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RNA replicon for improving gene expression and application thereof

A replicon, gene technology, applied in the field of genetic engineering, can solve problems such as restricting promotion and patient compliance, increasing treatment costs, and short mRNA expression time

Active Publication Date: 2022-04-12
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the mRNA expression time is relatively short, and multiple repeated administrations are usually required to effectively regulate gene expression and the efficacy of gene therapy, which limits its clinical promotion and patient compliance, and increases the cost of treatment

Method used

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  • RNA replicon for improving gene expression and application thereof
  • RNA replicon for improving gene expression and application thereof
  • RNA replicon for improving gene expression and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070]Example 1 Construction NSP1 G357C / G1569A / A1572C / C1575T-NSP2 T3922C mutant

[0071] T7-VEE (Nsp1GGAC-NSP2T) -GFP:

[0072] 1) T7-VEE carrier enzyme sink primer:

[0073] T7veebglii f 5'-aaaagcgcagtcaccaaaaaagatctagtggtgagcgccc-3 '(SEQ IDNO.2);

[0074] T7VEENDEI R 5'-AtcgatgctGaggcgcgcccccatatgctagac-3 '(SEQ ID No.3);

[0075] G357C mutant primer:

[0076] G357C F 5'-gaAaatgaaggagctcgccgccgtcatgagcgccc-3 '(SEQ ID NO.14);

[0077] G357C R 5'-gctcatgacgcgggcgctccttcttttttctTGTCC-3 '(SEQ ID NO.15);

[0078] G1569A / A1572C / C1575T mutant primer:

[0079] G1569A / A1572C / C1575T F

[0080] 5'-ggccccctctggaagccgatgtcgactgatgttacaagagag-3 '(SEQ ID NO.16);

[0081] G1569A / A1572C / C1575T R

[0082] 5'-TaacatcaagtcgcggggcttcccagagTGGGCTCCTCAACATC-3 '(SEQ ID No.17);

[0083] T3922C mutant primer:

[0084] T3922C F 5'-gccccgtacgcacaatccttacaagctttcatcaac-3 '(SEQ ID No.4);

[0085] T3922C R 5'-TgaaagctTgtaaggattgtgcgtacggcctTG-3 '(SEQ ID No.5);

[0086] PCR amplification syste...

Embodiment 2

[0091] Example 2 Construction NSP2 G3892C-NSP3 A4714G mutant

[0092] T7-VEE (NSP2G-NSP3A) -GFP:

[0093] 1) T7-VEE carrier enzyme cutting point primer: as in Example 1;

[0094] G3892C mutation primers:

[0095] G3892C F 5'-ctgttgtattcattcgggtacgatcgcaaggcccgtac-3 '(SEQ ID No.6);

[0096] G3892C R 5'-CCTTGCGATCGTACCGAATGAATACAAACAACTTC-3 '(SEQ ID NO.7);

[0097] A4714G mutant primer:

[0098] A4714G F 5'-Tatatcctcgggagaaggcatgagcagtattaggtcg-3 '(SEQ ID NO.8);

[0099] A4714G R 5'-TaatacTgctCatgccttctccgaggatatacatgc-3 '(SEQ ID No.9).

[0100] The PCR amplification system is in Example 1, with T7-VEE (WT) -GFP as a template, using T7veeBGLIIF and G3892CR primer PCR amplification of upstream fragment (1714bp) containing G3892C mutation (1714 bp), G3892CF and A4714GR PCR amplification containing G3892C / T3922C The mutation and the A4714G mutation (853 bp), A4714GF and T7VEENDEIR primer PCR amplified a downstream fragment (2850 bp), agarose gel electrophoresis, and colloated recover...

Embodiment 3

[0105] Example 3 Construction NSP1 G357C / G1569A / A1572C / C1575T-NSP2 A3821T / T3922C mutant

[0106] T7-VEE (Nsp1GGAC-NSP2AT) -GFP:

[0107] A3821T mutant primer:

[0108] A3821T F 5'-cattggtgcttatagcgcggggctgttcaagtttcccgggggtcaaac-3 '(SEQ IDNO.10);

[0109] T7VEESMAI R 5'-gcttaagttagtgcgggccccccggggTcgactctCTAG-3 '(SEQ ID NO.11). The PCR amplification system is in Example 1, with T7-VEE (Nsp1GGAC-NSP2T) -GFP as a template, using A3821TF and T7VeesmAIR primer PCR amplification of DNA fragment (4460 bp), agarose gel electrophoresis, and collagen receiving.

[0110] 2) SMAI enzyme cutting T7-VEE (Nsp1GGAC-NSP2T) -GFP plasmid vector: 10x buffer 3 μL, T7-VEE (Nsp1GGAC-NSP2T) -GFP 26 μL, SMAI 1 μL; 37 ° C, 2H, agarose grease Glue electrophoresis, collided 7062 bp fragment.

[0111] 3) Homologous recombination, reaction system: containing 44.6 ng of PCR amplification fragment containing A3821T mutation 44.6 ng, SMAI-cut T7-VEE (Nsp1GGAC-NSP2T) -GFP plasmid vector 70.62 ng, 2X Clone...

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Abstract

The invention discloses an RNA replicon for improving gene expression and application thereof. The RNA replicon comprises a 5'untranslated region and a 3 'untranslated region, the promoter comprises a non-structural protein gene coding region, a subgenome promoter and a target gene coding region. According to the invention, non-structural protein region mutant replicable RNA introduced through a PCR site-directed mutagenesis technology is transfected to a mammalian eukaryotic cell through Lipofectamine2000 or nanoparticles, so that expression of cytokines and chemotactic factors mediated by a downstream subgenome promoter of the mammalian eukaryotic cell and including GM-CSF, IFN-gamma, IL-2, IL-12 and IL-15 can be remarkably enhanced; the compound can be applied to treatment of tumors, infectious diseases, autoimmune diseases, hereditary diseases or cardiovascular diseases.

Description

Technical field [0001] The present invention belongs to the field of genetic engineering, and in particular to an RNA replicon and its applications thereof in increasing gene expression. Background technique [0002] Gene substances are typically delivered in the cells in the form of DNA or RNA encoding the transgenic gene. However, the DNA has a low delivery efficiency to the cellular nucleus and the safety of the use of drugs in the clinical application of gene therapy in DNA carrier in the clinic. The letter enables RNA molecules (mRNA) to improve the safety of gene therapy. However, mRNA has a short period of expression, which typically requires repeated dosing to effectively regulate the efficacy of gene expression and gene therapy, limiting its clinical promotion and patient compliance, and improving treatment costs. Reparable RNA (Reprna), also known as RNA replicon or self-amplification RNA, originating from a positive chain or negative chain RNA virus. Avirus replicon in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/40C12N15/41C12N15/43C12N15/45C12N15/86A61K48/00A61K47/26A61K45/06A61K39/00A61P35/00A61P31/00A61P37/02A61P43/00A61P9/00
CPCY02A50/30A61K39/00A61K45/06A61K47/26A61K48/00A61P9/00A61P31/00A61P35/00A61P37/02A61P43/00C07K14/105C07K14/115C07K14/08C07K14/085C12N15/86
Inventor 张元林贵斌
Owner SOUTH CHINA UNIV OF TECH