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Abstract
There is much debate on the adeno-associated virus (AAV) serotype that best targets specific retinal cell types and the route of surgical delivery—intravitreal or subretinal. This study compared three of the most efficacious AAV vectors known to date in a mouse model of retinal degeneration (rd1 mouse) and macaque and human retinal explants. Green fluorescent protein (GFP) driven by a ubiquitous promoter was packaged into three AAV capsids: AAV2/8(Y733F), AAV2/2(quad Y-F) and AAV2/2(7m8). Overall, AAV2/2(7m8) transduced the largest area of retina and resulted in the highest level of GFP expression, followed by AAV2/2(quad Y-F) and AAV2/8(Y733F). AAV2/2(7m8) and AAV2/2(quad Y-F) both resulted in similar patterns of transduction whether they were injected intravitreally or subretinally. AAV2/8(Y733F) transduced a significantly smaller area of retina when injected intravitreally compared with subretinally. Retinal ganglion cells, horizontal cells and retinal pigment epithelium expressed relatively high levels of GFP in the mouse retina, whereas amacrine cells expressed low levels of GFP and bipolar cells were infrequently transduced. Cone cells were the most frequently transduced cell type in macaque retina explants, whereas Müller cells were the predominant transduced cell type in human retinal explants. Of the AAV serotypes tested, AAV2/2(7m8) was the most effective at transducing a range of cell types in degenerate mouse retina and macaque and human retinal explants.
Highlights
Inherited retinal degenerations are a leading cause of blindness in the working-age population of industrialised countries.[1]
This study found that intravitreal and subretinal injections were effective for AAV2/2(quad Y-F) and AAV2/2(7m8), but the subretinal route was more effective for AAV2/8(Y733F)
Three recombinant associated virus (AAV) vectors were produced by packaging the same expression cassette, consisting of Green fluorescent protein (GFP) driven by a ubiquitous CAG synthetic promoter with an SV40 poly(A) sequence, into three different AAV capsids: AAV2/8(Y733F), AAV2/2(quad Y-F) and AAV2/2(7m8)
Summary
Inherited retinal degenerations are a leading cause of blindness in the working-age population of industrialised countries.[1] Gene therapy is a therapeutic approach that has great potential to slow or reverse blinding retinal degeneration by delivering a normal copy of a mutated gene[2,3] (gene supplementation), editing the mutated gene[4] (for example, using CRISPR/Cas9), knocking down the expression of a mutant allele using RNA interference[5] or expressing neuroprotective factors.[6]. Adeno-associated virus (AAV) is the vector of choice for most retinal gene therapy applications where the transgene is relatively small because of its established record of safety and efficacy in preclinical studies and clinical trials.[2,3,7,8] The efficacy of a vector is measured by both the efficiency with which the genetic cargo is delivered and its specificity for the target cell type (its tropism). Subsequent work showed further increased transduction efficiency and a wider tropism by mutating two to seven surface tyrosine residues of AAV2/2.(ref. 10)
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