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PMO Antibiotic Efficacy in Mammalian Cells

PMO Antibiotic Efficacy in Mammalian Cells. Georgi Mitev Dept. of Biochem/Biophys Mentor: Dr. Bruce Geller Department of Microbiology AVI Biopharma, Inc. Antibiotics Today. Antibiotics becoming less effective Novel antibiotics needed Example: Mycobacterium tuberculosis in America

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PMO Antibiotic Efficacy in Mammalian Cells

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  1. PMO Antibiotic Efficacy in Mammalian Cells Georgi Mitev Dept. of Biochem/Biophys Mentor: Dr. Bruce Geller Department of Microbiology AVI Biopharma, Inc.

  2. Antibiotics Today • Antibiotics becoming less effective • Novel antibiotics needed • Example: Mycobacterium tuberculosis in America • 1993-1996 3.9% of Multi Drug Resistant cases are Extremely Drug Resistant • 2001-2004 increased to 4.5%

  3. Phosphorodiamidate Morpholino Oligomers • 10 bases • Phophorodiamidate linkages • Not a substrate for nucleases

  4. PMO and DNA

  5. PMOs and gene expression • PMOs are selectively reactive • PMOs bond to RNA

  6. Gene Expression

  7. Introducing PMO • DNA is transcribed into RNA • RNA binds to complementary PMO • Translation is blocked • PMO specificity

  8. AVI Research • PMO pure culture E. coli and S. typhimurium • PMO targeted to AcpP gene

  9. E. coli pure culture growth with PMO treatments

  10. S. typhimurium pure culture growth with PMO treatments

  11. AVI Research • Must also inhibit in tissue culture

  12. E. coli tissue culture growth with PMO treatments

  13. My Project • E. coli grows extracellularly • Can the PMO also work on an intracellular pathogen, such as S. typhimurium, growing in mammalian cells? • PMO must penetrate both human cell and bacterial cell • Similar PMO targeted to intracellular Mycobacterium tuberculosis inhibited growth in tissue culture • Research could lead to therapeutics for other intracellular pathogens such as Chlamydia trachomatis or Neisseria gonorrhea

  14. Hypothesis • PMOs targeted to the AcpP gene in S. typhimurium will be able to inhibit growth intracellularly in tissue culture

  15. Research • Find suitable drug for new strain S. typhimurium 14028s • AcpP neutral PMO • AcpP 3+ PMO • Both are 11 bases long and targeted to start codon

  16. Minimal Inhibitory Concentration 1 • MIC for targeted neutral PMO 1.250 μM • MIC for targeted 3+ PMO 0.156 μM • Ampicillin has a MIC of about 30 μM

  17. Growth Curve 1 • Comparison of 2x MIC concentration of targeted neutral PMO and targeted 3+ PMO • Measured by optical density and viable cell count

  18. Growth Curve 1

  19. Growth Curve 1

  20. Growth Curve 2 • Comparison of drugs at same concentration

  21. Growth Curve 2

  22. Growth Curve 2

  23. Tissue culture 1 • Test Targeted 3+ PMO efficacy in mouse macrophage cells • S. typhmurium inoculation • Extracellularly sterilized with gentomycin 10 mg/mL • Treated at 0 h with 3.13 M 3+ PMO • Lysed and plated

  24. Tissue Culture 1

  25. Tissue Culture 2 • Test for ratio of bacterial cell to culture cells • Carry experiment out to 42 h • Test for gentomycin efficacy • Treated at each time point

  26. Tissue Culture 2

  27. Tissue Culture 2

  28. Tissue Culture 2

  29. Tissue Culture 2

  30. Further Research • Test M. avium • What is the most effective target site for the PMO? Stem vs. loop

  31. Acknowledgements • Funding • HHMI • AVI Biopharma • Dr. Bruce Geller • Shannon Oda • Brett Mellbye • Susan Puckett • Dr. Pat Iversen • Dr. Kevin Ahern

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