Braun S E, Pan D, Aronovich E L, Jonsson J J, McIvor R S, Whitley C B
Department of Pediatrics, University of Minnesota, Minneapolis 55455, USA.
Hum Gene Ther. 1996 Feb 10;7(3):283-90. doi: 10.1089/hum.1996.7.3-283.
To explore the feasibility of ex vivo lymphocyte gene therapy for mild Hunter syndrome (mucopolysaccharidosis type II), we evaluated retrovirus-mediated gene transfer of the iduronate-2-sulfatase (IDS) coding sequence into peripheral blood lymphocytes from enzyme-deficient individuals (PBLMPS). Moloney murine leukemia virus-derived retroviral vectors were constructed by inserting the IDS cDNA under transcriptional regulation of the long terminal repeat (LTR) (in vector L2SN) or the cytomegalovirus (CMV) early promoter (vector LNC2). High-titer virus-producer cells were generated using amphotropic PA317 packaging cells. After 3 days of in vitro stimulation of T lymphocytes with anti-CD3 antibody and interleukin-2 (IL-2), PBLMPS were transduced once on each of the next 3 days. Seven to 21 days later, cultured PBLMPS were evaluated for gene transfer and IDS specific activity. Heterogeneous populations of L2SN-transduced PBLMPS had high levels of IDS enzyme activity (456 U/mg per hr +/- SD 292) despite a gene transfer efficiency of 5% or less. Owing to overexpression of IDS in that percentage of PBLMPS successfully transduced, IDS activity was increased above the deficiency found in patients with Hunter syndrome (< 20 U/mg per hr) to a level comparable with that of normal individuals (mean activity of uncultured normal leukocytes 807 U/mg per hr; SD 252). Reduced 35SO4-glucosaminoglycan (GAG) accumulation was observed in PBLMPS that had been transduced with L2SN, or when PBLMPS were grown in medium that had been "conditioned" by growth of L2SN-transduced cells. This latter result indicated that metabolic cross-correction occurred by means of intercellular enzyme transfer. These studies of retrovirus-mediated expression and metabolic correction, finding near-normal levels of IDS in cultured PBLMPS and metabolic correction, demonstrate the potential for treatment of mild, nonneuropathic Hunter syndrome by means of ex vivo lymphocyte gene therapy.
为探究体外淋巴细胞基因治疗轻度亨特综合征（黏多糖贮积症II型）的可行性，我们评估了将艾杜糖-2-硫酸酯酶（IDS）编码序列通过逆转录病毒介导的基因转移导入酶缺陷个体的外周血淋巴细胞（PBLMPS）。通过将IDS cDNA插入长末端重复序列（LTR）（载体L2SN）或巨细胞病毒（CMV）早期启动子（载体LNC2）的转录调控下，构建莫洛尼鼠白血病病毒衍生的逆转录病毒载体。使用嗜异性PA317包装细胞产生高滴度病毒生产细胞。在用抗CD3抗体和白细胞介素-2（IL-2）对T淋巴细胞进行3天体外刺激后，在接下来的3天中每天对PBLMPS进行一次转导。7至21天后，对培养的PBLMPS进行基因转移和IDS比活性评估。尽管基因转移效率为5%或更低，但L2SN转导的PBLMPS异质群体具有高水平的IDS酶活性（456 U/mg每小时±标准差292）。由于在成功转导的那部分PBLMPS中IDS过表达，IDS活性从亨特综合征患者中发现的缺陷水平（<20 U/mg每小时）增加到与正常个体相当的水平（未培养正常白细胞的平均活性807 U/mg每小时；标准差252）。在用L2SN转导的PBLMPS中，或当PBLMPS在已被L2SN转导细胞生长“条件化”的培养基中生长时，观察到35SO4-葡糖胺聚糖（GAG）积累减少。后一结果表明通过细胞间酶转移发生了代谢交叉校正。这些关于逆转录病毒介导的表达和代谢校正的研究，在培养的PBLMPS中发现了接近正常水平的IDS并实现了代谢校正，证明了通过体外淋巴细胞基因治疗治疗轻度、非神经性亨特综合征的潜力。
