阳离子聚合物在非病毒基因转染中的研究进展

基因治疗为治疗先天性遗传疾病和严重后天获得性疾病提供了一条新途径.目前,基因载体分为两类:病毒载体和非病毒载体.病毒载体转染效率高,但由于某些病毒载体存在免疫原性、致癌性、宿主DNA插入整合等缺点,从而限制了它们的应用.非病毒载体具有价格低、制备简单、安全有效、无免疫原性等优点,成为基因载体研究的热点.阳离子多聚物是非病毒载体的典型代表.文中综述近年来阳离子多聚物作为基因载体的研究现状和进展,重点介绍了阳离子多聚物基因载体的分类和与DNA的相互作用和传递机制.     

Low-volume jet injection for efficient nonviral in vivo gene transfer

The transfer of naked deoxyribonucleic acid (DNA) represents an alternative to viral and liposomal gene transfer technologies for gene therapy applications. Various procedures are employed to deliver naked DNA into the desired cells or tissues in vitro and in vivo, such as by simple needle injection, particle bombardment, in vivo electroporation or jet injection. Among the various nonviral gene delivery technologies jet injection is gaining increasing acceptance because it allows gene transfer into different tissues with deeper penetration of the applied naked DNA. The versatile hand-held Swiss jet injector uses pressurized air to force small volumes of 3 to 10 μL of naked DNA into targeted tissues. The β-galactosidase (LacZ) reporter gene construct and tumor necrosis factor α gene-expressing vectors were successfully jet injected at a pressure of 3.0 bar into xenotransplanted human tumor models of colon carcinoma. Qualitative and quantitative expression analysis of jet injected tumor tissues revealed the efficient expression of these genes in the tumors. Using this Swiss jet-injector prototype repeated jet injections of low volumes (3–10 μL) into one target tissue can easily be performed. The key parameters of in vivo jet injection such as jet injection volume, pressure, jet penetration into the tumor tissue, DNA stability have been defined for optimized nonviral gene therapy. These studies demonstrate the applicability of the jet injection technology for the efficient and simultaneous in vivo gene transfer of two different plasmid DNAs into tumors. It can be employed for nonviral gene therapy of cancer using minimal amounts of naked DNA.     

Nonviral gene delivery: techniques and implications for molecular medicine

Medical research continues to illuminate the origins of many human diseases. Gene therapy has been widely proposed as a novel strategy by which this knowledge can be used to deliver new and improved therapies. Viral gene transfer is relatively efficient but there are concerns relating to the use of viral vectors in humans. Conversely, nonviral vectors appear safe but inefficient. Therefore, the development of an efficient nonviral vector remains a highly desirable goal. This review focuses on the numerous challenges preventing efficient nonviral gene transfer in vivo and discusses the many technologies that have been adopted to overcome these problems.     

Targeted vectors for gene therapy of cancer and retroviral infections

Gene therapy has developed to a technology which rapidly moved from the laboratory bench to the bedside in the clinic. This implies safe, efficient and targeted gene transfer systems for suitable application to the patient. Beside the development of such gene transfer vectors of viral or nonviral origin, improvement of cell type specific and inducible gene expression is pivotal for successful gene therapy leading to targeted gene action. Numerous gene therapy approaches for treatment of cancer and retroviral infections utilize cell type specific and/or regulatable promoter and enhancer sequences for the selective expression of therapeutic genes in the desired cell populations and tissues. In this article the recent developments and the potential of expression targeting are reviewed for gene therapy approaches of cancer and retroviral infections.     

Polymer‐coated viral vectors: hybrid nanosystems for gene therapy

The advantages and critical aspects of nanodimensional polymer‐coated viral vector systems potentially applicable for gene delivery are reviewed. Various viral and nonviral vectors have been explored for gene therapy. Viral gene transfer methods, although highly efficient, are limited by their immunogenicity. Nonviral vectors have a lower transfection efficiency as a result of their inability to escape from the endosome. To overcome these drawbacks, novel nanotechnology‐mediated interventions that involve the coating or modification of virus using polymers have emerged as a new paradigm in gene therapy. These alterations not only modify the tropism of the virus, but also reduce their undesirable interactions with the biological system. Also, co‐encapsulation of other therapeutic agents in the polymeric coating may serve to augment the treatment efficacy. The viral particles can aid endosomal escape, as well as nuclear targeting, thereby enhancing the transfection efficiency. The integration of the desirable properties of both viral and nonviral vectors has been found beneficial for gene therapy by enhancing the transduction efficiency and minimizing the immune response. However, it is essential to ensure that these attempts should not compromise on the inherent ability of viruses to target and internalize into the cells and escape the endosomes.     

Nonviral regenerative gene therapy of mammalian cutaneous damages

The rate and character of superficial tissue regeneration after wounds, burns, and other traumas depend on cell proliferation within the damaged area. The acceleration of wound healing via the stimulation of cell proliferation and extracellular matrix synthesis is one of the most important tasks of modern medicine. There are gene therapy approaches to wound treatment, such as the transfer of genes that encode mitogenic growth factors to the wound area. The most important step in the development of the gene therapy approaches is the design of gene delivery tools. Despite the high efficiency of viral vectors, the nonviral approaches have some advantages (low toxicity, low immunogenity, safety, and the absence of side effects). Among the nonviral gene delivery tools molecular conjugates are the most popular due to their efficiency, simplicity, and the capacity for targeted gene transfer. In the present work, we have developed two molecular conjugates, NLS-TSF7 and NLS-TSF12, which consist of the modified signal of the nuclear localization of the T-antigen of the SV40 virus (cationic part) and the peptide ligands of the mammalian transferrin receptor (ligand part). Those conjugates bind to plasmid DNA via the formation of polyelectrolytic complexes and are able to deliver plasmid DNA into cells that express transferrin receptors through receptor-mediated endocytosis. The transfer of the expression vector of the luciferase gene in the complex with the molecular conjugate NLS-TSF7 to murine surface tissues led to about the 100-fold increase of luciferase activity in comparison with the transfer of the free expression vector. The treatment of mice with incised wounds with complexes of the expression vector of the synthetic human gene that encodes insulin-like growth factor 1 with molecular conjugate NLS-TSF7 led to the acceleration of wound healing in comparison with mice treated with the free expression vector. The obtained results confirm the high efficiency of the developed approach to regenerative gene therapy for treating the superficial tissue damage of mammals.     

The evolution of heart gene delivery vectors

Gene therapy holds promise for treating numerous heart diseases. A key premise for the success of cardiac gene therapy is the development of powerful gene transfer vehicles that can achieve highly efficient and persistent gene transfer specifically in the heart. Other features of an ideal vector include negligible toxicity, minimal immunogenicity and easy manufacturing. Rapid progress in the fields of molecular biology and virology has offered great opportunities to engineer various genetic materials for heart gene delivery. Several nonviral vectors (e.g. naked plasmids, plasmid lipid/polymer complexes and oligonucleotides) have been tested. Commonly used viral vectors include lentivirus, adenovirus and adeno-associated virus. Among these, adeno-associated virus has shown many attractive features for pre-clinical experimentation in animal models of heart diseases. We review the history and evolution of these vectors for heart gene transfer.     

Recent progresses in gene delivery-based bone tissue engineering

Gene therapy has converged with bone engineering over the past decade, by which a variety of therapeutic genes have been delivered to stimulate bone repair. These genes can be administered via in vivo or ex vivo approach using either viral or nonviral vectors. This article reviews the fundamental aspects and recent progresses in the gene therapy-based bone engineering, with emphasis on the new genes, viral vectors and gene delivery approaches.     

Designing gene delivery vectors for cardiovascular gene therapy

Genetic therapy in the cardiovascular system has been proposed for a variety of diseases ranging from prevention of vein graft failure to hypertension. Such diversity in pathogenesis requires the delivery of therapeutic genes to diverse cell types in vivo for varying lengths of time if efficient clinical therapies are to be developed. Data from extensive preclinical studies have been compiled and a certain areas have seen translation into large-scale clinical trials, with some encouraging reports. It is clear that progress within a number of disease areas is limited by a lack of suitable gene delivery vector systems through which to deliver therapeutic genes to the target site in an efficient, non-toxic manner. In general, currently available systems, including non-viral systems and viral vectors such as adenovirus (Ad) or adeno-associated virus (AAV), have a propensity to transduce non-vascular tissue with greater ease than vascular cells thereby limiting their application in cardiovascular disease. This problem has led to the development and testing of improved vector systems for cardiovascular gene delivery. Traditional viral and non-viral systems are being engineered to increase their efficiency of vascular cell transduction and diminish their affinity for other cell types through manipulation of vector:cell binding and the use of cell-selective promoters. It is envisaged that future use of such technology will substantially increase the efficacy of cardiovascular gene therapy.     

Functional and biodegradable dendritic macromolecules with controlled architectures as nontoxic and efficient nanoscale gene vectors

Gene therapy has provided great potential to revolutionize the treatment of many diseases. This therapy is strongly relied on whether a delivery vector efficiently and safely directs the therapeutic genes into the target tissue/cells. Nonviral gene delivery vectors have been emerging as a realistic alternative to the use of viral analogs with the potential of a clinically relevant output. Dendritic polymers were employed as nonviral vectors due to their branched and layered architectures, globular shape and multivalent groups on their surface, showing promise in gene delivery. In the present review, we try to bring out the recent trend of studies on functional and biodegradable dendritic polymers as nontoxic and efficient gene delivery vectors. By regulating dendritic polymer design and preparation, together with recent progress in the design of biodegradable polymers, it is possible to precisely manipulate their architectures, molecular weight and chemical composition, resulting in predictable tuning of their biocompatibility as well as gene transfection activities. The multifunctional and biodegradable dendritic polymers possessing the desirable characteristics are expected to overcome extra- and intracellular obstacles, and as efficient and nontoxic gene delivery vectors to move into the clinical arena.     

Polymer based cardiovascular gene therapy

Therapeutic angiogenesis is a new potential treatment in cardiovascular disease. It is performed by the delivery of the angiogenic agents (protein, gene). Most important consideration for gene therapy is the construction of an effective therapeutic gene. Currently, VEGF is the most effective therapeutic gene for the neovascularization. We constructed the hypoxia-regulated VEGF plasmid using the Epo enhancer and RTP801 promoter. The efficiency of the pEpo-SV-VEGF and pRTP801-VEGF were evaluated by various methodsin vitro andin vivo. The results suggested that the hypoxia-inducible VEGF gene therapy system is effective and safe, which may be useful for the gene therapy of ischemic heart disease. Development of a safe and efficient gene carrier is another main requirement for successful gene therapy. Although viralbased gene delivery is currently the most effective way to transfer genes to cells, nonviral vectors are increasingly being considered forin vivo gene delivery. The advantages of nonviral gene therapy are lack of specific immunogenecity, simplicity of use, and ease of large-scale production. In addition, the simple conjugation of a targeting moiety to nonviral gene carrier can facilitate tissue-targeting gene delivery. We have developed two new gene carrier systems, TerplexDNA and WSLP (water soluble lipopolymer). These two are efficient carrier to ischemic myocardium and has low toxicity and high transfection efficiency. So it may allow for application ofin vivo gene therapy in the treatment of heart disease.     

The role of surface chemistry-induced cell characteristics on nonviral gene delivery to mouse fibroblasts

ABSTRACT: BACKGROUND: Gene delivery approaches serve as a platform to modify gene expression of a cell population with applications including functional genomics, tissue engineering, and gene therapy. The delivery of exogenous genetic material via nonviral vectors has proven to be less toxic and to cause less of an immune response in comparison to viral vectors, but with decreased efficiency of gene transfer. Attempts have been made to improve nonviral gene transfer efficiency by modifying physicochemical properties of gene delivery vectors as well as developing new delivery techniques. In order to further improve and understand nonviral gene delivery, our approach focuses on the cell-material interface, since materials are known to modulate cell behavior, potentially rendering cells more responsive to nonviral gene transfer. In this study, self-assembled monolayers of alkanethiols on gold were employed as model biomaterial interfaces with varying surface chemistries. NIH/3T3 mouse fibroblasts were seeded on the modified surfaces and transfected using either lipid- or polymer- based complexing agents. RESULTS: Transfection was increased in cells on charged hydrophilic surfaces presenting carboxylic acid terminal functional groups, while cells on uncharged hydrophobic surfaces presenting methyl terminations demonstrated reduced transfection for both complexing agents. Surface--induced cellular characteristics that were hypothesized to affect nonviral gene transfer were subsequently investigated. Cells on charged hydrophilic surfaces presented higher cell densities, more cell spreading, more cells with ellipsoid morphologies, and increased quantities of focal adhesions and cytoskeleton features within cells, in contrast to cell on uncharged hydrophobic surfaces, and these cell behaviors were subsequently correlated to transfection characteristics. CONCLUSIONS: Extracellular influences on nonviral gene delivery were investigated by evaluating the upregulation and downregulation of transgene expression as a function of the cell behaviors induced by changes in the cells' microenvronments. This study demonstrates that simple surface modifications can lead to changes in the efficiency of nonviral gene delivery. In addition, statistically significant differences in various surface-induced cell characteristics were statistically correlated to transfection trends in fibroblasts using both lipid and polymer mediated DNA delivery approaches. The correlations between the evaluated complexing agents and cell behaviors (cell density, spreading, shape, cytoskeleton, focal adhesions, and viability) suggest that polymer-mediated transfection is correlated to cell morphological traits while lipid-mediated transfection correlates to proliferative characteristics.     

Parvovirus vectors: use and optimisation in cancer gene therapy

With the increasing incidence and mortality of cancer worldwide, there is an urgent need for new therapeutic approaches. Gene therapy is one such approach and preliminary data are promising. Viral and nonviral vector systems for gene delivery are available, but most of the current systems suffer from disadvantages such as low transfection efficiencies, in vivo instability, targeting problems, mutagenic potential and immunogenicity. Viruses of the Parvoviridae family, which are characterised by their oncotropism, oncosuppression, long-term gene expression and human apathogenicity, potentially offer advantages as viral vectors. This article evaluates their usefulness in gene therapy strategies for cancer.     

肿瘤基因治疗的靶向策略

对肿瘤组织的靶向性可以提高基因治疗的效果 ,避免对正常组织的损伤 ,并且能降低作为载体的微生物对机体的危害。对于瘤内注射的给药方法 ,靶向性似乎显得不是特别重要 ,但是如果要系统给药 ,靶向性是很关键的一个问题。靶向基因治疗肿瘤可以通过靶向基因导入和靶向基因表达来实现。近年来 ,在靶向基因导入方面的研究有很多进展 ,例如 ,用双亲性的桥连分子协助腺病毒和逆转录病毒靶向转导 ;在各种病毒载体的衣壳蛋白中插入靶向性的小肽或较大的多肽靶向结构域 ;增殖病毒作为一种很有前途的抗肿瘤制剂可有效地靶向杀伤肿瘤细胞。受体介导的DNA或DNA 脂质体复合物的靶向系统和其他一些靶向性的有疗效的载体 ,如细菌 ,也处于研究中。其中的一些载体已经进入临床实验。为了实现基因的靶向可调控表达 ,组织或肿瘤特异性的启动子和人工合成的可调控表达系统被用来调控治疗基因的表达。反义核酸、核酶以及脱氧核酶 (DNAzyme)被用来靶向抑制与肿瘤发生密切相关基因的表达。     

Gene therapy: progress and challenges.

Gene therapy is the delivery of new genetic material into a patient's somatic cells for the treatment of disease and is made possible through the development of viral and non-viral gene transfer vectors. In the first five years of gene therapy, clinical studies failed to yield efficacy data with the vectors available at that time. The lack of consistent clinical benefit prompted the United States National Institute of Health Recombinant DNA Advisory Committee to evaluate gene therapy research and conclude that substantial improvements in gene transfer vectors were needed in the areas of vector safety and control of the level and duration of gene expression, and to increase the understanding of the biological interaction of gene transfer vectors with the host. We will describe the progress in development of gene delivery technology, focusing on improvements in vector safety, analysis of vector biodistribution and GMP manufacturing of viral and non-viral gene transfer systems over the last six years since the report. Whereas 5 years ago, investigators tested every vector for every potential disease indication, the accumulated database now enables investigators to select a single vector based upon it's known performance in a wide number of animal models and human clinical studies. We will also highlight several directions investigators have taken to improve the safety and efficacy of gene therapy vectors.     

The future of human gene therapy

Human gene therapy (HGT) is defined as the transfer of nucleic acids (DNA) to somatic cells of a patient which results in a therapeutic effect, by either correcting genetic defects or by overexpressing proteins that are therapeutically useful. In the past, both the professional and the lay community had high (sometimes unreasonably high) expectations from HGT because of the early promise of treating or preventing diseases effectively and safely by this new technology. Although the theoretical advantages of HGT are undisputable, so far HGT has not delivered the promised results: convincing clinical efficacy could not be demonstrated yet in most of the trials conducted so far, while safety concerns were raised recently as the consequence of the "Gelsinger Case" in Philadelphia. This situation resulted from the by now well-recognized disparity between theory and practice. In other words, the existing technologies could not meet the practical needs of clinically successful HGT so far. However, over the past years, significant progress was made in various enabling technologies, in the molecular understanding of diseases and the manufacturing of vectors. HGT is a complex process, involving multiple steps in the human body (delivery to organs, tissue targeting, cellular trafficking, regulation of gene expression level and duration, biological activity of therapeutic protein, safety of the vector and gene product, to name just a few) most of which are not completely understood. The prerequisite of successful HGT include therapeutically suitable genes (with a proven role in pathophysiology of the disease), appropriate gene delivery systems (e.g., viral and non-viral vectors), proof of principle of efficacy and safety in appropriate preclinical models and suitable manufacturing and analytical processes to provide well-defined HGT products for clinical investigations. The most promising areas for gene therapy today are hemophilias, for monogenic diseases, and cardiovascular diseases (more specifically, therapeutic angiogenesis for myocardial ischemia and peripheral vascular disease, restenosis, stent stenosis and bypass graft failure) among multigenic diseases. This is based on the relative ease of access of blood vessels for HGT, and also because existing gene delivery technologies may be sufficient to achieve effective and safe therapeutic benefits for some of these indications (transient gene expression in some but not all affected cells is required to achieve a therapeutic effect at relatively low [safe] dose of vectors). For other diseases (including cancer) further developments in gene delivery vectors and gene expression systems will be required. It is important to note, that there will not be a "universal vector" and each clinical indication may require a specific set of technical hurdles to overcome. These will include modification of viral vectors (to reduce immunogenicity, change tropism and increase cloning capacity), engineering of non-viral vectors by mimicking the beneficial properties of viruses, cell-based gene delivery technologies, and development of innovative gene expression regulation systems. The technical advances together with the ever increasing knowledge and experience in the field will undoubtedly lead to the realization of the full potential of HGT in the future.     

Novel Therapeutic Approaches for Various Cancer Types Using a Modified Sleeping Beauty-Based Gene Delivery System

Successful gene therapy largely depends on the selective introduction of therapeutic genes into the appropriate target cancer cells. One of the most effective and promising approaches for targeting tumor tissue during gene delivery is the use of viral vectors, which allow for high efficiency gene delivery. However, the use of viral vectors is not without risks and safety concerns, such as toxicities, a host immune response towards the viral antigens or potential viral recombination into the host''s chromosome; these risks limit the clinical application of viral vectors. The Sleeping Beauty (SB) transposon-based system is an attractive, non-viral alternative to viral delivery systems. SB may be less immunogenic than the viral vector system due to its lack of viral sequences. The SB-based gene delivery system can stably integrate into the host cell genome to produce the therapeutic gene product over the lifetime of a cell. However, when compared to viral vectors, the non-viral SB-based gene delivery system still has limited therapeutic efficacy due to the lack of long-lasting gene expression potential and tumor cell specific gene transfer ability. These limitations could be overcome by modifying the SB system through the introduction of the hTERT promoter and the SV40 enhancer. In this study, a modified SB delivery system, under control of the hTERT promoter in conjunction with the SV40 enhancer, was able to successfully transfer the suicide gene (HSV-TK) into multiple types of cancer cells. The modified SB transfected cancer cells exhibited a significantly increased cancer cell specific death rate. These data suggest that our modified SB-based gene delivery system can be used as a safe and efficient tool for cancer cell specific therapeutic gene transfer and stable long-term expression.     

基因治疗的输送屏障及输送载体的研究

基因治疗是将可具有治疗性的基因导入病变细胞以达到治疗遗传性疾病或获得性功能缺损疾病的治疗手段,是一种极具潜力的新型治疗方法。然而基因治疗面临着一系列一陆床应用障碍,其中缺乏理想的基因输送载体是首要问题。绝大多数基因治疗方案受困缺乏安全有效的基因输送手段,载体要达到目的地发挥作用,需要克服一系列复杂的体内生物屏障,包括细胞外屏障和细胞内屏障。目前基因输送载体主要分为病毒载体和非病毒载体,其中病毒载体天然进化至可进入宿主细胞,具有输送效率高,靶向性好的特点,但存在长期安全性的缺点。非病毒载体主要包括阳离子脂质体和阳离子聚合物,由于易于制备和无免疫原性、安全性好,被认为是更有潜力的输送载体,是目前研究的重点。本文结合基因治疗输送屏障的理论基础及临床研究,对基因输送载体系统的现状进行了综述。     

Gene transfer during surgical procedures with molecular surgical suture

Over the last decades, there has been an explosion of interest in plasmid DNA for gene therapy with reports of their efficacy in the fight against cancer, vascular diseases, and inherited diseases caused by specific gene defects (Srivastava, 2003). DNA plasmids present several advantages over the use of recombinant viruses concerning their production and safety issues. Plasmid DNA vectors can be constructed easily and economically, and they are free of size constraints imposed by viral packaging, obviating the need for an infectious vector and lessening the likelihood of toxicity and immunogenicity (Davis, 1993). Plasmids have a relative low cost, long shelf life and allow repetitive administration of the therapeutic gene without generating an immune response against the delivery vector (Donnelly, 2003). Finally, plasmids can be injected directly into tissues, such as heart (Sarkar, 2002), muscle (Neumeister, 2001, Dan, 2000) and tumors (De Marco, 2003, Sasaki, 2002).     

Persistence of recombinant adenovirus in vivo is not dependent on vector DNA replication.

Recombinant adenovirus vectors represent an efficient means of transferring genes into many different organs. The first-generation E1-deleted vector genome remains episomal and, in the absence of host immunity, persists long-term in quiescent tissues such as the liver. The mechanism(s) which allows for persistence has not been established; however, vector DNA replication may be important because replication has been shown to occur in tissue culture systems. We have utilized a site-specific methylation strategy to monitor the replicative fate of E1-deleted adenovirus vectors in vitro and in vivo. Methylation-marked adenovirus vectors were produced by the addition of a methyl group onto the N6 position of the adenine base of XhoI sites, CTCGAG, by propagation of vectors in 293 cells expressing the XhoI isoschizomer PaeR7 methyltransferase. The methylation did not affect vector production or transgene expression but did prevent cleavage by XhoI. Loss of methylation through viral replication restores XhoI cleavage and was observed by Southern analysis in a wide variety of, but not all, cell culture systems studied, including hepatoma and mouse and macaque primary hepatocyte cultures. In contrast, following liver-directed gene transfer of methylated vector in C57BL/6 mice, adenovirus vector DNA was not cleaved by XhoI and therefore did not replicate, even after a period of 3 weeks. Although replication may occur in some tissues, these results show that stabilization of the vector within the target tissue prior to clearance by host immunity is not dependent upon replication of the vector, demonstrating that the input transduced DNA genomes were the persistent molecules. This information will be useful for the design of optimal adenovirus vectors and perhaps nonviral episomal vectors for clinical gene therapy.