Postdoctoral Researcher University College London (UCL), United Kingdom
Abstract: Late-Onset Retinal Degeneration (L-ORD) is a rare, autosomal dominant disease caused by mutations in the C1QTNF5 gene, leading to vision loss through RPE dysfunction. As a late onset disease, there is a window of opportunity to treat patients prior to vision loss. Here, we investigate a dual cutting Non-Homologous End Joining (NHEJ) CRISPR/Cas9 approach, to specifically excise and switch off the mutated allele as a potential preventive measure for vision loss.
We used 3rd Generation Sequencing (Oxford Nanopore long-read sequencing) in a L-ORD patient cohort to reveal shared, mutant allele-specific Single Nucleotide Polymorphisms (SNPs) that could be targeted with CRISPR. We also generated induced Pluripotent Stem Cells (iPSC) from a L-ORD patient skin biopsy using episomal vectors. iPSCs generated were then nucleofected with spCas9 and combinations of two guides, and editing efficiency was examined by Sanger sequencing, ICE analysis and Snapgene. Dual edited clones were differentiated into iPSC-derived RPE and C1QTNF5 expression and localisation was studied.
Our results revealed the cohort of L-ORD patients had 24 shared SNPs, of which 3 created novel spCAS9 SiPAM sites (NGG) unique to the mutant allele. Using RNPs targeting these mutant-specific SiPAM sites, we successfully excised ~7.9kb within the C1QTNF5 gene. Isolation of clones demonstrated that the excision was specific to the mutated C1QTNF5 mutated allele. C1QTNF5-/+ iPSC successfully differentiated into RPE, and C1QTNF5 and collagen IV deposition in the basal lamina seen in the mutant iPSC-RPE cells was lost.
Our results show that the identification of allele-specific SiPAM sites on the mutated C1QTNF5 gene can be used for dual-guided CRISPR-Cas9 targeting and excising of the dominant C1QTNF5 allele, without affecting the WT allele. Most importantly, editing corrects the dominant negative phenotype of 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 using a range of delivery methods.
