Fusion protein delivery system and uses thereof
OwnerJohn C. Kappes, Xiaoyun Wuinventors
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Abstract
The present invention provides a composition of matter, comprising: DNA encoding a viral Vpx protein fused to DNA encoding a protein. In another embodiment of the present invention, there is provided a composition of matter, comprising: DNA encoding a viral Vpr protein fused to DNA encoding a protein. The present invention further provides DNA, vectors and methods for expressing a lentiviral pol gene in trans, independent of the lentiviral gag-pol. A gene transduction element is optionally delivered to a lentiviral vector according to the present invention. Also provided are various methods of delivering a virus inhibitory molecule to a target in an animal. Further provided is a pharmaceutical composition.
Claims40
A method for generating a recombinant viral protein-containing viral vector particle comprising:
- (a) providing a first nucleic acid segment encoding at least a recombinant protein wherein said protein comprises a fusion of a first and a second amino acid sequence, said first amino acid sequence comprising at least a functional portion of a Vpr or Vpx protein of a retrovirus, said retrovirus selected from the group consisting of HIV-1, HIV-2 or SIV, said second amino acid sequence comprising an amino acid sequence different from Vpr or Vpx sequence;
- (b) providing a second nucleic acid segment comprising retroviral nucleic acid sequences used for packaging, said nucleic acid segment encoding at least a functional portion of a Gag protein, said segment provided on a same or different nucleic acid strand as (a);
- (c) contacting said nucleic acids of (a) and (b) with a mammalian cell, said cell becoming transfected with said nucleic acids;
- and
- (d) producing viral particles from said cell, said particles containing said protein.
A method according to claim 1 15 further comprising providing a third nucleic acid segment for integration into nucleic acid of a host genome, said segment encoding at least one functional amino acid sequence, said segment further provided on a same or different nucleic acid strand as (a) and/or (b).
A method for generating a recombinant viral protein-containing viral vector particle comprising:
- (a) providing a first nucleic acid segment encoding at least a recombinant protein wherein said protein comprises a fusion of a first and a second amino acid sequence, said first amino acid sequence comprising at least a functional portion of a Vpr or Vpx protein of a retrovirus, said retrovirus selected from the group consisting of HIV-1, HIV-2 or SIV, said second amino acid sequence comprising an amino acid sequence different from Vpr or Vpx sequence;
- (b) providing a second nucleic acid segment comprising retroviral nucleic acid sequences used for packaging, said nucleic acid segment encoding at least a functional portion of a Gag protein and at least one functional Pol protein, said segment provided on a same or different nucleic acid strand as (a);
- (c) contacting said nucleic acids of (a) and (b) with a mammalian cell, said cell becoming transfected with said nucleic acids;
- and
- (d) producing viral particles from said cell, said particles containing said protein.
A method for generating a recombinant viral protein-containing viral vector particle comprising:
- (a) providing a first nucleic acid segment encoding at least a recombinant protein wherein said protein comprises a fusion of a first and a second amino acid sequence, said first amino acid sequence comprising at least a functional portion of a Vpr or Vpx protein of a retrovirus, said retrovirus selected from the group consisting of HIV-1, HIV-2 or SIV, said second amino acid sequence comprising an amino acid sequence different from Vpr or Vpx sequence;
- (b) providing a second nucleic acid segment comprising retroviral nucleic acid sequences used for packaging, said nucleic acid segment encoding at least a functional portion of a Gag protein and at least a functional portion of a Pro protein and nonfunctional reverse transcriptase and integrase genes, said segment provided on a same or different nucleic acid strand as (a);
- (c) contacting said nucleic acids of (a) and (b) with a mammalian cell, said cell becoming transfected with said nucleic acids;
- and
- (d) producing viral particles from said cell, said particles containing said protein.
A method for generating a recombinant viral protein-containing viral vector particle comprising:
- (a) providing a first nucleic acid segment encoding at least a viral reverse transcriptase;
- (b) providing a second nucleic acid segment comprising retroviral nucleic acid sequences used for packaging, said sequences comprising nucleic acid sequence encoding at least a Gag protein, said segment provided on a same or different nucleic acid strand as (a)
- (c) contacting said nucleic acids of (a) and (b) with a mammalian cell, said cell becoming transfected with said nucleic acids;
- and
- (d) producing viral particles from said cell, said particles containing said protein.
A method for generating a recombinant viral protein-containing viral vector particle comprising:
- (a) providing a first nucleic acid segment encoding at least a viral reverse transcriptase;
- (b) providing a second nucleic acid segment comprising retroviral nucleic acid sequences used for packaging, said sequences comprising nucleic acid sequence encoding a Gag protein and at least one functional Pol protein, said segment provided on a same or different nucleic acid strand as (a);
- (c) contacting said nucleic acids of (a) and (b) with a mammalian cell, said cell becoming transfected with said nucleic acids;
- and (d) producing viral particles from said cell, said particles containing said protein.
A method for generating a recombinant viral protein-containing viral vector particle comprising:
- (a) providing a first nucleic acid segment encoding at least a viral reverse transcriptase;
- (b) providing a second nucleic acid segment comprising retroviral nucleic acid sequences used for packaging, said sequences comprising nucleic acid sequence encoding a Gag protein and at least a functional portion of a Pro protein and nonfunctional reverse transcriptase and integrase genes, said segment provided on a same or different nucleic acid strand as (a);
- (c) contacting said nucleic acids of (a) and (b) with a mammalian cell, said cell becoming transfected with said nucleic acids;
- and
- (d) producing viral particles from said cell, said particles containing said protein.
A method for generating a recombinant viral protein-containing viral vector particle comprising:
- (a) providing a first nucleic acid sequence encoding a first polypeptide, said first polypeptide comprising at least a functional portion of a Vpr or Vpx amino acid sequence;
- (b) providing a second nucleic acid sequence fused to and in the same reading frame of said first nucleic acid sequence, said second nucleic acid sequence encoding a second polypeptide;
- (c) providing at least one of a promoter and a control element, said promoter and/or control element operatively linked to said first and said second nucleic acid sequences;
- (d) contacting said nucleic acids of (a), (b) and (c) with a mammalian cell, said cell becoming transfected with said nucleic acids;
- and
- (e) producing viral particles from said cell, said particles containing said protein.
A method according to claim 153 wherein said promoter and control element direct systhesis of said first and second polypeptides.
Dependencies are inferred from the claim text.