Previous experience with clinical bone marrow transplantation has shown that normal marrow is capable of providing life-long systemic metabolic correction for patients with MPS disease. However, translation of this principle to ex vivo hematopoietic stem cell (HSC) gene therapy has been limited by: (a) the inability of murine retroviruses to integrate into non- dividing cells; (b) lack of concentrated vector preparations to achieve a substantial MO1 and vector-cell """"""""approximation""""""""; and (c) immunologic selection against transgene-expressing cells. However, recent development of lentiviral vectors suggest an approach that may obviate these problems with broad implications for many applications of gene therapy. The overall objective of this project is to accomplish the requisite preclinical studies for ex vivo HSC gene therapy for Mucopolysaccharidosis (MPS) type I (i.e., Hurler syndrome and variants). However, recent development of lentiviral vectors suggest an approach that may obviate these problems with broad implications for many applications of gene therapy. The overall objective of this project is to accomplish the requisite preclinical studies for ex vivo HSC gene therapy for Mucopolysaccharidosis (MPS) type I (i.e., Hurler syndrome and variants). Toward this objective, conditions for ex vivo transduction of HSC. A lentiviral vector expressing GFP will ve evaluated in vitro with respect to optimizing transduction frequency and transgene of HSC. A lentiviral vector expressing GFP will be evaluated in vitro with respect to optimizing transduction frequency and transgene expression when targeted to murine bone marrow cells and to human G-CSF mobilized peripheral blood CD34+ cells. Subsequently, Specific Aim 2 will be to evaluate the potential for ex vivo bone marrow stem cell transduction, the potential for in vivo selection of transduced cells, as well as the degree of metabolic correction in a murine model of MPS disease. Lentiviral vectors will be constructed containing the cDNA for murine beta- glucuronidase (GUS) with, or without a genetically-engineered variant of dihydrofolate reductase (DHFR/tyr22) which has been found to optimize conditions of drug resistance in various murine test systems. Marrow from enzyme-deficient mice will be transduced ex vivo, and then administered to affected mice. Follow-up evaluations will study the response to genetically-modified HSC, specifically, determining the level of recombinant MPS enzyme activity achieved, the duration of expression, and clinical response. The animal studies will study the systemic response by enzymatic, molecular genetic, histopathologic and ultrastructural methods. The approach toward a human application with analogous lentiviral vectors for expression of alpha-L-iduronidase (IDUA). Aliquots of G-CSF mobilized CD34+ human peripheral blood progenitor cells will be treated with IDUA vector, on IDUA-DHFR vector, and studied for gene transfer and expression in cells grown in culture (i.e., short-term CFS and long-term LTCIC) or engrafted into NOD/SCID mice. These studies are directly aimed at developing a clinical trial of ex vivo HSC gene therapy for model disease MPS I. However, the studies will have much greater significance for the broader application of lentiviral vector systems and especially for application in HSC gene therapy.
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