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

摘要 血友病A（HA）是一种由凝血因子VIII（FVIII）缺乏引起的X连锁出血性疾病。 即使是适度增加凝血因子水平的治疗也与实质性改善有关 严重疾病的表型。目前HA的治疗是蛋白质替代疗法，然而， 这种疾病的治疗前景正在迅速改变。腺相关病毒的基因治疗方法 (AAV)FVIII的载体递送处于临床试验中。有趣的是，这些临床研究靶向FVIII的表达， 使用肝细胞特异性启动子元件，其基于如下假设： 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）。虽然临床前和临床研究已经证明肝细胞可以合成 由于FVIII的功能性，在肝细胞靶向表达后产生高水平的FVIII一直具有挑战性。在 在正在进行的HA的AAV临床研究中，有几个意想不到的观察结果。首次成功 AAV-hFVIII试验中，患者最多（>13例受试者），随访时间最长（>3年，正在进行中）， FVIII表达显著降低。迄今为止，三项试验的所有可用临床数据显示， 受试者之间具有显著变异性的载体剂量反应。重要的是，这些发现不是 在AAV-FIX临床研究中观察到，这表明递送存在额外的复杂性， FVIII的表达。临床研究中的这些非预期结果可能与FVIII部位相关 合成.因此，虽然HA基因治疗中的重大障碍已经克服，但未探索的机会 对于改善血友病患者的结果仍然存在。该提案的目标是靶向FVIII表达， 使用AAV载体研究LSEC中表达FVIII是否存在生物学差异， 研究AAV-FVIII递送至LSEC的功效。FVIII表达将靶向LSEC 使用新的启动子元件（特异性Aim 1），更特异性地将AAV靶向这些启动子元件的新的AAV衣壳， 将鉴别肝细胞（具体目标2）和肝细胞与LSEC衍生的FVIII之间的生物学差异 将研究AAV递送后的表达（具体目标3）。总之，这些研究将为 了解LSEC靶向AAV-FVII表达的生物学和功效，以支持开发 血友病A的治疗方法。

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.