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）2-珠蛋白链中的单个氨基酸取代引起。我们研究项目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，我们提出了一项 R21/R33 分阶段创新拨款申请，该申请使用一种新型非病毒基因疗法，该疗法将在转基因镰状小鼠中进行优化，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。该提案将测试以下假设：2 珠蛋白小基因可以转化人类 SCA 患者的 CD34 干细胞，并产生转基因的持续红系特异性表达，用于人类小型移植，并将改善患者的临床表型。在R21探索阶段，Specific Aim 1旨在优化睡美人转座子（SB-Tn）以及小鼠骨髓（BM）和造血干细胞（HSC）中的转染效率。在具体目标 2 中，我们将检查转化的镰状转基因小鼠 BM 和表型 c-kit Thy1 Lineage(Lin)-Sca1 (KTLS) 细胞的长期培养起始细胞 (LTC-IC)、LTC-IC 中 IHK 驱动的转基因表达的红系特异性和持久性，以及离体培养中的 SB-Tn 转座位点。随着R21研究的成功完成，我们将进入R33开发阶段，通过检查红细胞镰状化、Hb表达、溶血、氧化应激、血管炎症和血管闭塞来检查转基因镰状小鼠对干细胞基因治疗的生物学反应。 R33 阶段的具体目标 1 旨在测试源自外周血 CD34 群体的镰状人类祖细胞中的红细胞特异性 2-珠蛋白转基因。在此阶段，我们将利用癌症中心转化治疗核心系统，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 镰状小鼠中进行自体工程 HSC 的非烧蚀微型移植。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​​将在非消融性小型移植方案中使用来自动员（但不是通过 G-CSF）外周血的自体工程 CD34 细胞，在 5 名成人镰状细胞患者中测试最终构建体。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功效的长期评估相关的终点。此阶段的所有载体和细胞产品将通过我们机构的 NIH 批准的 GMP 明尼苏达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来自 SCA 患者的 CD34 干细胞，并在基因修饰的 HSC 降低强度自体移植后维持改善的镰状表型。 公共健康相关性：镰状细胞性贫血 (SCA) 是最常见的遗传性血液疾病之一，影响超过 72,000 名美国人和全世界数千人（NIH 出版物号 96-4057）。这种持续不断的疾病，表现为溶血性贫血和导致器官损伤的痛苦的血管闭塞危机，对生命质量和寿命产生重大影响，造成数百万人的护理费用和经济生产力损失。羟基脲、换血、预防性抗生素和干细胞移植等疗法改善了这些患者的前景。然而，尽管取得了这些进步，许多患者仍然经历痛苦的​​危机、急性胸部综合征和中风，所有这些都对生命质量和寿命造成累积损害。为了解决这些问题，我们提出将正常人 2-珠蛋白基因非病毒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。这些研究最初将在转基因镰状细胞小鼠中进行；如果成功，该技术将应用于人类镰状细胞病患者的干细胞。我们将使用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一种创新的基因治疗方法并在患者中进行评估，该方法有可能治疗和纠正 SCA。

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.