Genetic-engineered control of the immunogeneic state of vascular composite allografts during preservation

摘要 虽然对于由创伤和/或烧伤引起的严重缺损， 由于VCA在改善生活质量的治疗中存在复杂的伦理计算， 但代价是长期免疫抑制的副作用。从长远来看， 有希望的方法是通过骨髓共移植的混合嵌合体诱导耐受。在 短期内，监测免疫抑制的新战略，以便及时干预， VCA的免疫原性对于使有需要的患者更容易获得这种治疗是必要的。的 该应用程序的目标是开发一个功能保存平台， VCA移植物通过外源性施用基因修饰的细胞，其保存在临床上可行的 方案，并测试在排斥和耐受的啮齿动物模型中降低免疫原性的功效 诱导生物传感器细胞将以转录因子反应元件作为基因进行基因工程 启动子作为治疗诊断剂，同时驱动基于血液的生物标志物和 治疗性蛋白质以减弱排斥反应。我们将联合收割机与我们的灌注式过冷 保存技术，使细胞的植入，并提供一个时间窗口，使 在临床时间范围内利用工程化移植物。这一目标是根据我们的 先前的工作和初步结果，我们已经成功地工程细胞所需的反应， 特性，开发了一种离体大鼠肢体灌注系统，可以作为移植的平台 这些细胞生物传感器在移植前植入血管移植物中，并证明转导的细胞可以保留 在体内存活长达4个月。这项工作将导致一种智能嫁接技术， 测量局部组织信号传导将使得能够实际监测和诊断急性排斥事件， 改变移植患者的护理，因为药物治疗的依从性问题将大大减少， 将可能增加总体疗效并因此减少移植排斥。从长远来看，保护 这里开发的方法也可以在VCA移植中诱导耐受性，因此允许 因为它们的广泛使用目前是不可能的。

ABSTRACT While being an increasingly used option of reconstruction for severe defects arising from trauma and/or burn injuries, VCA remains limited in use due to the complex ethical calculus of a treatment that improves life quality dramatically, but at the cost of the side effects of long-term immunosuppression. In the longer term the most promising approach is the induction of tolerance by mixed chimerism by bone-marrow co-transplantation. In the shorter term, new strategies for monitoring immunosuppression to enable timely interventions, and reducing immunogenicity of VCAs are necessary for making this treatment more accessible to patients in need. The objective of this application is to develop a functional preservation platform that enables creating engineered VCA grafts by exogenous administration of genetically-modified cells, their preservation in a clinically practicable protocol, and testing the efficacy in reducing immunogenicity in rodent models of rejection and tolerance induction. Biosensor cells will be genetically engineered with a transcription factor response element as a gene promoter to serve as a theranostic, simultaneously driving the secretion of blood-based biomarker and a therapeutic protein to attenuate a rejection response. We will combine this with our perfusion-based supercooled preservation technologies to enable both engraftment of cells and to provide a time-window that makes the utilization of engineered grafts viable in clinical time frames. This objective has been formulated based on our prior work and preliminary results, where we have shown successful engineering of cells with required response characteristics, development of an ex vivo rat limb perfusion system that can serve as a platform for engrafting these cell biosensors into vascular grafts prior to transplant, and demonstrating that transducted cells can remain viable in vivo for up to 4 months. The proposed work would lead to a smart-graft technology that can sensitively measure local tissue signaling would enable practical monitoring and diagnosis of acute rejection episodes, can transform care of transplant patients since compliance issues in medication would be dramatically reduced, and will likely increase the overall efficacy and therefore reduce graft rejections. In the longer term, the preservation methods developed here could also be enabling for tolerance induction in VCA transplants, and therefore allow for their wide-spread use which is not possible currently.