Genetic and therapeutic studies of hemostatic and thrombotic disorders using zebrafish

项目总结/摘要 凝血系统的病理性失调是导致人类发病率的主要因素， 死亡，导致过度出血或凝血。在以下方面取得了重大进展： 凝血级联的遗传调节因子的鉴定，但许多未知的修饰基因有助于 在患有止血药物的患者和家庭中观察到的不同疾病严重程度和发病率， 血栓性疾病了解这些修饰可以帮助将病理学风险较高的患者分类为 并确定新的治疗靶点。虽然对出血性和 已经用血液制品和人源性或重组凝血剂制造了出血性疾病 由于各种因素，这些药物的保质期和储存条件有限，并且需要静脉输注。抗凝 近年来出现了许多新的直接作用口服抗凝剂的激增，但它们的机制， 作用仅限于凝血级联。在我们以前工作的基础上，该项目将利用 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，以发现止血调节基因， 可能改变人类凝血障碍的严重程度。我们研制了一组凝血因子 使用稳健的基因组编辑核酸酶（TALENS和CRISPR/Cas）突变斑马鱼，并进行化学 诱变实验已经鉴定了潜在的抑制突变株系， 血栓形成和止血调节基因。这组突变鱼也将用于无偏测定， 鉴定抑制出血或血栓形成新先导分子。本文中所述的方法 该提案将导致确定调节止血的关键非规范因素， 血小板生成，其中一些可能被证明是人类重要的遗传修饰因子，或将 提出了新的物种特异性但具有生物学洞察力的调节机制。这将有助于阐明 止血的调节机制，并且修饰剂也将是候选的诊断和治疗药物。 人血栓性和出血性疾病的靶点。这些目标将用于开发潜力 治疗出血和血栓形成的创新药物和新的治疗类别， 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.