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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.

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.

Applied Genetic Technologies Corporation is developing a recombinant adeno-associated virus (rAAV) vector for treatment of X-linked retinoschisis (XLRS), an inherited retinal disease characterized by splitting (schisis) of the layers of the retina, which causes poor vision. We report here results of a study evaluating the safety and biodistribution of rAAV2tYF-CB-hRS1 in RS1-deficient mice. Three groups of male RS1-deficient mice received an intravitreal injection in one eye of either vehicle, or rAAV2tYF-CB-hRS1 at one of two dose levels (1 × 10(9) or 4 × 10(9) vg/eye) and were sacrificed 30 or 90 days later. The intravitreal injection procedure was well tolerated in all groups, with no test article-related changes in ophthalmic examinations. Two low-dose vector-treated animals had minimally to mildly higher white blood cell counts at day 90. There were no other intergroup differences in hematology or clinical chemistry analyses and no test article-related gross necropsy observations. Microscopic pathology results demonstrated minimal to slight mononuclear cell infiltrates in 80% of vector-injected eyes at day 30 and 20% of vector-injected eyes at day 90. Immunohistochemistry studies showed RS1 labeling of the retina in all vector-treated eyes. At the day 90 sacrifice, there was a decrease in the severity of splitting/disorganization of the inner nuclear layer of the retina in high-dose vector-treated eyes. Biodistribution studies demonstrated vector DNA in vector-injected eyes but not in any nonocular tissue. These results support the use of rAAV2tYF-CB-hRS1 in clinical studies in patients with XLRS.

Induced pluripotent stem (iPS) cells have great potential for personalized regenerative medicine. Although several different methods for generating iPS cells have been reported, improvement of safety and efficiency is imperative. In this study, we tested the feasibility of using a triple tyrosine mutant AAV2 (Y444+500+730F) vector, designated AAV2.3m, to generate iPS cells. We developed a polycistronic rAAV2.3m vector expressing three reprogramming factors, Klf4, Oct4, and Sox2, and then used this vector to infect mouse adipose-derived mesenchymal stem cells (AT-MSCs) to induce the generation of iPS cells. We demonstrated that (1) the triple tyrosine mutant AAV2 vector is able to reprogram mouse adult adipose tissue-derived stem cells into the pluripotent state. Those rAAV2.3m-derived iPS (rAAV2.3m-iPS) cells express endogenous pluripotency-associated genes including Oct4, Sox2, and SSEA-1, and form teratomas containing multiple tissues in vivo; (2) c-myc, an oncogene, is dispensable in rAAV2.3m-mediated cellular reprogramming; and (3) transgene expression is undetectable after reprogramming, whereas vector DNA is detectable, indicating that transgenes are silenced. These results indicated the rAAV vector may have some advantages in generating iPS cells.

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.

A biodistribution and toxicology study was performed to test the acute toxicities of intradiaphragmatic injection of a recombinant adeno-associated virus (rAAV) 2/1-human acid alpha-Glucosidase (hGAA) driven by a cytomegalovirus (CMV) promoter (rAAV1-CMV-hGAA) in New Zealand white rabbits and in the rodent Pompe disease model by injecting at the right quadriceps. Studies performed using fluoroscopy and AAV2-GFP demonstrated spread upon intradiaphragmatic injection, and the ability of AAV to infect and express acid α-glucosidase (GAA) throughout the diaphragm. For the preclinical study, 10 rabbits (5 male, 5 female) were divided into two groups, vehicle control (Lactated Ringer's) and test article (1.5×10(12) vector genomes [vg] rAAV1-CMV-hGAA), and euthanized on day 21. After direct visualization, the left hemidiaphragm was injected at three locations. There was up to a 2,500-fold increase in circulating anti-AAV1 antibodies directed to the vector capsids. In addition, up to an 18-fold increase in antibodies against the GAA protein was generated. Injection sites maintained up to 1.0×10(5) vg/μg genomic DNA (gDNA), while uninjected sites had up to 1.0×10(4) vg/μg gDNA. Vector DNA was present in blood at 24 hr postinjection at up to 1.0×10(6) vg/μg gDNA, followed by a decrease to 1.0×10(3) vg/μg gDNA at euthanization on day 21. Nominal amounts of vector DNA were present in peripheral organs, including the brain, spinal cord, gonads, and skeletal muscle. Upon histopathological examination, fibroplasias of the serosal surface were noted at diaphragm injections sites of both groups. In addition, an increase in mononuclear cell infiltration in the diaphragm and esophagus in vector-dosed animals was found. Elevated creatine phosphokinase levels, an indicator of muscle repair, was observed in all animals postprocedure but persisted in vector-injected rabbits until euthanization. A follow-up study suggested that this was directed against the human transgene expression in a foreign species. Overall, this study demonstrates diffusion of vector throughout the diaphragm after localized injections.

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.

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.

Very long-chain acyl-coA dehydrogenase (VLCAD) is the rate-limiting step in mitochondrial fatty acid oxidation. VLCAD-deficient mice and patients clinical symptoms stem from not only an energy deficiency but also long-chain metabolite accumulations. VLCAD-deficient mice were treated systemically with 1 × 10(12) vector genomes of recombinant adeno-associated virus 9 (rAAV9)-VLCAD. Biochemical correction was observed in vector-treated mice beginning 2 weeks postinjection, as characterized by a significant drop in long-chain fatty acyl accumulates in whole blood after an overnight fast. Changes persisted through the termination point around 20 weeks postinjection. Magnetic resonance spectroscopy (MRS) and tandem mass spectrometry (MS/MS) revealed normalization of intramuscular lipids in treated animals. Correction was not observed in liver tissue extracts, but cardiac muscle extracts showed significant reduction of long-chain metabolites. Disease-specific phenotypes were characterized, including thermoregulation and maintenance of euglycemia after a fasting cold challenge. Internal body temperatures of untreated VLCAD(-/-) mice dropped below 20 °C and the mice became lethargic, requiring euthanasia. In contrast, all rAAV9-treated VLCAD(-/-) mice and the wild-type controls maintained body temperatures. rAAV9-treated VLCAD(-/-) mice maintained euglycemia, whereas untreated VLCAD(-/-) mice suffered hypoglycemia following a fasting cold challenge. These promising results suggest rAAV9 gene therapy as a potential treatment for VLCAD deficiency in humans.

Purpose: The authors previously showed that subretinal delivery of AAV5 vectors containing murine guanylate cyclase-1 (GC1) cDNA driven by either photoreceptor-specific (hGRK1) or ubiquitous (smCBA) promoters was capable of restoring cone-mediated function and visual behavior and preserving cone photoreceptors in the GC1 knockout (GC1KO) mouse for 3 months. Here, the authors compared therapy conferred by the aforementioned vectors to that achieved with the highly efficient capsid tyrosine mutant AAV8(Y733F) and asked whether long-term therapy is achievable in this model., Methods: AAV5-hGRK1-mGC1, AAV5-smCBA-mGC1, or AAV8(Y733F)-hGRK1-mGC1 was delivered subretinally to GC1KO mice between postnatal day (P)14 and P25. Retinal function was assayed by electroretinography. Localization of AAV-mediated GC1 expression and cone survival were assayed with immunohistochemistry, and the spread of vector genomes beyond the retina was quantified by PCR of optic nerve and brain tissue., Results: Cone function was restored with all vectors tested, with AAV8(Y733F) being the most efficient. Electroretinographic responses were clearly measurable out to 1 year after treatment. AAV-mediated expression of GC1 was found exclusively in photoreceptors out to 15 months after injection. Cones were preserved for at least 11 months after treatment. AAV5- and AAV8(733)-delivered vector genomes were recovered primarily from optic nerve of the treated eye and, in only instance, from brain (1 of 20 samples)., Conclusions: 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.

A canine model of Glycogen storage disease type Ia (GSDIa) is described. Affected dogs are homozygous for a previously described M121I mutation resulting in a deficiency of glucose-6-phosphatase-α. Metabolic, clinicopathologic, pathologic, and clinical manifestations of GSDIa observed in this model are described and compared to those observed in humans. The canine model shows more complete recapitulation of the clinical manifestations seen in humans including "lactic acidosis", larger size, and longer lifespan compared to other animal models. Use of this model in preclinical trials of gene therapy is described and briefly compared to the murine model. Although the canine model offers a number of advantages for evaluating potential therapies for GSDIa, there are also some significant challenges involved in its use. Despite these challenges, the canine model of GSDIa should continue to provide valuable information about the potential for generating curative therapies for GSDIa as well as other genetic hepatic diseases.

Mitochondrial beta-oxidation of fatty acids is required to meet physiologic energy requirements during illness and periods of fasting or physiologic stress, and is most active in liver and striated muscle. Acyl-CoA dehydrogenases of varying chain-length specificities represent the first step in the mitochondria for each round of beta-oxidation, each of which removes two-carbon units as acetyl-CoA for entry into the tricarboxylic acid cycle. We have used recombinant adeno-associated virus (rAAV) vectors expressing short-chain acyl-CoA dehydrogenase (SCAD) to correct the accumulation of fatty acyl-CoA intermediates in deficient cell lines. The rAAV-SCAD vector was then packaged into either rAAV serotype 1 or 2 capsids and injected intramuscularly into SCAD-deficient mice. A systemic effect was observed as judged by restoration of circulating butyryl- carnitine levels to normal. Total lipid content at the injection site was also decreased as demonstrated by noninvasive magnetic resonance spectroscopy (MRS). SCAD enzyme activity in the injected muscle was found at necropsy to be above the normal control mouse level. This study is the first to demonstrate the systemic correction of a fatty acid oxidation disorder with rAAV and the utility of MRS as a noninvasive method to monitor SCAD correction after in vivo gene therapy.

alpha1-Antitrypsin (AAT) deficiency is a single-gene disorder in which a mutation in the AAT (approved symbol SERPINA1) gene (PI*Z) leads to misfolding of the protein, loss of the protective antiprotease effect of AAT for the lungs, and a toxic effect on hepatocytes. Optimal therapy for AAT deficiency will require a high percentage of hepatocyte transduction to be effective for liver and lung disease. Recently, rAAV genomes pseudotyped with capsids from serotypes 7 and 8 showed efficient hepatic transduction. We hypothesized that upon portal vein injection to target hepatocytes, serotype 8 would better transduce target cells and therefore express hAAT in both a greater percentage of cells and greater amounts. AAV2 and pseudotyped vectors for serotypes 1, 5, and 8 carrying the human AAT transgene were injected at 1 x 10(10) particle doses into C57Bl/6 mice. Circulating hAAT from AAV2/8-injected animals showed a 2-log advantage over AAV2 and 3-log increase over AAV2/1 and 5 for the 24-week study. Most significantly, up to 40% of total liver cells stained positive for the transgene in AAV2/8 subjects while remaining primarily episomal. Therefore, pseudotyped AAV8 provides a vehicle to infect a high percentage of hepatocytes stably and thereby express therapeutic molecules to modify AAT PiZ transcripts.