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

带血管复合异体骨(VCA)移植是治疗畸形组织损伤、大量组织丢失或截肢的有效方法。不幸的是，传统的重建方法往往不能完全恢复面部或肢体严重损伤患者的功能。作为另一种选择，VCA移植是一种从捐赠者那里取出手臂等功能单元并移植到患者体内的过程，允许更大的功能恢复。尽管VCA的功能结果非常令人鼓舞，但VCA接受者一直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。此外，它们还显示出急性排斥事件的高发生率，并有长期后遗症和慢性排斥反应。人类白细胞抗原在同种异体移植后的免疫反应的激活中起着重要的作用，是急性和慢性排斥反应的触发因素。机器灌流是一种减少保存损伤、改善移植物评估和提高器官移植接受度的新方法。最近，我们展示了使用体外灌流策略对肺和肾等器官进行基因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，本项目旨在利用体外肢体机械灌流策略来预防缺血再灌注损伤所致的组织损伤，同时允许对肢体内皮细胞进行基因修饰，以提高MHC血型不合移植后的移植物存活率。这一建议是基于一只肢体移植的大鼠模型。大鼠后肢将被体外灌流慢病毒载体，编码针对MHC I和II类转录本的shRNAs，以稳定的方式沉默它们的表达。我们先前的研究表明，即使在没有免疫抑制的情况下，沉默MHC的表达也有助于提高移植后的存活率。为了评估体外机械灌流对肢体内皮细胞的影响，将测量灌流液中的细胞因子信号和组织损伤标志物。此外，我们小组以前建立的对实体器官进行基因修饰的方案将进行修改和改进，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和在没有免疫抑制的情况下免疫反应的强度。重要的是，将对与转基因VCA的产生和移植有关的安全方面进行广泛的表征和评估。该项目的成果将为确保VCA移植作为最佳重建策略的常规应用开辟一条新的道路，获得功能并消除免疫抑制的负担。

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.