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

• 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
• 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

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.

项目总结 肝移植是治疗肝功能衰竭的唯一方法，但由于大量的肝移植，许多患者在等待移植时死亡。 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 肝移植的采购和保存。IRI受损的移植物必须丢弃，因为它们 植入受体后出现功能障碍，导致患者死亡。缓解IRI是至关重要的 增加活体供肝的数量，提高患者的存活率。IRI机制涉及几个 小路。缺血损伤促进细胞损伤后，再灌注触发氧化应激和先天免疫 汇聚以激活细胞凋亡的途径，这是肝脏IRI的主要细胞死亡机制。肝细胞凋亡 胰岛素抵抗是由Fas受体(Fas)介导的，导致坏死和炎症，从而导致肝功能障碍。 在大鼠中，在缺血前减少Fas可以减少肝脏损伤。有可能减轻IRI诱导的肝脏 在移植过程中通过沉默肝细胞特异性Fas而导致的功能障碍。 该方案的目的是利用小干扰rna(Sirna)抑制IRI，提高肝移植质量。 用于移植。化学修饰的siRNA能够有效地、序列特异性地沉默任何靶基因 在活体内。修饰后的siRNAs与N-乙酰氨基半乳糖(GalNAc)结合后进入肝细胞。 GalNAc-siRNA技术是FDA批准的许多肝病药物的基础。在戴维斯博士的指导下。 Anastasia Khvorova(SiRNA)，Paulo Martins(肝脏移植)，Athma Pai(RNA序列)，Gyongyi Szabo(肝脏IRI)， Jacob Bledsoe(肝脏病理学)和Matthew Gounis(活体成像)，这个项目将在体内和EX开发 体内方法使用先前验证的针对Fas的GalNAc-siRNA来抑制IRI和保护肝功能。 目标1将确定在缺血前沉默Fas如何扰乱IRI通路(目标1.1)，并探讨沉默是否 Fas与其他介质联合使用可提供更好的保护，防止肝损伤(目标1.2)。GalNAc- 针对Fas的siRNA，单独或与已证实的氧化应激(HMGB1)和炎症(Tnfr1)结合 将介体注射到大鼠体内，并使用肝脏钳夹模型诱导IRI。再灌注后，靶点 将评估基因表达、肝脏损伤和IRI转录组的变化。目标2将决定如何 抑制肝移植中的Fas(体外)影响大鼠的移植结果。体外传递siRNA的实验研究 通过静态冷藏(SCS)或机器灌流(MP)保存，可导致移植肝摄取。大鼠 肝脏将被采购，针对Fas的GalNAc-siRNA将在SCS或MP期间交付。GalNAc-siRNA 摄取、Fas表达和IRI转录组的变化将在24小时内进行评估。这项实验将 然后重复，保存移植物以获得最大的GalNAc-siRNA摄取/效果，将移植物移植到大鼠体内， 并测量肝脏损伤/功能和受体存活率。研究结果将表征Fas沉默是如何 缺血前后影响肝脏IRI途径，确定体内和体外获得最大IRI的方法 抑制，并帮助开发治疗方法，以增加捐赠者池和改善患者存活率。