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Non-virus iPSCs induction composition and kit thereof

A composition and non-viral technology, applied in the field of bioengineering technology and regenerative medicine, can solve the problems of iPSCs cell tumorigenesis, achieve the effect of reducing clinical risk and shortening the induction culture time

Active Publication Date: 2016-08-17
OSINGLAY BIO PHARM CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it has been reported that the widely used episomal plasmid system for somatic cell reprogramming contains at least one of many high-risk oncogenes or factors, such as c-MYC, SV40-LT, TP53 inhibitors and other oncogenic factors. iPSCs obtained from these high-risk factors may have risks such as cell tumorigenesis

Method used

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  • Non-virus iPSCs induction composition and kit thereof
  • Non-virus iPSCs induction composition and kit thereof
  • Non-virus iPSCs induction composition and kit thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0138] This embodiment 1 provides a non-viral iPSCs induction method, comprising the following steps:

[0139] 1) constructing DNA sequences expressing reprogramming factors OCT4, SOX2, GLIS1, KLF4, L-MYC and hsa-miR-302s into episomal plasmids to obtain recombinant plasmids;

[0140] Wherein, the hsa-miR-302s is hsa-miR-302cluster, and its sequence is shown in SEQ ID No.12;

[0141] Among them, the reprogramming factors OCT4 and GLIS1 are connected through the P2A co-expression element and use the EF-1α promoter to initiate transcription, and KLF4 and SOX2 are connected through the P2A co-expression element and use the EF-1α promoter to initiate transcription, and will contain OCT4, The DNA sequences of the four genes GLIS1, KLF4, and SOX2 were jointly constructed into an episomal plasmid; the reprogramming factors L-MYC and hsa-miR-302s were transcribed through the EF-1α promoter and the CMV promoter, respectively, and their The DNA sequence is constructed into an episomal ...

Embodiment 2

[0152] This example is based on Example 1. During the induction culture process in step 2), a small molecular compound is added to stimulate the induced reprogramming process.

[0153] A method for inducing non-viral iPSCs, comprising the following steps:

[0154] 1) constructing DNA sequences expressing reprogramming factors OCT4, SOX2, GLIS1, KLF4, L-MYC and hsa-miR-302s into episomal plasmids to obtain recombinant plasmids;

[0155] Wherein, the hsa-miR-302s is hsa-miR-302cluster, and its sequence is shown in SEQ ID No.12;

[0156] Among them, the reprogramming factors OCT4 and GLIS1 are connected through the P2A co-expression element and use the EF-1α promoter to initiate transcription, and KLF4 and SOX2 are connected through the P2A co-expression element and use the EF-1α promoter to initiate transcription, and will contain OCT4, The DNA sequences of the four genes GLIS1, KLF4, and SOX2 were jointly constructed into an episomal plasmid; the reprogramming factors L-MYC an...

Embodiment 3

[0161] This example provides a non-viral iPSCs induction method. In this method, in addition to the reprogramming factors OCT4, SOX2, GLIS1, KLF4, L-MYC and hsa-miR-302s, the inhibitory factors c-MYC, SV40- The effect of LT or TP53 on iPSCs to study the effect of different reprogramming factors on iPSCs karyotype.

[0162] Among them, c-MYC, SV40-LT or TP53shRNA were respectively constructed into episomal plasmids, or TP53 inhibitor TP53siRNA was directly transfected into somatic cells, or Pifithrin-μ or Pifithrin-αhydrobromide was directly added to somatic cells.

[0163] A method for inducing non-viral iPSCs, comprising the following steps:

[0164] 1) The DNA sequences expressing reprogramming factors OCT4, SOX2, GLIS1, KLF4, L-MYC and hsa-miR-302s were constructed into episomal plasmids as the control group, that is, group 1; on the basis of the control group, the Add the inhibitory factors c-MYC, SV40-LT or TP53shRNA to the episomal plasmid as shown in the table below or...

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Abstract

The invention discloses a non-virus iPSCs induction composition and a kit thereof. Particularly, the non-virus iPSCs induction composition comprises multiple recombinant plasmids, and DNA sequences for expressing the reprogramming factors of POU5F1, SOX2, GLIS1, KLF4, MYCL and hsa-miR-302s are constructed to episomal plasmids to prepare the recombinant plasmids; the DNA sequence of hsa-miR-302s is a sequence including any one or two or more of hsa-miR-302a, hsa-miR-302b, hsa-miR-302c and hsa-miR-302d. High-risk reprogramming factors like c-MYC, SV40-LT and TP53 inhibiting factors are not added into the induction composition, the non-virus iPSCs induction composition is suitable for obtaining iPSCs with high safety in a non-integration induced reprogramming mode, and clinical risks are reduced to a large extent.

Description

technical field [0001] The invention relates to a non-viral iPSCs inducing composition and a kit thereof, belonging to the fields of bioengineering technology and regenerative medicine. Background technique [0002] Classical stem cell biology generally believes that cell differentiation is unidirectional and irreversible. In 1962, British scientist John B. Gurdon transplanted frog intestinal cell nuclei into enucleated frog egg cells and developed into a normal tadpole, which first confirmed that somatic cell nuclei can Similar techniques for reprogramming to a pluripotent cell state with early stages of embryonic development have also given rise to various cloned mammals. Forty years after Gurdon's results were reported, Japanese scientist Shinya Yamanaka successfully used four transcription factors (OCT4 (standard gene name: POU5F1), SOX2, KLF4, c-MYC, OSKM) carried by retroviruses in 2006 to successfully Mouse fibroblasts were reprogrammed into induced pluripotent stem ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85
CPCC12N5/0696C12N2501/065C12N2501/60C12N2501/602C12N2501/603C12N2501/604C12N2501/606C12N2501/65C12N2501/71C12N2501/727C12N15/85C12N2310/141C12N2510/00C12N2800/108C12N2830/85C12N5/0018C12N2506/45C12N2800/107C12N2830/60
Inventor 王淋立陈月花宋立兵关春燕
Owner OSINGLAY BIO PHARM CO LTD
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