# US 9,040,484 B2 · Production and delivery of a stable collagen

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- Number: US 9,040,484 B2
- Document ID: US9040484B2
- Status: Granted
- Kind: B2
- Application number: 14/112,444
- Filed: 2012-04-26
- Published: 2014-04-17
- Granted: 2015-05-26
- Earliest claimed: 2011-04-26
- Assignees: The Board of Trustees of the Leland Stanford Junior University; Department of Veterans Affairs
- Applicants: M. Peter Marinkovich; Alfred T. Lane; Jayakumar Rajadas
- Inventors: M. Peter Marinkovich; Alfred T. Lane; Jayakumar Rajadas
- Web page: https://patentgrep.com/patent/US9040484B2

## CPC

- C07K 14/78: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
- A61K 38/39: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
- A61M 37/0015: {by using microneedles}
- C12N 9/0071: {acting on paired donors with incorporation of molecular oxygen (1.14)}
- C12N 15/67: General methods for enhancing the expression
- A61M 2037/0023: {Drug applicators using microneedles}
- 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)
- A61M: DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY (introducing media into or onto the bodies of animals A61D7/00; means for inserting tampons A61F13/26; devices for administering food or medicines orally A61J; containers for collecting, storing or administering blood or medical fluids A61J1/05); DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY (surgery A61B; chemical aspects of surgical articles A61L); DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR {(Electrotherapy, e.g. producing anaesthesia by the use of alternating or intermittent currents A61N1/36021)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Improved methods are provided for the recombinant synthesis of collagen, particularly collagen VII, in host cell, and for therapeutic delivery of the same. The recombinant collagen is produced in a host cell that has increased levels of prolyl-4-hydroxylase, relative to basal cell levels. The collagen produced by the methods of the invention has increased numbers of modified proline residues, relative to a recombinant collagen produced in a host cell having basal levels of prolyl-4-hydroxylase. The increased proline modification provides for a collagen having increased stability, including increased in vivo stability.

## Claims

### Claim 1

A method of synthesizing collagen VII, the method comprising:
synthesizing recombinant collagen VII in a host cell that has been genetically modified to increase expression of prolyl-4-hydroxylase, wherein the genetic modification to increase expression of prolyl-4-hydroxylase comprises (i) introduction into the cell of an episomal vector comprising a genetic sequence encoding prolyl-4-hydroxylase operably linked to a promoter, or (ii) modifying the genomic sequence of an endogenous prolyl-4-hydroxylase in said cell to increase expression;
wherein recombinantly produced collagen VII has increased stability relative to a collagen produced in a comparable host cell lacking said genetic modification.

### Claim 2 (depends on 1)

The method of claim 1, wherein said episomal vector further comprises a genetic sequence encoding collagen VII operably linked to a promoter.

### Claim 3 (depends on 1)

The method of claim 1, wherein one or both of said collagen VII and said prolyl-4-hydroxylase are human.

### Claim 4 (depends on 1)

The method of claim 1, wherein said host cell is a mammalian cell.

### Claim 5 (depends on 1)

The method of claim 1, further comprising the step of isolating said collagen VII from said cell.

### Claim 6 (depends on 5)

The method of claim 5, further comprising the step of administering said collagen VII to an individual in need thereof.

### Claim 7 (depends on 6)

The method of claim 6, wherein said administration is intradermal.

### Claim 8 (depends on 6)

The method of claim 6, wherein said individual suffers from epidermolysis bullosa.

### Claim 9 (depends on 8)

The method of claim 8, wherein said epidermolysis bullosa is the result of a genetic defect in collagen VII.