Pan D, Jonsson J J, Braun S E, McIvor R S, Whitley C B
Gene Therapy Program, Department of Pediatrics, Institute of Human Genetics, University of Minnesota, 420 Delaware Street SE, Minneapolis, Minnesota, 55455, USA.
Mol Genet Metab. 2000 Jul;70(3):170-8. doi: 10.1006/mgme.2000.3012.
Expression of iduronate-2-sulfatase (IDS) from three different promoters in four retroviral vectors was studied in peripheral blood lymphocytes from patients with Hunter syndrome (PBL(MPS)), i.e., the LTR in vectors L2SN and L2, avian beta-actin promoter in LB2, and the CMV early promoter in LNC2. PBL(MPS) were exposed to packaging cell supernatant resulting in transduction frequencies ranging 10-fold from 5 to 49%. Surprisingly, IDS activities were equally high in all transduced lymphocyte populations: 515 U/mg/h in PBL(MPS)-L2SN, 734 in PBL(MPS)-LB2, 352 in PBL(MPS)-L2, and 389 in PBL(MPS)-LNC2 compared to controls (<10 in PBL(MPS)-LXSN or PBL(MPS)). The half-life of endocytosed IDS in PBL(MPS) was 1.9 days. However, the level of lymphocyte IDS activity from proviral expression was found to be only a fraction of the total, a large portion being derived from reuptake of enzyme from murine packaging cells, i.e., a "second source" of enzyme. Therefore, measurement of transgene lysosomal enzyme soon after exposure of target cells to vector supernatant may yield a gross overestimate of long-term transgene expression by transduced cells. Nevertheless, patient fibroblasts cocultured with transduced PBL(MPS) had reduced (35)SO(4)-GAG accumulation, levels similar to those of normal fibroblasts. These studies revealed a broadly applicable phenomenon: cells can be charged with a lysosomal enzyme to levels much higher than those found in nature. By "supercharging" cells with a lysosomal protein (or other molecule bearing the mannose-6-phosphate ligand), such cells may be exploited as vehicles for systemic delivery of therapeutic or diagnostic agents.
在患有亨特综合征患者的外周血淋巴细胞（PBL(MPS)）中研究了四种逆转录病毒载体中三种不同启动子驱动的艾杜糖醛酸-2-硫酸酯酶（IDS）的表达，即载体L2SN和L2中的LTR、LB2中的禽β-肌动蛋白启动子以及LNC2中的巨细胞病毒早期启动子。将PBL(MPS)暴露于包装细胞上清液，转导频率在5%至49%之间，相差10倍。令人惊讶的是，在所有转导的淋巴细胞群体中IDS活性同样高：与对照（PBL(MPS)-LXSN或PBL(MPS)中<10）相比，PBL(MPS)-L2SN中的IDS活性为515 U/mg/h，PBL(MPS)-LB2中为734，PBL(MPS)-L2中为352，PBL(MPS)-LNC2中为389。PBL(MPS)中内吞的IDS的半衰期为1.9天。然而，发现来自前病毒表达的淋巴细胞IDS活性水平仅占总量的一小部分，很大一部分来自从小鼠包装细胞重新摄取酶，即酶的“第二来源”。因此，在靶细胞暴露于载体上清液后不久测量转基因溶酶体酶可能会导致对转导细胞长期转基因表达的严重高估。尽管如此，与转导的PBL(MPS)共培养的患者成纤维细胞中(35)SO(4)-GAG积累减少，水平与正常成纤维细胞相似。这些研究揭示了一个广泛适用的现象：细胞可以被加载溶酶体酶至比自然界中发现的水平高得多的水平。通过用溶酶体蛋白（或其他带有甘露糖-6-磷酸配体的分子）“超负载”细胞，这样的细胞可以用作治疗或诊断剂全身递送的载体。
