RPE65-associated Leber Congenital Amaurosis Research Articles

A novel RPE65 variant p.(Ala391Asp) in Leber congenital amaurosis: a case report and literature review in Japan

IntroductionIn Japan, inherited retinal dystrophy caused by biallelic variants of the RPE65 gene is exceedingly rare. The purpose of this study was to describe a Japanese male patient with a novel variant in RPE65 associated with Leber congenital amaurosis (LCA).Case reportThe patient, diagnosed with LCA, exhibited infantile nystagmus and reported experiencing night blindness since early childhood. At 27 years of age, the patient underwent an ophthalmologically evaluation. Corrected visual acuity was Snellen equivalent 20/133 in the right eye and Snellen equivalent 20/100 in the left eye. Fundus examination revealed alterations in the retinal pigment epithelium characterized by hypopigmentation and narrowing of retinal vessels. Fundus autofluorescence imaging demonstrated a generally diminished autofluorescent signal. Full-field electroretinography identified a generalized dysfunction of both rod and cone systems in each eye. Whole exome sequencing identified a novel missense variant in RPE65 (NM_000329.3): c.1172C > A p.(Ala391Asp), which was classified as pathogenic, as well as a recurrent variant p.(Arg515Trp).ConclusionThis study provides valuable insights into the genotype–phenotype correlation of RPE65-associated LCA in Japanese patients, with critical implications for enhanced diagnostic accuracy and informed therapeutic decisions.

Real-world outcomes of voretigene neparvovec treatment in pediatric patients with RPE65-associated Leber congenital amaurosis.

To investigate real-world safety and efficacy of voretigene neparvovec gene therapy administration in pediatric patients with biallelic RPE65 disease-causing variants. A retrospective study of 27 eyes of 14 patients with RPE65-associated Leber congenital amaurosis examined postoperative complications and longitudinal changes in photoreceptor function following treatment with subretinal injection of voretigene neparvovec. Full-field stimulus threshold testing (FST), Goldmann visual fields (GVF), best-corrected visual acuity (BCVA), and central subfield thickness (CST) on optical coherence tomography (OCT) scans were collected preoperatively and up to 12months posttreatment. Baseline through 6-12month follow-up FST and GVF data were obtained for 13 eyes of 7 patients. FST improved for each eye after treatment with a mean improvement of 2.1 log-units (P < 0.001) and GVF improved for each eye with a mean improvement of 221 sum degrees (P < 0.001). BCVA improved from logMAR 0.98 at baseline to logMAR 0.83 at last follow-up (P < 0.001). Across 19 eyes of 10 patients included in CST analysis, there was a small but statistically significant 9-μ decrease in mean CST from baseline to last follow-up (P < 0.001). The most common postoperative issues included elevation in intraocular pressure (59%), persistent intraocular inflammation (15%), and vitreous opacities (26%) that resolved over a period of months. This report provides some of the earliest longitudinal real-world evidence of the pediatric safety and efficacy of voretigene neparvovec using multiple functional and structural measures of the retina. Outcomes demonstrate significant improvements in visual function consistent with clinical trial results.

A Virtual Reality Orientation and Mobility Test for Inherited Retinal Degenerations: Testing a Proof-of-Concept After Gene Therapy.

PurposeTo test the ability of a virtual reality (VR) orientation and mobility (O&M) protocol to serve a measure of functional vision for patients with inherited retinal degenerations (IRDs).MethodsA VR-O&M protocol designed using a commercially available VR hardware was tested in normally sighted control subjects (n=7; ages 10–35yo; Average 22.5yo) and patients with RPE65-associated Leber Congenital Amaurosis (n=3; ages 7–18yo; Average 12.7yo), in two of them before and after gene therapy. Patients underwent perimetry and full-field sensitivity testing. VR-O&M parameters correlated with the visual dysfunction.ResultsVisual acuities in RPE65 patients were on average worse than 20/200, dark-adapted sensitivity losses >5 log units, and fields constricted between 20° and 40°. Before treatment, patients required ~1000-fold brighter environment to navigate, had at least x4 more collisions, and were slower both to orient and navigate compared to control subjects. Improvements in cone- (by 1–2 L.u.) and rod-mediated (by >4 L.u.) sensitivities post-treatment led to fewer collisions (at least by half) at ~100-fold dimmer luminances, and to x4 times faster navigation times.ConclusionThis study provides proof-of-concept data in support for the use of VR-O&M systems to quantify the impact that the visual dysfunction and improvement of vision following treatments has on functional vision in IRDs. The VR-O&M was useful in potentially challenging scenarios such as in pediatric patients with severe IRDs.Translational RelevanceA VR-O&M test will provide much needed flexibility, both in its deployment as well as in the possibility to test various attributes of vision that may be impacted by gene therapy in the setting of translational studies.PrecisThis study provides proof-of-concept data in support for the use of a virtual reality orientation and mobility test to quantify the impact of the disease and of treatments thereof on functional vision in inherited retinal degenerations.

Introduction to the special issue on Ophthalmic Genetics: Vision in 2020.

In this special issue of the American Journal of Medical Genetics, Part C, we explore the ever-expanding field of Ophthalmic Genetics. The eye is unique among organs for its accessibility to physical examination, permitting exploration of every tissue by slit lamp microscopy, ophthalmoscopy, and imaging including color and autofluorescent photography, ultrasound, optical coherence tomography (OCT), electrophysiology, and adaptive optics confocal and scanning laser ophthalmoscopy. This accessibility permits a variety of surgical and nonsurgical treatments, including the first FDA-approved gene therapy, voretigene neparvovec-rzyl for RPE65-associated Leber Congenital Amaurosis. In this issue, we sought to provide a survey highlighting how heritable ophthalmic disorders are recognizable and accessible to clinical geneticists as well as ophthalmologists.

Generation and Characterization of Induced Pluripotent Stem Cells and Retinal Organoids From a Leber's Congenital Amaurosis Patient With Novel RPE65 Mutations.

RPE65-associated Leber congenital amaurosis (LCA) is one of highly heterogeneous, early onset, severe retinal dystrophies with at least 130 gene mutation sites identified. Their pathogenicity has not been directly clarified due to lack of diseased cells. Here, we generated human-induced pluripotent stem cells (hiPSCs) from one putative LCA patient carrying two novel RPE65 mutations with c.200T>G (p.L67R) and c.430T>C (p.Y144H), named RPE65-hiPSCs, which were confirmed to contain the same mutations. The RPE65-hiPSCs presented typical morphological features with normal karyotype, expressed pluripotency markers, and developed teratoma in NOD-SCID mice. Moreover, the patient hiPSCs were able to differentiate toward retinal lineage fate and self-form retinal organoids with layered neural retina. All major retinal cell types including photoreceptor and retinal pigment epithelium (RPE) cells were also acquired overtime. Compared to healthy control, RPE cells from patient iPSCs had lower expression of RPE65, but similar phagocytic activity and VEGF secretion level. This study provided the valuable patient specific, disease targeted retinal organoids containing photoreceptor and RPE cells, which would facilitate the study of personalized pathogenic mechanisms of disease, drug screening, and cell replacement therapy.

RNA-Based Therapeutic Strategies for Inherited Retinal Dystrophies.

Inherited retinal dystrophies (IRDs) are genetic diseases affecting 1 in every 3000 individuals worldwide. Nowadays, more than 250 genes have been associated with different forms of IRD. In the last decade, it has been shown that gene therapy is a promising approach to correct the genetic defects underlying IRD. In fact, voretigene neparvovec-rzyl (Luxturna™), the first commercialized gene therapy drug to treat RPE65-associated Leber congenital amaurosis, has opened new venues. However, IRDs are highly heterogeneous at genetic level making the design of novel strategies complicated. Unfortunately, the size of several frequently mutated genes is not suitable for the approved conventional therapeutic viral vectors; therefore, there is an urgent need for the development of alternatives, such as those targeting the pre-mRNA. In this mini-review, the potential of RNA-based strategies for IRDs is discussed.

Gene therapy for visual loss: Opportunities and concerns.

Many clinical trials using gene therapy have shown significant therapeutic benefits and exceptional safety records. Increasing evidence is verifying the long sought-after promise that gene therapy will genetically 'cure' some severely disabling diseases. In particular, the first gene therapy bioproduct for RPE65-associated Leber's congenital amaurosis, which was approved by the US Food and Drug Administration in 2017, has provided tremendous encouragement to the field of gene therapy. Recent developments in genome editing technologies have significantly advanced our capability to precisely engineer genomes in eukaryotic cells. Programmable nucleases, particularly the CRISPR/Cas system, have been widely adopted in studies applying genome engineering therapy to ocular diseases with the hope of managing these diseases. In this review article, we summarize the current approaches that have been developed in the area of gene therapy for ocular disease. We also discuss the challenges and opportunities facing gene therapy for ocular diseases, as well as its prospects.

A Cross-Sectional and Longitudinal Study of Retinal Sensitivity in RPE65-Associated Leber Congenital Amaurosis.

PurposeRPE65-associated Leber congenital amaurosis (RPE65-LCA) is an early-onset severe retinal dystrophy associated with progressive visual field loss. Phase I/II and III gene therapy trials have identified improved retinal sensitivity but little is known about the natural history of retinal sensitivity in RPE65-LCA.MethodsA total of 19 subjects (aged 9 to 23 years) undertook monocular full-field static perimetry of which 13 subjects were monitored longitudinally. Retinal sensitivity was measured as mean sensitivity (MS) and volumetrically quantified (in decibel-steradian) using visual field modeling and analysis software for the total (VTOT), central 30° (V30) and central 15° (V15) visual field. Correlation was evaluated between retinal sensitivity and age, best-corrected visual acuity (BCVA), contrast sensitivity, vision-related quality of life, and genotype. Test-retest reliability was also investigated.ResultsV30 was identified to have a strong, weak, and moderate correlation with age, BCVA and contrast sensitivity respectively. Furthermore, V30 was identified as having a weak linear relationship with the mobility and independence domains of the vision-related quality of life questionnaire. Longitudinal analysis demonstrated a slow loss of retinal sensitivity in this cohort. Subjects with at least one RPE65 nonsense variant appeared to show greater progressive loss of retinal sensitivity in the second decade of life than those without.ConclusionsVolumetric assessment of central 30° visual field sensitivity, V30, is a useful independent measure of retinal function and, in our data, represented the best metric to monitor deterioration of retinal sensitivity in RPE65-LCA. Furthermore, functional correlation with genotype may enable more informed prognostic counseling. (ClinicalTrials.gov number, NCT02714816.)

Retinal gene therapy.

Inherited retinal diseases are the leading cause of sight impairment in people of working age in England and Wales, and the second commonest in childhood. Gene therapy offers the potential for benefit. Pubmed and clinicaltrials.gov. Gene therapy can improve vision in RPE65-associated Leber Congenital Amaurosis (RPE65-LCA). Potential benefit depends on efficient gene transfer and is limited by the extent of retinal degeneration. The magnitude of vision improvement from RPE65-LCA gene therapy is suboptimal, and its durability may be limited by progressive retinal degeneration. The safety and potential benefit of gene therapy for inherited and acquired retinal diseases is being explored in a rapidly expanding number of trials. Developments in vector design and delivery will enable greater efficiency and safety of gene transfer. Optimization of trial design will accelerate reliable assessment of outcomes.

Severe Loss of Tritan Color Discrimination in RPE65 Associated Leber Congenital Amaurosis.

PurposeRPE65-associated Leber congenital amaurosis (RPE65-LCA) is a progressive severe retinal dystrophy with early profound dysfunction of rod photoreceptors followed by progressive cone photoreceptor degeneration. We aim to provide detailed information about how cone dysfunction affects color discrimination.MethodsSeven adults (aged 16–21) with RPE65-LCA underwent monocular color discrimination assessment using the Trivector and Ellipse versions of three computerized tests: Cambridge Colour Test (CCT), low vision version of the Cambridge Colour Test (lvvCCT), and the Universal Colour Discrimination Test (UCDT). For comparison, subjects were also tested using the American Optical Hardy Rand Rittler (AO-HRR) plates. Each assessment was repeated three times.ResultsThe Trivector version of the tests demonstrated that color discrimination along the tritan axis was undetectable in four subjects, and severely reduced in three subjects. These findings were confirmed by the Ellipse version of the tests. Color discrimination along the protan and deutan axes was evident but reduced in six of seven subjects. Four of seven subjects were unable to read any of the HRR plates.ConclusionsThe computerized color vision tests adopted in this study provide detailed information about color discrimination in adult RPE65-LCA patients. The condition is associated with severe impairment of color discrimination, particularly along the tritan axis indicating possible early involvement of S-cones, with additional protan and deutan loss to a lesser extent. This psychophysical assessment strategy is likely to be valuable in measuring the impact of therapeutic intervention on cone function.

Safety and Long-Term Efficacy of AAV4 Gene Therapy in Patients with RPE65 Leber Congenital Amaurosis.

The aim of this study was the evaluation of the safety and efficacy of unilateral subretinal injection of the adeno-associated vector (AAV) serotypes 2 and 4 (AAV2/4) RPE65-RPE65 vector in patients with Leber congenital amaurosis (LCA) associated with RPE65 gene deficiency. We evaluated ocular and general tolerance and visual function up to 1 year after vector administration in the most severely affected eye in nine patients with retinal degeneration associated with mutations in the RPE65 gene. Patients received either low (1.22× 1010 to 2× 1010 vector genomes [vg]) or high (between 3.27× 1010 and 4.8× 1010 vg) vector doses. An ancillary study, in which six of the original nine patients participated, extended the follow-up period to 2-3.5 years. All patients showed good ophthalmological and general tolerance to the rAAV2/4-RPE65-RPE65 vector. We observed a trend toward improved visual acuity in patients with nystagmus, stabilization and improvement of the visual field, and cortical activation along visual pathways during fMRI analysis. OCT analysis after vector administration revealed no retinal thinning, except in cases of macular detachment. Our findings show that the rAAV2/4.RPE65.RPE65 vector was well tolerated in nine patients with RPE65-associated LCA. Efficacy parameters varied between patients during follow-up.

Concise Review: Patient-Specific Stem Cells to Interrogate Inherited Eye Disease.

Whether we are driving to work or spending time with loved ones, we depend on our sense of vision to interact with the world around us. Therefore, it is understandable why blindness for many is feared above death itself. Heritable diseases of the retina, such as glaucoma, age-related macular degeneration, and retinitis pigmentosa, are major causes of blindness worldwide. The recent success of gene augmentation trials for the treatment of RPE65-associated Leber congenital amaurosis has underscored the need for model systems that accurately recapitulate disease. With the advent of patient-specific induced pluripotent stem cells (iPSCs), researchers are now able to obtain disease-specific cell types that would otherwise be unavailable for molecular analysis. In the present review, we discuss how the iPSC technology is being used to confirm the pathogenesis of novel genetic variants, interrogate the pathophysiology of disease, and accelerate the development of patient-centered treatments. Significance: Stem cell technology has created the opportunity to advance treatments for multiple forms of blindness. Researchers are now able to use a person's cells to generate tissues found in the eye. This technology can be used to elucidate the genetic causes of disease and develop treatment strategies. In the present review, how stem cell technology is being used to interrogate the pathophysiology of eye disease and accelerate the development of patient-centered treatments is discussed.

687. Therapeutic Correction of an LCA-Causing Splice Defect in the CEP290 Gene by CRISPR/Cas-Mediated Genome Editing

Leber congenital amaurosis (LCA) comprises a genetically heterogeneous group of early-onset retinal disorders characterized by severe loss of vision in the first years of life. Approximately 30% of LCA patients harbor mutations in the CEP290 gene, which codes for a centrosomal protein involved in ciliogenesis and ciliary trafficking in the photoreceptors. While AAV-mediated gene therapy is showing promise in clinical trials for RPE65-associated LCA, the size of CEP290 exceeds the limit for efficient packaging in AAV. Thus, alternative approaches to treat CEP290 associated LCA are needed. Here, we report a gene editing approach in which the CRISPR/Cas9 system is used to modify the endogenous CEP290 locus and restore normal function of the gene.The most common mutation in CEP290 is the IVS26 c. 2991+1655 A>G mutation. This point mutation in intron 26 of the gene generates a novel splice donor, resulting in aberrant splicing and the inclusion of an extra 128bp cryptic exon into the coding sequence of the transcript. Using the S. aureus CRISPR/Cas9 system, we employed a dual-cut approach in which two gRNAs are used to induce a pair of double strand breaks and excise the region of DNA containing the mutation. We screened gRNA pairs in primary fibroblasts derived from LCA patients harboring homozygous IVS26 mutations and identified pairs capable of generating targeted deletions with high efficiency. Using qRT-PCR, we showed that deletion of the mutation results in increased expression of wildtype CEP290 and concomitant decrease in expression of the aberrantly spliced mutant RNA species. Genome-wide specificity profiling allows for assessment of potential off-target modifications induced by the gRNAs and informs selection of optimal gRNA pairs for further study. Assays to assess ciliogenesis, as well as molecular characterization of gene editing in patient-derived photoreceptor precursor cells, demonstrate phenotypic correction in the relevant cell population. The use of the S. aureus CRISPR/Cas9 system enables efficient packaging of the Cas9 gene, as well as two gRNA genes, into a single AAV vector and provides a method for delivery of this system into patient photoreceptors.

S/MAR-containing DNA nanoparticles promote persistent RPE gene expression and improvement in RPE65-associated LCA

Mutations in genes in the retinal pigment epithelium (RPE) cause or contribute to debilitating ocular diseases, including Leber's congenital amaurosis (LCA). Genetic therapies, particularly adeno-associated viruses (AAVs), are a popular choice for monogenic diseases; however, the limited payload capacity of AAVs combined with the large number of retinal disease genes exceeding that capacity make the development of alternative delivery methods critical. Here, we test the ability of compacted DNA nanoparticles (NPs) containing a plasmid with a scaffold matrix attachment region (S/MAR) and vitelliform macular dystrophy 2 (VMD2) promoter to target the RPE, drive long-term, tissue-specific gene expression and mediate proof-of-principle rescue in the rpe65(-/-) model of LCA. We show that the S/MAR-containing plasmid exhibited reporter gene expression levels several fold higher than plasmid or NPs without S/MARs. Importantly, this expression was highly persistent, lasting up to 2 years (last timepoint studied). We therefore selected this plasmid for testing in the rpe65(-/-) mouse model and observe that NP or plasmid VMD2-hRPE65-S/MAR led to structural and functional improvements in the LCA disease phenotype. These results indicate that the non-viral delivery of hRPE65 vectors can result in persistent, therapeutically efficacious gene expression in the RPE.

Clinical gene therapy for the treatment of RPE65-associated Leber congenital amaurosis

Introduction: The positive results of pioneering clinical trials using gene therapy as treatment for patients with Leber congenital amaurosis (LCA) have ushered in a new era of molecular retinal therapeutics for LCA, other blinding retinal disorders and gene therapy applications.Areas covered: This review describes the role of retinal pigment epithelium-specific 65 kDa protein (RPE65) in the visual cycle and how RPE65 deficiency results in LCA; the extensive preclinical studies with recombinant adeno-associated virus (rAAV)-RPE65 gene vectors; and the human rAAV-RPE65 and related gene therapy clinical trials and studies. The literature search included a review of primary sources (e.g., journal articles) that reported study data results and key secondary sources such as meta-reviews available through PubMed, as well as reviews of clinical trial descriptions and results as reported in clinicaltrials.gov, conference publications and news releases.Expert opinion: LCA-RPE65 gene therapy is an example of successful, innovative, translational research. Further research is needed regarding how retinal gene therapy can be improved.

Leber congenital amaurosis due to RPE65 mutations and its treatment with gene therapy

Leber congenital amaurosis (LCA) is a rare hereditary retinal degeneration caused by mutations in more than a dozen genes. RPE65, one of these mutated genes, is highly expressed in the retinal pigment epithelium where it encodes the retinoid isomerase enzyme essential for the production of chromophore which forms the visual pigment in rod and cone photoreceptors of the retina. Congenital loss of chromophore production due to RPE65-deficiency together with progressive photoreceptor degeneration cause severe and progressive loss of vision. RPE65-associated LCA recently gained recognition outside of specialty ophthalmic circles due to early success achieved by three clinical trials of gene therapy using recombinant adeno-associated virus (AAV) vectors. The trials were built on multitude of basic, pre-clinical and clinical research defining the pathophysiology of the disease in human subjects and animal models, and demonstrating the proof-of-concept of gene (augmentation) therapy. Substantial gains in visual function of clinical trial participants provided evidence for physiologically relevant biological activity resulting from a newly introduced gene. This article reviews the current knowledge on retinal degeneration and visual dysfunction in animal models and human patients with RPE65 disease, and examines the consequences of gene therapy in terms of improvement of vision reported.

From the Laboratory to the Clinic: Molecular Genetic Testing in Pediatric Ophthalmology

To review the current state of molecular genetic testing as it relates to pediatric ophthalmology and to discuss its uses. Review and evaluation of available molecular genetic testing. Literature review and discussion of testing in practice based on the authors' clinical and laboratory experience. Fee-for-service testing for many genetic eye diseases now is available. A report is always generated for fee-for-service testing. Detection of DNA variants in genes known to cause eye disease must be interpreted taking into account the variability of the human genome, the presence of benign variants (polymorphisms), and the carrier frequency of recessive alleles. Negative results in genetic testing are helpful in some disorders for which most of the causative genes are known and many disease-causing variants have already been reported, but are less helpful in those that currently have many undiscovered causative genes or novel mutations. Research-based testing also is available, but does not always yield a result. Patients with RPE65-associated Leber congenital amaurosis may be eligible for the current gene therapy trial. Patients with a variety of disorders may benefit from improved surveillance if their genetic diagnosis is known. Entry into the genetic testing system often is via the patient's ophthalmologist. Collaboration with geneticists and genetic counselors, use of web sites to keep up with the ever-changing availability and detection rates, and knowledge of clinical trials, when combined with excellent clinical diagnosis, can improve diagnosis and allow eligible patients to participate in treatment trials.

Safety of Recombinant Adeno-Associated Virus Type 2–RPE65 Vector Delivered by Ocular Subretinal Injection

AAV2 delivery of the RPE65 gene to the retina of blind RPE65-deficient animals restores vision. This strategy is being considered for human trials in RPE65-associated Leber congenital amaurosis (LCA), but toxicity and dose efficacy have not been defined. We studied ocular delivery of AAV-2/2.RPE65 in RPE65-mutant dogs. There was no systemic toxicity. Ocular examinations showed mild or moderate inflammation that resolved over 3 months. Retinal histopathology indicated that traumatic lesions from the injection were common, but thinning within the injection region occurred only at the two highest vector doses. Biodistribution studies at 3 months postinjection showed no vector in optic nerve or visual centers in the brain and only isolated non-dose-related detection in other organs. We also performed biodistribution studies in normal rats at about 2 weeks and 2 months postinjection and vector was not widespread outside the injected eye. Dose–response results in RPE65-mutant dogs indicated that the highest 1.5-log unit range of vector doses proved efficacious. The efficacy and toxicity limits defined in this study lead to suggestions for the design of a subretinal AAV-2/2.RPE65 human trial of RPE65-associated LCA.

881. Safety, Efficacy and Biodistribution of Recombinant AAV2-RPE65 Vector Delivered by Ocular Subretinal Injection

Top of pageAbstract Introduction: AAV2 delivery of the RPE65 cDNA to the retina of blind RPE65-deficient dogs or rd12 mice restores vision as determined electrophysiologically and behaviorally. This strategy is being considered for human trials in RPE65-associated Leber congenital amaurosis, but safety and dose-efficacy within a non-toxic range with this vector have not been defined. Here we report the results of toxicology and biodistribution studies in two mammalian species, dogs and rats. Methods: Vector related pathology and spread after subretinal delivery of AAV2-CBA-RPE65 was studied in RPE65-mutant dogs and normal Spague-Dawley rats. Doses of vector delivered bracketed those intended for the clinical study. Results: There was no systemic toxicity at any vector dose in either species. In dogs ocular examinations showed mild or moderate inflammation that resolved over an initial 3-month period. Retinal histopathology indicated that traumatic lesions from the injection were common, but retinal thinning within the injection region only occurred at the highest vector doses. Biodistribution studies were also performed in RPE65-mutant dogs at various times after vector injection and in normal rats at about 2 weeks and 2 months post-injection. Vector DNA was not widespread outside the injected eye with blood and gonadal tissue consistently negative. Concomitant dose-response results in the RPE65-mutant dogs indicated that the highest 1.5 log unit range of vector doses was efficacious. Conclusions: Given the efficacy and toxicity limits defined in this study, a range for safe vector dose escalation of subretinal AAV2-CBA-RPE65 is suggested for initial human trials.