Peter Sonnentag

Safety and Biodistribution Evaluation of rAAV2tYF-CB-hRS1, a Recombinant Adeno-Associated Virus Vector Expressing Retinoschisin, in RS1-Deficient Mice

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.

Toxicology and Pharmacology of an AAV Vector Expressing Codon-optimized RPGR in RPGR-deficient Rd9 Mice

Applied Genetic Technologies Corporation (AGTC) is developing a recombinant adeno-associated virus (rAAV) vector AGTC-501, also designated AAV2tYF-GRK1-RPGRco, to treat retinitis pigmentosa (RP) in patients with mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. The vector contains a codon-optimized human RPGR cDNA (RPGRco) driven by a photoreceptor-specific promoter (G protein-coupled receptor kinase 1, GRK1) and is packaged in an AAV2 capsid with three surface tyrosine residues changed to phenylalanine (AAV2tYF). We conducted a safety and potency study of this vector administered by subretinal a injection in the naturally occurring RPGR-deficient Rd9 mouse model. Sixty Rd9 mice (20 per group) received a subretinal injection in the right eye of vehicle (control) or AAV2tYF-GRK1-RPGRco at one of two dose levels (4 × 108 or 4 × 109 vg/eye) and were followed for 12 weeks after injection. Vector injections were well tolerated, with no systemic toxicity. There was a trend towards reduced electroretinography b-wave amplitudes in the high vector dose group that was not statistically significant. There were no clinically important changes in hematology or clinical chemistry parameters and no vector-related ocular changes in life or by histological examination. Dose-dependent RPGR protein expression, mainly in the inner segment of photoreceptors and the adjacent connecting cilium region, was observed in all vector-treated eyes examined. Sequence integrity of the codon-optimized RPGR was confirmed by sequencing of PCR-amplified DNA, or cDNA reverse transcribed from total RNA extracted from vector-treated retinal tissues, and by sequencing of RPGR protein obtained from transfected HEK 293 cells. These results support the use of rAAV2tYF-GRK1-RPGRco in clinical studies in patients with XLRP caused by RPGR mutations.

299. Safety and Biodistribution Study of rAAV2tYF-PR1.7-hCNGB3 in CNGB3-Deficient Mice

Background: AGTC is developing a recombinant adeno-associated virus (rAAV) vector expressing the human CNGB3 gene, for treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity and absence of color discrimination. Here we report results of a toxicology and biodistribution study of this vector administered by subretinal injection in CNGB3-deficient mice. Methods: Three groups of CNGB3-deficient mice (n= 35 per sex per group) received a subretinal injection in one eye of 1 µL of vehicle (balanced salt solution with 0.014% Tween 20) or rAAV2tYF-PR1.7-hCNGB3 vector at a concentration of 1 × 1012 vg/mL (1 × 109 vg/eye) or 4 × 1012 vg/mL (4 × 109 vg/eye). The other eye was untreated. Ten animals/sex/group were used for toxicology evaluation with ophthalmic examinations and pathological evaluations, 10 animals/sex/group were used for biodistribution evaluation, and 15 animals/sex/group were used for efficacy evaluation. Half the animals in the biodistribution and toxicology groups were euthanized 4 weeks after vector administration and the remaining animals were euthanized 12 weeks after vector administration. For animals in the biodistribution groups, blood for qPCR analysis was obtained on Study Days 3, 8 and at euthanasia. At necropsy, samples of eyes, brain, heart, liver, gall bladder, kidneys, spleen, thymus, lungs, adrenals, ovaries, epididymides and testes were obtained for histopathology (for animals in the toxicology groups) or DNA PCR (for animals in the biodistribution groups). For animals scheduled for efficacy evaluations, electroretinography (ERG) testing included scotopic and photopic tests performed at Week 4, 8, and 12 on each eye and serum was collected at euthanasia for measurement of antibodies to AAV and hCNGB3. Results: There were no test article-related changes in clinical observations, body weights, food consumption, ocular examinations, clinical pathology parameters, organ weights, or macroscopic observations at necropsy. Cone-mediated ERG responses were detected after vector administration in the treated eyes in 90% of animals in the higher dose group, 31% of animals in the lower dose group, and none of the untreated or vehicle-treated eyes. Microscopic pathology results demonstrated minimal mononuclear cell infiltrates in the retina and vitreous of some animals at the interim euthanasia, and in the vitreous of some animals at the terminal euthanasia. Serum anti-AAV antibodies developed in most vector-injected animals. No animals developed antibodies to hCNGB3. Biodistribution studies demonstrated high levels of vector DNA in vector-injected eyes but little or no vector DNA in non-ocular tissue. Conclusions: Subretinal injection of rAAV2tYF-PR1.7-hCNGB3 in CNGB3-deficient mice was associated with no clinically important toxicology findings, rescue of cone-mediated ERG responses in vector-treated eyes, and vector DNA detection limited primarily to vector-injected eyes. These results support the use of rAAV2tYF-PR1.7-hCNGB3 in clinical studies in patients with achromatopsia caused by CNGB3 mutations. A Phase 1/2 clinical trial evaluating rAAV2tYF-PR1.7-hCNGB3 is scheduled to begin in 2016.

88. Safety and Biodistribution Study of rAAV2tYF-PR1.7-hCNGB3 in Nonhuman Primates

Background: AGTC is developing a recombinant AAV vector expressing the human CNGB3 gene for treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity and absence of color discrimination. Here we report results of a toxicology and biodistribution study of this vector administered by subretinal injection in cynomolgus macaques. Methods: Three groups of animals (n=2 males and 2 females per group) received a subretinal injection in one eye of 300 µL containing either vehicle or rAAV2tYF-PR1.7-hCNGB3 at one of two concentrations (4 × 1011 or 4 × 1012 vg/mL) and were evaluated for safety and biodistribution over a 3-month period prior to euthanasia. Toxicity assessment was based on mortality, clinical observations, body weights, ophthalmic examinations, intraocular pressure (IOP) measurements, electroretinography (ERG), visual evoked potentials (VEP), and clinical and anatomic pathology. Vector shedding and biodistribution was assessed by qPCR analyses. Immune responses to AAV and hCNGB3 were measured by ELISA, Elispot, or neutralization antibody assay for AAV2tYF. Results: There was no evidence of local or systemic toxicity and no changes in IOP, VEP responses, or hematology, coagulation or clinical chemistry parameters and no clinically important changes in ERG responses. Aqueous cells, sometimes with aqueous flare, were observed at the Day 3 evaluation in all groups and generally resolved or were at the mild (1+) levels by Week 4 and absent on Week 8 and thereafter except in one high dose animal. Posterior segment findings consisted of varying degrees of dose-related white vitreous cell, vitreous haze, white retinal perivascular sheathing, and white to grey-white subretinal infiltrates within and outside of the injection site. Vitreous haze resolved by Day 8 in eyes given vehicle control, by Week 4 in the low dose group and by Week 13 in the high dose group. Vitreous cells were observed at the mild (trace or 1+) level in the vehicle control group and resolved by Study Weeks 9 or 13 but persisted through Week 13 in a dose-related fashion in the low and high dose groups. Serum neutralizing antibodies against AAV2tYF were detected in all animals given vector. There were no T cell responses to AAV capsid peptides and no antibody or T cell responses to hCNGB3. Mononuclear cell infiltrates in the vitreous body/optic disc, of minimal intensity, in the vector-injected eye of all animals at both dose levels. All other tissues collected for histopathological examination showed no abnormalities. Results of biodistribution studies demonstrated that the vector did not spread widely or consistently outside the injected eye. High levels of vector DNA were found in vector-injected eyes but minimal or no vector DNA was found in any other tissue. Conclusions: Subretinal injection of rAAV2tYF-PR1.7-hCNGB3 at concentrations of 4 × 1011 or 4 × 1012 vg/mL was associated with a dose-related anterior and posterior segment inflammatory response that improved over time. There was no evidence of systemic toxicity and no changes in IOP, VEP responses, or hematology, coagulation or clinical chemistry parameters and no clinically important changes in ERG responses. These results support the use of rAAV2tYF-PR1.7-hCNGB3 in clinical studies in patients with achromatopsia. A Phase 1/2 clinical trial evaluating rAAV2tYF-PR1.7-hCNGB3 in patients with achromatopsia is scheduled to begin in 2016.

463. Safety and Biodistribution Study of rAAV2tYF-CB-hRS1 in RS1-deficient Mice

Intravitreal administration of rAAV2tYF-CB-hRS1 in RS1-deficient mice was well tolerated with minimal to slight ocular inflammatory cells detected by histopathology. RS1 expression demonstrated by immunohistochemistry was associated with decreased severity of splitting/disorganization of the inner nuclear layer of the retina at the higher dose level. These results support the use of rAAV2tYF-CB-hRS1 in clinical studies in patients with XLRS.

199. Initial Safety Evaluation of rAAV-hCNGB3 Vectors in Nonhuman Primates

Both AAV2tYF-hCNGB3 and AAV5-hCNGB3 were well tolerated after subretinal injection in in NHPs at a dose level of 4 × 10^10 or 4 × 10^11 vg/eye. Test article related findings included dose-dependent ocular inflammation that resolved by Study Week 12. No test article-related ERG or cortical visual evoked potential (VEP) effects were observed. In contrast to the severe inflammation noted in dogs receiving high dose of AAV5-hCNGB3 that usually resulted in involuntary early termination of the animals, the milder test article-related findings in macaques receiving AAV-hCNGB3 vectors that expresses a highly homologous xenogeneic human protein is helpful for guiding future development of rAAV-CNGB3 gene therapy for human patients.

663. Safety and Biodistribution Study of rAAV2tYF-CB-hRS1 in Nonhuman Primates

Background: AGTC is developing a recombinant AAV vector expressing retinoschisin (RS1) for treatment of humans with X-linked retinoschisis (XLRS). Here we report results of a toxicology and biodistribution study of this vector administered by intravitreal injection in cynomolgus macaques.Methods: Three groups of male cynomolgus macaques (n=3 per group) received an intravitreal injection in one eye of vehicle or rAAV2tYF-CB-hRS1 at one of two dose levels (4 × 10^10 or 4 × 10^11 vg/eye). Half of the animals were sacrificed 14 days after vector administration and the remaining animals were sacrificed 91 or 115 days after vector administration. At each sacrifice time point blood and tissue samples were collected for evaluation of safety and biodistribution.Results: There were no test article-related effects on intraocular pressure, electroretinography, visual evoked potentials, and clinical pathology parameters. Ocular exams demonstrated a dose-related ocular inflammatory response that improved over time. In the vehicle control group, aqueous cells ranged from trace to 4+ at 1 week after injection and resolved by Week 2. Vitreous cells ranged from trace to 2+ at 2 to 3 weeks, resolved by Week 4 in four animals and in the other two animals were resolved or resolving at Week 12. None of the animals in this group had vitreous haze. Ocular inflammation was greater in the vector-treated animals and was more pronounced at the higher dose. Aqueous cells ranged from 1+ to 4+ at Week 1 in all animals, resolved by Week 2, recurred at Week 2 to 4 in two animals in the low dose and three animals in the high dose group, and then resolved over the next several weeks. Vitreous cells developed in 5 of 6 animals in the low dose and all animals in the high dose group, and persisted at 2+ or greater for more than 4 weeks in two animals in the low dose and three animals in the high dose group. Vitreous haze developed at Week 4 in one animal in the low dose and three animals in the high dose group and resolved over the next several weeks. Microscopic pathology results demonstrated mononuclear infiltrates at the optic disc and/or around blood vessels in the ganglion cell layer, iris, and ciliary body of the injected eye was observed in 2 of 6 animals in the lower dose group and 4 of 6 animals in the higher dose group. Immunohistochemistry studies showed RS1 labelling of the ganglion cell layer at the foveal slope. At terminal sacrifice there was limited vector biodistribution outside the injected eye.Conclusions: Intravitreal administration of rAAV2tYF-CB-hRS1 in normal cynomolgus macaques was associated with dose-related anterior and posterior segment inflammatory response that improved over time. Histological examination showed mononuclear cell infiltrates that were more prevalent at the higher dose level and RS1 expression in the retinal ganglion cell ring was demonstrated by immunohistochemistry. Results from this study support the use of rAAV2tYF-CB-hRS1 in clinical studies in patients with XLRS.