Garcia-Bañuelos J, Siller-Lopez F, Miranda A, Aguilar L K, Aguilar-Cordova E, Armendariz-Borunda J
Institute of Molecular Biology in Medicine and Gene Therapy, CUCS, University of Guadalajara, Guadalajara, Mexico.
Gene Ther. 2002 Jan;9(2):127-34. doi: 10.1038/sj.gt.3301647.
Adenoviral vectors efficiently target normal liver cells; however, a clear-cut description of the safety boundaries for using adenovectors in hepatic cirrhosis has not been settled. With this in mind, we used a first-generation, replication-deficient adenoviral vector carrying the E. coli lacZ gene (Ad5betaGal) to monitor therapeutic range, biodistribution, toxicity and transduction efficiency in Wistar rats made cirrhotic by two different experimental approaches resembling alcoholic cirrhosis and biliary cirrhosis in humans. Further, we show proof of concept on fibrosis reversion by a 'therapeutic' Ad-vector (AdMMP8) carrying a gene coding for a collagen-degrading enzyme. Dose-response experiments with Ad5betaGal ranging from 1 x 10(8)-3 x 10(12) viral particles (vp) per rat (250 g), demonstrated that adenovirus-mediated gene transfer via iliac vein at 3 x 10(11 )vp/rat, resulted in an approximately 40% transduction in livers of rats made cirrhotic by chronic intoxication with carbon tetrachloride, compared with approximately 80% in control non-cirrhotic livers. In rats made cirrhotic by bile-duct obstruction only, 10% efficiency of transduction was observed. Biodistribution analyses showed that vector expression was detected primarily in liver and at a low level in spleen and kidney. Although there was an important increase in liver enzymes between the first 48 h after adenovirus injection in cirrhotic animals compared to non-transduced cirrhotic rats, this hepatic damage was resolved after 72-96 h. Then, the cDNA for neutrophil collagenase, also known as Matrix Metalloproteinase 8 (MMP8), was cloned in an Ad-vector and delivered to cirrhotic rat livers being able to reverse fibrosis in 44%. This study demonstrates the potential use of adenoviral vectors in safe transient gene therapy strategies for human liver cirrhosis.
腺病毒载体能有效地靶向正常肝细胞；然而，关于在肝硬化中使用腺病毒载体的安全界限尚无明确描述。考虑到这一点，我们使用携带大肠杆菌lacZ基因的第一代复制缺陷型腺病毒载体（Ad5βGal），来监测通过两种类似于人类酒精性肝硬化和胆汁性肝硬化的不同实验方法诱导肝硬化的Wistar大鼠的治疗范围、生物分布、毒性和转导效率。此外，我们展示了携带编码胶原降解酶基因的“治疗性”腺病毒载体（AdMMP8）逆转纤维化的概念验证。用Ad5βGal进行的剂量反应实验，每只大鼠（250克）的病毒颗粒（vp）范围为1×10⁸ - 3×10¹² ，结果表明，通过髂静脉以3×10¹¹ vp/大鼠进行腺病毒介导的基因转移，在经四氯化碳慢性中毒诱导肝硬化的大鼠肝脏中，转导率约为40%，而在对照非肝硬化肝脏中约为80%。在仅通过胆管阻塞诱导肝硬化的大鼠中，观察到转导效率为10%。生物分布分析表明，载体表达主要在肝脏中检测到，在脾脏和肾脏中水平较低。尽管与未转导的肝硬化大鼠相比，肝硬化动物在腺病毒注射后的前48小时内肝酶有显著升高，但这种肝损伤在72 - 96小时后得到缓解。然后，将也称为基质金属蛋白酶8（MMP8）的中性粒细胞胶原酶的cDNA克隆到腺病毒载体中，并递送至肝硬化大鼠肝脏，能够使44%的纤维化得到逆转。这项研究证明了腺病毒载体在人类肝硬化安全瞬时基因治疗策略中的潜在应用。
