Mascarenhas L, Stripecke R, Case S S, Xu D, Weinberg K I, Kohn D B
Divisions of Research Immunology/Bone Marrow Transplantation and Hematology/ Oncology, Department of Pediatrics, University of Southern California School of Medicine, Childrens Hospital Los Angeles, Los Angeles, CA, USA.
Blood. 1998 Nov 15;92(10):3537-45.
Autologous leukemia cells engineered to express immune-stimulating molecules may be used to elicit antileukemia immune responses. Gene delivery to human B-precursor acute lymphoblastic leukemia (ALL) cells was investigated using the enhanced green fluorescent protein (EGFP) as a reporter gene, measured by flow cytometry. Transfection of the Nalm-6 and Reh B-precursor ALL leukemia cell lines with an expression plasmid was investigated using lipofection, electroporation, and a polycationic compound. Only the liposomal compound Cellfectin showed significant gene transfer (3.9% to 12% for Nalm-6 cells and 3.1% to 5% for Reh cells). Transduction with gibbon-ape leukemia virus pseudotyped Moloney murine leukemia virus (MoMuLV)-based retrovirus vectors was investigated in various settings. Cocultivation of ALL cell lines with packaging cell lines showed the highest transduction efficiency for retroviral gene transfer (40.1% to 87.5% for Nalm-6 cells and 0.3% to 9% for Reh cells), followed by transduction with viral supernatant on the recombinant fibronectin fragment CH-296 (13% to 35.5% for Nalm-6 cells and 0.4% to 6% Reh cells), transduction on human bone marrow stroma monolayers (3.2% to 13.3% for Nalm-6 cells and 0% to 0.2% Reh cells), and in suspension with protamine sulfate (0.7% to 3.1% for Nalm-6 cells and 0% for Reh cells). Transduction of both Nalm-6 and Reh cells with human immunodeficiency virus-type 1 (HIV-1)-based lentiviral vectors pseudotyped with the vesicular stomatitis virus-G envelope produced the best gene transfer efficiency, transducing greater than 90% of both cell lines. Gene delivery into primary human B-precursor ALL cells from patients was then investigated using MoMuLV-based retrovirus vectors and HIV-1-based lentivirus vectors. Both vectors transduced the primary B-precursor ALL cells with high efficiencies. These studies may be applied for investigating gene delivery into primary human B-precursor ALL cells to be used for immunotherapy.
经过基因工程改造以表达免疫刺激分子的自体白血病细胞可用于引发抗白血病免疫反应。使用增强型绿色荧光蛋白（EGFP）作为报告基因，通过流式细胞术检测，研究了将基因导入人B前体急性淋巴细胞白血病（ALL）细胞的情况。使用脂质转染、电穿孔和一种聚阳离子化合物研究了用表达质粒转染Nalm-6和Reh B前体ALL白血病细胞系。只有脂质体化合物Cellfectin显示出显著的基因转移（Nalm-6细胞为3.9%至12%，Reh细胞为3.1%至5%）。在各种条件下研究了用长臂猿白血病病毒假型化的莫洛尼鼠白血病病毒（MoMuLV）为基础的逆转录病毒载体进行转导。ALL细胞系与包装细胞系共培养显示出逆转录病毒基因转移的最高转导效率（Nalm-6细胞为40.1%至87.5%，Reh细胞为0.3%至9%），其次是在重组纤连蛋白片段CH-296上用病毒上清液进行转导（Nalm-6细胞为13%至35.5%，Reh细胞为0.4%至6%），在人骨髓基质单层上进行转导（Nalm-6细胞为3.2%至13.3%，Reh细胞为0%至0.2%），以及与硫酸鱼精蛋白在悬浮液中进行转导（Nalm-6细胞为0.7%至3.1%，Reh细胞为0%）。用人免疫缺陷病毒1型（HIV-1）为基础的慢病毒载体进行转导，该载体用泡状口炎病毒-G包膜假型化，Nalm-6和Reh细胞的转导产生了最佳的基因转移效率，两种细胞系的转导率均超过90%。然后使用基于MoMuLV的逆转录病毒载体和基于HIV-1的慢病毒载体研究了将基因导入患者的原代人B前体ALL细胞的情况。两种载体都能高效转导原代B前体ALL细胞。这些研究可应用于研究将基因导入原代人B前体ALL细胞以用于免疫治疗。
