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 (a-thal) 是由于α-珠蛋白缺失或非缺失导致α-珠蛋白产生不足引起的。 内源性a-珠蛋白基因的缺失突变。在患有严重a-thal（a-thal没有合成或合成很少）的患者中 珠蛋白链），通过同种异体骨髓可以实现输血独立状态 移植，但这种方法仅限于部分患者，并且存在潜在的严重不良反应 效应，例如移植物排斥或移植物抗宿主病。没有严重致命性死亡的小鼠模型可用于 研究这种疾病并测试新疗法。我们提出的工作将通过以下方式解决这些知识差距 开发、表征和验证用于治疗严重 a-thal 的小鼠模型和基因治疗载体。 我们假设新的 a-thal 小鼠模型将定义控制红细胞合成的基本机制 存在过量 β-珠蛋白链时及其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严重α-地中海贫血的新型小鼠模型中表征这些特征。作为初步 研究中，我们通过移植两种细胞培育出不产生 a-珠蛋白链 (AG-KO) 的成年动物 AG-KO胎儿肝脏和条件AG-cKO造血干细胞进入野生型受体小鼠。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 这些红细胞输送氧气的能力受到限制，小鼠最终会出现类似低氧血症的情况， 显示脾肿大、肝脏和肾脏铁沉积以及血管闭塞事件。我们现在正在生成 仅表达 a-珠蛋白基因的一个拷贝的动物在最小的背景下表征了这种疾病 α-珠蛋白链的合成。大多数受 a-thal 影响的患者携带大量 a-珠蛋白基因缺失。 这些缺失对基于基因组编辑的基因治疗方法提出了严峻的挑战。 因此，我们假设严重的α-thal可以通过基因添加安全地挽救。在我们的第二个目标中，我们将 全面验证携带a-珠蛋白基因的慢病毒载体的安全性和逆转最严重疾病的能力 a-thal的形式。我们鉴定了 ALS20aI，其中 a-珠蛋白受到 ß-珠蛋白启动子及其基因座的控制 控制区域，作为最有效的载体。 ALS20αI 的一个拷贝可产生一定水平的外源 a-珠蛋白 与一种内源性a-珠蛋白基因产生的结果相当。事实上，ALS20aI 拯救了产生的动物 与 AG-KO 胎儿肝脏或有条件的 AG-cKO 造血干细胞相比，表明相对较低的载体 拷贝数可能会带来显着的治疗效果。我们将测试 ALS20aI 或其衍生物的能力 在小鼠造血干细胞和人源造血干细胞中表达最安全和最高水平的a-珠蛋白 合成低α-珠蛋白链或不合成α-珠蛋白链的红系细胞系。然后我们将评估构建体的能力 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 新型成年小鼠 a-thal 模型以及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.