Biology and Application of Platelet-Delivered Factor VIII

 描述（由申请方提供）：血友病A影响1：5000男性，是由于凝血因子（F）VIII缺乏所致。对这类患者的治疗已明显改善。他们护理中剩余的主要挑战之一是在20-30%的这些患者中产生FVIII抑制剂。许多这样的个体对免疫调节有反应，但有一个重要的亚组对这种治疗无反应，对FVIII旁路剂没有完全反应。此类患者通常会出现大关节损伤和复发性/危及生命的脑内出血。我们和其他人已经表明，血小板中异位表达的FVIII（pFVIII）储存在α颗粒中，并在血小板（Plt）活化后释放。该pFVIII在鼠模型中至少部分地受到保护而不受循环抑制剂的影响，因此其代表FVIII旁路剂的潜在替代治疗。然而，我们已经表明，pFVIII的空间/时间可用性与血浆FVIII不同，使其在某些情况下止血效果更好，而在其他情况下效果较差。我们还表明，在发育中的巨核细胞（Megs）中表达的异位FVIII可能对这些细胞有害，导致巨核细胞生成过程中细胞凋亡增加，影响小鼠骨髓移植（BMT）后Meg和Plt的恢复。我们还表明，在鼠止血研究中，增强FVIII单链稳定性的FVIII变体（FVIIIR 1645 H）显著增强pFVIII止血功效。基于我们的研究，我们谨慎地使用基于BMT的策略将pFVIII治疗转化为患者护理。我们提出了pFVIII Plt预防性输注策略。以下三个目的旨在测试pFVIII在抑制剂患者的临床相关止血挑战中的疗效，然后开发pFVIII Plt输注疗法作为此类患者护理的新方法。具体目标（SA）#1。定义pFVIII在临床相关止血模型中的疗效。pFVIII的疗效主要在鼠模型中进行了测试，这些鼠模型不会发生在抑制剂患者中观察到的大关节出血或脑内出血。我们建议使用我们的小组建立的自发性关节和脑内出血的FVIII空大鼠模型和脑内光化学损伤模型来测试pFVIII在这些目标设置中的疗效，特别是与并发抑制剂。SA编号。优化源自诱导多能干细胞（iPSC）的人Megs中的表达水平和pFVIII比活性。使用“安全港”、腺相关病毒位点1、插入方法以及慢病毒方法，我们将优化源自iPSC的Megs中FVIII的活性水平，并表征这些Megs引起的损伤。还将检查从这些Megs释放的Plt中的pFVIII水平及其体外止血功效。SA #3。定义pFVIII/iPSC衍生的Plt的止血功效。FVIII无效免疫缺陷NOD/SCID/IL 2 R γ缺陷（NSG）小鼠将输注来自特定目的2的衍生Plt，并在不存在和存在抑制剂以及存在共输注FVIII旁路剂的情况下测试这些Plt在保护小鼠方面的止血功效。

 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.