 (murine neuronal) cell lines. The three selected PE-Gold NAs showed significant BACE1 (β-site APP cleaving enzyme 1) gene silencing (50-60%). This work demonstrates the potential of PE-Gold NAs to deliver siRNA targeting BACE1 in neuronal cells. Finally, it was concluded that different polyelectrolytes used in the PE-Gold NAs achieve different gene silencing due to the variation in their delivery efficiencies. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Recombinant adenovirus-mediated gene transfer suppresses experimental arthritis

    Directory of Open Access Journals (Sweden)

    E. Quattrocchi

    2011-09-01

    Full Text Available Collagen Induced Arthritis (CIA is a widely studied animal model to develop and test novel therapeutic approaches for treating Rheumatoid Arthritis (RA in humans. Soluble Cytotoxic T-Lymphocyte Antigen 4 (CTLA4-Ig, which binds B7 molecule on antigen presenting cells and blocks CD28 mediated T-lymphocyte activation, has been shown to ameliorate experimental autoimmune diseases such as lupus, diabetes and CIA. Objective of our research was to investigate in vivo the effectiveness of blocking the B7/CD28 T-lymphocyte co-stimulatory pathway, utilizing a gene transfer technology, as a therapeutic strategy against CIA. Replication-deficient adenoviruses encoding a chimeric CTLA4-Ig fusion protein, or β-galactosidase as control, have been injected intravenously once at arthritis onset. Disease activity has been monitored by the assessment of clinical score, paw thickness and type II collagen (CII specific cellular and humoral immune responses for 21 days. The adenovirally delivered CTLA4-Ig fusion protein at a dose of 2×108 pfu suppressed established CIA, whereas the control β-galactosidase did not significantly affect the disease course. CII-specific lymphocyte proliferation, IFNg production and anti-CII antibodies were significantly reduced by CTLA4-Ig treatment. Our results demonstrate that blockade of the B7/CD28 co-stimulatory pathway by adenovirus-mediated CTLA4-Ig gene transfer is effective in treating established CIA suggesting its potential in treating RA.

  20. Development of Gold Nanoparticle towards Radioenhancement Therapy, Renal Clearance, siRNA Delivery and Light-Controlled Gene Silencing

    Science.gov (United States)

    Wang, Jianxin

    Gold nanoparticles (GNPs) have been widely studied and used in research for diagnostic, prophylactic or therapeutic purposes. However, they still face many technical challenges before they can be used to effectively address unmet biomedical needs. The theme of this dissertation is focused on addressing challenges of GNPs in clinical translation, and to improve their potential for application in radioenhancement therapy and siRNA delivery. We demonstrate the facile self-assembly of micellar gold nanocapsules using zwitterionic surfactants, with hydrodynamic diameters below 10 nm, which holds promise for good renal clearance to promote the excretion of GNPs in human body. We also prepared PEI- and PEG-coated GNPs and demonstrated their uptake into HeLa cells with exposure to soft X-rays (120 kVp), based on the consideration that the proximity of GNPs to nuclear DNA may be beneficial for enhancing low-energy ionizing radiotherapy. GNP-mediated siRNA delivery may be challenged by nonspecific siRNA desorption during circulation, which can cause off-target effects and immunogenicity. The use of gold nanorods (GNRs) for siRNA delivery also faces challenges like reduced dispersion stability during siRNA functionalization. We developed an effective way to load siRNA onto GNRs at high density, using oleylsulfobetaine (OSB) as an intermediate surfactant and dithiocarbamates (DTCs) as desorption-resistant anchors for siRNA. The GNR?siRNA complexes provided excellent control for laser-triggered gene silencing.

  1. A Promising Combo Gene Delivery System Developed from (3-Aminopropyl)triethoxysilane-Modified Iron Oxide Nanoparticles and Cationic Polymers

    International Nuclear Information System (INIS)

    Zhang Zubin; Song Lina; Dong Jinlai; Guo Dawei; Du Xiaolin; Cao Biyin; Zhang Yu; Gu Ning; Mao Xinliang

    2013-01-01

    (3-Aminopropyl)triethoxysilane-modified iron oxide nanoparticles (APTES-IONPs) have been evaluated for various biomedical applications, including medical imaging and drug delivery. Cationic polymers (CPs) such as Lipofectamine and TurboFect are widely used for research in gene delivery, but their toxicity and low in vivo efficiency limited their further application. In the present study, we synthesized water-soluble APTES-IONPs and developed a combo gene delivery system based on APTES-IONPs and CPs. This system significantly increased gene-binding capacity, protected genes from degradation, and improved gene transfection efficiency for DNA and siRNA in both adherent and suspension cells. Because of its great biocompatibility, high gene-carrying ability, and very low cytotoxicity, this combo gene delivery system will be expected for a wide application, and it might provide a new method for gene therapy.

  2. A Promising Combo Gene Delivery System Developed from (3-Aminopropyl)triethoxysilane-Modified Iron Oxide Nanoparticles and Cationic Polymers

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Zubin [Soochow University, Cyrus Tang Hematology Center (China); Song Lina; Dong Jinlai; Guo Dawei [School of Biological Science and Medical Engineering, Southeast University, State Key Laboratory of Bioelectronics and Jiangsu Key Laboratory for Biomaterials and Devices (China); Du Xiaolin; Cao Biyin [Soochow University, Cyrus Tang Hematology Center (China); Zhang Yu; Gu Ning [School of Biological Science and Medical Engineering, Southeast University, State Key Laboratory of Bioelectronics and Jiangsu Key Laboratory for Biomaterials and Devices (China); Mao Xinliang, E-mail: xinliangmao@suda.edu.cn [Soochow University, Cyrus Tang Hematology Center (China)

    2013-05-15

    (3-Aminopropyl)triethoxysilane-modified iron oxide nanoparticles (APTES-IONPs) have been evaluated for various biomedical applications, including medical imaging and drug delivery. Cationic polymers (CPs) such as Lipofectamine and TurboFect are widely used for research in gene delivery, but their toxicity and low in vivo efficiency limited their further application. In the present study, we synthesized water-soluble APTES-IONPs and developed a combo gene delivery system based on APTES-IONPs and CPs. This system significantly increased gene-binding capacity, protected genes from degradation, and improved gene transfection efficiency for DNA and siRNA in both adherent and suspension cells. Because of its great biocompatibility, high gene-carrying ability, and very low cytotoxicity, this combo gene delivery system will be expected for a wide application, and it might provide a new method for gene therapy.

  3. Solid lipid nanoparticles mediate non-viral delivery of plasmid DNA to dendritic cells

    Science.gov (United States)

    Penumarthi, Alekhya; Parashar, Deepti; Abraham, Amanda N.; Dekiwadia, Chaitali; Macreadie, Ian; Shukla, Ravi; Smooker, Peter M.

    2017-06-01

    There is an increasing demand for novel DNA vaccine delivery systems, mainly for the non-viral type as they are considered relatively safe. Therefore, solid lipid nanoparticles (SLNs) were investigated for their suitability as a non-viral DNA vaccine delivery system. SLNs were synthesised by a modified solvent-emulsification method in order to study their potential to conjugate with plasmid DNA and deliver them in vitro to dendritic cells using eGFP as the reporter plasmid. The DNA-SLN complexes were characterised by electron microscopy, gel retardation assays and dynamic light scattering. The cytotoxicity assay data supported their biocompatibility and was used to estimate safe threshold concentration resulting in high transfection rate. The transfection efficiency of these complexes in a dendritic cell line was shown to increase significantly compared to plasmid alone, and was comparable to that mediated by lipofectamine. Transmission electron microscopy studies delineated the pathway of cellular uptake. Endosomal escape was observed supporting the mechanism of transfection.

  4. Gene Delivery Particle Engineering Strategies for Shape-dependent Targeting of Cells and Tissues.

    Science.gov (United States)

    Kozielski, Kristen L; Sitti, Metin

    2017-01-01

    Successful gene delivery requires overcoming both systemic and intracellular obstacles before the nucleic acid cargo can successfully reach its tissue and subcellular target location. Materials & Methods: Non-viral mechanisms to enable targeting while avoiding off-target delivery have arisen via biological, chemical, and physical engineering strategies. Herein we will discuss the physical parameters in particle design that promote tissue- and cell-targeted delivery of genetic cargo. We will discuss systemic concerns, such as circulation, tissue localization, and clearance, as well as cell-scale obstacles, such as cellular uptake and nucleic acid packaging. In particular, we will focus on engineering particle shape and size in order to enhance delivery and promote precise targeting. We will also address methods to program or change particle shape in situ using environmentally triggered cues. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  5. Efficient Gene Delivery to Pig Airway Epithelia and Submucosal Glands Using Helper-Dependent Adenoviral Vectors

    Directory of Open Access Journals (Sweden)

    Huibi Cao

    2013-01-01

    Full Text Available Airway gene delivery is a promising strategy to treat patients with life-threatening lung diseases such as cystic fibrosis (CF. However, this strategy has to be evaluated in large animal preclinical studies in order to translate it to human applications. Because of anatomic and physiological similarities between the human and pig lungs, we utilized pig as a large animal model to examine the safety and efficiency of airway gene delivery with helper-dependent adenoviral vectors. Helper-dependent vectors carrying human CFTR or reporter gene LacZ were aerosolized intratracheally into pigs under bronchoscopic guidance. We found that the LacZ reporter and hCFTR transgene products were efficiently expressed in lung airway epithelial cells. The transgene vectors with this delivery can also reach to submucosal glands. Moreover, the hCFTR transgene protein localized to the apical membrane of both ciliated and nonciliated epithelial cells, mirroring the location of wild-type CF transmembrane conductance regulator (CFTR. Aerosol delivery procedure was well tolerated by pigs without showing systemic toxicity based on the limited number of pigs tested. These results provide important insights into developing clinical strategies for human CF lung gene therapy.

  6. Coating nanocarriers with hyaluronic acid facilitates intravitreal drug delivery for retinal gene therapy

    NARCIS (Netherlands)

    Martens, Thomas F.; Remaut, Katrien; Deschout, Hendrik; Engbersen, Johan F J; Hennink, Wim E.; Van Steenbergen, Mies J.; Demeester, Jo; De Smedt, Stefaan C.; Braeckmans, Kevin

    2015-01-01

    Retinal gene therapy could potentially affect the lives of millions of people suffering from blinding disorders. Yet, one of the major hurdles remains the delivery of therapeutic nucleic acids to the retinal target cells. Due to the different barriers that need to be overcome in case of topical or

  7. TET1-Mediated Hydroxymethylation Facilitates Hypoxic Gene Induction in Neuroblastoma

    Directory of Open Access Journals (Sweden)

    Christopher J. Mariani

    2014-06-01

    Full Text Available The ten-eleven-translocation 5-methylcytosine dioxygenase (TET family of enzymes catalyzes the conversion of 5-methylcytosine (5-mC to 5-hydroxymethylcytosine (5-hmC, a modified cytosine base that facilitates gene expression. Cells respond to hypoxia by inducing a transcriptional program regulated in part by oxygen-dependent dioxygenases that require Fe(II and α-ketoglutarate. Given that the TET enzymes also require these cofactors, we hypothesized that the TETs regulate the hypoxia-induced transcriptional program. Here, we demonstrate that hypoxia increases global 5-hmC levels, with accumulation of 5-hmC density at canonical hypoxia response genes. A subset of 5-hmC gains colocalize with hypoxia response elements facilitating DNA demethylation and HIF binding. Hypoxia results in transcriptional activation of TET1, and full induction of hypoxia-responsive genes and global 5-hmC increases require TET1. Finally, we show that 5-hmC increases and TET1 upregulation in hypoxia are HIF-1 dependent. These findings establish TET1-mediated 5-hmC changes as an important epigenetic component of the hypoxic response.

  8. Gelatin nanoparticles enhance delivery of hepatitis C virus recombinant NS2 gene.

    Science.gov (United States)

    Sabet, Salwa; George, Marina A; El-Shorbagy, Haidan M; Bassiony, Heba; Farroh, Khaled Y; Youssef, Tareq; Salaheldin, Taher A

    2017-01-01

    Development of an effective non-viral vaccine against hepatitis C virus infection is of a great importance. Gelatin nanoparticles (Gel.NPs) have an attention and promising approach as a viable carrier for delivery of vaccine, gene, drug and other biomolecules in the body. The present study aimed to develop stable Gel.NPs conjugated with nonstructural protein 2 (NS2) gene of Hepatitis C Virus genotype 4a (HCV4a) as a safe and an efficient vaccine delivery system. Gel.NPs were synthesized and characterized (size: 150±2 nm and zeta potential +17.6 mv). NS2 gene was successfully cloned and expressed into E. coli M15 using pQE-30 vector. Antigenicity of the recombinant NS2 protein was confirmed by Western blotting to verify the efficiency of NS2 as a possible vaccine. Then NS2 gene was conjugated to gelatin nanoparticles and a successful conjugation was confirmed by labeling and imaging using Confocal Laser Scanning Microscope (CLSM). Interestingly, the transformation of the conjugated NS2/Gel.NPs complex into E. coli DH5-α was 50% more efficient than transformation with the gene alone. In addition, conjugated NS2/Gel.NPs with ratio 1:100 (w/w) showed higher transformation efficiency into E. coli DH5-α than the other ratios (1:50 and 2:50). Gel.NPs effectively enhanced the gene delivery in bacterial cells without affecting the structure of NS2 gene and could be used as a safe, easy, rapid, cost-effective and non-viral vaccine delivery system for HCV.

  9. Monocyte Trafficking, Engraftment, and Delivery of Nanoparticles and an Exogenous Gene into the Acutely Inflamed Brain Tissue - Evaluations on Monocyte-Based Delivery System for the Central Nervous System.

    Directory of Open Access Journals (Sweden)

    Hsin-I Tong

    Full Text Available The ability of monocytes and monocyte-derived macrophages (MDM to travel towards chemotactic gradient, traverse tissue barriers, and accumulate precisely at diseased sites makes them attractive candidates as drug carriers and therapeutic gene delivery vehicles targeting the brain, where treatments are often hampered by the blockade of the blood brain barrier (BBB. This study was designed to fully establish an optimized cell-based delivery system using monocytes and MDM, by evaluating their homing efficiency, engraftment potential, as well as carriage and delivery ability to transport nano-scaled particles and exogenous genes into the brain, following the non-invasive intravenous (IV cell adoptive transfer in an acute neuroinflammation mouse model induced by intracranial injection of Escherichia coli lipopolysaccharides. We demonstrated that freshly isolated monocytes had superior inflamed-brain homing ability over MDM cultured in the presence of macrophage colony stimulating factor. In addition, brain trafficking of IV infused monocytes was positively correlated with the number of adoptive transferred cells, and could be further enhanced by transient disruption of the BBB with IV administration of Mannitol, Bradykinin or Serotonin right before cell infusion. A small portion of transmigrated cells was detected to differentiate into IBA-1 positive cells with microglia morphology in the brain. Finally, with the use of superparamagnetic iron oxide nanoparticles SHP30, the ability of nanoscale agent-carriage monocytes to enter the inflamed brain region was validated. In addition, lentiviral vector DHIV-101 was used to introduce green fluorescent protein (GFP gene into monocytes, and the exogenous GFP gene was detected in the brain at 48 hours following IV infusion of the transduced monocytes. All together, our study has set up the optimized conditions for the more-in-depth tests and development of monocyte-mediated delivery, and our data supported

  10. Molecular Imaging of Gene Expression and Efficacy following Adenoviral-Mediated Brain Tumor Gene Therapy

    Directory of Open Access Journals (Sweden)

    Alnawaz Rehemtulla

    2002-01-01

    Full Text Available Cancer gene therapy is an active area of research relying upon the transfer and subsequent expression of a therapeutic transgene into tumor cells in order to provide for therapeutic selectivity. Noninvasive assessment of therapeutic response and correlation of the location, magnitude, and duration of transgene expression in vivo would be particularly useful in the development of cancer gene therapy protocols by facilitating optimization of gene transfer protocols, vector development, and prodrug dosing schedules. In this study, we developed an adenoviral vector containing both the therapeutic transgene yeast cytosine deaminase (yCD along with an optical reporter gene (luciferase. Following intratumoral injection of the vector into orthotopic 9L gliomas, anatomical and diffusion-weighted MR images were obtained over time in order to provide for quantitative assessment of overall therapeutic efficacy and spatial heterogeneity of cell kill, respectively. In addition, bioluminescence images were acquired to assess the duration and magnitude of gene expression. MR images revealed significant reduction in tumor growth rates associated with yCD/5-fluorocytosine (5FC gene therapy. Significant increases in mean tumor diffusion values were also observed during treatment with 5FC. Moreover, spatial heterogeneity in tumor diffusion changes were also observed revealing that diffusion magnetic resonance imaging could detect regional therapeutic effects due to the nonuniform delivery and/or expression of the therapeutic yCD transgene within the tumor mass. In addition, in vivo bioluminescence imaging detected luciferase gene expression, which was found to decrease over time during administration of the prodrug providing a noninvasive surrogate marker for monitoring gene expression. These results demonstrate the efficacy of the yCD/5FC strategy for the treatment of brain tumors and reveal the feasibility of using multimodality molecular and functional imaging

  11. Hypoxia-Inducible Factor Directs POMC Gene to Mediate Hypothalamic Glucose Sensing and Energy Balance Regulation

    Science.gov (United States)

    Zhang, Hai; Zhang, Guo; Gonzalez, Frank J.; Park, Sung-min; Cai, Dongsheng

    2011-01-01

    Hypoxia-inducible factor (HIF) is a nuclear transcription factor that responds to environmental and pathological hypoxia to induce metabolic adaptation, vascular growth, and cell survival. Here we found that HIF subunits and HIF2α in particular were normally expressed in the mediobasal hypothalamus of mice. Hypothalamic HIF was up-regulated by glucose to mediate the feeding control of hypothalamic glucose sensing. Two underlying molecular pathways were identified, including suppression of PHDs by glucose metabolites to prevent HIF2α degradation and the recruitment of AMPK and mTOR/S6K to regulate HIF2α protein synthesis. HIF activation was found to directly control the transcription of POMC gene. Genetic approach was then employed to develop conditional knockout mice with HIF inhibition in POMC neurons, revealing that HIF loss-of-function in POMC neurons impaired hypothalamic glucose sensing and caused energy imbalance to promote obesity development. The metabolic effects of HIF in hypothalamic POMC neurons were independent of leptin signaling or pituitary ACTH pathway. Hypothalamic gene delivery of HIF counteracted overeating and obesity under conditions of nutritional excess. In conclusion, HIF controls hypothalamic POMC gene to direct the central nutrient sensing in regulation of energy and body weight balance. PMID:21814490

  12. Hypoxia-inducible factor directs POMC gene to mediate hypothalamic glucose sensing and energy balance regulation.

    Directory of Open Access Journals (Sweden)

    Hai Zhang

    2011-07-01

    Full Text Available Hypoxia-inducible factor (HIF is a nuclear transcription factor that responds to environmental and pathological hypoxia to induce metabolic adaptation, vascular growth, and cell survival. Here we found that HIF subunits and HIF2α in particular were normally expressed in the mediobasal hypothalamus of mice. Hypothalamic HIF was up-regulated by glucose to mediate the feeding control of hypothalamic glucose sensing. Two underlying molecular pathways were identified, including suppression of PHDs by glucose metabolites to prevent HIF2α degradation and the recruitment of AMPK and mTOR/S6K to regulate HIF2α protein synthesis. HIF activation was found to directly control the transcription of POMC gene. Genetic approach was then employed to develop conditional knockout mice with HIF inhibition in POMC neurons, revealing that HIF loss-of-function in POMC neurons impaired hypothalamic glucose sensing and caused energy imbalance to promote obesity development. The metabolic effects of HIF in hypothalamic POMC neurons were independent of leptin signaling or pituitary ACTH pathway. Hypothalamic gene delivery of HIF counteracted overeating and obesity under conditions of nutritional excess. In conclusion, HIF controls hypothalamic POMC gene to direct the central nutrient sensing in regulation of energy and body weight balance.

  13. Investigation of the mediating effects of IT governance-value delivery on service quality and ERP performance

    Science.gov (United States)

    Tsai, Wen-Hsien; Chou, Yu-Wei; Leu, Jun-Der; Chao Chen, Der; Tsaur, Tsen-Shu

    2015-02-01

    This study aimed to explore the mediating effects of IT governance (ITG)-value delivery in the relationships among the quality of vendor service, the quality of consultant services, ITG-value delivery and enterprise resource planning (ERP) performance. The sampling of this research was acquired from a questionnaire survey concerning ERP implementations in Taiwan. In this survey, 4366 questionnaires were sent to manufacturing and service companies listed in the TOP 5000: The Largest Corporations in Taiwan 2009. The results showed that an ERP system will exhibit a decreased error rate and improved performance if ERP system vendors and consultants provide good service quality. The results also demonstrated that significant relationships exist among the quality of vendor service, the quality of consultant services and value delivery. The contribution of this article is twofold. First, it found that value delivery provides an effective measure of ERP performance under an ITG framework. Second, it provides evidence of the partial mediating effects of value delivery between service quality and ERP performance. In other words, if enterprises want to improve ERP performance, they need to consider factors such as value delivery and the quality of a vendor/consultant's service.

  14. A magnetic nanoparticle-based multiple-gene delivery system for transfection of porcine kidney cells.

    Directory of Open Access Journals (Sweden)

    Yan Wang

    Full Text Available Superparamagnetic nanoparticles are promising candidates for gene delivery into mammalian somatic cells and may be useful for reproductive cloning using the somatic cell nuclear transfer technique. However, limited investigations of their potential applications in animal genetics and breeding, particularly multiple-gene delivery by magnetofection, have been performed. Here, we developed a stable, targetable and convenient system for delivering multiple genes into the nuclei of porcine somatic cells using magnetic Fe3O4 nanoparticles as gene carriers. After surface modification by polyethylenimine, the spherical magnetic Fe3O4 nanoparticles showed strong binding affinity for DNA plasmids expressing the genes encoding a green (DNAGFP or red (DNADsRed fluorescent protein. At weight ratios of DNAGFP or DNADsRed to magnetic nanoparticles lower than or equal to 10∶1 or 5∶1, respectively, the DNA molecules were completely bound by the magnetic nanoparticles. Atomic force microscopy analyses confirmed binding of the spherical magnetic nanoparticles to stretched DNA strands up to several hundred nanometers in length. As a result, stable and efficient co-expression of GFP and DsRed in porcine kidney PK-15 cells was achieved by magnetofection. The results presented here demonstrate the potential application of magnetic nanoparticles as an attractive delivery system for animal genetics and breeding studies.

  15. Comparison of Gene Delivery Methods for Transformation of Robinia pseudoacacia L.by Ti-plasmid

    OpenAIRE

    Ren, Chen; Koishiro, Gyokusen; Akira, Saito; Department of Forestry, Faculty of Agiculture, Kyushu University; Department of Forestry, Faculty of Agiculture, Kyushu University; Department of Forestry, Faculty of Agiculture, Kyushu University

    2000-01-01

    The bjnary vector plasmid plG121-Hm carrying the chimeric neomycin phosphotransferase (npt II),β-glucuronidase (gusA) andhygromycin phosphotransferase (hpt) genes was delivered into Robinia pseudoacacia L. callus using the Agrobacterium tuntefaciens-mediated transformation method and particle gun transformation method. It was determined that adding 10 mg/L of acetosyringone to Agrobacterium infection medium and callus-Agrobacterium co-culture medium could increase Agrobacterium-mediated trans...

  16. Recombinant adeno-associated viral (rAAV) vectors mediate efficient gene transduction in cultured neonatal and adult microglia

    Science.gov (United States)

    Su, Wei; Kang, John; Sopher, Bryce; Gillespie, James; Aloi, Macarena S.; Odom, Guy L.; Hopkins, Stephanie; Case, Amanda; Wang, David B.; Chamberlain, Jeffrey S.; Garden, Gwenn A.

    2015-01-01

    Microglia are a specialized population of myeloid cells that mediate CNS innate immune responses. Efforts to identify the cellular and molecular mechanisms that regulate microglia behaviors have been hampered by the lack of effective tools for manipulating gene expression. Cultured microglia are refractory to most chemical and electrical transfection methods, yielding little or no gene delivery and causing toxicity and/or inflammatory activation. Recombinant adeno-associated viral (rAAVs) vectors are non-enveloped, single-stranded DNA vectors commonly used to transduce many primary cell types and tissues. In this study, we evaluated the feasibility and efficiency of utilizing rAAV serotype 2 (rAAV2) to modulate gene expression in cultured microglia. rAAV2 yields high transduction and causes minimal toxicity or inflammatory response in both neonatal and adult microglia. To demonstrate that rAAV transduction can induce functional protein expression, we used rAAV2 expressing Cre-recombinase to successfully excise a LoxP-flanked miR155 gene in cultured microglia. We further evaluated rAAV serotypes 5, 6, 8, and 9, and observed that all efficiently transduced cultured microglia to varying degrees of success and caused little or no alteration in inflammatory gene expression. These results provide strong encouragement for the application of rAAV-mediated gene expression in microglia for mechanistic and therapeutic purposes. PMID:25708596

  17. Design and characterization of novel recombinant listeriolysin O-protamine fusion proteins for enhanced gene delivery.

    Science.gov (United States)

    Kim, Na Hyung; Provoda, Chester; Lee, Kyung-Dall

    2015-02-02

    To improve the efficiency of gene delivery for effective gene therapy, it is essential that the vector carries functional components that can promote overcoming barriers in various steps leading to the transport of DNA from extracellular to ultimately nuclear compartment. In this study, we designed genetically engineered fusion proteins as a platform to incorporate multiple functionalities in one chimeric protein. Prototypes of such a chimera tested here contain two domains: one that binds to DNA; the other that can facilitate endosomal escape of DNA. The fusion proteins are composed of listeriolysin O (LLO), the endosomolytic pore-forming protein from Listeria monocytogenes, and a 22 amino acid sequence of the DNA-condensing polypeptide protamine (PN), singly or as a pair: LLO-PN and LLO-PNPN. We demonstrate dramatic enhancement of the gene delivery efficiency of protamine-condensed DNA upon incorporation of a small amount of LLO-PN fusion protein and further improvement with LLO-PNPN in vitro using cultured cells. Additionally, the association of anionic liposomes with cationic LLO-PNPN/protamine/DNA complexes, yielding a net negative surface charge, resulted in better in vitro transfection efficiency in the presence of serum. An initial, small set of data in mice indicated that the observed enhancement in gene expression could also be applicable to in vivo gene delivery. This study suggests that incorporation of a recombinant fusion protein with multiple functional components, such as LLO-protamine fusion protein, in a nonviral vector is a promising strategy for various nonviral gene delivery systems.

  18. Gene delivery in conjunction with gold nanoparticle and tumor treating electric field

    Science.gov (United States)

    Tiwari, Pawan K.; Soo Lee, Yeon

    2013-08-01

    The advances in electrotherapy to treat the diseased biological cell instigate its extension in gene therapy through the delivery of gene into the nucleus. The objective of this study is to investigate the application of moderate intensity alternating electric field, also known as tumor treating electric field on a carrier system consisting of a charged gene complex conjugated to the surface of a gold nanoparticle. The gene delivery mechanism relies on the magnitude and direction of the induced electric field inside the cytoplasm in presence of carrier system. The induced electric field strength is significant in breaking the gene complex-gold nanoparticle bonding, and exerting an electric force pushing the charged gene into the nucleus. The electric force orientation is dependent on the aspect ratio (AR) of the gold nanoparticle and a relationship between them is studied via Maxwell two-dimensional (2D) finite element simulation analyzer. The development of charge density on the surface of carrier system and the required electric field strength to break the bonding are investigated utilizing the Gouy-Chapman-Grahame-Stern (GCGS) theoretical model. A carrier system having the aspect ratio of the gold nanoparticle in the range 1 < AR ≤ 5 and AR = 1 are substantial delivering cationic and anionic genes into the nucleus, respectively.

  19. An efficient parallel stochastic simulation method for analysis of nonviral gene delivery systems

    KAUST Repository

    Kuwahara, Hiroyuki

    2011-01-01

    Gene therapy has a great potential to become an effective treatment for a wide variety of diseases. One of the main challenges to make gene therapy practical in clinical settings is the development of efficient and safe mechanisms to deliver foreign DNA molecules into the nucleus of target cells. Several computational and experimental studies have shown that the design process of synthetic gene transfer vectors can be greatly enhanced by computational modeling and simulation. This paper proposes a novel, effective parallelization of the stochastic simulation algorithm (SSA) for pharmacokinetic models that characterize the rate-limiting, multi-step processes of intracellular gene delivery. While efficient parallelizations of the SSA are still an open problem in a general setting, the proposed parallel simulation method is able to substantially accelerate the next reaction selection scheme and the reaction update scheme in the SSA by exploiting and decomposing the structures of stochastic gene delivery models. This, thus, makes computationally intensive analysis such as parameter optimizations and gene dosage control for specific cell types, gene vectors, and transgene expression stability substantially more practical than that could otherwise be with the standard SSA. Here, we translated the nonviral gene delivery model based on mass-action kinetics by Varga et al. [Molecular Therapy, 4(5), 2001] into a more realistic model that captures intracellular fluctuations based on stochastic chemical kinetics, and as a case study we applied our parallel simulation to this stochastic model. Our results show that our simulation method is able to increase the efficiency of statistical analysis by at least 50% in various settings. © 2011 ACM.

  20. Sonoporation Increases Therapeutic Efficacy of Inducible and Constitutive BMP2/7 In Vivo Gene Delivery

    Science.gov (United States)

    Hofmann, Anna T.; Slezak, Paul; Schuetzenberger, Sebastian; Kaipel, Martin; Schwartz, Ernst; Neef, Anne; Nomikou, Nikolitsa; Nau, Thomas; van Griensven, Martijn; McHale, Anthony P.; Redl, Heinz

    2014-01-01

    Abstract An ideal novel treatment for bone defects should provide regeneration without autologous or allogenous grafting, exogenous cells, growth factors, or biomaterials while ensuring spatial and temporal control as well as safety. Therefore, a novel osteoinductive nonviral in vivo gene therapy approach using sonoporation was investigated in ectopic and orthotopic models. Constitutive or regulated, doxycycline-inducible, bone morphogenetic protein 2 and 7 coexpression plasmids were repeatedly applied for 5 days. Ectopic and orthotopic gene transfer efficacy was monitored by coapplication of a luciferase plasmid and bioluminescence imaging. Orthotopic plasmid DNA distribution was investigated using a novel plasmid-labeling method. Luciferase imaging demonstrated an increased trend (61% vs. 100%) of gene transfer efficacy, and micro-computed tomography evaluation showed significantly enhanced frequency of ectopic bone formation for sonoporation compared with passive gene delivery (46% vs. 100%) dependent on applied ultrasound power. Bone formation by the inducible system (83%) was stringently controlled by doxycycline in vivo, and no ectopic bone formation was observed without induction or with passive gene transfer without sonoporation. Orthotopic evaluation in a rat femur segmental defect model demonstrated an increased trend of gene transfer efficacy using sonoporation. Investigation of DNA distribution demonstrated extensive binding of plasmid DNA to bone tissue. Sonoporated animals displayed a potentially increased union rate (33%) without extensive callus formation or heterotopic ossification. We conclude that sonoporation of BMP2/7 coexpression plasmids is a feasible, minimally invasive method for osteoinduction and that improvement of bone regeneration by sonoporative gene delivery is superior to passive gene delivery. PMID:24164605

  1. The next step in gene delivery: molecular engineering of adeno-associated virus serotypes.

    Science.gov (United States)

    Wang, Jinhui; Faust, Susan M; Rabinowitz, Joseph E

    2011-05-01

    Delivery is at the heart of gene therapy. Viral DNA delivery systems are asked to avoid the immune system, transduce specific target cell types while avoiding other cell types, infect dividing and non-dividing cells, insert their cargo within the host genome without mutagenesis or to remain episomal, and efficiently express transgenes for a substantial portion of a lifespan. These sought-after features cannot be associated with a single delivery system, or can they? The Adeno-associated virus family of gene delivery vehicles has proven to be highly malleable. Pseudotyping, using AAV serotype 2 terminal repeats to generate designer shells capable of transducing selected cell types, enables the packaging of common genomes into multiple serotypes virions to directly compare gene expression and tropism. In this review the ability to manipulate this virus will be examined from the inside out. The influence of host cell factors and organism biology including the immune response on the molecular fate of the viral genome will be discussed as well as differences in cellular trafficking patterns and uncoating properties that influence serotype transduction. Re-engineering the prototype vector AAV2 using epitope insertion, chemical modification, and molecular evolution not only demonstrated the flexibility of the best-studied serotype, but now also expanded the tool kit for molecular modification of all AAV serotypes. Current AAV research has changed its focus from examination of wild-type AAV biology to the feedback of host cell/organism on the design and development of a new generation of recombinant AAV delivery vehicles. This article is part of a Special Section entitled "Special Section: Cardiovascular Gene Therapy". Copyright © 2010 Elsevier Ltd. All rights reserved.

  2. Local Delivery Is Critical for Monocyte Chemotactic Protein-1 Mediated Site-Specific Murine Aneurysm Healing

    Directory of Open Access Journals (Sweden)

    Siham Hourani

    2018-03-01

    Full Text Available BackgroundLocal delivery of monocyte chemotactic protein-1 (MCP-1/CCL2 via our drug-eluting coil has been shown to promote intrasaccular aneurysm healing via an inflammatory pathway.ObjectiveIn this study, we validate the importance of local MCP-1 in murine aneurysm healing. Whether systemic, rather than local, delivery of MCP-1 can direct site-specific aneurysm healing has significant translational implications. If systemic MCP-1 is effective, then MCP-1 could be administered as a pill rather than by endovascular procedure. Furthermore, we confirm that MCP-1 is the primary effector in our MCP-1 eluting coil-mediated murine aneurysm healing model.MethodsWe compare aneurysm healing with repeated intraperitoneal MCP-1 versus vehicle injection, in animals with control poly(lactic-co-glycolic acid (PLGA-coated coils. We demonstrate elimination of the MCP-1-associated tissue-healing response by knockout of MCP-1 or CCR2 (MCP-1 receptor and by selectively inhibiting MCP-1 or CCR2. Using immunofluorescent probing, we explore the cell populations found in healed aneurysm tissue following each intervention.ResultsSystemically administered MCP-1 with PLGA coil control does not produce comparable aneurysm healing, as seen with MCP-1 eluting coils. MCP-1-directed aneurysm healing is eliminated by selective inhibition of MCP-1 or CCR2 and in MCP-1-deficient or CCR2-deficient mice. No difference was detected in M2 macrophage and myofibroblast/smooth muscle cell staining with systemic MCP-1 versus vehicle in aneurysm wall, but a significant increase in these cell types was observed with MCP-1 eluting coil implant and attenuated by MCP-1/CCR2 blockade or deficiency.ConclusionWe show that systemic MCP-1 concurrent with PLGA-coated platinum coil implant is not sufficient to produce site-specific aneurysm healing. MCP-1 is a critical, not merely complementary, actor in the aneurysm healing pathway.

  3. Gene Expression by PBMC in Primary Sclerosing Cholangitis: Evidence for Dysregulation of Immune Mediated Genes

    Directory of Open Access Journals (Sweden)

    Christopher A. Aoki

    2006-01-01

    Full Text Available Primary sclerosing cholangitis (PSC is a chronic disease of the bile ducts characterized by an inflammatory infiltrate and obliterative fibrosis. The precise role of the immune system in the pathogenesis of PSC remains unknown. We used RNA microarray analysis to identify immune-related genes and pathways that are differentially expressed in PSC. Messenger RNA (mRNA from peripheral blood mononuclear cells (PBMC was isolated from both patients with PSC and age and sex matched healthy controls. Samples from 5 PSC patients and 5 controls were analyzed by microarray and based upon rigorous statistical analysis of the data, relevant genes were chosen for confirmation by RT-PCR in 10 PSC patients and 10 controls. Using unsupervised hierarchical clustering, gene expression in PSC was statistically different from our control population. Interestingly, genes within the IL-2 receptor beta, IL-6 and MAP Kinase pathways were found to be differently expressed in patients with PSC compared to controls. Further, individual genes, TNF-α induced protein 6 (TNFaip6 and membrane-spanning 4-domains, subfamily A (ms4a were found to be upregulated in PSC while similar to Mothers against decapentaplegic homolog 5 (SMAD 5 was downregulated. In conclusion, several immune-related pathways and genes were differentially expressed in PSC compared to control patients, giving further evidence that this disease is systemic and immune-mediated.

  4. Jumping the nuclear envelop barrier: Improving polyplex-mediated gene transfection efficiency by a selective CDK1 inhibitor RO-3306.

    Science.gov (United States)

    Zhou, Xuefei; Liu, Xiangrui; Zhao, Bingxiang; Liu, Xin; Zhu, Dingcheng; Qiu, Nasha; Zhou, Quan; Piao, Ying; Zhou, Zhuxian; Tang, Jianbin; Shen, Youqing

    2016-07-28

    Successful transfection of plasmid DNA (pDNA) requires intranuclear internalization of pDNA effectively and the nuclear envelope appears to be one of the critical intracellular barriers for polymer mediated pDNA delivery. Polyethylenimine (PEI), as the classic cationic polymer, compact the negatively charged pDNA tightly and make up stable polyplexes. The polyplexes are too large to enter the nuclear through nuclear pores and it is believed that the nuclear envelope breakdown in mitosis could facilitate the nuclear entry of polyplexes. To jump the nuclear envelope barrier, we used a selective and reversible CDK1 inhibitor RO-3306 to control the G2/M transition of the cell cycle and increased the proportion of mitotic cells which have disappeared nuclear envelope during transfection. Herein, we show that RO-3306 remarkably increases the transfection efficiency of PEI polyplexes through enhanced nuclear localization of PEI and pDNA. However, RO-3306 is less effective to the charge-reversal polymer poly[(2-acryloyl)ethyl(p-boronic acid benzyl)diethylammonium bromide] (B-PDEAEA) which responses to cellular stimuli and releases free pDNA in cytoplasm. Our findings not only offer new opportunities for improving non-viral based gene delivery but also provide theoretical support for the rational design of novel functional polymers for gene delivery. We also report current data showing that RO-3306 synergizes TRAIL gene induced apoptosis in cancer cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. A novel plasmid and SonoVue formulation plus ultrasound sonication for effective gene delivery in nude mice.

    Science.gov (United States)

    Wang, Xin-Lu; Zhao, Xiao-Yun; Li, Shuo; Jia, Chang-Jun; Jiang, Luo; Shi, Tei-Mei; Ren, Wei-Dong

    2013-10-11

    This study aimed to identify the effectiveness of gene therapy mediated by ultrasound-targeted SonoVue using the herpes simplex virus-thymidine kinase (TSV-TK) driven by the kinase insert domain receptor (KDR) promoter (KDR-TK) for the treatment of ovarian carcinomas in nude mice. The optimized conditions for gene transfection were also explored. In this study, we developed a novel technique to deliver a plasmid vector-carried gene into tumor xenografts in sixty nude mice. We first mixed plasmid DNA with SonoVue to form microbubbles and then treated the mice with ultrasound sonication to enhance plasmid gene transfection and expression in tumor xenografts. The data showed that injection of pBluescript-KDR-TK cDNA mixed with SonoVue into nude mice plus ultrasound sonication significantly (Group E) increased the transfection efficiency and expression of KDR-TK mRNA in tumor xenografts. The growth of tumor xenografts in nude mice was significantly suppressed in Group E compared to the other four control groups (Groups A, B, C, and D, namely, treatment with phosphate-buffered saline (PBS), KDR-TK+PBS, KDR-TK+SonoVue, KDR-TK+PBS+ultrasound sonication, respectively). TUNEL staining showed that SonoVue plus ultrasound sonication significantly induced apoptosis and reduced microvessel density (MVD). This study revealed that the formulation of plasmid with SonoVue plus ultrasound could provide efficient gene delivery into tumor xenografts. Increased gene expression was observed in vivo, which effectively reduced the tumor growth and MVD of tumor xenografts and induced apoptosis in tumor cells. Future clinical trials are necessary to further analyze the relevance of this technique. © 2013.

  6. Hydrogel Design for Supporting Neurite Outgrowth and Promoting Gene Delivery to Maximize Neurite Extension

    Science.gov (United States)

    Shepard, Jaclyn A.; Stevans, Alyson C.; Holland, Samantha; Wang, Christine E.; Shikanov, Ariella; Shea, Lonnie D.

    2012-01-01

    Hydrogels capable of gene delivery provide a combinatorial approach for nerve regeneration, with the hydrogel supporting neurite outgrowth and gene delivery inducing the expression of inductive factors. This report investigates the design of hydrogels that balance the requirements for supporting neurite growth with those requirements for promoting gene delivery. Enzymatically-degradable PEG hydrogels encapsulating dorsal root ganglia explants, fibroblasts, and lipoplexes encoding nerve growth factor were gelled within channels that can physically guide neurite outgrowth. Transfection of fibroblasts increased with increasing concentration of Arg-Gly-Asp (RGD) cell adhesion sites and decreasing PEG content. The neurite length increased with increasing RGD concentration within 10% PEG hydrogels, yet was maximal within 7.5% PEG hydrogels at intermediate RGD levels. Delivering lipoplexes within the gel produced longer neurites than culture in NGF-supplemented media or co-culture with cells exposed to DNA prior to encapsulation. Hydrogels designed to support neurite outgrowth and deliver gene therapy vectors locally may ultimately be employed to address multiple barriers that limit regeneration. PMID:22038654

  7. Efficient TALEN-mediated gene knockout in livestock.

    Science.gov (United States)

    Carlson, Daniel F; Tan, Wenfang; Lillico, Simon G; Stverakova, Dana; Proudfoot, Chris; Christian, Michelle; Voytas, Daniel F; Long, Charles R; Whitelaw, C Bruce A; Fahrenkrug, Scott C

    2012-10-23

    Transcription activator-like effector nucleases (TALENs) are programmable nucleases that join FokI endonuclease with the modular DNA-binding domain of TALEs. Although zinc-finger nucleases enable a variety of genome modifications, their application to genetic engineering of livestock has been slowed by technical limitations of embryo-injection, culture of primary cells, and difficulty in producing reliable reagents with a limited budget. In contrast, we found that TALENs could easily be manufactured and that over half (23/36, 64%) demonstrate high activity in primary cells. Cytoplasmic injections of TALEN mRNAs into livestock zygotes were capable of inducing gene KO in up to 75% of embryos analyzed, a portion of which harbored biallelic modification. We also developed a simple transposon coselection strategy for TALEN-mediated gene modification in primary fibroblasts that enabled both enrichment for modified cells and efficient isolation of modified colonies. Coselection after treatment with a single TALEN-pair enabled isolation of colonies with mono- and biallelic modification in up to 54% and 17% of colonies, respectively. Coselection after treatment with two TALEN-pairs directed against the same chromosome enabled the isolation of colonies harboring large chromosomal deletions and inversions (10% and 4% of colonies, respectively). TALEN-modified Ossabaw swine fetal fibroblasts were effective nuclear donors for cloning, resulting in the creation of miniature swine containing mono- and biallelic mutations of the LDL receptor gene as models of familial hypercholesterolemia. TALENs thus appear to represent a highly facile platform for the modification of livestock genomes for both biomedical and agricultural applications.

  8. A surface-mediated siRNA delivery system developed with chitosan/hyaluronic acid-siRNA multilayer films through layer-by-layer self-assembly

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Lijuan [Shanghai Key Laboratory of Magnetic Resonance, East China Normal University, Shanghai 200062 (China); Suzhou Novovita Bio-products Co., Ltd., Suzhou 215300 (China); Wu, Changlin, E-mail: Ph.Dclwu1314@sina.cn [Shanghai Key Laboratory of Magnetic Resonance, East China Normal University, Shanghai 200062 (China); Suzhou Novovita Bio-products Co., Ltd., Suzhou 215300 (China); Liu, Guangwan [Suzhou Novovita Bio-products Co., Ltd., Suzhou 215300 (China); Liao, Nannan [Shanghai Key Laboratory of Magnetic Resonance, East China Normal University, Shanghai 200062 (China); Suzhou Novovita Bio-products Co., Ltd., Suzhou 215300 (China); Zhao, Fang; Yang, Xuxia; Qu, Hongyuan [Suzhou Novovita Bio-products Co., Ltd., Suzhou 215300 (China); Peng, Bo [Shanghai Key Laboratory of Magnetic Resonance, East China Normal University, Shanghai 200062 (China); Chen, Li [Shanghai Key Laboratory of Magnetic Resonance, East China Normal University, Shanghai 200062 (China); Suzhou Novovita Bio-products Co., Ltd., Suzhou 215300 (China); Yang, Guang [Shanghai Key Laboratory of Magnetic Resonance, East China Normal University, Shanghai 200062 (China)

    2016-12-15

    Highlights: • We prepared Chitosan/Hyaluronic acid-siRNA multilayer as carrier to effectively load and protect siRNAs. • The stability and integrity of the siRNA was verified in the siRNA-loaded films. • The siRNA-loaded films showed good cells adhesion and gene silencing effect in eGFP-HEK 293T cells. • This is a new type of surface-mediated non-viral multilayer films. - Abstract: siRNA delivery remains highly challenging because of its hydrophilic and anionic nature and its sensitivity to nuclease degradation. Effective siRNA loading and improved transfection efficiency into cells represents a key problem. In our study, we prepared Chitosan/Hyaluronic acid-siRNA multilayer films through layer-by-layer self-assembly, in which siRNAs can be effectively loaded and protected. The construction process was characterized by FTIR, {sup 13}C NMR (CP/MAS), UV–vis spectroscopy, and atomic force microscopy (AFM). We presented the controlled-release performance of the films during incubation in 1 M NaCl solution for several days through UV–vis spectroscopy and polyacrylamide gel electrophoresis (PAGE). Additionally, we verified the stability and integrity of the siRNA loaded on multilayer films. Finally, the biological efficacy of the siRNA delivery system was evaluated via cells adhesion and gene silencing analyses in eGFP-HEK 293T cells. This new type of surface-mediated non-viral multilayer films may have considerable potential in the localized and controlled-release delivery of siRNA in mucosal tissues, and tissue engineering application.

  9. Application of quantum dots as vectors in targeted survivin gene siRNA delivery

    Directory of Open Access Journals (Sweden)

    Zhao JJ

    2013-04-01

    Full Text Available Jianjiang Zhao, Xiaoling Qiu, Zhiping Wang, Jie Pan, Jun Chen, Jiusong Han Department of Surgery, Guangdong Provincial Stomatological Hospital, Southern Medical University, Guangzhou, People's Republic of China Abstract: Gene silencing using short interfering RNA (siRNA is becoming an attractive approach for probing gene function in mammalian cells. This study evaluated the specificity and efficiency of quantum dots (QDs as non-viral gene vectors for delivery of survivin siRNA and downregulation of survivin gene expression in oral squamous cell carcinoma Tca8113 cells. Water-dispersible cationically-modified QDs were electrostatically attached to anionic siRNA molecules and complexed with siRNA for downregulating expression of the survivin gene. Cellular uptake and allocation of QD–siRNA complexes in Tca8113 cells were monitored using confocal laser scanning microscopy. Real-time polymerase chain reaction (PCR was used to quantify survivin messenger RNA (mRNA levels. CdSe QDs were observed with high intensity fluorescence under confocal laser scanning microscopy. Tca8113 cells were successfully transfected by QDs with survivin siRNA, and the red fluorescence from CdSe QDs and green fluorescein amidite fluorescence from siRNA could both be easily observed after 6 hours of incubation. The release of siRNA into the cytoplasm was verified through real-time PCR quantification that showed reduced survivin mRNA levels. In this study, survivin siRNA successfully complexed with water-soluble CdSe QDs and exhibited excellent fluorescent properties and downregulated the expression of the survivin gene in oral squamous cell carcinoma Tca8113 cells. QDs are a novel non-viral gene delivery vector. Keywords: quantum dots, survivin, siRNA delivery, transfection, Tca8113, tongue cancer cells

  10. Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system

    Directory of Open Access Journals (Sweden)

    Tuttle John

    2012-08-01

    Full Text Available Abstract Background We previously developed a virus-induced gene silencing (VIGS vector for cotton from the bipartite geminivirusCotton leaf crumple virus (CLCrV. The original CLCrV VIGS vector was designed for biolistic delivery by a gene gun. This prerequisite limited the use of the system to labs with access to biolistic equipment. Here we describe the adaptation of this system for delivery by Agrobacterium (Agrobacterium tumefaciens. We also describe the construction of two low-cost particle inflow guns. Results The biolistic CLCrV vector was transferred into two Agrobacterium binary plasmids. Agroinoculation of the binary plasmids into cotton resulted in silencing and GFP expression comparable to the biolistic vector. Two homemade low-cost gene guns were used to successfully inoculate cotton (G. hirsutum and N. benthamiana with either the CLCrV VIGS vector or the Tomato golden mosaic virus (TGMV VIGS vector respectively. Conclusions These innovations extend the versatility of CLCrV-based VIGS for analyzing gene function in cotton. The two low-cost gene guns make VIGS experiments affordable for both research and teaching labs by providing a working alternative to expensive commercial gene guns.

  11. Receptor-mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chloroplasts into the mouse circulatory system.

    Science.gov (United States)

    Limaye, Arati; Koya, Vijay; Samsam, Mohtashem; Daniell, Henry

    2006-05-01

    Oral delivery of biopharmaceutical proteins expressed in plant cells should reduce their cost of production, purification, processing, cold storage, transportation, and delivery. However, poor intestinal absorption of intact proteins is a major challenge. To overcome this limitation, we investigate here the concept of receptor-mediated oral delivery of chloroplast-expressed foreign proteins. Therefore, the transmucosal carrier cholera toxin B-subunit and green fluorescent protein (CTB-GFP), separated by a furin cleavage site, was expressed via the tobacco chloroplast genome. Polymerase chain reaction (PCR) and Southern blot analyses confirmed site-specific transgene integration and homoplasmy. Immunoblot analysis and ELISA confirmed expression of monomeric and pentameric forms of CTB-GFP, up to 21.3% of total soluble proteins. An in vitro furin cleavage assay confirmed integrity of the engineered furin cleavage site, and a GM1 binding assay confirmed the functionality of CTB-GFP pentamers. Following oral administration of CTB-GFP expressing leaf material to mice, GFP was observed in the mice intestinal mucosa, liver, and spleen in fluorescence and immunohistochemical studies, while CTB remained in the intestinal cell. This report of receptor-mediated oral delivery of a foreign protein into the circulatory system opens the door for low-cost production and delivery of human therapeutic proteins.

  12. Gene therapy for C-26 colon cancer using heparin-polyethyleneimine nanoparticle-mediated survivin T34A

    Directory of Open Access Journals (Sweden)

    Zhang L

    2011-10-01

    Full Text Available Ling Zhang1,*, Xiang Gao1,2,*, Ke Men1, BiLan Wang1, Shuang Zhang1, Jinfeng Qiu1, Meijuan Huang1, MaLing Gou1, Ning Huang2, ZhiYong Qian1, Xia Zhao1, YuQuan Wei11State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, 2Department of Pathophysiology, College of Preclinical and Forensic Medical Sciences, Sichuan University, Chengdu, People’s Republic of China*These authors contributed equally to this workBackground: Gene therapy provides a novel method for the prevention and treatment of cancer, but the clinical application of gene therapy is restricted, mainly because of the absence of an efficient and safe gene delivery system. Recently, we developed a novel nonviral gene carrier, ie, heparin-polyethyleneimine (HPEI nanoparticles for this purpose.Methods and results: HPEI nanoparticles were used to deliver plasmid-expressing mouse survivin-T34A (ms-T34A to treat C-26 carcinoma in vitro and in vivo. According to the in vitro studies, HPEI nanoparticles could efficiently transfect the pGFP report gene into C-26 cells, with a transfection efficiency of 30.5% ± 2%. Moreover, HPEI nanoparticle-mediated ms-T34A could efficiently inhibit the proliferation of C-26 cells by induction of apoptosis in vitro. Based on the in vivo studies, HPEI nanoparticles could transfect the Lac-Z report gene into C-26 cells in vivo. Intratumoral injection of HPEI nanoparticle-mediated ms-T34A significantly inhibited growth of subcutaneous C-26 carcinoma in vivo by induction of apoptosis and inhibition of angiogenesis.Conclusion: This research suggests that HPEI nanoparticle-mediated ms-T34A may have a promising role in C-26 colon carcinoma therapy.Keywords: gene therapy, mouse survivin-T34A, colon cancer, polyethyleneimine, nanoparticles, cancer therapy

  13. Gene delivery in malignant B cells using the combination of lentiviruses conjugated to anti-transferrin receptor antibodies and an immunoglobulin promoter.

    Science.gov (United States)

    Leoh, Lai Sum; Morizono, Kouki; Kershaw, Kathleen M; Chen, Irvin S Y; Penichet, Manuel L; Daniels-Wells, Tracy R

    2014-01-01

    We previously developed an antibody-avidin fusion protein (ch128.1Av) specific for the human transferrin receptor 1 (TfR1; CD71) to be used as a delivery vector for cancer therapy and showed that ch128.1Av delivers the biotinylated plant toxin saporin-6 into malignant B cells. However, as a result of widespread expression of TfR1, delivery of the toxin to normal cells is a concern. Therefore, we explored the potential of a dual targeted lentiviral-mediated gene therapy strategy to restrict gene expression to malignant B cells. Targeting occurs through the use of ch128.1Av or its parental antibody without avidin (ch128.1) and through transcriptional regulation using an immunoglobulin promoter. Flow cytometry was used to detect the expression of enhanced green fluorescent protein (EGFP) in a panel of cell lines. Cell viability after specific delivery of the therapeutic gene FCU1, a chimeric enzyme consisting of cytosine deaminase genetically fused to uracil phosphoribosyltransferse that converts the 5-fluorocytosine (5-FC) prodrug into toxic metabolites, was monitored using the MTS or WST-1 viability assay. We found that EGFP was specifically expressed in a panel of human malignant B-cell lines, but not in human malignant T-cell lines. EGFP expression was observed in all cell lines when a ubiquitous promoter was used. Furthermore, we show the decrease of cell viability in malignant plasma cells in the presence of 5-FC and the FCU1 gene. The present study demonstrates that gene expression can be restricted to malignant B cells and suggests that this dual targeted gene therapy strategy may help to circumvent the potential side effects of certain TfR1-targeted protein delivery approaches. Copyright © 2014 John Wiley & Sons, Ltd.

  14. Manipulating the membrane penetration mechanism of helical polypeptides via aromatic modification for efficient gene delivery.

    Science.gov (United States)

    Zheng, Nan; Song, Ziyuan; Yang, Jiandong; Liu, Yang; Li, Fangfang; Cheng, Jianjun; Yin, Lichen

    2017-08-01

    The delivery performance of non-viral gene vectors is greatly related to their intracellular kinetics. Cationic helical polypeptides with potent membrane penetration properties and gene transfection efficiencies have been recently developed by us. However, they suffer from severe drawbacks in terms of their membrane penetration mechanisms that mainly include endocytosis and pore formation. The endocytosis mechanism leads to endosomal entrapment of gene cargos, while the charge- and helicity-induced pore formation causes appreciable cytotoxicity at high concentrations. With the attempt to overcome such critical challenges, we incorporated aromatic motifs into the design of helical polypeptides to enhance their membrane activities and more importantly, to manipulate their membrane penetration mechanisms. The aromatically modified polypeptides exhibited higher cellular internalization level than the unmodified analogue by up to 2.5 folds. Such improvement is possibly because aromatic domains promoted the polypeptides to penetrate cell membranes via direct transduction, a non-endocytosis and non-pore formation mechanism. As such, gene cargos were more efficiently delivered into cells by bypassing endocytosis and subsequently avoiding endosomal entrapment, and the material toxicity associated with excessive pore formation was also reduced. The top-performing aromatic polypeptide containing naphthyl side chains at the incorporated content of 20mol% revealed notably higher transfection efficiencies than commercial reagents in melanoma cells in vitro (by 11.7 folds) and in vivo (by 9.1 folds), and thus found potential utilities toward topical gene delivery for cancer therapy. Cationic helical polypeptides, as efficient gene delivery materials, suffer from severe drawbacks in terms of their membrane penetration mechanisms. The main cell penetration mechanisms involved are endocytosis and pore formation. However, the endocytosis mechanism has the limitation of endosomal

  15. Retroviral-mediated gene therapy for the differentiation of primary cells into a mineralizing osteoblastic phenotype.

    Science.gov (United States)

    Phillips, Jennifer E; García, Andrés J

    2008-01-01

    Bone tissue engineering has emerged as a promising strategy for the repair of critical-sized skeletal fractures. However, the clinical application of this approach has been limited by the availability of a robust mineralizing cell source. Non-osteogenic cells, such as skin fibroblasts, are an attractive cell-source alternative because they are easy to harvest from autologous donor skin biopsies and display a high capacity for in vitro expansion. We have recently demonstrated that retroviral gene delivery of the osteoblastic transcription factor Runx2/Cbfa1 promotes osteogenic differentiation in primary dermal fibroblasts cultured in monolayer. Notably, sustained expression of Runx2 was not sufficient to promote functional osteogenesis in these cells, and co-treatment with the steroid hormone dexamethasone was required to induce deposition of biologically-equivalent matrix mineralization. On the basis of these results, we then investigated the osteogenic capacity of these genetically engineered fibroblasts when seeded on polymeric scaffolds in vitro and in vivo. These experiments demonstrated that Runx2-expressing fibroblasts seeded on collagen scaffolds produce significant levels of matrix mineralization after 28 days in vivo implantation in a subcutaneous, heterotopic site. Overall, these results offer evidence that transcription factor-based gene therapy may be a powerful strategy for the conversion of a non-osteogenic cellular phenotype into a mineralizing cell source for bone repair applications. This concept may also be applied to control functional differentiation in a broad range of cell types and tissue engineering applications. The chapter below outlines detailed methods for the isolation and ex vivo genetic modification of primary dermal fibroblasts using retroviral-mediated delivery of the Runx2 transgene in both monolayer culture and three-dimensional scaffolds.

  16. A star-shaped porphyrin-arginine functionalized poly(L-lysine) copolymer for photo-enhanced drug and gene co-delivery.

    Science.gov (United States)

    Ma, Dong; Lin, Qian-Ming; Zhang, Li-Ming; Liang, Yuan-Yuan; Xue, Wei

    2014-05-01

    The co-delivery of drug and gene has become the primary strategy in cancer and other disease therapy. To co-deliver hydrophobic drug and functional gene efficiently into tumor cells, a star-shaped copolymer (PP-PLLD-Arg) with a photochemical internalization effect consisting of a porphyrin (PP) core and arginine-functionalized poly(L-lysine) dendron (PLLD-Arg) arms has been designed, and used to co-deliver docetaxel (DOC) and MMP-9 shRNA plasmid for nasopharyngeal cancer therapy. It was found that PP-PLLD-Arg/MMP-9 nanocomplex showed the photo-enhanced gene transfection efficiency in vitro, and could mediate a significant reduce of MMP-9 protein expression in HNE-1 cells. For co-delivery analysis, the obtained PP-PLLD-Arg/DOC/MMP-9 complexes could induce a more significant apoptosis than DOC or MMP-9 used only, and decreased invasive capacity of HNE-1 cells. Moreover, the star-shaped copolymer exhibited better blood compatibility and lower cytotoxicity compared to PEI-25k in the hemolysis and MTT assays, and also showed a good biocompatibility in vivo. Therefore, PP-PLLD-Arg with suited irradiation is a promising non-toxic and photo-inducible effective drug and gene delivery strategy, which should be encouraged in tumor therapy. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Quantification of Electroporation-Mediated Propidium Iodide Delivery into 3T3 Cells

    Science.gov (United States)

    Sadik, Mohamed M.; Li, Jianbo; Shan, Jerry W.; Shreiber, David I.; Lin, Hao

    2010-11-01

    Electroporation is an effective means to deliver exogenous molecules into the cellular cytoplasm, while simultaneously maintaining cell viability and functionality. In this technique, an applied electric field transiently permeabilizes the cellular membrane to enable molecular exchange. The main objective of the current work is to identify the transport mechanisms involved during electroporation, and to quantify the amount of molecules delivered into the cellular cytoplasm. An optical diagnostic system is developed to examine the transport of Propidium Iodide (PI) into 3T3 mouse fibroblast cells. Upon entering the permeabilized cell, PI binds to DNA/RNA within the cytoplasm to emit fluorescence, which is measured to track the dynamic accumulation of the dye within the cell. The results show that the total fluorescence intensity increases with a decreasing buffer electrical conductivity. The data are compared with numerical simulations, which reveals good agreement. The experimental observations and numerical analysis demonstrate that: 1) Electrophoresis plays a dominant role in mediating the transport. 2) An electrokinetic mechanism, field-amplified sample stacking, controls the achievable delivery efficiency. The study in this work is an important step toward the quantification as well as the eventual improvement of this useful technique.

  18. Assessing the transport of receptor-mediated drug-delivery devices across cellular monolayers.

    Science.gov (United States)

    Brewer, Erik; Lowman, Anthony M

    2014-01-01

    Receptor-mediated endocytosis (RME) has been extensively studied as a method for augmenting the transport of therapeutic devices across monolayers. These devices range from simple ligand-therapeutic conjugates to complex ligand-nanocarrier systems. However, characterizing the uptake of these carriers typically relies on their comparisons to the native therapeutic, which provides no understanding of the ligand or cellular performance. To better understand the potential of the RME pathway, a model for monolayer transport was designed based on the endocytosis cycle of transferrin, a ligand often used in RME drug-delivery devices. This model established the correlation between apical receptor concentration and transport capability. Experimental studies confirmed this relationship, demonstrating an upper transport limit independent of the applied dose. This contrasts with the dose-proportional pathways that native therapeutics rely on for transport. Thus, the direct comparison of these two transport mechanisms can produce misleading results that change with arbitrarily chosen doses. Furthermore, transport potential was hindered by repeated use of the RME cycle. Future studies should base the success of this technology not on the performance of the therapeutic itself, but on the capabilities of the cell. Using receptor-binding studies, we were able to demonstrate how these capabilities can be predicted and potentially adopted for high-throughput screening methods.

  19. DMPD: Signalling pathways mediating type I interferon gene expression. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17904888 Signalling pathways mediating type I interferon gene expression. Edwards M...hways mediating type I interferon gene expression. PubmedID 17904888 Title Signalling pathways...R, Slater L, Johnston SL. Microbes Infect. 2007 Sep;9(11):1245-51. Epub 2007 Jul 1. (.png) (.svg) (.html) (.csml) Show Signalling pat

  20. A geminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing

    OpenAIRE

    Yin, Kangquan; Han, Ting; Liu, Guang; Chen, Tianyuan; Wang, Ying; Yu, Alice Yunzi L.; Liu, Yule

    2015-01-01

    CRISPR/Cas has emerged as potent genome editing technology and has successfully been applied in many organisms, including several plant species. However, delivery of genome editing reagents remains a challenge in plants. Here, we report a virus-based guide RNA (gRNA) delivery system for CRISPR/Cas9 mediated plant genome editing (VIGE) that can be used to precisely target genome locations and cause mutations. VIGE is performed by using a modified Cabbage Leaf Curl virus (CaLCuV) vector to expr...

  1. Towards prostate cancer gene therapy: Development of a chlorotoxin-targeted nanovector for toxic (melittin) gene delivery.

    Science.gov (United States)

    Tarokh, Zahra; Naderi-Manesh, Hossein; Nazari, Mahboobeh

    2017-03-01

    Prostate cancer is the second leading cause of death due to cancer in men. Owing to shortcomings in the current treatments, other therapies are being considered. Toxic gene delivery is one of the most effective methods for cancer therapy. Cationic polymers are able to form stable nanoparticles via interaction with nucleic acids electrostatically. Branched polyethylenimine that contains amine groups has notable buffering capacity and the ability to escape from endosome through the proton sponge effect. However, the cytotoxicity of this polymer is high, and modification is one of the applicable strategies to overcome this problem. In this study, PEI was targeted with chlorotoxin (CTX) via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) cross-linker. CTX can bind specifically to matrix metalloproteinase-2 that is overexpressed in certain cancers. Melittin as the major component of bee venom has been reported to have anti-cancer activity. This was thus selected to deliver to PC3 cell line. Flow cytometry analysis revealed that transfection efficiency of targeted nanoparticles is significantly higher compared to non-targeted nanoparticles. Targeted nanoparticles carrying the melittin gene also decreased cell viability of PC3 cells significantly while no toxic effects were observed on NIH3T3 cell line. Therefore, CTX-targeted nanoparticles carrying the melittin gene could serve as an appropriate gene delivery system for prostate and other MMP-2 positive cancer cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Selective atonal gene delivery improves balance function in a mouse model of vestibular disease.

    Science.gov (United States)

    Schlecker, C; Praetorius, M; Brough, D E; Presler, R G; Hsu, C; Plinkert, P K; Staecker, H

    2011-09-01

    Loss of balance is often due to loss of vestibular hair cells. In mammals, regeneration of functional hair cells in the mature sensory epithelium is limited; therefore, loss of sensory cells can lead to debilitating balance problems. Delivery of the transcription factor atonal (atoh1) after aminoglycoside ototoxicity has previously been shown to induce the transdifferentiation of supporting cells into new hair cells and restore function. A problem with mouse aminoglycoside models is that the partial loss of hair cells seen in human disease is difficult to establish consistently. To more closely mirror human clinical balance dysfunction, we have used systemic application of 3,3'-iminodipropionitrile (IDPN), a vestibulotoxic nitrile compound known to cause vestibular hair cell loss, to induce a consistent partial loss of vestibular hair cells. To determine if balance function could be restored, we delivered atoh1 using a new adenovirus vector, based on Ad28. The Ad28 adenovector is based on a human serotype with a low seroprevalence that appears to target gene delivery to vestibular supporting cells. To further provide cell type selectivity of gene delivery, we expressed atoh1 using the supporting cell-specific glial fibrillary acid protein promoter. Delivery of this vector to IDPN-damaged vestibular organs resulted in a significant recovery of vestibular hair cells and restoration of balance, as measured by time on rotarod compared with untreated controls.

  3. Targeted delivery of genes to endothelial cells and cell- and gene-based therapy in pulmonary vascular diseases.

    Science.gov (United States)

    Suen, Colin M; Mei, Shirley H J; Kugathasan, Lakshmi; Stewart, Duncan J

    2013-10-01

    Pulmonary arterial hypertension (PAH) is a devastating disease that, despite significant advances in medical therapies over the last several decades, continues to have an extremely poor prognosis. Gene therapy is a method to deliver therapeutic genes to replace defective or mutant genes or supplement existing cellular processes to modify disease. Over the last few decades, several viral and nonviral methods of gene therapy have been developed for preclinical PAH studies with varying degrees of efficacy. However, these gene delivery methods face challenges of immunogenicity, low transduction rates, and nonspecific targeting which have limited their translation to clinical studies. More recently, the emergence of regenerative approaches using stem and progenitor cells such as endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) have offered a new approach to gene therapy. Cell-based gene therapy is an approach that augments the therapeutic potential of EPCs and MSCs and may deliver on the promise of reversal of established PAH. These new regenerative approaches have shown tremendous potential in preclinical studies; however, large, rigorously designed clinical studies will be necessary to evaluate clinical efficacy and safety. © 2013 American Physiological Society. Compr Physiol 3:1749-1779, 2013.

  4. Low cytotoxicity fluorescent PAMAM dendrimer as gene carriers for monitoring the delivery of siRNA

    Energy Technology Data Exchange (ETDEWEB)

    Guan, Lingmei [Sichuan University, State Key Laboratory of Bio-resources and Eco-environment, The Ministry of Education, College of Life Sciences (China); Huang, Saipeng [Chinese Academy of Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Center for Molecular Sciences, Institute of Chemistry (China); Chen, Zhao [Xi’an Jiaotong University, School of Science (China); Li, Yanchao [Chinese Academy of Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Center for Molecular Sciences, Institute of Chemistry (China); Liu, Ke [Sichuan University, State Key Laboratory of Bio-resources and Eco-environment, The Ministry of Education, College of Life Sciences (China); Liu, Yang, E-mail: yliu@iccas.ac.cn; Du, Libo, E-mail: dulibo@iccas.ac.cn [Chinese Academy of Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Center for Molecular Sciences, Institute of Chemistry (China)

    2015-09-15

    Visual detection of gene vectors has attracted a great deal of attention due to the application of these vectors in monitoring and evaluating the effect of gene carriers in living cells. A non-viral vector, the fluorescent PAMAM dendrimer (F-PAMAM), was synthesized through conjugation of PAMAM dendrimers and fluorescein. In vitro and ex vivo experiments show that F-PAMAM exhibits superphotostability, low cytotoxicity and facilitates endocytosis by A549 cells. The vector has a high siRNA binding affinity and it increases the efficiency of cy5-siRNA delivery in A549 cells, in comparison with a cy5-siRNA monomer. Our results provide a new method for simultaneously monitoring the delivery of siRNA and its non-viral carriers in living cells.

  5. The effect of dexamethasone/cell-penetrating peptide nanoparticles on gene delivery for inner ear therapy

    Directory of Open Access Journals (Sweden)

    Yoon JY

    2016-11-01

    Full Text Available Ji Young Yoon,1 Keum-Jin Yang,2 Shi-Nae Park,3 Dong-Kee Kim,3 Jong-Duk Kim1 1Department of Chemical and Biomolecular Engineering, BK 21 Plus Program, Korea Advanced Institute of Science and Technology, Guseong-Dong, Yuseong-Gu, Daejeon, 2Clinical Research Institute, St Mary’s Hospital, Daejeon, 3Department of Otolaryngology – Head and Neck Surgery, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea Abstract: Dexamethasone (Dex-loaded PHEA-g-C18-Arg8 (PCA nanoparticles (PCA/Dex were developed for the delivery of genes to determine the synergistic effect of Dex on gene expression. The cationic PCA nanoparticles were self-assembled to create cationic micelles containing an octadecylamine (C18 core with Dex and an arginine 8 (Arg8 peptide shell for electrostatic complexation with nucleic acids (connexin 26 [Cx26] siRNA, green fluorescent protein [GFP] DNA or brain-derived neurotrophic factor [BDNF] pDNA. The PCA/Dex nanoparticles conjugated with Arg8, a cell-penetrating peptide that enhances permeability through a round window membrane in the inner ear for gene delivery, exhibited high uptake efficiency in HEI-OC1 cells. This potential carrier co-delivering Dex and the gene into inner ear cells has a diameter of 120–140 nm and a zeta potential of 20–25 mV. Different types of genes were complexed with the Dex-loaded PCA nanoparticle (PCA/Dex/gene for gene expression to induce additional anti-inflammatory effects. PCA/Dex showed mildly increased expression of GFP and lower mRNA expression of inflammatory cytokines (IL1b, IL12, and INFr than did Dex-free PCA nanoparticles and Lipofectamine® reagent in HEI-OC1 cells. In addition, after loading Cx26 siRNA onto the surface of PCA/Dex, Cx26 gene expression was downregulated according to real-time polymerase chain reaction for 24 h, compared with that using Lipofectamine reagent. After loading BDNF DNA into PCA/Dex, increased expression of BDNF was observed for 30

  6. Hydrodynamic gene delivery in human skin using a hollow microneedle device.

    Science.gov (United States)

    Dul, M; Stefanidou, M; Porta, P; Serve, J; O'Mahony, C; Malissen, B; Henri, S; Levin, Y; Kochba, E; Wong, F S; Dayan, C; Coulman, S A; Birchall, J C

    2017-11-10

    Microneedle devices have been proposed as a minimally invasive delivery system for the intradermal administration of nucleic acids, both plasmid DNA (pDNA) and siRNA, to treat localised disease or provide vaccination. Different microneedle types and application methods have been investigated in the laboratory, but limited and irreproducible levels of gene expression have proven to be significant challenges to pre-clinical to clinical progression. This study is the first to explore the potential of a hollow microneedle device for the delivery and subsequent expression of pDNA in human skin. The regulatory approved MicronJet600® (MicronJet hereafter) device was used to deliver reporter plasmids (pCMVβ and pEGFP-N1) into viable excised human skin. Exogenous gene expression was subsequently detected at multiple locations that were distant from the injection site but within the confines of the bleb created by the intradermal bolus. The observed levels of gene expression in the tissue are at least comparable to that achieved by the most invasive microneedle application methods e.g. lateral application of a microneedle. Gene expression was predominantly located in the epidermis, although also evident in the papillary dermis. Optical coherence tomography permitted real time visualisation of the sub-surface skin architecture and, unlike a conventional intradermal injection, MicronJet administration of a 50μL bolus appears to create multiple superficial microdisruptions in the papillary dermis and epidermis. These were co-localised with expression of the pCMVβ reporter plasmid. We have therefore shown, for the first time, that a hollow microneedle device can facilitate efficient and reproducible gene expression of exogenous naked pDNA in human skin using volumes that are considered to be standard for intradermal administration, and postulate a hydrodynamic effect as the mechanism of gene delivery. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. [The progesterone receptor gene polymorphism as factor of risk for the preterm delivery].

    Science.gov (United States)

    Oliveira, Tenilson Amaral; da Cunha, Danielle Renzoni; Policastro, Adriana; Traina, Évelyn; Gomes, Mariano Tamura; Cordioli, Eduardo

    2011-06-01

    to investigate the association between gene polymorphism of the progesterone receptor (PROGINS) and the risk of premature birth. In this case-control study, 57 women with previous premature delivery (Case Group) and 57 patients with delivery at term in the current pregnancy and no history of preterm delivery (Control Group) were selected. A 10 mL amount of peripheral blood was collected by venipuncture and genomic DNA was extracted followed by the polymerase chain reaction (PCR) under specific conditions for this polymorphism and 2% agarose gel electrophoresis. The bands were visualized with an ultraviolet light transilluminator. Genotype and allele PROGINS frequencies were compared between the two groups by the χ2 test, with the level of significance set at value p < 0.05. The Odds Ratio (OR) was also used, with 95% confidence intervals. PROGINS genotypic frequencies were 75.4% T1/T1, 22.8% T1/T2 and 1.8% T2/T2 in the Group with Preterm Delivery and 80.7% T1/T1, 19.3% T1/T2 and 0% T2/T2 in the term Delivery Group. There were no differences between groups when genotype and allele frequencies were analyzed: p = 0.4 (OR = 0.7) and p = 0.4 (OR = 0.7). the present study suggests that the presence of PROGINS polymorphism in our population does not constitute a risk factor for premature birth.

  8. Hydrodynamic gene delivery in human skin using a hollow microneedle device

    OpenAIRE

    Dul, M.; Stefanidou, M.; Porta, P.; Serve, J.; O'Mahony, Conor; Malissen, B.; Henri, S.; Levin, Y.; Kochba, E.; Wong, F. S.; Dayan, C.; Coulman, S. A.; Birchall, J. C.

    2017-01-01

    Microneedle devices have been proposed as a minimally invasive delivery system for the intradermal administration of nucleic acids, both plasmid DNA (pDNA) and siRNA, to treat localised disease or provide vaccination. Different microneedle types and application methods have been investigated in the laboratory, but limited and irreproducible levels of gene expression have proven to be significant challenges to pre-clinical to clinical progression. This study is the first to explore the potenti...

  9. Short Hairpin RNA (shRNA): Design, Delivery, and Assessment of Gene Knockdown

    Science.gov (United States)

    Moore, Chris B.; Guthrie, Elizabeth H.; Huang, Max Tze-Han; Taxman, Debra J.

    2013-01-01

    Shortly after the cellular mechanism of RNA interference (RNAi) was first described, scientists began using this powerful technique to study gene function. This included designing better methods for the successful delivery of small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) into mammalian cells. While the simplest method for RNAi is the cytosolic delivery of siRNA oligonucleotides, this technique is limited to cells capable of transfection and is primarily utilized during transient in vitro studies. The introduction of shRNA into mammalian cells through infection with viral vectors allows for stable integration of shRNA and long-term knockdown of the targeted gene; however, several challenges exist with the implementation of this technology. Here we describe some well-tested protocols which should increase the chances of successful design, delivery, and assessment of gene knockdown by shRNA. We provide suggestions for designing shRNA targets and controls, a protocol for sequencing through the secondary structure of the shRNA hairpin structure, and protocols for packaging and delivery of shRNA lentiviral particles. Using real-time PCR and functional assays we demonstrate the successful knockdown of ASC, an inflammatory adaptor molecule. These studies demonstrate the practicality of including two shRNAs with different efficacies of knockdown to provide an additional level of control and to verify dose dependency of functional effects. Along with the methods described here, as new techniques and algorithms are designed in the future, shRNA is likely to include further promising application and continue to be a critical component of gene discovery. PMID:20387148

  10. Polydnaviruses of Parasitic Wasps: Domestication of Viruses To Act as Gene Delivery Vectors

    Directory of Open Access Journals (Sweden)

    Michael R. Strand

    2012-01-01

    Full Text Available Symbiosis is a common phenomenon in which associated organisms can cooperate in ways that increase their ability to survive, reproduce, or utilize hostile environments. Here, we discuss polydnavirus symbionts of parasitic wasps. These viruses are novel in two ways: (1 they have become non-autonomous domesticated entities that cannot replicate outside of wasps; and (2 they function as a delivery vector of genes that ensure successful parasitism of host insects that wasps parasitize. In this review we discuss how these novelties may have arisen, which genes are potentially involved, and what the consequences have been for genome evolution.

  11. Comparative analysis of DNA nanoparticles and AAVs for ocular gene delivery.

    Directory of Open Access Journals (Sweden)

    Zongchao Han

    Full Text Available Gene therapy is a critical tool for the treatment of monogenic retinal diseases. However, the limited vector capacity of the current benchmark delivery strategy, adeno-associated virus (AAV, makes development of larger capacity alternatives, such as compacted DNA nanoparticles (NPs, critical. Here we conduct a side-by-side comparison of self-complementary AAV and CK30PEG NPs using matched ITR plasmids. We report that although AAVs are more efficient per vector genome (vg than NPs, NPs can drive gene expression on a comparable scale and longevity to AAV. We show that subretinally injected NPs do not leave the eye while some of the AAV-injected animals exhibited vector DNA and GFP expression in the visual pathways of the brain from PI-60 onward. As a result, these NPs have the potential to become a successful alternative for ocular gene therapy, especially for the multitude of genes too large for AAV vectors.

  12. Efficient and safe gene delivery to human corneal endothelium using magnetic nanoparticles.

    Science.gov (United States)

    Czugala, Marta; Mykhaylyk, Olga; Böhler, Philip; Onderka, Jasmine; Stork, Björn; Wesselborg, Sebastian; Kruse, Friedrich E; Plank, Christian; Singer, Bernhard B; Fuchsluger, Thomas A

    2016-07-01

    To develop a safe and efficient method for targeted, anti-apoptotic gene therapy of corneal endothelial cells (CECs). Magnetofection (MF), a combination of lipofection with magnetic nanoparticles (MNPs; PEI-Mag2, SO-Mag5, PalD1-Mag1), was tested in human CECs and in explanted human corneas. Effects on cell viability and function were investigated. Immunocompatibility was assessed in human peripheral blood mononuclear cells. Silica iron-oxide MNPs (SO-Mag5) combined with X-tremeGENE-HP achieved high transfection efficiency in human CECs and explanted human corneas, without altering cell viability or function. Magnetofection caused no immunomodulatory effects in human peripheral blood mononuclear cells. Magnetofection with anti-apoptotic P35 gene effectively blocked apoptosis in CECs. Magnetofection is a promising tool for gene therapy of corneal endothelial cells with potential for targeted on-site delivery.

  13. Gene delivery process in a single animal cell after femtosecond laser microinjection

    Science.gov (United States)

    Hosokawa, Yoichiroh; Iguchi, Seriya; Yasukuni, Ryohei; Hiraki, Yuji; Shukunami, Chisa; Masuhara, Hiroshi

    2009-09-01

    Microinjection of extracellular molecules into a single animal cell was performed by an amplified femtosecond laser irradiation. When a single-shot laser pulse was focused on the plasma membrane of a single fibroblast from the mouse cell line NIH3T3 with a high-numerical aperture objective lens, a transient hole with a diameter of 1 μm was formed. The delivery process of extracellular molecules immediately after the hole formation was monitored by a fluorescence staining with fluoresceinisothiocyanate-dextran (FITC-dextran). Then the gene expression was confirmed using a DNA plasmid of an enhanced green fluorescent protein (EGFP). The gene expression was observed when the laser pulse was focused first on the cellular membrane and then on the nuclear membrane, while the gene was not expressed when the laser was focused only on the cellular membrane. On the basis of these results, the efficiency of gene delivery by the femtosecond laser microinjection and the subsequent gene expression were clarified.

  14. Microneedle delivery of plasmid DNA to living human skin: formulation coating, skin insertion and gene expression

    Science.gov (United States)

    Pearton, Marc; Saller, Verena; Coulman, Sion A; Gateley, Chris; Anstey, Alexander V; Zarnitsyn, Vladimir; Birchall, James C

    2012-01-01

    Microneedle delivery of nucleic acids, in particular plasmid DNA (pDNA), to the skin represents a potential new approach for the clinical management of genetic skin diseases and cutaneous cancers, and for intracutaneous genetic immunization. In this study excised human skin explants were used to investigate and optimize key parameters that will determine stable and effective microneedle-facilitated pDNA delivery. These include (i) high dose-loading of pDNA onto microneedle surfaces, (ii) stability and functionality of the coated pDNA, (iii) skin penetration capability of pDNA-coated microneedles, and (iv) efficient gene expression in human skin. Optimization of a dip-coating method enabled significant increases in the loading capacity, up to 100 micrograms of pDNA per 5-microneedle array. Coated microneedles were able to reproducibly perforate human skin at low (coated pDNA was partially compromised on storage, although this was improved through the addition of saccharide excipients without detriment to the biological functionality of pDNA. The pDNA-coated microneedles facilitated reporter gene expression in viable human skin. The efficiency of gene expression from coated microneedles will depend upon suitable DNA loading, efficient and reproducible skin puncture and rapid in situ dissolution of the plasmid at the site of delivery. PMID:22516089

  15. Chitosan/TPP-hyaluronic acid nanoparticles: a new vehicle for gene delivery to the spinal cord.

    Science.gov (United States)

    Gwak, So-Jung; Jung, Jong Kwon; An, Sung Su; Kim, Hyo Jin; Oh, Jin Soo; Pennant, William A; Lee, Hye Yeong; Kong, Min Ho; Kim, Keung Nyun; Yoon, Do Heum; Ha, Yoon

    2012-01-01

    Gene delivery offers therapeutic promise for the treatment of neurological diseases and spinal cord injury. Several studies have offered viral vectors as vehicles to deliver therapeutic agents, yet their toxicity and immunogenicity, along with the cost of their large-scale formulation, limits their clinical use. As such, non-viral vectors are attractive in that they offer improved safety profiles compared to viruses. Poly(ethylene imine) (PEI) is one of the most extensively studied non-viral vectors, but its clinical value is limited y its cytotoxicity. Recently, chitosan/DNA complex nanoparticles have een considered as a vector for gene delivery. Here, we demonstrate that DNA nanoparticles made of hyaluronic acid (HA) and chitosan have low cytotoxicity and induce high transgene expression in neural stem cells and organotypic spinal cord slice tissue. Chitosan-TPP/HA nanoparticles were significantly less cytotoxic than PEI at various concentrations. Additionally, chitosan-TPP/HA nanoparticles with pDNA induced higher transgene expression in vitro for a longer duration than PEI in neural stem cells. These results suggest chitosan-TPP/HA nanoparticles may have the potential to serve as an option for gene delivery to the spinal cord.

  16. Biopolymer-Based Nanoparticles for Drug/Gene Delivery and Tissue Engineering

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    Keiji Numata

    2013-01-01

    Full Text Available There has been a great interest in application of nanoparticles as biomaterials for delivery of therapeutic molecules such as drugs and genes, and for tissue engineering. In particular, biopolymers are suitable materials as nanoparticles for clinical application due to their versatile traits, including biocompatibility, biodegradability and low immunogenicity. Biopolymers are polymers that are produced from living organisms, which are classified in three groups: polysaccharides, proteins and nucleic acids. It is important to control particle size, charge, morphology of surface and release rate of loaded molecules to use biopolymer-based nanoparticles as drug/gene delivery carriers. To obtain a nano-carrier for therapeutic purposes, a variety of materials and preparation process has been attempted. This review focuses on fabrication of biocompatible nanoparticles consisting of biopolymers such as protein (silk, collagen, gelatin, β-casein, zein and albumin, protein-mimicked polypeptides and polysaccharides (chitosan, alginate, pullulan, starch and heparin. The effects of the nature of the materials and the fabrication process on the characteristics of the nanoparticles are described. In addition, their application as delivery carriers of therapeutic drugs and genes and biomaterials for tissue engineering are also reviewed.

  17. Capillary electrophoresis analysis of poly(ethylene glycol) and ligand-modified polylysine gene delivery vectors.

    Science.gov (United States)

    Guo, Yan; Sun, Ye; Gu, Jianren; Xu, Yuhong

    2007-04-15

    Cationic polymers including polylysine (PLL) and polyethylenimine are being widely tested as gene delivery vectors in various gene therapy applications. In many cases, the polymers were further modified by hydrophilic polymer grafting or ligand conjugation, which had been shown to greatly affect the vector stability, delivery efficiency and specificity. The characterization of modified polycation is particularly critical for quality control and vector development. Here several different separation modes using capillary electrophoresis for the analytical characterization of the modified polymers are described. PLL molecules were grafted with poly(ethylene glycol) (PEG) chain or conjugated with epidermal growth factor and analyzed under various analytical conditions. Poly(N,N'-dimethylacrylamide)-coated capillary was used to analyze the modified PLL to reduce the interaction between the samples and the capillary wall. PLLs containing different numbers of conjugated ligands were well separated with the coating method but, for PLL-g-PEG, the separation was poor under the same conditions. A method using low buffer pH and hydroxypropylmethyl cellulose additive was developed. These methods are useful to characterize various polycations and important for the quality control and application of potential gene delivery vectors.

  18. Prevention of adverse events of interferon γ gene therapy by gene delivery of interferon γ-heparin-binding domain fusion protein in mice

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    Mitsuru Ando

    2014-01-01

    Full Text Available Sustained gene delivery of interferon (IFN γ can be an effective treatment, but our previous study showed high levels of IFNγ-induced adverse events, including the loss of body weight. These unwanted events could be reduced by target-specific delivery of IFNγ after in vivo gene transfer. To achieve this, we selected the heparin-binding domain (HBD of extracellular superoxide dismutase as a molecule to anchor IFNγ to the cell surface. We designed three IFNγ derivatives, IFNγ-HBD1, IFNγ-HBD2, and IFNγ-HBD3, each of which had 1, 2, or 3 HBDs, respectively. Each plasmid-encoding fusion proteins was delivered to the liver, a model target in this study, by hydrodynamic tail vein injection. The serum concentration of IFNγ-HBD2 and IFNγ-HBD3 after gene delivery was lower than that of IFNγ or IFNγ-HBD1. Gene delivery of IFNγ-HBD2, but not of IFNγ-HBD3, effectively increased the mRNA expression of IFNγ-inducible genes in the liver, suggesting liver-specific distribution of IFNγ-HBD2. Gene delivery of IFNγ-HBD2-suppressed tumor growth in the liver as efficiently as that of IFNγ with much less symptoms of adverse effects. These results indicate that the adverse events of IFNγ gene transfer can be prevented by gene delivery of IFNγ-HBD2, a fusion protein with high cell surface affinity.

  19. Mismatch-mediated error prone repair at the immunoglobulin genes.

    Science.gov (United States)

    Chahwan, Richard; Edelmann, Winfried; Scharff, Matthew D; Roa, Sergio

    2011-12-01

    The generation of effective antibodies depends upon somatic hypermutation (SHM) and class-switch recombination (CSR) of antibody genes by activation induced cytidine deaminase (AID) and the subsequent recruitment of error prone base excision and mismatch repair. While AID initiates and is required for SHM, more than half of the base changes that accumulate in V regions are not due to the direct deamination of dC to dU by AID, but rather arise through the recruitment of the mismatch repair complex (MMR) to the U:G mismatch created by AID and the subsequent perversion of mismatch repair from a high fidelity process to one that is very error prone. In addition, the generation of double-strand breaks (DSBs) is essential during CSR, and the resolution of AID-generated mismatches by MMR to promote such DSBs is critical for the efficiency of the process. While a great deal has been learned about how AID and MMR cause hypermutations and DSBs, it is still unclear how the error prone aspect of these processes is largely restricted to antibody genes. The use of knockout models and mice expressing mismatch repair proteins with separation-of-function point mutations have been decisive in gaining a better understanding of the roles of each of the major MMR proteins and providing further insight into how mutation and repair are coordinated. Here, we review the cascade of MMR factors and repair signals that are diverted from their canonical error free role and hijacked by B cells to promote genetic diversification of the Ig locus. This error prone process involves AID as the inducer of enzymatically-mediated DNA mismatches, and a plethora of downstream MMR factors acting as sensors, adaptors and effectors of a complex and tightly regulated process from much of which is not yet well understood. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  20. A surface-mediated siRNA delivery system developed with chitosan/hyaluronic acid-siRNA multilayer films through layer-by-layer self-assembly

    Science.gov (United States)

    Wu, Lijuan; Wu, Changlin; Liu, Guangwan; Liao, Nannan; Zhao, Fang; Yang, Xuxia; Qu, Hongyuan; Peng, Bo; Chen, Li; Yang, Guang

    2016-12-01

    siRNA delivery remains highly challenging because of its hydrophilic and anionic nature and its sensitivity to nuclease degradation. Effective siRNA loading and improved transfection efficiency into cells represents a key problem. In our study, we prepared Chitosan/Hyaluronic acid-siRNA multilayer films through layer-by-layer self-assembly, in which siRNAs can be effectively loaded and protected. The construction process was characterized by FTIR, 13C NMR (CP/MAS), UV-vis spectroscopy, and atomic force microscopy (AFM). We presented the controlled-release performance of the films during incubation in 1 M NaCl solution for several days through UV-vis spectroscopy and polyacrylamide gel electrophoresis (PAGE). Additionally, we verified the stability and integrity of the siRNA loaded on multilayer films. Finally, the biological efficacy of the siRNA delivery system was evaluated via cells adhesion and gene silencing analyses in eGFP-HEK 293T cells. This new type of surface-mediated non-viral multilayer films may have considerable potential in the localized and controlled-release delivery of siRNA in mucosal tissues, and tissue engineering application.

  1. Roles of Prolyl Isomerases in RNA-Mediated Gene Expression

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    Roopa Thapar

    2015-05-01

    Full Text Available The peptidyl-prolyl cis-trans isomerases (PPIases that include immunophilins (cyclophilins and FKBPs and parvulins (Pin1, Par14, Par17 participate in cell signaling, transcription, pre-mRNA processing and mRNA decay. The human genome encodes 19 cyclophilins, 18 FKBPs and three parvulins. Immunophilins are receptors for the immunosuppressive drugs cyclosporin A, FK506, and rapamycin that are used in organ transplantation. Pin1 has also been targeted in the treatment of Alzheimer’s disease, asthma, and a number of cancers. While these PPIases are characterized as molecular chaperones, they also act in a nonchaperone manner to promote protein-protein interactions using surfaces outside their active sites. The immunosuppressive drugs act by a gain-of-function mechanism by promoting protein-protein interactions in vivo. Several immunophilins have been identified as components of the spliceosome and are essential for alternative splicing. Pin1 plays roles in transcription and RNA processing by catalyzing conformational changes in the RNA Pol II C-terminal domain. Pin1 also binds several RNA binding proteins such as AUF1, KSRP, HuR, and SLBP that regulate mRNA decay by remodeling mRNP complexes. The functions of ribonucleoprotein associated PPIases are largely unknown. This review highlights PPIases that play roles in RNA-mediated gene expression, providing insight into their structures, functions and mechanisms of action in mRNP remodeling in vivo.

  2. Virus-induced gene silencing and Agrobacterium tumefaciens-mediated transient expression in Nicotiana tabacum.

    Science.gov (United States)

    Zhang, Zhao; Thomma, Bart P H J

    2014-01-01

    Virus-induced gene silencing (VIGS) is a rapid method for transient silencing of plant genes. In this chapter, we describe the methodology for Tobacco rattle virus (TRV)-based VIGS in Nicotiana tabacum. In combination with subsequent co-expression of the tomato immune receptor Ve1 and the corresponding Verticillium effector Ave1 through Agrobacterium tumefaciens-mediated transient transformation (agroinfiltration), we established a rapid system for assessing the requirement of candidate plant genes for Ve1-mediated immune signaling.

  3. Recombinant adeno-associated viral (rAAV) vectors mediate efficient gene transduction in cultured neonatal and adult microglia.

    Science.gov (United States)

    Su, Wei; Kang, John; Sopher, Bryce; Gillespie, James; Aloi, Macarena S; Odom, Guy L; Hopkins, Stephanie; Case, Amanda; Wang, David B; Chamberlain, Jeffrey S; Garden, Gwenn A

    2016-01-01

    Microglia are a specialized population of myeloid cells that mediate CNS innate immune responses. Efforts to identify the cellular and molecular mechanisms that regulate microglia behaviors have been hampered by the lack of effective tools for manipulating gene expression. Cultured microglia are refractory to most chemical and electrical transfection methods, yielding little or no gene delivery and causing toxicity and/or inflammatory activation. Recombinant adeno-associated viral (rAAVs) vectors are non-enveloped, single-stranded DNA vectors commonly used to transduce many primary cell types and tissues. In this study, we evaluated the feasibility and efficiency of utilizing rAAV serotype 2 (rAAV2) to modulate gene expression in cultured microglia. rAAV2 yields high transduction and causes minimal toxicity or inflammatory response in both neonatal and adult microglia. To demonstrate that rAAV transduction can induce functional protein expression, we used rAAV2 expressing Cre recombinase to successfully excise a LoxP-flanked miR155 gene in cultured microglia. We further evaluated rAAV serotypes 5, 6, 8, and 9, and observed that all efficiently transduced cultured microglia to varying degrees of success and caused little or no alteration in inflammatory gene expression. These results provide strong encouragement for the application of rAAV-mediated gene expression in microglia for mechanistic and therapeutic purposes. Neonatal microglia are functionally distinct from adult microglia, although the majority of in vitro studies utilize rodent neonatal microglia cultures because of difficulties of culturing adult cells. In addition, cultured microglia are refractory to most methods for modifying gene expression. Here, we developed a novel protocol for culturing adult microglia and evaluated the feasibility and efficiency of utilizing Recombinant Adeno-Associated Virus (rAAV) to modulate gene expression in cultured microglia. © 2015 International Society for

  4. Evolution of contrast agents for ultrasound imaging and ultrasound-mediated drug delivery

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    Vera ePaefgen

    2015-09-01

    Full Text Available Ultrasound is one of the most frequently used diagnostic methods. It is a non-invasive, comparably inexpensive imaging method with a broad spectrum of applications, which can be increased even more by using bubbles as contrast agents. There are various different types of bubbles: filled with different gases, composed of soft- or hard-shell materials, and ranging in size from nano- to micrometers. These intravascular contrast agents enable functional analyses, e.g. to acquire organ perfusion in real-time. Molecular analyses are achieved by coupling specific ligands to the bubbles’ shell, which bind to marker molecules in the area of interest. Bubbles can also be loaded with or attached to drugs, peptides or genes and can be destroyed by ultrasound pulses to locally release the entrapped agent. Recent studies show that ultrasound contrast agents are also valuable tools in hyperthermia-induced ablation therapy of tumors, or can increase cellular uptake of locally released drugs by enhancing membrane permeability. This review summarizes important steps in the development of ultrasound contrast agents and introduces the current clinical applications of contrast-enhanced ultrasound. Additionally, an overview of the recent developments in ultrasound probe design for functional and molecular diagnosis as well as for drug delivery is given.

  5. Is caesarean delivery associated with sensitization to food allergens and IgE-mediated food allergy: a systematic review.

    Science.gov (United States)

    Koplin, Jennifer; Allen, Katie; Gurrin, Lyle; Osborne, Nicholas; Tang, Mimi L K; Dharmage, Shyamali

    2008-12-01

    Several studies have shown differences in the composition of the gastrointestinal flora of children who develop sensitization to food allergens compared with non-allergic children. It has been hypothesized that changes in the gut microbiota resulting from caesarean section delivery could increase a child's risk of developing food allergy; however, studies examining the relationship between mode of delivery and food allergy have produced conflicting results. The objective of this review was to determine whether there is sufficient evidence to support an association between delivery by caesarean section and the development of sensitization to food allergens and immunoglobulin E (IgE) mediated food allergy. Using predefined inclusion and exclusion criteria, MEDLINE and PubMed were searched for studies investigating the relationship between caesarean section delivery and food allergy. The information on the quality of the studies and results were extracted and analysed systematically. The search identified four relevant studies as per our protocol. Symptomatic food allergy was used as the outcome in two studies and was found to occur more frequently in children born by caesarean section in one study while the second study found no association between food allergy diagnoses and mode of delivery. The other two studies measured levels of food antigen-specific IgE, with both studies showing an increase in sensitization to food allergens among children born by caesarean section. Overall, there is evidence that the risk of developing IgE-mediated sensitization to food allergens is increased among children delivered by caesarean section, however further studies using objectively diagnosed food allergy as the outcome are needed to verify whether this equates to an increase in confirmed food allergy. Future birth cohort studies should control for the effects of mode of delivery when investigating environmental modifiers of food allergy.

  6. Enhancing gene delivery of adeno-associated viruses by cell-permeable peptides

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    Yarong Liu

    2014-01-01

    Full Text Available Adeno-associated virus type 2 (AAV2 is considered a promising gene delivery vector and has been extensively applied in several disease models; however, inefficient transduction in various cells and tissues has limited its widespread application in many areas of gene therapy. In this study, we have developed a general, but efficient, strategy to enhance viral transduction, both in vitro and in vivo, by incubating viral particles with cell-permeable peptides (CPPs. We show that CPPs increase internalization of viral particles into cells by facilitating both energy-independent and energy-dependent endocytosis. Moreover, CPPs can significantly enhance the endosomal escape process of viral particles, thus enhancing viral transduction to those cells that have exhibited very low permissiveness to AAV2 infection as a result of impaired intracellular viral processing. We also demonstrated that this approach could be applicable to other AAV serotypes. Thus, the membrane-penetrating ability of CPPs enables us to generate an efficient method for enhanced gene delivery of AAV vectors, potentially facilitating its applicability to human gene therapy.

  7. CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes.

    Science.gov (United States)

    Ehrke-Schulz, Eric; Schiwon, Maren; Leitner, Theo; Dávid, Stephan; Bergmann, Thorsten; Liu, Jing; Ehrhardt, Anja

    2017-12-07

    The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system revolutionized the field of gene editing but viral delivery of the CRISPR/Cas9 system has not been fully explored. Here we adapted clinically relevant high-capacity adenoviral vectors (HCAdV) devoid of all viral genes for the delivery of the CRISPR/Cas9 machinery using a single viral vector. We present a platform enabling fast transfer of the Cas9 gene and gRNA expression units into the HCAdV genome including the option to choose between constitutive or inducible Cas9 expression and gRNA multiplexing. Efficacy and versatility of this pipeline was exemplified by producing different CRISPR/Cas9-HCAdV targeting the human papillomavirus (HPV) 18 oncogene E6, the dystrophin gene causing Duchenne muscular dystrophy (DMD) and the HIV co-receptor C-C chemokine receptor type 5 (CCR5). All CRISPR/Cas9-HCAdV proved to be efficient to deliver the respective CRISPR/Cas9 expression units and to introduce the desired DNA double strand breaks at their intended target sites in immortalized and primary cells.

  8. Evaluation of gene delivery strategies to efficiently overexpress functional HLA-G on human bone marrow stromal cells

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    Joana S Boura

    2014-01-01

    Full Text Available Mesenchymal stromal cells (MSC constitutively express low levels of human leukocyte antigen-G (HLA-G, which has been shown to contribute to their immunomodulatory and anti-inflammatory properties. Here, we hypothesized that overexpression of HLA-G on bone marrow-derived MSC would improve their immunomodulatory function, thus increasing their therapeutic potential. Therefore, we investigated which gene transfer system is best suited for delivering this molecule while maintaining its immunomodulatory effects. We performed a side-by-side comparison between three nonviral plasmid-based platforms (pmax-HLA-G1; MC-HLA-G1; pEP-HLA-G1 and a viral system (Lv-HLA-G1 using gene transfer parameters that yielded similar levels of HLA-G1-expressing MSC. Natural killer (NK cell–mediated lysis assays and T cell proliferation assays showed that MSC modified with the HLA-G1 expressing viral vector had significantly lower susceptibility to NK-lysis and significantly reduced T cell proliferation when compared to nonmodified cells or MSC modified with plasmid. We also show that, in plasmid-modified MSC, an increase in Toll-like receptor (TLR9 expression is the mechanism responsible for the abrogation of HLA-G1's immunomodulatory effect. Although MSC can be efficiently modified to overexpress HLA-G1 using viral and nonviral strategies, only viral-based delivery of HLA-G1 is suitable for improvement of MSC's immunomodulatory properties.

  9. A Qualitative Case Study Comparing a Computer-Mediated Delivery System to a Face-to-Face Mediated Delivery System for Teaching Creative Writing Fiction Workshops

    Science.gov (United States)

    Daniels, Mindy A.

    2012-01-01

    The purpose of this case study was to compare the pedagogical and affective efficiency and efficacy of creative prose fiction writing workshops taught via asynchronous computer-mediated online distance education with creative prose fiction writing workshops taught face-to-face in order to better understand their operational pedagogy and…

  10. Cell-permeable peptides improve cellular uptake and therapeutic gene delivery of replication-deficient viruses in cells and in vivo.

    Science.gov (United States)

    Gratton, Jean-Philippe; Yu, Jun; Griffith, Jason W; Babbitt, Roger W; Scotland, Ramona S; Hickey, Reed; Giordano, Frank J; Sessa, William C

    2003-03-01

    Small polybasic peptides derived from the transduction domains of certain proteins, such as the third alpha-helix of the Antennapedia (Antp) homeodomain, can cross the cell membrane through a receptor-independent mechanism. These cell-permeable molecules have been used as 'Trojan horses' to introduce biologically active cargo molecules such as DNA, peptides or proteins into cells. Using these cell-permeable peptides, we have developed an efficient and simple method to increase virally mediated gene delivery and protein expression in vitro and in vivo. Here, we show that cell-permeable peptides increase viral cell entry, improve gene expression at reduced titers of virus and improve efficacy of therapeutically relevant genes in vivo.

  11. Combined ultrasound-targeted microbubble destruction and polyethylenimine-mediated plasmid DNA delivery to the rat retina: enhanced efficiency and accelerated expression.

    Science.gov (United States)

    Li, Hongli; Qian, Jin; Yao, Chunfang; Wan, Caifeng; Li, Fenghua

    2016-04-01

    Gene therapy has potential in the treatment of refractory retinal diseases. It is important to develop an effective delivery system in the retina. The present study aimed to investigate the efficacy and safety of ultrasound (US)-targeted microbubble destruction (UTMD)-mediated polyethylenimine (PEI) to the rat retina. Gene transfer was examined by injecting PEI/plasmid DNA (pDNA) with or without microbubbles (MBs) into the subretinal space of rats that were then exposed to US. We investigated enhanced green fluorescent protein (eGFP) expression on flat fundus oculi and performed quantitative analysis. Hematoxylin and eosin staining was used to observe tissue damage. UTMD significantly enhanced PEI/pDNA transfection efficiency safely by increasing both the transgene expression per cell and the percentage of transfected cells of the retina. PEI/pDNA combined with UTMD significantly increased the number of DNA gene copies and the mRNA level in the retinal pigment epithelium (RPE) and neural retina, respectively, compared to PEI/pDNA alone. The present study demonstrates that enhanced and accelerated pDNA expression can be achieved in the retina/RPE cells in vivo by UTMD physical techniques combined with a PEI chemical vector. Our study provides useful information for further in vivo retinal gene therapy work. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  12. Intracellular Protein Delivery and Gene Transfection by Electroporation Using a Microneedle Electrode Array

    Science.gov (United States)

    Choi, Seong-O; Kim, Yeu-Chun; Lee, Jeong Woo; Park, Jung-Hwan

    2012-01-01

    The impact of many biopharmaceuticals, including protein- and gene-based therapies, has been limited by the need for better methods of delivery into cells within tissues. Here, we present intracellular delivery of molecules and transfection with plasmid DNA by electroporation using a novel microneedle electrode array designed for targeted treatment of skin and other tissue surfaces. The microneedle array is molded out of polylactic acid. Electrodes and circuitry required for electroporation are applied to the microneedle array surface by a new metal-transfer micromolding method. The microneedle array maintains mechanical integrity after insertion into pig cadaver skin and is able to electroporate human prostate cancer cells in vitro. Quantitative measurements show that increasing electroporation pulse voltage increases uptake efficiency of calcein and bovine serum albumin, whereas increasing pulse length has lesser effects over the range studied. Uptake of molecules by up to 50 % of cells and transfection of 12 % of cells with a gene for green fluorescent protein is demonstrated at high cell viability. We conclude that the microneedle electrode array is able to electroporate cells, resulting in intracellular uptake of molecules, and has potential applications to improve intracellular delivery of proteins, DNA and other biopharmaceuticals. PMID:22328093

  13. Chitosan tripolyphosphate (CS/TPP) nanoparticles: preparation, characterization and application for gene delivery in shrimp.

    Science.gov (United States)

    Vimal, S; Abdul Majeed, S; Taju, G; Nambi, K S N; Sundar Raj, N; Madan, N; Farook, M A; Rajkumar, T; Gopinath, D; Sahul Hameed, A S

    2013-12-01

    The present study examines the use of CS/TPP nanoparticles for gene delivery in different tissues of shrimp through oral route. The viral gene of WSSV was used to construct DNA vaccines using pcDNA 3.1, a eukaryotic expression vector and the constructs were named as pVP28. The CS/TPP nanoparticles were synthesized by ionic gelation process and these particles were characterized. The structure and morphology of the nanoparticles were studied by field emission scanning electron microscopy (FE-SEM) and FTIR (Fourier Transform Infrared Spectra). The cytotoxicity of CS/TPP nanoparticles was evaluated by MTT assay using fish cell line. The expression of gene was confirmed by Immuno-dot blot, ELISA and RT-PCR analyses. The results indicate that DNA can be easily delivered into shrimp by feeding with CS/TPP nanoparticles. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Comparison of different cationized proteins as biomaterials for nanoparticle-based ocular gene delivery.

    Science.gov (United States)

    Zorzi, Giovanni K; Párraga, Jenny E; Seijo, Begoña; Sanchez, Alejandro

    2015-11-01

    Cationized polymers have been proposed as transfection agents for gene therapy. The present work aims to improve the understanding of the potential use of different cationized proteins (atelocollagen, albumin and gelatin) as nanoparticle components and to investigate the possibility of modulating the physicochemical properties of the resulting nanoparticle carriers by selecting specific protein characteristics in an attempt to improve current ocular gene-delivery approaches. The toxicity profiles, as well as internalization and transfection efficiency, of the developed nanoparticles can be modulated by modifying the molecular weight of the selected protein and the amine used for cationization. The most promising systems are nanoparticles based on intermediate molecular weight gelatin cationized with the endogenous amine spermine, which exhibit an adequate toxicological profile, as well as effective association and protection of pDNA or siRNA molecules, thereby resulting in higher transfection efficiency and gene silencing than the other studied formulations. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Transcriptome analyses and virus induced gene silencing identify genes in the Rpp4-mediated Asian soybean rust resistance pathway

    Science.gov (United States)

    Rpp4 (Resistance to Phakopsora pachyrhizi 4) confers resistance to P. pachyrhizi, the causal agent of Asian soybean rust (ASR). By combining expression profiling and virus induced gene silencing (VIGS), we are developing a genetic framework for Rpp4-mediated resistance. We measured gene expression i...

  16. Host-Induced Gene Silencing of Rice Blast Fungus Magnaporthe oryzae Pathogenicity Genes Mediated by the Brome Mosaic Virus.

    Science.gov (United States)

    Zhu, Lin; Zhu, Jian; Liu, Zhixue; Wang, Zhengyi; Zhou, Cheng; Wang, Hong

    2017-09-26

    Magnaporthe oryzae is a devastating plant pathogen, which has a detrimental impact on rice production worldwide. Despite its agronomical importance, some newly-emerging pathotypes often overcome race-specific disease resistance rapidly. It is thus desirable to develop a novel strategy for the long-lasting resistance of rice plants to ever-changing fungal pathogens. Brome mosaic virus (BMV)-induced RNA interference (RNAi) has emerged as a useful tool to study host-resistance genes for rice blast protection. Planta-generated silencing of targeted genes inside biotrophic pathogens can be achieved by expression of M. oryzae -derived gene fragments in the BMV-mediated gene silencing system, a technique termed host-induced gene silencing (HIGS). In this study, the effectiveness of BMV-mediated HIGS in M. oryzae was examined by targeting three predicted pathogenicity genes, MoABC1, MoMAC1 and MoPMK1 . Systemic generation of fungal gene-specific small interfering RNA (siRNA) molecules induced by inoculation of BMV viral vectors inhibited disease development and reduced the transcription of targeted fungal genes after subsequent M. oryzae inoculation. Combined introduction of fungal gene sequences in sense and antisense orientation mediated by the BMV silencing vectors significantly enhanced the efficiency of this host-generated trans-specific RNAi, implying that these fungal genes played crucial roles in pathogenicity. Collectively, our results indicated that BMV-HIGS system was a great strategy for protecting host plants against the invasion of pathogenic fungi.

  17. Topical Non-Invasive Gene Delivery using Gemini Nanoparticles in Interferon-gamma-deficient Mice

    Energy Technology Data Exchange (ETDEWEB)

    Badea,I.; Wettig, S.; Verrall, R.; Foldvari, M.

    2007-01-01

    Cutaneous gene therapy, although a promising approach for many dermatologic diseases, has not progressed to the stage of clinical trials, mainly due to the lack of an effective gene delivery system. The main objective of this study was to construct and evaluate gemini nanoparticles as a topical formulation for the interferon gamma (IFN-{gamma}) gene in an IFN-{gamma}-deficient mouse model. Nanoparticles based on the gemini surfactant 16-3-16 (NP16-DNA) and another cationic lipid cholesteryl 3{beta}-(-N-[dimethylamino-ethyl] carbamate) [Dc-chol] (NPDc-DNA) were prepared and characterized. Zetasizer measurement indicated a bimodal distribution of 146 and 468 nm average particle sizes for the NP16-DNA ({zeta}-potential +51 mV) nanoparticles and monomodal distribution of 625 nm ({zeta}-potential +44 mV) for the NPDc-DNA. Circular dichroism studies showed that the gemini surfactant compacted the plasmid more efficiently compared to the Dc-chol. Small-angle X-ray scattering measurements revealed structural polymorphism in the NP16-DNA nanoparticles, with lamellar and Fd3m cubic phases present, while for the NPDc-DNA two lamellar phases could be distinguished. In vivo, both topically applied nanoparticles induced higher gene expression compared to untreated control and naked DNA (means of 0.480 and 0.398 ng/cm{sup 2} vs 0.067 and 0.167 ng/cm{sup 2}). However, treatment with NPDc-DNA caused skin irritation, and skin damage, whereas NP16-DNA showed no skin toxicity. In this study, we demonstrated that topical cutaneous gene delivery using gemini surfactant-based nanoparticles in IFN-{gamma}-deficient mice was safe and may provide increased gene expression in the skin due to structural complexity of NP16 nanoparticles (lamellar-cubic phases).

  18. Gene Expression Profile Changes After Short-activating RNA-mediated Induction of Endogenous Pluripotency Factors in Human Mesenchymal Stem Cells

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    Jon Voutila

    2012-01-01

    Full Text Available It is now recognized that small noncoding RNA sequences have the ability to mediate transcriptional activation of specific target genes in human cells. Using bioinformatics analysis and functional screening, we screened short-activating RNA (saRNA oligonucleotides designed to target the promoter regions of the pluripotency reprogramming factors, Kruppel-like factor 4 (KLF4 and c-MYC. We identified KLF4 and c-MYC promoter-targeted saRNA sequences that consistently induced increases in their respective levels of nascent mRNA and protein expression in a time- and dose-dependent manner, as compared with scrambled sequence control oligonucleotides. The functional consequences of saRNA-induced activation of each targeted reprogramming factor were then characterized by comprehensively profiling changes in gene expression by microarray analysis, which revealed significant increases in mRNA levels of their respective downstream pathway genes. Notably, the microarray profile after saRNA-mediated induction of endogenous KLF4 and c-MYC showed similar gene expression patterns for stem cell- and cell cycle-related genes as compared with lentiviral vector-mediated overexpression of exogenous KLF4 and c-MYC transgenes, while divergent gene expression patterns common to viral vector-mediated transgene delivery were also noted. The use of promoter-targeted saRNAs for the activation of pluripotency reprogramming factors could have broad implications for stem cell research.

  19. Adenoassociated Virus Serotype 9-Mediated Gene Therapy for X-Linked Adrenoleukodystrophy

    OpenAIRE

    Gong, Yi; Mu, Dakai; Prabhakar, Shilpa; Moser, Ann; Musolino, Patricia; Ren, JiaQian; Breakefield, Xandra O; Maguire, Casey A; Eichler, Florian S

    2015-01-01

    X-linked adrenoleukodystrophy (X-ALD) is a devastating neurological disorder caused by mutations in the ABCD1 gene that encodes a peroxisomal ATP-binding cassette transporter (ABCD1) responsible for transport of CoA-activated very long-chain fatty acids (VLCFA) into the peroxisome for degradation. We used recombinant adenoassociated virus serotype 9 (rAAV9) vector for delivery of the human ABCD1 gene (ABCD1) to mouse central nervous system (CNS). In vitro, efficient delivery of ABCD1 gene was...

  20. Mucin-mediated nanocarrier disassembly for triggered uptake of oligonucleotides as a delivery strategy for the potential treatment of mucosal tumours

    Science.gov (United States)

    Martirosyan, A.; Olesen, M. J.; Fenton, R. A.; Kjems, J.; Howard, K. A.

    2016-06-01

    This work demonstrates gastric mucin-triggered nanocarrier disassembly for release of antisense oligonucleotides and consequent unassisted cellular entry as a novel oral delivery strategy. A fluorescence activation-based reporter system was used to investigate the interaction and mucin-mediated disassembly of chitosan-based nanocarriers containing a 13-mer DNA oligonucleotide with a flanked locked RNA nucleic acid gapmer design. Gastric mucins were shown to trigger gapmer release from nanocarriers that was dependent on the interaction time, mucin concentration and N : P ratio with a maximal release at N : P 10. In contrast to siRNA, naked gapmers exhibited uptake into mucus producing HT-MTX mono-cultures and HT-MTX co-cultured with the carcinoma epithelial cell line Caco-2. Importantly, in vivo gapmer uptake was observed in epithelial tissue 30 min post-injection in murine intestinal loops. The findings present a mucosal design-based system tailored for local delivery of oligonucleotides that may maximize the effectiveness of gene silencing therapeutics within tumours at mucosal sites.This work demonstrates gastric mucin-triggered nanocarrier disassembly for release of antisense oligonucleotides and consequent unassisted cellular entry as a novel oral delivery strategy. A fluorescence activation-based reporter system was used to investigate the interaction and mucin-mediated disassembly of chitosan-based nanocarriers containing a 13-mer DNA oligonucleotide with a flanked locked RNA nucleic acid gapmer design. Gastric mucins were shown to trigger gapmer release from nanocarriers that was dependent on the interaction time, mucin concentration and N : P ratio with a maximal release at N : P 10. In contrast to siRNA, naked gapmers exhibited uptake into mucus producing HT-MTX mono-cultures and HT-MTX co-cultured with the carcinoma epithelial cell line Caco-2. Importantly, in vivo gapmer uptake was observed in epithelial tissue 30 min post-injection in murine intestinal

  1. Inhibition of Histone Deacetylation and DNA Methylation Improves Gene Expression Mediated by the Adeno-Associated Virus/Phage in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Amin Hajitou

    2013-10-01

    Full Text Available Bacteriophage (phage, viruses that infect bacteria only, have become promising vectors for targeted systemic delivery of genes to cancer, although, with poor efficiency. We previously designed an improved phage vector by incorporating cis genetic elements of adeno-associated virus (AAV. This novel AAV/phage hybrid (AAVP specifically targeted systemic delivery of therapeutic genes into tumors. To advance the AAVP vector, we recently introduced the stress-inducible Grp78 tumor specific promoter and found that this dual tumor-targeted AAVP provides persistent gene expression, over time, in cancer cells compared to silenced gene expression from the CMV promoter in the parental AAVP. Herein, we investigated the effect of histone deacetylation and DNA methylation on AAVP-mediated gene expression in cancer cells and explored the effect of cell confluence state on AAVP gene expression efficacy. Using a combination of AAVP expressing the GFP reporter gene, flow cytometry, inhibitors of histone deacetylation, and DNA methylation, we have demonstrated that histone deacetylation and DNA methylation are associated with silencing of gene expression from the CMV promoter in the parental AAVP. Importantly, inhibitors of histone deacetylases boost gene expression in cancer cells from the Grp78 promoter in the dual tumor-targeted AAVP. However, cell confluence had no effect on AAVP-guided gene expression. Our findings prove that combination of histone deacetylase inhibitor drugs with the Grp78 promoter is an effective approach to improve AAVP-mediated gene expression in cancer cells and should be considered for AAVP-based clinical cancer gene therapy.

  2. Ultrasound and microbubble-targeted delivery of therapeutic compounds : ICIN Report Project 49: Drug and gene delivery through ultrasound and microbubbles

    NARCIS (Netherlands)

    Juffermans, L J M; Meijering, D B M; van Wamel, A; Henning, R H; Kooiman, K; Emmer, M; de Jong, N; van Gilst, W H; Musters, R; Paulus, W J; van Rossum, A C; Deelman, L E; Kamp, O

    The molecular understanding of diseases has been accelerated in recent years, producing many new potential therapeutic targets. A noninvasive delivery system that can target specific anatomical sites would be a great boost for many therapies, particularly those based on manipulation of gene

  3. Optimizing hyaluronidase dose and plasmid DNA delivery greatly improves gene electrotransfer efficiency in rat skeletal muscle

    DEFF Research Database (Denmark)

    Åkerström, Thorbjörn; Vedel, Kenneth; Needham Andersen, Josefine

    2015-01-01

    Transfection of rat skeletal muscle in vivo is a widely used research model. However, gene electrotransfer protocols have been developed for mice and yield variable results in rats. We investigated whether changes in hyaluronidase pre-treatment and plasmid DNA delivery can improve transfection...... efficiency in rat skeletal muscle. We found that pre-treating the muscle with a hyaluronidase dose suitable for rats (0.56. U/g b.w.) prior to plasmid DNA injection increased transfection efficiency by >200% whereas timing of the pre-treatment did not affect efficiency. Uniformly distributing plasmid DNA...... with a homogenous distribution. We also show that transfection was stable over five weeks of regular exercise or inactivity. Our findings show that species-specific plasmid DNA delivery and hyaluronidase pre-treatment greatly improves transfection efficiency in rat skeletal muscle....

  4. Cell-type-specific gene delivery into neuronal cells in vitro and in vivo

    International Nuclear Information System (INIS)

    Parveen, Zahida; Mukhtar, Muhammad; Rafi, Mohammed; Wenger, David A.; Siddiqui, Khwaja M.; Siler, Catherine A.; Dietzschold, Bernhard; Pomerantz, Roger J.; Schnell, Matthias J.; Dornburg, Ralph

    2003-01-01

    The avian retroviruses reticuloendotheliosis virus strain A (REV-A) and spleen necrosis virus (SNV) are not naturally infectious in human cells. However, REV-A-derived viral vectors efficiently infect human cells when they are pseudotyped with envelope proteins displaying targeting ligands specific for human cell-surface receptors. Here we report that vectors containing the gag region of REV-A and pol of SNV can be pseudotyped with the envelope protein of vesicular stomatitis virus (VSV) and the glycoproteins of different rabies virus (RV) strains. Vectors pseudotyped with the envelope protein of the highly neurotropic RV strain CVS-N2c facilitated cell type-specific gene delivery into mouse and human neurons, but did not infect other human cell types. Moreover, when such vector particles were injected into the brain of newborn mice, only neuronal cells were infected in vivo. Cell-type-specific gene delivery into neurons may present quite specific gene therapy approaches for many degenerative diseases of the brain

  5. Development of Non-Viral, Trophoblast-Specific Gene Delivery for Placental Therapy.

    Directory of Open Access Journals (Sweden)

    Noura Abd Ellah

    Full Text Available Low birth weight is associated with both short term problems and the fetal programming of adult onset diseases, including an increased risk of obesity, diabetes and cardiovascular disease. Placental insufficiency leading to intrauterine growth restriction (IUGR contributes to the prevalence of diseases with developmental origins. Currently there are no therapies for IUGR or placental insufficiency. To address this and move towards development of an in utero therapy, we employ a nanostructure delivery system complexed with the IGF-1 gene to treat the placenta. IGF-1 is a growth factor critical to achieving appropriate placental and fetal growth. Delivery of genes to a model of human trophoblast and mouse placenta was achieved using a diblock copolymer (pHPMA-b-pDMAEMA complexed to hIGF-1 plasmid DNA under the control of trophoblast-specific promoters (Cyp19a or PLAC1. Transfection efficiency of pEGFP-C1-containing nanocarriers in BeWo cells and non-trophoblast cells was visually assessed via fluorescence microscopy. In vivo transfection and functionality was assessed by direct placental-injection into a mouse model of IUGR. Complexes formed using pHPMA-b-pDMAEMA and CYP19a-923 or PLAC1-modified plasmids induce trophoblast-selective transgene expression in vitro, and placental injection of PLAC1-hIGF-1 produces measurable RNA expression and alleviates IUGR in our mouse model, consequently representing innovative building blocks towards human placental gene therapies.

  6. Helios(®) Gene Gun-Mediated Transfection of the Inner Ear Sensory Epithelium: Recent Updates.

    Science.gov (United States)

    Belyantseva, Inna A

    2016-01-01

    The transfection of vertebrate inner ear hair cells has proven to be challenging. Therefore, many laboratories attempt to use and improve different transfection methods. Each method has its own advantages and disadvantages. A particular researcher's skills in addition to available equipment and the type of experiment (in vivo or in vitro) likely determine the transfection method of choice. Biolistic delivery of exogenous DNA, mRNA, or siRNA, also known as Helios(®) Gene Gun-mediated transfection, uses the mechanical energy of compressed helium gas to bombard tissue with micron- or submicron-sized DNA or RNA-coated gold particles, which can penetrate and transfect cells in vitro or in vivo. Helios(®) Gene Gun-mediated transfection has several advantages: (1) it is simple enough to learn in a short time; (2) it is designed to overcome cell barriers even as tough as plant cell membrane or stratum corneum in the epidermis; (3) it can transfect cells deep inside a tissue such as specific neurons within a brain slice; (4) it can accommodate mRNA, siRNA, or DNA practically of any size to be delivered; and (5) it works well with various cell types including non-dividing, terminally differentiated cells that are difficult to transfect, such as neurons or mammalian inner ear sensory hair cells. The latter advantage is particularly important for inner ear research. The disadvantages of this method are: (1) low efficiency of transfection due to many variables that have to be adjusted and (2) potential mechanical damage of the tissue if the biolistic shot parameters are not optimal. This chapter provides a step-by-step protocol and critical evaluation of the Bio-Rad Helios(®) Gene Gun transfection method used to deliver green fluorescent protein (GFP)-tagged full-length cDNAs of myosin 15a, whirlin, β-actin, and Clic5 into rodent hair cells of the postnatal inner ear sensory epithelia in culture.

  7. Agrobacterium-Mediated Transformation of Tomato Elicits Unexpected Flower Phenotypes with Similar Gene Expression Profiles

    OpenAIRE

    Wang, Yi-Hong; Campbell, Michael A.

    2008-01-01

    BACKGROUND: Genetic transformation mediated by Agrobacterium tumefaciens is known to cause unexpected phenotypes. Mutations of a specific set of homeotic genes can result in altered floral structure. METHODOLOGY/PRINCIPAL FINDINGS: Previously we identified two genes (LeTGA1 and SOLly GLB1) induced by nutrient availability in tomato. To further elucidate their function, we sought to knock out the genes using antisense RNAi. When antisense constructs for the two different tomato genes were each...

  8. Non-viral gene delivery strategies for gene therapy: a “ménage à trois” among nucleic acids, materials, and the biological environment

    International Nuclear Information System (INIS)

    Pezzoli, Daniele; Candiani, Gabriele

    2013-01-01

    Gene delivery is the science of transferring genetic material into cells by means of a vector to alter cellular function or structure at a molecular level. In this context, a number of nucleic acid-based drugs have been proposed and experimented so far and, as they act on distinct steps along the gene transcription–translation pathway, specific delivery strategies are required to elicit the desired outcome. Cationic lipids and polymers, collectively known as non-viral delivery systems, have thus made their breakthrough in basic and medical research. Albeit they are promising alternatives to viral vectors, their therapeutic application is still rather limited as high transfection efficiencies are normally associated to adverse cytotoxic side effects. In this scenario, drawing inspiration from processes naturally occurring in vivo, major strides forward have been made in the development of more effective materials for gene delivery applications. Specifically, smart vectors sensitive to a variety of physiological stimuli such as cell enzymes, redox status, and pH are substantially changing the landscape of gene delivery by helping to overcome some of the systemic and intracellular barriers that viral vectors naturally evade. Herein, after summarizing the state-of-the-art information regarding the use of nucleic acids as drugs, we review the main bottlenecks still limiting the overall effectiveness of non-viral gene delivery systems. Finally, we provide a critical outline of emerging stimuli-responsive strategies and discuss challenges still existing on the road toward conceiving more efficient and safer multifunctional vectors.

  9. Role of the Na(+)/K(+)-ATPase beta-subunit in peptide-mediated transdermal drug delivery.

    Science.gov (United States)

    Wang, Changli; Ruan, Renquan; Zhang, Li; Zhang, Yunjiao; Zhou, Wei; Lin, Jun; Ding, Weiping; Wen, Longping

    2015-04-06

    In this work, we discovered that the Na(+)/K(+)-ATPase beta-subunit (ATP1B1) on epidermal cells plays a key role in the peptide-mediated transdermal delivery of macromolecular drugs. First, using a yeast two-hybrid assay, we screened candidate proteins that have specific affinity for the short peptide TD1 (ACSSSPSKHCG) identified in our previous work. Then, we verified the specific binding of TD1 to ATP1B1 in yeast and mammalian cells by a pull-down ELISA and an immunoprecipitation assay. Finally, we confirmed that TD1 mainly interacted with the C-terminus of ATP1B1. Our results showed that the interaction between TD1 and ATP1B1 affected not only the expression and localization of ATP1B1, but also the epidermal structure. In addition, this interaction could be antagonized by the exogenous competitor ATP1B1 or be inhibited by ouabain, which results in the decreased delivery of macromolecular drugs across the skin. The discovery of a critical role of ATP1B1 in the peptide-mediated transdermal drug delivery is of great significance for the future development of new transdermal peptide enhancers.

  10. The Yersinia pestis Ail protein mediates binding and Yop delivery to host cells required for plague virulence.

    Science.gov (United States)

    Felek, Suleyman; Krukonis, Eric S

    2009-02-01

    Although adhesion to host cells is a critical step in the delivery of cytotoxic Yop proteins by Yersinia pestis, the mechanism has not been defined. To identify adhesins critical for Yop delivery, we initiated two transposon mutagenesis screens using the mariner transposon. To avoid redundant cell binding activities, we initiated the screen with a strain deleted for two known adhesins, pH 6 antigen and the autotransporter, YapC, as well as the Caf1 capsule, which is known to obscure some adhesins. The mutants that emerged contained insertions within the ail (attachment and invasion locus) gene of Y. pestis. A reconstructed mutant with a single deletion in the ail locus (y1324) was severely defective for delivery of Yops to HEp-2 human epithelial cells and significantly defective for delivery of Yops to THP-1 human monocytes. Specifically, the Yop delivery defect was apparent when cell rounding and translocation of an ELK-tagged YopE derivative into host cells were monitored. Although the ail mutant showed only a modest decrease in cell binding capacity in vitro, the KIM5 Deltaail mutant exhibited a >3,000-fold-increased 50% lethal dose in mice. Mice infected with the Deltaail mutant also had 1,000-fold fewer bacteria in their spleens, livers, and lungs 3 days after infection than did those infected with the parental strain, KIM5. Thus, the Ail protein is critical for both Y. pestis type III secretion in vitro and infection in mice.

  11. AAV-mediated targeting of gene expression to the peri-infarct region in rat cortical stroke model.

    Science.gov (United States)

    Mätlik, Kert; Abo-Ramadan, Usama; Harvey, Brandon K; Arumäe, Urmas; Airavaara, Mikko

    2014-10-30

    For stroke patients the recovery of cognitive and behavioral functions is often incomplete. Functional recovery is thought to be mediated largely by connectivity rearrangements in the peri-infarct region. A method for manipulating gene expression in this region would be useful for identifying new recovery-enhancing treatments. We have characterized a way of targeting adeno-associated virus (AAV) vectors to the peri-infarct region of cortical ischemic lesion in rats 2days after middle cerebral artery occlusion (MCAo). We used magnetic resonance imaging (MRI) to show that the altered properties of post-ischemic brain tissue facilitate the spreading of intrastriatally injected nanoparticles toward the infarct. We show that subcortical injection of green fluorescent protein-encoding dsAAV7-GFP resulted in transduction of cells in and around the white matter tract underlying the lesion, and in the cortex proximal to the lesion. A similar result was achieved with dsAAV7 vector encoding the cerebral dopamine neurotrophic factor (CDNF), a protein with therapeutic potential. Viral vector-mediated intracerebral gene delivery has been used before in rodent models of ischemic injury. However, the method of targeting gene expression to the peri-infarct region, after the initial phase of ischemic cell death, has not been described before. We demonstrate a straightforward and robust way to target AAV vector-mediated over-expression of genes to the peri-infarct region in a rat stroke model. This method will be useful for studying the action of specific proteins in peri-infarct region during the recovery process. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Adeno-associated viral vectors as agents for gene delivery : application in disorders and trauma of the central nervous system

    NARCIS (Netherlands)

    Ruitenberg, Marc J; Eggers, Ruben; Boer, Gerard J; Verhaagen, J.

    2002-01-01

    The use of viral vectors as agents for gene delivery provides a direct approach to manipulate gene expression in the mammalian central nervous system (CNS). The present article describes in detail the methodology for the injection of viral vectors, in particular adeno-associated virus (AAV) vectors,

  13. Theranostic nanoparticles based on bioreducible polyethylenimine-coated iron oxide for reduction-responsive gene delivery and magnetic resonance imaging

    Directory of Open Access Journals (Sweden)

    Li D

    2014-07-01

    Full Text Available Dan Li,1,* Xin Tang,2,* Benjamin Pulli,1 Chao Lin,2 Peng Zhao,2 Jian Cheng,2 Zhongwei Lv,1 Xueyu Yuan,1 Qiong Luo,1 Haidong Cai,1 Meng Ye1 1Department of Nuclear Medicine, Shanghai 10th People’s Hospital, 2Shanghai East Hospital, The Institute for Biomedical Engineering and Nanoscience, Tongji University School of Medicine, Tongji University, People’s Republic of China *These authors contributed equally to this work Abstract: Theranostic nanoparticles based on superparamagnetic iron oxide (SPIO have a great promise for tumor diagnosis and gene therapy. However, the availability of theranostic nanoparticles with efficient gene transfection and minimal toxicity remains a big challenge. In this study, we construct an intelligent SPIO-based nanoparticle comprising a SPIO inner core and a disulfide-containing polyethylenimine (SSPEI outer layer, which is referred to as a SSPEI-SPIO nanoparticle, for redox-triggered gene release in response to an intracellular reducing environment. We reveal that SSPEI-SPIO nanoparticles are capable of binding genes to form nano-complexes and mediating a facilitated gene release in the presence of dithiothreitol (5–20 mM, thereby leading to high transfection efficiency against different cancer cells. The SSPEI-SPIO nanoparticles are also able to deliver small interfering RNA (siRNA for the silencing of human telomerase reverse transcriptase genes in HepG2 cells, causing their apoptosis and growth inhibition. Further, the nanoparticles are applicable as T2-negative contrast agents for magnetic resonance (MR imaging of a tumor xenografted in a nude mouse. Importantly, SSPEI-SPIO nanoparticles have relatively low cytotoxicity in vitro at a high concentration of 100 µg/mL. The results of this study demonstrate the utility of a disulfide-containing cationic polymer-decorated SPIO nanoparticle as highly potent and low-toxic theranostic nano-system for specific nucleic acid delivery inside cancer cells. Keywords

  14. Design and development of hyaluronan-functionalized polybenzofulvene nanoparticles as CD44 receptor mediated drug delivery system

    Science.gov (United States)

    Licciardi, Mariano; Scialabba, Cinzia; Giammona, Gaetano; Paolino, Marco; Razzano, Vincenzo; Grisci, Giorgio; Giuliani, Germano; Makovec, Francesco; Cappelli, Andrea

    2017-06-01

    A tri-component polymer brush (TCPB ), composed of a polybenzofulvene copolymer bearing low molecular weight hyaluronic acid (HA) on the surface of its cylindrical brush-like backbone and oligo-PEG fractions, was employed in the preparation of 350 nm nanostructured drug delivery systems capable of delivering the anticancer drug doxorubicin. The obtained drug delivery systems were characterized on the basis of drug loading and release, dimensions and zeta potential, morphology and in vitro cell activity, and uptake on three different human cell lines, namely the bronchial epithelial 16HBE, the breast adenocarcinoma MCF-7, and the colon cancer HCT116 cells. Finally, the ability of doxorubicin-loaded TCPB nanoparticles (DOXO-TCPB) to be internalized into cancer cells by CD44 receptor mediated uptake was assessed by means of uptake studies in HCT cells. These data were supported by anti-CD44-FITC staining assay. The proposed TCPB nanostructured drug delivery systems have many potential applications in nanomedicine, including cancer targeted drug delivery.

  15. Homologous gene targeting of a carotenoids biosynthetic gene in Rhodosporidium toruloides by Agrobacterium-mediated transformation.

    Science.gov (United States)

    Sun, Wenyi; Yang, Xiaobing; Wang, Xueying; Lin, Xinping; Wang, Yanan; Zhang, Sufang; Luan, Yushi; Zhao, Zongbao K

    2017-07-01

    To target a carotenoid biosynthetic gene in the oleaginous yeast Rhodosporidium toruloides by using the Agrobacterium-mediated transformation (AMT) method. The RHTO_04602 locus of R. toruloides NP11, previously assigned to code the carotenoid biosynthetic gene CRTI, was amplified from genomic DNA and cloned into the binary plasmid pZPK-mcs, resulting in pZPK-CRT. A HYG-expression cassette was inserted into the CRTI sequence of pZPK-CRT by utilizing the restriction-free clone strategy. The resulted plasmid was used to transform R. toruloides cells according to the AMT method, leading to a few white transformants. Sequencing analysis of those transformants confirmed homologous recombination and insertional inactivation of CRTI. When the white variants were transformed with a CRTI-expression cassette, cells became red and produced carotenoids as did the wild-type strain NP11. Successful homologous targeting of the CrtI locus confirmed the function of RHTO_04602 in carotenoids biosynthesis in R. toruloides. It provided valuable information for metabolic engineering of this non-model yeast species.

  16. Amphiphilic block co-polymers: preparation and application in nanodrug and gene delivery.

    Science.gov (United States)

    Xiong, Xiao-Bing; Binkhathlan, Ziyad; Molavi, Ommoleila; Lavasanifar, Afsaneh

    2012-07-01

    Self-assembly of amphiphilic block co-polymers composed of poly(ethylene oxide) (PEO) as the hydrophilic block and poly(ether)s, poly(amino acid)s, poly(ester)s and polypropyleneoxide (PPO) as the hydrophobic block can lead to the formation of nanoscopic structures of different morphologies. These structures have been the subject of extensive research in the past decade as artificial mimics of lipoproteins and viral vectors for drug and gene delivery. The aim of this review is to provide an overview of the synthesis of commonly used amphiphilic block co-polymers. It will also briefly go over some pharmaceutical applications of amphiphilic block co-polymers as "nanodelivery systems" for small molecules and gene therapeutics. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  17. Integration of Drug, Protein, and Gene Delivery Systems with Regenerative Medicine

    Science.gov (United States)

    Lorden, Elizabeth R.; Levinson, Howard M.; Leong, Kam W.

    2013-01-01

    Regenerative medicine has the potential to drastically change the field of health care from reactive to preventative and restorative. Exciting advances in stem cell biology and cellular reprogramming have fueled the progress of this field. Biochemical cues in the form of small molecule drugs, growth factors, zinc finger protein transcription factors and nucleases, transcription activator-like effector nucleases, monoclonal antibodies, plasmid DNA, aptamers, or RNA interference agents can play an important role to influence stem cell differentiation and the outcome of tissue regeneration. Many of these biochemical factors are fragile and must act intracellularly at the molecular level. They require an effective delivery system, which can take the form of a scaffold (e.g. hydrogels and electrospun fibers), carrier (viral and nonviral), nano- and micro-particle, or genetically modified cell. In this review, we will discuss the history and current technologies of drug, protein and gene delivery in the context of regenerative medicine. Next we will present case examples of how delivery technologies are being applied to promote angiogenesis in non-healing wounds or prevent angiogenesis in age related macular degeneration. Finally, we will conclude with a brief discussion of the regulatory pathway from bench-to-bedside for the clinical translation of these novel therapeutics. PMID:25787742

  18. Ovarian Cancer Therapy by VSVMP Gene Mediated by a Paclitaxel-Enhanced Nanoparticle.

    Science.gov (United States)

    Long, Jianlin; Yang, Yuping; Kang, Tianyi; Zhao, Wei; Cheng, Hao; Wu, Yujiao; Du, Ting; Liu, Beibei; Li, Yang; Luo, Feng; Gou, Maling

    2017-11-15

    Nanoparticles have great promise for gene delivery. However, the transfection efficiency of nanoparticle-based gene delivery systems is always unsatisfied to meet the requirement of effective gene therapy. Herein, we used low-dosage paclitaxel to enhance a nanoscaled gene delivery system that was self-assembled from N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoniummethyl sulfate and monomethoxy poly(ethylene glycol)-poly(d,l-lactide) (DPP), creating a paclitaxel-encapsulated DPP (P-DPP) nanoparticle. The encapsulated low-dosage paclitaxel significantly improved the gene delivery efficiency of the DPP nanoparticles against multiple cancer cells, in some of which the transfection efficiency is as high as 92%. By the P-DPP nanoparticle, vesicular stomatitis virus matrix protein (VSVMP) that could induce cell apoptosis was delivered to treat ovarian cancer. The encapsulation of paclitaxel in DPP nanoparticles increased the expression of VSVMP, enhancing VSVMP to induce antiproliferation and apoptosis in SKOV3 ovarian cancer cells. Intraperitoneal administration of P-DPP-delivered VSVMP effectively inhibited the intraperitoneal metastasis of SKOV3 ovarian cancer, which was more efficient than DPP-delivered VSVMP. Moreover, it was found that the tumor cell apoptosis induction, tumor cell proliferation inhibition, and tumor angiogenesis suppression were involved in the anticancer mechanism of this nanocomplex. Our data suggest that the encapsulation of low-dosage paclitaxel can enhance the gene delivery efficiency of the DPP nanoparticles against multiple cancer cells and exert a synergistic anticancer effect with VSVMP gene in ovarian cancer treatment. The VSVMP gene therapy delivered by the paclitaxel-enhanced nanoparticle has potential application in ovarian cancer therapy.

  19. Counterselection and co-delivery of transposon and transposase functions for Sleeping Beauty-mediated transposition in cultured mammalian cells.

    Science.gov (United States)

    Converse, Andrea D; Belur, Lalitha R; Gori, Jennifer L; Liu, Geyi; Amaya, Felipe; Aguilar-Cordova, Estuardo; Hackett, Perry B; McIvor, R Scott

    2004-12-01

    Sleeping Beauty (SB) is a gene-insertion system reconstructed from transposon sequences found in teleost fish and is capable of mediating the transposition of DNA sequences from transfected plasmids into the chromosomes of vertebrate cell populations. The SB system consists of a transposon, made up of a gene of interest flanked by transposon inverted repeats, and a source of transposase. Here we carried out a series of studies to further characterize SB-mediated transposition as a tool for gene transfer to chromosomes and ultimately for human gene therapy. Transfection of mouse 3T3 cells, HeLa cells, and human A549 lung carcinoma cells with a transposon containing the neomycin phosphotransferase (NEO) gene resulted in a several-fold increase in drug-resistant colony formation when co-transfected with a plasmid expressing the SB transposase. A transposon containing a methotrexate-resistant dihydrofolate reductase gene was also found to confer an increased frequency of methotrexate-resistant colony formation when co-transfected with SB transposase-encoding plasmid. A plasmid containing a herpes simplex virus thymidine kinase gene as well as a transposon containing a NEO gene was used for counterselection against random recombinants (NEO+TK+) in medium containing G418 plus ganciclovir. Effective counterselection required a recovery period of 5 days after transfection before shifting into medium containing ganciclovir to allow time for transiently expressed thymidine kinase activity to subside in cells not stably transfected. Southern analysis of clonal isolates indicated a shift from random recombination events toward transposition events when clones were isolated in medium containing ganciclovir as well as G418. We found that including both transposon and transposase functions on the same plasmid substantially increased the stable gene transfer frequency in Huh7 human hepatoma cells. The results from these experiments contribute technical and conceptual insight into

  20. The Role of Particle-Mediated DNA Vaccines in Biodefense Preparedness

    National Research Council Canada - National Science Library

    Dean, Hansi J; Haynes, Joel; Schmaljohn, Connie

    2005-01-01

    Particle-mediated epidermal delivery (PMED) of DNA vaccines is based on the acceleration of DNA-coated gold directly into the cytoplasm and nuclei of living cells of the epidermis, facilitating DNA delivery and gene expression...

  1. Fourth Generation Phosphorus-Containing Dendrimers: Prospective Drug and Gene Delivery Carrier

    Science.gov (United States)

    Shcharbin, D.; Dzmitruk, V.; Shakhbazau, A.; Goncharova, N.; Seviaryn, I.; Kosmacheva, S.; Potapnev, M.; Pedziwiatr-Werbicka, E.; Bryszewska, M.; Talabaev, M.; Chernov, A.; Kulchitsky, V.; Caminade, A.-M.; Majoral, J.-P.

    2011-01-01

    Research concerning new targeting delivery systems for pharmacologically active molecules and genetic material is of great importance. The aim of the present study was to investigate the potential of fourth generation (P4) cationic phosphorus-containing dendrimers to bind fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS), anti-neoplastic drug cisplatin, anti-HIV siRNA siP24 and its capability to deliver green fluorescent protein gene (pGFP) into cells. The interaction between P4 and ANS (as the model drug) was investigated. The binding constant and the number of binding centers per one molecule of P4 were determined. In addition, the dendriplex between P4 and anti-HIV siRNA siP24 was characterized using circular dichroism, fluorescence polarization and zeta-potential methods; the average hydrodynamic diameter of the dendriplex was calculated using zeta-size measurements. The efficiency of transfection of pGFP using P4 was determined in HEK293 cells and human mesenchymal stem cells, and the cytotoxicity of the P4-pGFP dendriplex was studied. Furthermore, enhancement of the toxic action of the anti-neoplastic drug cisplatin by P4 dendrimers was estimated. Based on the results, the fourth generation cationic phosphorus-containing dendrimers seem to be a good drug and gene delivery carrier candidate. PMID:24310590

  2. Mapping the AAV capsid host antibody response towards the development of second generation gene delivery vectors

    Directory of Open Access Journals (Sweden)

    Yu-Shan eTseng

    2014-01-01

    Full Text Available The recombinant Adeno-associated virus (rAAV gene delivery system is entering a crucial and exciting phase with the promise of more than 20 years of intense research now realized in a number of successful human clinical trials. However, as a natural host to AAV infection, anti-AAV antibodies are prevalent in the human population. For example, ~70% of human sera samples are positive for AAV serotype 2 (AAV2. Furthermore, low levels of pre-existing neutralizing antibodies in the circulation are detrimental to the efficacy of corrective therapeutic AAV gene delivery. A key component to overcoming this obstacle is the identification of regions of the AAV capsid that participate in interactions with host immunity, especially neutralizing antibodies, to be modified for neutralization escape. Three main approaches have been utilized to map antigenic epitopes on AAV capsids. The first is directed evolution in which AAV variants are selected in the presence of monoclonal antibodies or pooled human sera. This results in AAV variants with mutations on important neutralizing epitopes. The second is epitope searching, achieved by peptide scanning, peptide insertion or site-directed mutagenesis. The third, a structure biology-based approach, utilizes cryo-electron microscopy and image reconstruction of AAV capsids complexed to fragment antibodies, which are generated from monoclonal antibodies, to directly visualize the epitopes. In this review, the contribution of these three approaches to the current knowledge of AAV epitopes and success in their use to create second generation vectors will be discussed.

  3. High content analysis platform for optimization of lipid mediated CRISPR-Cas9 delivery strategies in human cells.

    Science.gov (United States)

    Steyer, Benjamin; Carlson-Stevermer, Jared; Angenent-Mari, Nicolas; Khalil, Andrew; Harkness, Ty; Saha, Krishanu

    2016-04-01

    Non-viral gene-editing of human cells using the CRISPR-Cas9 system requires optimized delivery of multiple components. Both the Cas9 endonuclease and a single guide RNA, that defines the genomic target, need to be present and co-localized within the nucleus for efficient gene-editing to occur. This work describes a new high-throughput screening platform for the optimization of CRISPR-Cas9 delivery strategies. By exploiting high content image analysis and microcontact printed plates, multi-parametric gene-editing outcome data from hundreds to thousands of isolated cell populations can be screened simultaneously. Employing this platform, we systematically screened four commercially available cationic lipid transfection materials with a range of RNAs encoding the CRISPR-Cas9 system. Analysis of Cas9 expression and editing of a fluorescent mCherry reporter transgene within human embryonic kidney cells was monitored over several days after transfection. Design of experiments analysis enabled rigorous evaluation of delivery materials and RNA concentration conditions. The results of this analysis indicated that the concentration and identity of transfection material have significantly greater effect on gene-editing than ratio or total amount of RNA. Cell subpopulation analysis on microcontact printed plates, further revealed that low cell number and high Cas9 expression, 24h after CRISPR-Cas9 delivery, were strong predictors of gene-editing outcomes. These results suggest design principles for the development of materials and transfection strategies with lipid-based materials. This platform could be applied to rapidly optimize materials for gene-editing in a variety of cell/tissue types in order to advance genomic medicine, regenerative biology and drug discovery. CRISPR-Cas9 is a new gene-editing technology for "genome surgery" that is anticipated to treat genetic diseases. This technology uses multiple components of the Cas9 system to cut out disease-causing mutations

  4. Genomic signature and toxicogenomics comparison of polycationic gene delivery nanosystems in human alveolar epithelial A549 cells

    Directory of Open Access Journals (Sweden)

    J Barar

    2009-10-01

    Full Text Available "nBackground and the purpose of the study: Of the gene delivery systems, non-viral polycationic gene delivery nanosystems have been alternatively exploited as a relatively safe delivery reagents compared to viral vectors. However, little is known about the genomic impacts of these delivery systems in target cells/tissues. In this study, the toxicogenomics and genotoxicity potential of some selected polycationic lipid/polymer based nanostructures (i.e., Oligofectamine® (OF, starburst polyamidoamine Polyfect® (PF and diaminobutane (DAB dendrimers were investigated in human alveolar epithelial A549 cells. "nMethods: To study the nature and the ontology of the gene expression changes in A549 cells upon treatment with polycationic nanostructures, MTT assay and microarray gene expression profiling methodology were employed. For microarray analysis, cyanine (Cy3/Cy5 labeled cDNA samples from treated and untreated cells were hybridized on target arrays housing 200 genes. "nResults and major conclusions: The polycationic nanosystems induced significant gene expression changes belonging to different genomic ontologies such as cell defence and apoptosis pathways. These data suggest that polycationic nanosystems can elicit multiple gene expression changes in A549 cells upon their chemical structures and interactions with cellular/subcellular components. Such impacts may interfere with the main goals of the desired genemedicine.

  5. Single Tyrosine Mutation in AAV8 Vector Capsid Enhances Gene Lung Delivery and Does Not Alter Lung Morphofunction in Mice

    Directory of Open Access Journals (Sweden)

    Sabrina V. Martini

    2014-08-01

    Full Text Available Background/Aims: Vectors derived from adeno-associated viruses (AAVs are important gene delivery tools for treating pulmonary diseases. Phosphorylation of surface-exposed tyrosine residues from AAV2 capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. We evaluated the pulmonary transduction efficiency of AAV8 vectors containing point mutations in surface-exposed capsid tyrosine residues. Methods: Male C57BL/6 mice (20-25 g, n=24 were randomly assigned into three groups: control group animals received intratracheal (i.t. instillation of saline (50 μl, wild-type AAV8 group, and capsid mutant Y733F AAV8 group, which received (i.t. AAV8 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP. Four weeks after instillation, lung mechanics and morphometry, vector transduction (immunohistochemistry and mRNA expression of eGFP, and inflammatory cytokines and growth factor expression were analyzed. Results: Tyrosine-mutant AAV8 vectors displayed significantly increased transduction efficiency in the lung compared with their wild-type counterparts. No significant differences were observed in lung mechanics and morphometry between experimental groups. There was no evidence of inflammatory response in any group. Conclusion: AAV8 vectors may be useful for new therapeutic strategies for the treatment of pulmonary diseases.

  6. Gene delivery: A single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus

    OpenAIRE

    Zanta, Maria Antonietta; Belguise-Valladier, Pascale; Behr, Jean-Paul

    1999-01-01

    Translocation of exogenous DNA through the nuclear membrane is a major concern of gene delivery technologies. To take advantage of the cellular import machinery, we have synthesized a capped 3.3-kbp CMVLuciferase-NLS gene containing a single nuclear localization signal peptide (PKKKRKVEDPYC). Transfection of cells with the tagged gene remained effective down to nanogram amounts of DNA. Transfection enhancement (10- to 1,000-fold) as a result of the signal peptide was observed irrespective of ...

  7. A Review of Gene Delivery and Stem Cell Based Therapies for Regenerating Inner Ear Hair Cells

    Directory of Open Access Journals (Sweden)

    Michael S. Detamore

    2011-09-01

    Full Text Available Sensory neural hearing loss and vestibular dysfunction have become the most common forms of sensory defects, affecting millions of people worldwide. Developing effective therapies to restore hearing loss is challenging, owing to the limited regenerative capacity of the inner ear hair cells. With recent advances in understanding the developmental biology of mammalian and non-mammalian hair cells a variety of strategies have emerged to restore lost hair cells are being developed. Two predominant strategies have developed to restore hair cells: transfer of genes responsible for hair cell genesis and replacement of missing cells via transfer of stem cells. In this review article, we evaluate the use of several genes involved in hair cell regeneration, the advantages and disadvantages of the different viral vectors employed in inner ear gene delivery and the insights gained from the use of embryonic, adult and induced pluripotent stem cells in generating inner ear hair cells. Understanding the role of genes, vectors and stem cells in therapeutic strategies led us to explore potential solutions to overcome the limitations associated with their use in hair cell regeneration.

  8. Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation.

    Science.gov (United States)

    Shcherbak, N; Kishchenko, O; Sakhno, L; Komarnytsky, I; Kuchuk, M

    2013-01-01

    Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants.

  9. RNA-mediated gene silencing of superoxide dismutase (bcsod1) in Botrytis cinerea

    NARCIS (Netherlands)

    Patel, R.M.; Kan, van J.A.L.; Bailey, A.M.; Foster, G.D.

    2008-01-01

    Gene silencing is a powerful tool utilized for identification of gene function and analysis in plants, animals, and fungi. Here, we report the silencing of superoxide dismutase (bcsod1) in Botrytis cinerea through sense and antisense-mediated silencing mechanisms. Because superoxide dismutase (SOD)

  10. DNA-Mediated Gene Therapy in a Mouse Model of Limb Girdle Muscular Dystrophy 2B

    Directory of Open Access Journals (Sweden)

    Julia Ma

    2017-12-01

    Full Text Available Mutations in the gene for dysferlin cause a degenerative disorder of skeletal muscle known as limb girdle muscular dystrophy 2B. To achieve gene delivery of plasmids encoding dysferlin to hind limb muscles of dysferlin knockout mice, we used a vascular injection method that perfused naked plasmid DNA into all major muscle groups of the hind limb. We monitored delivery by luciferase live imaging and western blot, confirming strong dysferlin expression that persisted over the 3-month time course of the experiment. Co-delivery of the follistatin gene, which may promote muscle growth, was monitored by ELISA. Immunohistochemistry documented the presence of dysferlin in muscle fibers in treated limbs, and PCR confirmed the presence of plasmid DNA. Because dysferlin is involved in repair of the sarcolemmal membrane, dysferlin loss leads to fragile sarcolemmal membranes that can be detected by permeability to Evan’s blue dye. We showed that after gene therapy with a plasmid encoding both dysferlin and follistatin, statistically significant reduction in Evan’s blue dye permeability was present in hamstring muscles. These results suggest that vascular delivery of plasmids carrying these therapeutic genes may lead to simple and effective approaches for improving the clinical condition of limb girdle muscular dystrophy 2B.

  11. Negative regulation of RIG-I-mediated antiviral signaling by TRK-fused gene (TFG) protein

    International Nuclear Information System (INIS)

    Lee, Na-Rae; Shin, Han-Bo; Kim, Hye-In; Choi, Myung-Soo; Inn, Kyung-Soo

    2013-01-01

    Highlights: •TRK-fused gene product (TFG) interacts with TRIM25 upon viral infection. •TFG negatively regulates RIG-I mediated antiviral signaling. •TFG depletion leads to enhanced viral replication. •TFG act downstream of MAVS. -- Abstract: RIG-I (retinoic acid inducible gene I)-mediated antiviral signaling serves as the first line of defense against viral infection. Upon detection of viral RNA, RIG-I undergoes TRIM25 (tripartite motif protein 25)-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that TRK-fused gene (TFG) protein, previously identified as a TRIM25-interacting protein, binds TRIM25 upon virus infection and negatively regulates RIG-I-mediated type-I IFN signaling. RIG-I-mediated IFN production and nuclear factor (NF)-κB signaling pathways were upregulated by the suppression of TFG expression. Furthermore, vesicular stomatitis virus (VSV) replication was significantly inhibited by small inhibitory hairpin RNA (shRNA)-mediated knockdown of TFG, supporting the suppressive role of TFG in RIG-I-mediated antiviral signaling. Interestingly, suppression of TFG expression increased not only RIG-I-mediated signaling but also MAVS (mitochondrial antiviral signaling protein)-induced signaling, suggesting that TFG plays a pivotal role in negative regulation of RNA-sensing, RIG-I-like receptor (RLR) family signaling pathways

  12. Negative regulation of RIG-I-mediated antiviral signaling by TRK-fused gene (TFG) protein

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Na-Rae; Shin, Han-Bo; Kim, Hye-In; Choi, Myung-Soo; Inn, Kyung-Soo, E-mail: innks@khu.ac.kr

    2013-07-19

    Highlights: •TRK-fused gene product (TFG) interacts with TRIM25 upon viral infection. •TFG negatively regulates RIG-I mediated antiviral signaling. •TFG depletion leads to enhanced viral replication. •TFG act downstream of MAVS. -- Abstract: RIG-I (retinoic acid inducible gene I)-mediated antiviral signaling serves as the first line of defense against viral infection. Upon detection of viral RNA, RIG-I undergoes TRIM25 (tripartite motif protein 25)-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that TRK-fused gene (TFG) protein, previously identified as a TRIM25-interacting protein, binds TRIM25 upon virus infection and negatively regulates RIG-I-mediated type-I IFN signaling. RIG-I-mediated IFN production and nuclear factor (NF)-κB signaling pathways were upregulated by the suppression of TFG expression. Furthermore, vesicular stomatitis virus (VSV) replication was significantly inhibited by small inhibitory hairpin RNA (shRNA)-mediated knockdown of TFG, supporting the suppressive role of TFG in RIG-I-mediated antiviral signaling. Interestingly, suppression of TFG expression increased not only RIG-I-mediated signaling but also MAVS (mitochondrial antiviral signaling protein)-induced signaling, suggesting that TFG plays a pivotal role in negative regulation of RNA-sensing, RIG-I-like receptor (RLR) family signaling pathways.

  13. Identification of certain cancer-mediating genes using Gaussian ...

    Indian Academy of Sciences (India)

    2015-09-29

    Sep 29, 2015 ... the notion of fuzzy set theory, for validating the clusters obtained by a clustering algorithm applied on cancer gene expression data. GFI is then used for the identification of genes that have altered quite significantly from normal state to carcinogenic state with respect to their mRNA expression patterns.

  14. Response to delivery stress is not mediated by beta-endorphin (1-31).

    Science.gov (United States)

    Harbach, Heinz; Antrecht, Kerstin; Boedeker, Rolf-Hasso; Hempelmann, Gunter; Markart, Philipp; Matejec, Reginald; Muehling, Joerg; Welters, Ingeborg; Zygmunt, Marek

    2008-01-01

    The aim of the study was to determine the reaction of the melanotroph and corticotroph-type pituitary proopiomelanocortin (POMC) response to vaginal delivery and caesarean section stress. Furthermore, the relationship between the release of pituitary POMC fragments, gonadotropins and sexual steroids were examined. Blood samples were obtained from 10 women in labour on arrival in the birth room (t(A)), at cervix dilatation of 5 cm (t(B)) and immediately after spontaneous delivery (t(C)) and in 16 patients undergoing elective caesarean section before induction of anaesthesia (t(B)) and immediately after delivery (t(C)). Samples were analysed for cortisol, ACTH, authentic beta-endorphin, beta-endorphin immunoreactive material (IRM), acetyl-N-beta-endorphin IRM (NAC), beta-lipotropin (beta-LPH) IRM, oestradiol (E(2)), progesterone (P), prolactin (PRL), FSH and LH. NAC representing the melanotroph-type pituitary POMC system did not increase during the course of caesarean section or spontaneous labour. In contrast, a significant increase of beta-endorphin IRM, beta-LPH IRM and ACTH were observed, representing an activation of the corticotroph-type POMC system. Highly significant correlations between POMC fragment concentrations during caesarean section and spontaneous labour were also observed. Sexual steroids (E(2) and P) decreased significantly. Except for beta-endorphin IRM and E(2) in course of spontaneous delivery no significant correlation was observed between POMC fragment and gonadotropins or sexual steroids. Caesarean section and spontaneous delivery activated the corticotroph but not the melanotroph POMC system.

  15. Efficient Oligo nucleotide mediated CRISPR-Cas9 Gene Editing in Aspergilli

    DEFF Research Database (Denmark)

    Nødvig, Christina Spuur; Hoof, Jakob Blæsbjerg; Kogle, Martin Engelhard

    2018-01-01

    CRISPR-Cas9 technologies are revolutionizing fungal gene editing. Here we show that survival of specific Cas9/sgRNA mediated DNA double strand breaks (DSBs) depends on the non-homologous end-joining, NHEJ, DNA repair pathway and we use this observation to develop a tool to assess protospacer....... niger, and in A. oryzae indicating that this type of repair may be wide spread in filamentous fungi. Importantly, we demonstrate that by using single-stranded oligo nucleotides for CRISPR-Cas9 mediated gene editing it is possible to introduce specific point mutations as well gene deletions...

  16. A Rabbit Model for Testing Helper-Dependent Adenovirus-Mediated Gene Therapy for Vein Graft Atherosclerosis

    Directory of Open Access Journals (Sweden)

    Lianxiang Bi

    2017-12-01

    Full Text Available Coronary artery bypass vein grafts are a mainstay of therapy for human atherosclerosis. Unfortunately, the long-term patency of vein grafts is limited by accelerated atherosclerosis. Gene therapy, directed at the vein graft wall, is a promising approach for preventing vein graft atherosclerosis. Because helper-dependent adenovirus (HDAd efficiently transduces grafted veins and confers long-term transgene expression, HDAd is an excellent candidate for delivery of vein graft-targeted gene therapy. We developed a model of vein graft atherosclerosis in fat-fed rabbits and demonstrated long-term (≥20 weeks persistence of HDAd genomes after graft transduction. This model enables quantitation of vein graft hemodynamics, wall structure, lipid accumulation, cellularity, vector persistence, and inflammatory markers on a single graft. Time-course experiments identified 12 weeks after transduction as an optimal time to measure efficacy of gene therapy on the critical variables of lipid and macrophage accumulation. We also used chow-fed rabbits to test whether HDAd infusion in vein grafts promotes intimal growth and inflammation. HDAd did not increase intimal growth, but had moderate—yet significant—pro-inflammatory effects. The vein graft atherosclerosis model will be useful for testing HDAd-mediated gene therapy; however, pro-inflammatory effects of HdAd remain a concern in developing HDAd as a therapy for vein graft disease.

  17. Norepinephrine transport-mediated gene expression in noradrenergic neurogenesis.

    Science.gov (United States)

    Hu, Yao Fei; Caron, Marc G; Sieber-Blum, Maya

    2009-04-08

    We have identified a differential gene expression profile in neural crest stem cells that is due to deletion of the norepinephrine transporter (NET) gene. NET is the target of psychotropic substances, such as tricyclic antidepressants and the drug of abuse, cocaine. NET mutations have been implicated in depression, anxiety, orthostatic intolerance and attention deficit hyperactivity disorder (ADHD). NET function in adult noradrenergic neurons of the peripheral and central nervous systems is to internalize norepinephrine from the synaptic cleft. By contrast, during embryogenesis norepinephrine (NE) transport promotes differentiation of neural crest stem cells and locus ceruleus progenitors into noradrenergic neurons, whereas NET inhibitors block noradrenergic differentiation. While the structure of NET und the regulation of NET function are well described, little is known about downstream target genes of norepinephrine (NE) transport. We have prepared gene expression profiles of in vitro differentiating wild type and norepinephrine transporter-deficient (NETKO) mouse neural crest cells using long serial analysis of gene expression (LongSAGE). Comparison analyses have identified a number of important differentially expressed genes, including genes relevant to neural crest formation, noradrenergic neuron differentiation and the phenotype of NETKO mice. Examples of differentially expressed genes that affect noradrenergic cell differentiation include genes in the bone morphogenetic protein (BMP) signaling pathway, the Phox2b binding partner Tlx2, the ubiquitin ligase Praja2, and the inhibitor of Notch signaling, Numbl. Differentially expressed genes that are likely to contribute to the NETKO phenotype include dopamine-beta-hydroxylase (Dbh), tyrosine hydroxylase (Th), the peptide transmitter 'cocaine and amphetamine regulated transcript' (Cart), and the serotonin receptor subunit Htr3a. Real-time PCR confirmed differential expression of key genes not only in neural

  18. Norepinephrine transport-mediated gene expression in noradrenergic neurogenesis

    Directory of Open Access Journals (Sweden)

    Sieber-Blum Maya

    2009-04-01

    Full Text Available Abstract Background We have identified a differential gene expression profile in neural crest stem cells that is due to deletion of the norepinephrine transporter (NET gene. NET is the target of psychotropic substances, such as tricyclic antidepressants and the drug of abuse, cocaine. NET mutations have been implicated in depression, anxiety, orthostatic intolerance and attention deficit hyperactivity disorder (ADHD. NET function in adult noradrenergic neurons of the peripheral and central nervous systems is to internalize norepinephrine from the synaptic cleft. By contrast, during embryogenesis norepinephrine (NE transport promotes differentiation of neural crest stem cells and locus ceruleus progenitors into noradrenergic neurons, whereas NET inhibitors block noradrenergic differentiation. While the structure of NET und the regulation of NET function are well described, little is known about downstream target genes of norepinephrine (NE transport. Results We have prepared gene expression profiles of in vitro differentiating wild type and norepinephrine transporter-deficient (NETKO mouse neural crest cells using long serial analysis of gene expression (LongSAGE. Comparison analyses have identified a number of important differentially expressed genes, including genes relevant to neural crest formation, noradrenergic neuron differentiation and the phenotype of NETKO mice. Examples of differentially expressed genes that affect noradrenergic cell differentiation include genes in the bone morphogenetic protein (BMP signaling pathway, the Phox2b binding partner Tlx2, the ubiquitin ligase Praja2, and the inhibitor of Notch signaling, Numbl. Differentially expressed genes that are likely to contribute to the NETKO phenotype include dopamine-β-hydroxylase (Dbh, tyrosine hydroxylase (Th, the peptide transmitter 'cocaine and amphetamine regulated transcript' (Cart, and the serotonin receptor subunit Htr3a. Real-time PCR confirmed differential expression

  19. Membrane fusion inducers, chloroquine and spermidine increase lipoplex-mediated gene transfection

    International Nuclear Information System (INIS)

    Wong-Baeza, Carlos; Bustos, Israel; Serna, Manuel; Tescucano, Alonso; Alcantara-Farfan, Veronica; Ibanez, Miguel; Montanez, Cecilia; Wong, Carlos; Baeza, Isabel

    2010-01-01

    Gene transfection into mammalian cells can be achieved with viral and non-viral vectors. Non-viral vectors, such as cationic lipids that form lipoplexes with DNA, are safer and more stable than viral vectors, but their transfection efficiencies are lower. Here we describe that the simultaneous treatment with a membrane fusion inducer (chlorpromazine or procainamide) plus the lysosomotropic agent chloroquine increases lipoplex-mediated gene transfection in human (HEK293 and C-33 A) and rat (PC12) cell lines (up to 9.2-fold), as well as in situ in BALB/c mice spleens and livers (up to 6-fold); and that the polyamine spermidine increases lipoplex-mediated gene transfection and expression in cell cultures. The use of these four drugs provides a novel, safe and relatively inexpensive way to considerably increase lipoplex-mediated gene transfection efficiency.

  20. Environmental factors influencing gene transfer agent (GTA mediated transduction in the subtropical ocean.

    Directory of Open Access Journals (Sweden)

    Lauren D McDaniel

    Full Text Available Microbial genomic sequence analyses have indicated widespread horizontal gene transfer (HGT. However, an adequate mechanism accounting for the ubiquity of HGT has been lacking. Recently, high frequencies of interspecific gene transfer have been documented, catalyzed by Gene Transfer Agents (GTAs of marine α-Proteobacteria. It has been proposed that the presence of bacterial genes in highly purified viral metagenomes may be due to GTAs. However, factors influencing GTA-mediated gene transfer in the environment have not yet been determined. Several genomically sequenced strains containing complete GTA sequences similar to Rhodobacter capsulatus (RcGTA, type strain were screened to ascertain if they produced putative GTAs, and at what abundance. Five of nine marine strains screened to date spontaneously produced virus-like particles (VLP's in stationary phase. Three of these strains have demonstrated gene transfer activity, two of which were documented by this lab. These two strains Roseovarius nubinhibens ISM and Nitratireductor 44B9s, were utilized to produce GTAs designated RnGTA and NrGTA and gene transfer activity was verified in culture. Cell-free preparations of purified RnGTA and NrGTA particles from marked donor strains were incubated with natural microbial assemblages to determine the level of GTA-mediated gene transfer. In conjunction, several ambient environmental parameters were measured including lysogeny indicated by prophage induction. GTA production in culture systems indicated that approximately half of the strains produced GTA-like particles and maximal GTA counts ranged from 10-30% of host abundance. Modeling of GTA-mediated gene transfer frequencies in natural samples, along with other measured environmental variables, indicated a strong relationship between GTA mediated gene transfer and the combined factors of salinity, multiplicity of infection (MOI and ambient bacterial abundance. These results indicate that GTA-mediated

  1. Bacterial plasmid-mediated quinolone resistance genes in aquatic environments in China

    OpenAIRE

    Yan, Lei; Liu, Dan; Wang, Xin-Hua; Wang, Yunkun; Zhang, Bo; Wang, Mingyu; Xu, Hai

    2017-01-01

    Emerging antimicrobial resistance is a major threat to human?s health in the 21st century. Understanding and combating this issue requires a full and unbiased assessment of the current status on the prevalence of antimicrobial resistance genes and their correlation with each other and bacterial groups. In aquatic environments that are known reservoirs for antimicrobial resistance genes, we were able to reach this goal on plasmid-mediated quinolone resistance (PMQR) genes that lead to resistan...

  2. Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment.

    Directory of Open Access Journals (Sweden)

    Ruben R Bender

    2016-06-01

    Full Text Available Receptor-targeted lentiviral vectors (LVs can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 Å distance. Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4 was substantially enhanced by reducing receptor size to below 100 Å. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs.

  3. BRCA1-mediated repression of select X chromosome genes

    Directory of Open Access Journals (Sweden)

    Ropers H Hilger

    2004-09-01

    Full Text Available Abstract Recently BRCA1 has been implicated in the regulation of gene expression from the X chromosome. In this study the influence of BRCA1 on expression of X chromosome genes was investigated. Complementary DNA microarrays were used to compare the expression levels of X chromosome genes in 18 BRCA1-associated ovarian cancers to those of the 13 "BRCA1-like" and 14 "BRCA2-like" sporadic tumors (as defined by previously reported expression profiling. Significance was determined using parametric statistics with P

  4. Differentiation of Xylella fastidiosa strains via multilocus sequence analysis of environmentally mediated genes (MLSA-E).

    Science.gov (United States)

    Parker, Jennifer K; Havird, Justin C; De La Fuente, Leonardo

    2012-03-01

    Isolates of the plant pathogen Xylella fastidiosa are genetically very similar, but studies on their biological traits have indicated differences in virulence and infection symptomatology. Taxonomic analyses have identified several subspecies, and phylogenetic analyses of housekeeping genes have shown broad host-based genetic differences; however, results are still inconclusive for genetic differentiation of isolates within subspecies. This study employs multilocus sequence analysis of environmentally mediated genes (MLSA-E; genes influenced by environmental factors) to investigate X. fastidiosa relationships and differentiate isolates with low genetic variability. Potential environmentally mediated genes, including host colonization and survival genes related to infection establishment, were identified a priori. The ratio of the rate of nonsynonymous substitutions to the rate of synonymous substitutions (dN/dS) was calculated to select genes that may be under increased positive selection compared to previously studied housekeeping genes. Nine genes were sequenced from 54 X. fastidiosa isolates infecting different host plants across the United States. Results of maximum likelihood (ML) and Bayesian phylogenetic (BP) analyses are in agreement with known X. fastidiosa subspecies clades but show novel within-subspecies differentiation, including geographic differentiation, and provide additional information regarding host-based isolate variation and specificity. dN/dS ratios of environmentally mediated genes, though fastidiosa isolates. Discovering the genetic relationships between X. fastidiosa isolates will provide new insights into the epidemiology of populations of X. fastidiosa, allowing improved disease management in economically important crops.

  5. Improved production of heterologous lipase in Trichoderma reesei by RNAi mediated gene silencing of an endogenic highly expressed gene.

    Science.gov (United States)

    Qin, Li-Na; Cai, Fu-Rong; Dong, Xin-Rui; Huang, Zhen-Bang; Tao, Yong; Huang, Jian-Zhong; Dong, Zhi-Yang

    2012-04-01

    A lipase gene (Lip) of the Aspergillus niger was de novo synthesized and expressed in the Trichoderma reesei under the promoter of the cellobiohydrolase I gene (cbh1). RNAi-mediated gene silencing was successfully used to further improve the recombinant lipase production via down-regulation of CBHI which comprised more than 60% of the total extracellular proteins in T. reesei. The gene and protein expression of CBHI and recombinant lipase were analyzed by real-time PCR, SDS-PAGE and activity assay. The results demonstrated that RNAi-mediated gene silencing could effectively suppress cbh1 gene expression and the reduction of CBHI could result in obvious improvement of heterologous lipase production. The reconstructed strains with decreased CBHI production exhibited 1.8- to 3.2-fold increase in lipase activity than that of parental strain. The study herein provided a feasible and advantageous method of increasing heterologous target gene expression in T. reesei through preventing the high expression of a specific endogenenous gene by RNA interference. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Bioreducible liposomes for gene delivery: from the formulation to the mechanism of action.

    Directory of Open Access Journals (Sweden)

    Gabriele Candiani

    Full Text Available BACKGROUND: A promising strategy to create stimuli-responsive gene delivery systems is to exploit the redox gradient between the oxidizing extracellular milieu and the reducing cytoplasm in order to disassemble DNA/cationic lipid complexes (lipoplexes. On these premises, we previously described the synthesis of SS14 redox-sensitive gemini surfactant for gene delivery. Although others have attributed the beneficial effects of intracellular reducing environment to reduced glutathione (GSH, these observations cannot rule out the possible implication of the redox milieu in its whole on transfection efficiency of bioreducible transfectants leaving the determinants of DNA release largely undefined. METHODOLOGY/PRINCIPAL FINDINGS: With the aim of addressing this issue, SS14 was here formulated into binary and ternary 100 nm-extruded liposomes and the effects of the helper lipid composition and of the SS14/helper lipids molar ratio on chemical-physical and structural parameters defining transfection effectiveness were investigated. Among all formulations tested, DOPC/DOPE/SS14 at 25:50:25 molar ratio was the most effective in transfection studies owing to the presence of dioleoyl chains and phosphatidylethanolamine head groups in co-lipids. The increase in SS14 content up to 50% along DOPC/DOPE/SS14 liposome series yielded enhanced transfection, up to 2.7-fold higher than that of the benchmark Lipofectamine 2000, without altering cytotoxicity of the corresponding lipoplexes at charge ratio 5. Secondly, we specifically investigated the redox-dependent mechanisms of gene delivery into cells through tailored protocols of transfection in GSH-depleted and repleted vs. increased oxidative stress conditions. Importantly, GSH specifically induced DNA release in batch and in vitro. CONCLUSIONS/SIGNIFICANCE: The presence of helper lipids carrying unsaturated dioleoyl chains and phosphatidylethanolamine head groups significantly improved transfection efficiencies

  7. Enhancement of plasmid-mediated stable gene expression by ...

    African Journals Online (AJOL)

    WPRE) is a possible enhancer of gene expression in mammalian cells that promotes efficient export of unspliced (RNA) into the cytoplasm, as has been proved in transient transfection. In this study, WPRE was evaluated for enhancing stable ...

  8. Plasmid-Mediated Quinolone Resistance Genes in Escherichia coli ...

    African Journals Online (AJOL)

    Erah

    PMQR) genes and the prevalence of extended spectrum β-lactamase (ESBL) types in Escherichia coli clinical isolates. Methods: Sixty-one ESBL-producing urinary E. coli isolates were studied. An antibiotic susceptibility test was performed ...

  9. Chitosan-Graft-Polyethylenimine/DNA Nanoparticles as Novel Non-Viral Gene Delivery Vectors Targeting Osteoarthritis

    Science.gov (United States)

    Lv, Lulu; Zhao, Huiqing

    2014-01-01

    The development of safe and efficient gene carriers is the key to the clinical success of gene therapy. The present study was designed to develop and evaluate the chitosan-graft-polyethylenimine (CP)/DNA nanoparticles as novel non-viral gene vectors for gene therapy of osteoarthritis. The CP/DNA nanoparticles were produced through a complex coacervation of the cationic polymers with pEGFP after grafting chitosan (CS) with a low molecular weight (Mw) PEI (Mw = 1.8 kDa). Particle size and zeta potential were related to the weight ratio of CP:DNA, where decreases in nanoparticle size and increases in surface charge were observed as CP content increased. The buffering capacity of CP was significantly greater than that of CS. The transfection efficiency of CP/DNA nanoparticles was similar with that of the Lipofectamine™ 2000, and significantly higher than that of CS/DNA and PEI (25 kDa)/DNA nanoparticles. The transfection efficiency of the CP/DNA nanoparticles was dependent on the weight ratio of CP:DNA (w/w). The average cell viability after the treatment with CP/DNA nanoparticles was over 90% in both chondrocytes and synoviocytes, which was much higher than that of PEI (25 kDa)/DNA nanoparticles. The CP copolymers efficiently carried the pDNA inside chondrocytes and synoviocytes, and the pDNA was detected entering into nucleus. These results suggest that CP/DNA nanoparticles with improved transfection efficiency and low cytotoxicity might be a safe and efficient non-viral vector for gene delivery to both chondrocytes and synoviocytes. PMID:24392152

  10. Herpes simplex virus type 1 VP22-mediated intercellular delivery of PTEN increases the antitumor activity of PTEN in esophageal squamous cell carcinoma cells in vitro and in vivo.

    Science.gov (United States)

    Yu, Xian; Li, Tingting; Xia, Yifan; Lei, Jun; Wang, Yan; Zhang, Lijuan

    2016-05-01

    In the past decade, studies have revealed that the phosphatase and tensin homolog (PTEN) protein, a tumor suppressor, comprises a potential biological marker and therapeutic target for esophageal squamous cell carcinoma (ESCC). As such, the delivery of the PTEN gene represents a powerful strategy for ESCC therapy. The tegument protein VP22 of herpes simplex virus type 1 (HSV-1) has been reported to act as a transporter of heterologous proteins across the host cell membrane, thereby enhancing the biological functions of these proteins. In the present study, the intercellular delivery and antitumor activity of the fusion protein PTEN-VP22 were examined in the esophageal squamous cell carcinoma cell line Eca109 both in vitro and in vivo. VP22-mediated PTEN intercellular delivery was confirmed in the Eca109 cells by western blot analysis and by quantitation of immunofluorescence. VP22 alone did not exert antiproliferative effects or induce cell cycle arrest, induction of apoptosis, blockage of the Akt and focal adhesion kinase (FAK) pathways, tumor growth inhibition, or antiangiogenic effects in Eca109 cells. However, compared with PTEN alone, PTEN-VP22 exerted significantly higher antiproliferative effects and induced cell cycle arrest at G1 stage, apoptosis and antiangiogenic effects in Eca109 cells. Together, our findings demonstrate that VP22 alone does not exert antitumor activity directly; however, this protein mediates the intercellular delivery of PTEN and thereby increases its intracellular concentration to achieve a therapeutic steady state, leading to an overall increase in the antitumor activity of PTEN. This study provides further experimental data to confirm the potential of VP22-based intercellular delivery strategies for enhancing the efficacy of gene therapy for cancer treatment.

  11. Adenovirus-Mediated Gene Therapy Against Viral Biothreat Agents

    Science.gov (United States)

    2016-04-12

    economy. Vaccine development is an important strategy to thwart the threat of these viral biothreat agents. There is an urgent need to improve...Alberta, Tl A 8K6. Canada E-mail: josh. wu@drdc-rddc.gc.ca .• 78 JoshQ.H. Wu existing vaccines against these agents and to develop new ones. Gene...of vaccines against viral biothreat agents. Genes encoding protective antigens of viral biothreat agents can be carried by these viral vectors and

  12. Enhanced thermogenic program by non-viral delivery of combinatory browning genes to treat diet-induced obesity in mice.

    Science.gov (United States)

    Park, Hongsuk; Cho, Sungpil; Janat-Amsbury, Margit M; Bae, You Han

    2015-12-01

    Thermogenic program (also known as browning) is a promising and attractive anti-obesity approach. Islet amyloid polypeptide (IAPP) and irisin have emerged as potential browning hormones that hold high potential to treat obesity. Here, we have constructed a dual browning gene system containing both IAPP and irisin (derived from fibronectin type III domain containing 5; FNDC5) combined with 2A and furin self-cleavage sites. Intraperitoneal administration of the construct complexed with a linear polyethylenimine into diet-induced obese mice demonstrated the elevation of anti-obesogenic effects characterized as the decreased body weight, adiposity, and levels of glucose and insulin. In addition, the construct delivery increased energy expenditure and the expression of core molecular determinants associated with browning. The additional advantages of the dual browning gene construct delivery compared to both single gene construct delivery and dual peptide delivery can be emphasized on efficacy and practicability. Hence, we have concluded that dual browning gene delivery makes it therapeutically attractive for diet-induced obesity treatment. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Research of Ultrasound-Mediated Transdermal Drug Delivery System Using Cymbal-Type Piezoelectric Composite Transducer

    Science.gov (United States)

    Huan, Huiting; Gao, Chunming; Liu, Lixian; Sun, Qiming; Zhao, Binxing; Yan, Laijun

    2015-06-01

    Transdermal drug delivery (TDD) implemented by especially low-frequency ultrasound is generally known as sonophoresis or phonophoresis which has drawn considerable wide attention. However, TDD has not yet achieved its full potential as an alternative to conventional drug delivery methods due to its bulky instruments. In this paper, a cymbal-type piezoelectric composite transducer (CPCT) which has advantages over a traditional ultrasound generator in weight, flexibility, and power consumption, is used as a substitute ultrasonicator to realize TDD. First, theoretical research on a CPCT based on the finite element analysis was carried out according to which a series of applicable CPCTs with bandwidths of 20 kHz to 100 kHz were elaborated. Second, a TDD experimental setup was built with previously fabricated CPCTs aimed at the administration of glucose. Finally, the TDD performance of glucose molecule transport in porcine skin was measured in vitro by quantifying the concentration of glucose, and the time variation curves were subsequently obtained. During the experiment, the driving wave form, frequency, and power consumption of the transducers were selected as the main elements which determined the efficacy of glucose delivery. The results indicate that the effectiveness of the CPCT-based delivery is constrained more by the frequency and intensity of ultrasound rather than the driving waveform. The light-weight, flexibility, and low-power consumption of a CPCT can potentially achieve effective TDD.

  14. Microneedle-mediated transdermal delivery of nanostructured lipid carriers for alkaloids from Aconitum sinomontanum.

    Science.gov (United States)

    Guo, Teng; Zhang, Yongtai; Li, Zhe; Zhao, Jihui; Feng, Nianping

    2017-09-12

    A combination method using microneedle (MN) pretreatment and nanostructured lipid carriers (NLCs) was developed to improve the transdermal delivery of therapeutics. The MN treatment of the skin and co-administration of NLCs loaded with total alkaloids isolated from Aconitum sinomontanum (AAS-NLCs) significantly increased the skin permeation of the drugs. Fluorescence imaging confirmed that MNs could provide microchannels penetrating the stratum corneum, and delivery of NLCs through the channels led to their deeper permeation. In vivo studies showed that combination of AAS-NLCs with MNs (AAS-NLCs-MN) in transdermal delivery could improve the bioavailability and maintain stable drug concentrations in the blood. Moreover, AAS-NLCs-MN showed benefits in eliminating paw swelling, decreasing inflammation and pain, and regulating immune function in adjuvant arthritis rats. After administration of AAS-NLCs-MN, no skin irritation was observed in rabbits, and electrocardiograms of rats showed improved arrhythmia. These results indicated that the dual approach combining MN insertion and NLCs has the potential to provide safe transdermal delivery and to improve the therapeutic efficacy through sustained release of AAS.

  15. Efficient cytosolic delivery mediated by polymersomes facilely prepared from a degradable, amphiphilic, and amphoteric copolymer.

    Science.gov (United States)

    Huang, Zhonghui; Teng, Wei; Liu, Longshan; Wang, Lichun; Wang, Qinmei; Dong, Yugang

    2013-07-05

    To solve problems in polymersome preparation caused by liposolubility of copolymers and to improve the cytosolic delivery efficiency of polymersomes to drugs, a lipopolysaccharide-amine (LPSA) copolymer with amphotericity and amphiphilicity is developed. LPSA contains two hydrophilic oppositely charged blocks (anionic oxidized alginate (OA), cationic polyethyleneimine (PEI 1.8 k)) and one hydrophobic block (cholesteryl), where OA is the backbone and cholesteryl-grafted PEI is the side chain. The two hydrophilic blocks first guarantee that LPSA will dissolve in water, and then help polymersome formation via electrostatic interactions to generate water insoluble interpolyelectrolyte complexes, which supplement the hydrophobic part to reach the right hydrophilicity/hydrophobicity ratio, and thus realize a one-step self-assembly of polymersomes in water. Our results show LPSA nanopolymersomes (LNPs) have low cytotoxicity and degradability, and an excellent ability to enter cells. TEM observation demonstrates that LNPs are entrapped in endosomes after endocytosis, and are then released to cytosols because of their strong endosomal escape capacity. As an example of cytosolic delivery to bioactive molecules, pDNA is delivered in mesenchymal stem cells, and more than 95% of cells express a large target protein, indicating that LNPs have high cytosolic delivery efficiency. Our study provides a novel, easy, and universal method to design copolymers for the preparation of polymersomes as efficient cytosolic delivery nanocarriers.

  16. Oil-filled polymer microcapsules for ultrasound-mediated delivery of lipophilic drugs

    NARCIS (Netherlands)

    Kooiman, Klazina; Böhmer, Marcel R.; Emmer, M.; Vos, Henk; Chlon, Ceciel; Shi, William T.; Hall, Christopher S.; de Winter, Suzanne H.P.M.; Schroën, Karin; Versluis, Michel; de Jong, N.; van Wamel, Annemieke

    2009-01-01

    The use of ultrasound contrast agents as local drug delivery systems continues to grow. Current limitations are the amount of drug that can be incorporated as well as the efficiency of drug release upon insonification. This study focuses on the synthesis and characterisation of novel polymeric

  17. Ultrasound-mediated drug delivery to the brain : principles, progress and prospects

    NARCIS (Netherlands)

    Dasgupta, Anshuman; Liu, Mengjiao; Ojha, Tarun|info:eu-repo/dai/nl/41330874X; Storm, G|info:eu-repo/dai/nl/073356328; Kiessling, Fabian; Lammers, Twan|info:eu-repo/dai/nl/304824577

    2016-01-01

    The blood–brain barrier (BBB) limits drug delivery to the central nervous system. When combined with microbubbles, ultrasound can transiently permeate blood vessels in the brain. This approach, which can be referred to as sonoporation or sonopermeabilization, holds significant promise for shuttling

  18. Ultrasound-mediated drug delivery to the brain: principles, progress and prospects

    NARCIS (Netherlands)

    Dasgupta, I.; Liu, M.; Ojha, T.; Storm, Gerrit; Kiessling, F.; Lammers, Twan Gerardus Gertudis Maria

    2016-01-01

    The blood–brain barrier (BBB) limits drug delivery to the central nervous system. When combined with microbubbles, ultrasound can transiently permeate blood vessels in the brain. This approach, which can be referred to as sonoporation or sonopermeabilization, holds significant promise for shuttling

  19. Increasing the density of nanomedicines improves their ultrasound-mediated delivery to tumours

    Czech Academy of Sciences Publication Activity Database

    Mo, S.; Carlisle, R.; Laga, Richard; Myers, R.; Graham, S.; Cawood, R.; Ulbrich, Karel; Seymour, L.; Coussios, C. C.

    2015-01-01

    Roč. 210, 28 July (2015), s. 10-18 ISSN 0168-3659 R&D Projects: GA MŠk(CZ) EE2.3.30.0029 Institutional support: RVO:61389013 Keywords : delivery * density * tumour Subject RIV: CE - Biochemistry Impact factor: 7.441, year: 2015

  20. Nymphal RNAi: systemic RNAi mediated gene knockdown in juvenile grasshopper

    Directory of Open Access Journals (Sweden)

    Dong Ying

    2005-10-01

    Full Text Available Abstract Background Grasshopper serves as important model system in neuroscience, development and evolution. Representatives of this primitive insect group are also highly relevant targets of pest control efforts. Unfortunately, the lack of genetics or gene specific molecular manipulation imposes major limitations to the study of grasshopper biology. Results We investigated whether juvenile instars of the grasshopper species Schistocerca americana are conducive to gene silencing via the systemic RNAi pathway. Injection of dsRNA corresponding to the eye colour gene vermilion into first instar nymphs triggered suppression of ommochrome formation in the eye lasting through two instars equivalent to 10–14 days in absolute time. QRT-PCR analysis revealed a two fold decrease of target transcript levels in affected animals. Control injections of EGFP dsRNA did not result in detectable phenotypic changes. RT-PCR and in situ hybridization detected ubiquitous expression of the grasshopper homolog of the dsRNA channel protein gene sid-1 in embryos, nymphs and adults. Conclusion Our results demonstrate that systemic dsRNA application elicits specific and long-term gene silencing in juvenile grasshopper instars. The conservation of systemic RNAi in the grasshopper suggests that this pathway can be exploited for gene specific manipulation of juvenile and adult instars in a wide range of primitive insects.

  1. Quaternized chitosan/rectorite intercalative materials for a gene delivery system

    International Nuclear Information System (INIS)

    Wang Xiaoying; Du Yumin; Pei Xiaofeng; Li Yan

    2008-01-01

    Non-viral vectors have gained increasing attention in gene therapy because of their safety, but with the shortcoming of low transfection efficiency. We have developed a hybrid material as a novel non-viral vector, which combines the advantages of both biopolymer and clay in a gene delivery system. Quaternized chitosan was intercalated into the interlayers of rectorite to obtain a new polymer/layered silicate nanocomposite. In vitro and in vivo toxicity studies revealed that the nanocomposites were biocompatible and non-toxic. At the nanocomposite:pDNA mass ratio of 8:1, they achieved 100% pDNA adsorption capacity. In vitro cell transfection revealed a transfection efficiency of 32.1% at 96 h as shown by a flow-cytometric study, and the intensive green fluorescence protein (GFP) expression could last for up to 120 h. Furthermore, an in vivo transfection study showed that the most prominent GFP expression was observed in the gastric and duodenum mucosa, and good transfection efficiency was also obtained when injected into the muscle. All the results suggest that quaternized chitosan/rectorite nanocomposite is a novel and potential non-viral gene carrier

  2. Targeted gene delivery in the cricket brain, using in vivo electroporation.

    Science.gov (United States)

    Matsumoto, Chihiro Sato; Shidara, Hisashi; Matsuda, Koji; Nakamura, Taro; Mito, Taro; Matsumoto, Yukihisa; Oka, Kotaro; Ogawa, Hiroto

    2013-12-01

    The cricket (Gryllus bimaculatus) is a hemimetabolous insect that is emerging as a model organism for the study of neural and molecular mechanisms of behavioral traits. However, research strategies have been limited by a lack of genetic manipulation techniques that target the nervous system of the cricket. The development of a new method for efficient gene delivery into cricket brains, using in vivo electroporation, is described here. Plasmid DNA, which contained an enhanced green fluorescent protein (eGFP) gene, under the control of a G. bimaculatus actin (Gb'-act) promoter, was injected into adult cricket brains. Injection was followed by electroporation at a sufficient voltage. Expression of eGFP was observed within the brain tissue. Localized gene expression, targeted to specific regions of the brain, was also achieved using a combination of local DNA injection and fine arrangement of the electroporation electrodes. Further studies using this technique will lead to a better understanding of the neural and molecular mechanisms that underlie cricket behaviors. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Quaternized chitosan/rectorite intercalative materials for a gene delivery system

    Science.gov (United States)

    Wang, Xiaoying; Pei, Xiaofeng; Du, Yumin; Li, Yan

    2008-09-01

    Non-viral vectors have gained increasing attention in gene therapy because of their safety, but with the shortcoming of low transfection efficiency. We have developed a hybrid material as a novel non-viral vector, which combines the advantages of both biopolymer and clay in a gene delivery system. Quaternized chitosan was intercalated into the interlayers of rectorite to obtain a new polymer/layered silicate nanocomposite. In vitro and in vivo toxicity studies revealed that the nanocomposites were biocompatible and non-toxic. At the nanocomposite:pDNA mass ratio of 8:1, they achieved 100% pDNA adsorption capacity. In vitro cell transfection revealed a transfection efficiency of 32.1% at 96 h as shown by a flow-cytometric study, and the intensive green fluorescence protein (GFP) expression could last for up to 120 h. Furthermore, an in vivo transfection study showed that the most prominent GFP expression was observed in the gastric and duodenum mucosa, and good transfection efficiency was also obtained when injected into the muscle. All the results suggest that quaternized chitosan/rectorite nanocomposite is a novel and potential non-viral gene carrier.

  4. Novel pH-Sensitive Cationic Lipids with Linear Ortho Ester Linkers for Gene Delivery

    Science.gov (United States)

    Chen, Haigang; Zhang, Huizhen; Thor, Der; Rahimian, Roshanak; Guo, Xin

    2012-01-01

    In an effort to develop pH-sensitive lipoplexes for efficient gene delivery, we report three novel cationic lipids containing a linear ortho ester linker that conjugates either the headgroup (Type I) or one hydrocarbon chain (Type II) with the rest of the lipid molecule. The cationic lipids carry either an iodide or a chloride counterion. Compared to our previously reported cyclic ortho ester linker, the linear ortho ester linker facilitated the construction of cationic liposomes and lipoplexes with different helper lipids. The chloride counterion not only facilitated the hydration of the lipid films during liposome construction, but also enhanced the hydrolysis of the ortho ester linker in the lipoplexes. After incubation at endosomal pH 5.5, the Type I lipoplexes aggregated and destabilized the endosome-mimicking model liposomes, but not the Type II lipoplexes. The helper lipids (DOPE or cholesterol) of the lipoplexes enhanced the pH-sensitivity of the Type I lipoplexes. In CV-1 cells (monkey kidney fibroblast), the Type I ortho ester-based lipoplexes, especially those with the chloride counterion, significantly improved the gene transfection efficiency, in some cases by more than 100 fold, compared to their pH-insensitive counterparts consisting of DOTAP. The gene transfection efficiency of the ortho ester-based lipoplexes was well correlated with their rate of aggregation and membrane destabilization in response to the endosomal pH 5.5. PMID:22480493

  5. Efficient gene delivery to human umbilical cord mesenchymal stem cells by cationized Porphyra yezoensis polysaccharide nanoparticles.

    Science.gov (United States)

    Yu, Qingtong; Cao, Jin; Chen, Baoding; Deng, Wenwen; Cao, Xia; Chen, Jingjing; Wang, Yan; Wang, Shicheng; Yu, Jiangnan; Xu, Ximing; Gao, Xiangdong

    2015-01-01

    This study centered on an innovative application of Porphyra yezoensis polysaccharide (PPS) with cationic modification as a safe and efficient nonviral gene vector to deliver a plasmid encoding human Wnt3a (pWnt3a) into human umbilical cord mesenchymal stem cells (HUMSCs). After modification with branched low-molecular-weight (1,200 Da) polyethylenimine, the cationized PPS (CPPS) was combined with pWnt3a to form spherical nanoscale particles (CPPS-pWnt3a nanoparticles). Particle size and distribution indicated that the CPPS-pWnt3a nanoparticles at a CPPS:pWnt3a weight ratio of 40:1 might be a potential candidate for DNA plasmid transfection. A cytotoxicity assay demonstrated that the nanoparticles prepared at a CPPS:pWnt3a weight ratio of 40:1 were nontoxic to HUMSCs compared to those of Lipofectamine 2000 and polyethylenimine (25 kDa). These nanoparticles were further transfected to HUMSCs. Western blotting demonstrated that the nanoparticles (CPPS:pWnt3a weight ratio 40:1) had the greatest transfection efficiency in HUMSCs, which was significantly higher than that of Lipofectamine 2000; however, when the CPPS:pWnt3a weight ratio was increased to 80:1, the nanoparticle-treated group showed no obvious improvement in translation efficiency over Lipofectamine 2000. Therefore, CPPS, a novel cationic polysaccharide derived from P. yezoensis, could be developed into a safe, efficient, nonviral gene vector in a gene-delivery system.

  6. Phasevarions mediate random switching of gene expression in pathogenic Neisseria.

    Directory of Open Access Journals (Sweden)

    Yogitha N Srikhanta

    2009-04-01

    Full Text Available Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression. In Haemophilus influenzae, the random switching of the modA gene controls expression of a phase-variable regulon of genes (a "phasevarion", via differential methylation of the genome in the modA ON and OFF states. Phase-variable mod genes are also present in Neisseria meningitidis and Neisseria gonorrhoeae, suggesting that phasevarions may occur in these important human pathogens. Phylogenetic studies on phase-variable mod genes associated with type III restriction modification (R-M systems revealed that these organisms have two distinct mod genes--modA and modB. There are also distinct alleles of modA (abundant: modA11, 12, 13; minor: modA4, 15, 18 and modB (modB1, 2. These alleles differ only in their DNA recognition domain. ModA11 was only found in N. meningitidis and modA13 only in N. gonorrhoeae. The recognition site for the modA13 methyltransferase in N. gonorrhoeae strain FA1090 was identified as 5'-AGAAA-3'. Mutant strains lacking the modA11, 12 or 13 genes were made in N. meningitidis and N. gonorrhoeae and their phenotype analyzed in comparison to a corresponding mod ON wild-type strain. Microarray analysis revealed that in all three modA alleles multiple genes were either upregulated or downregulated, some of which were virulence-associated. For example, in N. meningitidis MC58 (modA11, differentially expressed genes included those encoding the candidate vaccine antigens lactoferrin binding proteins A and B. Functional studies using N. gonorrhoeae FA1090 and the clinical isolate O1G1370 confirmed that modA13 ON and OFF strains have distinct phenotypes in antimicrobial resistance, in a primary human cervical epithelial cell model of infection, and in biofilm formation. This study, in conjunction with our previous work in H. influenzae, indicates

  7. Phasevarions mediate random switching of gene expression in pathogenic Neisseria.

    Science.gov (United States)

    Srikhanta, Yogitha N; Dowideit, Stefanie J; Edwards, Jennifer L; Falsetta, Megan L; Wu, Hsing-Ju; Harrison, Odile B; Fox, Kate L; Seib, Kate L; Maguire, Tina L; Wang, Andrew H-J; Maiden, Martin C; Grimmond, Sean M; Apicella, Michael A; Jennings, Michael P

    2009-04-01

    Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes) that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression). In Haemophilus influenzae, the random switching of the modA gene controls expression of a phase-variable regulon of genes (a "phasevarion"), via differential methylation of the genome in the modA ON and OFF states. Phase-variable mod genes are also present in Neisseria meningitidis and Neisseria gonorrhoeae, suggesting that phasevarions may occur in these important human pathogens. Phylogenetic studies on phase-variable mod genes associated with type III restriction modification (R-M) systems revealed that these organisms have two distinct mod genes--modA and modB. There are also distinct alleles of modA (abundant: modA11, 12, 13; minor: modA4, 15, 18) and modB (modB1, 2). These alleles differ only in their DNA recognition domain. ModA11 was only found in N. meningitidis and modA13 only in N. gonorrhoeae. The recognition site for the modA13 methyltransferase in N. gonorrhoeae strain FA1090 was identified as 5'-AGAAA-3'. Mutant strains lacking the modA11, 12 or 13 genes were made in N. meningitidis and N. gonorrhoeae and their phenotype analyzed in comparison to a corresponding mod ON wild-type strain. Microarray analysis revealed that in all three modA alleles multiple genes were either upregulated or downregulated, some of which were virulence-associated. For example, in N. meningitidis MC58 (modA11), differentially expressed genes included those encoding the candidate vaccine antigens lactoferrin binding proteins A and B. Functional studies using N. gonorrhoeae FA1090 and the clinical isolate O1G1370 confirmed that modA13 ON and OFF strains have distinct phenotypes in antimicrobial resistance, in a primary human cervical epithelial cell model of infection, and in biofilm formation. This study, in conjunction with our previous work in H. influenzae, indicates that

  8. The Yersinia pestis Ail Protein Mediates Binding and Yop Delivery to Host Cells Required for Plague Virulence▿

    Science.gov (United States)

    Felek, Suleyman; Krukonis, Eric S.

    2009-01-01

    Although adhesion to host cells is a critical step in the delivery of cytotoxic Yop proteins by Yersinia pestis, the mechanism has not been defined. To identify adhesins critical for Yop delivery, we initiated two transposon mutagenesis screens using the mariner transposon. To avoid redundant cell binding activities, we initiated the screen with a strain deleted for two known adhesins, pH 6 antigen and the autotransporter, YapC, as well as the Caf1 capsule, which is known to obscure some adhesins. The mutants that emerged contained insertions within the ail (attachment and invasion locus) gene of Y. pestis. A reconstructed mutant with a single deletion in the ail locus (y1324) was severely defective for delivery of Yops to HEp-2 human epithelial cells and significantly defective for delivery of Yops to THP-1 human monocytes. Specifically, the Yop delivery defect was apparent when cell rounding and translocation of an ELK-tagged YopE derivative into host cells were monitored. Although the ail mutant showed only a modest decrease in cell binding capacity in vitro, the KIM5 Δail mutant exhibited a >3,000-fold-increased 50% lethal dose in mice. Mice infected with the Δail mutant also had 1,000-fold fewer bacteria in their spleens, livers, and lungs 3 days after infection than did those infected with the parental strain, KIM5. Thus, the Ail protein is critical for both Y. pestis type III secretion in vitro and infection in mice. PMID:19064637

  9. Phasevarion mediated epigenetic gene regulation in Helicobacter pylori.

    Directory of Open Access Journals (Sweden)

    Yogitha N Srikhanta

    Full Text Available Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression. In Haemophilus influenzae and pathogenic Neisseria, the random switching of the modA gene, associated with a phase-variable type III restriction modification (R-M system, controls expression of a phase-variable regulon of genes (a "phasevarion", via differential methylation of the genome in the modA ON and OFF states. Phase-variable type III R-M systems are also found in Helicobacter pylori, suggesting that phasevarions may also exist in this key human pathogen. Phylogenetic studies on the phase-variable type III modH gene revealed that there are 17 distinct alleles in H. pylori, which differ only in their DNA recognition domain. One of the most commonly found alleles was modH5 (16% of isolates. Microarray analysis comparing the wild-type P12modH5 ON strain to a P12ΔmodH5 mutant revealed that six genes were either up- or down-regulated, and some were virulence-associated. These included flaA, which encodes a flagella protein important in motility and hopG, an outer membrane protein essential for colonization and associated with gastric cancer. This study provides the first evidence of this epigenetic mechanism of gene expression in H. pylori. Characterisation of H. pylori modH phasevarions to define stable immunological targets will be essential for vaccine development and may also contribute to understanding H. pylori pathogenesis.

  10. Peptide-conjugated micelles as a targeting nanocarrier for gene delivery

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Wen Jen, E-mail: wjlin@ntu.edu.tw; Chien, Wei Hsuan [National Taiwan University, School of Pharmacy, Graduate Institute of Pharmaceutical Sciences (China)

    2015-09-15

    The aim of this study was to develop peptide-conjugated micelles possessing epidermal growth factor receptor (EGFR) targeting ability for gene delivery. A sequence-modified dodecylpeptide, GE11(2R), with enhancing EGF receptor binding affinity, was applied in this study as a targeting ligand. The active targeting micelles were composed of poly(d,l-lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) copolymer conjugated with GE11(2R)-peptide. The particle sizes of peptide-free and peptide-conjugated micelles were 277.0 ± 5.1 and 308.7 ± 14.5 nm, respectively. The peptide-conjugated micelles demonstrated the cellular uptake significantly higher than peptide-free micelles in EGFR high-expressed MDA-MB-231 and MDA-MB-468 cells due to GE11(2R)-peptide specificity. Furthermore, the peptide-conjugated micelles were able to encapsulate plasmid DNA and expressed cellular transfection higher than peptide-free micelles in EGFR high-expressed cells. The EGFR-targeting delivery micelles enhanced DNA internalized into cells and achieved higher cellular transfection in EGFR high-expressed cells.

  11. Nonviral Gene Delivery from Nonwoven Fibrous Scaffolds Fabricated by Interfacial Complexation of Polyelectrolytes

    Science.gov (United States)

    Lim, Shawn H.; Liao, I-Chien; Leong, Kam W.

    2008-01-01

    We investigated a novel nonwoven fibrous scaffold as a vehicle for delivery of DNA. Fibers were formed by polyelectrolyte complexation of water-soluble chitin and alginate, and PEI–DNA nanoparticles were encapsulated during the fiber drawing process. Nanoparticles released from the fibers over time retained their bioactivity and successfully transfected cells seeded on the scaffold in a sustained manner. Transgene expression in HEK293 cells and human dermal fibroblasts seeded on the transfecting scaffolds was significant even after 2 weeks of culture compared to 3-day expression in two-dimensional controls. Fibroblasts seeded on scaffolds containing DNA encoding basic fibroblast growth factor (bFGF) demonstrated prolonged secretion of bFGF at levels significantly higher than baseline. This work establishes the potential of this fibrous scaffold as a matrix capable of delivering genes to direct and support cellular development in tissue engineering. PMID:16497560

  12. Nano-niosomes in drug, vaccine and gene delivery: a rapid overview

    Directory of Open Access Journals (Sweden)

    Abbas Pardakhty

    2013-10-01

    Full Text Available   Abstract Niosomes, non-ionic surfactant vesicles (NSVs, are the hydrated lipids composed mainly of different classes of non-ionic surfactants, introduced in the seventies as a cosmetic vehicle. Nowadays, niosomes are used as important new drug delivery systems by many research groups and also they are effective immunoadjuvants which some commercial forms are available in the market. These vesicles recently used as gene transfer vectors as well. This review article presents a brief report about the achievements in the field of nanoscience related to NSVs. Different polar head groups from a vast list of various surfactants with one, two or three lipophilic alkyl, perfluoroalkyl and steroidal moieties may be utilized to form the proper vesicular structures for encapsulating both hydrophilic and hydrophobic compounds. The methods of niosome preparation, the vesicle stability related aspects and many examples of pharmaceutical applications of NSVs will be presented. The routes of administration of these amphiphilic assemblies are also discussed. 

  13. Graphene oxide-cationic polymer conjugates: Synthesis and application as gene delivery vectors.

    Science.gov (United States)

    Teimouri, Mohsen; Nia, Azadeh Hashem; Abnous, Khalil; Eshghi, Hossein; Ramezani, Mohammad

    2016-01-01

    Nanomedicine as the interface between nanotechnology and medical sciences is a new area that has attracted the attention of vast groups of researchers. Carbon nanomaterials are common platform for synthesis of nanoparticles for biomedical applications due to their low cytotoxicity and feasible internalization into mammalian cell lines (Yang et al., 2007; Arora et al., 2014; Oh and Park, 2014). Synthesis of vectors based on various cationic polymers polyethylenimine (PEI), polypropylenimine (PPI) and polyamidoamine (PAMAM) and their derivatives were considered as a strategy for transferring plasmid DNA and treatment of genetic diseases. Considering the low cytotoxicity of graphene, chemical modification of its surface has led to fabrication of novel gene delivery systems based on graphene and graphene oxide. Herein we report the synthesis of three groups of vectors based on conjugation of graphene oxide (GO) with alkylated derivatives of three different cationic polymers (polyethylenimine (PEI), polypropylenimine (PPI) and polyamidoamine (PAMAM)) through different linkers including surface carboxyl group, glycine and spermidine. Two main challenges in design of gene delivery vectors is decreasing cytotoxicity while improving the transfection efficiency. All synthesized vectors showed significantly lower cellular toxicity compared to bare polymer. A plasmid encoding green fluorescent protein (GFP) was used to evaluate the transfection efficiency of nanoparticles both qualitatively using live cell fluorescent imaging and quantitatively using flow cytometry and each vector was compared to its polymer base. Most successful conjugation strategy was observed in the case of PEI conjugates among which most efficient vector was PEI-GO conjugate bearing glycine linker. This vector was 9 fold more effective in terms of the percent of EGFP transfected cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Nonviral gene-delivery by highly fluorinated gemini bispyridinium surfactant-based DNA nanoparticles.

    Science.gov (United States)

    Fisicaro, Emilia; Compari, Carlotta; Bacciottini, Franco; Contardi, Laura; Pongiluppi, Erika; Barbero, Nadia; Viscardi, Guido; Quagliotto, Pierluigi; Donofrio, Gaetano; Krafft, Marie Pierre

    2017-02-01

    Biological and thermodynamic properties of a new homologous series of highly fluorinated bispyridinium cationic gemini surfactants, differing in the length of the spacer bridging the pyridinium polar heads in 1,1' position, are reported for the first time. Interestingly, gene delivery ability is closely associated with the spacer length due to a structural change of the molecule in solution. This conformation change is allowed when the spacer reaches the right length, and it is suggested by the trends of the apparent and partial molar enthalpies vs molality. To assess the compounds' biological activity, they were tested with an agarose gel electrophoresis mobility shift assay (EMSA), MTT proliferation assay and Transient Transfection assays on a human rhabdomyosarcoma cell line. Data from atomic force microscopy (AFM) allow for morphological characterization of DNA nanoparticles. Dilution enthalpies, measured at 298K, enabled the determination of apparent and partial molar enthalpies vs molality. All tested compounds (except that with the longest spacer), at different levels, can deliver the plasmid when co-formulated with 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE). The compound with a spacer formed by eight carbon atoms gives rise to a gene delivery ability that is comparable to that of the commercial reagent. The compound with the longest spacer compacts DNA in loosely condensed structures by forming bows, which are not suitable for transfection. Regarding the compounds' hydrogenated counterparts, the tight relationship between the solution thermodynamics data and their biological performance is amazing, making "old" methods the foundation to deeply understanding "new" applications. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Efficient gene delivery to human umbilical cord mesenchymal stem cells by cationized Porphyra yezoensis polysaccharide nanoparticles

    Directory of Open Access Journals (Sweden)

    Yu Q

    2015-11-01

    Full Text Available Qingtong Yu,1,2 Jin Cao,2 Baoding Chen,3 Wenwen Deng,2 Xia Cao,2 Jingjing Chen,2 Yan Wang,2 Shicheng Wang,2 Jiangnan Yu,2 Ximing Xu,2 Xiangdong Gao1 1School of Life Science and Technology, China Pharmaceutical University, Nanjing, 2Department of Pharmaceutics, School of Pharmacy and Center for Drug/Gene Delivery and Tissue Engineering, Jiangsu University, 3Department of Medical Ultrasound, Affiliated Hospital of Jiangsu University, Zhenjiang, People’s Republic of China Abstract: This study centered on an innovative application of Porphyra yezoensis polysaccharide (PPS with cationic modification as a safe and efficient nonviral gene vector to deliver a plasmid encoding human Wnt3a (pWnt3a into human umbilical cord mesenchymal stem cells (HUMSCs. After modification with branched low-molecular-weight (1,200 Da polyethylenimine, the cationized PPS (CPPS was combined with pWnt3a to form spherical nanoscale particles (CPPS-pWnt3a nanoparticles. Particle size and distribution indicated that the CPPS-pWnt3a nanoparticles at a CPPS:pWnt3a weight ratio of 40:1 might be a potential candidate for DNA plasmid transfection. A cytotoxicity assay demonstrated that the nanoparticles prepared at a CPPS:pWnt3a weight ratio of 40:1 were nontoxic to HUMSCs compared to those of Lipofectamine 2000 and polyethylenimine (25 kDa. These nanoparticles were further transfected to HUMSCs. Western blotting demonstrated that the nanoparticles (CPPS:pWnt3a weight ratio 40:1 had the greatest transfection efficiency in HUMSCs, which was significantly higher than that of Lipofectamine 2000; however, when the CPPS:pWnt3a weight ratio was increased to 80:1, the nanoparticle-treated group showed no obvious improvement in translation efficiency over Lipofectamine 2000. Therefore, CPPS, a novel cationic polysaccharide derived from P. yezoensis, could be developed into a safe, efficient, nonviral gene vector in a gene-delivery system. Keywords: Porphyra yezoensis, nanoparticle

  16. Mesoporous Silica Nanomaterials for Applications in Catalysis, Sensing, Drug Delivery and Gene Transfection

    Energy Technology Data Exchange (ETDEWEB)

    Radu, Daniela Rodica [Iowa State Univ., Ames, IA (United States)

    2004-01-01

    The central theme of this dissertation is represented by the versatility of mesoporous silica nanomaterials in various applications such as catalysis and bio-applications, with main focus on biological applications of Mesoporous Silica Nanospheres (MSN). The metamorphosis that we impose to these materials from catalysis to sensing and to drug and gene delivery is detailed in this dissertation. First, we developed a synthetic method that can fine tune the amount of chemically accessible organic functional groups on the pores surface of MSN by exploiting electrostatic and size matching between the cationic alkylammonium head group of the cetyltrimethylammonium bromide (CTAB) surfactant and various anionic organoalkoxysilane precursors at the micelle-water interface in a base-catalyzed condensation reaction of silicate. Aiming nature imitation, we demonstrated the catalytic abilities of the MSNs, We utilized an ethylenediamine functional group for chelating Cu2+ as a catalytic functional group anchored inside the mesopores. Thus, a polyalkynylene-based conducting polymer (molecular wire) was synthesized within the Cu-functionalized MSNs silica catalyst. For sensing applications, we have synthesized a poly(lactic acid) coated mesoporous silica nanosphere (PLA-MSN) material that serves as a fluorescence sensor system for detection of amino-containing neurotransmitters in neutral aqueous buffer. We exploited the mesoporosity of MSNs for encapsulating pharmaceutical drugs. We examined bio-friendly capping molecules such as polyamidoamine dendrimers of generations G2 to G4, to prevent the drug leaching. Next, the drug delivery system employed MSNs loaded with Doxorubicin, an anticancer drug. The results demonstrated that these nano-Trojan horses have ability to deliver Doxorubicin to cancer cells and induce their death. Finally, to demonstrate the potential of MSN as an universal cellular transmembrane nanovehicle, we anchored positively charged dendrimers on

  17. miRNA-mediated functional changes through co-regulating function related genes.

    Directory of Open Access Journals (Sweden)

    Jie He

    Full Text Available BACKGROUND: MicroRNAs play important roles in various biological processes involving fairly complex mechanism. Analysis of genome-wide miRNA microarray demonstrate that a single miRNA can regulate hundreds of genes, but the regulative extent on most individual genes is surprisingly mild so that it is difficult to understand how a miRNA provokes detectable functional changes with such mild regulation. RESULTS: To explore the internal mechanism of miRNA-mediated regulation, we re-analyzed the data collected from genome-wide miRNA microarray with bioinformatics assay, and found that the transfection of miR-181b and miR-34a in Hela and HCT-116 tumor cells regulated large numbers of genes, among which, the genes related to cell growth and cell death demonstrated high Enrichment scores, suggesting that these miRNAs may be important in cell growth and cell death. MiR-181b induced changes in protein expression of most genes that were seemingly related to enhancing cell growth and decreasing cell death, while miR-34a mediated contrary changes of gene expression. Cell growth assays further confirmed this finding. In further study on miR-20b-mediated osteogenesis in hMSCs, miR-20b was found to enhance osteogenesis by activating BMPs/Runx2 signaling pathway in several stages by co-repressing of PPARγ, Bambi and Crim1. CONCLUSIONS: With its multi-target characteristics, miR-181b, miR-34a and miR-20b provoked detectable functional changes by co-regulating functionally-related gene groups or several genes in the same signaling pathway, and thus mild regulation from individual miRNA targeting genes could have contributed to an additive effect. This might also be one of the modes of miRNA-mediated gene regulation.

  18. Amino acid-substituted gemini surfactant-based nanoparticles as safe and versatile gene delivery agents.

    Science.gov (United States)

    Singh, Jagbir; Yang, Peng; Michel, Deborah; Verrall, Ronald E; Foldvari, Marianna; Badea, Ildiko

    2011-05-01

    Gene based therapy represents an important advance in the treatment of diseases that heretofore have had either no treatment or cure. To capitalize on the true potential of gene therapy, there is a need to develop better delivery systems that can protect these therapeutic biomolecules and deliver them safely to the target sites. Recently, we have designed and developed a series of novel amino acid-substituted gemini surfactants with the general chemical formula C(12)H(25) (CH(3))(2)N(+)-(CH(2))(3)-N(AA)-(CH(2))(3)-N(+) (CH(3))(2)-C(12)H(25) (AA= glycine, lysine, glycyl-lysine and, lysyl-lysine). These compounds were synthesized and tested in rabbit epithelial cells using a model plasmid and a helper lipid. Plasmid/gemini/lipid (P/G/L) nanoparticles formulated using these novel compounds achieved higher gene expression than the nanoparticles containing the parent unsubstituted compound. In this study, we evaluated the cytotoxicity of P/G/L nanoparticles and explored the relationship between transfection efficiency/toxicity and their physicochemical characteristics (such as size, binding properties, etc.). An overall low toxicity is observed for all complexes with no significant difference among substituted and unsubstituted compounds. An interesting result revealed by the dye exclusion assay suggests a more balanced protection of the DNA by the glycine and glycyl-lysine substituted compounds. Thus, the higher transfection efficiency is attributed to the greater biocompatibility and flexibility of the amino acid/peptide-substituted gemini surfactants and demonstrates the feasibility of using amino acid-substituted gemini surfactants as gene carriers for the treatment of diseases affecting epithelial tissue.

  19. A Broad Range of Dose Optima Achieve High-level, Long-term Gene Expression After Hydrodynamic Delivery of Sleeping Beauty Transposons Using Hyperactive SB100x Transposase

    Directory of Open Access Journals (Sweden)

    Kelly M Podetz-Pedersen

    2016-01-01

    Full Text Available The Sleeping Beauty (SB transposon system has been shown to enable long-term gene expression by integrating new sequences into host cell chromosomes. We found that the recently reported SB100x hyperactive transposase conferred a surprisingly high level of long-term expression after hydrodynamic delivery of luciferase-encoding reporter transposons in the mouse. We conducted dose-ranging studies to determine the effect of varying the amount of SB100x transposase-encoding plasmid (pCMV-SB100x at a set dose of luciferase transposon and of varying the amount of transposon-encoding DNA at a set dose of pCMV-SB100x in hydrodynamically injected mice. Animals were immunosuppressed using cyclophosphamide in order to prevent an antiluciferase immune response. At a set dose of transposon DNA (25 µg, we observed a broad range of pCMV-SB100x doses (0.1–2.5 µg conferring optimal levels of long-term expression (>1011 photons/second/cm2. At a fixed dose of 0.5 μg of pCMV-SB100x, maximal long-term luciferase expression (>1010 photons/second/cm2 was achieved at a transposon dose of 5–125 μg. We also found that in the linear range of transposon doses (100 ng, co-delivering the CMV-SB100x sequence on the same plasmid was less effective in achieving long-term expression than delivery on separate plasmids. These results show marked flexibility in the doses of SB transposon plus pCMV-SB100x that achieve maximal SB-mediated gene transfer efficiency and long-term gene expression after hydrodynamic DNA delivery to mouse liver.

  20. Development of Methods for Carrier-Mediated Targeted Delivery of Antiviral Compounds Using Monoclonal Antibodies

    Science.gov (United States)

    1987-04-01

    Bagger Hansen, A« and Kofod, H. (eds.), Optimization of Drug Delivery. Munksgaard, Copenhagen, p. 255. 5. Hurwltz, E., Kashl, R., Arnon, R., Wllchek...solvent system, the spots ware datactad by apraylng with vanillin solution, which had bean prepared according to the anlsaldehyde recipe given under...alcohol/ammonium hydroxide/water, vanillin spray detection) Rf 0.10. Fractions 33 to 41 afforded 300 mg of material, which was predominately 43^with a

  1. Enhanced efficacy of sublingual immunotherapy by liposome-mediated delivery of allergen.

    Science.gov (United States)

    Aliu, Have; Rask, Carola; Brimnes, Jens; Andresen, Thomas Lars

    2017-01-01

    Immunotherapy by sublingual administration of allergens provides high patient compliance and has emerged as an alternative to subcutaneous immunotherapy for the treatment of IgE-associated allergic diseases. However, sublingual immunotherapy (SLIT) can cause adverse events. Development of allergen delivery systems enabling more efficient delivery and hence lower allergen load might reduce the adverse events. In the present study, we have investigated neutral and cationic liposomes as delivery systems of ovalbumin (OVA), as a model allergen, in an OVA-induced allergic airway inflammation model. We investigated the liposome carriers' ability to improve tolerance induction of antigens compared to the corresponding dose of free OVA. Mice were treated sublingually over 2 weeks with free or liposome encapsulated OVA followed by intraperitoneal injections and intranasal challenge. Mice sublingually treated with OVA-liposomes showed a significant reduction of airway eosinophilia and splenocyte proliferation in comparison to free OVA. A similar nonsignificant pattern was seen for OVA-specific IgE antibodies. In addition, reduced levels of interferon-γ and interleukin-5 were observed in spleen cell culture supernatants from OVA-liposome-treated mice compared to the sham-treated group. In conclusion, in vivo efficacy data showed that prophylactic SLIT with OVA-liposomes is significantly more effective in preventing allergic inflammation than the corresponding dose of free OVA.

  2. Poly[N-(2-aminoethyl)ethyleneimine] as a New Non-Viral Gene Delivery Carrier : The Effect of Two Protonatable Nitrogens in the Monomer Unit on Gene Delivery Efficiency

    NARCIS (Netherlands)

    Khazaie, Yahya; Novo, Luis; van Gaal, Ethlinn; Fassihi, Afshin; Mirahmadi-Zareh, Seyedeh Zohreh; Esfahani, Mohammad Hossein Nasr; van Nostrum, Cornelus F.; Hennink, Wim E.; Dorkoosh, Farid

    2014-01-01

    Purpose. The aim of this study was to investigate the in vitro gene delivery efficiency of poly[N-(2-aminoethyl)ethylene-imine](PAEEI), a polymer with a linear Polyethyleneimine (LPEI) backbone and with aminoethyl side groups that has two protonatable nitrogen atoms per monomer unit instead of one

  3. Identification of certain cancer-mediating genes using Gaussian ...

    Indian Academy of Sciences (India)

    The effectiveness of the methodology has been demonstrated on three gene expression cancer datasets dealing with human lung, colon and leukemia. ... Department of Computer Science and Engineering, Netaji Subhash Engineering College, Kolkata, India; Machine Intelligence Unit, Indian Statistical Institute, Kolkata, ...

  4. Plant–Agrobacterium interaction mediated by ethylene and super-Agrobacterium conferring efficient gene transfer

    OpenAIRE

    Nonaka, Satoko; Ezura, Hiroshi

    2014-01-01

    Agrobacterium tumefaciens has a unique ability to transfer genes into plant genomes. This ability has been utilized for plant genetic engineering. However, the efficiency is not sufficient for all plant species. Several studies have shown that ethylene decreased the Agrobacterium-mediated transformation frequency. Thus, A. tumefaciens with an ability to suppress ethylene evolution would increase the efficiency of Agrobacterium-mediated transformation. Some studies showed that plant growth-pro...

  5. A rapid pathway toward a superb gene delivery system: programming structural and functional diversity into a supramolecular nanoparticle library.

    Science.gov (United States)

    Wang, Hao; Liu, Kan; Chen, Kuan-Ju; Lu, Yujie; Wang, Shutao; Lin, Wei-Yu; Guo, Feng; Kamei, Ken-ichiro; Chen, Yi-Chun; Ohashi, Minori; Wang, Mingwei; Garcia, Mitch André; Zhao, Xing-Zhong; Shen, Clifton K-F; Tseng, Hsian-Rong

    2010-10-26

    Nanoparticles are regarded as promising transfection reagents for effective and safe delivery of nucleic acids into a specific type of cells or tissues providing an alternative manipulation/therapy strategy to viral gene delivery. However, the current process of searching novel delivery materials is limited due to conventional low-throughput and time-consuming multistep synthetic approaches. Additionally, conventional approaches are frequently accompanied with unpredictability and continual optimization refinements, impeding flexible generation of material diversity creating a major obstacle to achieving high transfection performance. Here we have demonstrated a rapid developmental pathway toward highly efficient gene delivery systems by leveraging the powers of a supramolecular synthetic approach and a custom-designed digital microreactor. Using the digital microreactor, broad structural/functional diversity can be programmed into a library of DNA-encapsulated supramolecular nanoparticles (DNA⊂SNPs) by systematically altering the mixing ratios of molecular building blocks and a DNA plasmid. In vitro transfection studies with DNA⊂SNPs library identified the DNA⊂SNPs with the highest gene transfection efficiency, which can be attributed to cooperative effects of structures and surface chemistry of DNA⊂SNPs. We envision such a rapid developmental pathway can be adopted for generating nanoparticle-based vectors for delivery of a variety of loads.

  6. Exogenous DNA Loading into Extracellular Vesicles via Electroporation is Size-Dependent and Enables Limited Gene Delivery.

    Science.gov (United States)

    Lamichhane, Tek N; Raiker, Rahul S; Jay, Steven M

    2015-10-05

    Extracellular vesicles (EVs) hold immense promise for utilization as biotherapeutics and drug delivery vehicles due to their nature as biological nanoparticles that facilitate intercellular molecular transport. Specifically, EVs have been identified as natural carriers of nucleic acids, sparking interest in their use for gene therapy and RNA interference applications. So far, small RNAs (siRNA and miRNA) have been successfully loaded into EVs for a variety of delivery applications, but the potential use of EVs for DNA delivery has scarcely been explored. Here, we report that exogenous linear DNA can be associated with EVs via electroporation in quantities sufficient to yield an average of hundreds of DNA molecules per vesicle. We determined that loading efficiency and capacity of DNA in EVs is dependent on DNA size, with linear DNA molecules less than 1000 bp in length being more efficiently associated with EVs compared to larger linear DNAs and plasmid DNAs using this approach. We further showed that EV size is also determinant with regard to DNA loading, as larger microvesicles encapsulated more linear and plasmid DNA than smaller, exosome-like EVs. Additionally, we confirmed the ability of EVs to transfer foreign DNA loaded via electroporation into recipient cells, although functional gene delivery was not observed. These results establish critical parameters that inform the potential use of EVs for gene therapy and, in agreement with other recent results, suggest that substantial barriers must be overcome to establish EVs as broadly applicable DNA delivery vehicles.

  7. Efficient gene delivery and selective transduction of astrocytes in the mammalian brain using viral vectors

    Directory of Open Access Journals (Sweden)

    Nicolas eMerienne

    2013-07-01

    Full Text Available Astrocytes are now considered as key players in brain information processing because of their newly discovered roles in synapse formation and plasticity, energy metabolism and blood flow regulation. However, our understanding of astrocyte function is still fragmented compared to other brain cell types. A better appreciation of the biology of astrocytes requires the development of tools to generate animal models in which astrocyte-specific proteins and pathways can be manipulated. In addition, it is becoming increasingly evident that astrocytes are also important players in many neurological disorders. Targeted modulation of protein expression in astrocytes would be critical for the development of new therapeutic strategies. Gene transfer is valuable to target a subpopulation of cells and explore their function in experimental models. In particular, viral-mediated gene transfer provides a rapid, highly flexible and cost-effective, in vivo paradigm to study the impact of genes of interest during CNS development or in adult animals. We will review the different strategies that led to the recent development of efficient viral vectors that can be successfully used to selectively transduce astrocytes in the mammalian brain.

  8. Amphiphilic polymers formed from ring-opening polymerization: a strategy for the enhancement of gene delivery.

    Science.gov (United States)

    Zhang, Yi-Mei; Huang, Zheng; Zhang, Ji; Wu, Wan-Xia; Liu, Yan-Hong; Yu, Xiao-Qi

    2017-03-28

    Cationic liposomes and polymers are both important candidates for use as non-viral gene vectors. However, both of them have special shortcomings and application limits. This work is devoted to the combination of advantages of liposomes and polymers. The ring-opening polymerization strategy was used for the preparation of amphiphilic polymers from cyclen-based cationic small lipids. The non-hydrophobic polymer and the corresponding lipids were also prepared for performing structure-activity relationship studies. Gel electrophoresis results reveal that both the lipopolymers and liposomes could effectively condense DNA into nanoparticles and protect DNA from degradation. Compared to polymers, the DNA binding ability of liposomes is more affected by hydrophobic tails. Under the same dosage, the synthetic polymers have stronger DNA binding ability than the liposomes. In vitro transfection experiments show that the polymers could give better transfection efficiency, which was much higher than those of the corresponding liposomes and non-hydrophobic polymer. The oleyl moiety is suitable for lipidic vectors, but things were different for polymers. Under optimized conditions, up to 14.2 times higher transfection efficiency than that for 25 kDa bPEI could be obtained. More importantly, the lipopolymers showed much better serum tolerance, which was further confirmed by protein adsorption, gel electrophoresis, flow cytometry, and CLSM assays. The results indicate that ring-opening polymerization is a promising strategy for the enhancement of the gene delivery efficiency and biocompatibility of cationic lipids.

  9. Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications.

    Science.gov (United States)

    Liu, Chang; Zhang, Li; Liu, Hao; Cheng, Kun

    2017-11-28

    The CRISPR-Cas9 genome-editing system is a part of the adaptive immune system in archaea and bacteria to defend against invasive nucleic acids from phages and plasmids. The single guide RNA (sgRNA) of the system recognizes its target sequence in the genome, and the Cas9 nuclease of the system acts as a pair of scissors to cleave the double strands of DNA. Since its discovery, CRISPR-Cas9 has become the most robust platform for genome engineering in eukaryotic cells. Recently, the CRISPR-Cas9 system has triggered enormous interest in therapeutic applications. CRISPR-Cas9 can be applied to correct disease-causing gene mutations or engineer T cells for cancer immunotherapy. The first clinical trial using the CRISPR-Cas9 technology was conducted in 2016. Despite the great promise of the CRISPR-Cas9 technology, several challenges remain to be tackled before its successful applications for human patients. The greatest challenge is the safe and efficient delivery of the CRISPR-Cas9 genome-editing system to target cells in human body. In this review, we will introduce the molecular mechanism and different strategies to edit genes using the CRISPR-Cas9 system. We will then highlight the current systems that have been developed to deliver CRISPR-Cas9 in vitro and in vivo for various therapeutic purposes. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Polyglycerol-functionalized nanodiamond as a platform for gene delivery: Derivatization, characterization, and hybridization with DNA

    Directory of Open Access Journals (Sweden)

    Li Zhao

    2014-03-01

    Full Text Available A gene vector consisting of nanodiamond, polyglycerol, and basic polypeptide (ND-PG-BPP has been designed, synthesized, and characterized. The ND-PG-BPP was synthesized by PG functionalization of ND through ring-opening polymerization of glycidol on the ND surface, multistep organic transformations (–OH → –OTs (tosylate → –N3 in the PG layer, and click conjugation of the basic polypeptides (Arg8, Lys8 or His8 terminated with propargyl glycine. The ND-PG-BPP exhibited good dispersibility in water (>1.0 mg/mL and positive zeta potential ranging from +14.2 mV to +44.1 mV at neutral pH in Milli-Q water. It was confirmed by gel retardation assay that ND-PG-Arg8 and ND-PG-Lys8 with higher zeta potential hybridized with plasmid DNA (pDNA through electrostatic attraction, making them promising as nonviral vectors for gene delivery.

  11. Repressor-mediated tissue-specific gene expression in plants

    Science.gov (United States)

    Meagher, Richard B [Athens, GA; Balish, Rebecca S [Oxford, OH; Tehryung, Kim [Athens, GA; McKinney, Elizabeth C [Athens, GA

    2009-02-17

    Plant tissue specific gene expression by way of repressor-operator complexes, has enabled outcomes including, without limitation, male sterility and engineered plants having root-specific gene expression of relevant proteins to clean environmental pollutants from soil and water. A mercury hyperaccumulation strategy requires that mercuric ion reductase coding sequence is strongly expressed. The actin promoter vector, A2pot, engineered to contain bacterial lac operator sequences, directed strong expression in all plant vegetative organs and tissues. In contrast, the expression from the A2pot construct was restricted primarily to root tissues when a modified bacterial repressor (LacIn) was coexpressed from the light-regulated rubisco small subunit promoter in above-ground tissues. Also provided are analogous repressor operator complexes for selective expression in other plant tissues, for example, to produce male sterile plants.

  12. Synthetic polyspermine imidazole-4, 5-amide as an efficient and cytotoxicity-free gene delivery system

    Directory of Open Access Journals (Sweden)

    Duan S

    2012-07-01

    Full Text Available Shi-Yue Duan, Xue-Mei Ge, Nan Lu, Fei Wu, Weien Yuan, Tuo JinSchool of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, People's Republic of ChinaAbstract: A chemically dynamic spermine-based polymer: polyspermine imidazole-4, 5-amide (PSIA, Mw > 7 kDa was designed, synthesized, and evaluated in terms of its ability to deliver nucleic acids. This polymer was made from an endogenous monomer professionally condensing genes in sperms, spermine, and a known safety drug metabolite, imidazole-4, 5-dicarboxylic acid, through a bis-amide bond conjugated with the imidazole ring. This polymer can condense pDNA at a W/W ratio above 10 to form polyplexes (100–200 nm in diameter, which is consistent with the observation by transmission electron microscopy (TEM, and the zeta potential was in the range of 10–20 mV. The pDNA packaged polymer was stable in phosphate buffer solution (PBS at pH 7.4 (simulated body fluid while the polyplexes were releasing pDNA into the solution at pH 5.8 (simulated endo-lysosomes due to the degradation of the bis-amide linkages in response to changes in pH values. PSIA-polyplexes were able to achieve efficient cellular uptake and luciferase gene silencing by co-transfection of pDNA and siRNA in COS-7 cells and HepG2 cells with negligible cytotoxicity. Biodistribution of Rhodamine B-labeled PSIA-polyplexes after being systemically injected in BALB/c nude-mice showed that the polyplexes circulated throughout the body, accumulated mainly in the kidney at 4 hours of sample administration, and moved to the liver and spleen after 24 hours. All the results suggested that PSIA offered a promising example to balance the transfection efficiency and toxicity of a synthetic carrier system for the delivery of therapeutic nucleic acids.Keywords: gene delivery, polyspermine, cytotoxicity, transfection efficiency, biodistribution

  13. Laminin Mediates Tissue-specific Gene Expression in Mammary Epithelia

    Energy Technology Data Exchange (ETDEWEB)

    Streuli, Charles H; Schmidhauser, Christian; Bailey, Nina; Yurchenco, Peter; Skubitz, Amy P. N.; Roskelley, Calvin; Bissell, Mina J

    1995-04-01

    Tissue-specific gene expression in mammary epithelium is dependent on the extracellular matrix as well as hormones. There is good evidence that the basement membrane provides signals for regulating beta-casein expression, and that integrins are involved in this process. Here, we demonstrate that in the presence of lactogenic hormones, laminin can direct expression of the beta-casein gene. Mouse mammary epithelial cells plated on gels of native laminin or laminin-entactin undergo functional differentiation. On tissue culture plastic, mammary cells respond to soluble basement membrane or purified laminin, but not other extracellular matrix components, by synthesizing beta-casein. In mammary cells transfected with chloramphenicol acetyl transferase reporter constructs, laminin activates transcription from the beta-casein promoter through a specific enhancer element. The inductive effect of laminin on casein expression was specifically blocked by the E3 fragment of the carboxy terminal region of the alpha 1 chain of laminin, by antisera raised against the E3 fragment, and by a peptide corresponding to a sequence within this region. Our results demonstrate that laminin can direct tissue-specific gene expression in epithelial cells through its globular domain.

  14. Intra-Arterial Delivery of AAV Vectors to the Mouse Brain After Mannitol Mediated Blood Brain Barrier Disruption

    Science.gov (United States)

    Santillan, Alejandro; Sondhi, Dolan; Dyke, Jonathan P.; Crystal, Ronald G.; Gobin, Y. Pierre; Ballon, Douglas J.

    2014-01-01

    The delivery of therapeutics to neural tissue is greatly hindered by the blood brain barrier (BBB). Direct local delivery via diffusive release from degradable implants or direct intra-cerebral injection can bypass the BBB and obtain high concentrations of the therapeutic in the targeted tissue, however the total volume of tissue that can be treated using these techniques is limited. One treatment modality that can potentially access large volumes of neural tissue in a single treatment is intra-arterial (IA) injection after osmotic blood brain barrier disruption. In this technique, the therapeutic of interest is injected directly into the arteries that feed the target tissue after the blood brain barrier has been disrupted by exposure to a hyperosmolar mannitol solution, permitting the transluminal transport of the therapy. In this work we used contrast enhanced magnetic resonance imaging (MRI) studies of IA injections in mice to establish parameters that allow for extensive and reproducible BBB disruption. We found that the volume but not the flow rate of the mannitol injection has a significant effect on the degree of disruption. To determine whether the degree of disruption we observed with this method was sufficient for delivery of nanoscale therapeutics, we performed IA injections of an adeno-associated viral vector containing the CLN2 gene (AAVrh.10CLN2), which is mutated in the lysosomal storage disorder Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL). We demonstrated that IA injection of AAVrh.10CLN2 after BBB disruption can achieve widespread transgene production in the mouse brain after a single administration. Further, we showed that there exists a minimum threshold of BBB disruption necessary to permit the AAV.rh10 vector to pass into the brain parenchyma from the vascular system. These results suggest that IA administration may be used to obtain widespread delivery of nanoscale therapeutics throughout the murine brain after a single

  15. Poly(amido amine)s as gene delivery vectors: effects of quaternary nicotinamide moieties in the side chains

    NARCIS (Netherlands)

    Mateos timoneda, Miguel; Lok, Martin C.; Hennink, Wim E.; Feijen, Jan; Engbersen, Johannes F.J.

    2008-01-01

    To evaluate the effect of quaternary nicotinamide pendant groups on gene delivery properties, a series of poly(amido amine) (co)polymers were synthesized by Michael addition polymerization of N, N-cystaminebisacrylamide with variable ratios of 1-(4-aminobutyl)-3-carbamoylpyridinium (Nic-BuNH2), and

  16. Ultrasound-mediated interferon β gene transfection inhibits growth of malignant melanoma

    International Nuclear Information System (INIS)

    Yamaguchi, Kazuki; Feril, Loreto B.; Tachibana, Katsuro; Takahashi, Akira; Matsuo, Miki; Endo, Hitomi; Harada, Yoshimi; Nakayama, Juichiro

    2011-01-01

    Highlights: → Successful ultrasound-mediated transfection of melanoma (C32) cells with IFN-β genes both in vitro and in vivo. → Ultrasound-mediated IFN-β transfection inhibited proliferation of melanoma cells in vitro. → Ultrasound-mediated IFN-β transfection inhibited melanoma tumor growth in vivo. -- Abstract: We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon β (IFN-β) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-β in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-β genes mixed with microbubbles. Successful sonotransfection with IFN-β gene in vitro was confirmed by ELISA, which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-β gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers.

  17. Nanolipoparticles-mediated MDR1 siRNA delivery: preparation, characterization and cellular uptake

    Directory of Open Access Journals (Sweden)

    Mahnaz Nourbakhsh

    2015-01-01

    Full Text Available Objective(s: Lipid-based nanoparticles (NLP are PEGylated carriers composed of lipids and encapsulated nucleic acids with a diameter less than 100 nm. The presence of PEG in the NLP formulation improves the particle pharmacokinetic behavior. The purpose of this study was to prepare and characterize NLPs containing MDR1 siRNA and evaluate their cytotoxicity and cellular uptake. MDR1 siRNA could be used in multidrug resistance reversal in cancer therapy. Materials and Methods: siRNAs were encapsulated into NLPs consisted of mPEG-DSPE/DOTAP/DOPE (10:50:40 molar ratio by the detergent dialysis method. The particle diameters of NLPs and their surface charge were measured using dynamic light scattering. siRNA encapsulation efficiency was determined by an indirect method via filtration and free siRNA concentration determination. NLPs cytotoxicity was investigated by MTT assay. The ability of NLPs for siRNA delivery checked in two human cell lines (MCF-7/ADR and EPP85-181/RDB by fluorescence microscopy and compared with oligofectamine. Results: NLPs containing MDR1 siRNA were prepared with the stable size of 80-90 nm and the zeta potential near to neutral. The siRNA encapsulation efficacy was more than 80%. These properties are suitable for in vivo siRNA delivery. NLPs cytotoxicity studies demonstrated they were non-toxic at the doses used. NLPs improved siRNA localization in both cell lines. Conclusion: NLPs containing MDR1 siRNA can be a good candidate for in vivo siRNA delivery studies.

  18. The Schizosaccharomyces pombe mam1 gene encodes an ABC transporter mediating secretion of M-factor

    DEFF Research Database (Denmark)

    Christensen, P U; Davey, William John; Nielsen, O

    1997-01-01

    In the fission yeast Schizosaccharomyces pombe, cells of opposite mating type communicate via diffusible peptide pheromones prior to mating. We have cloned the S. pombe mam1 gene, which encodes a 1336-amino acid protein belonging to the ATP-binding cassette (ABC) superfamily. The mam1 gene is only...... expressed in M cells and the gene product is responsible for the secretion of the mating pheromone. M-factor, a nonapeptide that is S-farnesylated and carboxy-methylated on its C-terminal cysteine residue. The predicted Mam1 protein is highly homologous to mammalian multiple drug-resistance proteins...... and to the Saccharomyces cerevisiae STE6 gene product, which mediates export of a-factor mating pheromone. We show that STE6 can also mediate secretion of M-factor in S. pombe....

  19. Recombinant human parvovirus B19 vectors: erythroid cell-specific delivery and expression of transduced genes.

    Science.gov (United States)

    Ponnazhagan, S; Weigel, K A; Raikwar, S P; Mukherjee, P; Yoder, M C; Srivastava, A

    1998-06-01

    A novel packaging strategy combining the salient features of two human parvoviruses, namely the pathogenic parvovirus B19 and the nonpathogenic adeno-associated virus type 2 (AAV), was developed to achieve erythroid cell-specific delivery as well as expression of the transduced gene. The development of such a chimeric vector system was accomplished by packaging heterologous DNA sequences cloned within the inverted terminal repeats of AAV and subsequently packaging the DNA inside the capsid structure of B19 virus. Recombinant B19 virus particles were assembled, as evidenced by electron microscopy as well as DNA slot blot analyses. The hybrid vector failed to transduce nonerythroid human cells, such as 293 cells, as expected. However, MB-02 cells, a human megakaryocytic leukemia cell line which can be infected by B19 virus following erythroid differentiation with erythropoietin (N. C. Munshi, S. Z. Zhou, M. J. Woody, D. A. Morgan, and A. Srivastava, J. Virol. 67:562-566, 1993) but lacks the putative receptor for AAV (S. Ponnazhagan, X.-S. Wang, M. J. Woody, F. Luo, L. Y. Kang, M. L. Nallari, N. C. Munshi, S. Z. Zhou, and A. Srivastava, J. Gen. Virol. 77:1111-1122, 1996), were readily transduced by this vector. The hybrid vector was also found to specifically target the erythroid population in primary human bone marrow cells as well as more immature hematopoietic progenitor cells following erythroid differentiation, as evidenced by selective expression of the transduced gene in these target cells. Preincubation with anticapsid antibodies against B19 virus, but not anticapsid antibodies against AAV, inhibited transduction of primary human erythroid cells. The efficiency of transduction of primary human erythroid cells by the recombinant B19 virus vector was significantly higher than that by the recombinant AAV vector. Further development of the AAV-B19 virus hybrid vector system should prove beneficial in gene therapy protocols aimed at the correction of inherited and

  20. Nonviral Gene Delivery of Growth and Differentiation Factor 5 to Human Mesenchymal Stem Cells Injected into a 3D Bovine Intervertebral Disc Organ Culture System

    Directory of Open Access Journals (Sweden)

    Christian Bucher

    2013-01-01

    Full Text Available Intervertebral disc (IVD cell therapy with unconditioned 2D expanded mesenchymal stem cells (MSC is a promising concept yet challenging to realize. Differentiation of MSCs by nonviral gene delivery of growth and differentiation factor 5 (GDF5 by electroporation mediated gene transfer could be an excellent source for cell transplantation. Human MSCs were harvested from bone marrow aspirate and GDF5 gene transfer was achieved by in vitro electroporation. Transfected cells were cultured as monolayers and as 3D cultures in 1.2% alginate bead culture. MSC expressed GDF5 efficiently for up to 21 days. The combination of GDF5 gene transfer and 3D culture in alginate showed an upregulation of aggrecan and SOX9, two markers for chondrogenesis, and KRT19 as a marker for discogenesis compared to untransfected cells. The cells encapsulated in alginate produced more proteoglycans expressed in GAG/DNA ratio. Furthermore, GDF5 transfected MCS injected into an IVD papain degeneration organ culture model showed a partial recovery of the GAG/DNA ratio after 7 days. In this study we demonstrate the potential of GDF5 transfected MSC as a promising approach for clinical translation for disc regeneration.

  1. Synthesis and Characterization of Cationic Glycidyl-Based Poly(aminoester-Folic Acid Targeting Conjugates and Study on Gene Delivery

    Directory of Open Access Journals (Sweden)

    Yu Che Hsiao

    2012-07-01

    Full Text Available A new poly(aminoester (EPAE-FA containing folic acid and amino groups in the backbone and side chain was synthesized. EPAE-FA self-assembled readily with the plasmid DNA (pCMV-βgal in HEPES buffer and was characterized by dynamic light scattering, zeta potential, fluorescence images, and XTT cell viability assays. To evaluate the transfection effect of graft ratio of FA on the EPAE system, EPAE-FA polymers with two different graft ratios (EPAE-FA12k and EPAE-FA14k were also prepared. This study found that all EPAE-FA polymers were able to bind plasmid DNA and yielded positively charged complexes with nano-sized particles ( < 200 nm. To assess the transfection efficiency mediated by EPAE and EPAE-FA polymers, we performed in vitro transfection activity assays using FR-negative (COS-7 and FR-positive (HeLa cells. The EPAE-FA12k/DNA and EPAE-FA14k/DNA complexes were able to transfect HeLa cell in vitro with higher transfection efficiency than PEI25k/DNA at the similar weight ratio. These results demonstrated that the introduction of FA into EPAE system had a significant effect on transferring ability for FR-positive cells (HeLa. Examination of the cytotoxicity of PEI25k and EPAE-FA system revealed that EPAE-FA system had lower cytotoxicity. In this paper, EPAE-FA seemed to be a novel cationic poly(aminoester for gene delivery and an interesting candidate for further study.

  2. Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer.

    OpenAIRE

    Chang, Keejong; Qian, Jin; Jiang, MeiSheng; Liu, Yi-Hsin; Wu, Ming-Che; Chen, Chi-Dar; Lai, Chao-Kuen; Lo, Hsin-Lung; Hsiao, Chin-Ton; Brown, Lucy; Bolen, James; Huang, Hsiao-I; Ho, Pei-Yu; Shih, Ping Yao; Yao, Chen-Wen

    2002-01-01

    Abstract Background Transgenic animals have become valuable tools for both research and applied purposes. The current method of gene transfer, microinjection, which is widely used in transgenic mouse production, has only had limited success in producing transgenic animals of larger or higher species. Here, we report a linker based sperm-mediated gene transfer method (LB-SMGT) that greatly improves the production efficiency of large transgenic animals. Results The linker protein, a monoclonal ...

  3. MicroRNA-Mediated Gene Silencing in Plant Defense and Viral Counter-Defense

    OpenAIRE

    Liu, Sheng-Rui; Zhou, Jing-Jing; Hu, Chun-Gen; Wei, Chao-Ling; Zhang, Jin-Zhi

    2017-01-01

    MicroRNAs (miRNAs) are non-coding RNAs of approximately 20–24 nucleotides in length that serve as central regulators of eukaryotic gene expression by targeting mRNAs for cleavage or translational repression. In plants, miRNAs are associated with numerous regulatory pathways in growth and development processes, and defensive responses in plant–pathogen interactions. Recently, significant progress has been made in understanding miRNA-mediated gene silencing and how viruses counter this defense ...

  4. Optimization of cationic lipid mediated gene transfer: structure-function, physico-chemical, and cellular studies.

    Science.gov (United States)

    Carrière, Marie; Tranchant, Isabelle; Niore, Pierre-Antoine; Byk, Gerardo; Mignet, Nathalie; Escriou, Virginie; Scherman, Daniel; Herscovici, Jean

    2002-01-01

    The rationale design aimed at the enhancement of cationic lipid mediated gene transfer is discussed. These improvements are based on the straight evaluation of the structure-activity relationship and on the introduction of new structures. Much attention have been given to the supramolecular structures of the lipid/DNA complexes, to the effect of serum on gene transfer and to the intracellular trafficking of the lipoplexes. Finally new avenue using reducible cationic lipids has been discussed.

  5. Ultrasound-mediated transdermal drug delivery: mechanisms, scope, and emerging trends.

    Science.gov (United States)

    Polat, Baris E; Hart, Douglas; Langer, Robert; Blankschtein, Daniel

    2011-06-30

    The use of ultrasound for the delivery of drugs to, or through, the skin is commonly known as sonophoresis or phonophoresis. The use of therapeutic and high frequencies of ultrasound (≥0.7MHz) for sonophoresis (HFS) dates back to as early as the 1950s, while low-frequency sonophoresis (LFS, 20-100kHz) has only been investigated significantly during the past two decades. Although HFS and LFS are similar because they both utilize ultrasound to increase the skin penetration of permeants, the mechanisms associated with each physical enhancer are different. Specifically, the location of cavitation and the extent to which each process can increase skin permeability are quite dissimilar. Although the applications of both technologies are different, they each have strengths that could allow them to improve current methods of local, regional, and systemic drug delivery. In this review, we will discuss the mechanisms associated with both HFS and LFS, specifically concentrating on the key mechanistic differences between these two skin treatment methods. Background on the relevant physics associated with ultrasound transmitted through aqueous media will also be discussed, along with implications of these phenomena on sonophoresis. Finally, a thorough review of the literature is included, dating back to the first published reports of sonophoresis, including a discussion of emerging trends in the field. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Graphene nanoribbons as a drug delivery agent for lucanthone mediated therapy of glioblastoma multiforme.

    Science.gov (United States)

    Chowdhury, Sayan Mullick; Surhland, Cassandra; Sanchez, Zina; Chaudhary, Pankaj; Suresh Kumar, M A; Lee, Stephen; Peña, Louis A; Waring, Michael; Sitharaman, Balaji; Naidu, Mamta

    2015-01-01

    We report use of PEG-DSPE coated oxidized graphene nanoribbons (O-GNR-PEG-DSPE) as agent for delivery of anti-tumor drug Lucanthone (Luc) into Glioblastoma Multiformae (GBM) cells targeting base excision repair enzyme APE-1 (Apurinic endonuclease-1). Lucanthone, an endonuclease inhibitor of APE-1, was loaded onto O-GNR-PEG-DSPEs using a simple non-covalent method. We found its uptake by GBM cell line U251 exceeding 67% and 60% in APE-1-overexpressing U251, post 24h. However, their uptake was ~38% and 29% by MCF-7 and rat glial progenitor cells (CG-4), respectively. TEM analysis of U251 showed large aggregates of O-GNR-PEG-DSPE in vesicles. Luc-O-GNR-PEG-DSPE was significantly toxic to U251 but showed little/no toxicity when exposed to MCF-7/CG-4 cells. This differential uptake effect can be exploited to use O-GNR-PEG-DSPEs as a vehicle for Luc delivery to GBM, while reducing nonspecific cytotoxicity to the surrounding healthy tissue. Cell death in U251 was necrotic, probably due to oxidative degradation of APE-1. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Microemulsions mediated effective delivery of methotrexate hydrogel: more than a tour de force in psoriasis therapeutics.

    Science.gov (United States)

    Amarji, Basant; Garg, Neeraj K; Singh, Bhupinder; Katare, Om Prakash

    2016-01-01

    Methotrexate (MTX), a well known drug for the treatment of cancer and rheumatoid arthritis, has gained prominence in the treatment of psoriasis over the period of years. However, the present mode of systemic administration through oral or parenteral route has always proposition, full of compromises. The toxicity of drug to the vital organs and physiological environment is the major concern. Also, its poor skin penetration is one major problem. Hence novel system based on lipid carriers has been considered here to overcome the barriers. Microemulsions (MEs) were prepared using pseudo-ternary phase diagram (PTPD) and they were characterized for various parameters such as size, shape (cryo-SEM), PDI, zeta potential, etc. The chosen MEs system (optimized) was then incorporated into secondary vehicles and characterized for rheological behavior, texture profile analysis, in vitro release, ex vivo permeation and drug distribution into different layers of skin. The developed formulations were further evaluated in ex vivo and in vivo such as cell line study, imiquimod-induced psoriatic model, allergic contact dermatitis, rat tail model (% orthokeratosis) and safety test (Draize test). The MEs based MTX gel has shown its potential in locating the drug at the desired domain of stratum corneum, epidermal and dermal layers of skin and reducing systemic absorption. Our results are suggestive of MEs potential as a novel carrier for topical delivery of MTX in topical therapeutic and safety approaches. In conclusion, developed MEs-based hydrogel has shown promising results in achieving effective delivery of MTX.

  8. Delivery of carboplatin by carbon-based nanocontainers mediates increased cancer cell death

    Energy Technology Data Exchange (ETDEWEB)

    Arlt, M; Fuessel, S; Kraemer, K; Wirth, M P [Department for Urology, University Hospital Carl Gustav Carus, Technische Universitaet Dresden, Fetscherstrasse 74, 01307 Dresden (Germany); Haase, D; Hampel, S; Oswald, S; Bachmatiuk, A; Klingeler, R; Ritschel, M; Leonhardt, A; Buechner, B [Leibniz Institute for Solid State and Materials Research (IFW), Helmholtzstrasse 20, 01069 Dresden (Germany); Schulze, R, E-mail: kai.kraemer@uniklinikum-dresden.de [Bioanalytical Chemistry, Technische Universitaet Dresden, Bergstrasse 66, 01069 Dresden (Germany)

    2010-08-20

    Since the activity of several conventional anticancer drugs is restricted by resistance mechanisms and dose-limiting side-effects, the design of nanocarriers seems to be an efficient and promising approach for drug delivery. Their chemical and mechanical stability and their possible multifunctionality render tubular nanomaterials, such as carbon nanotubes (CNTs) and carbon nanofibres (CNFs), promising delivery agents for anticancer drugs. The goal of the present study was to investigate CNTs and CNFs in order to deliver carboplatin in vitro. No significant intrinsic toxicity of unloaded materials was found, confirming their biocompatibility. Carboplatin was loaded onto CNTs and CNFs, revealing a loading yield of 0.20 mg (CNT-CP) and 0.13 mg (CNF-CP) platinum per milligram of material. The platinum release depended on the carrier material. Whereas CNF-CP marginally released the drug, CNT-CP functioned as a drug depot, constantly releasing up to 68% within 14 days. The cytotoxicity of CNT-CP and CNF-CP in urological tumour cell lines was dependent on the drug release. CNT-CP was identified to be more effective than CNF-CP concerning the impairment of proliferation and clonogenic survival of tumour cells. Moreover, carboplatin, which was delivered by CNT-CP, exhibited a higher anticancer activity than free carboplatin.

  9. Killing of intracellular Mycobacterium tuberculosis by receptor-mediated drug delivery

    International Nuclear Information System (INIS)

    Majumdar, S.; Basu, S.K.

    1991-01-01

    p-Aminosalicylic acid (PAS) conjugated to maleylated bovine serum albumin (MBSA) was taken up efficiently through high-affinity MBSA-binding sites on macrophages. Binding of the radiolabeled conjugate to cultured mouse peritoneal macrophages at 4 degrees C was competed for by MBSA but not by PAS. At 37 degrees C, the radiolabeled conjugate was rapidly degraded by the macrophages, leading to release of acid-soluble degradation products in the medium. The drug conjugate was nearly 100 times as effective as free PAS in killing the intracellular mycobacteria in mouse peritoneal macrophages infected in culture with Mycobacterium tuberculosis. The killing of intracellular mycobacteria mediated by the drug conjugate was effectively prevented by simultaneous addition of excess MBSA (100 micrograms/ml) or chloroquine (3 microM) to the medium, whereas these agents did not affect the microbicidal action of free PAS. These results suggest that (i) uptake of the PAS-MBSA conjugate was mediated by cell surface receptors on macrophages which recognize MBSA and (ii) lysosomal hydrolysis of the internalized conjugate resulted in intracellular release of a pharmacologically active form of the drug, which led to selective killing of the M. tuberculosis harbored by mouse macrophages infected in culture. This receptor-mediated modality of delivering drugs to macrophages could contribute to greater therapeutic efficacy and minimization of toxic side effects in the management of tuberculosis and other intracellular mycobacterial infections

  10. Chloroquine mediated modulation of Anopheles gambiae gene expression.

    Directory of Open Access Journals (Sweden)

    Patrícia Abrantes

    2008-07-01

    Full Text Available Plasmodium development in the mosquito is crucial for malaria transmission and depends on the parasite's interaction with a variety of cell types and specific mosquito factors that have both positive and negative effects on infection. Whereas the defensive response of the mosquito contributes to a decrease in parasite numbers during these stages, some components of the blood meal are known to favor infection, potentiating the risk of increased transmission. The presence of the antimalarial drug chloroquine in the mosquito's blood meal has been associated with an increase in Plasmodium infectivity for the mosquito, which is possibly caused by chloroquine interfering with the capacity of the mosquito to defend against the infection.In this study, we report a detailed survey of the Anopheles gambiae genes that are differentially regulated by the presence of chloroquine in the blood meal, using an A. gambiae cDNA microarray. The effect of chloroquine on transcript abundance was evaluated separately for non-infected and Plasmodium berghei-infected mosquitoes. Chloroquine was found to affect the abundance of transcripts that encode proteins involved in a variety of processes, including immunity, apoptosis, cytoskeleton and the response to oxidative stress. This pattern of differential gene expression may explain the weakened mosquito defense response which accounts for the increased infectivity observed in chloroquine-treated mosquitoes.The results of the present study suggest that chloroquine can interfere with several putative mosquito mechanisms of defense against Plasmodium at the level of gene expression and highlight the need for a better understanding of the impacts of antimalarial agents on parasite transmission.

  11. PLGA nanoparticle-mediated delivery of tumor antigenic peptides elicits effective immune responses

    Directory of Open Access Journals (Sweden)

    Ma W

    2012-03-01

    Full Text Available Wenxue Ma1, Mingshui Chen1, Sharmeela Kaushal1,2, Michele McElroy1,2, Yu Zhang3, Cengiz Ozkan3, Michael Bouvet1,2, Carol Kruse4, Douglas Grotjahn5, Thomas Ichim6, Boris Minev1,7,81Moores Cancer Center, University of California San Diego, 2Department of Surgery, University of California San Diego, 3Laboratory of Biomaterials and Nanotechnology, University of California Riverside, 4UCLA Division of Neurosurgery, Los Angeles, 5Chemistry Department, San Diego State University, San Diego, 6MediStem Inc. San Diego, 7UCSD Division of Neurosurgery, San Diego, 8Genelux Corporation, San Diego, CA, USA Abstract: The peptide vaccine clinical trials encountered limited success because of difficulties associated with stability and delivery, resulting in inefficient antigen presentation and low response rates in patients with cancer. The purpose of this study was to develop a novel delivery approach for tumor antigenic peptides in order to elicit enhanced immune responses using poly(DL-lactide-co-glycolide nanoparticles (PLGA-NPs encapsulating tumor antigenic peptides. PLGA-NPs were made using the double emulsion-solvent evaporation method. Artificial antigen-presenting cells were generated by human dendritic cells (DCs loaded with PLGA-NPs encapsulating tumor antigenic peptide(s. The efficiency of the antigen presentation was measured by interferon-γ ELISpot assay (Vector Laboratories, Burlingame, CA. Antigen-specific cytotoxic T lymphocytes (CTLs were generated and evaluated by CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Fitchburg, WI. The efficiency of the peptide delivery was compared between the methods of emulsification in incomplete Freund’s adjuvant and encapsulation in PLGA-NPs. Our results showed that most of the PLGA-NPs were from 150 nm to 500 nm in diameter, and were negatively charged at pH 7.4 with a mean zeta potential of -15.53 ± 0.71 mV; the PLGA-NPs could be colocalized in human DCs in 30 minutes of incubation. Human DCs

  12. Monocyte-mediated delivery of polymeric backpacks to inflamed tissues: a generalized strategy to deliver drugs to treat inflammation.

    Science.gov (United States)

    Anselmo, Aaron C; Gilbert, Jonathan B; Kumar, Sunny; Gupta, Vivek; Cohen, Robert E; Rubner, Michael F; Mitragotri, Samir

    2015-02-10

    Targeted delivery of drugs and imaging agents to inflamed tissues, as in the cases of cancer, Alzheimer's disease, Parkinson's disease, and arthritis, represents one of the major challenges in drug delivery. Monocytes possess a unique ability to target and penetrate into sites of inflammation. Here, we describe a broad approach to take advantage of the natural ability of monocytes to target and deliver flat polymeric particles ("Cellular Backpacks") to inflamed tissues. Cellular backpacks attach strongly to the surface of monocytes but do not undergo phagocytosis due to backpack's size, disk-like shape and flexibility. Following attachment of backpacks, monocytes retain important cellular functions including transmigration through an endothelial monolayer and differentiation into macrophages. In two separate in vivo inflammation models, backpack-laden monocytes exhibit increased targeting to inflamed tissues. Cellular backpacks, and their abilities to attach to monocytes without impairing monocyte functions and 'hitchhike' to a variety of inflamed tissues, offer a new platform for both cell-mediated therapies and broad targeting of inflamed tissues. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Nanolayered hybrid mediates synergistic co-delivery of ligand and ligation activator for inducing stem cell differentiation and tissue healing.

    Science.gov (United States)

    Kang, Heemin; Kim, Minkyu; Feng, Qian; Lin, Sien; Wei, Kongchang; Li, Rui; Choi, Chan Ju; Kim, Tae-Hyun; Li, Gang; Oh, Jae-Min; Bian, Liming

    2017-12-01

    Cellular behaviors, such as differentiation, are regulated by complex ligation processes involving cell surface receptors, which can be activated by various divalent metal cations. The design of nanoparticle for co-delivery of ligand and ligation activator can offer a novel strategy to synergistically stimulate ligation processes in vivo. Here, we present a novel layered double hydroxide (LDH)-based nanohybrid (MgFe-Ado-LDH), composed of layered MgFe hydroxide nanocarriers sandwiching the adenosine cargo molecule, maintained through an electrostatic balance, to co-deliver the adenosine (Ado) ligand from the interlayer spacing and the Mg 2+ ion (ligation activator) through the dissolution of the MgFe nanocarrier itself. Our findings demonstrate that the MgFe-Ado-LDH nanohybrid promoted osteogenic differentiation of stem cells through the synergistic activation of adenosine A2b receptor (A2bR) by the dual delivery of adenosine and Mg 2+ ions, outperforming direct supplementation of adenosine alone. Furthermore, the injection of the MgFe-Ado-LDH nanohybrid and stem cells embedded within hydrogels promoted the healing of rat tibial bone defects through the rapid formation of fully integrated neo-bone tissue through the activation of A2bR. The newly formed bone tissue displayed the key features of native bone, including calcification, mature tissue morphology, and vascularization. This study demonstrates a novel and effective strategy of bifunctional nanocarrier-mediated delivery of ligand (cargo molecule) and activation of its ligation to receptor by the nanocarrier itself for synergistically inducing stem cell differentiation and tissue healing in vivo, thus offering novel design of biomaterials for regenerative medicine. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. An injectable elastin-based gene delivery platform for dose-dependent modulation of angiogenesis and inflammation for critical limb ischemia.

    Science.gov (United States)

    Dash, Biraja C; Thomas, Dilip; Monaghan, Michael; Carroll, Oliver; Chen, Xizhe; Woodhouse, Kimberly; O'Brien, Timothy; Pandit, Abhay

    2015-10-01

    Critical limb ischemia is a major clinical problem. Despite rigorous treatment regimes, there has been only modest success in reducing the rate of amputations in affected patients. Reduced level of blood flow and enhanced inflammation are the two major pathophysiological changes that occur in the ischemic tissue. The objective of this study was to develop a controlled dual gene delivery system capable of delivering therapeutic plasmid eNOS and IL-10 in a temporal manner. In order to deliver multiple therapeutic genes, an elastin-like polypeptide (ELP) based injectable system was designed. The injectable system was comprised of hollow spheres and an in situ-forming gel scaffold of elastin-like polypeptide capable of carrying gene complexes, with an extended manner release profile. In addition, the ELP based injectable system was used to deliver human eNOS and IL-10 therapeutic genes in vivo. A subcutaneous dose response study showed enhanced blood vessel density in the treatment groups of eNOS (20 μg) and IL-10 (10 μg)/eNOS (20 μg) and reduced inflammation with IL-10 (10 μg) alone. Next, we carried out a hind-limb ischemia model comparing the efficacy of the following interventions; Saline; IL-10, eNOS and IL-10/eNOS. The selected dose of eNOS, exhibited enhanced angiogenesis. IL-10 treatment groups showed reduction in the level of inflammatory cells. Furthermore, we demonstrated that eNOS up-regulated major proangiogenic growth factors such as vascular endothelial growth factors, platelet derived growth factor B, and fibroblast growth factor 1, which may explain the mechanism of this approach. These factors help in formation of a stable vascular network. Thus, ELP injectable system mediating non-viral delivery of human IL10-eNOS is a promising therapy towards treating limb ischemia. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Transcriptome-Wide Analysis of Hepatitis B Virus-Mediated Changes to Normal Hepatocyte Gene Expression.

    Directory of Open Access Journals (Sweden)

    Jason Lamontagne

    2016-02-01

    Full Text Available Globally, a chronic hepatitis B virus (HBV infection remains the leading cause of primary liver cancer. The mechanisms leading to the development of HBV-associated liver cancer remain incompletely understood. In part, this is because studies have been limited by the lack of effective model systems that are both readily available and mimic the cellular environment of a normal hepatocyte. Additionally, many studies have focused on single, specific factors or pathways that may be affected by HBV, without addressing cell physiology as a whole. Here, we apply RNA-seq technology to investigate transcriptome-wide, HBV-mediated changes in gene expression to identify single factors and pathways as well as networks of genes and pathways that are affected in the context of HBV replication. Importantly, these studies were conducted in an ex vivo model of cultured primary hepatocytes, allowing for the transcriptomic characterization of this model system and an investigation of early HBV-mediated effects in a biologically relevant context. We analyzed differential gene expression within the context of time-mediated gene-expression changes and show that in the context of HBV replication a number of genes and cellular pathways are altered, including those associated with metabolism, cell cycle regulation, and lipid biosynthesis. Multiple analysis pipelines, as well as qRT-PCR and an independent, replicate RNA-seq analysis, were used to identify and confirm differentially expressed genes. HBV-mediated alterations to the transcriptome that we identified likely represent early changes to hepatocytes following an HBV infection, suggesting potential targets for early therapeutic intervention. Overall, these studies have produced a valuable resource that can be used to expand our understanding of the complex network of host-virus interactions and the impact of HBV-mediated changes to normal hepatocyte physiology on viral replication.

  16. RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe

    Energy Technology Data Exchange (ETDEWEB)

    Smialowska, Agata, E-mail: smialowskaa@gmail.com [Center for Biosciences, Department of Biosciences and Nutrition, Karolinska Institute, Huddinge 141-83 (Sweden); School of Life Sciences, Södertörn Högskola, Huddinge 141-89 (Sweden); Djupedal, Ingela; Wang, Jingwen [Center for Biosciences, Department of Biosciences and Nutrition, Karolinska Institute, Huddinge 141-83 (Sweden); Kylsten, Per [School of Life Sciences, Södertörn Högskola, Huddinge 141-89 (Sweden); Swoboda, Peter [Center for Biosciences, Department of Biosciences and Nutrition, Karolinska Institute, Huddinge 141-83 (Sweden); Ekwall, Karl, E-mail: Karl.Ekwall@ki.se [Center for Biosciences, Department of Biosciences and Nutrition, Karolinska Institute, Huddinge 141-83 (Sweden); School of Life Sciences, Södertörn Högskola, Huddinge 141-89 (Sweden)

    2014-02-07

    Highlights: • Protein coding genes accumulate anti-sense sRNAs in fission yeast S. pombe. • RNAi represses protein-coding genes in S. pombe. • RNAi-mediated gene repression is post-transcriptional. - Abstract: RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its role in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe.

  17. Microfluidic electroporation for delivery of small molecules and genes into cells using a common DC power supply.

    Science.gov (United States)

    Wang, Hsiang-Yu; Lu, Chang

    2008-06-15

    Electroporation is an efficient method of introducing foreign impermeant molecules such as drugs and genes into cells. Conventional electroporation has been based on the application of short electrical pulses (electropulsation). Electropulsation requires specialized equipment and cannot be integrated easily with techniques such as electrophoresis which is based on constant voltage. Here we demonstrate the delivery of small molecules and genes into cells, using a microfluidic electroporation technique based on constant direct current (DC) voltage that we developed earlier. We demonstrate the delivery of two molecules into Chinese hamster ovary (CHO-K1) cells: a membrane impermeable nucleic acid dye (SYTOX Green) and a plasmid vector carrying the gene for green fluorescent protein (pEGFP-C1). Our devices can exert field variations to flowing cells that are analogous to the application of single or multiple pulses by having different geometries. We investigate the effects of the electrical parameters and different geometries of the device on the transfection efficiency and cell viability. Our technique provides a simple solution to electroporation-based drug and gene delivery by eliminating the need for a pulse generator. We envision that these simple microscale electroporation devices will have the potential to work in parallel on a microchip platform and such technology will allow high-throughput functional screening of drugs and genes. (c) 2008 Wiley Periodicals, Inc.

  18. Coating barium titanate nanoparticles with polyethylenimine improves cellular uptake and allows for coupled imaging and gene delivery.

    Science.gov (United States)

    Dempsey, Christopher; Lee, Isac; Cowan, Katie R; Suh, Junghae

    2013-12-01

    Barium titanate nanoparticles (BT NP) belong to a class of second harmonic generating (SHG) nanoprobes that have recently demonstrated promise in biological imaging. Unfortunately, BT NPs display low cellular uptake efficiencies, which may be a problem if cellular internalization is desired or required for a particular application. To overcome this issue, while concomitantly developing a particle platform that can also deliver nucleic acids into cells, we coated the BT NPs with the cationic polymer polyethylenimine (PEI)-one of the most effective nonviral gene delivery agents. Coating of BT with PEI yielded complexes with positive zeta potentials and resulted in an 8-fold increase in cellular uptake of the BT NPs. Importantly, we were able to achieve high levels of gene delivery with the BT-PEI/DNA complexes, supporting further efforts to generate BT platforms for coupled imaging and gene therapy. Copyright © 2013 Elsevier B.V. All rights reserved.