Overcoming FVIII protein misfolding and cell toxicity

项目总结/摘要 凝血因子VIII（FVIII）缺乏导致出血性疾病血友病A（HA），一种X染色体连锁的 出血缺陷影响全球1/5，000的男性。虽然预防性治疗可以预防出血发作， 尽管使用FVIII制剂或绕过FVIII的新分子给药，但没有有效的长期治愈。 通过腺相关病毒（AAV）介导的肝细胞异位表达FVIII的基因治疗， 作为HA的有希望的治疗方法出现，但遇到了两个障碍：1）需要非常高的 较高载体剂量以驱动肝细胞中FVIII表达;和2）治疗性转基因表达下降 随着时间的推移，伴随着载体注射后的短暂肝损伤，这两者都可能直接与 FVIII易于错误折叠、聚集和滞留在内质网（ER）中的事实。我们 证明了增加的FVIII合成不等于增加的FVIII分泌。自隔离以来， 在1984年，我们发现了许多有害的细胞反应作为结果的异源性FVIII基因， 表达野生型FVIII或B结构域缺失的FVIII（BDD），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ER中形成毒性聚集体; 2）脂肪肝; 3)激活先天性炎症反应; 4）激活ER未折叠蛋白反应，导致 细胞死亡; 5）有害活性氧的产生;和6）肝细胞的潜在发展 癌（HCC）。此外，我们和Xiao实验室（项目2）发现了两种BDD变体， 并从细胞中分泌出来。“F309 S”BDD-FVIII在Phe 309处具有单个氨基酸改变为Ser，因此其 不在急诊室聚集。另一种BDD-FVIII变体“X5”，其将人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。我们假设： 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;和B.更有效折叠和分泌的新型FVIII分子 在体内表现出更低的毒性是HA基因治疗的改进选择。这些假设将在3 目标：1.描述AAV-BDD在小鼠肝细胞中表达诱导的细胞应答，并定义 其病理生理后果，尤其是HCC发展; 2.充分评估BDD的潜力 变体F309 S和X5作为HA基因治疗的更有效、安全和持久的选择;和3.表征 人源化小鼠中原代人肝细胞中FVIII表达后的先天性应答（核心2）。我们的- 对这些问题的深入理解应该鼓励设计更优化的HA 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）。完成本项目 将可能提供开发安全持久的HA长期治疗所需的基本信息。

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.