Efficiency of dual siRNA-mediated gene therapy for intervertebral disc degeneration (IVDD)
Efficiency of dual siRNA-mediated gene therapy for intervertebral disc degeneration (IVDD)
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DOI：
10.1016/j.spinee.2018.10.016
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发表时间：
2019-05-01
期刊：
影响因子：
4.5
通讯作者：
Subbaiah, G. P., V
中科院分区：
文献类型：
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作者：
Banala, Rajkiran Reddy;Vemuri, Satish Kumar;Subbaiah, G. P., V
背景：腰椎间盘退变是腰痛的常见原因之一。椎间盘退变的病理生理机制涉及髓核细胞的凋亡和细胞外基质的降解。Caspase 3在细胞凋亡中起核心作用，ADAMTS5 （a Disintegrin and Metalloproteinase with Thrombospondin motif 5）基因在ECM降解中起关键作用。因此，我们假设，如果能够沉默这两个基因，细胞凋亡和ECM降解都可以被阻止，从而阻止椎间盘退变的进展，甚至逆转椎间盘退变。目的：本研究的目的是在体外和动物椎间盘退变模型中证明针对Caspase 3和ADAMTS5基因设计的小干扰RNA （siRNA）的再生潜力。研究设计：先进行体外研究，再进行兔模型体内研究。方法：采用人肝细胞癌（Hep G2）细胞系进行体外研究，验证脂质体siRNA对Caspase 3和ADAMTS5基因表达的调控作用。随后，在兔环穿刺模型中，通过单独给药siRNA （Caspase 3和ADAMTS5）和联合Caspase3-ADAMTS5)来验证siRNA的有效性，以评估它们在下调退行性椎间盘基因表达方面的协同作用。首次穿刺一周后，在兔的环状椎间盘上注射siRNA制剂（单和双）和磷酸盐缓冲盐水。在siRNA治疗前后（1,4和8周）进行磁共振成像（MRI）扫描，以评估椎间盘退变的进展。采用组织病理学和实时聚合酶链反应（RT-PCR）评价其疗效。我们没有收到任何进行这项研究的资金，我们与任何研究人员或科学团体都没有利益冲突。结果：体外和体内研究均表明siRNA制剂对下调Caspase 3和ADAMTS5基因的表达有有益作用。MRI和组织病理学评价显示，磷酸缓冲盐水和AT5-siRNA注射椎间盘椎间盘退变是进行性的，而Caspase 3-siRNA和双siRNA （Cas3-AT5-siRNA）制剂的椎间盘在注射后4周和8周表现出恢复和再生的迹象。在体外和体内实验中，通过RT-PCR分析，对设计的siRNA抗Cas3和AT5的效果进行了评估，结果显示Caspase 3-siRNA组Caspase 3基因下调，而ADAMTS5-siRNA组ADAMTS5基因未出现显著下调（即以折线变化表示）。双siRNA （Cas3-AT5 siRNA）制剂组观察到协同效应。结论：本实验提示，siRNA干预可显著降低椎间盘细胞凋亡程度。临床意义：将siRNA直接送入椎间盘具有非手术治疗椎间盘退变的潜力。(C) 2018爱思唯尔公司版权所有。
BACKGROUND CONTEXT: One of the common causes of low back pain is intervertebral disc degeneration. The pathophysiology of disc degeneration involves apoptosis of nucleus pulposes cells and degradation of extra cellular matrix (ECM). Caspase 3 plays a central role in apoptosis and the ADAMTS5 (A Disintegrin and Metalloproteinase with Thrombospondin motifs 5) gene plays a critical role in ECM degradation. Hence, we hypothesized that if one can silence these two genes, both apoptosis and ECM degradation can be prevented, thereby preventing the progression and even reverse disc degeneration.PURPOSE: The purpose of this study is to demonstrate the regenerative potential of small interfering RNA (siRNA) designed against Caspase 3 and ADAMTS5 genes in an in vitro and animal model of disc degeneration.STUDY DESIGN: In vitro study followed by in vivo study in a rabbit model.METHODS: In vitro studies were done using the human hepatocellular carcinoma (Hep G2) cell line for validating the efficacy of liposomal siRNA in controlling the expression of genes (Caspase 3 and ADAMTS5). Later, siRNA's validation was done in a rabbit annular punctured model by administering siRNA's individually (Caspase 3 and ADAMTS5) and in combination Caspase3-ADAMTS5) for assessing their synergistic effect in down regulating the gene expression in the degenerative discs. Annular punctured intervertebral discs of the rabbit were injected with siRNA formulations (single and dual) and phosphate buffer saline, one week after initial puncture. Magnetic resonance imaging (MRI) scans were done before and after siRNA treatment (1, 4 and 8 weeks) for assessing the progression of disc degeneration. The histopathology and real time polymerase chain reaction (RT-PCR) studies were done for evaluating their efficacy. We did not receive any funding for conducting the study, and we do not have a conflict of interest with any researchers or scientific groups.RESULTS: The observations made from both in vitro and in vivo studies indicate the beneficial effects of siRNA formulation in down regulating the expression of Caspase 3 and ADAMTS5 genes. The MRI and histopathological evaluation showed that the disc degeneration was progressive in phosphate buffer saline and AT5-siRNA injected discs but the discs that received Caspase 3-siRNA and dual siRNA (Cas3-AT5-siRNA) formulation showed signs of recovery and regeneration 4 and 8 weeks after injection. The efficacy of siRNA designed against Cas3 and AT5 was also assessed in both in vitro and in vivo experiments by using RT-PCR analysis and the results showed downregulation of Caspase 3 gene in Caspase 3-siRNA group, but there was no significant downregulation of ADAMTS5 gene in ADAMTS5-siRNA group (ie, indicated by fold change). Synergistic effect was observed in the group that received dual siRNA (Cas3-AT5 siRNA) formulation.CONCLUSIONS: This experiment suggests that intervention by siRNA treatment significantly reduced the extent of apoptosis in the discs.CLINICAL SIGNIFICANCE: Delivery of siRNA directly into spinal discs has a potential in treating disc degeneration nonsurgically. (C) 2018 Elsevier Inc. All rights reserved.