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：
10537869
负责人：
Julianna E Buchwald
金额：
$ 3.15万
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/10537869
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 Infiltration Inflammation Inflammatory Innate Immune Response Ischemia Liver Liver Dysfunction Liver Failure Liver diseases Measures Mediating Mediator of activation protein Messenger RNA Modeling Necrosis Neutrophil Infiltration Organ Outcome Oxidative Stress Pathology Pathway interactions Patients Perfusion Pharmaceutical Preparations Process Proteins Rattus Reperfusion Injury Reperfusion Therapy Savings Small Interfering RNA Small RNA Technology Testing Time Traction Transaminases Transplantation Waiting Lists base 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 receptor expression 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.

项目摘要肝移植是肝衰竭的唯一治愈供体肝脏短缺。供体供应不足在缺血 - 再灌注损伤（IRI）进一步减少肝移植物的采购和保存。由于它们是植入接受者后功能失调，导致患者死亡。减轻IRI至关重要增加可行的供体肝脏的数量并改善患者的生存率。 IRI机制涉及几个途径。缺血性侮辱后，促进细胞损伤，再灌注会触发氧化应激和先天免疫融合到激活凋亡的途径，这是肝脏IRI的主要细胞死亡机制。肝细胞凋亡在IRI中，由FAS受体（FAS）介导，并导致坏死和炎症，从而引起肝功能障碍。在缺血之前，大鼠减少FA会减少肝脏损伤。有可能减轻伊利诱导的肝脏在移植过程中，通过沉默肝细胞特异性FAS的功能障碍。该提案的目的是使用小的干扰RNA（siRNA）抑制IRI并提高肝移植的质量用于移植。化学修饰的siRNA可实现任何靶基因的有效序列特异性沉默体内。与N-乙酰基乳酰胺（GALNAC）结合时，修饰的siRNA被递送至肝细胞。 Galnac-SiRNA技术是众多FDA批准的肝病药物的基础。在Drs的指导下。 Anastasia Khvorova（Sirna），Paulo Martins（肝移植），Athma Pai（RNA SEQ），Gyongyi Szabo（肝脏IRI），IRI），雅各布·布莱索（Jacob Bledsoe）（肝脏病理学）和马修·戈尼（Matthew Gounis）（体内成像），该项目将在体内发展和EX 使用先前验证的galnac-siRNA靶向FAS来抑制IRI和保护肝功能的体内接近。 AIM 1将确定缺血之前如何使FAS沉默FAS（AIM 1.1），并探索是否沉默 FAS与其他介体结合使用，可以更大的保护防止肝脏损害（AIM 1.2）。 galnac- 单独靶向FA的siRNA，或与经过验证的氧化应激（HMGB1）和炎症（TNFR1）结合使用（TNFR1）介体将被注入大鼠，并将使用肝夹模型诱导IRI。重新灌注后，目标将评估基因表达，肝损伤和IRI转录组变化。 AIM 2将决定如何在肝移植物中沉默的FA（离体）会影响大鼠的移植结局。在离体期间传递siRNA 通过静态冷藏（SC）或机器灌注（MP）保存，可导致肝脏移植物的摄取。鼠将采购肝脏，并在SCS或MP期间输送GALNAC-SIRNA靶向FA。 galnac-sirna 在24小时内，将评估摄取，FAS表达和IRI转录组变化。这个实验将然后重复，保留移植物，用于最大的galnac-sirna摄取/功效，将移植移植到大鼠中，并测量肝损伤/功能和受体存活。研究发现将表征FAS沉默如何前和后疾病后影响肝脏IRI途径，体内识别最大IRI的体内方法抑制作用，并有助于开发增加供体池并改善患者生存的疗法。