Precision Gene Editing on Osteogenesis Imperfecta

Project summary Osteogenesis imperfecta (OI) is the most common bone fragility disease, with an incidence of approximately 1 in 10,000-20,000 worldwide and 25,000–50,000 in the US. OI is an inherited genetic disorder, and symptoms include low bone mass, recurrent bone fractures following minor trauma, bowing of the long bones, vertebral compression, scoliosis, bone pain, stunted growth, and ligamentous and joint laxity. Up to 85% of OI patients have autosomal dominant mutations in either the COL1A1 or COL1A2 gene that encodes the pro-1(I) or pro- 2(I) polypeptide chains of type I collagen, the major structural protein of bone. Current drug treatments primarily focus on improving bone mineralization and show limited success because they cannot correct these mutations that disrupt collagen production. In addition, currently developing OI cell therapy has several challenges, such as limited mesenchymal stem cells from healthy donors, susceptibility to transplantation damage, bone targeting complexities, and immune rejection risks. To address these unmet needs, this proposal will develop and evaluate in vivo gene editing strategies to repair collagen mutations in mouse models of OI using bone-targeting recombinant adeno-associated virus (rAAV). Gene editing is one of the promising gene therapy approaches for rare diseases. However, it has not been reported that “gene editing based gene therapy for OI patients and its preclinical animal models. Recently, we confirmed that the CRISPR-Cas9-based gene editing approach could reverse the OI animal phenotypes with gene correction of the mutation site. However, the Cas9-based gene editing strategy induced a small percentage of off-target effects such as insertion, deletion, and replacement, which is an inevitable problem accompanied by the blunt-end double-strand DNA break (DSB) induced by the Cas9 protein and NHEJ, one of two DNA repairing processes of DSB. To avoid DSB-induced off-target effects, we will utilize 1) single-strand DNA break using a Cas9-nickase (Prime-Editing, PE), 2) staggered double-strand DNA break using Cas12a, 3) non-homologous end joining (NHEJ) inhibition, and 4) homology-directed repair (HDR) boosting. Successful completion of these aims will provide a proof-of-concept demonstration and compare the therapeutic efficacy of bone-targeting rAAV-based genome editing strategies to treat OI. Our skeletal gene therapy will not only provide OI patients with alternative treatment options but also develop a new platform that can be applied to treat other skeletal diseases.