Howrey R P, El-Alfondi M, Phillips K L, Wilson L, Rooney B, Lan N, Sullenger B, Smith C
Division of Hematology/Oncology, Department of Pediatrics, Duke University Medical Center, Durham, NC 27710, USA.
Gene Ther. 2000 Feb;7(3):215-23. doi: 10.1038/sj.gt.3301064.
A variety of gene therapy strategies are under development for the treatment of sickle cell anemia and other hemoglobinopathies. A number of alternative vectors have been developed to transfer and express the beta-globin gene and other therapeutic molecules, but none has resulted in efficient transduction and stable long-term expression in primary hematopoietic cells. One reason for this problem is that most vectors are initially evaluated in immortalized cell lines which may not faithfully recapitulate the biology of primary erythroid cells. In order to provide a more relevant system for efficiently evaluating alternative vector constructs for beta-globin disorders, we have developed (1) a simple method for generating primary human red blood cell (RBC) precursors in liquid culture established with mononuclear cells obtained from normal donors as well as patients with Hb SC disease; (2) a high titer retroviral vector which can be easily modified to optimize gene transfer and transgene expression; and (3) methods for transducing the RBC precursors at high efficiency. The development of simple and efficient methods and reagents for generating and transducing primary human RBC precursors provides a facile and effective means for screening alternative gene therapy strategies. Gene Therapy (2000) 7, 215-223.
目前正在开发多种基因治疗策略用于治疗镰状细胞贫血和其他血红蛋白病。已经开发了许多替代载体来转移和表达β-珠蛋白基因及其他治疗分子，但没有一种能在原代造血细胞中实现高效转导和稳定的长期表达。这个问题的一个原因是，大多数载体最初是在永生化细胞系中评估的，而这些细胞系可能无法如实地重现原代红细胞的生物学特性。为了提供一个更合适的系统来有效评估用于β-珠蛋白疾病的替代载体构建体，我们开发了：（1）一种简单的方法，利用从正常供体以及Hb SC病患者获得的单核细胞在液体培养中生成原代人类红细胞（RBC）前体；（2）一种高滴度逆转录病毒载体，可轻松修改以优化基因转移和转基因表达；（3）高效转导RBC前体的方法。开发用于生成和转导原代人类RBC前体的简单高效方法和试剂，为筛选替代基因治疗策略提供了一种简便有效的手段。《基因治疗》（2000年）7卷，215 - 223页 。
