Changhai Hospital of Traditional Chinese Medicine, Second Military Medical University, Shanghai 200433, China.
Department of Orthopaedics and Rehabilitation, University of Florida College of Medicine, Gainesville, Florida 32611, USA.
J Integr Med. 2014 Jan;12(1):20-34. doi: 10.1016/S2095-4964(14)60003-0.
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.
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.
We observed that treatment with pristimerin, at as low as 1 μmol/L concentration, significantly enhanced rAAV2 vector-mediated transgene expression in vitro, and intraperitoneal co-administration with pristimerin at 4 mg/(kg·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.
These studies suggest the potential beneficial use of pristimerin and pristimerin-containing herb extract in future liver-targeted gene therapy with rAAV vectors.
在本研究中，我们系统评估了两种来自中药的生物活性化合物——雷公藤红素和千金藤素，在体外人细胞系和体内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。
用 rAAV 载体感染人细胞系，分别进行模拟处理或用雷公藤红素或千金藤素处理。处理后检测转基因表达、核转位病毒基因组的百分比和细胞内蛋白质的泛素化。此外，非肥胖型糖尿病/严重联合免疫缺陷γ（NSG）小鼠尾静脉注射 rAAV 载体，并同时给予二甲基亚砜、雷公藤红素、千金藤素或阳性对照药硼替佐米。随时间检测和比较肝脏中的转基因表达。
我们观察到，以低至 1 μmol/L 的浓度用千金藤素处理可显著增强 rAAV2 载体介导的转基因表达，腹腔内联合给予千金藤素 4 mg/（kg·d）连续 3 天可显著促进鼠肝细胞中病毒转导。千金藤素还显著增强了酪氨酸突变 rAAV2 载体和携带超大转基因盒的 rAAV8 载体的转导效率。千金藤素介导 rAAV 载体转导效率增加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转位。
这些研究表明，千金藤素和含千金藤素的草药提取物在未来以 rAAV 载体为基础的肝靶向基因治疗中有潜在的有益作用。
