CRISPR editing of anti-anemia drug target rescues independent preclinical models of retinitis pigmentosa.

Retinitis pigmentosa (RP) is one of the most common forms of hereditary neurodegeneration. It is caused by one or more of at least 3,100 mutations in over 80 genes that are primarily expressed in rod photoreceptors. In RP, the primary rod-death phase is followed by cone death, regardless of the underlying gene mutation that drove the initial rod degeneration. Dampening the oxidation of glycolytic end products in rod mitochondria enhances cone survival in divergent etiological disease models independent of the underlying rod-specific gene mutations. Therapeutic editing of the prolyl hydroxylase domain-containing protein gene (PHD2, also known as Egln1) in rod photoreceptors led to the sustained survival of both diseased rods and cones in both preclinical autosomal-recessive and dominant RP models. Adeno-associated virus-mediated CRISPR-based therapeutic reprogramming of the aerobic glycolysis node may serve as a gene-agnostic treatment for patients with various forms of RP.

The Peripapillary Retina - A Common Juncture in Stargardt Disease and Idiopathic Intracranial Hypertension.

PURPOSE: To present the multimodal imaging and functional exam findings in a case of combined Stargardt disease and idiopathic intracranial hypertension. METHODS: The patient was evaluated with multimodal imaging including color fundus photography, short wavelength autofluorescence, spectral domain optical coherence tomography as well as functional testing such as Humphrey visual fields and full-field electroretinogram. RESULTS: A 35-year-old woman was referred for evaluation of bilateral transient visual obscurations over the course of 2 months. Optic disc edema was observed in both eyes as well as a bulls-eye maculopathy with pisciform flecks. Magnetic resonance imaging and subsequent lumbar puncture confirmed a diagnosis of idiopathic intracranial hypertension. Fundus autofluorescence demonstrated hyperautofluorescent flecks surrounding both the macula and the disc. Genetic testing and full-field electroretinogram confirmed a diagnosis of Stargardt disease. Notably, the peripapillary retina was not spared as is frequently seen in Stargardt disease, possibly due to the impact of disc edema in the area. The patient was treated with increasing doses of acetazolamide and familial testing for Stargardt disease was recommended. CONCLUSION: Both Stargardt disease and idiopathic intracranial hypertension are separately rare diseases with common anatomic intersection at the peripapillary retina. Stargardt disease typically spares the peripapillary retina; however, in the present case shows some evidence of peripapillary involvement. This finding suggests some relationship between disc edema due to idiopathic intracranial hypertension and the natural history of Stargardt disease.

RBP3-Retinopathy-Inherited High Myopia and Retinal Dystrophy: Genetic Characterization, Natural History, and Deep Phenotyping.

PURPOSE: To examine the genetic and clinical features and the natural history of RBP3-associated retinopathy. DESIGN: Multi-center international, retrospective, case series of adults and children, with moleculraly confirmed RBP3-asociated retinopathy. METHODS: The genetic, clinical, and retinal imaging findings, including optical coherence tomography (OCT) and fundus autofluorescence (FAF), were investigated both cross-sectionally and longitudinally. The results of international standard full-field electroretinography (ERG) and pattern electroretinography (PERG) were reviewed. RESULTS: We ascertained 12 patients (5 female and 7 male) from 10 families (4 patients previously reported). Ten novel disease-causing RBP3 variants were identified. Ten patients were homozygous. The mean age (±SD, range) of the group was 21.4 years (±19.1, 2.9-60.5 years) at baseline evaluation. All 12 patients were highly myopic, with a mean spherical equivalent of -16.0D (range, -7.0D to -33.0D). Visual acuity was not significantly different between eyes, and no significant anisometropia was observed. Mean best-corrected visual acuity (BCVA) was 0.48 logMAR (SD, ±0.29; range, 0.2-1.35 logMAR); at baseline. Eleven patients had longitudinal BCVA assessment, with a mean BCVA of 0.46 logMAR after a mean follow-up of 12.6 years. All patients were symptomatic with reduced VA and myopia by the age of 7 years old. All patients had myopic fundi and features in keeping with high myopia on OCT, including choroidal thinning. The 4 youngest patients had no fundus pigmentary changes, with the rest of the patients presenting with a variable degree of mid-peripheral pigmentation and macular changes. FAF showed variable phenotypes, ranging from areas of increased signal to advanced atrophy in older patients. OCT showed cystoid macular edema at presentation in 3 patients, which persisted during follow-up in 2 patients and resolved to atrophy in the third patient. The ERGs were abnormal in 9 of 9 cases, revealing variable relative involvement of rod and cone photoreceptors with additional milder dysfunction post-phototransduction in some. All but 1 patient had PERG evidence of macular dysfunction, which was severe in most cases. CONCLUSIONS: This study details the clinical and functional phenotype of RBP3-retinopathy in the largest cohort reported to date. RBP3-retinopathy is a disease characterized by early onset, slow progression over decades, and high myopia. The phenotypic spectrum and natural history as described herein has prognostic and counseling implications. RBP3-related disease should be considered in children with high myopia and retinal dystrophy.

A novel pathogenic CRB1 variant presenting as Leber Congenital Amaurosis 8 and evaluation of gene editing feasibility.

INTRODUCTION: Leber Congenital Amaurosis (LCA) is an inherited retinal disease that presents in infancy with severely decreased vision, nystagmus, and extinguished electroretinography findings. LCA8 is linked to variants in the Crumbs homolog 1 (CRB1) gene. CASE DESCRIPTION: We report a novel CRB1 variant in a 14-year-old male presenting with nystagmus, worsening vision, and inability to fixate on toys in his infancy. Color fundus photography revealed nummular pigments in the macula and periphery. Imaging studies revealed thickened retina on standard domain optical coherence tomography and widespread atrophy of the retinal pigment epithelium on autofluorescence. Full-field electroretinography revealed extinguished scotopic and significantly reduced photopic responses. Genetic testing demonstrated a novel homozygous variant, c.3057 T > A; p.(Tyr1019Ter), in the CRB1 gene. This variant is not currently amenable to base editing, however, in silico analysis revealed several potential prime editing strategies for correction. CONCLUSION: This case presentation is consistent with LCA8, suggesting pathogenicity of this novel variant and expanding our knowledge of disease-causing CRB1 variants.

Primary versus Secondary Elevations in Fundus Autofluorescence.

The method of quantitative fundus autofluorescence (qAF) can be used to assess the levels of bisretinoids in retinal pigment epithelium (RPE) cells so as to aid the interpretation and management of a variety of retinal conditions. In this review, we focused on seven retinal diseases to highlight the possible pathways to increased fundus autofluorescence. ABCA4- and RDH12-associated diseases benefit from known mechanisms whereby gene malfunctioning leads to elevated bisretinoid levels in RPE cells. On the other hand, peripherin2/RDS-associated disease (PRPH2/RDS), retinitis pigmentosa (RP), central serous chorioretinopathy (CSC), acute zonal occult outer retinopathy (AZOOR), and ceramide kinase like (CERKL)-associated retinal degeneration all express abnormally high fundus autofluorescence levels without a demonstrated pathophysiological pathway for bisretinoid elevation. We suggest that, while a known link from gene mutation to increased production of bisretinoids (as in ABCA4- and RDH12-associated diseases) causes primary elevation in fundus autofluorescence, a secondary autofluorescence elevation also exists, where an impairment and degeneration of photoreceptor cells by various causes leads to an increase in bisretinoid levels in RPE cells.

Venous Tortuosity in COL4A2-Associated Gould Syndrome.

Mutations in collagen-encoding genes have been linked to numerous systemic diseases. Specifically, pathologic alterations in COL4A2 have been linked to Gould syndrome, a hereditary angiopathy affecting the brain, kidneys, and eyes. However, the ocular phenotype associated with COL4A2-associated disease has yet to be fully characterized. In this report, we describe a novel variant in COL4A2 identified in a 48-year-old woman and her 15-year-old daughter. Funduscopic examination demonstrated significant venous and arteriolar tortuosity. Genetic testing revealed a novel variant, c.2321G>A:p.(Gly774Glu), in COL4A2. This vascular phenotype is similar to the familial retinal arterial tortuosity seen in COL4A2-associated Gould syndrome with additional venous involvement. [Ophthalmic Surg Lasers Imaging Retina 2023;54:536-539.].

Prime Editing Strategy to Install the PRPH2 c.828+1G>A Mutation.

Mutations in peripherin 2 (PRPH2) are associated with a spectrum of inherited retinal diseases (IRDs) including retinitis pigmentosa (RP) and macular degeneration. As PRPH2 is localized to cone and rod outer segments, mutations in PRPH2 lead the disorganization or absence of photoreceptor outer segments. Here, we report on a patient with PRPH2-linked RP who exhibited widespread RPE atrophy with a central area of macular atrophy sparing the fovea. In future studies, we plan to model the pathobiology of PRPH2-based RP using induced pluripotent stem cell (iPSC)-derived retinal organoids. To effectively model rare mutations using iPSC-derived retinal organoids, we first require a strategy that can install the desired mutation in healthy wild-type iPSC, which can efficiently generate well-laminated retinal organoids. In this study, we developed an efficient prime editing strategy for the installation of the pathogenic PRPH2 c.828+1 G>A splice-site mutation underlying our patient's disease.

Generation of an Avian Myeloblastosis Virus (AMV) Reverse Transcriptase Prime Editor.

Prime editing (PE) is a novel, double-strand break (DSB)-independent gene editing technology that represents an exciting avenue for the treatment of inherited retinal diseases (IRDs). Given the extensive and heterogenous nature of the 280 genes associated with IRDs, genome editing has presented countless complications. However, recent advances in genome editing technologies have identified PE to have tremendous potential, with the capability to ameliorate small deletions and insertions in addition to all twelve possible transition and transversion mutations. The current PE system is based on the fusion of the Streptococcus pyogenes Cas9 (SpCas9) nickase H840A mutant and an optimized Moloney murine leukemia virus (MMLV) reverse-transcriptase (RT) in conjunction with a PE guide RNA (pegRNA). In this study, we developed a prime editor based on the avian myeloblastosis virus (AMV)-RT and showed its applicability for the installation of the PRPH2 c.828+1G>A mutation in HEK293 cells.

Analysis of CRB1 Pathogenic Variants Correctable with CRISPR Base and Prime Editing.

The mouse and human retina contain three major Crumbs homologue-1 (CRB1) isoforms. CRB1-A and CRB1-B have cell-type-specific expression patterns making the choice of gene augmentation strategy unclear. Gene editing may be a viable alternative for the amelioration of CRB1-associated retinal degenerations. To assess the prevalence and spectrum of CRB1-associated pathogenic variants amenable to base and prime editing, we carried out an analysis of the Leiden Open Variation Database. Editable variants accounted for 54.5% for base editing and 99.8% for prime editing of all CRB1 pathogenic variants in the Leiden Open Variation Database. The 10 most common editable pathogenic variants for CRB1 accounted for 34.95% of all pathogenic variants, with the c.2843G>A, p.(Cys948Tyr) being the most common editable CRB1 variant. These findings outline the next step toward developing base and prime editing therapeutics as an alternative to gene augmentation for the amelioration of CRB1-associated retinal degenerations.

Rod Photoreceptor-Specific Ablation of Metformin Target, AMPK, in a Preclinical Model of Autosomal Recessive Retinitis Pigmentosa.

Retinal gene therapies have shown tremendous progress in the past decade, but the sheer number of disease-causing mutations makes their applicability challenging. In this study we test our hypothesis that retinitis pigmentosa-associated retinal degeneration can be prevented through AMP-activated protein kinase (AMPK)-associated metabolic pathway reprogramming using a gene-independent model of degeneration and rescue. We show that recue of photoreceptor structure and function is not achieved through our model of metabolic reprogramming. These results suggest that RP may not be treatable through AMPK pathway modulation-based therapies.

Generation of CRB1 RP Patient-Derived iPSCs and a CRISPR/Cas9-Mediated Homology-Directed Repair Strategy for the CRB1 c.2480G>T Mutation.

Mutations in the Crumbs-homologue-1 (CRB1) gene lead to a spectrum of severe inherited retinal diseases, including retinitis pigmentosa (RP). The establishment of a genotype-phenotype correlation in CRB1 patients has been difficult due to the substantial variability and phenotypic overlap between CRB1-associated diseases. This phenotypic modulation may be due to several factors, including genetic modifiers, deep intronic mutations, isoform diversity, and copy number variations. Induced pluripotent stem cell (iPSC)-derived patient retinal organoids are novel tools that can provide sensitive, quantitative, and scalable phenotypic assays. CRB1 RP patient iPSC-derived retinal organoids have shown reproducible phenotypes compared to healthy retinal organoids. However, having genetically defined iPSC isogenic controls that take into account potential phenotypic modulation is crucial. In this study, we generated iPSC from an early-onset CRB1 patient and developed a correction strategy for the c.2480G>T, p.(Gly827Val) CRB1 mutation using CRISPR/Cas9-mediated homology-directed repair.

A New Preclinical Model of Retinitis Pigmentosa Due to Pde6g Deficiency.

Purpose: Retinitis pigmentosa (RP) is the most common cause of inherited blindness, with onset occurring as early as 4 years of age in certain rare but severe forms caused by mutations in the gamma subunit of phosphodiesterase 6 (PDE6). Studies in humans and mice have shown that RP pathology begins with progressive photoreceptor death, which then drives changes in downstream neurons, neighboring retinal pigment epithelium (RPE), and vasculature. Here, we present the first detailed analysis of RP disease progression in Pde6g-deficient mice. Design: Experimental study of an RP mouse model. Subjects: We studied Pde6g-/- and Pde6g+/- mice at the age of 7, 16, 30, 44, and 56 days with n = 2 to 5 per group and time point. Methods: Photoreceptor degeneration and retinal remodeling were analyzed in retinal sections by immunofluorescence. Retinal blood vessel degradation was analyzed in flat-mounted retinas immunolabeled for isolectin GS-IB4. Protein expression was measured by immunoblot. Acellular capillaries were assessed in trypsin-digested and hematoxylin-eosin-stained retinas at postnatal day (P) 44. Retinal pigment epithelium cells were delineated in flat-mounted RPE-choroid-sclera by immunolabeling for the cell-adhesion protein ß-catenin. Main Outcome Measures: Immunofluorescence and morphometry (quantitative analysis of outer nuclear layer, dendrite area, vessel area, acellular vessels, RPE cell size, number of nuclei per RPE cell, RPE cell eccentricity, and RPE cell solidity). Results: This novel RP model exhibits early onset and rapid rod degeneration, with the vast majority gone by P16. This pathology leads to retinal remodeling, including changes of inner retinal neurons, early activation of glia cells, degradation of retinal vasculature, and structural abnormalities of the RPE. Conclusions: The pathology in our Pde6g-/- mouse model precisely mirrors human RP progression. The results demonstrate the significant role of the gamma subunit in maintaining phosphodiesterase activity and provide new insights into the disease progression due to Pde6g deficiency. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Spatiotemporal control of genome engineering in cone photoreceptors.

BACKGROUND: Cones are essential for color recognition, high resolution, and central vision; therefore cone death causes blindness. Understanding the pathophysiology of each cell type in the retina is key to developing therapies for retinal diseases. However, studying the biology of cone cells in the rod-dominant mammalian retina is particularly challenging. In this study, we used a bacterial artificial chromosome (BAC) recombineering method to knock in the "CreERT2" sequence into the Gnat2 and Arr3 genes, respectively and generated three novel inducible CreERT2 mice with different cone cell specificities. RESULTS: These models (Gnat2CreERT2, Arr3T2ACreERT2, and Arr3P2ACreERT2) express temporally controllable Cre recombinase that achieves conditional alleles in cone photoreceptors. Cre-LoxP recombination can be induced as early as postnatal day (PD) two upon tamoxifen injection at varying efficiencies, ranging from 10 to 15% in Gnat2CreERT2, 40% in Arr3T2ACreERT2, and 100% in Arr3P2ACreERT2. Notably, knocking in the P2A-CreERT2 cassette does not affect cone cell morphology and functionality. Most cone-phototransduction enzymes, including Opsins, CNGA3, etc. are not altered except for a reduction in the Arr3 transcript. CONCLUSIONS: The Arr3P2ACreERT2 mouse, an inducible cone-specific Cre driver, is a valuable line in studying cone cell biology, function, as well as its relationship with rod and other retinal cells. Moreover, the Cre activity can be induced by delivering tamoxifen intragastrically as early as PD2, which will be useful for studying retinal development or in rapid degenerative mouse models.

Translatability barriers between preclinical and clinical trials of AAV gene therapy in inherited retinal diseases.

Inherited retinal diseases (IRDs) are progressive degenerative diseases which cause gradual vision loss or complete blindness. As over 270 gene mutations have been identified in the underlying pathology of IRDs, gene therapy as a treatment modality has been an increasingly active realm of investigation. Currently, the most common vehicle of ocular gene delivery is the adeno-associated virus (AAV) vector. This is injected into the immune-privileged subretinal space to mediate transgene expression in retinal cells. Although numerous animal models of IRDs have demonstrated successful outcomes following AAV-mediated gene delivery, many of these studies fail to translate into successful outcomes in clinical trials. The purpose of this review is to A) comparatively assess preclinical and clinical IRD trials in which the success of AAV-mediated therapy failed to translate between animal and human participants B) discuss factors which may complicate the translatability of gene therapy in animals to results in humans.

CRISPR editing demonstrates rs10490924 raised oxidative stress in iPSC-derived retinal cells from patients with ARMS2/HTRA1-related AMD.

Genome-wide association studies (GWAS) have identified genetic risk loci for age-related macular degeneration (AMD) on the chromosome 10q26 (Chr10) locus and are tightly linked: the A69S (G>T) rs10490924 single-nucleotide variant (SNV) and the AATAA-rich insertion-deletion (indel, del443/ins54), which are found in the age-related maculopathy susceptibility 2 (ARMS2) gene, and the G512A (G>A) rs11200638 SNV, which is found in the high-temperature requirement A serine peptidase 1 (HTRA1) promoter. The fourth variant is Y402H complement factor H (CFH), which directs CFH signaling. CRISPR manipulation of retinal pigment epithelium (RPE) cells may allow one to isolate the effects of the individual SNV and thus identify SNV-specific effects on cell phenotype. Clustered regularly interspaced short palindromic repeats (CRISPR) editing demonstrates that rs10490924 raised oxidative stress in induced pluripotent stem cell (iPSC)-derived retinal cells from patients with AMD. Sodium phenylbutyrate preferentially reverses the cell death caused by ARMS2 rs10490924 but not HTRA1 rs11200638. This study serves as a proof of concept for the use of patient-specific iPSCs for functional annotation of tightly linked GWAS to study the etiology of a late-onset disease phenotype. More importantly, we demonstrate that antioxidant administration may be useful for reducing reactive oxidative stress in AMD, a prevalent late-onset neurodegenerative disorder.

Monitoring Lesion Area Progression in Stargardt Disease: A Comparison of En Face Optical Coherence Tomography and Fundus Autofluorescence.

Purpose: To compare longitudinal changes in en face spectral domain-optical coherence tomography (SD-OCT) measurements of ellipsoid zone (EZ) and retinal pigment epithelium (RPE) loss to changes in the hypoautofluorescent and hyperautofluorescent (AF) areas detected with short-wavelength (SW)-AF in ABCA4-associated retinopathy. Methods: SD-OCT volume scans were obtained from 20 patients (20 eyes) over 2.6 ± 1.2 years (range 1-5 years). The EZ, and RPE/Bruch's membrane boundaries were segmented, and en face slab images generated. SubRPE and EZ slab images were used to measure areas of atrophic RPE and EZ loss. These were compared to longitudinal measurements of the hypo- and abnormal AF (hypoAF and surrounding hyperAF) areas. Results: At baseline, the en face area of EZ loss was significantly larger than the subRPE atrophic area, and the abnormal AF area was significantly larger than the hypoAF area. The median rate of EZ loss was significantly greater than the rate of increase in the subRPE atrophic area (1.2 mm2/yr compared to 0.5 mm2/yr). The median rate of increase in the abnormal AF area was significantly greater than the increase in the hypoAF area (1.6 mm2/yr compared to 0.6 mm2/yr). Conclusions: En face SD-OCT can be used to quantify changes in RPE atrophy and photoreceptor integrity. It can be a complementary or alternative technique to SW-AF with the advantage of monitoring EZ loss. The SW-AF results emphasize the importance of measuring changes in the hypo- and abnormal AF areas. Translational Relevance: The findings are relevant to the selection of outcome measures for monitoring ABCA4-associated retinopathy.

Scleral Thickness in Autosomal Dominant Best Vitelliform Macular Dystrophy.

PURPOSE: To investigate the posterior and equatorial scleral thickness in patients with autosomal dominant Best disease, a condition that has chronic subretinal fluid. METHODS: Retrospective study involving patients with Best disease and age-matched controls. Participants were evaluated with contact B-scan ultrasonography and enhanced depth imaging optical coherence tomography to evaluate scleral thickness in the posterior pole and equator. Univariate analysis and generalized estimating equations were used. RESULTS: Of 9 patients with genetically proven Best disease and 23 age-matched controls, there was no significant difference in the age or the gender proportion between groups. Subfoveal choroidal thickness and axial length were not significantly different between groups. Both posterior scleral (OD; 1.38mm vs. 0.89mm, P<.001 and OS; 1.39mm vs. 0.83mm, P<.001) and equatorial scleral (OD; 0.61mm vs. 0.42mm, P=.003, and OS; 0.55mm vs. 0.41mm, P=.017) thicknesses were much greater in cases as compared with controls. Multivariate analysis showed male sex and having Best disease were each significant predictors of posterior scleral thickness and Best disease was the sole significant predictor for equatorial scleral thickness. CONCLUSION: BEST1 gene may have a developmental role leading to having a thicker sclera, influencing disease manifestation, and contributing to the accumulation of subretinal fluid in Best disease.

Development of a translatable gene augmentation therapy for CNGB1-retinitis pigmentosa.

In this study, we investigate a gene augmentation therapy candidate for the treatment of retinitis pigmentosa (RP) due to cyclic nucleotide-gated channel beta 1 (CNGB1) mutations. We use an adeno-associated virus serotype 5 with transgene under control of a novel short human rhodopsin promoter. The promoter/capsid combination drives efficient expression of a reporter gene (AAV5-RHO-eGFP) exclusively in rod photoreceptors in primate, dog, and mouse following subretinal delivery. The therapeutic vector (AAV5-RHO-CNGB1) delivered to the subretinal space of CNGB1 mutant dogs restores rod-mediated retinal function (electroretinographic responses and vision) for at least 12 months post treatment. Immunohistochemistry shows human CNGB1 is expressed in rod photoreceptors in the treated regions as well as restoration of expression and trafficking of the endogenous alpha subunit of the rod CNG channel required for normal channel formation. The treatment reverses abnormal accumulation of the second messenger, cyclic guanosine monophosphate, which occurs in rod photoreceptors of CNGB1 mutant dogs, confirming formation of a functional CNG channel. In vivo imaging shows long-term preservation of retinal structure. In conclusion, this study establishes the long-term efficacy of subretinal delivery of AAV5-RHO-CNGB1 to rescue the disease phenotype in a canine model of CNGB1-RP, confirming its suitability for future clinical development.