CAREER: Development of DNA nanocarriers to redirect immune cells toward chosen targets

PI: Stephan， Matthias提案号：1452492医学目前缺乏一种干预措施，可以迅速教育免疫系统消除病变细胞群，例如由癌症或病毒感染产生的细胞群。例如，尽管疫苗可以训练免疫系统在不损害健康组织的情况下选择性地破坏患病细胞，但它们可能需要几个月的时间才能引起反应，到那时病情可能变得致命。为了解决这个问题，这里提出的项目融合了材料科学、免疫学和基因治疗的概念，创造了可注射的试剂，可以快速重新编程循环免疫细胞，以识别和摧毁肿瘤。具体来说，它验证了一种假设，即靶向的、携带基因的纳米颗粒可以通过基因重新编程循环T细胞，使其表达识别肿瘤目标的受体，从而迅速消灭它们。基于实验室培养淋巴细胞的早期结果已经证实，一种新型纳米颗粒结构可以有效地将肿瘤反应受体基因转移到这些细胞中。该项目的结果可能是变革性的，因为它为开发一系列定制的基因修饰系统奠定了基础，这些系统可以迅速产生针对任何可靶向病原体的免疫力；使用这些，癌症或病毒感染的治疗可以在诊断后立即开始，只需应用适当的纳米颗粒试剂。此外，作为对人群进行免疫以预防罕见疾病的替代方案，免疫细胞的纳米粒子编程可以“按需”提供这种保护。因此，该项目可对疫情和流行病作出快速反应，从而对公共卫生产生影响。此外，研究者的化学家、生物工程师和免疫学家组成的跨学科团队将直接将这项研究整合到各种教学和教育推广计划中，为K-12学生和他们的老师提供基于现实世界的、最先进的学习经验，了解生物材料的发展如何影响医学。疫苗接种已发展成为现代医学最重要的疗法之一，纳米技术的进步提供了能够增强对疫苗的免疫反应的工具。然而，尽管疫苗可以产生具有特定特异性的T细胞，但它们产生的速度很慢，而且往往产生的数量不足以控制既定疾病。到目前为止，还没有一种方法可以迅速教育免疫系统摧毁患病细胞。该项目提出开发一种可注射的合成载体，它可以快速重新编程循环T细胞，以识别和破坏病变细胞。它将验证这样一种假设，即这些淋巴细胞可以通过靶向的、携带基因的纳米粒子进行基因重组，以表达疾病识别受体，使它们能够迅速排斥不稳定的细胞。初步的体外研究已经证实，通过适当设计的纳米颗粒可以有效地将白血病反应性T细胞受体（TCR）基因转移到淋巴细胞中。该项目的长期目标是开发一种广泛的可注射基因修饰系统，以定制治疗各种疾病。这一目标将通过三个具体目标来实现：(1)优化纳米级基因传递系统的设计，使其在增殖的T细胞中实现持久和稳健的转基因表达；(2)研究聚合纳米载体的内化如何在分子水平上影响T细胞；(3)测量将特定受体基因引入循环T细胞如何诱导小鼠疾病消退。编码Wilms肿瘤抗原特异性TCR的DNA将被包裹在脂质双层包被的多孔二氧化硅纳米颗粒中，这些纳米颗粒将通过偶联抗cd3抗体选择性地靶向循环T细胞。最初的实验将确定是否可以通过在TCR基因中插入一个支架/基质附着区（S/MAR）序列来实现稳定的受体表达，从而使质粒能够作为片段进行自我复制。核定位信号增加基因转移到T细胞的可能性也将被研究。详细的基因表达谱将阐明纳米颗粒摄取如何在分子水平上影响淋巴细胞。该技术的治疗潜力将在接受不同剂量和频率的DNA纳米载体全身注射的白血病小鼠中进行测试，外周血中重编程T细胞的百分比将通过流式细胞术进行量化。治疗组之间肿瘤进展的差异将通过连续生物发光肿瘤成像来测量。该项目产生的技术可能潜在地影响任何具有明确抗原用于受体靶向的疾病。它们基于一种新的方案，可以“按需”快速诱导主动免疫，所涉及的治疗方法可以快速分发、简单储存和易于应用。考虑到传统免疫疗法的缺点，这种方法可能会发展成为一个具有广泛应用的新的生物技术领域。这项研究也将直接整合到教育项目中，以提高人们对材料科学和免疫学领域就业机会的认识。

PI: Stephan, Matthias Proposal Number: 1452492Medicine currently lacks an intervention that can rapidly educate the immune system to eliminate diseased cell populations, such as those produced by cancer or virus infections. For example, although vaccines can train the immune system to selectively destroy diseased cells without damaging healthy tissue, they may require months to elicit responses, by which time the condition can become lethal. To address this problem, the project proposed here merges concepts from materials science, immunology, and gene therapy to create injectable reagents that can quickly reprogram circulating immune cells to recognize and destroy tumors. Specifically, it tests the hypothesis that targeted, gene-bearing nanoparticles can genetically reprogram circulating T cells to express receptors that recognize tumor targets, so they can rapidly eliminate them. Early results based on laboratory-raised lymphocytes already establish that a novel nanoparticle configuration can efficiently transfer tumor-reactive receptor genes into these cells. The results of the project could be transformative, by creating a basis for developing a repertoire of gene modification systems customized to rapidly generate immunity against any targetable pathogen; using these, treatment of cancer or viral infections could begin immediately following diagnosis by simply applying the appropriate nanoparticle reagents. Also, as an alternative to immunizing populations to protect against uncommon diseases, nanoparticle programming of immune cells could provide this protection "on demand." Thus, the project may impact public health by enabling rapid responses to outbreaks and epidemics. In addition, the investigator's interdisciplinary team of chemists, bioengineers and immunologists will directly integrate this research into a variety of teaching and educational outreach programs to provide K-12 students and their teachers with real-world-based, state-of-the-art learning experiences concerning how developments in biomaterials can impact medicine.Vaccination evolved into one of the most important therapies of modern medicine, and advances in nanotechnology have provided tools that can amplify immune responses to vaccines. However, even though vaccines can create T cells with defined specificities, they do so slowly and often produce insufficient numbers to control established disease. To date, no methodology is available that can rapidly educate the immune system to destroy diseased cells. This project proposes to develop an injectable synthetic vehicle that can quickly reprogram circulating T cells to recognize and destroy diseased cells. It will test the hypothesis that these lymphocytes can be genetically reconfigured by targeted, gene-bearing nanoparticles to express disease-recognizing receptors, enabling them to bring about rapid rejection of erratic cells. Pilot in vitro studies already establish that efficient transfer of leukemia-reactive T cell receptor (TCR) genes into lymphocytes can be achieved via appropriately engineered nanoparticles. The long-term project goal is to develop a broad repertoire of injectable gene modification systems customized to treat diverse conditions. This goal will be pursued via three specific aims: (1) optimize the design of a nanoscale gene delivery system that achieves persistent and robust transgene expression in proliferating T cells; (2) investigate how internalization of polymeric nanocarrriers affects T cells at the molecular level; and (3) measure how the introduction of specific receptor genes into circulating T cells induces disease regression in mice. DNA encoding a TCR specific for the Wilms tumor antigen will be encapsulated in lipid bilayer-coated porous silica nanoparticles, which will be selectively targeted to circulating T cells by coupling anti-CD3 antibodies onto them. Initial experiments will determine whether stable receptor expression can be achieved in dividing lymphocytes by inserting into the TCR gene a Scaffold/matrix attachment region (S/MAR) sequence, which enables plasmids to self-replicate as episomes. The possibility that nuclear localization signals will increase gene transfer into T cells will also be investigated. Detailed gene expression profiling will clarify how nanoparticle uptake affects lymphocytes at the molecular level. The therapeutic potential of the technology will be tested in leukemia-bearing mice receiving systemic injections of DNA nanocarriers at different doses and frequencies, and the percentage of reprogrammed T cells in the peripheral blood will be quantified by flow cytometry. Differences in tumor progression between treatment groups will be measured using serial bioluminescence tumor imaging. Technologies arising from the project could potentially impact any disease that has a defined antigen for receptor targeting. They are based on a novel scheme that can quickly induce active immunity "on demand", and the therapeutics involved are amenable to rapid distribution, simple storage and easy application. Considering the shortcomings of conventional immunotherapy, this approach could evolve into a new biotechnology arena with broad applications. The research will also be directly integrated into educational programs promoting awareness of career opportunities at the interface of materials science and immunology.