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

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

21. 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

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