GENE TARGETING STRATEGIES FOR THE TREATMENT OF OSTEOGENESIS IMPERFECTA

描述（由申请人提供）：成骨不全症（Osteogenesis Imperfecta, Ol）是一种由I型胶原基因COL1A1或COL1A2突变引起的遗传性疾病，可导致严重骨骼异常、骨折和过早死亡。严重的Ol通常是由破坏胶原蛋白三螺旋结构的显性突变引起的，因此有效的治疗需要消除显性突变等位基因。本研究的长期目标是开发一种基于基因修饰的自体间充质干细胞（MSCs）移植的治疗骨髓瘤的新方法，这种干细胞可以在体内产生成骨细胞。这些间充质干细胞将通过基于腺相关病毒（AAV）的载体进行基因修饰，该载体可以有效地靶向同源染色体基因，从而破坏突变的胶原基因。在COL1A1突变的Ol间充质干细胞中，AAV基因靶向载体破坏了多达90%的选定间充质干细胞中的COL1A1基因，然后产生正常胶原并形成骨骼，这是迄今为止发表的唯一成功的成人干细胞基因靶向的例子。

DESCRIPTION (provided by applicant): Osteogenesis Imperfecta (Ol) is a genetic disease caused by mutations in the type I collagen genes COL1A1 or COL1A2 that can result in major skeletal abnormalities, fractures, and premature death. Severe forms of Ol are typically caused by dominant mutations that disrupt the collagen triple helix, so an effective treatment will require the elimination of dominant, mutant alleles. The long-term objective of this proposal is to develop a novel approach for the treatment of Ol based on the transplantation of genetically modified autologous mesenchymal stem cells (MSCs) that produce bone-forming osteoblasts in vivo. These MSCs will be genetically modified by vectors based on adeno-associated virus (AAV) that can efficiently target homologous, chromosomal genes and thereby disrupt mutant collagen genes. In Ol MSCs with mutations in COL1A1, an AAV gene targeting vector disrupted the COL1A1 gene in up to 90% of selected MSCs, which then produced normal collagen and formed bone, in the only published example to date of successful gene targeting in adult human stem cells. Here these findings will be extended by targeting the COL1A2 gene in Ol MSCs with novel AAV vectors that do not encode foreign antigens, and demonstrating that these targeted cells form normal collagen and bone. Targeting frequencies will be determined in MSCs from multiple individuals to evaluate the effects of genetic variation on gene targeting and recombination. Transplantation methods for gene-targeted MSCs will be studied in a rabbit model to optimize long-term MSC engraftment rates and determine safety. These experiments are intended to develop an AAV targeting vector and MSC transplantation protocol that would be appropriate for clinical trials of Ol. These experiments are significant because they will develop a new and promising approach for the treatment of Ol. The research will also have broader implications, since gene targeting methods for human MSCs could potentially be applied to many diseases of bone, cartilage, muscle, and possibly other tissues.