Development of a CRISPR dCas9-KRAB based strategy for treatment for dominant retinal degenerative blindness

Abstract

Purpose : Inherited retinal disease is a major cause of blindness worldwide. The most common disease-causing mutation in patients with RHO-associated retinitis pigmentosa (RP) is a dominant gain-of-function missense variant at amino acid residue 23 (Pro23His). Treatment for this disease and other dominantly inherited retinal dystrophies will require knockdown or ablation of mutant allele expression. To that end the purpose of this study was to develop a CRISPR-Cas9-mediated transcriptional repression strategy capable of suppressing RHO expression.

Methods : We created CRISPR-Cas9 transcriptional knockdown reagents using catalytically inactive S. aureus Cas9 (dCas9) fused to the Krüppel associated box (KRAB) transcriptional repressor domain and evaluated four guides targeted to transcriptional regulatory elements in the promoter region of RHO. Using a reporter construct carrying GFP cloned downstream of the RHO promoter fragment (nucleotides -1403 to +73), we assayed GFP knockdown in cells treated with our RHO promoter-targeted CRISPR-dCas9 reagents. Following functional confirmation, reagents were packaged into AAV5 vectors and delivered to human retinal explants (N=3) and Pro23His mutant retinal degenerative swine (N=4). RHO knockdown was demonstrated via Western blot analysis and immunocytochemistry.

Results : Quantitative RT-PCR analysis of cells treated with CRISPR-dCas9 and reporter plasmids demonstrated ~74%-84% reduction in GFP expression when compared to control cells treated with reporter plasmid only. The guide with the greatest knockdown in this assay (sag94) was cloned into AAV5 cassette plasmids along with dCas9-KRAB and used in downstream ex vivo and in vivo experiments. Western blot analysis demonstrated significant RHO knockdown (50%) in human retinal explants treated with AAV5-RHOpCRISPRi vector compared to untreated controls. When we extended these studies into the Pro23His rhodopsin mutant swine model, we observed a 20% knockdown of rhodopsin in CRISPRi-treated retinae compared with untreated contralateral control retinae at two-weeks post injection.

Conclusions : We have generated a dCas9-KRAB gene repression system suitable for suppressing RHO expression in vivo. This work may provide a paradigm from which to develop CRISPR-dCas9 therapies to treat dominantly inherited retinal dystrophies.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.