Abstract
Purpose :
Age-related Macular Degeneration (AMD) is a major cause of sight loss in the developed world with known predisposing single nucleotide polymorphisms (SNPs). CRISPR-mediated genome editing enables the possibility of targeting negatively associated SNPs in an allele-specific manner, investigation into which requires a relevant cell model.
Methods :
Targeted PCRs identified a number of AMD-related SNPs in 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. 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 via BsmBI cloning sites and packaged into lentivirus. Targeting efficiency of the AMD-related SNPs was evaluated with TIDE analysis 30 days after lentivirus transduction of ARPE19 cells. The levels of different complement factor pathway proteins, where SNPs in genes associated with this pathway are believed to play a pivotal role in AMD pathology, were then evaluated by ELISA.
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 7-1%. For instance, rs1410996 and rs641153 SNPs in CFH and CFB genes revealed up to 7.8 and 17.4% targeting efficiency respectively. These samples further exhibited elevated levels of CFH and CFB proteins compared to the control.
Conclusions :
These results show a capability to manipulate the complement factor system in an AMD model with a therapeutic potential to target different disease predisposing SNPs.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.