Method for knocking out AAV receptor, HEK293 cell strain with AAV receptor knocked out and application

A cell line and cell technology, applied in the fields of genetic engineering and cytology, can solve the problems of low AAV yield, difficulty in scale expansion, and susceptibility to infection.

Active Publication Date: 2017-10-13
GUANGZHOU PACKGENE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The three-plasmid transfection method is simple and fast, but because HEK293 is an adherent cell, it is difficult to scale up this method
However, the AAV in the supernatant is secreted by HEK293 cells and will re-enter HEK293 cells because AAV easily infects HEK293 cells
After infection into HEK293 cells, AAV will undergo steps such as lysosome inclusion, digestion of the shell, and AAV genome escape from lysosomes...

Method used

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  • Method for knocking out AAV receptor, HEK293 cell strain with AAV receptor knocked out and application
  • Method for knocking out AAV receptor, HEK293 cell strain with AAV receptor knocked out and application

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Embodiment 1

[0042] Embodiment 1 AAV receptor knockout HEK293 cell line of the present invention and its preparation method

[0043] 1. HEK293 cell line knocked out of AAV receptor of the present invention

[0044] During the production of AAV by conventional HEK293 cells, AAV will be secreted into the supernatant, and the AAV in the supernatant can easily infect HEK293 cells and enter HEK293 cells again. After infection into HEK293 cells, AAV will undergo lysosome inclusion, The steps of digesting the shell and AAV genome escaping from lysosomes will eventually lead to the recycling of AAV that has been produced and secreted, and the total AAV yield will become lower.

[0045] KIAA0319L (hereinafter referred to as AAVR) is an essential receptor for AAV-infected cells. The receptor is located on the surface of the cell membrane, and the gene is located on chromosome 1p34.3. NCBI Reference Sequence: NM_024874.4. Once the receptor KIAA0319L (AAVR) is knocked out, the cell Basically cannot b...

Embodiment 2

[0072] Example 2 Preparation of AAV by AAV receptor knockout HEK293 cell line of the present invention

[0073] The method for preparing AAV by HEK293 cell line with AAV receptor knockout, the specific operation steps include:

[0074] Utilize the cell line ARKO-X88 of the present invention, adopt conventional AAV production method, namely as follows:

[0075] 1. Spread ARKO-X88 cells about 3x105 into a 24-well plate, and the cell density is about 60-70% when transfected the next day;

[0076] 2. Use the AAV helper vector pRC-DJ (vector source: on the basis of pAAV2 / 2 derived from the UPENNvector core of the University of Pennsylvania, U.S., and replace the AAV2 Cap2 sequence with the published AAV-DJ sequence) and the Ad helper vector pAd -deltaF6 (source of vector: PL-F-PVADF6 from UPENN vector core, University of Pennsylvania) and recombinant AAV vector pITR-CAG.EGFP (source of vector: PL-C-PV1045 pENN from UPENN vector core, University of Pennsylvania) AAV CB6 PI MASTE...

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Abstract

The invention relates to the field of gene engineering and cytology, in particular to an AAV receptor knocking out method, an HEK293 cell strain with an AAV receptor being knocked out and an application. The knocked-out AAV receptor is KIAA0319L. The knocking out method comprises the following steps: establishing a sgRNA carrier of a targeted AAVR genome sequence; carrying out transfection for a 293 T-cell, and culturing to obtain each clonal cell; carrying out Suveyor gene mutation analysis test; selecting AAVR mutant monoclone; then selecting an AAVR mutant cell strain with high yield of AAV; identifying a mutation condition of other potential mutant site; and enlarging the cell strain without other mutant sites, and establishing a library for production. The final yield of the AAV produced by the HEK293 cell strain of the knocked-out AAV receptor is about 50 percent higher than the yield of the conventional HEK293.

Description

technical field [0001] The invention relates to the fields of genetic engineering and cytology, in particular to a method for knocking out AAV receptors, HEK293 cell lines with knockout AAV receptors and applications thereof. Background technique [0002] At present, gene therapy is gradually becoming a reality from the dream of countless scientists in recent decades. The carrier of gene therapy has always been the key to the whole gene therapy. As a gene carrier, it needs to be safe, effective and specific. After years of evolution and elimination of various viral and non-viral vectors, recombinant adeno-associated virus (rAAV) vectors are currently recognized as gene therapy vectors that meet the above three criteria. Since the production rate of AAV has been increasing slowly for a long time, the popularity of clinical application is also restricted by this. With the launch of Glybera, the first gene therapy drug approved by the EU for the treatment of fatty acid enzyme...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C12N15/90C12N7/00C12R1/93
CPCC07K14/705C12N5/0603C12N5/0686C12N7/00C12N15/85C12N15/907C12N2510/00C12N2750/14151C12N2810/10
Inventor 李华鹏
Owner GUANGZHOU PACKGENE BIOTECH CO LTD
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