Targeted-delivery of small interference RNA against anthrax

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 DESCRIPTION: Targeted-delivery of small interference RNA against anthrax. Anthrax is a serious disease caused by Bacillus afgfnthracis, a bacterium that forms spores. Anthrax most commonly occurs in wild and domestic mammalian species; but can also occur in humans when they are exposed to infected animals or animal tissues, or when anthrax spores are dispersed as a bioterrorist weapon. The complicated immunization schedule with the licensed vaccine BioThrax calls for a new and easily administered anthrax vaccine. Since anthrax is a disease that rarely occurs naturally in humans, it is more realistic to develop a post exposure prophylaxis or therapy instead of mass immunization, as with the current vaccine. Following exposure, macrophages ingest anthrax spores and travel to the lymph node where these spores germinate. The B. anthracis bacteria are then released into the bloodstream and produce toxins that are key factors in the virulence of disease: protective antigen (PA), edema factor (EF), and lethal factor (LF). PA is the receptor binding toxin component that attaches to either of two host cell receptors: anthrax toxin receptor 1 (ANTXR1 or tumor endothelial marker 8/TEM8) and anthrax toxin receptor 2 (ANTXR2 or capillary morphogenesis protein 2/CMG2). After binding, PA is cleaved and the receptor-bound portions form a heptameric pore that binds EF or LF. The toxin complexes are endocytosed and delivered into the cytosol. The activities of LeTx and EdTx result in malfunction of the immune system, edema, shock, and death. Our preliminary study has shown that inhibition of ANTXR expression by RNA interference (RNAi) technology using specific anti-ANTXR small interference RNA (siRNA) could prevent cytotoxicity of anthrax toxins. We hypothesize that a detoxified anthrax toxin could be used as a delivery vehicle for anti-ANTXR siRNA. In order to evaluate this hypothesis, we propose the following three specific aims: Specific Aim 1: To generate detoxified anthrax toxins for ANTXR-targeted siRNA delivery. Specific Aim 2: To assess the inhibitory effect on anthrax toxin induced cytotoxicity in ANTXR-silenced cells. Specific Aim 3: To evaluate efficacy of the anti-ANTXR siRNA treatment in a mouse model of anthrax. We anticipate that the proposed host-targeted treatment strategy will prevent severe illness and death in patients exposed to both wild type and even antibiotic-resistant B. anthracis spores by natural infection or a bioterrorist attack. Furthermore, this technology can be developed as a platform to treat other antimicrobial-resistant pathogens that employ pore-forming toxins as virulence factors.