RNA-Based Strategies for Prevention and Treatment of Alphavirus and Hemorrhagic Fever Virus Infection

Background: Viral infections cause endemic and epidemic diseases worldwide. Alphaviruses, including Sindbis virus, Chikungunya virus, and Venezuelan equine encephalitis virus, are mosquito-vectored viruses that primarily cause either arthritogenic disease or acute encephalitis. Viral hemorrhagic fever (VHF) is caused by several distinct groups of viruses, including Andes virus (ANDV), a New World hantavirus transmitted to humans primarily through contact with long-tailed rice rats that can impact U.S. troops deployed stateside and overseas. These can be associated with high mortality and therefore threaten our deployed Warfighters. Given their zoonotic nature, they are generally confined to endemic areas where their hosts live, but with increased human migration and globalization, such diseases are no longer geographically limited and have a broader impact on the general public and military troops alike. Some milder symptoms of alphavirus infection include headaches, fevers, chills, rashes, joint pain, and vomiting, while more severe manifestations include encephalitis (inflammation of the brain), long-term abnormalities in the central nervous system (CNS), and death. ANDV can cause hantavirus cardiopulmonary syndrome. The few available vaccines and therapeutics have limited efficacy and are not FDA- approved. Conventional antivirals, such as monoclonal antibodies and vaccines, take years to develop, and neither alphavirus disease nor VHF is a current focus of major drug companies. Objective/Hypothesis: Alphaviruses and hemorrhagic fever viruses have RNA genomes that are replicated by error-prone polymerases and adapt to changing environments to cause emerging diseases in humans. To address the global burden of alphavirus disease and VHF, we propose to expand our next-generation RNA-based therapeutics platform to develop effective medical countermeasures as prophylactic or therapeutic drugs for these viruses. Our flexible, adaptable, and rapid platform of oligonucleotides (oligos) includes small interfering RNAs (siRNAs), antisense oligonucleotides (ASOs), and siRNASOs that combine both of these RNA species that will serve as countermeasures to proactively address existing diseases and limit emerging infections. For fiscal year 2023, we propose the following research focus areas to address these viral threats: (1) identify alphavirus targets and define critical host factors that confer sensitivity or promote resistance to these viruses, (2) develop refined RNA-based prevention and treatment strategies to combat these viruses and modulate host factors at the site of infection, and (3) test these strategies in advanced preclinical animal models of alphavirus and hemorrhagic fever virus infection with the inclusion of a Syrian hamster model of ANDV in preparation for IND applications with the FDA. Rationale: Oligo drugs for functional gene silencing have revolutionized human medicine, and several have been FDA-approved, with additional agents on the horizon. Our team has developed a transformative platform for next-generation oligo drug development using chemical modifications that have resulted in significant improvements, including (1) unprecedented duration of effect-up to 12 months following a single injection, (2) clean safety profile, (3) high specificity, and (4) high functionality. Oligo drugs are ideal as a quick-acting treatment following virus exposure, provided that safe, efficient delivery is achieved. The mechanism of action relies on rapid (within 12 hours) and persistent activation of the innate immune system. Once the viral sequence is known, advanced mapping tools can be used to design a virus-specific therapeutic within days. Manufacturers can scale up oligo drug production to treat millions of people within months of identifying new threats. Specific Aims: Aim 1. Identify top host factors required for alphavirus propagation using proteomics-based approaches and target with oligo-based therapeutics in the context of viral infection in relevant cell models. Aim 2. Design and develop custom oligo drugs that target the alphavirus genome in relevant cell lines. Aim 3. Optimize oligo-based therapeutics against alphaviruses in animal models of infection. Aim 4. Define host factors required for ANDV and test oligo-based therapeutics in an advanced animal model. Study Design: Using our well-established pipeline, we will design and synthesize oligo drugs targeting pro-viral host factors and maximally conserved regions of the representative alpha- and hantaviruses. Our oligo drugs will feature a chemical scaffold that supports functional, sustained delivery to the lung, musculoskeletal system, and CNS. We will screen 10-20 small RNAs for each viral gene and identify fully modified hyper-functional in vivo active compounds that decrease the viral load. To mitigate for viral escape mutations, we will screen multitargeting constructs for multiple viral genes or genome elements. We will target both previously identified and novel host factors required for infection. The top viral and host gene-targeting siRNAs/ASOs will be combined in a single multitargeting construct. We can develop formulations with broad-spectrum activity. We will assess outcomes in animal models, including viral load, inflammation/necrosis in vital organs, and survival. Relevance: Viruses pose a significant threat to our Service Members, but many have been relatively understudied by the scientific community. This lack of knowledge has created a significant gap in our ability to treat and prevent such infections. There is also precedence for viruses as biological weapons. Oligo drugs are ideal because a single dose can provide long-lasting protection, are well-suited for both prevention and treatment strategies, can be rapidly produced at a low cost, are stable at tropical temperatures where many of these infections occur, and do not require a cold chain. Once our platform is optimized, we can easily apply our strategy to target additional viral pathogens of interest. Less