重组腺相关病毒介导的骨保护素基因治疗抑制磨损碎片诱导的骨溶解

Douglas D. Robertson, Michael J. Mueller, Kirk E. Smith, P. Commean, Thomas Pilgram, Jeffrey E. Johnson
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引用次数: 150

摘要

背景:磨损碎片引起的骨溶解继发的骨科植入物无菌松动是一个严重的问题。骨保护素(OPG)是一种抑制破骨细胞活化和骨吸收的天然诱饵蛋白。本研究探讨了使用表达OPG的重组腺相关病毒载体进行基因治疗是否能抑制磨损碎片诱导的骨溶解。方法:构建共表达OPG的重组腺相关病毒(rAAV)载体(rAAV-OPG- ires - egfp)。同时制备了表达b-半乳糖苷酶(rAAV-LacZ)的对照载体。体外验证实验检测rAAV-OPG-IRES-EGFP转导效率、OPG在骨薄片中的表达水平和功能以及破骨细胞活性。rAAV-OPG-IRES-EGFP体内基因治疗对磨损碎片诱导的骨溶解的影响随后在小鼠颅骨模型中进行了评估,该模型在引入磨损碎片之前进行了单次肌内注射载体。第10天采用组织形态学法测定rAAV-OPG-IRES-EGFP基因治疗对磨损碎片诱导的破骨细胞生成和骨吸收的影响。结果:体外实验表明,rAAV-OPG-IRES-EGFP和rAAV-LacZ在1000次感染下,100%的人胚胎肾293细胞被转导。在rAAV-OPG-IRES-EGFP感染1000次的情况下,4天后培养基中OPG浓度达到135 ng/mL。通过骨片破骨细胞活性测定,我们发现rAAV-OPG-IRES-EGFP治疗与甲状旁腺激素刺激对照相比,吸收减少了7倍,与rAAV-LacZ对照相比减少了11倍。此外,在含有raav - opg - ires - egfp感染的成纤维细胞的共培养中,观察到RANKL和巨噬细胞集落刺激因子诱导的脾细胞破骨细胞生成减少了17倍。体内给药rAAV-OPG-IRES-EGFP导致注射部位肌细胞可检测到转导,血清OPG水平在第2天显著升高(p < 0.05)。在第6天达到最大浓度,然后在整个观察期趋于平稳。相比之下,在假处理、未感染钛刺激或raav - lacz感染的小鼠中无法检测到血清OPG。在对照小鼠中,钛植入导致矢状缝合线附近的破骨细胞平均数量增加了三倍,矢状缝合线的软组织平均面积增加了两倍。相比之下,在接受rAAV-OPG-IRES-EGFP治疗的钛植入动物中,破骨细胞数量保持在基础水平,矢状缝处软组织面积明显减少,表明钛颗粒对骨溶解的完全抑制。结论:单次肌内注射rAAV-OPG-IRES-EGFP载体可有效诱导肌细胞产生高水平的OPG。OPG有效抑制磨损碎片诱导的破骨细胞生成和骨溶解。临床意义:目前,还没有批准的药物治疗来预防或抑制假体周围骨溶解。虽然临床前研究已经确定了潜在的药物治疗(即双膦酸盐),但没有证据表明这些药物可以有效治疗患者的无菌性松动。这是体内OPG基因治疗可用于预防磨损碎片诱导的骨溶解的第一个证据。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Recombinant Adeno-Associated Virus-Mediated Osteoprotegerin Gene Therapy Inhibits Wear Debris-Induced Osteolysis
Background: Aseptic loosening of orthopaedic implants secondary to wear debris-induced osteolysis is a serious problem. Osteoprotegerin (OPG) is a natural decoy protein that inhibits osteoclast activation and bone resorption. This study investigated whether gene therapy using a recombinant adeno-associated viral vector that expresses OPG can inhibit wear debris-induced osteolysis.Methods: A recombinant adeno-associated virus (rAAV) vector co-expressing OPG (rAAV-OPG-IRES-EGFP) was generated. A control vector expressing b-galactosidase (rAAV-LacZ) was also prepared. In vitro validation experiments were performed to determine rAAV-OPG-IRES-EGFP transduction efficiency, OPG expression level and function in bone wafer, and osteoclastic activity.The effect of rAAV-OPG-IRES-EGFP in vivo gene therapy on wear debris-induced osteolysis was then evaluated in a mouse calvarial model in which a single intramuscular injection of the vector was administered prior to the introduction of the wear debris. The effects of the rAAV-OPG-IRES-EGFP gene therapy on wear debris-induced osteoclastogenesis and bone resorption were determined by histomorphometry on day 10.Results: In vitro experiments revealed that 100% of human embryonic kidney 293 cells were transduced at a multiplicity of infection of 1000 with both rAAV-OPG-IRES-EGFP and rAAV-LacZ. At a rAAV-OPG-IRES-EGFP multiplicity of infection of 1000, an OPG concentration of 135 ng/mL of culture media was achieved after four days. Using a bone-wafer assay for osteoclast activity, we found that treatment with rAAV-OPG-IRES-EGFP reduced resorption sevenfold compared with parathyroid hormone-stimulated controls and elevenfold compared with rAAV-LacZ controls. Furthermore, a seventeenfold decrease in RANKL and macrophage colony-stimulating factor-induced splenocyte osteoclastogenesis was observed in co-cultures containing rAAV-OPG-IRES-EGFP-infected fibroblasts.In vivo administration of rAAV-OPG-IRES-EGFP resulted in detectable transduction of myocytes at the injection site and a significant increase in expression of serum OPG levels by the second day (p < 0.05). Maximal concentrations were obtained on day 6 and then leveled off throughout the observation period. In contrast, serum OPG could not be detected in the sham-treated, uninfected titanium-stimulated, or rAAV-LacZ-infected mice. In the control mice, titanium implantation resulted in a threefold increase in the mean number of osteoclasts adjacent to the sagittal suture as well as a twofold increase in the mean area of soft tissue in the sagittal suture compared with the sham-treated mice. In contrast, osteoclast numbers remained at basal levels, and the area of soft tissue in the sagittal suture was markedly reduced in titanium-implanted animals that received rAAV-OPG-IRES-EGFP treatment, demonstrating a complete inhibition of osteolysis in response to titanium particles.Conclusions: A single intramuscular injection of the rAAV-OPG-IRES-EGFP vector can efficiently transduce myocytes to produce high levels of OPG. The OPG effectively inhibits wear debris-induced osteoclastogenesis and osteolysis.Clinical Relevance: Currently, there is no approved drug therapy to prevent or inhibit periprosthetic osteolysis. Although preclinical studies have identified potential drug therapies (i.e., bisphosphonates), there is no evidence that these drugs can effectively treat aseptic loosening in patients. This is the first evidence that in vivo OPG gene therapy can be used to prevent wear debris-induced osteolysis.
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