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RNA composition for inducing neuronal differentiation, differentiation method and application thereof

A composition and neuron technology, applied in the field of cell engineering, can solve the problems of restricting the development of mRNA technology, poor mRNA stability and immunogenicity, etc., and achieve the effects of improving translation efficiency and improving differentiation efficiency.

Pending Publication Date: 2022-07-05
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is well known that traditional mRNA has poor stability and strong immunogenicity, which greatly limits the development of mRNA technology.

Method used

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  • RNA composition for inducing neuronal differentiation, differentiation method and application thereof
  • RNA composition for inducing neuronal differentiation, differentiation method and application thereof
  • RNA composition for inducing neuronal differentiation, differentiation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Optimization of the transfection process of in vitro synthesized mRNA

[0035] This example is an optimization experiment of the transfection process of mRNA synthesized in vitro, including modification of mRNA and optimization of transfection conditions.

[0036] (1) In vitro synthesized mRNA will cause an immune response after transfection into cells. In order to improve the translation efficiency of transfected mRNA, luciferase luciferase mRNA is modified with different UTP and polyA tailed in turn, and then transfected into cells. The effect of modification method on mRNA translation efficiency is shown in Figure 1.

[0037] Depend on Figure 1a It can be seen that luciferase using Cleancap AG method capping is better than ARCA capping method; Figure 1b It can be seen that the expression efficiency of luciferase was significantly improved after adding poly A tail. Therefore, the modification method of CleanCap AG capping and poly-A tailing can signific...

Embodiment 2

[0041] Example 2 A method for inducing differentiation of cortical glutamatergic neurons

[0042] The present embodiment provides a method for inducing differentiation of cortical glutamatergic neurons, the method comprising the following steps in sequence:

[0043] S1. Synthesize the mRNAs of NEUROG1, NEUROG2, and NEUROD1 proteins, and perform CleancapAG capping and poly-A tailing modification on them to obtain mod-mRNA;

[0044] S2. Synthesize miR-9 and miR-124 respectively to obtain a MicroRNA composition;

[0045] S3. 600ng of mod-mRNA in S1 (including 200ng NEUROG1, 200ng NEUROG2 and 200ng NEUROD1) and 800ng of MicroRNA in S2 (including 400ng miR-9, 400ng miR-124) were transfected into human polynucleotides using 4ul transfection reagent lipotem. able stem cells, the cortical glutamatergic neurons can be obtained within seven days, such as Figure 4 shown.

Embodiment 3

[0046] Example 3 Cortical glutamatergic neuron type and functional verification

[0047] Activating the target gene is a signal that prompts the target cell type to be obtained, but whether the cells obtained by in vitro differentiation are consistent with or similar to in vivo functions needs to be verified in many ways. Type and function identification;

[0048] S1. RNA-seq

[0049] In order to identify the neuron types produced by in vitro differentiation, we took mRNA at different differentiation time points and performed RNA-seq to verify the effect of different transcription factors on neural differentiation. According to the expression of TUBB3, we screened the neural induction combination: NEUROG1, NEUROG2, NEUROD1 and miRNAs, the results are as Figure 5 , it can be seen from the analysis that in vitro differentiated cells exhibit different developmental stages. On the ninth day of differentiation, genes specifically expressed in glutamatergic neurons were signific...

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Abstract

The invention discloses an RNA (Ribonucleic Acid) composition for inducing neuronal differentiation. The RNA composition comprises a modified mRNA (Messenger Ribonucleic Acid) and a MicroRNA composition, the invention also provides a method for inducing differentiation of cortical glutamic acid energetic neurons by using the RNA composition, and pluripotent stem cells can be induced to be differentiated into neurons within seven days through transient overexpression of the mRNA and MicroRNA composition; the invention also provides an application of the RNA composition, and the RNA composition is used for inducing cortical glutamic acid neurons to differentiate. The transfection process and the mRNA delivery system are optimized, so that the translation efficiency of transfected mRNA and the expression quantity of mRNA in cells are remarkably improved. The method is suitable for inducing the differentiation of the cortical glutamic acid neurons, and the differentiated cortical glutamic acid neurons can be further applied to the construction of pathological models of human nerve-related diseases.

Description

technical field [0001] The invention belongs to the field of cell engineering and relates to a neuron differentiation method, in particular to an RNA composition mod-mRNA 3FM for inducing cortical glutamatergic neuron differentiation, a method and its application. Background technique [0002] In recent years, stem cell technology has developed rapidly, which is the research basis for developmental research, disease modeling and regenerative medicine, and opens up new directions for the study of human diseases. Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), have the potential to differentiate into neurons. The hPSCs directional differentiation technology provides a more realistic pathophysiological model for the study of neurodegenerative diseases, and the transplantation of differentiated nerve cells provides a new idea for the treatment of neurological diseases. [0003] Existing differentiati...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/113C12N15/87C12N5/10C12N5/0793
CPCC07K14/47C12N15/113C12N15/87C12N5/0696C12N5/0619C12N2310/141C12N2510/00C12N2506/45
Inventor 方芳夏尼诺朱玲玲
Owner UNIV OF SCI & TECH OF CHINA
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