In Vivo Gene Targeting to Treat Inherited Bone Disease

 描述(申请人提供)：骨干细胞的突变会损害骨骼的新陈代谢和再生。成骨不全(OI)是最常见的遗传性骨病，以骨脆性为特征，临床表现从轻微的骨折增加到严重的骨畸形和死亡。患有OI的个体具有改变前胶原结构或产生的突变，并导致异常的胶原纤维形成，扰乱骨骼的发育和更新。OI的治疗选择有限，不能减轻OI的并发症。我们最近的工作证明，AAV基因打靶载体可以破坏导致OI的突变胶原等位基因，靶细胞产生正常的胶原并形成骨。虽然这些结果表明，靶向频率接近1%的基因组编辑是有效的，但需要加强AAV基因靶向，以提供更强大的体内遗传操作。在这里，我们将扩展我们的成功，并提出三条研究路线，以开发一种遗传性骨病患者的治疗方法。首先，我们将使用CRISPR-AAV系统修改胶原基因，该系统将允许中断或纠正体内导致OI的基因。其次，我们将研究转基因骨干细胞在体内是否具有生长优势，以及转化生长因子-β信号如何影响骨骼疾病的骨外表现。最后，我们将在临床前兔模型中进行一系列实验，以建立 最佳的基因靶向传递方案。这一建议的成功完成将使人们更好地理解转化生长因子-β信号和骨中干细胞的动力学，并将AAV基因靶向推向临床试验。

 DESCRIPTION (provided by applicant): Mutations in bone stem cells can impair bone metabolism and regeneration. Osteogenesis imperfect (OI) is the most common form of genetic bone disease and characterized by bone fragility with clinical manifestations varying from a mild increase in fractures to severe bone deformities and death. Individuals with OI have mutations that alter procollagen structure or production and lead to abnormal collagen fibril formation disrupting bone development and renewal. Treatment options for OI are limited and do not alleviate the complications seen in OI. Our recent work has demonstrated that AAV gene targeting vectors can disrupt the mutant collagen alleles that cause OI and that targeted cells produced normal collagen and formed bone. While these results indicate the effectiveness of genomic editing with targeting frequencies approaching 1%, an enhancement of AAV gene targeting is required to provide more robust in vivo genetic manipulations. Here we will expand upon our successes and propose three lines of investigations to develop a therapeutic approach for patients with inherited bone disease. First we will modify collagen genes using a CRISPR-AAV system that will allow for the disruption or correction of genes that cause OI in vivo. Second, we will investigate whether genetically corrected bone stem cells have a growth advantage in vivo and how TGF-beta signaling affects the extra-skeletal manifestations of bone disease. Finally we will conduct a series of experiments in a preclinical rabbit model to establish the optimal gene targeting delivery protocol. Successful completion of this proposal will lead to an improved understand of TGF-beta signaling and stem cell kinetics in bone and advance AAV gene targeting toward a clinical trial.