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

摘要 A-地中海贫血(a-thal)是由于a-珠蛋白的缺失或非缺失而导致的 内源性α-珠蛋白基因的缺失突变。在重度甲状旁腺素(无或极少合成a- 珠蛋白链)，一种输血独立状态可以通过异基因骨髓实现 移植，但这种方法仅限于一些患者，并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。目前还没有严重甲型肝炎的小鼠模型可用来 研究这种疾病并测试新的治疗方法。我们提议的工作将通过以下方式解决这些知识差距 开发、鉴定和验证治疗重症贫血的小鼠模型和基因治疗载体。 我们假设，新的α-Thal小鼠模型将定义控制RBC合成的基本机制 以及它如何影响红细胞生成、铁代谢和凝血。在……里面 我们的第一个目标是，我们将在严重α-地中海贫血的新型小鼠模型中描述这些特征。作为初步准备 在研究中，我们通过移植两者产生了不产生a-珠蛋白链(AG-KO)的成年动物 将AG-KO胎肝和条件AG-CKO造血干细胞导入野生型受体小鼠。这些动物 表现为恶化，红细胞(RBC)仅表达b-珠蛋白链。由于严重的 由于这些红细胞输送氧气的能力有限，小鼠最终会死于类似低氧血症的情况， 表现为脾肿大、肝肾铁沉积和血管闭塞事件。我们现在正在生成 只表达一份a-珠蛋白基因的动物在最小的情况下表征这种疾病 α-珠蛋白链的合成。大多数受甲状旁腺白血病影响的患者携带大量的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来说是一个严重的挑战。 因此，我们假设通过基因添加可以安全地挽救严重的甲型肝炎。在我们的第二个目标中，我们将 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 A-thal的形式。我们鉴定了ALS20aI，其中a-珠蛋白受？-珠蛋白启动子及其基因座的控制 控制区作为最有效的媒介。一个拷贝的ALS20αI在一定水平上产生外源α-珠蛋白 与一个内源性a-珠蛋白基因产生的基因相当。事实上，ALS20aI拯救了产生的动物 用AG-KO胎肝或条件AG-CKO造血干细胞，提示载体相对较低 拷贝数可能会带来显著的治疗效果。我们将测试ALS20aI或其衍生品的能力 在小鼠造血干细胞和人源造血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a-珠蛋白链的红系细胞系。然后，我们将评估这些构造的能力 以挽救患者细胞中观察到的异常特征。因此，本研究的目标是开发 甲状旁腺白血病新的成年小鼠模型及有效的基因治疗方法。

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.