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Method for improving gene therapy efficiency by overexpressing adeno-associated virus receptors

An expression cassette and vector technology, applied in gene therapy, virus/phage, and other methods of inserting foreign genetic materials, etc., can solve the problems of lack of specificity, no application prospect of gene therapy, and lack of

Active Publication Date: 2019-02-15
EAST CHINA NORMAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In early studies, heparin sulfate was used to promote the recognition of AAV2 by cells and the infection efficiency of AAV9 was promoted by sialylated glycoprotein. Not specific
In addition, in the search for AAV secondary receptors, it was found that the FGFR1 receptor was used to increase the infection of AAV2, and the liver growth receptor (c-MET) was used to increase the infection of AAV2 and AAV3. There are no more clinical application prospects of gene therapy for other reasons
[0011] In summary, there is still a lack of satisfactory highly efficient AAV viral vector systems in this field to meet the stringent requirements of clinical gene therapy.

Method used

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  • Method for improving gene therapy efficiency by overexpressing adeno-associated virus receptors
  • Method for improving gene therapy efficiency by overexpressing adeno-associated virus receptors
  • Method for improving gene therapy efficiency by overexpressing adeno-associated virus receptors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0298] Example 1 Modified adeno-associated virus receptor, overexpressed in human HEK293T cells, and improved infection efficiency of adeno-associated virus carrying GFP

[0299] 1. Construct AAV expression vector according to the transformation strategy

[0300] To transform the adeno-associated virus receptor, the transformation scheme is as follows figure 1 shown. The cDNA sequence of gland-associated receptor (AU040320) was obtained from NCBI, and PKD domain 3 was deleted on the basis of the sequence, and the transmembrane segment was modified or replaced. After determining the modified adeno-associated virus receptor sequence, the modified adeno-associated virus receptor expression vector is constructed. The plasmid used in this example is an AAV expression vector, the promoter is PGK, and the PGK promoter promotes the expression of the transformed AAV receptor gene.

[0301] It can be seen that the basic composition of the adeno-associated virus receptor AAV expressio...

Embodiment 2

[0307] Example 2 Overexpression of the AAV receptor gene in mice to enhance the infection efficiency of the adeno-associated virus of GFP

[0308] 1. Packaging the adeno-associated virus of the AAV receptor gene, and detecting the titer of the virus obtained after packaging.

[0309] 2. Injection of adeno-associated virus into wild-type mice.

[0310] There were three groups of mice injected with adeno-associated virus, the first group was not injected with virus, the second group was injected with 2.5x10^10vg AAV-GFP, the third group was injected with 2.5x10^10vg AAV-GFP and 5x10^10vg AAV Receptor, the fourth group A total of 2.5x10^10vg AAV-GFP and 1x10^11vg AAV Receptor were injected.

[0311] 3. Detection of AAV-GFP infection on the liver

[0312] Two weeks after the virus injection, the mouse liver tissue samples were taken to make frozen tissue sections, the tissue sections were soaked in PBS for 10 minutes, incubated with DAPI for 5 minutes, after the incubation, the ...

Embodiment 3

[0313] Example 3 Overexpression of adeno-associated receptors improves the therapeutic efficiency of adeno-associated virus in the treatment of phenylketonuria

[0314] In this embodiment, taking PKU as an example, the improvement effect of the infection enhancing element of the present invention on the effect of gene therapy is verified. Phenylketonuria is an autosomal recessive genetic disease of phenylalanine metabolism disorder. According to the genetic disease mutation database, construct PAH with the largest number of patients with mutations R408W mutant mice.

[0315] The overall scheme includes the following steps:

[0316] (1) Packaging and purifying the AAV virus expressing the infection enhancing element of the present invention, and measuring the titer of the purified virus by qPCR.

[0317] (2) Design sgRNA for the gene sequence of the phenylketonuria (PKU) mouse model, and design a homologous recombination template according to the target position, clone the CRIS...

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Abstract

The present invention provides a method for improving the gene therapy efficiency by overexpressing adeno-associated virus receptors, and particularly provides an infection enhancing element having the following structure: Z1-Z2-Z3-Z4(I); Z1 is an optional signal peptide element; Z2 is a PKD-1 structural domain, a PKD-2 structural domain or a PKD-1-PKD-2 structural domain; Z3 is a no or PKD-3 structural domain; Z4 is a transmembrane element; '-' means a peptide bond connecting the above elements; with the additional condition that when Z3 is a PKD-3 element, Z4 is not a transmembrane element of an AAVR (Adeno-Associated Virus Receptor). The infection enhancing element provided by the invention can significantly improve the infection efficiency of an AAV virus vector system and also improvethe gene editing efficiency, and can significantly reduce the blood phenylalanine content of a phenylketonuria-treated mouse, thereby treating the phenylketonuria.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for improving gene therapy efficiency by overexpressing adeno-associated virus receptors. Background technique [0002] With the deepening of the understanding of disease pathogenesis and the deepening of genomics research, 3276 single-gene disorders have been confirmed to be related to diseases. For genetic disorder diseases, especially for genetic diseases, the existing treatment methods either cannot fundamentally (that is, DNA level) treat, or the treatment efficiency is low, so the treatment is not only time-consuming and labor-intensive, but the effect is not satisfactory, giving Barriers to patient and clinical care. [0003] Gene therapy, as a method to treat genetic diseases fundamentally, has been considered as an effective treatment for genetic diseases since it was proposed in the 1970s. Traditional gene therapy is divided into two types: gene replacement thera...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/864C12N15/90A61K48/00A61P3/00
CPCA61K48/0008A61K48/005C12N15/86C12N15/907C12N2750/14143C12N2800/107
Inventor 李大力尹树明邵婷婷王立人马列张晓辉刘明耀
Owner EAST CHINA NORMAL UNIV
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