Poxvirus expression system

• US 9,273,327 B2
• Filed: 04/15/2011
• Issued: 03/01/2016
• 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
• Primary Examiner(s): Blumel, Benjamin P
• Assistant Examiner(s): GILL, RACHEL B
• Application Number:
  US13/641,512
• Publication Number:
  US 20130195912A1
• Time in Patent Office: 1,782 Days
• US Class Current: 1/1
• 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 characterized 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 inserting the nucleic acid sequence that encodes the foreign peptide at a position downstream of a poxvirus promoter of an open reading frame (ORF) non-essential to the viability of a poxvirus, wherein said ORF corresponds to modified vaccinia virus Ankara (MVA) ORFs 176R, 041L, 005R, and 050L, or wherein said ORF corresponds to the orthologous vaccinia virus ORFs B8R, F11L, C11R, and E3L;

wherein following said insertion,

(i) the nucleic acid that encodes the foreign peptide is operably-linked to the poxvirus promoter of said non-essential ORF 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 said non-essential ORF.

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.

3. The method of claim 1, wherein following insertion of the nucleic acid sequence that encodes the foreign peptide at least part of the non-essential poxvirus ORF remains present in the poxvirus genome.

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

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

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

6. A poxvirus vector comprising:

at least one transgene;

wherein said transgene is located downstream of a poxvirus promoter and comprises a 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 in the poxvirus genome relative to said poxvirus promoter, as the ATG start codon of said non-essential poxvirus open reading frame;

wherein the poxvirus open reading frame is an open reading frame corresponding to modified vaccinia virus Ankara (MVA) open reading frames 176R, 041L, 005R and 050L, or an open reading frame corresponding to the orthologous vaccinia virus open reading frames B8R, F11L, C11R and E3L.

7. The poxvirus vector of claim 6, wherein at least part of the nucleic acid sequence encoding the non-essential poxvirus open reading frame is present.

8. The poxvirus vector of claim 6, wherein the poxvirus vector is derived from a poxvirus selected from:

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

9. An immunogenic composition comprising the poxvirus vector of claim 6, wherein said vector is attenuated.

10. A pharmaceutical composition comprising:

the poxvirus vector according to claim 6;

and a pharmaceutically acceptable carrier.