Targeting Photoreceptors via Intravitreal Delivery Using Novel, Capsid-Mutated AAV Vectors

Abstract

Development of viral vectors capable of transducing photoreceptors by less invasive methods than subretinal injection would provide a major advancement in retinal gene therapy. We sought to develop novel AAV vectors optimized for photoreceptor transduction following intravitreal delivery and to develop methodology for quantifying this transduction in vivo. Surface exposed tyrosine (Y) and threonine (T) residues on the capsids of AAV2, AAV5 and AAV8 were changed to phenylalanine (F) and valine (V), respectively. Transduction efficiencies of self-complimentary, capsid-mutant and unmodified AAV vectors containing the smCBA promoter and mCherry cDNA were initially scored in vitro using a cone photoreceptor cell line. Capsid mutants exhibiting the highest transduction efficiencies relative to unmodified vectors were then injected intravitreally into transgenic mice constitutively expressing a Rhodopsin-GFP fusion protein in rod photoreceptors (Rho-GFP mice). Photoreceptor transduction was quantified by fluorescent activated cell sorting (FACS) by counting cells positive for both GFP and mCherry. To explore the utility of the capsid mutants, standard, (non-self-complementary) AAV vectors containing the human rhodopsin kinase promoter (hGRK1) were made. Vectors were intravitreally injected in wildtype mice to assess whether efficient expression exclusive to photoreceptors was achievable. To restrict off-target expression in cells of the inner and middle retina, subsequent vectors incorporated multiple target sequences for miR181, an miRNA endogenously expressed in the inner and middle retina. Results showed that AAV2 containing four Y to F mutations combined with a single T to V mutation (quadY−F+T−V) transduced photoreceptors most efficiently. Robust photoreceptor expression was mediated by AAV2(quadY−F+T−V) −hGRK1−GFP. Observed off-target expression was reduced by incorporating target sequence for a miRNA highly expressed in inner/middle retina, miR181c. Thus we have identified a novel AAV vector capable of transducing photoreceptors following intravitreal delivery to mouse. Furthermore, we describe a robust methodology for quantifying photoreceptor transduction from intravitreally delivered AAV vectors.

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

“…It has been established that AAV2 accumulates at the vitreoretinal junction, presumably via its interaction with HS receptors within the ILM (23). This scenario is supported by the observation that AAV serotypes that bind sialic acid (e.g., AAV5), which is absent from the ILM, do not accumulate (23,30). AAV5 fails to transduce healthy/intact rodent retina when delivered by Ivt injection, despite being highly efficient in RPE and photoreceptors when delivered directly to the subretinal space (31)(32)(33).…”

“…The rational design of the AAV capsid to avoid proteosomal degradation enhances transduction efficiencies (30,(35)(36)(37)(38). This is accomplished by the substitution of surface-exposed residues that otherwise would be phosphorylated and thereby target the capsid for ubiquitin-dependent proteasomal degradation.…”

La conception de vecteurs adaptés à la thérapie génique oculaire

“…Lorsque les propriétés recherchées ne sont pas présentes dans les sérotypes naturels, l'ingénierie de la capside du virus peut être utilisée pour répondre à nos besoins en thérapie génique. d'améliorer cette efficacité [30]. Ces observations suggèrent que la poursuite de l'optimisation de la capside du virus adéno-associé, par modification des résidus d'acides aminés impliqués dans la phosphorylation, est une piste importante pour augmenter l'efficacité de transfert de gènes.…”

Restoration of synaptic function in sight for degenerative retinal disease

“…This route of injection is a routine and safe procedure in ophthalmic surgery that is performed under local anesthesia. While penetration of AAV vectors from the vitreous into the outer retinal layers has been a major obstacle in early studies, recent capsid modifications have yielded vectors with improved ability to efficiently transduce outer retinal cell types, including photoreceptors and bipolar cells in the mouse retina (26,27). It has yet to be shown whether penetration and efficacy of outer retinal cell transduction after intravitreal injection will show comparable results in the human retina and whether this route of injection will be suffi-…”