Postnatal Cell-Based Therapies for Hemophilia A

项目总结 尽管甲型血友病(HA)患者的治疗护理有了很大的发展，但仍然没有一种是治愈的，而且所有的 依赖终身、经常性、昂贵的FVIII输液(≥$600,000/年/患者)。此外, 30%的重度HA患者产生了对FVIII的抑制性抗体，这是 医管局的临床管理。因此，迫切需要开发新的疗法，能够提供更长的- 持久的/永久性的改善，和/或可以克服目前阻碍治疗的免疫障碍。这个 通过诸如骨髓基质细胞(MSC)的细胞平台输送FVIII是一种有吸引力的方法， 由于病毒转导后，MSC分泌高水平的载体编码的FVIII，这是无法区分的 从天然蛋白质中分离出来，不转化或发展为克隆显性。此外，在输液时，MSC 可以在实质和血管周围区域内的多个器官中长期驻留，将它们放置在 最理想的是将FVIII输送到循环中。MSC具有免疫调节/抗炎特性， 如果使用自体MSC，它可以使FVIII以耐受性方式传递。我们最近测试了 利用模拟抑制因子基因的绵羊品系表达FVIII的MSC的治疗潜力 形成(注射FVIII蛋白)和临床症状的严重形式的人HA，并显示 在没有受体预适应的情况下，单倍体相合的MSC的出生后给药被设计成 表达高水平的FVIII导致两只儿科HA绵羊完全纠正表型，逆转 现有的半关节，并恢复正常的身体活动。值得注意的是，这种表型修正是漫长的。 尽管在这些绵羊中存在高效价抑制物，但移植的MSC没有被清除 接受者的免疫系统，使他们能够在多个部位长期存在。这项先前的工作使我们能够 假设表达FVIII的MSC的输送导致血管周围植入和释放FVIII 蛋白质以可治愈HA的水平进入循环，并使用MSC稳定地提供高水平 进入循环的FVIII水平可能代表着一种新的手段，用于临床纠正HA患者的前驱... 现有的抑制剂。因此，我们建议：1)确定FVIII的植入地点和持续时间- 制备自体骨髓间充质干细胞，并测试该疗法在介导临床/表型改善方面的能力 有无预先存在的抑制剂的绵羊受体；2)测试是否具有免疫调节特性 MSC将使自体细胞能够以耐受性方式将FVIII呈递给FVIII-幼稚受体，从而 这些动物将终身不受抑制；以及3)调查FVIII通过 这种基于MSC的方法可以作为一种诱导免疫耐受的新方法。 现有的抑制剂，并将该方法与当前的免疫耐受诱导方案进行比较。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/治愈严重HA和克服FVIII抑制剂困扰患者的免疫障碍的策略。

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.