Compositions and methods for enhanced expression of recombinant polypeptides from a single vector using a peptide cleavage site

• US 20050042721A1
• Filed: 04/26/2004
• Published: 02/24/2005
• Est. Priority Date: 06/03/2003
• Status: Active Grant

• Current Assignee: BioSante Pharmaceuticals, Inc.
• Original Assignee: BioSante Pharmaceuticals, Inc.
• Inventors: Jooss, Karin, Fang, Jianmin, Simmons, Andrew, Qian, Jing-Jing
• Granted Patent: US 7,709,224 B2
• Time in Patent Office: Days
• US Class Current: 435/69.100
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C12N 15/8273 for drought, cold, salt resistance
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2800/60 Vectors containing traps for, e.g. exons, promoters
• CPC Class Codes: C12N 2830/00 Vector systems having a special element relevant for transcription
• CPC Class Codes: C12N 2830/15 chimeric enhancer/promoter combination
• CPC Class Codes: C12N 2830/42 being an intron or intervening sequence for splicing and/or stability of RNA
• CPC Class Codes: C12N 2830/48 regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE
• CPC Class Codes: C12N 2830/85 mammalian
• CPC Class Codes: C12N 2830/90 avian
• CPC Class Codes: C12N 2840/203 having an IRES

Abstract

Vector constructs for expression of two or more functional proteins or polypeptides under operative control of a single promoter and methods of making and using the same are described. The vectors comprise a self-processing cleavage site between each respective protein or polypeptide coding sequence. The vector constructs include the coding sequence for a self-processing cleavage site and may further include an additional proteolytic cleavage sequence which provides a means to remove the self processing peptide sequence from expressed protein(s) or polypeptide(s). The vector constructs find utility in methods for enhanced production of biologically active proteins and polypeptides in vitro and in vivo.

Claims

1. A vector for expression of a recombinant protein, comprising:

in the 5′

to 3′

direction, a promoter operably linked to a first protein or polypeptide coding sequence, a sequence encoding a self-processing cleavage site and a second protein or polypeptide coding sequence.

2. A vector according to claim 1, wherein the vector is selected from the group consisting of an adeno-associated virus (AAV) vector, a lentivirus vector, a retrovirus vector, a replication competent adenovirus vector, a replication deficient adenovirus vector, a gutless adenovirus vector, a herpes virus vector and a nonviral plasmid.

3. A vector according to claim 2, wherein the sequence encoding said self-processing cleavage site comprises a 2A sequence.

4. A vector according to claim 3, wherein said 2A sequence is a Foot and Mouth Disease Virus (FMDV) sequence.

5. A vector according to claim 4, wherein the 2A sequence encodes a peptide comprising amino acid residues NFDLLKLAGDVESNPGPFF (SEQ ID NO:

1).

6. A vector according to claim 3, wherein said first protein or polypeptide coding sequence encodes a Factor VIII heavy chain or fragment thereof.

7. A vector according to claim 3, wherein said first protein or polypeptide coding sequence encodes a Factor VIII light chain or fragment thereof.

8. A vector according to claim 6, wherein said coding sequence is the full length coding sequence for a Factor VIII heavy chain.

9. A vector according to claim 7, wherein said coding sequence is the full length coding sequence for a Factor VIII light chain.

10. A vector according to claim 1, further comprising an additional proteolytic cleavage site between said first protein or polypeptide coding sequence and said sequence encoding a self-processing cleavage site.

11. A vector according to claim 10, wherein said additional proteolytic cleavage site is a furin cleavage site with the consensus sequence RXK(R)R (SEQ ID NO:

10).

12. A vector according to claim 1, wherein the promoter is selected from the group consisting of an elongation factor 1-alpha promoter (EF1a) promoter, a phosphoglycerate kinase-1 promoter (PGK) promoter, a cytomegalovirus immediate early gene promoter (CMV), a chimeric liver-specific promoter (LSP) a cytomegalovirus enhancer/chicken beta-actin promoter (CAG), a tetracycline responsive promoter (TRE), a transthyretin promoter (TTR), a simian virus 40 promoter (SV40) and a CK6 promoter.

13. A vector according to claim 1, further comprising a signal sequence.

14. A vector according to claim 2, wherein said vector is an adeno-associated virus (AAV) vector.

15. A vector according to claim 3, wherein said first protein or polypeptide coding sequence and said second protein or polypeptide coding sequence are expressed in a substantially equimolar ratio.

16. A recombinant protein or polypeptide produced by a cell transfected with a vector of claim 5.

17. A recombinant protein or polypeptide produced by a cell transfected with a vector of claim 11.

18. A recombinant protein or polypeptide produced by a cell transfected with a vector of claim 14.

19. A host cell transfected with a vector of claim 5.

20. A host cell transfected with a vector of claim 11.

21. A host cell transfected with a vector of claim 14.

22. A method for producing a protein, polypeptide or fragment thereof, comprising the steps of:

(i) transducing a host cell with a vector according to claim 1;

and

(ii) expressing said recombinant protein or polypeptide in said transduced host cell, wherein said first protein or polypeptide coding sequence and said second protein or polypeptide coding sequence are expressed in a substantially equimolar ratio.

23. The method according to claim 22, wherein the sequence encoding said self-processing cleavage site comprises a 2A sequence.

24. The method according to claim 23, wherein said 2A sequence is a Foot and Mouth Disease Virus (FMDV) sequence.

25. The method according to claim 24, wherein the 2A sequence encodes an oligopeptide comprising amino acid residues NFDLLKLAGDVESNPGPFF (SEQ ID NO:

1).

26. The method according to claim 23, wherein said vector further comprises an additional proteolytic cleavage site between the coding sequence for said first protein or polypeptide chain and the sequence encoding said self-processing cleavage site.

27. The method according to claim 26, wherein said additional proteolytic cleavage site is a furin cleavage site with the consensus sequence RXK(R)R (SEQ ID NO:

10).