Vectors having enhanced expression, and methods of making and uses thereof

• US 6,004,777 A
• Filed: 03/12/1997
• Issued: 12/21/1999
• Est. Priority Date: 03/12/1997
• Status: Expired due to Term

• Current Assignee: Arizona Board of Regents (University of Arizona), Virogenetics Corporation
• Original Assignee: Arizona Board of Regents (University of Arizona), Virogenetics Corporation
• Inventors: Jacobs, Bertram L., Goebel, Scott J., Tartaglia, James, Paoletti, Enzo, Cox, William I., Pincus, Steven E., Gettig, Russell Robert
• Primary Examiner(s): Brusca, John S.
• Application Number:
  US08/815,809
• Time in Patent Office: 1,014 Days
• Field of Search: 435/69.1, 435/91.41, 435/320.1, 536/23.1, 536/23.72
• US Class Current: 435/69.1
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 15/85 for animal cells

Abstract

Disclosed and claimed are vectors having enhanced expression and methods for making and using them. Enhancement of expression is from substantially co-temporal expression of at least one first nucleic acid molecule and at least one second nucleic acid molecule. The second nucleic acid molecule encodes a translation factor. The contemporaneous expression can be from operably linking the first and second nucleic molecules to a single promoter, or from operably linking the first nucleic acid molecule to a first promoter and the second nucleic molecule to a second promoter wherein the first and second promoters function substantially contemporaneously. Thus, the first and second nucleic acid molecules can be at the same locus in the vector or at different loci. The second nucleic acid molecule can encode encode one translation factor or more than one translation factor. The translation factor can be a K3L open reading frame, an E3L open reading frame, a VAI RNA, an EBER RNA, a sigma 3 open reading frame, a TRBP open reading frame, or combinations thereof. The vector can be a poxvirus such as an attenuated poxvirus; for instance, a NYVAC vaccinia virus or an ALVAC canarypox virus.

Claims

1. A vector for enhanced expression of at least one first nucleic acid molecule in a cell, said vector modified to comprise nucleic acid sequences comprising the first nucleic acid molecule and at least one second nucleic acid molecule encoding a translation factor, wherein the vector is modified to comprise the nucleic acid sequences by at least one insertion into the vector, and there is substantially co-temporal expression of the first and second nucleic acid molecules, whereby expression of the second nucleic acid molecule enhances expression of the first nucleic acid molecule by enhancing translation, wherein the insertion is not present in the vector prior to the modification.

2. The vector of claim 1 wherein the first nucleic acid molecule is operably linked to a first promoter and the second nucleic acid molecule is operably linked to a second promoter, and the first and second promoters are functional substantially co-temporally.

3. The vector of claim 2 wherein the first and second nucleic acid molecules are at different loci within the vector.

4. The vector of claim 2 wherein the first and second nucleic acid molecules are at the same locus within the vector.

5. The vector of claim 1 wherein the first nucleic acid molecule and the second nucleic acid molecule are operably linked to a promoter.

6. The vector of claim 1 wherein the translation factor effects inhibition of eIF-2α

phosphorylation or inhibition of PKR phosphorylation or sequesters dsRNA.

7. The vector of claim 6 wherein said at least one second molecule is selected from the group consisting of:

a K3L open reading frame, an E3L open reading frame, a VAI RNA, an EBER RNA, a sigma 3 open reading frame, a TRBP open reading frame, and combinations thereof.

8. The vector of claim 1 wherein said first nucleic acid molecule is selected from the group consisting of nucleic acid molecules encoding:

an epitope of interest, a biological response modulator, a growth factor, a recognition sequence, a therapeutic gene, and a fusion protein.

9. The vector of claim 1 which is a recombinant virus.

10. The vector of claim 9 which is a recombinant poxvirus.

11. A method for preparing a vector as claimed in claim 1 comprising modifying the vector to comprise the at least one second nucleic acid molecule, and optionally also modifying the vector to comprise the first nucleic acid molecule, so that there is substantially co-temporal expression of the first and second nucleic acid molecules.

12. The method of claim 11 comprising operably linking the first nucleic acid molecule to a first promoter and the second nucleic acid molecule to a second promoter, wherein the first and second promoters are functional substantially co-temporally.

13. The method of claim 11 comprising operably linking the first and second nucleic acid molecules to a promoter.

14. A method of expressing a gene product in vitro comprising infecting, or transfecting, a suitable cell with a vector as claimed in claim 1.

15. A method of increasing expression of at least one first nucleic acid molecule by a vector comprising the first nucleic acid molecule, in a cell, comprising modifying the vector to comprise at least one second nucleic acid molecule encoding a translation factor, and expressing the vector in the cell;

wherein there is substantially co-temporal expression of the first and second nucleic acid molecules, whereby expression of the second nucleic acid molecule enhances expression of the first nucleic acid molecule by enhancing translation.