Self-replicating RNA (Ribonucleic Acid) for expressing differentiation-related transcription factor and application of self-replicating RNA in preparation of tumor treatment medicine

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to self-replicating RNA for expressing differentiation-related transcription factors and application of the self-replicating RNA in preparation of tumor treatment drugs. The invention relates to a technical means for inducing tumor cells to differentiate into mature cells, expression of important differentiation-related transcription factors in tumor cells is regulated by using messenger ribonucleic acid, malignant phenotypes of malignant solid tumor cells are inhibited, and the effect of treating malignant solid tumors is achieved, so that the messenger ribonucleic acid is applied to the preparation method and application of solid tumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to self-replicating RNA expressing differentiation-related transcription factors and its application in the preparation of tumor therapeutic drugs. It is a technical means to induce tumor cells to differentiate into mature cells. By using messenger ribonucleic acid to regulate the expression of important differentiation-related transcription factors in tumor cells, it inhibits the malignant phenotype of malignant solid tumor cells and achieves the effect of treating malignant solid tumors, thereby being applied to the preparation method and application of solid tumor drugs. Background Technology

[0002] The treatment of malignant solid tumors remains a significant challenge in clinical practice, especially for those that cannot be surgically eradicated, for which effective treatments are still lacking. Although radiotherapy, chemotherapy, targeted therapy, and immunotherapy have provided various treatment options in recent years, chemotherapy resistance often develops as the tumor progresses. Furthermore, many tumors lack effective targeted drug targets or are insensitive to immunotherapy. This is particularly true for some gastrointestinal tumors such as liver cancer and pancreatic cancer, as well as gliomas of the brain, where effective drug treatments are scarce, resulting in a poor prognosis for patients.

[0003] Tumor differentiation-inducing therapy promotes the differentiation of tumor cells into mature normal cells through various inducing factors, restoring their normal phenotype and function and inhibiting the proliferation of malignant tumor cells. This strategy breaks with conventional approaches to tumor treatment. A classic example is the use of all-trans retinoic acid (ATA) for differentiation therapy in acute promyelocytic leukemia, which has achieved good clinical efficacy and is widely used. However, differentiation-inducing therapy for malignant solid tumors remains a challenge in current tumor treatment. Recent studies have found that transcription factors related to organ differentiation, development, and functional maintenance can induce related tumor cells to differentiate into normal cells. Upregulating these differentiation-related transcription factors can inhibit tumor cell growth without significantly affecting the function of normal tissue cells, opening up new directions for tumor differentiation-inducing therapy, especially for the treatment of malignant solid tumors. Therefore, for tumors in different tissues, specific targeted regulation of proteins, molecules, and genes closely related to tumor cell differentiation is the core issue of tumor differentiation-inducing therapy. Utilizing genetic engineering techniques to target and regulate the expression of important differentiation function genes and induce tumor cells to differentiate into mature cell phenotypes may fundamentally reverse the progression of malignant tumors.

[0004] The hepatocyte nuclear factor (HNF) family, including HNF1, HNF3, HNF4, HNF6, and CCAAT / enhancer-binding protein (C / EBP), is a group of transcription factors that are predominantly expressed in the liver and regulate each other, playing a crucial role in liver development and function maintenance. HNF4α is expressed in tissues such as the liver, kidneys, pancreas, and intestines, but primarily in mature hepatocytes. In mature hepatocytes, HNF4α binds to the promoters of approximately 12% of intracellular genes, participating in the maintenance of important functions such as hepatocyte lipid metabolism, albumin synthesis, drug detoxification, energy metabolism, and bile acid synthesis. It is also an important gene for maintaining the hepatocyte epithelial phenotype. HNF4α also regulates the development of kidney and intestinal tissues and regulates insulin production. Studies on liver cancer have found that the dedifferentiation state of liver cancer is accompanied by the downregulation of a large number of hepatocyte nuclear factor expressions, among which the downregulation of HNF4α expression is a crucial step in liver cancer development. Previous studies have shown that HNF4α expression is decreased in various epithelial-derived tumors such as liver cancer (including hepatocellular carcinoma and intrahepatic cholangiocarcinoma), pancreatic cancer, colorectal cancer, and kidney cancer. Overexpression of HNF4α can inhibit tumor cell proliferation and metastasis and promote tumor cell apoptosis, suggesting that HNF4α is a potential target for tumor therapy (Differentiation therapy of hepatocellular carcinoma in mice with recombinantadenovirus carrying hepatocyte nuclear factor-4alpha gene. Hepatology. 2008Nov;48(5):1528-39).

[0005] HNF1α is another important hepatocyte nuclear factor that can bind to the cis-acting elements of at least 200 liver target genes. These target genes are involved in many important liver functions, such as glycogen synthesis and storage, gluconeogenesis, lipid metabolism, serum protein synthesis, and detoxification. Previous studies have shown that HNF1α expression is decreased in hepatocellular carcinoma. Upregulation of HNF1α in hepatocellular carcinoma cells can promote the expression of liver function genes in hepatocellular carcinoma cells, arrest the cell cycle of liver tumor cells in the G2 / M phase, thereby inhibiting tumor cell proliferation and significantly inhibiting tumor growth in vivo (Recombinant adenovirus carrying the hepatocyte nuclearfactor-1alpha gene inhibits hepatocellular carcinoma xenograft growth in mice. Hepatology, 2011, 54(6):2036-2047.). Recent studies have found that combined overexpression of a group of hepatocyte nuclear factors, including HNF4α, HNF1α, and FOXA3 (also known as HNF3γ), can successfully transform liver tumor cells into mature hepatocytes in vitro and in vivo. This further demonstrates that increasing the expression of hepatocyte nuclear factors such as HNF4α, HNF1α, and FOXA3 is an ideal strategy for inducing differentiation therapy for liver cancer. (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 a crucial role in mammalian pancreatic development and is involved in maintaining the expression of exocrine pancreatic-specific genes, including elastase 1 and amylase. Previous studies have shown that PTF1A expression is absent in ductal pancreatic cancer. Maintaining PTF1A expression can completely block the formation of pancreatic cancer cells, and restoring PTF1A expression can induce early-stage cancer cells to transform into normal pancreatic cells and inhibit the growth of late-stage pancreatic cancer cells. Therefore, it is a potential target for differentiation-induced therapy of 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), neurogenin 2 (NEUROG2), and ASCL1 are all transcription factors related to neuronal differentiation, participating in the induction of neuronal differentiation during early brain development. They 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 the differentiation of astrocytes 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, making them important targets for differentiation-induced therapy of gliomas. (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 tumor cells to mature further provide a basis for differentiation-induced tumor therapy. Currently, it is known that the functions of transcription factors are mainly performed in the cell nucleus. To achieve the goal of upregulating related transcription factors to treat tumors, we need to use vectors to mediate the expression of related genes in tumor cells and require effective means to maintain their expression in tumor cells. Previously used expression vectors, such as adenoviruses, adeno-associated viruses, lentiviruses, and plasmids, all have shortcomings in terms of safety, expression efficiency, and tissue accessibility. Our team previously held a patent that used an adenovirus vector to express HNF4α and HNF1α, demonstrating the inhibitory effect of HNF4α and HNF1α on liver cancer. Due to the rapid proliferation of tumors, tumor treatment requires repeated administration to ensure therapeutic efficacy. However, viral vectors can induce immune system rejection of the delivery system, and pre-existing immunity to adenovirus exists in the population, thus limiting the use of viral vectors to mediate the expression of target genes in tumor cells and the frequency of drug administration for tumor treatment. Furthermore, adenovirus vectors have shortcomings in safety and tissue targeting, and their preparation cost is high, limiting their potential for practical application in tumor treatment.

[0009] mRNA technology is a revolutionary gene delivery technology that has emerged in recent years. This technology synthesizes mRNA molecules with specific sequences through in vitro transcription and encapsulates them in lipid nanoparticles (LNPs) to transport them into human cells. The cells then rely on their own translation system to translate the mRNA into the target protein. Compared with existing viral vector-based delivery systems, this technology offers superior efficiency and safety. Furthermore, the tissue specificity of the delivery system can be increased by selecting and modifying the lipid nanoparticles. Currently known potential applications of mRNA technology mainly focus on vaccine development for infectious diseases, therapeutic tumor vaccines, protein replacement therapies for protein deficiencies caused by gene defects, and gene editing for treating hereditary diseases or modifying immune cells. However, because mRNA can only mediate transient high expression of the target gene in cells, it has significant limitations in expression duration and efficiency, failing to meet the need for 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 developed in recent years based on mRNA technology. It is a recombinant RNA with a viral structure. By modifying the bicistronic genome of alphaviruses, the sequences encoding the four non-structural proteins (NSPs) from the virus are retained, and the viral structural protein gene located after the viral subgenome promoter (SGP) is replaced with a heterologous gene of interest (GOI) encoding the target protein, thus constructing an RNA vector capable of self-amplification within cells to express the target gene. Currently, self-amplifying RNA vectors are mainly modified from Venezuelan equine encephalitis virus (VEEV), sindbisalphavirus (SIN), or Semliki Forest virus (SFV), with self-amplifying RNA derived from Venezuelan equine encephalitis virus being the most commonly used. Upon entering the host cell cytoplasm, the self-replicating RNA (SRNA) first translates into four non-structural proteins (NSP1, NSP2, NSP3, and NSP4). These proteins then polymerize through a complex, multi-step process to form an RNA-dependent RNA polymerase complex. The RNA polymerase complex first synthesizes a complementary negative-strand RNA intermediate from the positive-strand RNA, and then uses the intermediate as a template to synthesize two distinct positive-strand RNAs. The first positive-strand RNA is a copy of the original full-length genomic RNA; the second positive-strand RNA is a large amount of subgenomic RNA encoding the target gene. This mechanism enables the self-replicating RNA to achieve high-level and sustained expression of the target protein with low doses. While the self-replicating RNA retains the viral genome's self-amplification ability, it cannot express viral structural proteins and therefore cannot produce a complete virus capable of transmission. Its entry into cells relies on delivery via lipid nanoparticles, thus exhibiting good safety and potential tumor-targeting capabilities.

[0011] Currently, in cancer treatment, self-replicating RNA is considered applicable to the preparation of RNA vaccines and the mediating of tumor-killing cytokine expression in tumor cells. For example, Chinese invention patent application CN117280029A discloses a nucleic acid vector and its usage method, which modulates the tumor microenvironment by activating the relative expression of tumor-infiltrating lymphocytes and / or immunogenic cellular characteristics in the tumor microenvironment to achieve cancer treatment; Chinese invention patent application CN117279661A discloses a composition and method for inducing immune responses to ESR1, PI3K, HER2, and HER3, which treats cancer by inducing immune responses; Chinese invention patent application CN115968299A discloses a neoantigen expressed in multiple myeloma and its uses, which also uses self-replicating RNA as a vector to prepare tumor vaccines. The mechanism of action of tumor vaccines 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, activating T cells to attack tumor cells, thereby controlling or eliminating the tumor.

[0012] However, there are no reports of using self-replicating RNA to express transcription factors, especially those related to tissue and organ differentiation, in tumor cells to treat tumors. Summary of the Invention

[0013] The purpose of this invention is to provide a self-replicating RNA that expresses differentiation-related transcription factors and its application in the preparation of tumor therapeutic drugs. By utilizing the self-replicating RNA to express transcription factors in tumor cells, the differentiation of tumor cells into mature cells can be induced, the malignant phenotype of malignant solid tumor cells can be inhibited, and the therapeutic effect on malignant solid tumors can be achieved. This invention aims to address how to achieve long-term and specific high expression of related transcription factors in tumor cells and improve the tumor-suppressing effect.

[0014] The expression levels of relevant differentiation transcription factors involved in this invention are lower in tumor cells than in normal cells. The inventors' research found that self-replicating RNA, after entering tumor cells, maintains its distribution within the cells despite rapid tumor cell proliferation due to its self-replication characteristic, and can sustain protein expression for over 20-30 days. Therefore, it is highly suitable for mediating the expression of target genes in tumor cells. Simultaneously, animal experiments showed that tail vein injection of self-replicating RNA encapsulated in lipid nanoparticles can mediate the specific expression of target genes in tumor tissues, while the expression of target genes is virtually undetectable in other normal tissues, suggesting that self-replicating RNA has good tumor targeting properties. Based on these findings, the inventors propose using self-replicating RNA vectors to restore or overexpress HNF4α in tumor tissues, inducing tumor cells to differentiate into normal cells and inhibiting tumor cell growth, thereby achieving a therapeutic effect on tumors.

[0015] This invention utilizes self-replicating RNA technology to introduce relevant differentiation-inducing transcription factors into relevant tumor cells for overexpression. These factors are specifically and persistently expressed in tumor cells, thereby inducing the relevant tumor cells to differentiate into mature cells for tumor treatment.

[0016] The self-replicating RNA described above has an optimized polyA tail length, enabling it to maintain a longer-lasting, high-level expression of the target gene in vivo. The self-replicating RNA structure includes a 5' cap, a 5' UTR, four non-structural genes (NSP1-4), a 26S subgenomic promoter (SGP), the target gene (GOI), a 3' UTR, and a polyA tail. To optimize the performance of the self-replicating RNA, previous studies have explored various strategies to modify NSP1-4, introducing mutations to improve replication efficiency and immunogenicity, and increasing the expression intensity and duration of different target genes in cells. We found that self-replicating RNA vectors with different NSP1-4 mutations showed little difference in their efficiency in mediating HNF4α expression in tumor cells. On the other hand, previous studies have shown that the length and sequence of the polyA tail of mRNA affect mRNA stability and ribosomal translation; altering the polyA tail length can influence the expression duration and level of encoded proteins by regulating the rate of mRNA degradation and translation. However, it remains unclear whether the polyA tail of self-replicating RNA affects the expression duration and level of the target gene in tumor cells. 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. We found that self-replicating RNA with a polyA length between 35-100 nt could mediate relatively stable and long-term overexpression of HNF4α in tumor cells, with 50-70 nt showing better expression levels and duration. Self-replicating RNA with a polyA tail of 60-70 nt achieved the most stable and efficient expression.

[0017] This invention also investigated the effects of non-replicating HNF4α-mRNA, HNF4α-saRNA, and adenovirus-mediated HNF4α overexpression in tumor cells. The results showed that both HNF4α-saRNA and HNF4α-mRNA could upregulate HNF4α in liver cancer cells, but HNF4α-mRNA expression was lower and its duration was shorter. In contrast, HNF4α-saRNA expression was more than twice that of HNF4α-mRNA and its duration was longer. The upregulation effect of HNF4α-mRNA on HNF4α expression could only be maintained for 3 days, while HNF4α-saRNA could maintain HNF4α in liver cancer cells for at least 7 days. Furthermore, the upregulation efficiency of HNF4α expression by HNF4α-saRNA was no less than that of adenovirus, indicating that self-replicating RNA can mediate the expression of target genes in tumor cells more efficiently than non-replicating mRNA.

[0018] In a first aspect, the present invention provides a self-replicating RNA expressing a differentiation-related transcription factor, wherein the differentiation-related transcription factor is a transcription factor related to the differentiation and functional maintenance of tissues and organs, and the expression level of the transcription factor in tumor cells is lower than that in normal cells; the transcription factor is delivered into tumor cells via the self-replicating RNA and a delivery medium, and the transcription factor is expressed in tumor cells in a long-term and specific manner, inducing tumor cells to differentiate into normal mature cells, inhibiting tumor cell proliferation and / or inducing tumor cell apoptosis.

[0019] The term "long-term expression" refers to expression lasting at least seven days (including seven days).

[0020] The aforementioned specific high expression refers to the absence, low expression, or transient expression in normal cells.

[0021] The self-replicating RNA includes a 5' cap, a non-coding region, a non-structural gene, a 26S subgenome promoter, a 3' non-coding region, and a polyadenylated tail.

[0022] The self-replicating RNA has a 35-100 nt polyadenylate tail.

[0023] Preferably, the self-replicating RNA has a 40-90 nt polyadenylated tail.

[0024] More preferably, the self-replicating RNA has a 50-75 nt polyadenylated tail.

[0025] Optimal, the self-replicating RNA has a 60-70 nt polyadenylated tail.

[0026] The transcription factors mentioned are selected from HNF4α, HNF1α, FOXA3, PTF1A, NUROND1, Neurogenin-2, Ascl1, and other transcription factors related to tissue and organ differentiation and function maintenance.

[0027] HNF4α (GENBANK No.: NM_000457.6, as shown in SEQ ID NO:1);

[0028] HNF1α (GENBANK No.: NM_001306179.2, as shown in SEQ ID NO:2);

[0029] FOXA3 (GENBANK No.: NM_004497.3, as shown in SEQ ID NO:3);

[0030] PTF1A (GENBANK No.: NM_178161.3, as shown in SEQ ID NO:4);

[0031] NUROND1 (GENBANK No.: NM_002500.5, as shown in SEQ ID NO:5);

[0032] Neurogenin-2 (GENBANK number: NM_024019.4);

[0033] Ascl1 (GENBANK No.: NM_004316.4).

[0034] The delivery medium is selected from one or more of the following combinations: liposomes, viral replicon particles, lipid-based nanoparticles, polymer nanoparticles, physiological buffers, microspheres, immunostimulatory complexes, and conjugates of bioactive ligands.

[0035] In a preferred embodiment of the present invention, the sequence of the self-replicating RNA is shown in SEQ ID NO:6. This sequence is an optimization of the polyA tail of wild-type self-replicating RNA, preferably a 67nt polyA tail. The 67-nucleotide A tail of saRNA can increase the half-life of saRNA in vivo, promoting saRNA stability and translation.

[0036] In a preferred embodiment of the present invention, the sequence of the HNF4α self-replicating RNA includes any one of the following (a) to (c):

[0037] (a) RNA consisting of the nucleotide sequence shown in SEQ ID NO:7;

[0038] (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:7 and having activity expressed as HNF4α; and

[0039] (c) An RNA consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:7 and having activity expressed as HNF4α.

[0040] In a preferred embodiment of the present invention, the sequence of the HNF1α self-replicating RNA includes any one of the following (a) to (c):

[0041] (a) RNA consisting of the nucleotide sequence shown in SEQ ID NO:8;

[0042] (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:8 and having activity expressed as HNF1α; and

[0043] (c) An RNA consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:8 and having activity expressed as HNF1α.

[0044] In a preferred embodiment of the present invention, the sequence of the FOXA3 self-replicating RNA includes any one of the following (a) to (c):

[0045] (a) RNA consisting of the nucleotide sequence shown in SEQ ID NO:9;

[0046] (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:9 and having activity expressed as FOXA3; and

[0047] (c) An RNA consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:9 and having the activity of being expressed as FOXA3.

[0048] In a preferred embodiment of the present invention, the sequence of the PTF1A self-replicating RNA includes any one of the following (a) to (c):

[0049] (a) RNA consisting of the nucleotide sequence shown in SEQ ID NO:10;

[0050] (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

[0051] (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.

[0052] 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):

[0053] (a) RNA consisting of the nucleotide sequence shown in SEQ ID NO:11;

[0054] (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

[0055] (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.

[0056] 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.

[0057] The malignant solid tumors mentioned are liver cancer, pancreatic cancer, stomach cancer, intestinal cancer, kidney cancer, lung cancer, and glioma.

[0058] 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.

[0059] The transcription factors mentioned are selected from NUROND1, Neurogenin-2, and Ascl1, and the malignant solid tumor mentioned is glioma.

[0060] 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.

[0061] In a preferred embodiment of the present invention, the delivery medium is lipid-based nanoparticles (LNPs), the LNPs comprising ionizable lipids, 1,2-distearate-sn-propanetriyl-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), cholesterol and dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG 2000) or PEGylated lipids containing polyethylene glycol fragments. The preferred LNP composition includes a molar ratio of DSPC:cholesterol:DMG-PEG-2000:ionizable lipids, ranging from DSPC: 5%-20%, cholesterol: 30%-55%, PEG: 0.5%-3%, and ionizable lipids: 30%-60%, with a total lipid molar ratio of 100%, optionally 9.4:42.5:1.8:46.3 (DSPC:cholesterol:DMG-PEG 2000:ionizable lipids). The N:P ratio ranges from 5:1 to 10:1, optionally 6:1, and the LNP has a particle size of approximately 40-300 nm.

[0062] In a preferred embodiment of the present invention, when the transcription factor is HNF4α or HNF1α, the delivery medium is lipid-based nanoparticles (LNPs), and the lipids are selected from ALC-0315, SM-102 or DHA-1.

[0063] ALC-0315, SM-102, or DHA-1 have the following specific structures:

[0064] ALC-0315

[0065] SM-102

[0066] DHA-1

[0067] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the gene delivery system described above.

[0068] The technical effects of this invention are as follows: This invention utilizes a self-replicating RNA vector based on an mRNA technology platform, combined with lipid nanoparticle encapsulation and delivery, to highly express important differentiation-related transcription factors in tumor cells, inducing tumor cells to transform into normal cells, inhibiting the malignant phenotype of tumors, and achieving the goal of treating tumors. This is a novel approach to differentiation-induced tumor therapy. Based on our previous animal experiments and researcher-initiated clinical studies, we can confirm that this technology can effectively inhibit tumor growth in vivo, representing a new approach to tumor treatment and potentially opening up a new avenue for tumor therapy. Furthermore, this invention differs significantly from current applications of mRNA technology in tumor vaccines. RNA tumor vaccines utilize RNA technology to express relevant tumor antigens in antigen-presenting cells to stimulate the body to produce an immune response against these antigens, thereby controlling or eliminating tumors. In contrast, this invention utilizes RNA technology to mediate the expression of differentiation transcription factors in tumor cells, leveraging the differentiation-promoting effects of these transcription factors to inhibit the malignant phenotype of tumors. Although non-replicating mRNAs can mediate the transient expression of target genes in tumor cells, stable and efficient expression of differentiation-related transcription factors is required in tumor cells to induce differentiation of tumor cells into normal cells. Therefore, this invention proposes for the first time the use of self-replicating RNA technology to restore or overexpress differentiation-related transcription factors whose expression declines during tumorigenesis, thereby inhibiting tumor cell growth and achieving a therapeutic effect on tumors. Thus, the gene delivery system provided by this invention can induce the differentiation of malignant solid tumor cells into normal mature cells, inhibit tumor cell proliferation, and induce tumor cell apoptosis. Furthermore, this invention optimizes the self-replicating RNA vector used to obtain a delivery system that can stably and efficiently express target genes in tumor cells.

[0069] In summary, this invention utilizes self-replicating RNA to express differentiation-related transcription factors, resulting in not only high expression levels and efficiency but also longer duration of expression. For example, the preferred HNF4α-saRNA of this invention can maintain HNF4α in liver cancer cells for more than 7 days, with both expression levels and durations significantly higher than those of non-replicating HNF4α-mRNA. Compared with viral vectors, it has advantages such as good safety, strong tissue accessibility, and repeatable administration. Therapeutic effects have been observed in tumor-bearing animals and patients. Attached Figure Description

[0070] Figure 1 This is a schematic diagram of the structure of self-replicating RNAs expressing HNF4α with polyA tails of different lengths. GOI represents HNF4α; the lengths of the polyA tails are 30nt, 35nt, 40nt, 50nt, 60nt, 67nt, 75nt, 90nt, 100nt, and 110nt, respectively.

[0071] Figure 2Huh-7 cells were treated with lipid nanoparticles containing HNF4α-saRNALNPs with polyA tails of different lengths. Cell proteins were collected 3 and 7 days after transfection. Western blot was used to detect the expression level of HNF4α protein after different treatments to observe the effect of polyA length on the overexpression of HNF4α mediated by self-reinforced RNA in hepatocellular carcinoma cells.

[0072] Figure 3 Huh7 cells were treated with 2 ng / 200 cells of HNF4α-saRNA and HNF4α-mRNA lipid nanoparticles for 1, 3, and 7 days, respectively, and proteins were collected afterward. Huh7 cells were also infected with adenovirus Ad-HNF4α and control virus Ad-GFP for 1, 3, and 7 days, respectively, and proteins were collected afterward. Western blot was used to detect the expression level of HNF4α protein after different treatments. ImageJ software was used to calculate the relative expression level of HNF4α protein at each time point.

[0073] Figure 4 Huh-7 cells were treated with lipid nanoparticles containing different concentrations of HNF4α-saRNA and control GFP-saRNA for 1, 3, 5, and 7 days, and the expression level of HNF4α protein was detected by Western blot. GFP-saRNA served as a self-replicating mRNA control.

[0074] Figure 5 After treating Huh-7 cells with lipid nanoparticles of different concentrations of HNF4α-saRNA and GFP-saRNA, the changes in cell proliferation after treatment were detected by CCK8 assay.

[0075] Figure 6 The effect of HNF4α-saRNA and GFP-saRNA lipid nanoparticle treatment on the clone-forming ability of Huh7 liver cancer cells.

[0076] Figure 7 After treating Huh7 cells with lipid nanoparticles of different concentrations of HNF4α-saRNA and GFP-saRNA for 3 days, liver function-related genes were detected by RT-PCR.

[0077] Figure 8 After treating Huh7 cells with different concentrations of HNF4α-saRNA and GFP-saRNA lipid nanoparticles for 3 days, the expression levels of tumor cell stemness-related genes were detected by RT-PCR.

[0078] Figure 9After treating Huh7 cells with HNF4α-saRNA and GFP-saRNA lipid nanoparticles for 3 days, the glycogen storage in Huh7 cells was detected by periodate-Schiff (PAS) staining, and the PAS-positive areas in Huh7 cells were counted using ImageJ software.

[0079] Figure 10 After treating Huh7 cells with HNF4α-saRNA and GFP-saRNA lipid nanoparticles for 3 days, the uptake capacity of acetylated low-density lipoprotein (ac-LDL) in Huh7 cells was detected using Dil-ac-LDL fluorescent substrate, and the ac-LDL positive region in Huh7 cells was quantified using imageJ software.

[0080] Figure 11 After treating Huh7 cells with HNF4α-saRNA and GFP-saRNA lipid nanoparticles for 3 days, the content of senescence-related β-galactosidase in Huh7 cells was detected, and the β-galactosidase-positive region in Huh7 cells was quantitatively analyzed using ImageJ software.

[0081] Figure 12 After treating Huh7 cells with HNF4α-saRNA and GFP-saRNA lipid nanoparticles for 3 days, Annexin V / PI staining was used to detect cell apoptosis.

[0082] Figure 13 Flowchart of an experimental study on the treatment of Huh7 cell subcutaneous xenografts by intratumoral injection of HNF4α-saRNA and HNF4α-mRNA lipid nanoparticles.

[0083] Figure 14 Tumor proliferation curves in a Huh7 cell subcutaneous xenograft model treated with HNF4α-saRNA and HNF4α-mRNA are shown. GFP-saRNA is a self-replicating mRNA lipid nanoparticle control, and physiological saline is a solvent control.

[0084] Figure 15 Gross image of a Huh7 cell subcutaneous xenograft tumor treated with HNF4α-saRNA and HNF4α-mRNA.

[0085] Figure 16 The left graph shows the tumor weight and the right graph shows the tumor inhibition rate in a Huh7 cell subcutaneous xenograft model treated with HNF4α-saRNA and HNF4α-mRNA.

[0086] Figure 17 Western blot analysis was used to detect the expression level of HNF4α in tumor tissues of a Huh7 cell subcutaneous xenograft model treated with HNF4α-saRNA.

[0087] Figure 18 The expression changes of HNF4α and Ki67 in tumor tissue were detected by immunohistochemistry (left), and the statistical diagram of the positive staining area of ​​HNF4α and Ki67 in tumor tissue (right).

[0088] Figure 19 Flowchart of an experimental study on the treatment of Huh7 cell orthotopic liver tumors by tail vein injection of self-replicating HNF4α-saRNA and HNF4α-mRNA lipid nanoparticles.

[0089] Figure 20 The images show the in vivo fluorescence signals of HNF4α-saRNA and HNF4α-mRNA in mice before and after injection in a Huh7 cell orthotopic liver tumor model. GFP-sRNA is a self-replicating RNA lipid nanoparticle control, and physiological saline is a solvent control.

[0090] Figure 21 Statistical graph of in vivo fluorescence signals in mice at different time points during the treatment of Huh7 cell liver orthotopic tumors with HNF4α-saRNA and HNF4α-mRNA.

[0091] Figure 22 Gross image of the tumor in an experiment using HNF4α-saRNA and HNF4α-mRNA lipid nanoparticles to treat Huh7 cell liver orthotopic tumors.

[0092] Figure 23 In an experiment where HNF4α-saRNA and HNF4α-mRNA lipid nanoparticles were used to treat Huh7 cell liver orthotopic tumors, the tumor weight (left) and tumor inhibition rate (right) are statistical graphs.

[0093] Figure 24 HE staining was used to detect the morphology of tumor tissue, and immunohistochemistry was used to detect changes in Ki67 expression (left). The statistical graph of the area of ​​Ki67 positive staining in tumor tissue (right) is shown.

[0094] Figure 25 During the treatment of Huh7 cell liver orthotopic tumors with HNF4α-saRNA, tumor tissues were collected at different time points after treatment, and the expression level of HNF4α protein was detected by Western blot.

[0095] Figure 26 shows the changes in HNF4α expression detected by immunohistochemistry at different time points after treatment of Huh7 cell liver orthotopic tumors with HNF4α-saRNA (left) and the statistical diagram of the area of ​​HNF4α positive staining in the tumor tissue (right).

[0096] Figure 27During the treatment of Huh7 cell liver orthotopic tumors with HNF4α-saRNA, tumor tissues were collected at different time points after treatment, and the expression of liver function-related genes in the tumor tissues was detected by quantitative PCR.

[0097] Figure 28 shows the expression level of HNF4α protein in HuCC-T1 cholangiocarcinoma cells after 1 day and 3 days of treatment with different concentrations of HNF4α-saRNA and HNF4α-mRNA lipid nanoparticles, as detected by Western blot.

[0098] Figure 29 HNF4α-saRNA LNP inhibits the proliferation of HuCC-T1 cholangiocarcinoma cells.

[0099] Figure 30 The effects of HNF4α-mRNA LNP and HNF4α-saRNA LNP on the clonogenic ability of HuCC-T1 cholangiocarcinoma cells.

[0100] Figure 31 After transfecting HNF4α-saRNA LNP at different concentrations into HCT116 colon cancer cells for 1 day and 3 days, the expression level of HNF4α protein was detected by Western blotting.

[0101] Figure 32 shows the effects of HNF4α-mRNA LNP and HNF4α-saRNA LNP on the proliferation of HCT 116 colorectal cancer cells.

[0102] Figure 33 shows the effects of HNF4α-mRNA LNP and HNF4α-saRNA LNP on the clone-forming ability of HCT 116 colorectal cancer cells.

[0103] Figure 34 shows the expression level of HNF4α protein detected by Western blot after treating pancreatic cancer cells PANC1 with different concentrations of HNF4α-saRNA lipid nanoparticles for 1 day and 3 days.

[0104] Figure 35 During the treatment of Huh7 cell liver orthotopic tumors with HNF4α-saRNA, tumor tissues and other normal tissues were collected at different time points after treatment, and the levels of HNF4α mRNA (HNF4α) and self-replicating RNA vector (VEEV) were detected by quantitative PCR.

[0105] Figure 36To enhance MRI to show that the overall size of hepatocellular carcinoma lesions remained stable after HNF4α-saRNA LNP treatment. A. Target lesion of hepatocellular carcinoma 13 weeks before treatment (dashed box). B. Target lesion of hepatocellular carcinoma significantly larger at baseline than before treatment. C. Target lesion of hepatocellular carcinoma remained stable in size 10 weeks after treatment. D. Target lesion of hepatocellular carcinoma remained stable in size 23 weeks after treatment, with liquefactive necrosis appearing in some areas (arrow).

[0106] Figure 37 Chest CT scans show that hepatocellular carcinoma (HCC) lung metastases gradually shrink after HNF4α-saRNA LNP treatment. A. Baseline HCC lung metastases (arrow). B. Four weeks after treatment, HCC lung metastases slightly larger than baseline. C. Eight weeks after treatment, HCC lung metastases slightly smaller than four weeks prior. D. Eighteen weeks after treatment, HCC lung metastases further shrink, with the lesion diameter significantly smaller than baseline.

[0107] Figure 38 To enhance MRI and show the changes in hepatocellular carcinoma target lesions after HNF4α-saRNA LNP treatment. A. Baseline hepatocellular carcinoma target lesions (dashed line) show significant enhancement in the arterial phase. B. Four weeks after treatment, hepatocellular carcinoma target lesions are larger than baseline, and arterial phase enhancement is weaker than baseline. C. Nine weeks after treatment, hepatocellular carcinoma target lesions are further enlarged, and arterial phase enhancement is further weakened. D. Sixteen weeks after treatment, hepatocellular carcinoma target lesions continue to enlarge, arterial phase enhancement continues to weaken, and enhancement disappears in most areas (arrow).

[0108] Figure 39 After treating hepatocellular carcinoma Huh7 cells with different concentrations of HNF1α-saRNA LNP for 1 day and 3 days, the expression level of HNF4α protein was detected by Western blot.

[0109] Figure 40 Tumor proliferation curve (left) and relative tumor volume statistics (right) in a Huh7 cell subcutaneous xenograft model treated with HNF1α-saRNA; physiological saline was used as the solvent control.

[0110] Figure 41 Gross image of a Huh7 cell subcutaneous xenograft tumor treated with HNF1α-saRNA lipid nanoparticles.

[0111] Figure 42 In a Huh7 cell subcutaneous xenograft model treated with HNF1α-saRNA lipid nanoparticles, the tumor weight is shown in the left graph and the tumor inhibition rate is shown in the right graph.

[0112] Figure 43 After treating Huh7 hepatocellular carcinoma cells with different concentrations of FOXA3-saRNA LNP for 3 days, the expression level of FOXA3 protein was detected by Western blot.

[0113] Figure 44 After treating pancreatic cancer PANC1 cells with different concentrations of PTF1A-saRNA LNP for 1 day and 3 days, the expression level of PTF1A protein was detected by Western blot.

[0114] Figure 45 One day after transfecting BHK21 suckling hamster cells with NEUROD1-saRNA, the expression level of NEUROD1 protein was detected by Western blot.

[0115] Figure 46 Flowchart of an experimental study on subcutaneous implantation of Huh7 hepatocellular carcinoma tumors via tail vein injection of self-replicating HNF4α-saRNA and HNF4α / HNF1α / FOXA3-saRNA lipid nanoparticles.

[0116] Figure 47 Tumor proliferation curves in a subcutaneous xenograft model of hepatocellular carcinoma Huh7 cells treated with HNF4α-saRNA and HNF4α / HNF1α / FOXA3-saRNA.

[0117] Figure 48 A statistical graph showing the tumor weight of a subcutaneous hepatocellular carcinoma Huh7 cell xenograft model treated with HNF4α-saRNA and HNF4α / HNF1α / FOXA3-saRNA.

[0118] Figure 49 The expression levels of HNF4α in tumor tissues of a subcutaneous hepatocellular carcinoma Huh7 cell xenograft model treated with HNF4α-saRNA and HNF4α / HNF1α / FOXA3-saRNA were detected by ELISA.

[0119] Figure 50 Flowchart of an experiment on intratumoral injection of PTF1A-saRNA lipid nanoparticles for the treatment of subcutaneous xenografts of pancreatic cancer AsPC-1 cells.

[0120] Figure 51 Tumor proliferation curves in a subcutaneous xenograft model of pancreatic cancer AsPC-1 cells treated with intratumoral injection of PTF1A-saRNA lipid nanoparticles.

[0121] Figure 52 Statistical graph of tumor weight in a subcutaneous xenograft model of pancreatic cancer AsPC-1 cells treated with intratumoral injection of PTF1A-saRNA lipid nanoparticles.

[0122] Figure 53 Statistical graph of tumor inhibition rate in a subcutaneous xenograft model of pancreatic cancer AsPC-1 cells treated with intratumoral injection of PTF1A-saRNA lipid nanoparticles.

[0123] Figure 54 The expression level of PTF1A in tumor tissue of a subcutaneous xenograft model of pancreatic cancer AsPC-1 cells treated with PTF1A-saRNA was detected by Western blot.

[0124] Figure 55 Flowchart of an experimental procedure for intratumoral injection of NUROD1-saRNA lipid nanoparticles to treat subcutaneous xenografts of U87 cells in gliomas.

[0125] Figure 56 Tumor proliferation curves in a subcutaneous xenograft model of U87 cells in glioma treated with intratumoral injection of NUROD1-saRNA lipid nanoparticles.

[0126] Figure 57 Gross image of a tumor in a subcutaneous xenograft model of U87 cells used to treat gliomas by intratumoral injection of NUROD1-saRNA lipid nanoparticles.

[0127] Figure 58 Statistical graph of tumor weight in a subcutaneous xenograft model of U87 cells for glioma treated with intratumoral injection of NUROD1-saRNA lipid nanoparticles. Detailed Implementation

[0128] The specific embodiments provided by the present invention will be described in detail below with reference to examples. The advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0129] Unless otherwise described, embodiments of the present invention will employ conventional techniques of molecular biology, cell biology, and immunology, all of which are known to those skilled in the art. These techniques are fully described in the following literature: for example, *Molecular Cloning: A Laboratory Manual*, 4th edition (2017); *A Concise Laboratory Manual of Cell Biology* (2007); *A Concise Laboratory Manual of Immunology* (2010). Alternatively, the instructions provided by the reagent manufacturer may be followed.

[0130] Unless otherwise stated, percentages and parts are by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0131] All data in this paper were analyzed and plotted using GraphPad Prism 8 software. All error values ​​are presented as mean ± standard error (±SEM). A t-test was used to compare two groups of data. The statistical test was two-tailed, where p < 0.05 was considered statistically significant, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.

[0132] Example 1: Preparation of self-replicating RNA and LNP encapsulation

[0133] 1. Preparation of linear mRNA and self-replicating RNA

[0134] Self-replicating RNA (saRNA) is based on an engineered alphavirus genome containing genes encoding non-structural proteins that enable RNA replication, while the structural protein sequences are replaced by the target gene sequence. saRNA includes a 5' cap, a non-coding region (5'UTR), four non-structural genes (NSP1-4), a 26S subgenomic promoter, the target gene, a 3' non-coding region (3'UTR), and a polyadenylated tail. Linear RNA is designed based on the structure of eukaryotic mRNA, containing genes encoding the target protein, as well as the necessary cap structure, 5'UTR, 3'UTR, and poly(A). The preparation methods for non-replicating RNA and self-replicating RNA are consistent, starting with a linear template. To prepare a linear template for RNA transcription, plasmid DNA is restriction-digested using BspQI enzyme (New England Biolabs, R0712L) and purified using a PureLink® PCR purification kit (Invitrogen, K310002). 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) were mixed and incubated at 37°C for 2 hours for in vitro transcription of the template. After the transcription reaction, DNase I (1 U / μg DNA) was added and incubated at 37°C for 15 minutes to remove the DNA template. The RNA obtained from transcription was then purified and recovered using LiCl precipitation.

[0135] 2. LNP encapsulates RNA

[0136] Lipid nanoparticles were rapidly prepared by mixing an ethanol phase and an aqueous phase in a microfluidic device (INano™ L system, Micro&Nano). The aqueous phase was a 50 mM citrate buffer (pH 6.0) containing purified saRNA. The ethanol phase contained proprietary ionizable lipids: 1,2-distearate-sn-glycerophosphate-choline (DSPC) (Avanti, 850365P), cholesterol (Sigma-Aldrich, C8667), and 1,2-dicylo-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (NOF, GM020). The mRNA-LNPs were assembled in a molar ratio of 9.4:42.5:1.8:46.3 (DSPC:cholesterol:DMG-PEG 2000:DHA-1), N / P=6. The LNPs formed by this formulation were analyzed for particle size, PDI, RNA concentration, and encapsulation efficiency.

[0137] Example 2: Optimizing the length of the polyA tail of self-replicating RNA

[0138] According to the method of Example 1 and Figure 1 The diagram shows the preparation of self-replicating HNF4α-expressing RNAs (HNF4α-saRNA LNPs) wrapped with polyA tails of varying lengths. The HNF4α-saRNA LNPs were prepared from Huh7 liver cancer cells at a ratio of 3 x 10⁻⁶. 5 Cells were seeded into 6-well plates and cultured overnight. HNF4α-saRNA LNPs of different polyA lengths diluted with Opti-MEM were added. After 6 hours of culture, 1 mL of DMEM medium containing 20% ​​FBS was added. Transfected cells were lysed using RIPA buffer after 3 or 7 days, and Western blot analysis was performed to detect intracellular HNF4α protein levels. Figure 2 The results showed that HNF4α-saRNA with a polyA tail length of 35-100 nt mediated HNF4α overexpression in Huh-7 cells. Tumor cells transfected with self-replicating RNA with a polyA tail length of 40-90 nt showed higher HNF4α expression, while self-replicating RNA with a polyA tail length of 50-75 nt showed better HNF4α expression levels and duration. Self-replicating RNA with a polyA tail length of 60-70 nt (especially 67 nt) mediated the most efficient expression. The optimal self-replicating RNA sequence with the best polyA tail length after optimization is shown in SEQ ID NO:6, and the HNF4α-saRNA sequence is shown in SEQ ID NO:7 (this sequence was used in subsequent examples).

[0139] Example 3: Comparison of the ability of non-replicating HNF4α-mRNA, HNF4α-saRNA, and adenovirus AdHNF4α to mediate HNF4α overexpression in tumor cells

[0140] Huh7 liver cancer cells at 3x10 5 Cells were seeded into 6-well plates and cultured overnight. After washing with 1 ml of PBS, 1 ml of Opti-MEM diluted HNF4α-saRNA LNP, control GFP-saRNA LNP, and non-replicating HNF4α-mRNA LNP were added (final concentration: 2 ng RNA / 200 cells). After 6 hours of culture, 1 ml of DMEM medium containing 20% ​​FBS was added. Transfected cells were lysed using RIPA buffer at 1, 3, 5, or 7 days, and Western blot analysis was performed to detect intracellular HNF4α protein levels. Figure 3 The effect of adenovirus-mediated HNF4α overexpression was compared with that of Huh7 cells containing adenovirus AdHNF4α expressing HNF4α and its control virus AdGFP. Figure 3 The results showed that both HNF4α-saRNA and HNF4α-mRNA could upregulate HNF4α in liver cancer cells, but HNF4α-mRNA expression was lower and its expression duration was shorter; while HNF4α-saRNA expression was higher and its duration was longer, and its expression efficiency was no lower than that of adenovirus.

[0141] Example 4: HNF4α-saRNA treats liver cancer by inducing differentiation of liver cancer cells into hepatocytes.

[0142] 1. Different concentrations of HNF4α-saRNA LNP upregulate HNF4α expression in Huh-7 hepatocellular carcinoma cells.

[0143] Huh7 liver cancer cells at 3x10 5 Cells were seeded into 6-well plates and cultured overnight. After washing with 1 ml of PBS, HNF4α-saRNA LNP and control GFP-saRNA LNP, serially diluted with Opti-MEM, were added (final concentrations of 0.5, 1, and 2 ngRNA / 200 cells). After 6 hours of culture, 1 ml of DMEM medium containing 20% ​​FBS was added. Transfected cells were lysed using RIPA buffer at 1, 3, 5, or 7 days, and Western blot analysis was performed to detect intracellular HNF4α protein levels. Figure 4 The results showed that HNF4α-saRNA LNP upregulated HNF4α expression, while the control GFP-saRNA LNP did not affect HNF4α expression in liver cancer cells.

[0144] 2. HNF4α-saRNA LNP inhibits the growth of liver cancer cells.

[0145] We used Huh7 liver cancer cells to verify the inhibitory effect of the gene delivery system on malignant phenotypes such as tumor cell proliferation and colony formation. Huh7 cells were loaded at 3 × 10⁻⁶ cells per cell line. 3 Cells 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 PBS. Different concentrations of HNF4α-saRNA LNP diluted in 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. Results showed that HNF4α-saRNA LNP significantly inhibited the proliferation of liver cancer cells compared to control cells. Figure 5 ).

[0146] 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 / 200 cells in 96-well plates and cultured overnight. After transfection, HNF4α-saRNA LNP and control GFP-saRNA LNP (2 ng / 200 cells) were added. 24 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. Results are shown below. Figure 6 HNF4α-saRNA significantly inhibited the clonogenic ability of liver cancer cells.

[0147] 3. HNF4α-saRNA LNP induces differentiation of hepatocellular carcinoma cells into mature hepatocytes.

[0148] ① HNF4α-saRNA restores liver function gene expression in liver cancer cells

[0149] Huh-7 liver cancer cells were treated with 3x10 5Cells 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 HNF4α-regulated liver function-related genes and tumor stemness-related genes. Figure 7 ,8). The results showed that HNF4α-saRNA upregulated the expression of liver function-related genes and downregulated the expression of tumor stemness-related genes.

[0150] ②HNF4α-saRNA promotes glycogen storage and low-density lipoprotein uptake in liver cancer cells.

[0151] Glycogen storage and low-density lipoprotein uptake are important functions of normal liver cells. The inventors further examined these functions to determine whether liver cancer cells differentiate into mature liver cells.

[0152] Huh7 liver cancer cells were divided into 3x10 4 Cells were seeded in 24-well plates, and 1 ng of HNF4α-saRNA LNP and corresponding controls were added per 200 cells. Three days after LNP delivery, glycogen storage capacity was verified using a PAS reaction kit (Beyotime). Cells were fixed with 70% ethanol 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, and the cells were reacted in a humidified chamber in the dark for 10 minutes. The periodic acid solution was then removed, and the cells were immersed in PBS and washed on a shaker for 5 minutes. 100 μl of Schiff's reagent was added to each sample, and the cells were placed in a humidified chamber and stained in a 37°C oven in the dark for 1 hour. The staining solution was removed, and the cells were immersed in PBS and washed on a shaker for 5 minutes. Finally, 100 μl of hematoxylin staining solution was added to each sample, and staining was performed for 30 seconds. The staining solution was removed, and the cells were rinsed twice with PBS to remove any excess stain. The cells were photographed under a microscope, and the stained cells were counted. Figure 9 The results showed that HNF4α-saRNA promoted the glycogen storage capacity of liver cancer cells.

[0153] Huh7 liver cancer cells were divided into 3x10 4Cells were seeded in 24-well plates, and 1 ng of HNF4α-saRNA LNP and corresponding controls were added per 200 cells. Three days after LNP delivery, the cell culture medium was aspirated, cells were washed with PBS, and then 200 μL of Dilac-LDL (Invitrogen) was diluted 1:100 with DMEM and added to each well. After 3 hours, the DMEM containing Dilac-LDL was removed, cells were washed with PBS, and fixed with 4% paraformaldehyde for 15 minutes. After nuclear staining with DAPI, the cells were mounted, photographed using a confocal fluorescence microscope, and the fluorescence signal was statistically analyzed. Figure 10 The results showed that HNF4α-saRNA promoted the uptake of low-density lipoprotein in liver cancer cells.

[0154] ③HNF4α-saRNA promotes senescence in liver cancer cells

[0155] Huh7 liver cancer cells were divided into 3x10 4 Cells were seeded in 24-well plates, and 1 ng of HNF4α-saRNA LNP and corresponding controls were added per 200 cells. 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 that HNF4α-saRNA treatment significantly increased the number of senescent liver cancer cells.

[0156] ④ HNF4α-saRNA induces apoptosis in liver cancer cells

[0157] Huh7 liver cancer cells were divided into 3x10 5Cells were seeded in 24-well plates, and 1 ng of HNF4α-saRNA LNP and corresponding controls were added per 200 cells. 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 minutes. Cells were then analyzed using flow cytometry (Attune NxT, Invitrogen). Flow cytometry data were analyzed using FlowJo V10 software. Figure 12 The results showed that HNF4α-saRNA treatment significantly increased apoptosis in liver cancer cells.

[0158] 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.

[0159] 3. HNF4α-saRNA LNP inhibits the growth of subcutaneous tumors in mice.

[0160] 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 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. Figure 13 14). Five days after RNA injection, mice were sacrificed, and tumors were excised and weighed. Figure 15 ,16), and simultaneously calculate the tumor inhibition rate ( Figure 16 Partial tumor tissue was paraffin-embedded, and HNF4α expression was detected by Western blot and immunohistochemistry. Figure 17 , 18Ki67 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 and achieved a tumor inhibition rate of over 50%. Figure 16 While HNF4α-mRNA showed no significant inhibitory effect on tumor growth, Western blot and immunohistochemical results revealed that HNF4α-saRNA significantly increased the protein expression of HNF4α in tumor tissues, and significantly decreased 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.

[0161] 4. HNF4α-saRNA LNP inhibits the growth of hepatocellular carcinoma cells implanted in situ in the liver.

[0162] 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. 3 Small tumor fragments were transplanted subcapsularly into the livers of male nude bab / c mice. Tumor growth was monitored using an IVIS spectrum optical imaging system. Three days after transplantation, mice were evenly divided into four groups based on fluorescence signal intensity. Each group received a dose of 2 mg / kg via tail vein injection of HNF4α-saRNA LNP, GFP-saRNA LNP, HNF4α-mRNA LNP, and physiological saline (solvent control). Figure 19 The efficacy of HNF4α-saRNA was evaluated based on bioluminescence monitoring of tumor growth in mice. Figure 20 ), and simultaneously plot the tumor growth curve ( Figure 21 At the end of the experiment, the mice were sacrificed, the tumors were removed and weighed, and the tumor inhibition rate was calculated. Figure 22 , 23 Tumor tissue was embedded in paraffin, and its morphology was assessed by HE staining. Ki67 staining was used to assess the tumor's proliferative status. Figure 24 The results showed that tumor growth was reduced after tail vein injection of HNF4α-saRNA LNP, with a tumor inhibition rate exceeding 40% compared to control tumors, while HNF4α-mRNA LNP had no significant tumor-inhibiting effect. Simultaneously, tumor tissue was collected at different time points after LNP injection for Western blot and immunohistochemical analysis to detect HNF4α expression. Figure 25 , 26 Ki67 staining was used to assess the tumor's proliferative status. Figure 26 Quantitative PCR detection of hepatocyte differentiation-related indicators ( Figure 27The results showed that HNF4α-saRNA significantly upregulated the expression of HNF4α and liver function-related genes in tumor tissues. These results further indicate that HNF4α-saRNA can induce liver cancer cells to differentiate into normal hepatocytes, thereby inhibiting liver cancer growth.

[0163] Example 5: HNF4α-saRNA inhibits the proliferation of bile duct cancer cells

[0164] 1. Different concentrations of HNF4α-saRNA LNP upregulated HNF4α expression in HuCC-T1 cholangiocarcinoma cells.

[0165] HuCC-T1 cholangiocarcinoma cells were seeded at a density of 40%-50% into 6-well plates and cultured overnight. After washing with 1 ml of PBS, HNF4α-saRNA LNPs were serially diluted with Opti-MEM (final concentrations of 1 and 2 ng RNA / 200 cells). After 6 hours of culture, 1 ml of DMEM medium containing 20% ​​FBS was added. The transfected cells were lysed using RIPA buffer at 1 and 3 days later, and Western blot analysis was performed to detect intracellular HNF4α protein levels. Figure 28 The results showed that HNF4α-saRNA LNP upregulated the expression of HNF4α in bile duct cancer cells.

[0166] 2. HNF4α-saRNA LNP inhibits the growth of bile duct cancer cells.

[0167] HuCC-T1 bile duct cancer cells at 3×10 3 Cells were seeded at a density of 1 / well in 96-well plates and cultured overnight. After incubation, the supernatant was removed, and the cells were washed with 100 μl of PBS. Different concentrations of HNF4α-saRNA LNP diluted in Opti-MEM (final concentrations of 1 and 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 to bring the FBS concentration to 10%. 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, compared with control cells, HNF4α-saRNA LNP significantly inhibited the proliferation of cholangiocarcinoma cells. Figure 29 ).

[0168] 3. HNF4α-saRNA LNP inhibits clonal formation of cholangiocarcinoma cells.

[0169] HuCC-T1 bile duct cancer cells at 3×10 3Cells were seeded at a density of 96-well plates and cultured overnight. Afterward, HNF4α-saRNA LNP and HNF4α-mRNA LNP (2 ng / 200 cells) were added for transfection. 24 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 minutes, washed again with PBS, air-dried, and photographed. The number of colonies was counted using ImageJ to determine the effect of RNA on the colony-forming ability of tumor cells. Figure 30 The results showed that HNF4α-saRNA significantly inhibited the clonogenic ability of cholangiocarcinoma cells, while HNF4α-mRNA did not affect the clonogenic ability of tumor cells.

[0170] Example 6: HNF4α-saRNA inhibits the proliferation of colon cancer cells

[0171] 1. Different concentrations of HNF4α-saRNA LNP upregulate HNF4α expression in HCT-116 colorectal cancer cells.

[0172] HCT-116 colon cancer cells were seeded at a density of 40%-50% into 6-well plates and cultured overnight. After washing with 1 ml of PBS, HNF4α-saRNA LNPs were serially diluted with Opti-MEM (final concentrations of 1 and 2 ng RNA / 200 cells). After 6 hours of culture, 1 ml of DMEM medium containing 20% ​​FBS was added. The transfected cells were lysed using RIPA buffer at 1 and 3 days later, and Western blot analysis was performed to detect intracellular HNF4α protein levels. Figure 31 The results showed that HNF4α-saRNA LNP upregulated the expression of HNF4α in colorectal cancer cells.

[0173] 2. HNF4α-saRNA LNP inhibits the growth of bile duct cancer cells.

[0174] HCT-116 colon cancer cells at 3×10 3Cells were seeded at a density of 1 / well in 96-well plates and cultured overnight. After incubation, the supernatant was removed, and the cells were washed with 100 μl PBS. Different concentrations of HNF4α-saRNA LNP and HNF4α-mRNA LNP (final concentration 2 ng RNA / 200 cells) diluted with Opti-MEM were added to the cells. After 6 hours, an equal volume of 20% FBS was added to the culture medium to bring the FBS concentration to 10%. 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, compared with control cells, HNF4α-saRNA LNP significantly inhibited the proliferation of colorectal cancer cells. Figure 32 HNF4α-mRNA LNP had no significant effect on tumor cell proliferation.

[0175] 3. HNF4α-saRNA LNP inhibits colony formation of colorectal cancer cells.

[0176] HCT-116 colon cancer cells at 3×10 3 Cells were seeded at a density of 2 ng / well in 96-well plates and cultured overnight. Afterward, HNF4α-saRNA LNP and HNF4α-mRNA LNP (2 ng / 200 cells) were added for transfection. 24 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 again with PBS, 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. Figure 33 The results showed that HNF4α-saRNA significantly inhibited the clonogenic ability of colorectal cancer cells, while HNF4α-mRNA did not affect the clonogenic ability of tumor cells.

[0177] Example 7: HNF4α-saRNA upregulates the expression of HNF4α in pancreatic cancer cells

[0178] Human pancreatic cancer cells PANC1 were seeded at a density of 40%-50% into 6-well plates and cultured overnight. After washing with 1 ml of PBS, HNF4α-saRNA LNPs were serially diluted with Opti-MEM (final concentrations of 1 and 2 ng RNA / 200 cells). After 6 hours of culture, 1 ml of DMEM medium containing 20% ​​FBS was added. The transfected cells were lysed using RIPA buffer at 1 and 3 days later, and Western blot analysis was performed to detect intracellular HNF4α protein levels. Figure 34 The results showed that HNF4α-saRNA LNP upregulated the expression of HNF4α in pancreatic cancer cells.

[0179] Example 8: HNF4α-saRNA LNP specifically upregulates HNF4α expression in tumor tissues in vivo.

[0180] 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. 3 Small tumor fragments were transplanted subcapsularly into the livers of male Bab / c nude mice, and tumor growth was monitored using an IVIS spectrum optical imaging system. Once the tumors reached a suitable size, HNF4α-saRNA LNP was administered via tail vein injection at a dose of 2 mg / kg body weight. Organ tissues and tumor tissues were harvested from the mice 3 and 5 days post-injection. Quantitative PCR was used to detect the expression distribution of the alphavirus genome sequence (VEEV) and the LNP-mediated human HNF4α gene sequence in various organs and tumors. Figure 35 The results showed that 3 days after tail vein injection of HNF4α-saRNA LNP, trace amounts of VEEV and human HNF4α RNA were detectable in the heart, spleen, and kidneys; VEEV and HNF4α gene sequences were almost undetectable in the liver, lungs, and muscle tissues; however, high abundance of VEEV and HNF4α gene sequences was clearly detected in tumor tissues. 5 days after tail vein injection of HNF4α-saRNA LNP, except for extremely low expression in the heart, VEEV and human HNF4α RNA were undetectable in other organs; however, VEEV and HNF4α gene sequences were still clearly detectable in tumor tissues. Figure 35 This indicates that self-replicating RNA can mediate the expression of HNF4α in tumor cells in vivo.

[0181] Example 9: HNF4α-saRNA LNP treatment for patients with advanced liver cancer

[0182] The relevant CDMO company was commissioned to produce the HNF4α-saRNA LNP formulation in accordance with pharmaceutical standards and GMP standards. The relevant CRO company was commissioned to complete the single-dose toxicity test of the HNF4α-saRNA LNP formulation. The specific process and results are as follows: The rats were administered the drug once by tail vein injection, and the observation period was 14 days. A negative control group (0.9% sodium chloride injection), a carrier control group (LNP solution) and a test sample group (150 μg / rat) were set up. All animals survived to the planned dissection date. No gross necropsy abnormalities were found at the end of the observation period, and no histopathological changes related to the test sample were found.

[0183] Following the completion of acute toxicity studies, an investigator-initiated clinical trial was conducted, using hepatic artery catheterization for drug administration. Dose escalation trials at 25 μg, 50 μg, and 100 μg were performed (3 patients in each group). None of the 9 patients experienced dose-limiting toxicities or treatment-related grade 3 or higher adverse events, or serious adverse events. Four patients showed no significant progression of liver cancer lesions after more than 5 months of treatment, and one patient experienced a significant reduction in lung metastases. Figure 36 In another patient, focal necrosis was observed in the liver cancer lesion. Figure 37 In 3 patients, significant necrosis and markedly reduced enhancement were observed in the target lesions. Figure 38 Preliminary results show that the HNF4α-saRNA LNP preparation has good safety and significantly inhibits tumor growth in patients with advanced liver cancer.

[0184] Example 10: HNF1α-saRNA Inhibits the Development and Progression of Malignant Liver Cancer

[0185] The general steps are the same as in Example 1, and the HNF1α-saRNA sequence is shown in SEQ ID NO:8.

[0186] 2.1. Different concentrations of HNF4α-saRNA LNP upregulated HNF1α expression in Huh-7 hepatocellular carcinoma cells.

[0187] Huh7 liver cancer cells at 3x10 5 Cells were seeded into 6-well plates and cultured overnight. After washing with 1 ml of PBS, HNF1α-saRNA LNP was serially diluted with Opti-MEM (final concentration: 1 or 2 ng RNA / 200 cells). After 6 hours of culture, 1 ml of DMEM medium containing 20% ​​FBS was added. Transfected cells were lysed using RIPA buffer at 1, 3, or 7 days later, and Western blot analysis was performed to detect intracellular HNF1α protein levels. Figure 39 The results showed that HNF1α-saRNA LNP upregulated HNF1α expression.

[0188] 2. HNF1α-saRNA LNP inhibits the growth of subcutaneous tumors in mice.

[0189] 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 mm3 Mice were randomly divided into two groups (n=6 per group). One group received intratumoral injection of HNF1α-saRNA (5 μg / 75 μl / mouse, treatment group), while the other received intratumoral injection of saline (control group). Tumor volume was measured daily after injection, tumor growth curves were plotted, and relative tumor volume RTV (RTV) was calculated. Figure 40 Five days after RNA injection, mice were sacrificed, tumors were removed and weighed, and tumor inhibition rates were calculated. Figure 41 ,42). The results showed that HNF1α-saRNA significantly inhibited the growth of liver cancer implants.

[0190] Example 11: FOXA3-saRNA Upregulates FOXA3 Expression in Huh-7 Hepatocellular Carcinoma Cells

[0191] The general steps are the same as in Example 1, and the FOXA3-saRNA sequence is shown in SEQ ID NO:9.

[0192] Huh7 liver cancer cells at 3x10 5 Cells were seeded into 6-well plates and cultured overnight. After washing with 1 ml of PBS, serially diluted FOXA3-saRNA LNP and control GFP-saRNA LNP (final concentrations of 0.5, 1, and 2 ngRNA / 200 cells) were added using Opti-MEM. After 6 hours of culture, 1 ml of DMEM medium containing 20% ​​FBS was added. Three days later, 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 the control GFP-saRNA LNP did not affect FOXA3 expression in liver cancer cells.

[0193] Example 12: PTF1A-saRNA upregulates PTF1A expression in pancreatic cancer PANC1 cells.

[0194] The general steps are the same as in Example 1, and the PTF1A-saRNA sequence is shown in SEQ ID NO:10.

[0195] Pancreatic cancer cells PANC1 at a rate of 2.5 x 10⁻⁶ 5Cells were seeded into 6-well plates and cultured overnight. After washing with 1 ml of PBS, PTF1A-saRNA LNP and control GFP-saRNA LNP, diluted serially with Opti-MEM (final concentrations of 0.5, 1, and 2 ng RNA / 200 cells), were added. After 6 hours of culture, 1 ml of DMEM medium containing 20% ​​FBS was added. Three days later, transfected cells were lysed using RIPA buffer, and Western blot analysis was performed to detect intracellular PT protein levels in PTF1A cells. Figure 44 The results showed that PTF1A-saRNA LNP upregulated PTF1A expression, while the control GFP-saRNA LNP did not affect PTF1A expression in liver cancer cells.

[0196] Example 13 NEUROD1-saRNA-mediated overexpression of NEUROD1 in cells

[0197] The general steps are the same as in Example 1, and the NEUROD1-saRNA sequence is shown in SEQ ID NO:11.

[0198] BHK-21 kidney cells from neonatal hamsters were seeded at 3 x 10⁵ cells / well in 6-well plates in EMEM medium without antibiotics. After 24 h of complete adhesion, transient transfection was performed: 0.1 μg and 1 μg NEUROD1-saRNA were added to 100 μl of Opti-MEM as mixture 1 (MIX1), and 0.3 μl / 3 μl of Lipofectamine MessengerMAX (Themo) were added to 100 μl of Opti-MEM as mixture 2 (MIX2). MIX1 and MIX2 were incubated at room temperature for 10 min each, then mixed and incubated at room temperature for 20 min. The mixture was then added to the corresponding 6-well plates at 200 μl / well, and the plates were immediately placed in a CO₂ incubator. After 24 h, RIPA lysis buffer was collected, and Western blot was used to detect the intracellular NEUROD1 protein level. Figure 45 The results showed that NEUROD1-saRNA mediated the overexpression of NEUROD1.

[0199] Example 14: Mix HNF4α / HNF1α / FOXA3-saRNA LNP inhibits the growth of subcutaneous implanted tumors of mouse liver cancer cells.

[0200] Three saRNAs, HNF4α, HNF1α, and FOXA3, were mixed in equal mass ratios using a conventional LNP delivery system. The resulting mixture was then encapsulated using a microfluidic system to synthesize the drug mix HNF4α / HNF1α / FOXA3-saRNALNP. Six- to eight-week-old female nude mice (BALB / c immunodeficient strain) were purchased from Shanghai Lingchang Biotechnology Co., Ltd., and were housed under specific pathogen-free environmental conditions using a 12-hour on / off light cycle. 5 × 10 6 Huh-7 cells were subcutaneously injected into the right axilla of female 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 150-170 mm. 3 Mice were randomly divided into two groups (n=3 per group). Seven days apart, mice were injected twice via tail vein with saline (solvent control), 5 μg of single HNF4α-saRNA LNP, and 10 μg of mix HNF4α / HNF1α / FOXA3-saRNA LNP. Figure 46 The final volume was 200 µl. Tumor volume was measured every 1-2 days after injection, and the endpoint tumor volume in the saline (solvent control) group was 975.28 ± 72.28 mm. 3 The tumor volume in the 5 μg single HNF4α-saRNA LNP group reached 732.76 ± 96.40 mm. 3 The tumor volume in the 10μg mix HN4α / HNF1α / FOXA3-saRNA LNP group reached 517.36±14.84 mm. 3 Plotting tumor growth curves ( Figure 47 Two days after the last administration, the mice were sacrificed, and the tumors were excised and weighed. Figure 48 Simultaneously, the tumor inhibition rate was calculated. ELISA was used to detect the expression of human HNF4α in tumor tissue. Figure 49The results showed that intratumoral injection of 10 μg mix HNF4α / HNF1α / FOXA3-saRNALNP slowed tumor growth, with a tumor inhibition rate exceeding 40%, significantly higher than that of 5 μg single HNF4α-saRNALNP. ELISA results showed that both 5 μg single HNF4α-saRNALNP and 10 μg mix HNF4α / HNF1α / FOXA3-saRNALNP increased the expression of human HNF4α in tumors, with the expression of human HNF4α in tumors treated with 5 μg single HNF4α-saRNALNP being higher than that with 10 μg mix HNF4α / HNF1α / FOXA3-saRNALNP. These results indicate that mix HNF4α / HNF1α / FOXA3-saRNALNP can work synergistically to exert a tumor-suppressive effect on hepatocellular carcinoma xenografts.

[0201] Example 15: PTF1A-saRNA LNP inhibits the growth of subcutaneous implanted tumors of pancreatic cancer cells in mice.

[0202] Six- to eight-week-old female nude mice (BALB / c immunodeficient strain) were purchased from Shanghai Lingchang Biotechnology Co., Ltd., and were housed under specific pathogen-free environmental conditions using a 12-hour on / off light cycle. 5 × 10 6 ASPC-1 cells were subcutaneously injected into the right axilla of female 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 200-300 mm. 3 Mice were randomly divided into two groups (n=3 per group). Every 8 days, mice were injected three times into their tumors with 10 μg of PTF1A-saRNA LNP or physiological saline (solvent control). Figure 50 The final volume was 50 µl. Tumor volume was measured every 1-2 days after injection, and a tumor growth curve was plotted. Figure 51 One day after the last administration, mice were sacrificed, tumors were excised and weighed, and the tumor inhibition rate was calculated. Figure 52 , Figure 53 Western blot analysis of PTF1A expression in tumor tissue ( Figure 54 The results showed that intratumoral injection of PTF1A-saRNA slowed tumor growth and achieved a tumor inhibition rate of over 40%. Figure 53Western blot results showed that PTF1A-saRNA significantly increased the protein expression level of PTF1A in tumor tissue. These results indicate that PTF1A-saRNA can effectively induce the expression of PTF1A in cancer cells, thereby inhibiting the growth of mouse pancreatic cancer cell tumors.

[0203] Example 16 NeuroD1-saRNA LNP inhibits the growth of subcutaneous tumors in mice

[0204] Six- to eight-week-old female nude mice (BALB / c immunodeficient strain) were purchased from Shanghai Lingchang Biotechnology Co., Ltd., and were housed under specific pathogen-free environmental conditions using a 12-hour on / off light cycle. 5 × 10 6 U87 cells were subcutaneously injected into the right axilla of female 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 210-250 mm. 3 Mice were randomly divided into two groups (n=3 per group). Every 7 days, mice were injected twice into their tumors with 10 μg NeuroD1-saRNA LNP or physiological saline (solvent control). Figure 55 The final volume was 80 µl. Tumor volume was measured every 1-2 days after injection, and a tumor growth curve was plotted. Figure 56 Seven days after the last administration, mice were sacrificed, tumors were excised and weighed, and the tumor inhibition rate was calculated. Figure 57 , Figure 58 The results showed that intratumoral injection of NeuroD1-saRNA LNP slowed tumor growth and inhibited tumor weight by 44%. These results indicate that NeuroD1-saRNA LNP can effectively inhibit the growth of mouse glioma cell implants.

[0205] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A self-replicating RNA expressing a differentiation-related transcription factor, wherein the differentiation-related transcription factor is a transcription factor related to tissue and organ differentiation and function maintenance, and the expression level of the transcription factor in tumor cells is lower than that in normal cells; the transcription factor is delivered to tumor cells via self-replicating RNA and a delivery medium, and the transcription factor is expressed in tumor cells in a long-term and specific high-expression manner, inducing tumor cells to differentiate into normal mature cells, inhibiting tumor cell proliferation and / or inducing tumor cell apoptosis.

2. The self-replicating RNA expressing differentiation-related transcription factors according to claim 1, characterized in that, The self-replicating RNA has a 35-100 nt polyadenylate tail.

3. The self-replicating RNA expressing differentiation-related transcription factors according to claim 1, characterized in that, The self-replicating RNA has a 40-90 nt polyadenylated tail.

4. The self-replicating RNA expressing differentiation-related transcription factors according to claim 1, characterized in that, The self-replicating RNA has a 50-75 nt polyadenylate tail.

5. The self-replicating RNA expressing differentiation-related transcription factors according to claim 1, characterized in that, The self-replicating RNA has a 60-70 nt polyadenylate tail.

6. The self-replicating RNA expressing differentiation-related transcription factors according to any one of claims 1 to 5, characterized in that, The transcription factors mentioned are selected from HNF4α, HNF1α, FOXA3, PTF1A, NUROND1, Neurogenin-2, or Ascl1.

7. The self-replicating RNA expressing differentiation-related transcription factors according to claim 6, characterized in that, The delivery medium is selected from one or more of the following combinations: liposomes, viral replicon particles, lipid-based nanoparticles, polymer nanoparticles, physiological buffers, microspheres, immunostimulatory complexes, and conjugates of bioactive ligands.

8. The self-replicating RNA expressing differentiation-related transcription factors according to claim 1, characterized in that, The sequence of the self-replicating RNA is shown in SEQ ID NO:

6.

9. The self-replicating RNA expressing differentiation-related transcription factors according to claim 8, characterized in that, The sequence of the HNF4α self-replicating RNA includes any one of the following (a) to (c): (a) RNA consisting of the nucleotide sequence shown in SEQ ID NO:7; (b) An RNA consisting of a nucleotide sequence having one or more nucleotides deleted, substituted, added or inserted in the nucleotide sequence shown in SEQ ID NO:7 and having activity expressed as HNF4α; and (c) An RNA consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:7 and having activity expressed as HNF4α.

10. The self-replicating RNA expressing differentiation-related transcription factors according to claim 8, characterized in that, The sequence of the HNF1α self-replicating RNA includes any one of the following (a) to (c): (a) RNA consisting of the nucleotide sequence shown in SEQ ID NO:8; (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:8 and having activity expressed as HNF1α; and (c) An RNA consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:8 and having activity expressed as HNF1α.

11. The self-replicating RNA expressing differentiation-related transcription factors according to claim 8, characterized in that, The FOXA3 self-replicating RNA sequence includes any one of the following (a) to (c): (a) RNA consisting of the nucleotide sequence shown in SEQ ID NO:9; (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:9 and having activity expressed as FOXA3; and (c) An RNA consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:9 and having the activity of being expressed as FOXA3.

12. The self-replicating RNA expressing differentiation-related transcription factors according to claim 8, characterized in that, The PTF1A self-replicating RNA sequence includes any one of the following (a) to (c): (a) RNA consisting of the nucleotide sequence shown in SEQ ID NO:10; (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 (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.

13. The self-replicating RNA expressing differentiation-related transcription factors according to claim 8, characterized in that, The sequence of the NUROND1 self-replicating RNA includes any one of the following (a) to (c): (a) RNA consisting of the nucleotide sequence shown in SEQ ID NO:11; (b) An RNA consisting of a nucleotide sequence having one or more nucleotides deleted, substituted, added or inserted in the nucleotide sequence shown in SEQ ID NO:11 and having the activity of NUROND1 expression; and (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.

14. The use of self-replicating RNA expressing differentiation-related transcription factors as described in any one of claims 1 to 13 in the preparation of drugs for treating malignant solid tumors.

15. The use of the self-replicating RNA expressing differentiation-related transcription factors according to claim 14 in the preparation of drugs for treating malignant solid tumors, characterized in that, The malignant solid tumors mentioned are liver cancer, pancreatic cancer, stomach cancer, intestinal cancer, kidney cancer, lung cancer, and glioma.

16. A gene delivery system, wherein the gene delivery system comprises a self-replicating RNA expressing a differentiation-related transcription factor as described in any one of claims 1 to 14 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.

17. The gene delivery system according to claim 16, characterized in that, When the transcription factor is HNF4α or HNF1α, the delivery medium is lipid-based nanoparticles, and the lipids are selected from ALC-0315, SM-102 or DHA-1.

18. A pharmaceutical composition comprising the gene delivery system as described in claim 16 or 17.

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