CELL AND GENE THERAPY IN A MOUSE MODEL OF HUMAN OSTEOGEN

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Osteogenesis imperfecta (OI) is a group of heritable disorders of connective tissue whose common feature is bone fragility. Most forms of OI are the result of mutations in the genes that encode proalpha1 and proalpha2 polypeptide chains of type I collagen the major protein of bone. The long-term objective of the present proposal is to test the feasibility of using cell therapy or gene therapy for some forms of OI and other bone related diseases. The major focus of the proposal is to utilize a mouse model of human OI type III that has defective synthesis of proalpha2(I) chains to evaluate the feasibility of reversing OI defects by bone marrow stromal cell transplantation or by use of bone marrow stromal cells to deliver therapeutic genes to bone. The aims are: (1) to evaluate the potential of bone marrow stromal cells from normal donor mice transplanted into syngeneic OI mice to synthesize and deposit normal type I collagen in bone matrix of the recipient mice and (2), to test the feasibility of gene therapy by demonstrating that bone marrow stromal cells transduced with collagen genes will home to bone and express the genes in bone. As a prelude to this, bone marrow stromal cells will be established from the normal mice by flushing the marrow from femurs and tibias. The established bone marrow stromal cells will be transduced with a retroviral vector containing LacZ and neo genes (BAG-LacZ Neo) prior to transplanting them to the recipient mice to aid in cell tracking. The bone marrow stromal cells established from normal mice will be injected in the femurs of the irradiated or non-irradiated OI mice and the expression of the proalpha2(I) chains will be determined by immunofluorescence localization using a mouse alpha2(I) anti-serum and cyanogen bromide digestion of the bone collagen of the recipient mice. To test for the gene expression of collagen genes in bone by bone marrow stromal cells, bone marrow stromal cells transduced with the mouse proalpha2(I) cDNA will be injected in the femurs of the oim mice. The presence of the transduced cells in the recipient mice will be assessed by PCR and gene expression will be determined by immunofluorescence localization of the alpha2(I) chain in bone matrix. Future plans will involve determining the amount of collagen made by the transplanted cells in the bones of the recipient mice and the assessment of the bone quality by radiographic, histological and biomechanical analysis of the bones of the recipient mice.