单基因遗传病基因治疗中病毒型载体的应用

摘要：在单基因遗传病的基因治疗中,病毒型载体是较为常用的将目的基因导入靶细胞的有效运载工具之一,分为腺病毒载体、腺相关病毒载体、逆转录病毒载体及慢病毒载体等。在本文中,我们拟就单基因遗传病基因治疗中病毒型载体的应用现状和研究进展做较为系统地阐述。     

慢病毒载体与基因治疗

载体是供插入目的基因并将其导入宿主细胞内表达或复制的运载工具。常用的有SV40衍生载体、逆转录病毒载体、腺病毒载体、腺病毒相关病毒载体、细小病毒载体、疱疹病毒载体、痘苗病毒载体等。近几年来。慢病毒载体受到高度重视，慢病毒载体来源于人类免疫缺陷病毒HIV-1、HIV-2、SIV，具有转染分裂和非分裂的T细胞、树枝状细胞、造血干细胞及巨噬细胞^[1]，基因疗法是一种新方法。Hawley^[2]等的文章中指出。由于慢病毒载体可作用于细胞周期的G0／G1期。成为遗传性疾病治疗的有效手段。基因治疗的最初目的是传送特殊的基因至预定的靶细胞，并且直接表达该基因的性质，达到治疗效果。在治疗遗传性、代谢性、神经系统疾病、癌症及艾滋病方面有广泛的应用前景。     

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...     

腺病毒及腺病毒相关病毒载体与基因治疗

腺病毒及腺病毒相关病毒能感染增殖与静止的细胞,宿主范围广。其稳定、安全滴度高、转染效率高。腺病毒载体能承载较大的外源基因,腺病毒相关病毒载体能定向整合入靶细胞基因组内等特点,使腺病毒及腺病毒相关病毒载体成为体内用于基因治疗的理想运载系统。     

腺病毒相关病毒载体的安全性评价

腺病毒相关病毒（AAV）作为基因治疗的病毒载体之一，已经受到广泛地关注。与其它载体相比，重组AAV具有宿主范围广、免疫原性小和安全性较好等优点，然而对重组AAV的靶向整合、毒性、在体内分布情况、产生免疫应答反应以及是否诱发肿瘤等问题仍还不十分清楚。本文就腺病毒相关病毒载体的安全性作一较全面而系统地评价。     

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。     

血友病A基因治疗载体研究现状

血友病A是X染色体隐性遗传出血性疾病.其发病原因是患者血液中先天缺乏凝血因子F Ⅷ.用于血友病A基因治疗研究的载体有病毒载体和非病毒载体,目前研究较多的是病毒载体,主要有逆转录病毒载体和慢病毒载体,腺病毒载体及腺相关病毒载体等.非病毒载体主要有质粒、脂质体、转座子等.文章拟对血友病A基因治疗各载体的特点和研究进展作一综...     

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.     

腺病毒载体在肿瘤基因治疗和基因疫苗领域的应用研究进展

腺病毒载体是目前应用最为广泛的基因治疗病毒载体之一。相较其他病毒载体,腺病毒具有较高的基因转导效率,广泛的组织亲和性,不整合入宿主基因组等优点。腺病毒可以被改造为复制缺陷病毒载体和选择性复制的溶瘤病毒载体。腺病毒载体较强的免疫原性使其可以被应用于基因疫苗和抗癌治疗中。目前众多的临床试验都证实了腺病毒载体在上述治疗试验中的安全性和有效性。     

不同载体转基因效率及AAV2介导bFGF基因在肌腱中表达的研究

目的:比较腺病毒、腺相关病毒和脂质体-质粒三种载体在肌腱中的基因转染效率并观测腺相关病毒载体介导bFGF基因在肌腱愈合过程中表达.方法:用微量注射器将携带增强绿色荧光蛋白报告基因的三种载体注射入正常肌腱中,用荧光显微镜观测术后不同时间点肌腱内EGFP的表达.将AAV2-bFGF病毒颗粒注入损伤肌腱,使用免疫组化染色观测bFGF在肌腱内表达.结果三种不同的载体转染肌腱3天后有EGFP表达、7天最明显、14天时下降;21天时少有表达.在同期的时间点,注射腺病毒、腺相关病毒组肌腱内EGFP表达明显强于质粒载体,而腺病毒、腺相关病毒两组之间无较大区别.免疫组化显示注射AAV2-bFGF后的第2、3和4周的肌腱表达很强的bFGF.结论:腺病毒、腺相关病毒载体的基因转染效率高于脂质体-质粒载体,AAV2携带的外源性bFGF基因在肌腱细胞内较强表达,提示我们将来在体内转基因到肌腱的研究中能够选择腺病毒和腺相关病毒载体作为运载基因的工具.     

Viral vectors in cancer immunotherapy: which vector for which strategy?

Gene therapy involves the transfer of genetic information to a target cell to facilitate the production of therapeutic proteins and is now a realistic prospect as a cancer treatment. Gene transfer may be achieved through the use of both viral and non-viral delivery methods and the role of this method in the gene therapy of cancer has been demonstrated. Viruses represent an attractive vehicle for cancer gene therapy due to their high efficiency of gene delivery. Many viruses can mediate long term gene expression, while some are also capable of infecting both dividing and non-dividing cells. Given the broadly differing capabilities of various viral vectors, it is imperative that the functionality of the virus meets the requirements of the specific treatment. A number of immunogene therapy strategies have been undertaken, utilising a range of viral vectors, and studies carried out in animal models and patients have demonstrated the therapeutic potential of viral vectors to carry genes to cancer cells and induce anti-tumour immune responses. This review critically discusses the advances in the viral vector mediated delivery of immunostimulatory molecules directly to tumour cells, the use of viral vectors to modify tumour cells, the creation of whole cell vaccines and the direct delivery of tumour antigens in animal models and clinical trials, specifically in the context of the suitability of vector types for specific strategies.     

Current status of genetic modulation of growth factors in wound repair

Growth factors are members of a large functional group of polypeptide regulatory molecules secreted by different cells. They are important players in orchestrating all stages of wound healing exerting their influence through autocrine and paracrine fashions within sites of injury and repair. They are mitogen, chemotactic, they regulate cell-cell interactions and influence synthesis and composition of extracellular matrix components. The use of growth factors to stimulate wound healing is a promising therapeutic approach to repair chronic tissue defects. The delivery of genetic material offers an attractive treatment modality to produce an appropriate amount of growth factor proteins within the wound site. Gene therapy might become a significant treatment modality for those wound healing pathologies refractory to other wound management approaches. This review discusses several methods of growth factor gene transfer into wound tissue.     

单纯疱疹病毒扩增子载体转导的ATM基因在活体的异位表达(英文)

小脑性共济失调与血管扩张症 (A-T)是一种常染色体隐性遗传性疾病 ,具多效表型。此病的特征是小脑变性、免疫缺陷、生长迟缓、早熟性衰老、染色体不稳定、肿瘤易患体质和对辐射的过度敏感。目前尚未发现有效的方法阻断此病的进行性发展。基因治疗是此病的潜在希望。ATM是此病的致病基因 ,编码 ATM蛋白激酶。本研究将 ATM c DNA完整的读码框架克隆进 1型单纯疱疹病毒 (HSV-1)扩增子载体 (p TO-ATM)。培养的 A-T细胞经 p TO-ATM病毒载体转导后 ,用免疫组织化学方法证明了 ATM在这些细胞的异位表达。为了进一步研究在活体应用此载体系统的可能性 ,选择大鼠脑作为实验靶区。先用免疫组织化学方法观察了内源性 ATM在成年大鼠前脑和小脑的正常分布 ,发现在尾壳核有低水平的 ATM出现 ,而在其余脑区 ,ATM水平则显著地高于尾壳核区。将 p TO-ATM病毒载体注射到成年大鼠的一侧尾壳核 ,3 d后在注射区 (包括针道周围的区域 )观察到密集的 ATM免疫反应阳性神经元。本研究结果证明 ,HSV扩增子病毒载体可以稳定地运载相当大的 c DNA片段 ,转基因在离体和活体均能有效地表达。本研究为使用基因治疗的方法治疗 A-T病人的神经变性迈出了第一步     

A defective HIV-1 vector for gene transfer to human lymphocytes

Conclusions All practical development of HIV-1 vectors for in vivo testing will require high titers of recombinant virus free of helper virus. To date, an HIV-based vector system with an efficiency of gene transfer comparable to that achieved by the Mo-MuLV-based system has not yet been developed. Such development will require a better definition of the optimal placement of viral cis-acting sequences within the HIV-1 vector. The establishment of stable packaging cell lines should increase the efficiency of production of recombinant HIV-1 viruses for future use. T-lymphocytes are, in fact, an important potential target for therapeutic gene transfer. The possible advantages of such vectors over currently available retroviral vectors suggest that the continued study of HIV-1 vectors for gene transfer to human T-lymphocytes is warranted.Abbreviations HIV Human immunodeficiency virus - LTR Long terminal repeat     

超声清创系统结合负压创面疗法在骨外露创面中的应用

目的 研究超声清创系统结合负压创面疗法治疗骨外露创面的临床效果.方法 采用超声手术系统结合负压创面疗法治疗17例患者,共18个骨外露创面,骨外露创面面积平均（10.5±6.6）cm^2.观察坏死骨组织清除速度、清除效果,清创术后3周创面愈合率,植皮成活率,创面完全愈合时间.结果 超声清创系统可以快速而有效地清除坏死骨组织以及磨除骨皮质.清创术后3周创面愈合（62.3±25.1）%,未愈合创面肉芽组织长满创面,色鲜红.植皮全部成活率88.9%,从第一次清创至创面完全愈合时间为（28.1±9.1）d.结论 超声清创系统结合负压创面疗法治疗骨外露创面可有效清除坏死骨组织,促进肉芽组织迅速生长,缩小创面面积,从而加速创面愈合,是治疗骨外露创面的一种行之有效的方法.     

Possibilities of non-viral gene transfer to improve cutaneous wound healing

Enhancement of dermal and epidermal regeneration represents a crucial goal for the treatment of acute, e.g. burn and trauma wounds, and chronic wounds, e.g. diabetic, autoimmune, arterial and venous wounds. Studies defining molecular mechanisms of the complex cascade of wound healing have shown that growth factors represent a new therapeutic strategy. The clinical application of growth factors in the form of proteins has been shown to be of little benefit. Therefore new delivery systems and therapeutic strategies needed to be developed to improve dermal and epidermal regeneration, one of which is gene therapy. For successful gene delivery the selection of an appropriate vector has been shown to be paramount. Because Retroviruses, Adenoviruses and Adeno-Associated Viruses can cause immunologic reactions and mutations, non-viral delivery systems for gene therapy, such as liposomal gene transfer appear advantageous over viral gene therapy. This review discusses the success, potential and limitations of non-viral gene transfer to improve regeneration of dermal and epidermal structures.     

骨形成蛋白介导的基因治疗研究进展

基因治疗是指通过导入基因的功能片段改善机体生理状况或者治疗疾病.利用基因治疗可在骨缺损或骨折局部释放骨形成蛋白（bone morphogenetic proteins,BMP）,并且无需异体载体,方法包括体内法和离体法.BMP基因治疗可以促进骨和软骨形成、脊柱融合、颌面骨和牙齿修复、肌腱韧带形成,此外对椎间盘退变性疾病也可采用BMP基因治疗.总之BMP基因治疗方法经济有效,是有前景的治疗手段.     

Cancer is a functional repair tissue

When a wound occurs, growth and repair genes (GR genes, such as oncogenes, proto-oncogenes, etc.) in surrounding cells are activated and secretion of growth and repair factors (GR factors, such as growth, stem cell, and stimulating factors, etc.) is induced to heal the wound. However, if the wound is persistent due to chronic physical (radiation, electromagnetic field, trauma, particles, etc.), chemical (carcinogens, toxic chemicals, heavy metals etc.) or biological (aging, free radicals, inflammation, nutrient deficiency, bacteria and virus infections, stress, etc.) damage, amplification of GR gene activation in surrounding cells may lead to a clinical cancer. Based on the commonalities between cancer and wound healing, a new hypothesis of cancer is presented: malignancies are not passive mutated useless masses; rather, they are functional tissues produced by GR gene activation to secrete GR factors in an effort to heal persistent wounds in the body. Based on the hypothesis, current cancer treatments aimed at killing cancer cells only may be misguided. The logical extension of the hypothesis is that cancer treatment focused on wound healing by limiting causes of persistent wounds, providing repair cells, GR factors, and substrates required by repair cells may yield more fruitful results than treatments focused on killing cancer cells alone. Spontaneous regressions of cancer, although rare, may be successful examples of serendipitous spontaneous wound healing. Standard therapies aimed at killing cancer cells, should be limited to adjuvant status for limiting symptoms or buying time for completion of the wound healing process. Attempts to destroy cancer cells without healing underlying persistent wounds will allow for eventual recurrence.     

Efficient gene delivery into adult cardiomyocytes by recombinant Sindbis virus

Somatic gene therapy as a potential strategy for the treatment of myocardial diseases relies on an efficient gene transfer into cardiac muscle cells. The difficulty of delivering genes into adult cardiomyocytes exists not only in vivo but also in primary culture systems. Therefore, possibilities for ex vivo gene transfer and the in vitro study of physiological processes by reverse genetics are limited. We investigated the potential of an alphavirus-based vector system to transduce adult rat cardiomyocytes (ARC) in culture using a replication-deficient Sindbis virus (SIN) encoding beta-galactosidase (SIN-LacZ). Transduction efficiency depended on the virus concentration used, with expression of the reporter gene being detectable in up to 80% of cultured ARC as early as 24 h after infection. We observed a remarkably lower cytotoxicity of this viral vector in ARC than in other cells such as fibroblasts and neonatal cardiomyocytes. Additionally, no perceptible changes in the morphology of the nuclei or cytoskeleton were found in ARC 48 h after infection with SIN-LacZ. We conclude that SIN vectors are useful for gene delivery into adult cardiomyocytes and believe that improved versions of this viral system may be useful for cardiovascular gene therapy in the future.