Poxvirus Expression System

• US 20130195912A1
• Filed: 04/15/2011
• Published: 08/01/2013
• Est. Priority Date: 04/16/2010
• Status: Active Grant

• Current Assignee: Oxford University Innovation Limited (University of Oxford)
• Original Assignee: Isis Innovation Limited
• Inventors: Cottingham, Matthew Guy
• Granted Patent: US 9,273,327 B2
• Time in Patent Office: Days
• US Class Current: 424/199.1
• CPC Class Codes: A61K 2039/5256 expressing foreign proteins
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 15/8636 Vaccina virus vectors
• CPC Class Codes: C12N 2710/24143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2800/204 of bacterial origin, e.g. BAC
• CPC Class Codes: C12N 2800/50 Vectors for producing vectors
• CPC Class Codes: C12N 2830/34 being a transcription initiation element
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

There is provided a method for inserting a nucleic acid sequence that encodes a foreign peptide into a poxvirus genome, said method comprising: identifying in the poxvirus genome a poxvirus open reading frame wherein said open reading frame is characterised by an initial ATG start codon and wherein expression of said open reading frame is driven by an operably-linked poxvirus promoter located upstream of the open reading frame and wherein expression of said open reading frame provides a peptide that is non-essential to viability of the poxvirus; and inserting the nucleic acid sequence that encodes the foreign peptide at a position downstream of the poxvirus promoter; wherein following said insertion, (i) the nucleic acid that encodes the foreign peptide is operably-linked to the poxvirus promoter and expression of said nucleic acid is driven by said poxvirus promoter; and (ii) translation of the foreign peptide is initiated at an ATG start codon located at the same position as the ATG start codon of the poxvirus open reading frame. Also provided are a poxvirus vector and corresponding uses of the poxvirus vector in medicine.

Claims

1. A method for inserting a nucleic acid sequence that encodes a foreign peptide into a poxvirus genome, said method comprising:

A) identifying in the poxvirus genome a poxvirus open reading frame wherein said open reading frame is characterised by an initial ATG start codon and wherein expression of said open reading frame is driven by an operably-linked poxvirus promoter located upstream of the open reading frame and wherein expression of said open reading frame provides a peptide that is non-essential to viability of the poxvirus; and

B) inserting the nucleic acid sequence that encodes the foreign peptide at a position downstream of the poxvirus promoter;

wherein following said insertion,

(i) the nucleic acid that encodes the foreign peptide is operably-linked to the poxvirus promoter and expression of said nucleic acid is driven by said poxvirus promoter; and

(ii) translation of the foreign peptide is initiated at an ATG start codon located at the same position as the ATG start codon of the poxvirus open reading frame.

2. The method of claim 1, wherein the nucleic acid sequence that encodes a foreign peptide lacks any promoter capable of driving expression of the foreign peptide.

4. The method of claim 1, wherein following insertion of the nucleic acid sequence that encodes a foreign peptide at least part of the poxvirus open reading frame remains present in the poxvirus genome.

5. The method of claim 1, wherein the nucleic acid sequence that encodes a foreign peptide is inserted in frame with the initial ATG start codon.

6. The method of claim 1, wherein during step A) a poxvirus genome is identified that comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs:

1-10 and wherein the start codon of the open reading frame is located at positions 26-28 of any one of SEQ ID NOs;

1-10.

7. The method of claim 6, wherein the nucleic acid sequence that encodes a foreign peptide is inserted in frame with the ATG located at positions 26-28 of any one of SEQ ID NOs:

1-10.

8. The method of claim 1, wherein the nucleic acid sequence that encodes a foreign peptide is inserted by homologous recombination.

9. The method of claim 1, wherein the poxvirus is selected from:

vaccinia virus, modified vaccinia virus Ankara (MVA), NYVAC, fowlpox virus, and canarypox virus.

12. The method of claim 9, wherein the poxvirus is vaccinia virus.

13. The method of claim 12, wherein the poxvirus open reading frame is selected from the following vaccinia virus open reading frames:

B8R, F11L, K6L, A44L, E5R, C11R, and B2R.

14. A poxvirus vector obtainable by the method of claim 1.

15. The A poxvirus vector of claim 14, comprising:

at least one transgene;

wherein said transgene comprises a poxvirus promoter and a nucleic acid sequence that encodes a foreign peptide, and wherein said poxvirus promoter is located upstream of said nucleic acid sequence that encodes a foreign peptide;

wherein said poxvirus promoter is operably-linked to said nucleic acid sequence and expression of said nucleic sequence is driven by said poxvirus promoter;

wherein the poxvirus promoter is a promoter that drives the expression of an open reading frame that is non-essential to the viability of a naturally-occurring poxvirus; and

wherein said nucleic acid sequence that encodes a foreign peptide includes an ATG start codon located at the same position as the ATG start codon of said non-essential poxvirus open reading frame.

16. The poxvirus vector of claim 15, wherein at least part of the nucleic acid sequence encoding the non-essential poxvirus open reading frame is present.

17. The poxvirus vector of claim 15, wherein the poxvirus vector comprises nucleotides 1-25 of any one of SEQ ID NOs:

1-10, or nucleotides 1-25 of a nucleotide sequence having at least 80% sequence identity any one of SEQ ID NOs;

1-10;

preferably wherein the ATG start codon of the open reading frame is located at a position immediately downstream of said nucleotides 1-25.

18. The poxvirus vector of claim 17, wherein the nucleic acid sequence that encodes a foreign peptide is located in frame with (preferably immediately after) the ATG start codon located at a position immediately downstream of said nucleotides 1-25.

19. The poxvirus vector of claim 14, wherein the poxvirus is selected from:

vaccinia virus, modified vaccinia virus Ankara (MVA), NYVAC, fowlpox virus, and canarypox virus.

21. A poxvirus vector according to claim 14, for use in medicine, for use in stimulating or inducing an immune response in a subject, or for use in the treatment or prevention of at least one infectious disease.

23. The poxvirus vector according to claim 21, wherein said vector is administered as part of a ‘

prime-boost’

vaccination regime.

26. A pharmaceutical composition comprising:

a poxvirus vector according to claim 14; and

a pharmaceutically acceptable carrier.

3. (canceled)

10. (canceled)

11. The method of claim 10, wherein the poxvirus open reading frame is selected from the following MVA open reading frames:

176R, 041L, 027L, 157L, 052R, 005R, and 168R.

20. (canceled)

22. (canceled)

24. (canceled)

25. (canceled)

27. (canceled)