Capsid protein of adeno-associated virus (AAV), vector of AAV as well as construction method and application of vector of AAV

A capsid protein and viral vector technology, applied in the field of genetic engineering, can solve the problems of high packaging cost and stimulate human immune response, and achieve the effect of promoting clinical progress, reducing production cost and improving targeting.

Active Publication Date: 2020-04-03
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the transduction efficiency of rAAV is affected by the length of the target gene sequence, and the serotypes selected for different types of transduced cells and tissue organs are also different. In addition, the cost of AAV production and packaging is relatively high, and high-dose injection will stimulate human immunity. reaction

Method used

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  • Capsid protein of adeno-associated virus (AAV), vector of AAV as well as construction method and application of vector of AAV
  • Capsid protein of adeno-associated virus (AAV), vector of AAV as well as construction method and application of vector of AAV
  • Capsid protein of adeno-associated virus (AAV), vector of AAV as well as construction method and application of vector of AAV

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1, construction of adeno-associated virus vector

[0031] 1. Site-directed mutagenesis on the capsid protein of adeno-associated virus

[0032] 1.1. Determine that the plasmid used for site-directed mutation is the capsid protein necessary for packaging adeno-associated virus. In this embodiment, the capsid protein of adeno-associated virus DJ serotype is selected;

[0033] 1.2. Perform site-directed mutation on the adeno-associated virus VP1, and mutate the 269th amino acid from the original serine to threonine (S269T), design mutation primers for this site, and select a read length of about 20 bp before and after the mutation site , and replace the codon triplet sequence of the amino acid codon triplet at the original position with the codon triplet sequence of the target amino acid, and verify it on the primer design software, and slightly delete it according to the requirements of GC ratio, Tm value, primer dimer or hairpin structure, etc. , in this embodim...

Embodiment 2

[0043] Embodiment 2, the preparation of adeno-associated virus mutant

[0044] 1. Small-scale packaging virus

[0045] 1.1. Mix and package the correctly sequenced AAV capsid mutation plasmid, the auxiliary plasmid necessary for packaging AAV, and the target plasmid (pAAV-CMV-EGFP is used in this example) in a ratio of 1:1:2 Adeno-associated virus, the cells used for packaging in this example are HEK293. After 72 hours, use a fluorescent microscope to observe the transfection of the three plasmids and harvest the crude extract of the virus, and compare the expression of the viral GFP fluorescent protein packaged with the wild-type adeno-associated virus capsid protein. Evaluate its packaging efficiency;

[0046] 1.2. Use a 0.22 μm Sangon needle filter to filter and purify the packaged virus vector, and initially remove impurities;

[0047] 1.3. Quantitatively infect HEK293, Huh7, HepG2, NIH3T3 cells with the purified adeno-associated virus in small packets for screening, and...

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Abstract

The invention discloses a capsid protein of an adeno-associated virus (AAV) which has an amino acid sequence shown as SEQ ID NO: 1. The invention also discloses an isolated nucleic acid molecule whichencodes the capsid protein of the AAV. The invention further discloses a vector of the AAV which encodes the capsid protein of the AAV. The invention still further discloses a construction method andapplication of the vector of the AAV. According to the capsid protein of the AAV, modification is performed on the capsid protein of the AAV-DJ , so that the AAV-DJ has significantly improved transduction efficiency in cell lines, including HEK293, various liver cancer cells, mouse fibroblasts and the like, and mouse livers compared with wild-type AAV, so that immune response in subjects caused by high-dose injection of virus vectors is avoided; and moreover, production process of the AAV is optimized so as to reduce production cost. Thus, clinical progress of AAV gene therapy products is promoted

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an adeno-associated virus capsid protein, a vector, a construction method and application thereof. Background technique [0002] At present, gene therapy has become a research hotspot in the field of biomedicine. The most commonly used vectors in gene therapy include adenovirus, lentivirus and adeno-associated virus (adeno-associated virus, AAV, the same below). Wild-type adeno-associated virus (wtAAV, the same below) belongs to the Parvoviridae family, and is a single-stranded DNA virus with a diameter of 20nm. The viral genome of ITR, the same below), between ITR is an open reading frame ORF, comprising capsid protein Cap, replication protein Rep, packaging activator protein AAP. Wild-type adeno-associated virus is known to integrate into the human chromosome at the AAVS1 locus. The recombinant adeno-associated virus (rAAV, the same below) genome sequence only retains the I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/015C12N15/35C12N15/864C12N7/01A61K48/00A61P35/00
CPCC07K14/005C12N15/86C12N7/00A61K48/0025A61P35/00C12N2750/14121C12N2750/14122C12N2750/14143C12N2750/14132
Inventor 凌晨郑青云
Owner FUDAN UNIV
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