title: Lack of correlation between predicted and actual off-target effects of short-interfering RNAs targeting the human papillomavirus type 16 E7 oncogene.
abstract: BACKGROUND
  When designing therapeutic short-interfering RNAs (siRNAs), off-target effects (OTEs) are usually predicted by computational quantification of messenger RNAs (mRNAs) that contain matches to the siRNA seed sequence in their 3' UTRs. It is assumed that the higher the number of predicted transcriptional OTEs, the greater the size of the actual OTE signature and the more detrimental the phenotypic consequences in target-negative cells.
  
  
  METHODS
  We tested this general assumption by investigating the OTEs of potential therapeutic siRNAs targeting the human papillomavirus (HPV) type-16 E7 oncogene. We studied HPV-negative squamous epithelial cells, from normal cervix (NCx) and skin (HaCaT), which would be vulnerable to 'bystander' OTEs following transfection in vivo.
  
  
  RESULTS
  We observed no correlation between the number of computationally predicted OTEs and the actual number of seed-dependent OTEs (P=0.76). On average only 20.5% of actual transcriptional OTEs were seed-dependent (i.e., predicted). The unpredicted OTEs included stimulation of innate immune pathways, as well as indirect (downstream) effects of other OTEs, which affected important cancer-associated pathways. Although most significant OTEs observed were seen in both NCx and HaCaT cells, only 0-5.9% of differentially expressed genes overlapped between the two cell types.
  
  
  CONCLUSIONS
  These data do not support the assumption that actual OTEs correlate well with predicted OTEs.
authors.affiliation: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.
authors.forename: J E
authors.initials: JE
authors.lastname: Hanning
authors.forename: H K
authors.initials: HK
authors.lastname: Saini
authors.forename: M J
authors.initials: MJ
authors.lastname: Murray
authors.forename: S
authors.initials: S
authors.lastname: van Dongen
authors.forename: M P A
authors.initials: MP
authors.lastname: Davis
authors.forename: E M
authors.initials: EM
authors.lastname: Barker
authors.forename: D M
authors.initials: DM
authors.lastname: Ward
authors.forename: C G
authors.initials: CG
authors.lastname: Scarpini
authors.forename: A J
authors.initials: AJ
authors.lastname: Enright
authors.forename: M R
authors.initials: MR
authors.lastname: Pett
authors.forename: N
authors.initials: N
authors.lastname: Coleman
mesh_terms: D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000821:virology; D045744:Cell Line, Tumor; D002584:Cervix Uteri / Q000166:cytology; D004847:Epithelial Cells / Q000821:virology; D005260:Female; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D050725:Papillomavirus E7 Proteins / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering; D012867:Skin / Q000166:cytology; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000821:virology*
chemical_list: D050725:Papillomavirus E7 Proteins; D034741:RNA, Small Interfering; C059731:oncogene protein E7, Human papillomavirus type 16