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

DAVIES-SALA C; SOLER BISTUE A; MARTIN FA; VOZZA NF

Villa Carlos Paz

Congreso; Congreso de SAMIGE; 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 of 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?)-IbmRNA 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 with 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.The EGSs were designed to include a 13-nucleotide segment antisense to the target mRNA followed at the 3‟ end by the ACCA sequence that interacts with the UGG sequence within the RNA component of the E. coliRNase P. E. coliAS19 is a hyperpermeable strain and pFC9 is a plasmid that includes aac(6 ́)-Ib. To determine the effects of LNA/DNA EGSs on Ak resistance in vivo E. coliAS19(pFC19) cells were incubated in the presence of LNA/DNA EGSs for an hour before addition of Ak. In vitro RNase P cleavage experiments were carried out with EGSs with a variety of LNA/DNA configurations to identify those that induced RNAse P cleavage at highest efficiency. EGSs assayed included compounds in which all three or four deoxynucleotides at the ACCA 3‟-end were replaced in addition to other substitutions in the antisense region. It was established that four replacements at the 3‟ terminal ACCA showed higher efficiency of cleavage compared with those with three replacements. Likewise replacements on the 5 ́-end of EGSs were also tested while keeping 3‟end constant. Some particular LNA substitutions were able to enhance in vitro RNAse P cleavage of the aac(6 ́)-IbmRNA. Cultures exposed to selected antisense LNA/DNA EGSs showed a significant reduction in cell counts when compared to those that had been exposed to sense controls or random LNA/DNA EGSs. These results indicate that non-hydrolyzable DNA/LNA EGSs with the appropriate design interfere with Ak resistance expression in vivo.