Genetic and therapeutic studies of hemostatic and thrombotic disorders using zebrafish

项目摘要/摘要 凝血系统的病理性失调是人类发病率和 死亡，导致大量出血或凝血。在以下方面取得了重大进展： 凝血级联的遗传调节基因的鉴定，但许多未知的修饰基因参与了 在止血患者和家属中观察到不同的疾病严重程度和外显率 血栓性疾病。了解这些修饰因素有助于将病理风险较高的患者分类为 以及确定新的治疗靶点。尽管出血性和非传染性疾病治疗方面的重大改进 出血性疾病是用血液制品和人类衍生或重组凝血造成的 这些因素都限制了保质期和储存条件，需要静脉输液。抗凝 近年来出现了许多新的直接作用的口服抗凝剂的激增，但它们的作用机制 作用仅限于凝固级联。在我们以前工作的基础上，这个项目将利用 强大的遗传工具，包括基因组编辑核酸酶、下一代测序和斑马鱼。 我们将对基因组进行大规模的询问，以发现与止血调控基因 有可能改变人类凝血障碍的严重程度。我们已经开发出一组凝血因子 利用健壮的基因组编辑核酸酶(TALENS和CRISPR/CAS)突变斑马鱼并进行化学 已确定潜在抑制突变株系的诱变实验 血栓形成和止血修饰基因。这组突变鱼也将被用于无偏见的检测 确定抑制出血或血栓形成的新的先导分子。本文件中描述的方法 提案将导致确定调节止血和 血栓生成，其中一些很可能被证明是人类重要的遗传修饰因素，或者将 提出新的物种特有但具有生物洞察力的监管机制。这将为我们揭示 止血的调节机制和修饰物也将是候选的诊断和治疗 人类血栓性和出血性疾病的靶标。这些目标将被用来开发潜力 治疗出血和血栓形成的创新药物和新的治疗类别可能受益 一般人群，以及出血和血栓性疾病患者。

PROJECT SUMMARY/ABSTRACT Pathologic dysregulation of the coagulation system is a major contributor to human morbidity and mortality, resulting in either excessive bleeding or clotting. Significant progress has been made in the identification of genetic regulators of the coagulation cascade, but many unknown modifier genes contribute to the variable disease severity and penetrance observed among patients and families with hemostatic and thrombotic disorders. Understanding such modifiers could help classify patients at higher risk for pathology as well as identify novel therapeutic targets. Although major improvements to treatment of hemorrhagic and bleeding disorders have been made with blood products and human derived or recombinant coagulation factors, these have limited shelf life and storage conditions, and require intravenous infusion. Anticoagulation has seen a surge in recent years with many new direct acting oral anticoagulants, but their mechanisms of action are limited to the coagulation cascade. Building on our previous work, this project will take advantage of powerful genetic tools, including genome editing nucleases, next generation sequencing, and the zebrafish. We will conduct a large scale interrogation of the genome to discover hemostasis regulatory genes with the potential to modify the severity of human coagulation disorders. We have developed a panel of clotting factor mutant zebrafish using robust genome editing nucleases (TALENS and CRISPR/Cas) and conducted chemical mutagenesis experiments that have identified potential suppressor mutant lines harboring prospective thrombosis and hemostasis modifier genes. This panel of mutant fish will also be used for unbiased assays to identify novel lead molecules that suppress hemorrhage or thrombosis. The approaches described in this proposal will lead to the identification of the key non-canonical factors regulating hemostasis and thrombopoiesis, some of which will likely prove to be important genetic modifying factors in humans, or will suggest novel species-specific but biologically insightful regulatory mechanisms. This will shed light on the regulatory mechanisms of hemostasis, and the modifiers will also be candidate diagnostic and therapeutic targets for human thrombotic and hemorrhagic diseases. These targets will be utilized to develop potential innovative agents and new therapeutic classes for treatment of hemorrhage and thrombosis that could benefit the general population, as well as patients with bleeding and thrombotic disorders.