Controlling Inflammation and the Immune Response after Intravitreal Injection with Post-Processing of AAV Vectors and Modulation of Capsid and Viral Genome Titers

Abstract

Purpose : The relative contributions of AAV viral capsid and viral genome, dosed intravitreally, to ocular inflammation and immune responses are not well understood. We investigated their relative contributions by varying the empty/full capsid ratio and capsid and genome titers in non-human primates.

Methods : rAAV2tYF viral vector manufactured by AGTC was processed to enrich for full capsids (capsids with genome), for empty capsids (capsids without genome), or removal of capsids altogether (residual). Both eyes of Cynomolgus monkeys (Macaca fascicularis) were dosed intravitreally. Dosing was normalized based on capsid or viral genome titer. Ophthalmic exams (OE) were performed over 12 weeks to assess inflammation. Upon termination, eyes and cervical and axillary lymph nodes were collected. Eyes were processed for histopathology, in situ hybridization (ISH) for reporter expression, and cytokine analysis and RNAseq analysis of vitreous humor. Lymph nodes underwent immunophenotyping by flow cytometry, and serum was collected to assess neutralizing antibody titers.

Results : The inflammation profile (per OE) differed between the aqueous and vitreous compartments. Aqueous peak inflammation occurred 2-4 weeks after injection, and then decreased to a low level. Moreover, the presence of sufficient viral genomes was required to induce aqueous inflammation. Vitreous inflammation increased 2 weeks after injection and remained elevated. The presence of capsids, with or without genomes, sufficed to induce vitreous inflammation. Mononuclear cell infiltrate was noted for all groups with sufficient amount of viral genome. ISH showed increased expression signal in groups enriched for full capsids. Cytokine and RNAseq data differed between groups enriched for full or empty capsid. Immunophenotyping and NAb analysis showed similar profiles among dosing groups.

Conclusions : Both viral capsid and viral genome contributed to inflammation. The viral genome contributed more to inflammation in the anterior whereas the capsid contributed more to the cellular immune response in the vitreous. Both responses were localized primarily within the eye, with minimal systemic response. Importantly, enriching for full capsids in a vector preparation increased transduction and reduced the inflammation when normalized to vector genomes.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.