In vivo repopulation ability of genetically corrected bone marrow cells from Fanconi anemia patients
In vivo repopulation ability of genetically corrected bone marrow cells from Fanconi anemia patients
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DOI：
10.1073/pnas.0510613103
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发表时间：
2006-02-14
影响因子：
11.1
通讯作者：
Marty, M
中科院分区：
文献类型：
--
作者：
Cohen-Haguenauer, O;Péault, B;Marty, M
范可尼贫血（FA）是一种罕见的遗传性基因组不稳定综合征，是造血干细胞缺乏的最佳例子之一。虽然FA可能是一个很好的候选人骨髓（BM）遗传校正离体，敲除动物模型是不够的，以指导临床前的步骤，基因治疗的尝试已被证明是令人失望的。造成这些不良结果的原因是当置于培养物中时，BM细胞的特征性和显著的早期死亡。我们在这里表明，人类原代FA BM细胞的存活率可以通过使用限制氧化应激的特定培养条件来改善。再加上逆转录病毒介导的转移的主要互补组FANCA-cDNA，我们可以实现长期重建的干细胞室在体外和体内。基因校正的BM培养物生长>120天，并且在培养的细胞移植到NOD/SCID小鼠中后，在转导后6个月可以检测到携带FANCA转基因的克隆原性人细胞。相比之下，未转导的细胞在培养15天后死亡。出于伦理原因的必要性，在非常有限数量的原代BM细胞上进行实验。通过使用低细胞因子方案和符合监管要求的条件，基因校正的细胞的特遣队慢慢出现，体内植入的潜力未得到满足。干细胞的未来治疗应用可能会从这些数据中扩展。此外，我们提供了一个基因校正的人类原代细胞生长模型，该模型具有更好地描述DNA损伤和氧化应激在FA发病机制中的联合作用的潜力。
Fanconi anemia (FA) is a rare inherited genomic instability syndrome representing one of the best examples of hematopoietic stem cell deficiency. Although FA might be an excellent candidate for bone marrow (BM) genetic correction ex vivo, knockout animal models are not sufficient to guide preclinical steps, and gene therapy attempts have proven disappointing so far. Contributing to these poor results is a characteristic and dramatic early BM-cells die-off when placed in culture. We show here that human primary FA BM cell survival can be ameliorated by using specific culture conditions that limit oxidative stress. When coupled with retrovirus-mediated transfer of the main complementation group FANCA-cDNA, we could achieve long-term reconstitution of the stem cell compartment both in vitro and in vivo. Gene-corrected BM cultures grew for >120 days, and after cultured cell transplantation into NOD/SCID mice, clonogenic human cells carrying the FANCA transgene could be detected 6 months after transduction. By comparison, untransduced cells died in culture by 15 days. Of necessity for ethical reasons, experiments were conducted on a very limited number of primary BM cells. By using low cytokine regimen and conditions matching regulatory requirements, a contingent of gene-corrected cells slowly emerges with an unmet potential for in vivo engraftment. Future therapeutic applications of stem cells might be expanding from these data. In addition, we provide a model of gene-corrected human primary cell growth that carries the potential to better delineate the combined role of both DNA damage and oxidative stress in the pathogenesis of FA.