小环DNA载体介导的自体内合成生物蛋白复合制剂对肾脏移植后免疫排斥反应治疗效果

Hypertension and diabetes mellitus(DM) are the leading cause of end-stage renal disease (ESRD) worldwide. Despite strict blood glucose, hypertension, and metabolic abnormalities controls, the number of diabetic patients entering ESRD because of hypertension and DM remains extremely high. Many of these patients will need kidney transplantation, but new-onset diatetes after transplantation (NODAT) is a serious complication of solid organ transplantation that can adversely affect the survival of the transplant recipient and graft. Among the immunosuppressive regimens, high-dose steroids are associated with the highest incidence of NODAT, and Long-term treatment with CNI causes irreversible chronic nephropathy. Recently, it has been reported that minicircle vectors could allow the expression of transgenes using the protein synthesis system of the host. Here, we tested a novel strategy to permit the production of synthetic biologics using minicircle technology and evaluated their feasibility as a therapeutic tool in kidney transplantation model in rats. We engineered vectors to carry cassette sequences for tocilizumab [anti- CTLA4-Ig antibody] and/or etanercept [interleukin-6 receptor (sIL-6R)-Fc fusion protein], and then isolated minicircle vectors from the parent vectors. We verified the production of proteins from minicircles and their duration in cells and rats. We also evaluated whether these proteins were expressed at levels sufficient to ameliorate kidney transplantation model in rats. Each minicircle transfected into cells was detectable for at least 30 days. In rats, the drugs were mainly expressed in the liver and were detectable for at least 10 days after a single injection. These drugs were also detected in the blood. Treatment of rats with minicircles prolonged renal allograft survival, accumulating body of evidence demonstrates that suppression of CTLA4 or/and IL-6 expression is closely associated with the pathogenesis of allograft rejection, which accompanied by activation of Klotho/FGF23/AKT/FoxO signaling pathway. Nevertheless, the exact role for oxidative stress,Th/Treg, Klotho/FGF23/AKT/FoxO signaling pathway in allograft rejection is undetermined. This study was designed to investigate that blocking of CTLA4-Ig and/or IL-6R using minicircles encoding tocilizumab and/or etanercept was functionally active and relevant for preventing acute allograft rejection. Our finding may provide self-reproducing synthetic protein drugs produced using minicircle technology are potentially powerful tools for preventing allrograft rejection in kidney transplantation.

随社会发展和老龄化，高血压和糖尿病已是危害人类健康的主要罪魁祸首。以上疾病引起的终末期肾病人数剧增，且需行肾脏替代治疗。而肾脏替代治疗中最理想的治疗方案为肾脏移植, 但现阶段需终身服用免疫抑制剂药物。临床上常用的免疫抑制剂均可引起新发糖尿病。CTLA-4与IL-6对移植后免疫排斥反应具有治疗效果，但制备单克隆抗体所需耗材量大，生物利用度小，费用昂贵。小环DNA载体介导的自体内合成生物蛋白制剂可替代单克隆抗体。本实验拟通过体内、体外实验观察小环DNA载体介导的自体内合成生物蛋白复合制剂对免疫排斥反应治疗效应。相应治疗对Th1、Th2、Th17细胞与Treg细胞反应变化，进一步揭示与细胞凋亡和自噬的关系。阐明Klotho/FGF23/AKT/FoxO氧化应激损伤信号传导通路在免疫排斥反应中的作用机制。从而进一步揭示免疫排斥反应分子发病机制及治疗方案，为今后生物学制剂在临床治疗提供理论依据。

单克隆抗体等合成生物制剂在移植物排斥反应相关治疗方面取得了一定成功，但由于制备和合成相关单克隆抗体等生物制剂时纯化及提取成本高，故新型生物制剂的开发受到限制。我们利用小环DNA载体（MC）技术，该技术可以稳定而安全表达靶分子并从自体细胞中分泌，使本技术成为在体外和体内产生所需蛋白质的间接有效治疗方案。我们设计了一种小环MC载体，其中包含MC_anti-sCTLA4-Ig, MC_anti-sIL-6R以及MC_anti-sCTLA4-Ig-sIL-6双靶抗体。我们验证了MC在体外和体内大量产生CTLA4-Ig和/或IL-6蛋白。在皮肤移植模型中，通过单次静脉注射小环DNA载体介导的CTLA4-Ig和/或IL-6，进行治疗效果评估。使用CTLA4-Ig和/或IL-6的小环DNA载体治疗表现出延长皮肤移植生存时间，并改善肾脏组织学变化和免疫调节。这些发现表明，小环DNA载体介导技术获得的自体内合成CTLA4-Ig和/或IL-6蛋白生物制剂药物具有功能活性，可减少同种异体移植物相关排斥反应。小环DNA载体介导的CTLA4-Ig和/或IL-6的治疗组延长了移植后生存率，其伴随着Th1，Th2，Th17和Treg免疫指标相关调节，这些反应过程与Klotho/FGF23/AKT-FoxO细胞相关通路表达密切相关。小环DNA载体介导的CTLA4-Ig和/或IL-6治疗显著降低了氧化应激指标和细胞凋亡，细胞凋亡相关基因（active caspase-3, Bcl-2/Bax比值）对移植物存活进行了调节。以上发现表明，使用小环DNA载体介导自体合成CTLA4-Ig和/或IL-6具有功能活性，从而进一步揭示免疫排斥反应分子发病机制及治疗方案，为今后生物学制剂在移植排斥反应临床治疗提供理论依据。