Podevin Guillaume, Otta Edson, Nguyen Jean Michel, Pichard Virginie, Aubert Dominique, Moullier Philippe, Ferry Nicolas
Laboratoire de Thérapie génique, INSERM ERM 0105, CHU Hôtel Dieu, Blv. Jean Monet, 44093 Nantes cedex 1, France.
J Gene Med. 2004 Jan;6(1):16-21. doi: 10.1002/jgm.469.
Highly efficient retrovirus-mediated gene transfer into hepatocytes in vivo triggers an immune response directed against transduced hepatocytes. This effect may be due either to spreading of retroviral vectors in the blood stream with subsequent infection of antigen presenting cells (APCs) or to cross-presentation of the transgene product present as a contaminant in the viral stock. In order to decrease immune response, we evaluated the effect of asanguineous perfusion of the liver as well as purification of the viral stock on long-term transduction of hepatocytes using the nls-lacZ marker gene.
Animals were divided in four groups. In group 1, the viral supernatant was perfused in the regenerating liver after complete vascular exclusion of the organ. In group 2, using the same strategy, animals received retroviral supernatant that was passed through a beta-galactosidase affinity column to reduce beta-galactosidase contamination. In two control groups (respectively groups 3 and 4) the corresponding viral supernatants were delivered via peripheral injection.
In group 1, 23.1% of animals had no immune response 2 months after gene delivery vs. 33.4% in group 2, 4.3% in control group 3, and 0% in control group 4. Statistical analysis of the results demonstrated that only the difference between groups 2 and 3 was statistically significant. This indicated that both asanguineous perfusion together with passage through an affinity column were required to decrease significantly immune response.
Our present results suggest that both supernatant contamination and viral spreading contribute to immune response after retrovirus-mediated gene delivery to the liver.
高效逆转录病毒介导的基因在体内导入肝细胞会引发针对转导肝细胞的免疫反应。这种效应可能是由于逆转录病毒载体在血流中扩散，随后感染抗原呈递细胞（APC），或者是由于病毒原液中作为污染物存在的转基因产物的交叉呈递。为了降低免疫反应，我们使用核定位信号 - 乳糖酶基因（nls-lacZ）标记基因评估了肝脏无血灌注以及病毒原液纯化对肝细胞长期转导的影响。
将动物分为四组。在第1组中，在肝脏完全血管阻断后，将病毒上清液灌注到再生肝脏中。在第2组中，采用相同策略，动物接受经过β-半乳糖苷酶亲和柱处理以减少β-半乳糖苷酶污染的逆转录病毒上清液。在两个对照组（分别为第3组和第4组）中，通过外周注射给予相应的病毒上清液。
在第1组中，基因递送2个月后，23.1%的动物没有免疫反应，而第2组为33.4%，第3组对照组为4.3%，第4组对照组为0%。结果的统计分析表明，只有第2组和第3组之间的差异具有统计学意义。这表明无血灌注和通过亲和柱处理都需要显著降低免疫反应。
我们目前的结果表明，上清液污染和病毒扩散都有助于逆转录病毒介导的基因递送至肝脏后的免疫反应。
