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Brian P Rossmiller, Haoyu Mao, Alfred S Lewin; Gene therapy with self-complementary recombinant AAV in models of autosomal dominant retinitis pigmentosa caused by RHO mutations. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3304.
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Retinitis pigmentosa is the leading hereditary cause of blindness with 30-40% of cases attributable to autosomal dominant retinitis pigmentosa (ADRP). 30% of ADRP arises in the rhodopsin (RHO). We propose knocking down of endogenous RHO and replacing it with a “hardened” copy, RHO with nucleotide changes that preserve the amino acid sequence but decrease efficiency of knock-down. Here we report the use of a scAAV serotype 8 (Y733F) to express a hardened human rhodopsin (hRHO) and either miRNA or hammerhead ribozyme (rz) under the control of the human opsin proximal promoter (HOPS).
Four different knock-down methods were tested, Rz407 and Rz525, miRNA301 and shRNA301 against both the wild-type and hardened RHO target regions. The transfections were done in HEK293 cells (n=6) with (1) the target plasmid psiCheck2 dual luciferase containing RHO target region, (2) plasmid expressing the shRNA, miRNA or Rz against the target region and (3) a control miRNA. The reduction in expression of luciferase was measured at 24 and 48 hours post transfection. The mouse models of ADRP used included human P23H RHO transgenic (n=12), and Rho I307N (n=10). Both were given subretinal injections of AAV-HOP-hRHO and four different RNA knockdown agents (shRNA301, miRNA301, Rz407 or Rz525) in one eye and AAV-HOP-GFP in the other. The mice were followed using electroretinogram and optical coherence tomography.
The knock-down results show shRNA301 and Rz525 to cause the largest reduction of RHO mRNA. One month post injection, there was no statistically significant difference between P23H RHO eyes injected with AAV-hRHO-miRNA301 and those injected with AAV-HOPS-GFP. We noted a 50% increase in a-wave amplitudes in I307N Rho eyes injected with AAV-hRho-Rz407 and those injected with AAV-HOPS-GFP, but this difference did not reach statistical significance.
We have generated a series of combination RNA knockdown and replacement AAV vectors that may be useful for the treatment of ADRP. At early time points, our tests of these specific vectors have not been conclusive. The injected mice will be followed for longer intervals and additional mice will be added to the study to determine if the difference in visual function of the experimentally treated eyes versus the control is statistically significant.
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