Disease mechanism and therapies for retinal degeneration

National Eye Institute

Investigators

Linked publications, trials & patents

Abstract

1. Gene therapy via CRISPR/Cas9 mediated genome editing Mutations in the gene for Retinitis Pigmentosa GTPase Regulator (RPGR) cause the X-linked form of inherited retinal degeneration, and the majority are frameshift mutations in a highly repetitive, purine-rich region of RPGR known as the OFR15 exon. Truncation of the reading frame in this terminal exon ablates the functionally important C-terminal domain. We hypothesized that targeted excision in ORF15 by CRISPR/Cas9 and the ensuing repair by non-homologous end joining could restore RPGR reading frame in a portion of mutant photoreceptors thereby correcting gene function in vivo. We tested this hypothesis in the rd9 mouse, a naturally occurring mutant line that carries a frameshift mutation in RPGRORF15, through a combination of germline and somatic gene therapy approaches. In germline gene edited rd9 mice, probing with RPGR domain-specific antibodies demonstrated expression of full length RPGRORF15 protein. Hallmark features of RPGR mutation-associated early disease phenotypes, such as mislocalization of cone opsins, were no longer present. Subretinal injections of the same guide RNA (sgRNA) carried in AAV sgRNA and SpCAs9 expression vectors restored reading frame of RPGRORF15 in a subpopulation of cells with broad distribution throughout the retina, confirming successful correction of the mutation. These data suggest that a simplified form of genome editing mediated by CRISPR, as described here, could be further developed to repair RPGRORF15 mutations in vivo. Our study is the first to demonstrate template-free in vivo correction in mature photoreceptors as a proof-of-principle for future work that aims to correct disease-causing mutations in RPGRORF15 (published by Gumerson et al,). To gain greater insights into the molecular mechanism of the rescue, we produced iPSC lines from rd9 mouse tail fibroblasts (in collaboration with GEC). Initial differentiation attempts were unsuccessful due to apparent heterogeneity of the cloned cells, in which many appear to have differentiated into fibroblast like lineages. We then subcloned the iPSCs and chosen those with undifferentiated morphology. This turned out to be successful as the newly isolated clones appear to go into retinal lineages readily. Ongoing work is aimed at reproduce genome editing events in the rd9 mice, and identify the precise changes at the genome level to distinguish between two competing hypotheses: non-homologous end joining vs. microhomology mediated recombination. The latter is a plausible scenario given the highly repetitive nature of the ORF15 region and the potential for DNA looping. Such knowledge will help us further improve the efficiency of genome editing. To extend this work into a human disease setting, we have developed RPGR iPSC lines from patient donor fibroblast with RPGR ORF15 c.2715_2716insCCTC causing a fs. Six lines were generated and two of these were confirmed to have normal karyotypes. Separately, all six lines were shown to be efficient producers of retinal organoids. Focusing on lines with correct karyotypes, we have generated batches of organoids. Ongoing work is aimed at replicating the success in rd9 mouse, namely to allow expression of full length RPGR ORF15 in the mutant cells following CRISPR mediated gene editing. Ananya Samanta is leading these efforts with help from members of the gene therapy and stem cell groups. 2. Disease modeling of human Usher Syndrome type 1C in retinal organoids and therapeutic studies The ability to model human USH1 in vitro and to conduct therapeutic studies represent a potential breakthrough in this field. This is because no USH1 mouse models were ever shown to replicate the retinal phenotype despite the fact that they faithfully replicate the cochlear defects. The underlying reasons remain unclear and have a remained a puzzle to Usher disease research. From 4 patient and family control donor fibroblasts representing two different USH1C alleles (including the common Acadian allele) we have generated many batches of retinal organoids with staggered ages (up to 350 days). We have now identified a retinal phenotype consistent with USH1C disease. These include loss of outer segments, rhodopsin mislocalization, disorganization of outer limiting membranes and up regulation of GFAP, a generalized marker of retinal degeneration. The phenotype becomes manifest after Day 250 and appears to progress as the organoids age further. We show that the USH1C protein (harmonin) appears to be expressed primarily in Muller glia, indicating the primary pathogenic event is initiated in the cell type. Thus, modeling of USH1C allele in a dish is successful. Because competing hypotheses exist in the field with regard to cellular origin of USH1C and the target cell type for gene therapy, we have generated AAV carrying USH1C controlled by either Muller directed promoter or a general promoter, and will transduce organoids with the AAVs. This will help settle the debate regarding the targe cell type for gene based therapies. As efforts are currently underway to initiate clinical trials of USH1C, our work is a timely and critical addition to the literature. We anticipate a manuscript on this study to be completed in March. Current and planned work will examine the physiological role of USH1C in Muller glia, the disease mechanism, and potential small molecule drug screen for alleviating the disease in organoids. Interestingly, the common USH1C alleles appear to be hypomorphic, as the proteins are still expressed having skipped a few residues but remaining in frame. To define a genuine null phenotype which is likely to be more severe thus allowing a shorter experimental cycle (disease appearing earlier), we deleted the entire USH1C gene in iPSCs and generated organoids from the null cell lines. This work is ongoing. 3. Improving the yield, reproducibility and quality of retinal organoid generation from iPSCs Existing protocols for differentiation of retinal organoids from iPSCs can be quite laborious, time consuming, and give variable yields from different lines. Difficult lines often yield few or no organoids at all. Such complexities limit the use of this in vitro system in applications requiring large-scale production of organoids. Currently methods include isolation of presumed optic vesicle-like structures from adherent cultures by dissection, a labor-intensive and time-consuming step that requires extensive practice and/or training. We set out to improve these techniques so that retinal organoid production from iPSCs will be more To improve on this step, we have developed a simple and efficient method to generate ROs by scraping the entire adherent culture and growing the resulting cell aggregates in a free-floating condition. This improved, robust and efficient protocol should facilitate large-scale differentiation of pluripotent stem cells into retinal organoids in support of human disease modeling and therapy development. Secondly, although multiple differentiation protocols are currently in use, hPSCs exhibit tremendous variability in differentiation efficiency, with some cell lines consistently yielding few or even no ROs thus limiting their utility in research. To improve the efficiency and robustness of RO generation, we set out to identify factors that promote neural retina differentiation from hPSCs. We found here that nicotinamide treatment at the early stage of differentiation significantly improved RO yield across 8 hPSC lines from different donors. Importantly, nicotinamide treatment enabled efficient production of ROs from cell lines that would otherwise fail to generate meaningful number of ROs. Further analyses revealed that nicotinamide treatment promotes neural commitment of hPSCs at the exp