Transactivation of Fetal Hemoglobin Genes for Treatment*

描述（由申请人提供）： 目前镰状细胞病的基因治疗策略集中在转导 造血干细胞与含有抗镰状球蛋白的病毒载体 基因如γ-、0/5-或修饰的β-珠蛋白基因。虽然这是一个可行的 方法，治疗水平的珠蛋白mRNA的长期表达， 蛋白质从转导基因magamma是，难以实现。一个替代 一种方法是用一种转录因子基因编码 一种特异性增强内源性γ-或β-珠蛋白基因的蛋白质 表情在这种情况下，相对低水平的新转录因子 可以刺激高水平的γ-或δ-珠蛋白基因表达。 Sangamo BioSciences是一家率先使用锌指的公司 Cys 2-His 2型ZFP具有高特异性亲和力，几乎 任何感兴趣的调控序列。设计师ZFP具有更强大的 而非内生因素。ZFP被设计为结合到 几乎任何序列都具有高特异性和更大的亲和力， 内生因素ZFP也可以被设计成正常地集成功能 分布在几个内生因素之间，否则这些因素将不得不 可以组合地添加以实现期望的结果。 特异性和有效激活γ-和β-珠蛋白基因的ZFP 将在镰状细胞瘤小鼠模型中测试培养细胞中的表达 疾病这些小鼠从人类胎儿血红蛋白（HbF）转换为人类镰状血红蛋白。 血红蛋白（HbS）在出生后以类似于人类患者的方式 疾病尽管这些小鼠在出生时没有症状， 当转换发生时，成年动物会发育出大部分（如果不是全部） 疾病的病理学。ZFP特异有效地激活 γ-和β-珠蛋白基因表达将用慢病毒转导 载体导入分离自镰状小鼠的纯化造血干细胞中。 在将这些基因修饰的细胞移植到接受者体内后， 将对动物进行监测以长期纠正疾病。相对 低水平的这些有效的转录因子应该能够 激活γ-和β-珠蛋白基因的水平，将改善 在大多数器官系统中观察到的病理学。这些研究将提供一个坚实的 这为随后在人类患者中进行临床试验奠定了基础 毁灭性的疾病

DESCRIPTION (provided by applicant): Current gene therapy strategies for sickle cell disease focus on transduction of hematopoietic stem cells with viral vectors containing anti-sickling globin genes such as y-, 0/5- or modified P-globin genes. Although this is a viable approach, long term expression of therapeutic levels of globin mRNA and protein from transduced genes magamma be, difficult to achieve. An alternative approach is to transduce stem cells with a transcription factor gene encoding a protein that specifically enhances endogenous gamma- or beta-globin gene expression. In this case relatively low levels of a novel transcription factor may stimulate high levels of gamma- or 5-globin gene expression. Sangamo BioSciences is a company which has pioneered the use of zinc finger proteins (ZFPs) of the Cys2-His2 type with high specific affinities to almost any regulatory sequence of interest. Designer ZFPs have more robust characteristics than endogenous factors. ZFPs are designed to bind to virtually any sequence with high specificity and greater affinity than endogenous factors. ZFPs can also be designed to integrate functions normally distributed between several endogenous factors, which would otherwise have to be added combinatorially to effect a desired outcome. ZFPs that specifically and efficiently activate gamma- and beta-globin gene expression in cultured cells will be tested in a mouse model of sickle cell disease. These mice switch from human fetal hemoglobin (HbF) to human sickle hemoglobin (HbS) after birth in a manner similar to human patients with the disease. Although the mice are asymptomatic at birth, severe disease develops as the switch occurs, and adult animals develop most if not all of the pathology of the disease. ZFPs that specifically and efficiently activate gamma- and beta-globin gene expression will be transduced with lentiviral vectors into purified hematopoietic stem cells isolated from the sickle mice. After transplantation of these genetically modified cells into recipients, the animals will be monitored for long term correction of the disease. Relatively low levels of these potent transcription factors should be capable of activating gamma- and beta-globin genes to a level that will ameliorate the pathology observed in most organ systems. These studies will provide a solid foundation for subsequent clinical trials in human patients with this devastating disease.