Limb perfusion as a route for re-conditioning and genetic engineering: preventing damage and decreasing immunogenicity to support survival after allogeneic transplantation

肢体灌注作为修复和基因工程的途径：预防损伤并降低免疫原性以支持同种异体移植后的生存

• 批准号: 500341366
• 负责人: Professorin Dr. Constanca Figueiredo
• 金额: --
• 依托单位: Institut für Transfusionsmedizin und Transplantat Engineering
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/500341366?language=en
• 关键词: Limb; perfusion; route; re; conditioning

Vascularized composite allograft (VCA) transplantation is an established and valid treatment in the management of disfiguring tissue damages, massive tissue loss or amputation. Unfortunately, conventional reconstructive methods often fail in completely restoring function in patients with severe facial or limb injuries. As an alternative, VCA transplantation represents a process by which functional units such as an arm are retrieved from a donor and transplanted into the patient, allowing for the possibility of greater functional recovery. Despite the very encouraging functional outcomes, VCA recipients have been subject to a burden of immunosuppression comparable to that of solid organ transplantation, including cytomegalovirus infection and neoplasia. In addition, they have shown a high incidence of acute rejection episodes with long-term sequelae and chronic rejection. HLA play an important role in the activation of immune responses after allogeneic transplant settings and are the trigger for both acute and chronic rejection. Machine perfusion is a novel approach to decrease preservation injury, improve graft assessment, and increase organ acceptance for transplantation. Recently, we demonstrated the great potential of using ex vivo perfusion strategies to genetically modify organs such as the lung and kidney towards reduction of their immunogenicity and improving graft survival. Therefore, this project aims at using ex vivo limb machine perfusion strategies to prevent tissue damage due to ischemia reperfusion injury and simultaneously allowing for the genetic modification of the limb endothelium to improve graft survival after MHC incompatible transplantation. This proposal is based on a limb transplantation rat model. Rat hind limbs will be ex vivo perfused with lentiviral vectors encoding for shRNAs targeting MHC class I and II transcripts to silence their expression in a stable manner. Our previous studies showed that silencing MHC expression contributes to increase graft survival after transplantation even in the absence of immunosuppression. To assess the effect of ex vivo machine perfusion on the limb endothelium, cytokine signatures and tissue injury markers will be measured in the perfusates. Furthermore, the protocols previously established by our group to genetically modify solid organs will be adapted and improved to achieve an optimal transduction efficiency of the limb tissues. After transplantation experiments, graft survival and the strength of the immune response in absence of immunosuppression will be comprehensively monitored. Importantly, an extensive characterization and evaluation of safety aspects related to the generation and transplantation of genetically modified VCA will be performed. The results achieved by this project will define a new road to ensure the routinely application of VCA transplantation as optimal reconstructive strategy with gain of function and eliminating the burden of immunosuppression.

血管化复合同种异体移植（VCA）是一种公认的有效治疗毁容组织损伤，大面积组织缺损或截肢的方法。不幸的是，传统的重建方法往往无法完全恢复功能的患者严重的面部或肢体损伤。作为替代方案，VCA移植代表了一个过程，通过该过程，从供体取回功能单元（例如手臂）并移植到患者体内，从而允许更大的功能恢复的可能性。尽管功能结局非常令人鼓舞，但VCA接受者仍承受着与实体器官移植相当的免疫抑制负担，包括巨细胞病毒感染和肿瘤形成。此外，它们还显示出具有长期后遗症的急性排斥发作和慢性排斥的高发生率。HLA在同种异体移植后免疫应答的激活中起重要作用，并且是急性和慢性排斥反应的触发因素。机器灌注是一种新的方法，以减少保存损伤，改善移植评估，并增加器官移植的接受性。最近，我们证明了使用离体灌注策略对器官如肺和肾进行遗传修饰以降低其免疫原性和提高移植物存活率的巨大潜力。因此，本项目旨在使用离体肢体机器灌注策略来防止由于缺血再灌注损伤引起的组织损伤，同时允许对肢体内皮进行遗传修饰以提高MHC不相容移植后的移植物存活率。本研究以大鼠肢体移植模型为基础。将用编码靶向MHC I类和II类转录物的shRNA的慢病毒载体离体灌注大鼠后肢，以稳定的方式沉默其表达。我们以前的研究表明，沉默MHC表达有助于增加移植后的移植物存活，即使在没有免疫抑制。为了评估离体机器灌注对肢体内皮的影响，将测量灌注液中的细胞因子特征和组织损伤标志物。此外，我们小组先前建立的遗传修饰实体器官的方案将被调整和改进，以实现肢体组织的最佳转导效率。移植实验后，将全面监测移植物存活率和无免疫抑制情况下的免疫反应强度。重要的是，将对与转基因VCA的生成和移植相关的安全性方面进行广泛的表征和评价。本项目的研究结果将为VCA移植作为获得功能和消除免疫抑制负担的最佳重建策略的常规应用开辟一条新的道路。