Fine-tuning of therapeutic gene regulation is essential for functional improvement in FAM161A-deficient ciliopathy

Abstract

Purpose : Gene replacement is the most intuitive approach to recessive diseases, and has spearheaded the development of gene therapy. Defects in the FAM161A gene encoding for two isoforms of a ciliary protein have been proven to cause autosomal recessive retinitis pigmentosa (RP28). FAM161A is a protein expressed in the basal body and in the inner scaffold of the connecting cilium of photoreceptors. Upon FAM161A loss, the cilium structure disorganizes and the outersegments progressively collapse (Mercey et al. 2022). This study aims to determine the efficiency of these isoforms to restore the connecting cilia and improve retinal function after gene transfer.

Methods : We designed and produced AAV2/8 vectors expressing the human isoforms HL (with exon 4) or HS (without exon 4) under a composite IRBP-GRK1 promoter or promoters derived from the human FAM161A regulatory regions FCBR1-F0.4 or FCBR2-F0.4. We injected these vectors at 2 weeks of age in Fam161a^tmb/tmb mice, shortly after the start of cilium disorganization, and evaluate FAM161A expression, retinal function and structure up to 3 months post-injection.

Results : While IRBP-GRK and FCBR2-F0.4 promoters lead to FAM161A expression not only in the connecting cilium but also in the inner segment and photoreceptor cell body, FCBR1-F0.4 faithfully reproduces WT exclusive connecting cilium expression and reassemble the inner scaffold of connecting cilium as assessed by Ultrastructure expansion microscopy (U-ExM). Both IRBP-GRK1 and FCBR1-F0.4 vectors preserved retina thickness and outersegment structure. For IRBP-GRK vectors, only combined administration of HL and HS expressing vectors maintain an outer nuclear thickness similar to wild type condition, but not retinal function. Finally, only the combined administration and high doses of FCBR1-F0.4 HL and HS vectors is able to correctly reconstruct the cilium structure (evidenced by U-ExM), preserve the same number of connecting cilia as wild type retina and to improve retinal function up to 3 months post-injection.

Conclusions : Our work demonstrates that fine-tuning of the therapeutic gene regulation is essential for retinal function restoration. Not only the promoter, but also the dose and the isoforms are conditions which should be carefully addressed to securely propose gene therapy for diseases linked to structural proteins such as FAM161A.

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