Precision Apheresis: stem cell isolation from patients with sickle cell disease for gene therapy using high-throughput microfluidics

精密血浆分离术：使用高通量微流控技术从镰状细胞病患者中分离干细胞进行基因治疗

• 批准号: 10723247
• 负责人: Avanish Mishra
• 金额: $ 13.39万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-05 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10723247
• 关键词: Address; Advisory Committees; Affect; American; Animal Model; Antibodies; Autologous; Biomedical Engineering; Blood; Blood Cells; Blood Component Removal; Blood Platelets; Blood specimen; Boston; CD34 gene; Cancer Diagnostics; Cannulations; Catheters; Cell Separation; Cell Therapy; Cells; Characteristics; Circulation; Clinical; Collection; Committee Members; Computer Models; Disease; Dose; Engineering; Engraftment; Epitopes; Funding; Genes; Goals; Hematocrit procedure; Hematology; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Hemoglobinopathies; Hour; Human; Immunodeficient Mouse; Immunotherapy; Individual; Inherited; Interphase Cell; Kinetics; Label; Lead; Leukocytes; Magnetism; Mentors; Methods; Microfluidic Microchips; Microfluidics; Modeling; Molecular; Mus; Nature; Pathology; Patients; Pediatric Hospitals; Peripheral; Peripheral Blood Stem Cell; Persons; Platelet aggregation; Process; Research; Research Personnel; Research Proposals; Residual state; Resource-limited setting; Sickle Cell; Sickle Cell Anemia; Sorting; Stem cell transplant; Surface; Technology; Testing; Thrombophilia; Time; Training; Translating; Transplantation; United States; United States National Institutes of Health; Work; Xenograft procedure; blood product; cost; feasibility testing; gene therapy; in vivo; instrument; manufacture; microfluidic technology; migration; mouse model; nano; nanoparticle; programs; skills; stem cell biology; stem cell gene therapy; stem cell technology; stem cells; success; technology development; therapeutic gene; tissue culture

PROJECT SUMMARY Sickle Cell Disease (SCD) affects millions of people around the world. Recently, stem cell gene therapy has emerged as a potentially curative option for SCD. Obtaining a sufficient dose of hematopoietic stem and progenitor cells (HSPCs) from peripheral blood is paramount to the success of these gene therapies. However, the higher numbers of RBCs in apheresis products can adversely impact the yield of valuable hematopoietic stem cells during purification (~46% loss). There is, therefore, an immediate unmet need to develop an isolation technology that can efficiently recover hematopoietic stem cells from apheresis products, irrespective of their hematocrits. To address this challenge, I will develop a microfluidic HSPC isolation chip (HSPC-iChip) capable of recovering >95% CD34+ cells from full apheresis products (~300 mL) in an hour (Aim 1). I will bring advancements (in microfluidic technologies) from the field of cancer diagnostics to the field of hematology to accomplish this. My central hypothesis is that the HSPC-iChip can isolate highly viable and functional hematopoietic stem cells. To test this hypothesis, I will genetically edit the isolated stem cells and analyze engraftment, disease correction, and human hematopoiesis in NBSGW mice (Aim 1). Additionally, under the influence of centrifugal forces, the hypercoagulable state of sickle cell patients can lead to the formation of cell clusters. These clusters have been observed to destabilize the cell collection interface, requiring highly skilled apheresis operators for stem cell collection from sickle cell patients. Once the apheresis product is collected, subsequent purification of ~1% HSPCs from the rest of the cells further necessitates specialized instruments and consumables. This restricts a broader implementation of sickle cell gene therapy as most patients reside in low-resource settings where skilled labor, bio-cleanrooms, and financial capabilities are restricted. To address this challenge, in Aim 2, I will test the feasibility of a Precision Apheresis technology that can directly separate HSPCs from peripheral circulation in a single step based on their surface epitopes (CD34). The training objective of this project is to provide Dr. Mishra—who has a strong background in microfluidics and cell sorting—with additional scientific training from leading pioneers in therapeutic gene editing for hemoglobinopathies (Dr. Bauer, Boston Children's Hospital/Harvard), stem cell apheresis and pathology (Dr. Manis, Boston Children's Hospital/Harvard), clinical hematology (Dr. Azar, MGH/Harvard) high-throughput microfluidics (Dr. Toner, lead mentor, MGH/Harvard), animal models and advanced tissue culture (Dr. Haber, co-mentor, MGH/Harvard), nanoparticle kinetics (Dr. Bhatia, MIT), computational modeling of blood cells (Dr. Koumoutsakos, Harvard), and closed-loop mouse-chip models (Dr. Manalis, MIT). This additional training will prepare Dr. Mishra to lead an independent transdisciplinary research program in hematology, sickle cell disease, and bioengineering.

项目摘要 镰状细胞病（SCD）影响着全世界数百万人。最近，干细胞基因治疗已经 成为SCD的潜在治疗选择。获得足够剂量的造血干细胞， 来自外周血的造血祖细胞（HSPC）对于这些基因疗法的成功至关重要。然而，在这方面， 单采产品中较高数量的RBC可能对有价值的造血干细胞的产量产生不利影响， 纯化过程中的干细胞（约46%损失）。因此，有一个迫切的未满足的需要，发展一个隔离， 可以从单采产品中有效回收造血干细胞的技术，无论其 血细胞比容为了应对这一挑战，我将开发一种微流控HSPC分离芯片（HSPC-iChip）， 在1小时内从全部单采产物（~300 mL）中回收> 95%的CD34+细胞（目标1）。我必使 从癌症诊断领域到微流体技术领域的进展 血液学来实现这一点。我的中心假设是，HSPC-iChip可以分离出高度可行的， 功能性造血干细胞为了验证这一假设，我将对分离的干细胞进行基因编辑， 分析NBSGW小鼠中的植入、疾病矫正和人造血（Aim 1）。此外，根据 离心力的影响，镰状细胞患者的高凝状态可导致形成 细胞簇已经观察到这些簇使细胞收集界面不稳定，这需要高度的细胞分离。 熟练的单采操作员从镰状细胞患者中采集干细胞。一旦单采产品 收集，随后从其余细胞中纯化约1%的HSPC进一步需要专门的HSPC。 仪器和消耗品。这限制了镰状细胞基因治疗的广泛实施， 患者居住在低资源环境中，熟练劳动力、生物洁净室和财务能力 是受限制的。为了应对这一挑战，在目标2中，我将测试精密单采的可行性 一种可以直接从外周循环中分离HSPC的技术， 表面表位（CD34）。该项目的培训目标是提供米什拉博士-谁拥有强大的 微流控和细胞分选背景，以及来自领先先驱的额外科学培训， 血红蛋白病的治疗性基因编辑（Bauer博士，波士顿儿童医院/哈佛），干细胞 单采血液成分术和病理学（Manis博士，波士顿儿童医院/哈佛），临床血液学（Azar博士， MGH/哈佛）高通量微流体（Toner博士，首席导师，MGH/哈佛），动物模型和 高级组织培养（Haber博士，共同导师，MGH/哈佛），纳米颗粒动力学（Bhatia博士，麻省理工学院）， 血细胞的计算模型（Koumoutsakos博士，哈佛）和闭环小鼠芯片模型（Koumoutsakos博士，哈佛大学）。 Manalis，MIT）。这项额外的培训将使米什拉博士能够领导一项独立的跨学科研究。 血液学、镰状细胞病和生物工程专业。