In Vivo BM Stem Cell Gene Transfer for MPS type I

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DESCRIPTION (provided by applicant): HIV-based lentiviral vectors (LV) have demonstrated the capacity for in vivo gene transfer and gene expression in non-dividing cells after local injection. However, the potential of in vivo bone marrow stem cell gene transfer is virtually unexplored, except for results recently published by others and us and our preliminary data. The in vivo approach of bone marrow stem cell gene transfer could take full advantage of any source of stem cells present in bone cavity and avoid many of the difficulties encountered by ex vivo HSC gene transfer. The ultimate objective of this study is to determine if intra bone marrow injection of LV can efficiently transduce BM stem cells, and if the levels of gene transfer can achieve phenotypic correction for diseases such as Mucopolysaccharidosis type I (MPS I) that affects multiple organs including the brain, bone and hematopoietic system. A selectable marking SIN-LV containing eGFP and MGMT (P140K) will be used to inject normal adult mice (Specific Aim1). Gene transfer in HSC will be evaluated in BMT recipients and in vitro LTCIC cultures by CPU assay, FACS analysis, QPCR, clonality analysis and fluorescent microscopy. Transduction of MPC will be studied in stromal cell cultures using in vitro MPC differentiation assays and immuno-fluorescent microscopy, while systemic biodistribution will also be assessed including the gonad. We will also investigate the effects on transduction profiles and efficiency in BM stem cells by pretreatment of 5FU/SDF-1, myeloablation, tourniquet, re-administration of LV and MGMT-mediated in vivo selection. MPS I knock-out mice will be utilized to study the degrees of metabolic, neurological and skeletal corrections after intra bone marrow injection of a LV containing a human IDUA cDNA (Specific Aim 2). Three age groups of MPS I mice (young, adult and elderly) will be used to assess the aging effects on gene transfer patterns and the degree of correction on degree of correction on disease manifestations. Taken together, these data would not only open a door to a novel approach for treatment of human diseases, but also provide a new tool to study adult stem cell plasticity and the nature of hematopoiesis.