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Allele Dependent Silencing of Collagen Type I Using Small Interfering RNAs Targeting 3'UTR Indels: a Novel Therapeutic Approach in Osteogenesis Imperfecta
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Metabolic Bone Diseases.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Metabolic Bone Diseases.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Metabolic Bone Diseases.
Karolinska Institutet.
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2013 (English)In: International Journal of Medical Sciences, ISSN 1449-1907, Vol. 10, no 10, 1333-1343 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2013. Vol. 10, no 10, 1333-1343 p.
Keyword [en]
osteogenesis imperfecta, OI, allele-specific silencing, siRNA, collagen, COL1A1, COL1A2, indel, insertion/deletion, RNAi, mutation, gene-therapy, therapy
National Category
Endocrinology and Diabetes
Research subject
Genetics; Medical Genetics; Medicine
URN: urn:nbn:se:uu:diva-208936DOI: 10.7150/ijms.5774ISI: 000324411800011PubMedID: 23983594OAI: diva2:655404
Available from: 2013-10-11 Created: 2013-10-11 Last updated: 2013-12-05Bibliographically approved
In thesis
1. Osteogenesis Imperfecta: Genetic and Therapeutic Studies
Open this publication in new window or tab >>Osteogenesis Imperfecta: Genetic and Therapeutic Studies
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Osteogenesis imperfecta (OI) is a heterogeneous disease of connective tissue, the cardinal symptom being fractures and severity ranging from mild to lethal. Dominant mutations in collagen I, encoded by COL1A1 and COL1A2, cause >90% of cases.

To delineate genotype-phenotype correlations and pharmaco-genetic response, collagen I was sequenced in 150 unrelated Swedish families and clinical data were collected in Paper I. Mutation type, gene affected, and N- to C-terminal location correlated with phenotype and severity. Bisphosphonate response assessed by calculated yearly change in lumbar spine bone mineral density (BMD) was inversely related to age and BMD at treatment initiation. Mutations associated with a more severe phenotype exhibited an increased response after 2 years; however, all types of OI responded well.

To investigate the effect of naturally occurring variations in collagen I, the only common coding single nucleotide polymorphism (rs42524 in COL1A2) was genotyped in 2004 healthy men in Paper II. Heterozygous genotype was associated with decreased BMD and an increased risk of stroke.

An adolescent with repeated fractures despite a markedly high BMD harbored a unique C-terminal procollagen cleavage-site mutation in COL1A1, which motivated extensive investigations in concert with a similar COL1A2 case in Paper III. The probands were found to have impaired procollagen processing, incorporation of collagen with retained C-propeptide in matrix and increased mineral to matrix ratio, which demonstrates that C-propeptide cleavage is crucial to normal bone mineralization and structure.

Bisphosphonate therapy has insufficient effect in OI, and as classical OI is a dominant disorder severe cases would benefit from silencing of the mutated allele. In Paper IV and V small interfering RNAs (siRNAs) were used to allele-specifically target primary human bone cells heterozygous for I) a coding polymorphism in COL1A2 and II) insertion/deletions in the 3’UTR of COL1A1 and COL1A2. Results were promising with altered allele ratios and decreased mRNA levels in the predicted fashion.

To summarize, this thesis found that collagen I is crucial to bone and connective tissue and that collagen I mutations create markedly diverse phenotypes. Age, BMD and pharmaco-genetic effects influence the response to bisphosphonate therapy in individuals with OI; however, novel approaches are needed. Utilizing allele-specific siRNAs may be a way forward in the treatment of severe OI.


Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 96 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 936
OI, BMD, Genotype, Phenotype, Pharmaco-genetics, Bisphosphonate, Therapy, Gene-therapy, Mutation, Collagen, Collagen type I, Allele-specific silencing, siRNA, RNAi, COL1A1, COL1A2, Stroke, C-propeptide, Mineralization, Heterozygous disadvantage
National Category
Endocrinology and Diabetes
Research subject
Genetics; Medicine; Medical Genetics
urn:nbn:se:uu:diva-208942 (URN)978-91-554-8772-0 (ISBN)
Public defence
2013-11-29, Enghoffsalen, Ingång 50, Akademiska Sjukhuset, Uppsala, 09:15 (English)
Swedish Research Council
Available from: 2013-11-08 Created: 2013-10-11 Last updated: 2014-01-23

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Lindahl, KatarinaKindmark, AndreasLaxman, NavyaRubin, Carl-JohanLjunggren, Östen
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