Flasshove M, Banerjee D, Mineishi S, Li M X, Bertino J R, Moore M A
James Ewing Laboratory of Developmental Hematopoiesis, Sloan-Kettering Institute for Cancer Research, New York, NY 10021.
Blood. 1995 Jan 15;85(2):566-74.
Retroviral gene transfer into human myeloid precursor cells allows introduction of marker genes as well as genes conferring resistance to chemotherapeutic drugs. We transduced a human mutant dihydrofolate reductase (DHFR) cDNA into CD34 antigen-positive peripheral blood cells from patients with breast or ovarian cancer obtained after treatment with chemotherapy and granulocyte colony-stimulating factor (G-CSF). This mutant DHFR has been shown to confer resistance to methotrexate (MTX) in murine bone marrow. We established a transduction protocol that permitted ex vivo expansion and selection of transduced early progenitor cells. The number of progenitor cells from transduced CD34-positive cells increased 50-fold after cytokine prestimulation with interleukin-1 (IL-1), c-kit ligand (KL; stem cell factor), and IL-3 and 2 weeks in liquid culture. Transduced colony-forming unit-granulocyte-macrophage (CFU-GM), assayed directly after the transduction procedure, were protected completely against 2 x 10(-8) mol/L MTX, a concentration that significantly reduced the CFU-GM detected in the control population. Gene transfer of the mutant DHFR led to a twofold selective advantage for a pre-CFU population after exposure to MTX in liquid culture (P < .001). Polybrene, in contrast with protamine, significantly inhibited the expansion of progenitors. The presence of proviral DNA was monitored by polymerase chain reaction (PCR) and was detected in greater than 80% of CFU-GM and ex vivo expanded pre-CFU. We have demonstrated that human hematopoietic precursor cells can be expanded extensively after retroviral gene transfer. The same population of early progenitors can be selected ex vivo with low-dose MTX. As long-term expression of transduced genes in human hematopoietic cells remains a problem in vivo, these results may have implications for future clinical trials, especially for the introduction of nonselectable genes.
将逆转录病毒基因导入人髓系前体细胞，不仅可以引入标记基因，还能导入赋予对化疗药物抗性的基因。我们将人突变型二氢叶酸还原酶（DHFR）cDNA转导至经化疗和粒细胞集落刺激因子（G-CSF）治疗后获得的乳腺癌或卵巢癌患者的CD34抗原阳性外周血细胞中。这种突变型DHFR已被证明能使小鼠骨髓对甲氨蝶呤（MTX）产生抗性。我们建立了一种转导方案，该方案允许在体外扩增和选择转导的早期祖细胞。在用白细胞介素-1（IL-1）、c-kit配体（KL；干细胞因子）和IL-3进行细胞因子预刺激并在液体培养基中培养2周后，转导的CD34阳性细胞中的祖细胞数量增加了50倍。在转导过程后直接检测的转导集落形成单位-粒细胞-巨噬细胞（CFU-GM），对2×10⁻⁸mol/L的MTX具有完全抗性，该浓度显著降低了对照群体中检测到的CFU-GM。在液体培养基中暴露于MTX后，突变型DHFR的基因转移导致前CFU群体具有两倍的选择性优势（P <.001）。与鱼精蛋白相比，聚凝胺显著抑制祖细胞的扩增。通过聚合酶链反应（PCR）监测前病毒DNA的存在，并在超过80%的CFU-GM和体外扩增的前CFU中检测到。我们已经证明，逆转录病毒基因转移后人造血前体细胞可以大量扩增。同一群体的早期祖细胞可以用低剂量MTX在体外进行选择。由于转导基因在人造血细胞中的长期表达在体内仍然是一个问题，这些结果可能对未来的临床试验有影响，特别是对于引入不可选择的基因。
