纳米脂质载体crispr/cas9纠正MYOC基因突变青光眼的作用机制

Mutations in the myocilin gene are associated with juvenile and adult-onset primary open-angle glaucoma. Due to the mutation， myocilin protein misfolded, triggering the ER stress and leading to the apoptosis cell in trabecular meshwork, which can only be corrected or deleted by gene editing. Previously, we found lipid nanoparticles (LNP) can rapidly transfect trabecular meshwork through intracameral injection. Therefore, we hypothesize that the crispr/cas9 gene editing on MYOC mutation can reduce the myocilin protein misfolded. This study will use the lipid nanoparticles delivering gRNA/Cas9 to specifically recognize the MYOC sequence by double guide RNA and to knockdown the MYOC gene expression, comparing the efficiency of AAV and LNP delivery system in trabecular meshwork, detecting the ER stress protein expression and apoptosis markers after injection. The extracellular matrix（ECM）protein and micro-structure changes of trabecular meshwork were also investigated to explore the effect and interaction of wild type myocilin protein with ECM protein. This study can reveal the possible function of myocilin, which can provide the biological mechanism for MYOC gene therapy.

MYOC所致突变可导致开角型青光眼和青少年开角型青光眼，机制为MYOC突变蛋白在小梁网细胞中异常聚集，引发ER stress升高导致细胞凋亡，目前尚无有效基因治疗。我们前期研究发现纳米脂质载体（LNP）能够在前房快速转染。本研究提出“CRISPR/Cas9对MYOC基因编辑可减少突变所致蛋白错误折叠”的假设，拟通过LNP携带Cas9和双gRNA，特异性识别MYOC突变小鼠小梁网中MYOC序列并剪切敲除，对比AAV和LNP在小梁网的转染效率，检测ER stress相关蛋白和凋亡因子的表达改变，并检测与myocilin有关的小梁网细胞外基质（ECM）蛋白和小梁网显微结构变化。一方面验证小梁网细胞的外源基因干预效率，另一方面揭示MYOC剪切后小梁网ECM所受影响以及myocilin蛋白对ECM的潜在调控作用，为MYOC突变青光眼基因治疗提供分子生物学理论基础。

MYOC所致突变可导致开角型青光眼和青少年开角型青光眼，机制为MYOC突变蛋白在小梁网细胞中异常聚集，引发ER stress升高导致细胞凋亡，目前尚无有效基因治疗。本研究提出“CRISPR/Cas9对MYOC基因编辑可减少MYOC突变所致蛋白错误折叠”的假设, 通过构建hMYOC基因点突变青光眼小鼠模型N450Y和P370L，设计双gRNA特异性识别MYOC序列并敲除，使剪切效率到达61.9%。对比了慢病毒、脂质纳米颗粒，类病毒在小梁网细胞及组织的转染效率，开发及筛选适用于小梁网组织的安全性及有效性均较为理想的转染载体：类病毒VLP载体。验证了VLP介导的 CRISPR体系对于细胞系、MYOC点突变小鼠、及食蟹猴MYOC的基因剪切及沉默作用，其在食蟹猴剪切效率约为10%-20%。本研究为MYOC的基因治疗及小梁网组织的基因编辑临床应用提供了前期基础。