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