Historically, osteogenesis imperfecta (OI) has been the paradigm for the study of a dominantly inherited disease of connective tissue resulting from a mutation that disrupts the three dimensional structure of a major extracellular protein. The study of OI mutations has shown that the disease results not only from an improperly organized bone matrix that is continually undergoing remodeling (high turnover bone) but also from impaired matrix production due to the high intracellular content of misfolded collagen chains (low collagen secretion) and retarded osteoblast differentiation. While therapies directed at any of these mechanisms can improve disease severity, everyone agrees that correction of the underlying genetic mutation is the ultimate therapeutic goal. With advances in stem cell biology and in methods for homologous recombination at a defined genomic site, a potential roadmap for a gene/stem cell therapy for OI has become feasible. This application will test the practicalit of the roadmap as we envision it. Fibroblasts obtained from normal and OI subjects with genetically defined mutations resulting in the severest forms of the disease will be converted to iPS cell using methods that do not integrate the transcription factors into the host genome. To follow the osteogenic fate of these cells, a bone restricted reporter gene (Col2.3GFP) will be inserted into the AAVR1 locus using a proven Zn finger technology. Subsequently the genome of OI-iPS line will be cleaved near the site of the mutation in the Col1A1 gene using TALEN targeting proteins to direct correcting DNA into the host chromatin repair mechanism. To enhance the possibility the cleaved DNA will be repaired by the desired homology-driven mechanism rather than the non-homologous end joining (NHEJ) mechanism, transient co-transfection of genes encoding herpes derived recombination enhancement proteins (UL12 and ICP8) will be added to the targeting protocol to determine if a higher proportion of correctly targeted clones is obtained. Once corrected OI-iPS clonal cell lines are obtained, their ability to correct a skeletal repair defect as well as control iPS clones, and certainly better than the uncorrected OI-iPS clones, will be evaluated by fluorescence-based cryohistological, molecular expression and mechanical criteria. This proposal is a proof of principle that correction of OI mutations will restore normal osteoblast differentiation and fully functional bone matrix production, which will yield the most desired cellular tool to determine the best manner to deliver the cells back to the affected host and achieve the greatest clinical impact on skeletal health and function. In addition, the technical pathway will be applicable to many other dominantly inherited diseases by providing a rapid and relatively low cost method for gene correction in IPS cells that are still capable of multi-lineage differentiation.
A method for correcting the OI gene mutation in stem cells derived from affected adult individuals will be developed. It utilizes the latest techniques from stem cell biology and target gene correction, and it will evaluate a new approach to make the method more faithful and less expensive than the methods that are in current use. The other aspect of the grant is to demonstrate that the bone formed by these corrected stem cells in repairing bone defects in mice is equivalent to bone formed from normal stem cells and dramatically better that the bone formed from OI stem cells.