Collagen Gene Targeting with Adeno-Associated Virus Vectors Osteogenesis Imperfecta (OI) is a genetic disease caused by mutations in the type I collagen genes COLIAI or COL1 42 that can result in major skeletal abnormalities, deformities, frequent fractures, pain, and premature death. Severe forms of OI are typically caused by dominant mutations that disrupt the Collagen triple helix, so an effective treatment will require removal or correction of dominant, mutant alleles. The long-term objective of this proposal is to develop a novel approach for the treatment of OI based on the transplantation of genetically modified autologous mesenchymal stem cells (MSCs) that are expected to produce bone-forming osteoblasts in vivo. Adenoassociated virus (AAV) vectors have been shown to efficiently introduce specific genetic modifications into homologous chromosomal sequences, and here they will be used both to knockout and correct mutant, human COL 1A I alleles. Experiments will be performed with an existing collection of OI fibroblasts with defined COLIA1 mutations, and with an OI MSC bank established and characterized here. In one strategy, a single AAV targeting vector will be used to knockout any COL 1A allele, with the goal of converting severe OI due to helix-disrupting mutations to a mild form of OI due to a single null allele. In other strategies, AAV targeting vectors will be designed to correct specific COL 1A1 mutations and create wild-type alleles. Several approaches will be developed to select for cells that have undergone AAV-mediated gene targeting and express normal COL1 1A genes, with the goal of simplifying the ex vivo manipulations that would be used in future clinical trials. The proliferative and muItilineage potential of gene-targeted MSCs will be assessed by in vitro assays and also by an in vivo bone-forniing assay after implantation in immunodeficient mice. Mixtures of MSCs marked with reporter genes will be assayed together to assess proliferation and bone formation when MSCs with different COLIA] genotypes coexist. In order to promote the engraftment of genetically modified MSCs, we will use chemical inducers of dimerization to multimerize engineered growth factor receptors and provide an inducible cell proliferation switch to MSCs. This switch will provide a method for pharmacologically controlled in vivo expansion of MSCs after transplantation. These studies are intended to lay the groundwork for future clinical trials for OI and other diseases that could benefit from the transnlantation of autologous genetically modified MSCs.
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