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 患有各种疾病的患者。这个问题尤其导致了许多 个性化医疗方法旨在根据患者的具体情况制定治疗方案，以改善 结果。其中许多方法需要直接在 来自每位患者的原代细胞。然而，这对翻译成 临床上，因为这些细胞的收集、测试和操作通常非常昂贵、耗时 消耗和劳动密集型过程。 基因组工程方法的快速改进增强了人们对前景的乐观态度 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，传统的基因递送方法仍然需要 术前对患者血液进行费力且繁琐的白细胞去除术和靶细胞纯化程序 基因传递。此外，基因工程还存在显着的缺点，例如无常抑制 目标功能和不可预测的脱靶效应，并且需要能够 对靶细胞进行常规和重复测定。因此，需要有效的多基因递送方法来减少 通过在治疗细胞中共表达治疗和保护标记来实现脱靶毒性。 该项目旨在构建一个微流控原代细胞编辑平台（pCEP），以实现稳健、经济、 对从体液中纯化的细胞进行可扩展和直接的遗传修饰。 pCEP 将选择性地捕获初级 通过新型微尺度涡流从血液中靶向细胞，并有效地共同传递治疗基因和基因 通过捕获细胞的自动电穿孔来编辑机器。与病毒介导的传递不同，pCEP 采用物理基因注入机制，可提供更低的运营成本和更高的有效负载 能够直接提供良好生产规范 (GMP) 级遗传材料。多个基因 通过自动切换，可以以剂量控制的方式将感兴趣的物质顺序注射到细胞质中 交付解决方案。 pCEP方法在临床应用中的多功能性和可行性将得到验证 通过使用模型系统进行基因插入和删除，以实现不增殖的永生化 通过 hTERT 质粒注射产生体细胞并通过 CRISPR-cas9 产生 PD-1 敲除 T 淋巴细胞 分别进行基因编辑。我们预计 pCEP 将为基因组编辑提供自动化解决方案 直接从体液中靶向原代细胞，以及评估不可预见的简单方法 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.