A morpholino oligomer targeting highly conserved internal ribosome entry site sequence is able to inhibit multiple species of picornavirus

Members of the genera Enterovirus and Rhinovirus (family Picornaviridae) cause a wide range of human diseases. An established vaccine is available only for poliovirus, and no effective therapeutic is available for treatment of any pathogenic picornavirus. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded-DNA-like antisense agents that readily enter cells. A panel of PPMO was tested for antiviral activity against various picornaviruses. PPMO targeting conserved IRES sequence were highly active against human rhinovirus 14, coxsackievirus B2, and poliovirus 1 (PV1), reducing PV1 titers by up to 6 log10 in cell cultures. Comparative sequence analysis led us to design a PPMO (EnteroX) targeting 22 nucleotides of IRES sequence that are perfectly conserved across greater than 99% of all human enteroviruses and rhinoviruses. EnteroX reduced PV1 replication in cell-culture to an extent similar to that of other IRES-specific PPMO. Resistant PV1 arose in cell cultures after twelve passages in the presence of EnteroX, and were found to have two mutations within the EnteroX target sequence. Nevertheless, cPVR transgenic mice treated once daily by intraperitoneal injection with EnteroX either before and/or after intraperitoneal infection with 3x10(8) plaque forming units (3 times the 50% lethal dose) of PV1 had about 80% higher survival than controls. Viral titer in tissues taken at day 5 post-infection showed that animals in the EnteroX-treated group averaged over 3, 4, and 5 log10 less virus in small intestine, spinal cord and brain, respectively, than control animals. These results suggest that EnteroX may have broad therapeutic potential against entero- and rhinoviruses.