Document Type

Journal Article

Department/Unit

School of Chinese Medicine

Title

Therapeutic RNA interference targeting CKIP-1 with a cross-species sequence to stimulate bone formation

Language

English

Abstract

Objectives

Casein kinase 2 interacting protein 1 (CKIP-1) is a newly discovered intracellular negative regulator of bone formation without affecting bone resorption. In this study, we aimed to identify a cross-species siRNA sequence targeting CKIP-1 to facilitate developing a novel RNAi-based bone anabolic drug for reversing established osteoporosis.

Methods

Eight specifically designed cross-species CKIP-1 siRNA sequences were screened in human, rhesus, rat and mouse osteoblast-like cells in vitro to identify the optimal sequence with the highest knockdown efficiency. The effect of this optimal siRNA sequence on osteogenic differentiation and matrix mineralization was further examined in osteoblast-like cells across different species, followed by an immunogenicity assessment in human peripheral blood mononuclear cells in vitro. The intra-osseous localization and silencing efficiency of the optimal siRNA were examined in vivo using a biophotonic system and real-time polymerase chain reaction, respectively. The RNAi-mediated cleavage of the CKIP-1 transcript was confirmed by rapid amplification of the 5′ cDNA ends in vivo. Furthermore, the effect of the optimal siRNA sequence on osteogenic differentiation, bone turnover biomarkers, bone mass and micro-architecture parameters was investigated in healthy and osteoporotic rodents.

Results

The CKIP-1 siRNA sequence (si-3) was identified as the optimal sequence, which consistently maintained CKIP-1 mRNA/protein expression at the lowest level across species in vitro. The si-3 significantly increased mRNA expression levels of osteoblast phenotypic genes and matrix mineralization across species without inducing an immunostimulatory activity in vitro. The intra-osseous localization and RNAi-mediated CKIP-1 silencing with high efficiency were confirmed in vivo. Periodic intravenous injections of si-3 promoted mRNA expression of osteoblast phenotypic genes, enhanced bone formation, increased bone mass and elevated serum level of bone formation marker without raising urine level of bone resorption marker in the healthy rodents. Moreover, the si-3 treatment promoted bone formation, improved trabecular micro-architecture and reversed bone loss in the osteoporotic mice.

Conclusions

The identified optimal CKIP-1 siRNA sequence (si-3) could promote osteogenic differentiation across species in vitro, stimulate bone formation in the healthy rodents and reverse bone loss in the osteoporotic mice.

Keywords

Bone formation, CKIP-1, Cross-species, SiRNA sequences

Publication Date

2-2014

Source Publication Title

Bone

Volume

59

Start Page

76

End Page

88

Publisher

Elsevier

Peer Reviewed

1

Copyright

© 2013 Published by Elsevier Inc.

Funder

This study was supported by Contract Grant (Ribo-HK001) from Suzhou Ribo Life Science Co. Ltd., the Hong Kong General Research Fund (HKBU479111, HKBU478312 and HKBU261113), the Research Grants Council & Natural Science Foundation Council (N_HKBU435/12), Hong Kong Research Grants Council (RGC) Early Career Scheme (ECS) (489213) and the Natural Science Foundation Council (81272045). The authors thank Shanghai GenePharma Co., Ltd for the synthesis of the CKIP-1 siRNA sequences.

DOI

10.1016/j.bone.2013.11.007

Link to Publisher's Edition

http://dx.doi.org/10.1016/j.bone.2013.11.007

ISSN (print)

87563282

ISSN (electronic)

18732763

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