GENOME EDITING FOR DEVELOPING A TREATMENT FOR BETA GLOBIN DISORDERS

描述（由申请人提供）：β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珠蛋白基因突变引起的，导致β珠蛋白合成减少或消失。如果没有β珠蛋白链配对， 过量的α珠蛋白链易于氧化成血红素、沉淀和损伤红细胞前体以及成熟红细胞，导致无效的红细胞生成和严重贫血。患有最严重形式的β地中海贫血的患者需要终身输血和铁螯合治疗。目前唯一的治疗方法是用组织相容性供体细胞进行骨髓移植，这对许多成年患者来说是一种有限的选择。本文提出的努力旨在最终显著改善 所有没有移植选择的患者，通过确定最佳的靶向基因工程方法来这样做。我们将探索，并排，三个有前途的方法单独和/或在某些组合：1）重新激活发育沉默的γ珠蛋白与过量的α珠蛋白配对（SA#1）; 2）下调α珠蛋白合成（SA#2）;和3）驱动治疗性γ珠蛋白基因靶向插入β珠蛋白基因座（SA#3）。为此，我们计划利用开源靶向基因组工程平台TALE效应子核酸酶（TALEN）来编辑原代正常或患者动员的外周血（MPB）hCD 34+细胞的相关基因组基因座。我们预期的遗传靶点包括a）有效的γ珠蛋白阻遏物BCL 11 A及其增强子，以及KLF 1（3个靶位点），B）β珠蛋白基因座内推定的γ珠蛋白阻遏物结合位点（3个靶位点），c）α珠蛋白启动子中α珠蛋白反式激活因子KLF 4的结合位点d）α珠蛋白基因座中的α +地中海贫血相关基因组位点（4个靶位点），和d）β珠蛋白基因座内用于插入治疗性γ珠蛋白基因的两个推定位点（2个靶位点）。首先使用正常MPB hCD 34+细胞评估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。将通过将编辑的正常hCD 34+细胞移植到免疫缺陷型NOD/SCID IL 2 γ缺失小鼠中来研究编辑和球蛋白调节的持久性。基于正常hCD 34+细胞评价的最有前途的方法将应用于β地中海贫血hCD 34+细胞，其中将检查体外和体内红系参数的改善。我们的初步数据，在正常的3个选定的基因组位点和β地中海贫血MPB hCD 34+细胞，强大的γ珠蛋白再激活，持久的编辑和γ再激活外源受体的有效离体编辑是令人信服的，并表明这种新的方法有可能被开发成β地中海贫血的治愈性疗法。

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.