NRL-centered Rod Gene Regulatory Network identified by gene delivery in a patient iPSC-derived retinal organoid model of NRL-retinopathy

Abstract

Purpose : Neural retina leucine zipper (NRL) is an essential transcription factor for cell fate specification and functional maintenance of rod photoreceptors (PRs) in the mammalian retina. Mutations in NRL lead to retinal diseases. We delivered exogenous NRL to the developing human retinal organoid (RO) model of NRL-retinopathy, aiming to identify NRL-centered gene regulatory network (GRN) involved in PR specification and to optimize gene therapy of associated diseases.

Methods : We differentiated human iPSCs derived from a patient with peripheral field loss and nyctalopia and normal control into ROs. The patient carried a homozygous null mutation in the NRL gene (p.L75Pfs*19). We characterized NRL-L75Pfs patient ROs using immunohistochemistry, immunoblotting, and transcriptomic approaches and compared these to control ROs. We treated ROs at different stages of differentiation with two serotypes of AAV (AAV2/2 and 7m8) and evaluated the treatment effects by histological methods and single-cell sequencing. We are performing ATAC-seq, CUT&RUN and Hi-C and will integrate all ‘omics’ datasets to generate NRL-centered GRN.

Results : NRL-L75Pfs ROs form a PR population dominated by S-cones and lacking rods during development, manifesting as S-opsin present in most PRs, while rod specific gene expression was either reduced or abolished. Although mutant NRL transcripts are detectable in NRL-L75Pfs ROs, the corresponding NRL protein is completely absent. We found that AAV-mediated gene augmentation of NRL at differentiation day 120 can reduce the number of S-opsin+ cones and promote Rhodopsin-expressing rod generation in NRL-L75Pfs ROs. AAV treatment too early or too late failed to exhibit such an effect, suggesting that there is a critical window for NRL to affect cell fate specification of human PRs. Moreover, 7m8 AAV was more effective than AAV2/2 treatment during this process. Finally, single-cell analysis reveals the generation of a population of cells expressing rod-specific genes in the 7m8-treated patient ROs. We are using this and additional ‘omics’ data to establish GRNs that drive rod differentiation.

Conclusions : We have used an in vitro model of NRL loss-of-function disease to interrogate regulatory mechanisms underlying rod cell fate determination and developed an AAV treatment paradigm for exploring gene therapy for NRL-associated inherited retinal diseases.

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