A NOVEL STRATEGY TO INHIBIT SARS-COV-2 INFECTION AND COVID-19

We propose in this application a novel therapeutic approach for the eradication of CoV-2. We developed a new strategy, which consists of hijacking the viral replication machinery to trigger the death of CoV-2-infected cells, while preserving uninfected cells. We propose to administer intranasally human ACE2 transgenic mice a "tailored" RNA encoding the diphtheria toxin fragment A (DTA) called {CoV-2 Hijack DTA} that is only recognized and transcribed by the CoV-2 polymerase (Pol/RdRp) present in infected cells, triggering DTA expression and rapid death of infected cells. Since DTA cannot cross the cellular membrane, it cannot kill uninfected cells. Because RNA can be easily broken down in the body, it needs to be transported within a protective carrier. Noninvasive aerosol inhalation is a well-established method of drug delivery to the respiratory tract and represents an ideal route for nucleic-acid-based therapeutics as demonstrated in various clinical trials. We propose to design degradable polymer-lipid nanoparticles (LNPs) that can deliver RNAs by nebulization (inhalation) to the respiratory tract. We propose to synthesize hyperbranched poly-beta amino esters (hPBAEs) to enable nanoformulation by nebulizer of stable and concentrated polyplexes suitable for inhalation. This strategy should achieve uniform distribution of RNAs throughout lungs resulting in high levels of proteins of interest 24h post-inhalation of hPBAE polyplexes without local or systemic toxicity due to rapid degradation of hPBAE vectors. The safety and antiviral efficacy of nebulized {CoV-2 Hijack DTA} RNA LNPs stably protected by degradable hPBAEs will be analyzed in CoV-2 variant-infected mice. Our in vivo imaging IVIS Lumina S5 system permits a daily bioluminescence (NanoLuc-CoV-2) or fluorescence (mNeonGreen CoV-2) quantification of the {CoV-2 Hijack DTA} RNA LNPs-mediated killing of infected lungs in live mice. Viral and host events required for the beneficial therapeutic effect of {CoV-2 Hijack DTA} RNA LNPs will be investigated including caspase pathway activation, membrane permeability and chromosomal degradation of infected lung cells as well as the prevention of the development of interstitial pneumonia and cytokine cascade. In summary, this application proposes to demonstrate the safety and efficacy of this novel therapeutic approach that consists of hijacking the enzymatic viral replication machinery to trigger the specific killing of CoV-2-infected cells, but not of uninfected cells. Importantly, {CoV-2 Hijack DTA} RNA LNPs will be administered at different time points of the infection in order to compare the preventive and therapeutic efficacy of our novel approach.