Hemophilia A (HA) is a monogenic disease characterized by plasma clotting factor 8 (F8) deficiency due to F8 mutation. We have been attempting to cure HA permanently using a CRISPR-Cas9 gene-editing strategy. In this study, we induced targeted integration of BDDF8 (B-domain-deleted F8) gene into the albumin locus of HA mice by hydrodynamic tail vein injection of editing plasmid vectors. One week after treatment, a high F8 activity ranging from 70% to 280% of normal serum levels was observed in all treated HA mice but dropped to background levels 3–5 weeks later. We found that the humoral immune reaction targeting F8 is the predominant cause of the decreased F8 activity. We hypothesized that hydrodynamic injection-induced liver damage triggered the release of large quantities of inflammatory cytokines. However, coinjection of plasmids expressing a dozen immunomodulatory factors failed to curtail the immune reaction and stabilize F8 activity effectively. The spCas9 plasmid carrying a miR-142-3p target sequence alleviated the cellular immune response but could not deliver therapeutic efficacy. Strikingly, immunosuppressant cyclophosphamide virtually abolished the immune response, leading to a year-long stable F8 level. Our findings should have important implications in developing therapies in mouse models using the hydrodynamic gene delivery approach, highlighting the necessity of modulating the innate immune response triggered by liver damage.

中文翻译：

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血友病 A 的流体动力学基因治疗中免疫反应的调节

血友病 A (HA) 是一种单基因疾病，其特征是由于 F8 突变导致血浆凝血因子 8 (F8) 缺乏。我们一直在尝试使用 CRISPR-Cas9 基因编辑策略永久治愈 HA。在这项研究中，我们诱导了BDDF8的靶向整合(B-结构域缺失的 F8) 基因通过流体动力学尾静脉注射编辑质粒载体进入 HA 小鼠的白蛋白基因座。治疗一周后，在所有治疗的 HA 小鼠中观察到高 F8 活性，范围为正常血清水平的 70% 至 280%，但在 3-5 周后降至背景水平。我们发现针对 F8 的体液免疫反应是 F8 活性降低的主要原因。我们假设流体动力注射诱导的肝损伤触发了大量炎性细胞因子的释放。然而，共注射表达十几种免疫调节因子的质粒未能有效地减少免疫反应并稳定 F8 活性。携带 miR-142-3p 靶序列的 spCas9 质粒减轻了细胞免疫反应，但不能提供治疗效果。引人注目的是，免疫抑制剂环磷酰胺实际上消除了免疫反应，导致长达一年的稳定 F8 水平。我们的研究结果应该对使用流体动力学基因传递方法在小鼠模型中开发治疗具有重要意义，突出了调节由肝损伤引发的先天免疫反应的必要性。