Oral Presentation Australasian RNA Biology and Biotechnology Association 2025 Conference

Development of Intranasal Antiviral RNA Therapeutics targeting SARS-CoV-2 (DARTS) (130613)

Chantelle L Ahlenstiel 1 , Yuan Zhang 1 , Haiqao Wang 2 , Matt Johansen 3 , Camille Esneau 4 , Madeline Zhang 5 , Scott Ledger 1 , Ernest Moles 6 , Philip Hansbro 3 , Maria Kavallaris 6 , Nathan Bartlett 4 , Pall Thordarson 7 , Cees Van Rijn 5 , Daniela Traini 2 , Anthony D Kelleher 1
  1. Kirby Institute, Sydney, NSW, Australia
  2. Macquarie University, Macquarie Park, NSW, Australia
  3. Centre for Inflammation, Centenary Institute and University of Technology Sydney, Sydney, NSW, Australia
  4. Hunter Medical Research Institute , University of Newcastle, Newcastle, NSW, Australia
  5. University of Amsterdam, Amsterdam, Netherlands
  6. Children's Canter Institute, University of New South Wales, Sydney, NSW, Australia
  7. The UNSW RNA Institute,, Sydney, NSW, Australia

Introduction: RNA therapeutics are an exciting treatment modality. Antiviral short interfering (si)RNA therapeutics that are highly conserved have potential to treat a range of diverse viral infections. The current antiviral treatments of COVID are limited and have many side effects with drug-drug interactions due to intravenous or oral administration. We have developed broad spectrum antiviral siRNA targeting the SARS-CoV-2 Nsp1 region. Here we investigated their antiviral efficacy in vitro and in vivo following encapsulated with an optimised lipid nanoparticle (LNP) formulation and delivery using an intranasal spray device (MedSpray) for delivery directly to the respiratory tract.

Methods: siRNA-LNP complexes were generated using the Nanoassembr Ignite platform. The proprietary optimised LNP was based on the FDA-approved Alnylam (Onpattro) formulation. LNP QC included measuring encapsulation efficiency (EE%), nanosize, polydispersity and zeta potential. Off-target effects of four interferon stimulated genes (ISGs) were measured by RT-qPCR of mRNA levels. Antiviral efficacy was assessed using cell survival assays and viral mRNA levels by RT-qPCR in VeroE6 and Calu-3 cells. Aerosol performance was assessed via industry standard human respiratory models (Alberta idealised nasal inlet-AINI, and Next Generator Impactor-NGI) and novel nasal expansion chamber models. Preclinical safety and efficacy was assessed ex vivo using the air-liquid interface (ALI)-primary human bronchial epithelial cell (BEC) cultures model and in vivo the K18-ACE2 transgenic mouse model of SARS-Co-2 infection.

Results: siRNA-LNP formulations were <100 nm, with EE% of >80%, Polydispersity of <0.3, and a neutral charge zeta potential. No significant off-target effects were induced by the siRNA-LNP formulation. Virus suppression induced by siRNA-LNP in infected VeroE6 and Calu-3 cells was reported ranging between 1.94 and 3.03 log, compared to controls. Biodistribution studies reported aerosol deposition of siRNA-LNP (84.6%) in the nasopharynx region of the AINI-NGI respiratory model. Intranasal instillation of siRNA-LNP in the transgenic mouse model demonstrated the optimised LNP formulation was safe and non-inflammatory in the lung and improved the clinical outcome with decreased viral RNA levels in the lung by ~1/2 log, compared to control groups. This is comparable to current antivirals.

Discussion and Conclusions: This study demonstrates broad-spectrum siRNA encapsulated in an optimised LNP have potential to treat COVID infection without off-target effects in vitro and in vivo. Nasopharynx biodistribution post-device treatment offers a targeted therapeutic approach for siRNA-LNP in the upper respiratory tract to suppress SARS-CoV-2 infection. This platform technology is broadly applicable to other respiratory virus infections.