ADENO ASSOCIATED VIRUS MEDIATED TRANSDUCTION--HEMATOPOIETIC STEM/PROGENITOR CELLS

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The adeno-associated virus 2(AAV)-based vector system has attracted considerable attention as an alternative to the more commonly used retroviral vectors for its potential use in gene therapy primarily because AAV is a non-pathogenic virus for humans, and the wild-type (wt) AAV genome has been shown to integrate into the human chromosomal DNA in a site-specific manner. However, a number of questions related to the basic molecular biology of the wt AAV interactions with normal human diploid cells in general, and that of the recombinant AAV in particular, remain largely unexplored. We have initiated systematic studies to pursue answers to these questions, and obtained preliminary evidence that the wt AAV interacts with normal human diploid fibroblasts in a manner that is distinct from that with human aneuploid cells. The hypotheses to be tested in this subproject are that the wt AAV integrates into normal human hematopoietic cells site-specifically at a site distinct from that characterized in human aneuploid cells, and that recombinant AAV genomes lacking the viral coding sequences (rep and/or cap) integrate in diploid hematopoietic cells at sites different from that for the wt AAV genome. The following Specific Aims will be pursued: 1. Comparison of patterns of integration of the wt AAV genome in human aneuploid and diploid hematopoietic cells, including purified populations of primitive progenitor cells from normal human bone marrow and umbilical cord blood: Using Southern blot and polymerase-chain reaction (PCR) analyses, the patterns of integration of the wt AAV genome will be compared in human aneuploid and diploid hematopoietic cells. 2. Evaluation of the role of AAV-encoded proteins in the site-specific integration of the wt and the recombinant AAV genomes: Recombinant viral vectors containing either the viral rep or the cap gene sequences will be used to evaluate whether AAV-encoded proteins play a role in the site- specific integration. 3. Molecular cloning and characterization of the AAV-integration sites in diploid cells, and comparison with that from aneuploid cells: In addition to direct cloning into bacteriophage lambda and plasmid vectors, PCR-based cloning strategies will be employed to obtain DNA sequences that contain the AAV-integration sites from human hematopoietic cells. The primary structure and transcriptional potential of these sequences will also be determined. 4. Evaluation of AAV-mediated transduction in vivo, and the potential for long-term expression of the transduced genes in a murine model system: Murine hematopoietic stem and progenitor cells will be transduced ex vivo, and following in vivo reconstitution of recipient mice, the patterns of integration of the wt and the recombinant AAV genomes as well as safety and efficacy of the AAV-based vector system will be examined. These studies will provide new insights into the basic molecular biology of the AAV-normal diploid cell interactions, and also evaluate the in vivo efficacy and safety of the AAV-based vector system prior to its potential use in human gene therapy. These studies also relate to our long-term interests in parvoviruses and human disease.