ADIPOR1基因突变导致常显遗传视网膜色素变性的发病机制和基因编辑治疗研究

Retinitis pigmentosa （RP）is the most frequent subtype of inherited retinal disease, with no effective treatment. To date, more than eighty genes have been reported to be associated with RP, which account for approximately 50-60% of all RP cases, suggesting that a significant number of RP-related genes are yet to be discovered. Identify novel disease-causing genes and illustrate the pathogenesis of it, which will pave the way for RP patient’s genetic diagnosis, family plan and treatment in the future. Gene therapy is becoming a new promising treatment. From our previous studies ADIPOR1 gene mutation result in adRP, ADIPOR1 gene is a novel disease-causing gene for RP. The objective of this study is to reveal the pathogenesis mechanism of ADIPOR1 induced adRP, and to investigate the feasibility of gene editing therapy on ADIPOR1 mutation induced adRP. In the present study, we plan to (1) investigate the role of ADIPOR1, its ligand and its activating pathway in vitro; (2) make ADIPOR1 mouse model which carry the specific mutation (c.929A>G; p.Y310C), and explore the pathogenesis of RP on this model; (3) culture Embryonic Stem Cells derived from ADIPOR1 mouse (mADIPOR1-ESCs), design sgRNAs targeting to different regions of the mutation and a single-stranded oligodeoxynucleotide (ssODN) as a template for HDR, package SpCas9, ssODN and each of sgRNA into AAV vector and deliver them into mADIPOR1-ESCs, select the effective combination for in vivo experiments; (4) the ADIPOR1 mice will be randomly divided into two groups, in the first group we deliver the effective SpCas9, sgRNA and ssODN to the subretinal space of ADIPOR1 mice; in the other group we deliver the effective SpCas9 and sgRNA to the subretinal space of ADIPOR1 mice; we characterize the effects of gene therapy using genetic, morphology, biochemical and electrophysiological readouts. Our study try to reveal the pathogenesis of ADIPOR1 gene mutation induced adRP, and that gene editing therapy offer an efficient and persistent DNA level correction, which could ultimately offer therapeutic benefit to adRP in the future.

视网膜色素变性 (RP) 是我国致盲的主要病因之一，目前无有效疗法。在确定RP新的致病基因的基础上研究其发病机制，探索可能的治疗方案和预防措施，将对此病的诊断、预防和治疗产生革命性的影响。本课题组前期研究中发现ADIPOR1基因突变可导致常显遗传RP （adRP）, 是RP新的致病基因。在此基础上，本课题研究目的是探讨ADIPOR1 基因突变导致adRP的发病机制, 以及对其进行基因编辑治疗的可行性和应用前景。本课题将借助于ADIPOR1基因定点突变小鼠模型，结合细胞培养，通过基因转染等技术探讨ADIPOR1基因的功能、其配体和下游通路；应用CRISPR/Cas9等技术和AAV病毒载体探讨进行该基因定点编辑治疗的方法和治疗效果。以此论证ADIPOR1基因突变导致adRP的发病机制，以及应用基因编辑技术可实现目的基因DNA层面有效而持久的修复，为未来应用该技术临床治疗adRP提供理论依据。

视网膜色素变性(RP)是我国致盲的主要病因之一，目前无有效疗法。本课题研究目标是探讨ADIPOR1基因突变导致adRP的发病机制, 在此基础上探讨基因治疗的可行性和应用前景。鉴于ADIPOR1基因突变患者数量较少，考虑到未来的临床应用前景不大，慎重考虑后本课题选择对中国IRDs患者中最常见的CYP4V2和RHO基因进行发病机制探索，并在此基础上进行基因治疗药物研发。CYP4V2基因突变导致结晶样视网膜变性（BCD），是中国IRDs患者中最常见的致病基因之一。CYP4V2基因突变导致编码蛋白功能丧失，我们设计了基因替代治疗药物研发。目前已完成体外药效学、体内药效学、药代动力学、毒理学的临床前研究，已初步证明 rAAV2/8-hCYP4V2 基因替代治疗的有效性和安全性。并率先在全球范围内开展了CYP4V2基因治疗的第一个IIT临床试验（合作单位：同仁医院），临床试验结果显示药物具有良好安全性，并能显著改善患者视功能，该项目预计2022年度申报IND。RHO基因突变导致adRP，是RP中最常见的致病基因之一，该基因突变导致异常蛋白产生，通过基因编辑手段沉默突变链的表达，进而抑制突变蛋白的产生可达到有效治疗效果。鉴于中国与西方国家在RHO突变热点上有显著差异，本课题组前期研究中总结出中国RHO突变的热点，进行编辑治疗药物研发。已完成体外药效学、体内药效学、药代动力学、毒理学的临床前研究，已初步证明 rAAV2/8-Cas9-sgRNA-hRHO 基因定点编辑治疗的有效性和安全性，该项目正在准备IIT临床试验申请，预计2022年底申报IND。上述两个项目均已完成知识产权转化，转化金额共计1000万。为未来药物上市奠定坚实基础，为该类疾病患者带来曙光。