Targeted expression of factor VIII in liver sinusoidal endothelial cells for gene therapy for hemophilia A

摘要 血友病A(HA)是一种X连锁的出血性疾病，由凝血因子VIII(FVIII)缺乏引起。 旨在轻微提高凝血因子水平的治疗与实质性改善有关 严重疾病的表型。目前治疗HA的方法是蛋白质替代疗法，然而， 这种疾病的治疗格局正在迅速改变。腺相关病毒的基因治疗方法 (AAV)载体递送FVIII正在进行临床试验。有趣的是，这些临床研究针对的是FVIII的表达 使用肝细胞特异性启动子元件结合到肝细胞，这是基于FVIII是 在肝细胞中合成。然而，FVIII合成的主要部位被确定为肝窦。 内皮细胞(LSEC)。而临床前和临床研究表明，肝细胞可以合成 功能性FVIII，在肝细胞靶向表达后产生高水平的FVIII一直是一个挑战。在……里面 在正在进行的甲型肝炎病毒临床研究中，出现了几个意想不到的观察结果。第一次成功 患者最多(13名受试者)、随访时间最长(3年，正在进行)的AAV-hFVIII试验观察到 FVIII的表达显著降低。到目前为止，来自三个试验的所有可用的临床数据都显示缺乏 在受试者中具有显著变异性的媒介剂量反应。重要的是，这些发现并不是 在AAV-FIX临床研究中观察到的结果表明，交付和 FVIII的表达。临床研究中这些意想不到的发现可能与FVIII的位置有关 综合。因此，尽管HA的基因治疗已经克服了重大障碍，但尚未探索的机会 对于改善的血友病患者的结果仍然存在。这项提案的目标是针对FVIII表达 应用AAV载体研究LSECs和LSECs中FVIII的表达是否存在生物学差异 并研究AAV-FVIII对LSECs的作用。FVIII的表达将针对LSEC 利用新的启动子元件(特异性目标1)，新的AAV衣壳更特异地靶向这些AAV 将鉴定细胞(特定目标2)以及肝细胞和LSEC之间的生物学差异 将调查AAV交付后的表达情况(具体目标3)。总之，这些研究将提供基础 为了解LSEC靶向AAV-FVIII表达以支持发育的生物学和有效性 血友病A的这种治疗方法。

ABSTRACT Hemophilia A (HA) is an X-linked bleeding disorder caused by a deficiency in coagulation factor VIII (FVIII). Therapies aimed at even modest increases in clotting factor levels are associated with substantial improvement of the severe disease phenotype. The current treatment for HA is protein replacement therapy, however, the therapeutic landscape is rapidly changing for this disorder. Gene therapy approaches for adeno-associated viral (AAV) vector delivery of FVIII are in clinical trials. Interestingly, these clinical studies target expression of FVIII to hepatocytes using hepatocyte specific promoter elements which is based on the assumption that FVIII is synthesized in hepatocytes. However, the primary site of FVIII synthesis was identified as the liver sinusoidal endothelial cell (LSEC). While preclinical and clinical studies have demonstrated that hepatocytes can synthesize functional FVIII, it has been challenging to produce high levels of FVIII after hepatocyte targeted expression. In the ongoing AAV clinical studies for HA, there have been several unexpected observations. The first successful AAV-hFVIII trial with the most patients (>13 subjects) and the longest follow-up (>3 years, ongoing) observed a significant decrease in FVIII expression. All of the available clinical data from three trials to date shows a lack of a vector dose response with significant variability among subjects. Importantly, these findings were not observed in the AAV-FIX clinical studies suggesting that there is additional complexity to the delivery and expression of FVIII. These unanticipated findings in the clinical studies may be related to the site of FVIII synthesis. Thus, while significant hurdles have been overcome in gene therapy for HA, unexplored opportunities for improved hemophilia patient outcomes remain. The goal of this proposal is to target FVIII expression to LSECs using AAV vectors to study if there are biological differences in expressing FVIII in LSECs and hepatocytes and to study the efficacy of AAV-FVIII delivery to LSECs. FVIII expression will be targeted to LSECs using novel promoter elements (Specific Aim 1), novel AAV capsids that more specifically target AAV to these cells will be identified (Specific Aim 2) and the biological differences between hepatocyte and LSEC derived FVIII expression after AAV delivery will be investigated (Specific Aim 3). Together, these studies will provide the basis for understanding the biology and efficacy of LSEC targeted AAV-FVIII expression to support the development of this therapeutic approach for hemophilia A.