Overcoming FVIII protein misfolding and cell toxicity

项目摘要/摘要 凝血因子(FVIII)缺乏导致X染色体连锁的出血性疾病血友病A(HA) 全世界每5000名男性中就有1名患有出血性缺陷。虽然出血可以通过预防措施来预防 使用FVIII制剂或绕过FVIII的新分子，没有有效的长期治愈方法。 腺相关病毒(AAV)介导的肝细胞异位表达FVIII基因治疗 作为HA的一种很有前途的治疗方法，但遇到了两个障碍：1)非常 大剂量载体驱动FVIII在肝细胞中的表达；2)降低治疗性转基因表达 随着时间的推移伴随着载体注射后的一过性肝损害，这两种情况都可能直接与 FVIII在内质网(ER)容易发生错误折叠、聚集和滞留。我们 证明FVIII合成增加并不等同于FVIII分泌增加。由于隔离了 FVIII基因，我们已经发现了许多有害的细胞反应作为结果的异源 野生型FVIII或B结构域缺失的FVIII(BDD)的表达，目前临床基因中的FVIII衍生物 HA的治疗研究。这些反应包括：1)在内质网中形成有毒聚集体；2)肝骨病； 3)激活固有的炎症反应；4)激活内质网未折叠蛋白反应，导致 细胞死亡；5)有害的活性氧的产生；6)肝细胞的潜在发展 癌症(HCC)。此外，我们和肖实验室(项目2)发现了两个高效折叠的BDD变体 从细胞中分泌出来。“F309S”BDD-FVIII在Phe309位有一个单一的氨基酸改变为Ser，所以它确实是这样的 而不是在急诊室中聚集。另一个BDD-FVIII变异体“X5”，它将人类BDD中的5个氨基酸交换为 相应的猪残留物，也比野生型BDD-FVIII更有效地分泌。我们假设： A.通过AAV递送在肝细胞中表达的FVIII在内质网中形成聚集并激活应激反应 导致细胞死亡和潜在的肝细胞癌；以及b.更有效地折叠和分泌的新型FVIII分子 在体内表现出较少的毒性是HA基因治疗的改进选择。这些假设将在3个月内得到验证 目的：1.研究AAV-BDD在小鼠肝细胞中的表达及其诱导的细胞反应 它们的病理生理后果，特别是肝细胞癌的发展；2.充分评估BDD的潜力 变异体F309S和X5作为HA基因治疗更有效、更安全和更持久的选择；以及3.特征 人源化小鼠原代人肝细胞对FVIII表达的先天反应(核心2)。我们在- 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疾病的影响的理解 与体内AAV-BDD转导相关的炎症反应(项目3)。本项目竣工 可能会为开发一种安全持久的HA长期治疗方法提供必要的信息。

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.