Improved intravitreal AAV-mediated inner retinal gene transduction by surgical internal limiting membrane (ILM) peeling in cynomolgus monkeys

Abstract

Purpose : The adeno-associated virus (AAV) vector is an ideal tool for retinal gene therapy, and there are two administration routes, subretinal injection (SR) and intravitreal injection (IV). SR is used in many studies because of its efficiency of gene transduction of outer retina. However, SR induces an iatrogenic retinal detachment, which causes several adverse effects. On the other hand, IV is considered to be safer than SR, although the transduction efficiency in large animals was insufficient, as the vitreous and internal limiting membrane (ILM) acted as barriers to transduction. To overcome this issue, we performed vitrectomy (VIT) and ILM peeling before AAV vector injection.

Methods : Among the six eyes in the three female cynomolgus monkeys (aged 10-13 years old), two eyes received standard 3-port VIT (VIT group), two eyes received ILM peeling in addition to the standard 3-port VIT (VIT+ILM group), and the remaining two received no pretreatment (IV group) at 4 weeks before AAV injection. All eyes were penetrated by 30-G needle into the vitreous at the pars plana, and 50 μl of triple-mutated self-complementary AAV serotype 2 vector (1.9 × 10¹³ v.g./ml) encoding enhanced green fluorescent protein (GFP) was administered. Nineteen weeks after intravitreal injection of AAV vector, transduction efficiency was analyzed. To detect potential adverse effects, the retinas were monitored using color fundus photography, optical coherence tomography (OCT) and electroretinography (ERG).

Results : Little expression of GFP was detected in the IV and VIT groups. On the other hand, strong GFP expression was detected within the peeled ILM area in the VIT+ILM group. Intraocular inflammation was observed in 4 of 6 eyes, however the inflammation was transient and no morphological changes were detected on color fundus images or OCT. Transient reduction of ERG amplitude was detected during the period of intraocular inflammation.

Conclusions : These results indicate that surgical ILM peeling before intravitreal AAV vector administration would be safe and useful for efficient transduction of the nonhuman primate retina and provide therapeutic benefits for the treatment of glaucoma and retinal diseases.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.