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-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.

• Authors: Baosheng Guo, Baoting Zhang, Lizhen Zheng, Tao Tang, Jin Liu, Heng Wu, Zhijun Yang, Songlin Peng, Xiaojuan He, Hongqi Zhang, Kevin K M Yue, Fuchu He, Lingqiang Zhang, Ling Qin, Zhaoxiang Bian, Weihong Tan, Zicai Liang, Aiping Lu, Ge Zhang
• Journal: Bone (Bone) Vol. 59 Pg. 76-88 (Feb 2014) ISSN: 1873-2763 [Electronic] United States
• PMID: 24246247 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2013. Published by Elsevier Inc.

Chemical References

• Bone Morphogenetic Protein 2
• Carrier Proteins
• Intracellular Signaling Peptides and Proteins
• PLEKHO1 protein, human
• RNA, Messenger
• RNA, Small Interfering

Topics

• Animals
• Base Sequence
• Bone Matrix (metabolism)
• Bone Morphogenetic Protein 2 (metabolism)
• Calcification, Physiologic
• Carrier Proteins (genetics, metabolism)
• Cell Differentiation (genetics)
• Computational Biology
• Female
• Femur (diagnostic imaging, metabolism)
• Gene Knockdown Techniques
• Humans
• Immunization
• Intracellular Signaling Peptides and Proteins
• Macaca mulatta (genetics)
• Mice
• Osteoblasts (metabolism, pathology)
• Osteogenesis (genetics)
• Phenotype
• RNA Interference
• RNA, Messenger (genetics, metabolism)
• RNA, Small Interfering (metabolism)
• Rats
• Species Specificity
• Up-Regulation (genetics)
• X-Ray Microtomography