CN113444723A

CN113444723A - Interfering RNA for inhibiting vascular endothelial growth factor receptor 2 gene expression and application thereof

Info

Publication number: CN113444723A
Application number: CN202010229195.2A
Authority: CN
Inventors: 王根宇; 林美娜
Original assignee: Jenkem Technology Co Ltd
Current assignee: Jenkem Technology Co Ltd
Priority date: 2020-03-27
Filing date: 2020-03-27
Publication date: 2021-09-28

Abstract

The invention discloses an interfering RNA for inhibiting the expression of a vascular endothelial growth factor receptor 2 gene and application thereof. In particular, the interfering RNA is siRNA comprising GGAGUGAGAUGAAGAAAUU and/or AAUUUCUUCAUCUCACUCC nucleotide sequences, can effectively inhibit VEGFR2 gene expression, and provides a new choice for preventing or treating VEGFR2 related diseases (particularly VEGFR2 related cancers).

Description

Interfering RNA for inhibiting vascular endothelial growth factor receptor 2 gene expression and application thereof

Technical Field

The invention relates to the technical field of molecular biology and biological medicine, in particular to an interfering RNA (ribonucleic acid), especially siRNA (small interfering ribonucleic acid) for inhibiting the mRNA expression of a Vascular Endothelial Growth Factor Receptor 2 (VEGFR 2) and application thereof.

Background

Vascular endothelial growth factor receptors are cell surface receptors of the VEGF receptor subfamily consisting of three closely related receptor tyrosine kinases including VEGFR1(FLT, FLT1), VEGFR2(FLK-1, KDR) and VEGFR3(FLT4, PCL). The common feature is that there is a tyrosine kinase insert in the catalytic domain, the activity of which is activated by receptor and ligand binding, and which is phosphorylated by the receptor to cause many intracellular enzymatic and other reactions. However, VEGFR2(FLK-1, a receptor containing a kinase insert region: kinase insert domain-containing receptor, KDR) plays an important role in the growth and differentiation of cells, and is involved in the proliferation and angiogenesis of endothelial cells. It is highly expressed in embryonic vascular endothelial cells, while its expression in mature vascular endothelial cells is reduced. Binding of VEGFR2 to a ligand (e.g., VEGF a, C, D, or E) induces receptor dimerization and autophosphorylation of several tyrosine (Y) residues in the C-terminal domain of VEGFR 2. This autophosphorylation causes downstream activation of several signaling cascades, leading to endothelial cell proliferation and migration, increased vascular permeability, and angiogenesis. VEGF/VEGFR2 is aberrantly expressed, specifically up-regulated, in a variety of cancers and diseases, while inhibiting expression of VEGF/VEGFR2 is believed to ameliorate or treat diseases.

RNA interference (RNAi) is a new gene silencing technology developed in recent years, siRNA is an effector molecule of RNAi, and in vitro synthesis of siRNA is the latest development of RNA interference technology, and particularly in the aspect of specifically inhibiting gene expression of mammalian cells, a new way is created for gene therapy.

Disclosure of Invention

The invention provides an interfering RNA for inhibiting VEGFR2 mRNA expression.

Specifically, the interfering RNA comprises a sense strand and an antisense strand complementary paired to the sense strand in the reverse direction.

Specifically, the interfering RNA is selected from: siRNA, dsRNA, shRNA, airRNA, miRNA and combinations thereof.

In one embodiment of the present invention, the interfering RNA is siRNA.

In one embodiment of the present invention, the interfering RNA is chemically synthesized.

Specifically, the interfering RNA comprises the nucleotide sequence shown as SEQ ID NO: 1 or a nucleotide sequence represented by SEQ ID NO: 1, or a nucleotide sequence shown in the specification.

Specifically, the interfering RNA comprises the nucleotide sequence shown as SEQ ID NO: 2 or a nucleotide sequence represented by SEQ ID NO: 2, and (b) the nucleotide sequence shown in the figure.

Specifically, the sense strand of the interfering RNA comprises or consists of the following nucleotide sequence: 5'-GGAGUGAGAUGAAGAAAUU-3' are provided.

Specifically, the antisense strand of the above interfering RNA comprises or consists of the following nucleotide sequence: 5'-AAUUUCUUCAUCUCACUCC-3' are provided.

In one embodiment of the present invention, the end (e.g. 3' end) of the sense strand and/or the antisense strand of the interfering RNA (e.g. siRNA) molecule may further be provided with n dangling bases (Over-hang) to increase the activity of the interfering RNA. Wherein the pendant bases may be identical or different deoxynucleosides (e.g., deoxythymidine (dT), deoxycytidine (dC), deoxyuridine (dU), etc.), n is an integer of 1 to 10 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10), particularly an integer of 2 to 4; in one embodiment of the invention, n-2, the pendant base may be dTdT, dTdC or dUdU, or the like.

In one embodiment of the invention, the 3' ends of the sense and antisense strands of the interfering RNA molecule) are provided with a dangling base dTdT.

Specifically, the sense strand of the interfering RNA comprises or consists of the following nucleotide sequence: 5 '-GGAGUGAGAUGAAGAAAUU dTdT-3'.

Specifically, the antisense strand of the above interfering RNA comprises or consists of the following nucleotide sequence: 5 '-AAUUUCUUCAUCUCACUCC dTdT-3'.

In some embodiments, the interfering RNA molecules described above may further comprise at least one modified nucleotide, and the modified interfering RNA has better properties, such as higher stability, lower immunostimulatory properties, etc., than the corresponding unmodified interfering RNA.

The invention also provides a delivery system of the interfering RNA, which comprises the interfering RNA and a carrier.

Specifically, any vector suitable for delivering the above-described interfering RNA of the present invention to a target tissue or a target cell or the like can be used as the vector, such as those disclosed in the prior art (e.g., Chenzhonghua, Zhude Sheng, Li Jun, Huang Zhang Du. "research progress on non-viral siRNA vector". China pharmacological report 2015, 31 (7): 910-4; WangRui, Polygala japonica, Yang Jing. "research progress on siRNA-carrying Nanoprotein. pharmacy 2017, 28 (31): 4452 4455).

In one embodiment of the present invention, the vector is a viral vector, specifically, a lentivirus, retrovirus, adenovirus, herpes simplex virus, and the like.

In another embodiment of the present invention, the above-mentioned vector is a non-viral vector, such as liposome, polymer, polypeptide, antibody, aptamer, etc. or a combination thereof; wherein, the weight ratio of the interfering RNA to the non-viral vector can be 1:1-50 (such as 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1: 50).

Specifically, the liposome may be a cationic lipid (e.g., lipofectamine series, 1, 2-dioleoyl-3-trimethylammonium propane (DOTAP) available from invitrogen), a neutral ion liposome (e.g., Dioleoylphosphatidylcholine (DOPC), cholesterol, etc.), an anionic liposome (e.g., Dioleoylphosphatidylglycerol (DOPG), Dioleoylphosphatidylethanolamine (DOPE), etc.), or a mixture thereof.

Specifically, the polymer may be a synthetic polymer (e.g., polyethyleneimine, cyclodextrin, etc.) or a natural polymer (e.g., chitosan, telogen, etc.) or a mixture thereof.

Specifically, the polypeptide may be a Cell Penetrating Peptide (CPP) (e.g., protamine, Tat peptide, transportan peptide, pentatin peptide, oligo-arginine peptide, etc.).

Specifically, the antibody may be a single-chain antibody (e.g., scFv-tp, scFv-9R, etc.).

The invention also provides a pharmaceutical composition, which comprises the interfering RNA or the delivery system thereof and pharmaceutically acceptable auxiliary materials.

The invention also provides application of the interfering RNA or the delivery system and the pharmaceutical composition thereof in preparing a medicament for preventing and/or treating VEGFR2 related diseases.

The invention also provides application of the interfering RNA or the delivery system thereof in designing a medicament for preventing and/or treating VEGFR2 related diseases.

The invention also provides application of the interfering RNA or the delivery system and the pharmaceutical composition thereof in inhibiting the expression of the VEGFR2 gene in living cells.

The present invention also provides a method of inhibiting VEGFR2 gene expression in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of the above-described interfering RNA of the invention or a delivery system, pharmaceutical composition thereof.

The present invention also provides a method for preventing and/or treating VEGFR2 related diseases, which comprises the step of administering to a subject a therapeutically effective amount of the above-described interfering RNA or delivery system, pharmaceutical composition thereof of the present invention.

Specifically, the above diseases include, but are not limited to, cancer, ocular diseases, inflammation, and the like.

Specifically, the above cancers include, but are not limited to, lung cancer (e.g., non-small cell lung cancer), liver cancer, esophageal cancer, leukemia, cervical cancer, colorectal cancer, pancreatic cancer, kidney cancer, bladder cancer, breast cancer, prostate cancer, stomach cancer, oral epithelial cancer, ovarian cancer, head and neck cancer, brain tumor, glioma, and the like.

Specifically, the above-mentioned ocular diseases include, but are not limited to, proliferative diabetic retinopathy, diabetic macular edema, herpes simplex viral stromal keratitis, age-related macular degeneration, uveitis, rubeosis iridis, conjunctivitis, keratitis, blepharitis, hordeolum, chalazion, iritis, macular degeneration, retinopathy and the like.

The present invention also provides a method for introducing the above-described interfering RNA of the present invention into a cell, which comprises the step of contacting the cell with a delivery system for the interfering RNA.

Specifically, the above cell is in a subject.

Specifically, the above step of contacting the cell with the delivery system of interfering RNA is a step of administering the delivery system of interfering RNA into the body of the subject via a systemic route or a local route to contact the cell.

The inventors of the present invention, through design and synthesis, obtain an interfering RNA (e.g., siRNA), which can effectively inhibit VEGFR2 gene expression, and provide a new choice for preventing or treating VEGFR2 related diseases (especially VEGFR2 related cancers).

Drawings

FIG. 1 shows a mass spectrum of sense strand of siRNA synthesized in example 1.

FIG. 2 is a mass spectrum of the antisense strand of siRNA synthesized in example 1

FIG. 3 shows the results of the expression level of KDR mRNA in cells.

Detailed Description

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

The term "interfering RNA" or "RNAi" or "interfering RNA sequence" as used herein includes single-stranded RNA (e.g., mature miRNA, ssRNAi oligonucleotide, ssDNAi oligonucleotide) or double-stranded RNA (i.e., duplex RNA such as siRNA, dsRNA, shRNA, aiRNA, or precursor miRNA) that is capable of reducing or inhibiting expression of a target gene or sequence when the interfering RNA is in the same cell as the target gene or sequence (e.g., by mediating degradation and inhibition of translation of mRNA complementary to the interfering RNA sequence). Interfering RNA thus refers to single-stranded RNA complementary to a target mRNA sequence or double-stranded RNA formed from two complementary strands or from a single self-complementary strand. In particular, the interfering RNA molecules are chemically synthesized.

The phrase "inhibiting expression of a target gene" refers to the ability of an interfering RNA (e.g., siRNA) of the invention to silence, reduce, or inhibit expression of a target gene (e.g., VEGFR2 gene). To examine the extent of gene silencing, a test sample (e.g., a biological sample from a target organism expressing a target gene or a sample of cells expressing a target gene in culture) is contacted with an interfering RNA (e.g., siRNA) that silences, reduces, or inhibits expression of the target gene, the expression of the target gene in the test sample is compared to the expression of the target gene in samples not contacted with the interfering RNA (e.g., siRNA), and a control sample (e.g., a sample expressing the target gene) can be set to a value of 100%. In particular embodiments, silencing, inhibition, or reduction of expression of the target gene is achieved when the test sample has a value of about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 10%, 5%, or 0% relative to the control sample. Suitable assays include, but are not limited to, assaying protein or mRNA levels using techniques known to those skilled in the art, such as, for example, dot blot, Northern blot, real-time RT-PCR, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those skilled in the art.

Interfering RNAs include "small interfering RNAs" or "sirnas," where each strand of the siRNA molecule comprises nucleotides of about 15 to about 60 in length (e.g., nucleotides of about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 in length, or nucleotides of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 in length). In a specific embodiment, the siRNA is chemically synthesized. The siRNA molecules of the invention are capable of silencing expression of a target sequence in vitro and/or in vivo. In other embodiments, the siRNA comprises at least one modified nucleotide, e.g., the siRNA comprises one, two, three, four, five, six, seven, eight, nine, ten or more modified nucleotides in the double-stranded region.

As used herein, the term "dsRNA" or "precursor RNAi molecule" is intended to include any precursor molecule that is processed in vivo by an endonuclease to produce an active siRNA.

As used herein, the term "small hairpin RNA" or "short hairpin RNA" or "shRNA" includes short RNA sequences that produce tight hairpin turns (hairpin turns) that can be used to silence gene expression by RNA interference. The shRNA hairpin structure can be cleaved into siRNA by the cellular machinery.

Typically, micrornas (mirnas) are single-stranded RNA molecules of about 21-23 nucleotides in length that regulate gene expression.

In the present invention, the term "therapeutically effective amount" refers to the amount of a subject compound that will elicit the biological or medical response of a tissue, system or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term "therapeutically effective amount" includes the amount of the following active ingredients: when administered, it is sufficient to prevent the development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the active ingredient, the disease to be treated and its severity, as well as the age, weight, sex, etc. of the subject.

In the present invention, the subject may be a mammal, e.g., a human, monkey, dog, rabbit, mouse, rat, etc.; in one embodiment of the present invention, the subject is a human.

In the present invention, the terms "VEGFR 2" and "KDR" both represent Vascular Endothelial Growth Factor Receptor 2(Vascular Endothelial Growth Factor Receptor 2), and are used interchangeably.

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1: synthesis of siRNA

Mu. mol of general solid phase support 3 '-cholesterol modified CPG (product of Chemcene) and 2' -O-TBDMS protected RNA phosphoramidite monomer are weighed and dissolved in anhydrous acetonitrile solution to make the concentration reach 0.2M. 5-ethylthio-1H-tetrazole (a product of Chemcene) acetonitrile solution is prepared to be used as an activating agent (0.25M), 0.02M iodine pyridine/water solution is prepared to be used as an oxidizing agent, 3 percent trichloroacetic acid dichloromethane solution is prepared to be used as a deprotection reagent, and the deprotection reagent is placed at a reagent designated position corresponding to an ABI 394 model DNA/RNA automatic synthesizer. Setting a synthesis program, inputting a specified oligonucleotide base sequence, starting cyclic oligonucleotide synthesis, wherein the coupling time of each step is 6 minutes, and the coupling time of the galactose ligand corresponding to the L and S monomers is 10-20 minutes. After automatic circulation, the oligonucleotide solid phase synthesis is completed. The CPG was blown dry with dry nitrogen, transferred to a 5ml EP tube, 2ml of ammonia/ethanol solution (3/1) was added, and heated at 55 ℃ for 16-18 hours. Centrifuging at 10000rpm for 10min, collecting supernatant, and draining off concentrated ammonia water/ethanol to obtain white colloidal solid. The solid was dissolved in 200. mu.l of 1M TBAF THF and shaken at room temperature for 20 hours. 0.5ml of 1M Tris-HCl buffer (pH 7.4) was added, shaken at room temperature for 15 minutes, and then placed in a centrifugal pump to pump the mixture to 1/2, the volume of which was the original volume, and THF was removed. The solution was extracted 2 times with 0.5ml chloroform, 1ml of 0.1M TEAA loading solution was added, the mixed solution was poured onto a solid phase extraction column on an HTCS LC-MS system (Novatia)) system to complete mass spectrometric detection analysis. The mass spectra of the sense and antisense strands are shown in FIGS. 1 and 2, respectively. Nucleic acid molecular weights were calculated by normalization with Promass software after the primary scan. The method synthesizes two single chains respectively, after the mass spectrum identification is correct, the two single chains are mixed according to the equal molar ratio, the mixture is incubated at 70 ℃ for 10min, the mixture is placed at room temperature for 20min, and the mixture is annealed into double chains, namely the siRNA.

The siRNA sense strand sequence is: 5 '-GGAGUGAGAUGAAGAAAUU dTdT-3'

The siRNA antisense strand sequence is: 5 '-AAUUUCUUCAUCUCACUCC dTdT-3'.

Example 2: inhibitory Effect of siRNA

1. Cell culture

Cell name: 293T

a) 293T cells in DMEM complete Medium at 37 ℃ with 5% CO₂And (5) culturing by an incubator conventionally.

b) mu.L of OPTI-MEM medium was diluted to 5. mu.L of siRNA (or siRNA NC at a concentration of 20. mu.M), and 50. mu.L of OPTI-MEM medium was diluted to 3. mu.L of Lipofectamine^TM3000 transfection reagents, mixing the two, shaking gently, standing for 15 min. Also, Mock and Blank controls were set.

c) Add 108. mu.L of the mixture to each well.

d) 293T cells in logarithmic growth phase were taken at 1.2X 10 per well⁵Cells were seeded in 12-well plates in 892. mu.L volumes per well, resulting in a total volume of 1000. mu.L per well. The siRNA (or siRNA NC) transfection concentrations were all 100 nM.

e) 24h after transfection 12 well plates were incubated at 37 ℃ in 5% CO₂Taken out of the incubator and used for extracting RNA for subsequent detection

2. RNA extraction

a) Trizol lysis: thoroughly removing the cell culture solution, adding 1mL of Trizol TM Reagent, sucking and beating the cells for 3-5 times by a liquid transfer gun, fully cracking the cells, and standing the cells for 3-5 minutes at room temperature;

b) adding 0.2 volume (0.2mL/1mL Trizol) of chloroform, shaking by votex for 15s, and standing at room temperature for 5 min;

c) centrifuging at 12000rpm for 15min at 4 deg.C, allowing stratification to occur, and carefully pipetting the upper aqueous phase (the volume of the aqueous phase is about 60% of the volume of Magzol) into a new 1.5mL centrifuge tube;

d) adding isopropanol (about 0.6mL) with the same volume as the supernatant, and uniformly mixing by reversing the upper part and the lower part, and precipitating at-20 ℃ for more than 1 h;

e) centrifuging at 4 deg.C and 12000rpm for 30min to obtain white precipitate at the bottom of the tube, and removing the supernatant;

f) adding 1mL of 75% ethanol, slightly blowing and sucking to float the precipitate, and centrifuging at 12000rpm at 4 ℃ for 5 min;

g) repeating the step f;

h) removing supernatant, centrifuging for a short time, sucking with 10 μ L gun, opening the centrifuge tube cover, drying, and adding appropriate amount of RNase-free H when the precipitate is semi-transparent₂And dissolving the O.

I) RNA quality inspection, Nanodrop detection of RNA content, and 1% agarose gel electrophoresis detection of RNA integrity.

3. Q-PCR detection process

3.1 reverse transcription of RNA

a) Taking total RNA extracted from a sample as a template, establishing the following reaction system:

TABLE 1 reaction System

b) Mixing the above systems, centrifuging to collect liquid to tube bottom, at 42 deg.C for 60min, at 72 deg.C for 10 min; the product is the cDNA template.

3.2 quantitative PCR

a) The reaction system was set up as follows:

TABLE 2 reaction System

The primer sequences of KDR primer and internal standard gene GAPDH are shown in Table 3.

TABLE 3 primer sequences

b) PCR amplification was performed as follows:

pre-denaturation at 95 ℃ for 10min, then entering the following cycle:

*95℃ 10s

60℃ 20s

70℃ 10s

reading board

Return to run 40 cycles

Preparing a melting curve: read plate between 70 ℃ and 95 ℃ every 0.5 ℃ and stop for 5 s.

4. Inhibiting effect

The relative expression amount of KDR mRNA was calculated by Δ Δ Ct using GAPDH as an internal standard gene. Compared with the transfection NC siRNA, the KDR siRNA has 77% inhibition rate on KDR mRNA. The expression levels of mRNA in each group of cells are shown in Table 4, FIG. 3.

TABLE 4 mRNA expression levels

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and the like that are within the spirit and principle of the present invention are included in the present invention.

The foregoing embodiments and methods described herein may vary based on the abilities, experience, and preferences of those skilled in the art.

In the present invention, listing the steps of a method in only a certain order does not constitute any limitation on the order of the method steps.

Sequence listing

<110> Beijing Kekai science and technology GmbH

<120> interfering RNA for inhibiting vascular endothelial growth factor receptor 2 gene expression and application thereof

<130> 1

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 19

<212> DNA/RNA

<213> Artificial Sequence

<400> 1

ggagugagau gaagaaauu 19

<210> 2

<211> 19

<212> DNA/RNA

<213> Artificial Sequence

<400> 2

aauuucuuca ucucacucc 19

Claims

1. An interfering RNA comprising the sequence set forth as SEQ ID NO: 1 and/or SEQ ID NO: 2.

2. The interfering RNA of claim 1, wherein the interfering RNA is selected from the group consisting of: siRNA, dsRNA, shRNA, airRNA, miRNA and combinations thereof.

3. The interfering RNA of claim 1 wherein the interfering RNA is siRNA.

4. The interfering RNA of claim 3, wherein the interfering RNA sense strand comprises the nucleotide sequence: 5'-GGAGUGAGAUGAAGAAAUU-3', respectively; and/or the presence of a gas in the gas,

the antisense strand of the interfering RNA comprises the following nucleotide sequence: 5'-AAUUUCUUCAUCUCACUCC-3' are provided.

5. The interfering RNA of claim 1, wherein the interfering RNA further comprises a dangling base;

preferably, the number of dangling bases is 1 to 10, more preferably 2 to 4;

preferably, the pendant base is a deoxynucleoside;

preferably, the dangling base is located at the 3' end of the sense strand and/or the antisense strand of the RNA.

6. The interfering RNA of claim 5 wherein the overhang base is dTdT, dTdC or dUdU.

7. The interfering RNA of claim 5, wherein the sense strand of the interfering RNA comprises the nucleotide sequence: 5 '-GGAGUGAGAUGAAGAAAUU dTdT-3'; and/or the presence of a gas in the gas,

the antisense strand of the interfering RNA comprises the following nucleotide sequence: 5 '-AAUUUCUUCAUCUCACUCC dTdT-3'.

8. The interfering RNA of any one of claims 1-7, wherein the interfering RNA is chemically synthesized.

9. A delivery system for the interfering RNA of any one of claims 1-8, comprising the interfering RNA of any one of claims 1-8 and a carrier;

preferably, the vector is a viral vector or a non-viral vector.

10. A pharmaceutical composition comprising the interfering RNA of any one of claims 1-8 or the delivery system of claim 9, and a pharmaceutically acceptable excipient.

11. Use of the interfering RNA of any one of claims 1-8, the delivery system of claim 9, the pharmaceutical composition of claim 10 for the preparation of a medicament for the prevention and/or treatment of a disease associated with vascular endothelial growth factor receptor 2;

preferably, the disease is cancer, an ocular disease or inflammation.