Parvoviruses constitute a unique group of small, single-stranded DNA viruses that infect all vertebrates. One of the non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV) has gained attention as a useful alternative to the more commonly used retrovirus- and adenovirus-based vectors in human gene therapy. In contrast to AAV, which possesses a broad host-range, the pathogenic human parvovirus B19 has been shown to have a remarkably narrow tissue-tropism for erythroid progenitor cells presumably because the virus uses the blood group P antigen as a cell surface receptor. We have developed recombinant human parvovirus B19 vectors in which recombinant AAV genomes are encapsidated in parvovirus B19 capsids. Although the viral titers obtained are low, using these hybrid vectors, we have demonstrated that P antigen alone is not sufficient to impart erythroid cell-specificity to parvovirus B19. We have proposed a """"""""triple level of erythroid-specificity"""""""" model for parvovirus B19 infection in which P antigen is utilized as a primary receptor for virus binding, activated alpha5beta1 integrin as a cell surface co-receptor for viral entry, and a hitherto unknown cellular transcription factor for erythroid cell-specific expression from the viral promoter at map unit 6 (B19p6). Using a variety of molecular techniques and recombinant parvovirus vectors, we will test the following hypotheses: 1. The inclusion of appropriate transcriptional control elements and parvovirus B19 """"""""packaging signal"""""""" wilt lead to improved titers of recombinant parvovirus B19 vectors; 2. Mature RBCs are utilized for systemic dissemination and efficient entry of parvovirus B19 into primary human hematopoietic cells in the erythroid lineage, and 3. A putative transcription factor, which is present only in primary human erythroid progenitor cells, mediates lineage-restricted gene expression from the parvovirus B19p6 promoter. The following three Specific Aims will be pursued: 1. Development of strategies to generate high-titer recombinant parvovirus B19 vectors. 2. Elucidation of underlying mechanisms of parvovirus B19 infection of primary human erythroid progenitor ceils. 3. Elucidation of recombinant parvovirus vector-mediated transduction of primary human hematopoietic stem cells and erythroid lineage-restricted gene expression, and characterization of the putative cellular transcription factor specific for the B19p6 promoter. The knowledge gained from these studies will shed light on parvovirus Bl9-host cell interactions, and will lead to further improvements in recombinant parvovirus B19 vectors for their potential use in human gene therapy. ? ?
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