Arumugam Paritha I, Scholes Jessica, Perelman Natalya, Xia Ping, Yee Jiing-Kuan, Malik Punam
Division of Hematology-Oncology, The Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, California, USA.
Mol Ther. 2007 Oct;15(10):1863-71. doi: 10.1038/sj.mt.6300259. Epub 2007 Jul 10.
Effective gene therapy for beta-thalassemia major (beta-TM) requires consistent, high expression of human beta-globin (hbeta-globin) in red blood cells (RBCs). Several groups have now shown that lentiviral (LV) vectors stably transmit the hbeta/hgamma-globin genes and large elements of the locus control region, resulting in correction of the murine thalassemia intermedia (TI) phenotype and survival of mice with the TM phenotype. However, current LVs show variable hbeta/hgamma-globin expression and require a high number of vector copies/cell for a therapeutic effect. To address this, we designed LVs flanked by the chicken hypersensitive site-4 (cHS4) chromatin insulator element and compared them with their "un-insulated" counterparts. We observed a consistent twofold-higher hbeta expression from insulated vectors in single-copy mouse erythroleukemia cell clones, an increase that resulted from reduced position effect variegation (PEV) and increased probability of expression from individual integrants. This effect was confirmed in vivo: an approximately twofold increase in hbeta expression was seen in the RBC progeny of murine hematopoietic stem cells, with significantly higher numbers of hbeta-expressing cells in individual secondary spleen colony-forming units. In summary, cHS4-insulated hbeta-globin LVs showed distinct chromatin barrier activity, resulting in higher, consistent hbeta expression. These studies have important implications for vector design for clinical trials for gene therapy for hemoglobinopathies.
针对重型β地中海贫血（β-TM）的有效基因治疗需要在红细胞（RBC）中持续、高表达人β-珠蛋白（hβ-珠蛋白）。现在有几个研究小组表明，慢病毒（LV）载体能稳定传递hβ/hγ-珠蛋白基因以及基因座控制区的大片段元件，从而纠正小鼠中间型地中海贫血（TI）的表型，并使具有TM表型的小鼠存活。然而，目前的慢病毒载体显示出hβ/hγ-珠蛋白表达存在差异，且为达到治疗效果需要在每个细胞中有大量的载体拷贝数。为了解决这个问题，我们设计了两侧带有鸡高敏位点4（cHS4）染色质绝缘子元件的慢病毒载体，并将它们与其“未绝缘”的对应载体进行比较。我们观察到，在单拷贝小鼠红白血病细胞克隆中，绝缘载体的hβ表达始终高出两倍，这种增加是由于位置效应斑驳（PEV）减少以及单个整合体表达概率增加所致。这种效应在体内得到了证实：在小鼠造血干细胞的RBC后代中，hβ表达增加了约两倍，在单个二级脾集落形成单位中，表达hβ的细胞数量显著增多。总之，cHS4绝缘的hβ-珠蛋白慢病毒载体显示出独特的染色质屏障活性，从而导致更高、更一致的hβ表达。这些研究对于血红蛋白病基因治疗临床试验的载体设计具有重要意义。
