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Preparation method and system of recombinant adeno-associated virus and recombinant bacmid

A technology of recombinant baculovirus and virus, which is applied in the field of preparation of recombinant adeno-associated virus, can solve the problems of poor passaging stability of BEV and easy loss of foreign genes, etc., achieve good system compatibility, improve passaging stability, and improve overall stability sexual effect

Active Publication Date: 2021-03-26
JINFAN BIOMEDICAL TECH (WUHAN) CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the deficiencies or improvement needs of the prior art, the present invention provides a preparation method, system and recombinant bacmid of recombinant adeno-associated virus, by producing the necessary functional elements (Cap gene expression cassette, Rep gene expression frame and at least one of the core expression element ITR-GOI) with an exogenous target gene is inserted into the N-terminal or C-terminal of the essential gene of the recombinant baculovirus genome, aiming to solve the problem of inserting the essential functional element into the existing technology The non-essential locus of the recombinant baculovirus genome, the foreign gene is easily lost during the passage of the baculovirus, resulting in poor passage stability of the generated BEV

Method used

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  • Preparation method and system of recombinant adeno-associated virus and recombinant bacmid
  • Preparation method and system of recombinant adeno-associated virus and recombinant bacmid
  • Preparation method and system of recombinant adeno-associated virus and recombinant bacmid

Examples

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preparation example Construction

[0060] A method for preparing recombinant adeno-associated virus provided by the invention, such as figure 1 shown, including the following steps:

[0061] (1) Constructing a recombinant bacmid comprising a recombinant baculovirus genome for producing a recombinant adeno-associated virus; the recombinant baculovirus genome contains essential functional elements for producing the recombinant adeno-associated virus, and the essential functional elements include the Cap gene , Rep gene and core expression element ITR-GOI with exogenous target gene;

[0062] (2) Transfecting the recombinant bacmid comprising the recombinant baculovirus genome for producing recombinant adeno-associated virus obtained in step (1) into a host cell line for culture;

[0063] Wherein, at least one of the essential functional elements is inserted into the N-terminal or C-terminal of the essential gene locus of the recombinant baculovirus genome. The N-terminal or C-terminal of the essential gene locus...

Embodiment 1

[0103] rAAV was prepared using DH10Bac-Cap-Rep(Ac135)-Tn7(ITR-GOI).

[0104] This embodiment includes a homologous recombination vector and a shuttle vector: the homologous recombination vector targets the essential gene Ac135 gene, contains the expression cassettes of functional protein components necessary for the production of rAAV, the Chol expression cassette, and the ETL-EGFP expression cassette, wherein Chol The expression cassette is used as a screening marker for Red homologous recombination positive clones, and the ETL-EGFP expression cassette is used for TCID of BEV 50 Titer determination; the shuttle vector contains the core expression element ITR-GOI expression cassette of rAAV.

[0105] The method for preparing rAAV using the rAAV preparation system provided in this embodiment includes the following steps:

[0106] 1.1 Construction of a homologous recombination vector comprising the essential functional elements Cap and Rep gene expression cassettes described in...

Embodiment 2

[0128] In view of Example 1, we targeted the rAAV capsid protein gene expression cassette (Cap) and the Rep gene expression cassette (Rep) to the essential gene Ac135 gene, and obtained BEV stability and rAAV packaging rate better than placing Rep / Cap in Recombinant baculovirus bacmid at the PH locus of the non-essential gene. Therefore, in the following examples, Rep / Cap is placed next to the essential gene GP64, and the core expression element ITR-GOI expression cassette of rAAV is placed at the C-terminus of various essential genes of baculovirus to evaluate the stability of BEV Sex and packaging rate of rAAV.

[0129] rAAV was prepared using DH10Bac-ΔCC-Cap-Rep(GP64)-ITR-GOI(Ac135).

[0130] This example contains two homologous recombination vectors: one is a homologous recombination vector targeting the essential gene GP64 containing the Cap and Rep expression cassettes, the Chol expression cassette and the ETL-EGFP expression cassette containing the functional protein c...

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Abstract

The invention belongs to the field of gene therapy, and particularly relates to a preparation method and system of recombinant adeno-associated virus and recombinant bacmids. Firstly, constructing recombinant bacmids of a recombinant baculovirus genome containing necessary functional elements for producing the recombinant adeno-associated virus; wherein at least one necessary functional element isinserted into the N terminal or the C terminal of the necessary locus of the recombinant baculovirus genome; and then transfecting the obtained recombinant bacmids containing the recombinant baculovirus genome for producing the recombinant adeno-associated virus into a host cell line for culturing to prepare the recombinant adeno-associated virus. Compared with a recombinant baculovirus obtainedby preparing recombinant bacmids through traditional Tn7 recombination, the recombinant baculovirus obtained by inserting core elements containing Cap, Rep and ITR into the two sides of an essential gene of the baculovirus has the advantages that the production level of continuous passage rAAV in cells is more stable, and the rAAV yield is higher.

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. Background technique [0002] Recombinant adeno-associated virus (rAAV) is one of the most promising vectors in the field of gene therapy due to its wide host range, low immunogenicity, high safety, and ability to mediate long-term stable expression of foreign genes in animals. . [0003] With the approval of the first recombinant adeno-associated virus (rAAV)-mediated gene therapy drug, there is an increasing demand for large-scale AAV vector manufacturing technology (Yla-Herttuala, S., Mol Ther. 20, 1831-1832, 2012 ). However, large-scale production of rAAV remains a limiting factor in the development of this type of therapy. Therefore, in order to overcome this difficulty and the problem of biosafety, a production system utilizing the ability of baculovirus to infect inse...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866C12N15/35C12N7/01C12R1/93
CPCC12N15/86C07K14/005C12N7/00C12N2710/14043C12N2750/14122C12N2750/14152C12N2710/14143C12N2710/14144C12N2750/14143C12N2750/14151Y02A50/30C12N2710/14044
Inventor 不公告发明人
Owner JINFAN BIOMEDICAL TECH (WUHAN) CO LTD
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