Liu J M, Young N S, Walsh C E, Cottler-Fox M, Carter C, Dunbar C, Barrett A J, Emmons R
Hum Gene Ther. 1997 Sep 20;8(14):1715-30. doi: 10.1089/hum.1997.8.14-1715.
Fanconi Anemia (FA) is a rare genetic disorder characterized by progress pancytopenia, congenial abnormalities, and a predisposition to malignancy. Therapy is currently limited to allogeneic marrow transplantation; patients lacking a suitable donor usually die from aplasia or acute leukemia. Recently, mutation in a novel gene named FACC (Fanconi anemia C-complementing) has been identified as causing one type of FA. FACC mutations, which introduce splicing errors or stop codons, have been identified in approximately 15% of FA patients. We have recently been successful in functional complementation of four FA cell lines using retroviral vectors to transfer a copy of the normal FACC gene. We also analyzed the ability of our viral vectors to functionally correct hematopoietic progenitor cells from a patient bearing a splice donor mutation. As for the lymphoid cell lines, these CD34-enriched cells were extremely sensitive to MMC. After infection of these progenitor cells with viral vectors bearing normal FACC, the progenitors gave rise to increased numbers of colonies both in the absence and presence of up to 5nM MMC, whereas control cells were completely destroyed by 1nM MMC. In summary, we have demonstrated that: (1) retroviral vectors can be engineered to transfer a normal FACC gene to FA(C) lymphoid cell lines and primary hematopoietic cells; (2) introduction of a normal FACC gene into CD34+ progenitors markedly enhances their growth in the absence and presence of MMC. This study is designed to determine whether hematopoietic progenitors transduced with the normal FACC gene can be reinfused safely into FA(C) patients. CD34+ cells obtained from G-CSF mobilized peripheral blood will be transduced ex vivo over a 72-hour period in the presence of IL-3, IL-6, and Stem Cell Factor with the FACC retroviral vector. These transduced cells will be reinfused into FA(C) patients. Patients will be monitored for toxicities as well as evidence of successful gene transfer and expression. The procedure will be repeated up to a total of 4 times with each treatment 2-4 months apart. Theoretically, these rescued stem cells should have a selective growth advantage within the hypoplastic FA marrow environment in vivo.
范可尼贫血（FA）是一种罕见的遗传性疾病，其特征为进行性全血细胞减少、先天性异常以及易患恶性肿瘤。目前的治疗方法仅限于异基因骨髓移植；缺乏合适供体的患者通常死于再生障碍或急性白血病。最近，一种名为FACC（范可尼贫血C互补）的新基因中的突变已被确定为导致一种类型的FA的原因。在大约15%的FA患者中已发现FACC突变，这些突变会引入剪接错误或终止密码子。我们最近成功地使用逆转录病毒载体对四种FA细胞系进行了功能互补，以转移正常FACC基因的一个拷贝。我们还分析了我们的病毒载体对一名携带剪接受体突变患者的造血祖细胞进行功能校正的能力。至于淋巴样细胞系，这些富含CD34的细胞对丝裂霉素C（MMC）极其敏感。用携带正常FACC的病毒载体感染这些祖细胞后，无论有无高达5nM的MMC，祖细胞产生的集落数量都增加了，而对照细胞在1nM MMC作用下则完全被破坏。总之，我们已经证明：（1）可以构建逆转录病毒载体，将正常FACC基因转移到FA（C）淋巴样细胞系和原代造血细胞中；（2）将正常FACC基因引入CD34+祖细胞可显著增强其在有无MMC情况下的生长。本研究旨在确定用正常FACC基因转导的造血祖细胞是否能安全地回输到FA（C）患者体内。从粒细胞集落刺激因子（G-CSF）动员的外周血中获得的CD34+细胞将在白细胞介素-3（IL-3）、白细胞介素-6（IL-6）和干细胞因子存在的情况下，在72小时内用FACC逆转录病毒载体进行体外转导。这些转导的细胞将回输到FA（C）患者体内。将对患者进行毒性监测以及基因转移和表达成功的证据监测。该程序将总共重复进行4次，每次治疗间隔2至4个月。从理论上讲，这些挽救的干细胞在体内发育不全的FA骨髓环境中应具有选择性生长优势。
