No abstract

MERTK is an essential component of the signaling network that controls phagocytosis in retinal pigment epithelium (RPE), the loss of which results in photoreceptor degeneration. Previous proof-of-concept studies have demonstrated the efficacy of gene therapy using human MERTK (hMERTK) packaged into adeno-associated virus (AAV2) in treating RCS rats and mice with MERTK deficiency. The purpose of this study was to assess the safety of gene transfer via subretinal administration of rAAV2-VMD2-hMERTK in subjects with MERTK-associated retinitis pigmentosa (RP). After a preclinical phase confirming the safety of the study vector in monkeys, six patients (aged 14 to 54, mean 33.3 years) with MERTK-related RP and baseline visual acuity (VA) ranging from 20/50 to <20/6400 were entered in a phase I open-label, dose-escalation trial. One eye of each patient (the worse-seeing eye in five subjects) received a submacular injection of the viral vector, first at a dose of 150 µl (5.96 × 10(10)vg; 2 patients) and then 450 µl (17.88 × 10(10)vg; 4 patients). Patients were followed daily for 10 days at 30, 60, 90, 180, 270, 365, 540, and 730 days post-injection. Collected data included (1) full ophthalmologic examination including best-corrected VA, intraocular pressure, color fundus photographs, macular spectral domain optical coherence tomography and full-field stimulus threshold test (FST) in both the study and fellow eyes; (2) systemic safety data including CBC, liver and kidney function tests, coagulation profiles, urine analysis, AAV antibody titers, peripheral blood PCR and ASR measurement; and (3) listing of ophthalmological or systemic adverse effects. All patients completed the 2-year follow-up. Subretinal injection of rAAV2-VMD2-hMERTK was associated with acceptable ocular and systemic safety profiles based on 2-year follow-up. None of the patients developed complications that could be attributed to the gene vector with certainty. Postoperatively, one patient developed filamentary keratitis, and two patients developed progressive cataract. Of these two patients, one also developed transient subfoveal fluid after the injection as well as monocular oscillopsia. Two patients developed a rise in AAV antibodies, but neither patient was positive for rAAV vector genomes via PCR. Three patients also displayed measurable improved visual acuity in the treated eye following surgery, although the improvement was lost by 2 years in two of these patients. Gene therapy for MERTK-related RP using careful subretinal injection of rAAV2-VMD2-hMERTK is not associated with major side effects and may result in clinical improvement in a subset of patients.

Adeno-associated virus (AAV) has proven an effective gene delivery vehicle for the treatment of retinal disease. Ongoing clinical trials using a serotype 2 AAV vector to express RPE65 in the retinal pigment epithelium have proven safe and effective. While many proof-of-concept studies in animal models of retinal disease have suggested that gene transfer to the neural retina will also be effective, a photoreceptor-targeting AAV vector has yet to be used in the clinic, principally because a vector that efficiently but exclusively targets all primate photoreceptors has yet to be demonstrated. Here, we evaluate a serotype 5 AAV vector containing the human rhodopsin kinase (hGRK1) promoter for its ability to target transgene expression to rod and cone photoreceptors when delivered subretinally in a nonhuman primate (NHP). In vivo fluorescent fundus imaging confirmed that AAV5-hGRK1-mediated green fluorescent protein (GFP) expression was restricted to the injection blebs of treated eyes. Optical coherence tomography (OCT) revealed a lack of gross pathology after injection. Neutralizing antibodies against AAV5 were undetectable in post-injection serum samples from subjects receiving uncomplicated subretinal injections (i.e., no hemorrhage). Immunohistochemistry of retinal sections confirmed hGRK1 was active in, and specific for, both rods and cones of NHP retina. Biodistribution studies revealed minimal spread of vector genomes to peripheral tissues. These results suggest that AAV5-hGRK1 is a safe and effective AAV serotype/promoter combination for targeting therapeutic transgene expression protein to rods and cones in a clinical setting.

The authors demonstrate for the first time that long-term therapy (∼1 year) is achievable in a mammalian model of GC1 deficiency. These data provide additional justification for the development of an AAV-based gene therapy vector for the clinical treatment of Leber congenital amaurosis-1.

Gene therapy strategies for congenital myopathies may require repeat administration of adeno-associated viral (AAV) vectors in response to several limitations inherent to the clinical design: 1) administration of doses below therapeutic efficacy in patients enrolled in early phase clinical trials; 2) progressive reduction of the therapeutic gene expression over time as a result of increasing muscle mass in patients treated at a young age; and 3) a possibly faster depletion of pathogenic myofibers in this patient population. Immune responses triggered by the first vector administration, and to subsequent ones, represent a major obstacle for successful gene transfer in young patient population. Anti-capsid and anti-transgene product related humoral and cell-mediated responses have been previously observed in all preclinical models and human subjects who received gene therapy or ERT treatment for congenital myopathies. Immune responses may result in reduced efficacy of the gene transfer over time and/or may preclude for the possibility of re-administration of the same vector. This study presents a case of Pompe patient dosed with an AAV1-GAA vector after receiving Rituximab and Sirolimus to modulate the immune responses. A key finding of this single subject case report is the observation that B-cell ablation with rituximab prior to AAV vector exposure results in non-responsiveness to both capsid and transgene, therefore allowing the possibility of repeat administration in the future. This observation is significant for future gene therapy studies and establishes a clinically relevant approach to blocking immune responses to AAV vectors.

Proof of concept for MERTK gene replacement therapy has been demonstrated utilizing different viral vectors in the Royal College of Surgeon (RCS) rat, a well characterized model of recessive retinitis pigmentosa (RP) 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 AAV2 vector expressing human MERTK cDNA driven by the RPE-specific, VMD2 promoter. We demonstrate the potency of the vector in RCS rats by improved electroretinogram responses in treated eyes compared to 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 (BSS) 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 to controls. Evaluation of blood smears showed the lack of transformed cells in the. 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.

Our collaborative successful gene replacement therapy using AAV vectors expressing a variant of human RPGR-ORF15 in two canine models provided therapeutic proof of concept for translation into human treatment. The ORF15 sequence contained within this AAV vector, however, has ORF15 DNA sequence variations compared to the published sequence that are likely due to its unusual composition of repetitive purine nucleotides. This mutability is a concern for AAV vector production and safety when contemplating a human trial. In this study, we establish the safety profile of AAV-hIRBP-hRPGR and AAV-hGRK1-hRPGR vectors used in the initial canine proof-of-principle experiments by demonstrating hRPGR-ORF15 sequence stability during all phases of manipulation, from plasmid propagation to vector production to its stability in vivo after subretinal administration to animals. We also evaluate potential toxicity in vivo by investigating protein expression, retinal structure and function, and vector biodistribution. Expression of hRPGR is detected in the inner segments and synaptic terminals of photoreceptors and is restricted to the connecting cilium when the vector is further diluted. Treated eyes exhibit no toxicity as assessed by retinal histopathology, immunocytochemistry, optical coherence tomography, fundoscopy, electroretinogram, and vector biodistribution. Therefore, the hRPGR-ORF15 variant in our AAV vectors appears to be a more stable form than the endogenous hRPGR cDNA when propagated in vitro. Its safety profile presented here in combination with its proven efficacy supports future gene therapy clinical trials. INTRODUCTIONRetinitis pigmentosa (RP) is a common form of genetically heterogeneous, inherited retinal diseases characterized by progressive degeneration of rod and cone photoreceptor cells and affects 1 in 4000 individuals.1,2 The X-linked form of RP (XLRP), comprising an estimated 15% of total RP cases, is among the most severe forms.3 Mutations in the gene coding for RP GTPase regulator (RPGR) cause XLRP and account for over 70% of cases.

Allotopically expressed wild-type ND4 prevents the phenotype induced by G11778A mitochondrial DNA with a toxicology profile acceptable for testing in a phase I clinical trial.