proposed technical approach: new effort our approach uses the genetic transfer abilities of...
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Proposed Technical Approach: New effort
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.
Anthrax Bacterium releases 3 toxin components:
PA – Protective Antigen {PA63), cleaved (PA20)
EF – Edema Factor + LF – Lethal Factor
Anthrax Antagonist P1 dodecapeptide amino acid sequence: HTSTYWWLDGAP
The P1 dodecapeptide (HTSTYWWLDGAP) was synthesized and found to disrupt the binding of radiolabeled LFN to PA63 on Chinese hamster ovary (CHO) cells. (Mourez et al, 2001)
Task 1 – Create DNA Plasmid with ligated P1 Gene (x copies).
Task 2 – Design ex vivo macrophage electroporation protocol.
Task 3 – In vitro, in vivo and ex vivo testing.
BAA Number: 02-Q-4655
Mission Area: Expedient Biological Agent Battlefield Neutralization
Requirement Number: R-414 / ATL-414-MINDTEL-012-QC
Proposal Title: Post-Exposure Anthrax Neutralization – Macrophage Electroporation
MINDTEL, LLC. 11/01/2001
(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.
Rough Order of Magnitude Cost and Schedule:
Task 1 period of performance is 4 months, Tasks 2 and 3 begin after completion of Task 1 for a period of 8 months.
* Total performance period is 12 months
Task 1 ROM cost of $250,000
Task 2 + 3 ROM cost of $1,000,000
* Total cost is $1,250,000
Deliverables:
1. Electro-Pulse Generator: Electroporator
2. Macrophage Transfection Protocol
3. Anthrax Toxin Antagonist Gene Product – P1 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:
MindTel, LLC., Bill Rojas, Ph.D. - P.O. BOX 8995 - Rancho Santa Fe, CA. 92067, (858) 759-8808 - email: [email protected]
Post-Exposure Anthrax Neutralization – Macrophage ElectroporationOperational 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 P1 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 pH adjustment can be an effective treatment protocol.