title: Alleviation of shRNA off-target effects via rAAV vector-encoded sense strand decoys
creator: Mockenhaupt, Stefan
subject: ddc-570
subject: 570 Life sciences
description: The use of exogenous triggers of RNAi such as short hairpin RNAs (shRNAs) in combination with viral vector-mediated gene delivery holds great potential for applications in gene therapy, in biotechnological processes as well as in basic research. Ideally, shRNAs only exert their activity via the antisense strand that binds and silences the designated target mRNA. However, the undesired sense strand can also be activated and hence holds a certain silencing potential. In principle, this may contribute to erratic off-targeting which typically occurs via imperfect binding and subsequent inhibition of untargeted cellular mRNAs. The aim of this study was to assess whether shRNA sense strand activity plays an important role in producing off-target effects and –if so- to find novel means of counteracting this activity.  Functional characterization of relative strand activities of different shRNAs indeed revealed high levels of undesired sense strands activity for most RNAi triggers. This effect was found both upon plasmid transfection and rAAV2-mediated transduction which represents a therapeutically relevant system for gene delivery. We therefore devised a novel strategy for sense strand counteraction in which an shRNA is co-expressed with an inhibitor RNA transcript designed to stably sequester and inactivate the sense but not the antisense strand. Proof-of-concept for our approach was obtained in transfected human cells with an shRNA against hepatitis C virus (HCV). By using RNA-polymerase III-transcribed inhibitors known as tough decoys (TuDs) we could efficiently and specifically counteract the sense strand of the HCV-shRNA in luciferase and eGFP-based reporter assays. We then tested the shRNA-TuD combination in the context of a single self-complementary rAAV2 vector. TuD co-expression led to impairment of sense strand activity upon transduction of HEK293T and Huh7 cells. Inhibition of HCV replication in Huh7 cells was not altered indicating that the desired antisense strand activity was unaffected. Our strategy is hence compatible with rAAV-mediated gene delivery. Furthermore, expression profiling in Huh7 cells revealed that TuD co-expression specifically de-repressed endogenous off-target transcripts that carried seed matches to the shRNA sense strand leading to lower levels of perturbation of global gene expression. As expected, repression of transcripts carrying antisense strand seed matches remained unaffected. These results show that shRNA sense strands can indeed contribute to off-targeting and that TuD-mediated inhibition can be used to counteract this effect.    Besides our functional data, we also defined rules for TuD and shRNA design as well as promoter choice which allows implementation of the system for other shRNAs.   In this study, we provide new insights into the functionalities and relative activities of both strands of shRNAs. We furthermore present TuD-mediated selective counteraction of shRNA sense strands as a novel method to improve the functional strand bias and thus increase shRNA specificity. We are optimistic that our strategy will facilitate and further foster the clinical implementation of vector-based RNAi.  
date: 2012
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/13390/1/Mockenhaupt_PhD_thesis_final.pdf
identifier: DOI:10.11588/heidok.00013390
identifier: urn:nbn:de:bsz:16-opus-133900
identifier:   Mockenhaupt, Stefan  (2012) Alleviation of shRNA off-target effects via rAAV vector-encoded sense strand decoys.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/13390/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng