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.

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.

ENHANCED S-CONE SYNDROME: VISUAL FUNCTION, CROSS-SECTIONAL IMAGING, AND CELLULAR STRUCTURE WITH ADAPTIVE OPTICS OPHTHALMOSCOPY.

PURPOSE: To describe in detail the phenotype of a patient with enhanced S-cone syndrome. METHODS: We describe a 13-year-old boy who presented with blurred vision, vitreous cells, cystoid macular edema refractory to steroid treatment, and a negative uveitic workup. The patient underwent a complete ophthalmic examination, full-field electroretinograms (ffERG), automatic static perimetry and multimodal imaging with spectral domain optical coherence tomography, and adaptive optics scanning laser ophthalmoscopy (AOSLO). RESULTS: Spectral domain optical coherence tomography demonstrated cystoid macular edema and a hyperthick, delaminated midperipheral retina. Fluorescein angiography did not demonstrate macular leakage. Rod-mediated ffERGs were undetectable, and there was a supernormal response to short-wavelength stimuli compared with photopically matched longer wavelengths of light consistent with enhanced S-cone syndrome. Gene screening was positive for compound heterozygous mutations NR2E3: a known (c.119-2 A>C) and a novel (c.119-1G>A) mutation. By perimetry, sensitivities were normal or above normal for short-wavelength stimuli; there was no detectable rod-mediated vision. AOSLO demonstrated higher than normal cone densities in the perifoveal retina and evidence for smaller outer segment cone diameters. CONCLUSION: Evidence for supernumerary cones (at least twice the normal complement) by AOSLO and spectral domain optical coherence tomography was associated with supernormal S-cone sensitivities and electroretinogram responses confirming previous in vivo findings in postmortem human specimens. Smaller than normal cones in enhanced S-cone syndrome may represent "hybrid" photoreceptors analogous to the rd7/rd7 murine model of the disease.

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.

AMD-like retinopathy associated with intravenous iron.

Iron accumulation in the retina is associated with the development of age-related macular degeneration (AMD). IV iron is a common method to treat iron deficiency anemia in adults, and its retinal manifestations have not hitherto been identified. To assess whether IV iron formulations can be retina-toxic, we generated a mouse model for iron-induced retinal damage. Male C57BL/6J mice were randomized into groups receiving IV iron-sucrose (+Fe) or 30% sucrose (-Fe). Iron levels in neurosensory retina (NSR), retinal pigment epithelium (RPE), and choroid were assessed using immunofluorescence, quantitative PCR, and the Perls' iron stain. Iron levels were most increased in the RPE and choroid while levels in the NSR did not differ significantly in +Fe mice compared to controls. Eyes from +Fe mice shared histological features with AMD, including Bruch's membrane (BrM) thickening with complement C3 deposition, as well as RPE hypertrophy and vacuolization. This focal degeneration correlated with areas of high choroidal iron levels. Ultrastructural analysis provided further detail of the RPE/photoreceptor outer segment vacuolization and Bruch's membrane thickening. Findings were correlated with a clinical case of a 43-year-old patient who developed numerous retinal drusen, the hallmark of AMD, within 11 months of IV iron therapy. Our results suggest that IV iron therapy may have the potential to induce or exacerbate a form of retinal degeneration. This retinal degeneration shares features with AMD, indicating the need for further study of AMD risk in patients receiving IV iron treatment.

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.

Cone photoreceptor mosaic disruption associated with Cys203Arg mutation in the M-cone opsin.

Missense mutations in the cone opsins have been identified as a relatively common cause of red/green color vision defects, with the most frequent mutation being the substitution of arginine for cysteine at position 203 (C203R). When the corresponding cysteine is mutated in rhodopsin, it disrupts proper folding of the pigment, causing severe, early onset retinitis pigmentosa. While the C203R mutation has been associated with loss of cone function in color vision deficiency, it is not known what happens to cones expressing this mutant opsin. Here, we used high-resolution retinal imaging to examine the cone mosaic in two individuals with genes encoding a middle-wavelength sensitive (M) pigment with the C203R mutation. We found a significant reduction in cone density compared to normal and color-deficient controls, accompanying disruption in the cone mosaic in both individuals, and thinning of the outer nuclear layer. The C203R mosaics were different from that produced by another mutation (LIAVA) previously shown to disrupt the cone mosaic. Comparison of these mosaics provides insight into the timing and degree of cone disruption and has implications for the prospects for restoration of vision loss associated with various cone opsin mutations.

Age-dependent effects of RPE65 gene therapy for Leber's congenital amaurosis: a phase 1 dose-escalation trial.

BACKGROUND: Gene therapy has the potential to reverse disease or prevent further deterioration of vision in patients with incurable inherited retinal degeneration. We therefore did a phase 1 trial to assess the effect of gene therapy on retinal and visual function in children and adults with Leber's congenital amaurosis. METHODS: We assessed the retinal and visual function in 12 patients (aged 8-44 years) with RPE65-associated Leber's congenital amaurosis given one subretinal injection of adeno-associated virus (AAV) containing a gene encoding a protein needed for the isomerohydrolase activity of the retinal pigment epithelium (AAV2-hRPE65v2) in the worst eye at low (1.5 x 10(10) vector genomes), medium (4.8 x 10(10) vector genomes), or high dose (1.5 x 10(11) vector genomes) for up to 2 years. FINDINGS: AAV2-hRPE65v2 was well tolerated and all patients showed sustained improvement in subjective and objective measurements of vision (ie, dark adaptometry, pupillometry, electroretinography, nystagmus, and ambulatory behaviour). Patients had at least a 2 log unit increase in pupillary light responses, and an 8-year-old child had nearly the same level of light sensitivity as that in age-matched normal-sighted individuals. The greatest improvement was noted in children, all of whom gained ambulatory vision. The study is registered with ClinicalTrials.gov, number NCT00516477. INTERPRETATION: The safety, extent, and stability of improvement in vision in all patients support the use of AAV-mediated gene therapy for treatment of inherited retinal diseases, with early intervention resulting in the best potential gain. FUNDING: Center for Cellular and Molecular Therapeutics at the Children's Hospital of Philadelphia, Foundation Fighting Blindness, Telethon, Research to Prevent Blindness, F M Kirby Foundation, Mackall Foundation Trust, Regione Campania Convenzione, European Union, Associazione Italiana Amaurosi Congenita di Leber, Fund for Scientific Research, Fund for Research in Ophthalmology, and National Center for Research Resources.