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

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...

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, which will eventually lead to the recycling of AAV that has been produced and secreted, and the total AAV production will become lower.

[0005] It can be seen that the bottleneck of the production of AAV in the existing technology needs to be broken urgently. Improvement and innovation in the method to obtain a higher AAV production rate is a major problem that needs to be improved urgently at present in gene therapy.

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