Poller W, Schneider-Rasp S, Liebert U, Merklein F, Thalheimer P, Haack A, Schwaab R, Schmitt C, Brackmann H H
Medical University Clinic, University of Würtzburg, Germany.
Gene Ther. 1996 Jun;3(6):521-30.
Complex interactions between replication deficient adenoviral vectors (Ad5) and the immune system of the host influence the stability of transgenes in vivo. Vector-infected cells are attacked by diverse cellular immune mechanisms which limit transgene persistence. On the other hand, the products of several E3 region genes of wild-type adenovirus can suppress host immune reactions by interference with the expression of MHC class I molecules and by other mechanisms. We have developed an adenoviral vector for human factor IX (Ad5E3+FIX) which carries the E3 region of wild-type adenovirus, and an E3-deleted vector of otherwise similar structure (ad5 delta E3FIX). Intravenous injection of Ad5E3+FIX in C57BI/6 mice resulted in expression levels up to 6000 ng/ml of recombinant human factor IX in the mouse plasma and in enhanced transgene stability as compared with the vector Ad5 delta E3FIX. Whereas expression from E3-deleted vectors was essentially turned off 8 weeks after the gene transfer, the vector Ad5E3+FIX3+FIX supported transgene expression with therapeutic levels of human factor IX in the mouse plasma for > 4 months. The enhanced stability of the vector Ad5E3+FIX appears to be a consequence of efficient E3 region-mediated suppression of the host's antivector immune response. As an additional approach to improving transgene stability the influence of transient CD4+ T cell depletion of the host was investigated. CD4+ cytotoxic T lymphocytes contribute to the clearance of adenovirus-infected cells and play a pivotal role in the activation of CD8+ cytotoxic T cells and as helper T cells in the formation of human adenovirus neutralizing antibodies (HANA). Transient anti-CD4 treatment of the host limited to the time of vector injection resulted in a significant prolongation of transgene expression from the factor IX vector Ad5E3+FIX and a luciferase vector Ad5Luc. The combination of transient anti-CD4 treatment of the host and integration of a complete E3 region in an adenoviral vector resulted in markedly improved transgene stability after gene transfer to the liver (therapeutic factor IX levels for > 6 months).
复制缺陷型腺病毒载体（Ad5）与宿主免疫系统之间的复杂相互作用会影响体内转基因的稳定性。载体感染的细胞会受到多种细胞免疫机制的攻击，这些机制限制了转基因的持久性。另一方面，野生型腺病毒的几个E3区域基因的产物可通过干扰MHC I类分子的表达及其他机制来抑制宿主免疫反应。我们开发了一种用于人凝血因子IX的腺病毒载体（Ad5E3+FIX），它携带野生型腺病毒的E3区域，以及一种结构类似但E3缺失的载体（ad5 delta E3FIX）。将Ad5E3+FIX静脉注射到C57BI/6小鼠体内后，与载体Ad5 delta E3FIX相比，小鼠血浆中重组人凝血因子IX的表达水平高达6000 ng/ml，且转基因稳定性增强。虽然E3缺失载体的表达在基因转移8周后基本关闭，但载体Ad5E3+FIX3+FIX在小鼠血浆中支持人凝血因子IX治疗水平的转基因表达超过4个月。载体Ad5E3+FIX稳定性增强似乎是E3区域有效介导的宿主抗载体免疫反应抑制的结果。作为提高转基因稳定性的另一种方法，研究了宿主瞬时CD4+ T细胞耗竭的影响。CD4+ 细胞毒性T淋巴细胞有助于清除腺病毒感染的细胞，并且在激活CD8+ 细胞毒性T细胞以及作为辅助性T细胞在人腺病毒中和抗体（HANA）形成中起关键作用。仅在载体注射时对宿主进行瞬时抗CD4治疗导致凝血因子IX载体Ad5E3+FIX和荧光素酶载体Ad5Luc的转基因表达显著延长。宿主瞬时抗CD4治疗与在腺病毒载体中整合完整E3区域相结合，在基因转移至肝脏后导致转基因稳定性显著提高（治疗性凝血因子IX水平超过6个月）。
