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Method for increasing expression of survival motor neuron (SMN) protein based on gene editing technology, and application of method in spinal muscular atrophy (SMA) treatment

A protein expression and gene technology, applied in the field of genetic engineering, can solve the problems of high price, unacceptable for patients, and short-term functional SMN protein.

Active Publication Date: 2019-12-31
THE FIRST AFFILIATED HOSPITAL OF FUJIAN MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the ASO produced by it is degraded in the cell, and the expression of functional SMN protein is short-term
Taking clinical SMA treatment as an example, maintaining the expression of SMN protein requires regular and continuous supplementation of ASO, which requires intrathecal injection through lumbar puncture, which is difficult for patients to accept, which limits the relevant application of this method
In addition, the current price of ASO in the United States is as high as 750,000 U.S. dollars per person in the first year, and 375,000 U.S. dollars per year thereafter.

Method used

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  • Method for increasing expression of survival motor neuron (SMN) protein based on gene editing technology, and application of method in spinal muscular atrophy (SMA) treatment
  • Method for increasing expression of survival motor neuron (SMN) protein based on gene editing technology, and application of method in spinal muscular atrophy (SMA) treatment
  • Method for increasing expression of survival motor neuron (SMN) protein based on gene editing technology, and application of method in spinal muscular atrophy (SMA) treatment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Example 1, CRISPR / Cas9-mediated gene editing

[0105] SMN2 is a highly similar gene to SMN1, but due to the difference in C6T on exon 7 of SMN2, exon 7 is skipped during the splicing process, such as figure 1 A ~ B shown. The intron splicing silencers ISS-N1 and ISS+100 are located in exon 7. In order to prevent this exon skipping, the inventors used CRISPR / Cas9 to target ISS-N1 or ISS+100 in an attempt to destroy their structure. The gene splicing situation and the CRISPR / Cas9 editing strategy of the present invention are as follows: figure 1 shown.

[0106] In order to disrupt ISS-N1 or ISS+100, the inventors designed sgRNA at a position adjacent to or inside ISS-N1 or ISS+100. According to the position difference, the present inventors prepared a large number of sgRNAs to screen suitable sgRNAs, and found gRNAs suitable for destroying ISS-N1 or ISS+100 after repeated research and screening. The sequence of the sgRNA is as follows:

[0107] sgRNAs used to target...

Embodiment 2

[0115] Example 2. The splicing pattern of iPSC cell line SC-SMA that destroys ISS-N1 or ISS+100 is regulated and changed

[0116] In the monoclonal cell lines obtained in Example 1, the inventors detected two ISS-N1 disrupted cell lines, which were named SC-SMAI-2-1 (gene editing guided by sgRNA1), and SC-SMAI-2 -2 (gene editing guided by sgRNA1); and, an ISS+100 disrupted SC-SMAI-2-3 cell line (gene editing guided by sgRNA3) and mycoplasma-free. The present inventors performed RT-PCR analysis and qPCR analysis to determine the SMN2 gene splicing and SMN protein expression after destroying ISS-N1 or ISS+100 at the cellular level.

[0117] see results image 3 . As a result of RT-PCR analysis, the promotion of the full-length SMN2 (SMN-FL) transcript can be significantly seen ( image 3 , A). Moreover, the results of Western blot showed that the SMN protein was 1.7 times that of unedited ( image 3 , B).

Embodiment 3

[0118] Example 3, CRISPR / Cas9-mediated gene editing can prevent the occurrence of SMA

[0119] 1. Obtain mice with significant disruption of ISS-N1

[0120] Type III mice contain 4 copies of the SMN2 gene and no mouse Smn gene (Smn - / - ; SMN2 tg / tg ). Type III mice do not develop the SMA phenotype, but develop necrosis of the ears and shortening of the tail within a month or so.

[0121] The inventors used SpCas9, SaCas9mRNA and their corresponding sgRNA (sgRNA1, sgRNA2) to inject type III mice (Smn - / - ;SMN2 tg / tg ) and BH heterozygous mice (Smn - / + ) Fertilized eggs obtained from cages ( Figure 4 , A). As a result, there is a 50% chance of obtaining type I SMA mice (Smn - / - ;SMN2 tg / - ) and 50% likely to get heterozygous mice (Smn + / - ;SMN2 tg / - )( Figure 4 , A).

[0122] The average survival days of SMA mice was 12 days (n=35). The present inventors applied CRISPR / Cas9 gene editing technology to interfere with the splicing of SMN2 at the level of fertilized ...

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Abstract

The present invention provides a method for increasing expression of survival motor neuron (SMN) protein based on a gene editing technology, and an application of the method in spinal muscular atrophy(SMA) treatment. The method for increasing the expression of the SMN protein is achieved by using a CRISPR / Cas9 technology to destroy splicing silencer ISS-N1 or ISS+100 on intron 7 of SMN2. The provided method for increasing the expression of the SMN protein is modified from a DNA level, and effective, safe, efficient, economical and practical. At the same time, the present invention also provides a series of applications of new methods for increasing the SMN protein in the SMA treatment, including specific sgRNA sequences for gene editing, gene editing reagents, gene editing plasmids, geneediting cells and preparation methods thereof, gene editing animal preparation methods and gene editing medicines.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and more specifically, the invention relates to a method for increasing SMN protein expression based on gene editing technology and its application in SMA treatment. Background technique [0002] Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease that mainly affects the anterior horn of the spinal cord. Clinically, it mainly manifests as progressive atrophy and weakness of the limbs, and the mortality and disability rates are extremely high. It ranks first among fatal genetic diseases in infants and young children, with one out of every 6,000 newborns suffering from the disease. [0003] The causative gene of SMA is motor neuron survival gene (Survival motor neuron, SMN). Humans have two highly homologous SMN genes, namely SMN1 on the telomeric side and SMN2 on the centromere side. SMN1 encodes a full-length SMN protein is a functional SMN protein, while SMN2 mainly encode...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/90C12N5/10C12N15/113A61K47/54A61K38/46A61K48/00A61P21/00
CPCC12N15/85C12N15/907C12N9/22C12N5/0696C12N15/113A61K47/549A61K38/465A61K48/005A61P21/00C12N2310/10C12N2510/00Y02A50/30
Inventor 陈万金杨辉李锦晶林翔王柠
Owner THE FIRST AFFILIATED HOSPITAL OF FUJIAN MEDICAL UNIV
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