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Ryan F Boyd, Sanford L Boye, Shannon Boye, William W Hauswirth, Andras M Komaromy, Simon M Petersen-Jones, Joshua T Bartoe; Photoreceptor-Targeted Gene Delivery Using Intravitreally Administered AAV Vectors in Dogs. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3331.
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© ARVO (1962-2015); The Authors (2016-present)
Effective delivery of therapeutic transgenes to retinal photoreceptors using adeno-associated virus (AAV) vectors has traditionally required subretinal injection. Photoreceptor transduction ability following intravitreal AAV injection has improved in rodent models through the use of vectors containing capsid amino acid substitutions; but remains limited in large animal models. Our study compared the transduction efficiency of two novel GFP-expressing AAV vectors containing multiple capsid amino acid substitutions following intravitreal injection in dogs. The ability of two photoreceptor-specific promoters to drive targeted transgene expression was also evaluated.
Six normal dogs were used. All AAV vectors were intravitreally injected at the inner surface of the retina at a dose of 4 x 1011 vector genomes. Two AAV serotype 2/2 vectors were used, one with four capsid Tyr-to-Phe substitutions, and the other with four Tyr-to-Phe and one Thr-to-Val substitution. Each vector genome contained a GFP reporter gene driven by either the interphotoreceptor binding protein (IRBP) promoter or the transducin subunit alpha-2 (GNAT2) promoter with an IRBP enhancer; resulting in four different vector constructs. Each vector construct was injected into three eyes (4 vectors X 3 eyes = 6 dogs). In vivo fundoscopic imaging was performed for a period of eight weeks to monitor GFP expression. Immunohistochemistry was performed on retinal cryosections to evaluate the transduction efficiency and tropism of the vectors.
The two AAV vectors packaged with the IRBP promoter drove expression in rod and cone photoreceptors exclusively, without significant differences in the number of cells transduced. Overall retinal transduction efficiency for both AAV vectors containing the IRBP promoter was approximately 4% of cones and 2% of rods. A significantly higher level of photoreceptor transduction occurred in regions underlying retinal vasculature, with local transduction efficiencies of up to 31% of cones and 14% of rods. Vectors carrying the GNAT2 promoter produced cone-specific GFP expression, although this occurred at a low level that was not quantifiable.
AAV vectors packaged with the IRBP promoter limit intraocular transgene expression to retinal photoreceptors alone following intravitreal delivery in dogs. Overall retinal transduction is limited; but local regions exhibit increased efficiency.
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