The research proposed in Project 1 of this program will address four important aspects facing the development of adeno-associated virus as a gene transfer vector and its application to gene transfer in the treatment of mental retardation. )i) One of the most formidable problems facing the application of gene transfer techniques to gene therapy is that Maloney murine leukemia virus-derived retroviral vectors, the most well-developed and widely utilized system for high-efficiency and stable gene transfer into human cells, are dependent upon replication of the host cell for effective integration. Like retroviruses, adeno-associated virus also integrate into host cell chromosomal DNA, making it an attractive alternative to retroviruses as a vector system for gene therapy. Recently, it has been reported that AAV mediates high efficiency gene transfer and expression in quiescent cell populations, raising speculation that AAV is capable of integrating into non-dividing cells. However, direct and definitive molecular proof of chromosomal integration in non-dividing cell populations has been lacking. In this proposal, experiments are described for direct determination of AAV integration into chromosomal DNA of target cells after exposure to AAV vector under conditions which keep the target cells from replicating. (ii) It has also recently been reported that exposure of CD34+ enriched human bone marrow cells to AAV vectors results in a high proportion of transduced hematopoietic progenitors (CFU-GEMM). However, these transduction assays (for insertion of a gene mediating drug-resistant colony-formation) were conducted immediately after exposure to the virus and thus do not constitute determination of gene transfer into more primitive hematopoietic stem cells. Experiments are proposed herein to determine the efficiency of AAV-mediated gene transfer into long-term bone marrow culture initiating cells as a population more closely resembling primitive hematopoietic stem cells. (iii) The establishment of packaging cell lines expressing the AAV protein-encoding genes cap and rep has been problematic, most likely due to toxicity of the rep gene product. In this project, several approaches to establishing cell lines which conditionally express AAV proteins are proposed, including the use of adenovirus regulated AAV protein expression and also use of the E. coli lac operator/repressor system. Establishment of such packaging cell lines which can be quality-control tested will constitute and important advance in the application of AAV for gene therapy. (iv) Finally, the construction of AAV vectors for transduction of human iduronidase in hematopoietic cells is proposed, including gene transfer experiments and testing for in vitro expression of iduronidase activity in hematopoietic cultures established from Hunter patients. These experiments will thus address four of the central questions facing the application of AAV vectors to gene therapy of mental retardation, particularly by gene transfer into hematopoietic cells, and the potential of these vectors for resolving the challenge of gene transfer into human cells of clinical relevance.
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