联合CRISPR技术以及iPSC技术区别研究AMD高危序列ARMS2以及HTRA1基因型致病机理

Age-related macular degeneration (AMD) is a major cause of retinal damage. Genome-wide association studies (GWASs) and linkage analyses have provided strong risk factors for AMD. The two SNP are tightly linked and lie in the neighboring HTRA1 and ARMS2 genes. How these mutations might cause sight to deteriorate is unclear, the underlying molecular mechanisms of AMD are unknown. Recently, our unbiased proteome analysis suggested the antioxidant mechanisms of super oxide dismutase (SOD) are perturbed in affected cells and that, over time, this introduces reactive oxidative species (ROS) mediated cellular insults that eventually manifest as AMD. If ROS metabolism is indeed disrupted in AMD, then we might finally begin to address the causes of the disease. We developed a protocol that recapitulates aging in these patient-stem-cell-derived retinal cells by treated with A2E and blue light. We can manipulate the stem cell by using genome Gene-editing technology-CRISPR/Cas9, targeting disease-associated SNPs, to determine the individual contributions of each. Use the CRISPR/Cas9 system to convert HTRA1 and ARMS2 alleles from low-risk to high risk in patient-derived stem cells. Determine the individual contribution of human HTRA1 and ARMS2 alleles to AMD pathogenesis. Test whether CRISPR conversion from low to high-risk AMD alleles affects ROS levels in cells. Test the function of gene-edited patient-stem-cell-derived RPE by transplantation into mice. By applying these powerful methodologies, we believe we can finally identify the root causes of AMD and so begin to develop new therapies.

年龄相关性黄斑变性（AMD)是常见的与年龄相关不可逆致盲性眼病。全基因组关联分析（GWAS）发现ARMS2/HTRA1是AMD高危基因，作用机制尚不明确，而近期AMD药物临床研究对于疗效是否会因高危基因的差异而不同争议不断。我们前期研究发现这两个高危基因的iPS-RPE细胞经老化后SOD2表达、抗氧化能力等与野生型有差异，但因这两个基因紧密连接，常规基因编辑方法难以区别研究，而且野生型与高危型细胞其他基因背景信息不一致，干扰进一步通路分析。本项目我们将利用靶向基因修饰新技术CRISPR/case9，向野生型iPS细胞分别引入ARMS2以及HTRA1高危序列SNP，达到区别研究、排除背景信息干扰的目的，再与原细胞对比研究，全蛋白组学技术寻找高危基因特异差异蛋白，探索AMD致病机理；同时将基因编辑成功的iPS-RPE细胞进行视网膜下移植，验证CRISPR/case9技术的基因修饰安全性。

AMD是常见的与年龄相关的致盲性眼病，GWAS分析发现ARMS2和HTRA1基因是发病的高危因素，而这些易感基因作用机制尚不明确。前期研究发现高危型iPS-RPE细胞经老化后SOD2表达、抗氧化能力等与野生型有差异，但因基因紧密连接，难以分开研究。我们利用CRISPR/cas9基因编辑技术，向野生型iPS细胞分别引入ARMS2及HTRA1高危序列SNP，得到了ARMS2型iPS细胞系和HTRA1型iPS细胞系。根据RPE生理特点，加入吸烟血清作为氧化应激刺激因素，蛋白组学质谱对比分析ARMS2/HTRA1高危型及野生型RPE细胞，筛选得到了SOD2、Caveolin-1、ICAM1、complement component C9、Apolipoprotein E、Vitronectin等 6个具有明显差异的可信蛋白，通过WB蛋白表达验证发现Caveolin-1、ICAM1、complement component C9、Apolipoprotein E与质谱分析结果一致，在吸烟血清刺激组上调表达，而SOD2、Vitronectin与质谱分析结果相反，下调表达。吸烟血清刺激RPE细胞后吞噬功能增强，细胞间紧密连接被破坏。高危基因HTRA1的过表达也会引起Caveolin-1表达上调。以上说明高危型 RPE在吸烟应激下，Caveolin-1表达上调，进而调节细胞吞噬功能状态，而且会引起细胞间紧密连接破坏，或与高危基因型吸烟人群更易形成AMD相关。其中筛选出的Caveolin-1蛋白是与RPE代谢消化功能和A2E刺激相关的关键蛋白，我们利用A2E刺激RPE细胞来构建RPE细胞衰老模型，并探究与A2E相关的RPE细胞衰老相关蛋白质组学变化。A2E处理的RPE细胞的蛋白组学质谱鉴定出了RPE细胞衰老相关蛋白，即HMGB1与Caveolin-1。通过基因过表达/敲低实验证明了HMGB1与Caveolin-1在导致RPE细胞衰老方面的协同作用。甘草酸可用于人体抗衰老、抗炎、降压、增强肌体免疫力。将RPE细胞用甘草酸预处理后加入A2E刺激，发现甘草酸不仅能减轻A2E导致的RPE细胞衰老程度，还能降低HMGB1的分泌以及Caveolin-1的表达。综上表明A2E介导的HMGB1与Caveolin-1的表达与RPE衰老具有密切联系，而且甘草酸可能作为AMD疾病早期防治的潜在药物。