# US 2014/0155463 A1 · Spinal muscular atrophy treatment via targeting smn2 catalytic core

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- Number: US 2014/0155463 A1
- Document ID: US20140155463A1
- Status: Published application
- Kind: A1
- Application number: 14/134,057
- Filed: 2013-12-19
- Published: 2014-06-05
- Earliest claimed: 2010-04-28
- Assignees: Iowa State University Research Foundation, Inc.
- Applicants: Iowa State University Research Foundation, Inc.
- Inventors: Ravindra N. Singh; Natalia N. Singh
- Web page: https://patentgrep.com/patent/US20140155463A1

## 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)}
- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 25/00: Drugs for disorders of the nervous system
- C12N 2310/11: Antisense
- C12N 2310/315: Phosphorothioates
- C12N 2310/321: 2'-O-R Modification
- C12N 2310/3231: having an additional ring, e.g. LNA, ENA
- C12N 2320/30: Special therapeutic applications
- 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)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

The present invention is directed to methods and compositions for blocking the effect of the intronic inhibitory splicing region of intron 7 of the SMN2 gene. The compositions and methods of the instant invention include short oligonucleotide reagents (e.g., oligoribonucleotides) that effectively target sites in the SMN2 pre-mRNA, thereby modulating the splicing of SMN2 pre-mRNA to include exon 7 in the processed transcript. The short target regions are 8-mers and 5-mers and also include the identification of a single nucleotide base that is essential for initiating a long distance stearic inhibitory interactions as well as novel targets distant from intron 7 which block the intronic inhibitory splicing of the same. These short target regions and concomitant inhibitory blocking oligonucleotides are less expensive and easier to manufacture and are small enough to cross the blood brain barrier.

## Claims

### Claim 1

A method of enhancing the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in a cell or cell extract, comprising contacting the cell or cell extract with an oligonucleotide which is complementary to nucleotide 10C and 3 or more bases 5′ thereof of intron 7 of the SMN2 gene, such that the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in the cell or cell extract is enhanced.

### Claim 2 (depends on 1)

The method of claim 1 wherein said oligonucleotide has 8 or fewer nucleotides and which is complementary to nucleotides 7-14 of intron 7 of the SMN2 gene.

### Claim 3 (depends on 1)

The method of claim 1, wherein said oligonucleotide has a sequence complementary to the sequence 5′-CUGCCAGC-3′.

### Claim 4 (depends on 1)

The method of claim 1, wherein said oligonucleotide has a sequence complementary to the sequence 5′-CUGCC-3′.

### Claim 5 (depends on 1)

The method of claim 1, wherein the cell or cell extract is a spinal muscular atrophy (SMA) patient-derived neuronal cell, muscle cell or fibroblast, or extract thereof.

### Claim 6 (depends on 1)

The method of claim 1, wherein the cell or cell extract is selected from the group consisting of an embryonic stem cell, an embryonic stem cell extract, a neuronal stem cell and a neuronal stem cell extract.

### Claim 7 (depends on 1)

The method of claim 1, wherein the oligonucleotide is modified by the substitution of at least one nucleotide with a modified nucleotide, such that in vivo stability is enhanced as compared to a corresponding unmodified oligonucleotide.

### Claim 8 (depends on 7)

The method of claim 7, wherein the modified nucleotide is a sugar-modified nucleotide.

### Claim 9 (depends on 7)

The method of claim 7, wherein the modified nucleotide is a nucleobase-modified nucleotide.

### Claim 10 (depends on 9)

The method of claim 9, wherein the modified nucleotide is a 2′-deoxy ribonucleotide.

### Claim 11 (depends on 10)

The method of claim 10, wherein the 2′-deoxy ribonucleotide is 2′-deoxy adenosine or 2′-deoxy guanosine.

### Claim 12 (depends on 7)

The method of claim 7, wherein the modified nucleotide is a 2′-O-methyl ribonucleotide.

### Claim 13 (depends on 7)

The method of claim 7, wherein the modified nucleotide is selected from the group consisting of a 2′-fluoro, 2′-amino and 2′-thio modified ribonucleotide.

### Claim 14 (depends on 7)

The method of claim 7, wherein the modified nucleotide is selected from the group consisting of 2′-fluoro-cytidine, 2′-fluoro-uridine, 2′-fluoro-adenosine, 2′-fluoro-guanosine, 2′-amino-cytidine, 2′-amino-uridine, 2′-amino-adenosine, 2′-amino-guanosine and 2′-amino-butyryl-pyrene-uridine.

### Claim 15 (depends on 7)

The method of claim 7, wherein the modified nucleotide is selected from the group consisting of 5-bromo-uridine, 5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine, 5-fluoro-cytidine, and 5-fluoro-uridine, 2,6-diaminopurine, 4-thio-uridine, and 5-amino-allyl-uridine.

### Claim 16 (depends on 7)

The method of claim 7, wherein the modified nucleotide is a backbone-modified nucleotide.

### Claim 17 (depends on 16)

The method of claim 16, wherein the backbone-modified nucleotide contains a phosphorothioate group.

### Claim 18 (depends on 7)

The method of claim 7, wherein the modified nucleotide is a locked nucleic acid (LNA).

### Claim 19

A method of enhancing the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in an organism, comprising administering to the same an oligonucleotide which is complementary to nucleotide 10C and 3 or more bases 5′ thereof of intron 7 of the SMN2 gene, such that the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in the organism extract is enhanced.

### Claim 20 (depends on 19)

The method of claim 19, wherein the organism is a mammal.

### Claim 21 (depends on 20)

The method of claim 20, wherein the organism is a human.

### Claim 22 (depends on 21)

The method of claim 21, wherein the human has spinal muscular atrophy (SMA).

### Claim 23 (depends on 19)

The method of claim 19 wherein said oligonucleotide has 8 or fewer nucleotides and which is complementary to nucleotides 7-14 of intron 7 of the SMN2 gene.

### Claim 24 (depends on 23)

The method of claim 23, wherein said oligonucleotide has a sequence complementary to the sequence 5′-CUGCCAGC-3′.

### Claim 25 (depends on 23)

The method of claim 23, wherein said oligonucleotide has a sequence complementary to the sequence 5′-CUGCC-3′.

### Claim 26

A method of treating spinal muscular atrophy (SMA) in a patient, comprising administering to the patient an oligonucleotide which is complementary to nucleotide 10C and 3 or more bases 5′ thereof of intron 7 of the SMN2 gene, in a dose effective to enhance the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in cells of the patient, such that SMA in the patient is treated.

### Claim 27 (depends on 26)

The method of claim 26 wherein said oligonucleotide has 8 or fewer nucleotides and which is complementary to nucleotides 7-14 of intron 7 of the SMN2 gene.

### Claim 28 (depends on 27)

The method of claim 27, wherein said oligonucleotide has a sequence complementary to the sequence 5′-CUGCCAGC-3′.

### Claim 29 (depends on 27)

The method of claim 27, wherein said oligonucleotide has a sequence complementary to the sequence 5′-CUGCC-3′.