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

研究肝移植过程中 siRNA 介导的缺血再灌注损伤抑制作用

基本信息

批准号：
10740842
负责人：
Julianna E Buchwald
金额：
$ 3.25万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10740842
关键词：
Acetylgalactosamine Affect Apoptosis Biochemical Markers Blood Vessels CD95 Antigens Cell Death Cessation of life Chemicals Chemistry Clinical Closure by clamp Cryopreservation Euthanasia FDA approved Gene Expression Genes Goals Hepatic Tissue Hepatocyte Histology Hour Human In Vitro Individual Inflammation Inflammatory Inflammatory Infiltrate Innate Immune Response Ischemia Liver Liver Dysfunction Liver Failure Liver diseases Measures Mediating Mediator Messenger RNA Modeling Necrosis Neutrophil Infiltration Organ Outcome Oxidative Stress Pathology Pathway interactions Patients Perfusion Pharmaceutical Preparations Process Proteins Rattus Reperfusion Injury Reperfusion Therapy Small Interfering RNA Small RNA Technology Testing Traction Transaminases Transplantation Waiting Lists cell injury cytokine design end stage liver disease experimental study implantation improved in vivo in vivo imaging innate immune pathways knock-down liver function liver inflammation liver injury liver ischemia liver preservation liver transplantation mortality oxidative damage preservation receptor response to injury standard care subcutaneous therapy development transcriptome transcriptome sequencing uptake

项目摘要

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.

项目概要肝移植是治疗肝衰竭的唯一方法，但由于肝病巨大，许多患者在等待移植的过程中死亡。供体肝脏短缺。供体供应不足因缺血再灌注损伤（IRI）而进一步减少肝移植物的采购和保存。 IRI 损坏的移植物必须丢弃，因为它们植入受体后功能失调，导致患者死亡。迫切需要减轻 IRI 增加存活供体肝脏的数量并提高患者的生存率。 IRI机制涉及到几个方面途径。缺血性损伤促进细胞损伤后，再灌注会引发氧化应激和先天免疫汇聚激活细胞凋亡的途径，这是肝脏 IRI 中的主要细胞死亡机制。肝细胞凋亡 IRI 中的 IRI 由 Fas 受体 (Fas) 介导，导致坏死和炎症，从而导致肝功能障碍。在大鼠中，缺血前 Fas 的减少可减轻肝损伤。或许可以减轻 IRI 引起的肝脏损伤在移植过程中通过沉默肝细胞特异性Fas来抑制功能障碍。该提案的目标是利用小干扰RNA（siRNA）抑制IRI并提高肝移植质量用于移植。化学修饰的 siRNA 能够对任何靶基因进行有效的、序列特异性的沉默体内。当与 N-乙酰半乳糖胺 (GalNAc) 结合时，修饰的 siRNA 会被递送至肝细胞。 GalNAc-siRNA 技术是 FDA 批准的众多肝病药物的基础。在博士的指导下。 Anastasia Khvorova (siRNA)、Paulo Martins (肝脏移植)、Athma Pai (RNA seq)、Gyongyi Szabo (肝脏 IRI)、 Jacob Bledsoe（肝脏病理学）和 Matthew Gounis（体内成像），该项目将在体内和体外进行开发体内方法使用先前验证的 GalNAc-siRNA 靶向 Fas 来抑制 IRI 并保护肝功能。目标 1 将确定缺血前沉默 Fas 如何扰乱 IRI 通路（目标 1.1），并探讨沉默是否会影响 IRI 通路（目标 1.1）。 Fas 与其他介质结合可更好地防止肝损伤（目标 1.2）。 GalNAc- 单独靶向 Fas 的 siRNA 或与经过验证的氧化应激 (Hmgb1) 和炎症 (Tnfr1) 组合的 siRNA 介质将被注射到大鼠体内，并使用肝钳模型诱导 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 将决定如何肝移植物中的 Fas 沉默（离体）会影响大鼠的移植结果。离体期间递送 siRNA 通过静态冷藏（SCS）或机器灌注（MP）保存，可导致肝移植物的摄取。鼠将在 SCS 或 MP 期间采购肝脏并递送靶向 Fas 的 GalNAc-siRNA。 GalNAc-siRNA 将在 24 小时内评估摄取、Fas 表达和 IRI 转录组变化。这个实验将然后重复，保留移植物以获得最大的 GalNAc-siRNA 摄取/功效，将移植物移植到大鼠中，并测量肝损伤/功能和受体存活率。研究结果将描述 Fas 沉默的方式缺血前和缺血后影响肝脏 IRI 通路，确定最大 IRI 的体内和离体方法抑制，并帮助开发增加供体库并提高患者生存率的疗法。