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

摘要 α-地中海贫血（α-地中海贫血）是由于缺失或非缺失导致的α-珠蛋白产生不足而引起的。 内源性α-珠蛋白基因的缺失突变。在严重a-thal患者中（无或极少量a-thal合成）， 珠蛋白链），通过同种异体骨髓可实现输血独立状态 但是这种方法仅限于一些患者，并且受到潜在的严重不良反应的困扰。 影响，如移植物排斥或移植物抗宿主病。目前还没有严重的急性骨髓性白血病小鼠模型可用于 研究这种疾病并测试新的疗法。我们建议的工作将解决这些知识差距， 开发、表征和验证用于治疗严重骨髓炎的小鼠模型和基因治疗载体。 我们假设新的小鼠a-thal模型将确定控制红细胞合成的基本机制 在过量β-珠蛋白链的存在下，以及它如何影响红细胞生成、铁代谢和凝血。在 我们的第一个目标是在新的严重α-地中海贫血小鼠模型中表征这些特征。作为初步 在研究中，我们通过移植α-珠蛋白链和α-珠蛋白链，产生了不产生α-珠蛋白链（AG-KO）的成年动物。 将AG-KO胎肝和条件性AG-cKO造血干细胞移植到野生型受体小鼠中。这些动物 表现出恶化的表型，红细胞（RBC）仅表达b-珠蛋白链。由于严重 由于这些红细胞输送氧气的能力有限，小鼠最终死于类似低氧血症的疾病， 显示脾肿大、肝和肾铁沉积和血管闭塞事件。我们现在正在生成 仅表达一个拷贝的α-珠蛋白基因的动物，以在最小的 α-珠蛋白链的合成。大多数患有α-地中海的患者携带α-珠蛋白基因的大缺失。 这些缺失代表了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，严重的a-thal可以安全地挽救基因添加。在我们的第二个目标中， 充分验证携带a-珠蛋白基因的慢病毒载体的安全性和逆转最严重的 的形式。我们鉴定了ALS 20 α，其中α-珠蛋白在α-珠蛋白启动子及其基因座的控制下 控制区，作为最有效的载体。一个拷贝的ALS 20 αI产生的外源α-珠蛋白水平 与由一个内源性α-珠蛋白基因产生的相当。事实上，ALS 20可以拯救 与AG-KO胎肝或条件性AG-cKO造血干细胞，表明相对低的载体， 拷贝数的改变可能导致显著的治疗益处。我们将测试ALS 20及其衍生物的能力， 在小鼠造血干细胞和人源性造血干细胞中表达最安全和最高水平的α-珠蛋白， 合成低或不合成α-珠蛋白链的红系细胞系。然后，我们将评估这些结构的能力， 以挽救在A-Thal患者细胞中观察到的异常特征。因此，本研究的目标是开发 新的成年小鼠a-thal模型和针对该疾病的有效基因治疗方法。

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.