A novel thrombolytic targeting Von Willebrand Factor (VWF) to treat ischemic stroke

项目摘要 导致急性缺血性卒中（AIS）的动脉血栓形成是合并发病率和死亡率的主要原因。 全世界的死亡率。重组组织型纤溶酶原激活剂（rtPA）是唯一被批准用于治疗AIS的药物。 不幸的是，只有约5-10%的AIS患者实际接受rtPA治疗。rtPA治疗的风险 包括症状性颅内出血（ICH）显著增加， 接受药物治疗的患者。此外，rtPA在存在以下情况的患者中仅实现10%的再通： 大血管闭塞（LVO）卒中。这些凝块通常富含血小板， rtPA。迫切需要开发溶栓剂，其：1.靶向关键蛋白质， 中风服装结构，2.再通动脉闭塞，和3.安全性上级于rtPA。 血管性血友病因子（VWF）是AIS治疗的最佳靶点。VWF结合糖蛋白Ib Ⅶ（GPIb Ⅶ）， 血小板受体复合物GPIb-IX-V以及GPIIb-IIIa，导致血小板活化和聚集。 VWF也自缔合，延伸到血管腔中作为血小板和红细胞粘附的支架。 这些过程导致AIS患者中所见的动脉血栓形成。我们的初步资料表明， 来自中风患者的研究表明，大多数凝块具有富含VWF的血小板壳， 血栓核心，提供了与rtPA相关的动脉再通率差的解释。 适体是一种以阿糖胞苷为基础的药物，它以高亲和力和特异性抑制其靶蛋白。我们 已经分离并优化了结合并抑制VWF（DTRI-031）的RNA适体。我们还 设计了第二种寡核苷酸（DTRI-025），可在数分钟内完全逆转DTRI-031的活性。我们 在血栓形成的小型和大型动物模型中的数据表明DTRI-031既防止血栓形成， 形成和溶解完全形成的动脉闭塞优于rtPA。本提案的总体目标是：（1） 将AIS患者中升高的血浆VWF与凝血VWF相关联，以及2）证明DTRI-031对VWF的抑制可以 转化为AIS患者的有效治疗。 我们将检验以下假设：1）VWF是AIS治疗的最佳靶点。我们的初步数据显示 LVO AIS患者血浆VWF水平显著升高。我们的初步数据复制了这些 在小鼠中风模型中的发现。2)DTRI-031在体外有效溶解AIS血液样本中的凝块 患者和在中风的鼠模型中的体内。此外，DTRI-025在AIS中快速逆转DTRI-031活性， 在体外和体内。3)DTRI-031治疗通过增加AIS小鼠模型中的 再通，减少梗死体积，改善功能恢复。初步数据显示， DTRI-031处理的小鼠与对照相比具有减少的中风后梗塞体积。最后，DTRI-031具有 通过减少ICH、脑水肿形成和血脑屏障破坏改善安全性特征， AIS与rtPA相比。

PROJECT SUMMARY Arterial thrombosis resulting in acute ischemic stroke (AIS) is the leading cause of combined morbidity and mortality worldwide. Recombinant tissue plasminogen activator (rtPA) is the only drug approved to treat AIS. Unfortunately, only ~5-10% of patients who present with AIS actually receive rtPA. Risks of rtPA treatment include a significant increase in symptomatic intracranial hemorrhage (ICH), which occurs in up to 6.4% of patients who receive the drug. Moreover, rtPA only achieves 10% recanalization in patients who present with large vessel occlusion (LVO) stroke. These clots, which are commonly platelet-rich, are notoriously resistant to rtPA. A critical need exists to develop thrombolytic agents that: 1. target critical proteins involved in stroke clot architecture, 2. recanalize arterial occlusions, and 3. have a safety profile superior to rtPA. Von Willebrand Factor (VWF) is an optimal target for AIS treatment. VWF binds to glycoprotein Ib (GPIb) of the platelet receptor complex GPIb-IX-V as well as to GPIIb-IIIa, resulting in platelet activation and aggregation. VWF also self-associates, extending into the vessel lumen as a scaffold for platelet and red blood cell adhesion. These processes result in arterial thrombosis as seen in AIS patients. Our preliminary data of cerebral thrombi from stroke patients show that the majority of clots have a platelet shell rich with VWF encapsulating the thrombus core, providing an explanation for the poor arterial recanalization rate associated with rtPA. Aptamers are oligonucleotide-based drugs that inhibit their target proteins with high affinity and specificity. We have isolated and optimized an RNA aptamer that binds to and inhibits VWF (DTRI-031). We have also designed a second oligonucleotide (DTRI-025) that fully reverses DTRI-031 activity within minutes. Our data in small and large animal models of thrombosis demonstrates that DTRI-031 both prevents thrombus formation and lyses fully formed arterial occlusions better than rtPA. The overall goals of this proposal are to 1) correlate elevated plasma VWF to clot VWF in AIS patients and 2) demonstrate VWF inhibition by DTRI-031 can translate into an effective treatment for patients who present with AIS. We will test the hypotheses that 1) VWF is an optimal target for AIS treatment. Our preliminary data shows that LVO AIS patients have significantly elevated plasma VWF. Our preliminary data has replicated these findings in a murine model of stroke. 2) DTRI-031 effectively lyses clots in vitro in blood samples from AIS patients and in vivo in a murine model of stroke. Moreover, DTRI-025 rapidly reverses DTRI-031 activity in AIS in vitro and in vivo. 3) DTRI-031 treatment improves outcomes in a murine model of AIS by increasing recanalization, decreasing infarct volume, and improving functional recovery. Our preliminary data reveals that DTRI-031-treated mice have reduced post-stroke infarct volumes compared to control. Finally, DTRI-031 has an improved safety profile by decreasing ICH, cerebral edema formation and blood-brain-barrier breakdown after AIS compared to rtPA.