# US 2021/0340540 A1 · Chimeric double-stranded nucleic acid

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- Number: US 2021/0340540 A1
- Document ID: US20210340540A1
- Status: Published application
- Kind: A1
- Application number: 17/332,249
- Filed: 2021-05-27
- Published: 2021-11-04
- Earliest claimed: 2011-12-16
- Assignees: National University Corporation Tokyo Medical and Dental University; Osaka University
- Applicants: National University Corporation Tokyo Medical and Dental University; Osaka University
- Inventors: Takanori Yokota; Kazutaka Nishina; Satoshi Obika; Hidehiro Mizusawa
- Web page: https://patentgrep.com/patent/US20210340540A1

## CPC

- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 2310/11: Antisense
- C12N 2310/15: Nucleic acids forming more than 2 strands, e.g. TFOs
- C12N 2310/3181: Peptide nucleic acid, PNA
- C12N 2310/3231: having an additional ring, e.g. LNA, ENA
- C12N 2310/341: Gapmers, i.e. of the type ===---===
- C12N 2310/3513: Protein; Peptide
- C12N 2310/3515: Lipophilic moiety, e.g. cholesterol
- C12N 2310/53: partially self-complementary or closed
- C12N 2320/52: modulating the physical stability, e.g. GC-content
- 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

## Abstract

A method of reducing the level of a transcription product in a cell comprising contacting with the cell a composition comprising a double-stranded nucleic acid complex comprising a first nucleic acid strand annealed to a second nucleic acid strand, wherein: (i) the first nucleic acid strand hybridizes to the transcription product and comprises (a) a region consisting of at least 4 consecutive nucleotides that are recognized by RNase H when the strand is hybridized to the transcription product, (b) one or more nucleotide analogs located on 5′ terminal side of the region, (c) one or more nucleotide analogs located on 3′ terminal side of the region and (d) a total number of nucleotides and nucleotide analogs ranging from 8 to 35 nucleotides and (ii) the second nucleic acid strand comprises (a) nucleotides and optionally nucleotide analogs and (b) at least 4 consecutive RNA nucleotides.

## Claims

### Claim 1

A pharmaceutical composition which comprises a double stranded nucleic acid complex comprising a first nucleic acid strand annealed to a second nucleic acid strand, wherein:
(i) the first nucleic acid strand hybridizes to a transcription product of a target gene to modify the expression of the target gene by a non-RNase H-dependent antisense effect, wherein the first nucleic acid strand comprises
(a) consecutive DNA nucleotides, modified DNA nucleotides, nucleotide analogs and/or modified nucleotide analogs which have a sequence complementary to the transcription product and can hybridize to the transcription product, and
(b) the total number of the nucleotides, modified nucleotides and nucleotide analogs is 12 to 25 nucleotides, and
(ii) the second nucleic acid strand comprises
(a) consecutive RNA nucleotides or modified RNA nucleotides which have a sequence complementary to the first nucleic acid, and
the second nucleic acid strand comprises
(b) one or more modified nucleotides, nucleotide analogs and/or modified nucleotide analogs located on 5′ terminal side of the consecutive RNA nucleotides or modified RNA nucleotides, and/or
(c) one or more modified nucleotides, nucleotide analogs and/or modified nucleotide analogs located on 3′ terminal side of the consecutive RNA nucleotides or modified RNA nucleotides.

### Claim 2 (depends on 1)

The pharmaceutical composition of claim 1, wherein the non-RNase H-dependent antisense effect is the inhibition of the translation of the transcription product or a splicing function modifying effect.

### Claim 3 (depends on 1)

The pharmaceutical composition of claim 1, wherein the non-RNase H-dependent antisense effect is exon skipping.

### Claim 4 (depends on 1)

The pharmaceutical composition of claim 1, wherein the second nucleic acid strand further comprises a functional moiety having a targeted delivery function.

### Claim 5 (depends on 1)

The pharmaceutical composition of claim 1, wherein the function moiety is a lipid.

### Claim 6 (depends on 5)

The pharmaceutical composition of claim 5, wherein the lipid is selected from cholesterol, a fatty acid, a lipid-soluble vitamin, a glycolipid, and a glyceride.

### Claim 7 (depends on 5)

The pharmaceutical composition of claim 5, wherein the function moiety is selected from cholesterol, a tocopherol, and a tocotrienol.

### Claim 8 (depends on 6, 7)

The pharmaceutical composition of claim 6 or 7, wherein the function moiety is a peptide or a protein selected from a ligand of a receptor and an antibody.

### Claim 9 (depends on 1)

The pharmaceutical composition of claim 1, wherein the first nucleic acid strand comprises
(b) a 5′ wing region which comprises one or more nucleotide analogs or modified nucleotide analogs located 5′ terminal side of the consecutive DNA nucleotides, modified DNA nucleotides, nucleotide analogs and/or modified nucleotide analogs, and/or
(c) a 3′ wing region which comprises one or more nucleotide analogs or modified nucleotide analogs located 3′ terminal side of the consecutive DNA nucleotides, modified DNA nucleotides, nucleotide analogs and/or modified nucleotide analogs.

### Claim 10 (depends on 1)

The pharmaceutical composition of claim 1, wherein the second nucleic acid strand comprises:
(a) an RNA nucleotide or a modified RNA nucleotide and optionally a nucleotide analog, and optionally a DNA nucleotide; or
(b) a DNA nucleotide and/or a nucleotide analog; or
(c) PNA nucleotide.

### Claim 11 (depends on 1)

The pharmaceutical composition of claim 1, wherein the transcription product is a protein-coding mRNA transcription product.

### Claim 12 (depends on 1)

The pharmaceutical composition of claim 1, wherein the transcription product is a non-protein-coding transcription product.

### Claim 13 (depends on 1)

The pharmaceutical composition of claim 1, wherein the transcription product is a mRNA precursor that has not been spliced.

### Claim 14 (depends on 1)

The pharmaceutical composition of claim 1, wherein the total number of nucleotides, modified nucleotides, nucleotide analogs and/or modified nucleotide analogs in the first nucleic acid strand and the total number of RNA nucleotides, modified RNA nucleotides, DNA nucleotides, nucleotide analogs, and PNA nucleotides in the second nucleic acid strand are the same.

### Claim 15 (depends on 14)

The pharmaceutical composition of claim 14, wherein the total number of nucleotides, modified nucleotides and optionally comprised nucleotide analogs in the first nucleic acid strand and the total number of RNA nucleotides, modified RNA nucleotides, DNA nucleotides, nucleotide analogs, and PNA nucleotides in the second nucleic acid strand are different.

### Claim 16 (depends on 15)

The pharmaceutical composition of claim 15, wherein the total number of RNA nucleotides, modified RNA nucleotide, DNA nucleotides, nucleotide analogs, and PNA nucleotides in the second nucleic acid strand is greater than the total number of nucleotides, modified nucleotides and optionally comprised nucleotide analogs in the first nucleic acid strand.

### Claim 17 (depends on 16)

The pharmaceutical composition of claim 16, wherein the first nucleic acid strand comprises one or more nucleotide analogs located 5′ and/or 3′ to the consecutive DNA nucleotides, modified DNA nucleotides, nucleotide analogs and/or modified nucleotide analogs which have a sequence complementary to the transcription product.

### Claim 18 (depends on 17)

The pharmaceutical composition of claim 17, wherein the first nucleic acid strand comprises the 5′ wing region and the 3′ wing region, and wherein the 5′ wing region comprises at least 2 nucleotide analogs and the 3′ wing region comprises at least 2 nucleotide analogs.

### Claim 19 (depends on 18)

The pharmaceutical composition of claim 18, wherein the 5′ wing region and the 3′ wing region independently comprise 2 to 10 nucleotide analogs.

### Claim 20 (depends on 19)

The pharmaceutical composition of claim 19, wherein the 5′ wing region and the 3′ wing region independently comprise 2 to 3 nucleotide analogs.

### Claim 21 (depends on 1)

The pharmaceutical composition of claim 1, wherein the modified nucleotides of the first nucleic acid strand comprise at least one nucleotide comprising a 2′-O—CH3 group or a 2′-O—CH2CH2OCH3 (MOE) group.

### Claim 22 (depends on 1)

The pharmaceutical composition of claim 1, wherein the first nucleic acid strand comprises at least one nucleotide analog which is a bridged nucleotide.

### Claim 23 (depends on 22)

The pharmaceutical composition of claim 22, wherein the first nucleic acid strand comprises bridged nucleotides independently selected from LNA, cEt-BNA, amideBNA (AmNA), and cMOE-BNA.

### Claim 24 (depends on 14)

The pharmaceutical composition of claim 14, wherein the first nucleic acid strand comprises bridged nucleotides independently selected from a ribonucleotide in which the carbon atom at the 2′-position and the carbon atom at the 4′-position are bridged by 4′-(CH2) p-O-2′, 4′-(CH2) p-S-2′, 4′-(CH2) p-OCO-2, 4′-(CH2) n-N (R3)-O—(CH2) m-2′, where p, m and n represent an integer from 1 to 4, an integer from 0 to 2, and an integer from 1 to 3, respectively, and R3 represents a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group.

### Claim 25 (depends on 14)

The pharmaceutical composition of claim 14, wherein the first nucleic acid strand comprises nucleotide analogs independently selected from PNA, GNA, TNA, tcDNA, morpholino nucleic acid, 2′-O-methylated nucleic acid, 2′-O-methoxyethylated nucleic acid, and BNA.

### Claim 26 (depends on 1)

The pharmaceutical composition of claim 1, wherein at least one of the nucleotides or modified nucleotides of the first nucleic acid strand are phosphorothioated.

### Claim 27 (depends on 1)

The pharmaceutical composition of claim 1, wherein at least one of the nucleotide analogs of the first nucleic acid strand are phosphorothioated.

### Claim 28 (depends on 14)

The pharmaceutical composition of claim 14, wherein the first nucleic acid strand comprises the 5′ wing region and the 3′ wing region, and wherein
(i) the nucleotide analogs of the 5′ wing region are bridged nucleotides;
(ii) the nucleotide analogs of the 3′ wing region are bridged nucleotides; and
(iii) the consecutive nucleotides which have a sequence complementary to the transcription product are DNA nucleotides or modified DNA nucleotides.

### Claim 29 (depends on 28)

The pharmaceutical composition of claim 28, wherein the second nucleic acid strand comprises one or more phosphorothioated nucleotides located 5′ and/or 3′ to the consecutive RNA nucleotides or modified RNA nucleotides which have a sequence complementary to the first nucleic acid strand.

### Claim 30 (depends on 29)

The pharmaceutical composition of claim 29, wherein the second nucleic acid strand comprises
a 5′ wing region consisting of one or more nucleotide analogs located 5′ to the consecutive RNA nucleotides or modified RNA nucleotides which have a sequence complementary to the first nucleic acid, and/or
a 3′ wing region consisting of one or more nucleotide analogs located 3′ to the consecutive RNA nucleotides of modified RNA nucleotides which have a sequence complementary to the first nucleic acid.

### Claim 31 (depends on 30)

The pharmaceutical composition of claim 30, wherein the second nucleic acid strand comprises the 5′ wing region and the 3′ wing region, and wherein the 5′ wing region comprises at least 2 nucleotide analogs and the 3′ wing region of the first strand comprises at least 2 nucleotide analogs.

### Claim 32 (depends on 31)

The pharmaceutical composition of claim 31, wherein the 5′ wing region and the 3′ wing region independently comprise 2 to 10 nucleotide analogs.

### Claim 33 (depends on 31)

The pharmaceutical composition of claim 31, wherein the 5′ wing region and the 3′ wing region independently comprise 2 to 3 nucleotide analogs.

### Claim 34 (depends on 31, 32, 33)

The pharmaceutical composition of any one of claims 31 to 33, wherein the second nucleic acid strand comprises at least one RNA nucleotide comprising a 2′-O—CH3 group or a 2′-O—CH2CH2OCH3 (MOE) group.

### Claim 35 (depends on 31, 32, 33)

The pharmaceutical composition of any one of claims 31 to 33, wherein the second nucleic acid strand comprises at least one nucleotide analog which is a bridged nucleotide.

### Claim 36 (depends on 35)

The pharmaceutical composition of claim 35, wherein the second nucleic acid strand comprises bridged nucleotides independently selected from LNA, cEt-BNA, amideBNA (AmNA), and cMOE-BNA.

### Claim 37 (depends on 35)

The pharmaceutical composition of claim 35, wherein the second nucleic acid strand comprises bridged nucleotides independently selected from a ribonucleotide in which the carbon atom at the 2′-position and the carbon atom at the 4′-position are bridged by 4′-(CH2) p-O-2′, 4′-(CH2) p-S-2 ‘, 4′-(CH2) p-OCO-2, 4′-(CH2) n-N (R3)-O— (CH2) m-2’, where p, m and n represent an integer from 1 to 4, an integer from 0 to 2, and an integer from 1 to 3, respectively, and R3 represents a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group.

### Claim 38 (depends on 1)

A method for modifying the expression of a target gene by a non-RNase H-dependent antisense effect comprising contacting a cell with the pharmaceutical composition of claim 1.