En31FnCas9-based adenine base editors for precision editing in patient-specific iPSCs and correction of two pathogenic mutations linked to Leber Congenital Amaurosis (LCA)

Abstract

Purpose : This study aims to establish the proof-of-concept for correction of mutations in patient-specific induced pluripotent stem cell (PS-iPSC) lines, using a high fidelity Francisella novicida Cas9-based, adenine base editing system.

Methods : Edit efficiency of FnCas9 variants (en1, en15, en31) were assessed in comparison with spCas9, using CRISPR-gRNAs targeting the PAX6 gene in ARPE19 cells. Guide oligos targeting mutation loci in RD3 (c.296+1 G>A) and RPE65 (c.992 G>A) were cloned into en31ABEmax8.17, an en31FnCas9-based adenine base editor (ABE) and nucleofected in different PS-iPSCs. The edited cells were clonally expanded and screened for mutation correction by deep sequencing of target loci. Off-target edits and genomic integrity were assessed by digenome-seq (DGS) and whole genome-seq (WGS) respectively. The iPSC lines were differentiated to retinal organoids (ROs) and RPE cells. Transcript and protein expression were evaluated by RT-PCR, western blotting and immunocytochemistry.

Results : In ARPE19 cells, FnCas9 variants (en1, en15) efficiently edited PAX6 loci, resulting in loss of protein expression. In hiPSCs, en1FnCas9 (18.6%) and en15FnCas9 (23%) showed higher edit efficiency than spCas9 (13.8%) (N=3). Further, using the en31FnCas9-based ABE, we observed precise A>G base conversion and mutation correction at RD3 loci in 13% of cells, with a 21 bp guide with NGG PAM. Similarly, we observed precise editing at RPE65 loci in 21%, 13%, 8% of the cells, using 21 bp guides with NGG, GGA, AAG PAMs respectively (N=3). The edited clonal lines showed >99% mutation correction, with no undesirable bystander edits at the target loci. DGS analysis of the RPE65 guide has confirmed two guide-dependant off target edits, within intergenic regions. WGS analysis confirmed the absence of any gross chromosomal alterations. Characterization of the mature iPSC-RPE cells and ROs has confirmed restoration of RPE65 mRNA and protein expression in edited cells, comparable to that of control cells.

Conclusions : The en31FnCas9-based ABE system allows precise and efficient mutation correction in RD3 and RPE65 in iPSCs, for applications in autologous cell therapy. With PAM flexibility and variable guide lengths (g20-21), it offers high fidelity with improved edit efficiency and genome accessibility for therapeutic use.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.