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. In a recent survey of one thousand consecutive families clinically diagnosed with an inherited eye disease, the disease causing genotype was identified in 76% of cases. Of these, approximately 12% carried dominantly inherited variants, of which approximately 20% lay within the gene BEST1. Expression of BEST1is restricted to RPE cells, and dominant mutations in BEST1 cause a maculopathy characterized by excessive accumulation of autofluorescent material between the RPE cells and photoreceptor outer segments leading to formation of subretinal lesions and loss of central vision. Treatment for this and other dominantly inherited retinal dystrophies may benefit from 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 suppressingBEST1expression.

Methods : We created CRISPR-Cas9 transcriptional knockdown reagents using catalytically inactive S. pyogenes Cas9 (dCas9) fused to the Krüppel associated box (KRAB) transcriptional repressor domain and evaluated six guides targeted to transcriptional regulatory elements in the promoter region of BEST1. Using a reporter construct carrying GFP cloned downstream of the BEST1 promoter fragment (nucleotides -600 to +38), we assayed GFP knockdown in cells treated with our BEST1promoter-targeted CRISPR-dCas9 reagents. Following functional confirmation, reagents were packaged into lentiviral particles for targeting of patient derived RPE cells (iPSC-RPE).

Results : Quantitative RT-PCR analysis of cells treated with CRISPR-dCas9 and reporter plasmids demonstrated ~70%-79% reduction in GFP expression when compared to control cells treated with reporter plasmid only. Two guides with the greatest knockdown in this assay (g465 and g539) were cloned into lentiviral transgene cassette plasmids along with dCas9-KRAB, packaged into VSVG-pseudotyped lentiviral particles. To assess gene knockdown in a disease specific model system, iPSC derived RPE cells were generated.

Conclusions : We have generated a dCas9-KRAB gene repression system suitable for suppressing BEST1expression. 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 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.