MRNA (messenger ribonucleic acid) medicine for inhibiting tumor stem cells and reducing dryness of tumor cells and preparation method of mRNA medicine

Abstract

The invention provides an mRNA (messenger Ribonucleic Acid) medicine for inhibiting tumor stem cells and reducing the dryness of the tumor cells and a preparation method of the mRNA medicine. The mRNA medicine comprises any one or a combination of at least two of linear PTEN mRNA, self-replicating PTEN mRNA and annular PTEN mRNA; the linear PTEN mRNA comprises a 5 'UTR (Untranslated Region) sequence, a PTEN protein coding sequence and a 3' UTR sequence; the nucleotide sequence of the PTEN protein coding sequence comprises a sequence as shown in SEQ ID NO. 1. The mRNA nano-drug prepared by the invention not only has high transfection efficiency in tumor stem cells, but also can induce differentiation of the tumor stem cells and remarkably inhibit growth of the tumor stem cells; in drug-resistant tumor cells, the stemness of the drug-resistant tumor cells is inhibited, so that the sensitivity to chemotherapy, targeted antibodies, immunotherapy and the like is improved, and the killing effect of the drug is improved. The invention also shows an excellent anti-tumor effect in a human tumor cell line xenotransplantation model of the drug-resistant breast cancer in vivo, and provides a promising treatment strategy for the treatment of the drug-resistant breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an mRNA drug that inhibits tumor stem cells and reduces tumor cell stemness, and its preparation method. Specifically, it relates to an mRNA nanomedicine targeting PTEN and its application in inhibiting tumor stem cells or reducing tumor cell stemness, thereby improving the treatment of drug-resistant cancers. Background Technology

[0002] With advancements in medicine and biotechnology, various types of nucleic acid preparations are being used to treat a wide range of major diseases, including infectious diseases, cancer, and rare diseases. These nucleic acid preparations include DNA, antisense nucleic acids (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA), aptamers, and ribozymes. Among these preparations, mRNA, as an important therapeutic tool, has shown particularly disruptive advantages in the vaccine field due to its safety, rapid preparation, and drug-likeness.

[0003] In modern cancer treatment strategies, the emergence of drug resistance significantly limits the effectiveness of many treatment methods. Traditional chemotherapy and targeted therapy often face the problem of tumor cells evading drug effects. Cancer stem cells (CSCs) play a crucial role in these processes, and their high resistance to traditional treatments makes eradicating tumor cells particularly difficult. Cancer stem cells are a subpopulation of cells in tumor tissue, possessing the ability to self-renew and differentiate into multiple lineages, enabling them to promote tumor recurrence and spread after treatment. These cells are highly resistant to traditional chemotherapy, radiotherapy, and targeted therapy, thus becoming a major challenge in cancer treatment. In the field of cancer research, phosphorylase and tensin homolog deleted on chromosome ten (PTEN) is widely considered one of the most important tumor suppressor factors. PTEN regulates cell growth, division, and survival through multiple pathways, and its loss or downregulation is closely related to the occurrence and development of various solid tumors. In various solid tumors, the absence or dysfunction of PTEN is a key factor leading to enhanced tumor cell stemness. It has been confirmed that PTEN deficiency is directly linked to an increase in tumor stem cells. This increased tumor stemness not only promotes tumor growth and metastasis but also leads to drug resistance. Therefore, restoring PTEN can not only inhibit tumor growth and metastasis but also reverse tumor drug resistance, providing a new strategy for cancer treatment.

[0004] Therefore, there is an urgent need to provide an mRNA drug that inhibits tumor stem cells and reduces tumor cell stemness, thereby suppressing tumor stem cells, reducing tumor cell stemness, and reversing tumor resistance to existing treatments, especially for mRNA nanomedicines targeting solid tumors that are highly resistant to drugs and have a tendency to worsen due to PTEN deficiency. Summary of the Invention

[0005] To address the shortcomings of existing technologies and practical needs, this invention provides an mRNA drug that inhibits tumor stem cells and reduces tumor cell stemness, as well as its preparation method. This drug effectively inhibits tumor stem cells, significantly reduces tumor stemness, reverses drug resistance in various types of cancer cells, and significantly improves the efficacy of existing chemotherapy and targeted drugs.

[0006] To achieve this objective, the present invention employs the following technical solution: In a first aspect, the present invention provides an mRNA nanomedicine targeting PTEN, wherein the mRNA nanomedicine comprises any one or a combination of at least two of linear PTEN mRNA, self-replicating PTEN mRNA, or circular PTEN mRNA. The linear PTEN mRNA includes a 5'UTR sequence, a PTEN protein-coding sequence, and a 3'UTR sequence; The nucleic acid sequence encoding the PTEN protein includes the sequence shown in SEQ ID NO.1.

[0007] In this invention, the nucleic acid sequence of the PTEN protein coding sequence was codon-optimized based on the original nucleotide sequence with human as the target host, and the optimized PTEN protein coding sequence is shown in SEQ ID NO.1.

[0008] Preferably, the open reading frame sequence of the linear PTEN mRNA comprises any one or a combination of at least two of the following sequences: (1) A sequence as shown in SEQ ID NO.2; (2) A nucleic acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the sequence shown in SEQ ID NO.2; (3) Nucleic acid sequences that are inserted, substituted, added, or deleted based on SEQ ID NO.2; (4) Degenerate sequences of nucleic acid sequences as shown in (2) or (3).

[0009] SEQ ID NO.1:

[0010] SEQ ID NO.2:

[0011] Preferably, the linear PTEN mRNA contains any one of cap1, cap2, or cap3 cap structures.

[0012] Preferably, the poly-A tail of the linear PTEN mRNA is 100-130 nucleotides long (e.g., 100, 101, 110, 120 or 130).

[0013] Preferably, the method for synthesizing PTEN mRNA includes replacing conventional bases with chemically modified bases, such as N1-Me-Pseudo UTP instead of conventional UTP to reduce immunogenicity.

[0014] Preferably, the mRNA nanomedicine further includes a carrier.

[0015] Preferably, the carrier comprises any one or a combination of at least two of the following: lipid nanoparticles, exosomes, polymer-lipid hybrid nanoparticles, metal nanoparticles, inorganic non-metallic nanoparticles, adenoviruses, or retroviruses.

[0016] Preferably, the polymer-lipid hybrid nanoparticles comprise polymer molecules, ionizable cationic lipid molecules, and PEGylated lipid molecules.

[0017] Preferably, the ionizable cationic lipid molecule includes any one of ALC-0315, SM-102, lipid Lipid P1, lipid Lipid 11, or GO-C14.

[0018] Preferably, the PEGylated lipid molecules include DSPE-PEG and / or DMG-PEG, wherein the number average molecular weight of each PEG is independently 500-50000 (e.g., 1000, 1200, 1400, 2000, 4000, 4200, 4400, 4600, 4800 or 5000).

[0019] In a second aspect, the present invention provides a method for preparing the mRNA nanomedicine described in the first aspect, the preparation method comprising: PTEN mRNA, polymer molecules, and ionizable lipid molecules were mixed at a weight ratio of 1:(10-40):(10-40) to form the organic phase, while PEGylated lipid molecules formed the aqueous phase. The organic phase was kept at a temperature of 20-30°C. o Rapidly add it dropwise into the aqueous phase at temperature C, and then add it within 20-30 minutes. o Stir at 800-1000 rpm for 30-60 min at C, with the volume ratio of aqueous phase to organic phase being (30-60):1, to obtain mRNA nanomedicine.

[0020] The specific point values ​​in the range 10-40 above can be 10, 11, 12, 20, 30, 38, 39 or 40.

[0021] The above 20-30 o The specific point value in C can be selected as 20. o C, 22 o C, 25 o C or 28 o C or 30 o C.

[0022] The specific point value in the above 800-1000 rpm range can be selected as 800 rpm, 900 rpm, or 1000 rpm.

[0023] The specific time values ​​in the above 30-60 min range can be 30 min, 40 min, 50 min, or 60 min.

[0024] The specific point values ​​in the range of 30-60 can be selected as 30, 40, 50 or 60.

[0025] Thirdly, the present invention provides the application of the mRNA nanomedicine targeting PTEN as described in the first aspect in the preparation of products that inhibit tumor stem cells or reduce the stemness of tumor cells.

[0026] Preferably, the tumor includes any one of non-small cell lung cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, bladder cancer, or liver cancer.

[0027] The targeted drug used in this invention is the HER2 antibody trastuzumab, which is administered via intraperitoneal injection.

[0028] Preferably, the mRNA nanomedicine is administered via intratumoral injection or intravenous injection.

[0029] In this invention, the drug administration sequence is as follows: first, intratumoral injection of nanomedicine, followed by intraperitoneal injection of HER2 antibody 20-28 h (e.g., 20 h, 24 h, or 28 h).

[0030] Compared with the prior art, the present invention has the following beneficial effects: The mRNA nanomedicine prepared in this invention not only has high transfection efficiency in tumor stem cells, but also can induce their differentiation and significantly inhibit their growth; in drug-resistant tumor cells, it improves their sensitivity to chemotherapy, targeted antibodies, immunotherapy, etc. by inhibiting their stemness, thereby enhancing the drug killing effect; it also shows excellent anti-tumor effects in an in vivo xenograft model of drug-resistant breast cancer human tumor cell lines, providing a promising treatment strategy for the treatment of drug-resistant cancer. Attached Figure Description

[0031] Figure 1 The graph shows the expression results of linear mRNA, self-replicating PTEN mRNA and circular mRNA in Example 1, where 1 is PBS, 2 is linear PTEN mRNA, 3 is self-replicating PTEN mRNA and 4 is circular PTEN mRNA. Figure 2 Figure A shows the results of mRNA drug-induced differentiation and inhibition of breast cancer stem cell growth. Figure B shows the results of flow cytometry analysis of breast cancer stem cell differentiation after mRNA drug treatment, and the results of mRNA drug and mRNA drug combined with docetaxel inhibiting breast cancer stem cell growth. Figure 3 Figure 1 shows the expression results of PTEN and cell stem marker in HCC1569 and MDA-MB-468 cell lines after mRNA drug treatment; Figure 4 Figure 1 shows the flow cytometry results of changes in apoptosis induced by trastuzumab and docetaxel in HCC1569 and MDA-MB-468 cell lines after mRNA drug treatment. Figure 2 shows the results of HCC1569 cell line and Figure 3 shows the results of MDA-MB-468 cell line. Figure 5 Tumor growth curve of a HER2-positive breast cancer PDX model treated with trastuzumab after mRNA drug treatment; Figure 6 Tumor growth curve of a docetaxel-treated triple-negative breast cancer PDX model after mRNA drug treatment; Figure 7 Western Blot results of PDX models after treatment with mRNA drugs in combination with chemotherapy drugs or targeted drugs; Figure 8 Tumor growth curves for a murine triple-negative breast cancer subcutaneous tumor model treated with mRNA drugs in combination with PD-1 immune checkpoint inhibitors; Figure 9 The figures show the cell activity results of treating lung cancer A549-DDP cell line and prostate cancer PC3 cell line with mRNA drugs, respectively. Figure A shows the cell activity data of A549-DDP cells treated with mRNA drugs combined with cisplatin; Figure B shows the cell activity data of PC3 cells treated with mRNA drugs combined with doxorubicin. Detailed Implementation

[0032] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0033] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0034] To facilitate a clearer understanding of this invention, certain terms are first defined. As used herein, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All patents and publications mentioned herein are incorporated herein by reference in their entirety.

[0035] The term "PLGA" refers to polylactic acid-glycolic acid copolymer; The term "DMF" refers to N,N'-dimethylformamide; The term "G0-C14" refers to a cationic lipid molecule; The term "PEG" refers to polyethylene glycol; The term "DSPE" refers to 1,2-distearyl-sn-glycerol-3-phosphoethanolamine; The term "mRNA" refers to messenger RNA.

[0036] The term "PTEN mRNA" refers to messenger RNA encoding phosphorylase and tensin homolog deleted on chromosome Ten (PTEN).

[0037] The term "tumor stemness" refers to a characteristic of tumors that relates to the ability of tumor cells to self-renew and differentiate into multiple lineages, similar to that of stem cells.

[0038] The term "targeted therapy" refers to a class of drugs that are specifically designed to target specific biomarkers (usually proteins or receptors).

[0039] The term "chemotherapy drugs" refers to drugs used to treat cancer; they work by inhibiting the growth and proliferation of cancer cells.

[0040] The term "route of administration" refers to how a drug enters the body. Common routes of administration include oral, intravenous, intramuscular, subcutaneous, and transdermal administration.

[0041] The term "subcutaneous tumor model" refers to a tumor model established in an animal by injecting a suspension of tumor cells subcutaneously into a mouse or rat.

[0042] The term "PDX model" refers to a tumor model established by directly transplanting human tumor tissue or cells into immunodeficient mice.

[0043] Example 1 Preparation and translation of linear PTEN mRNA.

[0044] The PTEN protein coding sequence shown in SEQ ID NO.1 was artificially synthesized and cloned into the pUC57-kana vector containing a T7 promoter, 5'UTR, Kozak sequence, two tandem 3'UTRs, and a polyA tail sequence. The constructed expression plasmid was transformed into *E. coli* Stbl3, amplified, cultured, and purified. The plasmid was digested with the restriction endonuclease SpeI as a template for subsequent in vitro transcription. In vitro transcription was performed using T7 RNA polymerase to synthesize linear PTEN mRNA, while simultaneously modifying the Cap1 cap structure using a capping reagent. The transcription product was treated with DNase I to remove the DNA template, and then purified using magnetic beads to obtain high-purity mRNA. The mRNA concentration and purity were determined using a micro-nucleic acid analyzer.

[0045] The obtained PTEN mRNA was delivered to human HCC1569 cells and MDA-MB-468 cells via the nanoparticles (NPs) described in Example 2. Cells were collected after 48 h of culture following transfection. Cells were lysed using RIPA lysis buffer, and proteins were collected. PTEN protein expression levels were detected using Western blotting, and PTEN gene transcription levels were detected using RT-qPCR to evaluate the in vitro expression performance of the prepared mRNA. The final sequence of the linear PTEN mRNA is shown in SEQ ID NO.3.

[0046] SEQ ID NO.3:

[0047] In addition, the present invention also prepares circular PTEN mRNA and self-replicating PTEN mRNA with the same linear PTEN mRNA sequence as described above.

[0048] 2×10 5 Two 293T cells were seeded into 12-well plates and incubated overnight. Linear PTEN mRNA, self-replicating PTEN mRNA, and circular PTEN mRNA were added using Lipo2000 transfection reagent, and incubated for 72 h. After removing the culture medium, the cells were washed once with PBS. Cells were lysed for 30 min in RIPA lysis buffer containing 1 mM PMSF on ice. The supernatant was collected after centrifugation at 12000 rpm. Protein concentration was then determined using a BCA protein assay kit. Protein samples were separated by 8% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk powder for 1 h and then incubated overnight at 4°C with either PTEN antibody (1:1000) or GAPDH antibody (1:1500). After washing with TBST, the membranes were incubated with the corresponding secondary antibody at room temperature for 2 h. Finally, enhanced chemiluminescence was used for color development. Results are shown below. Figure 1 As shown, RNA was expressed in all 293T cells, with circular RNA showing the highest expression level.

[0049] Example 2 Preparation of drugs that inhibit tumor stem cells and reduce the linear mRNA of tumor cells.

[0050] Dissolve 5 mg of PLGA in 1 mL of DMF, and dissolve 2.5 mg of G0-C14 in 1 mL of DMF. Mix 50 μL of PLGA solution and 100 μL of G0-C14 solution, with the weight ratio of circular PTEN mRNA to PLGA / G0-C14 being 1:12.5:12.5, and set aside for later use.

[0051] The above solution was quickly added to 10 mL of aqueous solution containing 1 mg DSPE-PEG to immediately form an mRNA drug that inhibits tumor stem cells and reduces tumor cell stemness. The mRNA drug was then stirred at 800 rpm for 30 min at 25°C to stabilize it.

[0052] Washing and concentration of mRNA drugs: NPs were washed three times with ice-cold ultrapure water using an Amicon ultrafiltration tube (MWCO 100 kDa) to remove organic solvents and free compounds, and finally the mRNA drugs were concentrated into 1 mL of PBS solution.

[0053] mRNA drugs can be used immediately or stored at -80 °C for later use.

[0054] Example 3 Preparation of drugs that inhibit tumor stem cells and reduce the stemness of tumor cells with circular mRNA.

[0055] Dissolve 5 mg of PLGA in 1 mL of DMF, and dissolve 2.5 mg of lipid P1 in 1 mL of DMF. Take 50 μL of PLGA solution and 100 μL of Lipid P1 solution, and mix the circular PTEN mRNA with PLGA / lipid P1 at a weight ratio of 1:10:15 for later use.

[0056] The above solution was quickly added to 10 mL of aqueous solution containing 1 mg DSPE-PEG to immediately form a circular mRNA drug that inhibits tumor stem cells and reduces tumor cell stemness. The mRNA drug was then stirred at 800 rpm for 30 min at 25°C to stabilize it.

[0057] Washing and concentration of mRNA drugs: see Example 1 for details.

[0058] Example 4 Preparation of drugs that inhibit tumor stem cells and reduce the self-replication of tumor cell stem cells using mRNA.

[0059] Dissolve 5 mg of PLGA in 1 mL of DMF, and dissolve 2.5 mg of SM-102 in 1 mL of DMF. Take 50 μL of PLGA solution and 100 μL of SM-102 solution, and mix them with self-replicating PTEN mRNA at a weight ratio of 1:15:10 for later use.

[0060] The above solution was quickly added to 10 mL of aqueous solution containing 1 mg DSPE-PEG, immediately forming a circular mRNA drug that inhibits tumor stem cells and reduces tumor cell stemness. The mRNA drug was then stabilized by stirring at 800 rpm for 30 min at 25°C.

[0061] Washing and concentration of mRNA drugs: see Example 1 for details.

[0062] Example 5 PTEN mRNA drugs effectively inhibit prostate cancer tumor stem cells and reduce tumor cell stemness.

[0063] Cell culture: PC3 prostate cancer cell lines were cultured in RPMI 1640 medium until 70-80% confluence.

[0064] Transfection procedure: PC3 cells were transfected using the linear mRNA drug prepared in Example 1 at a concentration of 100 nM. Cells were cultured for 48 h after transfection.

[0065] Protein and gene expression analysis: Protein extraction was performed using RIPA buffer, and PTEN protein levels were analyzed by SDS-PAGE and Western blotting. Anti-PTEN antibody and corresponding HRP-labeled secondary antibody were used, with GAPDH as an internal control protein. PC3 cells treated with the PTEN mRNA drug of this invention showed a significant upregulation of PTEN protein expression. Compared with the untreated control group, the level of PTEN protein in the treated cells was significantly increased.

[0066] Tumor stem cell marker analysis: The protein level of the tumor stem cell marker CD44 was analyzed using Western blotting with the corresponding anti-CD44 antibody. After treatment with the mRNA drug of this invention, the expression of the tumor stem cell marker CD44 decreased at the protein level. The results showed that the expression level of the stem cell marker was significantly reduced compared with the untransfected control group.

[0067] Cell proliferation and viability assessment: The proliferation and viability of transfected PC3 cells were assessed using the CCK-8 assay kit according to the manufacturer's instructions. Results are as follows: Figure 9 As shown in Figure B, after treatment with the mRNA drug of this invention in combination with doxorubicin, the cell viability was significantly lower than that of the control group, indicating that the drug resistance of cells treated with the mRNA drug of this invention was significantly reduced.

[0068] Example 6 PTEN mRNA drugs effectively inhibit lung cancer tumor stem cells and reduce tumor cell stemness.

[0069] Cell culture: A549-DDP lung cancer cell line was cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin.

[0070] Transfection procedure: Lung cancer cells were transfected with the linear mRNA drug prepared in Example 1 when they reached 70-80% confluency, at a concentration of 100 nM. The cells were cultured for another 48 h after treatment.

[0071] Protein and gene expression analysis: Experimental procedures are detailed in Example 5. The treated A549 cells showed significant upregulation of PTEN protein expression, with a marked increase in PTEN expression levels compared to the untreated control group.

[0072] Tumor stemness marker analysis: Experimental procedures are detailed in Example 5. Results showed that in A549 cells treated with the PTEN mRNA drug of this invention, the expression level of CD133 was significantly lower than that of the control group, indicating inhibition of tumor stemness.

[0073] Cell proliferation and viability assessment: Experimental procedures are detailed in Example 5. Results are as follows. Figure 9 As shown in Figure A, the cell viability after treatment with the mRNA drug of the present invention in combination with cisplatin was significantly lower than that of the control group, indicating that the drug resistance of cells treated with the PTEN mRNA drug of the present invention was significantly reduced.

[0074] Example 7 PTEN mRNA drugs effectively inhibit breast cancer tumor stem cells and reduce tumor cell stemness, and their application in reversing resistance to chemotherapy and targeted drugs.

[0075] Cell culture: HCC1569 and MDA-MB-468 breast cancer cell lines were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, respectively.

[0076] Transfection procedure: See Example 5 for details. Protein and gene expression analysis: See Example 5 for detailed procedures. Results are as follows. Figure 3 As shown, HCC1569 and MDA-MB-468 cells treated with the PTEN mRNA drug of the present invention showed significant upregulation of PTEN protein expression, with a significantly increased PTEN expression level compared with the untreated control group.

[0077] Tumor stem marker detection: See Example 5 for detailed procedure. Results are as follows. Figure 3 As shown, the expression level of ALDH1A1 in HCC1569 and MDA-MB-468 cells transfected with PTEN mRNA was lower than that in the control group, indicating that the tumor stemness characteristics were inhibited to a certain extent.

[0078] Apoptosis analysis: Apoptosis analysis was performed on cells treated with the PTEN mRNA drug of this invention using the Annexin V-FITC / PI apoptosis detection kit. Flow cytometry was used for data collection and analysis. Results are as follows: Figure 4 As shown in Figures A and B, compared with untransfected drug-resistant cells, cells transfected with PTEN mRNA showed a higher apoptosis rate under both different drug treatments, indicating the reversal of resistance to chemotherapy and targeted drugs.

[0079] Example 8 PTEN mRNA drugs effectively inhibit human xenograft breast cancer stem cells and reduce tumor cell stemness, and their application in reversing resistance to chemotherapy and targeted drugs.

[0080] Human-derived tissue xenograft model experiment: Fresh tumor tissue samples were obtained from patients who underwent breast cancer surgery. All relevant ethical guidelines and regulations were followed during sample acquisition, and informed consent was obtained from the patients. Sample processing: Under aseptic conditions, the tumor tissue samples were washed in ice-cold PBS solution to remove blood and necrotic tissue. The cleaned tumor tissue was cut into small pieces of approximately 3-5 mm. Immunodeficient C-NKG mice were selected as recipients for xenografting. Under aseptic conditions, the cut tumor tissue pieces were implanted subcutaneously into the mice.

[0081] Tumor growth monitoring: Mouse body weight and tumor growth were monitored regularly. Tumor length and width were recorded using calipers, and tumor volume was calculated. Once the tumor reached a predetermined volume (100 mm), the tumor was monitored. 3 (This allows for the commencement of drug treatment trials.)

[0082] PTEN mRNA drug therapeutic effect: In a breast cancer PDX model treated with PTEN mRNA of this invention, the tumor growth rate was significantly slowed. This indicates that PTEN mRNA successfully enhanced the sensitivity of tumor cells to targeted and chemotherapeutic drugs. Figure 2 As shown in Figure A, the sorted breast cancer stem cells were treated with phosphate-buffered saline (PBFS) and PTEN mRNA, respectively. Compared to the PBFS-treated group, the number of tumor stem cells in the samples treated with PTEN mRNA was significantly reduced. This indicates that PTEN mRNA successfully inhibited breast cancer tumor stem cells. Figure 2 As shown in Figure B, the PTEN mRNA drug of this invention effectively reversed drug resistance in breast cancer and significantly enhanced the anti-tumor effect of docetaxel.

[0083] The efficacy of PTEN mRNA combined with trastuzumab / docetaxel: Results are as follows Figure 5 As shown, in the HER2-positive PDX model, the PTEN mRNA drug of this invention significantly enhanced the antitumor effect of trastuzumab. The results are as follows... Figure 6 As shown, in a triple-negative breast cancer PDX model, the PTEN mRNA drug of this invention significantly enhanced the antitumor effect of docetaxel.

[0084] Apoptosis and biomarker analysis: Western blotting analysis was also performed in the PDX model, and the results are as follows... Figure 7As shown, tumor tissues treated with PTEN mRNA exhibited increased expression of apoptosis markers, and the expression level of the tumor stemness-related marker ALDH1A1 was decreased in tumor tissues compared with the untreated control group. This indicates that the PTEN mRNA drug of the present invention can effectively inhibit breast cancer tumor stem cells and reduce tumor cell stemness.

[0085] Example 9 Application of PTEN mRNA drugs in reversing resistance to immune checkpoint inhibitors.

[0086] Subcutaneous 4T1 cell line model experiment: BALB / c mouse-derived 4T1 cells were inoculated into the mammary fat pads of BALB / c mice. Mouse body weight and tumor growth were monitored regularly. Tumor length and width were recorded using calipers, and tumor volume was calculated. Once the tumor reached a predetermined volume (100 mm²), the tumor was classified as a subcutaneous model. 3 (This allows for the commencement of drug treatment trials.)

[0087] Reversal of programmed death-ligand 1 antibody resistance: Results as follows Figure 8 As shown, in the 4T1 subcutaneous model, PTEN mRNA treatment significantly enhanced the antitumor effect of programmed death-ligand 1 (CDL-1) antibody. Compared with the control group treated with CDL-1 antibody alone, the tumor growth in mice treated with PTEN mRNA combined with CDL-1 antibody was significantly slowed, and the final tumor volume was significantly reduced. Compared with the control group treated with CDL-1 antibody alone, the combined treatment group showed a significant reduction in lung metastases.

[0088] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An mRNA nanomedicine targeting PTEN, characterized in that, The mRNA nanomedicine includes any one or a combination of at least two of linear PTEN mRNA, self-replicating PTEN mRNA, or circular PTEN mRNA. The linear PTEN mRNA includes a 5'UTR sequence, a PTEN protein-coding sequence, and a 3'UTR sequence; The nucleic acid sequence encoding the PTEN protein includes the sequence shown in SEQ ID NO.

1.

2. The mRNA nanomedicine targeting PTEN according to claim 1, characterized in that, The open reading frame sequence of the linear PTEN mRNA includes any one or a combination of at least two of the following sequences: (1) A sequence as shown in SEQ ID NO.2; (2) A nucleic acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the sequence shown in SEQ ID NO.2; (3) Nucleic acid sequences that are inserted, substituted, added, or deleted based on SEQ ID NO.2; (4) Degenerate sequences of nucleic acid sequences as shown in (2) or (3).

3. The mRNA nanomedicine targeting PTEN according to claim 1 or 2, characterized in that, The linear PTEN mRNA contains any one of cap1, cap2 or cap3 cap structures; Preferably, the poly-A tail of the linear PTEN mRNA is 100-130 nucleotides in length.

4. The mRNA nanomedicine targeting PTEN according to any one of claims 1-3, characterized in that, The mRNA nanomedicine also includes a carrier.

5. The mRNA nanomedicine targeting PTEN according to claim 4, characterized in that, The carrier includes any one or a combination of at least two of the following: lipid nanoparticles, exosomes, polymer-lipid hybrid nanoparticles, metal nanoparticles, inorganic non-metal nanoparticles, adenoviruses, or retroviruses.

6. The mRNA nanomedicine according to claim 5, characterized in that, The polymer-lipid hybrid nanoparticles include polymer molecules, ionizable cationic lipid molecules, and PEGylated lipid molecules; Preferably, the polymer molecule includes any one of PLGA, PLA, or PEI; Preferably, the ionizable cationic lipid molecule includes any one of ALC-0315, SM-102, lipid Lipid P1, lipid Lipid11, or GO-C14; Preferably, the PEGylated lipid molecules include DSPE-PEG and / or DMG-PEG, wherein the number-average molecular weight of each PEG is independently 500-50000.

7. The mRNA nanomedicine according to any one of claims 4-6, characterized in that, The mass ratio of PTEN mRNA to vector is 1:(1-100).

8. A method for preparing mRNA nanomedicine targeting PTEN as described in any one of claims 1-7, characterized in that, The method includes: mixing PTEN mRNA, polymer molecules, and ionizable lipid molecules in a weight ratio of 1:(10-40):(10-40) to form an organic phase, with PEGylated lipid molecules forming an aqueous phase, and the organic phase being kept at 20-30 °C. o Rapidly add it dropwise into the aqueous phase at temperature C, and then add it within 20-30 minutes. o Stir at 800-1000 rpm for 30-60 min at C, with the volume ratio of aqueous phase to organic phase being (30-60):1, to obtain mRNA nanomedicine.

9. The use of the mRNA nanomedicine targeting PTEN as described in any one of claims 1-7 in the preparation of products for inhibiting tumor stem cells or reducing tumor cell stemness.

10. The application according to claim 9, characterized in that, The tumor includes any one of non-small cell lung cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, bladder cancer, or liver cancer.

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