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.

Treatment Potential for LCA5-Associated Leber Congenital Amaurosis.

Purpose: To determine the therapeutic window for gene augmentation for Leber congenital amaurosis (LCA) associated with mutations in LCA5. Methods: Five patients (ages 6-31) with LCA and biallelic LCA5 mutations underwent an ophthalmic examination including optical coherence tomography (SD-OCT), full-field stimulus testing (FST), and pupillometry. The time course of photoreceptor degeneration in the Lca5gt/gt mouse model and the efficacy of subretinal gene augmentation therapy with AAV8-hLCA5 delivered at postnatal day 5 (P5) (early, n = 11 eyes), P15 (mid, n = 14), and P30 (late, n = 13) were assessed using SD-OCT, histologic study, electroretinography (ERG), and pupillometry. Comparisons were made with the human disease. Results: Patients with LCA5-LCA showed a maculopathy with detectable outer nuclear layer (ONL) in the pericentral retina and at least 4 log units of dark-adapted sensitivity loss. The Lca5gt/gt mouse has a similarly severe and rapid photoreceptor degeneration. The ONL became progressively thinner and was undetectable by P60. Rod- and cone-mediated ERGs were severely reduced in amplitudes at P30 and became nondetectable by P60. Subretinal AAV8-hLCA5 administered to Lca5gt/gt mice at P5 and P15, but not at P30, resulted in structural and functional rescue. Conclusions: LCA5-LCA is a particularly severe form of LCA that was recapitulated in the Lca5gt/gt mouse. Gene augmentation resulted in structural and functional rescue in the Lca5gt/gt mouse if delivered before P30. Retained photoreceptors were visible within the central retina in all patients with LCA5-LCA, at a level equivalent to that observed in rescued Lca5gt/gt mice, suggesting a window of opportunity for the treatment of patients with LCA5-LCA.

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.

FREQUENT SUBCLINICAL MACULAR CHANGES IN COMBINED BRAF/MEK INHIBITION WITH HIGH-DOSE HYDROXYCHLOROQUINE AS TREATMENT FOR ADVANCED METASTATIC BRAF MUTANT MELANOMA: Preliminary Results From a Phase I/II Clinical Treatment Trial.

PURPOSE: To assess the potential ocular toxicity of a combined BRAF inhibition (BRAFi) + MEK inhibition (MEKi) + hydroxychloroquine (HCQ) regime used to treat metastatic BRAF mutant melanoma. METHODS: Patients with stage IV metastatic melanoma and BRAF V600E mutations (n = 11, 31-68 years of age) were included. Treatment was with oral dabrafenib, 150 mg bid, trametinib, 2 mg/day, and HCQ, 400 mg to 600 mg bid. An ophthalmic examination, spectral domain optical coherence tomography, near-infrared and short-wavelength fundus autofluorescence, and static perimetry were performed at baseline, 1 month, and q/6 months after treatment. RESULTS: There were no clinically significant ocular events; there was no ocular inflammation. The only medication-related change was a separation of the photoreceptor outer segment tip from the apical retinal pigment epithelium that could be traced from the fovea to the perifoveal retina noted in 9/11 (82%) of the patients. There were no changes in retinal pigment epithelium melanization or lipofuscin content by near-infrared fundus autofluorescence and short-wavelength fundus autofluorescence, respectively. There were no inner retinal or outer nuclear layer changes. Visual acuities and sensitivities were unchanged. CONCLUSION: BRAFi (trametinib) + MEKi (dabrafenib) + HCQ causes very frequent, subclinical separation of the photoreceptor outer segment from the apical retinal pigment epithelium without inner retinal changes or signs of inflammation. The changes suggest interference with the maintenance of the outer retinal barrier and/or phagocytic/pump functions of the retinal pigment epithelium by effective MEK inhibition.