Infusing CRISPR/donor-loaded adeno-associated viral vectors (AAV/CRISPR) could enable in vivo hepatic gene editing to remedy hemophilia B (HB) with inherited deficiency of clotting factor IX (FIX). Yet, current regimens focus on correcting HB with simple mutations in the coding region of the F9, overlooking those carrying complicated mutations involving the regulatory region. Moreover, a possible adverse effect of treatment-related inflammation remains unaddressed. Here we report that a single DNA cutting-mediated long-range replacement restored the FIX-encoding function of a mutant F9 (mF9) carrying both regulatory and coding defects in a severe mouse HB model, wherein incorporation of a synthetic Alb enhancer/promoter-mimic (P2) ensured FIX elevation to clinically meaningful levels. Through single-cell RNA sequencing (scRNA-seq) of liver tissues, we revealed that a subclinical hepatic inflammation post-AAV/CRISPR administration regulated the vulnerability of the edited mF9-harboring host cells to cytotoxic T lymphocytes (CTLs) and the P2 activity in a hepatocytic subset–dependent manner via modulating specific sets of liver-enriched transcription factors (LETFs). Collectively, our study establishes an AAV/CRISPR-mediated gene-editing protocol applicable to complicated monogenetic disorders, underscoring the potentiality of improving therapeutic benefits through managing inflammation.

注入 CRISPR/供体负载的腺相关病毒载体 (AAV/CRISPR) 可以使体内肝脏基因编辑能够治疗具有遗传性凝血因子 IX (FIX) 缺陷的血友病 B (HB)。然而，目前的治疗方案侧重于纠正F9编码区中具有简单突变的 HB ，而忽略了那些携带涉及调节区的复杂突变的 HB。此外，治疗相关炎症的可能副作用仍未得到解决。在这里，我们报告说，在严重的小鼠 HB 模型中，单个 DNA 切割介导的远程替代恢复了突变F9 ( mF9 ) 的 FIX 编码功能，该模型在严重的小鼠 HB 模型中具有调节和编码缺陷，其中掺入了合成的Alb增强子/启动子模拟（P2）确保 FIX 升高到临床有意义的水平。通过肝组织的单细胞 RNA 测序 (scRNA-seq)，我们揭示了 AAV/CRISPR 给药后的亚临床肝脏炎症调节了编辑后的​​mF9宿主细胞对细胞毒性 T 淋巴细胞 (CTL) 和 P2 活性的脆弱性通过调节特定的富肝转录因子 (LETF) 组以依赖肝细胞亚群的方式进行。总的来说，我们的研究建立了适用于复杂单基因疾病的 AAV/CRISPR 介导的基因编辑协议，强调了通过管理炎症改善治疗益处的潜力。