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

摘要 同时作为创伤和/或烧伤引起的严重缺陷的一种日益使用的重建选择 由于改善生活质量的治疗的复杂伦理演算，VCA的使用仍然受到限制 戏剧性的，但代价是长期免疫抑制的副作用。从长远来看，大多数 有希望的方法是通过骨髓联合移植通过混合嵌合体诱导耐受。在 短期内，监测免疫抑制的新战略，以实现及时干预，并减少 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。这个 本应用程序的目标是开发一个功能保存平台，使创建工程化的 外源性应用转基因细胞移植VCA，其保存在临床上是可行的 方案，并在排斥和耐受的啮齿动物模型中测试其降低免疫原性的效果 归纳法。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 启动子作为风湿剂，同时驱动基于血液的生物标志物和 治疗蛋白，以减轻排斥反应。我们将把它与我们的基于灌流的过冷技术结合起来 保存技术，既能植入细胞，又能提供时间窗口，使 在临床时限内可行的工程移植物的利用。这一目标是根据我们的 之前的工作和初步结果，其中我们已经成功地设计了具有所需响应的细胞 一种可作为移植平台的体外大鼠肢体灌流系统的特点和研制 在移植前将这些细胞生物传感器植入血管移植物，并证明转导的细胞可以保留 体内存活时间长达4个月。这项拟议的工作将导致一种智能移植技术，这种技术可以敏感地 测量局部组织信号将使急性排斥反应的实际监测和诊断成为可能 改变对移植患者的护理，因为服药依从性问题将大大减少，以及 可能会提高整体疗效，从而减少移植物排斥反应。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，因此允许 用于它们的广泛使用，这在目前是不可能的。

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.