CN118086310B - SiRNA for inhibiting circATF IP gene expression, delivery system and application - Google Patents

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an siRNA for inhibiting circATF IP gene expression, a delivery system and application thereof. An siRNA for inhibiting circATF IP gene expression, said siRNA having a sense strand and an antisense strand, the nucleotide sequence of said sense strand comprising a modified or unmodified nucleotide sequence as set forth in SEQ ID No. 1; the nucleotide sequence of the antisense strand comprises a modified or unmodified nucleotide sequence shown as SEQ ID NO. 2. The invention can obviously inhibit circATF IP gene expression in the brain, blood and liver of mice by injecting 3 times of medicaments to mice through nasal administration aiming at the siRNA expressed by circATF IP target genes. Meanwhile, the delivery system for delivering circATF IP can be used as circATF IP inhibitor, can be prepared into medicines with one or more pharmaceutically acceptable auxiliary materials, and is applied to treating indications related to circATF IP gene expression, such as cardiovascular diseases, neurodegenerative diseases, cancers, autoimmune diseases and the like.

Description

SiRNA for inhibiting circATF IP gene expression, delivery system and application

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an siRNA for inhibiting circATF IP gene expression, a delivery system and application thereof.

Background

The circRNA is an RNA sequence formed in a cell by covalent binding (reverse splicing) of the 5 'and 3' ends of a linear RNA. In the past, it was thought that circRNA is only a byproduct generated in the RNA splicing process and does not have important biological functions, but many researches in recent years indicate that the circRNA can be combined with miRNA as a miRNA sponge (miRNA sponge), so that the circRNA cannot be combined with target mRNA to block RNA interference effect of the miRNA, and thus occurrence and development of various central nervous system diseases are regulated. It was found that circSTAG can achieve FAAH degradation in astrocytes and improve corticosterone-induced depression-like behavior by capturing alk bh5 and blocking its translocation to the nucleus. Meanwhile, research shows that circDYM can be used as miRNA-9 sponge to improve depression-like symptoms by inhibiting miRNA-9 to reduce microglial cell activity. The series of studies suggest that circRNA is highly involved in the occurrence and development of major diseases. Meanwhile, the circRNA structure does not have a5 'end or a 3' end, cannot be cut by exonuclease, and is more stable in cells than a plurality of linear RNAs. Therefore, many research teams consider that the characteristic of high nuclease tolerance of the circRNA exists in major organs and peripheral blood for a long time, and the circRNA can be used as a clinical biomarker for various diseases. However, therapeutic techniques directed to targeting pathogenic circrnas have not been developed.

Major Depressive Disorder (MDD) is a chronic mental disorder characterized by emotional dysfunction, with a high recurrence rate, and serious impairment of physical and psychological health. MDD cases have increased over 5000 tens of thousands. Current treatment methods for MDD include Selective Serotonin Reuptake Inhibitors (SSRI), alpha 2-receptor blockers, monoamine oxidase inhibitors (MAOi), selective Norepinephrine Reuptake Inhibitors (SNRI), selective Norepinephrine and Dopamine Reuptake Inhibitors (SNDRI), and other small molecule drugs that inhibit neurotransmitter transduction. However, intervention of neurotransmitter transduction alone is not effective in alleviating depression-like behavior. The literature suggests that circular RNAs (circrnas) can promote the development and progression of depression by regulating a variety of biological mechanisms at the transcriptome level. Therefore, the targeting circRNA can be used as a precise therapeutic means for depression. Studies suggest that circATF IP is significantly highly expressed in biological samples from depressed patients, however no drug targeting circATF IP has been developed yet.

Disclosure of Invention

Aiming at the defects of the prior art, the aim of the invention can be realized by the following technical scheme:

An siRNA for inhibiting circATF IP gene expression, said siRNA having a sense strand and an antisense strand, the nucleotide sequence of said sense strand comprising a modified or unmodified nucleotide sequence as set forth in SEQ ID No. 1; the nucleotide sequence of the antisense strand comprises a modified or unmodified nucleotide sequence shown as SEQ ID NO. 2.

Further, the modification comprises overhanging a deoxyribonucleotide dTdT at the 3' end of the sense strand and/or the antisense strand; or adding ribonucleotide UU at the 3' -end of the sense strand and/or the antisense strand.

Further, the modification also includes fluorine substitution modification, methoxy modification or thio skeleton modification of the nucleotide.

Further, the nucleotide sequence of the siRNA is as follows:

Sense strand: 5'-caccatcctttcaaactcctgtgAA-3';

antisense strand: 5'-TTCACAGGAGTTTGAAAGGATGGTGG x t-3';

wherein the lower case letters denote ribonucleic acid RNA, the upper case letters denote deoxyribonucleic acid DNA, and the "+" indicates that the phosphate groups between the deoxyribonucleotides on both sides of the symbol are phosphorothioate groups.

The delivery system for delivering siRNA comprises the siRNA and a lipid component, wherein the nitrogen-phosphorus ratio of the lipid component to the siRNA is (3-10): 1.

Further, the composition of the lipid component includes DOTAP, NT1-O14B, SM-102, DSPC, cholesterol, and PEG lipid.

A method of preparing a delivery system for delivering siRNA, the method comprising the steps of:

preparing an siRNA buffer solution from the buffer solution for siRNA;

preparing lipid component into lipid ethanol solution with absolute ethanol;

Mixing a lipid ethanol solution with a buffer solution to prepare an empty-load delivery system;

mixing the empty-load delivery system and the siRNA buffer solution according to the nitrogen-phosphorus ratio of lipid components to the siRNA of (3-10): 1, removing ethanol, diluting and adjusting the pH value to 6.5-7.4.

A pharmaceutical composition comprising the siRNA for inhibiting circATF IP gene expression described above and the delivery system described above.

The application of the siRNA in preparing a medicament for treating depression.

The invention has the beneficial effects that:

1. The invention can obviously inhibit circATF IP gene expression in the brain, blood and liver of mice by injecting 3 times of medicaments to mice through nasal administration aiming at the siRNA expressed by circATF IP target genes. Therefore, the delivery system for delivering circATF IP can be used as circATF IP inhibitor, can be prepared into medicines with one or more pharmaceutically acceptable auxiliary materials, and can be used for treating indications related to circATF IP gene expression, such as cardiovascular diseases, neurodegenerative diseases, cancers, autoimmune diseases and the like.

2. In some embodiments, the pharmaceutical compositions of the present invention may be administered by the nasal route of administration.

3. In some embodiments, after modelling the depression in mice by Lipopolysaccharide (LPS), 3 doses of drug are injected nasally, and within one week of the end of dose, the brain, heart, liver, spleen, lung, kidney are removed for RT-qPCR and flow analysis, orbit blood is collected, serum is prepared, and ELISA detection is performed. The result shows that the siRNA medicine can obviously inhibit the expression of circATF IP genes of the whole body and the brain of a mouse, relieve the depression-like behavior of the mouse, reduce the inflammatory reaction of the brain and the whole body nerve, and has statistical significance. The experimental result shows that siRNA aiming at circATF IP target gene expression is hopeful to be a medicament for treating depression.

Drawings

In order to more clearly illustrate the embodiments of the present disclosure or the prior art, the drawings that are required for the description of the embodiments or the prior art will be briefly described, and it will be apparent to those skilled in the art that other drawings can be obtained from these drawings without inventive effort.

FIG. 1 shows the physicochemical characterization (particle size, potential, coating ratio) of SALNP;

FIG. 2 is an in vivo level verification SALNP of the brain delivery efficiency of coated si-circATF7IP to mice;

FIG. 3 is an in vitro horizontal validation SALNP of the silencing effect of coating si-circATF7IP on circATF IP;

FIG. 4 is a graph showing the silencing effect of SALNP coated si-circATF7IP on circATF IP in brain, liver, and plasma of mice;

FIG. 5 is an in vivo level verification SALNP of the effect of coating si-circATF7IP on mouse depressive-like behavior;

FIG. 6 shows the effect of in vivo level detection SALNP on the modulation of mouse brain pro-inflammatory factors by coating si-circATF7 IP;

FIG. 7 is a graph showing the regulatory effect of in vivo level detection SALNP coated si-circATF7IP on microglia;

FIG. 8 shows the regulatory effect of the coating si-circATF7IP on astrocytes in vivo level detection SALNP.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Unless otherwise indicated, in the foregoing and in the following, lower case letters denote ribonucleic acid RNA, upper case letters denote deoxyribonucleic acid DNA, and the phosphate groups between the deoxyribonucleotides flanking the symbol are phosphorothioate groups.

Example 1

1. CircATF7IP human-mouse homologous sequence and siRNA design

Sequencing analysis is carried out on circATF IP to obtain circATF IP sequence fragment homologous to human and mouse, and DICER substrate sequence design and chemical modification are carried out according to the fragment to synthesize siRNA structure, and specific information is shown in table 1.

TABLE 1

2. In vivo level verification SALNP of brain delivery effects on si-circATF IP

Si-circATF IP was taken and prepared with sodium acetate buffer (ph=5.0) to a solution of siRNA sodium acetate at ph=5.0, siRNA concentration 10uM, sodium acetate concentration 25 mM;

Preparing DOTAP, SM-102, DSPC, cholesterol and DMG-PEG2000 into lipid ethanol solution with total lipid concentration of 4mg/mL by using absolute ethanol; wherein, the lipid component comprises DOTAP 1.5%, SM-102.50%, DSPC 8.5%, cholesterol 38.5%, and DMG-PEG 2000.1.5% by mol percentage. 0.1mL of the lipid ethanol solution was injected into 1mL of sodium acetate buffer using a 1mL BD Shu Rui insulin syringe to prepare an empty nanoparticle suspension (SALNP 3). Wherein SM-102 can be replaced by DLin-MC3-DMA or NT1-O14B and prepared as a different empty nanoparticle suspension (DLin-MC 3-DMA SALNP. Sup. 1, NT1-O14B SALNP. Sup. 2).

Injecting 0.2mL of the siRNA sodium acetate solution prepared in the step (1) into 0.2mL of the empty nanoparticle solution prepared in the step (2) by using a 1mL BD Shu Rui insulin syringe, and incubating for 1 minute under vortex stirring to obtain SALNP primary solution loaded with siRNA. Repeating the steps (1) - (3), and combining to obtain more SALNP primary liquid loaded with siRNA;

removing ethanol in SALNP primary solution by ultrafiltration with a ultrafiltration tube with molecular weight cut-off of 100kDa by using 13000g centrifugal force, adjusting the concentration of siRNA to 300uM by using enucleated enzyme water, and adjusting the pH to 7.0 by using 0.1M sodium hydroxide or hydrochloric acid solution to obtain SALNP suspension for entrapping siRNA.

The C57BL/6 mice were divided into 3 groups of SALNP1+si-circATF IP group, SALNP2+si-circATF IP group and SALNP3+si-circATF IP group, 5 mice each, and the different SALNP-entrapped siRNA suspensions prepared above were stained with DiR and the C57BL/6 mice were nasally administered at an injection dose of 0.5mg/kg. Mice were anesthetized with isoflurane 4 hours after injection and fluorescence intensity was observed SALNP in an IVIS Spectrum imaging instrument, after imaging, mice were sacrificed and organs (brain, heart, liver, spleen, lung, kidney) were taken for fluorescence imaging again.

In vivo level verification SALNP-si-circATF7IP silencing effect on circATF IP Gene expression

Taking SALNP-loaded siRNA suspension prepared above, and carrying out nasal administration on a C57BL/6 mouse, wherein the administration dosage is 0.1mg/kg, 0.3mg/kg and 1mg/kg, and the administration frequency is once every other day for three times. Mice were sacrificed on day 5 after dosing was completed and brains, livers, blood were taken for later use.

3. RNA extraction method (Trizol method)

Respectively loading brain tissue and liver tissue into a grinding tube for grinding, wherein 0.5mL of Trizol reagent is added into about 200mg of organ tissue during grinding;

taking 100uL of organ tissue fluid after grinding, adding 900uL of Trizol reagent, standing at room temperature for 10 minutes, then adding 0.2mL of chloroform, fully mixing, and standing on ice for 10 minutes to completely dissociate the nucleoprotein complex;

centrifuging at 12000rpm at 4deg.C for 15 min, taking a new EP tube, adding 500 μl isopropanol, and pre-cooling on ice;

after centrifugation, the upper aqueous phase (about 500. Mu.L) was transferred to the new EP tube containing 500. Mu.L of isopropyl alcohol described above and allowed to stand on ice for 10min;

Centrifuging at 12000rpm for 10 minutes;

the supernatant was removed and the RNA pellet was washed once with 1mL of 75% ethanol;

centrifuging at 12000rpm for 5 minutes; removing the supernatant, and air-drying or vacuum-drying the RNA precipitate for 5-10 minutes;

Dissolving RNA in 30-50 mu L DEPC treated deionized water (water is added for dissolving according to the precipitation amount of RNA);

NanoDrop1000 was used to determine the concentration and purity of total RNA in the sample.

4. Reverse transcription

Use of the kit for Nuo Wei Zan HISCRIPT Q RT SuperMix for qPCR (+ GDNA WIPER)

Genomic DNA removal.

Preparation of the mixtures as described in Table 2 in RNase-free centrifuge tubes

TABLE 2

Gently beating and mixing by a pipette.

The reaction procedure: incubate at 42℃for 2 min.

CDNA Synthesis

Preparation of the mixture as described in Table 3 in an RNase-free centrifuge tube

TABLE 3 Table 3

Gently beating and mixing by a pipette.

The reaction procedure: incubation was performed at 50℃for 15 min and at 85℃for 2 min to inactivate the reverse transcriptase.

Quantitative PCR

Preparing a reagent: 2X AceQ qPCR SYBR GREEN MASTER Mix, DEPC treated water, cDNA template, circRNA upstream and downstream primers.

Instrument: quantStudioTM 5 System (272531287).

Preparing a reaction solution: the reaction solutions were prepared as shown in Table 4.

TABLE 4 Table 4

Real-time qPCR amplification was performed according to the reaction conditions in table 5.

TABLE 5

After the completion of the reaction, the expression level was calculated using 2 ^-ΔΔCt. The results are shown in FIG. 4. As can be seen from FIG. 4, SALNP-si-circATF7IP was successful in knocking down circATF IP in brain, liver and plasma of mice.

In vivo level verification SALNP-si-circATF IP for treatment of depression

LPS depression modeling was performed on mice. Healthy mice were injected intraperitoneally with 1mg/kg LPS daily for 5 consecutive days. Depressed mice were screened for drug treatment according to the behavioural test. LPS depression model mice behavioural tests were performed in quiet room at low intensity light between 9:00 and 17:00 and behavioural scoring was performed by the same experimenter. Mice were housed in the room for at least 3 hours prior to testing. The behavioural test was done by analyzing the images by Plexon software system (Plexon Inc, dallas, TX, USA), specific test items include: sucrose preference test (Sucrose PREFERENCE TEST, SPT), tail suspension test (Tail suspension test, TST), open field test (Open FELID TEST, OFT), and Forced swim test (Forced SWIMMING TEST, FST).

The C57BL/6 depressed mice were divided into 6 groups (11 each) of physiological saline, SALNP-si-scramble, SALNP-si-circATF IP (0.1 mg/kg), SALNP-si-circATF IP (0.3 mg/kg), SALNP-si-circATF IP (1 mg/kg) and fluoxetine-administered group (20 mg/kg intraperitoneal injection), respectively. The administration frequency was once every 2 days, three times in total. The behavioural test described above was again performed 5 days after the end of the treatment, verifying the relief effect of SALNP-si-circATF7IP on depression-like symptoms.

The results are shown in FIG. 5.

As shown in FIG. 5, SALNP-si-circATF7IP significantly improved the mouse depressive-like behavior in the dose group of 0.1-1 mg/kg, and was statistically significant.

In the description of the present specification, the descriptions of the terms "one embodiment," "example," "specific example," and the like, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims.

Claims (9)

1. An siRNA for inhibiting circATF IP gene expression, wherein the siRNA has a sense strand and an antisense strand, the nucleotide sequence of the sense strand comprising a modified or unmodified nucleotide sequence set forth in SEQ ID No. 1; the nucleotide sequence of the antisense strand comprises a modified or unmodified nucleotide sequence shown as SEQ ID NO. 2.

2. The siRNA of claim 1, wherein the modification comprises overhanging a deoxyribonucleotide dTdT at the 3' end of the sense and/or antisense strand; or adding ribonucleotide UU at the 3' -end of the sense strand and/or the antisense strand.

3. The siRNA of claim 2, wherein the modification further comprises fluorine substitution modification, methoxy modification or thio backbone modification of the nucleotide.

4. The siRNA of claim 1, wherein the siRNA has a nucleotide sequence of:

Sense strand: 5'-caccatcctttcaaactcctgtgAA-3';

antisense strand: 5'-TTCACAGGAGTTTGAAAGGATGGTGG x t-3';

wherein the lower case letters denote ribonucleic acid RNA, the upper case letters denote deoxyribonucleic acid DNA, and the "+" indicates that the phosphate groups between the deoxyribonucleotides on both sides of the symbol are phosphorothioate groups.

5. A delivery system for delivering siRNA, comprising the siRNA of any one of claims 1 to 4 and a lipid component, wherein the ratio of nitrogen to phosphorus of the lipid component to the siRNA is (3 to 10): 1.

6. The delivery system for delivering siRNA of claim 5, wherein the composition of the lipid component comprises DOTAP, NT1-O14B, SM-102, DSPC, cholesterol, and PEG lipid.

7. A method of preparing a delivery system for delivering siRNA, the method comprising the steps of:

preparing the buffer solution for siRNA according to any one of claims 1-4 into an siRNA buffer solution;

preparing lipid component into lipid ethanol solution with absolute ethanol;

Mixing a lipid ethanol solution with a buffer solution to prepare an empty-load delivery system;

mixing the empty-load delivery system and the siRNA buffer solution according to the nitrogen-phosphorus ratio of lipid components to the siRNA of (3-10): 1, removing ethanol, diluting and adjusting the pH value to 6.5-7.4.

8. A pharmaceutical composition comprising an siRNA for inhibiting circATF IP gene expression according to any one of claims 1-4 and a delivery system according to any one of claims 5-6.

9. Use of an siRNA of any one of claims 1-4 in the manufacture of a medicament for treating depression.