Transcript 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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 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
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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
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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
