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 1993-1996 3.9% of Multi Drug Resistant cases are Extremely Drug Resistant 2001-2004 increased to 4.5% Phosphorodiamidate Morpholino Oligomers 10 bases Phophorodiamidate linkages Not a substrate for nucleases PMO and DNA PMOs and gene expression PMOs are selectively reactive PMOs bond to RNA Gene Expression Introducing PMO DNA is transcribed into RNA RNA binds to complementary PMO Translation is blocked PMO specificity AVI Research PMO pure culture E. coli and S. typhimurium PMO targeted to AcpP gene E. coli pure culture growth with PMO treatments S. typhimurium pure culture growth with PMO treatments AVI Research Must also inhibit in tissue culture E. coli tissue culture growth with PMO treatments 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 Hypothesis PMOs targeted to the AcpP gene in S. typhimurium will be able to inhibit growth intracellularly in tissue culture 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 Minimal Inhibitory Concentration 1 for targeted neutral PMO 1.250 μM MIC for targeted 3+ PMO 0.156 μM Ampicillin has a MIC of about 30 μM MIC Growth Curve 1 Comparison of 2x MIC concentration of targeted neutral PMO and targeted 3+ PMO Measured by optical density and viable cell count Growth Curve 1 Growth Curve 1 Growth Curve 2 Comparison of drugs at same concentration Growth Curve 2 Growth Curve 2 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 Tissue Culture 1 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 Tissue Culture 2 Tissue Culture 2 Tissue Culture 2 Tissue Culture 2 Further Research Test M. avium What is the most effective target site for the PMO? Stem vs. loop Acknowledgements Funding HHMI AVI Biopharma Dr. Bruce Geller Shannon Oda Brett Mellbye Susan Puckett Dr. Pat Iversen Dr. Kevin Ahern