Platelet Factor 8 As A Novel Therapy for Hemophilia A

项目3：PONCZ项目主任/首席调查员(最后，第一。中)：高。凯瑟琳A。 项目3：血小板因子8作为治疗血友病A的新疗法 调查员(S)姓名和职称： [IVIorTimer Poncz，M.D. 宾夕法尼亚大学儿科学教授。 费城儿童医院。 罗德尼·卡迈尔博士。 宾夕法尼亚大学儿科学副教授。 费城儿童医院。 工程项目3简介 血友病A是最常见的遗传性严重出血素质，其原因是 凝血因子V11(F8)。替代疗法对出血发作有效，但费用昂贵 仍然与出血后遗症有关。重要的是，这些患者中高达30%的人会产生抗体 抑制F8活性。在这一亚组患者中治疗出血并发症的策略可能是有问题的， 特别是在免疫治疗/耐受以消除抑制物不成功的情况下。我们是第一个描述 异位表达的无B区人(/？)血小板中的F8(p/7BF8)可有效纠正 F8缺陷(F8“”)小鼠的出血素质。我们还表明phBF8的效果是 面对循环抑制剂的血浆HFQ。根据我们的观察，phF8的这些优势有所减弱 P/？BF8/F8“”小鼠在输注/7F8后形成的血栓不如F8“”小鼠稳定 更容易进行栓塞术。因此，我们认为phF8可能具有临床实用价值，但需要加强 改善其止血功能的疗效。这项建议有三个具体目标：具体目标1： 了解并改善pF8/F8“”小鼠的血栓形成。 认为pF8变异体可以增加pF8的比活性和/或减少pF8对细胞的凋亡效应 巨核细胞生成和/或提高pF8的表达水平可以显著改善F8的止血结果。 老鼠。拟议的研究将确认这些不同的过程，并确定需要测试的pF8候选对象 大动物F8“”模型和如下所述的人-鼠异种移植模型。 目的2：PFS的大型动物研究。将研究在特定目标1中定义的最有效的pF8变体 使用基于慢病毒的pF8表达系统驱动犬F8巨核细胞的表达“” 模型作为人类研究的前奏。这些犬类研究将检验改善自发性的有效性。 出血发作和出血模型，包括角质层和FeCIa动静脉损伤模型 这些F8的“”狗。具体目标3：研究人造血细胞中的pF8。为了解决我们的 慢病毒策略将在人类造血细胞中发挥作用，我们建议还检查慢病毒的传递 将最好的pF8导入(3D34*)人造血细胞，并证明我们可以在 人与小鼠巨核细胞相似，pF8定位于α颗粒内。免疫受损的小鼠 也会与这些祖细胞重组，而F8抗原的水平和活性可以 在这些异种移植的小鼠中实现了血小板测量。 综上所述，我们先前的研究表明，pF8为治疗糖尿病提供了一种新的治疗策略 血友病A患者，特别是那些循环抑制药物有问题的患者，但这 入路也可能与血栓不稳定性和栓塞率增加有关。拟议的研究将 在小鼠研究中进一步分析影响pF8止血效果的F8的特征并进行这些 概念和pF8变体将用于大型动物和临床前的人-鼠异种移植研究。 小灵通398(06/09版)页面延续格式页面 166 项目3：庞茨首席调查员/项目主任(最后、第一、中)：HIGH，凯瑟琳·A。 从头到尾 初步预算期明细预算 仅直接成本

PROJECT 3: PONCZ Program Director/Principal Investigator (Last, First. Middle): High. Katherine A. PROJECT 3: PLATELET FACTOR 8 AS A NOVEL THERAPY FOR HEMOPHILIA A Names and titles of investigator(s): ¿ IVIortimer Poncz, M.D. Professor of Pediatrics, University of Pennsylvania. The Children's Hospital of Philadelphia. ¿ Rodney Camire, Ph.D. Associate Professor of Pediatrics, University of Pennsylvania. The Children's Hospital of Philadelphia. Description of Project 3 Hemophilia A is the most common inherited severe bleeding diathesis and is due to a deficiency/dysfunction of coagulation Factor Vlll (F8). Replacement therapy is effective for bleeding episodes, but is expensive and remains associated with bleeding sequelae. Importantly, up to 30% of these patients develop antibodies that inhibit F8 activity. Strategies to treat bleeding complications in this subgroup of patients can be problematic, especially if immunotherapy/tolerance to eliminate the inhibitor is unsuccessful. We were the first to describe the ability of ectopically expressed B-domainless human (/?) F8 in platelets (p/7BF8) to be effective at correcting the bleeding diathesis in F8-deficient (F8""") mice. We also showed that phBF8 is 100-fold more effective than plasma hFQ in the face of circulating inhibitors. These advantages of phF8 are tempered by our observation that the resulting clots in p/?BF8/F8""" mice are less stable than clots formed in F8"" mice after /7F8 infusions and embolize more easily. We therefore believe that phF8 may have clinical utility, but needs enhanced efficacy to improve its hemostatic function. There are 3 specific aims to this proposal: Specific Aim 1: Understand and improve clot formation in pF8/F8""" mice. We now present new findings since the prior submission that pF8 variants that increase specific activity and/or reduce the apoptotic effect of pF8 on megakaryopoiesis and/or increase pF8 expression levels can markedly improve hemostatic outcome in F8""" mice. Proposed studies will confirm these distinct processes and identify strong pF8 candidates to be tested in both a large animal F8""" model and in a human-mouse xenotransplant model as described below. Specific Aim 2: Large animal studies of pFS. The most effective pF8 variants defined in Specific Aim 1 will be studied using a lentiviral-based pF8 expression system to drive expression in the megakaryocytes of a canine F8"" model as a prelude to human studies. These canine studies will examine efficacy in improving spontaneous bleeding episodes and in bleeding models, including cuticular and FeCIa arterial and venous injury models, in these F8"" dogs. Specific Aim 3: Studies of pF8 in human hematopoietic cells. To address whether our lentiviral strategy will work in human hematopoietic cells, we propose to also examine lentiviral-delivery of the best pF8s into (3D34* human hematopoietic cells and demonstrate that we can achieve similar levels of F8 in human as in murine megakaryocytes and that the pF8 localizes within a-granules. Immune-compromised mice will also be reconstituted with these progenitor cells, and the level of F8 antigen and activity that can be achieved in platelets in these xenotransplanted mice measured. In summary, our prior studies have demonstrated that pF8 offers a novel therapeutic strategy for the care of patients with hemophilia A, especially for those patients with problematic circulating inhibitors, but this approach may also be associated with increased clot instability and embolization. The proposed studies will further analyze the features of F8 that can effect pF8 hemostatic efficacy in murine studies and carry these concepts and pF8 variants forward to large animal and pre-clinical human-mouse xenotransplant studies. PHS 398 (Rev. 06/09) Page Continuation Format Page 166 PROJECT 3: Poncz Principal Investigator/Program Director (Last, First, Middle): High, Katherine A. FROM THROUGH DETAILED BUDGET FOR INITIAL BUDGET PERIOD DIRECT COSTS ONLY