Marc C. Peden

Gene Therapy for Leber Congenital Amaurosis Caused by RPE65 Mutations: Safety and Efficacy in 15 Children and Adults Followed Up to 3 Years

OBJECTIVE To determine the safety and efficacy of subretinal gene therapy in the RPE65 form of Leber congenital amaurosis using recombinant adeno-associated virus 2 (rAAV2) carrying the RPE65 gene. DESIGN Open-label, dose-escalation phase I study of 15 patients (range, 11-30 years of age) evaluated after subretinal injection of the rAAV2- RPE65 vector into the worse-functioning eye. Five cohorts represented 4 dose levels and 2 different injection strategies. MAIN OUTCOME MEASURES Primary outcomes were systemic and ocular safety. Secondary outcomes assayed visual function with dark-adapted full-field sensitivity testing and visual acuity with Early Treatment Diabetic Retinopathy Study charts. Further assays included immune responses to the vector, static visual fields, pupillometry, mobility performance, and optical coherence tomography. RESULTS No systemic toxicity was detected; ocular adverse events were related to surgery. Visual function improved in all patients to different degrees; improvements were localized to treated areas. Cone and rod sensitivities increased significantly in the study eyes but not in the control eyes. Minor acuity improvements were recorded in many study and control eyes. Major acuity improvements occurred in study eyes with the lowest entry acuities and parafoveal fixation loci treated with subretinal injections. Other patients with better foveal structure lost retinal thickness and acuity after subfoveal injections. CONCLUSIONS Gene therapy for Leber congenital amaurosis caused by RPE65 mutations is sufficiently safe and substantially efficacious in the extrafoveal retina. There is no benefit and some risk in treating the fovea. No evidence of age-dependent effects was found. Our results point to specific treatment strategies for subsequent phases. APPLICATION TO CLINICAL PRACTICE Gene therapy for inherited retinal disease has the potential to become a future part of clinical practice. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00481546.

Preclinical potency and safety studies of an AAV2-mediated gene therapy vector for the treatment of MERTK associated retinitis pigmentosa.

Abstract Proof of concept for MERTK gene replacement therapy has been demonstrated using different viral vectors in the Royal College of Surgeon (RCS) rat, a well characterized model of recessive retinitis pigmentosa that contains a mutation in the Mertk gene. MERTK plays a key role in renewal of photoreceptor outer segments (OS) by phagocytosis of shed OS tips. Mutations in MERTK cause impaired phagocytic activity and accumulation of OS debris in the interphotoreceptor space that ultimately leads to photoreceptor cell death. In the present study, we conducted a series of preclinical potency and GLP-compliant safety evaluations of an adeno-associated virus type 2 (AAV2) vector expressing human MERTK cDNA driven by the retinal pigment epithelium-specific, VMD2 promoter. We demonstrate the potency of the vector in RCS rats by improved electroretinogram (ERG) responses in treated eyes compared with contralateral untreated controls. Toxicology and biodistribution studies were performed in Sprague-Dawley (SD) rats injected with two different doses of AAV vectors and buffer control. Delivery of vector in SD rats did not result in a change in ERG amplitudes of rod and cone responses relative to balanced salt solution control-injected eyes, indicating that administration of AAV vector did not adversely affect normal retinal function. In vivo fundoscopic analysis and postmortem retinal morphology of the vector-injected eyes were normal compared with controls. Evaluation of blood smears showed the lack of transformed cells in the treated eyes. All injected eyes and day 1 blood samples were positive for vector genomes, and all peripheral tissues were negative. Our results demonstrate the potency and safety of the AAV2-VMD2-hMERTK vector in animal models tested. A GMP vector has been manufactured and is presently in clinical trial.

Ab-Externo AAV-Mediated Gene Delivery to the Suprachoroidal Space Using a 250 Micron Flexible Microcatheter

Background The current method of delivering gene replacement to the posterior segment of the eye involves a three-port pars plana vitrectomy followed by injection of the agent through a 37-gauge cannula, which is potentially wrought with retinal complications. In this paper we investigate the safety and efficacy of delivering adeno-associated viral (AAV) vector to the suprachoroidal space using an ab externo approach that utilizes an illuminated microcatheter. Methods 6 New Zealand White rabbits and 2 Dutch Belted rabbits were used to evaluate the ab externo delivery method. sc-AAV5-smCBA-hGFP vector was delivered into the suprachoroidal space using an illuminated iTrackTM 250A microcatheter. Six weeks after surgery, the rabbits were sacrificed and their eyes evaluated for AAV transfection using immunofluorescent antibody staining of GFP. Results Immunostaining of sectioned and whole-mounted eyes demonstrated robust transfection in all treated eyes, with no fluorescence in untreated control eyes. Transfection occurred diffusely and involved both the choroid and the retina. No apparent adverse effects caused by either the viral vector or the procedure itself could be seen either clinically or histologically. Conclusions The ab externo method of delivery using a microcatheter was successful in safely and effectively delivering a gene therapy agent to the suprachoroidal space. This method presents a less invasive alternative to the current method of virally vectored gene delivery.

Intravitreal bevacizumab treatment of bilateral peripapillary choroidal neovascularization from optic nerve head drusen

Choroidal neovascular membranes are a rare cause of decreased vision in children with optic nerve head drusen. We present a case of bilateral choroidal neovascular membranes associated with optic nerve head drusen in a 5-year-old boy who was successfully treated with a combination of focal laser photocoagulation and intravitreal bevacizumab.

Short-term response of macular oedema to intravitreal bevacizumab

Background/aims: Bevacizumab has been shown to be an effective treatment of macular oedema. This study assesses the response of macular oedema to bevacizumab with specific focus on the first 24 h postinjection. Methods: Subjects with diabetic macular oedema (DMO) or exudative age-related macular degeneration (ARMD) received intravitreal bevacizumab injections. Early Treatment of Diabetic Retinopathy Study (ETDRS) visual acuity and OCT macular thickness measurements were obtained at baseline, 1, 3, 6, 24 and 48 h, 1 week and 1 month postinjection. Results: The average baseline OCT was 452.91 µm (SD 182.96, range 249.00 to 784.00). After injection, there was a statistically significant decrease in OCT thickness within 2 h with a plateau phase out to 24 h, followed by a significant drop between 24 and 48 h, and a return towards baseline between 1 week and 1 month. Average changes in ETDRS visual acuity were not statistically significant, though many patients experienced a positive outcome. Conclusion: While there is an immediate pressure-related effect, it appears that the anti-VEGF effects of bevacizumab require approximately 24 h to become active and persist for 2–3 weeks. These results suggest that injections at 2–3-week intervals might provide improved clinical outcomes, compared with the currently typical 4–6-week interval of injections.

Optic Neuropathy from Cobalt Toxicity in a Patient who Ingested Cattle Magnets.

Cobalt is a widely used in the industrial production of hard metals. Cobalt ingestion has been reported to cause widespread systemic toxicity, but its effects on vision have been sparsely reported. The authors report the case of a patient who ingested cattle magnets, which remained in his stomach for an unknown duration of time. These magnets largely consist of cobalt that gradually leached into his blood stream, resulting in protean systemic manifestations, which included optic atrophy.

Variegated yet Non-Random Rod and Cone Photoreceptor Disease Patterns in RPGR-ORF15-associated Retinal Degeneration

Mutations in the ORF15 exon of the RPGR gene cause a common form of X-linked retinitis pigmentosa, which often results in severe loss of vision. In dogs and mice, gene augmentation therapy has been shown to arrest the progressive degeneration of rod and cone photoreceptors. However, the distribution of potentially treatable photoreceptors across the human retinas and the rate of degeneration are not known. Here, we have defined structural and functional features of the disease in 70 individuals with ORF15 mutations. We also correlated the features observed in patients with those of three Rpgr-mutant (Rpgr-ko, Rd9, and Rpgr-cko) mice. In patients, there was pronounced macular disease. Across the retina, rod and cone dysfunction showed a range of patterns and a spectrum of severity between individuals, but a high symmetry was observed between eyes of each individual. Genotype was not related to disease expression. In the Rpgr-ko mice, there were intra-retinal differences in rhodopsin and cone opsin trafficking. In Rd9 and Rpgr-cko mice, retinal degeneration showed inter-ocular symmetry. Longitudinal results in patients revealed localized rod and cone dysfunction with progression rates of 0.8 to 1.3 log per decade in sensitivity loss. Relatively retained rod and cone photoreceptors in mid- and far-peripheral temporal-inferior and nasal-inferior visual field regions should be good targets for future localized gene therapies in patients.

Subperiosteal orbital hemorrhage complicating cardiac surgery.

Subperiosteal orbital hemorrhage (SPOH) following cardiac surgery has not been previously reported. We present a patient who developed diplopia and right eye proptosis immediately after cardiac surgery for a mitral valve repair and coronary artery bypass graft. A computed tomography (CT) study demonstrated a right superior SPOH. The diplopia and proptosis resolved spontaneously within 4 weeks. Follow-up CT showed complete resolution of the SPOH.

Breaking the Mold of Orbital Cellulitis

A 52-year-old, immune-suppressed man presented with painful proptosis. Orbital imaging revealed enhancement of his right inferior rectus muscle and mild ethmoidal sinus disease. Sinus washings and turbinectomy demonstrated Curvularia. Despite aggressive intravenous antimicrobials, the patient remained febrile. Repeat imaging demonstrated a well-defined intramuscular abscess without contiguous orbital or paranasal involvement. Following surgical drainage, the patient improved. Cultures of the material expressed from the abscess confirmed a co-infection with Fusarium. Although rare, fungal abscess of the extraocular muscle should be considered in patients (particularly if immunosuppressed) with extraocular muscle enlargement resistant to conventional antimicrobial therapy. Prompt diagnosis and treatment could potentially prevent further serious morbidity or mortality.

Alternative technique for implantation of a scleral-fixated intraocular lens

We describe an alternative method of implanting a scleral-fixated intraocular lens (IOL) that facilitates passage of the suture and improves control of suture placement. An ab externo approach introduces a loop of polypropylene (Prolene) through the sclera in a single 27-gauge puncture. The loop is used to secure the IOL haptic in a cow-hitch fashion, which minimizes the risk for the knot to unravel and the IOL to dislocate. After the IOL is placed in the eye, it is secured with the externalized curved suture needle. Knots are covered with a scleral flap. This technique improves efficiency and control in placing an IOL near the normal anatomical location of the crystalline lens when the absence of capsule support precludes nontethered placement of an IOL in the sulcus or capsular bag.