title: Lentiviral vectors encoding short hairpin RNAs efficiently transduce and knockdown LINGO-1 but induce an interferon response and cytotoxicity in central nervous system neurones.
abstract: BACKGROUND
  Knocking down neuronal LINGO-1 using short hairpin RNAs (shRNAs) might enhance axon regeneration in the central nervous system (CNS). Integration-deficient lentiviral vectors have great potential as a therapeutic delivery system for CNS injuries. However, recent studies have revealed that shRNAs can induce an interferon response resulting in off-target effects and cytotoxicity.
  
  
  METHODS
  CNS neurones were transduced with integration-deficient lentiviral vectors in vitro. The transcriptional effect of shRNA expression was analysed using quantitative real time-polymerase chain reaction and northern blots were used to assess shRNA production.
  
  
  RESULTS
  Integration-deficient lentiviral vectors efficiently transduced CNS neurones and knocked down LINGO-1 mRNA in vitro. However, an increase in cell death was observed when lentiviral vectors encoding an shRNA were applied or when high vector concentrations were used. We demonstrate that high doses of vector or the use of vectors encoding shRNAs can induce an up-regulation of interferon-stimulated genes (2',5'-oligoadenylate synthase 1 and protein kinase R although not myxovirus resistance 1) and a down-regulation of off-target genes (including p75(NTR) and Nogo receptor 1). Furthermore, the northern blot demonstrated that these negative consequences occur even when lentiviral vectors express low levels of shRNAs. Taken together, these results may explain why neurite outgrowth was not enhanced on an inhibitory substrate following transduction with lentiviral vectors encoding an shRNA targeting LINGO-1.
  
  
  CONCLUSIONS
  These findings highlight the importance of including appropriate controls to verify silencing specificity and the requirement to check for an interferon response when conducting RNA interference experiments. However, the potential benefits that RNA interference and viral vectors offer to gene-based therapies to CNS injuries cannot be overlooked and demand further investigation.
authors.affiliation: Neurorestoration Group, Wolfson Centre for Age-Related Diseases, King's College London, Guy's Campus, London, UK. thomas.hutson@kcl. ac.uk
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Hutson
authors.forename: Edmund
authors.initials: E
authors.lastname: Foster
authors.forename: John M
authors.initials: JM
authors.lastname: Dawes
authors.forename: Robert
authors.initials: R
authors.lastname: Hindges
authors.forename: Rafael J
authors.initials: RJ
authors.lastname: Yáñez-Muñoz
authors.forename: Lawrence D F
authors.initials: LD
authors.lastname: Moon
mesh_terms: D000818:Animals; D002490:Central Nervous System / Q000254:growth & development / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D018014:Gene Transfer Techniques / Q000009:adverse effects; D005822:Genetic Vectors; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007372:Interferons / Q000378:metabolism; D016086:Lentivirus; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D016501:Neurites / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000009:adverse effects / Q000235:genetics*; D051381:Rats; D012038:Regeneration / Q000235:genetics*
chemical_list: C482293:LINGO1 protein, human; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D007372:Interferons