Non-viral gene therapy for sickle cell anemia

描述（由申请人提供）：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治疗SCA患者的基因转移方法。为了实现我们的目标，我们提出了一项R21/R33阶段的创新拨款申请，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阶段旨在建立概念验证，可以在R33开发阶段实施。该提案将验证一个假设，即2-球蛋白迷你基因可以转化来自人类SCA患者的CD34+干细胞，并产生用于人类迷你移植的持续的红细胞特异性转基因表达，并将改善患者的临床表型。在R21探索阶段，Specific Aim 1被设计用于优化睡美人转座子（SB-Tn）和小鼠骨髓（BM）和造血干细胞（hsc）的转染效率。在特异性目标2中，我们将检测转化的镰刀转基因小鼠BM和表型c-kit+Thy1+Lineage(Lin)- sca1 + (KTLS)细胞的长期培养起始细胞（ltc - ic）， IHK驱动的转基因表达在ltc - ic中的红细胞特异性和持久性，以及在离体培养中的SB-Tn转位位点。随着R21研究的成功完成，我们将进入R33开发阶段，通过检测红细胞镰状细胞、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状小鼠对干细胞基因治疗的生物学反应。R33期特异性Aim 1旨在检测来自外周血CD34+人群的镰状人祖细胞中红细胞特异性2-珠蛋白转基因。在这个阶段，我们将利用癌症中心的转化治疗核心系统，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。在特异性目标2中，我们将在NOD-SCID和NY1DD镰状小鼠中进行自体工程化造血干细胞的非消融微型移植。在特异性目标3中，在获得所有必要的监管和监督批准后，我们将在5名成年人类镰状血癌患者中测试最终构建物，使用来自动员（但不是G-CSF）外周血的自体工程CD34+细胞，采用非消融微型移植方案。测试的终点将包括所有必要的监测和确保受试者的安全性，以及与工程植入稳定性和有效性的长期评估相关的终点。本阶段的所有载体和细胞产品都将通过本机构经美国国立卫生研究院批准的GMP明尼苏达州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，并在基因修饰的hsc的低强度自体移植后维持改善的镰状表型。

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.