Department of Neurosurgery, Medical University of Innsbruck, Innsbruck 6020, Austria.
Exp Biol Med (Maywood). 2023 Jul;248(13):1134-1144. doi: 10.1177/15353702231171905. Epub 2023 Jun 24.
Degenerative disk disease (DDD) that aggravates structural deterioration of intervertebral disks (IVDs) can be accompanied by painful inflammation and immunopathological progressions. Current surgical or pharmacological therapies cannot repair the structure and function of IVDs. Nucleus pulposus (NP) cells are crucial for the preservation or restoration of IVDs by balancing the anabolic and catabolic factors affecting the extracellular matrix. Imbalanced anabolic and catabolic factors cause increased degradation of aggrecan. Aggrecanases A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS)4 and ADAMTS5 are the main degrading enzymes of aggrecan. Previously, we characterized adeno-associated virus (AAV6) as the most suitable serotype with marked NP cellular tropism and demonstrated that ADAMTS4 could be silenced by self-complementary adeno-associated virus grade 6 small helix ribonucleic acid (scAAV6-shRNA) in NP cells of degeneration grade III, which resulted in enrichment of aggrecan. Nonetheless, neither scAAV6-shRNA-mediated inhibition of ADAMTS5 nor joint inhibitions of ADAMTS4 and ADAMTS5 have been investigated, although both enzymes are regulated by analogous proinflammatory cytokines and have the same cleavage sites in aggrecan. Therefore, we attempted scAAV6-shRNA-mediated inhibitions of both enzymes in NP cells of degeneration grade IV to increase efficacies in treatments of DDD. The degeneration grade of IVDs in patients was determined by magnetic resonance imaging (MRI) before surgical operations. After isolation and culturing of NP cells, cells were transduced with scAAV6-shRNAs targeting ADAMTS4 or ADAMTS5. Transduced cells were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR), fluorescence microscopy, flow cytometry-assisted cell sorting (FACS), MTT assay (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay), immunoblotting, and enzyme-linked immunosorbent assay (ELISA). Joint transduction of NP cells exhibited high transduction efficacies (98.1%), high transduction units (TU) (1381 TU/Cell), and no effect on cell viability or proliferation. Above all joint treatments resulted in effective knockdown of ADAMTS4 (92.8%) and ADAMTS5 (93.4%) along with additive enrichment of aggrecan (113.9%). Treatment effects were significant for more than 56 days after transduction ( < 0.001). In conclusion, scAAV6-shRNA-mediated combined molecular therapy could be very valuable for more effective, durable, and less immunogenic treatment approaches in DDD.
退行性椎间盘疾病（DDD）可加重椎间盘（IVD）的结构恶化，并伴有疼痛性炎症和免疫病理进展。目前的手术或药物治疗不能修复 IVD 的结构和功能。 核（NP）细胞对于通过平衡影响细胞外基质的合成代谢和分解代谢因素来维持或恢复 IVD 至关重要。 合成代谢和分解代谢因素的失衡导致聚集蛋白聚糖的降解增加。 解整合素和金属蛋白酶与血栓反应蛋白基序（ADAMTS）4 和 ADAMTS5 是聚集蛋白聚糖的主要降解酶。 先前，我们将腺相关病毒（AAV6）鉴定为具有明显 NP 细胞趋向性的最适合血清型，并证明 III 级退变的 NP 细胞中，自我互补的腺相关病毒 6 型小螺旋 RNA（scAAV6-shRNA）可以沉默 ADAMTS4，从而导致聚集蛋白聚糖的富集。 尽管两种酶均受类似的促炎细胞因子调节且在聚集蛋白聚糖中有相同的裂解位点，但尚未研究 scAAV6-shRNA 介导的 ADAMTS5 抑制或 ADAMTS4 和 ADAMTS5 的联合抑制。 因此，我们尝试在 IV 级退变的 NP 细胞中进行 scAAV6-shRNA 介导的双重抑制，以提高 DDD 治疗的疗效。 在手术前，通过磁共振成像（MRI）确定患者 IVD 的退变程度。 分离并培养 NP 细胞后，用 scAAV6-shRNA 转导针对 ADAMTS4 或 ADAMTS5 的 NP 细胞。 通过逆转录定量聚合酶链反应（RT-qPCR）、荧光显微镜、流式细胞术辅助细胞分选（FACS）、MTT 测定（3-（4,5-二甲基噻唑-2）-2,5-二苯基四唑溴化物测定）、免疫印迹和酶联免疫吸附测定（ELISA）分析转导细胞。 联合转导 NP 细胞显示出高转导效率（98.1%）、高转导单位（TU）（1381 TU/Cell），且对细胞活力或增殖无影响。 最重要的是，联合治疗可有效敲低 ADAMTS4（92.8%）和 ADAMTS5（93.4%），同时聚集蛋白聚糖的含量增加（113.9%）。 转导后超过 56 天的治疗效果显著（<0.001）。 总之，scAAV6-shRNA 介导的联合分子治疗对于 DDD 的更有效、更持久和免疫原性更低的治疗方法可能非常有价值。
