Pristimerin enhances recombinant adeno-associated virus vector-mediated transgene expression in human cell lines in vitro and murine hepatocytes in vivo
Pristimerin enhances recombinant adeno-associated virus vector-mediated transgene expression in human cell lines in vitro and murine hepatocytes in vivo
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DOI：
10.1016/s2095-4964(14)60003-0
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发表时间：
2014-01-01
影响因子：
4.8
通讯作者：
Ling, Chen
中科院分区：
文献类型：
--
作者：
Wang, Li-na;Wang, Yuan;Ling, Chen
目的：在本研究中，我们系统地评估了两种来自中药的生物活性化合物，celastrol和pristimerin在体外人和小鼠肝细胞中增强重组腺相关病毒（rAAV）血清型载体介导的转基因表达的能力。方法：用rAAV载体对人细胞系进行模拟处理，或用celastrol或pritimin处理。处理后检测转基因表达、核易位病毒基因组百分比和细胞内蛋白泛素化。此外，非肥胖糖尿病/严重联合免疫缺陷γ (NSG)小鼠尾部静脉注射rAAV载体，并与二甲亚砜、雷公藤红素、普瑞替木林或阳性对照体博替佐米共同给药。检测转基因在肝脏中的表达，并随时间进行比较。结果：我们观察到，低至1 μ mol/L浓度的pritimerin在体外显著增强rAAV2载体介导的转基因表达，并且以4 mg/（kg中心点d）腹腔共给药3 d显著促进病毒在小鼠肝细胞中的转导。酪氨酸突变体rAAV2载体和携带超大转基因盒的rAAV8载体的转导效率也被pritimerin显著提高。pritimerin介导rAAV载体转导效率提高的潜在分子机制包括抑制细胞内蛋白质的蛋白酶体降解和增强载体基因组的核易位。结论：这些研究提示春茉莉素和含春茉莉素的草药提取物在未来以rAAV载体进行肝脏靶向基因治疗中的潜在益处。
OBJECTIVE: In the present study, we systemically evaluated the ability of two bioactive compounds from traditional Chinese medicine, celastrol and pristimerin, to enhance recombinant adeno-associated virus (rAAV) serotype vector-mediated transgene expression both in human cell lines in vitro, and in murine hepatocytes in vivo.METHODS: Human cell lines were infected with rAAV vectors with either mock treatment or treatment with celastrol or pristimerin. The transgene expression, percentage of nuclear translocated viral genomes and the ubiquitination of intracellular proteins were investigated post-treatment. In addition, nonobese diabetic/severe combined immunodeficient gamma (NSG) mice were tail vain-injected with rAAV vectors and co-administered with either dimethyl sulfoxide, celastrol, pristimerin or a positive control, bortezomib. The transgene expression in liver was detected and compared over time.RESULTS: We observed that treatment with pristimerin, at as low as 1 mu mol/L concentration, significantly enhanced rAAV2 vector-mediated transgene expression in vitro, and intraperitoneal co-administration with pristimerin at 4 mg/(kg center dot d) for 3 d dramatically facilitated viral transduction in murine hepatocytes in vivo. The transduction efficiency of the tyrosine-mutant rAAV2 vectors as well as that of rAAV8 vectors carrying oversized transgene cassette was also augmented significantly by pristimerin. The underlying molecular mechanisms by which pristimerin mediated the observed increase in the transduction efficiency of rAAV vectors include both inhibition of proteasomal degradation of the intracellular proteins and enhanced nuclear translocation of the vector genomes.CONCLUSION: These studies suggest the potential beneficial use of pristimerin and pristimerin-containing herb extract in future liver-targeted gene therapy with rAAV vectors.