Back to results
US 6,555,342 B1Granted
Fusion protein delivery system and uses thereof
AssigneeThe UAB Research Foundation
InventorsJohn C. Kappes, Xiaoyun Wu
Life of this patent
Estimating the term…
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.
Claims108
A transretroviral vector system comprising:
- at least a first nucleic acid segment encoding at least one fusion protein comprising at least a functional portion of a first truncated Gag protein fused in frame to a functional portion of a reverse transcriptase polypeptide having reverse transcriptase activity fused in frame to a functional portion of an integrase polypeptide having integrase activity, said first nucleic acid segment capable of expression in a mammalian cell, said functional portion of said first Gag protein capable of providing for the incorporation of said fusion protein into a viral particle.
A transretroviral vector system comprising:
- (a) at least a first nucleic acid segment encoding at least one fusion protein comprising at least a functional portion of a first truncated Gag protein fused in frame to a functional portion of a reverse transcriptase polypeptide having reverse transcriptase activity fused in frame to a functional portion of an integrase polypeptide having integrase activity, said first nucleic acid segment capable of expression in a mammalian cell, said functional portion of said first Gag protein capable of providing for the incorporation of said fusion protein into a viral particle;
- and,
- (b) at least a second nucleic acid segment comprising a nucleic acid sequence encoding at least a functional portion of a second Gag polypeptide and a functional portion of a protease polypeptide, said second nucleic acid segment provided on the same or a different nucleic acid strand than said first nucleic acid segment, said second nucleic acid segment capable of expression in said mammalian cell, said functional portion of said second Gag polypeptide and said protease polypeptide are capable of supporting the formation of an infectious viral particle, and wherein said second nucleic acid segment does not encode a functional Reverse Transcriptase polypeptide or a functional Integrase polypeptide.
A transretroviral vector system comprising:
- (a) at least a first nucleic acid segment encoding at least one fusion protein comprising at least a functional portion of a first truncated Gag protein fused in frame to a functional portion of a reverse transcriptase polypeptide having reverse transcriptase activity fused in frame to a functional portion of an integrase polypeptide having integrase activity, said first segment capable of expression in a mammalian cell, said functional portion of said first Gag protein capable of providing for the incorporation of said fusion protein into a viral particle;
- (b) at least a second nucleic acid segment comprising a nucleic acid sequence encoding at least a functional portion of a second Gag polypeptide and a functional portion of a protease polypeptide, said second nucleic acid segment provided on the same or a different nucleic acid strand than said first segment, said second nucleic acid segment capable of expression in said mammalian cell, said functional portion of said second Gag polypeptide and said protease polypeptide are capable of supporting the formation of an infectious viral particle and wherein said second nucleic acid segment does not encode a functional Reverse Transcriptase polypeptide or a functional Integrase polypeptide;
- and
- (c) at least a third nucleic acid segment comprising a nucleic acid sequence encoding an envelope polypeptide, said third nucleic acid segment provided on a different or the same nucleic acid strand as said first and said second nucleic acid segments.
1 more element
A transretroviral vector system comprising:
- (a) at least a first nucleic acid segment encoding at least one fusion protein comprising at least a functional portion of a first truncated Gag protein fused in frame to a functional portion of a reverse transcriptase polypeptide having reverse transcriptase activity fused in frame to a functional portion of an integrase polypeptide having integrase activity, said first nucleic acid segment capable of expression in a mammalian cell, said functional portion of said first Gag protein capable of providing for the incorporation of said fusion protein into a viral particle;
- (b) at least a second nucleic acid segment comprising a nucleotide sequence encoding at least a functional portion of a second Gag polypeptide and a functional portion of a protease polypeptide, said second nucleic acid segment provided on the same or a different nucleic acid strand than said first segment, said second nucleic acid segment capable of expression in said mammalian cell, said functional portion of said second Gag polypeptide and said protease polypeptide are capable of supporting the formation of an infectious viral particle, and wherein said second nucleic acid segment does not encode a functional Reverse Transcriptase polypeptide or a functional Integrase polypeptide;
- (c) at least a third nucleic acid segment comprising a nucleic acid sequence encoding an envelope polypeptide, said segment provided on a different or same nucleic acid strand as said first and said second nucleic acid segments;
- and
- (d) at least a fourth nucleic acid segment comprising a nucleic acid sequence of interest and at least one cis acting nucleic acid sequence functioning in packaging, reverse transcription and integration of said fourth nucleic acid segment into the genome of a target cell.
2 more elements
A method for generating a transretroviral vector comprising:
- (a) providing at least a first nucleic acid segment encoding at least one fusion protein comprising at least a functional portion of a first truncated Gag protein fused in frame to a functional portion of a reverse transcriptase polypeptide having reverse transcriptase activity fused in frame to a functional portion of an integrase polypeptide having integrase activity, said first segment capable of expression in a mammalian cell, said functional portion of said Gag polypeptide capable of providing for the incorporation of said fusion protein into a viral particle;
- (b) providing at least a second nucleic acid segment comprising a nucleic acid sequence encoding at least a functional portion of a second Gag polypeptide and a functional portion of a protease polypeptide, said second nucleic acid segment provided on the same or a different nucleic acid strand than said first segment, said second nucleic acid segment capable of expression in a mammalian cell, said functional portion of said second Gag polypeptide and said protease polypeptide are capable of supporting the formation of an infectious viral particle, and wherein said second nucleic acid segment does not encode a functional Reverse Transcriptase polypeptide or a functional Integrase polypeptide;
- (c) providing at least a third nucleic acid segment comprising nucleic acid sequence encoding an envelope polypeptide, said segment provided on a different or same nucleic acid strand as said first or said second nucleic acid segments;
- (d) contacting said nucleic acid segments of (a), (b), and (c) with the mammalian cell, said mammalian cell becoming transfected with said first, said second, and said third nucleic acid segment;
- and,
- (e) producing viral particles from said mammalian cell, said viral particles containing said fusion protein.
3 more elements
A method for generating a transretroviral vector comprising:
- (a) providing at least a first nucleic acid segment encoding at least one fusion protein comprising at least a functional portion of a first truncated Gag protein fused in frame to a functional portion of a reverse transcriptase polypeptide having reverse transcriptase activity fused in frame to a functional portion of an integrase polypeptide having integrase activity, said segment capable of expression in a mammalian cell, said functional portion of said first Gag polypeptide capable of providing for the incorporation of said fusion protein into a viral particle;
- (b) providing at least a second nucleic acid segment encoding at least a functional portion of a second Gag polypeptide and a functional portion of a protease polypeptide, said second nucleic acid segment provided on the same or a different nucleic acid strand than said first segment, said second nucleic acid segment capable of expression in the mammalian cell, said functional portion of said second Gag polypeptide and said protease polypeptide are capable of supporting the formation of an infectious viral particle and wherein said second nucleic acid segment does not encode a functional Reverse Transcriptase polypeptide or a functional integrase polypeptide;
- (c) providing at least a third nucleic acid segment comprising a nucleic acid sequence of interest and at least one cis acting nucleic acid sequence for packaging, reverse transcription and integration of said third nucleic acid segment into the genome of a target cell;
- (d) contacting said nucleic acids of (a), (b), and (c) with the mammalian cell, said mammalian cell becoming transfected with said first, said second, and said third nucleic acid segment;
- and,
- (e) producing viral particles from said mammalian cell, said particles containing said fusion protein.
3 more elements
A method for generating a transretroviral vector comprising:
- (a) providing at least a first nucleic acid segment encoding at least one fusion protein comprising at least a functional portion of a first truncated Gag protein fused in frame to a functional portion of a reverse transcriptase polypeptide having reverse transcriptase activity fused in frame to a functional portion of an integrase polypeptide having integrase activity, said first nucleic acid segment capable of expression in a mammalian cell, said functional portion of said first Gag polypeptide capable of providing for the incorporation of said fusion protein into a viral particle;
- (b) providing at least a second nucleic acid segment comprising a nucleic acid sequence encoding at least a functional portion of a second Gag polypeptide and a functional portion of a protease polypeptide, said second nucleic acid segment provided on the same or a different nucleic acid strand than said first segment, said second segment capable of expression in said mammalian cell, said functional portion of said second Gag polypeptide and a protease polypeptide are capable of supporting the formation of an infectious viral particle and wherein said second nucleic acid segment does not encode a functional Reverse Transcriptase polypeptide or a functional Integrase polypeptide;
- (c) providing at least a third nucleic acid segment comprising a nucleic acid sequence encoding an envelope polypeptide, said segment provided on a different or same nucleic acid strand as said first or said second nucleic acid segments;
- (d) providing at least a fourth nucleic acid segment comprising a nucleic acid sequence of interest and at least one cis acting nucleic acid sequence functioning in packaging, reverse transcription and integration of said fourth nucleic acid segment into the genome of a target cell;
- (e) contacting said nucleic acid segment of (a), (b), (c) and (d) with the mammalian cell, said mammalian cell becoming tranfected with said nucleic acids;
- and,
- (f) producing viral particles from said mammalian cell, said particles containing said fusion protein.
4 more elements
A transretroviral vector system comprising:
- at least a first nucleic acid segment encoding a first fusion protein comprising at least a functional portion of a first truncated Gag protein fused in frame to a functional portion of a Reverse Transcriptase polypeptide having reverse transcriptase activity;
- and,
- at least a second nucleic acid segment encoding a second fusion protein comprising at least a functional portion of a second truncated Gag protein fused in frame to a functional portion of an Integrase polypeptide having integrase activity;
- wherein said first and said second nucleic acid segment capable of expression in a mammalian cell;
- said functional portion of said first and said second Gag protein is capable of providing for the incorporation of said first and said second fusion protein into a viral particle;
- and, said first and said second nucleic acid sequences provided on the same or different nucleic acid strand.
3 more elements
A transretroviral vector system comprising:
- (a) at least a first nucleic acid segment encoding a first fusion protein comprising at least a functional portion of a first truncated Gag protein fused in frame to a functional portion of a Reverse Transcriptase polypeptide having reverse transcriptase activity;
- and,
- at least a second nucleic acid segment encoding a second fusion protein comprising at least a functional portion of a second truncated Gag protein fused in frame to a functional portion of an Integrase polypeptide having integrase activity;
- wherein said first and said second nucleic acid segment capable of expression in a mammalian cell;
- said functional portion of said first and said second Gag protein are capable of providing for the incorporation of said first and said second fusion protein into a viral particle;
- and, said first and said second nucleic acid sequences provided on the same or different nucleic acid strand;
- (b) at least a third nucleic acid segment comprising a nucleic acid sequence encoding at least a functional portion of a third Gag polypeptide and a functional portion of a protease polypeptide, wherein,
- said third nucleic acid segment provided on the same or a different nucleic acid strand than said first or said second nucleic acid segment;
- said third nucleic acid segment capable of expression in said mammalian cell;
- said functional portion of said third Gag polypeptide and said protease polypeptide are capable of supporting the formation of an infectious viral particle;
- and, wherein said third nucleic acid segment does not encode a functional Reverse Transcriptase polypeptide or a functional Integrase polypeptide.
8 more elements
A transretroviral vector system comprising:
- (a) at least a first nucleic acid segment encoding a first fusion protein comprising at least a functional portion of a first truncated Gag protein fused in frame to a functional portion of a Reverse Transcriptase polypeptide having reverse transcriptase activity;
- and,
- at least a second nucleic acid segment encoding a second fusion protein comprising at least a functional portion of a second truncated Gag protein fused in frame to a functional portion of an Integrase polypeptide having integrase activity;
- wherein said first and said second nucleic acid segment capable of expression in a mammalian cell;
- said functional portion of said first and said second Gag protein capable of providing for the incorporation of said first and said second fusion protein into a viral particle;
- and, said first and said second nucleic acid sequences provided on the same or different nucleic acid strand;
- (b) at least a third nucleic acid segment comprising a nucleic acid sequence encoding at least a functional portion of a third Gag polypeptide and a functional portion of a protease polypeptide, wherein,
- said third nucleic acid segment provided on the same or a different nucleic acid strand than said first or said second nucleic acid segment;
- said third nucleic acid segment capable of expression in said mammalian cell;
- said functional portion of said third Gag polypeptide and said protease polypeptide are capable of supporting the formation of an infectious viral particle;
- and, wherein said third nucleic acid segment does not encode a functional Reverse Transcriptase polypeptide or a functional Integrase polypeptide;
- and,
- (c) at least a fourth nucleic acid segment comprising a nucleic acid sequence encoding an envelope polypeptide, said fourth nucleic acid segment provided on a different or the same nucleic acid strand as said first, said second, said third, or said fourth nucleic acid segment.
10 more elements
A transretroviral vector system comprising:
- (a) at least a first nucleic acid segment encoding a first fusion protein comprising at least a functional portion of a first truncated Gag protein fused in frame to a functional portion of a Reverse Transcriptase polypeptide having reverse transcriptase activity;
- and,
- at least a second nucleic acid segment encoding a second fusion protein comprising at least a functional portion of a second truncated Gag protein fused in frame to a functional portion of an Integrase polypeptide having integrase activity;
- wherein said first and said second nucleic acid segment capable of expression in a mammalian cell;
- said functional portion of said first and said second Gag protein capable of providing for the incorporation of said first and said second fusion protein into a viral particle;
- and, said first and said second nucleic acid sequences provided on the same or different nucleic acid strand;
- (b) at least a third nucleic acid segment comprising a nucleic acid sequence encoding at least a functional portion of a third Gag polypeptide and a functional portion of a protease polypeptide, wherein,
- said third nucleic acid segment provided on the same or a different nucleic acid strand than said first or said second nucleic acid segment;
- said third nucleic acid segment capable of expression in said mammalian cell;
- said functional portion of said third Gag polypeptide and said protease polypeptide are capable of supporting the formation of an infectious viral particle;
- and, wherein said third nucleic acid segment does not encode a functional Reverse Transcriptase polypeptide or a functional Integrase polypeptide;
- and,
- (c) at least a fourth nucleic acid segment comprising a nucleic acid sequence encoding an envelope polypeptide, said fourth nucleic acid segment provided on a different or the same nucleic acid strand as said first, said second, said third, or said fourth nucleic acid segment;
- and,
- (d) at least a fifth nucleic acid segment comprising a nucleic acid sequence of interest and at least one cis acting nucleic acid sequence functioning in packaging, reverse transcription and integration of said fifth nucleic acid segment into the genome of a target cell.
12 more elements
A method for generating a transretroviral vector comprising:
- (a) providing at least a first nucleic acid segment encoding a first fusion protein comprising at least a functional portion of a first truncated Gag protein fused in frame to a functional portion of a Reverse Transcriptase polypeptide having reverse transcriptase activity;
- and,
- providing at least a second nucleic acid segment encoding a second fusion protein comprising at least a functional portion of a second truncated Gag protein fused in frame to a functional portion of an Integrase polypeptide having integrase activity;
- wherein said first and said second nucleic acid segment capable of expression in a mammalian cell;
- said functional portion of said first and said second Gag protein capable of providing for the incorporation of said first and said second fusion protein into a viral particle;
- and, said first and said second nucleic acid sequences provided on the same or different nucleic acid strand;
- (b) providing at least a third nucleic acid segment comprising a nucleic acid sequence encoding at least a functional portion of a third Gag polypeptide and a functional portion of a protease polypeptide, wherein,
- said third nucleic acid segment provided on the same or a different nucleic acid strand than said first or said second nucleic acid segment;
- said third nucleic acid segment capable of expression in said mammalian cell;
- said functional portion of said third Gag polypeptide and said protease polypeptide are capable of supporting the formation of an infectious viral particle;
- and, wherein said third nucleic acid segment does not encode a functional Reverse Transcriptase polypeptide or a functional Integrase polypeptide;
- and,
- (c) providing at least a fourth nucleic acid segment comprising a nucleic acid sequence encoding an envelope polypeptide, said fourth nucleic acid segment provided on a different or the same nucleic acid strand as said first, said second, said third, or said fourth nucleic acid segment;
- and,
- (d) contacting said nucleic acid segments of (a), (b), and (c) with the mammalian cell, said mammalian cell becoming transfected with said first, said second, said third, and said fourth nucleic acid segment;
- and,
- (e) providing viral particles from said mammalian cell, said viral particles containing said first and said second fusion protein.
14 more elements
A method for generating a transretroviral vector system comprising:
- (a) providing at least a first nucleic acid segment encoding a first fusion protein comprising at least a functional portion of a first truncated Gag protein fused in frame to a functional portion of a Reverse Transcriptase polypeptide having reverse transcriptase activity;
- and,
- providing at least a second nucleic acid segment encoding a second fusion protein comprising at least a functional portion of a second truncated Gag protein fused in frame to a functional portion of an Integrase polypeptide having integrase activity;
- wherein said first and said second nucleic acid segment capable of expression in a mammalian cell;
- said functional portion of said first and said second Gag protein capable of providing for the incorporation of said first and said second fusion protein into a viral particle;
- and, said first and said second nucleic acid sequences provided on the same or different nucleic acid strand;
- (b) providing at least a third nucleic acid segment comprising a nucleic acid sequence encoding at least a functional portion of a third Gag polypeptide and a functional portion of a protease polypeptide, wherein,
- said third nucleic acid segment provided on the same or a different nucleic acid strand than said first or said second nucleic acid segment;
- said third nucleic acid segment capable of expression in said mammalian cell;
- said functional portion of said third Gag polypeptide and said protease polypeptide are capable of supporting the formation of an infectious viral particle;
- and, wherein said third nucleic acid segment does not encode a functional Reverse Transcriptase polypeptide or a functional Integrase polypeptide;
- (c) providing at least a fourth nucleic acid segment comprising a nucleic acid sequence of interest and at least one cis acting nucleic acid sequence functioning in packaging, reverse transcription and integration of said fourth nucleic acid segment into the genome of a target cell;
- (d) contacting said nucleic acids of (a), (b), and (c) with the mammalian cell, said mammalian cell becoming transfected with said first, said second, said third and said fourth nucleic acid segment;
- and,
- (e) providing viral particles from said mammalian cell, said particles containing said first and said second fusion protein.
12 more elements
A method for generating a transretroviral vector comprising:
- (a) providing at least a first nucleic acid segment encoding a first fusion protein comprising at least a functional portion of a first truncated Gag protein fused in frame to a functional portion of a Reverse Transcriptase polypeptide having reverse transcriptase activity;
- and,
- providing at least a second nucleic acid segment encoding a second fusion protein comprising at least a functional portion of a second truncated Gag protein fused in frame to a functional portion of an Integrase polypeptide having integrase activity;
- wherein said first and said second nucleic acid segment capable of expression in a mammalian cell;
- said functional portion of said first and said second Gag protein capable of providing for the incorporation of said first and said second fusion protein into a viral particle;
- and, said first and said second nucleic acid sequences provided on the same or different nucleic acid strand;
- (b) providing at least a third nucleic acid segment comprising a nucleic acid sequence encoding at least a functional portion of a third Gag polypeptide and a functional portion of a protease polypeptide, wherein,
- said third nucleic acid segment provided on the same or a different nucleic acid strand than said first or said second nucleic acid segment;
- said third nucleic acid segment capable of expression in said mammalian cell;
- said functional portion of said third Gag polypeptide and said protease polypeptide are capable of supporting the formation of an infectious viral particle;
- and, wherein said third nucleic acid segment does not encode a functional Reverse Transcriptase polypeptide or a functional Integrase polypeptide;
- (c) providing at least a fourth nucleic acid segment comprising a nucleic acid sequence encoding an envelope polypeptide, said segment provided on a different or same nucleic acid strand as said first, said second, or said third nucleic acid segments;
- (d) providing at least a fifth nucleic acid segment comprising a nucleic acid sequence of interest and at least one cis acting nucleic acid sequence functioning in packaging, reverse transcription and integration of said fifth nucleic acid segment into the genome of a target cell;
- (e) contacting said nucleic acids of (a), (b), (c), and (d) with the mammalian cell, said mammalian cell becoming transfected with said first, said second, said third, said fourth and said fifth nucleic acid segment;
- and,
- (f) providing viral particles from said mammalian cell, said particles containing said first and said second fusion protein.
13 more elements
Dependencies are inferred from the claim text.