Nine dogs with hemophilia A were treated with adeno-associated viral (AAV) gene therapy and followed for up to 10 years. Administration of AAV8 or AAV9 vectors expressing canine factor VIII (AAV-cFVIII) corrected the FVIII deficiency to 1.9–11.3% of normal FVIII levels. In two of nine dogs, levels of FVIII activity increased gradually starting about 4 years after treatment. None of the dogs showed evidence of tumors or altered liver function. Analysis of integration sites in liver samples from six treated dogs identified 1,741 unique AAV integration events in genomic DNA and expanded cell clones in five dogs, with 44% of the integrations near genes involved in cell growth. All recovered integrated vectors were partially deleted and/or rearranged. Our data suggest that the increase in FVIII protein expression in two dogs may have been due to clonal expansion of cells harboring integrated vectors. These results support the clinical development of liver-directed AAV gene therapy for hemophilia A, while emphasizing the importance of long-term monitoring for potential genotoxicity.

九只患有 A 型血友病的狗接受了腺相关病毒 (AAV) 基因治疗，并进行了长达 10 年的随访。施用表达犬因子 VIII (AAV-cFVIII) 的 AAV8 或 AAV9 载体可将 FVIII 缺陷纠正至正常 FVIII 水平的 1.9–11.3%。治疗后约 4 年开始，九只狗中有两只的 FVIII 活性水平逐渐升高。没有一只狗表现出肿瘤或肝功能改变的证据。对 6 只接受治疗的狗的肝脏样本中的整合位点进行分析，在 5 只狗的基因组 DNA 和扩展的细胞克隆中发现了 1,741 个独特的 AAV 整合事件，其中 44% 的整合位于涉及细胞生长的基因附近。所有恢复的整合载体均被部分删除和/或重新排列。我们的数据表明，两只狗中 FVIII 蛋白表达的增加可能是由于含有整合载体的细胞的克隆扩增。这些结果支持针对甲型血友病的肝脏定向 AAV 基因治疗的临床开发，同时强调长期监测潜在基因毒性的重要性。