CRISPR GENOME SURGERY IN THE RETINA IN LIGHT OF OFF-TARGETING.

PURPOSE: Recent concerns regarding the clinical utilization of clustered regularly interspaced short palindromic repeats (CRISPR) involve uncertainties about the potential detrimental effects that many arise due to unintended genetic changes, as in off-target mutagenesis, during CRISPR genome surgery. This review gives an overview of off-targeting detection methods and CRISPR's place in the clinical setting, specifically in the field of ophthalmology. RESULTS: As CRISPR utilization in the laboratory setting has increased, knowledge regarding CRISPR mechanisms including its off-target effects has also increased. Although a perfect method for achieving 100% specificity is yet to be determined, the past few years have seen many developments in off-targeting detection and in increasing efficacy of CRISPR tools. CONCLUSION: The CRISPR system has high potential to be an invaluable therapeutic tool as it has the ability to modify and repair pathogenic retinal lesions. Although it is not yet a perfect system, with further efforts to improve its specificity and efficacy along with careful screening of off-target mutations, CRISPR-mediated genome surgery potential can become maximized and applied to patients.

A novel de novo CAPN5 mutation in a patient with inflammatory vitreoretinopathy, hearing loss, and developmental delay.

Mutations that activate the protease calpain-5 (CAPN5) cause a nonsyndromic adult-onset autoinflammatory eye disease characterized by uveitis, altered synaptic signaling, retinal degeneration, neovascularization, and intraocular fibrosis. We describe a pediatric patient with severe inflammatory vitreoretinopathy accompanied by hearing loss and developmental delay associated with a novel, de novo CAPN5 missense mutation (c.865C>T, p.Arg289Trp) that shows greater hyperactivation of the calpain protease, indicating a genotype-phenotype correlation that links mutation severity to proteolytic activity and the possibility of earlier onset syndromic disease with auditory and neurological abnormalities.

Calpain-5 gene expression in the mouse eye and brain.

OBJECTIVE: Our objective was to characterize CAPN5 gene expression in the mouse central nervous system. Mouse brain and eye sections were probed with two high-affinity RNA oligonucleotide analogs designed to bind CAPN5 RNA and one scramble, control oligonucleotide. Images were captured in brightfield. RESULTS: CAPN5 RNA probes were validated on mouse breast cancer tumor tissue. In the eye, CAPN5 was expressed in the ganglion cell, inner nuclear and outer nuclear layers of the retina. Signal could not be detected in the ciliary body or the iris because of the high density of melanin. In the brain, CAPN5 was expressed in the granule cell layers of the hippocampus and cerebellum. There was scattered expression in pons. The visual cortex showed faint signal. Most signal in the brain was in a punctate pattern.

CRISPR-mediated Ophthalmic Genome Surgery.

PURPOSE OF REVIEW: Clustered regularly interspaced short palindromic repeats (CRISPR) is a genome engineering system with great potential for clinical applications due to its versatility and programmability. This review highlights the development and use of CRISPR-mediated ophthalmic genome surgery in recent years. RECENT FINDINGS: Diverse CRISPR techniques are in development to target a wide array of ophthalmic conditions, including inherited and acquired conditions. Preclinical disease modeling and recent successes in gene editing suggest potential efficacy of CRISPR as a therapeutic for inherited conditions. In particular, the treatment of Leber congenital amaurosis with CRISPR-mediated genome surgery is expected to reach clinical trials in the near future. SUMMARY: Treatment options for inherited retinal dystrophies are currently limited. CRISPR-mediated genome surgery methods may be able to address this unmet need in the future.

Structural modeling of a novel SLC38A8 mutation that causes foveal hypoplasia.

BACKGROUND: Foveal hypoplasia (FH) in the absence of albinism, aniridia, microphthalmia, or achromatopsia is exceedingly rare, and the molecular basis for the disorder remains unknown. FH is characterized by the absence of both the retinal foveal pit and avascular zone, but with preserved retinal architecture. SLC38A8 encodes a sodium-coupled neutral amino acid transporter with a preference for glutamate as a substrate. SLC38A8 has been linked to FH. Here, we describe a novel mutation to SLC38A8 which causes FH, and report the novel use of OCT-angiography to improve the precision of FH diagnosis. More so, we used computational modeling to explore possible functional effects of known SLC38A8 mutations. METHODS: Fundus autofluorescence, SD-OCT, and OCT-angiography were used to make the clinical diagnosis. Whole-exome sequencing led to the identification of a novel disease-causing variant in SLC38A8. Computational modeling approaches were used to visualize known SLC38A8 mutations, as well as to predict mutation effects on transporter structure and function. RESULTS: We identified a novel point mutation in SLC38A8 that causes FH. A conclusive diagnosis was made using OCT-angiography, which more clearly revealed retinal vasculature penetrating into the foveal region. Structural modeling of the channel showed the mutation was near previously published mutations, clustered on an extracellular loop. Our modeling also predicted that the mutation destabilizes the protein by altering the electrostatic potential within the channel pore. CONCLUSION: Our results demonstrate a novel use for OCT-angiography in confirming FH, and also uncover genotype-phenotype correlations of FH-linked SLC38A8 mutations.

Calpain-5 Expression in the Retina Localizes to Photoreceptor Synapses.

PURPOSE: We characterize calpain-5 (CAPN5) expression in retinal and neuronal subcellular compartments. METHODS: CAPN5 gene variants were classified using the exome variant server, and RNA-sequencing was used to compare expression of CAPN5 mRNA in the mouse and human retina and in retinoblastoma cells. Expression of CAPN5 protein was ascertained in humans and mice in silico, in mouse retina by immunohistochemistry, and in neuronal cancer cell lines and fractionated central nervous system tissue extracts by Western analysis with eight antibodies targeting different CAPN5 regions. RESULTS: Most CAPN5 genetic variation occurs outside its protease core; and searches of cancer and epilepsy/autism genetic databases found no variants similar to hyperactivating retinal disease alleles. The mouse retina expressed one transcript for CAPN5 plus those of nine other calpains, similar to the human retina. In Y79 retinoblastoma cells, the level of CAPN5 transcript was very low. Immunohistochemistry detected CAPN5 expression in the inner and outer nuclear layers and at synapses in the outer plexiform layer. Western analysis of fractionated retinal extracts confirmed CAPN5 synapse localization. Western blots of fractionated brain neuronal extracts revealed distinct subcellular patterns and the potential presence of autoproteolytic CAPN5 domains. CONCLUSIONS: CAPN5 is moderately expressed in the retina and, despite higher expression in other tissues, hyperactive disease mutants of CAPN5 only manifest as eye disease. At the cellular level, CAPN5 is expressed in several different functional compartments. CAPN5 localization at the photoreceptor synapse and with mitochondria explains the neural circuitry phenotype in human CAPN5 disease alleles.

Nonallele specific silencing of ataxin-7 improves disease phenotypes in a mouse model of SCA7.

Spinocerebellar ataxia type 7 (SCA7) is a late-onset neurodegenerative disease characterized by ataxia and vision loss with no effective treatments in the clinic. The most striking feature is the degeneration of Purkinje neurons of the cerebellum caused by the presence of polyglutamine-expanded ataxin-7. Ataxin-7 is part of a transcriptional complex, and, in the setting of mutant ataxin-7, there is misregulation of target genes. Here, we designed RNAi sequences to reduce the expression of both wildtype and mutant ataxin-7 to test if reducing ataxin-7 in Purkinje cells is both tolerated and beneficial in an animal model of SCA7. We observed sustained reduction of both wildtype and mutant ataxin-7 as well as a significant improvement of ataxia phenotypes. Furthermore, we observed a reduction in cerebellar molecular layer thinning and nuclear inclusions, a hallmark of SCA7. In addition, we observed recovery of cerebellar transcripts whose expression is disrupted in the presence of mutant ataxin-7. These data demonstrate that reduction of both wildtype and mutant ataxin-7 by RNAi is well tolerated, and contrary to what may be expected from reducing a component of the Spt-Taf9-Gcn5 acetyltransferase complex, is efficacious in the SCA7 mouse.