JPWO2020123327A5 - - Google Patents

Claims (26)

A piggyBac transposase comprising an amino acid substitution at one or more of positions 147, 176, 221, 247, 429, 533 and 573 of SEQ ID NO:2. 2. The piggyBac transposase of claim 1, comprising amino acid substitutions of isoleucine with leucine at one or more of positions 147, 176, 221, and 247 of SEQ ID NO:2. 2. The piggyBac transposase of claim 1, comprising amino acid substitutions of serine with threonine at one or more of positions 429, 533, and 573 of SEQ ID NO:2. Amino acid substitution of isoleucine with leucine at one or more of positions 147, 176, 221 and 247 of SEQ ID NO:2 and/or one or more of positions 429, 533 and 573 of SEQ ID NO:2 2. The piggyBac transposase of claim 1, comprising an amino acid substitution of threonine for serine at . The following amino acid substitution of isoleucine with leucine at position 147 of SEQ ID NO: 2, amino acid substitution of isoleucine with leucine at position 247 of SEQ ID NO: 2, and amino acid substitution of serine with threonine at position 533 of SEQ ID NO: 2 5. The piggyBac transposase of claim 4, comprising at least one of The following amino acid substitution of isoleucine with leucine at position 147 of SEQ ID NO: 2, amino acid substitution of isoleucine with leucine at position 247 of SEQ ID NO: 2, and amino acid substitution of serine with threonine at position 533 of SEQ ID NO: 2 5. The piggyBac transposase of claim 4, comprising at least two of: 2. Substitution of leucine for isoleucine at position 147 of SEQ ID NO:2, substitution of leucine for isoleucine at position 247 of SEQ ID NO:2, and substitution of threonine for serine at position 533 of SEQ ID NO:2. 4. The piggyBac transposase according to 4. The titer of the recombinant protein of interest expressed by cells transfected with the modified piggyBac transposase is higher than the titer of the same protein of interest expressed by cells transfected with wild-type piggyBac transposase or without piggyBac transposase. 2. The piggyBac transposase of claim 1, which is improved relative to potency. 9. The piggyBac transposase of claim 8, wherein said recombinant protein of interest is an antigen binding protein. 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. 11. A modified nucleic acid molecule encoding the piggyBac transposase of claim 1 or 10. 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. 13. A vector comprising said nucleic acid molecule encoding the piggyBac transposase of claim 11 or 12. 13. A vector comprising said nucleic acid molecule encoding the piggyBac transposase of claim 11 or 12, said vector encoding at least one protein of interest flanking at least the 5' and 3' inverted repeat elements of the piggyBac transposon. A vector further comprising a nucleic acid sequence. A cell transfected with a piggyBac transposase encoded by the nucleic acid sequence of claim 12. A cell transfected with the vector of claim 13. 17. The cell of claim 15 or 16, wherein said cell is a host cell. 17. The cell according to claim 15 or 16, wherein said cell is a CHO cell. 17. The cell of claim 15 or 16, wherein said cell is an immune cell. a modified nucleic acid molecule encoding the piggyBac transposase of claim 11 and at least one nucleic acid molecule encoding at least one protein of interest flanking at least the 5′ and 3′ inverted repeat elements of the piggyBac transposon; Cells co-transfected with the containing vector. 14. A vector comprising a modified nucleic acid molecule encoding the piggyBac transposase of claim 13 and at least one protein encoding at least one protein of interest flanked by at least the 5' and 3' inverted repeat elements of the piggyBac transposon. A cell co-transfected with a vector containing a nucleic acid molecule. 14. At least one nucleic acid comprising a modified nucleic acid molecule encoding the piggyBac transposase of claim 13 and encoding at least one protein of interest flanked by at least the 5' and 3' inverted repeat elements of the piggyBac transposon Cells co-transfected with a vector containing the molecule. The titer of the protein of interest expressed by said cells transfected with the modified piggyBac transposase is the same titer of the protein of interest expressed by cells transfected with wild-type piggyBac transposase or without piggyBac transposase. 23. The cell of any one of claims 15, 16, 20, 21 or 22, wherein the cell is improved as compared to titer. A method for improving the titer of a recombinant protein of interest expressed by a host cell comprising:
modifying a nucleic acid molecule encoding a piggyBac transposase to increase stability of said host cell;
co-transfecting said host cell with a vector comprising a modified nucleic acid molecule encoding said piggyBac transposase and a nucleic acid sequence encoding said protein of interest flanked by at least the 5′ and 3′ inverted repeat elements of the piggyBac transposon; to do;
culturing said cells to express said recombinant protein of interest;
the titer of said recombinant protein of interest expressed by said host cells transfected with the modified piggyBac transposase expressed by host cells transfected with wild-type piggyBac transposase or without piggyBac transposase; A method wherein the potency of the recombinant protein of interest is improved compared to. said host cell comprises a first vector comprising said modified nucleic acid molecule encoding said piggyBac transposase and nucleic acid encoding said protein of interest flanked by at least the 5′ and 3′ inverted repeat elements of the piggyBac transposon; 25. The method of claim 24, transfected with a second vector containing the sequence. said host cell comprising said modified nucleic acid molecule encoding said piggyBac transposase and a nucleic acid sequence encoding said protein of interest flanked by at least the 5′ and 3′ inverted repeat elements of the piggyBac transposon; 25. The method of claim 24, wherein the vector is transfected.