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US 7,622,300 B2Granted
Trans-lentiviral vector particles and transduction of eukaryotic cells therewith
OwnerJohn C. Kappes, Xiaoyun Wuinventors
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Abstract
Trans-lentiviral gene transfer vectors, vector systems, vector particles, and methods for transduction of primary nondividing cells using the same, are described. Further described are modifications to the gene transfer vectors that improve transduction efficiency and/or gene expression, e.g., by incorporating the cis-acting sequences, PPT-CTS and/or WPRE, and derivatives or analogs thereof.
Claims43
A transretroviral vector system comprising:
- a) at least a first polynucleotide sequence comprising at least one nucleotide sequence encoding a polypeptide selected from the group consisting of:
- i) a functional portion of a Reverse Transcriptase polypeptide;
- and,
- ii) a functional portion of an Integrase polypeptide;
- wherein said first polynucleotide sequence does not encode a functional Gag polypeptide;
- and,
- b) a second polynucleotide sequence comprising a nucleotide sequence encoding at least a functional portion of a Gag polypeptide and a functional portion of a protease polypeptide, and said second polynucleotide sequence does not encode at least one of a functional Reverse Transcriptase polypeptide and a functional Integrase polypeptide and said second polypeptide is capable of expression in a mammalian cell;
- and,
- c) a third polynucleotide sequence comprising a nucleic acid sequence encoding a functional portion of an envelope polypeptide capable of mediating recognition and entry of the viral particle into a target cell, wherein said third polynucleotide sequence does not encode a functional Gag-Pol precursor polypeptide;
- d) a fourth polynucleotide sequence comprising a heterologous nucleic acid sequence of interest and at least one nucleotide sequence is selected from the group consisting of a functional equivalent of a polypurine tract-central terminator sequence (PPT-CTS), a functional equivalent of Woodchuck Hepatitis Virus Post transcriptional Regulatory Element (WPRE), a PPT-CTS, and a WPRE;
- wherein the functional portions of the Reverse Transcriptase, Integrase, Gag and protease polypeptides are from a retrovirus and said transretroviral vector system produces a viral particle capable of introducing said heterologous nucleotide sequence of interest into the genome of the target cell.
8 more elements
A method for delivering a heterologous nucleic acid sequence of interest to a target cell comprising
- a) forming a transretroviral particle using a transretroviral vector system comprising a gene transfer vector having the heterologous nucleic acid sequence of interest operably linked to a promoter active in said target cell, said gene transfer vector further comprises at least one nucleotide sequence selected from the group consisting of a WPRE, a PPT-CTS, a functional equivalent of a WPRE or a functional equivalent of a PPT-CTS;
- b) providing the target cell;
- and,
- c) transducing said target cell with said transretroviral particle,
- wherein said heterologous nucleic acid sequence of interest is integrated into the genome of the target cell.
2 more elements
A method for delivering a heterologous nucleic acid sequence of interest to a non-dividing target cell comprising:
- a) forming a transretroviral particle using a transretroviral vector system comprising a gene transfer vector having the heterologous nucleic acid sequence of interest operably linked to a promoter active in said non-dividing target cell;
- b) providing the non-dividing target cell;
- and,
- c) transducing said non-dividing target cell with the transretroviral particle;
- wherein said heterologous nucleic acid sequence of interest is integrated into the genome of the target cell and said gene transfer vector further comprises at least one nucleotide sequence selected from the group consisting of a WPRE, a PPT-CTS, a functional equivalent of WPRE and a functional equivalent of PPT-CTS.
2 more elements
Dependencies are inferred from the claim text.