[go: up one dir, main page]

WO2025237287A1 - Arn auto-réplicatif exprimant un facteur de transcription lié à la différenciation et son utilisation dans la préparation d'un médicament thérapeutique antitumoral - Google Patents

Arn auto-réplicatif exprimant un facteur de transcription lié à la différenciation et son utilisation dans la préparation d'un médicament thérapeutique antitumoral

Info

Publication number
WO2025237287A1
WO2025237287A1 PCT/CN2025/094572 CN2025094572W WO2025237287A1 WO 2025237287 A1 WO2025237287 A1 WO 2025237287A1 CN 2025094572 W CN2025094572 W CN 2025094572W WO 2025237287 A1 WO2025237287 A1 WO 2025237287A1
Authority
WO
WIPO (PCT)
Prior art keywords
self
hnf4α
cells
sarna
tumor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/094572
Other languages
English (en)
Chinese (zh)
Inventor
徐涵江
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Cell Diff Medicine Ltd
Original Assignee
Shanghai Cell Diff Medicine Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Cell Diff Medicine Ltd filed Critical Shanghai Cell Diff Medicine Ltd
Publication of WO2025237287A1 publication Critical patent/WO2025237287A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • This invention belongs to the field of pharmaceutical technology, specifically relating to self-replicating RNA expressing differentiation-related transcription factors and its application in the preparation of tumor therapeutic drugs. It is a technical means to induce tumor cells to differentiate into mature cells. By using messenger ribonucleic acid to regulate the expression of important differentiation-related transcription factors in tumor cells, it inhibits the malignant phenotype of malignant solid tumor cells and achieves the effect of treating malignant solid tumors, thereby being applied to the preparation method and application of solid tumor drugs.
  • Tumor differentiation-inducing therapy promotes the differentiation of tumor cells into mature normal cells through various inducing factors, restoring their normal phenotype and function and inhibiting the proliferation of malignant tumor cells.
  • This strategy breaks with conventional approaches to tumor treatment.
  • a classic example is the use of all-trans retinoic acid (ATA) for differentiation therapy in acute promyelocytic leukemia, which has achieved good clinical efficacy and is widely used.
  • ATA all-trans retinoic acid
  • differentiation-inducing therapy for malignant solid tumors remains a challenge in current tumor treatment. Recent studies have found that transcription factors related to organ differentiation, development, and functional maintenance can induce related tumor cells to differentiate into normal cells.
  • Upregulating these differentiation-related transcription factors can inhibit tumor cell growth without significantly affecting the function of normal tissue cells, opening up new directions for tumor differentiation-inducing therapy, especially for the treatment of malignant solid tumors. Therefore, for tumors in different tissues, specific targeted regulation of proteins, molecules, and genes closely related to tumor cell differentiation is the core issue of tumor differentiation-inducing therapy. Utilizing genetic engineering techniques to target and regulate the expression of important differentiation function genes and induce tumor cells to differentiate into mature cell phenotypes may fundamentally reverse the progression of malignant tumors.
  • HNF hepatocyte nuclear factor
  • C/EBP CCAAT/enhancer-binding protein
  • HNF4 ⁇ also regulates the development of kidney and intestinal tissues and regulates insulin production.
  • Studies on liver cancer have found that the dedifferentiation state of liver cancer is accompanied by the downregulation of a large number of hepatocyte nuclear factor expressions, among which the downregulation of HNF4 ⁇ expression is a crucial step in the development of liver cancer.
  • Previous studies have shown that HNF4 ⁇ expression is decreased in various epithelial-derived tumors such as liver cancer (including hepatocellular carcinoma and intrahepatic cholangiocarcinoma), pancreatic cancer, colorectal cancer, and kidney cancer.
  • HNF4 ⁇ is a potential target for tumor therapy (Differentiation therapy of hepatocellular carcinoma in mice with recombinant adenovirus carrying hepatocyte nuclear factor-4alpha gene. Hepatology. 2008 Nov; 48(5):1528-39).
  • HNF1 ⁇ is another important hepatocyte nuclear factor that can bind to the cis-acting elements of at least 200 liver target genes. These target genes are involved in many important liver functions, such as glycogen synthesis and storage, gluconeogenesis, lipid metabolism, serum protein synthesis, and detoxification. Previous studies have shown that HNF1 ⁇ expression is decreased in hepatocellular carcinoma.
  • Upregulation of HNF1 ⁇ in hepatocellular carcinoma cells can promote the expression of liver function genes in hepatocellular carcinoma cells, arrest the cell cycle of liver tumor cells in the G2/M phase, thereby inhibiting tumor cell proliferation and significantly inhibiting tumor growth in vivo (Recombinant adenovirus carrying the hepatocyte nuclear factor-1alpha gene inhibits hepatocellular carcinoma xenograft growth in mice. Hepatology, 2011, 54(6):2036-2047.).
  • Recent studies have found that combined overexpression of a group of hepatocyte nuclear factors, including HNF4 ⁇ , HNF1 ⁇ , and FOXA3 (also known as HNF3 ⁇ ) can successfully transform liver tumor cells into mature hepatocytes in vitro and in vivo.
  • hepatocyte nuclear factors such as HNF4 ⁇ , HNF1 ⁇ , and FOXA3 are an ideal strategy for inducing differentiation therapy for liver cancer. (Conversion of hepatoma cells to hepatocyte-like cells by defined hepatocyte nuclear factors. Cell Res. 2019 Feb; 29(2):124-135.)
  • Pancreas-associated transcription factor 1a plays a crucial role in mammalian pancreatic development and is involved in maintaining the expression of exocrine pancreatic-specific genes, including elastase 1 and amylase.
  • Previous studies have shown that PTF1A expression is absent in ductal pancreatic cancer. Maintaining PTF1A expression can completely block the formation of pancreatic cancer cells, and restoring PTF1A expression can induce early-stage cancer cells to transform into normal pancreatic cells and inhibit the growth of late-stage pancreatic cancer cells. It is a potential target for differentiation-induced therapy of pancreatic cancer. (Prevention and Reversion of Pancreatic Tumorigenesis through a Differentiation-Based Mechanism. Dev Cell. 2019 Sep 23; 50(6):744-754.e4.)
  • Neuronal differentiation 1 (NEUROD1), neurogenin 2 (NEUROG2), and ASCL1 are all transcription factors related to neural differentiation, participating in the induction of neural differentiation during early brain development. They play an important role in early brain development by inducing neural stem cells to differentiate into neurons. Previous studies have shown that NEUROD1 can induce astrocytes in the brain to differentiate into neurons in various brain disease models such as Alzheimer's disease, Huntington's disease, stroke, and epilepsy. Overexpression of NEUROD1, NEUROG2, and ASCL1 in glioma cells can transform proliferative glioma cells into non-proliferative neurons, making them important targets for inducing differentiation therapy for gliomas. (Transcription factor-based gene therapy to treat glioblastoma through direct neuronal conversion. Cancer Biol Med. 2021 Mar 23; 18(3):860-74.)
  • adenovirus vectors have shortcomings in safety and tissue targeting, and their preparation cost is high, limiting their potential for practical application in tumor treatment.
  • mRNA technology is a revolutionary gene delivery technology that has emerged in recent years. This technology involves synthesizing mRNA molecules with specific sequences through in vitro transcription, encapsulating them in lipid nanoparticles (LNPs), and transporting the mRNA into human cells. The cells then rely on their own translation system to translate the mRNA into the target protein. Compared to existing viral vector-based delivery systems, this technology offers superior efficiency and safety. Furthermore, the selection and modification of the lipid nanoparticles can enhance the tissue specificity of the delivery system.
  • Currently known potential applications of mRNA technology primarily focus on vaccine development for infectious diseases, therapeutic tumor vaccines, protein replacement therapies for protein deficiencies caused by gene defects, and gene editing for treating hereditary diseases or modifying immune cells. However, because mRNA can only mediate transient high expression of the target gene in cells, it has significant limitations in expression duration and efficiency, failing to meet the need for long-term stable expression of target proteins in rapidly proliferating tumor cells.
  • RNA Self-amplifying RNA
  • saRNA self-replicating RNA
  • srRNA self-replicating RNA
  • RNA vectors are mainly modified from Venezuelan equine encephalitis virus (VEEV), Sindbis alphavirus (SIN), or Semliki Forest virus (SFV), with self-amplifying RNA derived from Venezuelan equine encephalitis virus being the most commonly used.
  • VEEV Venezuelan equine encephalitis virus
  • SIIN Sindbis alphavirus
  • SFV Semliki Forest virus
  • SRNA self-replicating RNA
  • NSP1, NSP2, NSP3, and NSP4 non-structural proteins
  • the RNA polymerase complex first synthesizes a complementary negative-strand RNA intermediate from the positive-strand RNA, and then uses the intermediate as a template to synthesize two distinct positive-strand RNAs.
  • the first positive-strand RNA is a copy of the original full-length genomic RNA; the second positive-strand RNA is a large amount of subgenomic RNA encoding the target gene.
  • This mechanism enables the self-replicating RNA to achieve high-level and sustained expression of the target protein with low doses. While the self-replicating RNA retains the viral genome's self-amplification ability, it cannot express viral structural proteins and therefore cannot produce a complete virus capable of transmission. Its entry into cells relies on delivery via lipid nanoparticles, thus exhibiting good safety and potential tumor-targeting capabilities.
  • RNA vaccines are considered applicable to the preparation of RNA vaccines and the mediating of tumor-killing cytokines expression in tumor cells.
  • Chinese invention patent application CN117280029A discloses a nucleic acid vector and its usage method, which modulates the tumor microenvironment by activating the relative expression of tumor-infiltrating lymphocytes and/or immunogenic cellular characteristics in the tumor microenvironment to achieve cancer treatment;
  • Chinese invention patent application CN117279661A discloses a composition and method for inducing immune responses to ESR1, PI3K, HER2, and HER3, which treats cancer by inducing immune responses;
  • Chinese invention patent application CN115968299A discloses a neoantigen expressed in multiple myeloma and its uses, which also uses self-replicating RNA as a vector to prepare tumor vaccines.
  • the mechanism of action of tumor vaccines is through antigen-presenting cells (APCs), such as dendritic cells.
  • APCs antigen-presenting cells
  • DCs express tumor antigens and present them to T cells of the immune system, activating T cells to attack tumor cells, thereby controlling or eliminating the tumor.
  • the purpose of this invention is to provide a self-replicating RNA that expresses differentiation-related transcription factors and its application in the preparation of tumor therapeutic drugs.
  • the self-replicating RNA By utilizing the self-replicating RNA to express transcription factors in tumor cells, the differentiation of tumor cells into mature cells can be induced, the malignant phenotype of malignant solid tumor cells can be inhibited, and the therapeutic effect on malignant solid tumors can be achieved.
  • This invention aims to address how to achieve long-term and specific high expression of related transcription factors in tumor cells and improve the tumor-suppressing effect.
  • the expression levels of relevant differentiation transcription factors involved in this invention are lower in tumor cells than in normal cells.
  • the inventors' research found that self-replicating RNA, after entering tumor cells, maintains its distribution within the cells despite rapid tumor cell proliferation due to its self-replication characteristic, and can sustain protein expression for over 20-30 days. Therefore, it is highly suitable for mediating the expression of target genes in tumor cells.
  • animal experiments showed that tail vein injection of self-replicating RNA encapsulated in lipid nanoparticles can mediate the specific expression of target genes in tumor tissues, while the expression of target genes is virtually undetectable in other normal tissues, suggesting that self-replicating RNA has good tumor targeting properties.
  • the inventors propose using self-replicating RNA vectors to restore or overexpress HNF4 ⁇ in tumor tissues, inducing tumor cells to differentiate into normal cells and inhibiting tumor cell growth, thereby achieving a therapeutic effect on tumors.
  • This invention utilizes self-replicating RNA technology to introduce relevant differentiation-inducing transcription factors into relevant tumor cells for overexpression. These factors are specifically and persistently expressed in tumor cells, thereby inducing the relevant tumor cells to differentiate into mature cells for tumor treatment.
  • the self-replicating RNA described above has an optimized polyA tail length, enabling it to maintain a longer-lasting, high-level expression of the target gene in vivo.
  • the self-replicating RNA structure includes a 5' cap, a 5' UTR, four non-structural genes (NSP1-4), a 26S subgenomic promoter (SGP), the target gene (GOI), a 3' UTR, and a polyA tail.
  • NSP1-4 non-structural genes
  • SGP 26S subgenomic promoter
  • GOI target gene
  • 3' UTR a polyA tail
  • Self-replicating RNA with a polyA length between 35-100 nt could mediate relatively stable and long-term overexpression of HNF4 ⁇ in tumor cells, with 50-70 nt showing better expression levels and duration.
  • Self-replicating RNA with a polyA tail of 60-70 nt achieved the most stable and efficient expression.
  • This invention also investigated the effects of non-replicating HNF4 ⁇ -mRNA, HNF4 ⁇ -saRNA, and adenovirus-mediated HNF4 ⁇ overexpression in tumor cells.
  • the results showed that both HNF4 ⁇ -saRNA and HNF4 ⁇ -mRNA could upregulate HNF4 ⁇ in liver cancer cells, but HNF4 ⁇ -mRNA expression was lower and its duration was shorter.
  • HNF4 ⁇ -saRNA expression was more than twice that of HNF4 ⁇ -mRNA and its duration was longer.
  • the upregulation effect of HNF4 ⁇ -mRNA on HNF4 ⁇ expression could only be maintained for 3 days, while HNF4 ⁇ -saRNA could maintain HNF4 ⁇ in liver cancer cells for at least 7 days.
  • HNF4 ⁇ -saRNA upregulation efficiency of HNF4 ⁇ expression by HNF4 ⁇ -saRNA was no less than that of adenovirus, indicating that self-replicating RNA can mediate the expression of target genes in tumor cells more efficiently than non-replicating mRNA.
  • the present invention provides a self-replicating RNA expressing a differentiation-related transcription factor, wherein the differentiation-related transcription factor is a transcription factor related to the differentiation and functional maintenance of tissues and organs, and the expression level of the transcription factor in tumor cells is lower than that in normal cells; the transcription factor is delivered into tumor cells via the self-replicating RNA and a delivery medium, and the transcription factor is expressed in tumor cells in a long-term and specific manner, inducing tumor cells to differentiate into normal mature cells, inhibiting tumor cell proliferation and/or inducing tumor cell apoptosis.
  • the differentiation-related transcription factor is a transcription factor related to the differentiation and functional maintenance of tissues and organs, and the expression level of the transcription factor in tumor cells is lower than that in normal cells
  • the transcription factor is delivered into tumor cells via the self-replicating RNA and a delivery medium, and the transcription factor is expressed in tumor cells in a long-term and specific manner, inducing tumor cells to differentiate into normal mature cells, inhibiting tumor cell proliferation and/or inducing tumor
  • long-term expression refers to expression lasting at least seven days (including seven days).
  • the aforementioned specific high expression refers to the absence, low expression, or transient expression in normal cells.
  • the self-replicating RNA includes a 5' cap, a non-coding region, a non-structural gene, a 26S subgenome promoter, a 3' non-coding region, and a polyadenylated tail.
  • the self-replicating RNA has a 35-100 nt polyadenylate tail.
  • the self-replicating RNA has a 40-90 nt polyadenylated tail.
  • the self-replicating RNA has a 50-75 nt polyadenylated tail.
  • the self-replicating RNA has a 60-70 nt polyadenylated tail.
  • the transcription factors mentioned are selected from HNF4 ⁇ , HNF1 ⁇ , FOXA3, PTF1A, NUROND1, Neurogenin-2, Ascl1, and other transcription factors related to tissue and organ differentiation and function maintenance.
  • HNF4 ⁇ (GENBANK No.: NM_000457.6, as shown in SEQ ID NO:1);
  • HNF1 ⁇ (GENBANK No.: NM_001306179.2, as shown in SEQ ID NO:2);
  • FOXA3 (GENBANK No.: NM_004497.3, as shown in SEQ ID NO:3);
  • PTF1A (GENBANK No.: NM_178161.3, as shown in SEQ ID NO:4);
  • NUROND1 (GENBANK No.: NM_002500.5, as shown in SEQ ID NO:5);
  • Neurogenin-2 (GENBANK number: NM_024019.4);
  • Ascl1 (GENBANK No.: NM_004316.4).
  • the delivery medium is selected from one or more of the following combinations: liposomes, viral replicon particles, lipid-based nanoparticles, polymer nanoparticles, physiological buffers, microspheres, immunostimulatory complexes, and conjugates of bioactive ligands.
  • the sequence of the self-replicating RNA is shown in SEQ ID NO:6.
  • This sequence is an optimization of the polyA tail of wild-type self-replicating RNA, preferably a 67nt polyA tail.
  • the 67-nucleotide A tail of saRNA can increase the half-life of saRNA in vivo, promoting saRNA stability and translation.
  • the sequence of the HNF4 ⁇ self-replicating RNA includes any one of the following (a) to (c):
  • RNA consisting of the nucleotide sequence shown in SEQ ID NO:7;
  • RNA consisting of a nucleotide sequence having one or more nucleotide deletions, substitutions, additions, or insertions in the nucleotide sequence shown in SEQ ID NO:7 and having activity expressed as HNF4 ⁇ ;
  • RNA consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:7 and having activity expressed as HNF4 ⁇ .
  • the sequence of the HNF1 ⁇ self-replicating RNA includes any one of the following (a) to (c):
  • RNA consisting of the nucleotide sequence shown in SEQ ID NO:8;
  • RNA consisting of a nucleotide sequence having one or more nucleotide deletions, substitutions, additions, or insertions in the nucleotide sequence shown in SEQ ID NO:8 and having activity expressed as HNF1 ⁇ ;
  • RNA consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:8 and having activity expressed as HNF1 ⁇ .
  • the sequence of the FOXA3 self-replicating RNA includes any one of the following (a) to (c):
  • RNA consisting of the nucleotide sequence shown in SEQ ID NO:9;
  • RNA consisting of a nucleotide sequence having one or more nucleotide deletions, substitutions, additions, or insertions in the nucleotide sequence shown in SEQ ID NO:9 and having activity expressed as FOXA3;
  • RNA consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:9 and having the activity of being expressed as FOXA3.
  • sequence of the PTF1A self-replicating RNA includes any one of the following (a) to (c):
  • RNA consisting of the nucleotide sequence shown in SEQ ID NO:10;
  • RNA consisting of a nucleotide sequence having one or more nucleotide deletions, substitutions, additions, or insertions in the nucleotide sequence shown in SEQ ID NO: 10 and having activity expressed as PTF1A;
  • RNA consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:10 and having the activity of being expressed as PTF1A.
  • the sequence of the NUROND1 self-replicating RNA includes any one of the following (a) to (c):
  • RNA consisting of the nucleotide sequence shown in SEQ ID NO:11;
  • RNA consisting of a nucleotide sequence having one or more nucleotide deletions, substitutions, additions, or insertions in the nucleotide sequence shown in SEQ ID NO: 11 and having NUROND1 expression activity;
  • RNA consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:11 and having the activity of being expressed as NUROND1.
  • the invention provides the use of the self-replicating RNA expressing differentiation-related transcription factors in the preparation of drugs for treating malignant solid tumors.
  • the malignant solid tumors mentioned are liver cancer, pancreatic cancer, stomach cancer, intestinal cancer, kidney cancer, lung cancer, and glioma.
  • the transcription factors are selected from HNF4 ⁇ , HNF1 ⁇ , FOXA3, and PTF1A, and the malignant solid tumors are malignant solid tumors derived from epithelial cells. These malignant solid tumors include liver cancer, pancreatic cancer, gastric cancer, intestinal cancer, kidney cancer, and lung cancer.
  • the transcription factors mentioned are selected from NUROND1, Neurogenin-2, and Ascl1, and the malignant solid tumor mentioned is glioma.
  • the present invention provides a gene delivery system comprising a self-replicating RNA expressing a differentiation-related transcription factor as described above and a delivery medium; the gene delivery system delivers a target gene into tumor cells, thereby achieving long-term and specific high expression of the transcription factor within the tumor cells, inducing the tumor cells to differentiate into normal mature cells, inhibiting tumor cell proliferation, and/or inducing tumor cell apoptosis.
  • the delivery medium is lipid-based nanoparticles (LNPs).
  • LNPs lipid-based nanoparticles
  • the LNP composition includes ionizable lipids, 1,2-distearate-sn-propanetriyl-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), cholesterol, and dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG 2000) or PEGylated lipids containing polyethylene glycol fragments.
  • DSPC 1,2-distearate-sn-propanetriyl-3-phosphocholine
  • DOPE 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine
  • DMG-PEG 2000 dimyristoylglycerol-polyethylene glycol 2000
  • PEGylated lipids containing polyethylene glycol fragments DMG-PEG 2000
  • the LNP composition includes a molar ratio of DSPC:cholesterol:DMG-PEG-2000:ionizable lipids, ranging from DSPC:5%-20%, cholesterol:30%-55%, PEG:0.5%-3%, and ionizable lipids:30%-60%, with the total lipid molar ratio being 100%.
  • An optional ratio is 9.4:42.5:1.8:46.3 (DSPC:cholesterol:DMG-PEG 2000:ionizable lipids).
  • the N:P ratio ranges from 5:1 to 10:1, with an optional 6:1 ratio, and LNPs have a particle size of approximately 40-300 nm.
  • the delivery medium is lipid-based nanoparticles (LNPs), and the lipids are selected from ALC-0315, SM-102 or DHA-1.
  • ALC-0315, SM-102, or DHA-1 have the following specific structures:
  • the present invention provides a pharmaceutical composition comprising the gene delivery system described above.
  • This invention utilizes a self-replicating RNA vector based on an mRNA technology platform, combined with lipid nanoparticle encapsulation and delivery, to highly express important differentiation-related transcription factors in tumor cells, inducing tumor cells to transform into normal cells, inhibiting the malignant phenotype of tumors, and achieving the goal of treating tumors.
  • This is a novel approach to differentiation-induced tumor therapy. Based on our previous animal experiments and researcher-initiated clinical studies, we can confirm that this technology can effectively inhibit tumor growth in vivo, representing a new approach to tumor treatment and potentially opening up a new avenue for tumor therapy.
  • this invention differs significantly from current applications of mRNA technology in tumor vaccines.
  • RNA tumor vaccines utilize RNA technology to express relevant tumor antigens in antigen-presenting cells to stimulate the body to produce an immune response against these antigens, thereby controlling or eliminating tumors.
  • this invention utilizes RNA technology to mediate the expression of differentiation transcription factors in tumor cells, leveraging the differentiation-promoting effects of these transcription factors to inhibit the malignant phenotype of tumors.
  • non-replicating mRNAs can mediate the transient expression of target genes in tumor cells, stable and efficient expression of differentiation-related transcription factors is required in tumor cells to induce differentiation of tumor cells into normal cells.
  • this invention proposes for the first time the use of self-replicating RNA technology to restore or overexpress differentiation-related transcription factors whose expression declines during tumorigenesis, thereby inhibiting tumor cell growth and achieving a therapeutic effect on tumors.
  • the gene delivery system provided by this invention can induce the differentiation of malignant solid tumor cells into normal mature cells, inhibit tumor cell proliferation, and induce tumor cell apoptosis.
  • this invention optimizes the self-replicating RNA vector used to obtain a delivery system that can stably and efficiently express target genes in tumor cells.
  • this invention utilizes self-replicating RNA to express differentiation-related transcription factors, resulting in not only high expression levels and efficiency but also longer duration of expression.
  • the preferred HNF4 ⁇ -saRNA of this invention can maintain HNF4 ⁇ for more than 7 days in liver cancer cells, with both expression levels and durations significantly higher than those of non-replicating HNF4 ⁇ -mRNA.
  • it has advantages such as good safety, strong tissue accessibility, and repeatable administration. Therapeutic effects have been observed in tumor-bearing animals and patients.
  • Figure 1 shows a schematic diagram of the structures of self-replicating RNAs expressing HNF4 ⁇ with polyA tails of different lengths.
  • GOI represents HNF4 ⁇ ; the lengths of the polyA tails are 30nt, 35nt, 40nt, 50nt, 60nt, 67nt, 75nt, 90nt, 100nt, and 110nt, respectively.
  • Figure 2 shows the effects of HNF4 ⁇ -saRNA lipid nanoparticles with different polyA tails on the overexpression of HNF4 ⁇ in liver cancer cells.
  • Huh-7 cells were treated with these nanoparticles and collected 3 and 7 days after transfection.
  • Western blot was used to detect the expression level of HNF4 ⁇ protein after different treatments to observe the effect of polyA length on the overexpression of HNF4 ⁇ mediated by saRNA in liver cancer cells.
  • Figure 3 shows the protein collection after Huh7 cells were treated with 2 ng/200 cells of HNF4 ⁇ -saRNA and HNF4 ⁇ -mRNA lipid nanoparticles for 1, 3, and 7 days, respectively.
  • Adenovirus Ad-HNF4 ⁇ and control virus Ad-GFP were also used to infect Huh7 cells for 1, 3, and 7 days, respectively, and proteins were collected after these treatments.
  • Western blot was used to detect the expression level of HNF4 ⁇ protein after different treatments.
  • ImageJ software was used to calculate the relative expression level of HNF4 ⁇ protein at each time point.
  • Figure 4 shows the HNF4 ⁇ protein expression level of Huh-7 cells after treatment with lipid nanoparticles of different concentrations of HNF4 ⁇ -saRNA and control GFP-saRNA for 1, 3, 5, and 7 days, as detected by Western blot.
  • GFP-saRNA is a self-replicating mRNA control.
  • Figure 5 shows the changes in cell proliferation after treatment with different concentrations of HNF4 ⁇ -saRNA and GFP-saRNA lipid nanoparticles, as detected by CCK8 assay.
  • Figure 6 shows the effect of HNF4 ⁇ -saRNA and GFP-saRNA lipid nanoparticle treatment on the clone-forming ability of Huh7 liver cancer cells.
  • Figure 7 shows the results of RT-PCR detection of liver function-related genes after Huh7 cells were treated with different concentrations of HNF4 ⁇ -saRNA and GFP-saRNA lipid nanoparticles for 3 days.
  • Figure 8 shows the expression levels of tumor cell stemness-related genes detected by RT-PCR after Huh7 cells were treated with different concentrations of HNF4 ⁇ -saRNA and GFP-saRNA lipid nanoparticles for 3 days.
  • Figure 9 shows the glycogen storage in Huh7 cells after 3 days of treatment with HNF4 ⁇ -saRNA and GFP-saRNA lipid nanoparticles, as detected by periodic acid-Schiff (PAS) staining. ImageJ software was used to count the PAS-positive areas in Huh7 cells.
  • PAS periodic acid-Schiff
  • Figure 10 shows the uptake capacity of acetylated low-density lipoprotein (ac-LDL) in Huh7 cells after 3 days of treatment with HNF4 ⁇ -saRNA and GFP-saRNA lipid nanoparticles.
  • the uptake capacity of ac-LDL in Huh7 cells was detected using Dil-ac-LDL fluorescent substrate.
  • the quantification of ac-LDL positive regions in Huh7 cells was performed using ImageJ software.
  • Figure 11 shows the content of senescence-related ⁇ -galactosidase in Huh7 cells after 3 days of treatment with HNF4 ⁇ -saRNA and GFP-saRNA lipid nanoparticles.
  • the quantification of ⁇ -galactosidase-positive regions in Huh7 cells was performed using ImageJ software.
  • Figure 12 shows the apoptosis status of Huh7 cells after treatment with HNF4 ⁇ -saRNA and GFP-saRNA lipid nanoparticles for 3 days, as detected by Annexin V/PI staining.
  • Figure 13 is a flowchart of the experimental procedure for treating Huh7 cell subcutaneous xenografts by intratumoral injection of HNF4 ⁇ -saRNA and HNF4 ⁇ -mRNA lipid nanoparticles.
  • Figure 14 shows the tumor proliferation curves in a Huh7 cell subcutaneous xenograft model treated with HNF4 ⁇ -saRNA and HNF4 ⁇ -mRNA.
  • GFP-saRNA is a self-replicating mRNA lipid nanoparticle control
  • physiological saline is a solvent control.
  • Figure 15 shows a gross image of a Huh7 cell subcutaneous xenograft tumor treated with HNF4 ⁇ -saRNA and HNF4 ⁇ -mRNA.
  • Figure 16 shows the tumor weight statistics (left) and tumor inhibition rate statistics (right) of the Huh7 cell subcutaneous xenograft model treated with HNF4 ⁇ -saRNA and HNF4 ⁇ -mRNA.
  • Figure 17 shows the expression level of HNF4 ⁇ in tumor tissue of a Huh7 cell subcutaneous xenograft model treated with HNF4 ⁇ -saRNA as detected by Western blot.
  • Figure 18 shows the changes in the expression of HNF4 ⁇ and Ki67 in tumor tissue detected by immunohistochemistry (left) and the statistical graph of the positive staining area of HNF4 ⁇ and Ki67 in tumor tissue (right).
  • Figure 19 is a flowchart of the experimental procedure for treating Huh7 cell liver orthotopic tumors by tail vein injection of self-replicating HNF4 ⁇ -saRNA and HNF4 ⁇ -mRNA lipid nanoparticles.
  • Figure 20 shows the in vivo fluorescence signals of HNF4 ⁇ -saRNA and HNF4 ⁇ -mRNA in mice before and after injection in a Huh7 cell orthotopic liver tumor model.
  • GFP-sRNA is a self-replicating RNA lipid nanoparticle control
  • physiological saline is a solvent control.
  • Figure 21 shows the in vivo fluorescence signal statistics of mice at different time points during the treatment of Huh7 cell liver orthotopic tumors with HNF4 ⁇ -saRNA and HNF4 ⁇ -mRNA.
  • Figure 22 shows a gross image of the tumor in the experiment of treating Huh7 cell liver orthotopic tumors with HNF4 ⁇ -saRNA and HNF4 ⁇ -mRNA lipid nanoparticles.
  • Figure 23 shows the tumor weight (left) and tumor inhibition rate (right) statistics in the experiment of treating Huh7 cell liver orthotopic tumors with HNF4 ⁇ -saRNA and HNF4 ⁇ -mRNA lipid nanoparticles.
  • Figure 24 shows the tumor tissue morphology detected by HE staining, the changes in Ki67 expression detected by immunohistochemistry (left), and the statistical graph of the area of Ki67 positive staining in the tumor tissue (right).
  • Figure 25 shows the expression level of HNF4 ⁇ protein detected by Western blot at different time points after treatment of Huh7 cell orthotopic liver tumors with HNF4 ⁇ -saRNA.
  • Figure 26 shows the changes in HNF4 ⁇ expression detected by immunohistochemistry at different time points after treatment of Huh7 cell orthotopic liver tumors with HNF4 ⁇ -saRNA (left) and the statistical graph of HNF4 ⁇ positive staining area in tumor tissue (right).
  • Figure 27 shows the expression of liver function-related genes in tumor tissues at different time points after treatment with HNF4 ⁇ -saRNA during the treatment of Huh7 cell orthotopic liver tumors.
  • Figure 28 shows the expression level of HNF4 ⁇ protein in HuCC-T1 cholangiocarcinoma cells after 1 day and 3 days of treatment with different concentrations of HNF4 ⁇ -saRNA and HNF4 ⁇ -mRNA lipid nanoparticles, as detected by Western blot.
  • Figure 29 shows that HNF4 ⁇ -saRNA LNP inhibits the proliferation of HuCC-T1 cholangiocarcinoma cells.
  • Figure 30 shows the effects of HNF4 ⁇ -mRNA LNP and HNF4 ⁇ -saRNA LNP on the clonogenic ability of HuCC-T1 cholangiocarcinoma cells.
  • Figure 31 shows the expression level of HNF4 ⁇ protein detected by Western blotting after HCT116 colon cancer cells were transfected with different concentrations of HNF4 ⁇ -saRNA LNP for 1 day and 3 days.
  • Figure 32 shows the effects of HNF4 ⁇ -mRNA LNP and HNF4 ⁇ -saRNA LNP on the proliferation of colorectal cancer cells HCT 116.
  • Figure 33 shows the effects of HNF4 ⁇ -mRNA LNP and HNF4 ⁇ -saRNA LNP on the clone-forming ability of HCT 116 colorectal cancer cells.
  • Figure 34 shows the expression level of HNF4 ⁇ protein detected by Western blot after treating pancreatic cancer cells PANC1 with different concentrations of HNF4 ⁇ -saRNA lipid nanoparticles for 1 day and 3 days.
  • Figure 35 shows the levels of HNF4 ⁇ mRNA (HNF4 ⁇ ) and self-replicating RNA vector (VEEV) at different time points after treatment of Huh7 cell liver orthotopic tumors with HNF4 ⁇ -saRNA.
  • HNF4 ⁇ HNF4 ⁇ mRNA
  • VEEV self-replicating RNA vector
  • Figure 36 shows the overall stability of hepatocellular carcinoma lesions after HNF4 ⁇ -saRNA LNP treatment on enhanced MRI.
  • Figure 37 shows a chest CT scan demonstrating the gradual shrinkage of hepatocellular carcinoma lung metastases after HNF4 ⁇ -saRNA LNP treatment.
  • A Baseline hepatocellular carcinoma lung metastases (arrow).
  • B Four weeks after treatment, the hepatocellular carcinoma lung metastases are slightly larger than at baseline.
  • C Eight weeks after treatment, the hepatocellular carcinoma lung metastases are slightly smaller than four weeks prior.
  • D Eighteen weeks after treatment, the hepatocellular carcinoma lung metastases have further shrunk, with the lesion diameter significantly smaller than the baseline level.
  • Figure 38 shows the changes in hepatocellular carcinoma target lesions after HNF4 ⁇ -saRNA LNP treatment on enhanced MRI.
  • Figure 39 shows the expression level of HNF4 ⁇ protein detected by Western blot after treating Huh7 liver cancer cells with different concentrations of HNF1 ⁇ -saRNA LNP for 1 day and 3 days, respectively.
  • Figure 40 shows the tumor proliferation curve (left) and relative tumor volume statistics (right) in the Huh7 cell subcutaneous xenograft model treated with HNF1 ⁇ -saRNA; physiological saline was used as the solvent control.
  • Figure 41 shows a gross image of a Huh7 cell subcutaneous xenograft tumor treated with HNF1 ⁇ -saRNA lipid nanoparticles.
  • Figure 42 shows the tumor weight (left) and tumor inhibition rate (right) statistics in the Huh7 cell subcutaneous xenograft model treated with HNF1 ⁇ -saRNA lipid nanoparticles.
  • Figure 43 shows the expression level of FOXA3 protein detected by Western blot after treating Huh7 liver cancer cells with different concentrations of FOXA3-saRNA LNP for 3 days.
  • Figure 44 shows the expression level of PTF1A protein detected by Western blot after treating pancreatic cancer PANC1 cells with different concentrations of PTF1A-saRNA LNP for 1 day and 3 days, respectively.
  • Figure 45 shows the expression level of NEUROD1 protein detected by Western blot one day after transfection of BHK21 hamster cells with NEUROD1-saRNA.
  • Figure 46 is a flowchart of an experimental study on subcutaneous implantation of Huh7 hepatocellular carcinoma cells by tail vein injection of self-replicating HNF4 ⁇ -saRNA and HNF4 ⁇ /HNF1 ⁇ /FOXA3-saRNA lipid nanoparticles.
  • Figure 47 shows the tumor proliferation curves in the subcutaneous xenograft model of hepatocellular carcinoma Huh7 cells treated with HNF4 ⁇ -saRNA and HNF4 ⁇ /HNF1 ⁇ /FOXA3-saRNA.
  • Figure 48 shows the tumor weight statistics of a subcutaneous xenograft model of hepatocellular carcinoma Huh7 cells treated with HNF4 ⁇ -saRNA and HNF4 ⁇ /HNF1 ⁇ /FOXA3-saRNA.
  • Figure 49 shows the expression levels of HNF4 ⁇ in tumor tissues of a subcutaneous hepatocellular carcinoma Huh7 cell xenograft model treated with HNF4 ⁇ -saRNA and HNF4 ⁇ /HNF1 ⁇ /FOXA3-saRNA by ELISA.
  • Figure 50 is a flowchart of the experimental procedure for treating subcutaneous xenografts of pancreatic cancer AsPC-1 cells by intratumoral injection of PTF1A-saRNA lipid nanoparticles.
  • Figure 51 shows the tumor proliferation curve in a subcutaneous xenograft model of pancreatic cancer AsPC-1 cells treated with intratumoral injection of PTF1A-saRNA lipid nanoparticles.
  • Figure 52 shows the tumor weight statistics in the AsPC-1 cell subcutaneous xenograft model of pancreatic cancer treated with intratumoral injection of PTF1A-saRNA lipid nanoparticles.
  • Figure 53 shows the tumor inhibition rate of a subcutaneous xenograft model of pancreatic cancer AsPC-1 cells treated with intratumoral injection of PTF1A-saRNA lipid nanoparticles.
  • Figure 54 shows the expression level of PTF1A in tumor tissue of the AsPC-1 cell subcutaneous xenograft model of pancreatic cancer treated with PTF1A-saRNA by Western blot.
  • Figure 55 is a flowchart of the experimental procedure for intratumoral injection of NUROD1-saRNA lipid nanoparticles to treat subcutaneous xenografts of U87 cells in gliomas.
  • Figure 56 shows the tumor proliferation curve in a subcutaneous xenograft model of U87 glioma cells treated with intratumoral injection of NUROD1-saRNA lipid nanoparticles.
  • Figure 57 shows a gross image of the tumor in a subcutaneous xenograft model of U87 glioma cells treated with intratumoral injection of NUROD1-saRNA lipid nanoparticles.
  • Figure 58 shows the tumor weight statistics in a subcutaneous xenograft model of U87 glioma cells treated with intratumoral injection of NUROD1-saRNA lipid nanoparticles.
  • embodiments of the present invention will employ conventional techniques of molecular biology, cell biology, and immunology, all of which are known to those skilled in the art. These techniques are fully described in the following literature: for example, *Molecular Cloning: A Laboratory Manual*, 4th edition (2017); *A Concise Laboratory Manual of Cell Biology* (2007); *A Concise Laboratory Manual of Immunology* (2010). Alternatively, the instructions provided by the reagent manufacturer may be followed.
  • saRNA Self-replicating RNA
  • saRNA is based on an engineered alphavirus genome containing genes encoding non-structural proteins that enable RNA replication, while the structural protein sequences are replaced by the target gene sequence.
  • saRNA includes a 5' cap, a non-coding region (5'UTR), four non-structural genes (NSP1-4), a 26S subgenomic promoter, the target gene, a 3' non-coding region (3'UTR), and a polyadenylated tail.
  • Linear RNA is designed based on the structure of eukaryotic mRNA, containing genes encoding the target protein, as well as the necessary cap structure, 5UTR, 3UTR, and poly(A).
  • RNA transcription plasmid DNA is restriction-digested using BspQI enzyme (New England Biolabs, R0712L) and then... PCR purification was performed using an Invitrogen (K310002) kit.
  • BspQI enzyme New England Biolabs, R0712L
  • 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 a cap analog (3'OMe, Trinlink, N-7413) were mixed and incubated at 37°C for 2 hours for in vitro transcription of the template. After transcription, DNase I (1 U/ ⁇ g DNA) was added and incubated at 37°C for 15 minutes to remove the DNA template. The transcribed RNA was then purified and recovered using LiCl precipitation.
  • Lipid nanoparticles were rapidly prepared by mixing an ethanol phase and an aqueous phase in a microfluidic device (INano TM L system, Micro&Nano).
  • the aqueous phase was a 50 mM citrate buffer (pH 6.0) containing purified saRNA.
  • the ethanol phase contained proprietary ionizable lipids: 1,2-distearate-sn-glycerophosphate-choline (DSPC) (Avanti, 850365P), cholesterol (Sigma-Aldrich, C8667), and 1,2-dicylo-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (NOF, GMO20).
  • the LNPs formed by this formulation were analyzed for particle size, PDI, RNA concentration, and encapsulation efficiency.
  • HNF4 ⁇ -saRNA LNP self-replicating HNF4 ⁇ -expressing RNA
  • Huh7 liver cancer cells were seeded at 3 x 105 cells/well in 6-well plates and cultured overnight.
  • HNF4 ⁇ -saRNA LNPs of different polyA lengths diluted with Opti-MEM were added, and after 6 hours of culture, 1 mL of DMEM medium containing 20% FBS was added.
  • Transfected cells were lysed using RIPA buffer after 3 or 7 days, and Western blot analysis was performed to detect intracellular HNF4 ⁇ protein levels ( Figure 2).
  • HNF4 ⁇ -saRNA with a polyA tail length of 35-100 nt mediated HNF4 ⁇ overexpression in Huh-7 cells.
  • Tumor cells transfected with self-replicating RNA with a polyA tail length of 40-90 nt showed higher HNF4 ⁇ expression, while self-replicating RNA with a polyA tail length of 50-75 nt showed better HNF4 ⁇ expression levels and duration.
  • Self-replicating RNA with a polyA tail length of 60-70 nt (especially 67 nt) mediated the most efficient expression.
  • the optimal self-replicating RNA sequence with the best polyA tail length after optimization is shown in SEQ ID NO:6, and the HNF4 ⁇ -saRNA sequence is shown in SEQ ID NO:7 (this sequence was used in subsequent examples).
  • Example 3 Comparison of the ability of non-replicating HNF4 ⁇ -mRNA, HNF4 ⁇ -saRNA, and adenovirus AdHNF4 ⁇ to mediate HNF4 ⁇ overexpression in tumor cells
  • Huh7 liver cancer cells were seeded at 3 x 105 cells/well in 6-well plates and cultured overnight. After washing with 1 ml of PBS, 1 ml of Opti-MEM diluted HNF4 ⁇ -saRNA LNP, control GFP-saRNA LNP, and non-replicating HNF4 ⁇ -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. Western blot analysis of intracellular HNF4 ⁇ protein levels was performed on transfected cells using RIPA lysis buffer at 1, 3, 5, or 7 days ( Figure 3).
  • Example 4 HNF4 ⁇ -saRNA treats liver cancer by inducing differentiation of liver cancer cells into hepatocytes.
  • HNF4 ⁇ -saRNA LNP upregulate HNF4 ⁇ expression in Huh-7 hepatocellular carcinoma cells.
  • Huh7 liver cancer cells were seeded at 3 x 105 cells/well in 6-well plates and cultured overnight. After washing with 1 ml of PBS, HNF4 ⁇ -saRNA LNP and control GFP-saRNA LNP (final concentrations of 0.5, 1, and 2 ng RNA/200 cells) diluted serially with Opti-MEM were added. After 6 hours of culture, 1 ml of DMEM medium containing 20% FBS was added. Western blot analysis of intracellular HNF4 ⁇ protein levels was performed on transfected cells at 1, 3, 5, or 7 days using RIPA lysis buffer ( Figure 4). The results showed that HNF4 ⁇ -saRNA LNP upregulated HNF4 ⁇ expression, while control GFP-saRNA LNP did not affect HNF4 ⁇ expression in liver cancer cells.
  • HNF4 ⁇ -saRNA LNP inhibits the growth of liver cancer cells.
  • Huh7 liver cancer cells The efficacy of a gene delivery system on malignant phenotypes such as tumor cell proliferation and colony formation was verified using Huh7 liver cancer cells.
  • Huh7 cells were seeded at a density of 3 ⁇ 103 cells/well in 96-well plates and cultured overnight. After incubation, the supernatant was removed, and the cells were washed with 100 ⁇ l of PBS.
  • Different concentrations of HNF4 ⁇ -saRNA LNP diluted with Opti-MEM and control GFP-saRNA LNP were added to the cells. Six hours later, an equal volume of 20% FBS was added to the culture medium to achieve a 10% FBS concentration.
  • Huh7 liver cancer cells were seeded at a density of 3 ⁇ 103 cells/well in 96-well plates and cultured overnight. Then, HNF4 ⁇ -saRNA LNP and control GFP-saRNA LNP (2 ng/200 cells) were added for transfection. After 24 hours, the cells were trypsinized and transferred to 60 mm culture dishes. The culture medium was changed every 3 days, and the clonogenic status of tumor cells was observed under a microscope. After clonogenic formation, the cells were washed twice with PBS, stained with crystal violet for 20 min, washed with PBS again, dried, and photographed. The number of clones was counted using ImageJ to determine the effect of RNA on the clonogenic ability of tumor cells. The results are shown in Figure 6; HNF4 ⁇ -saRNA significantly inhibited the clonogenic ability of liver cancer cells.
  • HNF4 ⁇ -saRNA LNP induces differentiation of liver cancer cells into mature hepatocytes.
  • Huh-7 liver cancer cells were seeded at 3 x 105 cells/well in 6-well plates and cultured overnight. Cells were washed with PBS, and different concentrations of HNF4 ⁇ -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 HNF4 ⁇ and the expression of tumor stemness-related genes ( Figures 7 and 8). The results showed that HNF4 ⁇ -saRNA upregulated the expression of liver function-related genes and downregulated the expression of tumor stemness-related genes.
  • 2HNF4 ⁇ -saRNA promotes glycogen storage and low-density lipoprotein uptake in liver cancer cells.
  • Glycogen storage and low-density lipoprotein uptake are important functions of normal liver cells.
  • the inventors further examined these functions to determine whether liver cancer cells differentiate into mature liver cells.
  • Hepatocellular carcinoma cells were seeded at 3 x 104 cells/well in 24-well plates. 1 ng of HNF4 ⁇ -saRNA LNP and corresponding controls were added to each well. Three days after LNP delivery, the glycogen storage capacity of the cells was verified using a PAS reaction kit (Beyotime). Cells were fixed with 70% ethanol for 10 minutes, then the periodic acid solution was removed and the cells were equilibrated to room temperature. 100 ⁇ l of periodic acid solution was added to each sample, and the cells were reacted in a humidified chamber in the dark for 10 minutes. The periodic acid solution was then removed, and the cells were immersed in PBS and washed on a shaker for 5 minutes.
  • HHC7 Hepatocellular carcinoma cells
  • Hepatocellular carcinoma cells were seeded at 3 x 104 cells/well in 24-well plates. 1 ng of HNF4 ⁇ -saRNA LNP per 200 cells and a corresponding control were added. Three days after LNP delivery, the cell culture medium was aspirated, cells were washed with PBS, and then 200 ⁇ L of Dil ac-LDL (Invitrogen) diluted 1:100 with DMEM was added to each well. After 3 hours, the DMEM containing Dil ac-LDL was removed, cells were washed with PBS, and fixed with 4% paraformaldehyde for 15 minutes.
  • Dil ac-LDL Invitrogen
  • 3HNF4 ⁇ -saRNA promotes senescence in liver cancer cells
  • HCCs Hepatocellular carcinoma cells of type Huh7 were seeded at 3 x 104 cells/well in 24-well plates, and 1 ng/200 cells of HNF4 ⁇ -saRNA LNP and corresponding controls were added. Three days after LNP delivery, HCC cell senescence was assessed using a Senescence ⁇ -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 ⁇ -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 HCC cells after HNF4 ⁇ -saRNA treatment.
  • HNF4 ⁇ -saRNA induces apoptosis in liver cancer cells
  • Hepatocellular carcinoma cells (HFC-7) were seeded at 3 x 105 cells/well in 24-well plates, and 1 ng/200 cells of HNF4 ⁇ -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 ⁇ l of binding buffer.
  • HNF4 ⁇ -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.
  • HNF4 ⁇ -saRNA LNP inhibits the growth of subcutaneous tumors in mice.
  • mice Five-week-old male nude mice (BALB/c immunodeficient strain) were purchased from Shanghai BK/KY Biotechnology Co., Ltd., and housed under specific pathogen-free environmental conditions using a 12-hour on/off light cycle.
  • HNF4 ⁇ -saRNA LNP 5 ⁇ g of HNF4 ⁇ -saRNA LNP, GFP-saRNA LNP, HNF4 ⁇ -mRNA LNP, or physiological saline (solvent control) was injected into the tumor, resulting in a final volume of 75 ⁇ l.
  • Tumor volume was measured daily after injection, and tumor growth curves were plotted ( Figures 13, 14).
  • Results showed that intratumoral injection of HNF4 ⁇ -saRNA slowed tumor growth, with a tumor inhibition rate exceeding 50% (Figure 16), while HNF4 ⁇ -mRNA had no significant inhibitory effect on tumor growth.
  • Western blotting and immunohistochemical results showed that HNF4 ⁇ -saRNA significantly increased the protein expression of HNF4 ⁇ in tumor tissue, and significantly decreased the expression of Ki67, a tumor cell proliferation marker.
  • HNF4 ⁇ -saRNA LNP inhibits the growth of hepatocellular carcinoma cells implanted in situ in the liver.
  • the Huh7 human hepatocellular carcinoma cell line stably expressing the luciferase gene was inoculated into the axilla of male nude mice. After tumor formation, the tumor fragments were removed, cut into 1 mm3 pieces, and transplanted into the subcapsular region of the liver of male nude bab/c mice. Tumor growth in the mice was monitored using an IVIS spectrum optical imaging system. Three days after transplantation, the mice were evenly divided into four groups based on the intensity of the fluorescence signal. Each group was administered HNF4 ⁇ -saRNA LNP, GFP-saRNA LNP, HNF4 ⁇ -mRNA LNP, and physiological saline (solvent control) via tail vein injection at a dose of 2 mg/kg, respectively ( Figure 19).
  • HNF4 ⁇ -saRNA The efficacy of HNF4 ⁇ -saRNA was evaluated by monitoring tumor growth using bioluminescence (Figure 20), and tumor growth curves were plotted (Figure 21). At the end of the experiment, the mice were euthanized, the tumors were excised and weighed, and the tumor inhibition rate was calculated ( Figures 22 and 23). The tumor tissue was embedded in paraffin, and the morphology of the tumor tissue was assessed by HE staining, while the proliferation status of the tumor was assessed by Ki67 staining ( Figure 24). The results showed that tumor growth was reduced after tail vein injection of HNF4 ⁇ -saRNA LNP, with a tumor inhibition rate exceeding 40% compared to the control tumor, while HNF4 ⁇ -mRNA LNP had no significant tumor-inhibiting effect.
  • HNF4 ⁇ -saRNA significantly upregulated the expression of HNF4 ⁇ and liver function-related genes in tumor tissues.
  • Example 5 HNF4 ⁇ -saRNA inhibits the proliferation of bile duct cancer cells
  • HNF4 ⁇ -saRNA LNP upregulate HNF4 ⁇ expression in HuCC-T1 cholangiocarcinoma cells.
  • HNF4 ⁇ -saRNA LNPs (final concentrations of 1 and 2 ng RNA/200 cells) were serially diluted with Opti-MEM. After 6 hours of culture, 1 ml of DMEM medium containing 20% FBS was added. Western blotting was performed on transfected cells using RIPA lysis buffer at 1 and 3 days to detect intracellular HNF4 ⁇ protein levels (Figure 28). The results showed that HNF4 ⁇ -saRNA LNPs upregulated HNF4 ⁇ expression in cholangiocarcinoma cells.
  • HNF4 ⁇ -saRNA LNP inhibits the growth of bile duct cancer cells.
  • Cholangiocarcinoma cells (HuCC-T1) were seeded at a density of 3 ⁇ 103 cells/well in 96-well plates and cultured overnight. After aspirating the culture supernatant, the cells were washed with 100 ⁇ l of PBS. Different concentrations of HNF4 ⁇ -saRNA LNP diluted in 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 HNF4 ⁇ -saRNA LNP significantly inhibited the proliferation of cholangiocarcinoma cells compared to control cells (Figure 29).
  • HNF4 ⁇ -saRNA LNP inhibits clonal formation of cholangiocarcinoma cells.
  • Example 6 HNF4 ⁇ -saRNA inhibits the proliferation of colon cancer cells
  • HNF4 ⁇ -saRNA LNP upregulate HNF4 ⁇ expression in HCT-116 colorectal cancer cells.
  • HNF4 ⁇ -saRNA LNPs final concentrations of 1 and 2 ng RNA/200 cells
  • Opti-MEM Opti-MEM
  • 1 ml of DMEM medium containing 20% FBS was added.
  • Western blotting was performed on transfected cells using RIPA lysis buffer at 1 and 3 days to detect intracellular HNF4 ⁇ protein levels (Figure 31). The results showed that HNF4 ⁇ -saRNA LNPs upregulated HNF4 ⁇ expression in colorectal cancer cells.
  • HNF4 ⁇ -saRNA LNP inhibits the growth of bile duct cancer cells.
  • HCT-116 Colorectal cancer cells HCT-116 were seeded at a density of 3 ⁇ 103 cells/well in 96-well plates and cultured overnight. After aspirating the culture supernatant, the cells were washed with 100 ⁇ l of PBS. Different concentrations of HNF4 ⁇ -saRNA LNP and HNF4 ⁇ -mRNA LNP (final concentration 2 ng RNA/200 cells) diluted with Opti-MEM were added to the cells. Six hours later, an equal volume of 20% FBS was added to the culture medium to bring the FBS concentration to 10%. Cell proliferation was measured daily using a cell counting kit-8 (CCK-8, Dojindo) to determine the effect of RNA on tumor cell proliferation. The results showed that HNF4 ⁇ -saRNA LNP significantly inhibited the proliferation of colorectal cancer cells compared to control cells ( Figure 32), while HNF4 ⁇ -mRNA LNP had no significant effect on tumor cell proliferation.
  • CCK-8 cell counting kit-8
  • HNF4 ⁇ -saRNA LNP inhibits colony formation of colorectal cancer cells.
  • HCT-116 Colorectal cancer cells HCT-116 were seeded at a density of 3 ⁇ 103 cells/well in 96-well plates and cultured overnight. After incubation, HNF4 ⁇ -saRNA LNP and HNF4 ⁇ -mRNA 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, dried, and photographed. The number of colonies was counted using ImageJ to determine the effect of RNA on the colony formation ability of tumor cells (Figure 33). The results showed that HNF4 ⁇ -saRNA significantly inhibited the colony formation ability of colorectal cancer cells, while HNF4 ⁇ -mRNA did not affect tumor cell colony formation.
  • Example 7 HNF4 ⁇ -saRNA upregulates the expression of HNF4 ⁇ in pancreatic cancer cells
  • HNF4 ⁇ -saRNA LNPs Human pancreatic cancer cells PANC1 were seeded at a density of 40%-50% into 6-well plates and cultured overnight. After washing with 1 ml of PBS, HNF4 ⁇ -saRNA LNPs (final concentrations of 1 and 2 ng RNA/200 cells) were serially diluted with Opti-MEM. After 6 hours of culture, 1 ml of DMEM medium containing 20% FBS was added. Western blotting was performed on transfected cells using RIPA lysis buffer at 1 and 3 days to detect intracellular HNF4 ⁇ protein levels (Figure 34). The results showed that HNF4 ⁇ -saRNA LNPs upregulated HNF4 ⁇ expression in pancreatic cancer cells.
  • Example 8 HNF4 ⁇ -saRNA LNP specifically upregulates HNF4 ⁇ expression in tumor tissues in vivo.
  • the Huh7 human hepatocellular carcinoma cell line stably expressing the luciferase gene, was inoculated into the axilla of male nude mice. After tumor formation, the tumor fragments were removed, cut into 1 mm3 pieces, and transplanted into the subcapsular region of the liver of male Bab/c nude mice. Tumor growth in the mice was monitored using an IVIS spectrum optical imaging system. Once the tumors reached a suitable size, HNF4 ⁇ -saRNA LNP was administered via tail vein injection at a dose of 2 mg/kg body weight. Organ tissues and tumor tissues were collected from the mice 3 and 5 days after injection.
  • VEEV alphavirus genome sequence
  • LNP-mediated human HNF4 ⁇ gene sequence in various organs and tumors
  • the results showed that 3 days after tail vein injection of HNF4 ⁇ -saRNA LNP, trace amounts of VEEV and human HNF4 ⁇ RNA were detectable in the heart, spleen, and kidneys; almost no VEEV and HNF4 ⁇ gene sequences were detected in the liver, lungs, and muscle tissues; however, high abundance of VEEV and HNF4 ⁇ gene sequences was clearly detected in tumor tissues.
  • HNF4 ⁇ -saRNA LNP Five days after tail vein injection of HNF4 ⁇ -saRNA LNP, VEEV and human HNF4 ⁇ RNA were undetectable in all organs except for trace amounts detected in the heart. However, VEEV and HNF4 ⁇ gene sequences were still clearly detected in tumor tissue (Figure 35). This indicates that self-replicating RNA can mediate HNF4 ⁇ expression in tumor cells in vivo.
  • Example 9 HNF4 ⁇ -saRNA LNP treatment for patients with advanced liver cancer
  • a CDMO company was commissioned to produce a GMP-compliant HNF4 ⁇ -saRNA LNP formulation according to pharmaceutical standards.
  • a CRO company was commissioned to conduct a single-dose toxicity test of the HNF4 ⁇ -saRNA LNP formulation.
  • the specific process and results are as follows: Rats were administered the drug once via tail vein injection, with an observation period of 14 days. A negative control group (0.9% sodium chloride injection), a carrier control group (LNP solution), and a test sample group (150 ⁇ g/rat) were set up. All animals survived to the planned dissection date. No gross necropsy abnormalities were observed at the end of the observation period, and no histopathological changes related to the test sample were observed.
  • Example 10 HNF1 ⁇ -saRNA inhibits the occurrence and development of malignant liver cancer.
  • HNF4 ⁇ -saRNA LNP upregulate HNF1 ⁇ expression in Huh-7 hepatocellular carcinoma cells.
  • Huh7 liver cancer cells were seeded at 3 x 105 cells/well in 6-well plates and cultured overnight. After washing with 1 ml of PBS, HNF1 ⁇ -saRNA LNP was serially diluted with Opti-MEM (final concentration: 1, 2 ng RNA/200 cells). After 6 hours of culture, 1 ml of DMEM medium containing 20% FBS was added. Transfected cells were lysed using RIPA buffer at 1, 3, or 7 days, and Western blot analysis was performed to detect intracellular HNF1 ⁇ protein levels (Figure 39). The results showed that HNF1 ⁇ -saRNA LNP upregulated HNF1 ⁇ expression.
  • HNF1 ⁇ -saRNA LNP inhibits the growth of subcutaneous tumors in mice.
  • mice Five-week-old male nude mice (BALB/c immunodeficient strain) were purchased from Shanghai BK/KY Biotechnology Co., Ltd., and housed under specific pathogen-free environmental conditions using a 12-hour on/off light cycle.
  • HNF1 ⁇ -saRNA 5 ⁇ g/75 ⁇ l/mouse, treatment group
  • the other received saline control group
  • Tumor volume was measured daily after injection, and tumor growth curves were plotted to calculate the relative tumor volume RTV ( Figure 40).
  • Five days after RNA injection the mice were sacrificed, the tumors were removed and weighed, and the tumor inhibition rate was calculated ( Figures 41, 42). The results showed that HNF1 ⁇ -saRNA significantly inhibited the growth of hepatocellular carcinoma implants.
  • Example 11 FOXA3-saRNA upregulates FOXA3 expression in Huh-7 hepatocellular carcinoma cells
  • Huh7 liver cancer cells were seeded at 3 x 105 cells/well in 6-well plates and cultured overnight. After washing with 1 ml of PBS, FOXA3-saRNA LNP and control GFP-saRNA LNP (final concentrations of 0.5, 1, and 2 ng RNA/200 cells) were added serially diluted with Opti-MEM. After 6 hours of culture, 1 ml of DMEM medium containing 20% FBS was added. After 3 days, the transfected cells were lysed using RIPA buffer, and Western blot was used to detect the intracellular FOXA3 protein level (Figure 43). The results showed that FOXA3-saRNA LNP upregulated FOXA3 expression, while control GFP-saRNA LNP did not affect FOXA3 expression in liver cancer cells.
  • Example 12 PTF1A-saRNA upregulates PTF1A expression in pancreatic cancer PANC1 cells
  • Pancreatic cancer cells PANC1 were seeded at 2.5 x 105 cells/well in 6-well plates and cultured overnight. After washing with 1 ml of PBS, PTF1A-saRNA LNP and control GFP-saRNA LNP (final concentrations of 0.5, 1, and 2 ng RNA/200 cells) were added serially diluted with Opti-MEM. After 6 hours of culture, 1 ml of DMEM medium containing 20% FBS was added. Three days later, transfected cells were lysed using RIPA buffer, and Western blot analysis was performed to detect the protein level of PT in PTF1A cells (Figure 44). The results showed that PTF1A-saRNA LNP upregulated PTF1A expression, while control GFP-saRNA LNP did not affect PTF1A expression in liver cancer cells.
  • BHK-21 hamster kidney cells were seeded at 3 x 105 cells/well in 6-well plates in EMEM medium without antibiotics. After 24 h of complete adhesion, transient transfection was performed: 0.1 ⁇ g and 1 ⁇ g NEUROD1-saRNA were added to 100 ⁇ l of Opti-MEM as mixture 1 (MIX1), and 0.3 ⁇ l/3 ⁇ l Lipofectamine MessengerMAX (Themo) were added to 100 ⁇ l of Opti-MEM as mixture 2 (MIX2). MIX1 and MIX2 were incubated at room temperature for 10 min each, then mixed and incubated at room temperature for 20 min. The mixture was then added to the corresponding 6-well plates at 200 ⁇ l/well.
  • MIX1 and MIX2 were incubated at room temperature for 10 min each, then mixed and incubated at room temperature for 20 min.
  • Example 14 Mixed HNF4 ⁇ /HNF1 ⁇ /FOXA3-saRNA LNP inhibits the growth of subcutaneous implanted tumors of mouse liver cancer cells.
  • saRNAs HNF4 ⁇ , HNF1 ⁇ , and FOXA3 were mixed in equal mass ratios using a conventional LNP delivery system.
  • the mixed saRNAs were then encapsulated using a microfluidic system to synthesize the drug mix HNF4 ⁇ /HNF1 ⁇ /FOXA3-saRNA LNP.
  • Six- to eight-week-old female nude mice BALB/c immunodeficient strain
  • Five ⁇ 106 Huh-7 cells were subcutaneously injected into the right axilla of the female mice.
  • the endpoint tumor volume was 975.28 ⁇ 72.28 mm3 in the saline (solvent control) group, 732.76 ⁇ 96.40 mm3 in the 5 ⁇ g single HNF4 ⁇ -saRNA LNP group, and 517.36 ⁇ 14.84 mm3 in the 10 ⁇ g mix HNF4 ⁇ /HNF1 ⁇ /FOXA3-saRNA LNP group.
  • Tumor growth curves were plotted (Figure 47). Two days after the last administration, mice were sacrificed, tumors were excised and weighed (Figure 48), and tumor inhibition rate was calculated.
  • ELISA was used to detect the expression of human HNF4 ⁇ in tumor tissue (Figure 49).
  • Results showed that intratumoral injection of 10 ⁇ g mix HNF4 ⁇ /HNF1 ⁇ /FOXA3-saRNA LNP slowed tumor growth, with a tumor inhibition rate exceeding 40%, significantly higher than that of 5 ⁇ g single HNF4 ⁇ -saRNA LNP.
  • Example 15 PTF1A-saRNA LNP inhibits the growth of subcutaneous implanted tumors of pancreatic cancer cells in mice.
  • Example 16 NeuroD1-saRNA LNP inhibits the growth of subcutaneous tumors in mice

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Biochemistry (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Physics & Mathematics (AREA)
  • Nanotechnology (AREA)
  • Microbiology (AREA)
  • Medical Informatics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne le domaine technique de la biomédecine, et concerne en particulier un ARN auto-réplicatif exprimant un facteur de transcription lié à la différenciation et son utilisation dans la préparation d'un médicament thérapeutique antitumoral. La présente invention concerne un moyen technique permettant d'induire la différenciation des cellules tumorales en cellules matures, qui utilise l'acide ribonucléique messager pour réguler et maîtriser l'expression de facteurs de transcription importants liés à la différenciation dans les cellules tumorales, inhibe le phénotype malin des cellules tumorales solides malignes et permet de traiter les tumeurs solides malignes, ce qui permet de l'appliquer aux méthodes de préparation et à l'utilisation de médicaments contre les tumeurs solides.
PCT/CN2025/094572 2024-05-16 2025-05-13 Arn auto-réplicatif exprimant un facteur de transcription lié à la différenciation et son utilisation dans la préparation d'un médicament thérapeutique antitumoral Pending WO2025237287A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202410607331.5 2024-05-16
CN202410607331 2024-05-16

Publications (1)

Publication Number Publication Date
WO2025237287A1 true WO2025237287A1 (fr) 2025-11-20

Family

ID=97649432

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2025/094572 Pending WO2025237287A1 (fr) 2024-05-16 2025-05-13 Arn auto-réplicatif exprimant un facteur de transcription lié à la différenciation et son utilisation dans la préparation d'un médicament thérapeutique antitumoral

Country Status (2)

Country Link
CN (1) CN120966829A (fr)
WO (1) WO2025237287A1 (fr)

Also Published As

Publication number Publication date
CN120966829A (zh) 2025-11-18

Similar Documents

Publication Publication Date Title
Yang et al. Efficient hepatic delivery and protein expression enabled by optimized mRNA and ionizable lipid nanoparticle
CN102695525B (zh) 用于蛋白质表达的具有未修饰和修饰核苷酸的组合的rna
WO2025236837A1 (fr) Système d'administration de gène et son utilisation dans la préparation d'un médicament thérapeutique antitumoral
EP3071696B1 (fr) Compositions c/ebp alpha et méthodes d'utilisation
CN116406258B (zh) 一种雾化吸入的载药纳米颗粒以及用于治疗肺纤维化的siRNA序列组及其设计方法
JP2015526432A (ja) インビボにおいて膵臓ベータ細胞形成を誘導するための方法及び組成物
WO2017190695A1 (fr) Arnsi supprimant l'expression du gène egfr, son précurseur et applications associées
CN106177990A (zh) 一种新的前体miRNA及其在肿瘤治疗中的应用
Xu et al. Application of mPEG-CS-cRGD/Bmi-1RNAi-PTX nanoparticles in suppression of laryngeal cancer by targeting cancer stem cells
CN114958850A (zh) 一种基因组件、含有此基因组件的递送系统及其应用
CN110575436A (zh) Nlrp3抑制剂组合物及其应用
Chen et al. Long‐Term Storage Stable Vesicle‐Like Nanoparticles of Lipid and Polymer for siRNA and mRNA‐Mediated Cancer Immunotherapy
WO2025237287A1 (fr) Arn auto-réplicatif exprimant un facteur de transcription lié à la différenciation et son utilisation dans la préparation d'un médicament thérapeutique antitumoral
CN116919917B (zh) 一种磁靶向、装载肝细胞响应型mRNA的工程化细胞外囊泡及其构建方法和应用
US11149271B2 (en) Method and pharmaceutical composition for treating cancer
CN113181137B (zh) 一种pH响应性纳米颗粒介导的RNA纳米药物及其制备方法
TW202409281A (zh) 表現副孢菌素（ｐａｒａｓｐｏｒｉｎ）之載體及醫藥組成物
CN101474413A (zh) Drr1基因在制备抗肿瘤药物中的用途
JP6076076B2 (ja) 組織再生促進剤
Qiu et al. Encapsulating extracellular vesicles with a minimal RISC complex as novel gene silencing tool
CN115990270B (zh) 一种抑制肿瘤干性的纳米载体及其制备方法和应用
WO2025153828A1 (fr) Thérapie de remplacement de protéine
CN120437310A (zh) 基于外泌体的5-fu靶向递送系统及其制备方法与应用
Zhang et al. Crispr Cas9 fluorine-containing PEI nanomedicine inhibits the growth of hepatocellular carcinoma cells by knocking out NEDD8-E2 conjugase UBC12 (UBE2M)
CN105457040A (zh) 一种纳米粒子组合物及其在抗肿瘤血管生成中的应用