# US 8,772,571 B2 · Compositions and methods for osteogenic gene therapy

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- Number: US 8,772,571 B2
- Document ID: US8772571B2
- Status: Granted
- Kind: B2
- Application number: 12/879,842
- Filed: 2010-09-10
- Published: 2011-01-06
- Granted: 2014-07-08
- Earliest claimed: 2005-06-14
- Assignees: The United States of America as represented by the Department of Veterans Affairs; Loma Linda University
- Applicants: Kin-Hing William Lau; David J. Baylink; Susan L. Hall; Shin-Tai Chen; Subburaman Mohan
- Inventors: Kin-Hing William Lau; David J. Baylink; Susan L. Hall; Shin-Tai Chen; Subburaman Mohan
- Web page: https://patentgrep.com/patent/US8772571B2

## CPC

- C07K 14/50: Fibroblast growth factor [FGF]
- A61K 38/1825: {Fibroblast growth factor [FGF]}
- A61P 19/08: for bone diseases, e.g. rachitism, Paget's disease
- A61P 19/10: for osteoporosis
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- A01K 2207/12: Animals modified by administration of exogenous cells
- A01K 2267/035: Animal model for multifactorial diseases
- C12N 2799/027: where the vector is derived from a retrovirus
- 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)
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS

## Abstract

The present disclosure provides compositions and methods for increasing bone growth and/or enhancing wound healing, for example, fracture repair. The disclosure provides recombinant nucleic acids useful for promoting bone growth. For example, the disclosure provides recombinant nucleic acids that encode a fibroblast growth factor-2 (FGF-2) analog. The disclosure also provides vectors and cells incorporating these nucleic acids, as well as FGF-2 analogs encode by them. The disclosure also provides a mouse system of bone marrow transplantation and methods for producing as well as methods for using the system. Methods for inducing division and/or inducing differentiation of a hematopoietic stem cell are also provided, as are methods for enhancing bone growth and/or wound repair (for example, fracture repair).

## Claims

### Claim 1

A method of identifying an agent that modulates bone growth, maintenance and repair, the method comprising:
harvesting at least one hematopoietic stem cell;
transducing and expressing in the at least one mouse hematopoietic stem cell at least one heterologous nucleic acid comprising a polynucleotide sequence that encodes an osteogenic growth factor comprising a secretable analog of human fibroblast growth factor-2 (FGF-2) encoded by a nucleic acid sequence comprising in a 5′ to 3′ direction: a polynucleotide sequence that encodes a bone morphogenetic protein 2/4 (BMP2/4) hybrid secretion signal peptide sequence and a polynucleotide sequence that encodes a mature human FGF-2 polypeptide comprising a mutation that results in the substitution of an alanine for a cysteine in amino acid position 70 of FGF-2 and a mutation that results in the substitution of an asparagine for a cysteine in amino acid position 88 of FGF-2;
introducing the at least one hematopoietic stem cell or a progeny thereof into an immunologically compatible mouse, wherein expression of the secretable analog of human FGF-2 produces an increase in serum level of FGF-2 as compared to immunologically compatible mice engrafted with hematopoietic stem cells transduced with the BMP2/4 hybrid secretion signal peptide sequence and a wild type FGF-2-control;
administering an agent to the mouse; and
screening for modulation of FGF-2 secretion, wherein an increase in FGF-2 secretion identifies an agent that increases bone growth.

### Claim 2 (depends on 1)

The method of claim 1, wherein the hematopoietic stem cell is a pluripotent hematopoietic stem cell.

### Claim 3 (depends on 2)

The method of claim 2, wherein the hematopoietic stem cell expresses CXCR4.

### Claim 4 (depends on 2)

The method of claim 2, wherein the hematopoietic stem cell is a Sca-1+ cell.

### Claim 5 (depends on 1)

The method of claim 1, wherein the at least one heterologous nucleic acid comprising the polynucleotide sequence that encodes an osteogenic growth factor is introduced into the hematopoietic stem cell by electroporation, biolistic transformation, lipid mediated transformation, or transfection of the cell with a viral vector comprising the heterologous nucleic acid.

### Claim 6 (depends on 5)

The method of claim 5, wherein the viral vector is an adenovirus vector, an adeno-associated virus vector, a murine leukemia virus vector or a lentivirus vector.

### Claim 7 (depends on 6)

The method of claim 6, wherein the viral vector is a replication defective viral vector.

### Claim 8 (depends on 1)

The method of claim 1, wherein the polynucleotide sequence that encodes an osteogenic growth factor is operably linked to a transcription control sequence comprising a regulatable promoter.

### Claim 9 (depends on 8)

The method of claim 8, wherein the transcription control sequence comprises a tetracycline regulatable promoter.