Circular RNA drug for treating heart failure and use thereof

Abstract

Provided is a class of circular RNAs, comprising mecciND2 and mecciNd2 having nucleotide sequences set forth in SEQ ID No. 1 and SEQ ID No. 2, respectively. The mecciND2 or the mecciNd2 is capable of inhibiting excessive opening of mitochondrial permeability transition pores, thereby significantly reducing ROS levels in myocardial cells, maintaining mitochondrial activity, and further protecting the myocardial cells. Intravenously injecting mecciNd2 into a mouse model of heart failure can effectively protect cardiac function and delay the progression of heart failure. The circular RNA is used as a drug for treating heart failure and exhibits good safety and specificity.

Description

Circular RNA drugs for treating heart failure and their applications

[0001] This invention claims priority to patent application No. 2024119129516, filed on December 24, 2024, entitled "Circular RNA Drug for Treating Heart Failure and Its Application", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biomedical technology, specifically relating to circular RNA drugs for treating heart failure and their applications. More specifically, it relates to the application of a class of circular RNAs with specific sequence properties that regulate the release of mitochondrial reactive oxygen species (ROS) as drugs for treating heart failure. Background Technology

[0003] Heart failure (HF) is the leading cause of death from cardiovascular disease. The fundamental problem of HF is a decline in the heart's pumping function. The causes of this decline are multifaceted, but can be summarized as impaired myocardial contractility and / or diastolic function, chronic overload of the heart, and restricted ventricular filling. Currently, HF remains incurable; severe cases require surgical intervention or even heart transplantation. Treatment for HF primarily focuses on improving symptoms by dilating blood vessels, lowering blood pressure, reducing preload and afterload, decreasing the heart's energy demands, and preventing further adverse myocardial remodeling. These drugs do not repair damaged myocardial cells at the molecular level or fundamentally restore myocardial function, and they can lead to drug resistance. While they may improve survival rates, HF patients still face high mortality rates and low quality of life.

[0004] Mitochondria are crucial for highly energy-intensive organs, especially the heart. The human heart consumes 15 to 20 times its own weight in ATP daily; therefore, mitochondria in the heart need to synthesize approximately 6 kg of ATP daily to maintain heart function. Cardiac cardiomyocytes are the cells with the highest mitochondrial content, and mitochondria in mature cardiac cardiomyocytes are relatively large, accounting for about 40% of the volume of adult mammalian cardiac cardiomyocytes. Mitochondrial dysfunction in cardiac cardiomyocytes is a key factor leading to heart failure; in heart failure, cardiac ATP production is reduced by about 30% compared to normal cardiac cardiomyocytes.

[0005] Targeting damaged mitochondria and restoring cardiomyocyte function are important goals in heart failure treatment, but currently there are no treatments specifically targeting mitochondrial dysfunction. A key cause of mitochondrial dysfunction in cardiomyocytes is abnormally increased ROS production, and specifically clearing excess ROS from mitochondria is one of the strategies in heart failure drug development. Currently, no antioxidant drugs have been developed for heart failure patients. Some studies have shown that broad-spectrum antioxidants such as vitamin C, vitamin E, and N-acetylcysteine ​​(NAC) may help treat heart disease. Some mitochondrial-targeting antioxidants, such as mitoTEMPO and mitoQ, have shown good therapeutic effects in animal studies for heart failure.

[0006] The mitochondrial permeability transition pore (mPTP) is the main channel for the release of mitochondrial reactive oxygen species (ROS). Located between the inner and outer mitochondrial membranes, it is a non-selective channel composed of protein complexes that allows molecules smaller than 1.5 kDa to enter and exit. Under normal conditions, the mPTP is closed to maintain mitochondrial membrane potential and normal function. In the heart experiencing heart failure, excessive opening of the mPTP leads to loss of mitochondrial membrane potential in cardiomyocytes, resulting in oxidative damage to the cardiomyocytes. Cyclic protein D (CypD) is a positive regulator of the mPTP, binding to the mPTP component protein ATP5B and promoting mPTP opening. The mitochondrial heat shock protein TRAP1 is a negative regulator of the mPTP, competitively binding to CypD and thus inhibiting mPTP opening. Currently, the mPTP is also an important target for heart failure drug development.

[0007] Circular RNAs (circRNAs) are a large class of RNA molecules whose 5' and 3' ends are covalently linked to form a closed circular structure. Recent studies have shown that circRNAs have important regulatory functions and are closely related to various cardiovascular diseases. Currently, it is known that circRNAs in animal cells can originate from the nuclear genome and mitochondrial genome; circRNAs encoded by the mitochondrial genome are called mecciRNAs (mitochondria-encoded circRNAs). Numerous studies have demonstrated that circRNAs, including mecciRNAs, play important regulatory roles in normal mitochondrial activity, including promoting mitochondrial protein transport and regulating mitochondrial ROS levels. These functions are closely related to the development and progression of heart failure. Currently, there are no circRNA drugs used in the clinical treatment of heart failure. Summary of the Invention

[0008] To address the aforementioned problems, this invention provides the application of a class of circular RNAs with specific sequence properties capable of regulating mitochondrial ROS release as drugs for treating heart failure. One of the sequence characteristics of this class of circular RNAs is their ability to bind to mPTP components and their regulatory proteins and regulate mPTP opening, thereby maintaining mitochondrial function in cardiomyocytes, reducing intracellular ROS levels, thus protecting cardiomyocytes and delaying the progression of heart failure.

[0009] On the one hand, there is the application of circular RNA in drugs for the prevention, adjunctive treatment, and / or treatment of heart failure.

[0010] Specifically, the circular RNA binds to the mPTP components ATP5B and TRAP1 proteins. More specifically, the circular RNA has a single-stranded region and contains CUAC, UACC, or ACUA sequences.

[0011] More specifically, the circular RNA is encoded by the mitochondrial genome.

[0012] Preferably, the sequence of the circular RNA is SEQ ID No. 1 and / or SEQ ID No. 2.

[0013] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0014] More specifically, the pharmaceutically acceptable excipients are selected from one or more combinations of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

[0015] Preferably, the pharmaceutically acceptable excipient is selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, magnesium stearate, and mineral oil.

[0016] Specifically, the drug may also include a viral vector.

[0017] More specifically, the viral vector may be one or more of adenovirus vectors, lentivirus vectors, and retrovirus vectors.

[0018] Specifically, the drug may also include an encapsulation carrier.

[0019] More specifically, the encapsulation carrier includes, but is not limited to, cholesterol, nanoparticles, or liposomes.

[0020] Preferably, the embedding carrier can be a liposome.

[0021] Specifically, the drug dosage form is tablet, liquid, capsule, powder, or granule.

[0022] Specifically, the administration method of the drug is selected from oral, intravenous, intradermal, or subcutaneous injection.

[0023] In another aspect, the present invention provides a medicament for the prevention, adjunctive treatment and / or treatment of heart failure, the medicament comprising a circular RNA having the sequence SEQ ID No. 1 and / or SEQ ID No. 2.

[0024] Specifically, the heart failure mentioned includes, but is not limited to, acute heart failure or chronic heart failure.

[0025] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0026] More specifically, the pharmaceutically acceptable excipients are selected from one or more combinations of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

[0027] Preferably, the pharmaceutically acceptable excipient is selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, magnesium stearate, and mineral oil.

[0028] Specifically, the drug may also include a viral vector.

[0029] More specifically, the viral vector may be one or more of adenovirus vectors, lentivirus vectors, and retrovirus vectors.

[0030] Specifically, the drug may also include an encapsulation carrier.

[0031] More specifically, the encapsulation carrier includes, but is not limited to, cholesterol, nanoparticles, or liposomes.

[0032] Preferably, the embedding carrier can be a liposome.

[0033] Specifically, the drug dosage form is tablet, liquid, capsule, powder, or granule.

[0034] Specifically, the administration method of the drug is selected from oral, intravenous, intradermal, or subcutaneous injection.

[0035] The technical effects achieved by this invention are as follows:

[0036] (1) Higher targeting and specificity: The circular RNA of this invention binds to ATP5B and TRAP1 proteins and regulates the levels of TRAP1 and other proteins in mitochondria, inhibiting the excessive opening of mPTP, regulating the release of ROS in cardiomyocytes, and significantly reducing the level of ROS in cardiomyocytes. The circular RNA of this invention has better targeting and specificity than broad-spectrum antioxidant therapy.

[0037] (2) Safety of circular RNA: Circular RNA is less likely to trigger an immune response, so it is safer in application.

[0038] (3) Multiple applicability: The circular RNA of the present invention is not only applicable to the treatment of heart failure, but also has the function of protecting mitochondria and relieving oxidative stress, and has the potential to be extended to other mitochondrial damage-related diseases.

[0039] (4) Clinical and economic value: Existing treatments mainly aim to relieve heart failure symptoms. The circular RNA of this invention targets damaged mitochondria, inhibits the release of excessive mitochondrial ROS, and helps restore the function of myocardial cells, providing patients with more effective treatment options, and is expected to reduce treatment costs and improve patients' quality of life. Attached Figure Description

[0040] Figure 1 shows the identification of mitochondrial circular RNAs that bind to TRAP1 and ATP5B proteins. In Figure 1, A represents TRAP1 RIP-seq performed in human 293T cells, with Western blot results demonstrating successful enrichment of TRAP1 protein by the specific antibody; B represents the statistical results of mitochondrial circular RNAs identified by TRAP1 RIP-seq; C represents ATP5B RIP-seq performed in human 293T cells, with Western blot results demonstrating successful enrichment of ATP5B protein by the specific antibody; and D represents the statistical results of mitochondrial circular RNAs identified by ATP5B RIP-seq.

[0041] Figure 2 shows the sequence characteristics of mitochondrial circular RNAs that bind to TRAP1 and ATP5B, predicted by HOMER software.

[0042] Figure 3 is a schematic diagram of the secondary structure of mecciND2 and mecciNd2 predicted using mfold.

[0043] Figure 4 shows the binding of mecciND2 and mecciNd2 synthesized in vitro to TRAP1 and the increase of TRAP1 content in mitochondria. In Figure 4, A shows that mecciND2 transfected into 293T cells specifically binds to TRAP1 protein; B shows the changes in mitochondrial TRAP1 protein levels after mecciND2 transfection as detected by APEX assay; C shows the changes in TRAP1 and CypD levels at the whole-cell and mitochondrial levels after mecciND2 transfection as detected by Western blot; ACTIN is the internal control protein at the whole-cell level; TOMM40 is the internal control protein at the mitochondrial level; D shows the quantitative results of proteins in Figure C; E shows that mecciNd2 transfected into HL-1 cells specifically binds to TRAP1 protein; F shows the changes in mitochondrial TRAP1 protein levels after mecciNd2 transfection as detected by APEX assay; G shows the changes in mitochondrial TRAP1 protein levels after mecciNd2 transfection as detected by Western blot. blot analysis of changes in TRAP1 and CypD levels in whole cells after transfection with mecciNd2; H represents the quantitative results of proteins in the G plot; ns indicates no significant difference, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001.

[0044] Figure 5 shows the detection of mPTP and ROS levels in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) transfected with mecciND2 after hypoxia or doxorubicin stimulation. In Figure 5, A represents the Calcein AM signal indicating the degree of mPTP closure; stronger green fluorescence indicates a higher degree of closure, and weaker green fluorescence indicates a higher degree of opening. B represents the DCFH-DA signal indicating cellular ROS levels; strong red signal indicates high ROS levels, and weak red signal indicates low ROS levels. Hoechst specifically labels the cell nucleus, and the white dashed box indicates the outline of the cardiomyocytes. Untreated cells in the figure represent cells cultured under normoxic conditions without doxorubicin stimulation. The scale bar in the figure is 5 μm. ns indicates no significant difference, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0045] Figure 6 shows the detection of mPTP and ROS levels in HL-1 cardiomyocytes transfected with mecciNd2 after hypoxia or doxorubicin stimulation. A and C represent the Calcein AM signal indicating the degree of mPTP closure; stronger green fluorescence indicates a higher degree of closure, and weaker green fluorescence indicates a higher degree of opening. B and D represent DCFH-DA indicating cellular ROS levels; strong red signal indicates high ROS levels, and weak red signal indicates low ROS levels. Hoechst specifically labeled the cell nucleus. The scale bar in the figure is 5 μm. ns indicates no significant difference, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0046] Figure 7 shows the therapeutic effect of mecciNd2 in an doxorubicin-induced mouse heart failure model. A is a flowchart of doxorubicin treatment and mecciNd2 injection; B shows the expression of mecciNd2 in the heart after injection; C shows the ejection fraction of the heart in each group of mice as detected by color Doppler ultrasound; D shows the cardiac appearance of representative individuals from each group (scale bar is 5 mm); E shows the H&E and Masson staining results of heart tissue sections from mice after treatment (scale bar is 50 μm); F shows the cross-section of cardiomyocytes based on H&E staining results; G shows the degree of myocardial fibrosis based on Masson staining results; ns indicates no significant difference, * indicates p < 0.05, *** indicates p < 0.001; the number of mice in each group was 6, 6, 6, and 6, respectively.

[0047] Figure 8 shows the therapeutic effect of mecciNd2 in a stress overload-induced mouse heart failure model; where A is the flowchart of the mouse experiment; TAC: aortic arch coarctation; B is the ejection fraction of the heart of each group of mice detected by color Doppler ultrasound; Sham: sham surgery; C is the appearance of the heart of a representative individual of each group of mice; the scale bar in the figure is 5 mm; D is the H&E and Masson staining results of heart tissue sections of mice after treatment; the scale bar in the figure is 50 μm; E is the cross-section of cardiomyocytes based on the H&E staining results; F is the myocardial fibrosis based on the Masson staining results; ns indicates no significant difference, ** indicates p<0.01, *** indicates p<0.001; the number of mice in each group was 4, 4, 6, and 6, respectively. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0049] the term:

[0050] 1. mPTP, mitochondrial permeability transition pore.

[0051] 2. ATP5B is a component of mPTP; TRAP1 is a negative regulator of mPTP; CypD is a positive regulator of mPTP.

[0052] 3. TAC, aortic arch coarctation surgery.

[0053] 4. Doxorubicin (DOX) is a chemotherapy drug that has cardiotoxicity and can lead to chronic heart failure with long-term use.

[0054] The nucleotide sequences of mecciND2 and mecciNd2 described in this invention are shown in SEQ ID No. 1 and SEQ ID No. 2, and their secondary structures have a large number of single-stranded regions and contain “CUAC”, “UACC” or “ACUA” sequences as potential TRAP1 binding sites, which can bind to mPTP components and the regulatory protein TRAP1.

[0055] Note: According to the WIPOST.26 standard, uracil “U” in the sequence is represented by “T” in the sequence listing.

[0056] Example 1: Identification and Characterization of Circular RNAs Binding to TRAP1

[0057] 1.1 Experimental Methods

[0058] (1) RNA-IP of TRAP1 or ATP5B

[0059] Mitochondrial extraction is described in detail in a published article (PMID: 33588027). Mitochondria were cross-linked with 1% formaldehyde and then lysed in ice-cold RIPA buffer. The RIPA buffer formulation was 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.1% SDS, with the addition of RNase inhibitors, 2 mM DTT, and protease inhibitors. TRAP1 (or ATP5B) specific antibody or IgG control antibody was added to the remaining supernatant, and the mixture was incubated at 4°C for 4 hours to complete antigen-antibody conjugation. Protein G magnetic bead suspension (Invitrogen, 10004D) was added to the antigen-antibody mixture and incubated at 4°C for 2 hours to allow the antigen-antibody complex to bind to the magnetic beads. The protein-antibody-magnetic bead complex was washed twice with RIPA buffer, followed by washing with high-salt RIPA buffer (containing 500 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.1% SDS, and RNase inhibitors, 2 mM DTT, and protease inhibitors. Wash twice with NaCl; take 1 / 5 of the magnetic bead sample, add SDS loading buffer, and boil in a water bath at 100℃ for 10 minutes for subsequent Western blot analysis. Add proteinase K to the remaining protein-antibody-magnetic bead complex and incubate at 55℃ for 30 minutes to digest the protein. RNA is then extracted using TRIzol reagent. The purified RNA can be used for real-time quantitative PCR (RT-qPCR) or high-throughput sequencing analysis.

[0060] (2) RNA-seq (transcriptome sequencing technology)

[0061] High-throughput sequencing was performed by Beijing Novogene Co., Ltd. The library construction type was RIP library, with mitochondrial RNA randomly fragmented for library construction. The sequencing platform was Novaseq 6000PE150, and the sequencing depth was 8G clean data. Sequencing data were analyzed using circular RNA analysis software CIRI2 or find_circ, with hg38 as the reference genome.

[0062] (3) Circular RNA motif prediction

[0063] The motif of the circular RNA sequence that binds to ATP5B or TRAP1 was predicted using HOMER software with default parameters.

[0064] (4) Prediction of secondary structure of circular RNA

[0065] Go to the mfold website (http: / / www.mfold.org), select "RNA Folding Form", put the sequences of mecciND2 and mecciNd2 into the input boxes respectively, select the "circular" format, and make predictions using the default parameters.

[0066] 1.2 Experimental Results

[0067] As shown in Figure 1, TRAP1 and ATP5B bind to hundreds of mitochondrial circular RNAs.

[0068] Figure 2 shows the circular RNA motif of TRAP1 and ATP5B binding predicted using HOMER software.

[0069] As shown in Figure 3, both mecciND2 and mecciNd2 have a large number of single-chain regions in their secondary structures.

[0070] Example 2: Detection of the interaction between circular RNA and TRAP1 protein

[0071] 2.1 Experimental Methods

[0072] 2.1.1 Group I self-splicing intron-mediated circular RNA circularization method

[0073] (1) Design of transcription template

[0074] The transcription template was designed based on the published article (PMID:33406226) and then chemically synthesized by the sequencing company. The template contains the inverted T4 td gene Group I intron, T7 promoter and circular RNA sequence.

[0075] (2) In vitro synthesis of circular RNA precursor

[0076] The linear DNA fragment of the circular RNA carries the T7 promoter, and the circular RNA precursor RNA is synthesized in vitro by transcription using the T7 transcription kit (Thermo Scientific, K0441).

[0077] Add the following components to a 50 μL reaction system:

[0078] T7-DNA template: 1 μg;

[0079] T7 RNA polymerase: 5 μL;

[0080] ATP, CTP, GTP, UTP (use a mixture of UTP / biotin-UTP if biotin labeling is required): 10 mM each;

[0081] The mixture was incubated at 37°C for 4 hours.

[0082] (3) Transcription product purification

[0083] DNase I was added to remove template DNA and terminate the transcription reaction. The linear RNA obtained from transcription was then purified using TRIzol reagent.

[0084] (4) In vitro cyclization reaction

[0085] The transcribed linear RNA was incubated at 55°C for 15 minutes in a cyclization buffer (15 mM MgCl2, 1 mM DTT, 50 mM Tris-HCl, pH 7.5) and 2 mM GTP.

[0086] (5) Separation and purification of cyclized products

[0087] The reaction products were separated on a 5% urea PAGE gel, and the corresponding bands of circular RNA were excised. The RNA was then eluted overnight in elution buffer (20 mM Tris-HCl, pH 7.5, 250 mM NaOAc, 1 mM EDTA, 0.25% SDS). The eluted RNA was purified using the RNA Clean & Concentrator-5 kit (Zymo Research, R1013) to obtain high-purity circular RNA, which can be used for subsequent experiments and analyses.

[0088] 2.1.2 Pull-down assay of biotin-labeled circular RNA on cytoplasmic TRAP1

[0089] (1) Cell transfection

[0090] Cells were seeded in 15 mm culture dishes and transfected with 20 pmol biotin-labeled circular RNA (or circ-ctrl as a control) and cultured for 24 hours.

[0091] (2) Cell treatment

[0092] Before cell collection, cells were placed in a UV crosslinker and crosslinked at an intensity of 120 mJ / cm². After crosslinking, cells were digested with trypsin and collected with PBS. The cell pellet was resuspended in 1 mL of ice-cold digitonin buffer (150 mM NaCl, 50 mM HEPES, pH 7.4, 50 μg / mL digitonin, protease inhibitor, RNase inhibitor), incubated at 4°C with rotation for 15 min, and then centrifuged at 2,000 × g. The supernatant was collected as the cytoplasmic fraction for subsequent pull-down experiments.

[0093] (3) Magnetic bead capture

[0094] Take 50 μL of streptavidin magnetic beads (Invitrogen, 11205D) and block them with 1% BSA and 500 ng / μL yeast tRNA for 30 minutes at room temperature. Add the blocked magnetic beads to the cytoplasmic fraction and incubate by rotation at room temperature for 1 hour. Collect and wash the magnetic beads using a magnetic separator. Finally, resuspend the magnetic beads in 100 μL of digitonin buffer, add SDS loading buffer, and boil at 100°C for 10 minutes for subsequent Western blot analysis.

[0095] 2.1.3 Aptitude Test for Chemical Proximity (APEX)

[0096] (1) Transfect 5 nM circular RNA into 293T or HL-1 cells that stably express APEX2 localized to the mitochondrial matrix and culture for 24 hours.

[0097] (2) The cells were placed in a culture medium containing 500 mM biotin-phenol (APExBIO, A8011) and incubated at 37°C for 30 minutes. Then, 1 mM H2O2 was added at room temperature for 1 minute. Immediately afterward, 2 mL of stop solution (10 mM ascorbic acid, 5 mM Trolox) was added, and the reaction was stopped for 1 minute.

[0098] (3) Lyse cells with 1 mL of RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, protease inhibitor, RNase inhibitor). Add 50 μL of M-280 streptavidin magnetic beads (Invitrogen, 11206D) and incubate at room temperature for 2 hours to allow the labeled protein to bind to the magnetic beads.

[0099] (4) Wash the magnetic beads three times with RIPA buffer, resuspend the magnetic beads in 60 μL of 2×SDS loading buffer containing 20 mM DTT and 2 mM biotin, and boil at 95 °C for 15 minutes. The final protein sample is used for Western blot detection.

[0100] 2.1.4 Western blot

[0101] The protein mixture obtained in 2.1.2 or 2.1.3 was separated by SDS-PAGE gel electrophoresis and then transferred to a nitrocellulose membrane. After blocking with 5% skim milk powder / TBST, primary and secondary antibodies were prepared and incubated, followed by development and statistical analysis of grayscale values ​​using ImageJ.

[0102] 2.2 Experimental Results

[0103] As shown in Figures 4A and 4E, mecciND2 and mecciNd2 transfected into cells bind to the TRAP1 protein.

[0104] As shown in B and F of Figure 4, mecciND2 and mecciNd2 lead to an increase in the level of TRAP1 protein in mitochondria.

[0105] As shown in C, D, G, and H in Figure 4, mecciND2 and mecciNd2 lead to a decrease in CypD protein levels in cells.

[0106] Example 3: Functional detection of circular RNA in cardiomyocytes

[0107] 3.1 Experimental Methods

[0108] (1) Transfection and treatment of cardiomyocytes: In vitro circularized mecciND2 or mecciNd2 was transfected into induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and mouse cardiomyocyte line HL-1 cells using Thermo's Lipofectamine 3000 product. Hypoxia treatment conditions were: 1% O2 / 5% CO2 / balance N2, 37℃, 24 hours; normoxic conditions served as a control. Doxorubicin treatment conditions were: 2 μM doxorubicin, 6 hours.

[0109] (2) mPTP and ROS detection: In this invention, commercially available mPTP and ROS detection kits were used to stain specific cells for live cell staining, live cell photography was performed using a laser confocal microscope, and fluorescence statistics were performed using ImageJ software.

[0110] 3.2 Experimental Results

[0111] As shown in Figures 5 and 6, when cardiomyocytes are under hypoxia or doxorubicin stimulation, transfection with mecciND2 and mecciNd2 can maintain the closure of mitochondrial mPTPs in cardiomyocytes, reduce ROS levels, and protect cardiomyocytes.

[0112] Example 4: The therapeutic effect of mecciNd2 on doxorubicin-induced heart failure

[0113] 4.1 Experimental Methods

[0114] Mice (SPF grade C57BL / 6, 8 weeks old, purchased from Spiford (Beijing) Biotechnology Co., Ltd.):

[0115] Doxorubicin was administered intraperitoneally (5 mg / kg each time), and simultaneously mecciNd2 or control circular RNA (7 μg each time) encapsulated in liposomes was injected via the tail vein. Injections were given weekly for a total of 4 weeks, as shown in Figure 7A. The control circular RNA contained a 264 nt GFP sequence (SEQ ID No. 3).

[0116] Note: According to the WIPOST.26 standard, uracil “U” in the sequence is represented by “T” in the sequence listing.

[0117] The experiment was divided into 4 groups, and the specific information is as follows:

[0118] Group 1: Injection of PBS and control circular RNA (labeled as PBS in the attached figure; circ-ctrl);

[0119] Group 2: Injection of PBS and mecciNd2 (labeled as PBS and mecciNd2 in the attached diagram);

[0120] Group 3: Injection of doxorubicin and control circular RNA (labeled as doxorubicin in the attached figure; circ-ctrl);

[0121] Group 4: Injection of doxorubicin and mecciNd2 (labeled as doxorubicin and mecciNd2 in the attached diagram).

[0122] In week 5, cardiac function in mice was assessed using color Doppler ultrasound. After euthanasia, the hearts were removed for measurement and imaging. Heart tissue was fixed in 4% paraformaldehyde, dehydrated using a gradient of alcohols, embedded in paraffin, and cut into 5 μm thick sections. The sections were dewaxed in xylene and then stained with hematoxylin and eosin to obtain HE-stained sections, or stained with Masson's staining solution.

[0123] 4.2 Experimental Results

[0124] As shown in Figure 7B, intravenous injection of mecciNd2 can increase the expression level of mecciNd2 in cardiac tissue by about 20 times one day after the injection, and the increased expression level of mecciNd2 can be maintained until the 5th day.

[0125] As shown in Figure 7C, compared with DOX mice injected with control circular RNA, DOX mice treated with mecciNd2 maintained normal left ventricular ejection fraction.

[0126] As shown in Figure 7D, the heart morphology of DOX mice injected with control circular RNA was significantly atrophied, while the heart morphology of DOX mice treated with mecciNd2 was similar to that of PBS mice.

[0127] As shown in Figures 7 (E to G), compared to PBS mice, DOX mice injected with control circular RNA exhibited cardiomyocyte atrophy and an increased proportion of myocardial fibrosis. Treatment with mecciNd2 significantly reduced cardiomyocyte atrophy and fibrosis in DOX mice.

[0128] Example 5: The therapeutic effect of mecciNd2 on TAC-induced heart failure

[0129] 5.1 Experimental Methods

[0130] Mice (SPF grade C57BL / 6, 8 weeks old, purchased from Spiford (Beijing) Biotechnology Co., Ltd.):

[0131] Aortic arch coarctation (TAC) surgery: Mice were anesthetized with afodin (250 mg / kg, intraperitoneal injection). A small incision was made at the sternum to open the thoracic cavity and expose the aortic arch. A 60-silk suture was passed through the aortic arch between the right brachiocephalic artery and the left common carotid artery. A 27G suture pad was placed parallel to the aortic arch, and the suture was tightened to ligate the aortic arch. The suture pad was then removed, forming aortic arch coarctation. The suture was tied again to secure the aortic arch, and the thoracic cavity was closed layer by layer. The sham surgery group underwent the same procedure, except that the sham surgery group only threaded the suture without ligating the aortic arch.

[0132] One week after TAC, mecciNd2 or control circular RNA (7 μg each time) encapsulated in liposomes was injected via the tail vein every three days for a total of three injections, as shown in Figure 8A. The control circular RNA contains a 264 nt GFP sequence, identical to the sequence in SEQ ID No. 3 in 4.1.

[0133] The experiment was divided into 4 groups, and the specific information is as follows:

[0134] Group 1: Perform sham surgery and inject control circular RNA (labeled as Sham; circ-ctrl in the attached figure);

[0135] Group 2: Perform sham surgery and inject mecciNd2 (labeled as Sham; mecciNd2 in the attached diagram);

[0136] Group 3: Performed TAC surgery and injected with control circular RNA (labeled TAC in the attached figure; circ-ctrl);

[0137] Group 4: Perform TAC surgery and inject mecciNd2 (labeled as TAC and mecciNd2 in the attached diagram).

[0138] Three weeks after the last injection, cardiac function in mice was assessed using color Doppler ultrasound. After euthanizing the mice, the hearts were removed for measurement and photography. The heart tissue was fixed with 4% paraformaldehyde, dehydrated using a gradient of alcohols, embedded in paraffin, and cut into 5 μm thick sections. The sections were dewaxed with xylene and then stained with hematoxylin and eosin to obtain HE-stained sections, or stained with Masson's staining solution.

[0139] 5.2 Experimental Results

[0140] As shown in Figure 8B, compared with TAC mice injected with control circular RNA, TAC mice treated with mecciNd2 maintained normal left ventricular ejection fraction.

[0141] As shown in Figure 8C, TAC mice injected with control circular RNA exhibited eccentric hypertrophy of the heart, while TAC mice treated with mecciNd2 showed no significant cardiac enlargement.

[0142] As shown in Figures 8 (D to F), compared with Sham mice, TAC mice injected with control circular RNA showed significantly increased cardiomyocyte area and a higher proportion of myocardial fibrosis. Treatment with mecciNd2 significantly reduced cardiomyocyte hypertrophy and fibrosis in TAC mice.

Claims

1. The use of circular RNA in drugs for the prevention, adjunctive treatment, and / or treatment of heart failure, characterized in that, The circular RNA binds to the mPTP components ATP5B and TRAP1 protein; the circular RNA has a single-stranded region and contains CUAC, UACC, or ACUA sequences.

2. The application according to claim 1, characterized in that, The circular RNA is encoded by the mitochondrial genome.

3. The application according to claim 2, characterized in that, The sequence of the circular RNA is SEQ ID No. 1 and / or SEQ ID No.

2.

4. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

5. The application according to claim 1, characterized in that, The drugs also include viral vectors.

6. The application according to claim 5, characterized in that, The viral vector is one or more of adenovirus vector, lentivirus vector, and retrovirus vector.

7. The application according to claim 1, characterized in that, The drug also includes an encapsulation carrier.

8. The application according to claim 7, characterized in that, The embedding carrier is cholesterol, nanoparticles, or liposomes.

9. The application according to claim 8, characterized in that, The embedding carrier is a liposome.

10. A drug for the prevention, adjunctive treatment, and / or treatment of heart failure, characterized in that, The drug comprises circular RNA with sequences of SEQ ID No. 1 and / or SEQ ID No.

2.

11. The medicament according to claim 10, characterized in that, The heart failure mentioned includes acute heart failure or chronic heart failure.

Images