Novel expression vectors and uses thereof

Abstract

A method for treating an HIV disease in a subject in need of said treatment, comprising administering to the subject a therapeutically effective amount of a DNA vaccine comprising an expression vector and a pharmaceutically acceptable excipient, where the expression vector comprises: (a) a heterologous promoter operatively linked to a DNA sequence encoding a nuclear-anchoring protein, where the nuclear-anchoring protein comprises: (i) a DNA binding domain which binds to a specific DNA binding sequence, and (ii) a functional domain of the Bovine Papilloma Virus Type 1 E2 protein, where the functional domain binds to a nuclear component; (b) a multimerized DNA sequence that forms a binding site for the nuclear anchoring protein; and (c) at least one expression cassette comprising a DNA sequence encoding a protein or peptide that stimulates an immune response specific to the protein or peptide; where the expression vector lacks an origin of replication functional in mammalian cells.

1 . A method for treating an HIV disease in a subject in need of said treatment, said method comprising:
 administering to said subject a therapeutically effective amount of a DNA vaccine comprising an expression vector and a pharmaceutically acceptable excipient, wherein said expression vector comprises:   (a) a heterologous promoter operatively linked to a DNA sequence encoding a nuclear-anchoring protein, wherein said nuclear-anchoring protein comprises:
 (i) a DNA binding domain which binds to a specific DNA binding sequence, and 
 (ii) a functional domain of the Bovine Papilloma Virus Type 1 E2 protein, wherein said functional domain binds to a nuclear component; 
   (b) a multimerized DNA sequence that forms a binding site for said nuclear anchoring protein; and   (c) at least one expression cassette comprising a DNA sequence encoding a protein or peptide that stimulates an immune response specific to the protein or peptide;   wherein said expression vector lacks an origin of replication functional in mammalian cells.   
     
     
         2 . The method of  claim 1 , wherein said nuclear component is selected from the group consisting of mitotic chromatin, the nuclear matrix, nuclear domain 10 (ND10), and nuclear domain PML oncogenic domain (POD). 
     
     
         3 . The method of  claim 1 , wherein said nuclear-anchoring protein is a chromatin-anchoring protein, and said functional domain binds mitotic chromatin. 
     
     
         4 . The method of  claim 1 , wherein said nuclear-anchoring protein comprises a hinge or linker region. 
     
     
         5 . The method of  claim 1 , wherein said nuclear-anchoring protein is a natural protein of viral origin. 
     
     
         6 . The method of  claim 1 , wherein said nuclear-anchoring protein is an artificial protein. 
     
     
         7 . The method of  claim 1 , wherein said expression cassette comprises a DNA sequence of HIV origin. 
     
     
         8 . The method of  claim 7 , wherein said DNA sequence of HIV origin encodes a non-structural regulatory protein of HIV, or an immunogenic fragment thereof. 
     
     
         9 . The method of  claim 8 , wherein said nonstructural regulatory protein of HIV is selected from the group consisting of Nef, Tat and Rev. 
     
     
         10 . The method of  claim 9 , wherein said nonstructural regulatory protein of HIV is Nef. 
     
     
         11 . The method of  claim 7 , wherein said DNA sequence of HIV origin encodes a structural protein of HIV, or an immunogenic fragment thereof. 
     
     
         12 . The method of  claim 11 , wherein said DNA sequence of HIV origin is HIV gp120/gp160. 
     
     
         13 . The method of  claim 1 , wherein said vector comprises:
 (a) a first expression cassette comprising a DNA sequence encoding Nef, Tat or Rev; and   (b) a second expression cassette comprising a DNA sequence encoding Nef, Tat or Rev.   
     
     
         14 . The method of  claim 1 , wherein said vector comprises:
 (a) a first expression cassette comprising a DNA sequence encoding Nef, Tat or Rev; and   (b) a second expression cassette comprising a DNA sequence encoding a structural protein of HIV.   
     
     
         15 . The method of  claim 1 , wherein:
 said DNA binding domain comprises the DNA binding domain of the Bovine Papilloma Virus Type 1 E2 protein; and   said multimerized DNA sequence comprises multimerized E2 binding sites.   
     
     
         16 . The method of  claim 1 , wherein:
 said nuclear-anchoring protein comprises the Bovine Papilloma Virus Type 1 E2 protein, and   said multimerized DNA sequence comprises multimerized E2 binding sites.   
     
     
         17 . The method of  claim 1 , wherein said expression cassette comprises a DNA sequence encoding a fusion protein comprising the following components:
 (A) Rev, Nef, Tat (RNT);   (B) opt 17/24; and   (C) Cytotoxic T cell epitopes (CTL).   
     
     
         18 . The method of  claim 17 , wherein the order of the components from the 5′ end to the 3′ end of said fusion protein is A+B+C. 
     
     
         19 . The method of  claim 17 , wherein the components A, B, and C comprise the sequences of SEQ ID NOS: 5, 13 and 10, respectively. 
     
     
         20 . A DNA vaccine comprising an expression vector and a pharmaceutically acceptable excipient, wherein said expression vector comprises:
 (a) a heterologous promoter operatively linked to a DNA sequence encoding a nuclear-anchoring protein, wherein said nuclear-anchoring protein comprises:
 (i) a DNA binding domain which binds to a specific DNA binding sequence, and 
 (ii) a functional domain of the Bovine Papilloma Virus Type 1 E2 protein, wherein said functional domain binds to a nuclear component; 
   (b) a multimerized DNA sequence that forms a binding site for said nuclear anchoring protein; and   (c) at least one expression cassette comprising a DNA sequence encoding a protein or peptide that stimulates an immune response specific to the protein or peptide;   wherein said expression vector lacks an origin of replication functional in mammalian cells; and   wherein said expression cassette comprises a DNA sequence encoding a fusion protein comprising the following components:   (A) Rev, Nef, Tat (RNT);   (B) opt 17/24; and   (C) Cytotoxic T cell epitopes (CTL).   
     
     
         21 . The DNA vaccine of  claim 20 , wherein the order of the components from the 5′ end to the 3′ end of said fusion protein is A+B+C. 
     
     
         22 . The DNA vaccine of  claim 20 , wherein the components A, B, and C comprise the sequences of SEQ ID NOS: 5, 13 and 10, respectively. 
     
     
         23 . The DNA vaccine of  claim 21 , wherein the components A, B, and C comprise the sequences of SEQ ID NOS: 5, 13 and 10, respectively.