PARVOVIRUS VECTORS FOR HUMAN GENE THERAPY

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DESCRIPTION: The adeno-associated virus 2 (AAV), a non-pathogenic human parvovirus, has been suggested as a potentially useful vector for human gene therapy. However, a number of fundamental questions related to AAV-host cell interactions remain largely unexplored. These include the identity of the cellular receptor for AAV, and the nature of interaction of AAV with normal human diploid cells, the most likely target for a natural AAV infection, and also a potential target for gene therapy with this vector system. Similarly, although a second parvovirus of human origin, designated parvovirus B19, has been shown to possess a remarkable tropism for human hematopoietic progenitor cell in the erythroid lineage, B19-based vectors capable of erythroid cell-specific delivery of genes have not been developed. Dr. Srivastava and his colleagues propose to identify the cellular receptor for AAV, and evaluate the virus host interaction using primary human hematopoietic stem and progenitor cells in human umbilical cord blood. The erythroid cell-tropism of parvovirus B19 will be exploited to develop novel B19-based vectors to achieve targeted delivery of human globin genes. The hypotheses to be tested in this proposal are: 1. That entry of AAV in human cells is specific and receptor-mediated. 2. That erythroid cell-specific delivery and high level expression of a transduced beta-globin gene in hematopoietic cells is feasible in vitro as well as in vivo. The four specific aims are: 1. Characterization of interaction of recombinant AAV vectors with hematopoietic stem and progenitor cells in human umbilical cord blood, and identification of the putative cellular receptor for AAV. 2. Evaluation of AAV-mediated transduction and erythroid cell-specific, high level expression of a normal human beta-globin gene in hematopoietic progenitor cells in cord blood. 3. Development of parvovirus B19-based vector and B19-mediated erythroid cell-targeted delivery and expression of a normal human beta-globin gene in hematopoietic progenitor cells in cord blood. 4. Investigation of AAV- and B19- mediated transduction ex vivo, and the potential for long-term in vivo expression of the transduced human beta-globin gene in beta-thalassemic murine and non-human primate models. These studies will provide new insights into the basic molecular biology of parvovirus-diploid cell interactions as a prelude to the development of safe and effective vectors, and help in evaluating the in vivo efficacy and safety prior to their potential use in gene therapy for human hemoglobinopathies in general, and sickle cell disease and beta-thalassemia in particular.