Peptide-conjugated phosphodiamidate morpholino oligonucleotide (PPMO)-based henipavirus therapeutics

SUMMARY Nipah virus (NiV) is a highly lethal zoonotic paramyxovirus from the Henipavirus genus that causes severe respiratory disease and encephalitis in humans. To date, no antivirals have been approved for treatment or prevention of these infections. We will develop and test peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO)-based compounds as anti-NiV therapeutics. Phosphorodiamidate morpholino oligomers (PMO) are water soluble, nucleic-acid-like antisense agents having nuclease resistance. They form stable duplexes with complementary RNA, affecting function. PMOs are FDA-approved to treat forms of Duchenne muscular dystrophy. PMOs can be conjugated to a cell-penetrating peptide to produce peptide-PMO (PPMO) which enter cells without the need for transfection. Aqueous solutions of PPMOs have shown considerable antiviral efficacy against a number of RNA viruses. We evaluated the anti-henipavirus potential of PPMOs using the non-pathogenic henipavirus Cedar virus (CedV) at BSL2. We designed PPMOs to target the start codons for the mRNAs encoding the three viral proteins essential for viral RNA synthesis- nucleoprotein (N), phosphoprotein (P) and large protein (L). These exhibited low micromolar activity versus CedV replication in Vero cells. A pilot test of PPMO targeting NiV N and P demonstrated anti-NiV activity in cell culture. That the P gene is a viable target is notable because the NiV P gene also encodes two critical virulence factors, V and W, which disable the type I interferon response. Because V and W share the same N-terminus as the P protein, an inhibitor of P translation will also block V and W expression. Therefore, a single P-targeting PPMO could disable viral RNA synthesis and simultaneously promote antiviral IFN-I responses, potentially enhancing antiviral activity. Building on these data, we will design PPMOs to target NiV N, P and L mRNAs. These will be tested for inhibition against live NiV at BSL4 and mechanism of action assessed by using a BSL2 NiV minigenome assay. We will test the hypotheses that antiviral activity of PPMOs correlates with suppression of translation of the targeted mRNA and determine whether targeting the P start codon will augment PPMO antiviral effects by suppressing expression of V and W. We will then test the best performing PPMO in vivo, using a hamster model. We will use airway administration because (1) Respiratory symptoms are a significant component of NiV infection. (2) In hamsters, infection can spread from the airway to the central nervous system (CNS) via the olfactory bulb. (3) Prior studies have used respiratory delivery and respiratory NiV challenge to test therapeutic candidates. (4) We recently demonstrated that PPMOs delivered directly to the airway at a 1 mg/kg dose reduced SARS-CoV-2 lung titers by >104 infectious units per gram lung tissue, and our Preliminary Data demonstrates that intranasal delivery to mice results in sustained PPMO effects throughout the upper airway and extending into the olfactory bulb. The in vivo studies will include assessment of tolerability, tissue distribution and efficacy against NiV of the top performing PPMO. We expect these efforts to yield a candidate PPMO for further development.