Lorico Aurelio, Bratbak Daniel, Meyer Johann, Kunke David, Krauss Stefan, Plott Walter E, Solodushko Victor, Baum Christopher, Fodstad Oystein, Rappa Germana
Department of Tumor Biology, Norwegian Radium Hospital, Montebello, Oslo 0310, Norway.
Hum Gene Ther. 2005 Jun;16(6):711-24. doi: 10.1089/hum.2005.16.711.
In most experimental gene therapy protocols involving stem/progenitor cells, only a small fraction of cells, often therapeutically inadequate, can be transduced and made to express the therapeutic gene. A promising strategy for overcoming this problem is the use of a dominant selection marker, such as a drug resistance gene. In this paper, we explore the potential of the heavy subunit of gamma-glutamylcysteine synthetase (gamma-GCSh) to act as a selection marker. We found that 3T3 fibroblasts transduced with the bicistronic retroviral vector SF91/GCSh-eGFP, encoding gamma-GCSh and the enhanced green fluorescent protein (eGFP), were highly resistant to L-buthionine-(S,R)-sulfoximine (BSO), a gamma-GCS inhibitor with a low clinical toxicity profile. The level of resistance was not proportional to the increase in intracellular glutathione. In fact, cells overexpressing both heavy and light gamma-GCS subunits had higher intracellular GSH levels, and a lower level of resistance to the cytotoxic activity of BSO, compared with cells overexpressing gamma-GCSh alone. 3T3 fibroblasts overexpressing gamma-GCSh could be selected from cultures containing both naive and gene-modified cells by application of exogenous BSO selection pressure for 4 days. Also, primary neural stem/progenitor cells derived from the lateral ventricles of mouse neonatal brains and primary hematopoietic stem/progenitor cells (HSCs/HPCs) from mouse bone marrow, transduced with the gamma-GCSh-eGFP vector, could be selected by BSO treatment in vitro. On ex vivo BSO selection and reimplantation into a syngeneic myeloablated host, donor HSCs/HPCs repopulated the marrow and continued to express the transgene(s). These results provide proof of principle that somatic stem/progenitor cells, transduced simultaneously with a potentially curative gene and gamma-GCSh, can be selected by treatment with BSO before in vivo transplantation.
在大多数涉及干细胞/祖细胞的实验性基因治疗方案中，只有一小部分细胞（通常在治疗上并不充分）能够被转导并表达治疗性基因。克服这一问题的一个有前景的策略是使用显性选择标记，例如耐药基因。在本文中，我们探究了γ-谷氨酰半胱氨酸合成酶重亚基（γ-GCSh）作为选择标记的潜力。我们发现，用编码γ-GCSh和增强型绿色荧光蛋白（eGFP）的双顺反子逆转录病毒载体SF91/GCSh-eGFP转导的3T3成纤维细胞，对L-丁硫氨酸-（S，R）-亚砜亚胺（BSO）具有高度抗性，BSO是一种临床毒性较低的γ-GCS抑制剂。抗性水平与细胞内谷胱甘肽的增加不成正比。事实上，与单独过表达γ-GCSh的细胞相比，同时过表达γ-GCS重亚基和轻亚基的细胞具有更高的细胞内谷胱甘肽水平，但对BSO细胞毒性活性的抗性水平较低。通过施加外源BSO选择压力4天，可以从含有未处理细胞和基因修饰细胞的培养物中筛选出过表达γ-GCSh的3T3成纤维细胞。此外，用γ-GCSh-eGFP载体转导的源自新生小鼠脑侧脑室的原代神经干细胞/祖细胞和来自小鼠骨髓的原代造血干细胞/祖细胞（HSCs/HPCs），可以在体外通过BSO处理进行筛选。在体外进行BSO选择并重新植入同基因骨髓清除的宿主后，供体HSCs/HPCs重新填充骨髓并继续表达转基因。这些结果提供了原理证明，即与潜在治疗性基因和γ-GCSh同时转导的体细胞干细胞/祖细胞，在体内移植前可以通过BSO处理进行筛选。
