Abe T, Ito M, Okamoto Y, Kim H J, Takaue Y, Yasutomo K, Makimoto A, Yamaue T, Kawano Y, Watanabe T, Shimada T, Kuroda Y
Department of Pediatrics, University of Tokushima, Japan.
Exp Hematol. 1997 Aug;25(9):966-71.
To help establish an effective gene therapy protocol for patients with congenital metabolic diseases, we evaluated retrovirus-mediated transduction and long-term (LT) expression of the NeoR gene in cryopreserved and thawed CD34+ cells purified from granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (PB) of infant and cord blood (CB). The results were compared with those in bone marrow (BM) CD34+ cells. The final purity of the CD34+-enriched fraction from PB, CB, and BM, based on FACS analysis, was 88 +/- 14%, 73 +/- 13%, and 68 +/- 19% (mean +/- SEM), respectively. Cells were then cultured for 96 hours with supernatant containing the vector in the presence of interleukin (IL)-3, IL-6, and stem cell factor (SCF). The average efficiency of gene transfer into mobilized PB (n = 5) or CB CD34+ cells (n = 6) was significantly higher than that into BM CD34+ cells, as measured by G418-resistant colony-forming units for granulocyte/macrophage (CFU-GM; 59% or 58% vs. 39%; p < 0.05) and PCR-positive CFU-GM (83% or 79% vs. 53%; p < 0.05). When the evaluation was made in an LT culture system with irradiated allogeneic marrow stroma, these efficiencies were, respectively, 74% or 61% vs. 34% (p < 0.005 or < 0.02) for G418-resistant CFU-GM at week 5 of long-term culture, and 88% or 83% vs. 63% (p < 0.05) for PCR-positive CFU-GM. Fluorometric examination was performed for cell-cycle analysis before and after culture, and the results showed that the fraction of cycling cells was largest in freshly prepared BM (18%), whereas only a small portion of PB (4.6%) and CB (2%) was cycling. However, this value was 17% in BM, 22% in PB, and 13% in CB after culture. These results suggest that mobilized PB from small children and CB cells are suitable and realistic targets for clinical gene therapy and that tandem transduction procedures can be achieved by combining CB and PB.
为了帮助建立针对先天性代谢疾病患者的有效基因治疗方案，我们评估了逆转录病毒介导的新霉素抗性基因（NeoR）在从婴儿粒细胞集落刺激因子（G-CSF）动员的外周血（PB）和脐血（CB）中纯化并冻存和解冻的CD34+细胞中的转导及长期（LT）表达情况。将结果与骨髓（BM）CD34+细胞中的结果进行比较。基于流式细胞术分析，来自PB、CB和BM的CD34+富集组分的最终纯度分别为88±14%、73±13%和68±19%（平均值±标准误）。然后，在白细胞介素（IL）-3、IL-6和干细胞因子（SCF）存在的情况下，将细胞与含有载体的上清液一起培养96小时。通过粒细胞/巨噬细胞的G418抗性集落形成单位（CFU-GM）测量，基因转移到动员的PB（n = 5）或CB CD34+细胞（n = 6）中的平均效率显著高于转移到BM CD34+细胞中的效率（59%或58%对39%；p < 0.05）以及PCR阳性CFU-GM（83%或79%对53%；p < 0.05）。当在具有辐照的同种异体骨髓基质的长期培养系统中进行评估时，对于长期培养第5周的G418抗性CFU-GM，这些效率分别为74%或61%对34%（p < 0.005或< 0.02），对于PCR阳性CFU-GM为88%或83%对63%（p < 0.05）。在培养前后进行荧光检测以进行细胞周期分析，结果显示，循环细胞比例在新鲜制备的BM中最大（18%），而PB（4.6%）和CB（2%）中只有一小部分处于循环状态。然而，培养后该值在BM中为17%，在PB中为22%，在CB中为13%。这些结果表明，幼儿动员的PB和CB细胞是临床基因治疗合适且现实的靶点，并且通过结合CB和PB可以实现串联转导程序。
