GENE REPLACEMENT THERAPY--HUMAN AND MOUSE MODELS

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Adenosine deaminase (ADA) deficiency, a rare autosomal recessive condition causing a form of severe combined immunodeficiency, provides an optimal model disease to develop strategies for gene therapy of diseases which may be ameliorated by gene transfer into hematopoietic tissue. Our recent success in obtaining stable long term expression of human ADA in mice, via retroviral gene transfer in hematopoietic cells, offers encouragement that the techniques can be further improved and applied to gene therapy of inherited human diseases. Human hematopoietic stem cell infection will be attempted with the vector that was successful in transducing long term expression in mice (delta N2ADA) and with new vectors which will be constructed. Enrichment for primitive stem cells from hum bone marrow will allow us to examine the infectability of those cells with retroviral vectors. For this purpose, positive selection with anti-CD34 monoclonal antibodies will be combined with negative selection using anti-CD33 and anti-Ia antibodies to enrich the target population for primitive hematopoietic progenitor cells. Infection of primitive stem cells will be evaluated in long term bone marrow culture and through transplantation of human hematopoietic progenitors in immuno-deficient mice. Questions regarding issues of safety and expansion of the technology to the number of cells required for hematology reconstitution will be addressed. Ornithine transcarbamylase (OTC) deficiency, the most common and severe human defect in ureagenesis, is an excellent model disease to develop and test gene therapy strategies aimed at correcting inborn errors of liver cell metabolism. We have recently identified the molecular basis of OTC deficiency in the sparse fur (spf) mouse, the mouse model for the human defect. Construction of retroviral and adeno-associated viral vectors will be undertaken and the vectors tested for their ability to infect and transduce OTC activity into spf hepatocytes in vitro and in vivo. The creation of transgenic spf mice which express human OTC cDNA in small bowel and are biochemically and phenotypically correct for OTC deficiency points to a new target tissue for gene therapy of this disease. Recombinant retrovirus and adeno-associated virus which encode for OTC will be used to infect spf small intestinal epithelial cells to determine if gene transfer and expression of OTC in this tissue is possible and if successful transduction of OTC activity in this tissue will correct the inborn error of these mice.