Adeno-associated virus vector and application thereof

A carrier and virus particle technology, which is applied in the fields of genetic engineering and gene therapy, can solve the problems that safe and effective carriers have not yet been identified

Pending Publication Date: 2020-06-30
HUIGENE THERAPEUTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Adeno-associated virus (AAV) vector-mediated gene therapy has been approved in the United States for the treatment of rare inherited eye diseases and inherited central nervous system disorders, but a safe and effective vector capable of targeting inner ear cells has not yet been identified

Method used

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  • Adeno-associated virus vector and application thereof
  • Adeno-associated virus vector and application thereof
  • Adeno-associated virus vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0137] Example 1: AAV-DJ mutant screening experiment design

[0138] The cap sequence (SEQ ID No: 1) of AAV-DJ (Institute of Neurology, Chinese Academy of Sciences, see Grimm D et al., J Virol.2008Jun; 82(12):5887-911) was genetically modified to screen for efficient infection support Cell AAV vector. Specifically, the inventors designed three AAV-DJ capsid protein mutants, each containing the following substitutions: S491A, S500A, and S666A, and the corresponding polynucleotides were obtained by PCR amplification (all alanines at the mutation site are Encoded by GCC, the nucleotides at the remaining positions are the same as the AAV-DJ cap gene sequence), and then the corresponding AAV-rep / cap mutant plasmid (see Figure 1B ). The virions of AAV-DJ and its mutants (3 species) were packaged using a three-plasmid system, and then injected into the cochlea of ​​P0 ICR mice. After 2-3 weeks, the materials were taken for fluorescence observation and phenotype of the support cell inf...

Embodiment 2

[0140] Example 2: Infection efficiency in mouse cochlea

[0141] AAV-DJ and its mutant viruses were injected into the cochlea of ​​PO ICR mice. Each mouse is injected 4×10 9 vgAAV virus. Similarly, 3 weeks after the injection, the cochlear basement membrane of the mouse was stripped and stained, and the proportion of Tdtomato positive cells in the supporting cells of the apical, middle and basal parts were counted.

[0142] The experimental results showed that in AAV-DJ mutants (S491A, S500A, and S666A), 55.7%, 51.42% and 45.0% of the supporting cells (sox2 positive) in the top part of the AAV-DJ mutants were Tdtomato positive, which was higher than the 39.6 of AAV-DJ. %, but no significant difference ( Figure 2A , image 3 ).

[0143] In the AAV-DJ mutants (S491A, S500A and S666A), 64.4%, 41.9% and 49.2% of the supporting cells (sox2 positive) in the middle part were Tdtomato positive, which was significantly higher than the 32.0% of AAV-DJ ( Figure 2B , image 3 ).

[0144] In t...

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Abstract

The present invention relates to an adeno-associated virus (AAV) capsid protein mutant, a polynucleotide containing a nucleic acid sequence encoding the AAV capsid protein mutant, a vector containingthe polynucleotide, and a host cell containing the vector. The invention also relates to an adeno-associated vector comprising the AAV capsid protein mutant, a recombinant adeno-associated virion which is constructed from the AAV vector and carries a gene expression cassette, a method for producing the adeno-associated virus vector or virion, and an application of the adeno-associated virus vectorand virion in treating diseases.

Description

Technical field [0001] The present invention relates to the fields of genetic engineering and gene therapy. Specifically, the present invention relates to an AAV capsid protein mutant, an adeno-associated virus (AAV) vector containing the capsid protein mutant, and a vector constructed from it carrying gene expression AAV virions of the cassette, methods for generating and using such adeno-associated virus vectors or virions, and the use of such adeno-associated virus vectors and virions in disease treatment. [0002] Sequence Listing [0003] This application contains a sequence listing, which is incorporated herein by reference. Background technique [0004] The cochlea of ​​the inner ear is our peripheral sound perception organ. The auditory cells of the cochlea play a very important role in our perception of peripheral sounds. They convert external sound waves into electrophysiological signals, which are then gradually transmitted to the auditory center of the brain through the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/015C12N15/35C12N15/864C12N7/01A61K48/00A61P27/16
CPCC07K14/005C12N15/86C07K14/47A61K48/0008A61P27/16C12N2750/14143C12N2750/14122C12N2750/14123C07K14/705C12N2750/14145A61K48/0033A61K48/0075A61K48/0041A61K48/005A61K48/0058C12N2750/14171
Inventor 姚璇施霖宇王少冉柏伟娅
Owner HUIGENE THERAPEUTICS CO LTD
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