Prenatal Cell and Gene Therapy for Hemophilia A

项目摘要 使用超声引导的微创方法进行子宫内干细胞移植(IUTx)已被证实 在大约50名人类患者中对14种不同的遗传疾病进行了测试，证明了这一过程对 对胎儿和母亲都有风险。血友病A(HA)是一种理想的IUTx治疗疾病，因为75% 的甲型肝炎病例可以通过产前筛查得到诊断。IUTx可以治愈，或者至少可以转化为严重的 HA转变为中度/轻度表型，显著减少了对FVIII输血的需要。重要的是，暴露在 载体编码的FVIII在早期免疫发育过程中会诱导耐受FVIII并避免形成 在抑制剂中，HA治疗中最严重和最具威胁的并发症。事实上，最近的研究表明， 无论是严重的还是非严重的HA患者，FVIII抑制剂的发展都与 有死亡的危险。使用相同的临床前动物模型，这允许描述在 临床IUTx，我们发现产前输注转导的骨髓基质祖细胞(MSPs) 导致强大的长期，多器官整合到实质和血管系统，长期释放 分泌的转基因产物进入循环，并发展免疫耐受。使用相同的 羊水来源的基质祖细胞(AFP)IUTx后，获得了类似的结果。 利用一种绵羊品系，该品系模仿重症肺炎的遗传、抑制剂形成和临床症状 人类HA的形式，我们证明了父亲(半相合)MSPs的出生后移植是工程的 表达高水平的FVIII导致两只儿科HA绵羊完成表型纠正，逆转 现有的半关节，并恢复正常的身体活动。然而，就像在人类体内输注FVIII一样 患者，这两只羊在出生后治疗后出现了FVIII抑制剂。因此，这项工作提供了 令人信服的证据表明，表达FVIII的基质细胞移植可以治愈HA，如果 可以避免抑制剂的形成。因此，我们假设使用基于IUTx的策略来治疗HA 将克服因子替代疗法后观察到的两大缺点和 目前治疗HA的基因治疗方法，即缺乏持续的FVIII表达和免疫学 对治疗性蛋白质的反应。我们建议：1)确定理想的细胞来源，以获得最高 长期植入水平和体内分布模式，使FVIII最大限度地释放到 循环；2)使用HA绵羊演示使用最优细胞源和FVIII的IUTx方法 转基因构建，可以治愈，或至少永久地将重度HA转变为轻度表型；3)测试 接受IUTx是否排除了出生后输注FVIII后抑制物的诱导和/或诱导了耐受 即使在出生后用FVIII佐剂进行挑战后，这种情况仍然存在。完成后，这些研究将证明 使用细胞作为FVIII传递平台的安全性和有效性，并展示IUTx治愈或 改善HA表型，克服目前阻碍临床HA治疗的免疫相关障碍。

Project Summary Intrauterine stem cell transplantation (IUTx) using a minimally invasive, ultrasound-guided approach has been performed in ~50 human patients for 14 different genetic disorders, proving that this procedure poses minimal risk to both the fetus and the mother. Hemophilia A (HA) is an ideal disease to be treated by IUTx, since 75% of HA cases can be diagnosed through prenatal screening. IUTx could be curative, or at least convert severe HA to a moderate/mild phenotype, significantly reducing the need for FVIII infusions. Importantly, exposure to vector-encoded FVIII during early immunologic development would induce tolerance FVIII and avoid formation of inhibitors, the most serious and threatening complication in HA treatment. Indeed, recent studies showed in both severe and non-severe HA patients, that the development of FVIII inhibitors is associated with increased risk of death. Using the same preclinical animal model, that allowed the delineation of the conditions used in clinical IUTx, we showed that prenatal infusion of transduced bone marrow stromal progenitor cells (MSPs) resulted in robust long-term, multi-organ integration into both parenchyma and vasculature, long-term release of secreted transgene products into the circulation, and development of immune tolerance. Using the same sheep model, similar results were obtained after IUTx of amniotic fluid-derived stromal progenitors (AFPs). Utilizing a line of sheep that emulates the genetics, inhibitor formation, and clinical symptoms of the severe form of human HA, we showed that the postnatal transplantation of paternal (haploidentical) MSPs 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. However, like with FVIII infusions in human patients, these two sheep developed FVIII inhibitors following postnatal treatment. This work thus provides compelling evidence that the transplantation of FVIII-expressing stromal cells could be curative for HA, if inhibitor formation could be avoided. Therefore, we hypothesize that using an IUTx-based strategy to treat HA will overcome the two major shortcomings that have been observed following factor replacement therapy and current gene therapy approaches to treat HA, namely a lack of sustained FVIII expression and immunologic responses to the therapeutic protein. We propose to: 1) determine the ideal cell source to obtain the highest long-term engraftment levels and the in vivo distribution patterns that maximize release of FVIII into the circulation; 2) use HA sheep to demonstrate that an IUTx approach using the optimal cell source and FVIII transgene construct, can be curative, or at least permanently convert severe HA to a mild phenotype; 3) test whether receiving IUTx precludes inhibitor induction following postnatal FVIII infusion and/or induces tolerance that persists even after postnatal challenge with FVIII in adjuvant. Upon completion, these studies will prove the safety and efficacy of using cells as a FVIII delivery platform, and demonstrate the ability of IUTx to cure or improve HA phenotype, and defeat the immune-related hurdles that currently hinder clinical HA treatment.