层层自组装纳米粒作为非病毒基因载体

基因治疗的效果严重依赖于基因载体。与传统包封技术相比,在自组装技术基础上发展起来的以DNA为聚阴离子,与荷正电的高分子材料在溶液中形成纳米粒的方法,已成为目前最重要的非病毒基因载体制备手段,具有良好的应用前景。采用层层自组装(layer-by-layer assembly,LbL)技术可提高基因装载率,其优势还在于纳米粒表面性质的可控性:在温和的条件下实现多种材料在载体表面的固定,实现载体多功能化等。本文将对近年来国内外有关层层自组装纳米粒作为非病毒基因载体的研究进展以及本课题组在此方向的研究进行简要综述。     

基因枪技术及其在基因治疗中的应用进展

目前基因转染载体主要分为病毒型载体和非病毒型载体,病毒载体虽转染率较高、表达时间长,但其安全性令人担忧,非病毒载体中基因枪的优势最为明显,临床化趋势最强。通过分析非病毒基因转染技术面临的障碍,介绍了基因枪技术的产生和原理及其显著的优势,并总结了当前基因枪技术在基因治疗中的应用,指出了基因枪技术发展面临的问题和发展方向。     

可降解的非病毒基因载体的研究进展

选择合适的基因载体是基因治疗成功与否的一个关键环节,合适的基因载体应该具备较高转染效率,较好的组织相容性以及生物安全性等特点。相较于传统的病毒基因载体,非病毒基因载体具有低免疫原性,易于制备以及载药量大等特点,是较为理想的基因载体。而寻求可降解的非病毒基因载体可使其更为安全深入的应用到体内以及临床,意义重大。本研究就对常见的几种可降解的基因载体的转染效果及相关改性做一综述,为研究可降解非病毒基因载体的新方法提供新思路。     

猪肾细胞脂质磁转染系统的构建与表征

磁性纳米基因载体是一种非病毒基因载体,经过功能性基团修饰后能够连接阳离子转染剂构建细胞转染系统。本文将磁转染技术结合常用的脂质体转染,形成了一种新型动物体细胞转染方法,即称脂质磁转染(Liposomal magnetofection,LMF)。这将为体细胞克隆培育转基因动物提供稳定遗传的细胞系。为构建脂质磁性纳米基因载体复合物系统,本研究利用一种磁性纳米基因载体通过分子自组装与脂质阳离子转染剂结合,用于携带外源基因转染动物体细胞。通过原子力显微镜(AFM)观测、ζ电位-粒度等分析表征手段,研究磁性纳米基因载体的形貌、粒径分布、负载及浓缩DNA的方式。结果表明,通过猪肾(PK)细胞的LMF实验,与脂质体(Lipofectamine2000)介导的转染比较,具有较高的转染率,更重要的是克服了脂质体转染瞬时表达的缺陷。MTT细胞毒性试验结果也显示该方法具有较低的细胞毒性。因此LMF是一种切实可行的高效低毒性的细胞转染方法。     

作为非病毒基因载体的环糊精及其衍生物

环糊精由于自身的生物相容性和结构易裁剪性,通过结构修饰、聚合或超分子组合等设计被逐渐应用于非病毒基因载体系统。本文将分别从环糊精、其小分子衍生物、含环糊精聚合物以及超分子结构综述国内外近几年的设计思路和研究进展,并探讨含环糊精及其衍生物的非病毒基因载体的"结构-安全性-基因转染效率"关系。     

质粒载体的基因治疗

质粒载体的基因治疗作为一类极有前途的非病毒基因治疗方法,具有低免疫反应,安全稳定,低成本,适合商业化生产等特点,本文综合了用于基因治疗的质粒载体的转染机制,转染方法,构建和大规模制备。     

新型PEI 衍生物在COS-7 细胞中的转染与毒性研究

目的:寻找一种新型的转染效率高,毒性低的非病毒基因载体.方法:通过化学方法合成Polyimine-MPEI,然后以不同质量比包裹绿色荧光蛋白质粒,检测在COS-7细胞中的转染效率和毒性.结果:在比例从5到100之间,转染效率均比较理想,能达到1.00E+07以上,Polyimine-MPEI的毒性也很小,细胞的生长率均在80％以上,明显高于PEI25KDa对照组.结论:Polyimine-MPEI是一个很有研究前景的聚合物载体,具有高转染效率低毒性的特点,可以通过延长反应时间,增加分子量,增大转染能力.     

聚乙二醇-聚乙烯亚胺纳米微囊介导质粒转染骨髓间质干细胞

骨髓间质干细胞基因修饰及细胞移植是再生医学领域新兴的治疗策略和研究热点. 利用非病毒基因载体聚乙二醇-聚乙烯亚胺共聚物介导质粒转染骨髓间质干细胞, 观察共聚物/质粒纳米微囊在骨髓间质干细胞的吸附、内吞以及表达效率. 结果表明, 聚乙二醇-聚乙烯亚胺纳米微囊能保护质粒免受核酸酶降解, 将质粒包裹为直径100~150 nm的微囊并吸附于骨髓间质干细胞表面, 经由细胞内吞, 转染效率约为11.7%, 比阳离子脂质体介导的转染效率高8.5%, 是用于干细胞转染的良好基因载体.     

PEG修饰的聚阳离子基因载体PEG-Polycarbam-SP在COS-7细胞中转染和毒性的研究

目的：寻找一种转染效率高,细胞毒性低的非病毒基因载体,研究以人体内源性精胺为单体,以乙二醇二氯甲酸酯作为连接剂,以聚乙二醇（PEG）作为亲水基团连接剂合成亲水修饰聚阳离子载体PEG-Polycarbam-SP的基因担载效率,以及对非洲绿猴肾癌细胞COS-7的转染活性和细胞毒性的影响。方法：琼脂糖凝胶电泳方法考察复合物的基因担载效率,检测基因复合物的粒径和电位,以荧光素酶质粒为报告基因,研究PEG-Polycarbam-SP/DNA的复合物在COS-7细胞的转染活性,用MTT方法研究PEG-Polycarbam-SP对COS-7细胞的毒性。结果：聚合物与质粒在质量比5以后形成的复合物粒径稳定在50nm左右,Zate电位在20mV左右。COS-7细胞实验显示PEG-Polycarbam-SP具有低于PEI 25kDa的细胞毒性,同时也具有高效输送DNA的能力。结论：PEG-Polycarbam-SP是一种新型的高效、低毒,在基因治疗领域有潜在应用价值的非病毒基因输送载体。     

非病毒基因治疗的研究进展

非病毒基因治疗是相对于病毒性基因治疗而言,指采用非病毒的载体进行的基因治疗。非病毒的基因载体比病毒性基因载体具有高安全性、低免疫原性及易于生产的特点。本文就非病毒基因治疗所采用的主要方法、面蜂的主要问题及发展方向作一概括的介绍。随着人类对疾病发病分子机制的深入研究及人类基因组计划的实施,非病毒基因治疗将在人类疾病的治疗中发挥重要作用。     

Non-viral and hybrid vectors in human gene therapy: an update

Non-viral DNA vectors have several advantages over viral vectors. For example, virus production is expensive and there are safety concerns regarding viral manipulations. In addition, the size of the delivered plasmid is limited by the size of the viral capsid, whereas this is not a problem with non-viral vectors. The major disadvantage of using non-viral DNA delivery vectors, compared with their viral counterparts, is the low transfection efficiency. This has resulted in low levels of usage in clinical trials. Consequently, the majority of research into non-viral gene therapy has been focused on developing more efficient vectors.     

Enhanced gene transfer into brain capillary endothelial cells using Antp-modified DNA-loaded nanoparticles

Brain capillary endothelial cells (BCECs) have been considered as one of the primary targets for cerebral gene therapy. However, the cells, well-known for their poor function of endocytosis, are difficult to be transfected by general non-viral vectors. The aim of this study was to enhance the efficiency of transfection and expression in BCECs of DNA/polymer nanoparticles with the modification of membrane-penetrating peptide, Antennapedia peptide (Antp) polyethylenimine (PEI) and polyamidoamine (PAMAM) were chosen to prepare Antp-modified DNA-loaded nanoparticles with a complex coacervation technique. After a 20-min transfection, the efficiency, in terms of transfection and expression, of DNA/PEI NP or DNA/PAMAM NP was enhanced significantly with the modification of Antp. After a 3-h transfection of DNA/Antp/PEI NP, there was no difference in cellular uptake but an enhancement in gene expression, compared to DNA/PEI NP alone. However, both the transfection and expression efficiency of DNA/PAMAM NP were enhanced using Antp. These observations suggest that Antp can increase the membrane-penetrating ability of DNA-loaded nanoparticles, which can be employed as novel non-viral gene vectors.     

Human-derived NLS enhance the gene transfer efficiency of chitosan

Nuclear import is considered as one of the major limitations for non-viral gene delivery systems and the incorporation of nuclear localization signals (NLS) that mediate nuclear intake can be used as a strategy to enhance internalization of exogenous DNA.In this work, human-derived endogenous NLS peptides based on insulin growth factor binding proteins (IGFBP), namely IGFBP-3 and IGFBP-5, were tested for their ability to improve nuclear translocation of genetic material by non-viral vectors. Several strategies were tested to determine their effect on chitosan mediated transfection efficiency: co-administration with polyplexes, co-complexation at the time of polyplex formation, and covalent ligation to chitosan. Our results show that co-complexation and covalent ligation of the NLS peptide derived from IGFBP-3 to chitosan polyplexes yields a 2-fold increase in transfection efficiency, which was not observed for NLS peptide derived from IGFBP-5.These results indicate that the integration of IGFBP-NLS-3 peptides into polyplexes has potential as a strategy to enhance the efficiency of non-viral vectors.     

Optimization of in vitro vascular cell transfection with non-viral vectors for in vivo applications

BACKGROUND: Syngeneic vascular cells are interesting tools for indirect gene therapy in the cardiovascular system. This study aims to optimize transfection conditions of primary cultures of vascular smooth muscle cells (VSMCs) using different non-viral vectors and zinc as an adjuvant and to implant these transfected cells in vivo. METHODS: Non-liposomal cationic vectors (FuGene 6), polyethylenimines (ExGen 500), and histidylated polylysine (HPL) were used as non-viral vectors in vitro with secreted alkaline phosphatase (SEAP) as reporter gene. Transfection efficiency was compared in cultured rat, rabbit and human VSMCs and fibroblasts. Zinc chloride (ZnCl2) was added to optimize transfection of rat VSMCs in vitro which were then seeded in vivo. RESULTS: Much higher SEAP levels were obtained in rabbit cells with FuGene 6 (p <0.0001) at day 2 than in equivalent rat and human cells. Rat VSMCs transfected in vitro with FuGene 6 and ExGen 500 expressed higher SEAP levels than with HPL. In rat VSMCs, SEAP secretion was more than doubled by addition of 250 microM ZnCl2 (p <0.0001) for all vectors. Seeding of syngeneic VSMCs transfected under optimized conditions (FuGene 6/pcDNA3-SEAP +250 microM ZnCl2) into healthy Lewis rats using various routes or into post-infarct myocardial scar resulted in a peak of SEAP expression at day 2 and detectable activity in the plasma for at least 8 days. CONCLUSIONS: FuGene 6 is an efficient non-viral transfection reagent for gene transfer in somatic smooth muscle cells in vitro and ZnCl2 enhances its efficiency. This increased expression of the transgene product is maintained after seeding in vivo.     

Improving NK1R-targeted gene delivery of stearyl-antimicrobial peptide CAMEL by conjugating it with substance P

Specificity is a crucial condition that hampers the application of non-viral vectors for cancer gene therapy. In a previous study, we developed an efficient gene vector, stearyl-CAMEL, using N-terminal stearylation of the antimicrobial peptide CAMEL. Substance P (SP), an 11-residue neuropeptide, rapidly enters cells after binding to the neurokinin-1 receptor (NK1R), which is expressed in many cancer cell lines. In this study, the NK1R-targeted gene vector stearyl-CMSP was constructed by conjugating SP to the C-terminus of stearyl-CAMEL. Our results indicated that stearyl-CMSP displayed significant transfection specificity for NK1R-expressing cells compared with that shown by stearyl-CAMEL. Accordingly, the stearyl-CMSP/p53 plasmid complexes had significantly higher antiproliferative activity against HEK293-NK1R cells than they did against HEK293 cells, while the stearyl-CAMEL/p53 plasmid complexes did not show this specificity in antiproliferative activity. Consequently, conjugation of the NK1R-targeted ligand SP is a simple and successful strategy to construct efficient cancer-targeted non-viral gene vectors.     

A comprehensive review on histone-mediated transfection for gene therapy

Histone has been considered to be an effective carrier in non-viral gene delivery due to its unique properties such as efficient DNA binding ability, direct translocation to cytoplasm and favorable nuclear localization ability. Meanwhile, the rapid development of genetic engineering techniques could facilitate the construction of multifunctional fusion proteins based on histone molecules to further improve the transfection efficiency. Remarkably, histone has been demonstrated to achieve gene transfection in a synergistic manner with cationic polymers, affording to a significant improvement of transfection efficiency. In the review, we highlighted the recent developments and future trends in gene delivery mediated by histones or histone-based fusion proteins/peptides. This review also discussed the mechanism of histone-mediated gene transfection and provided an outlook for future therapeutic opportunities in the viewpoint of transfection efficacy and biosafety.     

Design,preparation and application of nucleic acid delivery carriers

Gene delivery vectors must deliver their cargoes into the cytosol or the nucleus, where DNA or siRNA functions in vivo. Therefore it is crucial for the rational design of the nucleic acid delivery carriers. Compared with viral vectors, non-viral vectors have overcome some fatal defections in gene therapy. Whereas the most important issue for the non-viral vectors is the low transfection efficiency, which hinders the progress of non-viral carriers. Sparked by the structures of the virus and understanding of the process of virus infection, various biomimic structures of non-viral carriers were designed and prepared to improve the transfection issues in vitro and in vivo. However, less impressive results are achieved. In this review, we will investigate the evolution of the virus-mimicking carriers of nucleic acids for gene therapy, especially in cancer therapy; explore and discuss the relationship between the structures, materials and functions of the carriers, to provide guidance for establishing safe and highly efficient non-viral carriers for gene therapy.     

体内基因转染技术在肝再生研究中的应用

肝脏是一个特殊的器官,不仅因为它独特的解剖结构和生理特征,而且它还具有无限的再生能力。在各种动物模型中,应用病毒或非病毒载体将肝细胞生长因子等基因转入体内,能增强肝再生能力,这就是肝脏基因转染技术在肝再生研究中的应用。未来的研究目标就是消除病毒载体的毒副作用和增加非病毒载体的转染率,这也是目前肝内基因转染技术中面临的主要难题;另一个研究目标就是用受体介导基因靶向肝转染,使转入基因在肝细胞中特异高表达。这些研究成果将有助于肝再生基因机制研究,以及将来临床基因治疗提供参考。