All-in-one purification and engineering of therapeutic cell systems

描述（由申请人提供）：细胞和基因癌症治疗需要人移植物的体外细胞处理。这种处理至少需要三步——细胞富集、细胞分离（破坏）和基因转移——每一步都需要使用单独的技术。虽然这些技术在研究中可能令人满意，但在临床治疗中用处有限，因为它们的处理速度较低，在异质人移植物中转染和分离的效率和特异性也较低。最严重的问题是，由于目前的技术是通过多个步骤进行管理的，而不是单一的、多功能的、同时进行的程序，它们延长了治疗过程，并在临床治疗中引入了不必要的复杂性、劳动力和资源；所有这些限制导致宝贵细胞的大量损失。该项目的目标是开发一种通用、高通量和多功能的技术，该技术将同时(1)转染靶T细胞，(2)破坏不需要的T调节细胞，(3)在异质移植物中保存有价值的非靶细胞。这些功能都将具有单靶细胞特异性、高效、低毒，并将在一个单一的、高通量的程序中实现，以满足处理离体人体移植物的临床需求。提出的技术采用了一种新的细胞介质，称为等离子体纳米气泡（pnb）。pnb不是粒子，而是短暂的细胞内事件，一种蒸汽纳米气泡，在短短纳秒内膨胀和崩溃。PNB机制支持这样一种假设，即用低能量激光脉冲用两种不同类型的金纳米颗粒（以及两种不同的PNB）产生的PNB治疗人类移植物可以同时实现以下三个功能：(1)通过向游离的外部质粒注入小PNB转染CD3+ T细胞，(2)用大PNB机械地破坏CD25+ T调节细胞，(3)保存CD3-和CD25-辅助（非靶）细胞。我们大量的初步数据表明，PNB技术提供了这种前所未有的同时三合一功能，我们的团队将通过两个具体目标来验证这一假设：利用金纳米粒子偶联物优化靶向人外周血单个核细胞（PBMC），在激光脉冲的作用下，在CD3+ t细胞中同时产生小的注射性PNBs，在CD25+ t调节细胞中同时产生大的破坏性PNBs。目标2。采用优化后的PNB方法，在不损伤非靶细胞的情况下，同时实现CD3+ T细胞的基因转移和CD25+ T调节细胞的破坏，一次批量流动，具有高通量。多功能、精确和高通量的一体化PNB技术将极大地影响细胞和基因治疗以及其他依赖于异质细胞系统体外处理的临床应用。

DESCRIPTION (provided by applicant): Cell and gene cancer therapies require ex vivo cell processing of human grafts. Such processing requires at least three steps - cell enrichment, cell separation (destruction), and gene transfer - each of which requires the use of a separate technology. While these technologies may be satisfactory for research use, they are of limited usefulness in the clinical treatment setting because they have a low processing rate, as well as a low transfection and separation efficacy and specificity in heterogeneous human grafts. Most problematic, because current technologies are administered in multiple steps - rather than in a single, multifunctional, and simultaneous procedure - they lengthen treatment process and introduce an unnecessary level of complexity, labor, and resources into clinical treatment; all these limitations result in high losses of valuable cells. The project goal is to develop a universal, high-throughput, and multifunctional technology that will simultaneously (1) transfect target T cells, (2) destroy unwanted T regulatory cells, and (3) preserve valuable non-target cells in heterogeneous grafts. Each of these functions will have single target cell specificity, hih efficacy, and low toxicity, and will be achieved in a single, high-throughput procedure to meet the clinical requirements for processing human graft ex vivo. Proposed technology employs novel cellular agents, called plasmonic nanobubbles (PNBs). PNBs are not particles, but transient, intracellular events, a vapor nanobubbles that expand and collapse in mere nanoseconds. PNB mechanism supports the hypothesis that treating human grafts with PNBs generated by two different types of gold nanoparticles (and consequently two different PNBs) with a low-energy laser pulse will simultaneously accomplish three following functions: (1) Transfect CD3+ T cells by injecting free external plasmid with small PNBs, (2) Destroy CD25+ T-regulatory cells mechanically with large PNBs and (3) Preserve CD3- and CD25- accessory (non-target) cells. Our extensive preliminary data indicate that PNB technology offers this unprecedented, simultaneous three-in-one functionality, and our team will test this hypothesis through two specific aims: Aim 1. Optimize targeting of human peripheral blood mononuclear cells (PBMC) with gold nanoparticle conjugates to achieve simultaneous generation of small injective PNBs in CD3+ T-cells and large destructive PNBs in CD25+ T-regulatory cells in response to a laser pulse. Aim 2. Adopt the optimized PNB method to enable simultaneous gene transfer in CD3+ T cells and destruction of CD25+ T-regulatory cells without damaging non-target cells in one bulk flow procedure with high throughput. The multi-functionality, precision, and high throughput of all-in-one PNB technology will tremendously impact cell and gene therapies and other clinical applications that depend on ex vivo processing of heterogeneous cell systems.