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Establishment of recombinant adeno-associated virus carrier production process

A virus vector and production process technology, applied in the field of establishment of recombinant adeno-associated virus vector production process, can solve the problems of many influencing factors, complicated operation, low immunogenicity, etc., and achieve stable and lasting expression, low immunogenicity, high purity effect

Pending Publication Date: 2018-07-27
SHANGHAI GENECHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This undoubtedly increases the possibility of wild-type AAV production, and this method is complicated to operate and has many influencing factors, it is difficult to efficiently separate and purify rAAV, and it is not suitable for large-scale production
The traditional process of purifying rAAV uses two rounds of cesium chloride density gradient centrifugation. However, this method is time-consuming and labor-intensive. The prepared AAV vector contains more protein and DNA impurities, and the transduction efficiency in vivo and in vitro is low.
In addition, although the AAV vector itself has the characteristics of low immunogenicity, several studies have shown that injection of higher AAV vector doses can also cause a significantly enhanced host immune response

Method used

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  • Establishment of recombinant adeno-associated virus carrier production process
  • Establishment of recombinant adeno-associated virus carrier production process
  • Establishment of recombinant adeno-associated virus carrier production process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Preparation of rAAV vector particles

[0049] Such as image 3 Shown, the preparation method of a kind of rAAV vector particle in this implementation comprises the following steps:

[0050] (1) Culture of HEK293T cells:

[0051] Production of rAAV vector particles was performed in HEK293T. HEK293T cells were mixed at 1.4×10 per dish 6 The amount of cells was inoculated in a 10cm culture dish containing 15% (volume percent concentration) FBS DMEM medium, and placed at 37 ° C, 5% CO 2 cultured in an environment of saturated humidity. Transfection was performed 2 days after inoculation.

[0052] (2) Transfection of HEK293T cells:

[0053] Before transfection, the cell culture medium was replaced with 5 ml of DMEM medium containing 5% (volume percent concentration) FBS. The two helper vector plasmids pHelper( Figure 1A ), prAAV-RC ( Figure 1A ) and a shuttle vector plasmid prAAV-GFP ( Figure 1B ) were co-transfected into HEK293T cells at a mass ratio of...

Embodiment 2

[0060] Example 2 Preparation of rAAV vector particles

[0061] Such as image 3 Shown, the preparation method of a kind of rAAV vector particle in this implementation comprises the following steps:

[0062] (5) Culture of HEK293T cells:

[0063] Production of rAAV vector particles was performed in HEK293T. HEK293T cells were mixed at 1.4×10 per dish 6 The amount of cells was inoculated in a 10 cm culture dish containing 8% (volume percent concentration) FBS DMEM medium, and placed at 37 ° C, 5% CO 2 cultured in an environment of saturated humidity. Transfection was performed 2 days after inoculation.

[0064] (6) Transfection of HEK293T cells:

[0065] Before transfection, the cell culture medium was replaced with 5 ml of DMEM medium containing 2% (volume percent concentration) FBS. The two helper vector plasmids pHelper( Figure 1A ), prAAV-RC ( Figure 1A ) and a shuttle vector plasmid prAAV-GFP ( Figure 1B ) were co-transfected into HEK293T cells at a mass ratio of...

Embodiment 3

[0072] Example 3 Preparation of rAAV vector particles

[0073] Such as image 3 Shown, the preparation method of a kind of rAAV vector particle in this implementation comprises the following steps:

[0074] (9) Culture of HEK293T cells:

[0075] Production of rAAV vector particles was performed in HEK293T. HEK293T cells were mixed at 1.4×10 per dish 6 The amount of cells was inoculated in a 10cm culture dish containing 20% ​​(volume percent concentration) FBS DMEM medium, and placed at 37 ° C, 5% CO 2 cultured in an environment of saturated humidity. Transfection was performed 2 days after inoculation.

[0076] (10) Transfection of HEK293T cells:

[0077] Before transfection, the cell culture medium was replaced with 5 ml of DMEM medium containing 10% (volume percent concentration) FBS. The two helper vector plasmids pHelper( Figure 1A ), prAAV-RC ( Figure 1A ) and a shuttle vector plasmid prAAV-GFP ( Figure 1B ) were co-transfected into HEK293T cells at a mass rati...

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Abstract

The invention belongs to the technical field of biology and particularly relates to establishment of a recombinant adeno-associated virus carrier production process. The invention firstly provides a preparation method of a recombinant adeno-associated virus carrier, which includes the steps of: (1) transfecting a plasmid containing a genomic sequence of the recombinant adeno-associated virus carrier into a culture cell; (2) under proper conditions, culturing the cell obtained in the step (1) and collecting a cell culture supernatant, which refers to a mixture of the cell containing the recombinant adeno-associated virus carrier and the culture supernatant; (3) purifying and concentrating the cell culture supernatant in the step (2) to obtain the recombinant adeno-associated virus carrier.The preparation method is basically suitable for majority of rAAV serotypes and mutants. The recombinant adeno-associated virus carrier is high in titer and purity, is low in immunogenicity and is safe and high-effective.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the establishment of a recombinant adeno-associated virus vector production process. Background technique [0002] Adeno-associated virus (AAV) is a replication-defective parvovirus, whose replication requires the assistance of helper viruses such as adenovirus or herpes virus, and in the absence of helper virus, AAV can only perform limited replication , which enters a latent state after infecting cells, integrates into chromosomes or exists in the form of episomes. Therefore, AAV belongs to the genus Dependovirus of the family Parvoviridae. [0003] The core of AAV is a complementary single-stranded DNA. Its capsid protein is 20-hedral stereosymmetric. The diameter of the virus particle is between 20 and 26 nm. It is one of the smallest viruses among known viruses that infect humans. The two ends of the AAV genome are 145bp inverted terminal repeat sequences (inverted...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/864C12N7/00
CPCC12N7/00C12N15/86C12N2750/14143C12N2750/14151
Inventor 李敏姜军李亚楠吴庆沈浩
Owner SHANGHAI GENECHEM
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