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。     

阳离子多聚物纳米基因载体系统的研究进展

阳离子多聚物能与DNA通过静电吸附作用而自组装成纳米微粒，保护DNA防止被核酸酶降解。阳离子多聚物由于具有合成简便、储存稳定、目的基因容量大、特异靶向性强、免疫原性低等优点被用作非病毒基因载体。阳离子多聚物按特性可分为两类：人工合成型和天然生物型。常见的人工合成型阳离子多聚物基因载体主要有：多聚左旋赖氨酸[poly(L-Iysine)，PLL]，多聚乙烯亚胺(polyethyIenimine，PEI)和星状树突体[PnIyamidnamine(PAMAM)dendrimers]等：天然生物型阳离子多聚物基因载体主要有壳聚糖及其衍生物和明胶等。本文重点讨论了阳离子多聚物介导的基因导入细胞机理和基因进行靶向性转移的策略，详细论述了各种阳离子多聚物用作基因载体的性能特点，最后对非病毒基因载体的发展作出展望。     

非病毒载体的研究现状

非病毒载体在基因表达质粒、反义寡核苷酸或反义表达质粒真核细胞的靶向转移中 ,有着病毒载体不可替代的作用 ,对这方面的研究人们投入了很大的精力 ,以期在基因治疗方面有所突破。本文综述了近年来非病毒载体的研究现状 ,分别阐明了质粒 DNA肌肉注射、脂质体载体、多聚阳离子载体、多肽导向载体以及嵌合载体 ,指出了非病毒载体亟需发展之处以及病毒载体与非病毒载体联合发展的必要性     

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.     

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.     

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。     

基因治疗在创伤愈合中的应用

创伤修复是一个复杂的生物学过程,各种生长因子、炎症介质等在创面愈合过程中扮演着重要角色。基因治疗是现代生物治疗的一项重要技术,在创伤修复,尤其是难愈性创面的治疗中具有广阔的应用前景。基因治疗分为病毒载体导入系统（逆转录病毒、慢病毒、腺病毒、单纯疱疹病毒和腺病毒相关病毒等）和非病毒载体导入系统的基因感染（直接注射、显微注射、基因枪、电穿孔、脂质体和脂质体复合物、阳离子多聚物等）。本文对创伤修复及基因治疗在该领域的应用进行文献综述。     

siRNA非病毒纳米载体研究进展

当前,小分子干扰RNA的递送方式主要包括病毒载体载送、化学修饰载送、显微注射载送、非病毒纳米载体载送。非病毒纳米载体载送与其它载送方式相比具有简便、安全、易被患者接受等优点,但由于siRNA易水解以及被细胞摄取能力较差等原因,细胞呈递效率普遍较低。因此,近年来旨在优化siRNA、增加其应用范围而对其进行修饰的研究越来越多。本文就近期siRNA非病毒纳米载体研究进行综述,为其临床应用提供理论依据。     

腺病毒伴随病毒载体的研究进展

腺病毒伴随病毒载体的研究进展姚二梅冯泽华侯云德随着对人类基因愈加深入的了解，以及对真核基因改造和转移技术的发展，人类基因治疗已成为现实。基因治疗的关键问题就是要寻找有效的基因转移系统。在病毒介导和非病毒介导的两类基因转移系统中，感染性的重组病毒载体是...     

杆状病毒在基因治疗中的应用进展

基因治疗的核心技术之一是获得高效、安全的基因转移载体.目前的非病毒载体(脂质体、多聚物、纳米载体等)和病毒载体(腺病毒、腺相关病毒、疱疹病毒和逆转录病毒等)都还各自存在着某些缺陷,而杆状病毒由于安全性高、可插入基因片段大、操作性好等优点展现了在基因治疗中的巨大应用前景,开辟了杆状病毒应用的新领域[1].就杆状病毒作为基因载体在体内外和癌症基因治疗中的应用进展及其相关问题(如介导体内基因表达时遇到的障碍和解决措施等)作一综述.     

Gene transfer by adenovirus-mimetic peptides in the presence of a cationic lipid and/or adenovirus. Analysis of the contribution of the viral and nonviral components

Summary.  Peptide and cationic lipid-based gene transfer vectors have shown promise for gene therapy but are still less efficient than viral gene transfer vectors. We have examined the mechanism of gene transfer of different adenovirus-mimetic peptides in the presence and absence of a cationic lipid, lipofectamine and/or adenovirus with the aim of improving the design of nonviral vectors for efficient gene transfer. Three polylysine-adenovirus-mimetic peptides were synthesised and examined for their efficacy for gene transfer. Transfection levels in four cell lines: adenovirus permissive human tracheal epithelial (56FHTE8o⁻), human lung carcinoma (A549), human colon carcinoma (Caco-2) cells, and adenovirus low-permissive Chinese hamster ovary (CHO) cells, were examined. The polylysine-adenovirus-mimetic peptides increased the level of transfection of a reporter transgene in all cell lines. Transfection was substantially increased when an adenovirus was added to cells after pre-incubation with the vector complexes. Formulation of the peptide vector complexes with lipofectamine increased their transfection efficacy and the subsequent addition of an adenovirus increased transfection levels even further but only in permissive cells. Pre-incubation of cells with lipofectamine-peptide vector complexes increased cell binding of the adenovirus but uptake was only increased in intermediate- or non-permissive cells. The addition of lipofectamine increased transgene expression of a recombinant adenovirus in non-permissive cells but not in permissive cells. Enhancement with an adenovirus of peptide vector gene transfer is probably due to more efficient endosome escape while enhancement of gene transfer by peptide vectors complexed to lipofectamine is due to an increase in cellular binding and/or internalisation of the adenovirus. Received February 8, 2002; accepted August 23, 2002     

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.     

Improvement of nonviral gene therapy by Epstein-Barr virus (EBV)-based plasmid vectors

The nonviral gene transfer technologies include naked DNA administration, electrical or particle-mediated transfer of naked DNA, and administration of DNA-synthetic macromolecule complex vectors. Each method has its advantage, such as low immunogenicity, inexpensiveness, ease in handling, etc., but the common disadvantage is that the transfection efficiency has been relatively poor as far as conventional plasmid vectors are involved. To improve the nonviral gene transfer systems, Epstein-Barr virus (EBV)-based plasmid vectors (also referred to EBV-based episomal vectors) have been employed. These vectors contain the EBNA1 gene and oriP element that enable high transfer efficiency, strong transgene expression and long term maintenance of the expression. In the current article, I review recent preclinical gene therapy studies with the EBV plasmid vectors conducted against various diseases. For gene therapy against malignancies, drastic tumor suppression was achieved by gancyclovir administrations following an intratumoral injection with an EBV plasmid vector encoding the HSV1-TK suicide gene. Equiping the plasmid with carcinoembryonic antigen (CEA) promoter sequences enabled targeted killing of CEA-positive tumor cells, which was not accomplished by conventional plasmid vectors without the EBV genetic elements. Transfection with an apoptosis-inducing gene was also effective in inhibiting tumors. Interleukin (IL)-12 and IL-18 gene transfer, either local or systemic, induced therapeutic antitumoral immune responses including augmentation of the cytotoxic T lymphocyte (CTL) and natural killer (NK) activities, while an autologous tumor vaccine engineered to secrete Th1 cytokines via the EBV system also induced growth retardation of tumors. Non-EBV conventional plasmids were much less effective in eliciting these therapeutic outcomes. Intracardiomuscular transfer of the beta-adrenergic receptor gene induced a significant elevation in cardiac output in cardiomyopathic animals, suggesting the usefulness of the EBV system in treating heart failure. The EBV-based nonviral delivery also worked as genetic vaccine that triggered prophylactic cellular and humoral immunity against acute lethal viral infection. All the nonviral delivery vehicles so far tested showed an improved transfection rate when combined with the EBV-plasmids. Collectively, the EBV-based plasmid vectors may greatly contribute to nonviral gene therapy against a variety of disorders, including malignant, congenital, chronic and infectious diseases.     

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。     

Nonviral vectors for cancer gene therapy: prospects for integrating vectors and combination therapies

Gene therapy has the potential to improve the clinical outcome of many cancers by transferring therapeutic genes into tumor cells or normal host tissue. Gene transfer into tumor cells or tumor-associated stroma is being employed to induce tumor cell death, stimulate anti-tumor immune response, inhibit angiogenesis, and control tumor cell growth. Viral vectors have been used to achieve this proof of principle in animal models and, in select cases, in human clinical trials. Nevertheless, there has been considerable interest in developing nonviral vectors for cancer gene therapy. Nonviral vectors are simpler, more amenable to large-scale manufacture, and potentially safer for clinical use. Nonviral vectors were once limited by low gene transfer efficiency and transient or steadily declining gene expression. However, recent improvements in plasmid-based vectors and delivery methods are showing promise in circumventing these obstacles. This article reviews the current status of nonviral cancer gene therapy, with an emphasis on combination strategies, long-term gene transfer using transposons and bacteriophage integrases, and future directions.     

Nonviral gene therapy

The last 10 years have seen substantial progress in the development and application of nonviral vectors in gene therapy. Several novel nonviral methods have been developed that approach viruses with respect to transfection efficiency. A variety of nonviral delivery systems that can be used for gene therapy in different clinical settings are also available. In this review article, we will detail all of the major nonviral vectors that are currently used in gene therapy while highlighting some recent developments, particularly the progress towards the understanding of the cellular and in vivo barriers in gene transfer. Recent advancement in achieving sustained and regulated gene expression will also be addressed. Finally, this review will briefly cover targeted gene repair using nonviral delivery systems. Their impact in gene therapy will also be discussed.     

Thyroid hormone (T3)-modification of polyethyleneglycol (PEG)-polyethyleneimine (PEI) graft copolymers for improved gene delivery to hepatocytes

Targeting of gene vectors to liver hepatocytes could offer the opportunity to cure various acquired and inherited diseases. Efficient gene delivery to the liver parenchyma has been obscured from efficient targeting of hepatocytes. Here we show that the thyroid hormone, triiodothyronine (T3), can be used to improve the gene transfer efficiency of nonviral gene vectors to hepatocytes in vitro and to the liver of mice in vivo. T3 conjugated to the distal ends of fluorescent labeled PEG-g-dextran resulted in T3-specific cellular endosomal uptake into the hepatocellular cell line HepG2. PEG-g-PEI graft copolymers with increasing molar PEG-ratios were synthesized, complexed with plasmid DNA, and transfected into HepG2 or HeLa cells. Gene transfer efficiency decreased as the number of PEG blocks increased. T3 conjugation to PEI and the distal ends of PEG blocks resulted in T3 specific gene transfer in HepG2 cells as evidenced by reduction of gene transfer efficiency after pre-incubation of cells with excess of T3. In vivo application of T3-PEG-g-PEI based gene vectors in mice after tail vein injection resulted in a significantly 7-fold increase of gene expression in the liver compared with PEG-g-PEI based gene vectors.     

Integrase-defective lentiviral vectors--a stage for nonviral integration machineries

Gene vehicles derived from lentiviruses have become highly esteemed tools for gene transfer and genomic insertion in a wealth of cell types both in vivo and ex vivo. However, accumulating evidence of preferred insertion into actively transcribed genes, driven by biological properties of the parental human immunodeficiency virus type 1, has questioned the safety of this vector technology. As a consequence, integrase-defective lentiviral vectors [IDLVs], carrying an inactive integrase protein, have been developed and used with success for persistent in vivo gene transfer to quiescent or slowly dividing cells. We and others have shown that episomal DNA delivered by IDLVs may serve as a substrate for heterologous integration machineries, including recombinases and transposases, and homologous recombination triggered by nuclease-induced DNA damage. New vector systems that combine the best of lentiviral gene delivery and nonviral integration systems are under development. The first prototypes of such hybrid lentiviral vectors facilitate efficient gene transfer and show profiles of insertion that are not dictated by the biological constraints of the normal integration pathway and are, therefore, significantly different from the profile of conventional lentiviral vectors. The stage is set for further exploration of these vectors. In this review, we summarize the background and short history of hybrid IDLV-based vector systems and discuss their applicability in gene therapy and treatment of genetic disease.     

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.