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Recombinant adeno-associated viruses carrying designed SMN1 gene expression cassettes and application

A technology of gene and virus vector, applied in the application field of treating spinal muscular atrophy, can solve the problems of safety risk, high-efficiency expression, etc.

Active Publication Date: 2018-11-13
BEIJING GENECRADLE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Intravenous injection of AAV viral vectors can lead to high expression of foreign genes in the liver, which may pose safety risks

Method used

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  • Recombinant adeno-associated viruses carrying designed SMN1 gene expression cassettes and application
  • Recombinant adeno-associated viruses carrying designed SMN1 gene expression cassettes and application
  • Recombinant adeno-associated viruses carrying designed SMN1 gene expression cassettes and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Example 1 Plasmid vector construction

[0095] (1) Promoter design and synthesis

[0096] Reference (Niwa H, et al. Gene. 1991;108:193-200.), with the CAG promoter (promoted by CMV enhancer and chicken beta-actin) in the mammalian expression vector pCAGGS vector (GenBank: LT727518.1) Based on the composition of the promoter), considering the length of the sequence, the partial sequence of the chicken beta-actin promoter in the CAG promoter was deleted to obtain a truncated CAG promoter, which was named CA promoter. Next, a partial sequence of the 5' untranslated region of human SMN1 gene mRNA (GenBank: NM_001297715.1) was introduced at the 3' end of the CA promoter sequence, and it was named CAS promoter. The intron sequence from position 449 to position 532 in the human RNA polymerase II 14.5kDa subunit gene (GenBank: Z23102.1) was introduced into the 3' end of the CA promoter sequence, and it was named CAT promoter. The intron sequence from position 62804 to positio...

Embodiment 2

[0114] Example 2 Preparation and assay of recombinant AAV virus

[0115] References (Xiao X, et al . J Virol. 1998;72(3):2224-2232.), the three-plasmid packaging system was used to package the recombinant AAV virus, and the cesium chloride density gradient centrifugation method was used to separate, purify and package the AAV virus. Briefly, AAV vector plasmids (pAAV2neo-CA-Fluc, pAAV2neo-CAT-Fluc, pAAV2neo-CAP-Fluc, pAAV2neo-CAS-Fluc, pAAV2neo-CAR-Fluc, pscAAV-CA-SMN1, pscAAV-CA-coSMN1, pscAAV -CAR-SMN1, pscAAV-CAR-coSMN1, pscAAV-U-CAR-coSMN1, pscAAV-CAR-SMN1-122T, pscAAV-CAR-coSMN1-122T or pscAAV-U-CAR-coSMN1-122T), helper plasmid (pHelper ) and AAV Rep and Cap protein expression plasmids (pAAV-DJ, pAAV-R2C5, pAAV-R2C9 or pAAV-R2C10) were mixed according to the molar ratio of 1:1:1, and the calcium phosphate method was used to transfect HEK293 cells. After 48 hours of transfection, the cells and culture supernatant were harvested, and the recombinant AAV virus was isolated...

Embodiment 3

[0122] Example 3 In vivo and in vitro evaluation experiments of promoters

[0123] (1) In vitro evaluation experiment

[0124] Since the AAVDJ vector has high transduction activity to various cells in vitro (Grimm D, et al. JVirol. 2008; 82: 5887-5911.), we will contain the Fluc gene expression cassette regulated by different promoters ( pAAV2neo-CA-Fluc, pAAV2neo-CAT-Fluc, pAAV2neo-CAP-Fluc, pAAV2neo-CAS-Fluc, pAAV2neo-CAR-Fluc) were packaged into AAVDJ virus. HEK293 cells and GM03813 cell lines were selected for in vitro evaluation of the designed promoters. HEK293 cells were derived from human embryonic kidney cells. The GM03813 cell line was purchased from Coriell Cell Repository in the United States, and was derived from a fibroblast cell line from a patient with SMA type I (Coovert DD, et al. Hum Mol Genet.1997; 6: 1205-1214.). The expression activities of the designed promoters in the cells derived from SMA patients and non-SMA patients were evaluated respectively fo...

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Abstract

The invention provides a series of recombinant adeno-associated viruses carrying designed SMN1 gene expression cassettes. An in vivo experiment indicates that the recombinant adeno-associated virusescan be efficiently introduced into a central nervous system, SMN1 protein is continuously and stably expressed, the lifetime of a spinal muscular atrophy (SMA) model animal is prolonged, its weight isincreased, and its growth and development are restored. Results indicate that the recombinant adeno-associated viruses can be used for developing new SMN1 gene mutation caused spinal muscular atrophytreatment drugs.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant adeno-associated virus vector carrying an artificially designed SMN1 gene expression frame and its application in treating spinal muscular atrophy. Background technique [0002] Spinal muscular atrophy (SMA) is a group of autosomal recessive genetic diseases common in children and adolescents. Foreign literature suggests that the incidence rate is 1 / 6000 to 1 / 10000 (Nicole S, et al. Muscle&Nerve. 2002;26(1):4-13.), and the carrier frequency in the population is about 1 / 40 to 1 / 50 between. The incidence rate of population in southern my country is estimated to be 1 / 53000 (Chung B, et al. J Child Neurol. 2003; 18(3):217-219.). [0003] The disease is characterized by muscle atrophy and paralysis caused by degeneration of motor neurons in the anterior horn of the spinal cord (MaryamOskoui, et al. Neurotherapeutics. 2008; 5:499-506. Nicole S, et al. Muscle&Nerve. 2002;26(...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/113C12N15/864A61K48/00A61K31/7088A61P25/00A61P21/00
CPCA61K48/00A61P21/00A61P25/00A61K31/7088C12N15/86C07K14/47C12N2750/14143
Inventor 马文豪吴小兵田文洪段伟松
Owner BEIJING GENECRADLE PHARM CO LTD
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