非病毒基因载体材料的研究进展

非病毒材料可成为基因治疗中的基因载体，使目的基因持续有效地表达。非病毒基因载体主要有脂质体、人工合成聚合物载体、天然聚合物载体、局部基因释放载体等。其中壳聚糖及其衍生物是一种优良的基因释放载体，局部基因释放载体技术将基因治疗与组织工程结合起来，在组织修复与重建方面将发挥重要作用。     

病毒载体介导的基因转移研究进展

实现基因治疗的关键在于目的基因的高效转移并适度表达 ,这将直接影响其治疗效率和安全性。因此探索理想的基因转移技术是基因治疗的一项重要内容 ,目前人们的注意力更多地集中于病毒载体 ,传统的逆转录病毒载体不能转染非分裂细胞 ,且载导容量有限 ,促使人们在寻找改进措施的同时积极研制其它类型的病毒载体 ,本文将阐述这些病毒载体的特性、应用情况及研究进展。     

纳米基因载体的研究现状

基因治疗正成为当今医学研究的热门课题,载体问题是制约基因治疗成功的关键.目前,常用的载体包括病毒载体和非病毒载体,后者以其无病毒毒性和免疫原性等优越性而倍受瞩目.随着纳米生物技术的发展和多种纳米材料的涌现,以纳米颗粒为基础的非病毒基因载体倍受研究者推崇.综述了纳米基因载体的研究现状,重点总结阐述了阳离子聚合物、无机纳米颗粒及磁性纳米颗粒载体系统,并探讨了其在应用研究方面的现状和前景.     

高分子载体的基因释放与组织工程

以高分子为载体释放编码蛋白质质粒DNA实现组织重建的方法是在多学科、基因治疗和组织工程概念的基础上提出的。其技术的核心问题是以生物可降解高分子为载体的基因缓释；本将综述以高分子为载体的基因释放及其在组织工程应用的研究进展，并指出现存的问题。     

非病毒型纳米载体在基因治疗中的研究现状及展望

近 10年来 ,新型非病毒载体在基因治疗中日益受到欢迎。其主要代表为纳米载体 ,具有无毒性及免疫原性的优势 ,已作为高效阳离子载体用于基因转移。体外基因转移实验表明 ,纳米载体的基因转移率高于普通脂质体及其它阳离子多聚体 ,如多聚氮丙啶及聚赖氨酸。本文对纳米载体的结构特点、性能、基因转移机制进行综述 ,并将其在体内外基因转移效率与其它非病毒载体作以比较     

载体与基因转染的研究进展

基因载体是指将基因或其它核酸物质运载到细胞中的工具.其化学本质可以是蛋白质或多肽、核酸、脂类、糖类、其它有机分子或它们的复合物.基因传递系统是基因治疗的重要组成部分,也是目前基因治疗的瓶颈.现有的基因载体包括两类.即病毒载体和非病毒载体.病毒载体转染效率高,但由于其转染具有免疫原性和致突变性限制了它的应用;非病毒载体系统具有低毒、低免疫原性和相对靶向性等优点,是新兴发展起来的基因转移系统.就各种载体的最新研究进展作一综述.     

非病毒型纳米载体在基因治疗中的研究现状及展望

近10年来，新型非病毒载体在基因治疗中日益受到欢迎。其主要代表为纳米载体，具有无毒性及免疫原性的优势，已作为高效阳离子载体用于基因转移。体外基因转移实验表明，纳米载体的基因转移率高于普通脂质体及其它阳离子多聚体，如多聚氮丙及聚赖氨酸。本对纳米载体的结构特点，性能，基因转移机制进行综述，并将其在体内外基因转移效率与其它非病毒载体作以比较。     

基因转移的DNA病毒方法

在基因治疗中,作为基因转移的载体,迄今几乎都用逆转录病毒,但是在某些情况下(例如将外源基因导入非分裂状态的细胞),DNA病毒作为载体具有明显的优越性。本文对腺相关病毒、腺病毒和疱疹病毒作为外源基因载体进行了简要的讨论。     

靶向性肿瘤基因转移研究进展

肿瘤基因治疗安全性是治疗应用中需考虑的一个重要问题。利用异常表达的原癌基因启动子，病毒基因特异性启动子，组织特异性表达的调控序列等手段将治疗基因局限于肿瘤局部表达而特异性杀伤肿瘤是提高肿瘤基因治疗安全性的一个手段。本文主要从以上这几个方面侧重阐述了逆转录病毒、腺病毒载体靶向性研究进展     

聚乙烯亚胺用作基因转染的研究进展

 基因载体问题以及与载体相关的免疫反应、细胞毒性和安全性等问题，是基因治疗领域亟待解决的关键问题之一。聚乙烯亚胺（PEI）是阳离子聚合物非病毒载体的典型代表^[1]，是一种很早便为人所知并予以应用的有机大分子。目前，以 PEI 阳离子聚合物与 DNA 形成的 PEI/DNA 复合物已成为非病毒基因载体的研究热点。本文就近年来这方面的研究进展作简要综述。 1 PEI的特性 PEI 每 3 个原子中有 1 个胺基原子，使其具有较高正电荷密度。根据 pH 与质子作用之间的对应关系可得出：自由 PEI 的结构在生理条件下有 1/6 至 1/5 胺基发生质子化反应，从而使溶酶体肿胀破裂，从而起到“质子海绵”作用，使 PEI/DNA 复合物得以释放入胞质，很大程度上减少了 DNA 在吞噬泡内富集并进而被降解的作用，因而可以提高转染效率^[2]。     

层层自组装技术在基因活化生物材料表面工程的应用

细胞/基因活化生物材料是未来生物材料发展的方向和重要特征.以高分子材料为载体的基因释放在基因治疗、再生医学和组织工程中具有广泛的应用前景.综述了层层自组装(layer-by-layer self-assembly) 技术在先进生物材料表面工程中的应用,进而探讨了该技术在生物材料表面构建原位基因释放系统的潜在应用,对生物材料的研究具有重要的现实意义.     

Modulating polymer chemistry to enhance non-viral gene delivery inside hydrogels with tunable matrix stiffness

Non-viral gene delivery holds great promise for promoting tissue regeneration, and offers a potentially safer alternative than viral vectors. Great progress has been made to develop biodegradable polymeric vectors for non-viral gene delivery in 2D culture, which generally involves isolating and modifying cells in vitro, followed by subsequent transplantation in vivo. Scaffold-mediated gene delivery may eliminate the need for the multiple-step process in vitro, and allows sustained release of nucleic acids in situ. Hydrogels are widely used tissue engineering scaffolds given their tissue-like water content, injectability and tunable biochemical and biophysical properties. However, previous attempts on developing hydrogel-mediated non-viral gene delivery have generally resulted in low levels of transgene expression inside 3D hydrogels, and increasing hydrogel stiffness further decreased such transfection efficiency. Here we report the development of biodegradable polymeric vectors that led to efficient gene delivery inside poly(ethylene glycol) (PEG)-based hydrogels with tunable matrix stiffness. Photocrosslinkable gelatin was maintained constant in the hydrogel network to allow cell adhesion. We identified a lead biodegradable polymeric vector, E6, which resulted in increased polyplex stability, DNA protection and achieved sustained high levels of transgene expression inside 3D PEG-DMA hydrogels for at least 12 days. Furthermore, we demonstrated that E6-based polyplexes allowed efficient gene delivery inside hydrogels with tunable stiffness ranging from 2 to 175 kPa, with the peak transfection efficiency observed in hydrogels with intermediate stiffness (28 kPa). The reported hydrogel-mediated gene delivery platform using biodegradable polyplexes may serve as a local depot for sustained transgene expression in situ to enhance tissue engineering across broad tissue types.     

Physical Non-Viral Gene Delivery Methods for Tissue Engineering

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.     

基因治疗的新型载体研究进展

建立和发展一个安全及有效的载体系统对基因治疗是极其重要的。尽管病毒载体已经在临床上用于基因治疗，但其安全性仍然不确切。近年来，许多非病毒性基因载体系统已被广泛开展及应用。本综述将讨论一些新的基因载体系统，特别是本实验室开展研究的载体系统，包括细胞转导肽，电脉冲导入系统，壳聚糖载体等。     

Polymeric gene carriers

Polymeric gene carriers are a potential alternative to using viral vectors. Polymeric carriers have relatively low immunogenicity and cytotoxicity. In addition, polymeric carriers can accommodate large-size DNA, be conjugated with appropriate functionalities, and be administered repeatedly. In spite of these advantages, polymeric gene carriers have some limitations, such as low gene transfection efficiencies and relatively short duration of gene expression. Therefore, extensive research has been conducted toward the development of efficient polymeric carriers. In this review, we discuss current problems associated with polymeric gene carriers and various strategies against transfection barriers in particular, gene stabilization and protection, cellular targeting, endosomal escaping, nuclear targeting, unpackaging, and biocompatibility. Finally, requirements for future polymeric gene carriers are considered. With all these ongoing efforts, polymeric carriers have become one of the promising gene delivery methods for human gene therapy.     

Transductional targeting with recombinant adenovirus vectors

Replication-deficient adenoviruses are considered as gene delivery vectors for the genetic treatment of a variety of diseases. The ability of such vectors to mediate efficient expression of therapeutic genes in a broad spectrum of dividing and non-dividing cell types constitutes an advantage over alternative gene transfer vectors. However, this broad tissue tropism may also turn disadvantageous when genes encoding potentially harmful proteins (e.g. cytokines, toxic proteins) are expressed in surrounding normal tissues. Therefore, specific restrictions of the viral tropism would represent a significant technological advance towards safer and more efficient gene delivery vectors, in particular for cancer gene therapy applications. In this review, we summarize various strategies used to selectively modify the natural tropism of recombinant adenoviruses. The advantages, limitations and potential impact on gene therapy operations of such modified vectors are discussed.     

基因治疗中的核酸药物及非病毒递送载体的研究进展

基因治疗是针对基因异常相关疾病的终极治疗技术,各种具有不同机制的核酸药物的出现为基因治疗带来了更多的可能性。但是,由于存在体内稳定性差、难以高效进入靶细胞等问题,核酸药物需要载体的帮助而进入目标细胞并到达特定的胞内位置,因此,开发安全高效的核酸递送系统是基因治疗的基石。与病毒载体相比,非病毒载体具有更高的安全性,但转染效率较低。随着纳米技术的发展,非病毒载体的效率得到了显著的提升,进入临床研究的数量逐渐增多。本文简要介绍基因治疗中的核酸药物及其递送载体,对非病毒核酸药物递送技术的瓶颈及进展做综合评述。     

Human artificial chromosome vectors meet stem cells

The recent emergence of stem cell-based tissue engineering has now opened up new venues for gene therapy. The task now is to develop safe and effective vectors that can deliver therapeutic genes into specific stem cell lines and maintain long-term regulated expression of these genes. Human artificial chromosomes (HACs) possess several characteristics that require gene therapy vectors, including a stable episomal maintenance, and the capacity for large gene inserts. HACs can also carry genomic loci with regulatory elements, thus allowing for the expression of transgenes in a genetic environment similar to the chromosome. Currently, HACs are constructed by a two prone approaches. Using a top-down strategy, HACs can be generated from fragmenting endogenous chromosomes. By a bottom-up strategy, HACs can be created de novo from cloned chromosomal components using chromosome engineering. This review describes the current advances in developing HACs, with the main focus on their applications and potential value in gene delivery, such as HAC-mediated gene expression in embryonic, adult stem cells, and transgenic animals.