Novel PiggyBac transposition subsystem and application thereof

Abstract

The invention relates to a PiggyBac transposon system and application thereof, and particularly provides a nucleic acid construct which comprises the following elements: a transposon 3 '-terminal repetitive sequence, a first polyA sequence, an insulator sequence with a transcription termination function, a transposon 5'-terminal repetitive sequence, a transposase coding sequence and a promoter for controlling the expression of the transposase. The invention further provides a host cell or a pharmaceutical composition containing the nucleic acid construct and application of the host cell or the pharmaceutical composition.

Description

technical field [0001] The invention relates to the field of transposon carriers, in particular to a novel PiggyBac transposon system and its application. Background technique [0002] It is a necessary condition for the realization of many cell therapy technologies, including immune cell therapy, to introduce foreign genes into the target cells for stable expression so as to realize the transgenic transformation of the target cells. Compared with transient expression, stably expressed exogenous genes can be integrated into the genome of target cells, and can maintain stable expression for a long time even in the case of multiple cell passages or changes in culture conditions. Commonly used transgenic systems include viral-based vectors, eukaryotic expression plasmid vectors, and transposon vectors. Genetic modification of primary human T cells using non-viral vector-based approaches has proven to be extremely difficult. Therefore, worldwide, most laboratories are still us...

AI Technical Summary

Problems solved by technology

However, the binary system requires the PiggyBac transposase carrier and the auxiliary carrier to be transposed into the cell at the same time before transposition can occur, which requires high requirements for transfection and is difficult

At the same time, the mechanism of the PiggyBac transposition system, PiggyBac transposase, inserts transposition fragments into the genome through the "cut-paste" mechanism, and this process is reversible, so as long as the expression of PiggyBac enzyme continues , the transposition fragments that have been integrated into the genome may also be re-cut, causing genome instability and substantially reducing transposition efficiency

The transposition efficiency of the common PiggyBac binary transposition system in T cells is usually around 10%, which is relatively low

And WO2019046815A1 also records that plasmid DNA is highly toxic to T cells, and its toxicity to T cells is related to the amount of DNA used for electroporation

The binary system undoubtedly expands the amount of plasmid DNA required for electroporation, increases the toxicity to cells, especially T cells, and reduces the survival rate of T cells transfected with plasmid DNA

However, the expression cassette of the PiggyBac transposase and the expression cassette of the foreign gene share the same bidirectional polyA sequence, which will cause mutual influence between the two expression cassettes in opposite directions, and in some types of cells, the unary transposon vector mediates The integration efficiency still needs to be further improved

[0006] Therefore, there is still a lack of an efficient one-way transposon system that can efficiently integrate foreign genes while shutting down the integration function in time

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