Comparison of Efficacy of Various shRNAs Targeting Human RHO mRNA in Cultured Cells as Model Therapeutics for Autosomal Dominant Retinitis Pigmentosa

Abstract

Purpose : Compare efficacy and properties shRNAs designed against human rhodopsin (hRHO) mRNA in a human cell culture environment with the goal of developing a potential post-transcriptional gene silencing (PTGS) therapeutic agent for autosomal dominant retinitis pigmentosa (adRP).

Methods : The expression of full length hRHO including 5`UTR and 3`UTR (1^st polyA signal) was driven by CMV promoter and shRNAs with H1 promoter in HEK293S cells. hRHO mRNA was quantified using the DDCt method relative to actin mRNA control. Alexa647-labeled anti-hRHO-1D4 antibody was used to measure rhodopsin protein in cells using Quantitative Imaging platform (QIP). Data was statistically evaluated using ANOVA and post-hoc t-tests with criterion standard of significance (p≤0.05). Accessibility in regions around the binding sites in hRHO were determined by multiparameter prediction of mRNA accessibility (mppRNA). Potential off-site targeting by each shRNA was determined by BLAST-N for the full length and “seed” nucleotides.

Results : Various shRNAs from this lab and other published sources were compared for their capacity to suppress hRHO mRNA in HEK293S cells. Computationally predicted accessibility in the different targeting regions varied among shRNAs. There were few off-targets identified in the human transcriptome for the 19 nt full shRNA elements in BLAST-N. All shRNAs exerted statistically significant knockdown of hRHO mRNA relative to scrambled or irrelevant targeting element controls. We designed and characterized shRNA-725 and shRNA-906 and compared them to published shRNA-BB1 and shRNA-Q1 (O’Reilly M. et al., 2007). ShRNA-725 suppresses hRHO mRNA (p=6.53E-10) and protein (p=2.93E-7) by over 90% compared to shRNA-scramble. ShRNA-906 showed the most hRHO mRNA transcription inhibitory effect of over 95% (p=7.14E-4) compared to other shRNAs tested.

Conclusions : We identified two highly effective shRNAs (725 and 906) in predicted accessible regions of hRHO as lead candidate PTGS therapeutic agents to further evaluate in our humanized mouse model of adRP. There remains concern that “seed” sequences at 5’ positions 2-7 in any shRNA could cause a plethora of off-target effects (Gu S et al. 2014; Jackson A et al. 2006).

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.