Central Nervous System-Targeted Antisense Therapeutics for Powassan Virus Infection

Background: Powassan virus (POWV) is the only tick-borne flavivirus endemic to North America. It causes disability and death due to infection of the central nervous system, and the frequency of human cases has been increasing. No effective treatments or vaccines are currently available. Phosphorodiamidate morpholino oligomers (PMO) are single-stranded nucleic-acid-like antisense agents having nuclease resistance. They are water-soluble, and can interfere with gene expression through stable duplex formation with complementary RNA. PMOs can be conjugated to a cell-penetrating peptide to produce peptide-PMOs (PPMOs) which enter cells readily without the need for transfection. PPMOs exhibit promise against a variety of emerging RNA viruses. Because PMOs are approved for clinical use for Duchenne muscular dystrophy, with four FDA-approved drugs currently on the U.S. market, and because a PPMO is in clinical trials for the same condition, antiviral PPMOs are highly plausible as drugs to treat viral infections. PPMOs exert their antiviral activity by binding functionally important RNA elements. Because their mechanism is sequence based, PPMOs are direct acting antivirals that can be modified to target a virus of interest. A prior study demonstrated that targeting the 5' end of a related tick-borne encephalitic flavivirus genome RNA exerted an antiviral effect, reducing virus titer in cultured cells. Previous work also established that morpholino antiviral compounds directed against the mosquito transmitted flavivirus, Japanese encephalitis virus (JEV), are effective within the brain following intracerebral administration. This suggests that antisense antiviral targeting within the central nervous system (CNS) could be a general approach for addressing neurological disease associated with flavivirus infections. However, direct administration to the brain is not a viable option for PPMO drug development. Objective: In these studies, we will produce CNS-directed anti-POWV transferrin receptor-specific PMO conjugates that can be administered systemically and protect mice from disease. Specific Aims: Our specific aims are to: Aim 1: Produce PMO conjugates with CNS delivery and anti-POWV activity. Aim 2: Evaluate antiviral and therapeutic activity of a transferrin receptor-targeted PPMO against Powassan virus in vivo. Study Design: We will develop transferrin-conjugated morpholino oligos and transferrin receptor antibody-conjugated morpholino oligos. This innovative, previously unexplored approach is expected to direct PMOs to the CNS because transferrin receptor has been extensively explored as a means of delivering drug compounds into the brain. The transferrin receptor-specific PPMOs will be evaluated by use of EGFP-654 mice, a general antisense oligo (ASO) delivery model that reports on ASO tissue distribution. In parallel we will, guided by prior PPMO-flavivirus studies, develop anti-POWV PPMOs by targeting conserved elements near the 5' and 3' ends of the viral genomic RNA. These PMOs will be conjugated to our standard P7 cell permeable peptide and tested for antiviral activity in cell culture to identify the optimal antiviral sequence. Once an optimal CNS delivery strategy and an optimal antiviral sequence are identified, we will combine these. This PPMO will be evaluated in mice for tissue distribution and tolerability and then tested in a mouse model of POWV disease, comparing the CNS-directed PPMO to a P7-PMO conjugate and a non-targeting control PPMO. Impact: Successful completion of this effort will yield a novel candidate POWV therapeutic and an antiviral strategy that could be effective against other tick-borne viral encephalitides. Less