Correction of ^-globin Mutations in Human Somatic and iPS Cells

项目总结(见说明)： 项目2的目标是使用患者来源的体细胞及其诱导的多能干细胞(IPS)衍生品纠正镰状细胞病(SCD)和B地中海贫血(B-Thal)突变。这些细胞最终将转化为造血干细胞(HSCs)，以重建患者的造血系统。 作为传统的基于cDNA的遗传治疗的替代方案，IPS细胞或其体细胞前体将通过序列特异性基因打靶策略、小片段同源替换(SFHR)、三链形成寡核苷酸(TFO)介导的同源交换和经典的同源重组(HR)进行修饰。这些研究将在锌指核酸酶(ZFN)和巨核酸酶(MNS)存在和不存在的情况下进行，巨核酸酶和巨核酸酶(MNS)是有效的重组刺激物。我们实验室以前的研究表明，携带A&GT；T镰刀突变的寡/多核苷酸小DNA片段(B[S]-DFS)在显微注射到野生型人类造血干细胞(HSC)中时，会以至少7%的频率将内源性野生型珠蛋白(B[A]-珠蛋白)转化为镰状细胞病B[S]-珠蛋白。这个项目将在两个具体的目标中测试几个假设。目的1验证SDF修饰和TFO修饰的人SCD和B-Thal体细胞及其IPS细胞衍生物可以转化为克隆的等量IPS细胞的假设，这些细胞是存活的、核型稳定的，并且可以分化为可植入的造血祖细胞。目的2：将验证经典的HR是纠正B-珠蛋白珠蛋白基因特定基因组突变的可靠的替代方法。对于B[S]-珠蛋白纯合子或确定的B-地贫突变的体细胞和IPS细胞，将通过SDF、TFO和经典的HR介导的同源交换进行遗传校正，以产生B-珠蛋白杂合子IPS细胞的克隆群体。如果患者体细胞被用于矫正，当克隆群体被分离后，它们将被转化为IPS细胞。在存在和不存在ZFN或序列特异性MN的情况下，将通过电穿孔或显微注射的方式将SDF导入细胞。每个目标都将评估校正后的细胞的核型稳定性、遗传完整性、产生或维持IPS细胞表型的能力，以及在体外分化为HSC的能力。畸胎瘤的形成将通过核心C进行评估，校正后的基因表达模式也将在通过核心B和UCSF阵列核心中进行评估。虽然初步研究将使用逆转录病毒重新编程的IPS细胞进行，但随着新的针对患者的IPS细胞系通过项目1和核心B可用，它们将得到纠正和评估。最后，在项目2中生成的校正后的IPS像元将在项目3中评估其区分为 将移植到NOD-SCID小鼠体内进行造血重建的造血祖细胞。

PROJECT SUMMARY (See instructions): The goal of Project 2 is correction of the sickle cell disease (SCD) and B-thalassemia (B-thal) mutations, using patient derived somafic cells and their induced pluripotent stem (iPS) cell derivatives. These cells will ultimately be converted to hematopoietic stem cells (HSCs) to reconstitute patient hematopoietic systems. As an alternative to conventional cDNA-based genetic therapies, IPS cells or their somafic precursors, will be modified by a sequence-specific gene targeting strategy, small fragment homologous replacement (SFHR), triplex forming oligonucleotide (TFO)-mediated homologous exchange and by classical homologous recombination (HR). These studies will be carried out in the presence and absence of zinc finger nucleases (ZFNs) and meganucleases and meganucleases (MNs) potent stimulators of recombination. Previous studies from our lab have shown that oligo/polynucleotide small DNA fragments (SDFs) carrying the A>T sickle mutation (B[S]-DFs), when microinjected into wild-type human hematopoietic stem cells (HSCs), will convert the endogenous wild-type globin (B[A]-globin) into the sickle cell disease B[S]-globin at frequencies of at least 7%. This project will test several hypotheses in 2 Specific Aims. Aim 1 will test the hypotheses that SDF-modified and TFO-modified human SCD and B-thal somafic cells and their IPS cell derivatives can be converted to clonal isoaltes IPS cells that are viable, karyotypically stable, and can be differentiated into engraftable hematopoietic precursors. Aim 2: will test the hyopthesis that classical HR is a reliable alterative method for correcting specific genomic mutations in the B-globin globin gene. Somatic and IPS cells homozygous for the B[S]-globin or defined B-thal mutations, will be genetically corrected by SDF, TFO, and classical HR mediated homologous exchange, to generate clonal populations of B-globin heterozygote IPS cells. If patient somafic cells are used for correction, they will be converted to IPS cells when a clonal population has been isolated. The cells will be transfected with SDFs by electroporation or microinjection and in the presence and absence of ZFNs or sequence-specific MNs. Each Aim will evaluate the corrected cells in terms of their karyotypic stability, genetic integrity, the ability to generate or maintain IPS cell phenotype, and the ability to differentiate in vitro into HSC. Teratoma formation will be evaluated by Core C and gene expression patterns after correction will also be evaluated in through Core B ad the UCSF Array Core. While initial studies will be carried out using retrovirally reprogrammed IPS cells, as new patient-specific IPS cell lines become available through Project 1 and Core B, they will be corrected and evaluated. Finally, corrected IPS cells generated in Project 2 will be evaluated in Project 3 for their ability to differentiate into hematopoietic precursors that will engraft into NOD-SCID mice for hematopoietic reconstitution.