GENOME EDITING FOR DEVELOPING A TREATMENT FOR BETA GLOBIN DISORDERS

描述(申请人提供)：贝塔地中海贫血是世界上最常见的遗传性血液疾病之一。它是由人类β珠蛋白基因突变导致的，导致β珠蛋白合成减少或丧失。如果没有贝塔球蛋白链可以配对， 过量的α-珠蛋白链容易氧化成半染色质，沉淀和破坏红细胞前体以及成熟的红细胞，导致无效的红细胞生成和严重的贫血。患有最严重形式的贝塔地中海贫血的患者需要终生输血和铁离子螯合治疗。目前唯一的治疗方法是使用组织相容的供体细胞进行骨髓移植，这对许多成年患者来说是有限的选择。这里提出的努力旨在最终显著改善临床情况。 所有没有移植选择的患者通过确定一种最佳的靶向基因工程方法来做到这一点。我们将并排探索三种有希望的单独和/或结合使用的方法：1)重新激活发育迟缓的伽马珠蛋白，使其与过量的阿尔法珠蛋白配对(SA#1)；2)下调阿尔法珠蛋白的合成(SA#2)；以及3)将治疗性伽马珠蛋白基因定向插入β珠蛋白基因(SA#3)。为此，我们计划利用一个开源的靶向基因组工程平台，TALE效应核酸酶(TALENS)来编辑原始的正常或患者动员的外周血(MPB)hCD34+细胞的相关基因组位点。我们预期的遗传靶点包括a)有效的伽马珠蛋白抑制物BCL11A及其增强子，以及KLF1(3个靶点)，b)β珠蛋白基因座内假定的伽马珠蛋白抑制物结合位点(3个靶点)，c)α珠蛋白启动子中的α珠蛋白反式激活因子KLF4的结合位点(3个靶点)，d)阿尔法+地中海贫血相关的阿尔法珠蛋白基因座中的基因组位点(4个靶点)，以及d)β珠蛋白基因座中插入治疗性伽马珠蛋白基因的两个假定位点(2个靶点)。编辑的效率和对珠蛋白表达、红细胞生成和其他潜在副作用的一般影响将首先使用正常的MPB hCD34+细胞进行评估。编辑和珠蛋白调节的持久性将通过将编辑后的正常hCD34+细胞移植到免疫缺陷NOD/SCID IL2γ缺失小鼠中来研究。基于对正常hCD34+细胞的评估，最有希望的方法将应用于β地中海贫血hCD34+细胞，在那里将检查体外和体内红系参数的改善。我们的初步数据表明，在正常和β地中海贫血的MPB hCD34+细胞中，3个选定的基因组座位和1个选定的基因组座位的高效体外编辑、强大的伽马珠蛋白再激活以及外源性受体的持久编辑和伽马再激活是令人信服的，并表明这种新方法有可能发展成为治疗β地中海贫血的方法。

DESCRIPTION (provided by applicant): Beta thalassemia is one of the most prevalent forms of heritable blood disorders in the world. It is caused by mutations in human beta globin genes that result in reduced or abolished beta globin synthesis. Without beta globin chains to pair with, excess alpha globin chains are susceptible to oxidation to hemichromes, precipitate and damage red blood cell precursors as well as mature red blood cells, leading to ineffective erythropoiesis and profound anemia. Patients afflicted with the most severe forms of beta thalassemia require lifelong blood transfusions and iron chelation treatment. The only cure at present is BM transplantation with histocompatible donor cells, a limited option for many adult patients. The effort proposed here aim to ultimately, significantly improve the clinical picture in all patients with no transplant option by identifying an optimal targeted genetic engineering approach to do so. We will explore, side-by-side, three promising approaches alone and/or in certain combinations: 1) to reactivate developmentally silent gamma globin to pair with excess alpha globin (SA#1); 2) to downregulate alpha globin synthesis (SA#2); and 3) to drive targeted insertion of a therapeutic gamma globin gene into the beta globin locus (SA#3). For this purpose we plan to utilize an open-source targeted genome engineering platform, TALE effector nucleases (TALENs) to edit the relevant genomic loci of primary normal or patient mobilized peripheral blood (MPB) hCD34+ cells. Our intended genetic targets include a) potent gamma globin repressors BCL11A and its enhancer, as well as KLF1 (3 target sites), b) putative gamma globin repressor binding sites within the beta globin locus (3 target sites), c) the binding sites f alpha globin transactivator KLF4 in alpha globin promoters (3 target sites), d) alpha+ thalassemia- associated genomic sites in alpha globin locus (4 target sites), and d) two putative sites within beta globin locus for the insertion of a therapeutic gamma globin gene (2 target sites). The efficiency of editing and general effects on globin expression, erythropoiesis, and other potential side-effects will first be evaluated using normal MPB hCD34+ cells. The durability of editing and globin modulation will be studied by transplantation of edited normal hCD34+ cells into immunodeficient NOD/SCID IL2γnull mice. The most promising approaches based on the evaluation of normal hCD34+ cells will be applied to beta thalassemic hCD34+ cells where the improvement in erythroid parameters both in vitro, and in vivo, will be examined. Our preliminary data on efficient ex-vivo editing of 3 selected genomic loci in normal and 1 in beta thalassemic MPB hCD34+ cells, robust gamma globin reactivation, and durable editing and gamma reactivation in exogeneic recipients are compelling and suggest that this novel approach has the potential to be developed into curative therapies for beta thalassemia.