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Mutated piggybac transposase

A technology of transposase and amino acid, applied in the direction of transferase, enzyme, hydrolase, etc., can solve the problems of high R&D cost, delay and inefficiency, and long development lead time of biological drugs

Pending Publication Date: 2021-07-30
AMGEN INC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

High R&D costs and long lead times for biopharmaceuticals make it imperative to eliminate delays and inefficiencies in drug development and, more importantly, manufacturing

Method used

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  • Mutated piggybac transposase
  • Mutated piggybac transposase
  • Mutated piggybac transposase

Examples

Experimental program
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Effect test

example 1

[0187] Example 1 Identification and Testing of Mutations

[0188] The structure of piggyBac transposase and other related transposases is unknown. We predicted wild-type Trichoplusia piggyBac transposase (SEQ ID NO: 2) The secondary structure motif. see figure 1 . We found that the piggyBac transposase is predominantly an α-helical protein. We designed mutations that might stabilize the α-helix, and these mutations could improve the overall stability of the transposase, which in turn could positively affect the expression of the transposase. We identified I147, I176, I221, I247 as residues that could potentially be mutated to improve alpha-helix stability of piggyBac transposase.

[0189] We also identified putative N-linked glycosylation sites (ie, NXS / T motifs) in the transposase sequence. Since, in general, NXT motifs undergo more complete glycosylation compared to NXS motifs, we hypothesized that mutation of NXS motifs to NXT motifs might improve overall glycosylatio...

example 2

[0198] Example 2 Expression of double and triple transposase mutants in MSX-added GS KO host cells

[0199] Using electroporation (Bio-Rad, Hercules, CA) DNA piggyBac transposase encoding 1) double mutant "ILT" (ITR, SEQ ID NO: 11), 2) triple mutant Glutamine synthase knockout CHO cells (GSKO cells) were transfected with a "LLT" DNA piggyBac transposase (LLT, SEQ ID NO:9) and 3) a circular plasmid not encoding piggyBac transposase (None), all Both cases were combined with a circular plasmid containing the gene of interest and the 5' and 3' inverted repeat elements of the Trichoplusia piggyBac transposon. Three target genes were tested, bispecific T cell engager heterologous Fc (Tcell engager heteroFc), IgG-scFv and monoclonal antibody (mAb).

[0200] On day 3 after transfection (25), after initial pools had recovered >90% viability (0-25) or not at all (0), 25 μM of methionine sulfoximine (MSX) (EDM dense EDM Millipore, Burlington, Mass.). The addition of methionine sulfoxi...

example 3

[0205] Example 3 Transfection using DNA or mRNA piggyBac transposase.

[0206] The transposase used to integrate the gene of interest can be DNA or mRNA based. One concern with using transposases for DNA transcription is the potential for integration of the transposase gene into the genome, which could result in active transcription / translation of the transposase in the transfected cell line. This could lead to genome instability due to transposase activity at potential recessive transposase recognition sites. An alternative is to use mRNA for transfection since it does not integrate into the genome.

[0207] Unmodified mRNA transcripts using wild-type bases and with 25% pseudo-U and 5-methyl-C end-capping substitutions were prepared against the piggyBac transposase of double mutant "ILT" and triple mutant "LLT" modification of synthetic mRNA transcripts.

[0208] Two sets of experiments were performed to evaluate mRNA translation transposases in transfections. In a first ...

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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.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 777,325, filed December 10, 2018, and U.S. Provisional Application No. 62 / 925,516, filed October 24, 2019, which are incorporated by reference in their entirety and for all purposes It is hereby incorporated herein as if fully set forth herein. technical field [0002] The present invention relates to piggyBac transposases engineered to increase stability in cells, and the use of these engineered piggyBac transposases in stably transfecting cells, cell line development, genome modification, and improving recombinant protein titers, etc. use. [0003] sequence listing [0004] This application contains a sequence listing in computer readable form (file name: A-2320-WO-PCT_Sequence.txt, created on 11 / 5 / 2019, its size is 64KB), as a separate part of this disclosure, and by References are incorporated herein in their entirety. Background technique [0005] Due to their wide range of applications...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/85C12N15/90
CPCC12N2800/90C12N15/85C12N15/90C12N9/1241C12P21/02C12Y207/07C12N15/52A61K38/00A61K48/00A61K35/17C07K14/7051
Inventor N·J·阿拉瓦尔K·M·达里斯J·L·斯蒂芬斯H·T·N·黎N·B·塔拉文
Owner AMGEN INC
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