Gene Targeting Strategies for the Treatment of Osteogenesis Imperfecta

描述（由申请人提供）：成骨不全（OI）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。严重形式的OI通常由破坏胶原蛋白三螺旋的显性突变引起，因此有效的治疗将需要消除显性突变等位基因。该提案的长期目标是开发一种基于移植遗传修饰的自体间充质干细胞（MSC）的治疗OI的新方法，所述骨髓间充质干细胞在体内产生成骨细胞。这些MSC将通过基于腺相关病毒（AAV）的载体进行遗传修饰，所述载体可以有效地靶向同源染色体基因，从而破坏突变型胶原基因。在具有C0L1A1突变的01 MSC中，AAV基因靶向载体破坏了高达90%的所选MSC中的C0L1A1基因，其然后产生正常胶原并形成骨，这是迄今为止在成人干细胞中成功基因靶向的唯一公开实例。 在这里，这些发现将通过用不编码外源抗原的新型AAV载体靶向O1 MSC中的COL1A2基因来扩展，并证明这些靶向细胞形成正常的胶原蛋白和骨。将在来自多个个体的MSC中确定靶向频率，以评价遗传变异对基因靶向和重组的影响。将在兔模型中研究基因靶向MSC的移植方法，以优化长期MSC植入率并确定安全性。这些实验旨在开发适于Ol临床试验的AAV靶向载体和MSC移植方案。 这些实验是重要的，因为它们将开发一种新的和有前途的方法用于治疗OI。这项研究还将具有更广泛的意义，因为人类MSC的基因靶向方法可能适用于许多骨骼，软骨，肌肉和其他组织的疾病。

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.