Improvements in the design of non-hydrolyzable LNA/DNA antisense oligonucleotides that interfere with amikacin resistance in vivo

DAVIES SALA, C.; SOLER BISTUE, A J C; MARTÍN, F A; VOZZA, N; CARPIO DE; ZORREGUIETA, A; TOLMASKY, M E

Villa Carlos Paz

Congreso; VI Congreso Argentino de Microbiología General. Sociedad Argentina de Microbiología General; 2009

SAMIGE

The increase in antibiotic resistance among pathogenic bacteria is a topic of growing concern. The aac(6´)-Ib gene, which encodes an acetyltransferase that catalyzes the inactivation of several aminoglycosides of clinical relevance including amikacin (Ak), is rapidly spreading in the clinical setting among a variety of gram negative pathogens. Inhibition of aac(6´)-Ib expression could help extending the life of Ak as a viable treatment. EGS technology consists on the use of RNA oligonucleotides, known as external guide sequences (EGSs), that are complementary to a target RNA molecule and elicit RNAse P-mediated cleavage of the target mRNA.  EGSC3 is an EGS that induces aac(6’)-Ib mRNA cleavage by RNase P in vitro and reduces levels of expression in vivo leading to a decrease in the MIC of Ak. Since oligoribonucleotides are rapidly degraded by nucleases, a practical utilization of EGS technology requires the design of isosequential non-hydrolyzable analogs that mimic the effect of the RNA EGSs. Co-oligomers containing combinations of locked nucleotides and deoxynucleotides (LNA/DNA) showed EGS activity in vitro. In this work we assessed these EGSs in vivo for their ability to interfere with Ak resistance. In vitro experiments were also carried out to test whether replacements of DNA for LNA in different positions of the EGSs could further improve RNAse P cleavage efficiency