多肽作为非病毒转基因载体组件的研究进展

在转基因领域,非病毒转基因载体因为具有许多病毒载体无法比拟的优点而越来越受到重视。但是理想的非病毒转基因载体的构建需要一些功能多肽的参与。这些多肽作为转基因载体的组件,在骨架构建、靶向策略的实施、内体膜的破裂和载体的核转运方面发挥重要作用。就多肽在这些方面的进展作一综述。     

非病毒转基因载体的研究进展

高效的转基因载体是基因释放系统的核心。非病毒转基因载体具有低免疫原性和易制备等特性。本文就近年来非病毒转基因载体的发展作一综述     

非病毒转基因载体的研究进展

高效的转基因载体是基因释放系统的核心。非病毒转基因载体具有低免疫原性和易制备等特性。本就近年来非病毒转基因载体的发展作一综述。     

病毒载体致突变性的实验研究

目的 :观察逆转录病毒pLXSN与腺病毒LacZ作为转基因载体所构建的转基因细胞的致突变作用 ,为转基因肿瘤细胞作为瘤苗进行临床提供安全性检测参数。方法 :将病毒与细胞共培养 ,用细胞DNA通过遗传毒理学实验技术进行体内和体外的致变性实验研究。结果 :转基因细胞DNA及培养上清液未显出致突变作用。结论 :经过修饰的病毒作为转基因载体未见其致突变作用     

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。     

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。     

基因转染技术在肾脏病研究中的应用

基因转染技术在肾脏病机理研究及其防治中的应用已受到关注,并提出了针对肾小球、肾小管和肾间质的各种基因转染方法.成功地应用病毒载体和非病毒载体进行直接体内基因转染,并通过系膜细胞载体和小管上皮细胞载体系统进行间接体内基因转染,并获得较好效果,为研究肾脏病的发病机理和治疗方法奠定了基础.     

长循环脂质体的研究进展及其在核医学中的应用

脂质体作为药物载体具有很多优点,但是其主动靶向性和稳定性较差,聚乙二醇或与配体连接的聚乙二醇修饰的脂质体,即长循环脂质体,具有延长脂质体在血液循环中的半衰期、提高其稳定性、改变脂质体在体内的生物学分布,赋予脂质体靶向性的优点。本文主要综述了长循环脂质体的研究进展及其在核医学中的应用。     

携带乙型肝炎病毒感染性克隆的四种转基因载体在体内外抗原表达分析

目的 构建四种不同结构的1.3倍全基因乙型肝炎病毒(HBV)转基因载体,观察HBV抗原在体内外表达水平的差异.方法 克隆1.3倍的HBV基因至慢病毒转基因质粒pCS-CG并取代其中的EGFP表达盒,构建四种结构的pCS-HBV1.3质粒,体外转染Huh 7细胞和尾静脉高压水动力法注射C57BL/6小鼠后,ELISA方法分别检测细胞上清和小鼠血清中HBsAg和HBeAg的表达.结果 成功构建了四种不同结构的pCS-HBV1.3质粒,四种质粒HBV S抗原与e抗原在体内外的表达趋势一致,即乙肝抗原表达水平在正向插入的转基因质粒中高于反向插入;HBV抗原表达水平还与载体序列中RRE调控元件和cPPT调控元件前的基因序列长度有关.结论 筛选获得了可在体内外高效表达HBV抗原的转基因载体可应用于乙肝病毒体内外感染模型的建立与应用.     

聚乙二醇修饰的共聚物纳米粒研究进展

可生物降解的聚合物纳米粒作为药物输送载体有很多优势，如可控释，靶向等。但是，由于聚合物纳米粒经静脉经给药后，数秒或数分钟内会被皮网状系统清除而无法普遍应用，为了克服这一缺点，越来越多的研究者引入亲水性组分聚乙二醇（PEG）对聚合物进行修饰，以避免其被内皮肉状系统摄取。聚乙二醇的引入不仅会影响聚合物纳米粒的生物降解行为，而且会影响药物的释放，体内分布等行为，本文综述了聚乙二醇修饰的共聚物纳米粒的制备，稳定性，载体，体外释药，体内分布，毒性等方面的研究进展，并对其前景进行预测。     

Synthetic and natural polycations for gene therapy: state of the art and new perspectives

Currently, the major drawback of gene therapy is the gene transfection rate. The two main types of vectors that are used in gene therapy are based on viral or non-viral gene delivery systems. There are several non-viral systems that can be used to transfer foreign genetic material into the human body. In order to do so, the DNA to be transferred must escape the processes that affect the disposition of macromolecules. These processes include the interaction with blood components, vascular endothelial cells and uptake by the reticuloendothelial system. Furthermore, the degradation of therapeutic DNA by serum nucleases is also a potential obstacle for functional delivery to the target cell. Cationic polymers have a great potential for DNA complexation and may be useful as non-viral vectors for gene therapy applications. The objective of this review was to address the state of the art in gene therapy using synthetic and natural polycations and the latest strategies to improve the efficiency of gene transfer into the cell.     

纳米基因载体的研究进展

由于使用病毒载体难以避免机体对病毒微粒的免疫反应和由病毒介导的随机整合或野生型病毒重整等潜在危害,因此应用纳米技术进行新型非病毒基因载体的研究发展迅速。本文就纳米微粒应用于基因载体的研究作一综述。     

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.     

Muscular gene transfer using nonviral vectors

Skeletal muscle is a target tissue of choice for the gene therapy of both muscle and non-muscle disorders. Investigations of gene transfer into muscle have progressed considerably from the expression of plasmid reporter genes to the production of therapeutic proteins such as trophic factors, hormones, antigens, ion channels or cytoskeletal proteins. Viral vectors are intrinsically the most efficient vehicles to deliver genes into skeletal muscles. But, because viruses are associated with a variety of problems (such as immune and inflammatory responses, toxicity, limited large scale production yields, limitations in the size of the carried therapeutic genes), nonviral vectors remain a viable alternative. In addition, as nonviral vectors allow to transfer genetic structures of various sizes (including large plasmid DNA carrying full-length coding sequences of the gene of interest), they can be used in various gene therapy approaches. However, given the lack of efficiency of nonviral vectors in experimental studies and in the clinical settings, the overall outcome clearly indicates that improved synthetic vectors and/or delivery techniques are required for successful clinical gene therapy. Today, most of the potential muscle-targeted clinical applications seem geared toward peripheral ischemia (mainly through local injections) and cancer and infectious vaccines, and one locoregional administration of naked DNA in Duchenne muscular dystrophy. This review updates the developments in clinical applications of the various plasmid-based non-viral methods under investigation for the delivery of genes to muscles.     

Development of liposomes and pseudovirions with fusion activity for efficient gene delivery

For efficient gene delivery, chimeric vectors combining non-viral vectors with viral components have been developed. In particular, increasing attention has been paid to viral fusion activity. HVJ (hemagglutinating virus of Japan; Sendai virus) fuses with the cell membrane at neutral pH, and HN and F, fusion proteins of the virus, contribute to the cell fusion. For fusion-mediated gene transfer, DNA-loaded liposomes were fused with UV-inactivated HVJ to form the fusion liposome, HVJ-liposome. Fusion-mediated delivery protects the molecules incorporated in the liposome from degradation in endosomes and lysosomes before reaching the cytoplasm. Reconstituted pseudovirions of fusion-competent viruses such as HVJ and influenza virus have been also developed by a detergent-lysis and-removal method. A more direct and practical approach is the conversion of fusion-competent virions to non-viral gene delivery particles. Based on this concept, the HVJ envelope vector was developed using inactivated particles of HVJ and has been utilized for gene therapy experiments and functional screening for therapeutic genes. A tissue-targeting HVJ envelope vector was also constructed.     

Non-viral approach toward gene therapy of cystic fibrosis lung disease

Since Cystic Fibrosis (CF) is an autosomal recessive disorder due to mutations in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene, studies towards a gene therapy approach to its treatment followed immediately upon the cloning of the gene. It was demonstrated that the insertion of a single copy of the wild-type gene restored the normal phenotype in CF cells in vitro. Encouraging results were obtained in many in vivo model systems (CF transgenic mice) involving viral as well as non-viral vectors, which demonstrated the recovery of CFTR function in the airways. These results constituted the basis for human studies. Of those with a non-viral approach, a total of seven clinical trials using cationic lipids have reported data on efficiency, efficacy and safety. An effective gene transfer approach for the treatment of CF lung disease is not however imminent: low transfection efficiency and poor maintenance of gene expression are so far the main obstacles on this therapeutic path. On the other hand, no important adverse effects have been documented and repeated administration in humans is possible. The understanding of tissue and cellular barriers is a prerequisite for the development of more efficient non-viral gene therapy protocols for CF patients. While cationic lipids have been shown to be blocked by the mucous airway barrier and not be able to transfect differentiated respiratory epithelial cells, a new class of non-viral vectors, cationic polymers, are endowed with chemical and biological properties that make them more efficient in mediating gene transfer than lipids. Cationic polymers, such as polyethylenimine, are promising vectors for CF lung gene therapy.     

Transfection of insulin-secreting cell line and rat islets by functional polymeric gene vector

The use of genetically modified islets is a potential strategy for overcoming pitfalls that currently plague islet transplantation. This study employed functional polymeric vectors specifically designed to transfect insulin-secreting cells and results were compared to various non-viral vectors. The evaluation included transfection efficiency, experimental condition effects, gross morphological observation, cytotoxicity, apoptosis, gene distribution in treated islets, insulin secretion function and time-dependent gene expression pattern. Observations from this study suggest that 1) the experimental conditions for islet transfection should be optimized, 2) the cytotoxicity of sulfonylurea containing vectors differs between the RINm5F cell line and primary pancreatic islets, 3) the non-viral vectors were primarily located in the peripheral region of an islet where the initial cell toxicity/apoptosis was apparent, 4) the genetic modification of pancreatic islets with genes for secretory proteins is more feasible than for residing proteins, and 5) the gene construct selection may prolong the gene expression period and oscillating pattern as demonstrated in this study. This study provides some fundamental background information that will aid in further designing polymeric gene vectors for the optimal manipulation of pancreatic islets prior to transplantation.     

Production of non viral DNA vectors

After some decades of research, development and first clinical approaches to use DNA vectors in gene therapy, cell therapy and DNA vaccination, the requirements for the pharmaceutical manufacturing of gene vectors has improved significantly step by step. Even the expression level and specificity of non viral DNA vectors were significantly modified and followed the success of viral vectors. The strict separation of "viral" and "non viral" gene transfer are historic borders between scientist and we will show that both fields together are able to allow the next step towards successful prevention and therapy. Here we summarize the features of producing and modifying these non-viral gene vectors to ensure the required quality to modify cells and to treat human and animals.     

Immunological hurdles to lung gene therapy

Gene delivery has the potential to offer effective treatment to patients with life-threatening lung diseases such as cystic fibrosis, alpha1-antitrypsin deficiency and lung cancer. Phase I/II clinical trials have shown that, in principle, gene transfer to the lung is feasible and safe. However, gene expression from both viral and non-viral gene delivery systems has been inefficient. In addition to extra- and intracellular barriers, the host innate and acquired immune system represents a major barrier to successful gene transfer to the lung. Results from studies in experimental animals and clinical trials have shown that inflammatory, antibody and T cell responses can limit transgene expression duration and readministration of the gene transfer vector. We will review here how the development of pharmacological and/or immunological agents can modulate the host immune system and the limitations of these strategies. A better understanding of the immunological barriers which exist in the lung might allow for a more sustained expression of the transgene and importantly help overcome the problem of readministration of viral vectors.