Purchase this article with an account.
L. S. Carvalho, H. V. Tran, A. J. Smith, R. R. Ali; Development of Cone-Specific Promoters for Use Within Recombinant AAV Vectors. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3021.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To evaluate the efficacy of diverse cone-specific promoters with two different recombinant adeno-associated virus (rAVV) serotypes in targeting gene expression specifically to cone photoreceptors.
rAAV2/2 and rAAV2/8 vectors were constructed that contain a cone-specific promoter to direct eGFP expression The cone-specific promoters or promoter regions selected for this study were human Transducin (GNAT2), cone arrestin (CAR), CNGA3 and CNGB3. Each rAAV was injected into the subretinal space of adult wild type mice (C57/Bl6). Retinas were analysed by scanning laser ophthalmoscope and immunohistochemistry at 2, 3 and 6 weeks post-injections. Cone specific expression was shown by co-localization of eGFP fluorescence and cone-specific markers such as cone opsins and Transducin antibody staining.
rAAV2/8 transduction and could be detected by SLO imaging at 2 weeks post-injections while rAAV2 was visible from the 3 weeks time point irrespective of the type of promoter used. Immunohistochemistry results indicated that all the promoters tested here were capable of driving expression in both L/M- and S-cones using either AAV serotype, but with a higher expression was achieved using rAAV2/8. Expression levels of each promoter tested will also be quantified by real time PCR.
The results presented here indicate a considerable advantage of using rAAV8 for gene delivery in the eye in comparison to rAAV2 due to higher transduction efficiency and a more rapid onset of expression. Cone-specific transduction can be achieved using fragments of the GNAT2 and CAR promoters.
This PDF is available to Subscribers Only