Abstract
Purpose :
STGD1 a rare form of macular degeneration, has a high unmet need and no approved treatment yet. The purpose is to (1) show the effect of the deep-intronic variant c.4539+2001 G>A in intron 30 of ABCA4 in patient derived cell models on ABCA4 protein (2) use gene editing to rescue the aberrant splicing and restore protein production as a proof of concept to create a potential single dose therapeutic for patients with deep-intronic variants in ABCA4.
Methods :
The impact of deep-intronic mutations in intron 30 of ABCA4 was assessed based on observing aberrant transcripts by RT-PCR analysis in three compound heterozygous mutant STGD1 patient iPSC-derived RPE cells (c.4539+1106C>T, c.4539+2001G>A, c.4539+2028C>T) in comparison to healthy control RPE cells. Immunostaining of a patient-derived (c.4539+2001G>A, compound heterozygous) retinal organoid generated after 175 days of differentiation was performed to screen for ABCA4 protein localization. DAPI (blue) was used to stain the nuclei and anti-ABCA4 antibody was used to stain the ABCA4 protein.
Helex’s proprietary EPIC-Cure platform was used to design two gRNAs to excise a region in intron 30 that spans six mutations (c.4539+1100 A>G, c.4539+1106 C>T, c.4539+2001 G>A, c.4539+2028 C>T, c.4539+2064 C>T, and c.4539+2066 C>G). Patient derived fibroblasts (c.4539+2001 G>A) were transfected with gRNAs with Cas9 mRNA using Lipofectamine messenger max. Target-specific PCR amplification was performed on genomic DNA post transfection to estimate excision efficiency. Control and patient-derived cells were transdifferentiated for 10 days, treated with cycloheximide to block nonsense-mediated decay and RT-PCR was performed to check for aberrant transcripts.
Results :
RT-PCR analysis of STGD1 patient-derived fibroblasts and immunostaining of the patient-derived organoids displayed formation of aberrant transcript and loss of correct ABCA4 protein localization confirming the pathogenicity of the specific deep-intronic mutations. High efficiency target region excision was observed with Helex’s proprietary gRNAs. RT-PCR analysis of the transdifferentiated heterogenous edited population demonstrated significant reduction in aberrant mRNA transcript.
Conclusions :
Our work serves as a strong proof of concept to further develop a gene editing- based approach for patients with deep-intronic mutations in ABCA4 that lead to Stargardt disease.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.