title: Allele dependent silencing of collagen type I using small interfering RNAs targeting 3'UTR Indels - a novel therapeutic approach in osteogenesis imperfecta.
abstract: Osteogenesis imperfecta, also known as "brittle bone disease", is a heterogeneous disorder of connective tissue generally caused by dominant mutations in the genes COL1A1 and COL1A2, encoding the α1 and α2 chains of type I (pro)collagen. Symptomatic patients are usually prescribed bisphosphonates, but this treatment is neither curative nor sufficient. A promising field is gene silencing through RNA interference. In this study small interfering RNAs (siRNAs) were designed to target each allele of 3'UTR insertion/deletion polymorphisms (indels) in COL1A1 (rs3840870) and COL1A2 (rs3917). For both indels, the frequency of heterozygous individuals was determined to be approximately 50% in Swedish cohorts of healthy controls as well as in patients with osteogenesis imperfecta. Cultures of primary human bone derived cells were transfected with siRNAs through magnet-assisted transfection. cDNA from transfected cells was sequenced in order to measure targeted allele/non-targeted allele ratios and the overall degree of silencing was assessed by quantitative PCR. Successful allele dependent silencing was observed, with promising results for siRNAs complementary to both the insertion and non-insertion harboring alleles. In COL1A1 cDNA the indel allele ratios were shifted from 1 to 0.09 and 0.19 for the insertion and non-insertion allele respectively while the equivalent resulting ratios for COL1A2 were 0.05 and 0.01. Reductions in mRNA abundance were also demonstrated; in cells treated with siRNAs targeting the COL1A1 alleles the average COL1A1 mRNA levels were reduced 65% and 78% compared to negative control levels and in cells treated with COL1A2 siRNAs the average COL1A2 mRNA levels were decreased 26% and 49% of those observed in the corresponding negative controls. In conclusion, allele dependent silencing of collagen type I utilizing 3'UTR indels common in the general population constitutes a promising mutation independent therapeutic approach for osteogenesis imperfecta.
authors: Department of Medical Sciences, Uppsala University, Uppsala, Sweden. katarina.lindahl@medsci.uu.se
authors: Katarina
authors: K
authors: Lindahl
authors: Andreas
authors: A
authors: Kindmark
authors: Navya
authors: N
authors: Laxman
authors: Eva
authors: E
authors: Åström
authors: Carl-Johan
authors: CJ
authors: Rubin
authors: Östen
authors: Ö
authors: Ljunggren
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics*; D000483:Alleles; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics*; D000090602:Collagen Type I, alpha 1 Chain; D006801:Humans; D010013:Osteogenesis Imperfecta / Q000628:therapy*; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering / Q000235:genetics / Q000502:physiology
chemical_list: D020413:3' Untranslated Regions; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D034741:RNA, Small Interfering
keywords: collagen type I; osteogenesis imperfecta; siRNA
references: J Invest Dermatol. 2011 Oct;131(10):2079-86
references: 21716320
references: J Invest Dermatol. 2012 Jun;132(6):1627-35
references: 22402445
references: Dev Med Child Neurol. 2009 Jan;51(1):17-29
references: 19087101
references: Am J Hum Genet. 2004 May;74(5):917-30
references: 15077201
references: Curr Biol. 2009 May 12;19(9):774-8
references: 19361997
references: Stem Cells. 2007 Dec;25(12):3183-93
references: 17823236
references: Int J Med Sci. 2008;5(6):361-5
references: 19015742
references: Blood. 2009 Jul 9;114(2):459-68
references: 19414862
references: PLoS One. 2011;6(6):e21352
references: 21712955
references: Hum Mutat. 2007 Mar;28(3):209-21
references: 17078022
references: Proc Natl Acad Sci U S A. 2002 Jun 25;99(13):8932-7
references: 12084934
references: Exp Hematol. 2010 Jul;38(7):593-602
references: 20417683
references: Am J Hum Genet. 2004 Apr;74(4):752-60
references: 15024692
references: PLoS One. 2011;6(12):e28582
references: 22174841
references: Birth Defects Orig Artic Ser. 1979;15(5B):113-29
references: 393318
references: FASEB J. 2012 Feb;26(2):668-77
references: 22038052
references: J Med Genet. 1979 Apr;16(2):101-16
references: 458828
references: Cochrane Database Syst Rev. 2008 Oct 08;(4):CD005088
references: 18843680
references: Mol Ther. 2011 Dec;19(12):2178-85
references: 21971427
references: Genome Res. 2005 Nov;15(11):1584-91
references: 16251468
references: Nature. 2001 May 24;411(6836):494-8
references: 11373684
references: Nature. 2006 May 4;441(7089):111-4
references: 16565705
references: Annu Rev Genet. 2012;46:475-97
references: 23145505
references: Blood. 2001 Mar 1;97(5):1227-31
references: 11222364
references: Am J Hum Genet. 1992 Sep;51(3):508-15
references: 1353940
references: Matrix Biol. 1998 Jan;16(6):349-52
references: 9503369
references: J Bone Miner Res. 2005 Jun;20(6):977-86
references: 15883638
references: Nat Med. 1999 Jun;5(6):707-10
references: 10371512
references: J Invest Dermatol. 2008 Mar;128(3):594-605
references: 17914454
references: Nucleic Acids Res. 2004 Jul 22;32(13):e109
references: 15272050
references: Proc Natl Acad Sci U S A. 2005 Aug 23;102(34):12177-82
references: 16091473
references: J Clin Endocrinol Metab. 2009 Feb;94(2):400-9
references: 19033370
references: Curr Drug Targets. 2010 Mar;11(3):345-60
references: 20210759
references: Science. 1999 Oct 29;286(5441):950-2
references: 10542148
references: Eur J Pediatr. 1995 Feb;154(2):123-9
references: 7720740
references: Eur J Hum Genet. 2004 Oct;12(10):864-6
references: 15241481
references: Blood. 2008 Feb 1;111(3):1717-25
references: 17967940
references: Nat Biotechnol. 2006 Aug;24(8):1005-15
references: 16823371
references: Cancer Sci. 2006 Aug;97(8):689-96
references: 16863503
references: Exp Neurol. 2009 Jun;217(2):312-9
references: 19289118
references: J Bone Miner Res. 2007 Jun;22(6):832-40
references: 17371163
references: PLoS Genet. 2006 Sep 8;2(9):e140
references: 16965178
references: Transplantation. 2005 Jun 15;79(11):1607-14
references: 15940052
references: Proc Natl Acad Sci U S A. 2006 Apr 11;103(15):5953-8
references: 16585504