AAV-based gene editing of type 1 collagen mutation to treat osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a genetic disorder character-ized by bone fragility, low bone mass, fractures, and extraske-letal manifestations. Since OI is commonly caused by single-nucleotide mutation(s) in the COL1A1 or COL1A2 genes encoding type I collagens, we developed a genome-editing strategy to correct a Col1a2 mutation in an OIM mouse model resembling a severe dominant form of human type III OI. Us -ing a recombinant adeno-associated virus (rAAV), we delivered CRISPR-Cas9 to bone-forming osteoblast-lineage cells in the skeleton. Homology-directed repair (HDR)-mediated gene ed-iting efficiency in these cells was improved when CRISPR-Cas9 was coupled with a donor AAV vector containing a promoter-less partial mouse Col1a2 complementary DNA sequence. This approach effectively reversed the dysregulation of osteogenic differentiation by a Col1a2 mutation in vitro. Furthermore, systemic administration of dual rAAVs in OIM mice lowered bone matrix turnover rates by reducing osteoblast and osteo-clast development while improving the cellular network of me-chano-sensing osteocytes embedded in the bone matrix. This strategy significantly improved bone architecture/mass/miner-alization, skeletal deformities, grip strength, and spontaneous fractures. Our study is the first demonstration that HDR-medi-ated gene editing via AAV-mediated delivery effectively cor-rects a collagen mutation in OI osteoblasts and reverses skeletal phenotypes in OIM mice.