Abstract
Purpose: :
We are interested in characterizing and treating the rapidly degenerating autosomal dominant retinitis pigmentosa (ADRP) mouse model, T17M. Work by our lab has shown this strain to be highly susceptible to light damage [1]. I hypothesize that the knockdown of transducin-a, in rods, will reduce rod degeneration and preserve cone function. It is, therefore, the purpose of this study to determine if siRNA mediated knockdown of transducin-a can inhibit the phototransduction cascade in rod cells preventing apoptosis resulting from exposure to bright light.
Methods: :
A cell culture assay was conducted to determine the siRNA with greatest knockdown of the transducin-a gene (GNAT1). Two small hairpin RNAs (shRNAs) were designed to target homologous regions between mouse and dog GNAT1. Transfections were done in HEK293 cells in triplicate using CMV-GNAT1 and H1-shRNA GNAT1 given at 1:0, 1:2, 1:4, and 1:6 ratios. Using a CBA-GFP expression plasmid as carrier, the total amount of DNA transfected was constant. The amount of transducin produced was observed at 48 hours post transfection using western blot with a mouse transducin specific antibody. The percent knockdown was then calculated using tubulin as the loading control and normalized against the control ratio which received only CMV-GNAT1 and CBA-GFP plasmids.
Results: :
siRNA GNAT1(b) showed the greatest knockdown 61.89%, 29.62% and 27.09% at the 1:2, 1:4 and 1:6 ratios, respectively, after 48 hours.
Conclusions: :
We demonstrated siRNA GNAT1(b) to be a viable siRNA for testing in T17M RHO transgenic mice. Knockdown will also be tested at 72 hours and confirmed through RT-PCR analysis of mRNA levels. Following in vitro analysis, the siRNA will be incorporated into a plasmid with AAV terminal repeats for packaging in AAV serotype 8. As the siRNAs were designed to target mouse and dog GNAT1, they may also be useful in the T4R dog model of ADRP which is also extremely sensitive to light damage.1. White, D.A., et al., Increased sensitivity to light-induced damage in a mouse model of autosomal dominant retinal disease. Invest Ophthalmol Vis Sci, 2007. 48(5): p. 1942-51.
Keywords: retinitis • gene transfer/gene therapy • retina