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

项目概要/摘要（30行） 治疗反应的差异仍然是为个体选择理想疗法时的一个巨大障碍。 患有各种疾病的病人。这个问题，特别是，导致了许多的发展， 个性化的医疗方法，旨在根据患者的情况定制治疗，以改善 结果。这些方法中的许多方法需要直接对样品进行系统的和定量的测定。 原代细胞来源于每个患者。然而，这对翻译成 临床上，由于这些细胞的收集、测试和操作通常非常昂贵， 消耗和劳动密集型过程。 基因组工程方法的快速改进使人们对基因组工程的前景更加乐观， 个性化基因治疗，因为这些方法具有优越的上级模块性，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 在治疗之前对患者血液进行费力且繁琐的白细胞去除和靶细胞纯化程序 基因传递此外，基因工程也有明显的缺点，如非永久性抑制， 目标功能和不可预测的脱靶效应，并要求基因递送技术能够 对靶细胞的常规和重复测定。因此，需要有效的多基因递送方法来减少 通过在治疗性细胞中共表达治疗性和保护性标志物而产生脱靶毒性。 该项目旨在构建一个微流控原代细胞编辑平台（pCEP），用于强大，负担得起， 从体液中纯化的细胞的可扩展的和直接的遗传修饰。pCEP将选择性地捕获初级 通过新型微尺度涡旋从血液靶向细胞并有效地共同递送治疗基因和基因 通过捕获的细胞的自动电穿孔的编辑机器。与病毒介导的递送不同，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载荷， 直接提供良好生产规范（GMP）级遗传物质的能力。多个基因 可以通过自动切换 交付解决方案。将验证pCEP方法用于临床应用的多功能性和可行性 通过使用用于非增殖性细胞永生化的模型系统进行基因插入和缺失， 通过hTERT质粒注射的体细胞和通过CRISPR-cas9产生PD-1敲除的T淋巴细胞 基因编辑。我们设想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 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.