AAV A NOVEL VECTOR FOR GLOBIN GENE THERAPY

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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.