Abstract
Purpose :
Stargardt macular dystrophy (STGD1) is the most prevalent form of inherited macular dystrophy worldwide and is therefore highly clinically relevant. To date, a variety of gene supplementation approaches have been tested to create a therapy with some reaching clinical trials. Currently, however, no treatment exists. Newer technologies, such as RNA base editors, enable correction of pathogenic G>A and T>C mutation correction. ABCA4 has ~1200 known pathogenic mutations, of which ~63% are transition mutations amenable to this editing technology. Here, we investigated the use of a Cas13-ADAR RNA base editing system to correct an ABCA4 pathogenic stop mutation, c.206 G>A; p.W60*.
Methods :
These experiments used a dual luciferase reporter assay comparing expression of Firefly luciferin to constitutively expressed Renilla luciferin. Fragments of the ABCA4 gene containing the desired mutation, c.206 G>A; p.W60* were inserted in-frame between the two luciferase elements with flanking F2A sites. As the insert contains a stop codon in frame, Firefly is not expressed but when mutation correction occurs, Firefly is expressed. The assay involved a triple transfection of HEK293T cells with the Cas13b-ADAR construct, the luciferase reporter plasmid, and the gRNA plasmid, for which 15 guide variants were tested. The output is the ratio of Firefly to Renilla expression.
Results :
Initial findings indicate that all 15 guides show activity relative to the mutant plasmid and the non-targeting control (NTC), p<0.0001. Three guides showed statistically significant levels of editing, G6, G7, and G12, relative to the mutant plasmid. However, there was no significant difference between guides. When taken as a percentage of the wild-type (WT) plasmid ie no mutation, these guides showed between 28-34% of WT expression. Further, initial transcript analysis using EditR was performed, where 43-61% of transcripts showed the WT ‘G’ instead of an 'A' with guides 6, 7, and 12.
Conclusions :
Overall, previous meta-analyses show 63% of pathogenic mutations in ABCA4 are amenable to Cas13b RNA base editing, particularly as there is no PAM-site constraint. This is a first look at RNA base editing targeting a mutation in ABCA4. The findings indicate that RNA base editing shows potential in targeting an ABCA4 sequence, thus indicating a clinical potential for Stargardt disease.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.