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M. S. Gorbatyuk, A. Schwein, W. W. Hauswirth, A. S. Lewin; Delivery of an siRNA-Resistant Rhodopsin Gene as Part of an Allele Independent Gene Therapy for Autosomal Dominant Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1979.
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© ARVO (1962-2015); The Authors (2016-present)
The goal of this project is to develop a gene therapy applicable to heterogeneous rhodopsin (RHO) mutations responsible for autosomal dominant retinitis pigmentosa (ADRP). We have already shown that small interfering RNA 301 (siRNA301) delivered by Adeno-associated virus (AAV) suppresses rhodopsin synthesis in vivo. The purpose of this study is to create a RHO allele tolerant to siRNA301 and find optimal expression conditions to deliver the RHO301 gene to the ADRP retina.
We used the site directed mutagenesis to place mismatches in the RHO sequence. The transfection of HEK 293 cell with the plasmid carrying RHO301 gene under control of CMV promoter was performed. The RHO protein production was verified by immunostaining analysis. To confirm the siRNA resistance of the RHO301 gene we cotransfected cultured cells with plasmids carrying wild type or RHO301 genes and siRNA301. The RHO mRNA level was compared by quantitative RT-PCR. To deliver the RHO301 in vivo the gene was inserted in a self-complementary AAV (scAAV) vector under the control of a mouse opsin promoter. ADRP animal models such as P23H human transgenic mice and P23H transgenic rats were injected at postanatal day 15. Electroretinography (ERG) was used to detect changes in the vision of animals.
We constructed the RHO301 gene by placing five silent mutations in the mouse RHOcDNA. In a tissue culture test of RHO301 tolerance, siRNA301 did not degrade RHO301 mRNA, but the wild-type RHO was sensitive to this siRNA. After transfer to the mouse retina using scAAV5, the mouse opsin promoter efficiently drove RHO gene expression in photoreceptors. Scotopic ERG analysis of injected animals bearing a P23H transgene demonstrated the preservation of a- and b-waves amplitudes in RHO301 injected eyes by 40 and 28%, respectively.
SiRNA resistance of the RHO301 gene was achieved through 5 silent mutations. RHO cDNA was efficiently expressed in mice following AAV-gene transfer. Unexpectedly, increased expression of this rhodopsin led to partial protection from retinal degeneration in transgenic mice and rat models of ADRP.
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