AAV--A NOVEL VECTOR FOR GLOBIN GENE THERAPY

基因治疗的最大愿望之一是最终开发出 这项技术将提供一种可行方法， 缺陷和对抗传染病。我的实验室从事 研究缺陷型人类细小病毒腺病毒的分子生物学， 目的是开发一种安全、高效的病毒载体， 用于人类基因治疗的载体。 AAV是一种依赖性细小病毒。也就是说，它需要与 另一种病毒（腺病毒或疱疹病毒的某些成员 组）以便在培养的细胞中进行生产性感染。在 在裂解性感染中，AAV DNA复制为4.7内切酶双链 分子，并包装成病毒粒子作为线性单链的两个 极性在没有辅助病毒共感染的情况下，AAV 基因组通过其末端以位点特异性的方式整合到宿主基因组中。 并以潜伏状态存在，直到细胞被感染 辅助病毒。然后，AAV DNA被“拯救”，复制并 建立正常的生产性感染。AAV具有广泛的宿主范围 感染性（人、猴、小鼠等）当同时感染 合适的帮手事实上，与目前的逆转录病毒载体相比 AAV的这些特征在利用AAV作为免疫调节剂方面是相当令人感兴趣的。 病毒载体人AAV具有许多优点。其中包括： (1)它在人类中普遍存在，（2）AAV可以浓缩到滴度 超过每毫升10（9）个传染单位，以及（3） 完全非致病性整合病毒。正在进行的研究是 揭示了这种非致病性的人类病毒现在可以被 作为一种载体。 我们开发了一种包装系统， 将外源基因插入AAV病毒体。我们还确定 有效整合所需的基本顺式作用序列 转化到宿主细胞DNA中最后，我们描述了野生型整合 并发现了位点特异性整合的令人兴奋的结果。这 最后的观察清楚地将AAV作为真核病毒载体区分开来， 这是人类基因治疗的潜力。的总体目标 拟议的工作是充分测试AAV作为一种特定的 用于珠蛋白基因治疗的转导病毒载体。

One of the great aspirations of gene therapy is to eventually develop technology which will provide a feasible approach to correct genetic defects and combat infectious diseases. My laboratory is engaged in studying the molecular biology of the defective human parvovirus adeno- addressed virus (AAV) in hopes of developing a safe efficient viral vector for human gene therapy. AAV is a dependent parvovirus. That is, it requires coinfection with another virus (either adenovirus or certain members of the herpes virus group) in order to undergo a productive infection in cultured cells. In a lytic infection, AAV DNA replicates as a 4.7 kilobase double-stranded molecule and is packaged into virion as linear single strands of both polarities. In the absence of coinfection with helper virus, the AAV genome integrates via its ends into the host genome in a site specific manner and resides there in a latent state until the cell is infected with helper virus. Then the AAV DNA is "rescued", replicates and establishes a normal productive infection. AAV has a broad host range for infectivity (human, monkey, mouse, etc.) when coinfected with the appropriate helper. In fact, compared to the current retroviral vectors these features of AAV are of considerable interest in utilizing AAV as a viral vector. Human AAV has a number of advantages. Some of them are: (1) it is ubiquitous in humans, (2) AAV can be concentrated to titers exceeding 10(9) infectious units per milliliter, and (3) it is completely nonpathogenic integrating virus. Ongoing research is revealing that this nonpathogenic human virus is now accessible for utilization as a vector. We have developed a packaging system which allows for efficient encapsidation of foreign genes into AAV virions. We have also identified the essential cis-acting sequences required for efficient integration into host cell DNA. Finally, we have characterized wild type integration and uncovered the exciting result of site-specific integration. This last observation clearly sets apart AAV as a eucaryotic viral vector and it's potential for gene therapy in humans. The overall objective of the proposed work is to fully test the feasibility of AAV as a specific transducing viral vector for globin gene therapy.