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多能干（iPS）细胞衍生物纠正镰状细胞病（SCD）和B型地中海贫血（B-thal）突变。这些细胞最终将转化为造血干细胞（HSC），以重建患者的造血系统。 作为常规的基于cDNA的遗传疗法的替代方案，IPS细胞或其体细胞前体将通过序列特异性基因靶向策略、小片段同源置换（SFHR）、三链体形成寡核苷酸（TFO）介导的同源交换和通过经典同源重组（HR）来修饰。这些研究将在存在和不存在锌指核酸酶（ZFN）和大范围核酸酶和大范围核酸酶（MN）的情况下进行。我们实验室的先前研究已经表明，携带A>T镰状突变（B[S]-DF）的寡/多核苷酸小DNA片段（SDF），当显微注射到野生型人造血干细胞（HSC）中时，将以至少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。本项目将测试2个具体目标中的几个假设。目的1将检验SDF修饰和TF 0修饰的人SCD和B-地中海体细胞及其IPS细胞衍生物可以转化为克隆同种型IPS细胞的假设，所述克隆同种型IPS细胞是活的、核型稳定的，并且可以分化为可移植的造血前体细胞。目标二：将测试hypopthesis，经典的HR是一个可靠的替代方法，用于纠正特定的基因组突变的B-珠蛋白珠蛋白基因。对于B[S]-珠蛋白或确定的B-珠蛋白突变纯合的体细胞和IPS细胞将通过SDF、TFO和经典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。如果使用患者体细胞进行校正，则当分离克隆群体时，它们将转化为IPS细胞。通过电穿孔或显微注射，在存在和不存在ZFN或序列特异性MN的情况下，用SDF转染细胞。每个目标将从核型稳定性、遗传完整性、产生或维持IPS细胞表型的能力以及体外分化为HSC的能力方面评价校正细胞。畸胎瘤形成将通过Core C进行评价，校正后的基因表达模式也将通过Core B和UCSF Array Core进行评价。虽然将使用逆转录病毒重编程的IPS细胞进行初始研究，但随着新的患者特异性IPS细胞系通过项目1和核心B获得，将对其进行校正和评价。最后，将在项目3中评价项目2中产生的校正的IPS细胞分化为 造血前体将植入NOD-SCID小鼠用于造血重建。

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.