EXPRESSION OF TGF-BETA IN PLASTIDS

• US 20090328250A1
• Filed: 09/11/2007
• Published: 12/31/2009
• Est. Priority Date: 09/11/2006
• Status: Abandoned Application

• Current Assignee: Renovo Ltd. (Bentley Park Ltd.)
• Original Assignee: Renovo Ltd. (Bentley Park Ltd.)
• Inventors: Day, Anil, Gisby, Martin, Ferguson, Mark William James, Occleston, Nick, Mellors, Phillip, O'Kane, Sharon, Laverty, Hugh Gerard
• Application Number:
  US12/440,688
• Publication Number:
  US 20090328250A1
• Time in Patent Office: Days
• US Class Current: 800/278
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 19/10 for osteoporosis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/02 for peripheral neuropathies
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/495 Transforming growth factor [TGF]
• CPC Class Codes: C12N 15/8214 Plastid transformation
• CPC Class Codes: C12N 15/8257 for the production of primary gene products, e.g. pharmaceutical products, interferon

Abstract

Provided is a method for the expression of a TGF-β in a plant. A chimeric nucleic acid sequence comprising: (1) a first nucleic acid sequence capable of regulating the transcription in a plant cell of (2) a second nucleic acid sequence, encoding a TGF-β, and adapted for expression in the plant cell; and (3) a third nucleic acid sequence encoding a termination region functional in said plant cell is introduced into a plant cell and the plant cell grown to produce TGF-β. The nucleic acid sequence may preferably be adapted for expression in a plant chloroplast. It is preferred that the TGF-β is TGF-β3, whether full length or in the form of an active fragment.

Claims

1. A method for the expression of a TGF-β

in a plant, said method comprising;

(a) introducing into a plant cell a chimeric nucleic acid sequence comprising;

(1) a first nucleic acid sequence capable of regulating the transcription in a plant cell of

(2) a second nucleic acid sequence, encoding a TGF-β

, and adapted for expression in the plant cell; and

(3) a third nucleic acid sequence encoding a termination region functional in said plant cell; and

(b) growing said plant cell to produce said TGF-β

.

2. A method according to claim 1, wherein the nucleic acid sequence is selected from the group consisting of:

a nucleic acid sequence suitable to be expressed in a chloroplast of a plant cell and a nucleic acid sequence adapted to be expressed in a chloroplast of a plant cell.

4. A method according to claim 1, wherein the TGF-β

is a human TGF-β

.

5. A method according to claim 1, wherein the TGF-β

is TGF-β

3.

6. A method according to claim 1, wherein the TGF-β

comprises a TGF-β

active fragment selected from the group consisting of;

Sequence ID No. 1;

Sequence ID No. 2; and

Sequence ID No. 3.

7. A method according to claim 1, wherein the TGF-β

comprises the full length TGF-β

protein.

8. A method according to claim 1, wherein the TGF-β

comprises a TGF-β

proprotein.

9. A method according to claim 1, wherein the second nucleic acid sequence comprises at least one substitution selected from the group consisting of:

a UGC codon compared to the native DNA encoding the TGF-β

3 ;

a CUG codon compared to the native DNA encoding the TGF-β

;

a UAC codon compared to the native DNA encoding the TGF-β

;

a GUG codon compared to the native DNA encoding the TGF-β

;

a CCC codon compared to the native DNA encoding the TGF-β

;

a AAC codon compared to the native DNA encoding the TGF-β

; and

a GAC codon compared to the native DNA encoding the TGF-β

.

16. A method according to claim 1, wherein the first nucleic acid sequence comprises a plastid promoter selected from the group consisting of:

promoters expressing photosynthesis-related genes;

promoters expressing genetic system genes;

promoters expressing genes recognised by the plastid encoded plastid (PEP) RNA polymerase or nucleus-encoded plastid (NEP) RNA polymerase;

a plastid psbA promoter; and

a plastid 16S rrn promoter.

17. A method according to claim 1, wherein the first nucleic acid sequence comprises a promoter selected from the group consisting of:

a Chlamydomonas psbA promoter;

a bacterial trc promoter;

a bacteriophage T7 promoter; and

a 16srrn promoter.

21. A method according to claim 1, wherein the first nucleic acid sequence comprises a ribosome binding site (RBS) selected from the group consisting of:

i) a plastid RBS;

ii) a bacterial RBS; and

iii) a bacteriophage RBS.

23. A method according to claim 1, wherein the third nucleic acid sequence comprises a terminator selected from the group consisting of:

i) a plastid terminator;

ii) a bacterial terminator; and

iii) a bacteriophage terminator.

25. A method according to claim 1, wherein the chimeric nucleic acid sequence further comprises a nucleic acid sequence for selection of transformed cells.

26. A method according to any of claim 1, wherein the second nucleic acid sequence comprises Sequence ID No. 5, or a sequence having at least 22% codon identity with Sequence ID No. 5.

28. A method according to claim 1, further comprising dissolving the TGF-β

in a solvent capable of preferentially solubilising recombinant TGF-β

, but not plant cell components.

30. A method according to any of claim 1, further comprising diafiltration to concentrate a solution of the TGF-β

.

31. A method according to claim 1, further comprising folding the TGF-β

in the presence of CHES (2-(cyclohexylamino)ethanesulfonic acid), or a functional analogue thereof, such that active TGF-β

is produced.

33. A method according to claim 1, further comprising using the TGF-β

so expressed in the manufacture of a medicament.

34. A method according to claim 33, wherein the medicament is for the prevention of scarring or fibrosis.

35. A TGF-β

produced by the method of claim 1.

36. A TGF-β

according to claim 35, wherein the TGF-β

is TGF-β

3.

37. A TGF-β

according to claim 35, wherein the TGF-β

comprises a TGF-β

active fragment selected from the group consisting of;

Sequence ID No. 1;

Sequence ID No. 2; and

Sequence ID No. 3.

38. A TGF-β

according to claim 35, wherein the TGF-β

comprises a TGF-β

proprotein.

45. A medicament comprising a TGF-beta produced in accordance with claim 1.

3. (canceled)

10-15. -15. (canceled)

18-20. -20. (canceled)

22. (canceled)

24. (canceled)

27. (canceled)

29. (canceled)

32. (canceled)

39. A chimeric nucleic acid sequence comprising:

(1) a first nucleic acid sequence capable of regulating the transcription in a plant cell of

(2) a second nucleic acid sequence, encoding a TGF-β

, and adapted for expression in a plant cell; and

(3) a third nucleic acid sequence encoding a termination region functional in a plant cell.

40. The nucleic acid of claim 39, comprising a nucleic acid sequence selected from the group consisting of:

a nucleic acid sequence suitable to be expressed in a chloroplast of a plant cell and a nucleic acid sequence adapted to be expressed in a chloroplast of a plant cell.

42. A nucleic acid sequence according to claim 39, comprising a nucleic acid sequence of Sequence ID No. 5, or a sequence having at least 22% codon identity with Sequence ID No. 5.

43. A plant transformed with a nucleic acid according to claim 39.

44. A plant seed comprising a nucleic acid according to claim 39.

41. (canceled)