Disease mechanism and therapies for retinal degeneration

National Eye Institute

Investigators

Linked publications & trials

Abstract

1. A major goal of the RCBD Section continues to be generating stem cell-derived retinal photoreceptors that are well differentiated as indicated by the elaboration of outer segments. Such an in vitro retinal model system would be much ideally suited for retinal disease modeling, drug discoveries and provide donor tissues for cell transplantation therapies and could to some extent replace many of the vertebrate animal models currently in use. Given the dependence of photoreceptors on RPE, one of the critical components that is missing in current retinal culture systems is a functioning RPE layer that interact meaningfully with the photoreceptors. Although many investigators attempted this dating back decades, a successful outcome has been elusive, attesting to the enormous challenges that lie ahead. We have devoted considerable resources and efforts towards developing photoreceptor-RPE co-culture systems aiming at replicating the in vivo relationship whereby RPE provide the critical support needed for photoreceptor outer segments morphogenesis and maintenance. In the past year we have generated many batches of retinal organoids and RPE monolayer cultures derived from human iPSC lines and have attempted to co-culture to two tissues in close apposition. We have experimented with a variety of configurations and devices, with the aim of delivering media differentially to these two cell types to simulate the in vivo setting. We have further investigated different polymers that encapsulate the retinal organoids separately or together with the RPE. While we have yet to reach our original goal of growing functional outer segments, we have nevertheless gained valuable experience and learnt many of the pitfalls, which have been incorporated in improving experimental designs. Recent addition of new team members with strong expertise in biomaterial science and device fabrication will bring much needed new expertise to bear on this important research project. 2. We have completed a study of CRISPR mediated gene therapy in a mouse model of retinal degeneration. Mutations in the gene for Retinitis Pigmentosa GTPase Regulator (RPGR) cause the X-linked form of inherited retinal degeneration. This is a form of RP that is clinically highly significant because its prevalence and severity of disease. Currently there are several clinical trials of AAV-mediated gene replacement therapy ongoing with uncertain outcomes. As a potential alternative strategy to replacement gene therapy which are plagued by several unresolved issues, we sought to correct the mutation in situ via a simplified version of genome editing mediated by CRISPR. The rationale of the design takes advantage of the fact that a majority of RPGR disease causing mutations are frameshift alleles 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. While this falls short of complete rescue of all cells, it would still deliver meaningful alleviation of the disease and may significantly improve patients quality of life if successfully translated to the clinic. 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. We have now expanded the study into human retina using iPSC derived retinal organoids derived from patients. We have successfully produced retinal organoids from patients iPSCs and are in the process of testing CRISPR mediated genome editing in an attempt to restore full-length RPGR-ORF15 expression and rescue the disease phenotype. Further, in collaboration with GEC headed by Dr. Lijin Dong, we have reprogrammed rd9 mouse cells into iPSCs and are in the process of generating retinal organoids. With the mouse retinal organoids, we will test ways to increase the efficiency of the rescue. Based on data from in vivo rd9 mouse studies, we have proposed a hypothesis that microhomology mediated recombination, rather than non-homologous end joining, is a major mechanism that drives the repair, giving the highly repetitive nature of the DNA sequence in this region. Retinal organoids carrying the rd9 mouse mutation will afford us the opportunity to study the mechanism in detail, and apply any new insight thus gained in the human retina. 3. We have continued methodological improvements in the process of retinal organoid generation from mouse and human iPSCs. Retinal organoids recapitulate key features of retinogenesis and provide a promising platform to study retinal development and disease in a human context. However, barriers remain in their wider adoption. One issue is that current protocols remain complex, demand considerable expertise and training, and suffer from batch to batch inconsistencies, as well as line to line variations in their ability to produce retinal organoids at a high yield. Although multiple protocols are currently in use, hPSCs exhibit tremendous variability in differentiation efficiency, with some cell lines consistently yielding few or even no ROs, limiting their utility in research. Following our previous work on a simplified harvesting method, we now show that nicotinamide treatment at a defined developmental window significantly improves RO yield across 8 hPSC lines from different donors, including some that would otherwise fail to generate a meaningful number of RO using conventional methods. This treatment appears to promote neural commitment of hPSCs at the expense of non-neural ectodermal cell fate, which in turn increases eye field progenitor generation. Further analysis suggested that this effect is partially mediated through inhibition of BMP signaling. Our data encourage a broader use of human ROs for disease modeling applications that require the use of multiple patient-specific cell lines.