# US 2020/0399638 A1 · Oligonucleotides matching col7a1 exon 73 for epidermolysis bullosa therapy

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- Number: US 2020/0399638 A1
- Document ID: US20200399638A1
- Status: Published application
- Kind: A1
- Application number: 16/746,403
- Filed: 2020-01-17
- Published: 2020-12-24
- Earliest claimed: 2015-03-11
- Assignees: Wings Therapeutics, Inc.
- Applicants: Wings Therapeutics, Inc.
- Inventors: Elisabeth Marlene Haisma; Marko Potman; Wouter Beumer; Vera Brinks
- Web page: https://patentgrep.com/patent/US20200399638A1

## 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)}
- C12N 2310/11: Antisense
- C12N 2310/315: Phosphorothioates
- C12N 2310/321: 2'-O-R Modification
- C12N 2310/346: having a combination of backbone and sugar modifications
- C12N 2320/33: Alteration of splicing
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Antisense oligonucleotides capable of preventing or reducing exon 73 inclusion into the human COL7A mRNA are characterized in various ways: (a) the oligonucleotide's sequence includes at most two CpG sequences; (b) the oligonucleotide has a length of no more than 24 nucleotides; (c) the oligonucleotide is capable of annealing to the (SRp40/SC35 binding/ESE) element in exon73. These oligonucleotides can usefully be oligoribonucleotides with modified internucleosidic linkages e.g. phosphorothioate linkages.

## Claims

### Claim at position 1

1-15. (canceled)

### Claim 16

An antisense oligoribonucleotide capable of preventing or reducing exon 73 inclusion into a human collagen type VII alpha 1 chain (COL7A1) mRNA when the mRNA is produced by splicing from a pre-mRNA in a human cell, wherein the oligoribonucleotide comprises a nucleotide sequence that is complementary to a target sequence in a COL7A1 pre-mRNA corresponding to SEQ ID NO: 5, 24, 25, 26, 27, 28, 39, 40, 41, 42, 43, 29, or 35, and the oligoribonucleotide comprises at least one nucleotide analogue or equivalent.

### Claim 17 (depends on 16)

The antisense oligoribonucleotide of claim 16, wherein the nucleotide sequence in the oligoribonucleotide that is complementary to the target nucleotide sequence has a degree of complementarity to the target nucleotide sequence of more than 80%, such that the oligoribonucleotide can anneal to the target nucleotide sequence in a COL7A1 pre-RNA molecule under physiological conditions, thereby facilitating skipping of exon 73.

### Claim 18 (depends on 16)

The antisense oligoribonucleotide of claim 16, wherein the target nucleotide sequence corresponds to SEQ ID NO: 35.

### Claim 19 (depends on 16)

The antisense oligoribonucleotide of claim 16, wherein
(a) the oligoribonucleotide has no more than two CpG sequences;
(b) the oligoribonucleotide has a length of between 12 and 24 nucleotides;
(c) the oligoribonucleotide has a non-natural internucleoside linkage; and/or
(d) the nucleotide analogue or equivalent comprises (i) a modified ribose moiety comprising a lower 2′-O-alkyl modification, a 2′-O-alkyl-O-alkyl modification, or a 2′-methoxyethoxy modification; or (ii) a locked nucleic acid (LNA), in which the 2′-carbon atom is linked to the 3′ or 4′ carbon atom of the ribose ring, thereby forming a bicyclic ribose moiety.

### Claim 20 (depends on 19)

The antisense oligoribonucleotide of claim 19, wherein
(a) the oligoribonucleotide has no more than one CpG sequence;
(b) the oligoribonucleotide has a length of between 16 and 23 nucleotides;
(c) the oligoribonucleotide has a phosphorothioate internucleoside linkage; and/or
(d) the nucleotide analogue or equivalent comprises (i) a modified ribose moiety comprising a 2′-O-methyl modification; or (ii) an LNA, in which the 2′-carbon atom is linked to the 4′ carbon atom of the ribose ring by a 2′-O, 4′-C-ethylene bridge.

### Claim 21 (depends on 20)

The antisense oligoribonucleotide of claim 20, wherein
(a) all the internucleoside linkages of the oligoribonucleotide are phosphorothioate linkages; and/or
(b) all the nucleotides of the oligoribonucleotide are nucleotide analogues or equivalents comprising (i) a modified ribose moiety comprising a 2′-O-methyl modification; or (ii) an LNA, in which the 2′-carbon atom is linked to the 3′ or 4′ carbon atom of the ribose ring.

### Claim 22 (depends on 16)

The antisense oligoribonucleotide of claim 16, wherein the oligoribonucleotide is capable of reducing exon 73 inclusion in a HeLa cell, or a sample derived therefrom, by more than 70%.

### Claim 23 (depends on 22)

The antisense oligoribonucleotide of claim 22, wherein the oligoribonucleotide is capable of reducing exon 73 inclusion in a HeLa cell, or a sample derived therefrom, by more than 80%.

### Claim 24 (depends on 23)

The antisense oligoribonucleotide of claim 23, wherein the oligoribonucleotide is capable of reducing exon 73 inclusion in a HeLa cell, or a sample derived therefrom, by more than 90%.

### Claim 25 (depends on 16)

A composition comprising the antisense oligoribonucleotide of claim 16 and one or more of a carrier, excipient, stabilizer, transfection agent, diluent, gelling agent, and buffer.

### Claim 26 (depends on 25)

The composition of claim 25, which is a pharmaceutical composition for use in human therapy.

### Claim 27 (depends on 16)

A method for preventing or reducing exon 73 inclusion into a human COL7A1 mRNA when said mRNA is produced by splicing from an RNA transcript in a human cell, the method comprising providing to the cell the antisense oligoribonucleotide of claim 16, under conditions conducive to uptake of the oligoribonucleotide by the cell, and allowing splicing to take place.

### Claim 28 (depends on 16)

A method for treating a disease or condition associated with a mutated exon 73 in the COL7A1 gene in cells of a human subject, the method comprising administering to the human subject the antisense oligoribonucleotide of claim 16.

### Claim 29 (depends on 28)

The method of claim 28, wherein the disease or condition is dystrophic epidermolysis bullosa (DEB).

### Claim 30 (depends on 28)

The method of claim 28, wherein the antisense oligoribonucleotide is administered to the human subject as
(a) a gymnotic form of the oligoribonucleotide;
(b) a conjugated form of the oligoribonucleotide; or
(c) a vector capable of expressing the oligoribonucleotide.

### Claim 31 (depends on 30)

The method of claim 30, wherein
(a) a conjugated form of the oligoribonucleotide is administered to the human subject, and the oligoribonucleotide is linked to a moiety that enhances uptake of the oligoribonucleotide by the cells; or
(b) a vector capable of expressing the antisense oligoribonucleotide is administered to the human subject, and the vector is an adeno-associated virus (AAV) vector or a retroviral vector.

### Claim 32 (depends on 28)

The method of claim 28, wherein the administering is
(i) systemic or topical; and/or
(ii) repeated one or more times.

### Claim 33 (depends on 28)

The method of claim 28, wherein the antisense oligoribonucleotide comprises the nucleotide sequence of SEQ ID NO: 35.

### Claim 34 (depends on 28)

The method of claim 28, wherein the antisense oligoribonucleotide consists of the nucleotide sequence of SEQ ID NO: 35.

### Claim 35 (depends on 34)

The method of claim 34, wherein
(a) the oligoribonucleotide has a phosphorothioate internucleoside linkage; and/or
(b) the nucleotide analogue or equivalent comprises (i) a modified ribose moiety comprising a 2′-O-methyl modification; or (ii) an LNA, in which the 2′-carbon atom is linked to the 3′ or 4′ carbon atom of the ribose ring.