title: Antisense downregulation of mutant huntingtin in a cell model.
abstract: BACKGROUND
  Huntington's disease (HD) is an inherited neurodegenerative disorder which is caused by an expansion of a CAG repeat sequence in the HD gene. The repeat encodes an expanded polyglutamine tract in the protein huntingtin. The still unknown pathological mechanisms leading to death of specific neurons in the brains of HD patients correlate with the expression of mutant huntingtin. Therefore, we have studied whether mutant huntingtin expression can be downregulated by antisense technique.
  
  
  METHODS
  NT2 precursor cells and differentiated postmitotic NT2-N neurons, respectively, were transfected with plasmid constructs containing exon 1 of the HD gene with expanded CAG repeats in frame with the reporter protein EGFP. The transfected cell cultures were treated with a phosphorothioated antisense oligonucleotide (PS-ASHD/20+) or a control oligonucleotide either by cotransfection or by addition to the culture medium.
  
  
  RESULTS
  Expression of the fusion protein containing the mutant huntingtin fragment resulted in diffuse green fluorescence in the cytoplasm and formation of aggregates in some of the NT2 cells and NT2-N neurons. We obtained antisense sequence-specific inhibition of expression of the fusion protein and/or suppression of the aggregate formation in both cell types. In the NT2 cells the antisense effect was dependent on the way of administration of the oligo.
  
  
  CONCLUSIONS
  The PS-antisense oligo is effective in downregulation of mutant huntingtin, and the reduction of aggregate formation is a sensitive biological marker. The findings suggest that antisense knockdown of huntingtin could be a useful strategy for treatment of HD, and could also be suitable for studies of the normal and pathological function of huntingtin in different cellular model systems.
authors.affiliation: Section of Neurogenetics, Institute of Medical Biochemistry and Genetics, University of Copenhagen, Denmark. hasholt@imbg.ku.dk
authors.forename: Lis
authors.initials: L
authors.lastname: Hasholt
authors.forename: Kathrine
authors.initials: K
authors.lastname: Abell
authors.forename: Anne
authors.initials: A
authors.lastname: Nørremølle
authors.forename: Christine
authors.initials: C
authors.lastname: Nellemann
authors.forename: Kirsten
authors.initials: K
authors.lastname: Fenger
authors.forename: Sven Asger
authors.initials: SA
authors.lastname: Sørensen
mesh_terms: D045744:Cell Line, Tumor; D015536:Down-Regulation; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D000071058:Huntingtin Protein; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D009474:Neurons / Q000737:chemistry; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D020319:Oligodeoxyribonucleotides, Antisense / Q000235:genetics*; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
chemical_list: C086055:HTT protein, human; D000071058:Huntingtin Protein; D008164:Luminescent Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D010455:Peptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C097188:polyglutamine