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A kind of AAV virus capable of efficiently infecting immune cells and its preparation method and application

An immune cell and virus technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of low transduction efficiency, no AAV serotypes are found, and achieve low immunogenicity and human safety. , highly infectious effect

Active Publication Date: 2020-02-14
ICARTAB BIOMEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In recent years, studies have found that AAV2, AAV5 and AAV6 can effectively transduce human monocyte-derived DCs cells (Dendritic cells), and have no adverse effects on the morphology and functional characteristics of DC cells. Compared with other serotype AAV virus vectors, AAV5 shows a high propensity for human DC cells, but low transduction efficiency; while AAV6 has high transduction efficiency for mouse-derived DC cells
So far, no AAV serotype that can efficiently infect immune cells has been found. In order to obtain AAV virus variants with higher transduction efficiency and better use in clinical gene therapy, it is necessary to develop other serotypes of AAV virus

Method used

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  • A kind of AAV virus capable of efficiently infecting immune cells and its preparation method and application
  • A kind of AAV virus capable of efficiently infecting immune cells and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] (1) Directly synthesize the DNA sequence shown in SEQ ID NO:2 by chemical synthesis method, which is called hCap gene, and perform PacI-AscI double enzyme digestion to obtain the digested DNA fragment; use PacI-AscI double enzyme digestion pAAV-RC vector, recover the digested product, and obtain the digested vector; prepare the T4 ligation reaction system between the digested DNA fragment and the digested vector, and connect overnight at 16°C; thus clone the DNA sequence into the pAAV-RC vector, and After the sequence verification was correct, the ligated product (cloning product) was transformed into 30uL Stbl3 E. coli competent. Immediately after the transformation, add 1mL of preheated SOC medium, then transfer it to a 15mL centrifuge tube, and add 5mL of SOC medium, culture at 37 degrees 180RPM for 1 hour, transfer all the liquid to 800mL LB medium , adding ampicillin with a final concentration of 50ug / mL, and cultured in shake flasks at 37 degrees for 16 hours. Af...

Embodiment 2

[0032] (1) Directly synthesize the DNA sequence shown in SEQ ID NO:3 by chemical synthesis method, which is called bCap gene, and carry out PacI-AscI double enzyme digestion, clone it into pAAV-RC, after the sequence verification is correct, The ligated product was transformed into 30uL Stbl3 Escherichia coli competent by electroporation. Immediately after electroporation, add 1mL of preheated SOC medium, then transfer it to a 15mL centrifuge tube, add 5mL of SOC medium, culture at 37 degrees 180RPM for 5 hours, transfer all the liquid to 500mL LB medium Add ampicillin with a final concentration of 50ug / mL, and culture in shake flasks at 37 degrees for 18 hours. After the culture was over, the bacterial pellet was collected by centrifugation at 2000 g, and the plasmid DNA was isolated using a plasmid extraction kit, and the adeno-associated virus plasmid was obtained after sequencing to verify correctness. AAV virus that can efficiently infect immune cells.

Embodiment 3

[0034] (1) The hCap gene was directly synthesized by chemical synthesis, and subjected to PacI-AscI double enzyme digestion, and cloned into pAAV-RC, which was used for subsequent AAV virus packaging after being verified to be correct by sequencing. The ligated product was transformed into 30uL Stbl3 Escherichia coli competent by electroporation. Immediately after electroporation, add 1mL of preheated SOC medium, then transfer it to a 15mL centrifuge tube, add 5mL of SOC medium, culture at 37 degrees 180RPM for 5 hours, transfer all the liquid to 500mL LB medium Add ampicillin with a final concentration of 50ug / mL, and culture in shake flasks at 37 degrees for 18 hours. After the culture was over, the bacterial pellet was collected by centrifugation at 2000 g, and the plasmid DNA was isolated using a plasmid extraction kit, and the adeno-associated virus plasmid was obtained after sequencing to verify correctness.

[0035] (2) Prepare 20 15cm culture dishes and inoculate 4.5x...

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Abstract

The invention provides an AAV virus capable of efficiently infecting immune cells and a preparing method and application thereof. The prepared novel AAV virus with a new structure protein has efficient infection to the immune cells; the virus can serve as a gene carrier, constitute a recombinant adenovirus with a foreign gene, carry the foreign gene to the immune cells, and inject the foreign gene into the immune cells after efficient infection, and accordingly the effect of the foreign gene is exerted; the AAV virus is wide in adaptation to the foreign gene, does not need a complicated preparing process, has ultra-low immunogenicity, is safe to a human body, and provides a wider choice to gene treatment.

Description

technical field [0001] The invention relates to a new AAV serotype, in particular to an AAV virus capable of efficiently infecting immune cells and a preparation method and application thereof. Background technique [0002] With the rapid development of molecular biology, gene therapy has become one of the most effective methods for the treatment of human hereditary or acquired diseases. Compared with traditional treatment methods, gene therapy is closer to the natural state of human gene expression, and the key is how to safely and effectively introduce therapeutic genes into the human body and enable long-term expression of therapeutic genes. Compared with adenovirus and lentiviral vectors, adeno-associated virus (AAV) has shown significant advantages in many preclinical and clinical studies due to its high safety, low immunogenicity, and ability to mediate long-term stable expression of genes. potential in the field of gene therapy. [0003] Adeno-associated virus (AAV)...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/864A61K48/00
Inventor 岳庆李凡池季平
Owner ICARTAB BIOMEDICAL
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