Preclinical Evaluation of rAAV.sFlt-1 in Monkeys: Toxicity and Biodistribution

Abstract

Purpose: : To conduct toxicity and biodistribution studies in order to fulfil the requirements of the Australian Therapeutic Goods Administration (equivalent to FDA) authority to commence Phase I/II human trials.

Methods: : All animal experimentation was conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Monkeys (M. fascicularis) were subretinally injected with rAAV.sFlt-1 (n=5), rAAV.gfp (n=2) or sFLT protein (n=1). Animals were subjected to regular clinical and ophthalmic examinations which included slit lamp examination, electroretinography and fundus photography. Peripherial blood was collected at different time points. At the time of euthanasia, tissue samples were collected. Real-time PCR, ELISA and flow cytometry were used to establish recombinant virus and protein dissemination and immune response.

Results: : All monkeys were healthy throughout the course of the study. Fundoscopic examination revealed a normal fundus and clear vitreous. Slit lamp examination did not detect any abnormality in the anterior segment. There were no differences in a-wave and b-wave amplitudes taken before and after injection. Real-time PCR did not detect the presence of the AAV vector in the tissues sampled. ELISA did not detect any AAV2 capsid or increase in sFlt-1 levels outside of the eye. However, sFlt-1 levels were increased in the vitreous of the AAV.sFlt-1-injected eyes. Preliminary study of the immunogenicity of rAAV.sFlt-1 based on changes in the population of immune cell subsets by flow cytometry showed no significant changes in T-, B- and NK- cell and monocyte numbers.

Conclusions: : These preclinical studies have shown that subretinal administration of rAAV.sflt was well tolerated with no adverse effect on general health and visual function. There was a lack of any observed systemic toxicity or immunogenicity. In the light of these data and the long term efficacy of rAAV.sFlt-1 human trials are warranted.

Keywords: choroid: neovascularization • gene transfer/gene therapy • drug toxicity/drug effects