Mutated piggybac transposase
AssigneeAmgen Inc.
InventorsNeeraj J. AGRAWAL, Kristine M. DARIS, Jennitte L. STEVENS, Huong Thi Ngoc LE, Noelia Blanco TALAVAN
Life of this patent
Estimating the term…
Abstract
PiggyBac transposases engineered to increase stability in a cell. The engineered piggyBac transposases are useful for stably transforming cells, cell line development, genome modification, and improving titer of recombinant proteins, among other uses.
Claims56
A piggyBac transposase comprising an amino acid substitution at one or more of the positions 147, 176, 221, 247, 429, 533, and 573 of SEQ ID NO:2.
A piggyBac transposase engineered to increase stability in a host cell, wherein the piggyBac transposase comprises an amino acid substitution at one or more of the positions 147, 176, 221, 247, 429, 533, and 573 of SEQ ID NO:2.
A piggyBac transposase having the amino acid sequence of SEQ ID NO:
- 4, SEQ ID NO:
- 6, SEQ ID NO:
- 8, SEQ ID NO:
- 10, SEQ ID NO:
- 12, SEQ ID NO:
- 14, or SEQ ID NO:
- 16.
A piggyBac transposase encoded by the nucleic acid sequence of SEQ ID NO:
- 3, SEQ ID NO:
- 5, SEQ ID NO:
- 7, SEQ ID NO:
- 9, SEQ ID NO:
- 11, SEQ ID NO:
- 13, SEQ ID NO:
- 15, SEQ ID NO:
- 17, or SEQ ID NO:18.
A method for improving the titer of a recombinant protein of interest expressed by a host cell comprising
- engineering a nucleic acid molecule encoding a piggyBac transposase to increase stability in the host cell;
- co-transfecting the host cell with an engineered nucleic acid molecule encoding the piggyBac transposase and with a vector comprising the nucleic acid sequence encoding the protein of interest flanked by at least the 5′ and 3′ inverted repeat elements of a piggyBac transposon;
- and
- culturing the cells to express the recombinant protein of interest,
- wherein the titer of the recombinant protein of interest expressed by the host cell transfected with the engineered piggyBac transposase is improved compared to the titer of the recombinant protein of interest expressed by a host cell transfected with a wild type piggyBac transposase or no piggyBac transposase.
A method for increasing recombinant protein production in a mammalian cell culture expressing a recombinant protein comprising
- establishing a cell culture in a bioreactor using a host cell that has been co-transfected with nucleic acid molecule encoding a piggyBac transposase engineered to increase stability in the host cell, and a vector comprising the nucleic acid molecule encoding the protein of interest flanked by at least the inverted repeat elements of a piggyBac transposon;
- and
- expressing the recombinant protein of interest;
- wherein the titer of the recombinant protein of interest expressed by the host cell transfected with the engineered piggyBac transposase is improved compared to the titer of the recombinant protein of interest expressed by the host cell transfected with a wild type piggyBac transposase or no piggyBac transposase.
A method for producing an isolated, purified, recombinant protein of interest comprising
- establishing a cell culture in a bioreactor with a host cell expressing a recombinant protein of interest, wherein the cell line has been co-transfected with a nucleic acid sequence encoding a piggyBac transposase engineered to increase stability in the host cell, and a vector comprising the nucleic acid sequence encoding the protein of interest flanked by at least the 5′ and 3′ inverted repeat elements of a piggyBac transposon;
- culturing the cells to express the recombinant protein of interest;
- harvesting the recombinant protein of interest,
- processing the recombinant protein of interest through one or more unit operations, and
- obtaining an isolated, purified, recombinant protein of interest.
A kit for transfecting a cell comprising
- a nucleic acid sequence encoding a piggyBac transposase engineered to increase stability in the host cell;
- and
- a vector comprising at least the 5′ and 3′ inverted repeat elements of a piggyBac transposon into which at least one nucleic acid sequence encoding at least one protein of interest is inserted.
A method of generating non-viral genetically modified cells, comprising:
- (a) establishing a vector comprising at least one nucleic acid sequence encoding at least one protein of interest flanked by at least the 5′ and 3′ inverted repeat elements of a piggyBac transposon;
- (b) isolating native immune cells from a donor or subject;
- (c) transfecting the cells with the vector and a nucleic acid sequence encoding a piggyBac transposase engineered to increase stability in a cell and;
- (d) expanding the cells by cell culture into a larger population of non-viral, genetically modified cells.
A method of treating a subject with a non-viral genetically modified cell, comprising:
- (a) establishing a vector comprising least one nucleic acid sequence encoding at least one protein of interest flanked by at least the 5′ and 3′ inverted repeat elements of a piggyBac transposon;
- (b) isolating native immune cells from a subject or donor;
- (c) transfecting the cells with the vector and a nucleic acid sequence encoding a piggyBac transposase engineered to increase stability in a cell;
- (d) expanding the cells by cell culture into a larger population of genetically modified cells;
- (e) isolating the transformed cells from the cell culture to obtain a cell population comprising the genetically modified cells;
- and
- (f) reintroducing the non-viral, genetically modified cells into the subject.
Dependencies are inferred from the claim text.