DESCRIPTION (provided by applicant): The most common form of genetic bone disease is ontogenesis imperfect (OI). Mutations in the structural protein genes COL1A1 and COL1A2 result in dominant forms of OI and recently, mutations in a number of different genes have been identified that result in recessive forms of OI. One of these genes, LEPRE1, encodes a protein that forms a complex with two other proteins and together they modify collagen peptides. Mutations in LEPRE1 have been identified that result in recessive OI, and one of these mutations, IVS5+1G>T, has been found in the African American population at a high carrier frequency of 1 in 100 to 1 in 300. Treatment options for OI are limited and results from transplantation studies, while encouraging, achieved only very low engraftment rates. Here we develop an AAV-mediated gene targeting approach to correct the mutated LEPRE1 gene in patient cell lines, and assess the in vivo growth potential of genetically-corrected stem cells in our conditional, knock-in OI mouse model. Two independent aims will be investigated in this project. Aim 1: To correct LEPRE1 mutations in pluripotent stem cells using AAV gene targeting vectors. The modification of an endogenous gene allows for its regulation to be governed by internal elements ensuring appropriate gene expression. We will use adeno-associated viral vectors to alter the LEPRE1 locus in patient- specific cells and foamy viral vectors to reprogram targeted cells into induced pluripotent stem cells (iPSCs). These iPSCs can be used as a renewable source of stem cells for future transplantation experiments. Aim 2: Develop a regenerative medicine OI mouse model to assess transplantation parameters. Stem cell transplantation and engraftment into bone is poorly understood. Past transplantation efforts into bone have not yielded long-term, high level engraftment. By developing our conditional knock-in Lepre1 OI mouse model, we will be able to determine the growth advantage of normal collagen expressing cells, type of cell to deliver, and percent of engrafted cells needed to improve symptoms of OI. In addition, we will study the effects of genetically modified stem cells on bone morphology.
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