Empirical advantages of adeno associated viral vectors in vivo gene therapy for arthritis.
Empirical advantages of adeno associated viral vectors in vivo gene therapy for arthritis.
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发表时间：
2000-04
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通讯作者：
J. Goater;R. Müller;G. Kollias;G. Firestein;I. Sanz;R. O’Keefe;E. Schwarz
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作者：
J. Goater;R. Müller;G. Kollias;G. Firestein;I. Sanz;R. O’Keefe;E. Schwarz
目的评价腺相关病毒（腺相关病毒）载体在体内和体外关节细胞基因递送中的实用性，并评估其作为关节炎基因治疗载体的潜力。方法将表达细菌β-半乳糖苷酶（β-gal）基因的重组腺相关病毒（rAAV）载体（rAAV-CMV-LacZ）直接导入健康正常小鼠膝关节或肿瘤坏死因子α（hTNFa-Tg）过表达小鼠关节炎膝关节。通过免疫组织化学和化学发光法测定β-gal表达水平。通过流式细胞术测定该载体在体外对原代成纤维细胞样滑膜细胞（FLS）的转导效率。使用相同的方法测定UV和γ-照射以及TNF-α对转导效率的影响。结果我们在健康小鼠关节细胞中几乎没有发现rAAV转导的证据。在第3天在所有动物中检测到靶基因表达，并且在注射后21天恢复到基线水平之前在第7天达到峰值。相反，在hTNFa-Tg动物中，患病小鼠的滑膜细胞、关节软骨细胞和滑膜细胞被rAAV-CMV-LacZ转导。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，rAAV转导FLS可以增强预处理与紫外线或γ-照射和TNF-α刺激。结论rAAV载体在关节炎的体内基因治疗中具有以下优势：（1）rAAV在体内优先转导关节炎细胞; (2)rAAV在体内可同时转染FLS和软骨细胞。(3)FLS的rAAV转导可以通过用诱导DNA修复酶的试剂预处理来增强。
OBJECTIVE 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. METHODS 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. RESULTS 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. CONCLUSION 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.