共查询到20条相似文献,搜索用时 15 毫秒
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基因治疗为治疗先天性遗传疾病和严重后天获得性疾病提供了一条新途径.目前,基因载体分为两类:病毒载体和非病毒载体.病毒载体转染效率高,但由于某些病毒载体存在免疫原性、致癌性、宿主DNA插入整合等缺点,从而限制了它们的应用.非病毒载体具有价格低、制备简单、安全有效、无免疫原性等优点,成为基因载体研究的热点.阳离子多聚物是非病毒载体的典型代表.文中综述近年来阳离子多聚物作为基因载体的研究现状和进展,重点介绍了阳离子多聚物基因载体的分类和与DNA的相互作用和传递机制. 相似文献
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Nonviral vector–based gene therapy is a promising strategy for treating a myriad of diseases. Cell‐penetrating peptides are gaining increasing attention as vectors for nucleic acid delivery. However, most studies have focused more on the transfection efficiency of these vectors than on their specificity and toxicity. To obtain ideal vectors with high efficiency and safety, we constructed the vector stearyl‐TH by attaching a stearyl moiety to the N‐terminus of the acid‐activated cell penetrating peptide TH in this study. Under acidic conditions, stearyl‐TH could bind to and condense plasmids into nanoparticle complexes, which displayed significantly enhanced cellular uptake and transfection efficiencies. In contrast, stearyl‐TH lost the capacities of DNA binding and transfection at physiological pH. More importantly, stearyl‐TH and the complexes formed by stearyl‐TH and plasmids displayed no obvious toxicity at physiological pH. Consequently, the high transfection efficiency under acidic conditions and low toxicity make stearyl‐TH a potential nucleic acid delivery vector for gene therapy. 相似文献
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《Autophagy》2013,9(5):667-682
Cationic liposome (lipoplex) and polymer (polyplex)-based vectors have been developed for nonviral gene delivery. These vectors bind DNA and enter cells via endosomes, but intracellular transfer of DNA to the nucleus is inefficient. Here we show that lipoplex and polyplex vectors enter cells in endosomes, activate autophagy and generate tubulovesicular autophagosomes. Activation of autophagy was dependent on ATG5, resulting in lipidation of LC3, but did not require the PtdIns 3-kinase activity of PIK3C3/VPS34. The autophagosomes generated by lipoplex fused with each other, and with endosomes, resulting in the delivery of vectors to large tubulovesicular autophagosomes, which accumulated next to the nucleus. The tubulovesicular autophagosomes contained autophagy receptor protein SQSTM1/p62 and ubiquitin, suggesting capture of autophagy cargoes, but fusion with lysosomes was slow. Gene delivery and expression from both lipoplex and polyplex increased 8-fold in atg5?/? cells unable to generate tubulovesicular autophagosomes. Activation of autophagy and capture within tubulovesicular autophagosomes therefore provides a new cellular barrier against efficient gene transfer and should be considered when designing efficient nonviral gene delivery vectors. 相似文献
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Walther W Stein U Fichtner I Voss C Schmidt T Schleef M Nellessen T Schlag PM 《Molecular biotechnology》2002,21(2):105-115
Jet-injection has become an applicable technology among other established nonviral delivery systems, such as particle bombardment
or in vivo electroporation. The low-volume jet injector employed in this study uses compressed air to inject solutions of
1.5–10 μL containing naked DNA into the desired tissue. The novel design of this prototype makes multiple jet-injections possible.
Therefore, repeated jet-injections into one target tissue can be performed easily. This jet-injector hand-held system was
used for the direct in vivo gene transfer of plasmid DNA into tumors to achieve efficient expression of reporter genes (β-galactosidase,
green fluorescent protein [GFP]) and of therapeutic genes (TNF-α) in different tumor models. The study presented here revealed
the key parameters of efficient in vivo jet-injection (jet-injection volume, pressure, jet penetration, DNA stability) to
define the optimal conditions for a jet-injection-aided nonviral gene therapy. 相似文献
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Fazhan Wang Zhou Qin Hansi Lu Siyan He Jing Luo Chaohui Jin Xiangrong Song 《The journal of gene medicine》2019,21(7)
Gene therapy has recently witnessed accelerated progress as a new therapeutic strategy with the potential to treat a range of inherited and acquired diseases. Billions of dollars have been invested in basic and clinical research on gene medicine, with ongoing clinical trials focused on cancer, monogenic diseases, cardiovascular diseases and other refractory diseases. Advances addressing the inherent challenges of gene therapy, particularly those related to retaining the delivery efficacy and minimizing unwanted immune responses, provide the basis for the widespread clinical application of gene medicine. Several types of genes delivered by viral or non‐viral delivery vectors have demonstrated encouraging results in both animals and humans. As augmented by clinical indications, gene medicine techniques have rapidly become a promising alternative to conventional therapeutic strategies because of their better clinical benefit and lower toxicities. Their application in the clinic has been extensive as a result of the approval of many gene therapy drugs in recent years. In this review, we provide a comprehensive overview of the clinical translation of gene medicine, focusing on the key events and latest progress made regarding clinical gene therapy products. We also discuss the gene types and non‐viral materials with respect to developing gene therapeutics in clinical trials. 相似文献
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Lei Chen Huayu Tian Jie Chen Xuesi Chen Yubin Huang Xiabin Jing 《The journal of gene medicine》2010,12(1):64-76
Background
The application of polyethylenimine (PEI) in gene delivery has been severely limited by significant cytotoxicity that results from a nondegradable methylene backbone and high cationic charge density. It is therefore necessary to develop novel biodegradable PEI derivates for low‐toxic, highly efficient gene delivery.Methods
A series of novel cationic copolymers with various charge density were designed and synthesized by grafting different kinds of oligoethylenimine (OEI) onto a determinate multi‐armed poly(L ‐glutamic acid) backbone. The molecular structures of multi‐armed poly(L ‐glutamic acid)‐graft‐OEI (MP‐g‐OEI) copolymers were characterized using nuclear magnetic resonance, viscosimetry and gel permeation chromatography. Moreover, the MP‐g‐OEI/DNA complexes were measured by a gel retardation assay, dynamic light scattering and atomic force microscopy to determine DNA binding ability, particle size, zeta potential, complex formation and shape, respectively. MP‐g‐OEI copolymers were also evaluated in Chinese hamster ovary and human embryonic kidney‐293 cells for their cytotoxicity and transfection efficiency.Results
The particle sizes of MP‐g‐OEI/DNA complexes were in a range of 109.6–182.6 nm and the zeta potentials were in a range of 29.2–44.5 mV above the N/P ratio of 5. All the MP‐g‐OEI copolymers exhibited lower cytotoxicity and higher gene transfection efficiency than PEI25k in the absence and presence of serum with different cell lines. Importantly, the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay revealed that the cytotoxicity of MP‐g‐OEI copolymers varied with their molecular weight and charge density, and two of MP‐g‐OEI copolymers (OEI600‐MP and OEI1800‐MP) could achieve optimal transfection efficiency at a similar low N/P ratio as that for PEI25k.Conclusions
MP‐g‐OEI copolymers demonstrated considerable potential as nonviral vectors for gene therapy. Copyright © 2009 John Wiley & Sons, Ltd. 相似文献10.
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基于EBNA1和oriP的载体在基因治疗中的应用研究进展 总被引:2,自引:0,他引:2
非病毒载体用于基因治疗的主要问题是导入靶细胞的效率较低,目的基因表达水平低,疗效持续时间也较短。EBNA1和oriP元件使引入该元件的质粒在真核细胞内保持为游离体、转运入核、转录增强。质粒携带的目的基因能够获得较高的转染效率,高水平、持续的表达。基于EBNA1/oriP的质粒在肿瘤、单基因缺陷先天性疾病、TNF相关的炎性疾病的基因治疗中显示了良好的应用前景。EBNA1/oriP元件用于构建人工染色体,携带目的基因的调控序列,可望实现可调控的基因治疗。 相似文献
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We have previously established mouse genetic models and identified the genetic components of quantitative trait loci (QTL)
on mouse chromosomes that contribute to phenotypes such as bone size, bone density, and bone's anabolic response to mechanical
loading. However, these regions contain dozens of unknown genes that are needed for functional testing. In this study, we
provided a protocol of nucleoporation with high efficiency by using a commercial nucleofection buffer and Gene Pulser to deliver
a test gene into bone cells for functional studies. We cloned an osteoblast differentiation-specific geneosterix (Osx) from a mouse bone cDNA library into a pHGCX expression vector and used nucleoporation to deliver pHGCX/Flag-Osx into the
nuclei of MC3T3-E1 cells. We then examined the transfection efficiency transgene expression, and function. Our results have
demonstrated that nucleoporation can deliver a transgene into MC3T3-E1 osteoblast cells with approx 94% transfection efficiency,
and express a functional Flag-Osx fusion protein capable of inducing cell differentiation as measured by an incease in alkaline
phosphatase (ALP) activity. Therefore, this experimental system provides a rapid, safe, and efficient cell-based model of
high-throughput phenotypic screening to identify candidate genes from physically mapped regions that are important for osteoblast
differentiation. 相似文献
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Optimization of a nonviral transfection system to evaluate Cox‐2 controlled interleukin‐4 expression for osteoarthritis gene therapy in vitro 下载免费PDF全文
Annemarie Lang Johannes Neuhaus Moritz Pfeiffenberger Erik Schröder Igor Ponomarev Yvonne Weber Timo Gaber Michael F. G. Schmidt 《The journal of gene medicine》2014,16(11-12):352-363
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Kun Ma Minxin Hu Meng Xie Haijun Shen Liyan Qiu Weimin Fan Hongying Sun Shuqing Chen Yi Jin 《The journal of gene medicine》2010,12(8):669-680
Background
Nuclear membrane is one of the main barriers in polymer mediated intracellular gene delivery. To improve the transgenic activity and safety of nonviral vector, triamcinolone acetonide (TA) as a nuclear localization signal was conjugated with different molecular weight polyethylenimine (PEI).Methods
Different molecular weight PEI [600, 1800, 25 000 (25k)] was conjugated with TA to synthesize PEI‐TA by two‐step reaction. Their physicochemical characteristics, in vitro cytotoxicity and transfection efficiency were evaluated. To investigate the difference of transfection efficiency of various molecular weight PEI‐TA, their transfection mechanism was further investigated by confocal microscopy and competition assay. Transgenic expression in vivo was evaluated by injection into hepatic portal vein of mice.Results
All PEI‐TA could form nanosize polyplexes with DNA and their physicochemical properties resemble each other. Their cytotoxicities were negligible compared to PEI 25k. The order of transfection efficiency was PEI 1800‐TA > PEI 600‐TA > PEI 25k‐TA. A transfection mechanism study displayed that TA could inhibit considerably the transgenic activity of PEI 1800‐TA and PEI 600‐TA, but that of PEI 25k‐TA was not inhibited. It was suggested that PEI 1800‐TA and PEI 600‐TA might translocate into the nucleus. Confocal microscopy investigation verified this suggestion. The data strongly suggested that the transfection efficiency of PEI 1800‐TA in vivo was much higher than that of PEI 25k, which was consistent with the results obtained in vitro.Conclusions
Low molecular weight PEI‐TA could translocate into the nucleus efficiently. PEI 1800‐TA presented higher transgenic activity and it has a great potential for gene therapy as a nonviral carrier. Copyright © 2010 John Wiley & Sons, Ltd. 相似文献19.
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Rudolph C Ortiz A Schillinger U Jauernig J Plank C Rosenecker J 《The journal of gene medicine》2005,7(1):59-66
BACKGROUND: Polyethylenimine (PEI) has been successfully used for gene delivery to the lungs of mice via aerosol application using a whole body nebulization device. In this report we optimized the design of such an aerosol device. METHODS: Aerosol devices were constructed as either serial inhalation apparatus or as a whole body nebulization chamber connected to an aerosol spacer placed in a horizontal or vertical position. PEI-based gene vectors were nebulized using a standard jet nebulizer and luciferase gene expression of various tissues was examined. RESULTS: Using a whole body aerosol device resulted in luciferase gene expression in the lungs of mice at the same level as compared with a serial inhalation apparatus. Whereas gene expression was enhanced in the presence of 5% CO(2)-in-air, anesthesia of mice strongly decreased gene expression even when mice were intubated with an intravascular cannula. Reduction of the median mass aerodynamic diameter (MMAD) of the aerosol from 3.4 to 0.27 microm by interposition of an aerosol spacer increased gene expression significantly 3-fold. Drying of the aerosol by silica gel additionally increased gene delivery significantly 3-fold. Reporter gene expression mediated by branched PEI 25 kDa was 9- and 15-fold higher as compared with linear PEIs of 22 and 25 kDa, respectively, and was dependent on the DNA concentration. Gene expression was detectable as soon as 6 h after gene vector application and reached a maximum after 72 h but was still detectable after 14 days. The presence of Zn(2+) did not increase gene expression. CONCLUSION: We propose aerosol drying as a novel and simple method of optimizing PEI-based gene delivery to the lungs. 相似文献