Investigating siRNA-mediated inhibition of ischemia-reperfusion injury during the liver transplantation process

项目摘要 肝移植是治疗肝衰竭的唯一方法，但许多患者在等待移植时因大量的肝细胞坏死而死亡。 供体肝脏短缺缺血再灌注损伤（IRI）进一步减少了供体供应不足， 肝移植物的获取和保存。虹膜损伤的移植物必须丢弃，因为它们 植入受体后功能障碍，导致患者死亡。缓解IRI是迫切需要的， 增加可供肝脏的数量并提高患者的存活率。IRI机制涉及多个 途径。缺血损伤后促进细胞损伤，再灌注触发氧化应激和先天免疫 在肝脏IRI中，细胞凋亡是主要的细胞死亡机制。肝细胞凋亡 Fas受体介导IRI的发生，并导致坏死和炎症，从而导致肝功能障碍。 在大鼠中，缺血前Fas减少可减轻肝损伤。可能会减轻IRI诱导的肝脏 在移植过程中，通过沉默肝细胞特异性Fas来抑制功能障碍。 本研究的目的是利用小干扰RNA（siRNA）抑制IRI，提高移植肝质量 进行移植化学修饰的siRNA能够有效的，序列特异性沉默任何靶基因 in vivo.当与N-乙酰半乳糖胺（GalNAc）偶联时，修饰的SiRNA被递送到肝细胞。 GalNAc-siRNA技术是许多FDA批准的肝病药物的基础。在博士的指导下， Anastasia Khvorova（siRNA），Paulo Martins（肝移植），Athma派（RNA测序），Gyongyi Szabo（肝IRI）， Jacob布莱索（肝脏病理学）和Matthew Gounis（体内成像），该项目将在体内和体外开发 使用先前验证的靶向Fas的GalNAc-siRNA抑制IRI并保护肝功能的体内方法。 目的1将确定缺血前Fas沉默如何干扰IRI通路（目的1.1），并探索沉默是否 Fas与其他介质联合使用可更好地保护肝脏免受损伤（目的1.2）。GalNAc- 单独或与经验证的氧化应激（Hmgb 1）和炎症（Tnfr 1）组合靶向Fas的siRNA 将介体注射到大鼠中，并使用肝钳夹模型诱导IRI。再灌注后，目标 将评估基因表达、肝损伤和IRI转录组变化。目标2将决定如何 肝脏移植物中Fas的沉默（离体）影响大鼠的移植结果。在离体期间递送siRNA 通过静态冷藏（SCS）或机器灌注（MP）的保存导致摄取到肝移植物中。大鼠 将获得肝脏，并在SCS或MP期间递送靶向Fas的GalNAc-siRNA。GalNAc-siRNA 将在24小时内评估IRI摄取、Fas表达和IRI转录组变化。这个实验将 然后重复，保存移植物以获得最大GalNAc-siRNA摄取/功效，将移植物移植到大鼠中， 以及测量肝损伤/功能和受体存活率。研究结果将描述Fas沉默 缺血前和缺血后影响肝脏IRI通路，确定体内和体外最大IRI方法 抑制，并帮助开发增加供体库和提高患者生存率的疗法。

PROJECT SUMMARY Liver transplantation is the only cure for liver failure, but many patients die waiting for a transplant due to a vast shortage of donor livers. Insufficient donor supply is further diminished by ischemia-reperfusion injury (IRI) during procurement and preservation of liver grafts. IRI-damaged grafts must be discarded because they are dysfunctional following implantation into recipients, causing patient death. Mitigating IRI is critically needed to increase the number of viable donor livers and improve patient survival. The IRI mechanism involves several pathways. After ischemic insult facilitates cellular injury, reperfusion triggers oxidative stress and innate immune pathways that converge to activate apoptosis, the major cell death mechanism in liver IRI. Hepatocyte apoptosis in IRI is mediated by Fas receptor (Fas), and leads to necrosis and inflammation, which cause liver dysfunction. In rats, Fas reduction prior to ischemia reduces liver damage. It may be possible to mitigate IRI-induced liver dysfunction by silencing hepatocyte-specific Fas during the transplantation process. The goal of this proposal is to use small interfering RNA (siRNA) to inhibit IRI and improve quality of liver grafts for transplantation. Chemically-modified siRNAs enable potent, sequence-specific silencing of any target gene in vivo. Modified siRNAs are delivered to hepatocytes when conjugated with N-acetylgalactosamine (GalNAc). GalNAc-siRNA technology is the basis of numerous FDA-approved liver disease drugs. With guidance from Drs. Anastasia Khvorova (siRNA), Paulo Martins (liver transplant), Athma Pai (RNA seq), Gyongyi Szabo (liver IRI), Jacob Bledsoe (liver pathology), and Matthew Gounis (in vivo imaging), this project will develop in vivo and ex vivo approaches using previously-validated GalNAc-siRNA targeting Fas to inhibit IRI and protect liver function. Aim 1 will determine how silencing Fas prior to ischemia perturbs IRI pathways (Aim 1.1), and explore if silencing Fas in combination with other mediators confers greater protection against liver damage (Aim 1.2). GalNAc- siRNAs targeting Fas, alone, or in combination with validated oxidative stress (Hmgb1) and inflammation (Tnfr1) mediators, will be injected into rats, and IRI will be induced using a liver clamp model. Post-reperfusion, target gene expression, liver damage, and IRI transcriptome changes will be assessed. Aim 2 will determine how silencing Fas in liver grafts (ex vivo) affects transplant outcomes in rats. Delivering siRNA during ex vivo preservation, by either static cold storage (SCS) or machine perfusion (MP), leads to uptake into liver grafts. Rat livers will be procured and GalNAc-siRNA targeting Fas will be delivered during SCS or MP. GalNAc-siRNA uptake, Fas expression, and IRI transcriptome changes will be assessed over 24 hours. This experiment will then be repeated, preserving grafts for maximal GalNAc-siRNA uptake/efficacy, transplanting grafts into rats, and measuring liver damage/function and recipient survival. Study findings will characterize how Fas silencing pre- and post-ischemia affects liver IRI pathways, identify in vivo and ex vivo approaches for maximal IRI inhibition, and help develop therapies that increase the donor pool and improve patient survival.