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Srini Goverdhan, Alun R. Barnard, Mandeep Singh, Peter Charbel Issa, Robert E. MacLaren; Characterization Of A Novel Gene Therapy Reporter Vector (rAAV2/5-CBA-CNGA3-IRES-GFP) In A Mouse Model Of Achromatopsia. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1921.
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© ARVO (1962-2015); The Authors (2016-present)
AAV mediated gene therapy holds promise as a potential clinical treatment for a variety of inherited retinal degenerations. Quantifying the effects of gene transfer on cone photoreceptor function in rodent models of human disease can however be challenging as these cells are sparse. The CNGA3 (alpha 3 subunit of the cone cyclic nucleotide-gated cation channel) knock-out mouse has a loss of function and a slow degeneration, similar to a human cone dystrophy. The purpose of this study was to assess a new AAV bicistronic expression cassette that both replaces CNGA3 and expresses green fluorescent protein (GFP) in order to allow the morphology of transduced cones to be studied in detail.
AAV2/5 vectors were generated with the gene encoding GFP coupled bicistronically downstream to the gene encoding CNGA3 by an internal ribosome entry site (IRES) and driven by a chicken beta-actin (CBA) promoter. Mice received subretinal injections at 3 weeks with 2µl of rAAV2/5-CBA-CNGA3-IRES-GFP (5x1011 vector particles) in the study eye and 2µl of rAAV2/5-CBA-GFP (5x1011 vector particles) in the control eye. SLO autofluorecence retinal imaging (Heidelberg OCT) was assessed 4 weeks post-injection for both CNGA3-IRES-GFP treated and GFP control eyes.
SLO autofluorecence showed comparable GFP expression for both CNGA3-IRES-GFP treated and GFP control eyes, indicating that IRES-translation from this construct was at least as good as a standard Kozac consensus. Histology of retinal sections at 7 weeks allowed identification of cone photoreceptors by CNGA3 immunostaining in the outer segment and GFP in the cytoplasm and inner segment. The presence of cytoplasmic GFP allowed the morphology of transduced cones and in particular, the cone pedicle synapses, to be studied in detail. In the control eyes at this age only occasional surviving cone cells could be identified.
An IRES-GFP linked construct is useful for assessing the effects of gene therapy on cone survival in mice in vivo by allowing SLO imaging to confirm successful gene transfer. Histologically the presence of GFP assists in studying proximal structural changes in the cone pedicle synapse after successful gene transfer in individual cells, which would otherwise be difficult to trace from the distal outer segments, where transgenic CNGA3 protein is localized.
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