siRNA inhibiting EGFR expression as well as precursors and application of siRNA

A technology for expressing vectors and precursors, which is used in the field of siRNA and its precursors for inhibiting EGFR gene expression, and can solve problems such as short half-life

Pending Publication Date: 2019-07-05
JIANGSU MICROMEDMARK BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are still some problems with siRNA, for example: naked siRNA is easily degraded due to RNase A in serum and extremely high renal clearance rate, and its half-life is short

Method used

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  • siRNA inhibiting EGFR expression as well as precursors and application of siRNA
  • siRNA inhibiting EGFR expression as well as precursors and application of siRNA
  • siRNA inhibiting EGFR expression as well as precursors and application of siRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0170]Example 1 Intravenous injection of MLF plasmids expressing EGFR-inhibiting genes into an orthotopic lung cancer model can effectively inhibit tumors

[0171] The experimental results show that there is a well-functioning miRNA liver delivery system in vivo. After intravenous injection of MLF plasmid, the liver produces MLF-encapsulated exosomes and efficiently transports functional MLF to other tissues.

[0172] Intravenous injection of LLC cell-induced orthotopic lung cancer model expressing MLF that inhibits EGFR gene expression in Lewis lung cancer cells EGFR Plasmids to assess their value in the therapeutic field.

[0173] For specific results, see figure 1 shown. Nude mice were injected intravenously with LLC cells, and 30 days later, lung tumor formation was confirmed by microscopic C-scan analysis. Four randomly assigned groups of tumor-bearing mice were injected intravenously with PBS and MLF respectively. SCR Plasmid (5mg / kg), MLF EGFR Plasmid (5 mg / kg) or...

Embodiment 2

[0174] Example 2 by intravenous injection of MLF EGFR+RVG plasmid to make MLF EGFR targeting brain tissue

[0175] Due to the existence of the blood-brain barrier, 98% of drugs including molecular targeted drugs are prevented from entering the brain. Considering that EGFR also plays a key role in glioma formation, a plasmid was constructed to simultaneously express MLF EGFR Fragment and Lamp2b protein (a membrane protein widely expressed in exosomes) and fused with a rabies virus surface glycoprotein short peptide (RVG peptide binds to the acetylcholine receptor expressed by nerve cells) ( figure 2 A). Absorbed and processed MLF in the liver EGFR+RVG After the plasmid, the designed RVG tag expressed on the exosome membrane can guide the exosome to cross the BBB barrier and make it delivered to the MLF EGFR into nerve cells.

[0176] via MLF EGFR Plasmid or MLF EGFR+RVG In exosomes isolated from plasmid-transfected HEK293T cells, MLF EGFR significantly increased the l...

Embodiment 3

[0180] Example 3 Intravenous injection of MLF EGFR+RVG MLF EGFR Targeted delivery to the brain and halts glioblastoma growth

[0181] To evaluate its therapeutic potential in vivo, a mouse model of glioblastoma was established by intracranial implantation of bioluminescent U87MG cells. The successfully modeled mice were randomly divided into 4 groups, and 5 mg / kg of MLFSCR+RVG plasmid and MLF were injected intravenously within two weeks. EGFR Plasmid, MLF EGFR +RVG plasmid and MLF EGFR +TNC+RVG plasmid, a total of 7 times. On days 14 and 21 after intravenous injection, survival analysis was performed and tumor growth was assessed with bioluminescent imaging scans ( image 3 A). Injection of MLF SCR+RVG The overall survival time of mice injected with plasmid (median survival = 24 days) was significantly longer than that injected with MLF SCR +RVG Plasmid (median survival = 14 days) or MLF EGFR plasmid (median survival = 14 days) mice ( image 3 B). When TNC is scree...

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Abstract

The invention discloses a siRNA inhibiting EGFR expression as well as precursor sequence and an application of siRNA. The EGFR siRNA can highly inhibit EGFR gene expression, and an in-vivo experimentshows that the EGFR siRNA has a certain inhibition effect on EGER tumors with high expression. The precursors and carriers of siRNA can form stable siRNA in a host and come into effect.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to siRNA for inhibiting EGFR gene expression, its precursor and application. Background technique [0002] The human epidermal growth factor receptor (EGFR) family is a membrane receptor with tyrosine kinase activity. EGFR is widely distributed on the cell surface of mammalian epithelial cells, fibroblasts, glial cells, keratinocytes, etc., and the EGFR signaling pathway plays an important role in physiological processes such as cell growth, proliferation, and differentiation. Experimental studies have found that EGFR is overexpressed in various degrees in many human tumors, and it has been proved that EGFR is closely related to the degree of differentiation, malignancy and infiltration of tumors, sensitivity to radiotherapy and chemotherapy, tumor drug resistance and prognosis. The EGFR family is considered to be one of the ideal molecular targets for anti-tumor therapy. ...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K31/7105A61P35/00
CPCC12N15/1138A61K31/7105A61P35/00C12N2310/141
Inventor 张辰宇陈熹梁宏伟付正
Owner JIANGSU MICROMEDMARK BIOTECH
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