The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, Zhejiang, China.
The First Clinical Medical College of Zhejiang Chinese Medical University, Hangzhou, Zhejiang, China.
PLoS One. 2020 Dec 15;15(12):e0243359. doi: 10.1371/journal.pone.0243359. eCollection 2020.
Osteoarthritis (OA) is the most common arthropathy, characterized by progressive degeneration of the articular cartilage. Currently, there are no disease-modifying approaches for OA treatment. Adeno-associated virus (AAV)-mediated gene therapy has recently become a potential treatment for OA due to its exceptional characteristics; however, the tropism and transduction efficiency of different AAV serotypes to articular joints and the safety profile of AAV applications are still unknown. The present study aims to screen an ideal AAV serotype to efficiently transfer genes to arthritic cartilage. AAV vectors of different serotypes expressing eGFP protein were injected into the knee joint cavities of mice, with all joint tissues collected 30 days after AAV injection. The transduction efficiency of AAVs was quantified by assessing the fluorescent intensities of eGFP in the cartilage of knee joints. Structural and morphological changes were analyzed by toluidine blue staining. Changes to ECM metabolism and pyroptosis of chondrocytes were determined by immunohistochemical staining. Fluorescence analysis of eGFP showed that eGFP was expressed in the cartilage of knee joints injected with each AAV vector. Quantification of eGFP intensity indicated that AAV2, 7 and 8 had the highest transduction efficiencies. Both toluidine blue staining and Mankin score showed that AAV6 aggravated cartilage degeneration. The analysis of key molecules in ECM metabolism suggested that AAV5 and 7 significantly reduced collagen type II, while AAV9 increased ADAMTS-4 but decreased MMP-19. In addition, transduction with AAV2, 5, 7 and 8 had no obvious effect on pyroptosis of chondrocytes. Comprehensive score analysis also showed that AAV2 had the highest score in intra-articular gene transfer. Collectively, our findings point to AAV2 as the best AAV serotype candidate for gene transfer on arthritic cartilage, resulting in minimal impact to ECM metabolism and pyroptosis of chondrocytes.
骨关节炎（OA）是最常见的关节病，其特征为关节软骨进行性退化。目前，OA 尚无改变病情的治疗方法。腺相关病毒（AAV）介导的基因治疗由于其独特的特性，最近成为 OA 的一种潜在治疗方法；然而，不同 AAV 血清型对关节的亲嗜性和转导效率以及 AAV 应用的安全性概况仍不清楚。本研究旨在筛选一种理想的 AAV 血清型，以有效将基因转移至关节炎软骨。不同血清型的 AAV 载体表达 eGFP 蛋白，注入小鼠膝关节腔，在 AAV 注射后 30 天收集所有关节组织。通过评估膝关节软骨中 eGFP 的荧光强度来量化 AAV 的转导效率。通过甲苯胺蓝染色分析结构和形态变化。通过免疫组织化学染色测定 ECM 代谢和软骨细胞焦亡的变化。eGFP 的荧光分析显示，每个 AAV 载体注射的膝关节软骨中均表达 eGFP。eGFP 强度的定量表明 AAV2、7 和 8 具有最高的转导效率。甲苯胺蓝染色和 Mankin 评分均表明 AAV6 加重了软骨退化。ECM 代谢关键分子的分析表明，AAV5 和 7 显著降低了胶原 II 型，而 AAV9 增加了 ADAMTS-4 但降低了 MMP-19。此外，AAV2、5、7 和 8 的转导对软骨细胞的焦亡没有明显影响。综合评分分析还表明，AAV2 在关节内基因转移中得分最高。综上所述，我们的研究结果表明，AAV2 是关节炎软骨基因转移的最佳 AAV 血清型候选物，对 ECM 代谢和软骨细胞焦亡的影响最小。
