Novel PiggyBac transposition subsystem and application thereof

A technology of transposon and transposase, which is applied in the direction of introducing foreign genetic material by using vectors, cells modified by introducing foreign genetic material, gene therapy, etc. It can solve the problems of low efficiency, high toxicity of plasmid DNA, and expanding the amount of electroporation And other issues

Pending Publication Date: 2022-04-12
SHANGHAI GENCELLS THERAPEUTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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

Method used

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  • Novel PiggyBac transposition subsystem and application thereof
  • Novel PiggyBac transposition subsystem and application thereof
  • Novel PiggyBac transposition subsystem and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] Embodiment 1, the construction of carrier

[0131] pKB20: PiggyBac transposon 3' terminal repeat sequence (3'ITR) (SEQ ID NO: 1), multiple cloning site (SEQ ID NO: 2), bGH polyA signal sequence (SEQ ID NO: 3) in sequence ), enhancer motif sequence (SEQ IDNO:4), insulator sequence with transcription termination function (C2 transcription pause site, SEQ ID NO:5), PiggyBac transposon 5' terminal repeat sequence (5'ITR) ( The reverse complement of SEQ ID NO:6), the reverse complement of the PiggyBac transposase coding sequence (SEQ ID NO:7), the reverse complement of the 5'UTR sequence (SEQ ID NO:8), and the miniCMV promoter The reverse complementary sequence of the sequence (SEQ ID NO:9) was spliced ​​into a long sequence (SEQ ID NO:10), which was synthesized by Shanghai Jierui Biotechnology Co., Ltd., and AgeI and AscI restriction sites were added at both ends , loaded into pUC57 (purchased from Shanghai Jierui Biology), named pKB20. For a schematic diagram of the pl...

Embodiment 2

[0162] Example 2, the integration of pKB vector containing EGFP expression cassette in Jurkat cells

[0163] Prepare 5×10 6 Vigorously growing low-passage Jurkat cells were transfected with 5 μg of pKB20-EGFP, pKB201-EGFP, pKB202-EGFP, and pKC20-EGFP into the nucleus (according to the instrument operating instructions) through the Lonza2b-Nucleofector instrument. 37°C, 5% CO 2 Incubator culture. After the cells were confluent, they were subcultured at a ratio of 1:10. On the 7th day, the 10th day, and the 14th day after the electroporation, the fluorescence microscope photographed and observed; the 7th day, the 10th day, and the 14th day (after 3 passages) after the electroporation were used to detect the change in the proportion of EGFP-positive cells, Jurkat cells not transfected with the plasmid were used as a control for flow cytometry. Cell fluorescence intensity was observed by fluorescence microscope during the whole process of electroporation. Viable cell counts...

Embodiment 3

[0169] Example 3, PB transposase expression time curve after pKB vector electroporation into Jurkat cells

[0170] Take 5×10 6 Vigorously growing low-passage Jurkat cells were transfected into the nucleus with 5 μg of pKB20-EGFP, pKB201-EGFP and pKB202-EGFP (according to the instrument’s operating instructions) using the Lonza2b-Nucleofector instrument, 37°C, 5% CO 2 After culture, cells were collected at 6, 12, 24, 48, 96 hours and 15 days after electroporation, and RNA was extracted, and the expression level of PB transposase was quantitatively detected by RT-PCR using β-actin as an internal reference. The results were as follows Figure 5 shown.

[0171] Figure 5 It was shown that the expression level of PB transposase in Jurkat cells electroporated with pKB20-EGFP, pKB201-EGFP and pKB202-EGFP reached the peak at 6h, and then began to decrease significantly. The expression level of PB transposase in the electrotransfected pKB20-EGFP and pKB201-EGFP cells was signific...

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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...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10A61K35/17A61K35/15A61P35/00
CPCA61K35/15A61P35/00C12N15/85A61K35/17A61K48/00C12N15/90C12N5/0636C12N2510/00C12N15/113C12N15/63C12N9/1241A61P43/00C12N2800/90C12N2830/50
Inventor 金华君许馥慧黄晨马星明刘天怡
Owner SHANGHAI GENCELLS THERAPEUTICS CO LTD
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