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

Abstract

AGTC is developing rAAV2tYF-PR1.7-hCNGB3, 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. We report here results of a study evaluating the safety and biodistribution of rAAV2tYF-PR1.7-hCNGB3 in cynomolgus macaques. 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 over a 3 month period prior to being euthanized. Administration of rAAV2tYF-PR1.7-hCNGB3 was associated with a dose-related anterior and posterior segment inflammatory response that was greater than that observed in eyes injected with the vehicle control. Most manifestations of inflammation improved over time except that vitreous cells persisted in vector-treated eyes until the end of the study. One animal in the lower vector dose group was euthanized on Study Day 5 based on a clinical diagnosis of endophthalmitis. There were no test article-related effects on intraocular pressure, visual evoked potential responses, hematology or clinical chemistry parameters, or gross necropsy observations. Histopathological examination demonstrated minimal mononuclear infiltrates in all vector-injected eyes. Serum anti-AAV antibodies developed in all vector-injected animals. No animals developed antibodies to CNGB3. 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-PR1.7-hCNGB3 in clinical studies in patients with achromatopsia caused by CNGB3 mutations.

In vivo directed evolution of AAV in the primate retina

“…NHP#9 and the previously described murine variant 7m8( 7 ) were packaged with a gamma-synuclein gene (SNCG) promoter to drive tdTomato expression in RGCs( 15 ) and the pR1.7( 16 ) promoter to yield GFP expression in cones. Vectors encoding both these constructs were mixed in equal ratios (∼1.5E+12 vg/construct/eye) and injected intravitreally in a cynomolgous monkey.…”

Long-Term Safety Evaluation of Continuous Intraocular Delivery of Aflibercept by the Intravitreal Gene Therapy Candidate ADVM-022 in Nonhuman Primates

“…Other published preclinical studies of AAV-based gene therapies have also described vector-related inflammation. [40][41][42][43] Although it is generally accepted that the ocular inflammation observed is likely linked to a local immune response to the gene therapy, 44 it is difficult to pinpoint exactly which vector component is responsible because different AAV capsid serotypes and transgenes were used across these studies.…”

Selected Aspects of Ocular Toxicity Studies With a Focus on High-Quality Pathology Reports: A Pathology/Toxicology Consultant’s Perspective

“…54,79 For gene therapy ocular drugs, biodistribution profile of the vector and viral genome DNA with quantitative polymerase chain reaction in ocular tissues, including the target tissue (retina), biofluids, and systemic tissues for potential off-target biodistribution, is conducted in ocular toxicity studies. [80][81] Antidrug antibodies in serum, plasma or blood are important to evaluate in dose-range and pivotal toxicity studies by intraocular routes for biologic ocular drug products, including single-dose administration, to understand potential effects on toxicokinetics or correlation with ocular inflammation to understand if it may be related to a potential adaptive immune reaction to the test article. 10,[12][13]29 For gene therapy ocular drug products, blood ADA to the viral vector and genome are conducted to understand humoral immunity, along with evaluation of cell-mediated immunity assays using peripheral blood mononulcear cells or other assays.…”

“…10,[12][13]29 For gene therapy ocular drug products, blood ADA to the viral vector and genome are conducted to understand humoral immunity, along with evaluation of cell-mediated immunity assays using peripheral blood mononulcear cells or other assays. 80,81 For gene therapy with adeno-associated viral (AAV) vectors in cynomolgus monkeys, intravitreal injections are associated with a higher risk of humoral immune responses and a less favorable biodistribution profile with more viral shedding to blood and draining lymphatic tissue, increased off-target deposition, and less efficient gene transfer compared to subretinal delivery of AAV vectors. 82,83 Detection of ADA in VH or AH is not currently a regulatory expectation for ocular toxicity studies with biologics and gene or cell therapies, and serum, plasma or blood ADA is generally sufficient for evaluating correlations between immunogenicity and ocular immune complex-related effects observed by ophthalmic or microscopic examination.…”

“…54,79 For gene therapy ocular drugs, biodistribution profile of the vector and viral genome DNA with quantitative polymerase chain reaction in ocular tissues, including the target tissue (retina), biofluids, and systemic tissues for potential off-target biodistribution, is conducted in ocular toxicity studies. 80–81…”