Malik Punam, Arumugam Paritha I, Yee Jing-Kuan, Puthenveetil Geetha
Saban Research Institute, Division of Hematology-Oncology, Childrens Hospital Los Angeles, Department of Pediatrics, Los Angeles, California 90027, USA.
Ann N Y Acad Sci. 2005;1054:238-49. doi: 10.1196/annals.1345.030.
beta-Thalassemias are the most common single-gene disorders and are potentially amenable to gene therapy. While retroviral vectors carrying the human beta-globin cassette were notoriously unstable and expressed poorly, considerable progress has now been made using lentiviral vectors (LVs), which stably transmit the beta-globin expression cassette. Mouse studies using LVs have shown correction of the beta-thalassemia-intermedia phenotype and a partial, variable correction of the mouse beta-thalassemia major phenotype, despite the use of beta-globin-hypersensitive sites that are known to result in position-independent effects. Our group used the alpha-globin-hypersensitive site in self-inactivating (SIN) LVs with long-term expression in secondary mice that resisted methylation-associated proviral silencing. However, these vectors also suffered from chromatin position effects. We therefore flanked a SIN-lentiviral vector carrying the human beta-globin expression cassette with a chromatin insulator and studied expression in bone marrow from four patients with transfusion-dependent human thalassemia major. We demonstrated normal levels of human beta-globin expression in erythroid cells produced in in vitro cultures for unilineage erythroid differentiation. There was restoration of effective erythropoiesis and reversal of the abnormally elevated apoptosis that characterizes beta-thalassemia. The gene-corrected human beta-thalassemia progenitor cells were transplanted into immune-deficient mice, where they underwent normal erythroid differentiation, expressed normal levels of human beta-globin, and displayed normal effective erythropoiesis 3-4 months after xenotransplantation. Variability of beta-globin expression in erythroid colonies derived in vitro or from xenograft bone marrow was similar to that seen in normal control subjects. Results show genetic correction of primitive human progenitor cells and normalization of the human thalassemia major phenotype.
β地中海贫血是最常见的单基因疾病，有可能接受基因治疗。虽然携带人β珠蛋白盒的逆转录病毒载体稳定性差且表达不佳，但目前使用慢病毒载体（LVs）已取得了相当大的进展，慢病毒载体能稳定传递β珠蛋白表达盒。使用LVs的小鼠研究表明，尽管使用了已知会产生位置独立效应的β珠蛋白超敏位点，但中间型β地中海贫血表型得到了纠正，重度小鼠β地中海贫血表型得到了部分、可变的纠正。我们的研究小组在自我失活（SIN）LVs中使用α珠蛋白超敏位点，在对甲基化相关的原病毒沉默具有抗性的二代小鼠中实现了长期表达。然而，这些载体也受到染色质位置效应的影响。因此，我们在携带人β珠蛋白表达盒的SIN慢病毒载体两侧加上染色质绝缘子，并研究了4例依赖输血的重度人类地中海贫血患者骨髓中的表达情况。我们证明，在用于单系红细胞分化的体外培养中产生的红细胞中，人β珠蛋白表达水平正常。有效红细胞生成得以恢复，β地中海贫血特有的异常升高的细胞凋亡得以逆转。经基因校正的人类β地中海贫血祖细胞被移植到免疫缺陷小鼠体内，在异种移植后3 - 4个月，它们经历了正常的红细胞分化，表达正常水平的人β珠蛋白，并显示出正常的有效红细胞生成。体外衍生或来自异种移植骨髓的红细胞集落中β珠蛋白表达的变异性与正常对照受试者相似。结果显示原始人类祖细胞的基因校正和重度人类地中海贫血表型的正常化。
