Novel Antiviral Oligonucleotide Therapy for Rabies

Abstract

There are many neuropathic viruses that can cause viral encephalitis in humans that are mostly lethal and incurable. Among these viruses, zoonotic rabies virus (RABV) is invariably fatal to humans following the onset of clinical symptoms. Rabies encephalitis is an incurable neurotropic infection that remains a neglected global public health challenge. Post-exposure prevention is highly effective when administered before the rabies virus reaches the central nervous system but, there is no effective treatment after the clinical symptoms emerge. The blood-brain barrier restricts the penetration and delivery of most antiviral agents into infected neuronal cells, preventing treatment of rabies encephalitis. This significant therapeutic deficiency underscores the necessity for innovative treatment methodologies. Consequently, this dissertation details the development and assessment of novel antiviral oligonucleotide strategies, with the aim of impeding rabies virus replication and investigating potential therapeutic interventions for rabies encephalitis. RNA interference (RNAi) using synthetic short double stranded RNAs (siRNAs) targeting viral genes to prevent viral replication is a promising antiviral approach. This technique allows selective binding of a targeted viral messenger RNA to induce viral mRNA degradation. Our initial study in chapter 2 focused on small interfering RNAs (siRNAs) that have been chemically modified and tagged with cholesterol, designed to specifically target the rabies virus nucleoprotein (N) gene. These investigations assessed target silencing, dose-response relationships, cellular uptake, cell viability, and antiviral efficacy within neuronal cells in vitro. We hypothesized that chemically modified cholesterol conjugated antiviral siRNAs targeting rabies virus Nucleoprotein N will rapidly inhibit rabies viral replication in vitro. The findings indicated that cholesterol-conjugated siRNAs demonstrated the capacity for self-delivery into neuronal cells. Cell viability remained stable for each siRNA treated group indicating similar metabolic activity and cell numbers for all groups assessed, while not showing any cytotoxicity in cells. These chemically modified cholesterol conjugated antiviral siRNAs showed sequence-specific inhibition of the viral target rabies virus Nucleoprotein N gene. Furthermore, the treatment reduced the replication of the rabies virus within the infected cells. Extracellular vesicles (EVs) are micron size molecules derived from plasma membrane presents an innovative platform for developing novel therapeutics with their ability to shuttle molecules between cells and through crossing the blood brain barrier. In the following study in chapter 3, we investigated the potential of rabies virus glycoprotein (RVG)-modified extracellular vesicles (EVs) as a targeted delivery system for antiviral siRNA. After engineering, isolating, and characterizing RVG-modified EVs, siRNA targeting the rabies virus nucleoprotein N gene was introduced. The antiviral effectiveness of these modified EVs was then assessed in rabies virus (CVS-11) infected neuronal cells. We hypothesized that engineered extracellular vesicles expressing RVG will increase brain tropism and ultimately increase the efficiency of anti-viral siRNA delivery into the central nervous system to rapidly inhibit rabies virus replication. This project aimed to implement a novel antiviral siRNA delivery therapeutic approach to advance the viral suppression for rabies encephalitis. Our data showed successful modification of EVs with the RVG peptide and loading of antiviral siRNAs targeting rabies virus Nucleoprotein N. Moreover, we found that RVG modified EVs were able to deliver their therapeutic siRNAs to rabies virus infected neuronal cells in vitro. Our results suggest that antiviral siRNAs targeting rabies virus Nucleoprotein N induced more than 60% to 75% N gene interference in rabies virus infected neuronal cells. This dissertation presents initial findings that lend support to the application of novel antiviral approaches, particularly oligonucleotides, in combating rabies. Furthermore, it identifies cholesterol-conjugated siRNAs and RVG-modified EV-mediated siRNA delivery as potentially efficacious therapeutic modalities for rabies encephalitis.