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Adeno-associated viral vectors for treatment of Fabry disease and uses thereof

A carrier and virus technology, applied in the direction of virus/bacteriophage, using vectors to introduce foreign genetic material, viruses, etc., can solve the problems of being infected, the ratio is very different, and the half-life of AAV is reduced

Pending Publication Date: 2022-05-17
STAIDSON (BEIJING) BIOPHARMACEUTICALS CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, natural AAV targeting is limited, especially when using AAV vectors for systemic administration, the proportion of tissues that can effectively infect target cells varies greatly depending on the choice of serotype, and other non-targeted tissue cells have potential risk of infection
And because natural AAV has naturally infected humans and other primates, humans and other primates will produce neutralizing antibodies against natural AAV, which will greatly reduce the half-life of AAV and cannot maximize the utilization of AAV vectors

Method used

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  • Adeno-associated viral vectors for treatment of Fabry disease and uses thereof
  • Adeno-associated viral vectors for treatment of Fabry disease and uses thereof
  • Adeno-associated viral vectors for treatment of Fabry disease and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Embodiment 1 vector construction

[0114] 1.1 Construction of pSC-DC172-Gluc, pSC-DC190-Gluc and pSC-CMV-Gluc plasmids

[0115] Synthesize DC172 promoter sequence fragments, DC190 promoter sequence fragments, CMV promoter sequence fragments with XhoI and NotI restriction sites at both ends and luciferase Gluc with NotI and XbaI restriction sites at both ends respectively (Gaussia luciferase) sequence fragment. Wherein, the nucleotide sequence of the DC172 promoter sequence fragment is shown in SEQ ID NO:1, and the nucleotide sequence of the DC190 promoter sequence fragment is shown in SEQ ID NO:16.

[0116] The pSC-CMV-EGFP plasmid (such as figure 1 Shown in A) After double-digestion with XhoI and XbaI, it is ligated with DC172 double-digested with XhoI and NotI and Gluc fragments with double-digestion with NotI and XbaI to form pSC-DC172-Gluc plasmid (such as figure 1 Shown in B); connect with XhoI and NotI double-digested DC190 and NotI and XbaI double-digested Glu...

Embodiment 2

[0125] Example 2 Virus Packaging and Genome Titer Detection

[0126] In this example, HEK293 cells (purchased from ATCC, number CRL-1573) were used as the production cell line, and the conventional three-plasmid packaging system was used to produce the recombinant AAV virus vector. The experimental methods used are routine methods in this area (referring to XiaoXiao, Juan Li, and Richard Jude Samulski.Production of high-titer recombinantadeno-associated virus vectors in the absence of helperadenovirus.J.Virol.1998,72(3):2224 ).

[0127] Take an appropriate amount of purified AAV sample, prepare a DNase I digestion reaction mixture as shown in the following table (Table 1), incubate at 37°C for 30 minutes, and incubate at 75°C for 10 minutes to inactivate DNase I.

[0128] Table 1

[0129] AAV samples 5μl 10×DNase I buffer 5μl DNase I 1μl RNase-free water 39μl total 50μl

[0130] After the treated AAV purified sample was diluted to...

Embodiment 3

[0141] The selection of embodiment 3 candidate drugs

[0142] 3.1 Selection of AAV vectors among drug candidates

[0143] (1), AAV vector targeting selection

[0144] AAV8 is currently known as the most efficient viral vector for liver-targeted transduction. AAVX is a vector obtained by DNA shuffling. In this example, whether AAVX has a stronger targeting ability than AAV8 is tested through in vivo and in vitro liver targeting studies in humans or monkeys.

[0145] 3.1.1 Comparison of infection efficiency of AAV2 / X and AAV2 / 8 vectors on different human liver cell lines

[0146] First, the effects of recombinant viruses scAAV2 / 8-CMV-EGFP and scAAV2 / X-CMV-EGFP on five human liver cell lines (Huh6, 7402, Huh7, HepG2 and 7721 were detected by flow cytometry, among which Huh6 was obtained from Shanghai Xinyu Biotechnology Co., Ltd. Technology Co., Ltd., 7402 and 7721 were obtained from Beijing Zhao Derivatives, Huh7 was obtained from the Cancer Institute of Cancer Hospital, Chin...

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Abstract

The invention discloses a recombinant adeno-associated virus (AAV) vector, which comprises an AAVX capsid and a vector genome, and the vector genome comprises a nucleotide sequence for coding functional alpha-galactosidase A and a regulatory sequence for guiding the expression of the alpha-galactosidase A sequence in a host cell. The invention also discloses application of the recombinant adeno-associated virus vector to treatment of Fabry disease.

Description

technical field [0001] The invention relates to the field of gene therapy, in particular to an adeno-associated virus vector for treating Fabry disease and its application. Background technique [0002] Fabry disease is a lysosomal storage disease caused by the lack of α-galactosidase A (α-GalA, GLA) in lysosomes, which can lead to the systemic distribution of neutral glycosphingolipids Accumulation, of which ceramide trihexyl (Gb3) is the main accumulation, mainly in lysosome endothelial cells, vascular smooth muscle cells, kidney cells, cardiomyocytes and dorsal root ganglion cells. The first clinical symptoms are neuralgia associated with the small nerve fibers of the peripheral and autonomic nervous systems, hypohidrosis and tremors, thereby affecting the quality of life. As we age, fatal complications develop mainly in the kidneys, heart and brain. Since Fabry disease is an X chromosome-related disease, only male hemizygotes and female heterozygotes are affected. The ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/864C12N15/56C12N15/35C07K14/015C12N5/10A61K9/00A61K48/00A61P3/00
CPCC12N15/86C12N5/0686C12N9/2465C07K14/005C12Y302/01022A61K48/0008A61K48/005A61K9/0019A61P3/00C12N2750/14143C12N2750/14122C12N2510/00
Inventor 张婷婷王超李捧花
Owner STAIDSON (BEIJING) BIOPHARMACEUTICALS CO LTD
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