Lim Sun Woo, Shin Yoo Jin, Cui Sheng, Ko Eun Jeong, Yoo Seok Ho, Chung Byung Ha, Yang Chul Woo
Transplant Research Center, College of Medicine, The Catholic University of Korea, Seoul, Korea.
Convergent Research Consortium for Immunologic Disease, Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea.
Korean J Intern Med. 2022 Sep;37(5):1031-1049. doi: 10.3904/kjim.2021.299. Epub 2022 Jun 21.
BACKGROUND/AIMS: We previously proposed minicircle vector technology as the potential platform for the development and production of new biologics. In this study, we have designed a novel target molecule for the treatment of allograft rejection and evaluated its feasibility as the therapeutic agent in this disease using the minicircle vector system.
We engineered vectors to carry cassette sequences for anti-CD25, interleukin-10 (IL-10), and C-X-C motif chemokine receptor 3 (CXCR3) fusion protein, and then isolated minicircle vectors from the parent vectors. We verified the substantial production of anti-CD25/IL-10/CXCR3 fusion protein from minicircles and their duration in HEK293T cells and mice models. We also evaluated whether minicircle-derived anti-CD25/IL-10/CXCR3 has therapeutic effects in a skin allograft in mice model.
We confirmed the production of anti-CD25/IL-10/CXCR3 from minicircle by its significant availability in cells transfected with the minicircle and in its conditioned media. After a single injection of minicircle by hydrodynamic injection via mouse tail vein, luminescence or red fluorescence was maintained until 40 days in the liver tissue, suggesting the production of anti-CD25/IL-10/CXCR3 protein from minicircles via protein synthesis machinery in the liver. Mice treated with the minicircle encoding anti-CD25/IL-10/CXCR3 showed prolonged skin allograft survival times accompanied by improved immunologic regulation e.g., reduction of the lymphocyte population of Th1, Th2, and Th17 and an induction of regulatory T cells.
These findings implied that self-generated anti-CD25/IL-10/CXCR3 protein drug by minicircle technology is functionally active and relevant for reducing allograft rejection. The minicircle vector system may be useful for developing new biological drugs, avoiding manufacturing or practical problems.
背景/目的：我们之前提出微环载体技术可作为开发和生产新型生物制品的潜在平台。在本研究中，我们设计了一种用于治疗同种异体移植排斥反应的新型靶分子，并使用微环载体系统评估了其作为该疾病治疗剂的可行性。
我们构建载体以携带抗CD25、白细胞介素-10（IL-10）和C-X-C基序趋化因子受体3（CXCR3）融合蛋白的盒式序列，然后从亲本载体中分离出微环载体。我们验证了微环载体中抗CD25/IL-10/CXCR3融合蛋白的大量产生及其在HEK293T细胞和小鼠模型中的持续时间。我们还评估了微环衍生的抗CD25/IL-10/CXCR3在小鼠模型的皮肤同种异体移植中是否具有治疗作用。
我们通过微环转染的细胞及其条件培养基中的显著可用性证实了微环产生抗CD25/IL-10/CXCR3。通过小鼠尾静脉进行水动力注射单次注射微环后，肝脏组织中的发光或红色荧光一直维持到40天，表明肝脏中的蛋白质合成机制通过微环产生了抗CD25/IL-10/CXCR3蛋白。用编码抗CD25/IL-10/CXCR3的微环处理的小鼠显示皮肤同种异体移植存活时间延长，同时免疫调节得到改善，例如Th1、Th2和Th17淋巴细胞群体减少以及调节性T细胞的诱导。
这些发现表明，通过微环技术自身产生的抗CD25/IL-10/CXCR3蛋白药物具有功能活性，与减少同种异体移植排斥反应相关。微环载体系统可能有助于开发新的生物药物，避免制造或实际问题。
