Preparation method of ALS (Amyotrophic Lateral Sclerosis) patient specific motor neuron

A motor neuron and specific technology, applied in the biological field, can solve problems such as large side effects, covering up key factors, and inability to truly reflect the pathogenic mechanism

Active Publication Date: 2017-01-25
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The clinical symptoms of the two are similar, but the pathogenic mechanism is unclear, and there is no effective drug treatment
One of the reasons is species differences. Due to the different genetic backgrounds of humans and model organisms, drugs that are effective in treating ALS screened in model organisms cannot relieve ALS symptoms clinically, or have severe side effects.
Second, the ALS disease model generated by overexpressing the mutant gene cannot truly reflect the patient's physiological condition
The expression level of the exogenous overexpression mutation pathogenic gene is much higher than the expression level of the single point gene mutation in the patient, thus covering up some key factors of pathogenicity and not really reflecting the pathogenic mechanism. Drugs screened on this basis Can not effectively relieve the clinical symptoms of ALS patients

Method used

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  • Preparation method of ALS (Amyotrophic Lateral Sclerosis) patient specific motor neuron
  • Preparation method of ALS (Amyotrophic Lateral Sclerosis) patient specific motor neuron
  • Preparation method of ALS (Amyotrophic Lateral Sclerosis) patient specific motor neuron

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Example 1. Obtaining induced pluripotent stem cells (iPSC) derived from ALS patients

[0097] 1. Obtaining induced pluripotent stem cells derived from ALS patients

[0098] 1. Cell culture

[0099] Two lines of skin fibroblasts derived from ALS patients carrying different gene mutations: FUS G1566A fibroblasts (ND29563) and SOD1 A272C fibroblasts (ND29149) were expanded in fibroblast culture medium. After the cells were cultured to a suitable density, they were digested and counted, and 1.5×10 6 indivual.

[0100] 2. Preparation of electrotransfer solution

[0101] Mix 100 μl opti-MEM (product of life technology), 1.5 μg pCXLE-hOCT3 / 4-shp53-F, 1.5 μg pCXLE-hSK, 1.5 μg pCXLE-hUL and 1.5 μg pCXLE-EGFP to obtain electrotransfer solution, and place at 1.5 ml EP tube.

[0102] 3. Use the electrotransfer solution obtained in step 2 to resuspend the FUS G1566A fibroblasts (ND29563) and SOD1 A272C fibroblasts (ND29149) obtained in step 1, respectively, transfer them to the...

Embodiment 2

[0113] Example 2. Targeted correction of gene mutations carried by two ALS-iPSCs

[0114] Utilize CRISPR / Cas9 gene editing technology to two kinds of iPSC (FUS) obtained in embodiment 1 G1566A - iPSC and SOD1 A272C -iPSCs) carry out targeted elimination of gene mutations, and finally obtain iPSCs that eliminate gene mutations. Specific steps are as follows:

[0115] 1. Cell culture

[0116] The two iPSCs (FUS G1566A - iPSC and SOD1 A272C -iPSC), the medium is mTESR. When the clone density reaches 70%-80%, digest with TrypLE, count and collect 5×10 6 cells.

[0117] 2. Preparation of electrotransfer solution

[0118] Mix 8 μg of Cas9-GFP plasmid, 8 μg of the corresponding gene-corrected recombinant template and 4 μg of the corresponding gene-corrected gRNA-mCherry plasmid, and dilute to 100 μl with opti-MEM (life technology) to obtain the electrotransfer solution, which is placed in a 1.5ml EP tube .

[0119] When the relevant gene mutation to be corrected is FUS G156...

Embodiment 3

[0137] Example 3. Obtaining and identification of motor neurons carrying ALS pathogenic gene mutations and gene-corrected motor neurons

[0138] 1. Obtaining motor neurons carrying ALS pathogenic gene mutations and gene-corrected motor neurons

[0139] The two iPSCs (FUS) carrying gene mutations obtained in Example 1 were G1566A - iPSC and SOD1 A272C -iPSC) and two kinds of gene-corrected iPSCs obtained in Example 2 (FUS G1566G - iPSC and SOD1 A272A -iPSCs) were directedly induced to differentiate into motor neurons at the same time, and FUS were obtained respectively G1566A -MN,SOD1 A272C -MN, FUS G1566G -MN,SOD1 A272A -MN. Schematic diagram of the protocol for directed differentiation of iPSCs into motor neurons. image 3 Shown in A. The specific operation is as follows:

[0140] 1. Inoculate the four small iPSC clones obtained in Example 1 and Example 2 into a culture plate previously covered with MEFs treated with mitomycin inactivation, and culture them with cDF...

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Abstract

The invention disclsoes a preparation method of an ALS (Amyotrophic Lateral Sclerosis) patient specific motor neuron. The method comprises the following steps: (1) reprogramming an in-vitro fibroblast carrying ALS related genic mutation from an ALS patient source to obtain induced pluripotent stem cells carrying the ALS related genic mutation (ALS-iPSC); (2) carrying out gene editing on the ALS-iPSC and specifically eliminating the ALS related genic mutation to obtain gene corrected induced pluripotent stem cells (cALS-iPSC); (3) carrying out orient induction and differentiation on the cALS-iPSC and the ALS-iPSC respectively to obtain the ALS patient specific motor neuron carrying the ALS related genic mutation or without the ALS related genic mutation. The specific motor neuron carrying ALS pathogenic gene mutation, obtained by the preparation method, can be used as an effective platform for carrying out efficient and high-throughput individualized drug screening; the ALS patient specific motor neuron without the ALS related genic mutation is hopefully used for treating and preventing ALS. The preparation method of the ALS patient specific motor neuron lays the foundation for ALS disease researches, disease model development, disease pathogenesis researches and disease treatment.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for preparing specific motoneurons of ALS patients. Background technique [0002] Human amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease, the main pathological feature is motor neuron degeneration, leading to muscle atrophy, paralysis, and even affecting the respiratory system, endangering the patient's life . The onset of ALS is rapid, and most patients die after 3-5 years of onset. ALS is divided into two types, sporadic ALS (SALS) and familial ALS (FALS). The clinical symptoms of the two are similar, but the pathogenic mechanism is unclear, and there is no effective drug treatment. The etiology of SALS is complex and may be due to the combined effects of environmental and genetic factors. Studies have found that FALS is generally caused by heterozygous mutations in a certain gene, such as SOD1, FUS, C9ORF72, TARDBP, etc. SOD1 gene mutation is the fi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/85C12N5/10C12Q1/02A61K35/30A61P21/00A61P25/00
CPCA61K35/30C07K14/47C12N5/0619G01N33/5058
Inventor 刘光慧曲静王丽霞任若通
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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