Tarantal A F, O'Rourke J P, Case S S, Newbound G C, Li J, Lee C I, Baskin C R, Kohn D B, Bunnell B A
California Regional Primate Research Center, University of California at Davis, 95616-8542, USA.
Mol Ther. 2001 Feb;3(2):128-38. doi: 10.1006/mthe.2000.0255.
Many life-threatening conditions that can be diagnosed early in gestation may be treatable in utero using gene therapy. In order to determine in utero gene transfer efficiency and safety, studies were conducted with fetal rhesus monkeys as a model for the human. Included in these studies were Moloney murine leukemia virus (MLV)-based amphotropic retrovirus, vesicular stomatitis virus-G (VSV-G) pseudotyped MLV, and a VSV-G pseudotyped HIV-1-based vector, all expressing the enhanced green fluorescent protein (EGFP) as a reporter gene and driven by a cytomegalovirus-immediate early promoter (N = 16). Rhesus monkey fetuses were administered viral vector supernatant preparations by the intraperitoneal (ip) (N = 14) or intrahepatic (ih) (N = 2) routes via ultrasound guidance at 55 +/- 5 days gestation (late first trimester; term 165 +/- 10 days). Fetuses were monitored sonographically, specimens were collected prenatally and postnatally, and tissue harvests were performed at birth or 3 or 6 months postnatal age (3-10 months post-gene transfer). PCR analyses demonstrated that transduced cells were present at approximately 1.2% in peripheral blood mononuclear cells from fetuses administered amphotropic MLV, <0.5% in fetuses receiving MLV/VSV-G, and approximately 4.2% for the lentiviral vector, which decreased to 2% at birth. Hematopoietic progenitors showed that overall (mean of all time points assessed), approximately 25% of the collected colonies were positive for the EGFP transgene with the lentiviral vector, which was significantly greater than results achieved with the MLV-based vector systems (4-9%; P < or = 0.001-0.016). At necropsy, 0.001-10% of the total genomic DNA was positive for EGFP in most tissues for all groups. EGFP-positive fluorescent cells were found in cell suspensions of thymus, liver, spleen, lymph nodes, cerebral cortex, and bone marrow (0.5-6%). Overall, the results of these studies have shown: (1) healthy infants expressing vector sequences up to 10 months post-gene transfer, (2) fetal primate administration of retroviral vectors results in gene transfer to multiple organ systems, (3) the highest level of gene transfer to hematopoietic progenitors was observed with the lentiviral vector system, and (4) there was no evidence of transplacental transfer of vector sequences into the dams. The rhesus monkey is an important preclinical primate model system for exploring gene transfer approaches for future applications in humans.
许多在妊娠早期即可诊断的危及生命的疾病，或许可通过基因疗法在子宫内进行治疗。为了确定子宫内基因转移的效率和安全性，研究人员以恒河猴胎儿作为人类模型展开了研究。这些研究包括基于莫洛尼鼠白血病病毒（MLV）的嗜异性逆转录病毒、水泡性口炎病毒-G（VSV-G）假型化的MLV，以及一种VSV-G假型化的基于HIV-1的载体，所有这些载体均表达增强型绿色荧光蛋白（EGFP）作为报告基因，并由巨细胞病毒-立即早期启动子驱动（N = 16）。在妊娠55±5天（孕早期晚期；足月为165±10天）时，通过超声引导经腹腔（ip）（N = 14）或肝内（ih）（N = 2）途径给恒河猴胎儿施用病毒载体上清液制剂。对胎儿进行超声监测，在产前和产后采集标本，并在出生时或出生后3或6个月（基因转移后3 - 10个月）进行组织采集。PCR分析表明，在接受嗜异性MLV的胎儿外周血单个核细胞中，转导细胞约占1.2%，接受MLV/VSV-G的胎儿中占比<0.5%，慢病毒载体组约为4.2%，出生时降至2%。造血祖细胞研究显示，总体而言（所有评估时间点的平均值），慢病毒载体组所采集的集落中约25%的EGFP转基因呈阳性，这显著高于基于MLV的载体系统所取得的结果（4 - 9%；P≤0.001 - 0.016）。尸检时，所有组大多数组织中总基因组DNA的0.001 - 10%对EGFP呈阳性。在胸腺、肝脏、脾脏、淋巴结、大脑皮层和骨髓的细胞悬液中发现了EGFP阳性荧光细胞（0.5 - 6%）。总体而言，这些研究结果表明：（1）基因转移后长达10个月有健康婴儿表达载体序列；（2）向灵长类胎儿施用逆转录病毒载体可导致基因转移至多个器官系统；（3）慢病毒载体系统对造血祖细胞的基因转移水平最高；（4）没有证据表明载体序列经胎盘转移至母猴体内。恒河猴是一种重要且用于临床前研究的灵长类动物模型系统，可用于探索未来应用于人类的基因转移方法。
