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.

项目概要 最近，干细胞基因疗法影响了全世界数百万人。 获得足够剂量的造血干细胞成为 SCD 的潜在治疗选择。 来自外周血的祖细胞（HSPC）对于这些基因疗法的成功至关重要。 单采产品中红细胞数量较多可能会对宝贵造血的产量产生不利影响 纯化期间的干细胞（约 46% 损失），因此，迫切需要开发一种分离方法。 可以从单采产品中高效回收造血干细胞的技术，无论其性质如何 为了应对这一挑战，我将开发一种具有微流控 HSPC 隔离芯片 (HSPC-iChip) 功能。 一小时内从全单采产品（约 300 mL）中回收 >95% CD34+ 细胞（目标 1）。 从癌症诊断领域到微流体技术领域的进步 我的中心假设是 HSPC-iChip 可以分离出高度存活的和 为了检验这个假设，我将对分离的干细胞进行基因编辑，并 分析 NBSGW 小鼠的植入、疾病纠正和人类造血（目标 1）。 受离心力的影响，镰状细胞病患者的高凝状态可导致形成 已观察到这些细胞簇会破坏细胞收集界面的稳定性，需要高度的稳定性。 熟练的单采术操作员从镰状细胞患者采集干细胞。 收集后，随后从其余细胞中纯化约 1% 的 HSPC 进一步需要专门的 这限制了镰状细胞基因治疗的更广泛实施。 患者居住在资源匮乏的环境中，那里缺乏熟练劳动力、生物洁净室和经济能力 为了应对这一挑战，在目标 2 中，我将测试精密血浆分离术的可行性。 该技术可以根据 HSPC 的性质，一步直接将其从外周循环中分离出来。 该项目的培训目标是为Mishra博士提供强大的表面表位（CD34）。 微流体和细胞分选的背景——并接受来自领先先驱者的额外科学培训 血红蛋白病的治疗性基因编辑（鲍尔博士，波士顿儿童医院/哈佛大学），干细胞 血浆分离术和病理学（Manis 博士，波士顿儿童医院/哈佛大学），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 先进的组织培养（Haber 博士，麻省总医院/哈佛大学联合导师）、纳米粒子动力学（Bhatia 博士，麻省理工学院）、 血细胞计算模型（哈佛 Koumoutsakos 博士）和闭环小鼠芯片模型（Koumoutsakos 博士） 麻省理工学院马纳利斯）。这项额外培训将为米什拉博士领导一项独立的跨学科研究做好准备。 血液学、镰状细胞病和生物工程项目。