Rapid development of SARS-CoV-2 specific therapeutics that leverage virus specific RNA elements

Our goal is leverage our recent insights into coronavirus B conserved RNA structures, and ourdiscovery of formulations for high efficiency lung delivery, into the rapid development of SARS-CoV-2 specifictherapeutics. Using a novel suite of computational technology tools, we have identified predicted RNAsecondary structures in regions conserved across coronavirus B genomes including SARS-CoV-2. We havealso identified two tandem predicted microRNA 191 (miR191) binding sites within the 5'-most such structure.In our current grant on influenza A virus (IAV), we identified an RNA secondary structure conserved across allIAV isolates that is essential for in vitro packaging and in vivo disease, then designed short highly stable lockednucleic acid (LNA) oligonucleotides to bind and distort this RNA packaging signal, and demonstrated that asingle dose of our lead LNA can a) provide immediate 100% protection for over 14 days from a lethal inoculumof IAV, b) provide 100% survival when administered 3 days after a lethal IAV inoculum, and c) while sufficientlyattenuating the infection, enable the subsequent development of high level immunity. Moreover, we have alsorecently discovered that empty deproteinized pollen shells represent an outstanding vehicle for delivery ofLNAs to the lung with much greater efficacy and tolerability than current formulations for nucleic acid delivery.We now hypothesize that 1) our identified RNA secondary structures in SARS-CoV-2 represent idealcandidate targets for disrupting the virus lifecycle, via structure-specific LNAs; 2) the miR191 binding siteswithin the 5'-most conserved RNA secondary structure reflect an essential mechanism for regulatingtranslation of corona B viruses that is amenable to targeting by specifically designed LNAs; 3) our noveldeproteinized pollen formulation represents an ideal means of delivering such LNAs to both prevent and treatestablished SARS-CoV-2 infections. We will test these hypotheses via the following specific aims that are to: 1)Determine which LNA gapmers from a screening panel synthesized against our identified conserved RNAsecondary structure targets are most disruptive to the latter's integrity, as assessed by SHAPE, REVI, andMutate-and-Map; 2) Refine the sequence (total LNA length, fine nucleotide target position, and length of singlestranded DNA gapmer) of the top performing LNA and test a panel of LNA analogs to identify the most potentdisrupter of targeted SARS-CoV-2 conserved RNA secondary structure; 3) Determine the effect of LNAsdesigned to sequester miR191 in cells transfected with a SARS-CoV-2 5' terminal RNA segment linked to aluciferase reporter; 4) Determine the effect of the identified lead LNAs (targeting conserved SARS-CoV-2 RNAsecondary structure, and sequestering miR191) on cells infected with SARS-CoV-2 in vitro, and in vivo whendelivered intranasally by current lung-targeting transfection reagent (i.e.JetPEi) vs. pollen shells to SARS-CoV-2-infected mice. Successful accomplishment of our aims will yield proof-of-concept for an exciting new class ofanti- SARS-CoV-2 RNA therapeutics within the short time frame of this proposal.