Non-viral gene therapy for sickle cell anemia

描述(申请人提供)：镰状细胞性贫血(SCA)是最常见的遗传性血液病之一，由血红蛋白(Hb)的2-珠蛋白链中的单一氨基酸替代引起。我们研究计划的长期目标是开发一种安全有效的基因转移方法来治疗SCA患者。为了实现我们的目标，我们提出了R21/R33阶段性创新拨款申请，使用一种新的非病毒基因疗法，该疗法将在转基因镰刀鼠中进行优化，并最终在SCA患者中进行测试。R21阶段旨在建立可在R33开发阶段实施的概念验证。该提案将检验这样一个假设，即2-珠蛋白微型基因可以转化来自人类SCA患者的CD34干细胞，并产生转基因的持久红系特异性表达，用于人类微型移植，并将改善患者的临床表型。在R21的探索阶段，特异靶1被设计用于优化睡美人转座子(SB-TN)和在小鼠骨髓(BM)和造血干细胞(HSCs)中的转座效率。在具体目标2中，我们将检测转化的镰刀转基因小鼠BM和表型c-kit Thy1谱系(LIN)-Sca1(KTLS)细胞的长期培养启动细胞(LTC-ICs)，LTC-ICs中IHK驱动的转基因表达的红系特异性和持久性，以及体外培养中SB-TN转座的位置。随着R21研究的成功完成，我们将实施R33的开发阶段，并通过检测红细胞镰状、Hb表达、溶血、氧化应激、血管炎症和血管闭塞来检测转基因镰刀鼠对干细胞基因治疗的生物学反应。R33阶段的特定目标1旨在测试来自外周血CD34群体的镰状人类祖细胞中的红系特异性2-珠蛋白转基因。在这一阶段，我们将利用癌症中心的转化性治疗核心系统，开始将载体生成和细胞治疗过程从研究状态转移到GMP状态。在具体目标2中，我们将在NOD-SCID和NY1DD镰刀鼠身上进行自体、工程化HSCs的非消融性微型移植。在具体目标3中，在获得所有必要的监管和监督批准后，我们将在五名成年人类镰刀患者身上测试最终构建，使用动员(但不是G-CSF)外周血中的自体工程CD34细胞，进行非消融性微型移植方案。测试的终点将包括监测和确保受试者安全所需的所有终点，以及与工程植入稳定性和有效性的长期评估相关的终点。在这个阶段，所有载体和细胞产品都将通过我们机构获得NIH批准的明尼苏达州GMP分子和细胞治疗设施进行生产和质量控制。我们的最终目标是证明2-珠蛋白转基因可以转化SCA患者的CD34干细胞，并在降低强度的基因修饰的HSCs自体移植后保持改善的镰刀表型。 公共卫生相关性：镰状细胞性贫血(SCA)是最常见的遗传性血液病之一，影响着超过72,000名美国人和全球数千人(NIH出版物第96-4057号)。这种无情的疾病表现为溶血性贫血和导致器官损害的痛苦的血管闭塞危机，对生活质量和寿命有重大影响，造成数百万人的护理费用和经济生产力损失。包括羟基脲、交换输血、预防性抗生素和干细胞移植在内的治疗改善了这些患者的前景。然而，尽管取得了这些进展，许多患者继续经历痛苦的危象、急性胸部综合征和中风，所有这些都对生活质量和时间造成了累积的损失。为了解决这些问题，我们建议将正常的人类2-珠蛋白基因以非病毒方式转移到造血干细胞中，并将这些基因改变的干细胞重新引入骨髓。这些研究将首先在转基因镰状细胞小鼠身上进行；如果成功，这些技术将应用于来自人类镰状细胞患者的干细胞。我们将使用一种独特的非病毒基因治疗方法来避免与病毒载体基因转移相关的潜在副作用。该研究项目的最终目标是开发和评估一种创新的基因治疗方法，这种方法具有治疗和纠正SCA的潜力。

DESCRIPTION (provided by applicant): Sickle cell anemia (SCA) is one of the most common inherited hematological diseases, and caused by a single amino acid substitution in the 2-globin chain of hemoglobin (Hb). The long-range goal of our research program is to develop a safe and effective gene transfer approach for treating SCA patients. To accomplish our objective, we propose an R21/R33 phased innovation grant application using a novel non- viral gene therapy that will be optimized in transgenic sickle mice and ultimately tested in patients with SCA. The R21 phase is designed to establish proof-of-concept that can be implemented for the R33 developmental phase. The proposal will test the hypothesis that a 2-globin minigene can transform CD34+ stem cells from human SCA patients and generate persistent erythroid-specific expression of the transgene for use in human mini-transplants and will improve the clinical phenotype in patients. In the R21 exploratory phase, Specific Aim 1 is designed to optimize the Sleeping Beauty transposon (SB-Tn) and transfection efficiency in mouse bone marrow (BM) and hematopoietic stem cells (HSCs). In Specific Aim 2, we will examine transformed sickle transgenic mouse BM and phenotypic c-kit+Thy1+Lineage(Lin)-Sca1+ (KTLS) cells for long-term culture initiating cells (LTC-ICs), erythroid-specificity and persistence of IHK- driven transgene expression in LTC-ICs, and SB-Tn transposition sites in ex vivo culture. With successful completion of the R21 studies, we will implement the R33 development phase and examine the biological response of transgenic sickle mice to stem cell gene therapy by examining red cell sickling, Hb expression, hemolysis, oxidative stress, vascular inflammation and vaso-occlusion. Specific Aim 1 of the R33 phase is designed to test the erythroid-specific 2-globin transgene in sickle human progenitor cells derived from the peripheral blood CD34+ population. During this stage, we will begin the process of transferring the vector generation and cell therapy processes from research status to GMP status, taking advantage of the Cancer Centers Translational Therapy Core system. In Specific Aim 2, we will carry out non-ablative mini-transplant of autologous, engineered HSCs in NOD-SCID and NY1DD sickle mice. In Specific Aim 3, after obtaining all necessary regulatory and oversight approvals, we will test the final construct in five adult human sickle patients, using autologous engineered CD34+ cells from mobilized (but not by G-CSF) peripheral blood in a non-ablative mini-transplant protocol. Endpoints tested will include all those necessary to monitor and ensure subject safety, as well as those related to long-term assessment of engineered engraftment stability and efficacy. All vector and cell products at this stage will have been produced and quality controlled through our institution's NIH approved GMP Minnesota Molecular and Cellular Therapy facility. Our ultimate goal is to show that the 2-globin transgene can transform CD34+ stem cells from SCA patients and sustain an improved sickle phenotype after reduced intensity autologous transplantation of the gene-modified HSCs. PUBLIC HEALTH RELEVANCE: Sickle cell anemia (SCA) is one of the most common inherited hematological disorders and affects over 72,000 Americans and thousands of others worldwide (NIH Publication No. 96-4057). This relentless disease, manifested by hemolytic anemia and painful vaso-occlusive crises leading to organ damage, has a significant impact on the quality and length of life, costing millions in care and lost economic productivity. Therapies including hydroxyurea, exchange transfusion, prophylactic antibiotics, and stem cell transplants have improved the outlook for these patients. However, despite these advances, many patients continue to experience painful crises, acute chest syndromes, and strokes, all of which take their accumulative toll on the quality and length of life. To address these issues, we propose a non-viral transfer of the normal human 2-globin gene into hematopoietic stem cells and re-introduction of these genetically altered stem cells into bone marrow. These studies will initially be carried out in transgenic sickle cell mice; and if successful the techniques will be applied to stem cells from human sickle cell patients. We will use a unique non-viral gene therapy approach to avoid the potential side effects associated with viral vector gene transfer. The ultimate goal of this research project is to develop and evaluate in patients an innovative gene therapy approach that has the potential to treat and correct SCA.