Postnatal Cell-Based Therapies for Hemophilia A

项目摘要 尽管血友病A（HA）患者的治疗护理有了很大的发展，但仍然没有治愈性的， 依赖于终身、反复、昂贵的FVIII输注（≥ 600，000美元/年/患者）。此外，本发明还提供了一种方法， >30%的重度HA患者产生FVIII抑制性抗体，这是HA治疗中最严重的挑战。 HA的临床管理。因此，迫切需要开发新的疗法，可以提供更长的时间- 持久/永久的改善，和/或可以克服目前阻碍治疗的免疫障碍。的 通过细胞平台如骨髓基质细胞（MSC）递送FVIII，是一种有吸引力的方法， 因为在病毒转导后，MSC分泌高水平载体编码的FVIII， 从天然蛋白质，并不转化或进展到克隆优势。此外，在输注时，MSC 可以长期停留在实质和血管周围区域的多个器官中， 理想地用于将FVIII递送到循环中。MSC具有免疫调节/抗炎特性， 如果使用自体MSC，它可以使FVIII以致耐受性方式递送。我们最近测试了 利用模拟遗传学、抑制剂和免疫抑制剂的绵羊系的表达FVIII的MSC的治疗潜力 形成（给予FVIII蛋白）和严重形式的人HA的临床症状，并显示 在没有接受者预处理的情况下，出生后给予单倍体相合的MSC， 表达高水平的FVIII导致两只儿科HA羊的表型完全纠正，逆转了 存在关节积血，并恢复正常的体力活动。值得注意的是，这种表型校正是长期的， 尽管在这些绵羊中存在高滴度抑制剂，但这种情况持续存在，并且移植的MSC没有被清除。 接受者的免疫系统，使他们能够长期坚持在多个网站。这项先前的工作使我们 假设表达FVIII的MSC的递送导致血管周围植入和FVIII的释放 将蛋白质以可以治疗HA的水平进入循环，并且使用MSC稳定地递送高蛋白质， 水平的FVIII进入循环可能代表了一种新的手段，临床纠正HA患者的前 现有的抑制剂。因此，我们建议：1）确定FVIII植入的部位和持续时间- 产生自体MSC，并测试这种疗法介导临床/表型改善的能力， 有和没有预先存在的抑制剂的绵羊受体; 2）测试 MSC将使自体细胞能够以致耐受性方式将FVIII呈递给FVIII初治受体，使得 这些动物将终身无瘤;和3）研究FVIII的持续递送是否通过 这种基于MSC的方法可以用作在患有前 现有的抑制剂，并比较这种方法与目前的免疫耐受诱导方案。我们希望 这些研究将为临床上可行的治疗方法的开发提供必要的知识 治疗/治愈重度HA和克服受FVIII抑制剂困扰患者免疫障碍的策略。

PROJECT SUMMARY Despite great developments in treatment care for patients with Hemophilia A (HA), still none is curative, and all depend upon lifelong, recurrent, prohibitively expensive, FVIII infusions (≥$600,000/year/patient). In addition, >30% of patients with severe HA develop inhibitory antibodies to FVIII, which is the most serious challenge in the clinical management of HA. There is thus a critical need to develop novel therapies that can offer a longer- lasting/permanent improvement, and/or can defeat the immune hurdles that currently thwart treatment. The delivery of FVIII through a cellular platform, such as marrow stromal cells (MSC), is an appealing approach, since following viral transduction, MSC secrete high levels of vector-encoded FVIII that is indistinguishable from the native protein, and do not transform or progress to clonal dominance. In addition, upon infusion, MSC can lodge long-term in multiple organs within both the parenchyma and the perivascular zones, placing them ideally for delivering FVIII into the circulation. MSC have immunomodulatory/anti-inflammatory properties and, if autologous MSC are used, it may enable FVIII delivery in a tolerogenic fashion. We recently tested the therapeutic potential of FVIII-expressing MSC utilizing a line of sheep that emulates the genetics, inhibitor formation (to administered FVIII protein), and clinical symptoms of the severe form of human HA, and showed that, without recipient preconditioning, the postnatal administration of haploidentical MSC engineered to express high levels of FVIII led to complete phenotypic correction of two pediatric HA sheep, reversal of existing hemarthroses, and return to normal physical activity. Remarkably, this phenotypic correction was long lasting despite the presence of high-titer inhibitors in these sheep, and the engrafted MSC were not cleared by the recipient's immune system, enabling them to persist long-term in multiple sites. This prior work leads us to hypothesize that the delivery of FVIII-expressing MSC results in perivascular engraftment and release of FVIII protein into the circulation at levels that could be curative for HA, and that using MSC to stably deliver high levels of FVIII into the circulation may represent a novel means of clinically correcting HA patients with pre- existing inhibitors. Therefore we propose to: 1) Determine the sites and duration of engraftment of FVIII- producing autologous MSC, and test the ability of this therapy to mediate clinical/phenotypic improvement in sheep recipients with and without pre-existing inhibitors; 2) Test whether the immunomodulatory properties of MSC will enable autologous cells to present FVIII to the FVIII-naïve recipient in a tolerogenic fashion, such that these animals will be inhibitor-free for life; and 3) Investigate whether the continued delivery of FVIII through this MSC-based approach can be used as a novel means of inducing immune tolerance in animals with pre- existing inhibitors, and compare this method with current immune tolerance induction protocols. We hope that these studies will provide the necessary knowledge for the development of a clinically viable therapeutic strategy to treat/cure severe HA and overcome immunological hurdles in patients beset with FVIII inhibitors.