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Michael Massengill, Deep Patel, William A Beltran, Alfred S Lewin; Tandem Delivery of Short Hairpin RNAs and Rhodopsin cDNA to Combat Retinal Degeneration in Autosomal Dominant Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4082.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in rhodopsin (RHO) yield an inherited form of blindness known as autosomal dominant Retinitis Pigmentosa (adRP). We previously showed that delivery of a short hairpin RNA (shRNA) to degrade endogenous RHO and a degradation-resistant (hardened) RHO cDNA with recombinant adeno-associated virus (rAAV) preserves vision in an adRP mouse expressing P23H mutant RHO. To enhance the therapeutic efficacy of our strategy, we identified shRNAs with superior knockdown efficiency of RHO mRNA. The purpose of this study was to generate rAAV vectors expressing these shRNAs and a corresponding hardened RHO cDNA. We also sought to enhance expression of the hardened RHO cDNA by including the rhodopsin enhancer region (RER).
shRNAs were designed utilizing the Whitehead Institute’s siRNA Selection. Generation of hardened-human RHO cDNA was achieved with the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent). We measured expression of RHO mRNA by qRT-PCR. Wild-type C57BL6 were included for in vivo testing. Statistical significance (p<0.05) was determined via 2-way ANOVA.
Three shRNAs (131, 134, 820), each capable of targeting the degradation of human wild-type and mutant (T17M, P23H) RHO mRNA, were previously incorporated within a H1 promoter-shRNA cassette and cloned in to rAAV2 plasmids. Two hardened RHO cDNAs (RHO134, RHO820) were generated by creating silent mutations in the shRNA binding site. The hardened RHO cDNAs were cloned in to the rAAV2 plasmids already containing a shRNA. The resulting combination plasmid was able to simultaneously degrade GFP-tagged human RHO mRNA and express hardened RHO mRNA in vitro. To increase expression of the hardened RHO cDNA, we generated a rAAV2/AAV5 vector containing the rhodopsin enhancer region (RER) in addition to the RHO820 cDNA. Inclusion of the RER in the rAAV2/AAV5 vector did not significantly affect the production of RHO820 mRNA after subretinal injection in wild-type mice.
We generated rAAV vectors containing shRNAs and hardened RHO cDNA that will be tested in rapidly degenerating models of adRP, such as the T17M mouse and T4R dog. The RER did not significantly affect expression of the transgene when included in the rAAV2/AAV5 vector.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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