Proteasome inhibition enhances AAV-mediated transgene expression in human synoviocytes in vitro and in vivo
Proteasome inhibition enhances AAV-mediated transgene expression in human synoviocytes in vitro and in vivo
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DOI：
10.1016/j.ymthe.2004.10.020
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发表时间：
2005-04-01
影响因子：
12.4
通讯作者：
Hirsch, R
中科院分区：
文献类型：
--
作者：
Jennings, K;Miyamae, T;Hirsch, R
为探讨重组腺相关病毒(RAAV)载体在类风湿关节炎(RA)治疗中的应用价值，在CMV启动子的控制下，将携带小鼠IL-10基因的重组腺相关病毒(RAAV)感染类风湿关节炎(RA)患者的原代人成纤维样滑膜细胞(FLS)。加入蛋白酶体抑制剂羧基苯甲氧基-L-亮氨酰-L-亮氨酰-L亮氨酸可显著增强IL-10转基因的表达，且呈剂量依赖关系。表达的增加是短暂的，在3天达到峰值，7天恢复到接近基线水平。即使在感染rAAV后13天加入zLLL，也能观察到这种增强。ZLLL的作用不是mIL-10转基因或CMV启动子所特有的，因为类似的发现是在CMV启动子驱动的鸡β-肌动蛋白启动子或GFP控制下编码α1-抗胰蛋白酶的rAAV构建。再次暴露于zLLL可反复诱导转基因表达。转基因mRNA水平与蛋白质水平同步增加。给预先注射了rAAV感染的FLS的SCID小鼠注射zLLL，也可以在体内重复诱导转基因表达。这些数据表明，抑制蛋白酶体可以显著增强rAAV感染后人RA FLS的转基因表达，并为体内调节滑膜转基因表达提供了一种可能的途径。
To explore the potential applicability of recombinant adeno-associated virus (rAAV) vectors in the treatment of rheumatoid arthritis (RA), primary human fibroblast-like synoviocytes (FLS) derived from patients with RA were infected with rAAV encoding mouse IL-10 under the control of the CMV promoter. Addition of the proteasome inhibitor carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (zLLL) to the cultures dramatically enhanced expression of the IL-10 transgene, in a dose-dependent manner. The increased expression was transient, peaking at 3 days and returning to near baseline by 7 days. The enhancement was observed even when zLLL was added 13 days after infection with rAAV. The effect of zLLL was not specific to either the mIL-10 transgene or the CMV promoter, as similar findings were observed using an rAAV construct encoding alpha 1-anti-trypsin under the control of the chick beta-actin promoter or GFP, driven by the CMV promoter. Transgene expression could be repeatedly induced by reexposure to zLLL. Transgene mRNA levels increased in parallel with protein levels. Transgene expression could also be repeatedly induced in vivo by administering zLLL to SCID mice previously injected with rAAV-infected FLS. These data demonstrate that proteasome inhibition can dramatically enhance transgene expression in human RA FLS following infection with rAAV and suggest a possible approach to regulating synovial transgene expression in vivo.