Chemical modifications of antisense morpholino oligomers enhance their efficacy against ebolavirus infection

Phosphorodiamidate morpholino oligomers (PMO) are uncharged nucleic acid-like molecules designed to inactivate specific gene expression via antisense-based steric hindrance of mRNA translation. PMOs have been successful in knockdown of viral gene expression and replication in the case of acute viral infections in animal models and are well tolerated in human clinical trials. We propose that antisense PMOs represent a promising class of therapeutic agents to combat filoviral infections. Previously we have shown that mice treated with a PMO complementary to a region spanning the start codon of VP24 mRNA were protected against lethal Ebolavirus challenge. In the present study, we report on the ability of two additional VP24-specific PMOs to reduce cell-free translation of a VP24 reporter, to inhibit in vitro replication of Ebolavirus, and to protect mice against lethal challenge when delivered prior to infection. Additionally, structure-activity relationship evaluations were conducted to assess enhancement of antiviral efficacy associated with PMO chemical modifications that included conjugation with peptides of variable length and composition, positioning of conjugated peptides to either the 5' or 3' terminus, and charge modifications conferred by addition of piperazine moieties. Conjugation with arginine-rich peptides greatly enhanced the antiviral efficacy of VP24-specific PMOs in infected cells and in mice during lethal Ebolavirus challenge.