Abstract
Purpose :
Inherited retinal dystrophies (IRD) are a broad group of genetic eye diseases causing vision loss and other complications that have no treatment. Gene editing is one of the most promising strategies for future personalized and translational medicine and is suitable for single-nucleotide substitutions found in many of the IRD cases. This study aims to revert several IRD mutations and analyze different key factors in the successful correction of pathogenic variants. For that purpose we focused on enhancing CRISPR/TALEN guides, repair templates designs and testing different cell culture and electroporation conditions for efficient on-target edition.
Methods :
To correct the damaging variants we have used CRISPR/Cas9 and TALEN technologies in different human induced pluripotent stem cells (hiPSCs) obtained from IRD patients affected by Retinitis Pigmentosa (PDE6A, RHO and USH2A genes), Stargardt disease (ABCA4), Best disease (BEST1) or Achromatopsia (PDE6C). We have in silico designed several CRISPR (sgRNA) and TALEN guides, and repair templates considering some important parameters. Afterwards, we performed CRISPR or TALEN in hiPSCs trying different conditions for assay optimization. Finally, hiPSC clones were Sanger sequenced for on-target and off-targets screening.
Results :
We have repaired several IRD mutations in patient-derived hiPSCs with no detectable off-target aberrations, thus potentially reversing the diseased condition. We have found that, sgRNAs harboring the pathogenic variant effectively discriminate between the two alleles in heterozygous cases. Regarding repair templates, the introduction of a Cas9-silent mutation was important to ensure a single repair cycle avoiding DNA re-cut by Cas9. As well, the amount and stability of the oligonucleotide template are critical for efficient correction. Notably, we found that TALEN technology was significantly less efficient for gene editing than CRISPR/Cas9.
Conclusions :
sgRNAs, TALENs and repair templates designs are important to achieve precise and efficient single-nucleotide gene editing in hiPSCs. Research done on these therapeutic approximations is crucial for the development of potential IRDs’ treatment through permanent correction of the pathogenic variants.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.