This proposal will explore the possibility that patients with osteogenesis imperfecta (OI) can potentially be treated with their own osteoprogenitors by gene-engineening the cells from their bone marrow that are referred to as mesenchymal stem cells or marrow stromal cells (MSCs). We will use antibodies to surface epitopes we have recently identified to prepare clonal and homogeneous preparations of the earliest progenitors in cultures of MSCs that have an enhanced ability to undergo multilineage differentiation and that are rapidly self-renewing cells (RS cells). The RS cells will be used to test the hypothesis that they are similar to CD34 positive stem cells used for bone marrow transplants and that therefore they will be the most effective cells to provide long-term engraftment of osteoprogenitors into bone. We will also test the hypothesis that RS cells from patients with OI can be gene-engineered to correct the deleterious effect, of mutations in type I Collagen that produce the disease. The Specific Aims are (1) Use a series of antibodies to surface epitopes we have recently identified to prepare clonal and homogeneous preparation, of RS cells from cultures of human MSCs. In the process, test the hypothesis that RS cells can be further fractionated to obtain homogeneous preparations of stem cells that are even more effective as osteoprogenitors for engrafment into bone. (2) Define the osteogenic potential in vitro of the RS cell preparations by assays of the rates of mineralization and assays of expressed genes by mRNA microarrays and proteomics. (3) Determine the osteogenic potential in vivo of the RS cell preparations by assays of differentiation into bone after subcutaneous implantation in vehicles or after systemic infusion into immunodeficient mice. (4) Determine the feasibility of correcting the gene defect in a patients own RS cells, (a) by overexpression of a cDNA for the wildtype COL I A I gene, or (b) by replacing a mutated COLlAl gene by homologous recombination.
NIH ExPorter
NIH ExPorter