Department of Molecular Biology and Genomics, Institute for Molecular Biology and Gene Therapy, University of Guadalajara, Sierra Mojada 950, Colonia Independencia, Edificio Q. Tercer Piso, CP 44340, Guadalajara, Jalisco, Mexico.
Department of Health Sciences, University of Guadalajara, Av. Periférico Oriente 555, Ejido San José Tateposco, CP 48525, Tonalá, Jalisco, Mexico.
Mol Biotechnol. 2020 Apr;62(4):260-272. doi: 10.1007/s12033-020-00247-x.
Pre-existing immune response against adenovirus could diminish transgene expression efficiency when Ad is employed in humans as gene therapy vector. We previously used Ad-hΔuPA (Recombinant adenovirus expressing human urokinase-type plasminogen activator) as antifibrotic gene therapy in cirrhosis models and demonstrated its effectiveness. As a further clinical approach, transient Cyclosporine A (CsA) immunosuppression was induced in cirrhotic animals to determine whether Ad-hΔuPA administration retained efficacy. Adenovirus sensitization was achieved by systemic administration of non-therapeutic Ad-βGal (Recombinant adenovirus expressing beta-galactosidase) after 4 weeks of intraperitoneal carbon tetrachloride (CCl) regimen. Cirrhosis induction continued up to 8 weeks. At the end of CCl intoxication, immunosuppression was achieved with three CsA doses (40 mg/kg) as follows: 24 h before administration of Ad-hΔuPA, at the moment of Ad-hΔuPA injection and finally, 24 h after Ad-hΔuPA inoculation. At 2 and 72 h after Ad-hΔuPA injection, animals were sacrificed. Liver, spleen, lung, kidney, heart, brain, and testis were analyzed for Ad-biodistribution and transgene expression. In naïve animals, Ad-hΔuPA genomes prevailed in liver and spleen, while Ad-sensitized rats showed Ad genomes also in their kidney and heart. Cirrhosis and Ad preimmunization status notably diminished transgene liver expression compared to healthy livers. CsA immunosuppression in cirrhotic animals has no effect on Ad-hΔuPA biodistribution, but increments survival.
先前针对腺病毒的免疫反应可能会降低将腺病毒作为基因治疗载体应用于人体时的转基因表达效率。我们之前曾使用 Ad-hΔuPA（表达人尿激酶型纤溶酶原激活剂的重组腺病毒）作为肝硬化模型中的抗纤维化基因治疗方法，并证明了其有效性。作为进一步的临床方法，我们在肝硬化动物中诱导短暂的环孢素 A（CsA）免疫抑制，以确定 Ad-hΔuPA 给药是否仍具有疗效。在腹腔内四氯化碳（CCl）方案 4 周后，通过全身给予非治疗性 Ad-βGal（表达β-半乳糖苷酶的重组腺病毒）来实现腺病毒致敏。肝硬化诱导持续到 8 周。在 CCl 中毒结束时，通过给予 3 次 CsA（40mg/kg）来实现免疫抑制，具体如下：在给予 Ad-hΔuPA 之前 24 小时，在给予 Ad-hΔuPA 注射时，最后在 Ad-hΔuPA 接种后 24 小时。在给予 Ad-hΔuPA 后 2 和 72 小时，处死动物。分析肝脏、脾脏、肺、肾、心脏、大脑和睾丸中的腺病毒分布和转基因表达。在未致敏的动物中，Ad-hΔuPA 基因组主要存在于肝脏和脾脏中，而 Ad 致敏的大鼠的肾脏和心脏中也存在 Ad 基因组。与健康肝脏相比，肝硬化和 Ad 预免疫状态明显降低了转基因在肝脏中的表达。CsA 免疫抑制在肝硬化动物中对 Ad-hΔuPA 的分布没有影响，但增加了存活率。
