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Recombinant plasmid for inhibiting SOST gene expression, bone-targeted recombinant adeno-associated virus thereof and application of bone-targeted recombinant adeno-associated virus

A recombinant plasmid and gene expression technology, applied in the fields of application, virus, gene therapy, etc., can solve the complex problems of shRNA therapy, achieve good bone targeting properties, relieve osteoporosis, and reduce the concentration

Active Publication Date: 2022-01-11
郭保生 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Sclerostin is a glycoprotein encoded, expressed and translated by the sost gene, which can bind to the Lrp5 / 6 receptor on the cell membrane surface, inhibit the intracellular Wnt / β-catenin pathway, and have the effect of inhibiting bone formation, so the shRNA of the Sost gene can be used as a gene However, since Sost is mostly secreted by bone cells, and the shRNA treatment of bone cells is more complicated, there is no effective way to interfere with the Sost technology in bone cells at the animal level.
At the same time, because shRNA technology has been found to have certain hepatotoxicity in mice, it has not yet been used for clinical verification

Method used

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  • Recombinant plasmid for inhibiting SOST gene expression, bone-targeted recombinant adeno-associated virus thereof and application of bone-targeted recombinant adeno-associated virus
  • Recombinant plasmid for inhibiting SOST gene expression, bone-targeted recombinant adeno-associated virus thereof and application of bone-targeted recombinant adeno-associated virus
  • Recombinant plasmid for inhibiting SOST gene expression, bone-targeted recombinant adeno-associated virus thereof and application of bone-targeted recombinant adeno-associated virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] 1.1 Preparation of bone targeting virus

[0050] Adeno-associated virus type 2 / 9 (rAAV2 / 9) was used to transform the bone-targeting virus, briefly describe it, and construct the basic sequence encoding bone-targeting peptide DSS (AspSerSer) 6 The codon optimization of the DNA sequence of the AAV2 Rep gene and the AAV9 Cap gene was inserted into the codon of the AAV9 Cap gene to obtain the Q588 capsid (DSS-588).

[0051] Then construct the DSS-Nter capsid, first mutate the start codon of VP2 in pAAV2 / 9 (ACG→ACC), express only VP1 and VP3 (pAAV2 / 9.novp2), and in another plasmid, fuse the DSS sequence To the N-terminus of the AAV9-VP2 ORF, the Kozak sequence and the ATG initiation codon were placed directly upstream of the DSS sequence to allow CMV promoter-driven expression (pcDNA.DSS-VP2(AAV9)), and the constructed pAAV2 / 9. novp2 and pcDNA.DSS-VP2 (AAV9) were used for the production of rAAV. The specific transformation work was completed by Shanghai Union Biotechnology...

Embodiment 2

[0105] In order to verify the effectiveness of the bone-targeted virus in the process of infecting bone cells, the bone-targeted enveloped EGFP virus (AAV9-DSS-Nter-EGFP) ( figure 1 ), and control virus, and rAAV2 / 9-EGFP virus.

[0106] Injected into 10-week-old mice through the tail vein at a concentration of 4*10 11 . Two months later, the mice were subjected to in vivo fluorescence detection, histological GFP immunofluorescence staining, and quantitative detection of GFP protein in different tissues to clarify whether the bone-targeting virus can reach bone cells and its efficiency.

[0107] In vivo fluorescence detection found that the fluorescence intensity in bone tissue was significantly higher than that of ordinary type ( figure 2 ), the immunofluorescence detection of bone tissue sections found that after the bone-targeting virus was injected, the expression level of EGFP in bone cells was significantly higher than that in the control group ( image 3 ), extractin...

Embodiment 3

[0109] In order to verify and screen the available sost-shRNA plasmids, shSost was transferred into 293T cells, and the expression level of Sost mRNA in the cells was detected after 24 hours. Effective fragments were screened for bone-targeted virus packaging.

[0110] Through the Lipo2000 transfection system, we transfected the three Sost-shRNA constructs (see 1.3 in Example 1, and Y10680: GCCTCATCTGCCTACTTGT; Y10681: GCCTTCAGGAATGATGCCA; Y10682: CCATCCCTATGACGCCAAA) interference plasmids into 293T cells, and extracted after 24 hours The detection of cell mRNA found that the Sost-shRNA2 interference sequence (maker: Y20681) has the function of significantly inhibiting the expression of Sost in 293T cells ( Figure 5 ). Therefore, it was selected as the virus envelope plasmid for follow-up research.

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Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses a recombinant plasmid for inhibiting SOST gene expression and a bone-targeted recombinant adeno-associated virus and application of the bone-targeted recombinant adeno-associated virus. The bone-targeted recombinant adeno-associated virus, which can target bone cells and reduce the expression and the secretion of bone cell Sclerostin (Sost), and has anti-osteoporosis effect, is constructed, and has the administration route including intramuscular injection and intravenous infusion, is convenient to use, and provides the powerful evidence for the application in the treatment or prevention of osteoporosis.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a recombinant plasmid for inhibiting the expression of SOST gene and its bone-targeting recombinant adeno-associated virus and its application. Background technique [0002] Osteoporosis is a systemic bone metabolic disease that can lead to decreased bone mass, decreased bone density, and changes in bone microstructure. Osteoporosis can be divided into primary and secondary according to the etiology. Secondary is generally a bone disease caused by other diseases or drugs, while primary is a physiological decline that inevitably occurs with age, including three types of bone disease: menopausal, senile and idiopathic. quality loose. Postmenopausal osteoporosis mostly occurs in middle-aged and elderly women aged 55-65, while senile osteoporosis mostly occurs in the elderly over 70 years old, and idiopathic osteoporosis is more common in adolescents and adults, often accompan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/864C12N15/12A01K67/027C12N7/00A61K48/00A61K47/64A61K38/17
CPCC12N15/86C07K14/4703A01K67/0276C12N7/00A61K48/005A61K48/0008A61K48/0075A61K38/1709A61K47/64A61P19/10A01K2227/105C12N2750/14121C12N2750/14123C12N2750/14152Y02A50/30
Inventor 郭保生石天舒蒋青陈响
Owner 郭保生
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