Rivella S, Sadelain M
Department of Human Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Semin Hematol. 1998 Apr;35(2):112-25.
Gene addition strategies are rational approaches to the treatment of sickle cell anemia and thalassemia. The goal of such genetic treatments is to introduce a functional globin transcription unit in hematopoietic stem cells and express the transgene in a manner that is erythroid-specific, elevated, relatively constant from one cell to another, and sustained over time. Gene transfer is mediated by an expanding array of viral and nonviral vectors. High-titer retroviral vectors harboring the human beta-globin gene and the core sequences of the human beta-globin locus control region yield erythroid-specific gene expression in erythroid cell lines and in short-term murine bone marrow chimeras. However, we show that expression remains subject to position effect variegation and often decreases over time in vivo. Rather than a progressive transcriptional silencing in all cells, we ascribe the waning expression to the gradual emergence in blood of erythroid progeny derived from more and more primitive precursor cells in the months after transplantation. In our model, transgene expression is therefore determined by the integration site and the differentiation stage of the transduced cell at the time of integration. Globin expression is thus different in the progeny of a transduced erythroid progenitor cell and in the erythroid progeny of a transduced hematopoietic stem cell, reflecting the effect of flanking chromatin in differentiated cells and of chromatin remodeling at the site of integration in the progeny of multipotential cells. This model predicts that insulators and matrix attachment regions could be highly valuable to gene therapy in combination with potent transcriptional activators. When efficient gene transfer in hematopoietic stem cells is achieved at last, the challenge will be to regulate gene expression in vivo and overcome transgene variegation and transgene silencing.
基因添加策略是治疗镰状细胞贫血和地中海贫血的合理方法。此类基因治疗的目标是在造血干细胞中引入一个功能性珠蛋白转录单位，并以红细胞特异性、高水平、细胞间相对恒定且随时间持续表达的方式表达转基因。基因转移由一系列不断增加的病毒和非病毒载体介导。携带人β-珠蛋白基因和人β-珠蛋白基因座控制区核心序列的高滴度逆转录病毒载体在红细胞系和短期小鼠骨髓嵌合体中产生红细胞特异性基因表达。然而，我们发现表达仍受位置效应斑驳的影响，并且在体内通常会随时间下降。我们将表达减弱归因于移植后数月中，越来越原始的前体细胞衍生的红细胞后代在血液中逐渐出现，而不是所有细胞中进行性的转录沉默。因此，在我们的模型中，转基因表达由转导细胞在整合时的整合位点和分化阶段决定。因此，转导的红细胞祖细胞后代和转导的造血干细胞的红细胞后代中的珠蛋白表达不同，这反映了分化细胞中侧翼染色质的作用以及多能细胞后代整合位点处染色质重塑的作用。该模型预测，绝缘子和基质附着区域与强效转录激活剂结合对基因治疗可能非常有价值。当最终在造血干细胞中实现高效基因转移时，挑战将是在体内调节基因表达并克服转基因斑驳和转基因沉默。
