title: Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model.
abstract: X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton's tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTK transcripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we engineered a transgenic mouse that harbors a BAC with an authentic, mutated, splice-defective human BTK gene. BTK transgenic mice were bred onto a Btk knockout background to avoid interference of the orthologous mouse protein. Using this model, we determined that BTK-specific SCOs are able to correct aberrantly spliced BTK in B lymphocytes, including pro-B cells. Correction of BTK mRNA restored expression of functional protein, as shown both by enhanced lymphocyte survival and reestablished BTK activation upon B cell receptor stimulation. Furthermore, SCO treatment corrected splicing and restored BTK expression in primary cells from patients with XLA. Together, our data demonstrate that SCOs can restore BTK function and that BTK-targeting SCOs have potential as personalized medicine in patients with XLA.
authors.forename: Burcu
authors.initials: B
authors.lastname: Bestas
authors.forename: Pedro M D
authors.initials: PM
authors.lastname: Moreno
authors.forename: K Emelie M
authors.initials: KE
authors.lastname: Blomberg
authors.forename: Dara K
authors.initials: DK
authors.lastname: Mohammad
authors.forename: Amer F
authors.initials: AF
authors.lastname: Saleh
authors.forename: Tolga
authors.initials: T
authors.lastname: Sutlu
authors.forename: Joel Z
authors.initials: JZ
authors.lastname: Nordin
authors.forename: Peter
authors.initials: P
authors.lastname: Guterstam
authors.forename: Manuela O
authors.initials: MO
authors.lastname: Gustafsson
authors.forename: Shabnam
authors.initials: S
authors.lastname: Kharazi
authors.forename: Barbara
authors.initials: B
authors.lastname: Piątosa
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Roberts
authors.forename: Mark A
authors.initials: MA
authors.lastname: Behlke
authors.forename: Matthew J A
authors.initials: MJ
authors.lastname: Wood
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Gait
authors.forename: Karin E
authors.initials: KE
authors.lastname: Lundin
authors.forename: Samir
authors.initials: S
authors.lastname: El Andaloussi
authors.forename: Robert
authors.initials: R
authors.lastname: Månsson
authors.forename: Anna
authors.initials: A
authors.lastname: Berglöf
authors.forename: Jesper
authors.initials: J
authors.lastname: Wengel
authors.forename: C I Edvard
authors.initials: CI
authors.lastname: Smith
mesh_terms: D000077329:Agammaglobulinaemia Tyrosine Kinase; D000361:Agammaglobulinemia / Q000201:enzymology / Q000628:therapy*; D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D002478:Cells, Cultured; D040181:Genetic Diseases, X-Linked / Q000201:enzymology / Q000628:therapy*; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008822:Mice, Transgenic; D009000:Monocytes / Q000201:enzymology; D009841:Oligonucleotides / Q000235:genetics*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000502:physiology*; D012326:RNA Splicing
chemical_list: D009841:Oligonucleotides; D008156:Luciferases; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; C000625951:Btk protein, mouse
references.citation: J Exp Med. 2000 Dec 4;192(11):1611-24
references.pmid: 11104803
references.citation: RNA. 2008 Feb;14(2):336-46
references.pmid: 18073344
references.citation: Immunity. 1995 Sep;3(3):283-99
references.pmid: 7552994
references.citation: Cell. 1993 Jan 29;72(2):279-90
references.pmid: 8425221
references.citation: ACS Chem Biol. 2013 Apr 19;8(4):832-9
references.pmid: 23394403
references.citation: Nat Immunol. 2012 Oct;13(10):963-71
references.pmid: 22941246
references.citation: Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7582-7
references.pmid: 8755518
references.citation: Nucleic Acids Res. 2010 Nov;38(20):7100-11
references.pmid: 20615897
references.citation: Mol Ther Nucleic Acids. 2013 Aug 27;2:e117
references.pmid: 23982190
references.citation: Hum Mutat. 1999;13(4):280-5
references.pmid: 10220140
references.citation: Mol Ther. 2008 Sep;16(9):1624-9
references.pmid: 18545222
references.citation: Biochemistry. 1998 May 5;37(18):6235-9
references.pmid: 9572837
references.citation: Hum Mol Genet. 2008 Dec 15;17(24):3909-18
references.pmid: 18784278
references.citation: Methods Mol Biol. 2012;867:117-29
references.pmid: 22454058
references.citation: Hum Mutat. 2006 Dec;27(12):1209-17
references.pmid: 16969761
references.citation: Immunol Rev. 2009 Mar;228(1):58-73
references.pmid: 19290921
references.citation: Hum Mol Genet. 2011 Aug 15;20(16):3151-60
references.pmid: 21576124
references.citation: Org Biomol Chem. 2011 Jan 7;9(1):243-52
references.pmid: 21049102
references.citation: Immunity. 2010 Nov 24;33(5):713-22
references.pmid: 21093320
references.citation: Expert Rev Clin Immunol. 2013 Dec;9(12):1205-21
references.pmid: 24215410
references.citation: J Biol Chem. 1999 Dec 17;274(51):36193-9
references.pmid: 10593905
references.citation: Adv Immunol. 1995;59:135-223
references.pmid: 7484460
references.citation: Curr Gene Ther. 2012 Jun;12(3):161-78
references.pmid: 22533378
references.citation: Proc Natl Acad Sci U S A. 2008 Sep 30;105(39):14814-9
references.pmid: 18806224
references.citation: Clin Exp Immunol. 1993 May;92(2):200-4
references.pmid: 7683584
references.citation: Biochemistry. 2001 Dec 4;40(48):14645-54
references.pmid: 11724578
references.citation: Neth J Med. 2002 Apr;60(3):140-7
references.pmid: 12164371
references.citation: Medicine (Baltimore). 2008 Sep;87(5):253-258
references.pmid: 18794707
references.citation: PLoS Biol. 2007 Apr;5(4):e73
references.pmid: 17355180
references.citation: Clin Immunol. 2002 Sep;104(3):221-30
references.pmid: 12217331
references.citation: Medicine (Baltimore). 1996 Nov;75(6):287-99
references.pmid: 8982147
references.citation: Mol Ther. 2006 Oct;14(4):471-5
references.pmid: 16854630
references.citation: Proc Natl Acad Sci U S A. 2009 Jan 27;106(4):1205-10
references.pmid: 19164558
references.citation: Clin Immunol. 1999 Jun;91(3):243-53
references.pmid: 10370369
references.citation: Blood. 2004 Aug 15;104(4):1191-7
references.pmid: 15117762
references.citation: Q J Med. 1993 Jan;86(1):31-42
references.pmid: 8438047
references.citation: Nucleic Acids Res. 2003 Jul 1;31(13):3568-71
references.pmid: 12824367
references.citation: Biochim Biophys Acta. 1999 Dec 10;1489(1):141-58
references.pmid: 10807004
references.citation: Adv Genet. 2013;82:47-107
references.pmid: 23721720
references.citation: Biochem J. 2008 Jun 1;412(2):307-13
references.pmid: 18271753
references.citation: Nat Rev Drug Discov. 2012 Jan 20;11(2):125-40
references.pmid: 22262036
references.citation: Proc Natl Acad Sci U S A. 2000 May 9;97(10):5633-8
references.pmid: 10805816
references.citation: Ann N Y Acad Sci. 2006 Oct;1082:74-6
references.pmid: 17145928
references.citation: FEBS Lett. 2005 Mar 28;579(9):1900-3
references.pmid: 15792793
references.citation: Bioessays. 2001 May;23(5):436-46
references.pmid: 11340625
references.citation: Nucleic Acids Res. 2011 Sep 1;39(16):7077-91
references.pmid: 21609956
references.citation: Hum Gene Ther. 2013 May;24(5):479-88
references.pmid: 23521559
references.citation: Medicine (Baltimore). 1985 May;64(3):145-56
references.pmid: 2581110
references.citation: Proc Natl Acad Sci U S A. 2007 Apr 3;104(14):6007-12
references.pmid: 17389389
references.citation: RNA. 2008 May;14(5):802-13
references.pmid: 18369186
references.citation: Mol Ther. 2012 Feb;20(2):376-81
references.pmid: 22108858
references.citation: Science. 2002 Aug 9;297(5583):1007-13
references.pmid: 12114529
references.citation: Genes Dev. 2004 Jun 1;18(11):1241-50
references.pmid: 15145827
references.citation: J Immunol. 1994 Jan 15;152(2):557-65
references.pmid: 8283037
references.citation: EMBO Rep. 2011 Feb;12(2):136-41
references.pmid: 21212803
references.citation: Nucleic Acids Res. 2010 Jan;38(1):e3
references.pmid: 19854938
references.citation: Pediatr Res. 2002 Feb;51(2):159-68
references.pmid: 11809909
references.citation: Immunol Res. 2001;23(2-3):147-56
references.pmid: 11444380
references.citation: Curr Opin Mol Ther. 2009 Apr;11(2):124-32
references.pmid: 19330718
references.citation: Immunology. 2008 Feb;123(2):239-49
references.pmid: 17725607
references.citation: Rev Infect Dis. 1987 Mar-Apr;9(2):334-56
references.pmid: 3296100
references.citation: Chem Biodivers. 2010 Mar;7(3):536-42
references.pmid: 20232325
references.citation: Mol Ther. 2008 Jul;16(7):1316-22
references.pmid: 18461057
references.citation: Mol Cell Biol. 2013 Aug;33(16):3214-26
references.pmid: 23754751
references.citation: Eur J Immunol. 1993 Dec;23(12):3109-14
references.pmid: 8258324
references.citation: Nature. 1993 Jan 21;361(6409):226-33
references.pmid: 8380905
references.citation: Nucleic Acids Res. 1989 Aug 11;17(15):6129-41
references.pmid: 2771642
references.citation: Biochemistry. 2002 Aug 6;41(31):9973-81
references.pmid: 12146961
references.citation: Mol Ther. 2009 Mar;17(3):554-61
references.pmid: 19142179
references.citation: J Lipid Res. 2007 Apr;48(4):763-7
references.pmid: 17242417
references.citation: Chem Soc Rev. 2011 Dec;40(12):5680-9
references.pmid: 21556437
references.citation: Blood. 1998 Mar 15;91(6):2152-6
references.pmid: 9490703
references.citation: Cardiovasc Res. 2010 Feb 1;85(3):444-53
references.pmid: 19815563
references.citation: Chem Rev. 2007 Nov;107(11):4672-97
references.pmid: 17944519
references.citation: Pediatrics. 1998 Feb;101(2):276-84
references.pmid: 9445504
references.citation: Annu Rev Immunol. 2009;27:199-227
references.pmid: 19302039
references.citation: Cell. 2012 Mar 16;148(6):1085-8
references.pmid: 22424220
references.citation: Hum Mol Genet. 1995 Apr;4(4):693-700
references.pmid: 7633420