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Preparation method and system of recombinant adeno-associated virus (rAAV), and recombinant bacmid

A virus and bacmid technology, applied in the field of recombinant adeno-associated virus preparation, can solve the problems of difficulty in promotion, poor system flexibility and versatility, poor virus stability, etc., to improve the stability of passage, have flexibility and compatibility, good compatibility

Active Publication Date: 2019-04-12
WUHAN INST OF PHYSICS & MATHEMATICS CHINESE ACADEMY OF SCI
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Problems solved by technology

[0005] In view of the above defects or improvement needs of the prior art, the present invention provides a preparation method, system and recombinant bacmid of a recombinant adeno-associated virus (rAAV). The core expression elements are included in the shuttle plasmid, and the expression frames of the functional protein components required to produce rAAV are separately included on the shuttle plasmid and the baculovirus genome, thereby reducing the difficulty of constructing the shuttle plasmid and improving the overall efficiency of the gene therapy vector preparation system. Stability, compatibility and flexibility, thereby solving the difficulty of constructing shuttle plasmids in the existing recombinant adeno-associated virus baculovirus preparation system, poor system flexibility and versatility, poor virus stability, and difficult to promote technology question

Method used

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  • Preparation method and system of recombinant adeno-associated virus (rAAV), and recombinant bacmid
  • Preparation method and system of recombinant adeno-associated virus (rAAV), and recombinant bacmid
  • Preparation method and system of recombinant adeno-associated virus (rAAV), and recombinant bacmid

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preparation example Construction

[0043] The preparation method of recombinant adeno-associated virus (rAAV) provided by the invention comprises the following steps:

[0044] (1) Prepare the shuttle plasmid and its corresponding recombinant bacmid containing the baculovirus genome respectively;

[0045] The shuttle plasmid at least comprises rAAV core expression elements with heterologous functional gene fragments;

[0046] The shuttle plasmid is preferably a pFBD plasmid, which includes at least rAAV core expression elements with heterologous functional gene fragments; it may also include an expression cassette for some functional protein components required for rAAV production. The expression cassette of the functional protein component may be the expression cassette of the AAV functional protein component or the expression cassette of other functional protein components.

[0047] The recombinant bacmid containing the baculovirus genome contains expression cassettes for other functional protein components n...

Embodiment 1

[0087] Example 1. Preparation of rAAV using DH10Bac-Tn7-(ITR-GOI)-Cap-Δ(Chia-Cath)-Rep

[0088] When the shuttle plasmid pFBD contains the rAAV core expression element ITR-GOI and the AAV Cap gene expression cassette, the corresponding recombinant bacmid integrates the AAV Rep gene expression cassette and other functional protein component expression cassettes ( figure 2 A).

[0089] A preparation system for rAAV, comprising: a shuttle plasmid and its corresponding recombinant bacmid containing the baculovirus genome; the shuttle plasmid is based on the pfast.Bac.Dual plasmid, and its multiple cloning site is inserted with ITR-GOI sequence and Cap Gene, the Cap gene is placed downstream of the P10 promoter; the recombinant bacmid of the baculovirus genome is AcMNPV E2 with deletion of non-essential genes Chia gene and Cath gene, its gene sequence is as follows: Genbank accession No.KM667940.1, The Chia and Cath genes were deleted (105,353bp-108,025bp), and the Rep gene expre...

Embodiment 2

[0110] Example 2. Preparation of rAAV using DH10Bac-Tn7-(ITR-GOI)-Rep-Δ(Chia-Cath)-Cap

[0111] When the shuttle plasmid pFBD contains the core expression element ITR-GOI of rAAV and the Rep gene expression cassette of AAV, the corresponding recombinant bacmid needs to integrate the Cap gene expression cassette of AAV and the expression cassettes of other functional protein components ( image 3 A).

[0112] A preparation system for rAAV, comprising: a shuttle plasmid and its corresponding recombinant bacmid containing the baculovirus genome; the shuttle plasmid is based on the pfast.Bac.Dual plasmid, and its multiple cloning site is inserted with ITR-GOI sequence and Rep Gene, the Rep gene is placed downstream of the PH promoter; the recombinant bacmid of the baculovirus genome is AcMNPV E2 with deletion of non-essential genes Chia and Cath genes, its gene sequence is as follows: Genbank accession No.KM667940.1, its (Location range 105,353bp-108,025bp) Chia and Cath genes we...

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Abstract

The invention discloses a preparation method and system of recombinant adeno-associated virus (rAAV), and recombinant bacmid. The method comprises: (1) separately preparing shuttle plasmids and a corresponding recombinant bacmid containing baculovirus genome; (2) integrating a rAAV core expression element which has a heterologous functional gene fragment with other expression cassettes which produce functional protein components necessary for rAAV so as to obtain a recombinant bacmid containing recombinant baculovirus genome producing the rAAV; and (3) transfecting the obtained recombinant bacmid into a host cell line for culturing. The system comprises the shuttle plasmids and the corresponding recombinant bacmid containing the baculovirus genome. The recombinant bacmid comprises at leastone expression cassette that produces functional protein components necessary for rAAV. The system has flexibility, compatibility, and higher passage stability.

Description

technical field [0001] The invention belongs to the field of gene therapy, and more specifically relates to a preparation method, system and recombinant bacmid of a recombinant adeno-associated virus (rAAV). Background technique [0002] Recombinant adeno-associated virus (rAAV) has the characteristics of high safety, low immunogenicity, wide host range, and the ability to mediate long-term stable expression of foreign genes in animals. It is one of the most promising vectors in the field of gene therapy. [0003] At present, there are three main methods for large-scale production of rAAV using the baculovirus expression system: two baculovirus systems (Two Bac system), one baculovirus system (One Bac system) that relies on packaging cell lines, and one that does not rely on packaging cell lines. One Bac system based on shuttle plasmid. The main process of the two baculovirus system is to integrate the Rep gene and Cap gene of AAV into a baculovirus genome through Tn7 recom...

Claims

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Application Information

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IPC IPC(8): C12N15/866C12N7/01
CPCC12N7/00C12N15/86C12N2710/14043C07K14/005C12N2710/14122C12N2710/14144C12N2710/14151C12N2750/14151C12N2750/14143
Inventor 吴阳徐富强梅婷蒋良玉韩增鹏
Owner WUHAN INST OF PHYSICS & MATHEMATICS CHINESE ACADEMY OF SCI
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