September 2016
Volume 57, Issue 12
ARVO Annual Meeting Abstract  |   September 2016
Gene Therapy Utilizing Short Hairpin RNAs for Autosomal Dominant Retinitis Pigmentosa
Author Affiliations & Notes
  • Michael Massengill
    University of Florida, Gainesville, Florida, United States
  • William W Hauswirth
    University of Florida, Gainesville, Florida, United States
  • Alfred S Lewin
    University of Florida, Gainesville, Florida, United States
  • Footnotes
    Commercial Relationships   Michael Massengill, University of Florida (P); William Hauswirth, AGTC, Inc. (I), University of Florida (P); Alfred Lewin, AGTC, Inc. (C), University of Florida (P)
  • Footnotes
    Support  NIH grants: R24 EY022012-04 and P30-EY021721
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 139. doi:
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      Michael Massengill, William W Hauswirth, Alfred S Lewin; Gene Therapy Utilizing Short Hairpin RNAs for Autosomal Dominant Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2016;57(12):139.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose : Autosomal dominant RP (adRP) caused by mutant rhodopsin (RHO) is incurable. We previously showed that tandem delivery of a short hairpin RNA (shRNA) to degrade endogenous RHO and a hardened RHO cDNA with recombinant adeno-associated virus (rAAV) preserves vision in a mouse model of adRP with P23H mutant RHO. To date, our attempts to extend this methodology to rapidly degenerating mouse models of adRP with either T17M or I307N mutant RHO have been unsuccessful. The goal of this study was to design rAAV vectors containing shRNAs with high knockdown efficiency of RHO to extend our approach to other models of adRP with mutant RHO.

Methods : We designed ten siRNAs to target both human and dog RHO. Co-transfections of siRNAs or shRNA plasmids and a plasmid expressing GFP-tagged human RHO were performed with Lipofectamine in 293T cells. qRTPCR and flow cytometry (n=3) 48 hours post-transfection were utilized to measure percent knockdown of RHO. Non-targeting siRNAs or shRNA plasmids served as controls. Statistical significance (p<0.05) was determined via 1-way ANOVA followed by a Student-Newman-Keuls Test. Experiment1: siRNAs were transfected at concentrations of 10, 20, & 40nM with a plasmid encoding GFP-tagged human RHO (500nM). Experiment 2: Three siRNAs were selected and their sequences were expressed as small hairpin RNA (shRNA) driven by the H1 promoter. Each was transfected at 200, 400, and 800nM with GFP-tagged human RHO (400nM). Experiment 3: The H1-shRNA cassettes were cloned in rAAV plasmids and were transfected at 800nM with either GFP-tagged WT, T17M or P23H human RHO.

Results : The ten siRNAs demonstrated knockdown efficiencies that ranged between 0 and 70% at the protein level. Interestingly, knockdown at the RNA level was similar among siRNAs. We selected siRNAs 1, 13, and 15 for further analysis. When incorporated in to a H1-shRNA cassette, shRNAs 1, 13, and 15 demonstrated statistically significant knockdown of 23.3% ± 10.1, 52.8% ± 9.7, and 73.9% ± 5.5, respectively, at 400nM when compared to control. The H1-shRNA cassettes maintained their biological activity when cloned in to rAAV2 and were effective at targeting GFP-tagged human RHO with mutations causing adRP (T17M, P23H).

Conclusions : We generated rAAV vectors containing shRNAs with superior knockdown efficiency that will be tested in other animal models of adRP, such as the T17M mouse and T4R dog.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.


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