Abstract
Purpose :
Late-Onset Retinal Degeneration (L-ORD) is an autosomal dominant Inherited Degenerative Disease caused by mutations in the C1QTNF5 gene, which causes vision loss through RPE dysfunction. We investigate the use of a dual cutting Non-Homologous End Joining (NHEJ) CRISPR/Cas9 approach in patients’ iPSC to specifically excise and switch off the mutated allele as a potential preventive measure for vision loss.
Methods :
Induced Pluripotent Stem Cells (iPSC) were generated from a L-ORD patient biopsy using episomal vectors. We have identified CRISPR targetable Single Nucleotide Polymorphism-induced Protospacer Adjacent Motifs (Si-PAMs), unique to the C1QTNF5 dominant allele, in a cohort of L-ORD patients using long-range PCR and 3rd Generation Sequencing (Nanopore). iPSCs were nucleofected with ribonucleoprotein complexes containing spCas9 and combinations of two guides. Editing was examined in cells by Sanger sequencing, ICE analysis and alignment in Snapgene. Dual edited clones were differentiated into iPSC-derived RPE and C1QTNF5 expression and localisation was studied.
Results :
iPSC lines carrying the L-ORD S163R mutation were successfully generated and differentiated into iPSC-RPE. Long-range 3rd Generation Sequencing of C1QTNF5 gene in a cohort of three L-ORD patients revealed 24 novel SNPs, of which 3 created novel spCas9 SiPAM sites (NGG) on the dominant allele. Using RNPs targeting the mutated allele, we identified an excision of ~7.9kb within the C1QTNF5 gene. Analysis of clones demonstrated that the excision was specific to the mutated C1QTNF5 allele. C1QTNF5-/+ iPSC successfully differentiated into RPE, and C1QTNF5 and collagen IV increased deposition in the basal lamina seen in the mutant iPSC-RPE cells was lost (n=3).
Conclusions :
Our results show that the identification of allele-specific SiPAM sites on the mutated C1QTNF5 gene can be used for dual CRISPR-Cas9 targeting and excising large regions of the dominant C1QTNF5 allele, without affecting the WT allele. Most importantly, editing corrects the dominant negative C1QNTF5 deposition in L-ORD cells. With no treatments currently available for L-ORD, this therapeutic approach could be used to inactivate the faulty gene, preventing loss of vision in L-ORD patients. Future work will further characterise the functional implications of knocking out the mutant allele. We will also test our editing approach directly in iPSC-derived RPE.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.