# US 8,785,410 B2 · Antisense composition and method for treating muscle atrophy

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- Number: US 8,785,410 B2
- Document ID: US8785410B2
- Status: Granted
- Kind: B2
- Application number: 12/983,798
- Filed: 2011-01-03
- Published: 2011-07-07
- Granted: 2014-07-22
- Earliest claimed: 2005-02-09
- Assignees: Sarepta Therapeutics, Inc.
- Applicants: Patrick L. Iversen; Dwight D. Weller; Alan P. Timmins
- Inventors: Patrick L. Iversen; Dwight D. Weller; Alan P. Timmins
- Web page: https://patentgrep.com/patent/US8785410B2

## CPC

- C12N 15/1136: {against growth factors, growth regulators, cytokines, lymphokines or hormones}
- A61K 31/675: having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
- A61K 47/645: {Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT}
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07K 7/08: having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68)
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12N 2310/11: Antisense
- C12N 2310/31: of the backbone
- C12N 2310/3145: with the nitrogen in 3' or 5'-position
- C12N 2310/3233: Morpholino-type ring
- C12N 2310/3513: Protein; Peptide
- C12N 2320/30: Special therapeutic applications
- C12Q 2600/158: Expression markers
- Y10T 436/143333: Saccharide [e.g., DNA, etc.]
- 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
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- 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)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

A method and compound for treating skeletal muscle mass deficiency in a human subject are disclosed. The composition is an oligomer of morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, contains between 10-40 nucleotide bases, has a base sequence effective to hybridize to an expression-sensitive region of processed or preprocessed human myostatin RNA transcript, identified, in its processed form, by SEQ ID NO:6, and is capable of uptake by target muscle cells in the subject. In practicing the method, the compound is administered in an amount and at a dosage schedule to produce an overall reduction in the level of serum myostatin measured in the patient, and preferably to bring the myostatin level within the a range determined for normal, healthy individuals.

## Claims

### Claim 1

A method of treating skeletal muscle mass deficiency in a human subject comprising
(a) measuring blood or tissue levels of myostatin in the subject,
(b) administering to the subject, a myostatin-expression-inhibiting amount of a substantially uncharged antisense compound (i) composed of morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, (ii) capable of uptake by target muscle cells in the subject, (iii) containing between 10-40 nucleotide bases, and (iv) having a base sequence that is complementary to at least 12 contiguous bases in a target region of preprocessed human myostatin RNA transcript, identified by SEQ ID NO:13, wherein the antisense compound includes a base sequence as set forth in SEQ ID NO:3 and is capable of effectively blocking production of a full-length processed myostatin transcript from a preprocessed myostatin transcript,
(c) by said administering, forming within target muscle cells in the subject, a base-paired heteroduplex structure composed of human myostatin RNA transcript and the antisense compound and having a Tm of dissociation of at least 45° C., thereby inhibiting expression of myostatin in said cells,
(d) at a selected time following said administering, measuring a blood or tissue level of myostatin in the subject, and
(e) repeating said administering, using the myostatin levels measured in (d) to adjust the dose or dosing schedule of the amount of antisense compound administered, if necessary, so as to reduce measured levels of myostatin over those initially measured and to maintain such levels of myostatin measured in step (d) within a range determined for normal, healthy individuals.

### Claim 2 (depends on 1)

The method of claim 1, wherein the myostatin value measured in step (a) is above a selected threshold for normal healthy people.

### Claim 3 (depends on 1)

The method of claim 1, wherein the morpholino subunits in the compound administered are joined by phosphorodiamidate 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, amino or alkyl amino, including dialkylamino.

### Claim 4 (depends on 1)

The method of claim 1, wherein the compound is administered as a conjugate of the compound and an arginine-rich peptide effective to promote uptake of the compound into muscle target cells.

### Claim 5 (depends on 2)

The method of claim 2, in which at least 2 and no more than half of the total number of intersubunit linkages are positively charged at physiological pH.

### Claim 6 (depends on 5)

The method of claim 5, wherein said morpholino subunits are joined by phosphorodiamidate 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 for the uncharged linkages is alkyl, alkoxy, thioalkoxy, or an alkyl amino of the form wherein NR2, where each R is independently hydrogen or methyl, and for the positively charged linkages, X is 1-piperazine.

### Claim 7 (depends on 4)

The method of claim 4, wherein the arginine-rich peptide has one of the sequences identified as SEQ ID NOS:7-9, and the arginine-rich peptide is covalently coupled at its C terminus to the 5′ end of the antisense compound.

### Claim 8 (depends on 1)

The method of claim 1, wherein said administering and measuring is carried out for a selected period of at least 2 weeks.

### Claim 9 (depends on 1)

The method of claim 1, wherein said administering is by oral administration.

### Claim 10

An antisense composition for use in increasing skeletal muscle mass in a human subject, comprising a substantially uncharged antisense compound
(i) composed of morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit,
(ii) capable of uptake by target muscle cells in the subject,
(iii) containing between 10-40 nucleotide bases, and
(iv) having a base sequence that is complementary to at least 12 contiguous bases in a target region of preprocessed human myostatin transcript identified by SEQ ID NO:13,
wherein the antisense compound includes a base sequence as set forth in SEQ ID NO:3 and is capable of effectively blocking production of a full-length processed myostatin transcript from a preprocessed myostatin transcript.

### Claim 11 (depends on 10)

The composition of claim 10, wherein the morpholino subunits in the compound are joined by phosphorodiamidate 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, amino or alkyl amino, including dialkylamino.

### Claim 12 (depends on 10)

The composition of claim 10, which includes said compound conjugated to an arginine-rich peptide effective to promote uptake of the compound into target muscle cells.

### Claim 13 (depends on 10)

The composition of claim 10, in which at least 2 and no more than half of the total number of intersubunit linkages are positively charged at physiological pH.

### Claim 14 (depends on 13)

The composition of claim 13, wherein said morpholino subunits are joined by phosphorodiamidate 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 for the uncharged linkages is alkyl, alkoxy, thioalkoxy, or an alkyl amino of the form wherein NR2, where each R is independently hydrogen or methyl, and for the positively charged linkages, X is 1-piperazine.

### Claim 15 (depends on 12)

The composition of claim 12, wherein the arginine-rich peptide has one of the sequences identified as SEQ ID NOS:7-9.

### Claim 16 (depends on 12)

The composition of claim 12, arginine-rich peptide is covalently coupled at its C terminus to the 5′ end of the antisense compound.