ELOVL4 Knock-out Retinal Organoids show VLC-PUFA Elongation Dysfunction

Abstract

Purpose : Very long-chain polyunsaturated fatty acids (VLC-PUFAs) are non-dietary lipids that are exclusively found in the retina and a few other tissues in the body. These C26-C38 n-3 and n-6 lipids are synthesized from long-chain polyunsaturated fatty acid dietary precursors through the action of the ELOVL4 fatty acid elongase. VLC-PUFAs contribute to the maintenance of the highly curved photoreceptor outer segment membrane disks and facilitate photoreceptor synaptic transmission. Autosomal dominant mutations in ELOVL4 lead to a form of Stargardt macular dystrophy (STGD3) that shares many features with dry age-related macular degeneration (AMD). It has also been shown that VLC-PUFA profiles are distinctly abnormal in AMD donor retinas. These findings suggest that abnormalities of VLC-PUFA metabolism are intimately associated with macular degeneration and that strategies to increase VLC-PUFA levels by supplementation could help slow the degeneration process.Here, we use CRISPR/Cas-9 to knock out ELOVL4 in iPSCs, and then generate retinal organoids (ROs) as a human-tissue-based system to characterize the efficacy of exogenous VLC-PUFA supplementation as a means of delaying or preventing macular degeneration.

Methods : We generated isogenic ELOVL4 homozygous knockout and control iPSC lines using CRIPR/Cas-9 editing. Probes were designed to target sequences in the introns surrounding exon 1 resulting in a complete excision of a 1.5 kb fragment including the entire ELOVL4 exon 1. We used GC-MS to characterize the VLC-PUFA profile in fully mature (30-week-old) control ROs and show that ELOVL4^-/-ROs are functional knockouts with measurable defects in VLC-PUFA metabolism. We supplemented both control and ELOVL4^-/- ROs with C32 n-3 or C30 n-6 VLC-PUFAs to investigate the uptake and metabolism of exogenous VLC-PUFA.

Results : We observed that control ROs possessed the full suite of VLC-PUFAs, whereas we were unable to detect VLC-PUFAs longer than C30 in ELOVL4^-/- ROs. Both control and ELOVL4^-/- ROs were able to uptake exogenous VLC-PUFAs; however, only the control ROs were able to elongate past C32.

Conclusions : These results validate ROs as a model for ELOVL4 metabolism and establish that ELOVL4^-/- ROs supplemented with synthetic VLC-PUFAs could be used as a model to study the pathophysiology and treatment of diseases associated with ELOVL4 dysfunction.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.