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M. Natkunarajah, P. Trittibach, A. Smith, Y. Duran, J. McIntosh, A. Nathwani, R. Ali; Evaluation of Transduction Efficiency of Full Length and Self Complementary Recombinant AAV2-8 With Pseudotypes 2-2 and 2-5 in the Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1674. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Recombinant AAV (rAAV) mediated gene transfer has been successfully used to treat a number of animal models of retinal disease, and the vector has recently been approved for use in clinical trials of gene therapy for inherited retinal degeneration. In this study we have tested a novel AAV pseudotype, rAAV2-8, which has unknown intraocular cell tropism and have also compared self-complementary rAAV 2-2, 2-5 and 2-8 and their corresponding single stranded counterparts with regard to kinetics and efficiency of reporter gene expression.
We performed subretinal, anterior chamber and intravitreal injections of rAAV2-2, 2-5 and 2-8, and their self-complementary counterparts. All vectors contained the CMV promoter expressing green fluorescent protein (GFP). We compared the temporal and cell specific transduction profile using Image Pro Software to quantify GFP luminance on fundus photographs and retinal sections.
Following subretinal administration, rAAV2-8 and scAAV2-8 transduce RPE and photoreceptors more efficiently than rAAV2-2, scAAV2-2, rAAV2-5 and scAAV2-5. GFP expression was noted at day 1 post injection with either rAAV2-8 or scAAV2-8 vs. day 3 for all other vectors. Peak GFP luminance was around day 50 post injection, with scAAV2-8 displaying faster kinetics of expression than rAAV2-8. Expression of GFP using rAAV 2-2, sc AAV2-2, rAAV2-5 and scAAV2-5 showed much lower luminance levels by comparison. GFP expression from rAAV2-2 and scAAV2-2 (at 8weeks) and rAAV2-5 and scAAV2-5 (at 5 weeks) showed fewer photoreceptor and RPE cells transduced compared with rAAV2-8 and scAAV2-8 (at 5weeks). No cell transduction was visible using rAAV2-8 administration via an anterior chamber or intravitreal approach.ConclusionrAAV2-8 is more efficient and has a faster onset of reporter gene expression in RPE and photoreceptor cells than rAAV2-2 and 2-5. Self-complementary vectors showed faster expression profiles and higher transduction efficiency than their single stranded counterparts, confirming complementary strand synthesis being a rate limiting step in vivo. These results suggest that self-complementary vectors and AAV-2-8, in particular, may offer new possibilities for the treatment of retinal disorders by gene therapy.
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