Site-Specific Correction of Sickle Cell Disease Using Acoustofludic Gene Delivery

使用声流控基因传递对镰状细胞病进行位点特异性校正

• 批准号: 10023174
• 负责人: Jason Nathaniel Belling
• 金额: $ 3.23万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-16 至 2021-06-10
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10023174
• 关键词: Acoustics; Address; Adult; Affect; Autoimmune Diseases; Autologous; Autologous Transplantation; Biological Assay; CD34 gene; CRISPR/Cas technology; Cell Line; Cell Membrane Permeability; Cell Therapy; Cell membrane; Cell model; Cell physiology; Cells; Clinical; Clinical Management; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complex; DNA Sequence Alteration; Development; Devices; Disease; Electroporation; Engraftment; Erythrocytes; Erythroid; Flow Cytometry; Foundations; Frequencies; Gene Cluster; Gene Delivery; Gene-Modified; Generations; Genes; Goals; Heart Diseases; Hematological Disease; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hemoglobin; Hemoglobinopathies; Hereditary Disease; High Pressure Liquid Chromatography; Human Cell Line; Improve Access; Intervention; K-562; Lung diseases; Mechanics; Mediating; Medical; Methods; Microfluidics; Modeling; Mutation; Output; Patients; Peripheral Blood Mononuclear Cell; Permeability; Polymerase Chain Reaction; Process; Production; Property; Quality of life; Reaction; Recovery; Research; Ribonucleoproteins; Risk; Running; Sickle Cell; Sickle Cell Anemia; Site; Stem cell transplant; Structure; System; T-Lymphocyte; Techniques; Technology; Testing; Therapeutic; Toxic effect; Transfection; Translations; Xenograft procedure; base; beta Globin; bioinformatics tool; cell injury; clinical practice; clinical translation; clinically relevant; cost; design; gene correction; gene therapy; graft vs host disease; immunogenic; innovation; insertion/deletion mutation; interdisciplinary collaboration; lipofection; mouse model; new technology; next generation sequencing; processing speed; repaired; stem; stem cell population; stem cell therapy; stem cells; targeted nucleases; uptake; vector; voltage

Project Summary Sickle cell disease (SCD) is among the most common monogenetic inherited disorders. Clinical management of SCD is primarily supportive. However, in the most severe cases, the only definitive curative option for patients suffering from SCD is an allogeneically matched hematopoietic stem cell transplant. This hemoglobinopathy directly affects the structure and function of hemoglobin, leading to deficiencies of β-globin chains in the development of functional adult hemoglobin. Furthermore, the lack of fully matched donors for patients to receive a stem cell transplant runs the risk of adverse immunogenic reactions, such as auto-immune disorders or graft-versus-host disease. Recent efforts to address this disease and its clinical sequela have focused on gene therapies based on the transplantation of autologous gene-modified hematopoietic stem & progenitor cells (HSPC), where a patient's own cells are corrected and reinfused to enable production of fully functioning erythrocytes. However, non-viral strategies for the batch processing of stem cell gene therapies are known to be inefficient and are unable to meet clinical demands. We hypothesize that the optimization of an acoustofluidic therapeutic platform that physically permeabilizes cells for the delivery of CRISPR-Cas9 biomolecules will address this technologic gap. This high-throughput gene-delivery strategy will enable our long-term goal to generate gene-modified stem cell therapies quickly and efficiently for curing sickle cell disease. This physical permeabilization process renders target cells transiently permeable, enabling vector uptake while minimizing damage to the cell membrane and maintaining high levels of viability. In order to achieve our clinical target, our proposed specific aims include: 1) optimize acoustofluidic gene delivery in model cell lines harboring the sickle cell mutation and 2) evaluate site-specific correction of the sickle cell disease mutation in hematopoietic stem and progenitor cells. Given the utility of this acoustofluidic technology, there is a wide range of heart, lung, and blood disorders that can be addressed, overcoming the state of the art for gene delivery. We expect the generation of rapid and safe gene-modified stem cell therapies using our acoustofludic technology will greatly improve access to these medical interventions and the quality of life for patients with the most severe cases of SCD.

项目摘要 镰状细胞病（SCD）是最常见的单基因遗传性疾病之一。临床 SCD的管理主要是支持性的。然而，在最严重的情况下， SCD患者的治疗选择是同种异体匹配的造血干细胞 移植这种血红蛋白病直接影响血红蛋白的结构和功能，导致 功能性成人血红蛋白发育中β-珠蛋白链的缺陷。而且 缺乏完全匹配的供体供患者接受干细胞移植存在不良反应的风险。 免疫原性反应，如自身免疫性疾病或移植物抗宿主病。最近努力 针对这一疾病及其临床后遗症的基因治疗主要是基于 自体基因修饰的造血干细胞和祖细胞（HSPC）移植，其中 患者自身的细胞被校正并重新输注，以能够产生功能齐全的红细胞。 然而，已知用于干细胞基因疗法的批量处理的非病毒策略是可行的。 效率低，不能满足临床需要。我们假设， 声流体治疗平台，其物理地透化细胞以递送 CRISPR-Cas9生物分子将填补这一技术空白。这种高通量的基因传递 该战略将使我们的长期目标能够快速产生基因修饰的干细胞疗法， 有效地治疗镰状细胞病。这种物理透化过程使靶细胞 瞬时渗透，使载体摄取，同时最大限度地减少对细胞膜的损伤， 保持高水平的生存能力。为了达到我们的临床目标，我们提出了具体的 目标包括：1）优化携带镰刀的模型细胞系中的声流体基因递送 细胞突变和2）评估镰状细胞病突变的位点特异性校正， 造血干细胞和祖细胞。考虑到这种声流体技术的实用性， 克服了现有技术水平，可以解决各种心脏、肺和血液疾病 用于基因传递。我们期待着使用基因修饰的干细胞疗法， 我们的声流体技术将大大改善这些医疗干预的可及性， 最严重的SCD患者的生命周期。