Hairpin extensions enhance the efficacy of mycolyl transferase-specific antisense oligonucleotides targeting Mycobacterium tuberculosis.
We have investigated the efficacy of modifying gene-specific antisense phosphorothioate oligodeoxyribonucleotides (PS-ODNs) by the addition of 5' and 3' hairpin extensions. As a model system, we have targeted the Mycobacterium tuberculosis 30/32-kDa mycolyl transferase protein complex genes encoding three highly related enzymes (antigens 85 A, B, and C). Whereas the addition of a hairpin extension at only one end of the PS-ODNs did not improve their inhibitory capacity, the addition of hairpin extensions at both ends enhanced their capacity to inhibit M. tuberculosis multiplication in comparison with unmodified PS-ODNs. A combination of three 5'-, 3'-hairpin-modified PS-ODNs (HPS-ODNs) targeting each of the three mycolyl transferase transcripts inhibited bacterial growth in broth culture by approximately 1.75 log units (P < 0.0001) and in human THP-1 macrophages by approximately 0.4 log units (P < 0.0001), which to our knowledge has not previously been demonstrated for any PS-ODN; reduced target gene transcription by > or =90%; caused approximately 90% reduction in mycolyl transferase expression; and increased bacterial sensitivity to isoniazid by 8-fold. The growth-inhibitory effect of the HPS-ODNs was gene-specific. Mismatched HPS-ODNs had no growth-inhibitory capacity. This study demonstrates that 5'- and 3'-HPS-ODNs are highly efficacious against M. tuberculosis and supports the further development of antisense technology as a therapeutic modality against tuberculosis.
Harth
Günter
G
Division of Infectious Diseases, Department of Medicine, 37-121 Center for Health Sciences, School of Medicine, University of California-Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095-1688, USA.
Zamecnik
Paul C
PC
Tabatadze
David
D
Pierson
Katherine
K
Horwitz
Marcus A
MA
D000217:Acyltransferases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004353:Drug Evaluation, Preclinical; D004357:Drug Synergism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005798:Genes, Bacterial; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009000:Monocytes / Q000187:drug effects / Q000382:microbiology; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology* / Q000254:growth & development; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D000217:Acyltransferases; C112580:antigen 85A, Mycobacterium tuberculosis; C121433:antigen 85B, Mycobacterium tuberculosis; D007538:Isoniazid