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

项目摘要 镰状细胞病是最常见的单基因遗传病之一。临床 SCD的管理主要是支持的。然而，在最严重的情况下，唯一确定的 SCD患者的治疗选择是异基因相合的造血干细胞 移植。这种血红蛋白病直接影响到血红蛋白的结构和功能，导致 β-珠蛋白链在功能性成人血红蛋白发育中的缺陷。此外， 对于接受干细胞移植的患者来说，缺乏完全匹配的捐赠者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。最近的努力是 针对这种疾病及其临床后遗症的研究主要集中在基因治疗上。 自体基因修饰的造血干/祖细胞移植 患者自身的细胞会被纠正并重新注入，以产生功能齐全的红细胞。 然而，已知的干细胞基因治疗批量处理的非病毒策略是 效率低下，无法满足临床需求。我们假设优化一个 声流治疗平台，物理渗透细胞，用于输送 CRISPR-Cas9生物分子将解决这一技术差距。这种高通量的基因传递 战略将使我们的长期目标能够迅速和 治疗镰状细胞病的有效药物。这种物理渗透过程使目标细胞 瞬时渗透性，使载体能够摄取，同时将对细胞膜的损害降至最低 保持高水平的生存能力。为了实现我们的临床目标，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的声流控基因传递 细胞突变和2)评价镰状细胞病突变的定点纠正 造血干细胞和祖细胞。考虑到这种声流控技术的实用性，有 可以解决的各种心脏、肺和血液疾病，克服了最先进的状态 用于基因传递。我们期望通过使用基因修饰的干细胞疗法来产生快速和安全的疗法 我们的声流技术将极大地改善获得这些医疗干预的机会和质量 对于最严重的SCD患者来说，这是一种生命的保障。

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.