Salgar Shashikumar K, Yang Dinghua, Ruiz Phillip, Miller Joshua, Tzakis Andreas G
Department of Surgery, University of Miami, Miami, FL 33136, USA.
Hum Gene Ther. 2004 Feb;15(2):131-44. doi: 10.1089/104303404772679940.
The Epstein-Barr virus-encoded protein BCRF1 (viral interleukin [vIL]-10) is a biologically active homologue of cellular interleukin (IL)-10. In this study, a novel gene therapy approach to prolong allograft survival was designed. Autologous (syngeneic) hematopoietic progenitor/stem cell-enriched (HSC; lineage(-ve)) population derived from CBA/J mouse bone marrow were transduced with retrovirus encoding vIL-10 gene (vIL-10-HSC), ex vivo; vIL-10-HSC were injected (4-6 x 10(6) cells intravenously) into lethally (9.5 Gy) or sublethally (4 Gy) irradiated CBA/J mice. Six weeks after vIL-10-HSC administration, vascular heterotopic heart (C57BL/6) transplantation was performed. Ex vivo, the vIL-10-HSC produced 5.4 +/- 0.5 ng of vIL-10 protein/2 x 10(5) cells per 24 hr. In vivo, serum vIL-10 production was 187 +/- 205 pg/ml during 3-10 weeks after vIL-10-HSC administration. Cardiac allograft survival was prolonged (p < 0.004) in lethally (71 +/- 40 days) and sublethally (114 +/- 15 days) irradiated mice that received vIL-10-HSC compared to controls that received unengineered (UE) HSC or vector DNA-engineered HSC (12-16 days). However, secondary skin graft (C57BL/6) survival was not prolonged in cardiac allograft-tolerant animals. In the vIL-10-HSC-administered group, graft histopathology demonstrated mild arteritis/venulitis (grade 0.7) and rejection (grade 1.0). Intragraft expression of costimulatory molecules (B7.1, B7.2), cytokines (IL-2, IL-4, mIL-10, interferon [IFN]-gamma), and inducible nitric oxide synthase (iNOS) molecules was markedly lower in vIL-10-HSC-treated tolerant grafts that survived more than 100 days compared to vector DNA-HSC- or UE-HSC-treated controls. Furthermore, T lymphocytes derived from vIL-10-HSC-treated tolerant recipients demonstrated hyporeactivity to donor antigens in mixed lymphocyte cultures. Administration of autologous vIL-10-engineered HSC prior to organ transplantation prolonged cardiac allograft survival significantly.
爱泼斯坦-巴尔病毒编码的蛋白BCRF1（病毒白细胞介素[vIL]-10）是细胞白细胞介素（IL）-10的生物活性同源物。在本研究中，设计了一种延长同种异体移植物存活时间的新型基因治疗方法。将源自CBA/J小鼠骨髓的自体（同基因）富含造血祖细胞/干细胞的群体（HSC；谱系阴性）在体外用编码vIL-10基因的逆转录病毒（vIL-10-HSC）进行转导；将vIL-10-HSC（4 - 6×10⁶个细胞静脉内注射）注入接受致死剂量（9.5 Gy）或亚致死剂量（4 Gy）照射的CBA/J小鼠体内。在给予vIL-10-HSC六周后，进行血管异位心脏（C57BL/6）移植。在体外，vIL-10-HSC每24小时每2×10⁵个细胞产生5.4±0.5 ng的vIL-10蛋白。在体内，给予vIL-10-HSC后3 - 10周期间血清vIL-10的产生量为187±205 pg/ml。与接受未改造（UE）HSC或载体DNA改造的HSC（12 - 16天）的对照组相比，接受vIL-10-HSC的致死照射（71±40天）和亚致死照射（114±15天）的小鼠心脏同种异体移植物存活时间延长（p < 0.004）。然而，在心脏同种异体移植耐受的动物中，二次皮肤移植（C57BL/6）的存活时间并未延长。在给予vIL-10-HSC的组中，移植物组织病理学显示轻度动脉炎/静脉炎（0.7级）和排斥反应（1.0级）。与载体DNA-HSC或UE-HSC处理的对照组相比，在存活超过100天的vIL-10-HSC处理的耐受移植物中，共刺激分子（B7.1、B7.2）、细胞因子（IL-2、IL-4、mIL-10、干扰素[IFN]-γ）和诱导型一氧化氮合酶（iNOS）分子的移植物内表达明显较低。此外，源自vIL-10-HSC处理的耐受受体的T淋巴细胞在混合淋巴细胞培养中对供体抗原表现出低反应性。在器官移植前给予自体vIL-10改造的HSC可显著延长心脏同种异体移植物的存活时间。
