Development of a microfluidic primary cell editing platform (pCEP) for personal gene therapy

项目摘要/摘要(30行) 在为个体选择理想的治疗方法时，治疗反应的差异仍然是一个巨大的障碍 患有各种疾病的病人。这一问题尤其导致了许多 个性化医疗方法旨在根据患者的情况量身定做治疗方法，以改善 结果。这些方法中的许多都需要直接在 原代细胞来源于每个患者。然而，这给翻译成 临床，因为收集、测试和操作这些细胞通常是极其昂贵的，时间- 消耗和劳动密集型的过程。 基因组工程方法的快速进步增强了人们对 个性化基因治疗，因为这些方法具有卓越的模块化、特异性和能力 快速纠正致病基因。然而，传统的基因传递方法仍然需要 患者血液的白细胞分离和靶细胞纯化程序繁琐繁琐 基因传递。此外，基因工程还存在明显的缺陷，如非永久性抑制。 靶功能和不可预测的脱靶效应，并要求基因传递技术能够 对靶细胞进行常规和重复检测。因此，需要有效的多基因传递方法来减少 通过在治疗细胞中共表达治疗性和保护性标记物来实现靶外毒性。 该项目旨在构建一个微流控初级细胞编辑平台(PCEP)，用于强大、负担得起的 对从体液中提纯的细胞进行可伸缩和直接的基因改造。PCEP将选择性地捕获主要 通过新的微尺度漩涡从血液中提取靶细胞，并高效地共传递治疗基因和基因 通过自动电穿孔捕获的细胞来编辑机器。与病毒介导的传递不同，pCEP 采用物理基因注入机制，提供更低的运营成本和更高的有效负载 能够直接提供良好的制造规范(GMP)级遗传材料。多个基因 可以通过自动切换将兴趣以剂量控制的方式顺序地注射到胞质中 交付解决方案。PCEP方法的通用性和临床应用的可行性将得到验证 通过使用模型系统执行基因插入和删除以实现非增殖的不朽 HTERT注射体细胞和CRISPR-Cas9产生PD-1基因敲除T淋巴细胞 分别是基因编辑。我们设想pCEP将为基因组编辑提供一个自动化的解决方案 直接从体液中定位原代细胞，以及一种简单易行的方法来评估不可预见的情况 新开发的基因编辑技术对人类细胞的不利影响。

Project Summary/Abstract (30 lines) Variation in treatment response remains a formidable obstacle when selecting ideal therapies for individual patients suffering from various maladies. This problem, in particular, has led to the development of numerous personalized medicine approaches aimed at tailoring therapy on a patient-by-patient basis in order to improve outcomes. Many of these approaches require systematic and quantitative assays to be performed directly on primary cells derived from each patient. However, this poses a particular challenge for translation into the clinic, as the collection, testing, and manipulation of these cells are typically extremely expensive, time- consuming and labor-intensive processes. Rapid improvements in genomic engineering methods have bolstered optimism for the prospect of personalized gene therapy because these methods possess superior modularity, specificity, and capability for rapid correction of disease-conferring genes. However, conventional gene delivery methods continue to require laborious and cumbersome leukapheresis and target-cell purification procedures of patients' blood prior to gene delivery. Moreover, gene engineering suffers from notable shortcomings, such as impermanent inhibition of target functions and unpredictable off-target effects, and demands gene delivery techniques capable of routine and repeated assays on target cells. Efficient multigene delivery methods are thus desirable to reduce off-target toxicity by co-expressing therapeutic and protective markers in therapeutic cells. This project aims to construct a microfluidic primary cell editing platform (pCEP) for robust, affordable, scalable and direct genetic modification of cells purified from bodily fluids. pCEP will selectively trap primary target cells from blood via novel microscale vortices and efficiently co-deliver therapeutic genes and gene editing machinery via automated electroporation of the captured cells. Unlike virus-mediated delivery, pCEP employs a physical gene injection mechanism that offers lower operational costs and higher payloads with the ability to directly deliver Good Manufacturing Practice (GMP)-grade genetic materials. Multiple genes of interest can be sequentially injected into the cytosol in a dose-controlled manner by automated switching of delivery solutions. The versatility and feasibility of the pCEP approach for clinical applications will be validated by performing gene insertion and deletion using model systems for the immortalization of non-proliferating somatic cells via hTERT plasmid injection and production of PD-1 knockout T-lymphocytes via CRISPR-cas9 gene editing, respectively. We envision that pCEP will provide an automated solution for genomic editing of target primary cells directly from bodily fluids as well as a simple and facile means to assess unforeseen adverse effects of newly developed gene-editing techniques for human cells.