EVALUATION OF CELLULAR GENE THERAPY FOR OI

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DESCRIPTION (Taken from the application): Ontogenesis imperfecta (0I) 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 proposal is to develop strategies using cell therapy or gene therapy for the treatment of some forms of OI and other bone related diseases. The focus of this research proposal is to utilize a mouse model of human OI (oim) that has defective synthesis of proalpha2(I) chains to evaluate the feasibility of reversing OI defects and other bone related disease by either bone marrow stromal cell transplantation or delivery of normal collagen 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 engraft, synthesize and deposit normal type I collagen in bone matrix of the recipient mice and (2), to test the feasibility of gene therapy by evaluating the potential of bone marrow stromal to be transduced with collagens and to deliver 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) antiserum and cyanogen bromide digestion of the bone collagen of the recipient mice. To test for the collagen gene expression by bone marrow stromal cells, the cells will be transduced with an adenovirus containing the mouse proalpha2(I) collagen gene and the transduced cells will be injected in the femurs of the o=s mice. The alpha2(I) collagen expression will be determined by immunofluorescence localization using the mouse proalpha2(I) and the cyanogen bromide digestion of the tissue. Future plans will involve determination of 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.