AAV--A NOVEL VECTOR FOR GLOBIN GENE THERAPY

AAV——珠蛋白基因治疗的新型载体

• 批准号: 2224421
• 负责人: RICHARD J SAMULSKI
• 金额: $ 16.37万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-01 至 1997-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2224421
• 关键词: adeno associated virus group; clone cells; disease /disorder model; gene therapy; genetic transduction; genetically modified animals; globin; laboratory mouse; plasmids; sickle cell anemia; thalassemia; transcription factor; transfection /expression vector

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.

基因疗法的最大愿望之一是最终发展 将提供可行方法来纠正遗传的技术 缺陷和战斗传染病。我的实验室参与了 研究缺陷的人细节病毒腺病毒的分子生物学 寻求病毒（AAV），希望开发安全的有效病毒 人类基因疗法的载体。 AAV是依赖的细小病毒。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）为了在培养细胞中经历生产性感染。在 裂解感染，AAV DNA复制为4.7千座双链 分子并将其包装成Virion作为两者的线性单链 极性。在没有辅助病毒的没有共同感染的情况下，AAV 基因组通过其末端整合到特定地点的宿主基因组中 方式并居住在潜在状态，直到细胞感染 辅助病毒。然后，AAV DNA被“救出”，重复和 建立正常的生产感染。 AAV的主机范围很广 与感染性（人类，猴子，小鼠等）共同感染时 合适的助手。实际上，与当前的逆转录病毒载体相比 AAV的这些特征在利用AAV作为一个 病毒载体。人AAV具有许多优势。其中一些是： （1）它在人类中无处不在，（2）AAV可以集中在滴度上 每毫升超过10（9）个传染性单位，（3）是 完全非致病性整合病毒。正在进行的研究是 揭示这种非致病性人类病毒现在可以使用 用作向量。 我们已经开发了一个包装系统，该系统允许有效 外源基因封装到AAV病毒中。我们还确定了 有效整合所需的基本顺式作用序列 进入宿主细胞DNA。最后，我们表征了野生型集成 并发现了特定地点集成的令人兴奋的结果。这 上次观察显然将AAV分开为桉树病毒载体和 这是人类基因疗法的潜力。总体目标 拟议的工作是充分测试AAV作为特定的可行性 环球蛋白基因疗法的转导病毒载体。