pmo antibiotic efficacy in mammalian cells georgi mitev dept. of biochem/biophys mentor: dr. bruce...
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PMO Antibiotic Efficacy in Mammalian Cells
Georgi MitevDept. of Biochem/Biophys
Mentor: Dr. Bruce GellerDepartment of MicrobiologyAVI Biopharma, Inc.
Antibiotics Today
Antibiotics becoming less effective Novel antibiotics needed Example: Mycobacterium tuberculosis in
America1993-1996 3.9% of Multi Drug Resistant cases are
Extremely Drug Resistant 2001-2004 increased to 4.5%
Phosphorodiamidate Morpholino Oligomers10 basesPhophorodiamidate linkagesNot a substrate for nucleases
PMO and DNA
PMOs and gene expression
PMOs are selectively reactivePMOs 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 PMOAcpP 3+ PMO Both are 11 bases long and targeted to
start codon
Minimal Inhibitory Concentration 1
MIC for targeted neutral PMO 1.250 μMMIC for targeted 3+ PMO 0.156 μMAmpicillin has a MIC of about 30 μM
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 inoculationExtracellularly sterilized with gentomycin
10 mg/mLTreated at 0 h with 3.13 M 3+ PMOLysed 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. aviumWhat is the most effective target site for
the PMO? Stem vs. loop
Acknowledgements
FundingHHMIAVI Biopharma
Dr. Bruce Geller Shannon Oda Brett Mellbye Susan Puckett Dr. Pat Iversen Dr. Kevin Ahern