Abstract
Purpose:
We are interested in characterizing and treating the rapidly degenerating autosomal dominant retinitis pigmentosa (ADRP) mouse model, T17M RHO. Photoreceptors in this transgenic strain are highly susceptible to light damage. I hypothesize that the knockdown of transducin 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 can inhibit the phototransduction cascade in rod cells preventing apoptosis resulting from exposure to bright light.
Methods:
gene (GNAT1). Two small hairpin RNAs (shRNAs) were designed to target homologous regions between mouse and dog GNAT1. Transfections were done in HEK293 cells (n=6) using CMV-GNAT1 and H1-shRNA GNAT1 given at 1:0, 1:2, 1:4, and 1:6 ratios. Using a plasmid with a control miRNA, total amount of DNA transfected at each ratio was kept constant. The amount of transducin produced was observed at 24 and 72 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. Following the in vitro knock down assay, each shRNA was inserted into an a plasmid containing GFP under the expression of a mouse opsin promoter and packaged into adeno associated virus serotype 8 with a capsid modification, AAV8 (Y733F).
Results:
siRNA GNAT1(a) showed the greatest knockdown with 76%, 71.4% and 69.1% of the GNAT mRNA remaining at the 1:2, 1:4 and 1:6 ratios, respectively, after 72 hours.
Conclusions:
We demonstrated siRNA GNAT1(a) to be a viable siRNA for testing in T17M RHO transgenic mice. Both shRNAs (a) and (b) targeting GNAT1 are being packaged into AAV8 (Y733F) for in vivo analysis in mice. Once we verify reduction of transducin-α in these mice, we will expose them to clinical light levels and measure retinal preservation. Since the siRNAs also target dog transducin, they will be tested in the T4R dog model of ADRP which is also unusually sensitive to light damage.
Keywords: 702 retinitis •
538 gene transfer/gene therapy