GENE TARGETING STRATEGIES FOR THE TREATMENT OF OSTEOGENESIS IMPERFECTA

描述(由申请人提供)：这是R01 AR048328的竞争性修订申请，以回应NIH通知NOT-OD-09-058：“NIH宣布恢复法案资金可用于竞争性修订申请”。成骨不全(OI)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。我们先前的研究表明，基于腺相关病毒(AAV)的基因打靶载体可以有效地干扰OI患者骨髓间充质干细胞(MSCs)中突变的COL1A1和COL1A2基因，然后这些MSCs产生正常的胶原并形成骨。在这次的R01中，我们获得了三个特定目标的资金：开发针对突变胶原基因的改良AAV载体，确定基因多态对靶向的影响，以及移植MSCs并使用兔模型测量植入。这些目标旨在开发一种治疗策略，包括分离患者的MSCs，通过基因打靶消除突变的胶原基因，以及移植这些自体的、靶向的MSCs。虽然这仍然是一种有希望的方法，但它的主要缺点是在体外扩增过程中可以获得的MSCs的数量。在这一竞争性修订应用中，我们建议通过从OI MSCs获得诱导多能干细胞(IPSCs)来克服这一潜在限制，IPSCs是永生的，能够在体内分化为MSCs并形成骨。我们将从OI MSCs中分离出IPSCs，通过AAV介导的基因打靶来纠正其中的胶原突变，并证明这些IPSCs仍然可以形成骨。 与公共卫生相关：这些实验旨在开发一种新的治疗模式，即通过基因靶向纠正患者特有的多能干细胞(IPSCs)，然后将其返回给患者。他们将确定纠正IPSCs基因突变并使用这些细胞生成骨骼的可行性，这对治疗成骨不全等遗传性疾病具有重大意义。

DESCRIPTION (provided by applicant): This is a competitive revision application for R01 AR048328 in response to NIH Notice NOT-OD-09- 058: "NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications". Osteogenesis Imperfecta (OI) 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. We showed previously that gene targeting vectors based on adeno-associated virus (AAV) can efficiently disrupt mutant COL1A1 and COL1A2 genes in mesenchymal stem cells (MSCs) from individuals with OI, and that these MSCs then produced normal collagen and formed bone. In this R01, we received funding for three specific aims: to develop improved AAV vectors that target mutant collagen genes, to determine the effects of genetic polymorphisms on targeting, and to transplant MSCs and measure engraftment using a rabbit model. These Aims are intended to develop a therapeutic strategy consisting of isolation of a patient's MSCs, gene targeting to eliminate the mutant collagen gene, and transplantation of these autologous, targeted MSCs. While this remains a promising approach, its major shortcoming is the number of MSCs that can be obtained during ex vivo expansion. In this competitive revision application we propose to overcome this potential limitation by deriving induced pluripotent stem cells (iPSCs) from OI MSCs, which are immortal and capable of differentiating into MSCs and forming bone in vivo. We will derive iPSCs from OI MSCs, correct the collagen mutations in them by AAV-mediated gene targeting, and demonstrate these iPSCs can still form bone. PUBLIC HEALTH RELEVANCE: These experiments are meant to develop a new therapeutic paradigm, in which patient-specific pluripotent stem cells (iPSCs) are corrected by gene targeting and then returned to the patient. They will establish the feasibility of correcting genetic mutations in iPSCs and using these cells to generate bone, with major significance for the treatment of genetic diseases such as osteogenesis imperfecta.