The Human Rhodopsin Kinase Promoter in an AAV5 Vector Confers Rod- and Cone-Specific Expression in the Primate Retina

Abstract

Adeno-associated virus (AAV) has proven an effective gene delivery vehicle for the treatment of retinal disease. Ongoing clinical trials using a serotype 2 AAV vector to express RPE65 in the retinal pigment epithelium have proven safe and effective. While many proof-of-concept studies in animal models of retinal disease have suggested that gene transfer to the neural retina will also be effective, a photoreceptor-targeting AAV vector has yet to be used in the clinic, principally because a vector that efficiently but exclusively targets all primate photoreceptors has yet to be demonstrated. Here, we evaluate a serotype 5 AAV vector containing the human rhodopsin kinase (hGRK1) promoter for its ability to target transgene expression to rod and cone photoreceptors when delivered subretinally in a nonhuman primate (NHP). In vivo fluorescent fundus imaging confirmed that AAV5-hGRK1-mediated green fluorescent protein (GFP) expression was restricted to the injection blebs of treated eyes. Optical coherence tomography (OCT) revealed a lack of gross pathology after injection. Neutralizing antibodies against AAV5 were undetectable in post-injection serum samples from subjects receiving uncomplicated subretinal injections (i.e., no hemorrhage). Immunohistochemistry of retinal sections confirmed hGRK1 was active in, and specific for, both rods and cones of NHP retina. Biodistribution studies revealed minimal spread of vector genomes to peripheral tissues. These results suggest that AAV5-hGRK1 is a safe and effective AAV serotype/promoter combination for targeting therapeutic transgene expression protein to rods and cones in a clinical setting.

AAV-Mediated Gene Therapy in the Guanylate Cyclase (RetGC1/RetGC2) Double Knockout Mouse Model of Leber Congenital Amaurosis

“…The only reports of AAV8-mediated photoreceptor expression in NHP indicated that AAV8 at (1 · 10 10 vg) did not efficiently transduce foveal, parafoveal, or perifoveal cones following subretinal injection (Vandenberghe et al, 2011a(Vandenberghe et al, 2011b. In contrast, AAV5 efficiently transduces cones in all these areas of NHP retina at comparable vector concentration (Boye et al, 2012). Hence, assuming a subretinal delivery route, the evidence to date would support the use of AAV5 in conjunction with the GRK1 promoter for driving GC1 expression in a clinical setting.…”

“…Multiple studies have shown that the human rhodopsin kinase (hGRK1) promoter drives efficient and rod/cone-specific transgene expression in mice (Khani et al, 2007;Boye et al, 2010Boye et al, , 2011Pawlyk et al, 2010;Sun et al, 2010;Mihelec et al, 2011) and most recently in nonhuman primates (Boye et al, 2012). Notably, within the NHP retina, the hGRK1 promoter was active in foveal cones (Boye et al, 2012). Viral serotype choice is dependent upon the route of administration as transduction profiles differ greatly depending on where the vector is deposited (Vandenberghe and Auricchio, 2012).…”

Impact of Heparan Sulfate Binding on Transduction of Retina by Recombinant Adeno-Associated Virus Vectors

“…Mice were injected intravitreally with 4 ϫ 10 10 vg (high dose) or 4 ϫ 10 9 vg (low dose) or subretinally with 1 ϫ 10 10 vg (high dose) or 2 ϫ 10 9 vg (low dose). Although C57Bl/6J mice do not have sortable photoreceptors and, thus, FACS cannot be used as described above, hGRK1 has been shown to restrict transgene expression to rods and cones following subretinal injection (14,57). Representative fundus images and retinal cross sections revealed a clear dose response for each vector following either injection route (Fig.…”

“…Data described above revealed that both AAV2(quadY-FϩT-V) and AAV2(4pMut) transduce rod photoreceptors following Ivt injection. We next quantified the relative performance of these variants by utilizing a construct containing the photoreceptor-specific human rhodopsin kinase (hGRK1) promoter driving mCherry in C57Bl/6J mice (14,57). Mice were injected intravitreally with 4 ϫ 10 10 vg (high dose) or 4 ϫ 10 9 vg (low dose) or subretinally with 1 ϫ 10 10 vg (high dose) or 2 ϫ 10 9 vg (low dose).…”

“…However, the majority of inherited retinal diseases are caused by defects in photoreceptors, highlighting the need to identify serotypes capable of transducing this cell type. We and others have shown that various subretinally delivered AAV serotypes are capable of efficient transduction of photoreceptors in nonhuman primates (NHP) (14)(15)(16)(17)(18)(19). However, subretinal injection of AAV2 under the fovea of some LCA2 patients led to central retinal thinning and loss of visual acuity (20).…”