GENE TARGETING STRATEGIES FOR THE TREATMENT OF OSTEOGENESIS IMPERFECTA

描述(申请人提供)：成骨不全(Ol)是一种遗传性疾病，由I型胶原基因COL1A1或COL1A2突变引起，可导致严重的骨骼异常、骨折和过早死亡。严重的OL通常是由破坏胶原三螺旋的显性突变引起的，因此有效的治疗将需要消除显性突变等位基因。这一建议的长期目标是开发一种基于体内产生成骨细胞的转基因自体间充质干细胞(MSCs)移植的治疗OL的新方法。这些MSCs将被基于腺相关病毒(AAV)的载体进行基因改造，这种载体可以有效地靶向同源的染色体基因，从而破坏突变的胶原基因。在具有COL1A1突变的Ol MSCs中，AAV基因打靶载体在高达90%的选定MSCs中干扰了COL1A1基因，然后这些细胞产生正常的胶原并形成骨，这是迄今为止在成人干细胞中成功进行基因打靶的唯一已发表的例子。 在这里，通过用不编码外源抗原的新型AAV载体靶向Ol MSCs中的COL1A2基因，并证明这些靶细胞形成正常的胶原和骨骼，这些发现将得到扩展。将确定来自多个个体的MSCs的靶向频率，以评估遗传变异对基因靶向和重组的影响。基因靶向MSCs的移植方法将在兔模型中进行研究，以优化长期MSC植入率并确定安全性。这些实验旨在开发一种适合于OL临床试验的AAV靶向载体和MSC移植方案。 这些实验意义重大，因为它们将开发一种新的、有希望的治疗OL的方法。这项研究还将具有更广泛的意义，因为针对人类MSCs的基因靶向方法可能会应用于骨、软骨、肌肉，可能还有其他组织的许多疾病。

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.