Development and validation of novel mouse models and expression vectors for characterizing severe alpha-thalassemia pathophysiology and evaluating gene therapy approaches.

开发和验证新型小鼠模型和表达载体，用于表征严重α-地中海贫血病理生理学和评估基因治疗方法。

• 批准号: 10659630
• 负责人: STEFANO RIVELLA
• 金额: $ 60.43万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10659630
• 关键词: Adult; Adverse effects; Affect; Allogeneic Bone Marrow Transplantation; Allogenic; Anemia; Animals; Area; Blood Transfusion; Cell Line; Cells; Cessation of life; Clinical; Clinical assessments; Coagulation Process; Deposition; Development; Disease; Emigrations; Erythrocytes; Erythroid; Erythroid Cells; Erythropoiesis; Erythropoietin; Event; Failure to Thrive; Fetal Liver; Functional disorder; Gene Deletion; Gene Transduction Agent; Genes; Genetic; Genetic Diseases; Geographic Locations; Goals; Graft Rejection; Hematocrit procedure; Hematology; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hemoglobin concentration result; Hemoglobinopathies; Hepatosplenomegaly; Human; Hypoxemia; Individual; Inherited; Iron; Iron Overload; Kidney; Knock-out; Knowledge; Lentivirus Vector; Life; Liver; Locus Control Region; Longevity; Modeling; Modification; Multiple Organ Failure; Mus; Mutation; Organ; Oxygen; Pathology; Patients; Periodicals; Phenotype; Production; Proteins; Publishing; Safety; Sickle Cell Anemia; Splenomegaly; Techniques; Testing; Therapeutic; Transplantation; Validation; Work; alpha Globin; alpha-Thalassemia; beta Globin; beta Thalassemia; curative treatments; design; disease phenotype; ethnic minority; experience; expression vector; gene therapy; genome editing; graft vs host disease; hepcidin; improved; insight; interest; iron metabolism; mature animal; mouse model; novel; novel strategies; novel therapeutics; prevent; promoter; racial minority; socioeconomics; therapeutic gene; tool; vector

ABSTRACT a-Thalassemia (a-thal) is caused by insufficient production of the a-globin protein due to either deletional or non- deletional mutations of endogenous a-globin genes. In patients with severe a-thal (no or minimal synthesis of a- globin chains), a blood transfusion independent-state is achievable through allogeneic bone marrow transplantation, but this approach is limited to only some patients and is plagued by potential serious adverse effects, such as graft rejection or graft-versus-host disease. No mouse models of severe a-thal are available to study this disease and to test new therapies. Our proposed work will address these knowledge gaps by developing, characterizing, and validating mouse models and gene therapy vectors for treating severe a-thal. We hypothesize that new mouse models of a-thal will define the basic mechanism that governs RBC synthesis in the presence of excess ß-globin chains and how it affects erythropoiesis, iron metabolism and coagulation. In our first aim we will characterize these features in novel mouse models of severe a-thalassemia. As preliminary studies, we generated adult animals that do not produce a-globin chains (AG-KO) through transplantation of both AG-KO fetal liver and conditional AG-cKO hematopoietic stem cells into wild-type recipient mice. These animals demonstrate a worsening phenotype, with red blood cells (RBC) that express only b-globin chains. Due to severe limitation of these RBC to deliver oxygen, the mice eventually succumb to a condition resembling hypoxemia, showing splenomegaly, liver and kidney iron deposition, and vaso-occlusive events. We are now generating animals that only express one copy of the a-globin gene to characterize this disease in the context of minimal synthesis of a-globin chains. Most of the patients affected by a-thal carry large deletions of the a-globin genes. These deletions represent a serious challenge for gene therapy approaches based on genome editing. Therefore, we hypothesize that severe a-thal can be safely rescued by gene addition. In our second aim we will fully validate lentiviral vectors carrying the a-globin gene for their safety and ability to reverse the most severe forms of a-thal. We identified ALS20aI, in which a-globin is under control of the ß-globin promoter and its locus control region, as the most efficient vector. One copy of ALS20αI yields exogenous a-globin at a level comparable to that produced by one endogenous a-globin gene. Indeed, ALS20aI rescues animals generated with AG-KO fetal liver or conditional AG-cKO hematopoietic stem cells, suggesting that a relatively low vector copy number could result in dramatic therapeutic benefits. We will test ALS20aI or its derivatives for their ability to express the safest and highest level of a-globin in mouse hematopoietic stem cells and human-derived erythroid cell lines that synthesize low or no a-globin chains. We will then evaluate the constructs for their ability to rescue the abnormal features observed in a-thal patient cells. Thus, the goals of this study are to develop novel adult mouse models of a-thal and an effective gene therapy approach for this disease.

抽象的 a- thalassymia（A-thal）是由于缺失或非 - 内源性A-珠蛋白基因的缺失突变。在严重A-THAL的患者中（无或最少合成A- 球蛋白链），通过同种异体骨髓可以实现输血独立国家 移植，但是这种方法仅限于某些患者，并且受到潜在严重的敌人的困扰 效果，例如图形排斥或图形 - 抗宿主疾病。没有严重A-thal的鼠标模型可用于 研究这种疾病并测试新疗法。我们提出的工作将通过 开发，表征和验证小鼠模型和基因治疗载体，以治疗严重的A-THAL。 我们假设A-THAL的新鼠标模型将定义控制RBC合成的基本机制 在存在过多的β-球蛋白链的情况下，它如何影响促红细胞生成，铁代谢和凝结。 我们的第一个目标我们将在新型的严重A-甲性贫血的新型小鼠模型中表征这些特征。作为初步 研究，我们产生了不产生A-氯珠蛋白链（AG-KO）的成年动物 AG-KO胎儿肝脏和有条件的Ag-CKO造血干细胞成野生型受体小鼠。这些动物 表现出令人担忧的表型，红细胞（RBC）仅表达B-珠蛋白链。由于严重 这些RBC的限制供应氧气，小鼠有时会屈服于类似低氧血症的疾病， 显示脾肿大，肝脏和肾脏铁沉积以及血管熟悉的事件。我们现在正在生成 仅表达A-珠蛋白基因的一个副本以在最小的背景下表征该疾病的动物 A-珠蛋白链的合成。受A-thal影响的大多数患者携带大量缺失A-珠蛋白基因。 这些缺失是基于基因组编辑的基因治疗方法的严重挑战。 因此，我们假设可以通过添加Gene可以安全地挽救严重的A-THAL。在我们的第二个目标中，我们将 完全验证携带A-Globin基因的慢病毒载体，以扭转最严重的能力 我们确定了ALS20AI，其中A-Globin正在控制β-珠蛋白启动子及其基因座 控制区域，是最有效的向量。一个ALS20αI的副本可在水平上产生外源A-珠蛋白 与一个内源性a-蛋白基因产生的相当。确实，Als20ai回应产生的动物 与AG-KO胎儿肝或有条件的Ag-CKO造血干细胞，表明相对较低的载体 拷贝数可能会带来巨大的理论收益。我们将测试ALS20AI或其衍生物的能力 表达小鼠造血干细胞中最安全和最高水平的A-珠蛋白和人类衍生的 红细胞链合成的红细胞系线细胞系。然后，我们将评估其能力的结构 挽救在A-THAL患者细胞中观察到的异常特征。那是这项研究的目标是发展 A-thal的新型成年小鼠模型和该疾病的有效基因治疗方法。