CRISPR Base Editing; A feasible treatment for inherited retinopathy

Abstract

Purpose : CRISPR base editing provides an opportunity for a scarless cure of many inherited retinopathies. Leber Congenital Amaurosis 16 (LCA16) is a severe form of inherited retinal dystrophy caused by several reported mutations in KCNJ13 that codes for an inwardly rectifying potassium channel Kir7.1. One of the most deleterious is a nonsense mutation W53X (c.158G>A; tGg>tAg) resulting in a truncated Kir7.1 protein, notably within the retinal pigmented epithelium (RPE). Here we aim to test whether a CRISPR-base editor (BE) can correct the W53X mutation, ex vivo, in LCA16-patient-derived fibroblasts as a proof-of-principle of a in vivo somatic editing therapeutic approach.

Methods : Two different base editing approaches [ribonucleoprotein (RNP) complex or plasmid] were used to correct W53X mutation. The RNP constituted sgRNA (C*U*A*AUGGAUAUGCGCUAGCG, Synthego) and ABE7.10 protein (Aldevron) in 1:1 molar ratio. For plasmid based approach, we used CRISPR-BE plasmid (Addgene#112101) and sgRNA plasmid (Addgene#126445) in 3:1 ratio. The fibroblasts were nucleofected to deliver the BEs. We delivered a second dose of BEs at 72 hours post treatment. The editing efficiency at the sgRNA on-target site was assessed by PCR on genomic DNA (isolated at 120 hours post treatment), subjected to deep sequencing using Illumina sequencing platforms. Untreated cells or cells that had only partial components of the BE delivered (e.g., sgRNA only; sgRNA plasmid only; ABE7.10 protein only; editor plasmid only) served as experimental controls.

Results : W53X patient-derived fibroblasts treated with one dose of plasmid BEs showed 7.72% efficiency in editing which was increased to 27.88% after the second nucleofection. Our control fibroblasts had about 1% indels arising due to baseline errors during PCR or sequencing. A higher editing efficiency was observed for plasmid mediated delivery as compared to RNP delivery (~1%). Notably, we did notice the low rates of off target G>A and C>T changes (<1%) similar to controls.

Conclusions : Our results indicate that the base editing has low indel editing outcomes (~1%).
Since LCA16-carrier individuals have no phenotype, ~25% editing efficiency will likely result in a functional recovery of the Kir7.1 channel in the RPE with likely therapeutic efficacy.
The study demonstrates a proof-of-principle to genetically correct loss-of-function point mutations in the endogenous pathogenic genes that cause blinding eye diseases.

This is a 2020 ARVO Annual Meeting abstract.