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US 6,958,226 B1Granted
Packaging cells comprising codon-optimized gagpol sequences and lacking lentiviral accessory proteins
AssigneeChildren's Medical Center Corporation
InventorsJohn T. Gray, Jeng-Shin Lee, Richard C. Mulligan
Life of this patent
Estimating the term…
Abstract
Novel packaging cell lines useful for generating viral accessory protein independent HIV-derived retroviral vector particles, methods of constructing such packaging cell lines and methods of using the viral accessory protein independent HIV-derived retroviral vector particles are disclosed.
Claims22
A packaging cell line comprising:
- a) a mammalian cell;
- b) a first retroviral nucleotide sequence in the cell which comprises a codon optimized coding sequence for a HIV gagpol and lacks coding sequences for HIV accessory proteins, Rev response element and constitutive transport elements;
- c) a second retroviral nucleotide sequence in the cell which comprises the coding sequence for a heterologous envelope protein;
- and
- d) a third retroviral nucleotide sequence in the cell which comprises a DNA sequence of interest and HIV cis-acting sequences required for packaging, reverse transcription and integration, wherein said packaging cell line produces a HIV-derived retroviral vector particle.
2 more elements
A packaging cell line comprising:
- a) a mammalian cell;
- b) a first retroviral nucleotide sequence in the cell which comprises a codon optimized coding sequence for a HIV gagpol and lacks coding sequences for HIV accessory proteins, Rev response element and constitutive transport elements;
- and
- a second retroviral nucleotide sequence in the cell which comprises a DNA sequence of interest and HIV cis-acting sequences required for packaging, reverse transcription and integration.
1 more element
A packaging cell line comprising:
- a) a mammalian cell;
- b) a first retroviral nucleotide sequence in the cell which comprises a codon optimized coding sequence for a HIV gagpol and lacks coding sequences for HIV accessory proteins, Rev response element and constitutive transport elements;
- and
- c) a second retroviral nucleotide sequence in the cell which comprises the coding sequence for a heterologous envelope protein.
1 more element
A method of producing a packaging cell line which produces a HIV-derived retroviral vector particle, comprising co-transfecting mammalian host cells with:
- a) a first plasmid comprising a codon optimized DNA sequence which encodes HIV gagpol proteins and lacks DNA sequences encoding HIV accessory proteins, Rev response element and constitutive transport elements;
- b) a second plasmid comprising a DNA sequence which encodes a heterologous envelope protein;
- and
- c) a third plasmid comprising a DNA sequence of interest and HIV cis-acting sequences required for packaging, reverse transcription and integration, thereby producing a packaging cell line which produces a HIV-derived retroviral vector particle.
1 more element
A method of producing a HIV-derived retroviral vector particle comprising the steps of:
- a) co-transfecting mammalian host cells with:
- i) a first plasmid comprising a codon optimized DNA sequence which encodes HIV gagpol proteins and lacks DNA sequences encoding HIV accessory proteins, Rev response element and constitutive transport elements;
- ii) a second plasmid comprising a DNA sequence which encodes a heterologous envelope protein;
- and
- iii) a third plasmid comprising a DNA sequence of interest and HIV cis-acting sequences required for packaging, reverse transcription and integration,
- b) maintaining the transfected cells under conditions suitable for virus particle production;
- and
- c) recovering virus particle produced in step b).
5 more elements
A packaging cell line comprising:
- a) a mammalian cell;
- b) a first retroviral nucleotide sequence in the cell which comprises a codon optimized coding sequence for a lentivirus gagpol and lacks coding sequences for lentivirus accessory proteins, Rev response element and constitutive transport elements;
- c) a second retroviral nucleotide sequence in the cell which comprises the coding sequence for a heterologous envelope protein;
- and
- d) a third retroviral nucleotide sequence in the cell which comprises a DNA sequence of interest and lentivirus cis-acting sequences required for packaging, reverse transcription and integration,
- wherein said packaging cell line produces a lentivirus-derived retroviral vector particle.
3 more elements
A packaging cell line comprising:
- a) a mammalian cell;
- b) a first retroviral nucleotide sequence in the cell which comprises a codon optimized coding sequence for lentivirus gagpol and lacks coding sequences for lentivirus accessory proteins, Rev response element and constitutive transport elements;
- and
- c) a second retroviral nucleotide sequence in the cell which comprises a DNA sequence of interest and lentivirus cis-acting sequences required for packaging, reverse transcription and integration.
1 more element
A packaging cell line comprising:
- a) a mammalian cell;
- b) a first retroviral nucleotide sequence in the cell which comprises a codon optimized coding sequence for lentivirus gagpol and lacks coding sequences for lentivirus accessory proteins, Rev response element and constitutive transport elements;
- and
- c) a second retroviral nucleotide sequence in the cell which comprises the coding sequence for a heterologous envelope protein.
1 more element
A method of producing a packaging cell line which produces a lentivirus-derived retroviral vector particle, comprising co-transfecting mammalian host cells with:
- a) a first plasmid comprising a codon optimized DNA sequence which encodes lentivirus gagpol proteins and lacks DNA sequences encoding lentivirus accessory proteins, Rev response element and constitutive transport elements;
- b) a second plasmid comprising a DNA sequence which encodes a heterologous envelope protein;
- and
- c) a third plasmid comprising a DNA sequence of interest and lentivirus cis-acting sequences required for packaging, reverse transcription and integration, thereby producing a packaging cell line which produces a lentivirus-derived retroviral vector particle.
1 more element
A method of producing a lentivirus-derived retroviral vector particle comprising the steps of:
- a) co-transfecting mammalian host cells with:
- i) a first plasmid comprising a codon optimized DNA sequence which encodes lentivirus gagpol proteins and lacks DNA sequences encoding lentivirus accessory proteins, Rev response element and constitutive transport elements;
- ii) a second plasmid comprising a DNA sequence which encodes a heterologous envelope protein;
- and
- iii) a third plasmid comprising a DNA sequence of interest and lentivirus cis-acting sequences required for packaging, reverse transcription and integration,
- b) maintaining the transfected cells under conditions suitable for virus particle production;
- and
- c) recovering virus particle produced in step b).
5 more elements
Dependencies are inferred from the claim text.