In Vivo Gene Targeting to Treat Inherited Bone Disease

 描述（申请人提供）：骨干细胞突变可损害骨代谢和再生。成骨不全（OI）是遗传性骨病的最常见形式，其特征在于骨脆性，临床表现从轻度骨折增加到严重骨畸形和死亡不等。患有OI的个体具有改变前胶原结构或产生的突变，并导致异常胶原纤维形成，破坏骨发育和更新。OI的治疗选择有限，不能缓解OI中观察到的并发症。我们最近的工作表明，AAV基因靶向载体可以破坏导致OI的突变胶原等位基因，靶向细胞产生正常胶原和形成骨。虽然这些结果表明靶向频率接近1%的基因组编辑的有效性，但需要增强AAV基因靶向以提供更稳健的体内遗传操作。在这里，我们将扩大我们的成功，并提出三条调查线，以开发一种治疗方法，遗传性骨病患者。首先，我们将使用CRISPR-AAV系统修饰胶原基因，这将允许破坏或纠正体内引起OI的基因。其次，我们将研究遗传校正的骨干细胞是否在体内具有生长优势，以及TGF-β信号传导如何影响骨骼疾病的骨骼外表现。最后，我们将在临床前兔模型中进行一系列实验，以建立 最佳的基因靶向递送方案。该提案的成功完成将导致对骨中TGF-β信号传导和干细胞动力学的更好理解，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.