BAA Number 02 Q4655 Mission Area Expedient Biological

BAA Number: 02 -Q-4655 Mission Area: Expedient Biological Agent Battlefield Neutralization MINDTEL, LLC. 11/01/2001 Requirement Number: R-414 / ATL-414 -MINDTEL-012 -QC Proposal Title: Post-Exposure Anthrax Neutralization – Macrophage Electroporation (Left Image) Blue stained macrophage and red blood cells. Tiny purple platelets are also visible. (Right Image) Schematic effects of electroporation on macrophage membrane surface showing how gene product enters cell. Operational Capability to be Provided: - Develop procedures to disrupt Anthrax toxin assembly on macrophage cell membrane with 24 hours of treatment. - Model Anthrax infection. Protective Antigen (PA) = pre-pore at the cell surface. Two enzymes = Edema Factor (EF) and Lethal Factor (LF), bind to the pre-pore and are internalized. Once in the endosome, the PA component transforms into a pore, EF and LF reach the cytoplasm. Completed toxin enters cells, destroys key enzymes and kills macrophages. Anthrax inhalation (@10, 000 spores) = massive internal bleeding and shock. Lethal effects are cytokines, released by dying macrophages reduce clotting abilities. Antibiotics alone are useless within 1 -6 days after symptoms appear, even if all the bacteria are killed, as many toxins have already been released. - Block combined toxic effects of the 3 components: bind a small fragment of a designed P 1 polypeptide (12 amino-acids long) onto the PA toxin. This polypeptide neutralizes the ability of the complex to form a pore and subsequent infection. This approach with a strict regimen of antibiotics and proper blood p. H adjustment can be an effective treatment protocol. Proposed Technical Approach: New effort Rough Order of Magnitude Cost and Schedule: Our approach uses the genetic transfer abilities of electroporation to transfect macrophages with the DNA recombinant plasmids to counteract Anthrax toxins. Short electro-magnetic pulses (Electroporation) are able to produce small pores in the membranes of human macrophage cells. This provides a method for producing life-saving polypeptides within 24 hours of treatment. Task 1 period of performance is 4 months, Tasks 2 and 3 begin after completion of Task 1 for a period of 8 months. Anthrax Bacterium releases 3 toxin components: PA – Protective Antigen {PA 63), cleaved (PA 20) EF – Edema Factor + LF – Lethal Factor Anthrax Antagonist P 1 dodecapeptide amino acid sequence: HTSTYWWLDGAP The P 1 dodecapeptide (HTSTYWWLDGAP) was synthesized and found to disrupt the binding of radiolabeled LFN to PA 63 on Chinese hamster ovary (CHO) cells. (Mourez et al, 2001) Task 1 – Create DNA Plasmid with ligated P 1 Gene (x copies). Task 2 – Design ex vivo macrophage electroporation protocol. Task 3 – In vitro, in vivo and ex vivo testing. * Total performance period is 12 months Task 1 ROM cost of $250, 000 Task 2 + 3 ROM cost of $1, 000 * Total cost is $1, 250, 000 Deliverables: 1. Electro-Pulse Generator: Electroporator 2. Macrophage Transfection Protocol 3. Anthrax Toxin Antagonist Gene Product – P 1 ligated to plasmid 4. Data deliverables: monthly status report, final report, test plans, test reports, specifications, computer program end items, user’s manual, drawings, transition plan. Corporate Information: Mind. Tel, LLC. , Bill Rojas, Ph. D. - P. O. BOX 8995 - Rancho Santa Fe, CA. 92067, (858) 759 -8808 - email: billrojas@hotmail. com