Gene delivery system and use thereof in preparation of tumor therapeutic drug

Abstract

The present invention pertains to the technical field of biomedicine, and specifically relates to a gene delivery system and use thereof in the preparation of a tumor therapeutic drug. The present invention relates to a technical means for inducing differentiation of tumor cells into mature cells, which utilizes messenger ribonucleic acid to regulate the expression of HNF4α in tumor cells, inhibits the malignant phenotype of malignant solid tumor cells, and achieves the effect of treating malignant solid tumors, thereby being applied to a preparation method and use of a solid tumor drug.

Description

Gene delivery system and its application in preparing tumor treatment drugs TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a gene delivery system and its application in preparing tumor treatment drugs. BACKGROUND

[0002] The treatment of malignant solid tumors is one of the current clinical difficulties, especially for malignant solid tumors that cannot be eradicated by surgery, and there is still a lack of effective treatment methods in clinic. Although in recent years, radiotherapy, chemotherapy, targeted therapy and immunotherapy have provided various means for the treatment of tumors, but with tumor progression, there will be drug resistance, and a large number of tumors lack effective targeted drug targets or are not sensitive to immunotherapy, especially some digestive tract tumors such as liver cancer and pancreatic cancer, which lack effective drug treatment methods, and the prognosis of patients is not optimistic.

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

[0004] The hepatocyte nuclear factor (HNF) family includes HNF1, HNF3, HNF4, HNF6 and CCAAT / enhancer binding protein (C / EBP), which is a group of transcription factors that are dominantly expressed in the liver and are mutually regulated, and plays a key role in liver development and function maintenance. Among them, hepatocyte nuclear factor 4 alpha (HNF4α) is a transcription factor belonging to the nuclear receptor family, which is expressed in tissues such as liver, kidney, pancreas, and intestine, but mainly expressed in mature hepatocytes. In mature hepatocytes, HNF4α can bind to about 12% of the gene promoters in the cell, participate in maintaining important functions of hepatocytes such as lipid metabolism, albumin synthesis, drug detoxification, energy metabolism, and bile acid synthesis, and is also an important gene for maintaining the epithelial phenotype of hepatocytes. HNF4α also regulates the development of kidney and intestinal tissues, and regulates insulin production in pancreatic tissue. Previous studies have shown that HNF4α expression is decreased in various epithelial-derived tumors such as liver cancer (including hepatocellular carcinoma, intrahepatic cholangiocarcinoma), pancreatic cancer, intestinal cancer, and kidney cancer, and overexpression of HNF4α can inhibit tumor cell proliferation, metastasis, and promote tumor cell apoptosis, suggesting that HNF4α is a potential target for tumor treatment. HNF4α has 9 subtypes, and different subtypes depend on the transcriptional regulation of its selective promoters P1 and P2. The HNF4α subtypes regulated by the P1 promoter (HNF4α1-HNF4α6 subtypes) are mainly expressed in the liver and kidney, and the HNF4α subtypes regulated by the P2 promoter (HNF4α7-HNF4α9 subtypes) are mainly expressed in the pancreas and stomach tissues, and the HNF4α subtypes regulated by both P1 and P2 promoters are expressed in the intestine. The inventors' team previously owned the invention patent "HNF4α induced differentiation treatment of human malignant solid tumors" (Chinese patent number: ZL200810034200.3), which used an adenovirus vector to mediate overexpression of HNF4α isoform 2 (HNF4α2) in hepatocellular carcinoma cells, and confirmed the therapeutic effect of HNF4α on tumors.

[0005] In order to achieve tumor treatment, it is necessary to ensure long-term stable and efficient expression of HNF4α in tumor cells; due to the rapid proliferation ability of tumors, repeated administration is required for tumor treatment to ensure treatment effect. However, due to the pre-existing immunity to adenovirus in the human population, and the fact that adenovirus vectors can induce an anti-viral response from the human immune system, thereby rejecting the expression of the target gene in tumor cells mediated by the viral vector, the frequency of administration of adenovirus vectors for overexpression of HNF4α for tumor treatment is limited. On the other hand, previous studies have also shown that adenovirus vectors have defects in safety and tissue targeting, and their preparation cost is relatively high, so the actual application potential of adenovirus vectors for overexpression of HNF4α for tumor treatment is low.

[0006] mRNA technology is a revolutionary gene delivery technology developed in recent years. This technology synthesizes mRNA molecules with specific sequences by in vitro transcription, and uses lipid nanoparticles (LNPs) to encapsulate mRNA for transportation into human cells. The mRNA is translated into target proteins by the cell's own translation system, which is more efficient and safer than the current viral vector-based delivery system. If combined with the screening and modification of nano-lipid particles, the tissue specificity of the delivery system can be increased. The potential application of mRNA technology currently known mainly focuses on vaccine development for infectious diseases, therapeutic tumor vaccines, protein replacement therapy for protein deficiency caused by genetic defects, and gene editing for genetic diseases or immune cell modification. However, mRNA can only mediate transient high expression of target genes in cells, which has significant shortcomings in expression duration and efficiency, and cannot meet the demand for long-term stable expression of target proteins in rapidly proliferating tumor cells.

[0007] Self-amplifying RNA (saRNA, or self-replicating RNA, srRNA) is a gene delivery vector based on mRNA technology developed in recent years, which is a recombinant RNA with viral structure. By modifying the bi-cistronic genome of alphaviruses, the sequence encoding four non-structural proteins (NSPs) from the virus is retained, and the viral structural protein gene located after the subgenome promoter (SGP) is replaced by a heterologous gene (gene of interest, GOI) encoding the protein of interest, to construct a RNA vector that can express the gene of interest with self-amplification ability in cells. At present, the self-amplifying RNA vector is mainly modified from Venezuelan equine encephalitis virus (VEEV), sindbis alphavirus (SIN) or Semliki Forest virus (SFV), among which the self-amplifying RNA derived from Venezuelan equine encephalitis virus is the most commonly used. The self-amplifying RNA enters the cytoplasm of the host cell, first translates the four non-structural proteins (NSP1, NSP2, NSP3, NSP4), and then polymerizes to form an RNA-dependent RNA polymerase complex through a complex, multi-step process. The RNA polymerase complex first synthesizes a complementary negative-strand RNA intermediate from the positive-strand RNA, and then synthesizes two different positive-strand RNAs using the latter as a template. The first positive-strand RNA is a copy of the original full-length genomic RNA; the second positive-strand RNA is a large number of subgenomic RNAs encoding the gene of interest. This mechanism enables self-amplifying RNA to achieve high-level and persistent expression of the target protein at a low dose of self-amplifying RNA. Self-amplifying RNA retains the self-amplification ability of the viral genome, but cannot express the structural proteins of the virus, and therefore cannot produce complete viruses with the ability to spread. Its entry into cells depends on the delivery of lipid nanoparticles, so it has good safety and potential tumor targeting ability.

[0008] Currently in tumor treatment, self-replicating RNA is considered to be applicable to the preparation of RNA vaccine and the expression of tumor killing cytokines in tumor cells. For example, Chinese invention patent application CN117280029A discloses a nucleic acid vector and a use method, which modulates the tumor microenvironment by activating the relative expression of tumor infiltrating lymphocytes and / or the immunogenic cell characteristics in the tumor microenvironment to achieve the treatment of cancer; Chinese invention patent application CN117279661A discloses a composition and method for inducing ESR1, PI3K, HER2 and HER3 immune response, which is a method for treating cancer by inducing immune response; Chinese invention patent application CN115968299A discloses a new antigen expressed in multiple myeloma and its use, which also uses self-replicating RNA as a carrier to prepare a tumor vaccine. The principle of the tumor vaccine is to express tumor antigens in antigen-presenting cells (APCs), such as dendritic cells (DCs), and present them to T cells of the immune system, activate T cells to attack tumor cells, and then control or eliminate tumors.

[0009] However, there is no report on using self-replicating RNA to express transcription factors, especially transcription factors related to tissue and organ differentiation, in tumor cells for tumor treatment. SUMMARY

[0010] The purpose of the present application is to use self-replicating RNA to induce tumor cells to differentiate into mature cells by overexpressing HNF4a in tumor cells, thereby inhibiting the growth of tumor cells and providing a new means for tumor treatment.

[0011] Hepatocyte nuclear factor HNF4a has lower expression in related tumor cells than in normal cells. The inventors found that self-replicating RNA can be distributed in tumor cells without being reduced by rapid proliferation of tumor cells based on its self-replication characteristics, and can express proteins for more than 20-30 days, so it is very suitable for mediating the expression of target genes in tumor cells. Animal experiments show that tail vein injection of self-replicating RNA encapsulated in lipid nanoparticles can mediate the specific expression of target genes in tumor tissues, while the expression of target genes in other normal tissues is basically not detected, indicating that self-replicating RNA has good tumor targeting. Based on these findings, the inventors propose to use self-replicating RNA vectors to restore or overexpress HNF4a in tumor tissues, induce tumor cells to differentiate into normal cells, inhibit the growth of tumor cells, and thus achieve the effect of treating tumors.

[0012] The present application introduces HNF4a into tumor cells by self-replicating RNA technology to overexpress in tumor cells, and expresses specifically and long-term in tumor cells, so as to achieve the purpose of treating tumors by inducing related tumor cells to differentiate into mature cells.

[0013] The self-replicating RNA optimizes the length of the polyA tail, which can maintain longer and higher level expression of the target gene in vivo. The structure of the self-replicating RNA includes a 5' cap, a non-coding region (5' UTR), four non-structural genes (NSP1-4), a 26S subunit group promoter (SGP), a target gene (GOI), a 3' non-coding region (3' UTR), and a polyA tail. In order to optimize the performance of the self-replicating RNA, previous studies have tried various strategies to modify NSP1-4 by introducing mutations to improve the replication efficiency and immunogenicity of the self-replicating RNA, and to increase the expression intensity and duration of different target genes in cells. We found that the efficiency of different NSP1-4 mutant self-replicating RNA vectors in mediating HNF4α expression in tumor cells was not significantly different. On the other hand, previous studies have shown that the length and sequence of the polyA tail of mRNA affect the stability and ribosome translation of mRNA, and changing the length of the polyA tail can affect the expression duration and expression level of the encoded protein by regulating the degradation rate and translation level of mRNA. However, it is not clear whether the polyA of the self-replicating RNA affects the expression duration and expression level of the target gene in tumor cells. In order to obtain a self-replicating RNA vector that can mediate stable and efficient expression of HNF4α in tumor cells, we optimized the length of the polyA of the self-replicating RNA used, and found that the self-replicating RNA with a polyA length of 35-100 nt can mediate relatively stable and long-term overexpression of HNF4α in tumor cells, among which the expression level and duration of 60-75 nt are better, and the polyA tail of 67 nt can obtain the most stable and efficient expression.

[0014] The present application investigates the effect of non-replicating HNF4α-mRNA and HNF4α-saRNA and adenovirus-mediated overexpression of HNF4α in tumor cells, and the results show that both HNF4α-saRNA and HNF4α-mRNA can up-regulate HNF4α in hepatocellular carcinoma cells, but the expression level of HNF4α-mRNA is lower and the expression duration is shorter; while the expression level of HNF4α-saRNA is more than 2 times higher than that of HNF4α-mRNA, and the maintenance time is longer, the effect of HNF4α-mRNA on up-regulating HNF4α expression can only be maintained for 3 days, while HNF4α-saRNA can maintain HNF4α in hepatocellular carcinoma cells for at least 7 days, and the expression efficiency of HNF4α-saRNA in up-regulating HNF4α is not lower than that of adenovirus.

[0015] In a first aspect of the present application, a gene delivery system is provided, which is composed of a nucleic acid vector and a delivery vehicle; 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 proliferation of tumor cells or / and induces apoptosis of tumor cells.

[0016] The HNF4α has a GENBANK number of NM_000457.6, and is shown in SEQ ID NO: 1.

[0017] The self-replicating RNA comprises a 5' cap, a 5' non-coding region, a non-structural gene, a 26S subunit group promoter, a 3' non-coding region, and a poly(A) tail.

[0018] The self-replicating RNA has a poly(A) tail of 35-100 nt.

[0019] More preferably, the self-replicating RNA has a poly(A) tail of 60-75 nt.

[0020] Most preferably, the self-replicating RNA has a poly(A) tail of 67 nt.

[0021] In a preferred embodiment of the present application, the sequence of the self-replicating RNA is shown in SEQ ID NO: 3, which is an optimized poly(A) tail of the wild-type self-replicating RNA, and the preferred poly(A) tail is 67 nt; the saRNA with a 67-nt poly(A) tail can increase the half-life of the saRNA in vivo, and promote the stability and translation of the saRNA.

[0022] The gene delivery system, wherein the delivery vehicle is selected from one or more of the following in combination: liposomes, viral replicon particles, lipid-based nanoparticles, polymeric nanoparticles, physiological buffers, microspheres, immunostimulatory complexes, conjugates of biologically active ligands.

[0023] More preferably, the delivery vehicle is a lipid-based nanoparticle (LNP), and the lipids in the LNP comprise:

[0024] 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) at a molar ratio of 5%-20%;

[0025] Cholesterol at a molar ratio of 30%-55%;

[0026] Dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG 2000) or a pegylated lipid containing a polyethylene glycol fragment at a molar ratio of 0.5%-3%;

[0027] Ionizable lipid (ALC-0315, SM-102, DHA-1, etc.), molar ratio 30%-60%.

[0028] In a preferred embodiment of the present application, the sum of the molar ratios of the lipids is 100%; the composition of the lipids is DSPC: cholesterol: DMG-PEG 2000: ionizable lipid molar ratio 9.4: 42.5: 1.8: 46.3.

[0029] The N:P ratio in the LNP ranges from 5:1 to 10:1, preferably 6:1.

[0030] The LNP has a particle size of about 40-300 nm.

[0031] In a second aspect of the present application, the use of the above-mentioned gene delivery system in the preparation of a drug for treating malignant solid tumors is provided.

[0032] More preferably, the malignant solid tumor is an epithelial cell-derived malignant solid tumor.

[0033] More preferably, the malignant solid tumor is liver cancer, pancreatic cancer, gastric cancer, intestinal cancer, kidney cancer, lung cancer.

[0034] In a third aspect of the present application, a pharmaceutical composition is provided, which comprises the above-mentioned gene delivery system.

[0035] Technical effects of the present application: The present application uses self-replicating RNA vectors based on mRNA technology platform combined with lipid nanoparticle encapsulation delivery means to highly express important differentiation-related gene HNF4a in tumor cells, induce tumor cells to transform into normal cells, inhibit tumor malignant phenotype, and achieve the purpose of treating tumors. This is a new means of inducing differentiation to treat tumors. According to the results of our previous animal experiments and the clinical research initiated by researchers, it can be confirmed that this technical means can effectively inhibit the growth of tumors in vivo, and is a new scheme for tumor treatment, which is expected to open up a new track for tumor treatment. On the other hand, the present application is significantly different from the current application of mRNA technology in tumor vaccines. RNA tumor vaccines use RNA technology to express related tumor antigens in antigen-presenting cells to stimulate the body to produce an immune response against these antigens, thereby playing a role in controlling or eliminating tumors; while the present application uses RNA technology to mediate the expression of transcription factor HNF4a in tumor cells, and plays the role of HNF4a promoting differentiation, thereby inhibiting the malignant phenotype of tumors. Although non-replicating mRNA can also mediate the transient expression of target genes in tumor cells, in order to induce tumor cells to differentiate into normal cells, HNF4a needs to be expressed stably and efficiently in tumor cells. Therefore, the present application first proposes to use self-replicating RNA technology to restore or overexpress HNF4a, which is decreased in the process of tumor occurrence, to inhibit tumor cell growth and achieve the effect of treating tumors. Therefore, the gene delivery system provided by the present application can induce malignant solid tumor cells to differentiate into normal mature cells, inhibit the proliferation of tumor cells, and induce tumor cell apoptosis. At the same time, after optimizing the self-replicating RNA vector used in the present application, a delivery system that can stably and efficiently express HNF4a in tumor cells is obtained.

[0036] In summary, the present application uses self-replicating RNA to express HNF4a, which not only has high expression amount and high expression efficiency, but also has longer maintenance time. For example, the preferred HNF4a-saRNA of the present application can maintain HNF4a in hepatocellular carcinoma cells for more than 7 days, and the expression amount and expression time are much higher than non-replicating HNF4a-mRNA. Compared with viral vectors, it has the advantages of good safety, strong tissue accessibility, and repeatable drug administration, and therapeutic effects have been observed in tumor-bearing animals and patients. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is a schematic diagram of the structure of self-replicating RNA expressing HNF4a with different lengths of polyA tail. GOI is HNF4a; the length of polyA tail is 30nt, 35nt, 40nt, 50nt, 60nt, 67nt, 75nt, 90nt, 100nt, and 110nt, respectively.

[0038] Figure 2 is a western blot for detecting the expression level of HNF4a protein after different treatments, to observe the effect of polyA length on the overexpression of HNF4a in hepatoma cells mediated by self-replicating RNA.

[0039] Figure 3 is a western blot for detecting the expression level of HNF4a protein after different treatments, to observe the effect of polyA length on the overexpression of HNF4a in hepatoma cells mediated by self-replicating RNA.

[0040] Figure 4 is a western blot for detecting the expression level of HNF4a protein after different treatments, to observe the effect of polyA length on the overexpression of HNF4a in hepatoma cells mediated by self-replicating RNA.

[0041] Figure 5 is a CCK8 assay for detecting the proliferation of Huh-7 cells after different treatments.

[0042] Figure 6 is a CCK8 assay for detecting the proliferation of Huh-7 cells after different treatments.

[0043] Figure 7 is a RT-PCR for detecting the expression level of liver function related genes after different treatments.

[0044] Figure 8 is a RT-PCR for detecting the expression level of tumor cell stemness related genes after different treatments.

[0045] Figure 9 is a PAS staining for detecting the glycogen storage in Huh-7 cells after different treatments.

[0046] Figure 10 is the acetylated low density lipoprotein (ac-LDL) uptake ability of Huh7 cells treated with HNF4a-saRNA and GFP-saRNA lipid nanoparticles for 3 days, using Dil-ac-LDL fluorescent substrate to detect the ac-LDL uptake ability of Huh7 cells, and image J software to count the ac-LDL positive area in Huh7 cells.

[0047] Figure 11 is the detection of the senescence-related β-galactosidase content in Huh7 cells treated with HNF4a-saRNA and GFP-saRNA lipid nanoparticles for 3 days, and image J software to count the β-galactosidase positive area in Huh7 cells.

[0048] Figure 12 is the detection of apoptosis in Huh7 cells treated with HNF4a-saRNA and GFP-saRNA lipid nanoparticles for 3 days by Annexin V / PI staining.

[0049] Figure 13 is the experimental flow chart of intratumoral injection of HNF4a-saRNA and HNF4a-mRNA lipid nanoparticles to treat Huh7 cell subcutaneous xenograft tumor.

[0050] Figure 14 is the tumor proliferation curve in Huh7 cell subcutaneous xenograft tumor model treated with HNF4a-saRNA and HNF4a-mRNA. GFP-saRNA is the self-replicating mRNA lipid nanoparticle control, and normal saline is the vehicle control.

[0051] Figure 15 is the gross appearance of Huh7 cell subcutaneous xenograft tumor treated with HNF4a-saRNA and HNF4a-mRNA.

[0052] Figure 16 is the tumor weight statistical chart (left) and tumor inhibition rate statistical chart (right) of Huh7 cell subcutaneous xenograft tumor model treated with HNF4a-saRNA and HNF4a-mRNA.

[0053] Figure 17 is the Western blot detection of the expression amount of HNF4a in the tumor tissue of Huh7 cell subcutaneous xenograft tumor model treated with HNF4a-saRNA.

[0054] Figure 18 is the detection of the expression changes of HNF4a and Ki67 in tumor tissue by immunohistochemistry (left), and the statistical chart of HNF4a and Ki67 positive staining area in tumor tissue (right).

[0055] Figure 19 is the experimental flow chart of tail vein injection of self-replicating HNF4a-saRNA and HNF4a-mRNA lipid nanoparticles to treat Huh7 cell liver orthotopic xenograft tumor.

[0056] Figure 20 is a graph of the in vivo fluorescence signal of mice before and after injection of HNF4a-saRNA and HNF4a-mRNA in a Huh7 cell liver orthotopic xenograft model. GFP-saRNA is a self-replicating RNA lipid nanoparticle control, and saline is a vehicle control.

[0057] Figure 21 is a graph of the in vivo fluorescence signal of mice at different time points during treatment of Huh7 cell liver orthotopic xenografts with HNF4a-saRNA and HNF4a-mRNA.

[0058] Figure 22 is a graph of the tumor weight (left) and tumor inhibition rate (right) in a Huh7 cell liver orthotopic xenograft experiment treated with HNF4a-saRNA and HNF4a-mRNA lipid nanoparticles.

[0059] Figure 23 is a graph of the tumor weight (left) and tumor inhibition rate (right) in a Huh7 cell liver orthotopic xenograft experiment treated with HNF4a-saRNA and HNF4a-mRNA lipid nanoparticles.

[0060] Figure 24 is a graph of the HE staining to detect the morphology of tumor tissue, immunohistochemical staining to detect the expression of Ki67 (left), and a graph of the Ki67 positive staining area in tumor tissue (right).

[0061] Figure 25 is a graph of the expression level of HNF4a protein detected by western blot in tumor tissue taken at different time points after treatment during treatment of Huh7 cell liver orthotopic xenografts with HNF4a-saRNA.

[0062] Figure 26 is a graph of the expression of HNF4a detected by immunohistochemical staining (left) and a graph of the HNF4a positive staining area in tumor tissue (right) in tumor tissue taken at different time points after treatment during treatment of Huh7 cell liver orthotopic xenografts with HNF4a-saRNA.

[0063] Figure 27 is a graph of the expression of liver function related genes in tumor tissue detected by quantitative PCR in tumor tissue taken at different time points after treatment during treatment of Huh7 cell liver orthotopic xenografts with HNF4a-saRNA.

[0064] Figure 28 is a graph of the expression level of HNF4a protein detected by western blot in cholangiocarcinoma HuCC-T1 cells treated with different concentrations of HNF4a-saRNA and HNF4a-mRNA lipid nanoparticles for 1 day and 3 days.

[0065] Figure 29 is a graph of the inhibition of the proliferation of cholangiocarcinoma HuCC-T1 cells by HNF4a-saRNA LNP.

[0066] Figure 30 shows the effect of HNF4a-mRNA LNP and HNF4a-saRNA LNP on the colony formation ability of cholangiocarcinoma cell HuCC-T1.

[0067] Figure 31 shows the expression level of HNF4a protein detected by western blot 1 day and 3 days after the treatment of intestinal cancer cell HCT116 with different concentrations of HNF4a-saRNA LNP.

[0068] Figure 32 shows the effect of HNF4a-mRNA LNP and HNF4a-saRNA LNP on the proliferation of intestinal cancer cell HCT116.

[0069] Figure 33 shows the effect of HNF4a-mRNA LNP and HNF4a-saRNA LNP on the colony formation ability of intestinal cancer cell HCT116.

[0070] Figure 34 shows the expression level of HNF4a protein detected by western blot 1 day and 3 days after the treatment of pancreatic cancer cell PANC1 with different concentrations of HNF4a-saRNA lipid nanoparticles.

[0071] Figure 35 shows the mRNA level of HNF4a (HNF4a) and self-replicating RNA vector (VEEV) detected by quantitative PCR in tumor tissues and other normal tissues taken at different time points during the treatment of Huh7 cells with HNF4a-saRNA.

[0072] Figure 36 shows the overall stability of liver cancer lesions after the treatment with HNF4a-saRNA LNP as shown by enhanced MRI. A. Liver cancer target lesions (dashed line box) before treatment for 13 weeks. B. Liver cancer target lesions were significantly larger than before treatment at the baseline stage. C. Liver cancer target lesions maintained stable in size after treatment for 10 weeks. D. Liver cancer target lesions maintained stable in size after treatment for 23 weeks, with partial areas showing liquefactive necrosis (arrow).

[0073] Figure 37 shows the gradual shrinkage of liver cancer lung metastasis lesions after the treatment with HNF4a-saRNA LNP as shown by chest CT. A. Liver cancer lung metastasis lesions (arrow) at the baseline stage. B. Liver cancer lung metastasis lesions were slightly larger than at the baseline after treatment for 4 weeks. C. Liver cancer lung metastasis lesions were slightly smaller than 4 weeks before after treatment for 8 weeks. D. Liver cancer lung metastasis lesions were further reduced in size, with the lesion diameter being significantly smaller than the baseline level after treatment for 18 weeks.

[0074] Figure 38. Enhanced MRI shows changes in the target lesion of liver cancer after HNF4a-saRNA LNP treatment. A. The target lesion of liver cancer (dotted line) is significantly enhanced in the arterial phase at baseline. B. The target lesion of liver cancer is larger than baseline and the arterial phase enhancement is weaker than baseline 4 weeks after treatment. C. The target lesion of liver cancer is further enlarged and the arterial phase enhancement is further weakened 9 weeks after treatment. D. The target lesion of liver cancer continues to enlarge and the arterial phase enhancement continues to weaken, with most of the region losing enhancement (arrow) 16 weeks after treatment. DETAILED DESCRIPTION

[0075] The specific embodiments of the present application will be described in detail below with examples. The advantages and features of the present application will become more apparent with the description. However, these examples are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements all fall within the protection scope of the present application.

[0076] Unless otherwise described, the embodiments of the present application will employ conventional techniques of molecular biology, cell biology and immunology, which are known to those skilled in the art. These techniques are fully described in, for example, Molecular Cloning: A Laboratory Manual, 4th Edition (2017); Short Protocols in Cell Biology: A Compendium of Methods from Cell Biology, 2007; Short Protocols in Immunology: A Compendium of Methods from Immunology, 2010. Alternatively, they can be performed according to the instructions provided by the reagent manufacturers.

[0077] Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as understood by those skilled in the art. In addition, any method and material similar or equivalent to those described can be applied in the present application. The preferred methods and materials described herein are only exemplary.

[0078] All data in this paper were analyzed and plotted using GraphPad Prism 8 software. All data error values were shown as mean ± standard error (±SEM). The comparison between two groups of data used t-test, and the statistical test was two-tailed, where p<0.05 was considered statistically significant, * represented p<0.05, ** represented p<0.01, and *** represented p<0.001.

[0079] Example 1: Preparation of self-replicating RNA and LNP encapsulation

[0080] 1. Preparation of linear mRNA and self-replicating RNA

[0081] Self-replicating RNA (saRNA) is based on an engineered alphavirus genome containing genes encoding non-structural proteins that are capable of RNA replication, with the structural protein sequences replaced by a gene of interest. The saRNA includes a 5' cap, non-coding region (5' UTR), four non-structural genes (NSP1-4), a 26S subgenomic promoter, a gene of interest, a 3' non-coding region (3' UTR), and a poly(A) tail. Linear RNAs are designed according to eukaryotic mRNA structure, containing a gene encoding a protein of interest, as well as the necessary cap structure, 5' UTR, 3' UTR, and poly(A). Non-replicating and self-replicating RNAs are prepared identically, starting from linear templates. To prepare linear templates for RNA transcription, plasmid DNA is restriction digested with BspQI enzyme (New England Biolabs, R0712L) and purified using the QIAquick® 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) are mixed together in a cocktail and the mixture is incubated at 37°C for 2 hours for in vitro transcription from the template. After the transcription reaction is complete, DNase I (1 U / μg DNA) is added and incubated at 37°C for 15 minutes to remove the DNA template, and then the transcribed RNA is purified using the LiCl precipitation method.

[0082] 2. LNP-encapsulated RNA

[0083] Lipid nanoparticles are prepared by mixing the ethanol phase and the aqueous phase in a microfluidic device (INano TM ​LNP formulations were prepared by rapid mixing in a QG-Plus® L system, Micro & Nano). The aqueous phase was 50 mM citrate buffer (pH 6.0) containing purified saRNA. The ethanol phase contained proprietary ionizable lipids, 1,2-distearoyl-sn-glycero- phosphocholine (DSPC) (Avanti, 850365P), cholesterol (Sigma-Aldrich, C8667), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (NOF, GM020). mRNA-LNPs were assembled with a molar ratio of 9.4:42.5:1.8:46.3 (DSPC:cholesterol:DMG-PEG 2000:LP-1), N / P = 6. The LNP formulations were subjected to particle size, PDI, RNA concentration, and encapsulation efficiency tests.

[0084] Example 2: Optimization of the length of polyA tail of self-replicating RNA

[0085] LNP-encapsulated self-replicating RNAs expressing HNF4a with different lengths of polyA tail were prepared according to the method of Example 1 and as shown in Figure 1. Liver cancer cells Huh7 were seeded at 3x10 5 Cells were seeded at 6-well plates and cultured overnight, and then different polyA length of HNF4a-saRNA LNPs diluted with Opti-MEM were added, respectively. After 6 hours of culture, 1 mL of DMEM medium containing 20% FBS was added. The transfected cells were collected with RIPA lysis buffer for Western blot detection of intracellular HNF4a protein levels (Figure 2) after 3 or 7 days. The results showed that HNF4a-saRNAs with polyA tail lengths of 35-100 nt can mediate overexpression of HNF4a in Huh-7 cells. Tumor cells transfected with self-replicating RNAs with polyA tail lengths of 40-90 nt had higher HNF4a expression, and self-replicating RNAs with polyA tail lengths of 60 nt-75 nt had better HNF4a expression levels and longer expression times. Self-replicating RNA with a polyA tail of 67 nt mediated the most efficient expression. The self-replicating RNA sequence with the best experimental results after optimization of the polyA tail length is shown as SEQ ID NO: 2, and the HNF4a-saRNA sequence is shown as SEQ ID NO: 3 (the sequence is used in subsequent examples).

[0086] Example 3: Comparison of the ability of non-replicating HNF4a-mRNA, HNF4a-saRNA, and adenovirus AdHNF4a to mediate overexpression of HNF4a in tumor cells

[0087] Liver cancer cells Huh7 were seeded at 3x10 5Cells were seeded into 6-well plates and cultured overnight, washed with 1 ml PBS, and then 1 ml Opti-MEM diluted HNF4a-saRNA LNP, control GFP-saRNA LNP and non-replicating HNF4a-mRNA LNP (final concentration of 2 ng RNA / 200 cells) were added respectively. After 6 hours of incubation, 1 mL DMEM medium containing 20% FBS was added. The transfected cells were collected with RIPA lysis buffer at 1, 3, 5 or 7 days for Western blot to detect the protein level of HNF4a in cells (Figure 3). Compared with the effect of adenovirus-mediated overexpression of HNF4a on Huh7 cells to which adenovirus expressing HNF4a (AdHNF4a) and its control virus (AdGFP) were added (Figure 3). The results show that both HNF4a-saRNA and HNF4a-mRNA can up-regulate HNF4a in hepatoma cells, but the expression amount of HNF4a-mRNA is lower and the expression time is shorter; while the expression amount of HNF4a-saRNA is higher and the maintenance time is longer, and its expression efficiency is not lower than that of adenovirus.

[0088] Example 4: HNF4a-saRNA treats hepatoma by inducing hepatoma cells to differentiate into hepatocytes

[0089] 1. Different concentrations of HNF4a-saRNA LNP up-regulate the expression of HNF4a in hepatoma Huh-7 cells

[0090] Hepatoma cells Huh7 were seeded at 3x10 5 Cells were seeded into 6-well plates and cultured overnight, washed with 1 ml PBS, and then 1 ml Opti-MEM diluted HNF4a-saRNA LNP, control GFP-saRNA LNP (final concentration of 0.5, 1, 2 ng RNA / 200 cells) were added respectively. After 6 hours of incubation, 1 mL DMEM medium containing 20% FBS was added. The transfected cells were collected with RIPA lysis buffer at 1, 3, 5 or 7 days for Western blot to detect the protein level of HNF4a in cells (Figure 4), and the results show that HNF4a-saRNA LNP up-regulates the expression of HNF4a, while control GFP-saRNA LNP does not affect the expression of HNF4a in hepatoma cells.

[0091] 2. HNF4a-saRNA LNP inhibits the growth of hepatoma cells

[0092] Hepatoma cells Huh7 were used to verify the inhibition of the gene delivery system on the malignant phenotype such as tumor cell proliferation and clonal formation. Huh7 cells were seeded at 3x10 3Cells were seeded at different densities in 96-well plates and cultured overnight. Afterward, the supernatant was removed, and the cells were washed with 100 μl of PBS. Different concentrations of HNF4α-saRNA LNP diluted with Opti-MEM and control GFP-saRNA LNP were added to the cells. After 6 hours, an equal volume of 20% FBS was added to the culture medium to achieve a 10% FBS concentration. Cell proliferation was measured daily using a cell counting kit-8 (CCK-8, Dojindo) to determine the effect of RNA on tumor cell proliferation. The results showed that HNF4α-saRNA LNP significantly inhibited the proliferation of liver cancer cells compared to control cells (Figure 5).

[0093] Next, the inventors conducted experiments on the effect of the gene delivery system on the clonogenic ability of tumor cells. They used 3 × 10⁻⁶ gene delivery systems to deliver Huh7 liver cancer cells. 3 Cells were seeded at a density of 2 ng / well in 96-well plates and cultured overnight. Afterward, HNF4α-saRNA LNP and control GFP-saRNA LNP (2 ng / 200 cells) were added for transfection. Twenty-four hours later, cells were trypsinized and transferred to 60 mm culture dishes. The culture medium was changed every 3 days, and the colony formation of tumor cells was observed under a microscope. After colony formation, cells were washed twice with PBS, stained with crystal violet for 20 min, washed with PBS again, air-dried, and photographed. The number of colonies was counted using ImageJ to determine the effect of RNA on the colony formation ability of tumor cells. The results are shown in Figure 6. HNF4α-saRNA significantly inhibited the colony formation ability of liver cancer cells.

[0094] 3. HNF4α-saRNA LNP induces differentiation of hepatocellular carcinoma cells into mature hepatocytes.

[0095] ①HNF4α-saRNA restores liver function gene expression in liver cancer cells

[0096] Huh-7 liver cancer cells were treated with 3x10 5 Cells were seeded into 6-well plates and cultured overnight. Cells were washed with PBS, and different concentrations of HNF4α-saRNA LNP and GFP-saRNA LNP diluted with Opti-MEM were added. After 6 hours of culture, 1 mL of DMEM medium containing 20% ​​FBS was added. Cells were harvested 1, 3, or 7 days after transfection to extract RNA. Real-time quantitative PCR was used to detect the expression of liver function-related genes regulated by HNF4α and the expression of tumor stemness-related genes (Figures 7 and 8). The results showed that HNF4α-saRNA upregulated the expression of liver function-related genes and downregulated the expression of tumor stemness-related genes.

[0097] ②HNF4α-saRNA promotes glycogen storage and low-density lipoprotein uptake in liver cancer cells.

[0098] Glycogen storage and low density lipoprotein uptake ability are important functions of normal hepatocytes, the inventors further confirmed whether the liver cancer cells were differentiated into mature hepatocytes by examining these functions.

[0099] Liver cancer cells Huh7 were seeded at 3x10 4 Cells were seeded at 24-well plate at 200 cells / well, 1 ng / 200 cells of HNF4a-saRNA LNP and corresponding control were added. Glycogen storage ability of cells was verified by PAS reaction kit (Bi Yun Tian) 3 days after LNP delivery. 70% ethanol was added to fix cells for 10 minutes, then the periodic acid solution was removed and equilibrated to room temperature, 100 μl of periodic acid solution was added to each sample, reacted for 10 minutes in a wet box in the dark, then the periodic acid solution was removed, soaked in PBS and placed in a shaker for 5 minutes. 100 μl of Schiff reagent was added to each sample, placed in a wet box, and stained in a 37°C oven for 1 hour in the dark. The staining solution was removed, soaked in PBS and placed in a shaker for 5 minutes. Finally, 100 μl of hematoxylin staining solution was added to each sample and stained for 30 seconds. The staining solution was removed, rinsed with PBS twice, and the background was washed off. Photographed under a microscope and counted the stained cells (Figure 9). The results showed that HNF4a-saRNA promoted the glycogen storage ability of liver cancer cells.

[0100] Liver cancer cells Huh7 were seeded at 3x10 4 Cells were seeded at 24-well plate at 200 cells / well, 1 ng / 200 cells of HNF4a-saRNA LNP and corresponding control were added. 3 days after LNP delivery, the cell culture medium was removed, the cells were washed with PBS, then 200 ul of Dil ac-LDL (Invitrogen) was added to each well after diluting with DMEM at a ratio of 1:100. After 3 hours, the DMEM containing Dil ac-LDL was removed, the cells were washed with PBS, and 4% paraformaldehyde was added to fix for 15 minutes. After DAPI staining, the slide was mounted, photographed using a confocal fluorescence microscope, and the fluorescence signal was counted (Figure 10). The results showed that HNF4a-saRNA promoted the low density lipoprotein uptake ability of liver cancer cells.

[0101] ③HNF4a-saRNA promotes liver cancer cell senescence

[0102] Liver cancer cells Huh7 were seeded at 3x10 4Cells were seeded in 24-well plates, and 1 ng / 200 cells of HNF4α-saRNA LNP and corresponding controls were added. Three days after LNP delivery, HCC cell senescence was assessed using a Senescence β-Galactosidase staining kit (Beyotime, China). Cells were fixed with 4% formaldehyde for 15 minutes and then incubated overnight at 37°C with fresh senescence-associated β-galactosidase staining solution. Senescent cells were observed and captured under a microscope. The number of senescent cells was counted using image analysis software (Image-Pro Plus 6.0, Media Cybernetics) (Figure 11). The results showed a significant increase in senescent hepatocellular carcinoma cells after HNF4α-saRNA treatment.

[0103] ④ HNF4α-saRNA induces apoptosis in liver cancer cells

[0104] Huh7 liver cancer cells were divided into 3x10 5 Cells were seeded in 24-well plates, and 1 ng / 200 cells of HNF4α-saRNA LNP and corresponding controls were added. Three days after LNP delivery, apoptotic cells were detected using the APC Annexin V / PI apoptosis kit (Biolegend, China) according to the manufacturer's instructions. Cells were digested with 0.25% trypsin and washed twice with PBS. Cells were then transferred to tubes and resuspended in 100 μl of binding buffer. 400 μl of binding buffer containing 5 μl APC Annexin V and 5 μl proiodine was added to each tube, and the cells were incubated in the dark at 25°C for 15 min. Cells were then analyzed using flow cytometry (Attune NxT, Invitrogen). Flow cytometry data were analyzed using FlowJo V10 software (Figure 12). The results showed a significant increase in apoptosis in HNF4α-saRNA-treated liver cancer cells.

[0105] 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.

[0106] 3. HNF4α-saRNA LNP inhibits the growth of subcutaneous tumors in mice.

[0107] Five-week-old male nude mice (BALB / c immunodeficient strain) were purchased from Shanghai BK / KY Biotechnology Co., Ltd., and were housed under specific pathogen-free environmental conditions using a 12-hour on / off light cycle. 1×10 6Huh-7 cells were subcutaneously injected into the right axilla of male nude mice. The tumor size was measured in two dimensions using calipers, and the volume was calculated using the following formula: Volume = Length × (Width) 2 ×1 / 2. When the average tumor volume reaches approximately 100 mm... 3 Mice were randomly divided into four groups (n=7 per group). 5 μg of HNF4α-saRNA LNP, GFP-saRNA LNP, HNF4α-mRNA LNP, or physiological saline (solvent control) was injected into the tumor of each mouse, with a final volume of 75 μl. Tumor volume was measured daily after injection, and tumor growth curves were plotted (Figures 13 and 14). Five days after RNA injection, mice were sacrificed, and tumors were excised and weighed (Figures 15 and 16), while the tumor inhibition rate was calculated (Figure 16). Some tumor tissues were paraffin-embedded, and HNF4α expression was detected by Western blotting and immunohistochemistry (Figures 17 and 18). Ki67 staining was used to assess the proliferation status of tumor cells (Figure 17). The results showed that intratumoral injection of HNF4α-saRNA slowed tumor growth, with a tumor inhibition rate exceeding 50% (Figure 16), while HNF4α-mRNA had no significant inhibitory effect on tumor growth. Western blot and immunohistochemical results showed that HNF4α-saRNA significantly increased the protein expression of HNF4α in tumor tissues, while significantly decreasing the expression of Ki67, a tumor cell proliferation marker. These results indicate that HNF4α-saRNA can effectively induce the expression of HNF4α in cancer cells, thereby inhibiting the growth of mouse hepatocellular carcinoma implants.

[0108] 4. HNF4α-saRNA LNP inhibits the growth of hepatocellular carcinoma cells implanted in situ in the liver.

[0109] The Huh7 human hepatocellular carcinoma cell line, which stably expresses the luciferase gene, was inoculated into the axilla of male nude mice. After tumor formation, the tumor was removed and cut to a depth of 1 mm. 3Small pieces of tumor were transplanted under the liver capsule of male nude bab / c mice. The growth of the tumor in the mice was monitored using the IVIS spectrum optical imaging system. Three days after transplantation, the mice were evenly divided into four groups according to the strength of the fluorescence signal, and were injected with HNF4a-saRNA LNP, GFP-saRNA LNP, HNF4a-mRNA LNP, and normal saline (vehicle control) at a dose of 2 mg / kg of body weight via the tail vein (FIG. 19). The growth of the tumor in the mice was monitored according to bioluminescence, and the efficacy of the HNF4a-saRNA was evaluated (FIG. 20). The tumor growth curve was plotted (FIG. 21). The mice were sacrificed at the end of the experiment, and the tumors were excised and weighed to calculate the tumor inhibition rate (FIGS. 22 and 23). The tumor tissue was paraffin-embedded, and HE staining was used to evaluate the morphology of the tumor tissue, and Ki67 staining was used to evaluate the proliferation state of the tumor (FIG. 24). The results showed that the growth of the tumor was reduced after the injection of HNF4a-saRNA LNP, and the tumor inhibition rate was more than 40% compared with the control tumor, while HNF4a-mRNA LNP had no significant effect on the inhibition of the tumor. Western blot and immunohistochemical detection of HNF4a expression were performed on the tumor tissue collected at different time points after the injection of LNP (FIGS. 25 and 26), and Ki67 staining was used to evaluate the proliferation state of the tumor (FIG. 26). Quantitative PCR was used to detect liver cell differentiation-related indicators (FIG. 27). The results showed that HNF4a-saRNA significantly upregulated the expression of HNF4a and liver function-related genes in the tumor tissue. These results further indicated that HNF4a-saRNA can induce the differentiation of hepatocarcinoma cells into normal liver cells, thereby inhibiting the growth of hepatocarcinoma.

[0110] Example 5: HNF4a-saRNA inhibits the proliferation of cholangiocarcinoma cells

[0111] 1. Different concentrations of HNF4a-saRNA LNP upregulate the expression of HNF4a in cholangiocarcinoma HuCC-T1 cells

[0112] Cholangiocarcinoma HuCC-T1 cells were seeded in N6 plates at a density of 40%-50% and cultured overnight. After washing with 1 ml of PBS, HNF4a-saRNA LNP was added in series with Opti-MEM (final concentration of 1, 2 ng RNA / 200 cells). After 6 hours of culture, 1 mL of DMEM medium containing 20% FBS was added. The transfected cells were collected with RIPA lysis buffer at 1 and 3 days for Western blot detection of the intracellular HNF4a protein level (FIG. 28). The results showed that HNF4a-saRNA LNP upregulated the expression of HNF4a in cholangiocarcinoma cells.

[0113] 2. HNF4a-saRNA LNP inhibits the growth of cholangiocarcinoma cells

[0114] HuCC-T1 bile duct cancer cells at 3×10 3 Cells were seeded at a density of 1 / 2 well in 96-well plates and cultured overnight. Afterward, the supernatant was aspirated, and the cells were washed with 100 μl of PBS. Different concentrations of HNF4α-saRNA LNP diluted with Opti-MEM (final concentrations of 1 and 2 ng RNA / 200 cells) were added to the cells. Six hours later, an equal volume of 20% FBS was added to the culture medium for each cell type, bringing 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 HNF4α-saRNA LNP significantly inhibited the proliferation of cholangiocarcinoma cells compared to control cells (Figure 29).

[0115] 3. HNF4α-saRNA LNP inhibits clonal formation of cholangiocarcinoma cells.

[0116] HuCC-T1 bile duct cancer cells at 3×10 3 Cells were seeded at a density of 1 / 2 well in 96-well plates and cultured overnight. Transfection was then performed using HNF4α-saRNA LNP and HNF4α-mRNA LNP (2 ng / 200 μL). After 24 hours, 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. Once colonies formed, 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 formation ability of tumor cells (Figure 30). The results showed that HNF4α-saRNA significantly inhibited the colony formation ability of cholangiocarcinoma cells, while HNF4α-mRNA did not affect tumor cell colony formation.

[0117] Example 6: HNF4α-saRNA inhibits the proliferation of colon cancer cells

[0118] 1. Different concentrations of HNF4α-saRNA LNP upregulate HNF4α expression in HCT-116 colorectal cancer cells.

[0119] Intestinal cancer cell HCT-116 was seeded in N6 well plate at 40%-50% density and cultured overnight, washed with 1ml PBS, then added HNF4a-saRNA LNP diluted with Opti-MEM (final concentration 1, 2ng RNA / 200 cells) respectively. After 6 hours of culture, 1ml DMEM medium containing 20% FBS was added. The transfected cells were collected with RIPA lysis buffer for Western blot to detect the protein level of HNF4a in cells (Figure 31), and the results showed that HNF4a-saRNA LNP up-regulated the expression of HNF4a in intestinal cancer cells.

[0120] 2. HNF4a-saRNA LNP inhibits the growth of cholangiocarcinoma cells

[0121] Intestinal cancer cell HCT-116 was seeded in N6 well plate at 40%-50% density and cultured overnight, washed with 1ml PBS, then added HNF4a-saRNA LNP diluted with Opti-MEM (final concentration 1, 2ng RNA / 200 cells) respectively. After 6 hours of culture, 1ml DMEM medium containing 20% FBS was added. The transfected cells were collected with RIPA lysis buffer for Western blot to detect the protein level of HNF4a in cells (Figure 31), and the results showed that HNF4a-saRNA LNP up-regulated the expression of HNF4a in intestinal cancer cells. 3 Cells were seeded in 96-well plates at a density of 200 cells / well and cultured overnight, then the culture supernatant was removed and the cells were washed with 100ul PBS. Different concentrations of HNF4a-saRNA LNP and HNF4a-mRNA LNP diluted with Opti-MEM (final concentration 2ng RNA / 200 cells) were added to the cells, and after 6 hours, an equal volume of 20% FBS was added to the culture medium of the corresponding cells to make the FBS concentration in the culture medium 10%. Cell counting kit-8 (CCK-8, Dojindo) was used to determine cell proliferation every day to determine the effect of RNA on the proliferation ability of tumor cells. The results showed that HNF4a-saRNA LNP significantly inhibited the proliferation of intestinal cancer cells compared with control cells (Figure 32), while HNF4a-mRNA LNP had no obvious effect on the proliferation of tumor cells.

[0122] 3. HNF4a-saRNA LNP inhibits the formation of intestinal cancer cell clones

[0123] Intestinal cancer cell HCT-116 was seeded in N6 well plate at 40%-50% density and cultured overnight, washed with 1ml PBS, then added HNF4a-saRNA LNP diluted with Opti-MEM (final concentration 1, 2ng RNA / 200 cells) respectively. After 6 hours of culture, 1ml DMEM medium containing 20% FBS was added. The transfected cells were collected with RIPA lysis buffer for Western blot to detect the protein level of HNF4a in cells (Figure 31), and the results showed that HNF4a-saRNA LNP up-regulated the expression of HNF4a in intestinal cancer cells. 3 Cells were seeded in 96-well plates at a density of 200 cells / well and cultured overnight, then the culture supernatant was removed and the cells were washed with 100ul PBS. Different concentrations of HNF4a-saRNA LNP and HNF4a-mRNA LNP diluted with Opti-MEM (final concentration 2ng RNA / 200 cells) were added to the cells, and after 6 hours, an equal volume of 20% FBS was added to the culture medium of the corresponding cells to make the FBS concentration in the culture medium 10%. Cell counting kit-8 (CCK-8, Dojindo) was used to determine cell proliferation every day to determine the effect of RNA on the proliferation ability of tumor cells. The results showed that HNF4a-saRNA LNP significantly inhibited the proliferation of intestinal cancer cells compared with control cells (Figure 32), while HNF4a-mRNA LNP had no obvious effect on the proliferation of tumor cells.

[0124] Example 7: HNF4a-saRNA up-regulate the expression of HNF4a in pancreatic cancer cells

[0125] Human pancreatic cancer cells PANC1 were seeded into 6-well plates at 40-50% confluency and cultured overnight, washed with 1 ml PBS, and then added with HNF4a-saRNA LNP diluted in Opti-MEM (final concentration of 1, 2 ng RNA / 200 cells) respectively. After 6 hours of incubation, 1 mL DMEM medium containing 20% FBS was added. The transfected cells were collected with RIPA lysis buffer at 1 and 3 days for Western blot to detect the protein level of HNF4a in the cells (Figure 34), and the results showed that HNF4a-saRNA LNP up-regulated the expression of HNF4a in pancreatic cancer cells.

[0126] Example 8: HNF4a-saRNA LNP specifically up-regulate the expression of HNF4a in tumor tissues in vivo

[0127] Human hepatocarcinoma cell line Huh7 stably expressing luciferase gene was inoculated into the armpit of male nude mice, and after the tumor was formed, the tumor mass was taken out and cut into 1 mm 3 small pieces, which were transplanted under the liver capsule of male Bab / c nude mice. The growth of the tumor in the mice was monitored using the IVIS spectrum optical imaging system. When the tumor grew to an appropriate size, HNF4a-saRNA LNP was injected into the tail vein of the mice at a dose of 2 mg / kg body weight, and the organs and tumor tissues of the mice were taken out at 3 and 5 days after injection, respectively. Quantitative PCR was used to detect the expression and distribution of alphavirus genome sequence (VEEV) and LNP-mediated human HNF4a gene sequence in the organs and tumor (Figure 35). The results showed that 3 days after the injection of HNF4a-saRNA LNP into the tail vein, VEEV and human HNF4a RNA were detected in the heart, spleen, and kidney in trace amounts, and almost no VEEV and HNF4a gene sequence was detected in the liver, lung, and muscle tissues, but high levels of VEEV and HNF4a RNA were detected in the tumor tissue. 5 days after the injection of HNF4a-saRNA LNP into the tail vein, no VEEV and human HNF4a RNA was detected in the organs except for trace amounts of expression in the heart, but VEEV and HNF4a gene sequence was still detected in the tumor tissue (Figure 35). This indicated that self-replicating RNA can mediate the expression of HNF4a in tumor cells in vivo.

[0128] Example 9: HNF4a-saRNA LNP treatment of patients with advanced liver cancer

[0129] The CDMO company was commissioned to produce HNF4a-saRNA LNP formulations in accordance with pharmaceutical standards and in compliance with GMP standards. The CRO company was commissioned to complete a single-dose toxicity test of the HNF4a-saRNA LNP formulation. The specific process and results are as follows: the rats were injected once in the tail vein, and the observation period was 14 days. The negative control group (0.9% sodium chloride injection), the carrier control group (LNP solution), and the test product group (150 μg per rat) were set up. All animals survived to the scheduled autopsy day, and no gross autopsy abnormalities or histopathological changes related to the test product were observed at the end of the observation period.

[0130] After completing the acute toxicity test, a researcher-initiated clinical trial was conducted using hepatic arterial cannulation for administration. Dose escalation tests of 25 μg, 50 μg, and 100 μg (3 cases each) were completed. None of the 9 patients experienced dose-limiting toxicity or treatment-related adverse events of grade 3 or above or serious adverse events. Four patients were treated for more than 5 months, and there was no obvious progression of liver cancer lesions. In one of these patients, lung metastases were significantly reduced (Figure 36), and in another patient, focal necrosis of liver cancer lesions was observed (Figure 37). In 3 patients, target lesions showed obvious necrosis, and the enhancement was significantly reduced (Figure 38). Preliminary results show that the HNF4a-saRNA LNP formulation has good safety and significantly inhibits tumor growth in patients with advanced liver cancer.

[0131] The preferred embodiments of the present application have been specifically described above, but the present application is not limited to the described embodiments, and those skilled in the art can make various equivalent modifications or replacements without departing from the spirit of the present application. These equivalent modifications or replacements are all included in the scope defined by the claims of the present application.

Claims

1. A gene delivery system, which is composed of a nucleic acid vector and a delivery vehicle; the nucleic acid vector is a self-replicating RNA carrying HNF4α, the gene delivery system delivers HNF4α into tumor cells, induces tumor cells to differentiate into normal mature cells, inhibits proliferation of tumor cells or / and induces apoptosis of tumor cells.

2. The gene delivery system of claim 1, wherein, The self-replicating RNA carries a poly-A tail of 35-100 nt.

3. The gene delivery system of claim 1, wherein, The self-replicating RNA carries a poly-A tail of 60-75 nt.

4. The gene delivery system of claim 1, wherein, The self-replicating RNA carries a poly-A tail of 67 nt.

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 gene delivery system according to any one of claims 1 to 5, wherein, The delivery vehicle is selected from the following one or more in combination: liposome, viral replicon particle, lipid-based nanoparticle, polymeric nanoparticle, physiological buffer, microsphere, immunostimulatory complex, conjugate of biologically active ligand.

7. The gene delivery system of claim 6, wherein, The delivery vehicle is a lipid-based nanoparticle, wherein the lipids include: 1, 2-distearoyl-sn-glycero-3-phosphocholine or 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, molar ratio 5%-20%; cholesterol, molar ratio 30%-55%; dimyristoylglycerol-polyethylene glycol 2000 or pegylated lipid containing polyethylene glycol fragment, molar ratio 0.5%-3%; ionizable lipid, molar ratio 30%-60%.

8. The gene delivery system of claim 7, wherein, The N: P ratio in the lipid-based nanoparticle ranges from 5: 1 to 10: 1, and the particle size of the nanoparticle is 40-300 nm. 9.Use of the gene delivery system according to any one of claims 1 to 8 in the preparation of a medicament for treating malignant solid tumors of epithelial origin.

10. Use of the gene delivery system according to claim 9 for the manufacture of a medicament for the treatment of a malignant solid tumor, characterized in that, The malignant solid tumor is liver cancer, pancreatic cancer, intestinal cancer, kidney cancer, gastric cancer, lung cancer. 11.A pharmaceutical composition comprising the gene delivery system according to any one of claims 1 to 7.

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