Characterization of the limitations to gene transfer and associated inflammation following intravitreal AAV injection in nonhuman primates

Abstract

Purpose : For gene therapy applications in the primate retina, intravitreal injections of adeno-associated virus (AAV) have been shown to transduce a limited amount of retina, mainly in the perifoveal region. The internal limiting membrane (ILM) has been previously hypothesized to be a barrier to broader transduction. Also, the inflammation associated with intravitreal injections has not been well described.

Methods : Rhesus macaques (N=13 eyes, total) were injected with AAV.CMV.GFP.WPRE via a regular intravitreal injection (N=3, 10¹¹ particles, 2/2 or Anc80 capsids) or via a subretinal injection (N=8, 10¹⁰ particles, 2/5, 2/9, or Anc80 capsids). In two additional eyes, the ILM was first surgically removed followed by concentrated application of 10¹¹ particles of AAV 2/2 onto the peeled area. This “peel and puddle” procedure consisted of a vitrectomy, posterior hyaloid separation, ILM staining, ILM peeling over the majority of the macula, fluid-air exchange, and deposition of concentrated virus on the peeled area with 30 minutes of supine positioning.

Results : The brightness and extent of GFP reporter gene expression was much greater with the peel/puddle technique than with a regular intravitreal injection. In the peel/puddle eyes, fluorescence was noted in an area that matched the extent of the ILM peel, as shown by imaging and histology. In all eyes, inflammation in the first 2 weeks was mild or moderate and self-resolving. At weeks 3-5, all of the regular intravitreal eyes (N=3) and the peel/puddle eyes (N=2) developed vitritis. The most severe eye also developed hyphema and cataract. In all other eyes, the inflammation was well controlled with 4mg of intravitreal Triescence. Similar amounts of late inflammation were seen between the intravitreal and peel/puddle eyes. None of the eyes injected with 10¹⁰ particles subretinally developed vitritis.

Conclusions : The vitreous, ILM, and/or physical parameters of the vector delivery significantly limit gene transfer to the retina. Surgical minimization of these barriers demonstrate highly increased levels and distribution of transduction. Importantly, our studies demonstrate intravitreal injection of AAV to potentially lead to clinically significant inflammation at doses normally tolerated in the subretina.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

 

GFP expression across the macula using the peel/puddle technique