Macular retina-targeting AAV capsid identified through multi-species screening in mice, rabbits, pigs, and monkeys

Abstract

Purpose : Adeno-associated virus vector is the main in vivo gene delivery method. To improve tissue tropism, capsid engineering through rational design, directed evolution, and artificial intelligence has attracted much attention and has been performed, resulting in the usage of engineered capsids in clinical trials. However, the lack of cross-species translatability of novel capsids limits its broader application and creates significant challenges for clinical development.

Methods : In this study, a novel capsid (AAV2.N54) with improved tropism to deep regions of retinas was identified through multi-species screening in mice, pigs, rabbits, and monkeys. In screening studies, fundus images, immunohistochemistry, and confocal scanning laser ophthalmoscopy were performed to assess the tropism of 18 candidate capsids and controls after intravitreal administration.

Results : Across all four species, AAV2.N54 showed improved tropism over wildtype AAV2 and a previously described engineered capsid AAV2.7m8, evidenced by efficient delivery to the macular retina and the ability to detarget the ciliary body. AAV2.N54 also had a solid safety profile with minimal inflammation, similar to controls. The transgene expression cassette for treating wet AMD was optimized through in vitro screening and measurement of protein levels by ELISA. Then, AAV2.N54-Aflibercept was assessed in disease models of mice and rabbits. In the laser-induced choroidal neovascularization (LCNV) mouse model, fluorescein angiography and flatmount analysis showed that AAV2.N54-Aflibercept prophylactically reduced retina lesions, with improved efficacy than the commercial protein drug Eylea. In the DL-AAA rabbit disease model, AAV2.N54-Aflibercept demonstrated remarkable therapeutic efficacy in reducing retina leakage. In addition, a GLP toxicity study in NHPs was performed, demonstrating the safety profile of intravitreal dosing of AAV2.N54-Aflibercept at the dose up to 5.7x10¹¹ vg/eye. Based on these results, GMP production has been finished, and an investigational new drug (IND) application will be submitted soon.

Conclusions : An engineered capsid with improved delivery to the macular retina and cross-species translatability was identified, and a novel gene therapy for treating wet AMD was developed.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.