Goater J, Müller R, Kollias G, Firestein G S, Sanz I, O'Keefe R J, Schwarz E M
Department of Medicine, University of Rochester Medical Center, NY 14642, USA.
J Rheumatol. 2000 Apr;27(4):983-9.
To evaluate the utility of the adeno associated viral (AAV) vector for gene delivery to joint cells in vivo and in vitro, and to assess its potential as a vector for arthritis gene therapy.
A recombinant AAV (rAAV) vector expressing the bacterial beta-galactosidase (beta-gal) gene (rAAV-CMV-LacZ) was directly introduced into healthy-normal mouse knees, or arthritic knees in mice overexpressing tumor necrosis factor-alpha (hTNFalpha-Tg). Beta-gal expression levels were determined by immunohistochemistry and chemiluminescence. The transduction efficiency of this vector on primary fibroblast-like synoviocytes (FLS) in vitro was determined by FACS. The effects of UV and gamma-irradiation as well as TNF-alpha on transduction efficiency were determined using the same methods.
We found little evidence of rAAV transduction in the joint cells of healthy mice. Target gene expression was detected in all animals at Day 3, and peaked at Day 7 before returning to baseline levels 21 days after injection. In contrast, synoviocytes, articular chondrocytes, and meniscal cells of diseased mice were transduced by rAAV-CMV-LacZ in hTNFalpha-Tg animals. Transduction efficiencies correlated with joint damage, and target gene expression was up to 10-fold greater than that seen in the normal mice. In vitro, we found that rAAV transduction of FLS can be enhanced by pretreatment with UV or gamma-irradiation and TNF-alpha stimulation.
We find that rAAV vectors have several empirical advantages for in vivo gene therapy for arthritis: (1) rAAV preferentially transduces arthritic joint cells in vivo. (2) rAAV can transduce both FLS and chondrocytes in vivo. (3) rAAV transduction of FLS can be augmented by pretreatment with agents that induce DNA repair enzymes.
评估腺相关病毒（AAV）载体在体内和体外向关节细胞进行基因传递的效用，并评估其作为关节炎基因治疗载体的潜力。
将表达细菌β-半乳糖苷酶（β-gal）基因的重组腺相关病毒（rAAV）载体（rAAV-CMV-LacZ）直接注射到健康正常小鼠的膝关节，或过表达肿瘤坏死因子-α（hTNFα-Tg）的小鼠的关节炎膝关节中。通过免疫组织化学和化学发光法测定β-gal的表达水平。通过流式细胞术（FACS）测定该载体在体外对原代成纤维样滑膜细胞（FLS）的转导效率。使用相同方法确定紫外线和γ射线照射以及肿瘤坏死因子-α对转导效率的影响。
我们发现健康小鼠关节细胞中几乎没有rAAV转导的证据。在第3天，所有动物均检测到靶基因表达，并在第7天达到峰值，然后在注射后21天恢复到基线水平。相比之下，在hTNFα-Tg动物中，rAAV-CMV-LacZ可转导患病小鼠的滑膜细胞、关节软骨细胞和半月板细胞。转导效率与关节损伤相关，靶基因表达比正常小鼠高10倍。在体外，我们发现用紫外线或γ射线照射以及肿瘤坏死因子-α刺激预处理可增强FLS的rAAV转导。
我们发现rAAV载体在关节炎体内基因治疗方面具有几个经验优势：（1）rAAV在体内优先转导关节炎关节细胞。（2）rAAV在体内可转导FLS和软骨细胞。（3）用诱导DNA修复酶的试剂预处理可增强FLS的rAAV转导。
