# US 2010/0169989 A1 · Use of Follistatin-Like Related Gene (FLRG) to Increase Muscle Mass

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- Number: US 2010/0169989 A1
- Document ID: US20100169989A1
- Status: Published application
- Kind: A1
- Application number: 12/529,179
- Filed: 2008-03-06
- Published: 2010-07-01
- Earliest claimed: 2007-03-07
- Assignees: The Johns Hopkins University
- Inventors: Se-Jin Lee
- Web page: https://patentgrep.com/patent/US20100169989A1

## CPC

- A61K 38/16: Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof {(enzyme inhibitors A61K38/005)}
- A01K 67/0275: Genetically modified vertebrates, e.g. transgenic
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- C07K 14/4703: {Inhibitors; Suppressors}
- C07K 14/475: Growth factors; Growth regulators
- C07K 14/71: for growth factors; for growth regulators
- A01K 2217/05: Animals comprising random inserted nucleic acids (transgenic)
- A01K 2217/054: inducing loss of function
- A01K 2217/15: Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
- A01K 2217/206: Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
- A01K 2227/105: Murine
- A01K 2267/035: Animal model for multifactorial diseases
- A01K 2267/0362: Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- 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)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- 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)

## Abstract

The present relates to use of follistatin-like related gene (FLRG) to increase muscle mass in a subject. As such, methods of ameliorating the severity of a pathologic condition characterized, at least in part, by a decreased amount, development or metabolic activity of muscle are provided. In addition transgenic non-human mammals expressing FLRG and having increased muscle mass as compared to a corresponding mammal having a myostatin-null mutation or a decreased level of myostatin are provided.

## Claims

### Claim 1

A method of increasing muscle tissue growth in a subject comprising:
(a) administering to the subject a therapeutically effective amount of Follistatin-Like Related Gene (FLRG); and
(b) comparing an increase in muscle tissue growth in the subject to muscle tissue growth of a corresponding subject treated with a specific inhibitor of myostatin expression or activity,
wherein the increase in muscle tissue growth following administration of FLRG is greater as compared to the muscle tissue growth of the corresponding subject treated with an inhibitor of myostatin expression or activity, which is indicative of increasing the growth of muscle tissue in the subject as a result of FLRG administration.

### Claim 2 (depends on 1)

The method of claim 1, wherein the subject is a mammal.

### Claim 3 (depends on 2)

The method of claim 2, wherein the mammal is ovine, porcine, bovine, murine or human.

### Claim 4 (depends on 1)

The method of claim 1, wherein the muscle tissue growth is at least two-fold greater than the muscle tissue growth of the corresponding subject treated with a specific inhibitor of myostatin expression or activity in the absence of another agent.

### Claim 5

A method of increasing the growth of muscle tissue in a subject comprising administering to the subject a therapeutically effective amount of an inhibitor of myostatin expression or activity in combination with a therapeutically effective amount of FLRG either prior to, simultaneously with or following the inhibitor of myostatin expression or activity, thereby increasing muscle tissue growth in the subject.

### Claim 6 (depends on 5)

The method of claim 5, wherein the inhibitor of myostatin expression or activity is a myostatin prodomain comprising amino acid residues from about 20 to 262 of a promyostatin polypeptide as set forth in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20.

### Claim 7 (depends on 5)

The method of claim 5, wherein the inhibitor of myostatin expression or activity is an antibody, or functional fragment thereof, which binds to myostatin.

### Claim 8 (depends on 7)

The method of claim 7, wherein the antibody is a monoclonal antibody or a polyclonal antibody.

### Claim 9 (depends on 8)

The method of claim 8, wherein the monoclonal antibody is a humanized monoclonal antibody or a chimeric monoclonal antibody or fragment thereof.

### Claim 10 (depends on 5)

The method of claim 5, wherein the inhibitor of myostatin expression or activity is an antisense or interfering RNA nucleic acid.

### Claim 11 (depends on 5)

The method of claim 5, wherein the inhibitor of myostatin expression or activity is a polynucleotide encoding a dominant negative myostatin polypeptide or a polynucleotide encoding a truncated myostatin polypeptide.

### Claim 12 (depends on 5)

The method of claim 5, wherein the muscle tissue growth is at least two-fold greater than the muscle tissue growth of the corresponding subject treated with a specific inhibitor of myostatin expression or activity in the absence of another agent.

### Claim 13 (depends on 5)

The method of claim 5, wherein the subject is a mammal.

### Claim 14 (depends on 13)

The method of claim 13, wherein the mammal is ovine, porcine, bovine, murine, or human.

### Claim 15

A method of ameliorating the severity of a pathologic condition characterized, at least in part, by a decreased amount, development or metabolic activity of muscle comprising:
(a) contacting a muscle cell of a subject in need thereof with an FLRG polynucleotide or polypeptide, and
(b) comparing an increase in muscle tissue growth in the subject to muscle tissue growth of a corresponding subject treated with a specific inhibitor of myostatin expression or activity,
wherein the increase in muscle tissue growth following administration of FLRG is greater as compared to the muscle tissue growth in the subject treated with an inhibitor of myostatin expression or activity, which is indicative of increasing the growth of muscle tissue in the subject as a result of FLRG administration, thereby ameliorating the severity of the pathologic condition.

### Claim 16 (depends on 15)

The method of claim 15, wherein the pathological condition is a wasting disorder.

### Claim 17 (depends on 16)

The method of claim 16, wherein the wasting disorder is selected from the group consisting of cachexia and anorexia.

### Claim 18 (depends on 16)

The method of claim 16, wherein the wasting disorder is selected from the group consisting of a muscular dystrophy and a neuromuscular disease.

### Claim 19 (depends on 15)

The method of claim 15, wherein the pathological condition is a metabolic disorder.

### Claim 20 (depends on 19)

The method of claim 19, wherein the metabolic disorder is selected from the group consisting of obesity and type II diabetes.

### Claim 21

A method of ameliorating the severity of a pathologic condition characterized, at least in part, by a decreased amount, development or metabolic activity of muscle in a subject comprising contacting a muscle cell of a subject in need thereof with an inhibitor of myostatin expression or activity in combination with FLRG either prior to, simultaneous with or following the inhibitor of myostatin expression or activity.

### Claim 22 (depends on 21)

The method of claim 21, wherein the inhibitor of myostatin expression or activity is a myostatin prodomain comprising amino acid residues from about 20 to 262 of a promyostatin polypeptide as set forth in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20.

### Claim 23 (depends on 21)

The method of claim 21, wherein the inhibitor of myostatin expression or activity is an antibody, or functional fragment thereof, which binds to myostatin.

### Claim 24 (depends on 21)

The method of claim 21, wherein the antibody is a monoclonal antibody or a polyclonal antibody.

### Claim 25 (depends on 24)

The method of claim 24, wherein the monoclonal antibody is a humanized monoclonal antibody or a chimeric monoclonal antibody or fragment thereof.

### Claim 26 (depends on 21)

The method of claim 21, wherein the inhibitor of myostatin expression or activity is an antisense or interfering RNA nucleic acid.

### Claim 27 (depends on 21)

The method of claim 21, wherein the inhibitor of myostatin expression or activity is a polynucleotide encoding a dominant negative myostatin polypeptide or a polynucleotide encoding a truncated myostatin polypeptide.

### Claim 28 (depends on 21)

The method of claim 21, wherein the pathological condition is a wasting disorder.

### Claim 29 (depends on 28)

The method of claim 28, wherein the wasting disorder is selected from the group consisting of cachexia and anorexia.

### Claim 30 (depends on 28)

The method of claim 28, wherein the wasting disorder is selected from the group consisting of a muscular dystrophy and a neuromuscular disease.

### Claim 31 (depends on 21)

The method of claim 21, wherein the pathological condition is a metabolic disorder.

### Claim 32 (depends on 31)

The method of claim 31, wherein the metabolic disorder is selected from the group consisting of obesity and type II diabetes.

### Claim 33

A transgenic non-human mammal whose genome contains a nucleic acid sequence comprising FLRG and a regulatory element comprising a muscle-specific promoter operably linked and integrated into the genome of the mammal, wherein the nucleic acid sequence is expressed so as to result in elevated levels of FLRG and an increase in muscle mass in the mammal as compared to a corresponding mammal whose genome contains a myostatin-null mutation or a decreased level of myostatin as compared with normal basal levels.

### Claim 34 (depends on 30)

The transgenic non-human mammal of claim 30, wherein the muscle tissue growth of the transgenic non-human mammal is at least two-fold greater than the muscle tissue growth of the corresponding mammal whose genome lacks FLRG and contains a myostatin-null mutation or a decreased level of myostatin as compared with normal basal level.

### Claim 35 (depends on 33)

The transgenic non-human mammal of claim 33, wherein the regulatory element comprises a myosin light chain promoter and an enhancer.

### Claim 36 (depends on 33)

The transgenic non-human mammal of claim 33, wherein the regulatory element comprises a myosin light chain promoter and myosin light chain 1/3 enhancer.

### Claim 37 (depends on 33)

The transgenic non-human mammal of claim 33, wherein the genome of the corresponding mammal contains a truncated Activin Type II receptor gene that encodes a truncated dominant negative Activin Type II receptor lacking kinase activity.

### Claim 38 (depends on 37)

The transgenic non-human mammal of claim 37, wherein the Activin Type II receptor is an Activin RIIA or an Activin RIIB.

### Claim 39 (depends on 33)

The transgenic non-human mammal of claim 33, wherein the mammal is murine, ovine, porcine, or bovine.

### Claim 40

A transgenic non-human mammal whose genome contains a myostatin-null mutation or a decreased level of myostatin as compared with normal basal levels, a nucleic acid sequence comprising FLRG, and a regulatory element comprising a muscle-specific promoter operably linked and integrated into the genome of the mammal, wherein the nucleic acid sequence is expressed so as to result in elevated levels of FLRG and an increase in muscle mass in the mammal as compared to a corresponding mammal whose genome contains a myostatin-null mutation or a decreased level of myostatin alone.

### Claim 41 (depends on 37)

The transgenic non-human mammal of claim 37, wherein the muscle tissue growth of the transgenic non-human mammal is at least two-fold greater than the muscle tissue growth of the corresponding mammal whose genome lacks FLRG and contains a myostatin-null mutation or a decreased level of myostatin as compared with normal basal level.

### Claim 42 (depends on 40)

The transgenic non-human mammal of claim 40, wherein the regulatory element comprises a myosin light chain promoter and an enhancer.

### Claim 43 (depends on 40)

The transgenic non-human mammal of claim 40, wherein the regulatory element comprises a myosin light chain promoter and myosin light chain 1/3 enhancer.

### Claim 44 (depends on 40)

The transgenic non-human mammal of claim 40, wherein either or both of the genomes of the transgenic non-human mammal and the corresponding mammal contains a truncated Activin Type II receptor gene that encodes a truncated dominant negative Activin Type II receptor lacking kinase activity.

### Claim 45 (depends on 44)

The transgenic non-human mammal of claim 44, wherein the Activin Type II receptor is an Activin RIIA or an Activin RIIB

### Claim 46 (depends on 40)

The transgenic non-human mammal of claim 40, wherein the mammal is murine, ovine, porcine, or bovine.