Production and Delivery of a Stable Collagen

Patent metadata

Field	Value
Publication number	US-2015232535-A1
Application number	US-201514700951-A
Country	US
Kind code	A1
Filing date	Apr 30, 2015
Priority date	Apr 26, 2011
Publication date	Aug 20, 2015
Grant date	—

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.

First claim

What is claimed is:

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 recombinantly produced collagen VII has increased stability relative to a collagen produced in a comparable host cell lacking said genetic modification.

2 . The method of claim 1 , wherein the genetic modification to increase expression of prolyl-4-hydroxylase comprises introduction into the cell of an episomal vector comprising a genetic sequence encoding prolyl-4-hydroxylase operably linked to a promoter.

3 . The method of claim 2 , wherein said episomal vector further comprises a genetic sequence encoding collagen VII operably linked to a promoter.

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

5 . The method of claim 1 , wherein the genetic modification to increase expression of prolyl-4-hydroxylase comprises modifying the genomic sequence of an endogenous prolyl-4-hydroxylase in said cell to increase expression.

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

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

8 . The method of claim 7 , further comprising the step of administering said collagen VII to an individual in need thereof.

9 . The method of claim 8 , wherein said administration is intradermal.

10 . The method of claim 8 , wherein said individual suffers from epidermolysis bullosa.

11 . The method of claim 10 , wherein said epidermolysis bullosa is the result of a genetic defect in collagen VII.

12 . A pharmaceutical composition comprising a purified collagen VII produced by the methods of claim 1 ; and
a pharmaceutically acceptable excipient.

13 . A method of delivering a therapeutic protein to the dermal layer of the skin, the method comprising:
fabricating an array of biodegradable or biocompatible microneedles, wherein said microneedles comprise said therapeutic protein; and contacting the skin of an affected individual with said array with a pressure sufficient to deliver said microneedles to the dermal layer of the skin.

14 . The method of claim 13 , wherein said therapeutic protein is collagen VII.

15 . The method of claim 14 , wherein said collagen VII is produced by the method of synthesizing recombinant collagen VII in a host cell that has been genetically modified to increase expression of prolyl-4-hydroxylase;
wherein recombinantly produced collagen VII has increased stability relative to a collagen produced in a comparable host cell lacking said genetic modification.

16 . The method of claim 15 , wherein said individual suffers from epidermolysis bullosa.

17 . An array of biodegradable or biocompatible microneedles, wherein said microneedles comprise a therapeutic protein.

18 . The array of claim 17 , wherein said therapeutic protein is collagen VII.

19 . The array of claim 18 , wherein said collagen VII is produced by the method of synthesizing recombinant collagen VII in a host cell that has been genetically modified to increase expression of prolyl-4-hydroxylase;
wherein recombinantly produced collagen VII has increased stability relative to a collagen produced in a comparable host cell lacking said genetic modification.

Assignees

• Univ Leland Stanford Junior
• Us Dept Veterans Affairs

Inventors

• Marinkovich M Peter
• Lane Alfred T
• Rajadas Jayakumar

Classifications

• A61M37/0015

  by using microneedles · CPC title

• A61M2037/0023

  Drug applicators using microneedles · CPC title

• C07K14/78 Primary

  Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG] · CPC title

• A61K38/39

  Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG] · CPC title

• C12N9/0071

  acting on paired donors with incorporation of molecular oxygen (1.14) · CPC title