# US 8,129,352 B2 · Antisense antiviral compound and method for treating ssRNA viral infection

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- Number: US 8,129,352 B2
- Document ID: US8129352B2
- Status: Granted
- Kind: B2
- Application number: 11/226,995
- Filed: 2005-09-14
- Published: 2006-03-23
- Granted: 2012-03-06
- Earliest claimed: 2004-09-16
- Assignees: AVI BioPharma, Inc.
- Inventors: Patrick L. Iversen; David A. Stein
- Web page: https://patentgrep.com/patent/US8129352B2

## CPC

- C12N 15/1131: {against viruses}
- A61P 31/12: Antivirals
- A61P 31/14: for RNA viruses
- C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 2310/11: Antisense
- C12N 2310/314: Phosphoramidates
- C12N 2310/3233: Morpholino-type ring
- C12N 2310/3513: Protein; Peptide
- C12N 2310/531: Stem-loop; Hairpin
- C12N 2320/11: for the determination of target sites, i.e. of active nucleic acids
- C12N 2770/00011: Details
- C12N 2770/10011: Arteriviridae
- C12N 2770/12011: Astroviridae
- C12N 2770/16011: Caliciviridae
- C12N 2770/20011: Coronaviridae
- C12N 2770/24011: Flaviviridae
- C12N 2770/28011: Hepeviridae
- C12N 2770/32011: Picornaviridae
- C12N 2770/36011: Togaviridae
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The invention provides antisense antiviral compounds and methods of their use and production in inhibition of growth of viruses of the Flaviviridae, Picornoviridae, Caliciviridae, Togaviridae, Arteriviridae, Coronaviridae, Astroviridae and Hepeviridae families in the treatment of a viral infection. The antisense antiviral compounds are substantially uncharged morpholino oligonucleotides having a sequence of 12-40 subunits, including at least 12 subunits having a targeting sequence that is complementary to a region associated with stem-loop secondary structure within the 5′-terminal end 40 bases of the positive-sense RNA strand of the virus.

## Claims

### Claim 1

A method of inhibiting replication of a Flaviviridae positive strand RNA virus selected from yellow fever virus, Dengue virus, tick born encephalitis virus, and West Nile virus, comprising:
administering to mammalian host cells a virus-inhibitory amount of an antisense oligonucleotide analog characterized by (i) a nuclease-resistant backbone, (ii) being capable of uptake by mammalian host cells, (iii) containing up to 25 nucleotide bases, (iv) being composed of morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, and (v) having a targeting sequence that comprises the sequence set forth in SEQ ID NO:47, 49, 51, or 55, wherein the antisense oligonucleotide analog disrupts a stem-loop structure in the 5′-terminal region of the RNA virus and inhibits virus production.

### Claim 2 (depends on 1)

The method of claim 1, wherein the heteroduplex has a Tm of dissociation of at least 45° C.

### Claim 3 (depends on 1)

The method of claim 1, wherein the intersubunit linkages are uncharged.

### Claim 4 (depends on 1)

The method of claim 1, wherein the morpholino subunits are joined by intersubunit linkages, in accordance with the structure:
where Y1=O, Z=O, Pj is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide; and X is alkyl, alkoxy, thioalkoxy; or —NR2, where each R is independently hydrogen or lower alkyl.

### Claim 5 (depends on 4)

The method of claim 4, wherein X is —NR2, wherein R is independently hydrogen or methyl.

### Claim 6 (depends on 1)

The method of claim 1, wherein the antisense oligonucleotide is attached to an arginine-rich polypeptide effective to enhance the uptake of the compound into host cells.

### Claim 7 (depends on 6)

The method of claim 6, wherein the arginine-rich polypeptide has the sequence of SEQ ID NO: 122.

### Claim 8 (depends on 1)

The method of claim 1, wherein the administration is to a mammalian subject in a therapeutically effective amount to treat a Flaviviridae virus infection.

### Claim 9 (depends on 8)

The method of claim 8, wherein the Flaviviridae virus is Dengue virus.

### Claim 10 (depends on 1)

The method of claim 1, wherein the administration is by intravenous or oral administration.

### Claim 11 (depends on 1)

The method of claim 1, wherein the antisense oligonucleotide analog administered is contained in a cocktail of antisense oligonucleotides directed against two or more Flaviviridae positive-strand RNA viruses.

### Claim 12

A method of treating an infection by a Flaviviridae positive strand RNA virus selected from yellow fever virus, Dengue virus, tick born encephalitis virus, and West Nile virus, comprising:
administering to a subject a therapeutically effective amount of an antisense morpholino oligonucleotide, wherein the morpholino oligonucleotide has
(i) a targeting sequence that comprises the sequence set forth in SEQ ID NO: 47, 49, 51, or 55,
(ii) a subunit length of up to 25 subunits, wherein morpholino subunits of the oligomer are joined by intersubunit linkages in accordance with the structure:
wherein Y1=O, Z=O, Pj is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide; and X is —NR2, where each R is independently hydrogen or methyl; and
wherein the antisense morpholino oligomer forms a heteroduplex with the target region to disrupt the stem loop structure and inhibit Flaviviridae virus production.

### Claim 13 (depends on 12)

The method of claim 12, wherein the Flaviviridae virus is a Dengue virus and the targeting sequence comprises SEQ ID NO:51.

### Claim 14 (depends on 12)

The method of claim 12, wherein the Flaviviridae is a yellow fever virus and the targeting sequence comprises SEQ ID NO:49.

### Claim 15 (depends on 12)

The method of claim 12, wherein the Flaviviridae is a tick borne encephalitis virus and the targeting sequence comprises SEQ ID NO:55.

### Claim 16 (depends on 12)

The method of claim 12, wherein the Flaviviridae is a West Nile virus and the targeting sequence comprises SEQ ID NO:47.

### Claim 17 (depends on 13)

The method of claim 13, wherein the antisense oligonucleotide has a subunit length of about 22 subunits.

### Claim 18 (depends on 17)

The method of claim 17, wherein the antisense oligonucleotide is 22 subunits in length and is 100% complementary to SEQ ID NO:6.

### Claim 19 (depends on 14)

The method of claim 14, wherein the antisense oligonucleotide is attached to an arginine-rich polypeptide effective to enhance the uptake of the compound into host cells.

### Claim 20 (depends on 19)

The method of claim 19, wherein the arginine-rich polypeptide has the sequence set forth in SEQ ID NO: 122.