Mesenchymal stem cells (MSCs) are adult cells with the capacity to differentiate into multiple cell types, including bone, fat, cartilage, and muscle cells. In order to effectively utilize autologous MSCs in cell-based therapies, precise genetic manipulations are required to eliminate the effects of disease-causing mutations. We previously used adeno-associated virus (AAV) vectors to target and inactivate mutant COL1A1 genes in MSCs from individuals with the brittle bone disorder, osteogenesis imperfecta (OI). Here we have used AAV vectors to inactivate mutant COL1A2 genes in OI MSCs, thereby demonstrating that both type I collagen genes responsible for OI can be successfully targeted. We incorporated improved vector designs so as to minimize the consequences of random integration, facilitate the removal of potential antigens, and avoid unwanted exon skipping. MSCs targeted at mutant COL1A2 alleles produced normal type I procollagen and formed bone, thereby demonstrating their therapeutic potential.