Biology and Application of Platelet-Delivered Factor VIII

 描述(申请人提供)：血友病A影响1：5000男性，是由于凝血因子(F)VIII缺乏。对这类患者的治疗已有显著改善。在他们的护理中仍然存在的主要挑战之一是在这些患者中开发FVIII的抑制剂。许多这样的患者对免疫调节有反应，但有相当一部分人对这种治疗顽固不化，对FVIII旁路药物没有完全反应。这类患者通常会出现大的关节损伤和反复/危及生命的脑内出血。我们和其他人已经证明，在血小板(PFVIII)中异位表达的FVIII(PFVIII)储存在阿尔法颗粒中，并在血小板(Plt)激活后释放。在小鼠模型中，这种pFVIII S至少部分地被循环抑制剂保护，因此它代表了一种潜在的替代FVIII旁路药物的治疗方法。然而，我们已经证明，pFVIII的时空可用性不同于血浆FVIII，这使得它在某些情况下更有效，而在其他情况下则不那么有效。我们还表明，在发育中的巨核细胞(MEGS)中表达的异位FVIII对这些细胞有害，导致巨核细胞在巨核生成过程中凋亡增加，影响小鼠骨髓移植(BMT)后Meg和Plt的恢复。我们还表明，在小鼠止血研究中，增强FVIII单链稳定性的FVIII变体(FVIIIR1645H)显著增强了pFVIII的止血效果。基于我们的研究，我们建议使用基于骨髓移植的策略将pFVIII疗法转化为患者护理。相反，我们建议采用pFVIII Plt预防性输注策略。以下三个目的旨在测试pFVIII在临床相关止血挑战中对使用抑制剂的患者的有效性，然后开发pFVIII Plt输注疗法作为治疗此类患者的一种新方法。特定目的(SA)#1.确定pFVIII在临床相关止血模型中的有效性。PFVIII的有效性主要在没有出现大量关节出血或脑内出血的小鼠模型中进行测试，这些模型在服用抑制剂的患者中可见。我们建议使用我们小组建立的自发性关节和脑内出血的FVIII空白大鼠模型和脑内光化学损伤模型来测试pFVIII在这些靶点设置中的有效性，特别是在同时使用抑制剂的情况下。SA#.优化诱导多能干细胞(IPSCs)来源的人MEGs的表达水平和pFVIII特异性活性。利用“安全港”、腺相关病毒位点1、插入方法和慢病毒方法，我们将优化IPSCs来源的MEGs中FVIII的水平活性，并表征这些MEGs所造成的损伤。还将检测pFVIII水平及其在这些MEGs释放的Plt中的体外止血效果。SA#3.确定pFVIII/IPSC衍生的Plts的止血效果。Fvia零免疫缺陷NOD/SCID/IL2Rγ缺陷(NSG)小鼠将被输注来自特定Aim 2的派生Plts，这些Plts在没有和存在抑制剂以及存在共注入的Fviii旁路药物的情况下将被测试对保护小鼠的止血效果。

 DESCRIPTION (provided by applicant): Hemophilia A affects 1:5000 males and is due to deficiencies of the coagulation factor (F) VIII. Therapy for such patients has markedly improved. One of the remaining major challenges in their care is the development of inhibitors to FVIII in 20-30% of these patients. Many of such individuals respond to immunomodulation, but a significant subset are recalcitrant to such therapy and are not fully responsive to FVIII bypass agents. Such patients often develop large joint damage and recurrent/life-threatening intracerebral bleeds. We and others have shown that FVIII ectopically expressed in platelets (pFVIII) is stored in alpha granules and released following platelet (Plt) activation. This pFVIII s at least partially protected from circulating inhibitors in murine models so that it represents a potential alternative care to FVIII bypass agents. However, we have shown that the spatial/temporal availability of pFVIII differs from plasma FVIII making it more hemostatically effective in some settings and less so in others. We have also shown that ectopic FVIII expressed in developing megakaryocytes (Megs) can be deleterious to these cells resulting in increased apoptosis during megakaryopoiesis, affecting Meg and Plt recovery post-bone marrow transplantation (BMT) in mice. We also have shown that a FVIII variant that enhances FVIII single-chain stability (FVIIIR1645H) markedly enhances pFVIII hemostatic efficacy in murine hemostasis studies. Based on our studies, we caution translating pFVIII therapy to patient care using a BMT-based strategy. We propose instead a pFVIII Plt prophylactic infusion strategy. The following three aims propose to test the efficacy of pFVIII in clinically relevant hemostatic challenges for patients with inhibitors and then develop the pFVIII Plt infusion therapy as a novel approach in the care of such patients. Specific Aim (SA) #1. Define the efficacy of pFVIII in clinically relevant models of hemostasis. pFVIII efficacy has been tested mostly in murine models that do not develop large joint bleeds or intracerebral bleeds seen in patients with inhibitors. We propose using a FVIII null rat model of spontaneous joint and intracerebral bleeds established by our group and an intracerebral photochemical injury model to test the efficacy of pFVIII in these target settings, especially with concurrent inhibitors. SA#. Optimize expression levels and pFVIII specific activity in human Megs derived from induced pluripotent stem cells (iPSCs). Using a "safe harbor", adeno-associated virus site 1, insertion approach as well as a lentiviral approach, we will optimize the level activity of FVIII in Megs derived from iPSCs and characterize the injury incurred by these Megs. pFVIII levels and its in vitro hemostatic efficacy in Plts released from these Megs will also be examined. SA#3. Define the hemostatic efficacy of the pFVIII/iPSC-derived Plts. FVIII null immuno-deficient NOD/SCID/IL2Rγ-deficient (NSG) mice will be infused with derived Plts from Specific Aim 2, and the hemostatic efficacy of these Plts in protecting the mice will be tested in the absence and presence of inhibitors and presence of co-infused FVIII bypass agents.