Adeno-associated viral vector for treating III A or III B type mucopolysaccharidosis and application

A virus and recombinant vector technology, applied in the direction of virus/phage, single-stranded DNA virus, introduction of foreign genetic material using vectors, etc., can solve the problems of low efficiency of the central nervous system, little research on gene therapy drugs, and insufficient supply. , to achieve the effect of effective treatment

Active Publication Date: 2020-09-29
STAIDSON BEIJING BIOPHARMACEUTICALS CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of enzyme replacement therapy is that, first of all, it cannot provide enough enzymes to decompose the patient's mucopolysaccharide, and can only reduce the accumulation of mucopolysaccharide in the patient's body, control and improve the development of the disease
Second, the enzymes in the current enzyme replacement therapy cannot pass through the blood brain barrier (BBB) ​​and bone cells, and are not effective in improving central nervous system lesions (cns pathway) and bone lesions.
Although some MPSⅢA or MPSⅢB gene therapy drugs have been clinically used in the world, there are still many shortcomings, such as the low efficiency of AAV vector delivery to the central nervous system and the weak expression level of the promoter.
At present, there is no related gene therapy drug on the market, and there are very few domestic researches on related gene therapy drugs

Method used

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  • Adeno-associated viral vector for treating III A or III B type mucopolysaccharidosis and application
  • Adeno-associated viral vector for treating III A or III B type mucopolysaccharidosis and application
  • Adeno-associated viral vector for treating III A or III B type mucopolysaccharidosis and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] Embodiment 1 vector construction

[0131] Utilizing the promoter sequence of the present invention, a viral packaging vector is constructed according to conventional methods in the art.

Embodiment 2

[0132] Example 2 Virus Packaging and Genome Titer Detection

[0133] The present invention adopts insect cell SF9 (purchased from ATCC, its number is CRL-1711) as a production cell line, and BestBac baculovirus packaging system to produce recombinant AAV virus vector. For the experimental method used, refer to the operation manual of BestBac Baculovirus Packaging System of ExpressionSystem Company.

[0134] 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.

[0135] Table 1

[0136] AAV samples 5ul 10×DNaseI buffer 5ul DNaseI 1ul RNase-free water 39ul total 50ul

[0137] After the treated AAV purified sample was diluted to an appropriate multiple, the Q-PCR reaction system was configured with reference to the following table (Table 2).

[0138] Table 2

[01...

Embodiment 3

[0150] Example 3 Candidate drug in vitro expression and enzyme activity detection

[0151] 3.1 Detection of reporter gene expression level in vitro

[0152] In order to confirm the effect of ssAAV2 / 9-mediated exogenous gene expression, the packaged ssAAV2 / 9-CAG-EGFP virus was infected with 293T cells at different multiplicity of infection (MOI), respectively, and the infection MOI was 30000 and 90000, respectively, and detected after 24 hours The expression level of green fluorescence, the results are as follows figure 1 As shown, the results showed that ssAAV2 / 9-mediated green fluorescent protein was well expressed in a dose-dependent manner.

[0153] 3.2 In vitro expression level detection of candidate drug target genes

[0154] Next, the expression of the target gene mediated by the ssAAV2 / 9 system was detected, and the constructed and packaged recombinant viruses ssAAV2 / 9-CAG-SGSH and ssAAV2 / 9-CAG-NAGLU were respectively infected with 293T cells at different MOIs, among ...

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Abstract

The invention discloses a recombinant nucleic acid molecule. The recombinant nucleic acid molecule comprises a promotor and a nucleotide sequence for coring an SGSH protein or an NAGLU protein, whichare operable and sequentially connected. The invention further discloses a recombinant adeno-associated virus. The recombinant adeno-associated virus comprises an AAV capsid and a carrier genome, wherein the carrier genome comprises a nucleotide sequence for coding a functional SGSH protein or the NAGLU protein, and an expression control sequence for leading the nucleotide sequence of the SGSH protein or the nucleotide sequence of the NAGLU protein to express host cells. The invention further discloses an application of a recombinant adeno-associated virus to effectively treatment of III A orIII B type mucopolysaccharidosis.

Description

technical field [0001] The invention relates to the field of gene therapy, in particular to an adeno-associated virion carrier for treating type IIIA or type IIIB mucopolysaccharidosis and its application. Background technique [0002] Mucopolysaccharidosis (MPS) is a kind of acidic mucopolysaccharide (also known as glycosaminoglycan, GAG) cannot be degraded or degraded incompletely due to the lack or activity reduction of related acid hydrolase in lysosomes, resulting in GAG and It is a monogenic hereditary metabolic disease that causes severe disability and death caused by the accumulation of intermediate metabolites in the body. [0003] According to the defects of lysosomal decomposing enzymes and the different types of mucopolysaccharides stored, MPS can be divided into 7 large and 17 subtypes, including: MPS type I (including three subtypes of IH, IS, and IH / S), MPS type II (including two subtypes IIA and IIB), MPS type III (including four subtypes IIIA, IIIB, IIIC, a...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/864A61K48/00A61K38/47A61P3/00
CPCC12N9/14C12Y310/01001C12N9/2402C12Y302/0105C12N15/86A61K48/005A61K38/47A61P3/00C12N2750/14143Y02A50/30
Inventor 李萃李捧花
Owner STAIDSON BEIJING BIOPHARMACEUTICALS CO LTD
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