Granted Patent
B2
US 12,742,171 · App. 17/832,409 · Granted Sep 22, 2026
Integration of nucleic acid constructs into eukaryotic cells with a transposase from oryzias
Inventors:
Jeremy Minshull (Los Altos, CA); Sridhar Govindarajan (Los Altos, CA); Maggie Lee (San Jose, CA)
Assignee:
DNA TWOPOINTO INC.
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Abstract
The present invention provides polynucleotide vectors for high expression of heterologous genes. Some vectors further comprise novel transposons and transposases that further improve expression. Further disclosed are vectors that can be used in a gene transfer system for stably introducing nucleic acids into the DNA of a cell. The gene transfer systems can be used in methods, for example, gene expression, bioprocessing, gene therapy, insertional mutagenesis, or gene discovery.
Claims (15)
1 . A method of integrating a transposon into a eukaryotic cell, the method comprising (a) introducing into the cell a transposon comprising SEQ ID NO: 7 and SEQ ID NO: 8 as left and right ends respectively flanking a heterologous polynucleotide encoding a polypeptide; (b) introducing into the cell a transposase, the sequence of which is at least 90% identical to SEQ ID NO:782, wherein the transposase transposes the transposon to produce a genome comprising SEQ ID NO:7 and SEQ ID NO:8 flanking the heterologous polynucleotide at left and right ends respectively; and (c) expressing the polypeptide from the heterologous polynucleotide within the genome.
2 . The method of claim 1 , wherein the polypeptide is a chimeric antigen receptor.
3 . The method of claim 1 , wherein the polypeptide comprises a chain of an antibody.
4 . The method of claim 1 , further comprising purifying the polypeptide.
5 . The method of claim 1 , further comprising incorporating the purified polypeptide into a pharmaceutical composition.
6 . The method of claim 1 , wherein the cell is a mammalian cell.
7 . The method of claim 1 , wherein the cell is a human cell.
8 . The method of claim 1 , wherein the cell is a rodent cell.
9 . The method of claim 1 , wherein the transposase is introduced as a polynucleotide encoding the transposase.
10 . The method of claim 9 , wherein the polynucleotide is an mRNA.
11 . The method of claim 1 , wherein the transposase is introduced as a protein.
12 . The method of claim 1 , wherein the transposase has a sequence which comprises any of SEQ ID NOS:782 or 805-908.
13 . The method of claim 1 , wherein the transposon further comprises a sequence at least 90% identical to SEQ ID NO:12 immediately adjacent to the left transposon end and between the left transposon end and the heterologous polynucleotide and a sequence at least 90% identical to SEQ ID NO:15 immediately adjacent to the right transposon end and between the right transposon end and the heterologous polynucleotide.
14 . The method of claim 1 , wherein the heterologous polynucleotide encodes two open reading frames, each operably linked to a separate promoter, one encoding the polypeptide, and the other a second polypeptide.
15 . The method of claim 14 , wherein the open reading frames encode heavy and light chains of an antibody.
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Jun 9, 2022
From: MINSHULL, JEREMY; GOVINDARAJAN, SRIDHAR; LEE, MAGGIE
To: DNA TWOPOINTO INC.
Reel/Frame 060149/0994 →
Continuity (6)
Provisional Application
62982186
· Feb 27, 2020
Provisional Application
62873338
· Jul 12, 2019
Provisional Application
62831092
· Apr 8, 2019
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