Research Project
6555506
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.
