Efficient Hepatocyte Engraftment and Long-Term Transgene Expression After Reversible Portal Embolization in Nonhuman Primates
Efficient Hepatocyte Engraftment and Long-Term Transgene Expression After Reversible Portal Embolization in Nonhuman Primates
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DOI：
10.1002/hep.22739
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发表时间：
2009-03-01
期刊：
影响因子：
13.5
通讯作者：
Weber, Anne
中科院分区：
文献类型：
--
作者：
Dagher, Ibrahim;Tuan Huy Nguyen;Weber, Anne
离体基因治疗作为肝移植治疗肝脏代谢性疾病的替代方法的可行性需要在大型动物模型中进行分析。这种方法需要合适的基因转移载体和有效的肝细胞移植。慢病毒载体具有使未分裂的分化细胞（如肝细胞）增殖的能力，并且门静脉阻塞增加肝细胞植入。我们研究了可逆性门静脉栓塞结合离体慢病毒介导的基因转移是否是非人灵长类动物肝细胞成功植入的有效方法，以及转基因是否在原位移植肝细胞中长期表达。左叶切除后分离猴肝细胞，用可吸收材料栓塞动物的左和右门静脉前支。用Hoechst染料标记分离的肝细胞，或用在人载脂蛋白A-II启动子控制下表达绿色荧光蛋白的慢病毒在悬浮液中转导，并通过肠系膜下静脉移植。整个过程耐受良好。栓塞的肝脏在2周内进行了血运重建。未栓塞肝脏的体积从栓塞前的38.7% +/- 0.8%增加到栓塞后的55.9% +/- 1%，肝细胞显著增殖（栓塞后第3天为10.5% +/- 0.4%）。Hoechst标记肝细胞移植后的肝脏再增殖率为7.4% ± 1.2%。考虑到猴肝细胞群的平均转导效率为34%，采用转导肝细胞的肝脏再增殖率为2.1% +/- 0.2%，与Hoechst标记细胞获得的比例相似。转基因表达在移植后16周持续存在。结论：我们已经开发了一种新的方法，以提高肝细胞植入和表达转基因在非人灵长类动物的长期。该策略可适用于临床应用。（《肝脏病学》2009年;49：950-959。）
The feasibility of ex vivo gene therapy as an alternative to liver transplantation for the treatment of liver metabolic diseases needs to be analyzed in large animal models. This approach requires appropriate gene transfer vectors and effective hepatocyte engraftment. Lentiviral vectors have the ability to transduce nondividing differentiated cells, such as hepatocytes, and portal vein occlusion increases hepatocyte engraftment. We investigated whether reversible portal vein embolization combined with ex vivo lentivirus-mediated gene transfer is an effective approach for successful hepatocyte engraftment in nonhuman primates and whether the transgene remains expressed in the long term in transplanted hepatocytes in situ. Simian hepatocytes were isolated after left lobe resection, and the left and right anterior portal branches of animals were embolized with absorbable material. Isolated hepatocytes were labeled with Hoechst dye or transduced in suspension with lentiviruses expressing green fluorescent protein under the control of the human apolipoprotein A-II promoter and transplanted via the inferior mesenteric vein. The whole procedure was well tolerated. The embolized liver was revascularized within 2 weeks. The volume of nonembolized liver increased from 38.7% +/- 0.8% before embolization to 55.9% +/- 1% after embolization and hepatocytes significantly proliferated (10.5% +/- 0.4% on day 3 after embolization). Liver repopulation after transplantation with Hoechst-labeled hepatocytes was 7.4% +/- 1.2%. Liver repopulation was 2.1% +/- 0.2% with transduced hepatocytes, a proportion similar to that obtained with Hoechst-labeled cells, given that the mean transduction efficacy of simian hepatocyte population was 34%. Transgene expression persisted at 16 weeks after transplantation. Conclusion: We have developed a new approach to improve hepatocyte engraftment and to express a transgene in the long term in nonhuman primates. This strategy could be suitable for clinical applications. (HEPATOLOGY 2009;49: 950-959.)