Aptamer Screening for Bioterrorism Agent Therapeutics

描述（由申请人提供）：在darpa资助的德克萨斯大学医学分部（UTMB）的一个项目中，组合选择和基于结构的设计方法目前被用于开发一种新的治疗策略，以调节关键蛋白的差异表达，以应对潜在的生物恐怖主义（BT）病原体。专有的硫代酸或硫代酸修饰的双链DNA诱饵（即硫代适配体）可以调节宿主蛋白质组，特别是免疫应答细胞因子和关键转录因子，如NF-KB，在BT病原体的攻击过程中。UTMB研究结果显示，硫胺受体与NE-KB受体之间存在特异性相互作用，并对沙粒病毒引起的出血热（一种潜在的生物恐怖主义威胁）模型中的动物存活产生连带影响。无细胞和体内试验可用于测量适体有效性，但目前所有适体组合文库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价值，因为它具有更大的吞吐量潜力（与体内模型相比）和更多的生理反应（与无细胞测定相比）。然而，这种模型的一些关键挑战已经出现：能够(i)有效地将硫适体传递到细胞中，（ii）识别已经吸收了适体的细胞，以及（iii）在硫适体传递和BT病原体攻击后，在体外跟踪大量这些细胞。此外，生物安全控制问题与操纵受污染细胞有关。Oncosis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10105美元的速度扫描细胞，并在密封容器内以每秒10103美元的速度对特定的单个细胞进行激光照射。激光照射特定细胞可导致各种结果，包括细胞死亡（导致细胞纯化）、光化学激活、光注射。等。光注射是一种多用途的方法，可将各种物质（如质粒）装入细胞。蛋白质等)，其中激光是用来增加细胞膜的通透性，使物质进入细胞。渗透性的增加是短暂的，除此之外细胞不会受到伤害。据推测，LEAP平台的光注入和高通量原位扫描能力可用于克服上述硫帕坦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检测感染的细胞，从而在Pichinde病毒感染的巨噬细胞中进行硫腺苷筛选作为模型系统。

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.