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Nucleotide sequence for repairing DMD gene mutation and system

A nucleic acid sequence and gene technology, which is applied in the field of nucleic acid sequence and system for repairing DMD gene mutations, can solve problems such as ineffective packaging, achieve high correction efficiency, improve effective editing efficiency, and low off-target efficiency

Pending Publication Date: 2020-09-29
广东赤萌医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The full length of the SpCas9 nucleic acid sequence (including nuclear localization signal) is 4200bp, and the full length exceeds 4500bp after adding the promoter and polyA, which cannot effectively package AAV

Method used

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  • Nucleotide sequence for repairing DMD gene mutation and system
  • Nucleotide sequence for repairing DMD gene mutation and system
  • Nucleotide sequence for repairing DMD gene mutation and system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 Screening of gRNA target sequences in introns 49, 50, and 51 of Dys gene

[0066] 1.1 gRNA preparation

[0067] (1) Referring to the Sa.CRISPR / Cas9 target site design rules, design 21nt gRNA target sequences for the sequences of introns 49, 50, and 51 of the Dys gene respectively. The target sequences of the gRNA are shown in Table 1, Table 2, Shown in Table 3;

[0068] (2) Synthesize the sense strand and the antisense strand of the DNA sequence of the gRNA target sequence according to Table 1 (add cacc to the 5'-end of the sense strand, if the first nucleotide at the 5'-end of the sense strand is not guanine G, then Add caccG at the 5'-end of the sense strand; add aaac at the 5'-end of the antisense strand, if the first nucleotide at the 5'-end of the sense strand is not guanine G, then add aaac at the 3'- end plus C);

[0069] (3) The above-mentioned gRNA sense strand and antisense strand were mixed, treated at 90°C, cooled naturally to room temperature f...

Embodiment 2

[0111] Example 2 Sequencing Analysis Dystrophin Gene Mutation Efficiency and Mutation Type

[0112] Select the gRNA with higher mutation efficiency in Example 1, and further analyze the mutation efficiency and mutation type. With SEQ ID NO:10, SEQ ID NO:23 and SEQ ID NO:43; SEQ ID NO:105 and SEQ ID NO:107; SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:167 and SEQ ID Take NO:179 as an example to perform the following experimental operations.

[0113] 2.1 T carrier connection

[0114] (1) With the purified PCR product obtained in 1.5 (4) of Example 1, measure the concentration for subsequent use;

[0115] (2) Use Kangwei Century Master PCR Mix to add an adenine (A) to the 3'-end of the PCR product: take 2 μg of the PCR product in 3.1 and mix it with the Master PCR Mix at a ratio of 1:1 (V:V), and store at 72°C React for 30 minutes;

[0116] (3) Use the reaction product in 1% agarose gel electrophoresis 4.2, cut the gel to recover the target fragment and measure the concentration o...

Embodiment 3

[0137] Example 3 Screening of gRNA target sequence combinations in introns 49, 50, and 51 of Dys gene

[0138] 3.1 Design of gRNA target sequence combination ( Figure 43 )

[0139] For DMD mutants with deletion of exon 50, a plan to knock out exon 51 can be adopted, and the gRNA target sequences of intron 50 and intron 51 can be selected for combination;

[0140] For DMD mutants with deletion of exon 51, a plan to knock out exon 50 can be adopted, and the gRNA target sequences of intron 49 and intron 50 can be selected for combination;

[0141] For DMD mutants with mutations or deletions in exons 50-51, a plan to simultaneously knock out exons 50 and 51 can be adopted, and the gRNA target sequences of intron 49 and intron 51 can be selected for combination;

[0142] Table 5 Dys gene exon 50-51 upstream and downstream Sa-gRNA combinations

[0143] gRNA for intron 49 gRNA for intron 51 Group NO:1 SEQ ID NO: 23 SEQ ID NO: 153

[0144] Table 6 Sa-gR...

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Abstract

The invention discloses a nucleotide sequence for repairing DMD gene mutation and a system. A gene editing system is capable of knocking out a Dystrophin genomic region between the 31792310th site andthe 31854834th site, or between the 31747866th site and the 31838091th site, or between the 31747866th site and the 31854834th site of an X chromosome, and preferably, the gene editing system is capable of knocking out a Dystrophin genomic region between the 31815201th site and the 31846518th site, between the 31769972th site to the 31815200th site, or the 31769972th site to the 31846518th site of the X chromosome. The geneediting system can target to more than 17% of DMD patients, including EX51 (deficiency) 4%, EX50 (deficiency) 13%, EX45-50 (deficiency), EX48-50 (deficiency), EX50 (deficiency), EX51 (deficiency), EX52 (deficiency) and EX50 or EX51 exon area point mutation and other forms of mutations, and the adaptability is higher.

Description

technical field [0001] The invention relates to the field of gene therapy, in particular to nucleic acid sequences and systems for repairing DMD gene mutations. Background technique [0002] Duchenne muscular dystrophy syndrome (Duchenne muscular dystrophy, DMD), also known as pseudohypertrophic muscular dystrophy, is a single gene genetic disease of X chromosome recessive inheritance, caused by mutations in the dystrophin gene (Dystrophin, Dys) Sincerely. The incidence rate of DMD is 1 / 3500, and it has the characteristics of young age of onset, insidious onset, long course of disease and high case fatality rate. The patient developed muscle weakness or atrophy due to the continuous degeneration of skeletal muscles, and completely lost the ability to walk when he was 7 to 12 years old, and died of respiratory failure or heart failure when he was around 20 years old. At present, there is no effective cure for the disease, and clinically, symptomatic treatment and rehabilita...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N15/864
CPCC12N15/864C12N15/902C12N2750/14143
Inventor 孔祥东杨宇霞陶米林祝海宝刘洁喻小鲁陈梦麟达琦罗思施陈雪峰张楠
Owner 广东赤萌医疗科技有限公司
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