Codelivery of Small-Molecule Inhibitors to Enhance RNA-Encoded Antibody Production

Topic Area: Emerging Infectious Diseases Background: In the event of an infectious disease outbreak, particularly in a region of strategic importance, Department of Defense Service members are often deployed to distribute and administer countermeasures. While the rapid response effort is critical to halt disease spread, the first response teams also risk disease from transmission. This vulnerability was evident during the 2013-2016 Ebola outbreak in West Africa, when the World Health Organization reported that 240 health care workers became infected and 120 died of disease by late August in 2014. Countermeasures that provide immediate neutralizing protection will be crucial in protecting first responders from the threat of both natural and weaponized biological threats. The flexibility of ribonucleic acid (RNA)-based gene expression makes it a promising rapid response platform to deliver antibodies that neutralize the target pathogen. Problem: Since the concentration of antibody in serum correlates with protection from disease, the key challenge in RNA-encoded antibody therapy is the ability to generate protective level of antibodies from the limited number of cells accessed by a simple intramuscular injection. Alphavirus genomes, such as the clinical stage TC-83 genome, provide an attractive template for synthesis of modified replicon RNA (repRNA) constructs that use viral replication machinery to rapidly express large amounts of encoded genes. However, the replication process quickly becomes self-limiting when the total dose is concentrated over a limited number of cells, stimulating interferon (IFN)-activated antiviral responses that partially or completely shut down protein production from the repRNA. Proposed Research: This proposal aims to develop a dual cargo nanoparticle vehicle that co-delivers small molecule inhibitors of IFN induction or signaling with antibody-expressing repRNA. In Specific Aim 1, we will use High Content Analysis (HCA) system to screen a library of compounds with known IFN inhibitory activity for the ability to enhance protein translation from repRNA delivered using a Nanostructure Lipid Carrier (NLC). Primary criteria for down-selection will be the ability of compounds to enhance protein expression; we will also measure suppression in cytokine transcription as a potential correlate for expression. In Specific Aim 2, lead candidates will be formulated in the lipophilic core of NLCs and monitored for physicochemical stability and in vitro bioactivity. Finally, in Specific Aim 3, stable and active compounds formulated with NLC will be evaluated for effect on antibody expression from a co-delivered repRNA in immune competent mice. Hypothesis: Localized co-delivery of small molecule IFN inhibitors with NLC-formulated repRNA will significantly enhance protein production compared to NLC-formulated repRNA alone. Innovation: RNA-encoded antibodies offer a promising rapid response countermeasure against emerging infectious diseases, but challenges with efficient expression and safe delivery pose significant barriers to clinical viability. Current approaches proposing RNA-encoded antibodies are delivered intravenously (IV), so as to transfect a large number of protein producing liver cells. IV infusions are cumbersome and require clinical settings, which may not be a possibility for Armed Forces in emergency situations. Moreover, IV delivery of RNA using cationic lipid vehicles increases risk to severe anaphylactic reactions. The proposed research focuses exclusively on developing intramuscular (IM) injectable RNA-encoded antibodies to facilitate deployment and ease of administration. However, IM injection is target cell limited, resulting in suboptimal antibody production. Existing approaches to enhance expression have focused on modifications to the repRNA backbone, which include insertion of viral structural genes evolved to resist IFN-mediated shutdown. This approach risks genome packaging that can become a potential safety issue. The proposed research strategy focuses on leveraging the delivery vehicle to enhance expression. This approach is universal and independent of the RNA sequence. Successful demonstration of this platform technology will motivate development of thermostable formulations capable of carrying on the field in pre-filled injectors, providing Armed Forces effective countermeasures against potential bioterror threats. Less