CRISPR/Cas9-mediated genome editing for correction of inherited retinal disease mutations.

Abstract

Purpose : Viral-mediated gene therapy is an exciting development towards curing inherited blindness. However, some genes exceed the carrying capacity of viral vectors or require precise regulation of expression that is difficult to achieve using heterologous systems. To address these issues a precise genome editing technology would be useful. The purpose of this study was to employ CRISPR/Cas9 genome editing to develop strategies for three major classes of disease-causing mutations: 1) dominant gain-of-function, 2) deep intronic and 3) exonic mutations.

Methods : We designed mutation-specific sgRNA oligos and cloned them into bicistronic constructs expressing a chimeric small guide and tracrRNA transcript under control of the human Pol III U6 promoter upstream of a chicken beta-actin promoter driving a human codon-optimized Cas9. Donor homology-dependent repair (HDR) constructs were cloned carrying ~500 bp of homologous WT sequence. Constructs were delivered via transfection or electroporation. Editing efficiency was determined by T7E1 assay, TA-cloning and Sanger sequencing. Mutations targeted in this study: RHO (P23H), PAX6 (Q344X), USH2A (IVS40), and MAK (Alu insertion).

Results : To treat patients with dominant disease, sgRNAs targeting the mutant vs wildtype allele were designed (i.e. guides contained a single variation within the seed region or PAM site). T7E1 assay and Sanger sequencing showed that a sgRNA targeting the P23H mutation in RHO confers a 3-fold increase in targeting specificity (7% vs. 22%, respectively). A similar result was detected for the Q344X mutation in PAX6 (1% vs. 30%, respectively). To edit intronic recessive alleles, we demonstrated that co-delivery of two sgRNAs targeting upstream and downstream of the IVS40 mutation in USH2A successfully modified the locus (21% and 19%, respectively). To demonstrate the utility of our HDR strategy to correct an exonic mutation, we delivered an sgRNA targeting the MAK Alu insertion in exon 9 and a donor template carrying the corrected sequence. This construct restored WT transcript in iPSC-derived retinal precursor cells from a patient with MAK-associated RP.

Conclusions : Successful gene editing targeting three different classes of Mendelian mutations at several disease-causing loci brings us closer to the goal of providing genetically corrected autologous cell-based therapies for patients affected with inherited retinal degenerative disease.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.