Adeno-associated virus mutant applicable to specific infection of U87-MG cells

A mutant and heterologous technology, applied in the field of screening of mutants of adeno-associated virus, can solve the problems of low efficiency and poor specificity of glioma cell lines, and achieve significant tumor inhibition effect

Active Publication Date: 2022-03-22
OBIO TECH SHANGHAI CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing AAV serotype (AAV2 / 2) infects glioma cell line (U87-MG) with low efficiency and poor specificity. Infection works better

Method used

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  • Adeno-associated virus mutant applicable to specific infection of U87-MG cells
  • Adeno-associated virus mutant applicable to specific infection of U87-MG cells
  • Adeno-associated virus mutant applicable to specific infection of U87-MG cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 Peptide mutant library preparation

[0035] 1.1 Chemically synthesize the following two fragments of AAV2 / 2-7mer-NNS:

[0036] 5'GGTCTCGCCTCCAGAGAGGCAACNNSNNNNNNSNNSNNNSNNSAGACAAGCAGCTACCGGGAGACC3' (SEQ ID NO: 1)

[0037] 5'GGTCTCCCGGTAGCTGCTTGTCTSNNNNSNNNSNNNSNNNSNNNNGTTGCCTCTCTGGAGGCGAGACC3' (SEQ ID NO: 2)

[0038] 1.2 Add 10 μL each of the synthesized AAV2 / 2-7mer-NNS forward and reverse primers (the final concentration of the primers is 10 mM) to obtain the AAV2 / 2-7mer-NNS template by annealing. The annealing program is: 95°C, 5min; 95°C, 1min; 92min, 1min; 4°C, 60min. Among them, in the second and third steps, each cycle lowers 3° C., a total of 25 cycles.

[0039] 1.3 Plasmid pAAV-short UBC-mScarlet-polyA-P40-AAV2-Cap-FLEX-SV40 polyA (its structure and insertion site are as follows figure 1 shown) with BsaI for single enzyme digestion, the enzyme digestion system (50uL) is shown in Table 1.

[0040] Table 1

[0041]

[0042] After digestion at...

Embodiment 2

[0048] Example 2 library virus packaging and screening

[0049]2.1 AAV peptide mutant library virus packaging: 1.5*10^7 293AAV packaging cells per dish were inoculated into a 15cm cell culture dish, cultured for 18-24h, and transfection began after the cells adhered to the wall. Use PEI transfection reagent to transfer pAAV-short UBC-mScarlet-polyA-P40-AAV2-Cap-FLEX-SV40polyA–insertion expression vector library containing AAV2 / 2-7mer-NNS insert fragment, package Rep plasmid, and pHelper helper plasmid into In 293AAV cells, 72 hours after transfection, count the ratio of vector library cells in AAV-293 cells under a fluorescent microscope to determine the virus packaging efficiency. After the virus packaging is completed, blow the cells repeatedly with the tip of the pipette to completely detach all the cells from the culture dish, and collect all cell samples.

[0050] 2.2 Purification of the virus: The collected cell samples were repeatedly frozen and thawed at -80°C and 37°...

Embodiment 3

[0071] Screening and verification of embodiment 3AAV2 / 2 mutants

[0072] 3.1 Construction of AAV2 / 2 mutants

[0073] Using the natural serotype AAV2 / 2 as the vector, insert the candidate mutant AAV2 / 2-GLxx and other fragments at the 587-588 amino acid position to obtain a new serotype vector AAV2 / 2-GLxx, etc. According to the high-throughput results, construct More than 30 mutants were identified.

[0074] 3.2 Use the shuttle vector pAAV-CAG-mCherry-WPRE to package AAV2 / 2-GLxx as serotype vectors to obtain AAV viruses of various mutant serotypes.

[0075] Figure 7 Vector map for the shuttle vector pAAV-CAG-mCherry-WPRE

[0076] 3.3 Use WPRE primers to measure the virus titer of the above viruses, and use the test to confirm the expression of VP1, VP2, and VP3 of the viruses.

[0077] 3.4 AAV2 / 2-GLxx and other viruses of pAAV-CAG-mCherry-WPRE were mixed with MOI (multiplicity of infection) 1*10 5 293T, NIH3T3 and U87-MG cells were infected respectively, and the fluorescen...

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Abstract

The invention provides an adeno-associated virus mutant suitable for specific infection of U87-MG cells. According to the invention, the AAV serotype mutant capable of efficiently and specifically infecting U87-MG is obtained through library screening by utilizing a peptide fragment mutation library of AAV 2/2. An in-vitro killing experiment proves that the AAV serotype mutant obtained by screening can specifically infect and kill U87-MG cells, and has wide application in the aspects of in-vitro research on the occurrence and development mechanism of glioma and development of glioma treatment drugs.

Description

technical field [0001] The invention relates to virus packaging and screening, in particular to the screening of adeno-associated virus mutants. Background technique [0002] Glioma (Glioma) is a common primary malignant brain tumor of the central nervous system. It has the characteristics of high recurrence, poor prognosis, and high mortality. It is one of the deadliest tumors at present. At present, there is no particularly effective treatment strategy for glioma, and the existing core treatment methods are still conventional maximal excision of the tumor, as well as radiotherapy and chemotherapy. However, due to the characteristics of malignant invasive growth of glioma, conventional surgery, chemotherapy and radiotherapy can only slightly improve the quality of life and slightly prolong the survival period of some patients, and have no effect on the five-year survival rate of glioma after diagnosis. Significant improvement, and easy to relapse. [0003] Adeno-associate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K19/00C12N15/62C12N15/864C12N5/10A61K38/16A61P35/00
CPCC07K7/06C07K14/005C12N15/86A61P35/00C12N2750/14122C12N2750/14143C07K2319/00A61K38/00
Inventor 杨佳丽杨兴林潘讴东马佩敏
Owner OBIO TECH SHANGHAI CORP LTD
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