A functionally active retrovirus vector for gene therapy in Fanconi anemia group C.
A functionally active retrovirus vector for gene therapy in Fanconi anemia group C.
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用于范可尼贫血 C 组基因治疗的功能活性逆转录病毒载体。
DOI：
10.1182/blood.v84.2.453.bloodjournal842453
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发表时间：
1994
期刊：
影响因子：
20.3
通讯作者：
J. Liu
中科院分区：
文献类型：
--
作者：
C. Walsh;M. Grompe;E. Vanin;M. Buchwald;N. Young;A. Nienhuis;J. Liu
范可尼贫血（FA）是一种罕见的遗传性疾病，其特征是进行性全血细胞减少，先天性异常，并易患恶性肿瘤。最近，一个新的基因FACC（Fanconi贫血C互补）的突变已被确定为导致一种类型的FA。在这里，我们报告成功的功能互补的四个FA（C）细胞系使用逆转录病毒载体转移正常FACC基因的拷贝。FA细胞表型的标志是对交联剂如丝裂霉素C（MMC）的极端敏感性。由FACC病毒载体转导的细胞系的特征在于它们在MMC浓度下生长的能力，所述MMC浓度比亲本对照的抑制浓度高几个数量级。分析遗传校正的细胞系对MMC诱导的染色体断裂的敏感性，发现其已正常化。这两种不同的测定证实了我们的逆转录病毒载体能够将功能性FACC基因转移到从FA（C）患者建立的淋巴细胞系中。我们接下来分析了我们的病毒载体在功能上校正来自携带剪接供体突变的患者的造血祖细胞的能力。通过免疫亲和柱纯化祖细胞以富集具有高CD 34表达的细胞。与FA淋巴样细胞系相似，该患者的CD 34富集细胞对MMC极其敏感。用携带正常FACC的病毒载体感染这些祖细胞后，在不存在和存在≤ 5 nmol/L MMC的情况下形成的集落数量增加，但即使在不存在MMC的情况下，未感染的细胞也没有形成集落。聚合酶链扩增用于确认前病毒DNA整合。因此，逆转录病毒载体可以被工程化以将正常的FACC基因转移到携带四种不同FACC突变的淋巴样细胞系和原代造血细胞。通过基因转导拯救的FA干细胞应该在体内发育不良的FA骨髓环境中具有选择性生长优势。这些实验表明，基因治疗可能是FA的有效治疗策略。
Fanconi anemia (FA) is a rare genetic disorder characterized by progressive pancytopenia, congenital abnormalities, and a predisposition to malignancy. Recently, mutation in a novel gene named FACC (Fanconi anemia C complementing) has been identified as causing one type of FA. Here, we report successful functional complementation of four FA(C) cell lines using a retroviral vector to transfer a copy of the normal FACC gene. The hallmark of the FA cell phenotype is extreme sensitivity to cross-linking agents such as mitomycin C (MMC). Cell lines transduced by FACC viral vectors were distinguished by their ability to grow at concentrations of MMC several orders of magnitude higher than those concentrations inhibitory of parental controls. The genetically corrected cell lines were analyzed for susceptibility to MMC-induced chromosomal breakage and were found to have been normalized. These two different assays confirmed that our retroviral vectors were capable of transferring a functional FACC gene to lymphoid cell lines established from FA(C) patients. We next analyzed the ability of our viral vectors to functionally correct hematopoietic progenitor cells from a patient bearing a splice donor mutation. Progenitor cells were purified by an immunoaffinity column to enrich for cells with high CD34 expression. Similar to FA lymphoid cell lines, this patient's CD34-enriched cells were extremely sensitive to MMC. After infection of these progenitor cells with viral vectors bearing normal FACC, increased numbers of colonies formed both in the absence and presence of < or = 5 nmol/L MMC, but no colonies formed from uninfected cells, even in the absence of MMC. Polymerase chain amplification was used to confirm proviral DNA integration. Thus, retroviral vectors can be engineered to transfer a normal FACC gene to lymphoid cell lines and primary hematopoietic cells bearing four different FACC mutations. FA stem cells rescued by gene transduction should have a selective growth advantage within the hypoplastic FA marrow environment in vivo. These experiments suggest that gene therapy may be an effective treatment strategy for FA.
范可尼贫血基因FACC的突变分析。
影响因子：
9.8
作者：
Verlander,PC;Lin,JD;Udono,MU;Zhang,Q;Gibson,RA;Mathew,CG;Auerbach,AD
通讯作者：
Auerbach,AD