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体移植物的体外细胞处理。这种处理至少需要三个步骤--细胞浓缩、细胞分离(销毁)和基因转移--每一步都需要使用单独的技术。虽然这些技术对于研究用途可能是令人满意的，但它们在临床治疗环境中的用处有限，因为它们的处理率很低，而且在异种人体移植物中的转染和分离效率和特异性都很低。最有问题的是，因为目前的技术是通过多个步骤实施的，而不是以单一、多功能和同时进行的程序实施的，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；所有这些限制都会导致宝贵细胞的高度损失。该项目的目标是开发一种通用、高通量和多功能的技术，同时(1)转染目标T细胞，(2)摧毁不需要的T调节细胞，(3)在异种移植物中保存有价值的非目标细胞。这些功能中的每一个都将具有单一靶细胞特异性、HIH疗效和低毒性，并将通过单一的、高通量的程序实现，以满足体外处理人类移植物的临床要求。所提出的技术使用了被称为等离子体纳米气泡(PNBs)的新型细胞因子。PNB不是粒子，而是瞬时的细胞内事件，是一种蒸气纳米气泡，在短短几纳秒内膨胀和崩溃。PNB机制支持这样的假设，即用两种不同类型的金纳米颗粒(从而产生两种不同的PNBs)产生的PNBs在低能量激光脉冲作用下，将同时完成以下三个功能：(1)通过注射游离的外源小PNBs来转染CD3+T细胞；(2)用大的PNBs机械地破坏CD25+T调节细胞；(3)保存CD3-和CD25-辅助(非靶)细胞。我们广泛的初步数据表明，PNB技术提供了前所未有的同时三位一体的功能，我们的团队将通过两个特定目标来测试这一假设：目标1.使用金纳米颗粒结合物优化人外周血单个核细胞(PBMC)的靶向，以实现在CD3+T细胞中同时产生小的注射性PNBs，在CD25+T-调节细胞中响应激光脉冲同时产生大的破坏性PNBs。目的2.采用优化的PNB方法，使CD3+T细胞的基因转移和CD25+T-调节细胞的破坏同时进行，且不损伤非靶细胞。多功能、精确度和高通量的一体化PNB技术将极大地影响细胞和基因治疗以及其他依赖于异种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.