Gene Editing for CEP290-Associated Retinal Degeneration.

BACKGROUND: CEP290-associated inherited retinal degeneration causes severe early-onset vision loss due to pathogenic variants in CEP290. EDIT-101 is a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing complex designed to treat inherited retinal degeneration caused by a specific damaging variant in intron 26 of CEP290 (IVS26 variant). METHODS: We performed a phase 1-2, open-label, single-ascending-dose study in which persons 3 years of age or older with CEP290-associated inherited retinal degeneration caused by a homozygous or compound heterozygous IVS26 variant received a subretinal injection of EDIT-101 in the worse (study) eye. The primary outcome was safety, which included adverse events and dose-limiting toxic effects. Key secondary efficacy outcomes were the change from baseline in the best corrected visual acuity, the retinal sensitivity detected with the use of full-field stimulus testing (FST), the score on the Ora-Visual Navigation Challenge mobility test, and the vision-related quality-of-life score on the National Eye Institute Visual Function Questionnaire-25 (in adults) or the Children's Visual Function Questionnaire (in children). RESULTS: EDIT-101 was injected in 12 adults 17 to 63 years of age (median, 37 years) at a low dose (in 2 participants), an intermediate dose (in 5), or a high dose (in 5) and in 2 children 9 and 14 years of age at the intermediate dose. At baseline, the median best corrected visual acuity in the study eye was 2.4 log10 of the minimum angle of resolution (range, 3.9 to 0.6). No serious adverse events related to the treatment or procedure and no dose-limiting toxic effects were recorded. Six participants had a meaningful improvement from baseline in cone-mediated vision as assessed with the use of FST, of whom 5 had improvement in at least one other key secondary outcome. Nine participants (64%) had a meaningful improvement from baseline in the best corrected visual acuity, the sensitivity to red light as measured with FST, or the score on the mobility test. Six participants had a meaningful improvement from baseline in the vision-related quality-of-life score. CONCLUSIONS: The safety profile and improvements in photoreceptor function after EDIT-101 treatment in this small phase 1-2 study support further research of in vivo CRISPR-Cas9 gene editing to treat inherited retinal degenerations due to the IVS26 variant of CEP290 and other genetic causes. (Funded by Editas Medicine and others; BRILLIANCE ClinicalTrials.gov number, NCT03872479.).

Vitreoretinal lymphoma presenting as frosted branch angiitis in a patient with diffuse large B-cell lymphoma.

Purpose: To describe the evaluation, diagnosis, and treatment of vitreoretinal lymphoma presenting as frosted branch angiitis in a patient with diffuse large B-cell lymphoma (DLBCL). Observations: A 57-year-old woman with a history of non-Hodgkin lymphoma and recent DLBCL relapse presented with frosted branch angiitis that raised suspicion for an infectious retinitis but was found to be vitreoretinal lymphoma. Conclusions and Importance: This case primarily highlights the importance of considering vitreoretinal lymphoma on the differential diagnosis of etiologies of frosted branch angiitis. Despite suspicion for vitreoretinal lymphoma, it is also important to treat empirically for infectious etiologies of retinitis in cases of frosted branch angiitis. In this case where the diagnosis was ultimately vitreoretinal lymphoma, weekly alternating intravitreal injections of methotrexate and rituximab led to improvement in visual acuity and retinal infiltration.

Adeno-Associated Virus Serotype 2-hCHM Subretinal Delivery to the Macula in Choroideremia: Two-Year Interim Results of an Ongoing Phase I/II Gene Therapy Trial.

PURPOSE: To assess the safety of the subretinal delivery of a recombinant adeno-associated virus serotype 2 (AAV2) vector carrying a human choroideremia (CHM)-encoding cDNA in CHM. DESIGN: Prospective, open-label, nonrandomized, dose-escalation, phase I/II clinical trial. PARTICIPANTS: Fifteen CHM patients (ages 20-57 years at dosing). METHODS: Patients received uniocular subfoveal injections of low-dose (up to 5 × 1010 vector genome [vg] per eye, n = 5) or high-dose (up to 1 × 1011 vg per eye, n = 10) of a recombinant adeno-associated virus serotype 2 (AAV2) vector carrying a human CHM-encoding cDNA (AAV2-hCHM). Patients were evaluated preoperatively and postoperatively for 2 years with ophthalmic examinations, multimodal retinal imaging, and psychophysical testing. MAIN OUTCOME MEASURES: Visual acuity, perimetry (10-2 protocol), spectral-domain OCT (SD-OCT), and short-wavelength fundus autofluorescence (SW-FAF). RESULTS: We detected no vector-related or systemic toxicities. Visual acuity returned to within 15 letters of baseline in all but 2 patients (1 developed acute foveal thinning, and 1 developed a macular hole); the rest showed no gross changes in foveal structure at 2 years. There were no significant differences between intervention and control eyes in mean light-adapted sensitivity by perimetry or in the lateral extent of retinal pigment epithelium relative preservation by SD-OCT and SW-FAF. Microperimetry showed nonsignificant (< 3 standard deviations of the intervisit variability) gains in sensitivity in some locations and participants in the intervention eye. There were no obvious dose-dependent relationships. CONCLUSIONS: Visual acuity was within 15 letters of baseline after the subfoveal AAV2-hCHM injections in 13 of 15 patients. Acute foveal thinning with unchanged perifoveal function in 1 patient and macular hole in 1 patient suggest foveal vulnerability to the subretinal injections. Longer observation intervals will help establish the significance of the minor differences in sensitivities and rate of disease progression observed between intervention and control eyes.

Restoration of Vision and Retinal Responses After Adeno-Associated Virus-Mediated Optogenetic Therapy in Blind Dogs.

Purpose: Optogenetic gene therapy to render remaining retinal cells light-sensitive in end-stage retinal degeneration is a promising strategy for treatment of individuals blind because of a variety of different inherited retinal degenerations. The clinical trials currently in progress focus on delivery of optogenetic genes to ganglion cells. Delivery of optogenetic molecules to cells in the outer neural retina is predicted to be even more advantageous because it harnesses more of the retinal circuitry. However, this approach has not yet been tested in large animal models. For this reason, we evaluated the safety and efficacy of optogenetic therapy targeting remaining diseased cone photoreceptors in the Rcd1 dog model of retinitis pigmentosa. Methods: Imaging and measures of retinal function and functional vision were carried out, as well as terminal studies evaluating multi-electrode array recordings and histology. Results: Animals remained healthy and active throughout the study and showed improved retinal and visual function as assessed by electroretinography and visual-evoked potentials, improved navigational vision, and improved function of cone photoreceptors and the downstream retinal circuitry. Conclusions: The findings demonstrate that an optogenetic approach targeting the outer retina in a blind large animal model can partially restore vision. Translational Relevance: This work has translational relevance because the approach could potentially be extrapolated to treat humans who are totally blind because of retinal degenerative disease.

Short-term Assessment of Subfoveal Injection of Adeno-Associated Virus-Mediated hCHM Gene Augmentation in Choroideremia Using Adaptive Optics Ophthalmoscopy.

Importance: Subretinal injection for gene augmentation in retinal degenerations forcefully detaches the neural retina from the retinal pigment epithelium, potentially damaging photoreceptors and/or retinal pigment epithelium cells. Objective: To use adaptive optics scanning light ophthalmoscopy (AOSLO) to assess the short-term integrity of the cone mosaic following subretinal injections of adeno-associated virus vector designed to deliver a functional version of the CHM gene (AAV2-hCHM) in patients with choroideremia. Design, Setting, and Participants: This longitudinal case series study enrolled adult patients with choroideremia from February 2015 to January 2016 in the US. To be included in the study, study participants must have received uniocular subfoveal injections of low-dose (5 × 1010 vector genome per eye) or high-dose (1 × 1011 vector genome per eye) AAV2-hCHM. Analysis began February 2015. Main Outcomes and Measures: The macular regions of both eyes were imaged before and 1 month after injection using a custom-built multimodal AOSLO. Postinjection cone inner segment mosaics were compared with preinjection mosaics at multiple regions of interest. Colocalized spectral-domain optical coherence tomography and dark-adapted cone sensitivity was also acquired at each time point. Results: Nine study participants ranged in age from 26 to 50 years at the time of enrollment, and all were White men. Postinjection AOSLO images showed preservation of the cone mosaic in all 9 AAV2-hCHM-injected eyes. Mosaics appeared intact and contiguous 1 month postinjection, with the exception of foveal disruption in 1 patient. Optical coherence tomography showed foveal cone outer segment shortening postinjection. Cone-mediated sensitivities were unchanged in 8 of 9 injected and 9 of 9 uninjected eyes. One participant showed acute loss of foveal optical coherence tomography cone outer segment-related signals along with cone sensitivity loss that colocalized with disruption of the mosaic on AOSLO. Conclusions and Relevance: Integrity of the cone mosaic is maintained following subretinal delivery of AAV2-hCHM, providing strong evidence in support of the safety of the injections. Minor foveal thinning observed following surgery corresponds with short-term cone outer segment shortening rather than cone cell loss. Foveal cone loss in 1 participant raises the possibility of individual vulnerability to the subretinal injection.

Clinical Perspective: Treating RPE65-Associated Retinal Dystrophy.

Until recently, there was no approved treatment for a retinal degenerative disease. Subretinal injection of a recombinant adeno-associated virus (AAV) delivering the normal copy of the human RPE65 cDNA led to reversal of blindness first in animal models and then in humans. This led to the first US Food and Drug Administration (FDA)-approved gene therapy product for a genetic disease, voretigene neparvovec-rzyl (Luxturna). Luxturna was then approved by the European Medicines Association and is now available in the US through Spark Therapeutics and worldwide through Novartis. Not only has treatment with Luxturna changed the lives of people previously destined to live a life of blindness, but it has fueled interest in developing additional gene therapy reagents targeting numerous other genetic forms of inherited retinal disease. This review describes many of the considerations for administration of Luxturna and describes how lessons from experience with Luxturna could lead to additional gene-based treatments of blindness.

Cytomegalovirus retinitis in an immunocompetent host after complicated cataract surgery.

PURPOSE: To report a case of an immunocompetent patient who developed cytomegalovirus (CMV) retinitis after complicated cataract surgery resulting in aphakia. OBSERVATIONS: A 67-year-old man with type 2 diabetes developed retinitis six months after cataract surgery that resulted in aphakia. Anterior chamber DNA testing was positive for CMV. Comprehensive systemic work-up revealed no immune insufficiency. The retinitis was successfully treated with intravitreal foscarnet and extended oral valgancyclovir treatment, however, he subsequently developed rhegmatogenous retinal detachment. CONCLUSION AND IMPORTANCE: CMV retinitis may occur in immunocompetent patients in the setting of aphakia and prolonged topical steroid use.

Subretinal Injection of Voretigene Neparvovec-rzyl in a Patient With RPE65-Associated Leber's Congenital Amaurosis.

Leber's congenital amaurosis (LCA) is a rare inherited retinal degeneration (IRD) that causes severe vision loss, nyctalopia, and nystagmus within the first few years of life. RPE65 gene mutations cause approximately 6% of LCA cases and have become the target for therapy since voretigene neparvovec-rzyl became the first U.S. Food and Drug Administration-approved gene therapy product for IRDs in 2017. The surgery involves pars plana vitrectomy with subretinal injection of a viral vector that carries a functional copy of the RPE65 gene. Intraoperative optical coherence tomography is a useful adjunctive tool to confirm the injection has reached the subretinal space.

Safety of Same-Eye Subretinal Sequential Readministration of AAV2-hRPE65v2 in Non-human Primates.

We have demonstrated safe and effective subretinal readministration of recombinant adeno-associated virus serotype (rAAV) to the contralateral eye in large animals and humans even in the setting of preexisting neutralizing antibodies (NAbs). Readministration of AAV to the same retina may be desirable in order to treat additional areas of the retina not targeted initially or to boost transgene expression levels at a later time point. To better understand the immune and structural consequences of subretinal rAAV readministration to the same eye, we administered bilateral subretinal injections of rAAV2-hRPE65v2 to three unaffected non-human primates (NHPs) and repeated the injections in those same eyes 2 months later. Ophthalmic exams and retinal imaging were performed after the first and second injections. Peripheral blood monocytes, serum, and intraocular fluids were collected at baseline and post-injection time points to characterize the cellular and humoral immune responses. Histopathologic and immunohistochemical studies were carried out on the treated retinas. Ipsilateral readministration of AAV2-hRPE65v2 in NHPs did not threaten the ocular or systemic health through the time span of the study. The repeat injections were immunologically and structurally well tolerated, even in the setting of preexisting serum NAbs. Localized structural abnormalities confined to the outer retina and retinal pigmented epithelium (RPE) after readministration of the treatment do not differ from those observed after single or contralateral administration of an AAV carrying a non-therapeutic transgene in NHPs and were not observed in a patient treated with the nearly identical, FDA-approved, AAV2-hRPE65v2 vector (voretigene neparvovec-rzyl), suggesting NHP-specific abnormalities.

Utility of In Vitro Mutagenesis of RPE65 Protein for Verification of Mutational Pathogenicity Before Gene Therapy.

Importance: Next-generation sequencing can detect variants of uncertain significance (VUSs), for some of which gene therapy would not be advantageous. Therefore, the pathogenicity of compound heterozygous or homozygous variants should be confirmed before bilateral vitrectomy and administration of voretigene neparvovec-rzyl. Objective: To describe an in vitro mutagenesis assay for assessing the pathogenicity of variants in the RPE65 gene. Design, Setting, and Participants: This case series was conducted at 2 tertiary referral centers. Clinical history, imaging, and electrophysiologic testing results were reviewed from September 5, 2008, to December 31, 2019. Participants were 4 pediatric patients with Leber congenital amaurosis who were evaluated for or met the inclusion criteria for phase 1 to 3 clinical trials or were referred for voretigene neparvovec-rzyl treatment. Main Outcomes and Measures: A functional assay was used to confirm the pathogenicity of novel RPE65 VUSs in 4 patients with Leber congenital amaurosis. Results: Four patients with Leber congenital amaurosis had VUSs in RPE65. Patients 1 and 2 were siblings with the homozygous VUS c.311G>T p.(G104V). Patient 3 was a compound heterozygote with 1 known pathogenic allele, c.1202_1203insCTGG p.(Glu404AlafsTer4), and 1 VUS, c.311G>T p.(G104V), which segregated to separate alleles. Patient 4 was also a compound heterozygote with 1 pathogenic variant, c.11 + 5G>A, and 1 variant in trans, c.1399C>T p.(P467S). In vitro mutagenesis revealed that the G104V and P467S RPE65 proteins were catalytically inactive (0% isomerase activity). Patients 1 and 2 were excluded from participation in a phase 1 trial owing to high Adeno-associated virus 2 capsid-neutralizing antibodies. Patients 3 (G104V) and 4 (P467S) underwent successful surgical gene therapy with voretigene neparvovec-rzyl, and their response to lower white light intensity and visual field increased in fewer than 30 days after gene therapy intervention. Conclusions and Relevance: Findings from this study suggest that, in patients with missense mutations in RPE65, functional assays of protein function can be performed to assess the pathogenicity of variants in both compound heterozygous and homozygous cases. Given the potential risks of gene therapy operations, in vitro RPE65 activity testing should be considered to avoid the possibility of treating a false genotype.

Efficacy, Safety, and Durability of Voretigene Neparvovec-rzyl in RPE65 Mutation-Associated Inherited Retinal Dystrophy: Results of Phase 1 and 3 Trials.

PURPOSE: To report the durability of voretigene neparvovec-rzyl (VN) adeno-associated viral vector-based gene therapy for RPE65 mutation-associated inherited retinal dystrophy (IRD), including results of a phase 1 follow-on study at year 4 and phase 3 study at year 2. DESIGN: Open-label phase 1 follow-on clinical trial and open-label, randomized, controlled phase 3 clinical trial. PARTICIPANTS: Forty subjects who received 1.5×1011 vector genomes (vg) of VN per eye in at least 1 eye during the trials, including 11 phase 1 follow-on subjects and 29 phase 3 subjects (20 original intervention [OI] and 9 control/intervention [CI]). METHODS: Subretinal injection of VN in the second eye of phase 1 follow-on subjects and in both eyes of phase 3 subjects. MAIN OUTCOME MEASURES: End points common to the phase 1 and phase 3 studies included change in performance on the Multi-Luminance Mobility Test (MLMT) within the illuminance range evaluated, full-field light sensitivity threshold (FST) testing, and best-corrected visual acuity (BCVA). Safety end points included adverse event reporting, ophthalmic examination, physical examination, and laboratory testing. RESULTS: Mean (standard deviation) MLMT lux score change was 2.4 (1.3) at 4 years compared with 2.6 (1.6) at 1 year after administration in phase 1 follow-on subjects (n = 8), 1.9 (1.1) at 2 years, and 1.9 (1.0) at 1 year post-administration in OI subjects (n = 20), and 2.1 (1.6) at 1 year post-administration in CI subjects (n = 9). All 3 groups maintained an average improvement in FST, reflecting more than a 2 log10(cd.s/m2) improvement in light sensitivity at 1 year and subsequent available follow-up visits. The safety profile was consistent with vitrectomy and the subretinal injection procedure, and no deleterious immune responses occurred. CONCLUSIONS: After VN gene augmentation therapy, there was a favorable benefit-to-risk profile with similar improvement demonstrated in navigational ability and light sensitivity among 3 groups of subjects with RPE65 mutation-associated IRD, a degenerative disease that progresses to complete blindness. The safety profile is consistent with the administration procedure. These data suggest that this effect, which is nearly maximal by 30 days after VN administration, is durable for 4 years, with observation ongoing.

Amelioration of Neurosensory Structure and Function in Animal and Cellular Models of a Congenital Blindness.

Most genetically distinct inherited retinal degenerations are primary photoreceptor degenerations. We selected a severe early onset form of Leber congenital amaurosis (LCA), caused by mutations in the gene LCA5, in order to test the efficacy of gene augmentation therapy for a ciliopathy. The LCA5-encoded protein, Lebercilin, is essential for the trafficking of proteins and vesicles to the photoreceptor outer segment. Using the AAV serotype AAV7m8 to deliver a human LCA5 cDNA into an Lca5 null mouse model of LCA5, we show partial rescue of retinal structure and visual function. Specifically, we observed restoration of rod-and-cone-driven electroretinograms in about 25% of injected eyes, restoration of pupillary light responses in the majority of treated eyes, an ∼20-fold decrease in target luminance necessary for visually guided behavior, and improved retinal architecture following gene transfer. Using LCA5 patient-derived iPSC-RPEs, we show that delivery of the LCA5 cDNA restores lebercilin protein and rescues cilia quantity. The results presented in this study support a path forward aiming to develop safety and efficacy trials for gene augmentation therapy in human subjects with LCA5 mutations. They also provide the framework for measuring the effects of intervention in ciliopathies and other severe, early-onset blinding conditions.

Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial.

BACKGROUND: Phase 1 studies have shown potential benefit of gene replacement in RPE65-mediated inherited retinal dystrophy. This phase 3 study assessed the efficacy and safety of voretigene neparvovec in participants whose inherited retinal dystrophy would otherwise progress to complete blindness. METHODS: In this open-label, randomised, controlled phase 3 trial done at two sites in the USA, individuals aged 3 years or older with, in each eye, best corrected visual acuity of 20/60 or worse, or visual field less than 20 degrees in any meridian, or both, with confirmed genetic diagnosis of biallelic RPE65 mutations, sufficient viable retina, and ability to perform standardised multi-luminance mobility testing (MLMT) within the luminance range evaluated, were eligible. Participants were randomly assigned (2:1) to intervention or control using a permuted block design, stratified by age (<10 years and ≥10 years) and baseline mobility testing passing level (pass at ≥125 lux vs <125 lux). Graders assessing primary outcome were masked to treatment group. Intervention was bilateral, subretinal injection of 1·5 × 1011 vector genomes of voretigene neparvovec in 0·3 mL total volume. The primary efficacy endpoint was 1-year change in MLMT performance, measuring functional vision at specified light levels. The intention-to-treat (ITT) and modified ITT populations were included in primary and safety analyses. This trial is registered with ClinicalTrials.gov, number NCT00999609, and enrolment is complete. FINDINGS: Between Nov 15, 2012, and Nov 21, 2013, 31 individuals were enrolled and randomly assigned to intervention (n=21) or control (n=10). One participant from each group withdrew after consent, before intervention, leaving an mITT population of 20 intervention and nine control participants. At 1 year, mean bilateral MLMT change score was 1·8 (SD 1·1) light levels in the intervention group versus 0·2 (1·0) in the control group (difference of 1·6, 95% CI 0·72-2·41, p=0·0013). 13 (65%) of 20 intervention participants, but no control participants, passed MLMT at the lowest luminance level tested (1 lux), demonstrating maximum possible improvement. No product-related serious adverse events or deleterious immune responses occurred. Two intervention participants, one with a pre-existing complex seizure disorder and another who experienced oral surgery complications, had serious adverse events unrelated to study participation. Most ocular events were mild in severity. INTERPRETATION: Voretigene neparvovec gene replacement improved functional vision in RPE65-mediated inherited retinal dystrophy previously medically untreatable. FUNDING: Spark Therapeutics.

Management of Presumed Endogenous Fungal Endophthalmitis in a Child With Acute Lymphoblastic Leukemia.

The authors describe a case of presumed endogenous fungal endophthalmitis in an immunocompetent pediatric patient with acute lymphoblastic leukemia. A 15-year-old boy with a history of high-risk B-cell acute lymphoblastic leukemia status post-chemotherapy presented with acute changes in vision in his left eye. Fundus examination revealed a white bi-lobed chorioretinal lesion with overlying vitritis and associated subretinal fluid. Magnetic resonance imaging of the brain revealed small ring-enhancing lesions in the right parietal and left occipital lobes. Blood, cerebrospinal fluid, aqueous, and vitreous cultures were all negative. Bone marrow and vitreous cytology were negative for malignant cells. The patient was treated for presumed fungal endophthalmitis with systemic and intravitreal voriconazole, followed by pars plana vitrectomy with intravitreal voriconazole and amphotericin B injections. The chorioretinal lesion resolved and visual acuity recovered to 20/20. Chorioretinal infiltrates in a patient with leukemia may require treatment even in the absence of a definitive diagnostic test result. Intervention should be guided by risk analysis and clinical judgment. [J Pediatr Ophthalmol Strabismus. 2017;54:e42-e46.].

The Role of the Human Visual Cortex in Assessment of the Long-Term Durability of Retinal Gene Therapy in Follow-on RPE65 Clinical Trial Patients.

PURPOSE: Gene therapy (GT) has offered immense hope to individuals who are visually impaired because of RPE65 mutations. Although GT has shown great success in clinical trials enrolling these individuals, evidence for stability and durability of this treatment over time is still unknown. Herein we explored the value of functional magnetic resonance imaging (fMRI) as an objective measure to assess independently the longevity of retinal GT. DESIGN: Individuals with RPE65 mutations who underwent GT in their worse-seeing eye in a phase 1 clinical trial received a second subretinal injection in their contralateral eye in a follow-on clinical trial. Functional magnetic resonance imaging (MRI) was performed longitudinally to assess brain responses of patients with RPE65 mutations after stimulation of their most recently treated eye before and 1 to 3 years after GT. PARTICIPANTS: Seven participants with RPE65 mutations who were part of the follow-on clinical trial gave informed consent to participate in a longitudinal neuroimaging fMRI study. METHODS: All participants underwent fMRI using a 3-Tesla MRI system and a 32-channel head coil. Participants' cortical activations were assessed using a block design paradigm of contrast reversing checkerboard stimuli delivered using an MRI-compatible video system. MAIN OUTCOME MEASURES: The primary parameters being measured in this study were the qualitative and quantitative fMRI cortical activations produced by our population in response to the visual task. RESULTS: Functional MRI results showed minimal or no cortical responses before GT. Significant increase in cortical activation lasting at least 3 years after GT was observed for all participants. Repeated measures analysis showed significant associations between cortical activations and clinical measures such as full-field light sensitivity threshold for white, red, and blue colors; visual field; and pupillary light reflex. CONCLUSIONS: Participants with RPE65 mutations showed intact visual pathways, which became responsive and strengthened after treatment. Functional MRI results independently revealed the efficacy and durability of a 1-time subretinal injection. The fMRI results paralleled those recently reported during the long-term clinical evaluations of the same patients. Results from this study demonstrated that fMRI may play an important role in providing complementary information to patients' ophthalmic clinical evaluation and has usefulness as an outcome measure for future retinal intervention studies.

Intravitreal Bevacizumab for the Treatment of Vitreous Hemorrhage Due to Proliferative Diabetic Retinopathy.

PURPOSE: To evaluate the outcomes of intravitreal bevacizumab (IVB) use in patients with a vitreous hemorrhage (VH) secondary to proliferative diabetic retinopathy (PDR). DESIGN: Retrospective, interventional case series. METHODS: Patients who presented to Scheie Eye Institute between January 2008 and January 2015 with a new VH secondary to PDR and treated with IVB were included. Exclusion criteria consisted of IVB treatment prior to the study, a history of pars plana vitrectomy (PPV), and less than 1 year of follow-up. Outcomes of interest were additional treatments including PPV, injections, and panretinal photocoagulation (PRP), as well as visual acuity at baseline and at 1 year. RESULTS: Of the 111 eligible eyes, 55 (49.5%) had PRP, 35 (31.6%) were managed with injections alone, and 21 (18.9%) had PPV after 1 year. The overall average number of injections during this time was 2 (range, 1-9), and 13 (11.7%) eyes were managed with a single injection alone. Of the 69 eyes with 2 years of follow-up, 43 (62.3%) had PRP, 16 (23.2%) were treated with injections alone, and 10 (14.5%) had PPV. CONCLUSIONS: This study underscores the potentially important role that IVB injections have in the management of patients with VH secondary to PDR. The results indicate that a proportion of patients may be treated with a minimal amount of intervention requiring 1 or 2 anti-vascular endothelial growth factor injections only. Also, the rate of PPV at 2 years (27.9%, n = 31) suggests that most patients may be managed nonsurgically.

Evaluation of Dose and Safety of AAV7m8 and AAV8BP2 in the Non-Human Primate Retina.

Within the next decade, we will see many gene therapy clinical trials for eye diseases, which may lead to treatments for thousands of visually impaired people around the world. To target retinal diseases that affect specific cell types, several recombinant adeno-associated virus (AAV) serotypes have been generated and used successfully in preclinical mouse studies. Because there are numerous anatomic and physiologic differences between the eyes of mice and "men" and because surgical delivery approaches and immunologic responses also differ between these species, this study evaluated the transduction characteristics of two promising new serotypes, AAV7m8 and AAV8BP2, in the retinas of animals that are most similar to those of humans: non-human primates (NHPs). We report that while AAV7m8 efficiently targets a variety of cell types by subretinal injection in NHPs, transduction after intravitreal delivery was mostly restricted to the inner retina at lower doses that did not induce an immune response. AAV8BP2 targets the cone photoreceptors efficiently but bipolar cells inefficiently by subretinal injection. Additionally, transduction by both serotypes in the anterior chamber of the eye and the optic pathway of the brain was observed post-intravitreal delivery. Finally, we assessed immunogenicity, keeping in mind that these AAV capsids may be used in future clinical trials. We found that AAV8BP2 had a better safety profile compared with AAV7m8, even at the highest doses administered. These studies underscore the differences in AAV transduction between mice and primates, highlighting the importance of careful evaluation of therapeutic vectors in NHPs prior to moving to clinical trials.

Safety and durability of effect of contralateral-eye administration of AAV2 gene therapy in patients with childhood-onset blindness caused by RPE65 mutations: a follow-on phase 1 trial.

BACKGROUND: Safety and efficacy have been shown in a phase 1 dose-escalation study involving a unilateral subretinal injection of a recombinant adeno-associated virus (AAV) vector containing the RPE65 gene (AAV2-hRPE65v2) in individuals with inherited retinal dystrophy caused by RPE65 mutations. This finding, along with the bilateral nature of the disease and intended use in treatment, prompted us to determine the safety of administration of AAV2-hRPE65v2 to the contralateral eye in patients enrolled in the phase 1 study. METHODS: In this follow-on phase 1 trial, one dose of AAV2-hRPE65v2 (1.5 × 10(11) vector genomes) in a total volume of 300 µL was subretinally injected into the contralateral, previously uninjected, eyes of 11 children and adults (aged 11-46 years at second administration) with inherited retinal dystrophy caused by RPE65 mutations, 1.71-4.58 years after the initial subretinal injection. We assessed safety, immune response, retinal and visual function, functional vision, and activation of the visual cortex from baseline until 3 year follow-up, with observations ongoing. This study is registered with ClinicalTrials.gov, number NCT01208389. FINDINGS: No adverse events related to the AAV were reported, and those related to the procedure were mostly mild (dellen formation in three patients and cataracts in two). One patient developed bacterial endophthalmitis and was excluded from analyses. We noted improvements in efficacy outcomes in most patients without significant immunogenicity. Compared with baseline, pooled analysis of ten participants showed improvements in mean mobility and full-field light sensitivity in the injected eye by day 30 that persisted to year 3 (mobility p=0.0003, white light full-field sensitivity p<0.0001), but no significant change was seen in the previously injected eyes over the same time period (mobility p=0.7398, white light full-field sensitivity p=0.6709). Changes in visual acuity from baseline to year 3 were not significant in pooled analysis in the second eyes or the previously injected eyes (p>0.49 for all time-points compared with baseline). INTERPRETATION: To our knowledge, AAV2-hRPE65v2 is the first successful gene therapy administered to the contralateral eye. The results highlight the use of several outcome measures and help to delineate the variables that contribute to maximal benefit from gene augmentation therapy in this disease. FUNDING: Center for Cellular and Molecular Therapeutics at The Children's Hospital of Philadelphia, Spark Therapeutics, US National Institutes of Health, Foundation Fighting Blindness, Institute for Translational Medicine and Therapeutics, Research to Prevent Blindness, Center for Advanced Retinal and Ocular Therapeutics, Mackall Foundation Trust, F M Kirby Foundation, and The Research Foundation-Flanders.

Detailed functional and structural phenotype of Bietti crystalline dystrophy associated with mutations in CYP4V2 complicated by choroidal neovascularization.

PURPOSE: To describe in detail the phenotype of a patient with Bietti crystalline dystrophy (BCD) complicated by choroidal neovascularization (CNV) and the response to intravitreal Bevacizumab (Avastin®; Genentech/Roche). METHODS: A 34-year-old woman with BCD and mutations in CYP4V2 (c.802-8_806del13/p.H331P:c992A>C) underwent a complete ophthalmic examination, full-field flash electroretinography (ERG), kinetic and two-color dark-adapted perimetry, and dark-adaptometry. Imaging was performed with spectral domain optical coherence tomography (SD-OCT), near infrared (NIR) and short wavelength (SW) fundus autofluorescence (FAF), and fluorescein angiography (FA). RESULTS: Best-corrected visual acuity (BCVA) was 20/20 and 20/60 for the right and left eye, respectively. There were corneal paralimbal crystal-like deposits. Kinetic fields were normal in the peripheral extent. Retinal crystals were most obvious on NIR-reflectance and corresponded with hyperreflectivities within the RPE on SD-OCT. There was parafoveal/perifoveal hypofluorescence on SW-FAF and NIR-FAF. Rod > cone sensitivity loss surrounded fixation and extended to ~10° of eccentricity corresponding to regions of photoreceptor outer segment-retinal pigmented epithelium (RPE) interdigitation abnormalities. The outer nuclear layer was normal in thickness. Recovery of sensitivity following a ~76% rhodopsin bleach was normal. ERGs were normal. A subretinal hemorrhage in the left eye co-localized with elevation of the RPE on SD-OCT and leakage on FA, suggestive of CNV. Three monthly intravitreal injections of Bevacizumab led to restoration of BCVA to baseline (20/25). CONCLUSION: crystals in BCD were predominantly located within the RPE. Photoreceptor outer segment and apical RPE abnormalities underlie the relatively extensive retinal dysfunction observed in relatively early-stage BCD. Intravitreal Bevacizumab was effective in treating CNV in this setting.