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US-12618063-B2 - Pan-genotypic agents against influenza virus and methods of using the same

US12618063B2US 12618063 B2US12618063 B2US 12618063B2US-12618063-B2

Abstract

Methods of inhibiting influenza A virus in a sample are provided. Aspects of the methods include contacting a sample comprising viral RNA (vRNA) having a PSL2 motif with an effective amount of an agent that specifically binds the PSL2 motif to inhibit the influenza A virus. Also provided are methods of treating or preventing influenza A virus infection in a subject. Also provided are methods for screening a candidate agent for the ability to inhibit influenza A virus in a cell, the method comprising: contacting a sample with a candidate agent; and determining whether the candidate agent specifically binds to the PSL2 motif of vRNA. Also provided are compounds and pharmaceutical compositions comprising an oligonucleotide sequence complementary to a PB2 vRNA region that find use in the subject methods.

Inventors

  • Jeffrey S. Glenn
  • Rachel Hagey Saluti
  • Edward A. Pham

Assignees

  • THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY

Dates

Publication Date
20260505
Application Date
20240716

Claims (18)

  1. 1 . A composition comprising: an antiviral oligonucleotide compound or salt thereof, comprising an oligonucleotide sequence 5′ CGACCAAAAGAATTC 3′ (SEQ ID NO:56).
  2. 2 . The composition of claim 1 , wherein the oligonucleotide comprises an internucleoside linkage selected from: phosphorothioate, phosphorodithioate, phosphoramidate and thiophosphoramidate linkages.
  3. 3 . The composition of claim 2 , wherein the oligonucleotide comprises one or more chiral internucleoside linkages.
  4. 4 . The composition of claim 1 , wherein the oligonucleotide comprises one or more 2′-modified nucleotides.
  5. 5 . The composition of claim 1 , wherein the oligonucleotide comprises at least 5 deoxyribonucleotide units and is capable of recruiting an RNase.
  6. 6 . A composition comprising: an antiviral oligonucleotide compound or salt thereof, comprising an oligonucleotide sequence LNA14: 5′CGACcaaaagaATTC 3′ (SEQ ID NO:81), wherein capitalized letters denote LNA nucleotides and lowercase letters denote DNA nucleotides.
  7. 7 . The composition of claim 6 , wherein the oligonucleotide comprises an internucleoside linkage selected from: phosphorothioate, phosphorodithioate, phosphoramidate and thiophosphoramidate linkages.
  8. 8 . The composition of claim 7 , wherein the oligonucleotide comprises one or more chiral internucleoside linkages.
  9. 9 . A method of treating or preventing a virus infection in a subject, the method comprising: administering to a subject in need thereof a pharmaceutical composition comprising an effective amount of the composition according to claim 1 .
  10. 10 . The method of claim 9 , wherein the subject is at risk of influenza A virus infection and the administering protects the subject against infection for 1 week or more.
  11. 11 . The method of claim 9 , wherein the administering comprises weekly, biweekly, or monthly administration of an effective dose of the oligonucleotide compound.
  12. 12 . The method of claim 9 , wherein the pharmaceutical composition further comprises an additional active agent selected from a second oligonucleotide active agent and an antiviral drug.
  13. 13 . The method of claim 9 , wherein the subject has been diagnosed with or is suspected of having an influenza A virus infection.
  14. 14 . A method of treating or preventing a virus infection in a subject, the method comprising: administering to a subject in need thereof a pharmaceutical composition comprising an effective amount of the composition according to claim 6 .
  15. 15 . The method of claim 14 , wherein the subject is at risk of influenza A virus infection and the administering protects the subject against infection for 1 week or more.
  16. 16 . The method of claim 14 , wherein the administering comprises weekly, biweekly, or monthly administration of an effective dose of the oligonucleotide compound.
  17. 17 . The method of claim 14 , wherein the pharmaceutical composition further comprises an additional active agent selected from a second oligonucleotide active agent and an antiviral drug.
  18. 18 . The method of claim 14 , wherein the subject has been diagnosed with or is suspected of having an influenza A virus infection.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. patent application Ser. No. 17/716,497 filed Apr. 8, 2022, which application is a continuation of U.S. patent application Ser. No. 16/792,103, filed Feb. 14, 2020, now issued as U.S. Pat. No. 11,339,392, which application is a continuation-in-part of U.S. patent application Ser. No. 16/081,818, filed Aug. 31, 2018, now issued as U.S. Pat. No. 10,597,658, which application is a 371 National Phase Application of PCT/US2017/020241, filed Mar. 1, 2017, which application claims the benefit of U.S. Provisional Patent Application No. 62/302,548, filed Mar. 2, 2016, all of which are incorporated herein by reference in their entirety. GOVERNMENT RIGHTS This invention was made with government support under grant number AI109662 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (STAN-1292CIPCON2_S15-471_SEQ_LIST.xml; Size: 364,230 bytes; and Date of Creation: Sep. 12, 2024) is herein incorporated by reference in its entirety. INTRODUCTION Influenza A virus (IAV) is a segmented RNA virus that causes significant morbidity and mortality worldwide. All currently approved IAV antiviral drugs are targeted against viral proteins, are subtype limited, and are challenged by rising antiviral resistance against all drug class members. The IAV genome consists of eight single-stranded negative-sense viral RNA (vRNA) segments that encode a minimum of 14 known viral proteins. The vRNA, together with nucleoprotein (NP) and the heterotrimeric polymerase complex, comprised of PB2, PB1, and PA proteins, forms the complete viral ribonucleoprotein (vRNP). To be fully infectious, IAV virions must incorporate at least one of each segment's vRNP. Each vRNP interacts with at least one other partner to form a supramolecular complex likely maintained by intersegment RNA-RNA and/or protein-RNA interactions hypothesized to guide the packaging process. SUMMARY Aspects of the present disclosure provide pan-genotypic compositions designed to disrupt an RNA structural element of IAV, called Packaging Stem-Loop 2 (PSL2), within the 5′ packaging signal region of genome segment PB2. Disruption of PSL2 structure dramatically inhibits IAV. PSL2 is conserved across all tested influenza A subtypes. Methods of inhibiting influenza A virus in a sample are provided. Aspects of the methods include contacting a sample comprising viral RNA (vRNA) having a PSL2 motif with an effective amount of an agent that specifically binds the PSL2 motif to inhibit the influenza A virus. In some cases, the vRNA is isolated from a virion or a cell. In some cases, the vRNA is in a virion. In some cases, the vRNA is in an infected cell. Also provided are methods of treating or preventing influenza A virus infection in a subject. Also provided are methods for screening a candidate agent for the ability to inhibit influenza A virus in a cell, the method comprising: contacting a sample with a candidate agent; and determining whether the candidate agent specifically binds to the PSL2 motif of vRNA. Also provided are compounds and pharmaceutical compositions comprising an oligonucleotide sequence complementary to a PB2 vRNA region that find use in the subject methods. BRIEF DESCRIPTION OF THE FIGURES The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way. FIG. 1A-FIG. 1F depicts RNA secondary structures of wild-type PB2 (SEQ ID NO: 1) and packaging mutant vRNAs, PB2m757 (SEQ ID NO: 2), m745 (SEQ ID NO: 3), 1918 pandemic (H1N1) (SEQ ID NO: 4), High-path avian (H5M=N1) (SEQ ID NO: 5) and 2009 swine (H1N1) (SEQ ID NO: 6). FIG. 2, panels A and B, depicts the reactivity of full-length PB2 vRNA. FIG. 3A-FIG. 3E depicts the disruption of wild-type reactivity (SEQ ID NO: 7) by packaging-defective mutations, PB2m744b (SEQ ID NO: 8), PB2m745 (SEQ ID NO: 9), PB2m55c (SEQ ID NO: 10) and PB2m757 (SEQ ID NO: 11). FIG. 4 depicts the conservancy of nucleotide sequence containing the PSL2 structure. FIG. 5A-FIG. 5D depicts the 2-dimensional Mutate-and-Map analysis of PSL2 RNA secondary structure (FIG. 5C, SEQ ID NO: 12). FIG. 6, panels A-B, depicts the design of compensatory mutations to previously described PR8 PB2 mutants (panel A, SEQ ID NO: 13). FIG. 7, panels A-D, depicts synonymous mutation of single highly conserved codons of the PR8 PB2 vRNA (panel A, SEQ ID NOs: 14-15; panel B, SEQ ID NOs: 16-23 top to bottom). FIG. 8 depicts a table showing PB2 packaging mutant nomenclature and corresponding sites of mutation. FIG. 9A-FIG. 9C depicts the effect of synonymous mutation on PSL2 structure, PB2m731 (SEQ ID NO: 24), PB2m751 (SEQ ID NO: 25) and PB2m748 (SEQ ID NO: 26). FIG. 10, panels A-I, depicts the effect of compensatory mutations in PR8 PB2