Gene treatment medicament capable of inhabiting immunoreaction caused by transgenosis products and preparation method thereof

A gene drug and immune response technology, applied in gene therapy, drug combination, pharmaceutical formula, etc., can solve the problem of no solution for cellular immune response, achieve the effect of reducing cellular immune toxicity and ensuring safety

Inactive Publication Date: 2011-07-13
HUAQIAO UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still no feasible solution to the cellular immune response caused by the transgene product after rAAV vector transduction of target cells

Method used

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  • Gene treatment medicament capable of inhabiting immunoreaction caused by transgenosis products and preparation method thereof
  • Gene treatment medicament capable of inhabiting immunoreaction caused by transgenosis products and preparation method thereof
  • Gene treatment medicament capable of inhabiting immunoreaction caused by transgenosis products and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Preparation of rAAV / OVA-155T

[0021] ① Design of miR-155 tight binding sequence 155T

[0022] The tight binding sequence 155T: 5'-CCCCTATCACAATTAGCATTAA-3' was designed referring to the miR-155 sequence published on the relevant website (eg www.mirbase.org).

[0023] ② Construction of vector plasmid pAAV-GFP-155T

[0024] Design four tandem 155T sequences as: 5'-TCTAGAGTCG

[0025] CGAT CGGT GTGA CTCGAG-3'. In the box is the tight binding sequence 155T.

[0026] After the above sequence is double-digested with XhoI and XbaI, it is connected with the same double-digested rAAV vector plasmid, (the gene expression cassette of the vector plasmid contains promoter, transgene, transgene expression regulatory element, MicroRNA binding sequence, polyA Tail, AAV2 ITR (inverted terminal repeat sequence) at both ends of the gene expression frame, see figure 1 shown); the vector plasmid pAAV-GFP-155T was obtained. In pAAV-GFP-155T, four 155T sequences are connected i...

Embodiment 2

[0032] Preparation of rAAV / OVA-155TN

[0033] ① Design of miR-155 non-tight binding sequence 155TN

[0034] Only 8 bases at the 3' end of the miR-155 tight binding sequence 155T in Example 1 were retained, and the remaining bases were randomly changed to obtain the miR-155 non-tight binding sequence 155TN: 5'-XmAGCATTAA-3'. Where X represents a random base, and m is the number of random bases. In this embodiment, ATCCATCGACCGAA is used for Xm, and m is 14.

[0035] ② Construction of vector plasmid pAAV-GFP-155T

[0036] According to the method of Example 1, the four 155TN sequences were connected in series to the 3'UTR region of the rAAV vector plasmid transgene expression cassette, (the gene expression cassette of the vector plasmid contains a promoter, a transgene, and a transgene expression regulatory element in sequence , MicroRNA binding sequence, polyA tail, AAV2 ITR (inverted terminal repeat sequence) at both ends of the gene expression frame, see figure 1 shown); the...

Embodiment 3

[0042] Preparation of rAAV / OVA-155TN(1) and rAAV / OVA-155TN(8)

[0043] ①Construction of vector plasmids pAAV-GFP-155TN(1) and pAAV-GFP-155TN(8)

[0044] According to the method of Example 1, one 155TN sequence or eight 155TN sequences were connected in series to the 3'UTR region of the rAAV vector plasmid transgene expression cassette, (the gene expression cassette of the vector plasmid contains promoter, transgene, Transgene expression regulatory elements, MicroRNA binding sequence, polyA tail, AAV2 ITR (inverted terminal repeat sequence) at both ends of the gene expression frame, see figure 1 shown); the vector plasmids pAAV-GFP-155TN (1) and pAAV-GFP-155TN (8) were obtained respectively.

[0045] ②Construction of vector plasmids pAAV-OVA-155TN(1) and pAAV-OVA-155TN(8)

[0046] Ovalbumin (OVA) cDNA was used instead of GFP cDNA to obtain vector plasmids pAAV-OVA-155TN(1) and pAAV-OVA-155TN(8), respectively.

[0047] ③ Preparation of rAAV-OVA-155TN (1) and rAAV-OVA-155TN (8...

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Abstract

The invention discloses a gene treatment medicament capable of inhabiting immunoreaction caused by transgenosis products and a preparation method thereof, wherein the 3'-UTR (untranslated region) area of a transgenosis expression frame of a gene medicament carrier plasmid contains a combination sequence of miR-155, and the base sequence of the combination sequence is CCCCTATCACAATTAGCATTAA. According to the invention, on the basis of taking the problem, which can not be solved in the prior art, of cell immunoreaction caused by transgenosis products of rAAV carriers as a target, a technical solution that MicroRNA performs target silence on transgenosis expression in specific cells is utilized, thus the carrier not only can effectively express transgenosis in vivio, but also can inhabit immunoreaction caused by the transgenosis products of the rAAV carriers, thus providing a guarantee for security of the gene medicament based on the rAAV carriers.

Description

technical field [0001] The present invention relates to the fields of molecular pharmacology, molecular medicine and disease prevention and treatment, in particular to a gene therapy drug based on recombinant adeno-associated virus (rAAV), especially a gene therapy drug capable of inhibiting the immune response caused by a transgene product and a preparation method thereof. The drug can effectively suppress the generation of immune response caused by the product of the transgene. Background technique [0002] Gene therapy provides a seemingly perfect solution for the treatment of diseases caused by single gene defects, but the clinical research of gene therapy since 1990 has been facing various technical challenges and difficulties, including one subject in the clinical trial die. With the success of gene therapy for several major diseases, gene therapy has made a major breakthrough, so it was rated as one of the top ten scientific advances in 2009 by Science magazine [Albe...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61P37/00
Inventor 刁勇许瑞安
Owner HUAQIAO UNIVERSITY
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