Therapeutic RNA interference targeting CKIP-1 with a cross-species sequence to stimulate bone formation
Therapeutic RNA interference targeting CKIP-1 with a cross-species sequence to stimulate bone formation
复制标题
具有跨物种序列的针对 CKIP-1 的治疗性 RNA 干扰可刺激骨形成
DOI：
10.1016/j.bone.2013.11.007
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发表时间：
2014-02-01
期刊：
影响因子：
4.1
通讯作者：
Zhang, Ge
中科院分区：
文献类型：
--
作者：
Guo, Baosheng;Zhang, Baoting;Zhang, Ge
目的：酪蛋白激酶2相互作用蛋白1 （CKIP-1）是一种新发现的细胞内骨形成负调控因子，不影响骨吸收。在这项研究中，我们旨在鉴定一个针对CKIP-1的跨物种siRNA序列，以促进开发一种新的基于rnai的骨合成代谢药物，以逆转已建立的骨质疏松症。方法：在人、恒河鼠、大鼠和小鼠成骨样细胞中筛选8个专门设计的跨物种CKIP-1 siRNA序列，以确定敲除效率最高的最佳序列。在不同物种的成骨细胞样细胞中进一步研究了该最佳siRNA序列对成骨分化和基质矿化的影响，随后在体外的人外周血单核细胞中进行了免疫原性评估。利用生物光子系统和实时聚合酶链反应分别检测了最佳siRNA的骨内定位和沉默效率。rnai介导的CKIP-1转录物的裂解通过体内5' cDNA末端的快速扩增得到证实。此外，研究了最佳siRNA序列对健康和骨质疏松啮齿动物成骨分化、骨转换生物标志物、骨量和微结构参数的影响。结果：CKIP-1 siRNA序列（si-3）被确定为最优序列，该序列在体外持续将CKIP-1 mRNA/蛋白表达维持在最低水平。si-3在体外不诱导免疫刺激活性的情况下显著提高了成骨细胞表型基因mRNA表达水平和基质矿化水平。体内实验证实了骨内定位和rnai介导的CKIP-1高效沉默。定期静脉注射si-3可促进健康鼠成骨细胞表型基因mRNA表达，促进骨形成，增加骨量，提高血清骨形成标志物水平，但不提高尿骨吸收标志物水平。此外，si-3治疗促进骨质疏松小鼠骨形成，改善小梁微结构，逆转骨质流失。结论：经鉴定的最佳CKIP-1 siRNA序列（si-3）可促进体外物种间成骨分化，刺激健康啮齿动物成骨，逆转骨质疏松小鼠骨质流失。(C) 2013出版的爱思唯尔公司。
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. (C) 2013 Published by Elsevier Inc.