Eyeworld

EW RETINA 64 July 2017 by Liz Hillman EyeWorld Staff Writer said, imagining that if the CRISPR/ Cas-9 AAV-vector therapy was to come to fruition, it wouldn't be administered to patients until they started experiencing loss of rod cells. The takeaway from the study at the moment though is: "… loss of NRL in adult photoreceptors is not expected to lead to change in cell fate or reprogramming; instead the expression of many rod genes will be reduced or even abrogated in the absence of NRL, and several cone genes will likely be derepressed because of loss of NRL and dramat- ically decreased NR2E3 expression. We propose these dysmorphic and dysfunctional rods can overcome rod-specific degenerative mecha- nisms and survive longer in diseased retina. Our studies, therefore, pro- vide novel insights into regulation of rod photoreceptor homeostasis and suggest paradigms for treatment of retinal degenerative diseases." As there is currently no treat- ment for retinal pigmentosa, for example, "the need is great and pressing," Kang Zhang, MD, PhD, said in a press release statement. Dr. Zhang is senior author of the Cell Research study, and chief of oph- thalmic genetics, founding director of the Institute for Genomic Medi- cine, and co-director of biomaterials and tissue engineering, Institute of Engineering in Medicine, University of California, San Diego School of Medicine. "In addition, our approach of reprogramming mutation-sensitive cells to mutation-resistant cells may have broader application to other human diseases, including cancer," Dr. Zhang said. EW References 1. Yu W, et al. NRL knockdown by AAV-deliv- ered CRISPR/Cas9 prevents retinal degenera- tion in mice. Nat Commun. 2017;8:14716. 2. Zhu J, et al. Gene and mutation indepen- dent therapy via CRISPR-Cas9 mediated cellular reprogramming in rod photoreceptors. Cell Res. 2017;27:830–833. Editors' note: The sources have no financial interests related to their comments. Contact information Swaroop: balintfyj@nei.nih.gov Wu: balintfyj@nei.nih.gov Gene Therapy Core, National Eye Institute. There are a few unknowns that investigators will be trying to better understand in the coming years as research moves forward. One ques- tion is whether these gene-edited, inactive rod cells that are keeping cone cells alive will continue doing so for the life of the patient. Another question is whether Cas9, a bacte- rial protein, will illicit an immune response. There is the potential for off-targeting, Dr. Wu said. "We hope that the specific Cas9 vector we delivered only works at the NRL gene; however, it is not 100% guaranteed. We need to develop more specific tools to make sure it only inactivates the gene we target," he said. Researchers are also working on developing a system that doesn't work persistently. "It would be better if we can find a system that can be regulat- ed so that the Cas9 activity can be turned down at some point," Dr. Wu said. As Dr. Swaroop put it, "if we can develop a transitory system where you have a gene, it goes in, does its job, and then gets inactivated, bingo. Then there's no persistence of that activity in the long term … that to me is the extremely attractive part of the CRISPR/Cas9 study." Dr. Swaroop also said researches are working on non-CRISPR-mediat- ed methods to suppress NRL. "It depends on which one moves faster. If we can get a CRISPR/ Cas9 system, which is only active transiently and then gets inactivat- ed, maybe that will be out sooner, or we find some other inhibitor, some other gene that can turn off NRL, perhaps that will go faster," Dr. Swaroop said. He estimated that the research is 2 to 3 years out from soft studies and 5 years before Phase 1, Phase 2 clinical trials in humans. "We are very cautious. I know patients are always interested in getting the treatment quickly, but we have to balance that with safety. I don't want someone to be inject- ing anything in my eye unless I'm absolutely sure. I don't want people to lose their vision while they might still have good vision," Dr. Swaroop Separately, in many retinal diseases, it has become clear that the loss of rod cells can lead to cone cell death. Dr. Swaroop said that rod cells may provide a scaffold and/or produce protective factors that help keep cone cells alive and function- al. Previous research suggested that knocking out NRL to prevent rod cell death—though the cells are no longer functional as rods, nor are they differentiated enough to function as cones—seems to keep surrounding cone cells alive. "The question is, can we keep these rods alive even if they're not functional and maybe the cones will then live," Dr. Swaroop said. According to the Yu et al. Nature Communications study, it had yet to be reported whether in vivo deliv- ery of the CRISPR/Cas9 system to post-mitotic photoreceptors would be effective. The research yielded several findings: • Yu et al. demonstrated efficacy and potential benefit of AAV- CRISPR/Cas9-mediated gene disruption in post-mitotic mouse photoreceptors in vivo. • Three different mouse models for retinal degeneration were tested and NRL ablation using the AAV-CRISPR/Cas-9 technique preserved rods, though function- ally inactive, and resulted in a "relatively intact ONL." • These dysfunctional rods success- fully prevented secondary cell death of surrounding cone photo- receptors. Research by Zhu et al. showed inactivating NRL and NR2E3, a downstream transcription factor, using CRISPR/Cas9 in two retinitis pigmentosa animal models had sim- ilar results as Yu et al. "By reprogramming rod to cone-like photoreceptors in situ by inactivating NRL or NR2E3, we show an increase in cone-like cells with re- markable concomitant preservation of both cone and rod photoreceptors and retinal tissue, with restoration of visual function," Zhu et al. wrote. Using the AAV vector to deliver CRISPR/Cas9 turns off rod func- tion in only cells that are infected, limited to the number of particles of the virus that's injected, said the senior author of the Yu et al. study, Zhijian Wu, PhD, head of Ocular CRISPR/Cas9 used to reprogram rod photoreceptors, prevent secondary cell death of cone cells T wo new studies describe a similar gene therapy tech- nique that prevents retinal degeneration in mice. The research published in Nature Communications and Cell Research describes the use of CRISPR/ Cas9—a gene-editing technology consisting of clustered, regularly in- terspaced, short palindromic repeat (CRISPR) and the bacterial protein Cas9 to disrupt a gene at a specific location—delivered via an ade- no-associated virus (AAV) to target the gene known to control rod cell formation. 1,2 The research suggests, after safe- ty and efficacy testing and refine- ment, specific CRISPR/Cas9 vectors could be injected in patients with inherited retinal diseases to turn off the gene that leads to rod photo- receptor differentiation before the disease-causing genetic defect causes rod cells to die, resulting in cone cell death and subsequent central vision loss. About 2 decades ago, the gene NRL was discovered to be essential for producing rod photoreceptors by Anand Swaroop, PhD, chief, Neuro- degeneration and Repair Laboratory, National Eye Institute, Bethesda, Maryland, and an author on the study in Nature Communications. Researchers knew that removing this gene in germline of mice prevented rod photoreceptor formation and instead only cones formed. Later studies revealed that if NRL was forced to express in cone precursors, they would become rods. What's more, Dr. Swaroop ex- plained that this gene continues to be expressed throughout the life of the rod photoreceptor. "We have shown, over the years, that almost all genes that are required for rod function are either directly or indirectly regulated by NRL. So, once we had that back- ground, it was very clear that if we can suppress NRL, rods will not remain rods," Dr. Swaroop said. Studies describe promising research in gene editing to prevent retinal degeneration Research highlight

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