Overcoming FVIII protein misfolding and cell toxicity

克服 FVIII 蛋白错误折叠和细胞毒性

基本信息

批准号：
10560541
负责人：
RANDAL J. KAUFMAN
金额：
$ 57.7万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
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：
2022-02-05 至 2027-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10560541
关键词：
Address Adverse effects Affect Amino Acids Apoptotic B-domain-deleted factor VIII Biochemical Process Blood Coagulation Disorders Blood Coagulation Factor Bypass Cell Culture Techniques Cell Death Cell physiology Cell secretion Cells Cellular Stress Cessation of life Chronic Clinical Coagulation Process Complement Factor B Coupled Defect Dependovirus Development Dose Ectopic Expression Endoplasmic Reticulum Engineering Exhibits Factor IX Factor VIII Family suidae Fatty Liver Gene Transfer Genes Goals Grant Hemophilia A Hemorrhage Hepatocarcinogenesis Hepatocyte High Fat Diet Human Individual Inflammatory Response Injections Knowledge Lead Link Mediating Messenger RNA Monitor Mus Population Post-Translational Protein Processing Preparation Primary carcinoma of the liver cells Process Production Protein Biosynthesis Proteins Reactive Oxygen Species Safety Stress Tail Testing Therapeutic Time Toxic effect Translating Translations Transplantation Variant Veins X Chromosome biological adaptation to stress design digital enhancing factor gene therapy gene therapy clinical study human disease human model humanized mouse improved in vivo insight interest liver injury male mouse model novel plasmid DNA prevent prophylactic protein misfolding response ribosome profiling single-cell RNA sequencing systemic inflammatory response therapeutic transgene transcriptomics transgene expression vector

项目摘要

PROJECT SUMMARY/ABSTRACT Deficiency of clotting factor VIII (FVIII) causes the bleeding disorder hemophilia A (HA), an X chromosome-linked bleeding defect affecting 1/5,000 males world-wide. Although bleeding episodes are prevented by prophylactic administration of FVIII preparations or novel molecules that bypass FVIII, there is no effective long-term cure. Gene therapy through adeno-associated virus (AAV)-mediated ectopic FVIII expression in hepatocytes has emerged as a promising therapeutic approach for HA, but has met with two hurdles: 1) A requirement for very high vector doses to drive FVIII expression in hepatocytes; and 2) Declining therapeutic transgene expression over time accompanied with transient liver damage after vector injection, both of which may be directly linked to the fact that FVIII is prone to misfolding, aggregation and retention in the endoplasmic reticulum (ER). We demonstrated that increased FVIII synthesis does not equate to increased FVIII secretion. Since isolation of the FVIII gene in 1984, we have discovered many detrimental cellular responses as a consequence of heterologous expression of wildtype FVIII or B-domain deleted FVIII (BDD), presently the FVIII derivative in clinical gene therapy studies for HA. These responses include: 1) Formation toxic aggregates in the ER; 2) Hepatosteatosis; 3) Activation of an innate inflammatory response; 4) Activation of the ER unfolded protein response that leads to cell death; 5) Production of detrimental reactive oxygen species; and 6) Potential development of hepatocellular carcinoma (HCC). Moreover, we and the Xiao lab (Project 2) discovered two BDD variants that efficiently fold and are secreted from the cell. “F309S” BDD-FVIII has a single amino acid change at Phe309 to Ser so it does not aggregate in the ER. Another BDD-FVIII variant “X5”, which exchanges 5 amino acids in human BDD for corresponding porcine residues, is also secreted more efficiently than wildtype BDD-FVIII. We hypothesize that: a. FVIII expressed in hepatocytes by AAV delivery forms aggregates in the ER and activates stress responses resulting in cell death and potential HCC; and b. Novel FVIII molecules that more efficiently fold and are secreted will exhibit less toxicity in vivo are improved options for HA gene therapy. These hypotheses will be tested in 3 aims: 1. Delineate the cellular responses induced by AAV-BDD expression in hepatocytes of mice and define their pathophysiological consequences, especially HCC development; 2. Fully evaluate the potential of BDD variants F309S and X5 as more efficacious, safe and durable options for HA gene therapy; and 3. Characterize innate responses upon FVIII expression in primary human hepatocytes in humanized mice (Core 2). Our in- depth understanding of these issues should encourage the design of more optimal AAV-BDD vectors for HA gene therapy (Project 2) and increase our understanding of the impact of cellular stress and systemic inflammatory responses associated with AAV-BDD transduction in vivo (Project 3). Completion of this project will likely provide essential information required for development of a safe and durable long-term cure for HA.

项目概要/摘要凝血因子 VIII (FVIII) 缺乏会导致出血性疾病 A 型血友病 (HA)，这是一种 X 染色体连锁的疾病尽管出血事件可以通过预防措施预防，但出血缺陷影响着全世界 1/5,000 的男性。给予 FVIII 制剂或绕过 FVIII 的新型分子，没有有效的长期治疗方法。通过腺相关病毒 (AAV) 介导的肝细胞异位 FVIII 表达进行基因治疗成为一种有前途的 HA 治疗方法，但遇到了两个障碍：1）需要非常高剂量载体驱动肝细胞中的 FVIII 表达；2) 治疗性转基因表达下降随着时间的推移，载体注射后会出现短暂的肝损伤，这两者都可能与事实上，FVIII 容易在内质网 (ER) 中错误折叠、聚集和滞留。自分离以来，FVIII 合成的增加并不等于 FVIII 分泌的增加。 1984年，我们发现FVIII基因后，我们发现了许多由异源基因引起的细胞反应野生型 FVIII 或 B 结构域缺失的 FVIII (BDD) 的表达，目前是临床基因中的 FVIII 衍生物 HA 的治疗研究包括：1) ER 中形成毒性聚集体；2) 肝脂肪变性； 3) 激活先天性炎症反应；4) 激活内质网未折叠蛋白反应，从而导致细胞死亡；5) 活性氧的产生；以及 6) 肝细胞的潜在发育此外，我们和肖实验室（项目 2）发现了两种可有效折叠的 BDD 变体。并从细胞中分泌出来。BDD-FVIII 在 Phe309 处有一个氨基酸变为 Ser，因此它确实如此。另一种 BDD-FVIII 变体“X5”不会在 ER 中聚集，该变体将人 BDD 中的 5 个氨基酸交换为相应的猪残基，也比野生型 BDD-FVIII 更有效地分泌。 a. 通过 AAV 递送在肝细胞中表达的 FVIII 在 ER 中形成聚集体并激活应激反应导致细胞死亡和潜在的 HCC；以及 b. 更有效地折叠和分泌的新型 FVIII 分子。 HA 基因治疗的改进选择将表现出较低的体内毒性，这些假设将在 3 中得到检验。目的： 1. 描述小鼠肝细胞中 AAV-BDD 表达诱导的细胞反应并定义 2.充分评估BDD的潜力变体 F309S 和 X5 作为 HA 基因治疗的更有效、安全和持久的选择；以及 3. 表征人源化小鼠原代人肝细胞中 FVIII 表达的先天反应（核心 2）。对这些问题的深入理解应该鼓励为 HA 设计更优化的 AAV-BDD 载体基因治疗（项目 2）并增加我们对细胞应激和系统性影响的了解与 AAV-BDD 体内转导相关的炎症反应（项目 3）。可能会提供开发安全、持久的 HA 长期治疗方法所需的重要信息。