MRNA nano-drug as well as preparation method and application thereof

Abstract

The invention provides an mRNA (messenger Ribonucleic Acid) nano-drug as well as a preparation method and application thereof, and particularly relates to an mRNA nano-drug aiming at double targets of HER2 and p53 and application of the mRNA nano-drug to improvement of HER2 low-expression cancer targeted therapy. The mRNA nano-drug comprises HER2mRNA, p53mRNA and a co-delivery system for loading the HER2mRNA and the p53mRNA according to different proportions. The mRNA nano-drug prepared by the invention not only remarkably improves the killing effect of an HER2 targeted drug on HER2 low-expression and HER2 negative tumor cells in vitro, but also shows an excellent anti-tumor effect in an in-vivo HER2 negative breast cancer human tumor cell line xenotransplantation model and an HER2 low-expression breast cancer human tumor xenotransplantation model; and a promising treatment strategy is provided for HER2 low expression and HER2 negative cancers.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to an mRNA nanomedicine and its preparation method and application, and specifically relates to an mRNA nanomedicine targeting HER2 and p53 dual targets and its application in improving the targeted treatment of HER2-low-expressing (and HER2-negative) cancers. Background Art

[0002] The human epidermal growth factor receptor 2 (HER2) gene is a member of the epidermal growth factor receptor (EGFR) family and plays a key role in regulating cell growth, proliferation, and differentiation. However, its overexpression drives excessive cell proliferation and survival, thereby promoting tumorigenesis. HER2 overexpression occurs in a variety of malignant tumors, especially breast cancer, making it an effective target for tumor treatment. Over the years, a series of therapeutic strategies targeting HER2 have been developed, including monoclonal antibodies, small molecule inhibitors, vaccines, and chimeric antigen receptor T cell (CAR-T) therapy. Although these approaches have changed the treatment landscape for HER2-positive cancers, they have limited benefits for patients with HER2-low or HER2-negative cancers. Due to the lack of effective treatment options, patients with HER2-low or HER2-negative cancers often have a poor prognosis, which highlights the importance of developing new therapies for this type of cancer.

[0003] The p53 gene is often called the "guardian of the genome" and is critical for maintaining genomic stability. However, p53 is one of the most commonly mutated genes in cancer, and the loss of functional p53 can lead to uncontrolled cell proliferation, driving the occurrence and development of tumors. HER2-low-expressing and HER2-negative cancers have a low response rate to HER2-targeted therapy and a poor prognosis, and restoring wild-type p53 function provides an innovative intervention. In addition, combining the restoration of wild-type p53 function with strategies to regulate HER2 expression can make these cancers more sensitive to HER2-targeted therapy. This comprehensive approach creates new treatment opportunities for patients with HER2-low-expressing and HER2-negative cancers by simultaneously targeting the tumor-driving mechanisms of HER2 and p53.

[0004] mRNA nanomedicines are an innovative therapeutic approach that utilizes mRNA (mRNA) loaded into nanoparticles to deliver genetic instructions for synthesizing therapeutic proteins directly into target cells. This technology has attracted considerable attention due to its wide range of applications, including cancer vaccines and protein replacement therapies. Compared to plasmid DNA, mRNA not only avoids the potential genotoxicity associated with integration into the host genome but also reduces long-term accumulation in the body. These properties make mRNA an ideal platform for therapeutic intervention, providing precision and controllability to therapeutic strategies.

[0005] Therefore, there is an urgent need to provide an mRNA nanodrug that can significantly increase HER2 targeting and restore wtp53 function. Summary of the Invention

[0006] In view of the current situation that patients with HER2 low-expressing and HER2-negative cancers have a low response rate to HER2 targeted therapy and lack effective treatment strategies, the purpose of the present invention is to provide an mRNA nanodrug and its preparation method and application, which can significantly increase the HER2 target and restore wtp53 function, thereby improving the responsiveness of HER2 low-expressing and HER2-negative cancers to HER2 targeted therapy, and providing a safe and effective treatment strategy for HER2 low-expressing and HER2-negative cancers.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides an mRNA nanomedicine targeting both HER2 and p53, wherein the mRNA nanomedicine comprises: HER2 mRNA and p53 mRNA;

[0009] The HER2 mRNA includes a 5'UTR sequence, a HER2-Flag protein coding sequence and a 3'UTR sequence;

[0010] The p53 mRNA includes a 5'UTR sequence, a p53 protein coding sequence and a 3'UTR sequence;

[0011] The nucleic acid sequence of the HER2-Flag protein coding sequence includes the sequence shown in SEQ ID NO.1;

[0012] The nucleic acid sequence of the p53 protein coding sequence includes the sequence shown in SEQ ID NO.3.

[0013] In the present invention, the nucleic acid sequences of the HER2-Flag protein coding sequence and the p53 protein coding sequence are codon-optimized based on the original nucleotide sequences with humans as the target host. The optimized HER2-Flag protein coding sequence and the p53 protein coding sequence are shown in SEQ ID NO.1 and SEQ ID NO.3, respectively.

[0014] Preferably, the open reading frame sequence of the HER2 mRNA includes any one or a combination of at least two of the following sequences:

[0015] (1) the sequence shown in SEQ ID NO. 2;

[0016] (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%, or 99% identity to the sequence shown in SEQ ID NO. 2;

[0017] (3) Nucleic acid sequences that are inserted, substituted, added, or deleted based on SEQ ID NO. 2;

[0018] (4) A degenerate sequence of the nucleic acid sequence shown in (2) or (3).

[0019] Preferably, the open reading frame sequence of the p53 mRNA includes any one or a combination of at least two of the following sequences:

[0020] (1) the sequence shown in SEQ ID NO. 4;

[0021] (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%, or 99% identity to the sequence shown in SEQ ID NO. 4;

[0022] (3) Nucleic acid sequences that are inserted, substituted, added or deleted based on SEQ ID NO. 4;

[0023] (4) A degenerate sequence of the nucleic acid sequence shown in (2) or (3).

[0024] SEQ ID NO.1:

[0025]

[0026] SEQ ID NO.2:

[0027]

[0028] SEQ ID NO.3:

[0029]

[0030] SEQ ID NO.4:

[0031]

[0032] Preferably, the HER2 mRNA and p53 mRNA each independently contain any one of cap1, cap2 or cap3 cap structures.

[0033] Preferably, the poly-A tails of the HER2 mRNA and p53 mRNA are each independently 100-130 nucleotides in length.

[0034] Preferably, the method for synthesizing HER2 mRNA and p53 mRNA comprises using chemically modified bases to replace conventional bases, such as N1-Me-Pseudo UTP to replace conventional UTP to reduce immunogenicity.

[0035] Preferably, the nanomedicine is characterized by a co-delivery system of HER2 mRNA and / or p53 mRNA, wherein the mass ratio of HER2 mRNA to p53 mRNA is (1-10):(1-10), for example, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10 can be selected.

[0036] Preferably, the mRNA nanomedicine further includes a carrier.

[0037] Preferably, the carrier comprises any one or a combination of at least two of lipid nanoparticles, exosomes, polymer nanoparticles, metal nanoparticles, inorganic non-metallic nanoparticles, adenovirus or retrovirus.

[0038] Preferably, the lipid nanoparticles include ionizable cationic lipid molecules, neutral helper lipid molecules, cholesterol-like lipid molecules and PEGylated lipid molecules.

[0039] Preferably, the ionizable cationic lipid molecule includes any one of ALC-0315, SM-102, lipid Lipid P1 or lipid Lipid11.

[0040] Preferably, the neutral helper lipid molecule includes any one of DOPE, DOPC or DSPC.

[0041] Preferably, the cholesterol lipid molecule comprises cholesterol and / or cholesterol hemisuccinate.

[0042] Preferably, the PEGylated lipid molecules include DSPE-PEG and / or DMG-PEG, wherein the number average molecular weight of PEG is 1000-5000 (eg, 1000, 1200, 1400, 2000, 4000, 4200, 4400, 4600, 4800 or 5000).

[0043] Preferably, the molar ratio of the ionizable cationic lipid molecules, neutral auxiliary lipid molecules, cholesterol lipid molecules and PEGylated lipid molecules is (40-45):(10-12):(38.5-40):(1.5-2).

[0044] The specific point values among the above 40 to 45 can be selected as 40, 41, 42, 43, 44 or 45.

[0045] The specific point values among the above 10 to 12 can be selected as 10, 11 or 12.

[0046] The specific point values among the above 38.5 to 40 can be selected as 38.5, 39, 39.5 or 40.

[0047] The specific point values among the above 1.5 to 2 can be selected as 1.5, 1.6, 1.7, 1.8, 1.9 or 2.

[0048] In a second aspect, the present invention provides a method for preparing the mRNA nanodrug according to the first aspect, the method comprising:

[0049] HER2 mRNA and p53 mRNA are used as the aqueous phase, and ionizable cationic lipid molecules, neutral auxiliary lipid molecules, cholesterol lipid molecules and PEGylated lipid molecules are mixed in a molar ratio of (40-45):(10-12):(38.5-40):(1.5-2) as the organic phase. The aqueous phase and the organic phase are mixed using microfluidics at a volume ratio of 1:(1-10) and a flow rate of 10-20 mL / min to obtain the mRNA nanomedicine.

[0050] The specific point values among the above 40 to 45 can be selected as 40, 41, 42, 43, 44 or 45.

[0051] The specific point values among the above 10 to 12 can be selected as 10, 11 or 12.

[0052] The specific point values among the above 38.5 to 40 can be selected as 38.5, 39, 39.5 or 40.

[0053] The specific point values among the above 1.5 to 2 can be selected as 1.5, 1.6, 1.7, 1.8, 1.9 or 2.

[0054] The specific point values from 1 to 10 above can be selected as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0055] The specific point value in the above 10-20 mL / min can be selected as 10 mL / min, 12 mL / min, 14 mL / min, 16 mL / min, 18 mL / min or 20 mL / min.

[0056] In a third aspect, the present invention provides the use of the mRNA nanomedicine targeting the dual targets of HER2 and p53 as described in the first aspect in the preparation of a product for treating HER2-low-expressing cancer or HER2-negative cancer.

[0057] Preferably, the HER2-low expressing cancer or HER2-negative cancer includes any one of breast cancer, lung cancer, ovarian cancer, colon cancer or bladder cancer.

[0058] The HER2 targeted drug used in the present invention is HER2 antibody - trastuzumab, which is administered by intraperitoneal injection.

[0059] Preferably, the administration of the mRNA nanomedicine includes intratumoral injection.

[0060] The order of administration in the present invention is to first inject the nanomedicine into the tumor, and then inject the HER2 antibody (trastuzumab) into the abdominal cavity 20-28 hours (eg, 20 hours, 24 hours or 28 hours).

[0061] Compared with the prior art, the present invention has the following beneficial effects:

[0062] The mRNA nanomedicine of the present invention can not only significantly improve the killing effect of HER2-targeted drugs (such as trastuzumab) on HER2-low-expressing (primary cells from triple-negative breast cancer patients) and HER2-negative (HCC1937, H1299) tumor cells in vitro, but also demonstrates excellent anti-tumor effects in the human tumor cell line xenograft model (CDX model) of HER2-negative breast cancer and the human tumor xenograft model (PDX model) of HER2-low-expressing breast cancer in vivo, providing a promising treatment strategy for HER2-low-expressing and HER2-negative cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 This figure shows the results of validating the stable and efficient expression of HER2 mRNA and p53 mRNA;

[0064] Figure 2 This is the result of the inhibitory effect of HER2 and p53 combined with HER2-enhancing antibody (trastuzumab) on HCC1937 cells;

[0065] Figure 3This is the result of flow cytometry analysis of the apoptosis level of HCC1937 cells after treatment with various drugs;

[0066] Figure 4 This is a graph showing the survival rate of primary breast cancer cells (p53 mutation) after drug treatment;

[0067] Figure 5 This is the survival rate result of HCC1937 cells (p53 deficiency) after drug treatment;

[0068] Figure 6 This is a graph showing the survival rate of H1299 cells (p53 deficiency) after drug treatment;

[0069] Figure 7 This is the average tumor growth curve result of tumor-bearing mice;

[0070] Figure 8 This is the survival curve of tumor-bearing mice;

[0071] Figure 9 This is the average tumor growth curve result of PDX mice;

[0072] Figure 10 This is the survival curve result of PDX mice. DETAILED DESCRIPTION

[0073] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0074] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0075] The term “DOPE” refers to dioleoylphosphatidylethanolamine;

[0076] The term “DOPC” refers to 1,2-dioleoyl- sn -glycero-3-phosphocholine;

[0077] The term “DSPC” refers to distearoylphosphatidylcholine;

[0078] The term “PEG” refers to polyethylene glycol;

[0079] The term “DSPE” refers to 1,2-distearoyl- sn -glycero-3-phosphoethanolamine;

[0080] The term "DMG" refers to dioleoylglycerol.

[0081] Example 1

[0082] Preparation and translation of HER2 mRNA and p53 mRNA.

[0083] (1) The nucleic acid sequence encoding the HER2-Flag protein shown in SEQ ID No. 1 and the nucleic acid sequence encoding the p53 protein shown in SEQ ID No. 3 were artificially synthesized, and the sequences were cloned into the T7 promoter of the pUC57-kana vector. The vector contains sequences encoding a 5'UTR, a Kozak sequence, two end-to-end 3'UTRs, and a polyA tail. The nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID No. 3 was cloned into the multiple cloning site between the Kozak sequence and the two end-to-end 3'UTRs to construct a plasmid for in vitro transcription.

[0084] (2) The constructed plasmid was transformed into Escherichia coli Stbl3, cultured and amplified, and the plasmid was extracted.

[0085] (3) Use the restriction endonuclease SpeI to digest the extracted plasmid into a linear molecule (the constructed plasmid has a unique SpeI restriction site immediately after the polyA tail).

[0086] (4) Using the plasmid linearized in step (3) as a template, the in vitro T7 transcription kit of Novizan was used to synthesize the nucleic acid sequence of HER2 mRNA with an open reading frame of SEQ ID No. 2 and / or the p53 mRNA with an open reading frame of SEQ ID No. 4, and simultaneously adding the Cap1 cap structure to the HER2 mRNA and p53 mRNA using a co-transcriptional capping method.

[0087] (5) Use DNase I to remove the DNA template from the HER2 mRNA and p53 mRNA stock solutions.

[0088] (6) Use magnetic bead purification method to obtain the desired mRNA.

[0089] (7) Use an ultra-micro nucleic acid analyzer to detect the concentration and purity of mRNA.

[0090] (8) Verification of in vitro expression of mRNA: Human 293T cells were used as expression vectors and mRNA was transfected using Lipofectamine Messenger MAX Reagent (Invitrogen, Cat#1168-027). After culturing for 48 h, cells were lysed and collected using RIPA. Protein immunoassays were used to detect the expression levels of HER2 and p53 proteins to determine whether the mRNA could be translated into protein.

[0091] (9) Figure 1 As shown, when 1 μg of HER2 mRNA was used to transfect 293T cells, the expression level of HER2 protein in the cells was significantly increased; when 1 μg of p53 mRNA was used to transfect 293T cells, the expression level of p53 protein in the cells was significantly increased. The results showed that the prepared HER2 mRNA and p53 mRNA can be stably expressed in the host cells. The sequence of HER2 mRNA is shown in SEQ ID NO.5, and the sequence of p53 mRNA is shown in SEQ ID NO.6.

[0092] SEQ ID NO.5:

[0093]

[0094] SEQ ID NO.6:

[0095]

[0096] Example 2

[0097] The killing effect of HER2 and p53 combined with HER2-enhancing antibody (trastuzumab) on HCC1937 cells was evaluated in vitro.

[0098] Lipid nanoparticles (LNPs) were used as carriers to load the HER2 mRNA and p53 mRNA obtained in Example 1 to prepare mHER2@LNP and mP53@LNP, respectively, to evaluate whether the combination of HER2 and p53 dual targets can enhance the killing effect of HER2 antibody (trastuzumab) on HCC1937 cells.

[0099] (1) Cytotoxicity test: HCC1937 cells were seeded into 96-well plates at a density of 8,000 cells / well and incubated overnight. The old culture medium was replaced with fresh culture medium containing specific concentrations of PBS, mHER2@LNP, mP53@LNP, and mHER2@LNP+mP53@LNP (the concentration of HER2 mRNA was 0.4 μg / mL and the concentration of p53 mRNA was 0.2 μg / mL). After culturing for 24 h, the old culture medium was removed and culture medium containing PBS or trastuzumab (20 μg / mL) was added. After culturing for 48 h, the culture medium was removed, the cells were washed once with PBS, and the cell viability was detected using the CCK8 kit. Figure 2 As shown, the use of HER2 antibody (trastuzumab) alone has no inhibitory effect on HCC1937 cells, while the combination of mHER2@LNPs and mP53@LNPs can make the inhibition rate of HER2 antibody (trastuzumab) on HCC1937 cells reach more than 70%. The results show that the combination of HER2 and p53 can significantly enhance the inhibitory effect of HER2 antibody on HCC1937 cells.

[0100] (2) Flow cytometry analysis of cell apoptosis: HCC1937 cells were plated at 2×10 5 The cells were seeded into 12-well plates at a density of 100 cells / well and incubated overnight. The old culture medium was replaced with fresh culture medium containing specific concentrations of PBS, mHER2@LNP, mP53@LNP, and mHER2@LNP+mP53@LNP (the concentration of HER2 mRNA was 0.4 μg / mL and the concentration of p53 mRNA was 0.2 μg / mL). After culturing for 24 hours, the old culture medium was removed and culture medium containing trastuzumab (20 μg / mL) was added. After culturing for 48 hours, the culture medium was removed and washed once with PBS. The cells were digested with EDTA-free trypsin, and after collecting the cells, the cell apoptosis was detected using the Annexin V-FITC / PI apoptosis detection kit. The results are shown in Figure 2. Figure 3As shown, trastuzumab treatment alone did not change the level of cell apoptosis, while the combination of mHER2@LNPs and mP53@LNPs increased the level of cell apoptosis to 6 times the original level. The results showed that the combination of HER2 and p53 can significantly promote the apoptosis of HER2-negative breast cancer cells.

[0101] Example 3

[0102] Preparation and physicochemical characterization of mHER2 / mP53@LNP for co-delivery of HER2 mRNA and p53 mRNA.

[0103] Preparation of mHER2@LNP, mP53@LNP, and mHER2 / mP53@LNP: Ionizable lipid, cholesterol, DOPE, and DMG-PEG2000 were dissolved in ethanol at a molar ratio of 40:10:48.5:1.5 as the organic phase, and mRNA was dissolved in 0.1 mM hydrochloric acid (pH 4) as the aqueous phase. The organic and aqueous phases were mixed using a microfluidic chip at a volume ratio of 1:3 at a total flow rate of 15 mL / min. The RNA:ionizable lipid mass ratio was 1:10, and the RNA was a mixture of HER2 mRNA and p53 mRNA at varying ratios. The resulting LNPs were collected and centrifuged through Amicon Ultra-15 ultrafiltration tubes (NMWL = 100 kDa, Millipore), washed with DNA- and RNase-free water, and finally diluted with PBS buffer containing 10% sucrose to a theoretical RNA concentration of 200 μg / mL for storage.

[0104] In the RNA mixture, the ratios of HER2 mRNA to p53 mRNA were 1:0, 0:1, and optimized 1:2, 1:1, and 2:1. Particle size, potential, and polydispersity index (PDI) were determined using a Malvern particle size analyzer, and encapsulation efficiency was measured using the Quant-iT RiboGreen kit. The physical and chemical characterization results of the mHER2 / mP53@LNPs are shown in Table 1.

[0105] Table 1

[0106]

[0107] As can be seen from Table 1, mHER2 / mP53@LNPs co-delivering HER2 mRNA and p53 mRNA in different ratios (1:2, 1:1, 2:1) have similar physicochemical properties to mHER2@LNPs or mP53@LNPs loaded with HER2 mRNA or p53 mRNA, respectively. The particle size is 136-165 nm, the PDI is less than 0.23, the Zeta potential is 14.1-29.2 mV, and the encapsulation efficiency is greater than 95%. The results show that the mHER2 / mP53@LNPs prepared by optimizing the ratio of HER2 mRNA to p53 mRNA can achieve the co-delivery of HER2 mRNA and p53 mRNA while maintaining its excellent physicochemical properties.

[0108] Example 4

[0109] mHER2 / mP53@LNP, which co-delivers HER2 mRNA and p53 mRNA, enhances the killing effect of HER2 antibody (trastuzumab) on tumor cells in vitro.

[0110] Patient-derived primary breast cancer cells (HER2 low expression, p53 mutation), HCC1937 cells (HER2 negative, p53 deletion), and H1299 cells (HER2 negative, p53 deletion) were seeded into 96-well plates at a density of 8,000 cells / well and incubated overnight. The old culture medium was replaced with fresh culture medium containing specific concentrations of PBS, mHER2@LNP, mP53@LNP, mHER2@LNP+mP53@LNP, and mHER2 / mP53@LNP, respectively. After 24 hours of culture, the old culture medium was removed and culture medium containing PBS or trastuzumab (20 μg / mL) was added. After 48 hours of culture, the culture medium was removed and CCK8 working solution was added. The absorbance was measured at a wavelength of 450 nm using a microplate reader. For patient-derived primary breast cancer cells (low HER2 expression, p53 mutation), mHER2 / mP53@LNP with a ratio of mHER2:mP53=1:2 was used, the HER2 mRNA concentration was 0.4μg / mL, and the p53 mRNA concentration was 0.8μg / mL; for HCC1937 cells, mHER2 / mP53@LNP with a ratio of mHER2:mP53=2:1 was used, the HER2 mRNA concentration was 0.4μg / mL, and the p53 mRNA concentration was 0.2μg / mL; for H1299 cells, mHER2 / mP53@LNP with a ratio of mHER2:mP53=1:1 was used, the HER2 mRNA concentration was 0.4μg / mL, and the p53 mRNA concentration was 0.4μg / mL.

[0111] The results are as follows Figure 4-Figure 6As shown in the results, trastuzumab alone had no inhibitory effect on HCC1937 cells (HER2 negative, P53 deleted), H1299 cells (HER2 negative, P53 deleted), and patient-derived primary breast cancer cells (HER2 low expression, P53 mutation). However, the inhibition rates of trastuzumab combined with mHER2 / mP53@LNP on the three cell lines increased to 81.3%, 70.4%, and 59.4%, respectively. In addition, compared with the mHER2@LNP+mP53@LNP+trastuzumab group that delivered HER2 mRNA and p53 mRNA separately, the mHER2 / mP53@LNP+trastuzumab group of the co-delivery system not only maintained the inhibitory effect of the combined HER2 and p53 dual targets in HCC1937 cells and H1299 cells, but also showed a better inhibitory effect in primary breast cancer cells (low HER2 expression, P53 mutation). The results showed that the optimized co-delivery system amplified the advantages of the combined HER2 and p53 dual targets and further improved the sensitivity of p53-mutated breast cancer to HER2 targeted therapy.

[0112] Example 5

[0113] Evaluation of the therapeutic efficacy of HER2 mRNA combined with p53 mRNA-enhancing HER2 antibody (trastuzumab) in a HER2-negative breast cancer xenotumor model.

[0114] A p53-deficient, HER2-negative HCC1937 xenograft model was constructed using athymic nude mice for in vivo anti-tumor studies. Specifically, mice received intratumoral injections of mHER2@LNP or mHER2@LNP+mP53@LNP (HER2 mRNA 5μg / mouse, p53 mRNA 2.5μg / mouse) every three days for a total of six treatments. Trastuzumab (200μg / mouse) was injected intraperitoneally 24 hours after each LNP injection. Mice treated with PBS served as a control group. The tumor volume and survival of the mice were monitored, and tumor growth and survival curves were plotted.

[0115] The results are as follows Figure 7 As shown in the figure, tumors in tumor-bearing mice treated with PBS and trastuzumab alone grew rapidly, while treatment with the combined HER2 and p53 mHER2@LNP+mP53@LNP+trastuzumab group completely stopped tumor growth within 30 days after inoculation. Figure 8 It can be seen that compared with trastuzumab treatment alone, the survival time of mice treated with mP53@LNP+mHER2@LNP+trastuzumab was significantly prolonged (P<0.05).

[0116] Example 6

[0117] mHER2 / mP53@LNP enhances the therapeutic effect of HER2 antibody (trastuzumab) on PDX models derived from patients with HER2-low expressing breast cancer.

[0118] Tumor tissue from breast cancer patients with low HER2 expression and p53 mutation was transplanted into the back of immunodeficient mice to construct a PDX model with low HER2 expression. 3 Tumor-bearing mice were injected intratumorally with mHER2@LNP or mHER2 / mP53@LNP (HER2 mRNA 5 μg / mouse, p53 mRNA 10 μg / mouse) every three days. Trastuzumab 200 μg / mouse was then injected intraperitoneally 24 hours after LNP injection for a total of four treatments. Mice treated with PBS served as a control group. Tumor volume and survival were monitored, and tumor growth and survival curves were plotted.

[0119] The results are as follows Figure 9 As shown in the results, in a human tumor xenograft (PDX) model with low HER2 expression, trastuzumab alone had no tumor inhibitory effect, but combined with mHER2 / mP53@LNP, it showed a significant tumor inhibitory effect (P<0.001). Figure 10 As shown, among all treatment groups, mHER2 / mP53@LNP+trastuzumab prolonged the survival of tumor-bearing mice the most (P<0.01). Therefore, the results indicate that the synergistic effect of the HER2 / p53 dual-target therapy amplified by the co-delivery system (mHER2 / mP53@LNP) significantly enhanced the therapeutic effect of HER2 antibody (trastuzumab) on PDX models of HER2-low expressing breast cancer.

[0120] In summary, the present invention provides a strategy that can significantly improve targeted therapy for HER2-low-expressing and HER2-negative cancers by co-delivering HER2 mRNA and p53 mRNA. First, by optimizing the mRNA sequence and improving the synthesis process, HER2 mRNA and p53 mRNA with stable and efficient expression were prepared. Second, by optimizing the ratio of HER2 mRNA to p53 mRNA, an mHER2 / mP53 co-delivery system with excellent physicochemical properties was prepared. Finally, by co-delivering HER2 mRNA and p53 mRNA, not only was the killing effect of HER2 antibody (trastuzumab) on HER2-low-expressing (or negative) tumor cells significantly improved in cell experiments, but also significantly enhanced the anti-tumor effect of HER2 antibody (trastuzumab) in HER2-negative breast cancer CDX models and HER2-low-expressing breast cancer PDX models, and significantly improved the survival of tumor-bearing mice. The present invention significantly improves the anti-tumor effect of HER2-targeted therapy in HER2-low-expressing (or negative) cancers, providing a promising therapeutic strategy for the treatment of HER-2-low-expressing (or negative) cancers.

[0121] The applicant states that the present invention is intended to illustrate the detailed methods of the present invention through the above-described embodiments, but the present invention is not limited to the above-described detailed methods, that is, it does not mean that the present invention must rely on the above-described detailed methods in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for various raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

Claims

1. An mRNA nanomedicine targeting HER2 and p53 dual targets, characterized in that: The mRNA nanomedicine includes: HER2 mRNA and p53 mRNA; The HER2 mRNA includes a 5'UTR sequence, a HER2-Flag protein coding sequence and a 3'UTR sequence; The p53 mRNA includes a 5'UTR sequence, a p53 protein coding sequence and a 3'UTR sequence; The nucleic acid sequence of the HER2-Flag protein coding sequence includes the sequence shown in SEQ ID NO.1; The nucleic acid sequence of the p53 protein coding sequence includes the sequence shown in SEQ ID NO.

3.

2. The mRNA nanomedicine targeting HER2 and p53 dual targets according to claim 1, characterized in that: The open reading frame sequence of the HER2 mRNA includes any one or a combination of at least two of the following sequences: (1) the sequence 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%, or 99% identity to 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) a degenerate sequence of the nucleic acid sequence shown in (2) or (3); Preferably, the open reading frame sequence of the p53 mRNA includes any one or a combination of at least two of the following sequences: (1) the sequence shown in SEQ ID NO. 4; (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%, or 99% identity to the sequence shown in SEQ ID NO. 4; (3) Nucleic acid sequences that are inserted, substituted, added or deleted based on SEQ ID NO. 4; (4) A degenerate sequence of the nucleic acid sequence shown in (2) or (3).

3. The mRNA nanomedicine targeting HER2 and p53 dual targets according to claim 1 or 2, characterized in that: The HER2 mRNA and p53 mRNA each independently contain any one of cap1, cap2 or cap3 cap structures; Preferably, the poly-A tails of the HER2 mRNA and p53 mRNA are each independently 100-130 nucleotides in length.

4. The mRNA nanomedicine targeting HER2 and p53 dual targets according to any one of claims 1 to 3, characterized in that: The nanomedicine is characterized by a co-delivery system of HER2 mRNA and / or p53 mRNA, wherein the mass ratio of HER2 mRNA to p53 mRNA is (1-10):(1-10).

5. The mRNA nanomedicine targeting HER2 and p53 dual targets according to any one of claims 1 to 4, characterized in that: The mRNA nanomedicine further includes a carrier; Preferably, the carrier comprises any one or a combination of at least two of lipid nanoparticles, exosomes, polymer nanoparticles, metal nanoparticles, inorganic non-metallic nanoparticles, adenovirus or retrovirus.

6. The mRNA nanomedicine according to claim 5, characterized in that The lipid nanoparticles include ionizable cationic lipid molecules, neutral auxiliary lipid molecules, cholesterol lipid molecules and PEGylated lipid molecules; Preferably, the ionizable cationic lipid molecule comprises any one of ALC-0315, SM-102, lipid Lipid P1 or lipid Lipid11; Preferably, the neutral helper lipid molecule comprises any one of DOPE, DOPC or DSPC; Preferably, the cholesterol lipid molecule comprises cholesterol and / or cholesterol hemisuccinate; Preferably, the PEGylated lipid molecules include DSPE-PEG and / or DMG-PEG, wherein the number average molecular weight of PEG is 1000-5000.

7. The mRNA nanodrug according to claim 6, characterized in that The molar ratio of the ionizable cationic lipid molecules, the neutral auxiliary lipid molecules, the cholesterol lipid molecules and the PEGylated lipid molecules is (40-45): (10-12): (38.5-40): (1.5-2).

8. A method for preparing the mRNA nanodrug according to any one of claims 1 to 7, characterized in that: The method comprises: HER2 mRNA and p53 mRNA are used as the aqueous phase, and ionizable cationic lipid molecules, neutral auxiliary lipid molecules, cholesterol lipid molecules and PEGylated lipid molecules are mixed in a molar ratio of (40-45):(10-12):(38.5-40):(1.5-2) as the organic phase. The aqueous phase and the organic phase are mixed using microfluidics at a volume ratio of 1:(1-10) and a flow rate of 10-20 mL / min to obtain the mRNA nanomedicine.

9. Use of the mRNA nanodrug targeting both HER2 and p53 according to any one of claims 1 to 7 in the preparation of a product for treating HER2-low-expressing cancer or HER2-negative cancer.

10. The use according to claim 9, characterized in that The HER2 low-expressing cancer or HER2-negative cancer includes any one of breast cancer, lung cancer, ovarian cancer, colon cancer or bladder cancer.

Images