Several years of stable rAAV mediated transgene expression following subretinal injection in dog and primate does not show any adverse effect on retinal morphology and function

Abstract

Abstract: : Purpose : To evaluate the effect of long term and stable rAAV mediated transgene expression on retinal morphology and function in dog and primate. Methods : Following a single subretinal injection of rAAV–2, –4 or –5, carrying an CMV.gfp genome, three dogs and one primate were examined for long term studies. Green fluorescent protein (GFP) protein expression in these animals was monitored by fluorescent retinal imaging over several years. The retina of these long–term transgene expressing dogs and primate was evaluated by fluorescein and indocyanin green angiography. Additionally, retinal function was tested using simultaneous bilateral full–field flash electroretinography (ERG). Results : The AAV–2 injected dog displayed stable GFP expression for 3 years, the AAV–4 and AAV–5 dogs expressed GFP for almost two years, while AAV–4 injected primate presented stable transgene expression for more than 18 months post–injection. Optimized angiography exams were performed in these large animals and did not show any abnormalities of the retina. For the fluorescein angiography, the transduced retina showed normal angiogram except for the presence of an hyperfluorescent dot due to a window defect at the needle intrusion site. Functional evaluation of retinas expressing GFP for several months showed normal ERG amplitude responses which were similar to those of the non injected contralateral eye. Conclusions: Several years of transgene expression following subretinal injection of rAAV vectors does not seem to alter the structure nor the function of the retina in dog and primate models. Additionally, long–term transgene expressing animals did not exhibit other adverse effects such as tumour formation due to possible insertional mutagenesis. These long–term safety data on rAAV transgene expression in large animals are encouraging for futur ocular gene therapy applications.

Keywords: gene transfer/gene therapy • retina • retinal pigment epithelium