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. 2016 Feb;23(2):223-30.
doi: 10.1038/gt.2015.96. Epub 2015 Oct 15.

Photoreceptor-targeted gene delivery using intravitreally administered AAV vectors in dogs

R F Boyd  1 , D G Sledge  2 , S L Boye  3 , S E Boye  3 , W W Hauswirth  3 , A M Komáromy  1 , S M Petersen-Jones  1 , J T Bartoe  1
Affiliations

Photoreceptor-targeted gene delivery using intravitreally administered AAV vectors in dogs

R F Boyd et al. Gene Ther. 2016 Feb.

Erratum in

Abstract

Delivery of therapeutic transgenes to retinal photoreceptors using adeno-associated virus (AAV) vectors has traditionally required subretinal injection. Recently, photoreceptor transduction efficiency following intravitreal injection (IVT) has improved in rodent models through use of capsid-mutant AAV vectors; but remains limited in large animal models. Thickness of the inner limiting membrane (ILM) in large animals is thought to impair retinal penetration by AAV. Our study compared two newly developed AAV vectors containing multiple capsid amino acid substitutions following IVT in dogs. The ability of two promoter constructs to restrict reporter transgene expression to photoreceptors was also evaluated. AAV vectors containing the interphotoreceptor-binding protein (IRBP) promoter drove expression exclusively in rod and cone photoreceptors, with transduction efficiencies of ~4% of cones and 2% of rods. Notably, in the central region containing the cone-rich visual streak, 15.6% of cones were transduced. Significant regional variation existed, with lower transduction efficiencies in the temporal regions of all eyes. This variation did not correlate with ILM thickness. Vectors carrying a cone-specific promoter failed to transduce a quantifiable percentage of cone photoreceptors. The newly developed AAV vectors containing the IRBP promoter were capable of producing photoreceptor-specific transgene expression following IVT in the dog.

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Conflict of interest statement

CONFLICT OF INTEREST

The remaining authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Photoreceptor transduction efficiency following IVT of AAV2 (quad Y-F+T-V) IRBP and AAV2 (quad Y-F) IRBP. (a) Schematic demonstrating the visual streak (lime green oval) over which the cannula was targeted during injection, and regions from which sagittal cryosections were collected (blue vertical bars). Numbers (1–9) approximate regions from which individual transduction efficiencies were determined. Transduction efficiency was determined by dividing the number of GFP-positive rod or cone photoreceptors by the total number of outer nuclear layer cells in retinal images taken through a × 40 microscope objective. (b) Overall (values determined from regions 1–9 combined) mean transduction efficiency for eyes injected with AAV2 (quad Y-F+T-V) IRBP or AAV2 (quad Y-F) IRBP. Dark gray bars represent mean transduction efficiencies for cones and light gray bars represent rods; line bars represent s.e.m. No statistical differences were observed when the two vectors were compared (cones: P =0.43; rods: P=0.42). (c) Regional mean transduction efficiency for eyes injected with AAV2 (quad Y-F+T-V) IRBP and AAV2 (quad Y-F) IRBP. Statistical comparison data for retinal regions are shown in Table 2.
Figure 2
Figure 2
Localization of maximal photoreceptor transduction following IVT of AAV2 (quad Y-F+T-V) IRBP and AAV2 (quad Y-F) IRBP. Representative confocal scanning laser ophthalmoscopy images obtained at 6 weeks (a and b) and 8 weeks (c and d) following IVT demonstrate increased GFP fluorescence along the major retinal vasculature (solid arrows). Autofluorescence of the canine tapetum is responsible for the increased signal in the superior half of the images. Immunohistochemistry on cryosections shows a high level of rod and cone photoreceptor transduction directly underlying major retinal blood vessels for both AAV2 (quad Y-F) IRBP (e) and AAV2 (quad Y-F+T-V) IRBP (f). Scale bar, 50 µm. GCL, ganglion cell layer; hCAR, human cone arrestin; INL, inner nuclear layer; N, nasal; ONL, outer nuclear layer; RPE, retinal pigmented epithelium; T, temporal.
Figure 3
Figure 3
Photoreceptor-specific transduction following IVT of AAV2 (quad Y-F) IRBP and AAV2 (quad Y-F+T-V) IRBP. Representative photomicrographs demonstrate GFP expression was limited to rod and cone photoreceptors 8 weeks post-IVT (a, d). Transduction of L/M- and S-cone photoreceptor subtypes is demonstrated (white arrows) in panels (b, e), and (c, f), respectively. Co-labeling with the L/M-opsin and S-opsin antibodies is implied by approximation of a GFP-positive inner segment with an opsin-labeled outer segment in this confocal microscopy section. Scale bar, 50 µm. GCL, ganglion cell layer; hCAR, human cone arrestin; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigmented epithelium.
Figure 4
Figure 4
Assessment of regional retinal inner limiting membrane thickness. Representative photomicrographs of paraffin section from eyecups of normal dogs labeled with anti-laminin antibody (a, b) demonstrates thickness of the ILM (open arrows); subjectively, ILM was thinner in regions overlying large retinal vessels (solid arrow). There was limited variability of ILM thickness (c) across retinal regions 1–9 as defined in Figure 1a. Linear correlation analysis showed no correlation of ILM thickness with percent transduction of either cone (d) or rod photoreceptors (e). GCL, ganglion cell layer; ILM, inner limiting membrane; INL, inner nuclear layer; ONL, outer nuclear layer.
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