Chimeric retrovial expression vectors and particles containing a simple retroviral long terminal repeat, BLV or HIV coding regions and cis-acting regulatory sequences, and an RNA translational enhancer with internal ribsome entry site

• US 5,770,428 A
• Filed: 04/03/1996
• Issued: 06/23/1998
• Est. Priority Date: 02/17/1993
• Status: Expired due to Fees

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Boris-Lawrie, Kathleen A.
• Primary Examiner(s): Achutamurthy, Ponnathapura
• Assistant Examiner(s): Parkin, Jeffrey S.
• Application Number:
  US08/627,845
• Time in Patent Office: 811 Days
• Field of Search: 435/69.1, 435/69.3, 435/70.1, 435/172.3, 435/235.1, 435/236, 435/320.1, 424/187.1, 424/188.1, 424/199.1, 424/207.1, 424/208.1
• US Class Current: 435/235.1
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2740/13022 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2740/16043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2830/48 regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE
• CPC Class Codes: C12N 2830/50 regulating RNA stability, not being an intron, e.g. poly A signal
• CPC Class Codes: C12N 2840/203 having an IRES

Abstract

This invention is directed toward novel replication competent retroviral expression vectors and particles containing a single chimeric viral genome composed of a simple retroviral (e.g., spleen necrosis virus SNV!) long terminal repeat (LTR), complex retroviral (e.g., Bovine Leukemia Virus BLV! or Human Immunodeficiency Virus HIV!) gag, pol, and env coding regions, complex retroviral cis-acting sequences (e.g., the encapsidation signal E!, primer binding site PBS!, polypurine tract PPT!, and att sites), and an RNA translational enhancer containing an internal ribosome entry site (IRES). These chimeric viral genomes express the BLV or HIV gag, pol, and env genes in a tax/rex- or tat-/rev-independent manner, respectively. Moreover, the inclusion of an IRES site in the modified viral genome facilitates the expression of the env gene products. These retroviral vectors and particles provide a source of viral antigens that can be employed in both diagnostic assays and as immunogens for the production of specific immunological reagents.

Claims

1. A recombinant retroviral expression vector containing a single replication competent hybrid retroviral genome comprising the following elements:

i) a simple retrovirus (S-type) long terminal repeat (LTR);

ii) the bovine leukemia virus (BLV) gag, pol, and env genes;

iii) the bovine leukemia virus (BLV) encapsidation signal (E), primer binding site (PBS), polypurine tract (PPT), and att site; and

,

iv) a nucleic acid encoding for an RNA translational enhancer containing an internal ribosome entry site (IRES) and positioned on the vector 3'"'"' to the gag and pol genes and 5'"'"' to the env gene;

wherein said expression vector is capable of producing retroviral particles in at least one of a tax- or rex-independent manner.

2. The recombinant retroviral expression vector of claim 1 wherein said vector does not contain an S-type att site and is incapable of encoding for the tax or rex gene products.

3. A replication competent retrovirus particle containing a single hybrid retroviral genome comprising the following elements:

i) a simple retrovirus (S-type) long terminal repeat (LTR);

ii) the bovine leukemia virus (BLV) gag, pol, and env genes;

iii) the bovine leukemia virus (BLV) encapsidation signal (E), primer binding site (PBS), polypurine tract (PPT), and att site; and

,

iv) a nucleic acid encoding for an RNA translational enhancer containing an internal ribosome entry site (IRES) and positioned on the vector 3'"'"' to the gag and pol genes and 5'"'"' to the env gene;

wherein said retroviral genome is capable of being expressed in at least one of a tax- or rex-independent manner.

4. The replication competent retrovirus particle of claim 3 wherein said single hybrid retroviral genome does not contain an S-type att site and is incapable of encoding for the tax or rex gene products.

5. A recombinant retroviral expression vector containing a single replication competent hybrid retroviral genome comprising the following elements:

i) a simple retrovirus (S-type) long terminal repeat (LTR);

ii) the human immunodeficiency virus (HIV) gag, pol, and env genes;

iii) the human immunodeficiency virus (HIV) encapsidation signal (E), primer binding site (PBS), polypurine tract (PPT), and att site; and

,

iv) a nucleic acid encoding for an RNA translational enhancer containing an internal ribosome entry site (IRES) and positioned on the vector 3'"'"' to the gag and pol genes and 5'"'"' to the env gene;

wherein said expression vector is capable of producing retroviral particles in at least one of a tat- or rev-independent manner.

6. The recombinant retroviral expression vector of claim 5 wherein said vector does not contain an S-type att site and is incapable of encoding for the tat or rev gene products.

7. The recombinant retroviral expression vector of claim 5 wherein said vector comprises the following additional elements:

v) a rev gene positioned on the vector 3'"'"' to the gag, pol, and env genes; and

,

vi) a nucleic acid encoding for an RNA translational enhancer containing an internal ribosome entry site (IRES) and positioned on the vector 3'"'"' to the gag, pol, and env genes and 5'"'"' to the rev gene.

8. A replication competent retrovirus particle containing a single hybrid retroviral genome comprising the following elements:

i) a simple retrovirus (S-type) long terminal repeat (LTR);

ii) the human immunodeficiency virus (HIV) gag, pol, and env genes;

iii) the human immunodeficiency virus (HIV) encapsidation signal (E), primer binding site. (PBS), polypurine tract (PPT), and att site; and

,

iv) a nucleic acid encoding for an RNA translational enhancer containing an internal ribosome entry site (IRES) and positioned on the vector 3'"'"' to the gag and pol genes and 5'"'"' to the env gene;

wherein said retroviral genome is capable of being expressed in at least one of a tat- or rev-independent manner.