Gene delivery system and its application in the production of tumor therapeutic agents

Abstract

The aim of the present invention is to provide a new method for tumor treatment by mediating overexpression of HNF4α in tumor cells using self-replicating RNA and inducing the differentiation of tumor cells into mature cells, thereby suppressing the growth of tumor cells. [Solution] A gene delivery system consisting of a nucleic acid vector and a delivery vehicle, wherein the nucleic acid vector is a self-replicating RNA carrying HNF4α, the gene delivery system delivers HNF4α into tumor cells and induces the differentiation of tumor cells into normal mature cells, the self-replicating RNA is based on an engineered alphavirus genome and has a 35-100 nt polyadenylic acid tail, and the delivery vehicle is a lipid nanoparticle.

Description

[Technical field]

[0001] The present invention relates to the field of biomedicine technology, specifically to a gene delivery system and its application in the preparation of tumor therapeutic drugs. [Background technology]

[0002] The treatment of malignant solid tumors is currently one of the clinical difficulties, and the current situation is that there is a lack of effective clinical treatment methods, especially for malignant solid tumors that cannot be cured by surgery.In recent years, radiochemotherapy, targeted therapy and immunotherapy etc. have provided various methods for tumor treatment, but with tumor progression, chemotherapy drug resistance often occurs, and many tumors lack effective targeted drug targets or have low sensitivity to immunotherapy, especially some gastrointestinal tumors, such as liver cancer and pancreatic cancer, lack effective drug treatment methods, and the prognosis of patients is not optimistic.

[0003] Tumor differentiation induction therapy is an approach that uses various induction factors to differentiate tumor cells into mature normal cells, restore their normal phenotype and function, and suppress the proliferation of malignant tumor cells, and this approach has broken the general strategy of tumor treatment. A typical example is the treatment of acute promyelocytic leukemia using all-trans retinoic acid differentiation, which has now shown good therapeutic effects in clinical practice and has been widely applied. However, differentiation induction therapy for malignant solid tumors remains a challenge in tumor treatment. Recent studies have shown that transcription factors related to organ differentiation, development, and functional maintenance can induce the differentiation of related tumor cells into normal cells, and these differentiation-related transcription factors can be upregulated to suppress the growth of tumor cells, but without significantly affecting the function of normal tissue cells. This has opened a new avenue for tumor differentiation induction, especially for the treatment of malignant solid tumors. Therefore, targeting and regulating proteins, molecules and genes closely related to tumor cell differentiation specifically for tumors of different tissues is the core issue of tumor differentiation induction therapy, and it may be possible to fundamentally reverse the progression of malignant tumors by targeting and regulating the expression of important differentiation function genes through genetic engineering means and inducing the differentiation of tumor cells into mature cell phenotypes.

[0004] The hepatocyte nuclear factor (HNF) family includes HNF1, HNF3, HNF4, HNF6, and CCAAT / enhancer binding protein (C / EBP), which are a group of transcription factors that are predominantly expressed in the liver and are mutually regulated, and play an important role in liver development and function maintenance. Here, hepatocyte nuclear factor 4α (HNF4α) is a transcription factor belonging to the nuclear receptor family, and is expressed in tissues such as the liver, kidney, pancreas, and intestine, but is mainly expressed in mature hepatocytes. In mature hepatocytes, HNF4α can bind to approximately 12% of the gene promoters in the cells, and is involved in the maintenance of important functions such as hepatocyte fat metabolism, albumin synthesis, drug detoxification, energy metabolism, and bile acid synthesis, and is also an important gene for maintaining the hepatocyte epithelial phenotype. HNF4α regulates the development of kidney and intestinal tissues as well as the production of insulin in pancreatic tissue. Previous studies have shown that the expression of HNF4α is decreased in many epithelial-derived tumors, such as liver cancer (including hepatocellular carcinoma and cholangiocarcinoma), pancreatic cancer, intestinal cancer, and renal cancer, while overexpression of HNF4α can inhibit tumor cell proliferation and metastasis and promote tumor cell apoptosis. This indicates that HNF4α is a potential target for tumor therapy. There are a total of nine subtypes of HNF4α, which subtype depends on the transcriptional regulation of its selective promoters P1 and P2. HNF4α subtypes regulated by the P1 promoter (HNF4α1-HNF4α6 subtypes) are mainly expressed in the liver and kidney, while HNF4α regulated by the P2 promoter (HNF4α7-HNF4α9 subtypes) are mainly expressed in pancreatic and gastric tissues, and HNF4α subtypes regulated by the P1 and P2 promoters exist simultaneously in the intestine. The inventors' team previously held an invention patent entitled "Treatment of human malignant solid tumors by inducing differentiation of HNF4α" (Chinese patent number: ZL200810034200.3), which revealed the therapeutic effect of HNF4α on tumors through overexpression of HNF4α isomer 2 (HNF4α2) in liver cancer cells mediated by adenovirus vectors.

[0005] To realize tumor treatment, it is necessary to ensure that HNF4α is stably and efficiently expressed in tumor cells for a long time, and tumor treatment requires repeated drug administration to ensure therapeutic effect due to the rapid growth ability of tumors. The population has pre-existing immunity to adenovirus, and the adenovirus vector itself can induce an antiviral response in the human immune system, thereby rejecting the expression of the target gene mediated by the viral vector in tumor cells, limiting the frequency of drug administration in tumor treatment by overexpression of HNF4α in adenovirus vectors. Meanwhile, previous studies have also shown that the practicality of tumor treatment by overexpression of HNF4α in adenovirus vectors is low due to the safety and tissue tropism defects of adenovirus vectors, and the high cost of their preparation.

[0006] mRNA technology is an innovative gene delivery technology that has been developed in recent years. This technology synthesizes mRNA molecules with specific sequences by in vitro transcription, transports the mRNA into human cells by coating with lipid nanoparticles (LNPs), and translates the mRNA into target proteins using the cell's own translation system. It has superior efficiency and safety compared to existing delivery systems based on virus carriers, and when combined with screening and modification of nano lipid particles, the tissue specificity of the delivery system can be improved. The currently known potential application directions of mRNA technology are mainly focused on the development of vaccines for infectious diseases, therapeutic cancer vaccines, protein replacement therapy for protein deficiency due to gene deficiency, and gene editing for the treatment of genetic diseases or the modification of immune cells. However, mRNA can only mediate temporary and significant expression of genes in cells, and has obvious defects in expression time and expression efficiency, and cannot meet the demand for relatively long-term and stable expression of target proteins in tumor cells with rapid proliferation ability.

[0007] Self-replicating RNA (saRNA, also called srRNA) is a gene delivery vector based on the recently developed mRNA technology, and is a recombinant RNA with a viral structure. By modifying the double transgenome of alphaviruses, an RNA vector capable of expressing a target gene with self-replicating ability in cells is constructed by retaining the four nonstructural protein (NSPs) sequences of the virus-derived code and replacing the viral structural protein gene located behind the viral subgenome promoter (SGP) with a heterologous gene encoding a protein of interest (GOI). Currently, self-replicating RNA vectors are mainly modified from Venezuelan equine encephalitis virus (VEEV), sindbis alphavirus (SIN) or Semliki Forest virus (SFV), among which the self-replicating RNA derived from Venezuelan equine encephalitis virus is the most common. Once the self-replicating RNA enters the host cytoplasm, it first translates four nonstructural proteins (NSP1, NSP2, NSP3, and NSP4), which are then polymerized in a complex, multi-step process to form an RNA-dependent RNA polymerase complex. The RNA polymerase complex synthesizes a complementary negative-strand RNA intermediate from the positive-strand RNA and then uses the latter as a template to synthesize two different positive-strand RNAs. The first positive-strand RNA is a copy of the original full-length genomic RNA, and the second positive-strand RNA is a subgenomic RNA that encodes a large amount of target genes. This mechanism allows the self-replicating RNA to achieve high levels of continuous target protein expression with low doses of self-replicating RNA. Although the self-replicating RNA retains the self-replicating ability of the viral genome, it cannot express the viral structural proteins, and therefore cannot generate a complete virus with propagation ability, and it relies on lipid nanoparticle delivery for cell entry, which gives it excellent safety and potential tumor targeting ability.

[0008] At present, in tumor treatment, self-replicating RNA is believed to be applicable to prepare RNA vaccines and mediate the expression of tumor-killing cytokines in tumor cells.For example, Chinese invention patent application CN117280029A discloses a nucleic acid vector and its use method for regulating tumor microenvironment by the relative expression of activated tumor-infiltrating lymphocytes and / or immunogenic cellular features in tumor microenvironment to treat cancer; Chinese invention patent application CN117279661A discloses a composition and method for inducing ESR1, PI3K, HER2 and HER3 immune response, which treats cancer by inducing immune response; Chinese invention patent application CN115968299A discloses a novel antigen expressed in multiple myeloma and its use, which uses self-replicating RNA as a vector to prepare cancer vaccine. The mechanism of cancer 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 the T cells to launch attacks on tumor cells, and further controlling or eliminating the tumor.

[0009] However, there have been no reports of the use of self-replicating RNA to express transcription factors, particularly transcription factors associated with tissue and organ differentiation, in tumor cells to treat tumors. Summary of the Invention

[0010] The object of the present invention is to provide a new means for tumor treatment by mediating overexpression of HNF4α in tumor cells using self-replicating RNA, thereby inducing differentiation of tumor cells into mature cells, thereby suppressing tumor cell growth.

[0011] The expression level of hepatocyte nuclear factor HNF4α in the relevant tumor cells is lower than that in normal cells. The inventor's research has found that self-replicating RNA is very suitable for mediating the expression of target genes in tumor cells, because after entering the tumor cells, the distribution in the tumor cells does not decrease with the rapid proliferation of the tumor cells, and the protein can be continuously expressed for more than 20-30 days based on the property of self-replication. In addition, animal experiments have shown that the specific expression of the target gene in tumor tissue can be mediated by tail vein injection of lipid nanoparticle-coated self-replicating RNA, and the expression of the target gene is hardly detected in other normal tissues, so that the self-replicating RNA has good tumor tropism. Based on these findings, the inventor of the present application proposed to use a self-replicating RNA vector to restore or overexpress HNF4α in tumor tissue, and induce the differentiation of tumor cells into normal cells, thereby suppressing the growth of tumor cells and treating tumors.

[0012] The present invention uses self-replicating RNA technology to introduce HNF4α into tumor cells, overexpress it, and induces the differentiation of related tumor cells into mature cells through specific and long-term expression in tumor cells, thereby achieving the purpose of treating tumors.

[0013] The self-replicating RNA can optimize the length of the polyA tail and maintain the target gene at a high level of expression in the body for a longer period of time. The structure of the self-replicating RNA includes one 5'-end cap (5'Cap), a non-coding region (5'UTR), four non-structural genes (NSP1-4), one 26S subgenomic promoter (SGP), a target gene (GOI), a 3'-end non-coding region (3'UTR) and one polyadenylic acid tail (polyA). In order to optimize the performance of the self-replicating RNA, we tried to modify NSPS1-4 in various ways, such as improving the replication efficiency and immunogenicity of the self-replicating RNA by introducing mutations and increasing the expression strength and duration of different target genes in cells. We found that the self-replicating RNA vectors with different NSP1-4 mutations did not have a significant difference in the efficiency of mediating HNF4α expression in tumor cells. Meanwhile, studies have shown that the length and sequence of the polyadenylic acid tail (poly A) of mRNA affect the stability of mRNA and ribosome translation, and changing the length of the poly A tail affects the expression time and expression level of the encoded protein by regulating the degradation rate and translation level of mRNA. However, it is still unclear whether the poly A of the self-replicating RNA affects the expression time and expression level of the target gene in tumor cells. In order to obtain a self-replicating RNA vector that can mediate stable and efficient expression of HNF4α in tumor cells, the poly A length of the self-replicating RNA used was optimized, and it was found that self-replicating RNA with a poly A length between 35-100 nt can mediate relatively stable and long-term overexpression of HNF4α in tumor cells, of which the expression amount and expression time of 60-75 nt are relatively good, and the most stable and efficient expression can be obtained when the poly A tail is at 67 nt.

[0014] The present invention considers the effect of overexpressing HNF4α in tumor cells via non-replicating HNF4α-mRNA, HNF4α-saRNA and adenovirus, respectively, and finds that both HNF4α-saRNA and HNF4α-mRNA can upregulate HNF4α in liver cancer cells, but the expression level of HNF4α-mRNA is low and the expression time is short, while the expression level of HNF4α-saRNA is more than twice that of HNF4α-mRNA and the maintenance time is long. The upregulation of HNF4α by HNF4α-mRNA can only be maintained for 3 days, while in liver cancer cells, HNF4α-saRNA can maintain HNF4α for at least 7 days, and the expression efficiency of HNF4α-saRNA upregulation is higher than that of adenovirus.

[0015] A first aspect of the present invention provides a gene delivery system comprising a nucleic acid vector and a delivery vehicle, wherein the nucleic acid vector is a self-replicating RNA carrying HNF4α, and the gene delivery system delivers HNF4α into tumor cells, induces differentiation of tumor cells into normal mature cells, inhibits the proliferation of tumor cells, and / or induces apoptosis of tumor cells.

[0016] The HNF4α is shown in GENBANK No: NM_000457.6, SEQ ID NO: 1. The self-replicating RNA comprises a 5' cap, a 5' non-coding region, a nonstructural gene, a 26S subgenomic promoter, a 3' non-coding region and a polyadenylic acid tail.

[0017] The self-replicating RNA has a polyadenylic acid tail of 35-100 nt.

[0018] More preferably, the self-replicating RNA has a polyadenylic acid tail of 60-75 nt.

[0019] Most preferably, said self-replicating RNA has a polyadenylic acid tail of 67 nt.

[0020] In a preferred embodiment of the present invention, the sequence of the self-replicating RNA is as shown in SEQ ID NO:3, and this sequence is optimized to have a polyA tail of preferably 67 nt of the wild-type self-replicating RNA, and the saRNA having a 67 nucleotide A tail can increase the half-life of the saRNA in the body and promote the stability and translation of the saRNA.

[0021] In the gene delivery system, the delivery vehicle is one or a combination of a liposome, a viral replicon particle, a lipid nanoparticle, a polymer nanoparticle, a physiological buffer, a microsphere, an immune stimulating complex, and a bioactive ligand complex.

[0022] Preferably, the delivery vehicle is a lipid nanoparticle (LNP), and the lipid in the LNP is 1,2-distearoyl-sn-propyltriyl-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in a molar ratio of 5%-20%; Cholesterol with a molar ratio of 30%-55%; a pegylated lipid containing dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG 2000) or polyethylene glycol fragments at a molar ratio of 0.5%-3%; and an ionizable lipid (such as ALC-0315, SM-102, DHA-1, etc.) at a molar ratio of 30%-60%.

[0023] In a preferred embodiment of the invention, the total lipid molar ratio is 100% and the lipid composition is in the following molar ratio: DSPC:cholesterol:DMG-PEG 2000:ionizable lipid: 9.4:42.5:1.8:46.3.

[0024] The range of N:P ratio in LNPs is 5:1-10:1, preferably 6:1.

[0025] LNPs have a particle size of about 40-300 nm.

[0026] A second aspect of the present invention provides an application of the above gene delivery system in the preparation of a therapeutic drug for a malignant solid tumor.

[0027] Preferably, the malignant solid tumor is a malignant solid tumor of epithelial cell origin.

[0028] More preferably, the malignant solid tumor is liver cancer, pancreatic cancer, gastric cancer, intestinal cancer, renal cancer, or lung cancer.

[0029] A third aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned gene delivery system.

[0030] The technical effects of the present invention are as follows: The present invention uses a self-replicating RNA vector based on the mRNA technology platform in combination with a lipid nanoparticle-coated delivery means to highly express an important differentiation-related gene HNF4α in tumor cells, induce the transformation of tumor cells into normal cells, suppress the malignant phenotype of tumors, and achieve the purpose of tumor treatment. This is a new means of tumor treatment by differentiation induction, and the results of previous animal experiments and clinical research by researchers have confirmed that this technical means can effectively suppress tumor growth in the body, and is expected to open up a new avenue for tumor treatment as a new approach to tumor treatment. Meanwhile, there is a clear difference between the present invention and the current application of mRNA technology in cancer vaccines. RNA cancer vaccines use RNA technology to express relevant tumor antigens in antigen-presenting cells to stimulate the body to generate an immune response against these antigens, and further control or eliminate tumors, while the present invention uses RNA technology to mediate the expression of the transcription factor HNF4α in tumor cells, and the differentiation-promoting effect of HNF4α further suppresses the malignant phenotype of tumors. Although non-replicating mRNA can mediate the temporary expression of target genes in tumor cells, relatively stable and efficient expression of HNF4α in tumor cells is required to induce the differentiation of tumor cells into normal cells. Therefore, the present invention is the first to propose that HNF4α, whose expression decreases during the tumor development process, can be restored or overexpressed using self-replicating RNA technology to suppress the growth of tumor cells and achieve the effect of tumor treatment. Therefore, the gene delivery system provided by the present invention can induce the differentiation of malignant solid tumor cells into normal mature cells, suppress the proliferation of tumor cells, and induce apoptosis of tumor cells. In addition, the present invention has optimized the self-replicating RNA vector used, and has obtained a delivery system that can express HNF4α in tumor cells relatively stably and efficiently.

[0031] As described above, the present invention uses self-replicating RNA to express HNF4α, which has high expression level, high expression efficiency, and longer maintenance time.For example, the preferred HNF4α-saRNA of the present invention can maintain HNF4α in liver cancer cells for more than 7 days, and both the expression level and expression time are much higher than non-replicating HNF4α-mRNA.It has the advantages of being safer than viral vectors, being able to be incorporated into a wide range of tissue types, and being able to be administered repeatedly, and has been observed to have therapeutic effects in cancer-bearing animals and patients. [Brief description of the drawings]

[0032] [Figure 1] Schematic diagram of the self-replicating RNA expressing HNF4α with different lengths of polyA tail. The GOI is HNF4α, and the lengths of the polyA tails are 30nt, 35nt, 40nt, 50nt, 60nt, 67nt, 75nt, 90nt, 100nt, and 110nt, respectively. [Diagram 2] We treated Huh-7 cells with HNF4α self-replicating RNA lipid nanoparticles (HNF4α-saRNA LNPs) with different lengths of polyA tails, collected the cellular proteins 3 and 7 days after transfection, and detected the expression levels of HNF4α protein after different treatments by western blot to observe the effect of polyA length on the self-replicating RNA-mediated overexpression of HNF4α in liver cancer cells. [Diagram 3] Huh7 cells were treated with 2ng / 200 cells of HNF4α-saRNA and HNF4α-mRNA lipid nanoparticles for 1 day, 3 days, and 7 days, respectively, and proteins were collected. Huh7 cells were infected with adenovirus Ad-HNF4α and control virus Ad-GFP for 1 day, 3 days, and 7 days, respectively, and proteins were collected. Western blot was used to detect the expression levels of HNF4α protein after different treatments. The relative expression levels of HNF4α protein at each time point were statistically calculated using ImageJ software. [Figure 4]The expression levels of HNF4α protein were detected by western blotting after treating Huh-7 cells with different concentrations of HNF4α-saRNA and control GFP-saRNA lipid nanoparticles for 1, 3, 5, and 7 days. GFP-saRNA is a self-replicating RNA control. [Diagram 5] This was obtained by treating Huh-7 cells with different concentrations of HNF4α-saRNA and GFP-saRNA lipid nanoparticles, and then detecting changes in cell proliferation after treatment with CCK8. [Figure 6] This shows the effect of treatment with HNF4α-saRNA and GFP-saRNA lipid nanoparticles on the clone formation ability of liver cancer cells Huh7. [Figure 7] The results were obtained by treating Huh7 cells with different concentrations of HNF4α-saRNA and GFP-saRNA lipid nanoparticles for 3 days, and then detecting liver function-related genes by RT-PCR. [Figure 8] The results were obtained by treating Huh7 cells with different concentrations of HNF4α-saRNA and GFP-saRNA lipid nanoparticles for 3 days, and then detecting the expression levels of aridity-related genes in tumor cells by RT-PCR. [Figure 9] After treating Huh7 cells with HNF4α-saRNA and GFP-saRNA lipid nanoparticles for 3 days, glycogen storage in Huh7 cells was detected by periodic acid-Schiff (PAS) staining, and the PAS-positive areas in Huh7 cells were statistically analyzed using imageJ software. [Figure 10] After treating Huh7 cells with HNF4α-saRNA and GFP-saRNA lipid nanoparticles for 3 days, the ability of Huh7 cells to take up acetylated low-density lipoprotein (ac-LDL) was detected using Dil-ac-LDL fluorescent substrate, and the ac-LDL positive area in Huh7 cells was quantified and statistically analyzed using imageJ software. [Figure 11]After treating Huh7 cells with HNF4α-saRNA and GFP-saRNA lipid nanoparticles for 3 days, the content of senescence-associated β-galactosidase in Huh7 cells was detected, and the β-galactosidase-positive area in Huh7 cells was quantified and statistically analyzed using imageJ software. [Figure 12] This was obtained by treating Huh7 cells with HNF4α-saRNA and GFP-saRNA lipid nanoparticles for 3 days and then detecting apoptosis by Annexin V / PI staining. [Figure 13] This is an experimental flowchart for treating subcutaneously implanted Huh7 cancer cells by injecting HNF4α-saRNA and HNF4α-mRNA lipid nanoparticles into the tumor. [Figure 14] Tumor growth curves in a model of treatment of subcutaneously implanted Huh7 cancer cells with HNF4α-saRNA and HNF4α-mRNA. GFP-saRNA is the control for self-replicating mRNA lipid nanoparticles, and saline is the solvent control. [Figure 15] FIG. 1 is a schematic diagram showing the treatment of subcutaneously transplanted Huh7 cancer cells with HNF4α-saRNA and HNF4α-mRNA. [Figure 16] Weight statistics (left) and tumor inhibition rate statistics (right) of a model of treatment of subcutaneously transplanted Huh7 cancer cells with HNF4α-saRNA and HNF4α-mRNA. [Figure 17] Western blot analysis shows the expression level of HNF4α in tumor tissue of a model of subcutaneously transplanted Huh7 cancer treated with HNF4α-saRNA. [Figure 18] The changes in expression of HNF4α and Ki67 in tumor tissues detected by immunohistochemistry (left), and a statistical diagram of the areas of HNF4α and Ki67 positive staining in tumor tissues (right). [Figure 19] This is a flowchart of an experiment to treat orthotopic hepatic tumors of Huh7 cells by tail vein injection with HNF4α-saRNA and HNF4α-mRNA lipid nanoparticles. [Figure 20]Figure 1 shows mouse biofluorescence signals before and after injection of HNF4α-saRNA and HNF4α-mRNA in a hepatic orthotopic tumor model of Huh7 cells. GFP-saRNA is the control for self-replicating RNA lipid nanoparticles, and saline is the solvent control. [Figure 21] Statistical graphs of mouse biofluorescence signals at different time points during treatment of hepatic orthotopic tumors of Huh7 cells with HNF4α-saRNA and HNF4α-mRNA. [Figure 22] Schematic diagram of tumors in an experiment of treating orthotopically seeded hepatic tumors of Huh7 cells with HNF4α-saRNA and HNF4α-mRNA lipid nanoparticles. [Diagram 23] Statistical graphs of tumor weight (left) and tumor inhibition rate (right) in a treatment experiment of orthotopically seeded liver tumors of Huh7 cells with HNF4α-saRNA and HNF4α-mRNA lipid nanoparticles. [Figure 24] Detection of tumor tissue morphology by HE staining, detection of Ki67 expression changes by immunohistochemistry (left), and a statistical diagram of Ki67-positive staining area in tumor tissue (right). [Diagram 25] During the treatment of orthotopic liver tumors of Huh7 cells with HNF4α-saRNA, tumor tissues were taken at different time points after treatment, and the expression levels of HNF4α protein were detected by Western blot. [Figure 26] During the process of treating orthotopic liver tumors of Huh7 cells with HNF4α-saRNA, tumor tissues were taken at different time points after treatment, and changes in HNF4α expression were detected by immunohistochemistry (left), and the statistical graph of the area of ​​HNF4α-positive staining in the tumor tissue (right) was also shown. [Figure 27] During the process of treating orthotopic disseminated liver tumors of Huh7 cells with HNF4α-saRNA, tumor tissues were taken at different time points after treatment, and the expression of liver function-related genes in the tumor tissues was detected by quantitative PCR. [Figure 28]The results were obtained by detecting the expression level of HNF4α protein by western blot after treating cholangiocarcinoma cells HuCC-T1 with different concentrations of HNF4α-saRNA and HNF4α-mRNA lipid nanoparticles for 1 and 3 days. [Figure 29] We show that HNF4α-saRNA LNP inhibits the proliferation of cholangiocarcinoma cells HuCC-T1. [Diagram 30] The effect of HNF4α-mRNA LNPs and HNF4α-saRNA LNPs on the clonogenic potential of cholangiocarcinoma cells, HuCC-T1, is shown. [Diagram 31] The expression levels of HNF4α protein were detected by Western blot 1 and 3 days after treating intestinal cancer cells with different concentrations of HNF4α-saRNA LNPs. [Diagram 32] The effect of HNF4α-mRNA LNPs and HNF4α-saRNA LNPs on the proliferation of intestinal cancer cells HCT116 is shown. [Diagram 33] 1 shows the effect of HNF4α-mRNA LNPs and HNF4α-saRNA LNPs on the clonogenicity of intestinal cancer cells HCT116. [Diagram 34] The results were obtained by detecting the expression level of HNF4α protein by western blot after treating pancreatic cancer cells PANC1 with different concentrations of HNF4α-saRNA lipid nanoparticles for 1 and 3 days. [Diagram 35] During the process of treating orthotopic liver tumors of Huh7 cells with HNF4α-saRNA, tumor tissues and other normal tissues were taken at different time points after treatment, and the levels of HNF4α mRNA (HNF4α) and self-replicating RNA vector (VEEV) were detected by quantitative PCR. [Diagram 36]Enhanced MRI showing overall stable liver cancer target lesions after HNF4α-saRNA LNP treatment. A. Liver cancer target lesion (dotted box) 13 weeks until treatment. B. Liver cancer target lesions have clearly increased in size compared to pre-treatment during baseline. C. Ten weeks after treatment, liver cancer target lesion size has remained stable. D. 23 weeks after treatment, liver cancer target lesion size has remained stable, with some areas of liquefaction necrosis (arrows). [Figure 37] Chest CT scans showing the gradual shrinkage of hepatocellular carcinoma lung metastasis lesions after HNF4α-saRNA LNP treatment. A. Baseline hepatocellular carcinoma lung metastasis lesions (arrows). B. Four weeks after treatment, the hepatocellular carcinoma lung metastasis lesions slightly increased compared to baseline. C. Eight weeks after treatment, the hepatocellular carcinoma lung metastasis lesions slightly decreased compared to week four. D. Eighteen weeks after treatment, the hepatocellular carcinoma lung metastasis lesions further shrunk, and the diameter of the lesions was obviously smaller than the baseline level. [Figure 38] Enhanced MRI showing changes in liver cancer target lesions after HNF4α-saRNA LNP treatment. A. The liver cancer target lesions (dashed lines) were clearly more enhanced in the arterial phase than in the baseline phase. B. Four weeks after treatment, the liver cancer target lesions were larger compared to baseline, and arterial phase enhancement was weaker compared to baseline. C. Nine weeks after treatment, the liver cancer target lesions further increased, and arterial phase enhancement was further weakened. D. Sixteen weeks after treatment, the liver cancer target lesions continued to increase, while arterial phase enhancement continued to decrease, with most areas losing enhancement (arrows). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Hereinafter, the mode for carrying out the invention will be described in detail with reference to the embodiments. The advantages and features of the present invention will be made clear by the description. However, these embodiments are merely illustrative and do not limit the scope of the present invention. Those skilled in the art may make modifications or substitutions to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, and it should be understood that all of these modifications and substitutions are within the protection scope of the present invention.

[0034] Unless otherwise specified, the embodiments of the present invention employ conventional techniques well known in molecular biology, cell biology, and immunology. These techniques are fully described, for example, in Guidelines for Molecular Cloning Experiments, 4th Edition (2017), Guidelines for Precise Cell Biology Experiments (2007), and Guidelines for Precise Immunology Experiments (2010). Alternatively, they may be performed according to instructions provided by the reagent manufacturer.

[0035] Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described herein are merely illustrative.

[0036] All data herein are analyzed and plotted using GraphPad Prism 8 software. All data error values ​​are expressed as mean ± standard error (± SEM). Comparison between two sets of data was performed using t-test, and the statistical test was two-tailed, in which p<0.05 was considered to have statistical significance, * represents p<0.05, ** represents p<0.01, and *** represents p<0.001.

[0037] Example 1: Preparation of self-replicating RNA and LNP coating 1. Preparation of Linear mRNA and Self-replicating RNA The self-replicating RNA (saRNA) is based on an engineered alphavirus genome that contains genes encoding nonstructural proteins, which can perform RNA replication, and the structural protein sequence is replaced by the target gene sequence. The saRNA contains one 5'-end cap, a non-coding region (5'UTR), four non-structural genes (NSP1-4), one 26S subgenomic promoter, a target gene, a 3'-end non-coding region (3'UTR) and one polyadenylate tail. The linear RNA is designed according to eukaryotic mRNA structure and contains a gene encoding a target protein, as well as the essential cap structure, 5'UTR, 3'UTR and poly(A). The non-replicating RNA and the self-replicating RNA were prepared in the same manner. To prepare linear templates for RNA transcription, plasmid DNA was digested and cleaved with BspQI enzyme (New England Biolabs, R0712L) and purified using the PureLink® PCR Purification Kit (Invitrogen, K310002) to recover the linear templates. Transcription was performed using a mixture of T7 RNA polymerase (Promega, P1300), 1000U / ml RNase inhibitor (New England Biolabs, M0314L), 2U / ml inorganic pyrophosphatase (New England Biolabs, M2403L), 5mM NTPs (New England Biolabs, N0466S), and cap analog (3' OMe, Trinlink, N-7413), and the mixture was mixed and incubated at 37°C for 2 hours to perform in vitro transcription of the template. After the transcription reaction was completed, DNase I (1U / μg DNA) was added to remove the DNA template and incubated at 37°C for 15 minutes, and the RNA obtained by transcription was purified and collected by LiCl precipitation.

[0038] 2. LNP-coated RNA The lipid nanoparticles were mixed in the ethanol and aqueous phases using a microfluidic device (INano TMThe mRNA-LNPs were prepared by rapid mixing in a 50 mM citrate buffer (pH 6.0) containing purified saRNA. The ethanol phase contained the proprietary ionizable lipids distearoylphosphatidylcholine (DSPC) (Avanti, 850365P), cholesterol (Sigma-Aldrich, C8667) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (NOF, GM020). The molar ratio of DSPC:cholesterol:DMG-PEG2000:LP-1 was 9.4:42.5:1.8:46.3, N / P=6, for the assembly of mRNA-LNPs. The particle size, PDI, RNA concentration and coverage of the LNPs formed were then measured.

[0039] Example 2: Optimization of self-replicating RNA polyA tail length As described in Example 1 and shown in Figure 1, self-replicating RNA expressing HNF4α with different lengths of polyA tails coated on the LNP (HNF4α-saRNA LNP) was prepared. 5Cells / well were seeded into 6-well plates and cultured overnight, and HNF4α-saRNA LNPs with different polyA lengths diluted in Opti-MEM were added, and after 6 hours of culture, 1 mL of 20% FBS-containing DMEM medium was added. After 3 or 7 days, the transfected cells were collected for RIPA lysates and the protein level of HNF4α in the cells was detected by Western blot (Figure 2). The results showed that HNF4α-saRNA with a polyA tail length of 35-100 nt could mediate overexpression of HNF4α in Huh-7 cells, and the tumor cell HNF4α expression level of the transfected self-replicating RNA with a polyA tail length of 40-90 nt was high, while the HNF4α expression level and expression time of the self-replicating RNA with a polyA tail length of 60 nt-75 nt were better, and the self-replicating RNA with a polyA tail length of 67 nt could mediate the most efficient expression. The self-replicating RNA sequence with the best polyA tail length optimization experimental effect is shown in SEQ ID NO:2, and the HNF4α-saRNA sequence is shown in SEQ ID NO:3 (all subsequent examples use this sequence).

[0040] Example 3: Comparison of the ability of non-replicating HNF4α-mRNA, HNF4α-saRNA, and adenovirus AdHNF4α to mediate overexpression of HNF4α in tumor cells Liver cancer cells Huh7 3x10 5Cells / well 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 (final concentration 2 ng RNA / 200 cells) were added. After 6 hours of culture, 1 mL of DMEM medium containing 20% ​​FBS was added. Transfected cells were harvested after 1, 3, 5, or 7 days with RIPA lysis solution, and the protein level of HNF4α in the cells was detected by Western blot (Figure 3). The mediation effect of overexpression of HNF4α by adenovirus was compared with that of Huh7 cells treated with adenovirus AdHNF4α expressing HNF4α and its control virus AdGFP (Figure 3). As shown in the results, both HNF4α-saRNA and HNF4α-mRNA can upregulate HNF4α in liver cancer cells, but the expression level of HNF4α-mRNA is low and the expression time is short, while the expression level of HNF4α-saRNA is high and the maintenance time is long, and its expression efficiency is higher than that of adenovirus.

[0041] Example 4: Treatment of liver cancer by inducing differentiation of liver cancer cells into liver cells with HNF4α-saRNA 1. Different concentrations of HNF4α-saRNA LNPs upregulated the expression of HNF4α in hepatocellular carcinoma Huh7 cells. Liver cancer cells Huh7 3x10 5 Cells / well were seeded into 6-well plates and cultured overnight. After washing with 1 ml of PBS, HNF4α-saRNA LNPs and control GFP-saRNA LNPs serially diluted in 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. Transfected cells were harvested after 1, 3, 5, or 7 days with RIPA lysis solution, and the protein level of HNF4α in the cells was detected by Western blot (Figure 4). The results showed that HNF4α-saRNA LNPs upregulated the expression of HNF4α, while control GFP-saRNA LNPs did not affect the expression of HNF4α in liver cancer cells.

[0042] 2. Inhibition of liver cancer cell growth by HNF4α-saRNA LNPs The suppression of malignant phenotypes, such as tumor cell proliferation and clone formation, by the gene delivery system was examined using liver cancer cell Huh7. Huh7 cells were cultured at 3 × 10 3 The cells were seeded into 96-well plates at a density of 100 cells / well, and after overnight culture, the medium supernatant was aspirated and the cells were washed with 100 μl of PBS. Different concentrations of HNF4α-saRNA LNPs and control GFP-saRNA LNPs diluted with Opti-MEM were added to the cells, and after 6 hours, the same volume of 20% FBS was added to the culture medium of the corresponding cells so that the FBS concentration in the culture medium was 10%. The cell proliferation status was measured daily using a cell counting kit-8 (CCK-8, Dojindo) to clarify the effect of RNA on tumor cell proliferation ability. As a result, compared with the control cells, HNF4α-saRNA LNPs significantly suppressed the proliferation of liver cancer cells (Figure 5).

[0043] Next, the inventors performed an experiment to examine the effect of the gene delivery system on tumor cell clonogenicity. 3 The cells were seeded in a 96-well plate at a density of 1000 cells / well, cultured overnight, and then transfected with HNF4α-saRNA LNP and control GFP-saRNA LNP (2ng / 200cells). After 24 hours, trypsin-digested cells were transferred to a 60mm culture dish and cultured, changing the culture medium every 3 days, and the clonal formation status of tumor cells was observed under a microscope. After clonal formation, the cells were washed twice with PBS, stained with crystal violet for 20min, washed with PBS, dried and photographed, and the number of clones was counted using Image J to clarify the effect of RNA on the clonal formation ability of tumor cells. As shown in Figure 6, HNF4α-saRNA significantly suppressed the clonal formation ability of liver cancer cells.

[0044] 3.HNF4α-saRNA LNP induces differentiation of liver cancer cells into mature hepatocytes (1) Restoration of liver function gene expression in liver cancer cells by HNF4α-saRNA

[0045] Liver cancer cells Huh-7 at 3x10 5 Cells / well 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 in Opti-MEM were added. After 6 hours of culture, 1 mL of DMEM medium containing 20% ​​FBS was added. Cell extract RNA was collected 1, 3 or 7 days after cell transfection, and the expression of liver function-related genes and tumor stemness-related genes regulated by HNF4α were detected by real-time fluorescent quantitative PCR (Figure 7, Figure 8). The results showed that HNF4α-saRNA upregulated the expression of liver function-related genes and downregulated the expression of tumor stemness-related genes.

[0046] (2) HNF4α-saRNA enhances glycogen storage and low-density lipoprotein uptake in hepatocellular carcinoma cells

[0047] Glycogen storage and low-density lipoprotein uptake are important functions of normal liver cells, and the inventors will further examine these functions to clarify whether liver cancer cells can differentiate into mature liver cells.

[0048] Liver cancer cells Huh7 3x10 4Cells / well were seeded in a 24-well plate, and 1ng / 200 cells of HNF4α-saRNA LNPs and corresponding controls were added. Three days after LNP delivery, cellular glycogen storage capacity was verified by PAS reaction kit (Biyuntian). After adding 70% ethanol to fix the cells for 10 minutes, the periodic acid solution was removed and equilibrated to room temperature. 100μl of periodic acid solution was dropped onto each sample, and reacted in a wet box for 10 minutes in the dark, after which the periodic acid solution was removed, immersed in PBS and washed on a shaker for 5 minutes. 100μl of Schiff's reagent was dropped onto each sample, then placed in a wet box, placed in a 37℃ oven and stained for 1 hour in the dark. The staining solution was removed, immersed in PBS and washed on a shaker for 5 minutes. Finally, 100μl of hematoxylin staining solution was dropped onto each sample and stained for 30 seconds. The staining solution was removed, and the sample was rinsed twice with PBS to remove any floating color. The stained cells were photographed under a microscope and counted (Figure 9). The results showed that HNF4α-saRNA could promote the glycogen storage ability of liver cancer cells.

[0049] Liver cancer cells Huh7 3x10 4 Cells / well were seeded into 24-well plates, and 1ng / 200 cells of HNF4α-saRNA LNPs and corresponding controls were added. Three days after LNP delivery, the cell medium was aspirated and the cells were washed with PBS, after which Dil ac-LDL (Invitrogen) was diluted 1:100 with DMEM and 200μl was added to each well. After 3h, Dil ac-LDL-containing DMEM was removed, the cells were washed with PBS, and fixed with 4% paraformaldehyde for 15 minutes. After nuclear staining with DAPI, blocking was performed, and images were taken with a confocal fluorescence microscope, and the fluorescent signals were statistically analyzed (Figure 10). The results showed that HNF4α-saRNA could promote the low-density lipoprotein uptake ability of liver cancer cells.

[0050] (3) HNF4α-saRNA promotes senescence in liver cancer cells

[0051] Liver cancer cells Huh7 3x10 4Cells / well were seeded into 24-well plates, and 1ng / 200 cells of HNF4α-saRNA LNPs and corresponding controls were added. Three days after LNP delivery, the senescence of HCC cells was evaluated using a Senescence β-Galactosidase staining kit (Beyotime, China). Cells were fixed with 4% formaldehyde for 15 min and then incubated overnight at 37°C in fresh senescence-associated β-galactosidase staining solution. Senescent cells were observed and captured under a microscope. The number of senescent cells was calculated using image analysis software (Image-Pro Plus 6.0, Media Cybernetics) (Figure 11). The results showed that senescent liver cancer cells were significantly increased after HNF4α-saRNA treatment.

[0052] (4) Induction of apoptosis in liver cancer cells by HNF4α-saRNA

[0053] Liver cancer cells Huh7 3x10 5 Cells / well were seeded into 24-well plates and 1ng / 200 cells of HNF4α-saRNA LNPs and corresponding controls were added. Three days after LNP delivery, detection of apoptotic cells was performed using the APC Annexin V / PI Apoptosis Kit (Biolegend, China) according to the manufacturer's instructions. Cells were digested with 0.25% trypsin and then washed twice with PBS. Then, cells were transferred to test tubes and suspended in 100μl of binding buffer. 400μl of binding buffer containing 5μl of APC Annexin V and 5μl of propyliodamine was added to each test tube and incubated at 25℃ for 15min in the dark. Then, cells were analyzed using a flow cytometer (Attune NxT, Invitrogen). Flow cytometer data were analyzed using FlowJo V10 software (Figure 12). The results showed that apoptosis of liver cancer cells treated with HNF4α-saRNA was significantly increased.

[0054] These results indicate that HNF4α-saRNA can promote the differentiation of liver cancer cells into mature liver cells, restore liver cell function, and induce senescence and apoptosis in liver cancer cells.

[0055] 3.HNF4α-saRNA LNP suppresses the growth of subcutaneously transplanted cancer in mice

[0056] Five-week-old male nude mice (BALB / c immunodeficiency model) were purchased from Shanghai BK / KY Biotechnology Co., Ltd. and raised under specific pathogen-free environmental conditions. The lights were switched on and off for 12 hours. 6 Huh-7 cells were injected subcutaneously into the right flank of male nude mice, and tumor size was measured in two dimensions using calipers, volume = length × (width). 2 The volume was calculated by multiplying the volume by 1 / 2. The average tumor volume was approximately 100 mm 3When the tumor reached 100 μg, the mice were randomly divided into 4 groups (7 mice per group). 5 μg of HNF4α-saRNA LNP, GFP-saRNA LNP, HNF4α-mRNA LNP, or saline (solvent control) was injected into the tumor of the mice, with a final volume of 75 μl. After injection, the tumor volume was measured once a day, and a tumor growth curve was drawn (Figures 13 and 14). Five days after RNA injection, the mice were killed, the tumors were excised and weighed (Figures 15 and 16), and the tumor inhibition rate was calculated (Figure 16). Some tumor tissues were embedded in paraffin, and the expression of HNF4α was detected by Western-blot and immunohistochemistry (Figures 17 and 18), and the proliferation state of tumor cells was evaluated by Ki67 staining (Figure 17). As a result, after HNF4α-saRNA was injected into the tumor, tumor growth was slowed and the tumor inhibition rate exceeded 50% (Figure 16), but HNF4α-mRNA did not have a significant inhibitory effect on tumor growth. Western blot and immunohistochemistry results showed that HNF4α-saRNA significantly increased the protein expression of HNF4α in tumor tissues, and significantly decreased the expression of tumor cell proliferation indicator Ki67. These results clearly show that HNF4α-saRNA can effectively induce the expression of HNF4α in cancer cells, and further suppress the growth of mouse liver cancer disseminated tumors.

[0057] 4. HNF4α-saRNA LNP suppresses the growth of hepatocellular carcinoma cells disseminated in the liver

[0058] The human liver cancer cell line Huh7, which stably expresses the luciferase gene, was inoculated into the armpits of male nude mice. After tumor formation, the tumor mass was removed and cut into 1 mm 3The tumors were cut into blocks and transplanted under the Glisson capsule of male nude bab / c mice. The tumor growth status in the mice was monitored using the IVIS spectrum optical imaging system. Three days after transplantation, the mice were divided into 4 groups according to the intensity of the fluorescent signal, and HNF4α-saRNA LNP, GFP-saRNA LNP, HNF4α-mRNA LNP, and saline (solvent control) were administered via tail vein injection at a dose of 2 mg / kg body weight (Figure 19). The tumor growth status of the mice was monitored based on bioluminescence to evaluate the therapeutic effect of HNF4α-saRNA (Figure 20), and a tumor growth curve was drawn (Figure 21). After the experiment, the mice were killed, the tumors were excised and weighed, and the tumor inhibition rate was calculated (Figures 22 and 23). The tumor tissues were embedded in paraffin, and the tumor tissue morphology was evaluated by HE staining, and the tumor growth status was evaluated by Ki67 staining (Figure 24). The results showed that after tail vein injection of HNF4α-saRNA LNP, tumor growth was reduced and the tumor inhibition rate exceeded 40% compared with the control tumor, while HNF4α-mRNA LNP had no significant tumor inhibition effect. In addition, tumor tissues were collected at different time points after LNP injection to detect the expression status of HNF4α by western blot and immunohistochemistry (Figures 25 and 26), the tumor growth status was evaluated by Ki67 staining (Figure 26), and hepatocyte differentiation-related indicators were detected by quantitative PCR (Figure 27). The results showed that HNF4α-saRNA significantly upregulated the expression of HNF4α and liver function-related genes in tumor tissues. These results further demonstrated that HNF4α-saRNA can induce the differentiation of liver cancer cells into normal liver cells and further inhibit the growth of liver cancer.

[0059] Example 5: Inhibition of proliferation of cholangiocarcinoma cells by HNF4α-saRNA 1. Different concentrations of HNF4α-saRNA LNPs up-regulate the expression of HNF4α in cholangiocarcinoma HuCC-T1 cells.

[0060] Cholangiocarcinoma cells HuCC-T1 were seeded in 6-well plates at 40%-50% density and cultured overnight. After washing with 1ml of PBS, HNF4α-saRNA LNPs serially diluted in Opti-MEM (final concentrations of 1, 2ng RNA / 200 cells) were added. After 6 hours of culture, 1mL of DMEM medium containing 20% ​​FBS was added. The transfected cells were collected with RIPA lysis solution after 1 and 3 days and the protein level of HNF4α in the cells was detected by Western blot (Figure 28), which showed that HNF4α-saRNA LNPs upregulated the expression of HNF4α in cholangiocarcinoma cells.

[0061] 2.HNF4α-saRNA LNPs suppress the growth of cholangiocarcinoma cells.

[0062] Bile duct cancer cells HuCC-T1 were cultured at 3 × 10 3 The cells were seeded in a 96-well plate at a density of 100 cells / well and cultured overnight. The medium supernatant was aspirated and the cells were washed with 100 μl of PBS. Different concentrations of HNF4α-saRNA LNPs (final concentrations of 1 and 2 ng RNA / 200 cells) diluted with Opti-MEM were added to the cells, and after 6 hours, the same volume of 20% FBS was added to the culture medium of the corresponding cells so that the FBS concentration in the culture medium was 10%. The cell proliferation status was measured daily using a cell counting kit-8 (CCK-8, Dojindo) to clarify the effect of RNA on tumor cell proliferation ability. As a result, compared with the control cells, HNF4α-saRNA LNPs significantly suppressed the proliferation of cholangiocarcinoma cells (Figure 29).

[0063] 3.HNF4α-saRNA LNP suppresses clonogenesis of cholangiocarcinoma cells.

[0064] Bile duct cancer cells HuCC-T1 were cultured at 3 × 10 3The cells were seeded in a 96-well plate at a density of 1000 cells / well, cultured overnight, and then transfected with HNF4α-saRNA LNP and HNF4α-mRNA LNP (2ng / 200cells). After 24 hours, trypsin-digested cells were transferred to a 60mm culture dish and cultured, with the culture medium replaced every 3 days, and the clonal formation status of tumor cells was observed under a microscope. After clonal formation, the cells were washed twice with PBS, stained with crystal violet for 20 minutes, washed with PBS, dried, photographed, and the number of clones was counted with Image J to clarify the effect of RNA on the clonal formation ability of tumor cells (Figure 30). As a result, HNF4α-saRNA significantly suppressed the clonal formation ability of cholangiocarcinoma cells, but HNF4α-mRNA did not affect the clonal formation of tumor cells.

[0065] Example 6: Inhibition of intestinal cancer cell proliferation by HNF4α-saRNA 1. Different concentrations of HNF4α-saRNA LNPs upregulate the expression of HNF4α in intestinal cancer HCT-116 cells.

[0066] Intestinal cancer cells HCT-116 were seeded in 6-well plates at 40%-50% density and cultured overnight. After washing with 1ml of PBS, HNF4α-saRNA LNPs serially diluted in Opti-MEM (final concentrations of 1, 2ng RNA / 200 cells) were added. After 6 hours of culture, 1mL of DMEM medium containing 20% ​​FBS was added. The transfected cells were collected with RIPA lysis solution after 1 and 3 days and the protein level of HNF4α in the cells was detected by Western blot (Figure 31), which showed that HNF4α-saRNA LNPs upregulated the expression of HNF4α in intestinal cancer cells.

[0067] 2. Inhibition of cholangiocarcinoma cell growth by HNF4α-saRNA LNPs

[0068] Intestinal cancer cells HCT-116 were cultured at 3 × 10 3After seeding at a cell / well density in a 96-well plate and culturing overnight, the medium supernatant was aspirated and the cells were washed with 100 μl of PBS. Different concentrations of HNF4α-saRNA LNPs and HNF4α-mRNA LNPs (final concentration 2 ng RNA / 200 cells) diluted with Opti-MEM were added to the cells, and after 6 hours, the same volume of 20% FBS was added to the culture medium of the corresponding cells so that the FBS concentration in the culture medium was 10%. The cell proliferation status was measured daily using a cell counting kit-8 (CCK-8, Dojindo) to clarify the effect of RNA on tumor cell proliferation ability. As a result, compared with the control cells, HNF4α-saRNA LNPs significantly suppressed the proliferation of intestinal cancer cells (Figure 32), but HNF4α-mRNA LNPs had no obvious effect on tumor cell proliferation.

[0069] 3. Suppression of clonal formation of intestinal cancer cells by HNF4α-saRNA LNP

[0070] Intestinal cancer cells HCT-116 were cultured at 3 × 10 3 The cells were seeded in a 96-well plate at a density of 1000 cells / well, cultured overnight, and then transfected with HNF4α-saRNA LNP and HNF4α-mRNA LNP (2ng / 200cell). After 24 hours, trypsin-digested cells were transferred to a 60mm culture dish and cultured, with the culture medium replaced every 3 days, and the clonal formation status of tumor cells was observed under a microscope. After clonal formation, the cells were washed twice with PBS, stained with crystal violet for 20min, washed with PBS, dried, photographed, and the number of clones was counted with Image J to clarify the effect of RNA on the clonal formation ability of tumor cells (Figure 33). As a result, HNF4α-saRNA significantly suppressed the clonal formation ability of intestinal cancer cells, but HNF4α-mRNA did not affect the clonal formation of tumor cells.

[0071] Example 7: Upregulation of HNF4α expression in pancreatic cancer cells by HNF4α-saRNA Human pancreatic cancer cells PANC1 were seeded in a 6-well plate at 40%-50% density and cultured overnight. After washing with 1 ml of PBS, HNF4α-saRNA LNPs serially diluted in Opti-MEM (final concentrations of 1 and 2 ng RNA / 200 cells) were added. After 6 hours of culture, 1 mL of DMEM medium containing 20% ​​FBS was added. Transfected cells were collected with RIPA lysis solution after 1 and 3 days and the protein level of HNF4α in the cells was detected by Western blot (Figure 34), which showed that HNF4α-saRNA LNPs upregulated the expression of HNF4α in pancreatic cancer cells.

[0072] Example 8: HNF4α-saRNA LNP specifically upregulates HNF4α expression in tumor tissue in vivo The human liver cancer cell line Huh7, which stably expresses the luciferase gene, was inoculated into the armpits of male nude mice. After tumor formation, the tumor mass was removed and cut into 1 mm pieces. 3The tumors were cut into blocks and implanted under the Glisson capsule of male Bab / c nude mice, and the tumor growth status in the mice was monitored using the IVIS spectrum optical imaging system. After the tumors had grown to an appropriate size, HNF4α-saRNA LNP was administered via tail vein injection at a dose of 2 mg / kg body weight, and the mouse organ tissues and tumor tissues were taken 3 and 5 days after injection. The expression distribution of alphavirus genome sequence (VEEV) and LNP-mediated human HNF4α gene sequence in each organ and tumor was detected by quantitative PCR (Figure 35). As a result, 3 days after HNF4α-saRNA LNP was administered via tail vein injection, VEEV and human HNF4α RNA were detected in trace amounts in the heart, spleen, and kidney, and VEEV and HNF4α gene sequence were hardly detected in the liver, lung, and muscle tissues, but high abundance of VEEV and HNF4α RNA was clearly detected in the tumor tissue. Five days after HNF4α-saRNA LNP was administered via tail vein injection, VEEV and human HNF4α RNA were not detected in other organs except for a trace amount in the heart, but VEEV and HNF4α gene sequences were clearly detected in tumor tissues (Figure 35), indicating that self-replicating RNA can mediate the expression of HNF4α in tumor cells in vivo.

[0073] Example 9: Treatment of late stage liver cancer in patients with HNF4α-saRNA LNPs We commissioned a relevant CDMO to produce a HNF4α-saRNA LNP formulation that meets GMP standards in accordance with pharmaceutical standards, and commissioned a relevant CRO to complete a single-dose toxicity test of the HNF4α-saRNA LNP formulation. The specific process and results are as follows: Rats were given a single dose via tail vein injection, with an observation period of 14 days, and a negative control group (0.9% sodium chloride injection), vector control group (LNP solution), and test article group (150μg / animal) were set up. All animals were allowed to survive until the scheduled date of autopsy, and almost no anatomical abnormalities were observed at the end of the observation period, and no histopathological changes related to the test article were observed.

[0074] After completing the acute toxicity test, a clinical trial initiated by the researchers was conducted, in which the drug was administered via hepatic artery cannula, and a dose escalation test of 25μg, 50μg, and 100μg was completed (3 cases per group). None of the 9 patients experienced dose-limiting toxicity or treatment-related adverse events of grade 3 or higher or serious adverse events. Four patients were treated for more than 5 months, and the liver cancer lesions did not progress significantly, one of which had a significant reduction in the lung metastatic lesions (Figure 36), another patient had focal necrosis in the liver cancer lesions (Figure 37), and three patients had significant necrosis in the target lesions and a significant reduction in enhancement (Figure 38). The initial results showed that the HNF4α-saRNA LNP formulation had good safety and could significantly inhibit tumor growth in patients with late-stage liver cancer.

[0075] Although the preferred embodiments of the present invention have been described in detail above, the present invention is not limited to the above embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention, and all of these equivalent modifications or substitutions are included in the scope limited by the claims of the present invention.

Claims

1. A gene delivery system comprising a nucleic acid vector and a delivery vehicle, the nucleic acid vector being a self-replicating RNA carrying HNF4α, the gene delivery system delivering HNF4α into tumor cells and inducing the differentiation of the tumor cells into normal mature cells, the self-replicating RNA being based on the genome of an artificial alphavirus and having a 35-100 nt polyadenylic acid tail, the delivery vehicle being a lipid nanoparticle; A nucleic acid molecule having a structure including a 5' capping structure, a 5' UTR, a promoter, HNF4α, a 3' UTR, and a polyadenylic acid tail is prepared, and the nucleic acid molecule is mixed with DSPC: cholesterol: DMG-PEG2000 in a specific molar ratio to form an LNP, and the two are mixed to prepare an LNP-coated RNA; The self-replicating RNA mediates overexpression of HNF4α in tumor cells, thereby inducing differentiation of tumor cells into mature cells, thereby suppressing tumor cell growth. A gene delivery system comprising:

2. The gene delivery system described in claim 1, characterized in that the specific molar ratio of DSPC:cholesterol:DMG-PEG2000 is 9.4:42.5:1.

8.

3. The gene delivery system of claim 1, wherein the self-replicating RNA has a 60-75 nt polyadenylic acid tail.

4. The gene delivery system of claim 1, wherein the self-replicating RNA has a 67 nt polyadenylic acid tail.

5. The gene delivery system of claim 1, wherein the sequence of the self-replicating RNA is shown in SEQ ID NO:

3.

6. The delivery vehicle is a lipid-based nanoparticle, wherein the lipid is 1,2-distearoyl-sn-propyltriyl-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine in a molar ratio of 5%-20%; Cholesterol with a molar ratio of 30%-55%; A PEGylated lipid containing dimyristoylglycerol-polyethylene glycol 2000 or polyethylene glycol fragments in a molar ratio of 0.5%-3%; The gene delivery system according to any one of claims 1 to 5, further comprising an ionizable lipid in a molar ratio of 30% to 60%.

7. The gene delivery system of claim 6, wherein the N:P range in the lipid-based nanoparticles is 5:1 to 10:1, and the particle size of the nanoparticles is 40-300 nm.

8. A pharmaceutical composition comprising the gene delivery system according to any one of claims 1 to 5.

9. The pharmaceutical composition of claim 8 for use as a treatment for malignant solid tumors derived from epithelial cells.

10. The pharmaceutical composition according to claim 9, characterized in that the malignant solid tumor is liver cancer, pancreatic cancer, intestinal cancer, or gastric cancer.

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