Rosenzweig M, MacVittie T J, Harper D, Hempel D, Glickman R L, Johnson R P, Farese A M, Whiting-Theobald N, Linton G F, Yamasaki G, Jordan C T, Malech H L
New England Regional Primate Research Center, Harvard Medical School, Southborough, MA, USA.
Blood. 1999 Oct 1;94(7):2271-86.
Optimization of mobilization, harvest, and transduction of hematopoietic stem cells is critical to successful stem cell gene therapy. We evaluated the utility of a novel protocol involving Flt3-ligand (Flt3-L) and granulocyte colony-stimulating factor (G-CSF) mobilization of peripheral blood stem cells and retrovirus transduction using hematopoietic growth factors to introduce a reporter gene, murine CD24 (mCD24), into hematopoietic stem cells in nonhuman primates. Rhesus macaques were treated with Flt3-L (200 microgram/kg) and G-CSF (20 microgram/kg) for 7 days and autologous CD34(+) peripheral blood stem cells harvested by leukapheresis. CD34(+) cells were transduced with an MFGS-based retrovirus vector encoding mCD24 using 4 daily transductions with centrifugations in the presence of Flt3-L (100 ng/mL), human stem cell factor (50 ng/mL), and PIXY321 (50 ng/mL) in serum-free medium. An important and novel feature of this study is that enhanced in vivo engraftment of transduced stem cells was achieved by conditioning the animals with a low-morbidity regimen of sublethal irradiation (320 to 400 cGy) on the day of transplantation. Engraftment was monitored sequentially in the bone marrow and blood using both multiparameter flow cytometry and semi-quantitative DNA polymerase chain reaction (PCR). Our data show successful and persistent engraftment of transduced primitive progenitors capable of giving rise to marked cells of multiple hematopoietic lineages, including granulocytes, monocytes, and B and T lymphocytes. At 4 to 6 weeks posttransplantation, 47% +/- 32% (n = 4) of granulocytes expressed mCD24 antigen at the cell surface. Peak in vivo levels of genetically modified peripheral blood lymphocytes approached 35% +/- 22% (n = 4) as assessed both by flow cytometry and PCR 6 to 10 weeks posttransplantation. In addition, naïve (CD45RA(+) and CD62L(+)) CD4(+) and CD8(+) cells were the predominant phenotype of the marked CD3(+) T cells detected at early time points. A high level of marking persisted at between 10% and 15% of peripheral blood leukocytes for 4 months and at lower levels past 6 months in some animals. A cytotoxic T-lymphocyte response against mCD24 was detected in only 1 animal. This degree of persistent long-lived, high-level gene marking of multiple hematopoietic lineages, including naïve T cells, using a nonablative marrow conditioning regimen represents an important step toward the ultimate goal of high-level permanent transduced gene expression in stem cells.
造血干细胞动员、采集及转导的优化对于成功的干细胞基因治疗至关重要。我们评估了一种新方案的效用，该方案涉及使用Flt3配体（Flt3-L）和粒细胞集落刺激因子（G-CSF）动员外周血干细胞，并利用造血生长因子进行逆转录病毒转导，以将报告基因小鼠CD24（mCD24）导入非人灵长类动物的造血干细胞中。恒河猴接受Flt3-L（200微克/千克）和G-CSF（20微克/千克）治疗7天，然后通过白细胞分离术采集自体CD34(+)外周血干细胞。在无血清培养基中，使用基于MFGS的逆转录病毒载体编码mCD24，在存在Flt3-L（100纳克/毫升）、人干细胞因子（50纳克/毫升）和PIXY321（50纳克/毫升）的情况下，通过每天4次转导并离心，对CD34(+)细胞进行转导。本研究的一个重要且新颖的特点是，在移植当天通过亚致死剂量照射（320至400厘戈瑞）的低发病率方案对动物进行预处理，从而实现了转导干细胞在体内的增强植入。使用多参数流式细胞术和半定量DNA聚合酶链反应（PCR）对骨髓和血液中的植入情况进行了连续监测。我们的数据显示，转导的原始祖细胞成功且持续植入，能够产生多个造血谱系的显著细胞，包括粒细胞、单核细胞以及B和T淋巴细胞。在移植后4至6周，47%±32%（n = 4）的粒细胞在细胞表面表达mCD24抗原。通过流式细胞术和PCR评估，移植后6至10周，基因修饰外周血淋巴细胞的体内峰值水平接近35%±22%（n = 4）。此外，在早期时间点检测到的标记CD3(+) T细胞的主要表型为初始（CD45RA(+)和CD62L(+)）CD4(+)和CD8(+)细胞。在一些动物中，外周血白细胞的高水平标记在4个月内持续保持在10%至15%之间，6个月后水平较低。仅在1只动物中检测到针对mCD24的细胞毒性T淋巴细胞反应。使用非清髓性骨髓预处理方案对包括初始T细胞在内的多个造血谱系进行这种程度的持久、高水平基因标记，代表了朝着干细胞中高水平永久转导基因表达的最终目标迈出的重要一步。
