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

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-bas...

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Published in:Bone (New York, N.Y.) N.Y.), 2014-02, Vol.59, p.76-88
Main Authors: Guo, Baosheng, Zhang, Baoting, Zheng, Lizhen, Tang, Tao, Liu, Jin, Wu, Heng, Yang, Zhijun, Peng, Songlin, He, Xiaojuan, Zhang, Hongqi, Yue, Kevin K.M, He, Fuchu, Zhang, Lingqiang, Qin, Ling, Bian, Zhaoxiang, Tan, Weihong, Liang, Zicai, Lu, Aiping, Zhang, Ge
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Biological and medical sciences
Bone formation
Bone Matrix - metabolism
Bone Morphogenetic Protein 2 - metabolism
Calcification, Physiologic
Carrier Proteins - genetics
Carrier Proteins - metabolism
Cell Differentiation - genetics
CKIP-1
Computational Biology
Cross-species
Female
Femur - diagnostic imaging
Femur - metabolism
Fundamental and applied biological sciences. Psychology
Gene Knockdown Techniques
Humans
Immunization
Intracellular Signaling Peptides and Proteins
Macaca mulatta - genetics
Mice
Orthopedics
Osteoblasts - metabolism
Osteoblasts - pathology
Osteogenesis - genetics
Phenotype
Rats
RNA Interference
RNA, Messenger - genetics
RNA, Messenger - metabolism
RNA, Small Interfering - metabolism
siRNA sequences
Species Specificity
Up-Regulation - genetics
Vertebrates: anatomy and physiology, studies on body, several organs or systems
X-Ray Microtomography
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Summary: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.
ISSN:8756-3282
1873-2763
DOI:10.1016/j.bone.2013.11.007