基于非病毒载体的mRNA疫苗递送系统研究进展

mRNA疫苗是一类新型核酸疫苗。它具有通用性、开发迅速以及制造成本低等优点,目前已被广泛用于传染性疾病防治和癌症治疗的研究。但mRNA在体内的不稳定性和低表达限制其在临床的应用,因此开发出能高效转染mRNA的递送载体十分关键。目前的基因递送载体分为病毒载体和非病毒载体。本文对现有递送mRNA的非病毒载体进行综述,并对mRNA疫苗及其非病毒载体的研究前景进行展望。     

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。     

抗体/腺病毒复合物为载体的基因定位递送

临床基因治疗方案中基因载体在疾病部位的靶向递送仍是急待解决的问题。本研究将腺病毒与一种生物素化的特异性抗腺病毒六邻体的多克隆IgG结合，固定于结合了亲和素的胶原蛋白膜上，成功获得腺病毒载体基因靶向定位递送体系。体外稳定性研究结果表明该体系中病毒载体可有效保持活性。通过这种特异性抗体偶联方式，将携带单纯疱疹胸苷激酶（HSVtk）编码基因片断的腺病毒结合在胶原膜上转染大鼠平滑肌细胞（A10），加入更昔洛韦（ganciclovir）后，只有生长在胶原膜上及膜邻近50μm内的细胞被杀死。在使用非特异性抗体的对照实验中，整个培养基范围内的细胞几乎全被杀死。以绿色荧光蛋白（GFP）为报告基因对猪的心肌进行转基因实验，结果显示注射特异性抗体偶联病毒的胶原凝胶比直接注射病毒悬液获得更高效的心室基因表达。所有研究结果表明，通过生物素和特异性抗体使病毒载体固定在胶原蛋白基质上，可达到有效的局部定位基因表达，避免向非病灶部位的扩散，是基因治疗中一种极具发展潜力的载体定位递送方法。     

非病毒载体在siRNA体内递送中的应用

RNAi是一种由siRNA触发的特异基因沉默机制。自其发现以来,RNAi技术迅速发展为药物靶标确认和功能基因组学研究的新策略,并有望成为一种疾病基因治疗学方法。然而,由于siRNA体内不稳定的理化性质,递送siRNA成为RNAi发展的最大障碍。因此研发能够保护siRNA、促进细胞吸收、正确细胞定位和包内体逃逸,结合具有高生物相容性与减少非特异沉默和毒性反应的体内递送系统是RNAi成功用于临床治疗的关键。本文就体内非病毒载体方式递送siRNA的研究进展进行综述。     

十二烷基化壳聚糖基因支架血管内基因转运的实验研究

目的目前基因治疗方面存在的重要问题是缺乏有效的基因转运体系可以足量、安全地将治疗基因(无论病毒载体或非病毒载体)运送至体内靶细胞并提高其转染效率,以得到基因的高效表达。心血管内基因治疗的特殊性还在于很难把基因专一递送至血管组织的病灶处而不进入血液循环系统。实验研究提出了一种携带质粒DNA的烷基化壳聚糖纳米粒的血管内支架,可以有效地将质粒DNA递送至血管壁靶细胞并达到了高效转染的效果。     

基因治疗中非病毒载体的研究进展

基因治疗中非病毒载体技术近年来发展迅速。与病毒载体相比,非病毒载体具有简便、安全、毒性小等特点。该方法大体可分为两种:物理方法途径和化学载体转移方式。本文综述了非病毒载体的分类及其研究进展。     

基因治疗中非病毒载体的研究进展

基因治疗中非病毒载体技术近年来发展迅速。与病毒载体相比，非病毒载体具有简便、安全、毒性小等特点。该方法大体可分为两种：物理方法途径和化学载体转移方式。本文综述了非病毒载体的分类及其研究进展。     

非病毒型载体介导基因转染

基因载体是制约基因转移技术发展的关键。近年来,非病毒载体由于其安全、低毒、低免疫原性等特点而备受青睐。文章以脂质体和聚乙烯亚胺为代表,介绍了非病毒载体的性质、介导转染的机制。随着人们对细胞转染机制了解的深入以及生物材料科学的迅速发展,非病毒型载体将有望实现高效、低毒、靶向特异等特点,从而成为基因治疗中的理想载体。     

质粒型甲病毒复制子载体系统的构建及其功能鉴定

目的 构建质粒型甲病毒复制子载体系统,并对其功能进行鉴定.方法 在XJ-160病毒感染性cDNA克隆的基础上,将病毒非结构基因序列分为三个片段扩增,分步克隆至真核表达载体pVAX1 CMV启动子下游,并用多克隆位点序列替代病毒的结构基因构建XJ-160病毒质粒型复制子载体.将病毒核蛋白基因及包膜糖蛋白基因分别克隆至pVAX1 CMV启动子下游构建载体的两个辅助质粒.通过绿色荧光蛋白报告基因及海肾荧光素酶报告基因的表达检验该质粒型载体系统的功能特性.结果 成功构建了包括复制子载体和辅助质粒的质粒型甲病毒复制子载体系统,且该复制子载体系统可成功表达绿色荧光蛋白报告基因及海肾荧光素酶报告基因.结论 本研究构建了能够表达外源基因的质粒型甲病毒复制子载体系统,为目标基因表达、重组病毒颗粒制备等奠定了基础.     

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.     

Electrotransfer as a non viral method of gene delivery

Over the last few decades, various vectors have been developed in the field of gene therapy. There still exist a number of important unresolved problems associated with the use of viral as well as non viral vectors. These techniques can suffer from secondary toxicity or low gene transfer efficiency. Therefore an efficient and safe method of DNA delivery still needs to be found for medical applications. DNA electrotransfer is a physical method that consists of the local application of electric pulses after the introduction of DNA into the extra cellular medium. As electrotransfer has proven to be one of the most efficient and simple non viral methods of delivery, it may provide an important alternative technique in the field of gene therapy. The present review focuses on questions related to the mechanism of DNA electrotransfer, i.e. the basic physical processes responsible for the electropermeabilisation of lipid membranes. It also addresses the current limitations of the method as applied to DNA transfer, in particular its efficiency in achieving in vitro gene expression in cells and also its potential use for in vivo gene delivery.     

Progress in cationic lipid-mediated gene transfection: a series of bio-inspired lipids as an example

Over the last several years, various gene delivery systems have been developed for gene therapy applications. Although viral vector-based gene therapy has led to the greatest achievements in animal and human studies, synthetic non-viral vectors have also been developed as they offer several advantages over viral systems, including lower immunogenicity and greater nucleic acid packaging capacity. Nevertheless, the transfection efficiency of the current non-viral gene carriers still needs to be improved, especially as regards direct in vivo transfection. In particular, cationic lipid/nucleic acid complexes (termed lipoplexes) have been the subject of intensive investigation with a view to optimize their performance and to better understand their mechanisms of action, and consequently to design new approaches to overcome the critical barriers of cationic liposome-mediated gene delivery. A possible strategy may rely on considering the membrane constituents and properties of the vast variety of living organisms as a source of inspiration for the design of biocompatible, non-toxic and effective novel artificial liposomal systems. Thus, the present forward-looking review provides an overview of the progress already made during the last years in the field of cationic lipid-mediated gene transfection and also focuses on a series of novel bio-inspired lipids for both in vitro and in vivo gene transfection.     

基因强化骨组织工程中的病毒载体

背景：不同的基因输送策略也被应用到骨组织工程中以修复破坏的骨组织,作为最有效率的基因转运载体,病毒载体在骨组织工程中的应用方兴未艾。 目的：系统回顾和讨论目前基因强化骨组织工程中常用的病毒载体相关应用。 方法：利用PubMed数据库对2002年1月至2015年1月的相关文献进行了检索,检索的文章主要聚焦在病毒载体基因转导方法和其在骨组织工程中的应用。对腺病毒、反转录病毒、腺相关病毒和嵌合病毒在骨组织工程的相关应用及不足进行了讨论。总共24篇相关文献被纳入此篇综述。 结果与结论：总结了近年来病毒载体联合基因治疗促进骨组织再生的研究工作。讨论了包括装载目的基因的病毒载体联合种子细胞例如间充质干细胞植入支架材料修复骨缺损。研究表明,基因强化的骨组织工程比传统组织工程具有更多的优点;病毒载体介导的基因转染效率比普通载体更高;病毒载体介导的基因强化骨组织工程用于人体的安全性仍需要漫长的临床观察研究。病毒载体系统仍然是最有效的将外源基因转入种子细胞的手段之一。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Construction of retroviral vectors with enhanced efficiency of transgene expression

Retroviral vectors have been widely used in gene therapy due to their simple genomic structure and high transduction efficiency. We report a construction of Moloney murine sarcoma virus (MoMSV) and Moloney murine leukemia virus (MoMLV) hybrid-based retroviral vectors with significantly improved efficiency of transgene expression after stable incorporation into the host genome. In these vectors, the residual gag gene coding sequence located in the extended region of packaging signal was removed. These vectors, therefore, contain no coding sequence for the gag, pol, or env gene that can be used for homologous recombination with sequences introduced in the packaging system for a recombinant competent retrovirus (RCR) generation. A strong splice acceptor site obtained from the exon/intron junction of either the chimpanzee EF1-alpha gene or the human CMV major immediate early gene was placed downstream of the MoMSV packaging signal (Psi), significantly improving the efficiency of transgene expression. The 5' LTR U3 sequence was replaced with an extended human CMV major immediate early gene enhancer/promoter for a strong expression of full-length messages from the viral backbone, helping to maintain high levels of viral titer. These newly developed retroviral vectors should facilitate RCR-free gene transfer with significantly improved efficacy in clinical gene therapy trials.     

聚乙烯亚胺纳米粒制备及其应用的研究进展

基因治疗已经成为现代医疗中极具发展潜力的治疗手段。选择合适的运转载体是基因治疗成功与否的关键之一。尽管病毒载体转染效率很高,但是其免疫源性和安全性等问题限制了它的应用。通过化学反应制备而成的纳米粒作为非病毒转染载体有效解决了上述问题,从而提高了基因转染效率。近年来,大量的研究表明以聚乙烯亚胺为基础的纳米粒已经成为基因转...     

Stimuli-responsive biodegradable poly(methacrylic acid) based nanocapsules for ultrasound traced and triggered drug delivery system

Ultrasound contrast agents (UCAs) have been investigated for echogenic intravenous drug delivery system. Due to the traditional UCAs with overlarge size (micro-scale), their reluctant accumulation in target organs and the instability have presented severe obstacles to the accurate response to the ultrasound and severely limited their further clinical application. Furthermore, elimination of drug carriers from the biologic system after their carrying out the diagnostic or therapeutic functions is one important aspect to be considered. The drug carriers with large sizes, avoiding renal filtration, will lead to increasing toxicity. In this present paper, we design and develop a new type of triple-stimuli responsive (ultrasound/pH/GSH) biodegradable nanocapsules, in which fill up with perfluorohexane, and the DOX-loaded PMAA with disulfide crosslinking forms the wall. These soft nanocapsules with uniform size of 300 nm can easily enter the tumor tissues via EPR effects. The PMAA shell has high DOX-loading content (36 wt%) and great drug loading efficiency (93.5%), the PFH filled can effectively enhance US imaging signal through acoustic droplet vaporization (ADV), ensuring diagnostic and image-guided therapeutic applications. What is more, the disulfide-crosslinked PMAA shell is biodegradable and thus safe for normal organisms. These merits enabled us optimize the balance of diagnostic, therapeutic and biodegradable functionalities in a multifunctional theranostic nanoplatform.     

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.     

Heparan sulphate proteoglycans and viral vectors : ally or foe?

The attachment of viruses to the host cell surface is a critical stage that will largely condition cell permissivity and productive infection. The understanding of such mechanisms is therefore essential for gene therapy applications involving viruses, as this step will influence both targeting and delivery efficiency of the gene of interest. Viral attachment depends upon the recognition and binding of viral envelope/capsid proteins to specific cellular receptors that can be from very diverse origins. Amongst them are heparan sulphate proteoglycans (HSPGs), a family of glycoproteins which, through the large binding properties of their heparan sulphate (HS) polysaccharide chains, serve as attachment receptor for a great number of viruses. The aim of this review is to provide an update on the multiple roles of HSPGs during viral infection, with a special focus on viruses used as gene delivery vectors. Consequences of HS binding for gene therapy applications will be assessed, as well as the various strategies that have been developed to potentiate the advantages or to overcome the drawbacks resulting from viral vector interaction with HS.