GENOME EDITING FOR DEVELOPING A TREATMENT FOR BETA GLOBIN DISORDERS

用于开发β珠蛋白疾病治疗方法的基因组编辑

• 批准号: 8757148
• 负责人: THALIA STAMATOYANNOPOULOS
• 金额: $ 33.6万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8757148
• 关键词: 3' Untranslated Regions; Adult; Adverse effects; Allogenic; Anemia; Autologous; Binding; Binding Sites; Blood Transfusion; Bone Marrow Transplantation; CD34 gene; Candidate Disease Gene; Cells; Clinical; Comorbidity; Complement; Complex; Custom; DNA Double Strand Break; Data; Disease; Down-Regulation; Engraftment; Enhancers; Ensure; Erythrocytes; Erythroid; Erythroid Cells; Erythropoiesis; Evaluation; Exons; Future; Gene Targeting; Genes; Genetic; Genetic Engineering; Genome; Genome engineering; Genomics; Globin; Hematological Disease; Hematopoietic Stem Cell Transplantation; Hemoglobin; Hemolysis; Hereditary Disease; Human; Hybrids; IL2 gene; In Vitro; Inherited; Insertional Mutagenesis; Introns; Iron Chelation; Iron Overload; K562 Cells; Knockout Mice; Lead; Life; Mediating; Mus; Mutation; Nonhomologous DNA End Joining; Normal Cell; Organ; Patients; Pattern; Phenotype; Procedures; Production; Reporting; Side; Site; Stem cells; Surface; Technology; Thalassemia; Therapeutic; Trans-Activators; Transduction Gene; Transfusion; Transgenes; Transplantation; alpha Globin; alpha-1 Globin; alpha-Thalassemia; base; beta Globin; beta Thalassemia; design; erythroid differentiation; gamma Globin; gene correction; gene therapy; hemichrome; improved; in vivo; insertion/deletion mutation; leukemogenesis; novel; novel strategies; nuclease; open source; oxidation; peripheral blood; promoter; public health relevance; repaired; site-specific integration; volunteer

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 IL2?null 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.

描述（由申请人提供）：β地中海贫血是世界上最普遍的可遗传血液疾病形式之一。它是由人β球蛋白基因突变引起的，导致β球蛋白合成减少或废除。没有beta globin链可以配对， 多余的α球蛋白链易于氧化，氧化对半色素，沉淀和损害红细胞前体以及成熟的红细胞，导致无效的红细胞生成和深远的贫血。患有最严重形式的β丘脑贫血形式的患者需要终身输血和铁螯合治疗。目前，唯一的治愈方法是用组织兼容的供体细胞进行BM移植，这是许多成年患者的有限选择。这里提出的努力旨在最终，显着改善 所有没有移植选择的患者都通过确定最佳的靶向基因工程方法来做到这一点。我们将单独和/或某些组合中并排探索三种有前途的方法：1）重新激活发育中的无声伽马球蛋白与多余的alpha globin配对（SA＃1）； 2）下调α球蛋白合成（SA＃2）; 3）驱动将治疗性γ球蛋白基因插入β球蛋白基因座（SA＃3）。为此，我们计划利用开源的靶向基因组工程平台，故事效应核酸酶（TALENS）编辑原发性正常或患者动员外周血（MPB）HCD34+细胞的相关基因组基因座。我们预期的遗传靶标包括a）有效的γ球蛋白阻遏物BCL11A及其增强剂以及KLF1（3个目标位点），b）假定的γ球蛋白抑制剂beta球蛋白基因座（3个目标位点）内的假定γ球蛋白压抑剂结合位点，c）Alpha GlobIn blablf4 blaplf4 aLPha aLPHA INPHA inpha aLPHA INPHA）Alpha inpha） alpha球蛋白基因座（4个靶位点）中的丘脑症与基因组相关位点，d）β球蛋白基因座中的两个推定位点，用于插入治疗性伽玛球蛋蛋白基因（2个靶位点）。首先使用正常的MPB HCD34+细胞评估编辑和对球蛋白表达，红细胞生成和其他潜在副作用的一般影响的效率。通过将正常的HCD34+细胞移植到免疫缺陷的NOD/SCID IL2？NULL小鼠中，将研究编辑和球蛋白调制的耐用性。基于正常HCD34+细胞的评估的最有希望的方法将应用于β丘脑血症HCD34+细胞，其中将检查体外和体内红细胞参数的改善。 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.