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Construction method and delivery system of plaque corneal dystrophy animal model and gene therapy vector

A technology of malnutrition and gene therapy, applied in gene therapy, plant gene improvement, botanical equipment and methods, etc., can solve problems such as new treatment methods have not been effectively developed, plaque corneal dystrophy, etc.

Pending Publication Date: 2022-07-22
EYE INST OF SHANDONG FIRST MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In summary, new treatments for plaque corneal dystrophy other than surgery have not been effectively developed

Method used

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  • Construction method and delivery system of plaque corneal dystrophy animal model and gene therapy vector
  • Construction method and delivery system of plaque corneal dystrophy animal model and gene therapy vector
  • Construction method and delivery system of plaque corneal dystrophy animal model and gene therapy vector

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preparation example Construction

[0030] The invention provides a preparation method for an animal model of plaque corneal dystrophy, comprising the following steps: knocking out the Chst5 gene of the animal corneal endothelium by means of anterior chamber injection or inserting the nucleoside at position 149 of the second exon of the Chst5 gene Acid is mutated from G to A.

[0031] The present invention does not specifically limit the type of the animal model, and it is preferably a mouse model. When constructing the mouse model in the present invention, the Chst5 gene involved is preferably a mouse source, and its gene accession number is NM_019950.

[0032] The present invention can realize the construction of a mouse model by a gene knockout method, and the knockout method preferably includes designing a shRNA sequence for the Chst5, and inserting the shRNA sequence into the BsmBI restriction enzyme cleavage site of the adeno-associated virus vector where an adeno-associated virus knockout plasmid was con...

Embodiment 1

[0040] Construction of a mouse model of plaque corneal dystrophy 1

[0041] The shRNA oligonucleotide sequence 5'-accggacgctgccctttgccaagattttcaagagaaatcttggcaaagggcagcgtttttt-3' of mouse Chst5 (NM_019950) was inserted into the BsmBI restriction enzyme cleavage site of the adeno-associated virus GV478 vector to construct an adeno-associated virus knockout plasmid (AAV-siChst5). The same plasmid was constructed using the sequence of SEQ ID NO. 8: 5'-cgctgagtacttcgaaatgtc-3' as a negative control.

[0042] 1 μL concentration of 10 12 VG / mL of the above adeno-associated virus knockout plasmid was injected into the anterior chamber of mice through the cornea using a 36G stromal injection needle ( Figure 4 ).

[0043] For the gene knockout effect, use the primers shown in SEQ ID NO.5 and SEQ ID NO.6 to perform PCR gene expression verification, and the results are as follows Image 6 As shown, endothelial knockout of Chst5 can be achieved by anterior chamber injection, and anter...

Embodiment 2

[0046] Construction of Plaque Corneal Dystrophy Mouse Model 2

[0047] The nucleotide G at position 149 of the second exon of the mouse Chst5 (NM_019950) gene was changed to A by CRISPR-Cas9. That is: TGGCGCTCG->TGGCACTCG.

[0048] The gRNA sequences of Chst5-g1: and Chst5-g2 were designed near the mutation position and screened by target activity detection. Donor DNA Chst5-g1-g2-Oligo was designed and constructed according to the highly active gRNA target.

[0049] The DNA of the target gene is cut by CRISPR technology, and the homologous template Donor with point mutation is provided at the same time, and the base replacement is realized in a specific exon through the homologous recombination repair of DNA to achieve the purpose of point mutation.

[0050] Cas9 nickase mRNA, Cas9 target gRNA and Donor DNA were co-microinjected into mouse zygotes. The mice were genotyped two weeks after they were born, and PCR was used to detect whether the first mice with gene point mutat...

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Abstract

The invention provides a plaque corneal dystrophy animal model, a construction method of a gene therapy vector and a delivery system, and relates to the technical field of model construction. According to the animal model, animal Chst5 genes are knocked out from corneal endothelium in an anterior chamber injection mode or through Chst5R50H point mutation, and compared with an existing animal model, the eye symptom similarity is higher. The invention also provides a gene therapy vector which is realized by injecting the adeno-associated virus vector into the anterior chamber to carry the wild type mouse Chst5 gene. In the embodiment of the invention, the treatment of plaque corneal dystrophy can be realized by injecting the gene therapy vector into the anterior chamber of the mouse model.

Description

technical field [0001] The invention belongs to the technical field of model construction, and in particular relates to a plaque corneal dystrophy animal model and a construction method and delivery system of a gene therapy vector. Background technique [0002] Corneal dystrophy is a type of hereditary corneal disease, which is more common in young people. The degree of corneal opacity gradually increases with age, and eventually vision loss occurs. There is currently no effective drug treatment for corneal dystrophy. For corneal dystrophy involving the superficial layer, keratectomy can be performed on the lesion site, and corneal transplantation can only be performed for patients with lesions involving the deep layer of the cornea. Plaque corneal dystrophy is an onset of both eyes. The onset is more than 10 years old. It mainly affects the corneal stromal layer. There is still a possibility of recurrence after corneal transplantation, which will cause serious trouble to th...

Claims

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Application Information

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IPC IPC(8): C12N15/864C12N15/89C12N15/113C12N15/54C12N15/10A01K67/027A61K48/00A61K38/45A61P27/02
CPCC12N15/86C12N15/89C12N15/1137C12N9/13C12N15/102A01K67/0276A01K67/0275A61K48/0008A61K48/005A61K38/45A61P27/02C12Y208/02017C12N2750/14143C12N2800/107C12N2310/14C12N2310/20A01K2217/075A01K2207/05A01K2227/105A01K2267/0306C12N2310/531C12Q2521/327C12Q2525/151C12Q2525/161
Inventor 谢立信张碧凝周庆军戚本祥
Owner EYE INST OF SHANDONG FIRST MEDICAL UNIV
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