Aptamer Screening for Bioterrorism Agent Therapeutics

描述(由申请人提供)：目前正在使用组合选择和基于结构的设计方法来开发一种新的治疗策略，以调节关键蛋白质的差异表达，以应对潜在的生物恐怖主义(BT)病原体，该计划由DARPA资助，位于德克萨斯大学医学分校(UTMB)。专有的硫代硫酸盐或二硫代磷酸修饰的双链DNA诱饵(即硫代适配子)可以在与Bt病原体的攻击过程中调节宿主蛋白质组，特别是免疫反应细胞因子和关键转录因子如核因子-KB。UTMB结果显示，在一种潜在的生物恐怖主义威胁--阿拉伯病毒诱导的出血热模型中，硫代适配子和NE-KB Dirners之间存在特异性相互作用，并伴随着对动物存活的影响。无细胞和体内试验可用于测量适体的有效性，但目前所有适体组合文库的选择都是使用无细胞重组蛋白系统在体外进行的。基于细胞的体外筛选模型将具有重要的价值，因为它具有更大的吞吐潜力(与体内模型相比)和更多的生理反应(与无细胞检测相比)。然而，这种模型已经出现了一些关键的挑战：(I)高效地将硫代适配子输送到细胞内，(Ii)识别已经摄取适体的细胞，以及(Iii)在硫代适配子输送和Bt病原体挑战之后，在体外跟踪大量这些细胞。此外，生物安全遏制问题与操纵受污染的细胞有关。Oncosis已经开发了一种基于激光的系统，用于高速原位细胞扫描和操作。该系统以每秒105的速度扫描细胞，并以每秒103的速度对密封容器中的特定单个细胞进行激光照射。激光照射特定细胞可导致多种结果，包括细胞死亡(导致细胞纯化)、光化学激活、光注射。光注射是一种将多种物质(例如，质粒)加载到细胞中的通用程序。蛋白质等)，由此激光被用来增加细胞膜的通透性，允许物质进入细胞。渗透性的增加是暂时的，细胞在其他方面是无害的。据推测，LEAP平台的光注入和高通量原位扫描能力可以用来克服上述硫代适配子筛选中的三个关键挑战。因此，LEAP平台代表了一种改进的方法，可以在封闭系统平台内安全地操作和分析受感染的细胞，从而能够以Pichinde病毒感染的巨噬细胞为模型系统进行硫代适配子筛选。

DESCRIPTION (provided by applicant): Combinatorial selection and structure-based design methods are currently being used to develop a novel therapeutic strategy to modulate the differential expression of key proteins in response to potential bioterrorism (BT) pathogens under a DARPA-funded program at University of Texas-Medical Branch (UTMB). Proprietary phosphorothioate- or phosphorodithioate-modified duplex DNA decoys (i.e., thioaptamers) can modulate host proteomes, particularly the immune response cytokines and key transcription factors such as NF-KB, during challenge with BT pathogens. UTMB results showed specific interactions between thioaptamers and NE-KB dirners with concomitant influences on animal survival in a model of arenavirus-induced hemorrhagic fever, a potential bioterrorism threat. Cell-free and in vivo assays are available for measuring aptamer effectiveness, but presently all aptamer combinatorial library selection is performed in vitro using a cell-free recombinant protein system. A cell-based in vitro screening model would have significant value because of the greater throughput potential (as compared to in vivo models) and the more physiological response (as compared to cell-free assays). However, some key challenges to such a model have arisen: the ability to (i) efficiently deliver thioaptamers into cells, (ii) identify cells having taken up aptamer, and (iii) follow significant numbers of these cells in vitro following thioaptamer delivery and BT pathogen challenge. Additionally, biosafety containment concerns are associated with the manipulation of contaminated cells. Oncosis has developed a laser-based system for high-speed in situ cell scanning and manipulation. The system scans cells at >105 per second and laser-irradiates specific individual cells at >103 per second within a sealed container. Laser irradiation of specific cells can lead to various outcomes, including cell death (leading to cell purification), photochemical activation, optoinjection. etc. Optoinjection is a versatile procedure for loading cells with a variety of substances (e.g. plasmids. proteins, etc.), whereby a laser is used to increase the cell membrane permeability, allowing the substance to enter the cell. The increased permeability is transient, and the cells are otherwise unharmed. It is hypothesized that the optoinjection and high-throughput in situ scanning capabilities of the LEAP platform could be used to overcome the three key challenges in thioaptamer screening described above. The LEAP platform therefore represents an improved methodology to safely manipulate and assay infected cells within a closed-system platform, enabling thioaptamer screening in the Pichinde virus-infected macrophage as a model system.