Development of a Temperature-Stable Broad-Spectrum Antiviral for Respiratory Viral Pathogens

Background: Upon infection, retinoic acid-inducible gene I (RIG-I) recognizes viral nucleic acid as non-self, triggering innate antiviral immune responses to a suite of viruses - including influenza A virus, respiratory syncytial virus, and many others. Preclinical studies using synthetic pathogen-associated molecular pattern (PAMP) RNA motifs show that targeting RIG-I facilitates local innate immune activation for restriction of virus replication, enhancement of vaccine efficacy, and augmentation of immune-mediated tumor suppression. Our product, RAR (RIG-I-activating RNA), is an RNA PAMP inducing RIG-I signaling to drive the development of effective antibody and cellular immune responses for control of viral infection. Our technology is distinct from traditional antivirals that block viral replication directly (and as such can eventually generate antiviral resistance). RAR circumvents these issues by triggering a multimodal innate antiviral immunity that is similar, if not identical, to that naturally triggered by an RNA virus. Rationale for Hypothesis and Proposed Project: Despite detailed information on mechanism and antiviral activity, progressing RIG-I agonists into effective products has been hindered by the lack of safe and effective delivery systems. HDT Bio has solved this issue with our lead RNA delivery formulation, LION, a nanoparticle proven to effectively protect RNA for safe in vivo delivery. We have used LION-formulated RAR (HDT-201) to successfully prevent infection and disease from a broad spectrum of viruses both in vitro and in vivo, including influenza and SARS-CoV-2. Critically, HDT-201 is highly stable. Ongoing stability studies show HDT-201 retains activity for a minimum of 3 months at room temperature. Our Target Product Profile is to deliver HDT-201 in a pressurized metered-dose inhaler (pMDI) either as a pre-exposure prophylactic, in situations of high risk for exposure, or as a post-exposure therapeutic by administration immediately following a diagnosis of respiratory viral infection or upon presentation with mild viral-associated symptoms. We hypothesize we can develop a temperature-stable inhalable HDT-201 product, which will trigger innate antiviral responses to inhibit viral replication, reduce viral loads, inhibit virus transmission, and prevent progression to severe disease. Specific Aim 1: Develop HDT-201 into a pMDI for inhalation delivery. Specific Aim 2: Finalize Investigational New Drug (IND)-enabling preclinical and pharmacokinetic/pharmacodynamic studies. Option Specific Aim 1: Perform Good Laboratory Practice (GLP) toxicology study in rats. Study Design: In Specific Aim 1, HDT-201 will be spray-dried and formulated in an HFA propellant-based suspension formulation and loaded into a canister and valve to make prototype pMDI units to be used to complete method development work and to conduct studies assessing various critical properties of the formulation. Optimal parameters will be selected to generate pMDI units capable of delivering HDT-201 at the anticipated dose range, while retaining biological activity required to be efficacious. Short- and long-term physical and biochemical stability and potency will be tested at various storage conditions. In Specific Aim 2, we will finalize preclinical IND-enabling studies. We will perform time-course studies to assess RNA transcription profile kinetics of RAR-mediated innate immune activation, as well as determine levels of key cytokines and chemokines in bronchoalveolar lavage fluid and whole lungs using ELISA and flow cytometry. We will perform biodistribution studies following lung delivery of HDT-201 and conduct preliminary toxicology studies to inform dosing for a GLP toxicology study of HDT-201. As we are developing HDT-201 as a broad-spectrum antiviral, we will assess ex- actuated HDT-201 for efficacy in three animal models of respiratory disease - SARS-CoV-2, influenza, and respiratory syncytial virus. We will determine the minimum required dose of HDT-201 and determine the optimal dosing time and interval to prevent disease. In Option Specific Aim 1, we will perform a GLP toxicology study in rats in preparation for submission of an Investigational New Drug application to the U.S. Food and Drug Administration (FDA). Doses will be applied via an inhalation chamber, once every day for a total of 4 doses (days, 1, 2, 3, and 4). Health monitoring will occur twice daily for mortality, abnormalities, and clinical signs of pain or distress. Samples will be collected for bioanalysis and toxicokinetics, as well as histology and innate immune activation in the lung. Impact and Translation: Military personnel are deployed throughout the globe, often far from medical facilities in areas endemic for deadly human viruses. The spread of viral diseases, whether unintentionally or intentionally via biological terrorism, poses a significant risk to military health and readiness. The HDT-201 inhaler could become a standard part of a Soldier's kit allowing for rapid responses to intentional or unintentional outbreaks, interrupting the transmission chain and allowing military members to remain in action. HDT Bio Corporation is poised to move quickly to advance HDT-201. RAR has undergone process development for Good Manufacturing Practice (GMP) production, while LION is currently being evaluated in a Phase II/III trial in India. Upon completion of the proposed project herein, we will quickly advance into clinical trials to significantly impact the readiness of military personnel and civilians to respond to infectious disease outbreaks or biological terrorism. Less