title: Self-duplexing CUG repeats selectively inhibit mutant huntingtin expression.
abstract: Huntington's disease (HD) is a neurodegenerative genetic disorder caused by the expansion of the CAG repeat in the translated sequence of the HTT gene. This expansion generates a mutant huntingtin protein that contains an abnormally elongated polyglutamine tract, which, together with mutant transcript, causes cellular dysfunction. Currently, there is no curative treatment available to patients suffering from HD; however, the selective inhibition of the mutant allele expression is a promising therapeutic option. In this study, we developed a new class of CAG repeat-targeting silencing reagents that consist of self-duplexing CUG repeats. Self-duplex formation was induced through one or several U-base substitutions. A number of self-duplexing guide-strand-only short interfering RNAs have been tested through transfection into cells derived from HD patients, showing distinct activity profiles. The best reagents were highly discriminatory between the normal and mutant HTT alleles (allele selectivity) and the HTT transcript and other transcripts containing shorter CAG repeats (gene selectivity). We also demonstrated that the self-duplexing CUG repeat short interfering RNAs use the RNA interference pathway to elicit silencing, and repeat-targeting reagents showed similar activity and selectivity when expressed from short hairpin RNA vectors to achieve more durable silencing effects.
authors.affiliation: Department of Molecular Biomedicine, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Fiszer
authors.forename: Marta
authors.initials: M
authors.lastname: Olejniczak
authors.forename: Paulina
authors.initials: P
authors.lastname: Galka-Marciniak
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Mykowska
authors.forename: Wlodzimierz J
authors.initials: WJ
authors.lastname: Krzyzosiak
mesh_terms: D000483:Alleles; D002478:Cells, Cultured; D005822:Genetic Vectors; D006801:Humans; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000235:genetics*; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry*; D012091:Repetitive Sequences, Nucleic Acid
chemical_list: C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D034741:RNA, Small Interfering
references.citation: ScientificWorldJournal. 2011 Jan 05;11:102-17
references.pmid: 21218267
references.citation: Cell. 2012 Aug 31;150(5):895-908
references.pmid: 22939619
references.citation: Proc Natl Acad Sci U S A. 2003 Aug 19;100(17):9779-84
references.pmid: 12902540
references.citation: J Biol Chem. 2010 Apr 23;285(17):12755-64
references.pmid: 20159983
references.citation: Nature. 2009 Jul 23;460(7254):479-86
references.pmid: 19536157
references.citation: Nature. 2010 Aug 12;466(7308):835-40
references.pmid: 20703300
references.citation: Nature. 2001 May 24;411(6836):494-8
references.pmid: 11373684
references.citation: Biochemistry. 2010 Oct 12;49(40):8669-79
references.pmid: 20681613
references.citation: Mol Cell. 2002 Sep;10(3):537-48
references.pmid: 12408822
references.citation: Cell. 2002 Sep 6;110(5):563-74
references.pmid: 12230974
references.citation: RNA Biol. 2006 Apr;3(2):82-9
references.pmid: 17114944
references.citation: BMC Mol Biol. 2012 Mar 07;13:6
references.pmid: 22397573
references.citation: J Biol Chem. 1997 Dec 5;272(49):31079-85
references.pmid: 9388259
references.citation: Nat Biotechnol. 2005 Nov;23(11):1440-4
references.pmid: 16258545
references.citation: Biol Chem. 2011 Apr;392(4):315-25
references.pmid: 21294677
references.citation: Nat Biotechnol. 2009 May;27(5):478-84
references.pmid: 19412185
references.citation: Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W577-81
references.pmid: 15980540
references.citation: Proc Natl Acad Sci U S A. 2008 Apr 15;105(15):5868-73
references.pmid: 18398004
references.citation: Genes Dev. 2003 Feb 15;17(4):438-42
references.pmid: 12600936
references.citation: PLoS One. 2011;6(9):e24308
references.pmid: 21909428
references.citation: Chem Biol. 2010 Nov 24;17(11):1183-8
references.pmid: 21095568
references.citation: Ann N Y Acad Sci. 2009 Sep;1175:24-31
references.pmid: 19796074
references.citation: J Biol Chem. 2003 Nov 7;278(45):44312-9
references.pmid: 12952966
references.citation: Nat Med. 2004 Aug;10(8):816-20
references.pmid: 15235598
references.citation: Nucleic Acids Res. 2010 Jan;38(1):1-16
references.pmid: 19843612
references.citation: RNA. 2008 Feb;14(2):263-74
references.pmid: 18094121
references.citation: Nucleic Acids Res. 2011 Jul;39(13):5578-85
references.pmid: 21427085
references.citation: Nucleic Acids Res. 2010 Jul;38(12):4027-39
references.pmid: 20215431
references.citation: Biochemistry. 1999 Oct 26;38(43):14214-23
references.pmid: 10571995
references.citation: Biochem Biophys Res Commun. 2004 Apr 9;316(3):680-7
references.pmid: 15033453
references.citation: Nucleic Acids Res. 2003 May 1;31(9):2401-7
references.pmid: 12711685
references.citation: BMC Mol Biol. 2011 Apr 11;12:14
references.pmid: 21481235
references.citation: Science. 2004 Sep 3;305(5689):1437-41
references.pmid: 15284456
references.citation: Nucleic Acids Res. 2012 Jan;40(1):11-26
references.pmid: 21908410
references.citation: Cell. 2004 Jan 23;116(2):281-97
references.pmid: 14744438
references.citation: Nature. 2010 Jun 10;465(7299):818-22
references.pmid: 20505670
references.citation: Gene Ther. 2009 Jul;16(7):827-9
references.pmid: 19404320
references.citation: J Mol Med (Berl). 2013 Jun;91(6):683-91
references.pmid: 23512265
references.citation: Hum Mol Genet. 2002 Jan 15;11(2):175-84
references.pmid: 11809726
references.citation: RNA Biol. 2011 Jul-Aug;8(4):565-71
references.pmid: 21593608
references.citation: Drug Discov Today. 2012 May;17(9-10):443-50
references.pmid: 22285529
references.citation: Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):7195-200
references.pmid: 12782788
references.citation: Nat Rev Genet. 2011 Jan;12(1):19-31
references.pmid: 21116305