Murray L, Travis M, Luens-Abitorabi K, Olsson K, Plavec I, Forestell S, Hanania E G, Hill B
SyStemix, a Novartis Company, Palo Alto, CA 94304, USA.
Hum Gene Ther. 2000 Sep 20;11(14):2039-50. doi: 10.1089/10430340050143453.
Absence of durable high-level expression of transgenes from Moloney murine leukemia (Mo-MuLV) retroviral vectors has been a hurdle in bringing effective gene therapy to the clinic. In this study we have analyzed transgene expression among the progeny of mobilized hematopoietic stem cells (HSCs), comparing Mo-MuLV and mouse stem cell virus (MSCV) vectors, with or without addition of a scaffold attachment region (SAR) from the human interferon beta gene. Retroviral (RV) vector supernatant quality was assessed by comparing NGFR transgene expression by HEL cells, and transgene delivery and expression by CD34(+) cells 72 hr after transduction, using real-time PCR and FACS analysis. This is the first description of the effect of SAR within both Mo-MuLV and MSCV vector backbones on long-term RV transgene expression among in vivo HSC progeny in HSC repopulation assays (SCID-hu bone and NOD/SCID). After transduction of mobilized CD34(+) cells with MSCV-SAR vector, transgene expression was observed among a mean of 10% of donor HSC progeny in the SCID-hu bone (range, 0.6-43%). The predominant effect of SAR was to increase the mean fluorescence intensity (MFI) of transgene expression among HSC progeny in both in vivo bone repopulation models (three- to fourfold), and after long-term stromal cultures (twofold).
莫洛尼鼠白血病病毒（Mo-MuLV）逆转录病毒载体缺乏持久的高水平转基因表达，这一直是将有效的基因治疗应用于临床的障碍。在本研究中，我们分析了动员的造血干细胞（HSC）子代中的转基因表达情况，比较了Mo-MuLV和小鼠干细胞病毒（MSCV）载体，以及添加或不添加来自人干扰素β基因的支架附着区域（SAR）的情况。通过比较HEL细胞中NGFR转基因表达，以及转导后72小时CD34(+)细胞的转基因递送和表达情况，利用实时PCR和FACS分析评估逆转录病毒（RV）载体上清液质量。这是首次描述在HSC再填充试验（SCID-hu骨和NOD/SCID）中，Mo-MuLV和MSCV载体骨架内的SAR对体内HSC子代中长期RV转基因表达的影响。用MSCV-SAR载体转导动员的CD34(+)细胞后，在SCID-hu骨中平均10%的供体HSC子代中观察到转基因表达（范围为0.6 - 43%）。在体内骨再填充模型中，SAR的主要作用是使HSC子代中转基因表达的平均荧光强度（MFI）增加（三到四倍），在长期基质培养后增加两倍。
