Novel recombinant Adeno-Associated Virus transfects photoreceptor cells following intravitreal injection in sheep.

Abstract

Purpose : Subretinal (SR) delivery of recombinant Adeno-Associated Viruses (AAV) resulted in successful treatment of several inherited retinal degenerations in humans and animal models. However, due to potential complications, drawbacks and challenges of SR injections, there is an impetus to develop less invasive delivery methods such as intravitreal (IVT) injections. In the current study we evaluated whether an engineered AAV2-7m8 vector, with improved retinal penetration properties, can transduce cones following IVT injection in sheep.

Methods : Surgeries were performed on seven AAV2 seronegative, ophthalmologically normal five-month-old Afec-Assaf sheep. We used an engineered AAV2-7m8 vector that differs from its parental serotype AAV2 by a peptide insertion. The vector, carrying GFP under the control of a ubiquitous (CAG) promoter, was injected IVT in five eyes. The same vector, carrying GFP under the control of a cone specific (pr1.7) promoter, was injected IVT in 6 eyes. SR injections of AAV2-7m8 with both promoters served as positive controls. GFP expression was evaluated in vivo every four weeks by fundus photography using a fluorescein angiography lens. Four months post-treatment sheep were sacrificed and GFP expression confirmed by immunohistochemistry (IHC).

Results : Four weeks post-treatment, in vivo imaging of SR-injected eyes demonstrated substantial fluorescence indicating high GFP expression. Eyes injected IVT with AAV2-7m8-CAG-GFP began showing fluorescence on in vivo imaging eight weeks post-treatment, while in eyes treated IVT with AAV2-7m8-pr1.7-GFP first signs of GFP expression were evident 12 weeks post-treatment. IHC confirmed retinal GFP expression in SR positive controls and IVT-treated eyes. IVT injection of AAV2-7m8-CAG-GFP resulted in GFP expression in the ganglion cell layer, while its SR injection resulted in expression in RPE cells and photoreceptors. Both SR and IVT injections of AAV2-7m8-pr1.7-GFP resulted in GFP expression in cones. In vivo imaging and IHC demonstrated a more robust GFP expression in SR than in IVT injected eyes.

Conclusions : IVT-injected AAV2-7m8 successfully penetrated the ovine retina, leading to photoreceptor transduction. The efficacy of IVT delivery of AAV2-7m8 as a potential treatment of achromatopsia will be further investigated in our day blind sheep model.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.