Abstract
Purpose :
Age-related Macular Degeneration (AMD) is a major cause of sight loss in the developed world with associated single nucleotide polymorphisms (SNPs) in the complement system. CRISPR-mediated genome editing enables the possibility of targeting disease associated SNPs in an allele-specific manner, investigation into which requires a relevant cell model. The ARPE19 cell line, a spontaneously immortalised model that grows in a stable monolayer, expresses retinal pigment epithelium (RPE) specific markers and exhibits a typical RPE morphology, including the formation of tight junctions, represents a reliable model for CRISPR targeting of AMD risk SNPs.
Methods :
PCR amplifications of genomic DNA from ARPE-19 cells were used to screen for AMD-related SNPs in the complement factor cascade, Selected SNPs were then targeted to assess the allele-specific activity of CRISPR constructs. Guide RNA sequences were cloned into a CMV-driven SpCas9 vector and packaged into lentivirus. Targeting efficiency of the AMD-related SNPs was evaluated with TIDE analysis. The levels of different complement proteins, with associated AMD-SNPs present, were then assessed by ELISA. Finally, next-generation sequencing (NGS) analyses were used to evaluate allele specificity of the targeted SNPs.
Results :
Six different AMD-related SNPs (rs1061170, rs1410996, rs380390, rs641153, rs541862 and rs147259257) in three complement factor genes (CFH, CFB and C3) were successfully targeted with CRISPR. TIDE analysis of AMD-related SNPs in the ARPE19 cells revealed variable targeting efficiencies ranging between 3 to 31%. Corresponding NGS analysis also revealed variable allele specificity outcomes, for instance, rs1410996 SNPs in CFH gene revealed near complete allele specificity when targeting the pathogenic form, with only 1% of the wild type allele was shown to be targeted, comparted to 19% of the pathogenic form, suggesting achieving allele specificity is indeed possible with carful manipulation and design of the CRISPR targeting strategy.
Conclusions :
These results show a possibility of allele specific targeting of the complement factor system in an AMD model, with therapeutic potential to target different disease predisposing SNPs.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.