Salman Ahmed, Song Won Kyung, Storm Tina, McClements Michelle E, MacLaren Robert E
Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK.
Oxford Eye Hospital, Oxford University Hospitals NHS Foundation Trust, Oxford, UK.
Gene Ther. 2025 Mar;32(2):132-141. doi: 10.1038/s41434-025-00522-z. Epub 2025 Mar 18.
Age-related Macular degeneration (AMD) is a major cause of vision loss and is linked to several predisposing single nucleotide polymorphisms (SNPs). CRISPR-mediated genome editing offers the potential to target negatively associated SNPs in an allele-specific manner, necessitating the need for a relevant cell model. The ARPE-19 cell line, with its stable monolayer growth and retinal pigment epithelium (RPE) characteristics, serves as an ideal model for AMD studies. Chronic inflammation and complement system dysregulation are implicated in AMD pathogenesis. Most genetic variations associated with AMD are in complement genes, suggesting their regulatory role. In this study, we conducted targeted PCRs to identify AMD-related SNPs in ARPE-19 cells and used CRISPR constructs to assess allele-specific activity. Guide RNA sequences were cloned into an EF-1-driven SpCas9 vector and packaged into lentivirus. Targeting efficiencies were evaluated with TIDE analysis, and allele-specificity was measured with NGS analysis 30 days post-transduction. Our results showed varying targeting efficiencies depending on guide RNA efficacy. For example, TIDE analysis of CFH SNPs rs1061170 and rs1410996 revealed efficiencies of 35.5% and 33.8%, respectively. CFB SNP rs4541862 showed efficiencies from 3% to 36.7%, and rs641153 ranged from 3.4% to 23.8%. Additionally, allele-specific targeting of AMD-related SNPs rs1061170, rs1410996, rs4541862, and rs641153 ranged from 48% to 52% in heterozygous differentiated ARPE-19 cells. These findings demonstrate the potential to manipulate the complement system in an AMD model by targeting disease-associated SNPs in an allele-specific manner, offering a promising therapeutic approach.
年龄相关性黄斑变性（AMD）是视力丧失的主要原因，与多种易感性单核苷酸多态性（SNP）相关。CRISPR介导的基因组编辑提供了以等位基因特异性方式靶向负相关SNP的潜力，因此需要一个相关的细胞模型。ARPE - 19细胞系具有稳定的单层生长和视网膜色素上皮（RPE）特征，是AMD研究的理想模型。慢性炎症和补体系统失调与AMD发病机制有关。大多数与AMD相关的基因变异存在于补体基因中，表明它们具有调节作用。在本研究中，我们进行了靶向PCR以鉴定ARPE - 19细胞中与AMD相关的SNP，并使用CRISPR构建体评估等位基因特异性活性。引导RNA序列被克隆到EF - 1驱动的SpCas9载体中并包装成慢病毒。转导30天后，用TIDE分析评估靶向效率，用NGS分析测量等位基因特异性。我们的结果表明，根据引导RNA的效率，靶向效率有所不同。例如，对CFH SNP rs1061170和rs1410996的TIDE分析显示效率分别为35.5%和33.8%。CFB SNP rs4541862的效率为3%至36.7%，rs641153的效率为3.4%至23.8%。此外，在杂合分化的ARPE - 19细胞中，与AMD相关的SNP rs1061170、rs1410996、rs4541862和rs641153的等位基因特异性靶向范围为48%至52%。这些发现证明了通过以等位基因特异性方式靶向疾病相关SNP来操纵AMD模型中补体系统的潜力，提供了一种有前景的治疗方法。
