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A gene-vector-mediated sgRNA based on a CRISPR/Cas9 gene editing system and uses of the sgRNA

A gene therapy and recombinant vector technology, applied in the fields of recombinant expression vectors and gene therapy methods, can solve problems such as loss of function

Inactive Publication Date: 2017-12-15
THE SECOND HOSPITAL OF HEBEI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the neurotoxicity of mutant SOD1 is an acquired toxicity and is not related to the loss of function of SOD1 mutation [15]

Method used

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  • A gene-vector-mediated sgRNA based on a CRISPR/Cas9 gene editing system and uses of the sgRNA
  • A gene-vector-mediated sgRNA based on a CRISPR/Cas9 gene editing system and uses of the sgRNA
  • A gene-vector-mediated sgRNA based on a CRISPR/Cas9 gene editing system and uses of the sgRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 AAV9-SaCas9-SOD1 sgRNA cloning and virus packaging

[0061] According to the SaCas9 PAM sequence (NNGRRT) and the design principle of the optimal 21-nt length, five SOD1 sgRNAs were designed, digested with Bsa I, and cloned into the AAV-SaCas9-sgRNA vector (Addgene, px61591). AAV-SaCas9-SOD1 sgRNAs virus was packaged by three-plasmid co-transfection method, purified and concentrated, and its titer was determined by qPCR.

[0062] SOD1 sgRNA design and editing efficiency results:

[0063] Figure 1(A) shows the sequence design of five SOD1 sgRNAs;

[0064] Figure 1(B) shows that five SOD1 sgRNAs can mediate the cleavage of SOD1 by saCas9 to varying degrees, among which sgRNA1 and sgRNA5 have the best effect;

[0065] Figure 1(E) shows that six vectors have different delivery efficiencies for sgRNA5, among which adeno-associated virus has the best effect.

Embodiment 2

[0066] Example 2 Detection of gene editing efficiency of SOD1 sgRNA by flow cytometry

[0067] Transfect the SOD1-GFP plasmid into 293T cells, collect the cells after 48 hours, digest the cells with 0.25% trypsin, and centrifuge to obtain a pellet. Resuspend the cells in medium to 1 x 10 7 cells / ml cell suspension, GFP-positive cells were sorted by flow cytometry (FACSAria, BD), and the SOD1-GFP stable cell line was obtained. According to the Lipofectamine 2000 operation manual, transfect SaCas9-LacZ, SaCas9-sgRNA1 or SaCas9-sgRNA5 DNA into cell lines stably expressing SOD1-GFP, and use flow cytometry to detect the fluorescence intensity of GFP after 48 hours to determine the effect of sgRNA on SOD1 gene Editing-mediated efficiency. It can be seen that, compared with the control group sgLacZ, after transfection of sgRNA1 or sgRNA5 of SOD1, the fluorescence intensity decreased significantly.

[0068] The results of gene editing efficiency of SOD1 sgRNA detected by flow cyto...

Embodiment 3

[0070] Example 3 Western Blot detection of gene editing efficiency of SOD1 sgRNA

[0071] Total protein in the spinal cord was extracted using a total protein extraction kit (Applygen Technologies Inc., P1250). After the concentration was determined, 50 µg of protein was loaded on each sample for SDS-PAGE gel electrophoresis, transferred to PVDF membrane, blocked, and incubated overnight at 4ºC with beta-actin antibody (1:500, Proteintech) and hSOD1 antibody (1:1, Abcam) After that, wash thoroughly, incubate with fluorescently labeled secondary antibody for 1 hour at room temperature, and then use Odyssey infrared imaging system to detect the corresponding bands. It can be seen that the protein expression level of SOD1 decreased significantly after transfection of SOD1-sgRNA1 or SOD1-sgRNA5.

[0072] sgRNA1 and sgRNA5 can mediate the gene editing results of SaCas9 on SOD1 in vitro:

[0073] Figure 1(D) shows that after transfection of SOD1-sgRNA1 or SOD1-sgRNA5 in 293T cells...

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Abstract

A gene therapy method, which is mediated with a gene vector (including, but not limited to 9 type adeno-associated viruses), directly edits a superoxide dismutase 1 (SOD1, for short) mutant gene based on a CRISPR / Cas9 gene editing system, and treats amyotrophic lateral sclerosis (ALS, for short) in vitro and in transgenic mice, is provided by the invention. A provided recombinant virus has bioactivity, and is hoped to be a candidate virus for ALS gene therapy.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to an AAV9 viral vector-mediated sgRNA based on the CRISPR / Cas9 gene editing system and its application, including a composition with the sgRNA, a recombinant expression vector and a gene therapy method. Background technique [0002] Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, as a type of motor neuron disease (ALS / MND), and is the most common adult-onset motor neuron disease. ALS is a common degenerative disease of the nervous system like Alzheimer's disease and Parkinson's disease, but its development speed and nature are more serious than the other two degenerative diseases. Patients are characterized by degeneration of motor neurons in the spinal cord, brainstem, and cerebral cortex, manifested as progressive muscle weakness, and mostly die of respiratory failure; the onset occurs in adults, and the median survival period is only 3-5 years[1]. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/864C12N15/90C12N9/22C12N15/66A61K48/00A61K31/7105A61P25/02A61P21/00
CPCA61K31/7105C12N9/0089C12N9/22C12N15/1137C12N15/66C12N15/86C12N15/907C12N2750/14143C12N2810/10C12Y115/01001
Inventor 段伟松吴小兵李春岩
Owner THE SECOND HOSPITAL OF HEBEI MEDICAL UNIV
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