Bunnell B A, Metzger M, Byrne E, Morgan R A, Donahue R E
Clinical Gene Therapy Branch, National Center for Human Genome Research, Bethesda, MD 20850, USA.
Blood. 1997 Mar 15;89(6):1987-95.
High efficiency retroviral-mediated gene transfer to rhesus CD4+ peripheral blood lymphocytes (PBL) was accomplished using an optimized transduction protocol using a gibbon ape leukemia virus (GaLV) envelope-containing packaging cell line PG13. Engineered CD4+ PBL were administered to three nonmyeloablated animals in three or four separate infusions over 9 months. Polymerase chain reaction (PCR) demonstrated in vivo reconstitution of the genetically engineered CD4+ PBL at levels between 1% and 10% of the circulating leukocytes. This level of gene marking indicates that up to 30% of endogenous circulating CD4+ cells can be genetically engineered. The high levels of marked lymphocytes persist for the first 3 weeks following reinfusion then decline to < or = 0.1% over the next 21 weeks. Lymph node (LN) biopsies were performed to determine if the engineered CD4+ lymphocytes could traffic to lymphoid tissues. Marked lymphocytes were detected in LN biopsies 100 days following reinfusion of the transduced cells. Expression of retroviral vector-derived sequences was detected by reverse transcriptase (RT)-PCR analysis from CD4-enriched lymphocytes that were activated by culturing in the presence of recombinant interleukin-2 (rlL-2). A humoral immune response to fetal bovine serum (FBS) was detected in all animals following the second administration of the culture expanded CD4+ lymphocytes. No antibody response was detected to the neomycin-resistance (Neo(R)) transgene, the murine retroviral group-specific antigen (gag), or GaLV envelope (env) proteins.
利用含有长臂猿白血病病毒（GaLV）包膜的包装细胞系PG13，通过优化的转导方案，实现了高效逆转录病毒介导的基因向恒河猴CD4⁺外周血淋巴细胞（PBL）的转移。将经过基因工程改造的CD4⁺ PBL在9个月内分三次或四次单独输注给三只未进行骨髓消融的动物。聚合酶链反应（PCR）证明，基因工程改造的CD4⁺ PBL在体内得以重建，其水平在循环白细胞的1%至10%之间。这种基因标记水平表明，高达30%的内源性循环CD4⁺细胞能够被基因工程改造。大量标记的淋巴细胞在重新输注后的前3周持续存在，然后在接下来的21周内降至≤0.1%。进行淋巴结（LN）活检以确定经过基因工程改造的CD4⁺淋巴细胞是否能够迁移至淋巴组织。在转导细胞重新输注100天后的LN活检中检测到了标记的淋巴细胞。通过逆转录酶（RT）-PCR分析，从在重组白细胞介素-2（rlL-2）存在下培养而被激活的富含CD4的淋巴细胞中检测到了逆转录病毒载体衍生序列的表达。在第二次给予经培养扩增的CD4⁺淋巴细胞后，在所有动物中均检测到了对胎牛血清（FBS）的体液免疫反应。未检测到针对新霉素抗性（Neo(R)）转基因、鼠逆转录病毒群特异性抗原（gag）或GaLV包膜（env）蛋白的抗体反应。
