Molecular Hematopoiesis Section, Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Hum Gene Ther. 2010 Jun;21(6):695-703. doi: 10.1089/hum.2009.191.
The risk of genotoxicity of retroviral vector-delivered gene therapy targeting hematopoietic stem cells (HSCs) has been highlighted by the development of clonal dominance and malignancies in human and animal gene therapy trials. Large-animal models have proven invaluable to test the safety of retroviral vectors, but the detection of clonal dominance may require years of follow-up. We hypothesized that hematopoietic stress may accelerate the proliferation and therefore the detection of abnormal clones in these models. We administered four monthly busulfan (Bu) infusions to induce hematopoietic stress in a healthy rhesus macaque previously transplanted with CD34+ cells transduced with retroviral vectors carrying a simple marker gene. Busulfan administration resulted in significant cytopenias with each cycle, and prolonged pancytopenia after the final cycle with eventual recovery. Before busulfan treatment there was highly polyclonal marking in all lineages. After Bu administration clonal diversity was markedly decreased in all lineages. Unexpectedly, we found no evidence of selection of the MDS1/EVI1 clones present before Bu administration, but a clone with a vector integration in intron 1 of the histone deacetylase-7 (HDAC7) gene became dominant in granulocytes over time after Bu administration. The overall marking level in the animal was increased significantly after Bu treatment and coincident with expansion of the HDAC7 clone, suggesting an in vivo advantage for this clone under stress. HDAC7 expression was upregulated in marrow progenitors containing the vector. Almost 5 years after Bu administration, the animal developed progressive cytopenias, and at autopsy the marrow showed complete lack of neutrophil or platelet maturation, with a new population of approximately 20% undifferentiated blasts. These data suggest that chemotherapeutic stress may accelerate vector-related clonal dominance, even in the absence of drug resistance genes expressed by the vector. This model may both accelerate the detection of abnormal clones to facilitate analysis of genotoxicity for human gene therapy, and help assess the safety of administering myelotoxic chemotherapeutic agents in patients previously engrafted with vector-containing cells.
逆转录病毒载体靶向造血干细胞（HSCs）的基因治疗的遗传毒性风险已被人类和动物基因治疗试验中克隆优势和恶性肿瘤的发展所凸显。大型动物模型已被证明对于测试逆转录病毒载体的安全性非常有价值，但克隆优势的检测可能需要数年的随访。我们假设造血应激可能会加速这些模型中异常克隆的增殖，从而更容易检测到它们。我们给一只先前接受过用携带简单标记基因的逆转录病毒载体转导的 CD34+细胞移植的健康恒河猴进行了四次每月一次的白消安（Bu）输注，以诱导造血应激。每个周期的 Bu 给药都会导致明显的细胞减少，最后一个周期后出现长期的全血细胞减少，最终恢复。在 Bu 治疗前，所有谱系中都存在高度多克隆标记。Bu 给药后，所有谱系中的克隆多样性明显降低。出乎意料的是，我们没有发现 Bu 给药前存在的 MDS1/EVI1 克隆被选择的证据，但在 Bu 给药后，一个在组蛋白去乙酰化酶-7（HDAC7）基因内含子 1 中整合载体的克隆在粒细胞中逐渐占优势。Bu 治疗后，动物的总体标记水平显著升高，同时 HDAC7 克隆扩张，表明该克隆在应激下具有体内优势。骨髓祖细胞中含有载体的 HDAC7 表达上调。Bu 给药后近 5 年，动物出现进行性细胞减少症，尸检时骨髓显示完全缺乏中性粒细胞或血小板成熟，出现大约 20%的未分化原始细胞的新群体。这些数据表明，化疗应激可能会加速与载体相关的克隆优势，即使载体不表达耐药基因。该模型既可以加速异常克隆的检测，以促进人类基因治疗的遗传毒性分析，又可以帮助评估在先前植入含有载体细胞的患者中给予骨髓抑制化疗药物的安全性。
