Retinal Ciliopathies and Potential Gene Therapies: A Focus on Human iPSC-Derived Organoid Models

Abstract

The human photoreceptor function is dependent on a highly specialised cilium. Perturbation of cilial function can often lead to death of the photoreceptor and loss of vision. Retinal ciliopathies are a genetically diverse range of inherited retinal disorders affecting aspects of the photoreceptor cilium. Despite advances in the understanding of retinal ciliopathies utilising animal disease models, they can often lack the ability to accurately mimic the observed patient phenotype, possibly due to structural and functional deviations from the human retina. Human-induced pluripotent stem cells (hiPSCs) can be utilised to generate an alternative disease model, the 3D retinal organoid, which contains all major retinal cell types including photoreceptors complete with cilial structures. These retinal organoids facilitate the study of disease mechanisms and potential therapies in a human-derived system. Three-dimensional retinal organoids are still a developing technology, and despite impressive progress, several limitations remain. This review will discuss the state of hiPSC-derived retinal organoid technology for accurately modelling prominent retinal ciliopathies related to genes, including RPGR, CEP290, MYO7A, and USH2A. Additionally, we will discuss the development of novel gene therapy approaches targeting retinal ciliopathies, including the delivery of large genes and gene-editing techniques.

Keywords: CEP290; CRISPR/Cas9; MYO7A; RPGR; USH2A; adeno-associated virus (AAV); cilium; gene therapy; retinal ciliopathy; retinal organoid.

Figure 1

The structure of human cone and rod photoreceptors and connecting cilium. Both cone and rod photoreceptors are divided into two distinct sections, the inner segment, and the disc containing outer segment. The region connecting these distinct compartments is known as the connecting cilium or transition zone (CC/TZ). The outer segment lacks the capacity for protein synthesis and is dependent on protein trafficking across the CC/TZ and subsequent transport along the distal axoneme. Numerous proteins are required for cilial function and maintenance, such as RPGR and CEP290 localized in the CC/TZ or usherin located in the periciliary membrane (PCM). Black arrows depict anterograde and retrograde cilial transport.

Figure 2

Alternative approaches for the delivery for large cilial genes. (A) Dual AAV trans-splicing: Two independently delivered viral genomes containing either the 5′ or 3′ coding sequence (CDS) recombined via concatemerization or homologous recombination mediated by the homology recombination domains (HRDs). The inclusion of splice donor (SD) and splice acceptor (SA) sequences permits RNA splicing to produce a mature mRNA encoding for full-length protein. (B) Protein trans-splicing: The CDS is split across two independent viral vectors resulting in the expression of either N- or C-terminal polypeptides of the desired full-length protein. Adjoined to these peptides are either the N- or C-terminal of a split-intein; post-translationally, the split-intein spontaneously recombines and self-excises, resulting in full-length protein. (C) mRNA trans-splicing: dual AAVs result in the transcription of two independent mRNAs encoding each half of the desired sequence, and the inclusion of complementary binding domains (BDs) allows for recombination. Flanking SD and SA sites facilitate RNA splicing and the removal of the BD sequences, resulting in a mature, full-length mRNA. (D) High-capacity adenovirus has a large cargo capacity of up to 36 kb; this facilitates the delivery of large genes in their entirety with a single vector. Promoter (blue arrow); polyadenylation signal (pA) (created with BioRender.com).