Abstract
Purpose :
Developing a gene therapy for autosomal dominant Retinitis Pigmentosa (adRP) is extremely challenging, since suppression of the mutant protein may be required, in addition to delivery of a healthy gene. Furthermore, overexpression of many IRD genes can also lead to retinal degeneration, therefore it is necessary to maintain wild-type protein expression at endogenous levels. Around 35% of RP is caused by autosomal dominant mutations, with mutations in RP1 accounting for around 5-10% of cases. The CRISPR/Cas9 system can distinguish target sequences differing by a single base pair, thus we propose to develop a haplotype based approach to specifically ablate mutant RP1 alleles which bear any dominant frameshift or nonsense mutation in exon 4
Methods :
Three common RP1 haplotypes carrying distinct SNPs were identified from the 1000 Genome Project. sgRNAs for SNPs on each of the three haplotypes were synthesized and cotransfected with Cas9 expressing plasmids into human cell lines that are heterozygous or homozygous for the RP1 haplotypes. Editing efficiency and allele-specificity was determined by PCR and amplicon sequencing. The most efficient sgRNAs were then tested in combination with a non-allele specific sgRNA targeting intron 3. Dual DSBs and deletion of a fragment of RP1 genome were analysed and quantified by qPCR. The expression level of the targeted RP1 allele was quantified by qPCR in modified RP1-expressing Hap1 cells.
Results :
A non-allele specific sgRNA with high editing efficiency was tested in pair-wise combinations with allele specific sgRNAs targeting intron 1, or exon 4. These dual Cas9/sgRNAs were able to delete 3.5-6 kb of genomic DNA, encompassing either exons 2 and 3, or a large portion of exon 4 of RP1 in an allele specific manner. Deletion of the large fragment of RP1 genome led to reduction of RP1 expression in edited Hap1-EF1a-RP1 cells
Conclusions :
This proof-of-concept study is an important step towards the application of CRISPR/Cas9 gene editing technology in the treatment of patients with RP1 associated dominant IRDs. Our haplotype-based approach greatly enhances the feasibility of using the CRISPR-Cas9 system because a small number of sgRNAs are necessary to specifically target relatively large sets of mutant alleles. Furthermore, it paves the avenue of its application in human RP1 patients
This is a 2021 ARVO Annual Meeting abstract.