Safety and Biodistribution Evaluation in Cynomolgus Macaques of rAAV2tYF-CB-hRS1, a Recombinant Adeno-Associated Virus Vector Expressing Retinoschisin

Abstract

Applied Genetic Technologies Corporation is developing rAAV2tYF-CB-hRS1, a recombinant adenoassociated virus (rAAV) vector for treatment of X-linked retinoschisis (XLRS), an inherited retinal disease characterized by splitting (schisis) of retinal layers causing poor vision. We report here results of a study evaluating the safety and biodistribution of rAAV2tYF-CB-hRS1 in normal cynomolgus macaques. Three groups of male animals (n = 6 per group) received an intravitreal injection in one eye of either vehicle, or rAAV2tYF-CB-hRS1 at one of two dose levels (4 · 10 10 or 4 · 10 11 vg/eye). Half the animals were sacrificed after 14 days and the others after 91 or 115 days. The intravitreal injection procedure was well tolerated in all groups. Serial ophthalmic examinations demonstrated a dose-related anterior and posterior segment inflammatory response that improved over time. There were no test article-related effects on intraocular pressure, electroretinography, visual evoked potential, hematology, coagulation, clinical chemistry, or gross necropsy observations. Histopathological examination demonstrated minimal or moderate mononuclear infiltrates in 6 of 12 vector-injected eyes. Immunohistochemical staining showed RS1 labeling of the ganglion cell layer at the foveal slope in vector-injected eyes at both dose levels. Serum anti-AAV antibodies were detected in 4 of 6 vector-injected animals at the day 15 sacrifice and all vector-injected animals at later time points. No animals developed antibodies to RS1. Biodistribution studies demonstrated high levels of vector DNA in the injected eye but minimal or no vector DNA in any other tissue. These results support the use of rAAV2tYF-CB-hRS1 in clinical studies in patients with XLRS.

Safety and Biodistribution Evaluation in Cynomolgus Macaques of rAAV2tYF-PR1.7-hCNGB3, a Recombinant AAV Vector for Treatment of Achromatopsia

“…The highest levels were in the animal euthanized on study day 5, which is consistent with other studies that have shown higher levels of vector DNA in animals euthanized 1 or 4 weeks after vector administration compared with animals euthanized 3 months after vector administration. 33,37,38 In other studies, dose-dependent restoration of cone photoreceptor function after subretinal injection of rAAV2tYF-PR1.7-hCNGB3 was demonstrated in CNGB3 knockout mice and CNGB3 mutant dogs. In the mouse model, cone ERG responses developed in 31% of eyes treated at a vector concentration of 1 · 10 12 VG/ml and 90% of eyes treated at a vector concentration of 4.2 · 10 12 VG/ ml.…”

“…32 The ocular inflammation observed in this study is similar to the ocular inflammation seen in nonhuman primates after intravitreal injection of rAAV2tYF-CB-hRS1, an AAV vector expressing retinoschisin (RS1) and packaged in AAV2tYF capsids. 33 Routine postoperative medication in this study consisted of topical atropine, antibiotics, and corticosteroids during the first 10 days after surgery and additional dilating and antiinflammatory medications at later time points, based on the clinical evaluation by the attending veterinarian. Previous studies in which humans received subretinal injections of AAV vector expressing RPE65 or REP1 employed a variety of postoperative dilating, antibiotic, and antiinflammatory regimens, including oral corticosteroids in some studies.…”

“…40 The method used to produce and purify rAAV 2tYF-PR1.7-hCNGB3 is the same as the method used for rAAV2tYF-CB-hRS1. 33 This method resulted in a product with purity >90% and low or undetectable levels of process residuals, similar to the low levels of process residuals seen in toxicology and clinical batches of an rAAV1-CB-hAAT product that had a favorable safety profile when administered by intramuscular injection in a clinical trial at doses up to 6 · 10 12 VG/kg (4.2 · 10 14 total VG in a 70-kg person) in subjects with a 1 -antitrypsin deficiency. 41,42 In conclusion, subretinal injection of rAAV2tYF-PR1.7-hCNGB3 at a concentration of 4 · 10 11 or 4 · 10 12 VG/ml was associated with a dose-related anterior and posterior segment inflammatory response that improved over time except that vitreous cells persisted longer than other manifestations of ocular inflammation.…”

Safety and Biodistribution Evaluation in Cynomolgus Macaques of rAAV2tYF-PR1.7-hCNGB3, a Recombinant AAV Vector for Treatment of Achromatopsia

“…The highest levels were in the animal euthanized on study day 5, which is consistent with other studies that have shown higher levels of vector DNA in animals euthanized 1 or 4 weeks after vector administration compared with animals euthanized 3 months after vector administration. 33,37,38 In other studies, dose-dependent restoration of cone photoreceptor function after subretinal injection of rAAV2tYF-PR1.7-hCNGB3 was demonstrated in CNGB3 knockout mice and CNGB3 mutant dogs. In the mouse model, cone ERG responses developed in 31% of eyes treated at a vector concentration of 1 · 10 12 VG/ml and 90% of eyes treated at a vector concentration of 4.2 · 10 12 VG/ ml.…”

“…40 The method used to produce and purify rAAV 2tYF-PR1.7-hCNGB3 is the same as the method used for rAAV2tYF-CB-hRS1. 33 This method resulted in a product with purity >90% and low or undetectable levels of process residuals, similar to the low levels of process residuals seen in toxicology and clinical batches of an rAAV1-CB-hAAT product that had a favorable safety profile when administered by intramuscular injection in a clinical trial at doses up to 6 · 10 12 VG/kg (4.2 · 10 14 total VG in a 70-kg person) in subjects with a 1antitrypsin deficiency. 41,42 In conclusion, subretinal injection of rAAV2tYF-PR1.7-hCNGB3 at a concentration of 4 · 10 11 or 4 · 10 12 VG/ml was associated with a dose-related anterior and posterior segment inflammatory response that improved over time except that vitreous cells persisted longer than other manifestations of ocular inflammation.…”