Non-viral gene therapy for sickle cell anemia

镰状细胞性贫血的非病毒基因治疗

• 批准号: 7900987
• 负责人: ROBERT P HEBBEL
• 金额: $ 37.74万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7900987
• 关键词: Address; Adult; Adverse effects; Affect; African; American; Amino Acid Substitution; Antibiotics; Applications Grants; Area; Autologous; Autologous Transplantation; Benign; Biological; Blood; Blood Cells; Blood Vessels; Bone Marrow; Bone Marrow Cells; CD34 gene; CSF3 gene; Cancer Center; Caring; Cell Communication; Cell Lineage; Cell Therapy; Cells; Chromosomal Insertion; Clinical; DNA; Data; Detection; Development; Diabetes Mellitus; Disease; Dorsal; DsRed; Economics; Elements; Endothelial Cells; Engineering; Engraftment; Ensure; Epigenetic Process; Epitopes; Erythrocytes; Erythroid; Erythroid Cells; Eukaryotic Initiation Factors; Exhibits; Five-Year Plans; Gene Expression; Gene Transfer; Gene-Modified; Generations; Genes; Genome; Genomics; Globin; Goals; Hematological Disease; Hematopoietic; Hematopoietic stem cells; Hemoglobin; Hemolysis; Hemolytic Anemia; High Pressure Liquid Chromatography; Human; Hybrids; Inbred ICR Mice; Individual; Inherited; Institution; Link; Living Costs; Longevity; Measures; Mediating; Methodology; Minnesota; Modeling; Modification; Molecular; Monitor; Mus; Non obese; Organ; Outcome; Oxidative Stress; Pain; Patient Monitoring; Patients; Phase; Phenotype; Pluripotent Stem Cells; Population; Procedures; Process; Productivity; Proteins; Proto-Oncogene Protein c-kit; Protocols documentation; Publications; Quality Control; Recovery; Relative (related person); Reporter; Repression; Research; Research Personnel; Research Project Grants; Rewards; Risk; Safety; Severe Combined Immunodeficiency; Sickle Cell; Sickle Cell Anemia; Site; Skin; Sleeping Beauty; Solid; Somatic Cell; Sorting - Cell Movement; Specificity; Staging; Stem cell transplant; Stem cells; Stroke; System; Techniques; Technology; Testing; Thalassemia; Therapeutic; Time; Transcript; Transfection; Transfusion; Transgenes; Transgenic Animals; Transgenic Mice; Transgenic Organisms; Transplantation; Transposase; United States National Institutes of Health; Viral; Viral Vector; Western Blotting; Wild Type Mouse; acute chest syndrome; base; beta Globin; cell transformation; cell type; chromatin modification; clinical phenotype; design; design and construction; erythroid differentiation; experience; gene correction; gene therapy; high risk; human stem cells; hydroxyurea; improved; in vivo; innovation; member; mouse model; non-viral gene therapy; novel; novel strategies; peripheral blood; preference; programs; promoter; prophylactic; protein expression; public health relevance; response; sickling; transgene expression; vascular inflammation; vector

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.

描述（由申请人提供）：镰状细胞贫血（SCA）是最常见的遗传性血液学疾病之一，是由血红蛋白（HB）的2-球蛋白链中的单个氨基酸取代引起的。我们的研究计划的远程目标是开发一种治疗SCA患者的安全有效的基因转移方法。为了实现我们的目标，我们建议使用新型的非病毒基因疗法进行R21/R33分阶段创新赠款应用，该疗法将在转基因镰刀小鼠中进行优化，并最终在SCA患者中进行了测试。 R21阶段旨在建立可以在R33发育阶段实现的概念验证。该提案将检验以下假设：2-团体蛋白可以转化人类SCA患者的CD34+干细胞，并产生转基因的持续性红细胞特异性表达，以用于人类迷你移植剂中，并将改善患者的临床表型。在R21探索阶段，特定的目标1旨在优化小鼠骨髓（BM）和造血干细胞（HSC）中的睡美人转座子（SB-TN）和转染效率。在特定目标2中，我们将检查转变的镰状转基因小鼠BM和表型C-KIT+THY1+谱系（LIN）-SCA1+（KTLS）细胞，用于长期培养启动细胞（LTC-ISS），红脑特异性和IHK-DRIVEN型转移表达的rtc-sics和SB-TN培养物和SB-tn crubtion in vos and sb-tn crubition和sb-tn crubition in vy n vers and sb-tn。通过成功完成R21研究，我们将实施R33发育阶段，并通过检查红细胞疾病，HB表达，溶血，氧化应激，血管炎症和血管coc液来检查转基因小鼠对干细胞基因治疗的生物学反应。 R33期的具体目标1旨在测试源自外周血CD34+种群的镰状祖细胞中红细胞特异性2-珠蛋白转基因。在此阶段，我们将通过癌症中心转化治疗核心系统将媒介产生和细胞疗法过程从研究状态转移到GMP状态。在特定的目标2中，我们将在点点scID和NY1DD镰状小鼠中进行非全能的自动型HSC的迷你移植。在特定的目标3中，在获得所有必要的调节和监督批准后，我们​​将使用自体工程CD34+细胞在非平淡的微型小型植物方案中使用自体工程CD34+细胞测试最终结构。测试的端点将包括监控和确保主题安全的所有必要条件，以及与工程植入稳定性和功效的长期评估相关的内容。在此阶段，所有载体和细胞产品都将被生产，并通过我们机构批准的明尼苏达州分子和细胞治疗设施来控制质量。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，并在降低强度自体移植后，维持改善的镰状表型。 公共卫生相关性：镰状细胞贫血（SCA）是最常见的血液学疾病之一，影响了全世界72,000多名美国人和其他成千上万的美国人（NIH出版物号96-4057）。这种无情的疾病是由溶血性贫血表现出来的，疼痛的血管狭窄危机导致器官损害，对生活的质量和寿命产生了重大影响，损失了数百万的护理和失去的经济生产力。包括羟基脲，交换输血，预防性抗生素和干细胞移植的疗法改善了这些患者的前景。然而，尽管有这些进展，许多患者仍会经历痛苦的​​危机，急性胸部综合征和中风，所有患者都会累积他们的生活质量和生活时间。为了解决这些问题，我们提出了将正常人2-珠蛋白基因的非病毒转移到造血干细胞中，并将这些遗传学改变的干细胞重新引入到骨髓中。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的是在患者中开发和评估一种创新的基因治疗方法，具有治疗和纠正SCA的潜力。