Self-replicating RNA expressing differentiation-related transcription factor and use thereof in preparation of tumor therapeutic drug

Abstract

The present invention pertains to the technical field of biomedicine, and particularly relates to a self-replicating RNA expressing a differentiation-related transcription factor and use thereof in the preparation of a tumor therapeutic drug. The present invention is a technical means for inducing tumor cells to differentiate into mature cells, which utilizes messenger ribonucleic acid to regulate and control the expression of important differentiation-related transcription factors in tumor cells, inhibits the malignant phenotype of malignant solid tumor cells, and achieves the effect of treating malignant solid tumors, thereby being applied to the preparation methods and use of solid tumor drugs.

Description

Self-replicating RNA expressing differentiation-related transcription factors and application thereof in preparation of tumor treatment drugs TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to self-replicating RNA expressing differentiation-related transcription factors and application thereof in preparation of tumor treatment drugs, which is a technical means for inducing tumor cells to differentiate into mature cells, and utilizes messenger ribonucleic acid to regulate the expression of important differentiation-related transcription factors in tumor cells, so as to inhibit the malignant phenotype of malignant solid tumor cells and achieve the effect of treating malignant solid tumors, thereby being applied to the preparation method and application of solid tumor drugs. BACKGROUND

[0002] The treatment of malignant solid tumors is one of the difficulties in current clinical practice, especially for malignant solid tumors that cannot be completely removed by surgery, and there is still a lack of effective treatment means in clinical practice. Although in recent years, radiotherapy, chemotherapy, targeted therapy and immunotherapy have provided various means for treating tumors, tumor progression often leads to drug resistance, and a large number of tumors lack effective target drug targets or are not sensitive to immunotherapy, especially some digestive system tumors such as liver cancer, pancreatic cancer, and brain glioma, which lack effective drug treatment means, and the prognosis of patients is not optimistic.

[0003] Tumor differentiation therapy is a strategy that breaks the conventional thinking of tumor treatment, which promotes tumor cells to differentiate into mature normal cells, restores their normal phenotype and function, and inhibits the proliferation of malignant tumor cells. The classic example is the use of all-trans retinoic acid to differentiate acute promyelocytic leukemia, which has achieved good clinical results and has been widely used. However, the induced differentiation therapy for malignant solid tumors is still a difficulty in current tumor treatment. Recent studies have found that transcription factors related to organ differentiation and function maintenance can induce related tumor cells to differentiate into normal cells, and up-regulating these differentiation-related transcription factors can inhibit tumor cell growth, but has no significant effect on the function of normal tissue cells, which opens up a new direction for tumor induced differentiation, especially for the treatment of malignant solid tumors. Therefore, for different types of tumors, specific targeting of proteins, molecules and genes closely related to tumor cell differentiation is the core problem of tumor induced differentiation therapy, and targeted regulation of important differentiation function gene expression using genetic engineering technology can induce tumor cells to differentiate into mature cell phenotype, which may fundamentally reverse the progression of malignant tumors.

[0004] The hepatocyte nuclear factor (HNF) family includes HNF1, HNF3, HNF4, HNF6 and CCAAT / enhancer binding protein (C / EBP), which is a group of transcription factors that are predominantly expressed in the liver and are mutually regulated, playing a key role in liver development and function maintenance. Among them, HNF4α is expressed in liver, kidney, pancreas, intestine and other tissues, but mainly expressed in mature hepatocytes. In mature hepatocytes, HNF4α can bind to about 12% of the gene promoters in the cell, participate in maintaining the important functions of hepatocytes such as lipid metabolism, albumin synthesis, drug detoxification, energy metabolism, and bile acid synthesis, and is also an important gene for maintaining the epithelial phenotype of hepatocytes. HNF4α also regulates the development of kidney and intestinal tissues, and regulates the production of insulin. Studies on liver cancer have found that the dedifferentiation state of liver cancer is accompanied by a large number of down-regulation of hepatocyte nuclear factor expression, among which the down-regulation of HNF4α expression is an important link in the occurrence of liver cancer. Previous studies have shown that HNF4α expression is decreased in liver cancer (including hepatocellular carcinoma, intrahepatic cholangiocarcinoma), pancreatic cancer, intestinal cancer, kidney cancer and other tumors of epithelial origin, and overexpression of HNF4α can inhibit tumor cell proliferation, metastasis, and promote tumor cell apoptosis, suggesting that HNF4α is a potential target for tumor therapy (Differentiation therapy of hepatocellular carcinoma in mice with recombinant adenovirus carrying hepatocyte nuclear factor-4alpha gene. Hepatology. 2008 Nov; 48(5): 1528-39).

[0005] HNF1a is another important hepatocyte nuclear factor that binds to the cis-acting elements of at least 200 liver target genes involved in many important functions of the liver, such as glycogen synthesis and storage, gluconeogenesis, lipid metabolism, serum protein synthesis and detoxification. Previous studies have shown that HNF1a expression is decreased in hepatocellular carcinoma, and upregulating HNF1a in hepatoma cells can promote the expression of liver function genes in hepatoma cells, arrest the cell cycle of hepatoma cells in the G2 / M phase, and thus inhibit tumor cell proliferation and significantly inhibit tumor growth in vivo (Recombinant adenovirus carrying the hepatocyte nuclear factor-1 alpha gene inhibits hepatocellular carcinoma xenograft growth in mice. Hepatology, 2011, 54(6): 2036-2047.). Recent studies have found that overexpression of a group of hepatocyte nuclear factors including HNF4a, HNF1a and FOXA3 (also known as HNF3y) can successfully transform hepatoma cells into mature functional hepatocytes in vitro and in vivo, further indicating that increasing the expression of hepatocyte nuclear factors such as HNF4a, HNF1a and FOXA3 is an ideal strategy for inducing differentiation therapy for hepatocellular carcinoma. (Conversion of hepatoma cells to hepatocyte-like cells by defined hepatocyte nuclear factors. Cell Res. 2019 Feb; 29(2): 124-135.)

[0006] Pancreas associated transcription factor 1a (PTF1A) plays an important role in the development of mammalian pancreas and is involved in maintaining the expression of exocrine pancreas-specific genes, including elastase 1 and amylase. Previous studies have shown that PTF1A is deleted in ductal pancreatic cancer, and maintaining PTF1A expression can completely block the formation of pancreatic cancer cells. Restoring PTF1A expression can induce early cancer cells to transform into normal pancreatic cells and inhibit the growth of advanced pancreatic cancer cells, making PTF1A a potential target for differentiation therapy for pancreatic cancer. (Prevention and Reversion of Pancreatic Tumorigenesis through a Differentiation-Based Mechanism. Dev Cell. 2019 Sep 23; 50(6): 744-754.e4.)

[0007] Neuronal differentiation 1 (NEUROD1) and Neurogenin 2 (NEUROG2), ASCL1 are transcription factors related to neural differentiation, which are involved in inducing neural differentiation during early brain development and play an important role in early brain development by inducing neural stem cells to differentiate into neurons. Previous studies have shown that NEUROD1 can induce astrocytes in the brain to differentiate into neurons in various brain disease models such as Alzheimer's disease, Huntington's disease, stroke, and epilepsy. Overexpression of NEUROD1, NEUROG2 and ASCL1 in glioma cells can transform proliferative glioma cells into non-proliferative neurons, which are important targets for inducing differentiation therapy for brain glioma. (Transcription factor-based gene therapy to treat glioblastoma through direct neuronal conversion. Cancer Biol Med. 2021 Mar 23; 18(3): 860-74.)

[0008] These studies on differentiation-related transcription factors inducing related tumors to mature cells further provide a basis for inducing differentiation therapy for tumors. Currently, the function of transcription factors is mainly completed in the nucleus. In order to achieve the purpose of up-regulating related transcription factors to treat tumors, we need to use vector-mediated expression of related genes in tumor cells, and need effective means to maintain their expression in tumor cells. The expression vectors used in the past, such as adenovirus, adeno-associated virus, lentivirus and plasmid, all have defects in safety, expression efficiency and tissue accessibility. The patent previously owned by the inventor's team uses adenovirus vector to express HNF4α and HNF1α, which confirms the inhibitory effect of HNF4α and HNF1α on hepatocellular carcinoma. Due to the rapid proliferation ability of tumors, repeated administration is required for tumor treatment to ensure treatment effect. However, viral vectors can induce the immune system to reject the delivery system, and there is also pre-existing immunity to adenovirus in the human population, which limits the use of viral vectors to mediate the expression of target genes in tumor cells and the frequency of administration for tumor treatment. At the same time, adenovirus vectors have defects in safety and tissue targeting, and the preparation cost is relatively high, so the potential for practical application in tumor treatment is low.

[0009] mRNA technology is a revolutionary gene delivery technology developed in recent years. This technology synthesizes mRNA molecules with specific sequences by in vitro transcription, and uses lipid nanoparticles (LNPs) to encapsulate mRNA for transport into human cells, relying on the cell's own translation system to translate mRNA into target proteins. Compared with the existing delivery system based on viral vectors, it has good efficiency and safety; if combined with the screening and modification of nano-lipid particles, the tissue specificity of the delivery system can be increased. The potential application direction of the currently known mRNA technology mainly focuses on vaccine development for infectious diseases, therapeutic tumor vaccines, protein replacement therapy for protein deficiency caused by genetic defects, and gene editing to treat genetic diseases or modify immune cells. However, since mRNA can only mediate the transient high expression of the target gene in cells, there are significant deficiencies in expression duration and efficiency, which cannot meet the demand for relatively long-term stable expression of target proteins in rapidly proliferating tumor cells.

[0010] Self-amplifying RNA (saRNA, or self-replicating RNA, srRNA) is a gene delivery vector based on mRNA technology developed in recent years, which is a recombinant RNA with viral structure. By modifying the bi-cistronic genome of alphaviruses, the sequence encoding four non-structural proteins (NSPs) from the virus is retained, and the viral structural protein gene located after the subgenome promoter (SGP) is replaced by a heterologous gene (gene of interest, GOI) encoding the protein of interest, to construct a RNA vector that can express the gene of interest with self-amplification ability in cells. At present, the self-amplifying RNA vector is mainly modified from Venezuelan equine encephalitis virus (VEEV), sindbis alphavirus (SIN) or Semliki Forest virus (SFV), among which the self-amplifying RNA derived from Venezuelan equine encephalitis virus is the most commonly used. The self-amplifying RNA enters the cytoplasm of the host cell, first translates the four non-structural proteins (NSP1, NSP2, NSP3, NSP4), and then polymerizes to form an RNA-dependent RNA polymerase complex through a complex, multi-step process. The RNA polymerase complex first synthesizes a complementary negative-strand RNA intermediate from the positive-strand RNA, and then synthesizes two different positive-strand RNAs using the latter as a template. The first positive-strand RNA is a copy of the original full-length genomic RNA; the second positive-strand RNA is a large number of subgenomic RNAs encoding the gene of interest. This mechanism enables self-amplifying RNA to achieve high-level and persistent expression of the target protein at a low dose of self-amplifying RNA. Self-amplifying RNA retains the self-amplification ability of the viral genome, but cannot express the structural proteins of the virus, and therefore cannot produce complete viruses with the ability to spread. Its entry into cells depends on the delivery of lipid nanoparticles, so it has good safety and potential tumor targeting ability.

[0011] Currently in tumor treatment, self-replicating RNA is considered to be applicable to the preparation of RNA vaccines and the expression of tumor-killing cytokines in tumor cells. For example, Chinese invention patent application CN117280029A discloses a nucleic acid vector and a use method, which modulates the tumor microenvironment by activating the relative expression of tumor-infiltrating lymphocytes and / or the immunogenic cell characteristics in the tumor microenvironment to achieve the treatment of cancer; Chinese invention patent application CN117279661A discloses a composition and method for inducing ESR1, PI3K, HER2 and HER3 immune response, which is a method for treating cancer by inducing immune response; Chinese invention patent application CN115968299A discloses a new antigen expressed in multiple myeloma and its use, which also uses self-replicating RNA as a carrier to prepare a tumor vaccine. The working principle of the tumor vaccine is to express tumor antigens in antigen-presenting cells (APCs), such as dendritic cells (DCs), and present them to T cells of the immune system, activate T cells to attack tumor cells, and then control or eliminate tumors.

[0012] (DCs) and present them to T cells of the immune system, activate T cells to attack tumor cells, and then control or eliminate tumors.

[0013] However, there is no report on using self-replicating RNA to express transcription factors, especially tissue and organ differentiation-related transcription factors, in tumor cells to treat tumors. SUMMARY

[0014] The purpose of the present application is to provide a self-replicating RNA expressing differentiation-related transcription factors and its application in the preparation of tumor treatment drugs. By using self-replicating RNA to express transcription factors in tumor cells, the differentiation of tumor cells into mature cells is induced, the malignant phenotype of malignant solid tumor cells is inhibited, and the effect of treating malignant solid tumors is achieved. The present application needs to solve the problem of how to long-term and specifically express related transcription factors in tumor cells and improve the effect of inhibiting tumors.

[0015] The expression amount of the related differentiation transcription factor in the related tumor cells is lower than that in normal cells. The inventors found that after self-replicating RNA enters tumor cells, based on its self-replication characteristics, the distribution in tumor cells does not decrease with the rapid proliferation of tumor cells, and the protein can be expressed for more than 20-30 days, so it is very suitable for mediating the expression of target genes in tumor cells. Animal experiments show that tail vein injection of lipid nanoparticles encapsulated self-replicating RNA can mediate the specific expression of target genes in tumor tissues, while the expression of target genes in other normal tissues is basically not detected, indicating that self-replicating RNA has good tumor targeting. Based on these findings, the inventors propose to use self-replicating RNA vectors to restore or overexpress HNF4a in tumor tissues, induce tumor cells to differentiate into normal cells, inhibit tumor cell growth, and thus achieve the effect of treating tumors.

[0016] The present application introduces relevant induced differentiation transcription factors into relevant tumor cells by self-replicating RNA technology to overexpress in tumor cells, and to achieve the purpose of inducing tumor cells to differentiate into mature cells to treat tumors.

[0017] The length of the polyA tail of the self-replicating RNA is optimized to maintain longer and higher level expression of the target gene in vivo. The structure of the self-replicating RNA includes a 5' cap, a non-coding region (5' UTR), four non-structural genes (NSP1-4), a 26S subgenomic promoter (SGP), a target gene (GOI), a 3' non-coding region (3' UTR), and a polyA tail. In order to optimize the performance of the self-replicating RNA, previous studies have tried various strategies to modify NSP1-4 by introducing mutations to improve the replication efficiency and immunogenicity of the self-replicating RNA, and to increase the expression intensity and duration of different target genes in cells. We found that the efficiency of different NSP1-4 mutant self-replicating RNA vectors in mediating HNF4α expression in tumor cells was not significantly different. On the other hand, previous studies have shown that the length and sequence of the polyA tail of mRNA affect the stability and ribosome translation of mRNA, and changing the length of the polyA tail can affect the expression duration and expression level of the encoded protein by regulating the degradation rate and translation level of mRNA. However, it is not clear whether the polyA of the self-replicating RNA affects the expression duration and expression level of the target gene in tumor cells. In order to obtain a self-replicating RNA vector that can mediate stable and efficient expression of the target gene in tumor cells, we optimized the polyA length of the self-replicating RNA used with HNF4α as the target gene, and found that the self-replicating RNA with a polyA length of 35-100 nt can mediate stable and long-term overexpression of HNF4α in tumor cells, among which the expression level and duration of 50-70 nt are better, and the self-replicating RNA with a polyA tail of 60-70 nt can achieve the most stable and efficient expression.

[0018] Meanwhile, the application also investigates the effect of non-replicating HNF4a-mRNA and HNF4a-saRNA and adenovirus-mediated HNF4a overexpression in tumor cells, and the results show that both HNF4a-saRNA and HNF4a-mRNA can up-regulate HNF4a in hepatoma cells, but the expression amount of HNF4a-mRNA is lower and the expression time is shorter; the expression amount of HNF4a-saRNA is more than twice that of HNF4a-mRNA, and the maintenance time is long, the effect of HNF4a-mRNA on up-regulating HNF4a expression can only be maintained for 3 days, while HNF4a-saRNA can maintain HNF4a in hepatoma cells for at least 7 days, and the expression efficiency of HNF4a-saRNA on up-regulating HNF4a is not lower than that of adenovirus, which indicates that self-replicating RNA can more efficiently mediate the expression of target genes in tumor cells than non-replicating mRNA.

[0019] In the first aspect of the application, a self-replicating RNA for expressing a differentiation-related transcription factor is provided, wherein the differentiation-related transcription factor is a transcription factor related to the differentiation and function maintenance of tissues and organs, and the expression amount of the transcription factor in tumor cells is lower than that in normal cells; the transcription factor is delivered into tumor cells by self-replicating RNA and a delivery vehicle, and the transcription factor is long-acting and specifically highly expressed in tumor cells, so as to induce the differentiation of tumor cells into normal mature cells, inhibit the proliferation of tumor cells or / and induce the apoptosis of tumor cells.

[0020] The long-acting expression refers to expression for at least 7 days (including 7 days).

[0021] The specific high expression refers to no expression or low expression or short-term expression in normal cells.

[0022] The self-replicating RNA comprises a 5' cap, a non-coding region, a non-structural gene, a 26S subgenomic promoter, a 3' non-coding region and a poly(A) tail.

[0023] The self-replicating RNA has a 35-100 nt poly(A) tail.

[0024] Preferably, the self-replicating RNA has a 40-90 nt poly(A) tail.

[0025] More preferably, the self-replicating RNA has a 50-75 nt poly(A) tail.

[0026] Most preferably, the self-replicating RNA has a 60-70 nt poly(A) tail.

[0027] The transcription factor is selected from the group consisting of HNF4a, HNF1a, FOXA3, PTF1A, NUROND1, Neurogenin-2, Ascl1, and other tissue organ differentiation and function maintenance related transcription factors.

[0028] HNF4a (GENBANK No. NM_000457.6, as shown in SEQ ID NO: 1);

[0029] HNF1a (GENBANK No. NM_001306179.2, as shown in SEQ ID NO: 2);

[0030] FOXA3 (GENBANK No. NM_004497.3, as shown in SEQ ID NO: 3);

[0031] PTF1A (GENBANK No. NM_178161.3, as shown in SEQ ID NO: 4);

[0032] NUROND1 (GENBANK No. NM_002500.5, as shown in SEQ ID NO: 5);

[0033] Neurogenin-2 (GENBANK No. NM_024019.4);

[0034] Ascl1 (GENBANK No. NM_004316.4).

[0035] The delivery vehicle is selected from the group consisting of one or more of the following: liposomes, viral replicon particles, lipid-based nanoparticles, polymeric nanoparticles, physiological buffers, microspheres, immunostimulatory complexes, conjugates of biologically active ligands.

[0036] In a preferred embodiment of the present application, the sequence of the self-replicating RNA is as shown in SEQ ID NO: 6. This sequence is an optimized polyA tail for the wild-type self-replicating RNA, preferably a 67nt polyA tail, which can increase the half-life of the saRNA in vivo, and promote the stability and translation of the saRNA.

[0037] In a preferred embodiment of the present application, the sequence of the HNF4a self-replicating RNA comprises any one of the following (a) to (c):

[0038] (a) RNA consisting of the nucleotide sequence shown in SEQ ID NO: 7;

[0039] (b) a nucleotide sequence having one or a plurality of deletions, substitutions, additions, or insertions of nucleotides in the nucleotide sequence represented by SEQ ID NO: 7 and having an activity of expressing HNF4α; and

[0040] (c) a nucleotide sequence having 90% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 7 and having an activity of expressing HNF4α.

[0041] In one preferred embodiment of the present application, the sequence of the HNF1α self-replicating RNA includes any one of the following (a) to (c):

[0042] (a) an RNA consisting of the nucleotide sequence represented by SEQ ID NO: 8;

[0043] (b) a nucleotide sequence having one or a plurality of deletions, substitutions, additions, or insertions of nucleotides in the nucleotide sequence represented by SEQ ID NO: 8 and having an activity of expressing HNF1α; and

[0044] (c) a nucleotide sequence having 90% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 8 and having an activity of expressing HNF1α.

[0045] In one preferred embodiment of the present application, the sequence of the FOXA3 self-replicating RNA includes any one of the following (a) to (c):

[0046] (a) an RNA consisting of the nucleotide sequence represented by SEQ ID NO: 9;

[0047] (b) a nucleotide sequence having one or a plurality of deletions, substitutions, additions, or insertions of nucleotides in the nucleotide sequence represented by SEQ ID NO: 9 and having an activity of expressing FOXA3; and

[0048] (c) a nucleotide sequence having 90% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 9 and having an activity of expressing FOXA3.

[0049] In one preferred embodiment of the present application, the sequence of the PTF1A self-replicating RNA includes any one of the following (a) to (c):

[0050] (a) an RNA consisting of the nucleotide sequence represented by SEQ ID NO: 10;

[0051] (b) An RNA consisting of a nucleotide sequence having one or more nucleotide deletions, substitutions, additions, or insertions in the nucleotide sequence shown in SEQ ID NO:10 and having activity expressed as PTF1A; and

[0052] (c) An RNA consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:10 and having the activity of being expressed as PTF1A.

[0053] In a preferred embodiment of the present invention, the sequence of the NUROND1 self-replicating RNA includes any one of the following (a) to (c):

[0054] (a) RNA consisting of the nucleotide sequence shown in SEQ ID NO:11;

[0055] (b) RNA consisting of a nucleotide sequence having one or more nucleotide deletions, substitutions, additions, or insertions in the nucleotide sequence shown in SEQ ID NO:11 and having NUROND1 expression activity; and

[0056] (c) An RNA consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:11 and having the activity of being expressed as NUROND1.

[0057] In a second aspect, the invention provides the use of the self-replicating RNA expressing differentiation-related transcription factors in the preparation of drugs for treating malignant solid tumors.

[0058] The malignant solid tumors mentioned are liver cancer, pancreatic cancer, stomach cancer, intestinal cancer, kidney cancer, lung cancer, and glioma.

[0059] The transcription factors are selected from HNF4α, HNF1α, FOXA3, and PTF1A, and the malignant solid tumors are malignant solid tumors derived from epithelial cells. These malignant solid tumors include liver cancer, pancreatic cancer, gastric cancer, intestinal cancer, kidney cancer, and lung cancer.

[0060] The transcription factors mentioned are selected from NUROND1, Neurogenin-2, and Ascl1, and the malignant solid tumor mentioned is glioma.

[0061] In a third aspect, the present invention provides a gene delivery system comprising a self-replicating RNA expressing a differentiation-related transcription factor as described above and a delivery medium; the gene delivery system delivers a target gene into tumor cells, thereby achieving long-term and specific high expression of the transcription factor within the tumor cells, inducing the tumor cells to differentiate into normal mature cells, inhibiting tumor cell proliferation, and / or inducing tumor cell apoptosis.

[0062] In one preferred embodiment of the present application, the delivery vehicle is a lipid-based nanoparticle (LNP) having a composition comprising an ionizable lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and dimyristylglycerol-polyethylene glycol 2000 (DMG-PEG 2000) or a pegylated lipid containing a polyethylene glycol moiety. Preferably, the LNP has a composition comprising DSPC: cholesterol: DMG-PEG-2000: ionizable lipid in a molar ratio ranging from 5%-20% DSPC, 30%-55% cholesterol, 0.5%-3% PEG, and 30%-60% ionizable lipid, with the total of the molar ratios of the lipids being 100%, and optionally 9.4:42.5:1.8:46.3 (DSPC: cholesterol: DMG-PEG 2000: ionizable lipid). The LNP has a N:P ratio ranging from 5:1 to 10:1, and optionally 6:1, and a particle size of about 40-300 nm.

[0063] In one preferred embodiment of the present application, the delivery vehicle is a lipid-based nanoparticle (LNP) having a composition comprising an ionizable lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and dimyristylglycerol-polyethylene glycol 2000 (DMG-PEG 2000) or a pegylated lipid containing a polyethylene glycol moiety. Preferably, the LNP has a composition comprising DSPC: cholesterol: DMG-PEG-2000: ionizable lipid in a molar ratio ranging from 5%-20% DSPC, 30%-55% cholesterol, 0.5%-3% PEG, and 30%-60% ionizable lipid, with the total of the molar ratios of the lipids being 100%, and optionally 9.4:42.5:1.8:46.3 (DSPC: cholesterol: DMG-PEG 2000: ionizable lipid). The LNP has a N:P ratio ranging from 5:1 to 10:1, and optionally 6:1, and a particle size of about 40-300 nm.

[0064] ALC-0315, SM-102, or DHA-1, and the specific structures are as follows:

[0065] In a fourth aspect of the present application, a pharmaceutical composition is provided, which comprises the gene delivery system as described above.

[0066] Technical effects of the present application: The present application uses self-replicating RNA vectors based on mRNA technology platform combined with lipid nanoparticle encapsulation delivery means to highly express important differentiation-related transcription factors in tumor cells, induce tumor cells to transform into normal cells, inhibit tumor malignant phenotype, and achieve the purpose of treating tumors. This is a new means of inducing differentiation to treat tumors. According to the results of our previous animal experiments and the clinical research initiated by researchers, it can be confirmed that this technical means can effectively inhibit the growth of tumors in vivo, and is a new scheme for tumor treatment, which is expected to open up a new track for tumor treatment. On the other hand, the present application is significantly different from the current application of mRNA technology in tumor vaccines. RNA tumor vaccines use RNA technology to express related tumor antigens in antigen-presenting cells to stimulate the body to produce an immune response against these antigens, thereby playing a role in controlling or eliminating tumors; while the present application uses RNA technology to mediate the expression of differentiation transcription factors in tumor cells, and plays a role in promoting differentiation of related transcription factors, thereby inhibiting the malignant phenotype of tumors. Although non-replicating mRNA can also mediate the transient expression of target genes in tumor cells, in order to induce tumor cells to differentiate into normal cells, differentiation-related transcription needs to be expressed stably and efficiently in tumor cells. Therefore, the present application first proposes to use self-replicating RNA technology to restore or overexpress differentiation-related transcription factors that are down-regulated during tumor development, inhibit tumor cell growth, and achieve the effect of treating tumors. Therefore, the gene delivery system provided by the present application can induce malignant solid tumor cells to differentiate into normal mature cells, inhibit the proliferation of tumor cells, and induce tumor cell apoptosis. At the same time, after optimizing the self-replicating RNA vector used in the present application, a delivery system that can stably and efficiently express target genes in tumor cells is obtained.

[0067] In summary, the present application uses self-replicating RNA to express differentiation-related transcription factors, which not only has high expression amount and high expression efficiency, but also has longer maintenance time. For example, the preferred HNF4a-saRNA of the present application can maintain HNF4a in hepatoma cells for more than 7 days, and the expression amount and expression time are much higher than that of non-replicating HNF4a-mRNA. Compared with viral vectors, it has the advantages of good safety, strong tissue accessibility, and repeatable administration, and therapeutic effects have been observed in tumor-bearing animals and patients. BRIEF DESCRIPTION OF DRAWINGS

[0068] Figure 1 is a schematic diagram of self-replicating RNA expressing HNF4a with different lengths of polyA tail. GOI is HNF4a; the length of polyA tail is 30nt, 35nt, 40nt, 50nt, 60nt, 67nt, 75nt, 90nt, 100nt, and 110nt, respectively.

[0069] Figure 2 is a western blot for detecting the expression level of HNF4a protein after different treatments, to observe the effect of polyA length on the overexpression of HNF4a in hepatoma cells mediated by self-amplifying RNA.

[0070] Figure 3 is a western blot for detecting the expression level of HNF4a protein after different treatments, to observe the effect of polyA length on the overexpression of HNF4a in hepatoma cells mediated by self-amplifying RNA.

[0071] Figure 4 is a western blot for detecting the expression level of HNF4a protein after different treatments, to observe the effect of polyA length on the overexpression of HNF4a in hepatoma cells mediated by self-amplifying RNA.

[0072] Figure 5 is a CCK8 assay for detecting the proliferation of Huh-7 cells after different treatments.

[0073] Figure 6 is a CCK8 assay for detecting the proliferation of Huh-7 cells after different treatments.

[0074] Figure 7 is a RT-PCR for detecting the expression level of liver function related genes after different treatments.

[0075] Figure 8 is a RT-PCR for detecting the expression level of tumor cell stemness related genes after different treatments.

[0076] Figure 9 is a PAS staining for detecting the glycogen storage in Huh-7 cells after different treatments.

[0077] Figure 10 is the acetylated low density lipoprotein (ac-LDL) uptake ability of Huh7 cells treated with HNF4a-saRNA and GFP-saRNA liposome nanoparticles for 3 days, using Dil-ac-LDL fluorescent substrate to detect the ac-LDL uptake ability of Huh7 cells, and image J software to count the ac-LDL positive area in Huh7 cells.

[0078] Figure 11 is the detection of the content of senescence-related β-galactosidase in Huh7 cells treated with HNF4a-saRNA and GFP-saRNA liposome nanoparticles for 3 days, and image J software to count the β-galactosidase positive area in Huh7 cells.

[0079] Figure 12 is the detection of apoptosis in Huh7 cells treated with HNF4a-saRNA and GFP-saRNA liposome nanoparticles for 3 days by Annexin V / PI staining.

[0080] Figure 13 is the experimental flow chart of intratumoral injection of HNF4a-saRNA and HNF4a-mRNA liposome nanoparticles to treat Huh7 cell subcutaneous xenograft tumor.

[0081] Figure 14 is the tumor proliferation curve in Huh7 cell subcutaneous xenograft tumor model treated with HNF4a-saRNA and HNF4a-mRNA. GFP-saRNA is the self-replicating mRNA liposome nanoparticle control, and normal saline is the solvent control.

[0082] Figure 15 is the gross appearance of Huh7 cell subcutaneous xenograft tumor treated with HNF4a-saRNA and HNF4a-mRNA.

[0083] Figure 16 is the tumor weight statistical chart (left) and tumor inhibition rate statistical chart (right) of Huh7 cell subcutaneous xenograft tumor model treated with HNF4a-saRNA and HNF4a-mRNA.

[0084] Figure 17 is the Western blot detection of the expression amount of HNF4a in the tumor tissue of Huh7 cell subcutaneous xenograft tumor model treated with HNF4a-saRNA

[0085] Figure 18 is the detection of the expression changes of HNF4a and Ki67 in tumor tissue by immunohistochemistry (left), and the statistical chart of HNF4a and Ki67 positive staining area in tumor tissue (right).

[0086] Figure 19 is the experimental flow chart of tail vein injection of self-replicating HNF4a-saRNA and HNF4a-mRNA liposome nanoparticles to treat Huh7 cell liver orthotopic xenograft tumor.

[0087] Figure 20 is a graph of the in vivo fluorescence signal of mice before and after injection of HNF4a-saRNA and HNF4a-mRNA in a Huh7 cell liver orthotopic xenograft model. GFP-sRNA is a self-replicating RNA lipid nanoparticle control, and saline is a vehicle control.

[0088] Figure 21 is a graph of the in vivo fluorescence signal of mice at different time points during treatment of Huh7 cell liver orthotopic xenografts with HNF4a-saRNA and HNF4a-mRNA.

[0089] Figure 22 is a graph of the tumor weight (left) and tumor inhibition rate (right) in a Huh7 cell liver orthotopic xenograft experiment treated with HNF4a-saRNA and HNF4a-mRNA lipid nanoparticles.

[0090] Figure 23 is a graph of the tumor weight (left) and tumor inhibition rate (right) in a Huh7 cell liver orthotopic xenograft experiment treated with HNF4a-saRNA and HNF4a-mRNA lipid nanoparticles.

[0091] Figure 24 is a graph of the tumor weight (left) and tumor inhibition rate (right) in a Huh7 cell liver orthotopic xenograft experiment treated with HNF4a-saRNA and HNF4a-mRNA lipid nanoparticles.

[0092] Figure 25 is a graph of the tumor weight (left) and tumor inhibition rate (right) in a Huh7 cell liver orthotopic xenograft experiment treated with HNF4a-saRNA and HNF4a-mRNA lipid nanoparticles.

[0093] Figure 26 is a graph of the tumor weight (left) and tumor inhibition rate (right) in a Huh7 cell liver orthotopic xenograft experiment treated with HNF4a-saRNA and HNF4a-mRNA lipid nanoparticles.

[0094] Figure 27 is a graph of the tumor weight (left) and tumor inhibition rate (right) in a Huh7 cell liver orthotopic xenograft experiment treated with HNF4a-saRNA and HNF4a-mRNA lipid nanoparticles.

[0095] Figure 28 is a graph of the tumor weight (left) and tumor inhibition rate (right) in a Huh7 cell liver orthotopic xenograft experiment treated with HNF4a-saRNA and HNF4a-mRNA lipid nanoparticles.

[0096] Figure 29 is a graph of the tumor weight (left) and tumor inhibition rate (right) in a Huh7 cell liver orthotopic xenograft experiment treated with HNF4a-saRNA and HNF4a-mRNA lipid nanoparticles.

[0097] Figure 30 shows the effect of HNF4a-mRNA LNP and HNF4a-saRNA LNP on the colony formation ability of cholangiocarcinoma cell HuCC-T1.

[0098] Figure 31 shows the expression level of HNF4a protein detected by western blot after 1 day and 3 days of transfection of intestinal cancer cell HCT116 with different concentrations of HNF4a-saRNA LNP.

[0099] Figure 32 shows the effect of HNF4a-mRNA LNP and HNF4a-saRNA LNP on the proliferation of intestinal cancer cell HCT 116.

[0100] Figure 33 shows the effect of HNF4a-mRNA LNP and HNF4a-saRNA LNP on the colony formation ability of intestinal cancer cell HCT 116.

[0101] Figure 34 shows the expression level of HNF4a protein detected by western blot after 1 day and 3 days of treatment of pancreatic cancer cell PANC1 with different concentrations of HNF4a-saRNA lipid nanoparticles.

[0102] Figure 35 shows the mRNA level of HNF4a (HNF4a) and self-replicating RNA vector (VEEV) detected by quantitative PCR after treatment of Huh7 cells with HNF4a-saRNA during the process of liver orthotopic implantation tumor.

[0103] Figure 36 shows the overall stability of liver cancer lesions after treatment with HNF4a-saRNA LNP as shown by enhanced MRI. A. Liver cancer target lesions (dashed line box) before treatment for 13 weeks. B. Liver cancer target lesions were significantly larger than before treatment at the baseline period. C. Liver cancer target lesions maintained stable in size after treatment for 10 weeks. D. Liver cancer target lesions maintained stable in size after treatment for 23 weeks, with partial area showing liquefactive necrosis (arrow).

[0104] Figure 37 shows the gradual shrinkage of liver cancer lung metastasis lesions after treatment with HNF4a-saRNA LNP as shown by chest CT. A. Liver cancer lung metastasis lesions (arrow) at the baseline period. B. Liver cancer lung metastasis lesions were slightly larger than the baseline after treatment for 4 weeks. C. Liver cancer lung metastasis lesions were slightly smaller than 4 weeks before after treatment for 8 weeks. D. Liver cancer lung metastasis lesions were further reduced in size after treatment for 18 weeks, with the lesion diameter being significantly smaller than the baseline level.

[0105] Figure 38 is the enhanced MRI showing the changes of the target lesion of liver cancer after HNF4a-saRNA LNP treatment. A. The arterial phase of the target lesion of liver cancer (dotted line) is obviously enhanced at baseline. B. The target lesion of liver cancer is larger than baseline and the arterial phase is weaker than baseline after 4 weeks of treatment. C. The target lesion of liver cancer is further enlarged and the arterial phase is further weakened after 9 weeks of treatment. D. The target lesion of liver cancer continues to enlarge and the arterial phase continues to weaken, with most of the region losing enhancement (arrow) after 16 weeks of treatment.

[0106] Figure 39 is the Western blot detection of the expression level of HNF4a protein after different concentrations of HNF1a-saRNA LNP treating liver cancer Huh7 cells for 1 day and 3 days, respectively.

[0107] Figure 40 is the tumor proliferation curve (left) and the relative tumor volume histogram (right) in the Huh7 cell subcutaneous xenograft model treated with HNF1a-saRNA. Saline is the vehicle control.

[0108] Figure 41 is the gross picture of the Huh7 cell subcutaneous xenograft tumor treated with HNF1a-saRNA lipid nanoparticles.

[0109] Figure 42 is the tumor weight histogram (left) and tumor inhibition rate histogram (right) in the Huh7 cell subcutaneous xenograft model treated with HNF1a-saRNA lipid nanoparticles.

[0110] Figure 43 is the Western blot detection of the expression level of FOXA3 protein after different concentrations of FOXA3-saRNA LNP treating liver cancer Huh7 cells for 3 days, respectively.

[0111] Figure 44 is the Western blot detection of the expression level of PTF1A protein after different concentrations of PTF1A-saRNA LNP treating pancreatic cancer PANC1 cells for 1 day and 3 days, respectively.

[0112] Figure 45 is the Western blot detection of the expression level of NEUROD1 protein after transfecting BHK21 cells with NEUROD1-saRNA for 1 day.

[0113] Figure 46 is the experimental flow chart of the Huh7 cell subcutaneous xenograft experiment treated with self-replicating HNF4a-saRNA and HNF4a / HNF1a / FOXA3-saRNA lipid nanoparticles by tail vein injection.

[0114] Figure 47 is the tumor proliferation curve in the Huh7 cell subcutaneous xenograft model treated with HNF4a-saRNA and HNF4a / HNF1a / FOXA3-saRNA.

[0115] Figure 48 is a graph showing the tumor weight of the Huh7 hepatocarcinoma subcutaneous xenograft model treated with HNF4a-saRNA and HNF4a / HNF1a / FOXA3-saRNA.

[0116] Figure 49 is a graph showing the expression of HNF4a in the tumor tissue of the Huh7 hepatocarcinoma subcutaneous xenograft model treated with HNF4a-saRNA and HNF4a / HNF1a / FOXA3-saRNA.

[0117] Figure 50 is a graph showing the experimental procedure of the AsPC-1 pancreatic cancer subcutaneous xenograft model treated with PTF1A-saRNA liposome nanoparticles.

[0118] Figure 51 is a graph showing the tumor proliferation curve of the AsPC-1 pancreatic cancer subcutaneous xenograft model treated with PTF1A-saRNA liposome nanoparticles.

[0119] Figure 52 is a graph showing the tumor weight of the AsPC-1 pancreatic cancer subcutaneous xenograft model treated with PTF1A-saRNA liposome nanoparticles.

[0120] Figure 53 is a graph showing the tumor inhibition rate of the AsPC-1 pancreatic cancer subcutaneous xenograft model treated with PTF1A-saRNA liposome nanoparticles.

[0121] Figure 54 is a graph showing the expression of PTF1A in the tumor tissue of the AsPC-1 pancreatic cancer subcutaneous xenograft model treated with PTF1A-saRNA.

[0122] Figure 55 is a graph showing the experimental procedure of the U87 glioma subcutaneous xenograft model treated with NUROD1-saRNA liposome nanoparticles.

[0123] Figure 56 is a graph showing the tumor proliferation curve of the U87 glioma subcutaneous xenograft model treated with NUROD1-saRNA liposome nanoparticles.

[0124] Figure 57 is a graph showing the tumor gross of the U87 glioma subcutaneous xenograft model treated with NUROD1-saRNA liposome nanoparticles.

[0125] Figure 58 is a graph showing the tumor weight of the U87 glioma subcutaneous xenograft model treated with NUROD1-saRNA liposome nanoparticles. DETAILED DESCRIPTION

[0126] The present application will be described in detail below with reference to the embodiments. The advantages and features of the present application will become more apparent with the description. However, these embodiments are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements fall within the protection scope of the present application.

[0127] Unless otherwise described, the embodiments of the present application will employ conventional techniques of molecular biology, cell biology and immunology, which are known to those skilled in the art. These techniques are fully described in, for example, Molecular Cloning: A Laboratory Manual, 4th Edition (2017); Short Protocols in Cell Biology: A Compendium of Methods from Tissue Culture to Neuron Electrophysiology (2007); Short Protocols in Immunology: A Compendium of Methods from Tissue Culture to Neuron Electrophysiology (2010). Alternatively, they can be performed according to the instructions provided by the reagent manufacturers.

[0128] Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as familiar to those skilled in the art. In addition, any method and material similar or equivalent to those described can be applied in the present application. The preferred methods and materials described herein are only exemplary.

[0129] All data in the present application were analyzed and plotted using GraphPad Prism 8 software. All data error values were shown as mean ± standard error (±SEM). The comparison between two groups of data used t-test, and the statistical test was two-tailed, where p<0.05 was considered statistically significant, * represented p<0.05, ** represented p<0.01, and *** represented p<0.001.

[0130] Example 1: Preparation of self-replicating RNA and LNP encapsulation

[0131] 1. Preparation of linear mRNA and self-replicating RNA

[0132] Self-replicating RNA (saRNA) is based on an engineered alphavirus genome containing genes encoding non-structural proteins that are capable of RNA replication, with the structural protein sequences replaced by a gene of interest. The saRNA includes a 5' cap, non-coding region (5' UTR), four non-structural genes (NSP1-4), a 26S subgenomic promoter, a gene of interest, a 3' non-coding region (3' UTR), and a poly(A) tail. Linear RNAs are designed based on eukaryotic mRNA structure, containing a gene encoding a protein of interest, as well as the necessary cap structure, 5UTR, 3UTR, and poly(A). Both non-replicating and self-replicating RNAs are prepared from linear templates, starting with plasmid DNA that is restriction digested with BspQI enzyme (New England Biolabs, R0712L) and purified using a PCR purification kit (Invitrogen, K310002) for linear templates for RNA transcription. T7 RNA polymerase (Promega, P1300), 1000 U / ml RNase inhibitor (New England Biolabs, M0314L), 2 U / ml inorganic pyrophosphatase (New England Biolabs, M2403L), 5 mM NTPs (New England Biolabs, N0466S), and cap analog (3' OMe, Trinlink, N-7413) are mixed together in a cocktail for transcription, and the cocktail is mixed and incubated at 37°C for 2 hours for in vitro transcription from the template. After the transcription reaction is complete, DNase I (1 U / μg DNA) is added and incubated at 37°C for 15 minutes to remove the DNA template, and the transcribed RNA is then purified using LiCl precipitation.

[0133] 2. LNP-encapsulated RNA

[0134] Lipid nanoparticles are prepared by mixing ethanol and water phases in a microfluidic device (INano TM ​LNP encapsulated saRNAs were prepared in a rapid mixing setup (QG-1, Anton Paar, L-system, Micro & Nano) according to the manufacturer’s instructions. The aqueous phase was 50 mM citrate buffer (pH 6.0) containing purified saRNA. The ethanol phase contained proprietary ionizable lipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (Avanti, 850365P), cholesterol (Sigma-Aldrich, C8667), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (NOF, GM020). The mRNA-LNPs were assembled with a molar ratio of 9.4:42.5:1.8:46.3 (DSPC:cholesterol:DMG-PEG 2000:DHA-1), N / P = 6. The LNP formed from this formulation was subjected to particle size, PDI, RNA concentration, and encapsulation efficiency tests.

[0135] Example 2: Optimization of the length of polyA tail of self-replicating RNA

[0136] LNP encapsulated self-replicating RNAs expressing HNF4a with different lengths of polyA tail were prepared according to the method of Example 1 and as shown in Figure 1. Hepatoma cells Huh7 were seeded at 3x10 5 Cells were seeded at 6-well plates and cultured overnight, and then different polyA length of HNF4a-saRNA LNPs diluted with Opti-MEM were added respectively. After 6 hours of culture, 1 mL of DMEM medium containing 20% FBS was added. The transfected cells were collected with RIPA lysis buffer for Western blot detection of intracellular HNF4a protein levels (Figure 2) after 3 or 7 days. The results showed that HNF4a-saRNAs with polyA tail length of 35-100 nt can mediate overexpression of HNF4a in Huh-7 cells, and tumor cells transfected with self-replicating RNAs with polyA tail length of 40-90 nt have higher expression of HNF4a, and self-replicating RNAs with polyA tail length of 50 nt-75 nt have better expression of HNF4a and longer expression time, and self-replicating RNAs with polyA tail length of 60-70 nt (especially 67 nt) can mediate the highest efficient expression. The self-replicating RNA sequence with the best experimental effect after optimization of polyA tail length is shown as SEQ ID NO: 6, and the HNF4a-saRNA sequence is shown as SEQ ID NO: 7 (the sequence is used in subsequent examples).

[0137] Example 3: Comparison of the ability of non-replicating HNF4a-mRNA, HNF4a-saRNA and adenovirus AdHNF4a to mediate overexpression of HNF4a in tumor cells

[0138] Hepatoma cells Huh7 were seeded at 3x10 5Cells were seeded into 6-well plates and cultured overnight, washed with 1 ml PBS, and then 1 ml Opti-MEM diluted HNF4a-saRNA LNP, control GFP-saRNA LNP and non-replicating HNF4a-mRNA LNP (final concentration of 2 ng RNA / 200 cells) were added respectively. After 6 hours of incubation, 1 mL DMEM medium containing 20% FBS was added. The transfected cells were collected with RIPA lysis buffer at 1, 3, 5 or 7 days for Western blot to detect the protein level of HNF4a in cells (Figure 3). Compared with the effect of adenovirus-mediated overexpression of HNF4a on Huh7 cells by adding adenovirus expressing HNF4a AdHNF4a and its control virus AdGFP (Figure 3). The results show that both HNF4a-saRNA and HNF4a-mRNA can up-regulate HNF4a in hepatoma cells, but the expression of HNF4a-mRNA is lower and shorter in duration; while the expression of HNF4a-saRNA is higher and longer in duration, and its expression efficiency is not lower than that of adenovirus.

[0139] Example 4: HNF4a-saRNA treats hepatoma by inducing hepatoma cells to differentiate into hepatocytes

[0140] 1. Different concentrations of HNF4a-saRNA LNP up-regulate the expression of HNF4a in hepatoma Huh-7 cells

[0141] Hepatoma cells Huh7 were seeded at 3x10 5 Cells were seeded into 6-well plates and cultured overnight, washed with 1 ml PBS, and then 1 ml Opti-MEM diluted HNF4a-saRNA LNP, control GFP-saRNA LNP (final concentration of 0.5, 1, 2 ng RNA / 200 cells) were added respectively. After 6 hours of incubation, 1 mL DMEM medium containing 20% FBS was added. The transfected cells were collected with RIPA lysis buffer at 1, 3, 5 or 7 days for Western blot to detect the protein level of HNF4a in cells (Figure 4), and the results show that HNF4a-saRNA LNP up-regulates the expression of HNF4a, while control GFP-saRNA LNP does not affect the expression of HNF4a in hepatoma cells.

[0142] 2. HNF4a-saRNA LNP inhibits the growth of hepatoma cells

[0143] Hepatoma cells Huh7 were used to verify the inhibition of the gene delivery system on the malignant phenotype such as tumor cell proliferation and clonal formation. Huh7 cells were seeded at 3x10 3Cells were seeded at different densities in 96-well plates and cultured overnight. Afterward, the supernatant was removed, and the cells were washed with 100 μl of PBS. Different concentrations of HNF4α-saRNA LNP diluted with Opti-MEM and control GFP-saRNA LNP were added to the cells. After 6 hours, an equal volume of 20% FBS was added to the culture medium to achieve a 10% FBS concentration. Cell proliferation was measured daily using a cell counting kit-8 (CCK-8, Dojindo) to determine the effect of RNA on tumor cell proliferation. The results showed that HNF4α-saRNA LNP significantly inhibited the proliferation of liver cancer cells compared to control cells (Figure 5).

[0144] Next, the inventors conducted experiments on the effect of the gene delivery system on the clonogenic ability of tumor cells. They used 3 × 10⁻⁶ gene delivery systems to deliver Huh7 liver cancer cells. 3 Cells were seeded at a density of 2 ng / well in 96-well plates and cultured overnight. Afterward, HNF4α-saRNA LNP and control GFP-saRNA LNP (2 ng / 200 cells) were added for transfection. Twenty-four hours later, cells were trypsinized and transferred to 60 mm culture dishes. The culture medium was changed every 3 days, and the colony formation of tumor cells was observed under a microscope. After colony formation, cells were washed twice with PBS, stained with crystal violet for 20 min, washed with PBS again, air-dried, and photographed. The number of colonies was counted using ImageJ to determine the effect of RNA on the colony formation ability of tumor cells. The results are shown in Figure 6. HNF4α-saRNA significantly inhibited the colony formation ability of liver cancer cells.

[0145] 3. HNF4α-saRNA LNP induces differentiation of liver cancer cells into mature hepatocytes.

[0146] ①HNF4α-saRNA restores liver function gene expression in liver cancer cells

[0147] Huh-7 liver cancer cells were treated with 3x10 5 Cells were seeded into 6-well plates and cultured overnight. Cells were washed with PBS, and different concentrations of HNF4α-saRNA LNP and GFP-saRNA LNP diluted with Opti-MEM were added. After 6 hours of culture, 1 mL of DMEM medium containing 20% ​​FBS was added. Cells were harvested 1, 3, or 7 days after transfection to extract RNA. Real-time quantitative PCR was used to detect the expression of liver function-related genes regulated by HNF4α and the expression of tumor stemness-related genes (Figures 7 and 8). The results showed that HNF4α-saRNA upregulated the expression of liver function-related genes and downregulated the expression of tumor stemness-related genes.

[0148] ②HNF4α-saRNA promotes glycogen storage and low-density lipoprotein uptake in liver cancer cells.

[0149] Glycogen storage and low density lipoprotein uptake ability are important functions of normal hepatocytes, the inventors further confirmed whether the liver cancer cells were differentiated into mature hepatocytes by examining these functions.

[0150] Liver cancer cells Huh7 were seeded at 3x10 4 Cells were seeded at 24-well plate at 200 cells / well, 1 ng / 200 cells of HNF4a-saRNA LNP and corresponding control were added. Glycogen storage ability of cells was verified by PAS reaction kit (Bi Yun Tian) 3 days after LNP delivery. 70% ethanol was added to fix cells for 10 minutes, then the periodic acid solution was removed and equilibrated to room temperature, 100 μl of periodic acid solution was added to each sample, reacted for 10 minutes in a wet box in the dark, then the periodic acid solution was removed, soaked in PBS and placed in a shaker for 5 minutes. 100 μl of Schiff reagent was added to each sample, placed in a wet box, and stained in a 37°C oven for 1 hour in the dark. The staining solution was removed, soaked in PBS and placed in a shaker for 5 minutes. Finally, 100 μl of hematoxylin staining solution was added to each sample and stained for 30 seconds. The staining solution was removed, rinsed with PBS twice, and the background was washed off. Photographed under a microscope and counted the stained cells (Figure 9). The results showed that HNF4a-saRNA promoted the glycogen storage ability of liver cancer cells.

[0151] Liver cancer cells Huh7 were seeded at 3x10 4 Cells were seeded at 24-well plate at 200 cells / well, 1 ng / 200 cells of HNF4a-saRNA LNP and corresponding control were added. 3 days after LNP delivery, the cell culture medium was removed, the cells were washed with PBS, then 200 ul of Dil ac-LDL (Invitrogen) was added to each well after diluting with DMEM at a ratio of 1:100. After 3 hours, the DMEM containing Dil ac-LDL was removed, the cells were washed with PBS, and 4% paraformaldehyde was added to fix for 15 minutes. After DAPI staining, the slide was mounted, photographed using a confocal fluorescence microscope, and the fluorescence signal was counted (Figure 10). The results showed that HNF4a-saRNA promoted the low density lipoprotein uptake ability of liver cancer cells.

[0152] ③HNF4a-saRNA promotes liver cancer cell senescence

[0153] Liver cancer cells Huh7 were seeded at 3x10 4Cells were seeded in 24-well plates, and 1 ng / 200 cells of HNF4α-saRNA LNP and corresponding controls were added. Three days after LNP delivery, HCC cell senescence was assessed using a Senescence β-Galactosidase staining kit (Beyotime, China). Cells were fixed with 4% formaldehyde for 15 minutes and then incubated overnight at 37°C with fresh senescence-associated β-galactosidase staining solution. Senescent cells were observed and captured under a microscope. The number of senescent cells was counted using image analysis software (Image-Pro Plus 6.0, Media Cybernetics) (Figure 11). The results showed a significant increase in senescent hepatocellular carcinoma cells after HNF4α-saRNA treatment.

[0154] ④ HNF4α-saRNA induces apoptosis in liver cancer cells

[0155] Huh7 liver cancer cells were divided into 3x10 5 Cells were seeded in 24-well plates, and 1 ng / 200 cells of HNF4α-saRNA LNP and corresponding controls were added. Three days after LNP delivery, apoptotic cells were detected using the APC Annexin V / PI apoptosis kit (Biolegend, China) according to the manufacturer's instructions. Cells were digested with 0.25% trypsin and washed twice with PBS. Cells were then transferred to tubes and resuspended in 100 μl of binding buffer. 400 μl of binding buffer containing 5 μl APC Annexin V and 5 μl proiodine was added to each tube, and the cells were incubated in the dark at 25°C for 15 min. Cells were then analyzed using flow cytometry (Attune NxT, Invitrogen). Flow cytometry data were analyzed using FlowJo V10 software (Figure 12). The results showed a significant increase in apoptosis in HNF4α-saRNA-treated liver cancer cells.

[0156] The above results indicate that HNF4α-saRNA can promote the transdifferentiation of liver cancer cells into mature hepatocytes, restore hepatocyte function, and further induce senescence and apoptosis in liver cancer cells.

[0157] 3. HNF4α-saRNA LNP inhibits the growth of subcutaneous tumors in mice.

[0158] Five-week-old male nude mice (BALB / c immunodeficient strain) were purchased from Shanghai BK / KY Biotechnology Co., Ltd., and were housed under specific pathogen-free environmental conditions using a 12-hour on / off light cycle. 1×10 6Huh-7 cells were subcutaneously injected into the right axilla of male nude mice. The tumor size was measured in two dimensions using calipers, and the volume was calculated using the following formula: Volume = Length × (Width) 2 ×1 / 2. When the average tumor volume reaches approximately 100 mm... 3 Mice were randomly divided into four groups (n=7 per group). 5 μg of HNF4α-saRNA LNP, GFP-saRNA LNP, HNF4α-mRNA LNP, or physiological saline (solvent control) was injected into the tumor of each mouse, with a final volume of 75 μl. Tumor volume was measured daily after injection, and tumor growth curves were plotted (Figures 13 and 14). Five days after RNA injection, mice were sacrificed, and tumors were excised and weighed (Figures 15 and 16), while the tumor inhibition rate was calculated (Figure 16). Some tumor tissues were paraffin-embedded, and HNF4α expression was detected by Western blotting and immunohistochemistry (Figures 17 and 18). Ki67 staining was used to assess the proliferation status of tumor cells (Figure 17). The results showed that intratumoral injection of HNF4α-saRNA slowed tumor growth, with a tumor inhibition rate exceeding 50% (Figure 16), while HNF4α-mRNA had no significant inhibitory effect on tumor growth. Western blot and immunohistochemical results showed that HNF4α-saRNA significantly increased the protein expression of HNF4α in tumor tissues, while significantly decreasing the expression of Ki67, a tumor cell proliferation marker. These results indicate that HNF4α-saRNA can effectively induce the expression of HNF4α in cancer cells, thereby inhibiting the growth of mouse hepatocellular carcinoma implants.

[0159] 4. HNF4α-saRNA LNP inhibits the growth of hepatocellular carcinoma cells implanted in situ in the liver.

[0160] The Huh7 human hepatocellular carcinoma cell line, which stably expresses the luciferase gene, was inoculated into the axilla of male nude mice. After tumor formation, the tumor was removed and cut to a depth of 1 mm. 3Small pieces of tumor were transplanted under the liver capsule of male nude bab / c mice. The growth of the tumor in the mice was monitored using the IVIS spectrum optical imaging system. Three days after transplantation, the mice were evenly divided into four groups according to the strength of the fluorescence signal, and were injected with HNF4a-saRNA LNP, GFP-saRNA LNP, HNF4a-mRNA LNP, and normal saline (vehicle control) at a dose of 2 mg / kg via the tail vein (Figure 19). The growth of the tumor in the mice was monitored according to bioluminescence, and the efficacy of the HNF4a-saRNA was evaluated (Figure 20). The tumor growth curve was plotted (Figure 21). The mice were sacrificed at the end of the experiment, and the tumors were excised and weighed to calculate the tumor inhibition rate (Figures 22 and 23). The tumor tissue was paraffin-embedded, and HE staining was used to evaluate the morphology of the tumor tissue, and Ki67 staining was used to evaluate the proliferation state of the tumor (Figure 24). The results showed that the growth of the tumor was reduced after the injection of HNF4a-saRNA LNP, and the tumor inhibition rate was more than 40% compared with the control tumor, while HNF4a-mRNA LNP had no significant effect on the inhibition of the tumor. Western blot and immunohistochemical detection of HNF4a expression were performed on the tumor tissue collected at different time points after the injection of LNP (Figures 25 and 26), and Ki67 staining was used to evaluate the proliferation state of the tumor (Figure 26). Quantitative PCR was used to detect the expression of liver cell differentiation-related indicators (Figure 27). The results showed that HNF4a-saRNA significantly upregulated the expression of HNF4a and liver function-related genes in the tumor tissue. These results further indicated that HNF4a-saRNA can induce the differentiation of hepatoma cells into normal liver cells, thereby inhibiting the growth of hepatoma.

[0161] Example 5: HNF4a-saRNA inhibits the proliferation of cholangiocarcinoma cells

[0162] 1. Different concentrations of HNF4a-saRNA LNP upregulate the expression of HNF4a in cholangiocarcinoma HuCC-T1 cells

[0163] Cholangiocarcinoma HuCC-T1 cells were seeded in a 6-well plate at a density of 40%-50% and cultured overnight. After washing with 1 ml of PBS, HNF4a-saRNA LNP was added in series with Opti-MEM (final concentration of 1, 2 ng RNA / 200 cells). After 6 hours of culture, 1 mL of DMEM medium containing 20% FBS was added. The transfected cells were collected with RIPA lysis buffer at 1 and 3 days for Western blot detection of the intracellular HNF4a protein level (Figure 28). The results showed that HNF4a-saRNA LNP upregulated the expression of HNF4a in cholangiocarcinoma cells.

[0164] 2. HNF4a-saRNA LNP inhibits the growth of cholangiocarcinoma cells

[0165] Cholangiocarcinoma cells HuCC-T1 were seeded at 3 x 105cells / well in 96-well plates and cultured overnight. The culture medium was removed and the cells were washed with 100 μΐ PBS. Different concentrations of HNF4a-saRNA LNP diluted in Opti-MEM (final concentration: 1, 2 ng RNA / 200 cells) were added to the cells. After 6 hours, an equal volume of 20% FBS was added to the culture medium of the corresponding cells to make the FBS concentration in the culture medium 10%. Cell proliferation was determined using Cell Counting Kit-8 (CCK-8, Dojindo) every day to determine the effect of RNA on the proliferation ability of tumor cells. The results showed that HNF4a-saRNA LNP significantly inhibited the proliferation of cholangiocarcinoma cells compared with the control cells (Figure 29). 3 Cells were seeded at a density of 200 cells / well in 96-well plates and cultured overnight. After the culture medium was removed, the cells were washed with 100 μΐ PBS. Different concentrations of HNF4a-saRNA LNP diluted in Opti-MEM (final concentration: 1, 2 ng RNA / 200 cells) were added to the cells. After 6 hours, an equal volume of 20% FBS was added to the culture medium of the corresponding cells to make the FBS concentration in the culture medium 10%. Cell proliferation was determined using Cell Counting Kit-8 (CCK-8, Dojindo) every day to determine the effect of RNA on the proliferation ability of tumor cells. The results showed that HNF4a-saRNA LNP significantly inhibited the proliferation of cholangiocarcinoma cells compared with the control cells (Figure 29).

[0166] 3. HNF4a-saRNA LNP inhibits cholangiocarcinoma cell colony formation

[0167] Cholangiocarcinoma cells HuCC-T1 were seeded at 3 x 105cells / well in 96-well plates and cultured overnight. The culture medium was removed and the cells were washed with 100 μΐ PBS. Different concentrations of HNF4a-saRNA LNP diluted in Opti-MEM (final concentration: 1, 2 ng RNA / 200 cells) were added to the cells. After 6 hours, an equal volume of 20% FBS was added to the culture medium of the corresponding cells to make the FBS concentration in the culture medium 10%. Cell proliferation was determined using Cell Counting Kit-8 (CCK-8, Dojindo) every day to determine the effect of RNA on the proliferation ability of tumor cells. The results showed that HNF4a-saRNA LNP significantly inhibited the proliferation of cholangiocarcinoma cells compared with the control cells (Figure 29). 3 Cells were seeded at a density of 200 cells / well in 96-well plates and cultured overnight. After the culture medium was removed, the cells were washed with 100 μΐ PBS. Different concentrations of HNF4a-saRNA LNP diluted in Opti-MEM (final concentration: 1, 2 ng RNA / 200 cells) were added to the cells. After 6 hours, an equal volume of 20% FBS was added to the culture medium of the corresponding cells to make the FBS concentration in the culture medium 10%. Cell proliferation was determined using Cell Counting Kit-8 (CCK-8, Dojindo) every day to determine the effect of RNA on the proliferation ability of tumor cells. The results showed that HNF4a-saRNA LNP significantly inhibited the proliferation of cholangiocarcinoma cells compared with the control cells (Figure 29).

[0168] Example 6: HNF4a-saRNA inhibits the proliferation of intestinal cancer cells

[0169] 1. Different concentrations of HNF4a-saRNA LNP up-regulate the expression of HNF4a in intestinal cancer cells HCT-116 cells

[0170] Intestinal cancer cell HCT-116 was seeded at 40%-50% density into 6-well plates and cultured overnight, washed with 1 ml PBS, and then added with HNF4a-saRNA LNP diluted with Opti-MEM (final concentration of 1, 2 ng RNA / 200 cells) respectively. After 6 hours of culture, 1 mL of DMEM medium containing 20% FBS was added. The transfected cells were collected with RIPA lysis buffer for Western blot detection of intracellular HNF4a protein level (Figure 31) at 1 and 3 days, and the results showed that HNF4a-saRNA LNP up-regulated the expression of HNF4a in intestinal cancer cells.

[0171] 2. HNF4a-saRNA LNP inhibits the growth of cholangiocarcinoma cells

[0172] Intestinal cancer cell HCT-116 was seeded at 3x10 3 Cells were seeded at a density of 200 cells / well in 96-well plates and cultured overnight, and then the culture supernatant was aspirated and the cells were washed with 100 μl PBS. Different concentrations of HNF4a-saRNA LNP and HNF4a-mRNA LNP diluted with Opti-MEM (final concentration of 2 ng RNA / 200 cells) were added to the cells, and after 6 hours, an equal volume of 20% FBS was added to the culture medium of the corresponding cells to make the FBS concentration in the culture medium 10%. Cell counting kit-8 (CCK-8, Dojindo) was used to determine cell proliferation every day to determine the effect of RNA on the proliferation ability of tumor cells. The results showed that HNF4a-saRNA LNP significantly inhibited the proliferation of intestinal cancer cells compared with control cells (Figure 32), while HNF4a-mRNA LNP had no obvious effect on the proliferation of tumor cells.

[0173] 3. HNF4a-saRNA LNP inhibits the colony formation of intestinal cancer cells

[0174] Intestinal cancer cell HCT-116 was seeded at 3x10 3 Cells were seeded at a density of 200 cells / well in 96-well plates and cultured overnight, and then the culture supernatant was aspirated and the cells were washed with 100 μl PBS. Different concentrations of HNF4a-saRNA LNP and HNF4a-mRNA LNP diluted with Opti-MEM (final concentration of 2 ng RNA / 200 cells) were added to the cells, and after 6 hours, an equal volume of 20% FBS was added to the culture medium of the corresponding cells to make the FBS concentration in the culture medium 10%. Cell counting kit-8 (CCK-8, Dojindo) was used to determine cell proliferation every day to determine the effect of RNA on the proliferation ability of tumor cells. The results showed that HNF4a-saRNA LNP significantly inhibited the proliferation of intestinal cancer cells compared with control cells (Figure 32), while HNF4a-mRNA LNP had no obvious effect on the proliferation of tumor cells.

[0175] Example 7: HNF4a-saRNA up-regulate the expression of HNF4a in pancreatic cancer cells

[0176] Human pancreatic cancer cells PANC1 were seeded into 6-well plates at 40-50% confluency and cultured overnight, washed with 1ml PBS, then added with HNF4a-saRNA LNP diluted in Opti-MEM (final concentration of 1, 2 ng RNA / 200 cells) respectively. After 6 hours, 1ml DMEM medium containing 20% FBS was added. The transfected cells were collected with RIPA lysis buffer at 1 and 3 days for Western blot to detect the protein level of HNF4a in cells (Figure 34), and the results showed that HNF4a-saRNA LNP up-regulated the expression of HNF4a in pancreatic cancer cells.

[0177] Example 8: HNF4a-saRNA LNP specifically up-regulate the expression of HNF4a in tumor tissue in vivo

[0178] Human hepatocarcinoma cell line Huh7 stably expressing luciferase gene was inoculated into the armpit of male nude mice, and after the tumor was formed, the tumor mass was taken out and cut into 1mm 3 small pieces, which were transplanted under the liver capsule of male Bab / c nude mice. The growth of tumor in mice was monitored using IVIS spectrum optical imaging system. When the tumor grew to a suitable size, HNF4a-saRNA LNP was injected into the tail vein of the mice at a dose of 2mg / kg body weight, and the organs and tumor tissues of the mice were taken out at 3 days and 5 days after injection. Quantitative PCR was used to detect the expression and distribution of alphavirus genome sequence (VEEV) and LNP-mediated human HNF4a gene sequence in various organs and tumors (Figure 35). The results showed that 3 days after tail vein injection of HNF4a-saRNA LNP, VEEV and human HNF4a RNA were detected in the heart, spleen and kidney in trace amounts, and VEEV and HNF4a gene sequences were hardly detected in liver, lung and muscle tissues, but high abundance of VEEV and HNF4a gene sequences were detected in tumor tissues. 5 days after tail vein injection of HNF4a-saRNA LNP, VEEV and human HNF4a RNA were not detected in other organs except for trace amounts of expression in the heart, but VEEV and HNF4a gene sequences were still detected in tumor tissues (Figure 35). This indicated that self-replicating RNA can mediate the expression of HNF4a in tumor cells in vivo.

[0179] Example 9: HNF4a-saRNA LNP treatment of patients with advanced liver cancer

[0180] The CDMO company was commissioned to produce HNF4a-saRNA LNP formulations that met the GMP standards according to pharmaceutical standards, and the CRO company was commissioned to complete the single-dose toxicity test of the HNF4a-saRNA LNP formulation. The specific process and results are as follows: the rats were injected once in the tail vein, and the observation period was 14 days. The negative control group (0.9% sodium chloride injection), the carrier control group (LNP solution), and the test product group (150 μg per rat) were set up. All animals survived to the planned autopsy day, and no gross autopsy abnormalities or histopathological changes related to the test product were observed at the end of the observation period.

[0181] After completing the acute toxicity test, a researcher-initiated clinical trial was conducted using hepatic arterial cannulation for administration. Dose escalation tests of 25 μg, 50 μg, and 100 μg (3 cases each) were completed. None of the 9 patients experienced dose-limiting toxicity or treatment-related adverse events of grade 3 or above and serious adverse events. Four patients were treated for more than 5 months, and there was no significant progression of liver cancer lesions. In one of the patients, lung metastases were significantly reduced (Figure 36), and in another patient, liver cancer lesions showed focal necrosis (Figure 37). In 3 patients, target lesions showed obvious necrosis, and the enhancement was significantly reduced (Figure 38). Preliminary results showed that the HNF4a-saRNA LNP formulation had good safety and significantly inhibited tumor growth in patients with advanced liver cancer.

[0182] Example 10 HNF1a-saRNA Inhibits the Development of Malignant Liver Cancer

[0183] The general procedure was the same as in Example 1, and the HNF1a-saRNA sequence is shown in SEQ ID NO: 8.

[0184] 2. 1. Different concentrations of HNF4a-saRNA LNP up-regulate the expression of HNF1a in liver cancer Huh-7 cells

[0185] Liver cancer cells Huh7 were seeded at 3x10 5 Cells were seeded in 6-well plates and cultured overnight, washed with 1 ml of PBS, and then HNF1a-saRNA LNP was added in Opti-MEM for serial dilution (final concentration of 1, 2 ng RNA / 200 cells). After 6 hours of culture, 1 mL of DMEM medium containing 20% FBS was added. The transfected cells were collected with RIPA lysis buffer at 1, 3, or 7 days for Western blot to detect the protein level of HNF1a in cells (Figure 39), and the results showed that HNF1a-saRNA LNP up-regulated the expression of HNF1a.

[0186] 2. HNF1a-saRNA LNP inhibits the growth of mouse subcutaneous xenografts

[0187] Five-week-old male nude mice (BALB / c immunodeficient strain) were purchased from Shanghai BK / KY Biotechnology Co., Ltd., and were housed under specific pathogen-free environmental conditions using a 12-hour on / off light cycle. 1×10 6 Huh-7 cells were subcutaneously injected into the right axilla of male nude mice. The tumor size was measured in two dimensions using calipers, and the volume was calculated using the following formula: Volume = Length × (Width) 2 ×1 / 2. When the average tumor volume reaches approximately 100 mm... 3 Mice were randomly divided into two groups (n=6 per group). HNF1α-saRNA (5 μg / 75 μl / mouse, treatment group) and saline (control group) were injected intratumorally, respectively. Tumor volume was measured daily after injection, tumor growth curves were plotted, and the relative tumor volume RTV was calculated (Figure 40). Five days after RNA injection, mice were sacrificed, tumors were removed and weighed, and tumor inhibition rate was calculated (Figures 41, 42). The results showed that HNF1α-saRNA significantly inhibited the growth of liver cancer implanted tumors.

[0188] Example 11: FOXA3-saRNA Upregulates FOXA3 Expression in Huh-7 Hepatocellular Carcinoma Cells

[0189] The general steps are the same as in Example 1, and the FOXA3-saRNA sequence is shown in SEQ ID NO:9.

[0190] Huh7 liver cancer cells at 3x10 5 Cells were seeded into 6-well plates and cultured overnight. After washing with 1 ml of PBS, FOXA3-saRNA LNP and control GFP-saRNA LNP (final concentrations of 0.5, 1, and 2 ng RNA / 200 cells) were added serially diluted with Opti-MEM. After 6 hours of culture, 1 ml of DMEM medium containing 20% ​​FBS was added. After 3 days, transfected cells were lysed using RIPA buffer, and Western blot analysis was performed to detect intracellular FOXA3 protein levels (Figure 43). The results showed that FOXA3-saRNA LNP upregulated FOXA3 expression, while control GFP-saRNA LNP did not affect FOXA3 expression in hepatocellular carcinoma cells.

[0191] Example 12: PTF1A-saRNA upregulates PTF1A expression in pancreatic cancer PANC1 cells.

[0192] The general steps are the same as in Example 1, and the PTF1A-saRNA sequence is shown in SEQ ID NO:10.

[0193] Pancreatic cancer cells PANC1 at a rate of 2.5 x 10⁻⁶ 5Cells were seeded in 6-well plates and cultured overnight, washed with 1 ml PBS, then added PTF1A-saRNA LNP, control GFP-saRNA LNP (final concentration of 0.5, 1, 2 ng RNA / 200 cells) diluted in Opti-MEM respectively. After 6 hours of culture, 1 mL DMEM medium containing 20% FBS was added. The transfected cells were collected with RIPA lysis buffer after 3 days for Western blot to detect the intracellular PT protein level of PTF1A (Figure 44), the results showed that PTF1A-saRNA LNP LNP up-regulated the expression of PTF1A, while the control GFP-saRNA LNP did not affect the expression of PTF1A in hepatoma cells.

[0194] Example 13 NEUROD1-saRNA mediates overexpression of NEUROD1 in cells

[0195] The general procedure is the same as Example 1, and the NEUROD1-saRNA sequence is shown in SEQ ID NO: 11.

[0196] BHK-21 cells were seeded in 6-well plates at 3x105 cells / well in EMEM medium without antibiotics, and transient transfection was performed after 24 h of complete adhesion: 0.1 μg, 1 μg NEUROD1-saRNA was added to 100 μl Opti-MEM as MIX1, 0.3 μl / 3 μl Lipofectamine MessengerMAX (Thermo) was added to 100 μl Opti-MEM as MIX2, MIX1 and MIX2 were placed at room temperature for 10 min respectively, then MIX1 and MIX2 were mixed, and placed at room temperature for 20 min, then added to the corresponding 6-well plates, 200 μl / well, and then the six-well plates were placed in a carbon dioxide incubator, and after 24 h, RIPA lysis buffer was used to collect the cells for Western blot to detect the intracellular NEUROD1 protein level (Figure 45), the results showed that NEUROD1-saRNA mediated overexpression of NEUROD1.

[0197] Example 14 mix HNF4α / HNF1α / FOXA3-saRNA LNP inhibits the growth of subcutaneous xenografts of mouse hepatoma cells

[0198] Huh-7 cells were subcutaneously injected into the right axillary of female nude mice (BALB / c immunodeficient strain) purchased from Shanghai Shengao Biotechnology Co., Ltd. and raised in a specific pathogen-free environment. A 12-hour on-off light cycle was used. 5 x 10 6 Huh-7 cells were subcutaneously injected into the right axillary of female nude mice (BALB / c immunodeficient strain) purchased from Shanghai Shengao Biotechnology Co., Ltd. and raised in a specific pathogen-free environment. A 12-hour on-off light cycle was used. 5 x 10 2 Huh-7 cells were subcutaneously injected into the right axillary of female nude mice (BALB / c immunodeficient strain) purchased from Shanghai Shengao Biotechnology Co., Ltd. and raised in a specific pathogen-free environment. A 12-hour on-off light cycle was used. 5 x 10 3 When the average tumor volume reached about 150-170 mm 3 , the mice were randomly divided into 2 groups (3 animals in each group). The saline (vehicle control) group was injected twice with 5 μg single HNF4α-saRNA LNP, 10 μg mix HNF4α / HNF1α / FOXA3-saRNA LNP (Figure 46) via the tail vein at an interval of 7 days, with a final volume of 200 μl. The tumor volume was measured every 1-2 days after injection. The saline (vehicle control) group reached a tumor volume of 975.28 ± 72.28 mm 3 , the 5 μg single HNF4α-saRNA LNP group reached a tumor volume of 732.76 ± 96.40 mm 3 , and the 10 μg mix HN4α / HNF1α / FOXA3-saRNA LNP group reached a tumor volume of 517.36 ± 14.84 mm, tumor growth curves were plotted (Figure 47). Two days after the last administration, mice were sacrificed, tumors were excised and weighed (Figure 48), and tumor inhibition rates were calculated. ELISA was used to detect the expression of human HNF4a in tumor tissues (Figure 49). The results showed that intratumoral injection of 10 pg mix HNF4a / HNF1a / FOXA3-saRNA LNP slowed down tumor growth, and the tumor inhibition rate was more than 40%, which was significantly higher than that of 5 pg single HNF4a-saRNA LNP. ELISA results showed that both 5 pg single HNF4a-saRNA LNP and 10 pg mix HNF4a / HNF1a / FOXA3-saRNA LNP could increase the expression of human HNF4a in tumors, and the expression of human HNF4a in tumors treated with 5 pg single HNF4a-saRNA LNP was higher than that treated with 10 pg mix HNF4a / HNF1a / FOXA3-saRNA LNP. These results indicated that mix HNF4a / HNF1a / FOXA3-saRNA LNP could synergistically inhibit the growth of hepatocarcinoma cell xenografts.

[0199] Example 15 PTF1A-saRNA LNP Inhibits the Growth of Subcutaneous Xenografts of Mouse Pancreatic Cancer Cells

[0200] 6-8-week-old female nude mice (BALB / c immunodeficient strain) were purchased from Shanghai Sino-British Biotechnology Co., Ltd. and were raised in a specific pathogen-free environment under a 12-hour on-off light cycle. 5 x 10 6 ASPC-1 cells were subcutaneously injected into the right axillary of female nude mice, and the tumor size was measured in two dimensions using a vernier caliper. The volume was calculated by the following formula: volume = length x (width) 2 x 1 / 2. When the average volume of tumors reached about 200-300 mm 3When the average tumor volume reached about 210-250 mm3, the mice were randomly divided into 2 groups (3 animals per group). The mice were intratumorally injected with 10 μg of PTF1A-saRNA LNP, saline (vehicle control) every 8 days for 3 times, with a final volume of 50 μl. The tumor volume was measured every 1-2 days after injection, and the tumor growth curve was plotted (Figure 51). One day after the last administration, the mice were sacrificed, and the tumors were excised and weighed, and the tumor inhibition rate was calculated (Figure 52, Figure 53). The expression of PTF1A in tumor tissue was detected by western-blot (Figure 54). The results showed that the tumor growth was slowed down after intratumoral injection of PTF1A-saRNA, and the tumor inhibition rate was more than 40% (Figure 53). The western-blot results showed that PTF1A-saRNA significantly increased the protein expression of PTF1A in tumor tissue. These results indicated that PTF1A-saRNA can effectively induce the expression of PTF1A in cancer cells, thereby inhibiting the growth of mouse pancreatic cancer cell xenografts.

[0201] Example 16 NeuroD1-saRNA LNP Inhibits the Growth of Mouse Subcutaneous Xenografts

[0202] 6-8 week old female nude mice (BALB / c immunodeficient strain) were purchased from Shanghai Sino-British Biological Technology Co., Ltd. and raised in a specific pathogen-free environment, with a 12-hour on-off light cycle. 5 x 106U87 cells were injected subcutaneously into the right axillary of female nude mice, and the tumor size was measured in two dimensions using a vernier caliper. The volume was calculated by the following formula: volume = length x (width) 6 U87 cells were injected subcutaneously into the right axillary of female nude mice, and the tumor size was measured in two dimensions using a vernier caliper. The volume was calculated by the following formula: volume = length x (width) 2 ×1 / 2. When the average tumor volume reached about 210-250 mm 3 When the average tumor volume reached about 210-250 mm3, the mice were randomly divided into 2 groups (3 animals per group). The mice were intratumorally injected with 10 μg of PTF1A-saRNA LNP, saline (vehicle control) every 8 days for 3 times, with a final volume of 50 μl. The tumor volume was measured every 1-2 days after injection, and the tumor growth curve was plotted (Figure 51). One day after the last administration, the mice were sacrificed, and the tumors were excised and weighed, and the tumor inhibition rate was calculated (Figure 52, Figure 53). The expression of PTF1A in tumor tissue was detected by western-blot (Figure 54). The results showed that the tumor growth was slowed down after intratumoral injection of PTF1A-saRNA, and the tumor inhibition rate was more than 40% (Figure 53). The western-blot results showed that PTF1A-saRNA significantly increased the protein expression of PTF1A in tumor tissue. These results indicated that PTF1A-saRNA can effectively induce the expression of PTF1A in cancer cells, thereby inhibiting the growth of mouse pancreatic cancer cell xenografts.

[0203] The preferred embodiments of the present application have been specifically described above, but the present application is not limited to the embodiments described above, and those skilled in the art can make various equivalent modifications or replacements without departing from the spirit of the present application. These equivalent modifications or replacements are all included in the scope defined by the claims of the present application.

Claims

1. A self-replicating RNA expressing a differentiation-associated transcription factor, the differentiation-associated transcription factor being a transcription factor associated with tissue organ differentiation and function maintenance, the transcription factor being expressed at a lower amount in a tumor cell than in a normal cell; the transcription factor being delivered into the tumor cell by the self-replicating RNA and a delivery vehicle, the transcription factor being expressed at a high level in the tumor cell for a long time and specifically, inducing the tumor cell to differentiate into a normal mature cell, inhibiting proliferation of the tumor cell or / and inducing apoptosis of the tumor cell.

2. The self-replicating RNA expressing a differentiation-related transcription factor according to claim 1, characterized in that, The self-replicating RNA has a poly(A) tail of 35-100 nt.

3. The self-replicating RNA expressing a differentiation-related transcription factor according to claim 1, characterized in that, The self-replicating RNA has a poly(A) tail of 40-90 nt.

4. The self-replicating RNA expressing a differentiation-related transcription factor according to claim 1, characterized in that, The self-replicating RNA has a poly(A) tail of 50-75 nt.

5. The self-replicating RNA expressing a differentiation-related transcription factor according to claim 1, characterized in that, The self-replicating RNA has a poly(A) tail of 60-70 nt.

6. The self-replicating RNA expressing a differentiation-related transcription factor according to any one of claims 1 to 5, characterized in that, The transcription factor is selected from the group consisting of HNF4a, HNF1a, FOXA3, PTF1A, NUROND1, Neurogenin-2, and Ascll.

7. The self-replicating RNA expressing a differentiation-related transcription factor according to claim 6, characterized in that, The delivery vehicle is selected from the group consisting of one or more of the following in combination: liposome, viral replicon particle, lipid-based nanoparticle, polymeric nanoparticle, physiological buffer, microsphere, immunostimulatory complex, conjugate of a biologically active ligand.

8. The self-replicating RNA expressing a differentiation-related transcription factor of claim 1, wherein, The sequence of the self-replicating RNA is set forth in SEQ ID NO:

6.

9. The self-replicating RNA expressing a differentiation-related transcription factor according to claim 8, characterized in that, The sequence of the HNF4a self-replicating RNA includes any one of the following (a) to (c): (a) an RNA consisting of the nucleotide sequence set forth in SEQ ID NO: 7; (b) an RNA consisting of a nucleotide sequence having one or more deletions, substitutions, additions, or insertions of nucleotides in the nucleotide sequence set forth in SEQ ID NO: 7 and having activity in HNF4a expression; and (c) an RNA consisting of a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 7 and having activity in HNF4a expression. The sequence of the HNF1a self-replicating RNA includes any one of the following (a) to (c):

10. The self-replicating RNA expressing a differentiation-related transcription factor according to claim 8, characterized in that, (a) an RNA consisting of the nucleotide sequence set forth in SEQ ID NO: 8; (b) an RNA consisting of a nucleotide sequence having one or more deletions, substitutions, additions, or insertions of nucleotides in the nucleotide sequence set forth in SEQ ID NO: 8 and having activity in HNF1a expression; and (c) an RNA consisting of a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 8 and having activity in HNF1a expression. The sequence of the FOXA3 self-replicating RNA includes any one of the following (a) to (c): (a) an RNA consisting of the nucleotide sequence set forth in SEQ ID NO: 9; 11. The self-replicating RNA expressing a differentiation-related transcription factor of claim 8, wherein, (b) an RNA consisting of a nucleotide sequence having one or more deletions, substitutions, additions, or insertions of nucleotides in the nucleotide sequence set forth in SEQ ID NO: 9 and having activity in FOXA3 expression; and (c) an RNA consisting of a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 9 and having activity in FOXA3 expression. ​ (c) a RNA consisting of a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 9 and having an activity of FOXA3 expression.

12. The self-replicating RNA expressing a differentiation-related transcription factor of claim 8, wherein, The sequence of the PTF1A self-replicating RNA includes any one of the following (a) to (c): (a) a RNA consisting of the nucleotide sequence set forth in SEQ ID NO: 10; (b) a RNA consisting of a nucleotide sequence having a deletion, substitution, addition, or insertion of one or more nucleotides in the nucleotide sequence set forth in SEQ ID NO: 10 and having an activity of PTF1A expression; and (c) a RNA consisting of a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 10 and having an activity of PTF1A expression.

13. The self-replicating RNA expressing a differentiation-related transcription factor of claim 8, wherein, The sequence of the NUROND1 self-replicating RNA includes any one of the following (a) to (c): (a) a RNA consisting of the nucleotide sequence set forth in SEQ ID NO: 11; (b) a RNA consisting of a nucleotide sequence having a deletion, substitution, addition, or insertion of one or more nucleotides in the nucleotide sequence set forth in SEQ ID NO: 11 and having an activity of NUROND1 expression; and (c) a RNA consisting of a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 11 and having an activity of NUROND1 expression.

14. Use of the self-replicating RNA expressing a differentiation-related transcription factor according to any one of claims 1 to 13 in the manufacture of a medicament for treating a malignant solid tumor. The malignant solid tumor is liver cancer, pancreatic cancer, gastric cancer, intestinal cancer, kidney cancer, lung cancer, brain glioma.

15. Use of the self-replicating RNA expressing a differentiation-related transcription factor according to claim 14 for the manufacture of a medicament for the treatment of a malignant solid tumor.

16. A gene delivery system consisting of the self-replicating RNA expressing a differentiation-related transcription factor according to any one of claims 1 to 14 and a delivery vehicle; the gene delivery system delivers a target gene into a tumor cell, in which the transcription factor is expressed at a high level and specifically for a long time, inducing differentiation of the tumor cell into a normal mature cell, inhibiting proliferation of the tumor cell or / and inducing apoptosis of the tumor cell. When the transcription factor is HNF4a or HNF1a, the delivery vehicle is a lipid-based nanoparticle, and the lipid is selected from ALC-0315, SM-102, or DHA-1.

17. The gene delivery system of claim 16, wherein, 18. A pharmaceutical composition comprising the gene delivery system according to claim 16 or 17. ​

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