Abstract
Purpose :
No therapies exist for ABCA4-related-retinopathy. Of the 2200 variants, over 60% are missense and can cause protein misfolding. The correction of aberrant ABCA4 folding and traffic with small molecules is a potential therapeutic approach, as shown with CFTR in cystic fibrosis. Here we developed a novel in vitro platform to screen folding modulators for their ability to enhance ABCA4 traffic to the plasma membrane.
Methods :
A cell-based bioluminescence protein-fragment proximity complementation assay was developed for the robust detection of ABCA4 to the plasma membrane in transiently transfected HEK293. It consists of a two-luciferase-subunit system: one fused with the N-terminus of RP2 for localisation at the cytoplasmic face of the plasma membrane, the other linked to a protein of interest. Rhodopsin (RHO) served as positive control, while ER-retained P23H-RHO as negative control. Trafficking to the cell surface was compared between misfolded ABCA4 missense variants and WT-ABCA4. Candidate compounds known to rescue other ABC transporters were tested for their effect on ABCA4 trafficking. CRISPR gene editing of control iPSCs was used to produce isogenic iPSC ABCA4 misfolding variants to study their impact in retinal organoids.
Results :
The assay confirmed plasma membrane traffic with high levels of luminescence for control RHO, compared to very low levels for P23H-RHO. Treating P23H-RHO with 9-cis-retinal provided proof of concept that the luminescence system could detect pharmacological rescue. ABCA4 T983A and R2077W variants exhibited nearly 50% impaired traffic to the plasma membrane compared to WT-ABCA4. The CFTR correctors VX-809 and VX-661, kosmotrope TMAO, HDACi 4-PBA, and AMPK activators Metformin and AICAR were tested. 4-PBA and AICAR showed a positive dose-response effect resulting in increased luminescence for the ABCA4 variants. Control iPSCs were successfully gene edited to produce homozygous T983A and R2077W variant lines. In retinal organoids, these variants showed a reduction (≥ 60%) in protein levels and absence of ABCA4 from the photoreceptor outer segments.
Conclusions :
The proximity complementation assay can identify compounds that rescue missense variant ABCA4 traffic. Retinal organoids can be used to screen the effect of potential therapies facilitating the discovery of breakthrough agents for rescuing ABCA4 protein defects and mitigating ABCA4-related retinopathy.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.