Abstract
Purpose :
The apolipoprotein E allele ε2 (APOE2) has been identified as a genetic risk factor for age-related macular degeneration (AMD). Its roles in lipid metabolism, drusen formation and complement activation make it a potential therapeutic target. APOE2 differs from the common (wildtype) APOE3 allele by a single nucleotide polymorphism (SNP), a C>T transition at chr19:g.45412079. This SNP is potentially amenable to an A>G edit on the non-coding strand using an adenine base editor (ABE). Herein we develop an APOE2 base editing approach deliverable by virus-like nanoparticles.
Methods :
Sequence analysis of APOE2 revealed one viable spacer option for the NGG PAM of Strep. pyogenes Cas9 ABE. Performance between four ABE variants [ABE8e, ABE8e(TadA-8e V106W), ABEmax and ABEmax-P2A-GFP] was compared by co-transfecting HEK293T cells with plasmids expressing APOE2, the ABE and guide RNA (gRNA). After 5 days, DNA was extracted and editing rate quantified by EditR. The ABE-gRNA ribonucleoprotein (RNP) complex was packaged into engineered virus-like particles (eVLP) and tested in HEK293T cells.
Results :
In vitro on-target A>G editing efficiency at the APOE2 SNP was similar between the four ABEs, ranging from 38.0% (±10.5%) for ABE8e to 43.0% (±4.2%) for ABEmax-P2A-GFP. No off-target editing of a bystander adenine base was detected. When ABE-gRNA RNP was packaged into eVLPs and applied to HEK cells transfected with APOE2 plasmid, 1% on-target editing of APOE2 SNP and no bystander edits were detected among unsorted cells.
Conclusions :
CRISPR-mediated conversion of AMD-associated APOE2 variant to normal APOE3 is achieved using an adenine base editing construct. Limited editing rate when delivered using non-integrating virus-like nanoparticles is likely due to a low rate of transduction (<5%), which requires optimisation prior to in vivo studies.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.