MRNA (messenger Ribonucleic Acid) for coding mouse development pluripotent associated protein 3 mSTELLA and application of mRNA

Abstract

The invention provides mRNA (messenger ribonucleic acid) for coding mouse development pluripotent related protein 3mSTELLA and application of the mRNA. The mRNA is used for coding any one of the following proteins: (1) wild type mSTELLA protein; (2) a protein having more than 80% of identity with the wild type mSTELLA protein; (3) a protein containing amino acid sequences from the 80th site to the 119th site of the wild type mSTELLA protein; and (4) a protein having 80% or more identity with the amino acid sequences from the 80th site to the 119th site of the wild-type mSTELLA protein, or the like. The mRNA expresses the coding protein in vivo and in vitro, inhibits tumor specific DNA methylation, and has a tumor treatment effect, so that a new method is provided for tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an mRNA encoding a mouse developmental pluripotency-related protein 3mSTELLA and its applications. Background Technology

[0002] DNA methylation, the covalent binding of methyl groups to DNA molecules under the catalysis of DNA methyltransferases (DNMTs), is a crucial component of epigenetic regulation. It plays a vital role in regulating gene expression, maintaining genome stability, and embryonic development, and is closely related to the occurrence and development of various tumors. Studies have shown that DNMT expression levels in most tumor cells are higher than in normal cells. DNMT overexpression leads to abnormal hypermethylation and gene silencing of CpG islands in the promoter regions of tumor suppressor genes (TSGs), a phenomenon considered one of the most important epigenetic alterations in tumorigenesis. Therefore, inhibiting the activity of DNA methyltransferases can regulate DNA methylation levels and induce epigenetic reprogramming, thereby inhibiting tumor cell growth or killing tumor cells. The reversibility of epigenetic modifications, in particular, makes DNA methylation inhibitors significant in cancer therapy.

[0003] While DNA methylation inhibitors have shown some clinical efficacy, there is still room for improvement. For example, nucleoside inhibitors suffer from low bioavailability, poor stability, and high cytotoxicity, limiting their clinical application; while low-toxicity non-nucleoside inhibitors exhibit poor DNA methyltransferase inhibitory activity and selectivity. Therefore, there is an urgent need to develop highly efficient, highly selective, and low-toxicity DNA methylation inhibitors.

[0004] DNA methylation repressor proteins (DNA MRSPs) are a class of natural proteins that regulate the DNA methylation process. They function through various mechanisms, including directly or indirectly inhibiting the action of DNA methyltransferases (DNMTs), thereby preventing them from adding methyl groups to DNA. Therefore, mimicking or promoting the demethylation function of DNA MRSPs could be a novel strategy for cancer therapy. UHRF1, a protein containing ubiquitin-like PHD and ring finger domains, specifically recognizes hemimethylated DNA and recruits DNA methyltransferase 1 (DNMT1) to the DNA strand, adding a methylation label to the newly synthesized DNA strand, ensuring the correct transmission of DNA methylation patterns during cell division. Mouse developmental pluripotency-associated protein 3 (STELLA / DPPA3 / PGC7) competitively binds to UHRF1 and promotes its transport from the nucleus to the cytoplasm, directly or indirectly interfering with the interaction between UHRF1 and DNMT1, leading to a decrease in DNA methylation levels. It is a natural inhibitor of UHRF1, thus antagonizing DNA methylation. Therefore, mSTELLA plays a key role in regulating DNA methylation, and its expression and activity are potential targets for cancer treatment.

[0005] With the widespread application of COVID-19 mRNA vaccines, emerging therapies based on mRNA technology have developed rapidly, sparking research and innovation in RNA biomedicine. Compared to traditional therapies, mRNA technology can alter gene expression in a transient and controllable manner without changing the genome, rapidly achieving protein translation and demonstrating broad clinical application prospects in many disease areas. Preparing mRNA encoding mSTELLA to express the natural UHRF1 antagonist protein and reverse tumor-specific DNA hypermethylation represents a novel approach to cancer treatment. Simultaneously, the demethylation effect of this method also provides new strategies for diseases caused by DNA hypermethylation, cell reprogramming, and the preparation of engineered immune cells. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide an mRNA encoding the mouse developmental pluripotency-associated protein 3 mSTELLA and its applications. This mRNA expresses its encoded protein in vitro and in vivo, inhibiting DNA methylation and achieving methylation reprogramming, thereby exerting a therapeutic effect on tumors.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides an mRNA encoding a mouse developmental pluripotency-associated protein 3 mSTELLA, said mRNA encoding any one of the following proteins:

[0009] (1) Wild-type mSTELLA protein, the amino acid sequence of which is shown in SEQ ID NO.1;

[0010] (2) A mutant protein that has more than 80% identity with the wild-type mSTELLA protein shown in SEQ ID NO.1 and has a similar DNA demethylation function;

[0011] (3) A recombinant protein containing the amino acid sequence from position 80 to position 119 of the wild-type mSTELLA protein shown in SEQ ID NO.1 and having a DNA demethylation function;

[0012] The amino acid sequence from position 80 to position 119 is: QSAFPKRRVRTLLSVLKDPIAKMRRLVRIEQRQKRLEGNE, where Q is located at the N-terminus and E is located at the C-terminus.

[0013] (4) A recombinant protein containing more than 80% identity with the amino acid sequence from position 80 to position 119 of the wild-type mSTELLA protein shown in SEQ ID NO.1 and having a similar DNA demethylation function.

[0014] This invention designs and prepares mRNA encoding mouse developmental pluripotency-associated protein 3 (STELLA / DPPA3 / PGC7, referred to as mSTELLA in this invention), and further discovers that amino acids from position 80 to position 119 of wild-type mSTELLA protein can effectively exert demethylation function, which is of great significance for the development of corresponding therapeutic drugs to reverse tumor-specific DNA hypermethylation and other fields.

[0015] Preferably, the mRNA contains an open reading frame (ORF), and the nucleic acid sequence of the ORF includes any of the following:

[0016] (1) The sequence shown in SEQ ID NO.2;

[0017] (2) A degenerate or complementary sequence of the sequence shown in SEQ ID NO.2;

[0018] (3) It has more than 80% identity with the sequence shown in SEQ ID NO.2 and encodes a protein with a similar DNA demethylation function;

[0019] (4) A sequence comprising the sequence shown in SEQ ID NO.3 and encoding a recombinant protein having a DNA demethylation-like function;

[0020] (5) A sequence comprising more than 80% identity with the sequence shown in SEQ ID NO.3 and encoding a recombinant protein having a similar DNA demethylation function. Preferably, the 5' end of the mRNA further comprises a cap structure and / or a 5' UTR, and the 3' end further comprises a 3' UTR and / or a polyA tail.

[0021] In this invention, further designing cap structures, 5'UTR, 3'UTR, or polyA tails helps to achieve stable and efficient protein expression from mRNA.

[0022] In some implementations, the cap structure can be added to the 5' end of the mRNA via co-transcription or a capping kit.

[0023] In a preferred embodiment, capping is performed via co-transcription, and the cap structure is specifically m. 7 G5'ppp5'(2'-OMe)NpG. Where m 7 G is N7-methylguanine nucleoside, p is phosphate, ppp is triphosphate, 2'-OMe is 2'-methoxy modification, and N is any nucleoside, such as adenine nucleoside (A), guanine nucleoside (G), cytosine nucleoside (C) and uracil nucleoside (U), or other naturally occurring nucleosides and modified nucleosides.

[0024] Preferably, the open reading frame (ORF) contains mRNA encoding mSTELLA.

[0025] In some embodiments, the open reading frame (ORF) encodes the wild-type protein of mSTELLA (SEQ ID NO.1) or its homologs, fragments, or variants. The variant encoded by the mRNA is a protein having at least 80%, 85%, 90%, 95%, or 98% sequence identity with the wild-type protein and having similar DNA demethylation function.

[0026] In some embodiments, the open reading frame (ORF) encodes a recombinant protein containing the amino acid sequence from positions 80 to 119 of mSTELLA (specifically, the amino acid sequence: QSAFPKRRVRTLLSVLKDPIAKMRRLVRIEQRQKRLEGNE, where Q is at the N-terminus and E is at the C-terminus) with DNA-like demethylation function. The mRNA-encoded variant comprises a recombinant protein having at least 80%, 85%, 90%, 95%, or 98% identity with the wild-type mSTELLA amino acid sequence from positions 80 to 119 and having DNA-like demethylation function.

[0027] In some embodiments, the mRNA is a degenerate or complementary sequence encoding the above-mentioned protein sequence, or an mRNA sequence having at least 80%, 85%, 90%, 95%, or 98% identity, or a sequence encoding a protein with a DNA demethylation function.

[0028] In this document, unless otherwise stated, the amino acid positions of mSTELLA are numbered according to the wild-type protein sequence as shown in SEQ ID NO.1.

[0029] Preferably, the open reading frame (ORF) further includes encoding a polypeptide or protein fused with the target protein for expression; the polypeptide or protein is used for the expression, transport, detection, or tracing of the target protein.

[0030] Preferably, the polypeptide or protein includes a tag protein and / or a protein localization sequence.

[0031] Preferably, the tag protein is selected from any one or a combination of at least two of the following: Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, or SUMO tag.

[0032] Preferably, the protein localization sequence is a nuclear localization sequence (NLS).

[0033] Preferably, the nuclear localization sequence is selected from any one or a combination of at least two of the following: NLS of SV40 large T antigen, NLS of histones H1, H2A, H2B, H3 or H4, NLS of cyclin A, B or D, NLS of ribonucleic acid (RNA) polymerase I or II, NLS of nuclear factor κB (NF-κB), NLS of bovine serum albumin (BSA), NLS of proliferating cell nuclear antigen (PCNA), NLS of adenovirus E1A, cAMP response element binding protein (CREB), NLS of HIV-1 viral protein Rev, NLS1 or NLS2 of yeast nuclear localization sequence, MDMKKKDP containing the “MDM” motif, or KIKKAV sequence without arginine.

[0034] Preferably, the 5'UTR is selected from the 5'UTR of the following genes or their homologs, fragments or variants: β-globin (HBB) gene, heat shock protein 70 (Hsp70) gene, axial dynein heavy chain 2 (DNAH2) gene or 17β-hydroxysteroid dehydrogenase 4 (HSD17B4) gene.

[0035] In some implementations, the variant sequence of the 5'UTR may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the wild-type 5'UTR sequence of the corresponding gene.

[0036] In some embodiments, the 5'UTR comprises a 5'UTR derived from the 17β-hydroxysteroid dehydrogenase 4 (HSD17B4) gene or a homology, fragment, or variant thereof.

[0037] Preferably, the 5'UTR further includes a KOZAK sequence.

[0038] In some embodiments, the 5'UTR comprises a 5'UTR derived from the 17β-hydroxysteroid dehydrogenase 4 (HSD17B4) gene or a homology, fragment or variant thereof, and / or a KOZAK sequence.

[0039] Preferably, the 5'UTR comprises a nucleotide sequence as shown in SEQ ID NO.4 and / or SEQ ID NO.5.

[0040] Preferably, the 3'UTR is selected from the 3'UTR of the following genes or their homologs, fragments or variants: albumin (ALB) gene, α-globin gene, β-globin (HBB) gene, tyrosine hydroxylase gene, heat shock protein 70 (Hsp70) gene or lipoxygenase gene and collagen α gene.

[0041] In some embodiments, the variant sequence of the 3'UTR may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the wild-type 3'UTR sequence of the corresponding gene.

[0042] Preferably, the 3'UTR is a 3'UTR derived from the albumin (ALB) gene or its homolog, fragment, or mutant.

[0043] Preferably, the 3'UTR comprises a nucleotide sequence as shown in SEQ ID NO.6.

[0044] Preferably, the length of the polyA tail is 100-200 nucleotides, for example, it can be 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 nucleotides.

[0045] In some implementations, the polyA tail is 100-150 nucleotides in length.

[0046] In some implementations, the polyA tail is 120 nucleotides in length.

[0047] In some embodiments, the mRNA sequence encoding the mouse developmental pluripotency-associated protein 3mSTELLA, having a cap structure, 5'UTR, 3'UTR, or poly A tail, is shown in any one of SEQ ID NO. 7-10, and the corresponding DNA sequence is shown in SEQ ID NO. 11-14. The mRNA sequence of the truncated mSTELLA is shown in SEQ ID NO. 20 (truncated mSTELLA 51-150) or SEQ ID NO. 21 (truncated mSTELLA 70-150).

[0048] Preferably, the mRNA includes chemically modified nucleosides.

[0049] In some implementations, one or more nucleotides in the mRNA are modified. For example, one or more nucleotides (e.g., all nucleotides) in the mRNA may be independently replaced with naturally occurring or synthetically produced nucleotide analogs.

[0050] Preferably, the chemically modified nucleoside is selected from: pseudouridine, 5-methylcytidine, 5-methyluridine, 2-thiouridine, and N6-methyladenosine, wherein the artificially synthesized nucleotide analogue is selected from any one or a combination of at least two of N1-methylpseudouridine, 5-ethynyluridine, α-thioadenosine triphosphate, α-thiocytidine triphosphate, α-thioguanosine triphosphate, α-thiouridine triphosphate, α-thiopseudouridine triphosphate, or α-thiopseudo-N1-methylpseudouridine triphosphate.

[0051] In some implementations, the naturally occurring nucleotide analogue is selected from any one of pseudouridine, 2-thiouridine, 5-methyluridine, 5-methylcytidine, or N6-methyladenosine.

[0052] In some implementations, the synthetically produced nucleotide analogue is selected from N1-methylpseuuridine or 5-ethynyluridine.

[0053] In some embodiments, the artificially synthesized nucleotide analogue is selected from any one of α-adenosine triphosphate, α-cytidine triphosphate, α-guanosine triphosphate, α-uridine triphosphate, α-pseudouridine triphosphate, or α-N1-methylpseudouridine triphosphate.

[0054] In some implementations, one or more uridine triphosphates in the mRNA are each independently replaced with pseudouridine triphosphate, 2-thiouridine triphosphate, 5-methyluridine triphosphate, N1-methylpseudouridine triphosphate, 5-ethynyluridine triphosphate, α-thiouridine triphosphate, α-thiouridine pseudouridine triphosphate, or α-thiouridine N1-methylpseudouridine; and / or one or more cytidine triphosphates are each independently replaced with 5-methylcytidine triphosphate or α-thiocytidine triphosphate; and / or one or more adenosine triphosphates may be each independently replaced with N6-methyladenosine triphosphate or α-thioadenosine triphosphate.

[0055] In some implementations, one or more uridine triphosphates in the mRNA are each independently replaced with pseudouridine triphosphate, N1-methylpseudouridine triphosphate, 5-ethynyluridine triphosphate, α-thiouridine triphosphate, α-thiouridine pseudouridine triphosphate, or α-thiouridine N1-methylpseudouridine triphosphate.

[0056] In some implementations, one or more cytidine triphosphates in the mRNA are each independently replaced with 5-methylcytidine triphosphate or α-cytidine thiothiophosphate.

[0057] In this invention, the method for preparing the mRNA encoding mouse developmental pluripotency-associated protein 3 mSTELLA, as described in the first aspect, includes:

[0058] (A) mRNA is synthesized by transcription using a DNA sequence as a template; wherein the DNA sequence encodes wild-type mSTELLA protein, mutant mSTELLA protein, recombinant protein containing the amino acid sequence from position 80 to position 119 of mSTELLA, or recombinant protein containing more than 80% identity with the amino acid sequence from position 80 to position 119 of mSTELLA protein and having a similar DNA demethylation function.

[0059] (B) The mRNA is sequence optimized and / or chemically modified to obtain mRNA encoding wild-type protein, mutant protein or recombinant protein.

[0060] In a second aspect, the present invention provides a DNA that encodes the mRNA of the mouse developmental pluripotency-associated protein 3mSTELLA as described in the first aspect. In some embodiments, the DNA of the present invention is used for in vitro transcription to prepare the mRNA of the present invention.

[0061] Thirdly, the present invention provides a recombinant plasmid comprising the DNA sequence described in the second aspect.

[0062] Preferably, the backbone of the recombinant plasmid is pCDNA3.1 plasmid.

[0063] Preferably, the recombinant plasmid further comprises a start sequence (Ori), a T7 promoter, a 5'UTR, and a 3'UTR.

[0064] Preferably, the starting sequence is pUC Ori, and the sequence of pUC Ori includes the nucleotide sequence shown in SEQ ID NO.15.

[0065] Preferably, the sequence of the T7 promoter comprises the nucleotide sequence shown in SEQ ID NO.16.

[0066] Preferably, the 5'UTR is selected from the 5'UTR of the following genes or their homologs, fragments or variants: β-globin (HBB) gene, heat shock protein 70 (Hsp70) gene, axial dynein heavy chain 2 (DNAH2) gene or 17β-hydroxysteroid dehydrogenase 4 (HSD17B4) gene.

[0067] In some implementations, the variant sequence of the 5'UTR may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the wild-type 5'UTR sequence of the corresponding gene.

[0068] In some embodiments, the 5'UTR comprises a 5'UTR derived from the 17β-hydroxysteroid dehydrogenase 4 (HSD17B4) gene or a homology, fragment, or variant thereof.

[0069] Preferably, the 5'UTR further includes a KOZAK sequence.

[0070] Preferably, the 5'UTR comprises a 5'UTR derived from the 17β-hydroxysteroid dehydrogenase 4 (HSD17B4) gene or a homology, fragment or variant thereof, and / or a KOZAK sequence.

[0071] Preferably, the 5'UTR comprises a nucleotide sequence as shown in SEQ ID NO.4 and / or SEQ ID NO.5.

[0072] Preferably, the 3'UTR is selected from the 3'UTR of the following genes or their homologs, fragments or variants: albumin (ALB) gene, α-globin gene, β-globin (HBB) gene, tyrosine hydroxylase gene, heat shock protein 70 (Hsp70) gene, lipoxygenase gene or collagen α gene.

[0073] In some embodiments, the variant sequence of the 3'UTR may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the wild-type 3'UTR sequence of the corresponding gene.

[0074] Preferably, the 3'UTR is a 3'UTR derived from the albumin (ALB) gene or its homolog, fragment, or mutant.

[0075] Preferably, the 3'UTR comprises a nucleotide sequence as shown in SEQ ID NO.6.

[0076] Preferably, the recombinant plasmid further comprises polyA, a resistance gene promoter, and a resistance gene.

[0077] Preferably, the length of the polyA is 100-200 nucleotides, for example, it can be 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200, etc.

[0078] In some implementations, the polyA tail is 100-150 nucleotides in length.

[0079] In some implementations, the polyA tail is 120 nucleotides in length.

[0080] Preferably, the resistance gene promoter is an ampicillin resistance gene promoter.

[0081] Preferably, the resistance gene is an ampicillin resistance gene and / or a neomycin resistance gene.

[0082] Preferably, the recombinant plasmid comprises a nucleotide sequence as shown in SEQ ID NO.17.

[0083] Fourthly, the present invention provides the application of the mRNA encoding mouse developmental pluripotency-associated protein 3 mSTELLA as described in the first aspect in the preparation of mouse developmental pluripotency-associated protein 3 mSTELLA.

[0084] In this invention, an in-depth analysis of mouse developmental pluripotency-related protein 3 mSTELLA was conducted, and a corresponding mRNA was developed and designed. Furthermore, it was discovered that a shorter mRNA (containing only amino acids encoding positions 80 to 119) can also effectively express a protein with similar functions, providing more possibilities for mRNA sequence design.

[0085] Fifthly, the present invention provides a protein-carrier particle, the protein-carrier particle comprising any one of the following proteins and a carrier material encapsulating the protein:

[0086] The protein includes any one or a combination of at least two of the proteins derived from the translation of the mRNA encoding mouse developmental pluripotency-associated protein 3 mSTELLA as described in the first aspect.

[0087] In some implementations, the carrier material may be selected from protein, peptide nanoparticles (PNP), lipid nanoparticles (LNP), polymeric materials, or inorganic nanoparticles.

[0088] In a preferred embodiment, the carrier material is polypeptide nanoparticles (PNP).

[0089] In some embodiments, the PNP may comprise one or more of phospholipid compounds, polyethylene glycol-modified lipids, cholesterol and its derivatives, fatty acid-coupled peptides, or fluorescent dyes. For example, the PNP may comprise any one of phospholipid compounds, polyethylene glycol-modified lipids, cholesterol and its derivatives, fatty acid-coupled peptides, or fluorescent dyes, wherein any two, any three, any four, or all five are combined.

[0090] In a sixth aspect, the present invention provides an mRNA-carrier particle comprising the mRNA encoding mouse developmental pluripotency-associated protein 3 mSTELLA as described in the first aspect and a carrier material encapsulating the mRNA.

[0091] In some implementations, the carrier material may be selected from protein, peptide nanoparticles (PNP), lipid nanoparticles (LNP), exosomes, polymer materials or inorganic nanoparticles.

[0092] In a preferred embodiment, the carrier material is LNP.

[0093] In some implementations, the LNP may comprise one or more of ionized lipids, polyethylene glycol-modified lipids, cholesterol and its derivatives, or phospholipids. For example, the LNP may comprise any one of ionized lipids, polyethylene glycol-modified lipids, cholesterol and its derivatives, or phospholipids, wherein any two, any three, or a combination of all four.

[0094] In a seventh aspect, the present invention provides a DNA-carrier particle, the DNA-carrier particle comprising the DNA described in the second aspect and a carrier material encapsulating the DNA.

[0095] In some implementations, the carrier material may be selected from protein, peptide nanoparticles (PNP), lipid nanoparticles (LNP), exosomes, adenovirus (AdV) vectors, adeno-associated virus (AAV) vectors, lentiviral vectors, retroviral vectors, polymeric materials, or inorganic nanoparticles.

[0096] In a preferred embodiment, the carrier material is an adeno-associated virus (AAV) vector and a lentiviral vector.

[0097] In some embodiments, the adeno-associated virus (AAV) vector includes an AAV genome, inverted terminal repeats (ITRs), a promoter, a regulatory sequence, a polyA signal, and a selection marker.

[0098] In some embodiments, the lentiviral vector includes a transfer plasmid, a packaging plasmid, and an envelope plasmid containing a target protein.

[0099] Eighthly, the present invention provides a method for treating tumors, the method comprising administering to a subject an effective amount of any one or a combination of at least two of the following: the mRNA encoding mouse developmental pluripotency-associated protein 3 mSTELLA as described in the first aspect of the present invention; the DNA as described in the second aspect; the recombinant plasmid as described in the third aspect; the protein-carrier particle as described in the fifth aspect; the mRNA-carrier particle as described in the sixth aspect; or the DNA-carrier particle as described in the seventh aspect.

[0100] As used herein, the term "treatment" refers to a reduction in the risk of acquiring or developing a disease or condition, i.e., preventing the development of at least one clinical symptom of the disease in a subject who was susceptible to the disease or had not been exposed to the pathogen prior to the onset of the disease. For example, treatment may include: (i) preventing the disease, disorder, and / or condition in patients who may be susceptible to the disease, disorder, and / or condition but have not yet been diagnosed with it; (ii) suppressing the disease, disorder, and / or condition, i.e., preventing its development; or (iii) alleviating the disease, disorder, and / or condition, i.e., causing the remission of the disease, disorder, and / or condition.

[0101] As used herein, the term "effective amount" refers to the amount of a compound that is sufficient to achieve such treatment or prevention when administered to a subject for the treatment or prevention of a disease. "Effective amount" can vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject being treated. "Therapeutic effective amount" refers to an effective amount for therapeutic treatment. "Prophylactic effective amount" refers to an effective amount for prophylactic treatment.

[0102] In some embodiments of the invention, the therapeutically effective amount is an effective amount for tumor treatment. The therapeutically effective amount is determined by a diagnostic participant, such as someone skilled in the art, using known techniques and observations in similar circumstances. In determining the therapeutically effective amount or dosage, the diagnostic participant should consider many factors, including but not limited to: animal species, weight, age, health status, disease acquired, severity of disease, individual response, specific compound used, specific composition used, route of administration, biocompatibility of the formulation, duration of treatment, excretion rate of the specific compound used, selected dose range, drugs used in combination with or concurrently with the specific compound used, and similar factors well known in the medical field, and other relevant circumstances.

[0103] In some embodiments, the effective therapeutic dose is 0.0001-500 mg / kg body weight per day.

[0104] In some embodiments, the effective therapeutic dose is 0.01-300 mg / kg body weight.

[0105] In this invention, the total daily dose can be a single dose or an equal portion. If necessary, the effective daily dose can be divided into multiple doses for administration. Therefore, a single-dose composition can contain such an amount or an approximation thereof that constitutes the daily dose.

[0106] Of course, it should be understood that the effective therapeutic dose in this invention can be reasonably adjusted based on the subject's health status, disease progression, species origin, and other information, and can be converted into equal or approximate doses between different species based on common knowledge in the field.

[0107] As used herein, the term "administration" means the physical introduction of a drug agent into a subject using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes, such as by injection or infusion.

[0108] As used herein, the terms “subject,” “individual,” and “patient” are well-known in the art and are used interchangeably to refer to any subject requiring treatment, particularly a mammalian subject. Examples include, but are not limited to, humans and other primates, including non-human primates such as chimpanzees and other ape and monkey species. Non-human mammals may be any, but are not limited to, mice, rats, guinea pigs, hamsters, pigs, dogs, sheep, monkeys, rabbits, cats, cattle, or horses. The terms individual, subject, and patient do not, in themselves, indicate a specific age, sex, race, etc.

[0109] In an embodiment of the method of the present invention, the disease is a tumor, such as colorectal cancer, breast cancer, lung cancer, leukemia, or lymphoma.

[0110] In a ninth aspect, the present invention provides the use of the mRNA encoding mouse developmental pluripotency-associated protein 3 mSTELLA as described in the first aspect, the DNA as described in the second aspect, the recombinant plasmid as described in the third aspect, the protein-carrier particle as described in the fifth aspect, the mRNA-carrier particle as described in the sixth aspect, or the DNA-carrier particle as described in the seventh aspect in the preparation of a drug.

[0111] In some embodiments of the present invention, the drug is an anti-tumor drug.

[0112] In some embodiments of the present invention, the tumor includes solid tumors and / or non-solid tumors.

[0113] In some embodiments of the present invention, the tumor includes any one of colorectal cancer, breast cancer, lung cancer, leukemia, or lymphoma.

[0114] In some embodiments of the present invention, the drug further contains a second active ingredient.

[0115] In some embodiments of the present invention, the second active ingredient includes an immune checkpoint inhibitor.

[0116] In some embodiments of the present invention, the immune checkpoint inhibitor includes at least one of anti-PD1 antibody, anti-PD-L1 antibody, or anti-CTLA4 antibody.

[0117] In some embodiments of the present invention, the drug further contains pharmaceutically acceptable excipients.

[0118] In some embodiments of the present invention, the pharmaceutically acceptable excipients include, but are not limited to, at least one of fillers, binders, disintegrants, lubricants, etc.

[0119] In a tenth aspect, the present invention provides a tumor therapeutic drug comprising at least one or a combination of at least two of the following: mRNA encoding mouse developmental pluripotency-associated protein 3 as described in the first aspect; DNA as described in the second aspect; a recombinant plasmid as described in the third aspect; a protein-carrier particle as described in the fifth aspect; an mRNA-carrier particle as described in the sixth aspect; or a DNA-carrier particle as described in the seventh aspect.

[0120] Preferably, the tumor treatment drug further includes an immune checkpoint inhibitor.

[0121] Preferably, the immune checkpoint inhibitor includes at least one antibody selected from anti-PD1 antibody, anti-PD-L1 antibody, and anti-CTLA4 antibody.

[0122] Eleventhly, the present invention provides a method for treating tumors in combination with immune checkpoint inhibitors, the method comprising administering to a subject an effective amount of at least one or a combination of at least two of the following: the mRNA encoding mouse developmental pluripotency-associated protein 3 as described in the first aspect of the present invention; the DNA as described in the second aspect; the recombinant plasmid as described in the third aspect; the protein-carrier particle as described in the fifth aspect; the mRNA-carrier particle as described in the sixth aspect; or the DNA-carrier particle as described in the seventh aspect; and administering immune checkpoint inhibitors simultaneously or sequentially.

[0123] In some embodiments of the present invention, the immune checkpoint inhibitor includes at least one antibody selected from anti-PD1 antibody, anti-PD-L1 antibody, and anti-CTLA4 antibody.

[0124] In some embodiments of the present invention, the tumor includes any one of colorectal cancer, breast cancer, lung cancer, leukemia, or lymphoma.

[0125] In a twelfth aspect, the present invention provides the use of at least one or a combination of at least two of the following: the mRNA encoding mouse developmental pluripotency-associated protein 3 mSTELLA as described in the first aspect; the DNA as described in the second aspect; the recombinant plasmid as described in the third aspect; the protein-carrier particle as described in the fifth aspect; the mRNA-carrier particle as described in the sixth aspect; or the DNA-carrier particle as described in the seventh aspect, in the preparation of a therapeutic agent for diseases caused by DNA hypermethylation or related disease conditions.

[0126] In some embodiments of the present invention, diseases caused by DNA hypermethylation include any one of autoimmune diseases, nervous system diseases, mental diseases, reproductive diseases, cardiovascular diseases, metabolic diseases, and chronic inflammatory diseases.

[0127] In some embodiments of the present invention, diseases caused by DNA hypermethylation include any one of systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, Alzheimer's disease, depression, bipolar disorder, autism spectrum disorder, infertility, atherosclerosis, type 1 and type 2 diabetes, obesity, thalassemia, myelodysplastic syndrome, and inflammatory bowel disease.

[0128] In some embodiments of the present invention, disease-related conditions caused by DNA hypermethylation include reproductive disorders, recurrent miscarriages, abnormal embryonic development, birth defects, abnormal growth and development, neurodevelopmental disorders, and obesity.

[0129] In a thirteenth aspect, the present invention provides the application of at least one or a combination of at least two of the following in the process of cell reprogramming: the mRNA encoding mouse developmental pluripotency-associated protein 3 mSTELLA as described in the first aspect; the DNA as described in the second aspect; the recombinant plasmid as described in the third aspect; the protein-carrier particle as described in the fifth aspect; the mRNA-carrier particle as described in the sixth aspect; or the DNA-carrier particle as described in the seventh aspect.

[0130] In some embodiments of the present invention, the cell reprogramming process includes inducing pluripotent stem cells, regulating gene expression, altering cell fate, and improving reprogramming efficiency.

[0131] In some embodiments of the present invention, the cell reprogramming process is used to promote tissue repair and regeneration.

[0132] In a fourteenth aspect, the present invention provides the use of at least one or a combination of at least two of the following in the preparation of engineered immune cells: the mRNA encoding mouse developmental pluripotency-associated protein 3 mSTELLA as described in the first aspect, the DNA as described in the second aspect, the recombinant plasmid as described in the third aspect, the protein-carrier particle as described in the fifth aspect, the mRNA-carrier particle as described in the sixth aspect, or the DNA-carrier particle as described in the seventh aspect.

[0133] In some embodiments of the present invention, the immune cells are T cells or NK cells.

[0134] In some embodiments of the present invention, the engineered immune cells are prepared for cell immunotherapy.

[0135] Compared with the prior art, the present invention has the following beneficial effects:

[0136] This invention provides an mRNA encoding the mouse developmental pluripotency-associated protein 3 mSTELLA, its preparation method, its composition, and its applications. This invention designs and produces mRNA encoding mSTELLA, and increases the stability of the mRNA molecule and improves the efficiency of protein translation by adding 5'UTR and 3'UTR upstream and downstream of the open reading frame (ORF), optimizing the coding sequence, and chemically modifying it. This invention also provides the DNA sequence and plasmid for preparing mSTELLA mRNA. The mRNA-carrier particles, protein-carrier particles, and DNA-carrier particles provided by this invention can express the mSTELLA protein with DNA methylation inhibition function in vitro and in vivo, inhibiting cellular DNA methylation, thereby achieving the treatment of diseases caused by DNA hypermethylation or related diseases, and also providing a new strategy for cell reprogramming and engineered immune cell preparation. Attached Figure Description

[0137] Figure 1 A schematic diagram illustrating the interaction between the UHRF1 TTD-PHD domain and mSTELLA;

[0138] Figure 2 The image shows the results of immunoprecipitation between UHRF1 and the Flag-labeled mSTELLA mutant in 293T cells.

[0139] Figure 3The interaction between UHRF1 and amino acids 80-119 of mSTELLA in 293T cells is shown in Figure A, which is a schematic diagram of the recombination of amino acids 75-121 of human hSTELLA protein with the corresponding amino acids 80-119 of mouse mSTELLA protein. Figure B is a diagram of the results of immunoprecipitation between UHRF1 and Flag-labeled mSTELLA and the human recombinant protein hSTELLA-m80-119 containing amino acids 80-119 of mSTELLA.

[0140] Figure 4 This is a schematic diagram of the plasmid structure of pCDNA3.1-mSTELLA.

[0141] Figure 5 The image shows the electrophoresis results of the mRNA obtained through plasmid transcription.

[0142] Figure 6 The image shows the results of Western blot analysis of mSTELLA protein expressed by mSTELLAmRNA.

[0143] Figure 7 The figure shows the results of cancer-specific promoter DNA demethylation analysis on days 2, 6, and 10 after administration of the combination of LNP, LNP-mSTELLA mRNA, and LNP-mSTELLA-h75-121 mRNA to HCT116 cells.

[0144] Figure 8 The results show the tumor inhibitory effects of mRNA lipid nanoparticles. Figure A shows the cell growth curves of HCT116 cells after treatment with PBS, LNP, LNP-mSTELLA mRNA, and LNP-mSTELLA-h75-121 mRNA. Figure B shows the inhibitory effects of LNP-NC, LNP-mSTELLA 51-150 mRNA, LNP-mSTELLA 70-150 mRNA, and LNP-mSTELLA mRNA on the proliferation of HCT116 cells, compared to the PBS group.

[0145] Figure 9 The upregulation of colorectal cancer-associated tumor suppressor genes TSGs by LNP-mSTELLA 51-150 mRNA, LNP-mSTELLA 70-150 mRNA and LNP-mSTELLA mRNA.

[0146] Figure 10 Figure 1 shows the results of colony formation experiments on HCT116 cells after treatment with PBS, LNP, LNP-mSTELLA mRNA and LNP-mSTELLA-h75-121 mRNA.

[0147] Figure 11 The graphs show the tumor-inhibiting effects of LNP-mSTELLA mRNA in the HCT116 mouse xenograft tumor model. Figure A shows the tumor volume change, and Figure B shows the body weight comparison between the groups on day 16.

[0148] Figure 12 Figure 1 shows the changes in DNA methylation and related genes in HCT116 and RKO cells transfected with lentiviruses that stably express hSTELLA and mSTELLA. Figure A shows the results of cancer-specific promoter DNA demethylation analysis, and Figure B shows the relationship between gene upregulation and promoter DNA demethylation.

[0149] Figure 13 The figures show the results of xenograft lentivirus transfection of HCT116 cells and RKO cells stably expressing hSTELLA and mSTELLA in mouse models. Figures A and B show the change in xenograft tumor volume in mice over time. Figures C and D show the comparison of tumor volume of HCT116 mouse xenografts on day 25 and RKO mouse xenografts on day 19. Figures E and F show the survival curves of mouse xenografts. Detailed Implementation

[0150] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0151] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0152] The sequences involved in this invention are shown in Table 1.

[0153] Table 1

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163] Example 1: Confirming the key site of mSTELLA protein as a methylation inhibitor - I. Structural basis of the interaction between mSTELLA and UHRF1

[0164] 1. Co-crystallization of the TTD-PHD domains of mSTELLA and UHRF1

[0165] A truncated peptide from positions 85-119 of the mSTELLA protein was mixed with the TTD-PHD domain of the UHRF1 protein at a molar ratio of 1.5:1 and concentrated to 20 mg / mL. Crystals were grown at 293 K using a sitting-drop vapor diffusion method. Crystals of TTD-PHD and the truncated mSTELLA peptide were grown in 0.6 M NaCl, 0.1 M MES, pH 6.5, and 20% PEG4000.

[0166] 2. Collection and processing of eutectic diffraction data

[0167] The obtained crystals were cryoprotected in a mother liquor containing 30% glycerol, and data were collected at 100 K. Diffraction data for TTD-PHD and the truncated mSTELLA peptide were collected on the 02U1 beamline of the Shanghai Synchrotron Radiation Facility. The data were then automatically processed by the Aquariumpipeline and corrected using Aimless. The molecular structures of TTD-PHD and the truncated mSTELLA peptide were resolved using a molecular substitution method based on the TTD-PHD / H3K9me3 (PDB: 3ASK) structural model. Structural refinement was performed using Refmac5.

[0168] Figure 1 The mSTELLA truncated peptide exhibits an L-shaped fold consisting of a short α-helix (hereinafter referred to as αS) and a long α-helix (hereinafter referred to as αL) that binds to UHRF1. Figure 1 (B). Among them, the N-terminal motif of the truncated peptide 88 VRT 90 Occupying the acidic pocket of the PHD domain, R89 forms hydrogen bonds with D334 and D337 residues of UHRF1, while V88 and αS primarily mediate hydrophobic interactions. Figure 1 (C). The P98 residue of the mSTELLA truncated peptide causes a bend at the αS and αL junction, while partially inserting into the aromatic cage composed of F152, Y188, and Y191 residues of the UHRF1 TTD domain. Figure 1In the middle (D), protein binding is enhanced through non-covalent interactions. Simultaneously, αL inserts into the cavity between the PHD and TTD domains, where positively charged residues K101, R104, R107, and R111 form polar interactions with D307, C316, D337, and E335 residues of the UHRF1 PHD domain, while L105 and I108 interact with UHRF1 residues C302, C316, V315, and M338. Figure 1 (A). Furthermore, the crystal structure also reveals hydrophobic contacts between I99 and M102 in αL and M148, F152, F237, and P300 in the UHRF1 TTD-Linker region, as well as hydrogen bonds between residue R103 of αL and D230 of UHRF1. Figure 1 (D).

[0169] The above protein structure results show that V88, R89, T90, P98, I99, K101, M102, R103, R104, L105, R107, I108 and R111 residues of the mSTELLA protein are key sites for the interaction between mSTELLA and UHRF1.

[0170] II. Immunoprecipitation verification of mSTELLA as a key site for methylation inhibitor

[0171] HEK293T human renal epithelial cells derived from ATCC were cultured in DMEM medium supplemented with 10% v / v fetal bovine serum (FBS) and 1% antibiotics in an incubator at 37°C and 5% CO2.

[0172] Flag-tagged mSTELLA point mutants (V88A, R89A / T90A, K101A, R104A, L105A, R107A, R111A, I99A, M102A, R103A, I99A / M102A / R103A) were generated using the ClonExpress II One Step Cloning Kit (Vazyme) and transfected into HEK293T cells in exponential growth phase. Forty-eight hours after transfection, cells were collected, washed with pre-chilled PBS, and resuspended in lysis buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2.5 mM EDTA (pH 8.0), 1% IGEPEAL (v / v)) supplemented with phosphatase and protease inhibitors. The suspension was incubated on ice for 30 minutes and then sonicated to ensure complete cell lysis. Cell lysates were centrifuged at 12000g for 20 minutes at 4°C, and the supernatant was diluted in IP dilution buffer (20mM Tris-HCl (pH 8.0), 150mM NaCl, 2.5mM EDTA (pH 8.0)) for immunoprecipitation. The diluted supernatant was incubated overnight at 4°C with anti-UHRF1 antibody (Thermo Fisher Scientific, PA5-29884) or anti-FLAG antibody (Sigma-Aldrich, F1804). The antibody-protein complex was incubated with Protein A / G magnetic beads (DynaBeads) at 4°C for 3 hours. The magnetic beads were washed three times for 5 minutes each time with washing buffer (20mM Tris-HCl (pH 8.0), 150mM NaCl, 2.5mM EDTA (pH 8.0), 0.5% IGEPEAL (v / v)). The complex was eluted and denatured with SDS-Laemmli buffer, followed by SDS-PAGE separation and immunoblotting with indicator antibodies.

[0173] Figure 2 These are the results of co-immunoprecipitation (Co-IP) experiments. The results show that mutations in V88A, R89A / T90A, I99A, M102A, I99A / M102A / R103A, R104A, L105A, R107A, and R111 significantly disrupt the binding of mSTELLA-UHRF1, which are key sites for mSTELLA protein to act as a methylation inhibitor. This result is consistent with the crystal structure shown.

[0174] III. Immunoprecipitation verification of recombinant protein containing amino acid sequence 80-119 of mSTELLA

[0175] HEK293T human renal epithelial cells derived from ATCC were cultured in DMEM medium supplemented with 10% v / v fetal bovine serum (FBS) and 1% antibiotics in an incubator at 37°C and 5% CO2.

[0176] Based on the similarity and conservation of sequence length and secondary structure, amino acids 75-121 of the human hSTELLA protein were interchanged with amino acids 80-119 of the corresponding mouse mSTELLA protein, forming the mouse recombinant protein mSTELLA-h75-121 (SEQ ID NO.23) containing amino acids 75-121 of the human hSTELLA protein and the recombinant protein hSTELLA-m80-119 (SEQ ID NO.24) containing amino acids 80-119 of the mSTELLA protein. The Flag-labeled hSTELLA-m80-119 plasmid was constructed and transfected into HEK293T cells in the exponential growth phase. Simultaneously, the Flag-labeled wild-type mSTELLA plasmid was transfected as a control. The binding of the recombinant protein hSTELLA-m80-119 to UHRF1 was verified using the aforementioned immunoprecipitation method.

[0177] Figure 3 The results of the co-immunoprecipitation (Co-IP) experiment showed that the recombinant protein hSTELLA-m80-119, containing amino acids 80-119 of mSTELLA, significantly enhanced the binding of mSTELLA to UHRF1. Therefore, the amino acid sequence 80-119 of mSTELLA is a key region for the binding of mSTELLA protein to UHRF1 and is a crucial sequence for mSTELLA protein to function as a methylation inhibitor. Furthermore, the mouse recombinant mSTELLA protein (mSTELLA-h75-121, SEQ ID NO. 23), obtained by replacing the corresponding sequence of amino acids 75-121 of human hSTELLA, lacks demethylation function, further demonstrating that amino acids 80-119 of mSTELLA are essential for mSTELLA to exert its demethylation function. Figure 7-8 and Figure 10-11 ).

[0178] Example 2: Design and preparation of mSTELLA mRNA

[0179] I. Design of mRNA encoding mouse developmental pluripotency-associated protein 3 mSTELLA

[0180] 1. Sequence design for encoding mSTELLA

[0181] Based on the wild-type mSTELLA sequence, a DNA coding sequence was designed. After adding sequences such as 5'UTR, 3'UTR, poly A tail, and protein localization sequences (SEQ ID NO. 11) to the DNA coding sequence, homologous recombination was performed and inserted into the pCDNA3.1 plasmid to construct the recombinant plasmid pCDNA3.1-mSTELLA (SEQ ID NO. 17) containing the protein coding sequence.

[0182] The pCDNA3.1-mSTELLA plasmid contains elements including a start sequence (SEQ ID NO.15), a T7 promoter sequence (SEQ ID NO.16), a 5' UTR sequence (SEQ ID NO.4 and SEQ ID NO.5), a 3' UTR sequence (SEQ ID NO.6), a polyA tail, an ampicillin resistance gene promoter, an ampicillin resistance gene, and / or a neomycin resistance gene. These non-coding structures regulate the stability, translation efficiency, and immunogenicity of the mRNA transcribed from the pCDNA3.1-mSTELLA plasmid in vitro.

[0183] Sequencing confirmed the accuracy of the constructed plasmid sequence, and the successfully constructed plasmid structure is shown below. Figure 4 As shown.

[0184] 2. Sequence optimization of mRNA encoding mSTELLA

[0185] By optimizing the mRNA sequence, we can increase its yield during transcription, enhance its stability within cells, and improve translation efficiency.

[0186] Employ publicly available mRNA sequence optimization platforms, such as the LinearDesign sequence design algorithm and GenSmart. TM Codon optimization, using tools such as VectorBuilder and Java CodonAdaptation Tool, optimizes the coding sequence of mRNA by employing host-preferred codons, simplifying mRNA secondary structure, and increasing CG content. The optimized sequences are shown in SEQ ID NO. 18 and 19.

[0187] II. mRNA Preparation

[0188] Based on the design of the mSTELLA mRNA described above, the constructed plasmid containing the relevant elements was transformed into *E. coli* for amplification. The purified plasmid was linearized using restriction endonucleases. In vitro transcription was performed using T7 RNA polymerase with the linearized plasmid as a template. The standard co-transcription system included the following components: 7.5 mM ATP, 7.5 mM GTP, 7.5 mM CTP, 7.5 mM UTP, 7.5 mM Cap1 analogue, IVT buffer (40 mM Tris-HCl (25℃, pH 8.0), 20 mM MgCl2, 2.5 mM TCEP, 2 mM spermidine), 2 μg T7 RNA polymerase, 1 μg linearized DNA template, RNase inhibitor (2 U / μL), and inorganic pyrophosphatase (0.03 U / 20 μL). After incubation at 37℃ for 2–4 h, the mRNA product was obtained.

[0189] Then DNase I was added to the reaction system to digest the DNA template.

[0190] mRNA was purified using either lithium chloride precipitation or column purification. The specific method for lithium chloride precipitation was as follows: Add 2.5M LiCl solution to the digested reaction system, vortex to mix, and incubate at -20°C for 30 min. Then, centrifuge at 15000g for 15 min at 4°C, carefully discarding the supernatant. Wash the precipitate twice with 70% ethanol, air dry, and finally dissolve the mRNA in enzyme-free water. Column purification was performed according to the manufacturer's instructions.

[0191] Alternatively, capping and / or tailing kits can be used to cap and / or tail mRNA separately. Specific operating procedures should be followed according to the kit instructions.

[0192] pCDNA3.1-mSTELLA plasmid was transcribed in vitro to obtain mSTELLAmRNA, which was then translated into mSTELLA protein at the cellular and animal levels. The quality of the mRNA was assessed using agarose gel electrophoresis and capillary electrophoresis. Figure 5 Agarose gel electrophoresis results showed that the transcribed mRNA band was single and undegraded.

[0193] Using the above method, mRNAs as shown in SEQ ID NO.20, SEQ ID NO.21, and SEQ ID NO.22 were also obtained, named mSTELLA 51-150, mSTELLA 70-150, and mSTELLA-h75-121, respectively. The corresponding encoded protein sequences are truncated mSTELLA 51-150, truncated mSTELLA 70-150, and a recombinant protein (mSTELLA-h75-121) containing amino acids 75-121 of human hSTELLA without demethylation function (SEQ ID NO.23).

[0194] Example 3: Preparation of lipid nanoparticles of mRNA

[0195] Lipid nanoparticles containing mRNA were prepared using the mRNA synthesized in Example 2. Cationic lipids, distearate phosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000) were dissolved in ethanol at a molar ratio of 48.5:10:40:1.5. The lipid mixture was rapidly mixed with mRNA dissolved in 50 mM sodium citrate buffer (pH 5.0) at a volume ratio of 1:3. The lipid ethanol solution and the mRNA sodium citrate buffer were mixed using a microfluidic mixer to obtain lipid nanoparticles. Ethanol was then removed by dialysis, followed by ultrafiltration concentration and filtration through a 0.22 μm filter to obtain LNP-mRNA nanoparticles, which were stored at 4°C for use. The particle size, polydispersity index (PDI), and encapsulation efficiency data of the formulation are shown in Table 2.

[0196] Table 2

[0197]

[0198] Example 4: Cellular Level Evaluation

[0199] 1. Cell culture:

[0200] The human colorectal cancer cell line HCT116 was derived from ATCC and cultured in McCoy's 5A Modified Medium supplemented with 10% v / v fetal bovine serum (FBS) at 37°C and 5% CO2.

[0201] 2. Cell transfection:

[0202] The day before transfection, cells were digested with trypsin, counted, and seeded into culture plates to ensure a cell density of 80% at transfection. LNP-mRNA encapsulated in lipid nanoparticles was diluted with culture medium and added to the cell culture plates for transfection. The culture plates were then incubated at 37°C with 5% CO2. Samples were collected on days 2 (D2), 6 (D6), and 10 (D10), and appropriate detection methods were selected according to the experimental objectives.

[0203] 3. Western blot for protein immunoblotting

[0204] Cells were washed twice with pre-chilled PBS, homogenized using a homogenization column with 4% SDS cell lysis buffer, and then the total protein content was determined using the Pierce BCA protein assay (Thermo Fisher Scientific). Samples were separated by 10% SDS-PAGE gel, transferred to a PVDF membrane (Sigma-Aldrich), and blocked with 5% skim milk powder dissolved in TBST. The PVDF membrane was incubated overnight at 4°C with antibodies in the following ratio: anti-STELLA (1:300, Sigma-Aldrich, MAB4388) and anti-β-actin (1:5000, Sigma-Aldrich, A5441). The next day, the PVDF membrane was washed five times in TBST for 5 minutes each time. The membrane was then incubated with horseradish peroxidase (HRP)-labeled secondary antibody, reacted with ECL substrate (Pierce), and developed.

[0205] The expression levels of mSTELLA protein and mSTELLA-h75-121 recombinant protein by LNP-encapsulated mSTELLA mRNA and mSTELLA-h75-121 mRNA in HCT1116 cells are shown in the figure. Figure 6 The results showed that LNP can effectively deliver mRNA into cells, and the mRNA is translated and expressed at a high level within the cells to express the target protein.

[0206] 4. Cancer-specific promoter DNA methylation analysis

[0207] Genomic DNA methylation analysis was performed using the Promega Wizard Genomic DNA Purification Kit, following the manufacturer's instructions. The extracted genomic DNA underwent bisulfite conversion, deaminated unmethylated cytosine (C) to uracil (U), while methylated cytosine remained unchanged. The methylation status of whole-genome DNA was determined using the Illumina Infinium Methylation EPIC BeadChip methylation site detection chip. Raw data were preprocessed and normalized using R and the Bioconductor minfi package. Probes with p-values ​​> 0.01 were excluded from subsequent analyses, and methylation β-values ​​for probes were defined. Probes were mapped to the human genome GRCh37 / hg19 version and annotated using the Illumina HumanMethylation EPICanno.ilm10b2.hg19 package, and correlated with promoters, CpG islands, and cancer-specific methylation patterns.

[0208] Following the above method, HCT116 cells treated with LNP empty vector, LNP-mSTLELLA mRNA, and LNP-mSTLLA-h75-121 mRNA were analyzed for methylation on days 2, 6, and 10. The experimental results are as follows: Figure 7 As shown, HCT116 cells treated with LNP-mSTELLA mRNA showed a significant decrease in cancer-specific promoter methylation on day 2, and this decrease continued over time. In contrast, neither the LNP empty vector group nor the LNP-mSTELLA-h75-121 mRNA control group showed any demethylation effect.

[0209] It is evident that LNP-mSTELLA mRNA exhibits significant cancer-specific promoter demethylation at the cellular level. However, the recombinant protein mSTELLA-h75-121, containing amino acids 75-121 of human hSTELLA, lacks demethylation function, indicating that amino acid sequences 80-119 of mSTELLA are the key region for mSTELLA protein binding to UHRF1 and are crucial for mSTELLA protein's role as a methylation inhibitor.

[0210] 5. Cell proliferation detection

[0211] Prepare HCT116 cell suspension, according to a plating density of 1×10⁶ cells / mL. 4Cells were seeded in 6-well plates and incubated overnight at 37°C with 5% CO2. Cells were transfected with cell culture medium containing PBS, LNP empty vector, LNP-mSTELLA mRNA, and LNP-mSTELLA-h75-121 mRNA. Cells were counted daily from day 1 to day 5 post-treatment to assess cell proliferation.

[0212] Figure 8 Figure A shows that compared with the PBS group, LNP-mSTELLA mRNA significantly reduced cell proliferation, while LNP empty vector and LNP-mSTELLA-h75-121 mRNA expressing recombinant protein without demethylation function showed no significant difference from the PBS group. In the figure, ****P<0.0001, and ns indicates no significance.

[0213] It is evident that LNP-mSTELLAmRNA can inhibit the proliferation of tumor cells through demethylation.

[0214] Using the same method, cells were transfected with cell culture media containing PBS, LNP-mSTELLA 51-150 mRNA, LNP-mSTELLA 70-150 mRNA, and LNP-mSTELLA mRNA, with LNP-coated luciferase mRNA serving as a negative control (LNP-NC). Cell counts were performed on day 3 post-treatment. The effect of empty LNP vectors and different mRNAs on cell proliferation was evaluated by calculating the cell proliferation percentage (%) as (number of cells in the test group - number of cells in the PBS group) / number of cells in the PBS group × 100%.

[0215] Figure 8 Figure B shows that, compared to mSTELLA mRNA expressing the full-length protein sequence, truncated mSTELLA mRNAs (positions 51-150 and 70-150) also inhibited cell proliferation. This further demonstrates that amino acid positions 80-119 of mSTELLA are the key region for mSTELLA protein binding to UHRF1 and are crucial for mSTELLA's role as a methylation inhibitor. Designing shorter mRNAs (containing amino acids encoding positions 80-119) can also effectively express proteins with similar demethylation functions.

[0216] 6. Upregulation of tumor suppressor genes TSGs by mSTELLA mRNA

[0217] Cell cultures containing LNP-mSTELLA 51-150 mRNA, LNP-mSTELLA 70-150 mRNA, and LNP-mSTELLA mRNA were transfected, with LNP-coated luciferase mRNA serving as a negative control (LNP-NC). Transcriptional levels of colorectal cancer-related tumor suppressor genes (TSGs) were detected by real-time quantitative PCR on day 10 post-treatment. These included the WNT signaling pathway antagonist SFRP1, the apoptosis-regulating deubiquitinating enzyme UCHL1, and the angiogenesis regulator FBLN2.

[0218] Total RNA was extracted using RNAiso Plus reagent (Takara), and then 1 μg of total RNA was mixed with PrimeScript. TM cDNA was synthesized using RT Master Mix (Takara). Differential expression of SFRP1, UCHL1, and FBLN2 was detected using the SYBR Green PCR Kit (Takara) with cDNA samples by qPCR. β-actin was selected as the normalized internal reference gene for qPCR detection. The fold change compared to the control group was calculated using the ΔΔCt method.

[0219] Figure 9 The results showed that both full-length mSTELLA mRNA and truncated mSTELLA mRNA 51-150 and mSTELLA mRNA 70-150 exhibited excellent demethylation effects, and the transcriptional levels of tumor suppressor genes SFRP1, UCHL1 and FBLN2 were significantly upregulated after treatment.

[0220] 7. Cloning experiments

[0221] Similar to cell proliferation assays, clonogenic assays are also commonly used to assess the proliferative capacity of cells.

[0222] Single-cell suspensions of HCT116 cells were seeded into six-well plates with pre-treated bottoms at a density of 1000 cells per well and cultured at 37°C and 5% CO2. Cells were transfected with cell culture medium containing PBS, LNP empty vector, LNP-mSTELLA mRNA, and LNP-mSTELLA-h75-121 mRNA. After 10 days, the culture medium was removed, and the cells were stained with 0.1% crystal violet (dissolved in sterile water containing 20% ​​methanol) for visualization and quantification. Figure 10The results of the colony formation assay showed that tumor cells treated with LNP-mSTELLA mRNA exhibited significantly reduced proliferation and difficulty in forming cell colonies, while tumor cells treated with LNP-mSTELLA-h75-121 mRNA showed no significant difference in proliferation compared to those treated with PBS and LNP empty vector. Data in the figure are expressed as mean ± standard deviation (n = 3), ****P < 0.0001, ns indicates no statistical significance.

[0223] Example 5: In vivo evaluation - mouse tumor experiment

[0224] Experimental mice: 5-6 week old NOD-Prkdcem26Cd52Il2rgem26Cd22 / Gpt(NCG) mice

[0225] Experimental methods: NCG mice were housed under standardized conditions in a specific pathogen-free (SPF) grade animal facility. 1×10⁶ mice were... 6 HCT116 cells were mixed with Matrigel (1:1) in 0.1 mL PBS and subcutaneously injected into one side of the abdomen of each mouse. Once the tumor was palpable, mice were randomly assigned to five groups, receiving peritumoral injections of 30 μg and 90 μg of LNP-mSTELLA mRNA, while control groups included a lipid nanoparticle LNP empty vector group and 30 μg and 90 μg of LNP-mSTELLA-h75-121 mRNA expressing a non-demethylated functional mutant. Every other day, mice received either 0.1 mL of the LNP-mRNA formulation or the LNP empty vector via peritumoral subcutaneous injection for three weeks, with an additional five weeks of observation for the LNP-mSTELLA mRNA group after the administration of the drug. Tumor size was measured every other day, and tumor volume was calculated using the formula 0.5 × (length × width × height). Tumors were classified as tumors when their volume reached 2000 mm². 3 Or, at the end of the LNP-mSTELLAmRNA observation period (whichever comes first), the mice were euthanized.

[0226] The results are as follows Figure 11 As shown in Figure A, the data in the figure are expressed as mean ± standard error (n = 8), ****P < 0.0001, and ns indicates insignificance. An "×" indicates that at least one mouse had a tumor volume exceeding 2,000 mm². 3 The experiment was stopped at that time. The vertical dashed line indicates the time when drug administration was stopped. Both the LNP empty vector group and the 30 μg LNP-mSTELLA-h75-121 mRNA control group showed tumor volumes reaching 2000 mm² in mice on day 16. 3 The experiment was terminated. In the control group (90 μg LNP-mSTELLA-h75-121 mRNA), the tumor volume reached 2000 mmHg on day 20. 3The experiment was terminated. In contrast, the tumor volume in the LNP-mSTELLAmRNA 30μg group and the LNP-mSTELLAmRNA 90μg group did not reach 2000 mmHg until day 40 and day 42, respectively. 3 The experiment was terminated, and mSTELLAmRNA significantly inhibited tumor growth.

[0227] Meanwhile, there was no significant difference in body weight among the different treatment groups, and the LNP-mRNA carrier particle composition showed no significant side effects. Figure 11 (Figure B in the middle)

[0228] As can be seen, the lipid nanoparticles carrying mRNA of the present invention have good delivery efficiency, efficiently express mSTELLA in mice, and have a good inhibitory effect on tumors.

[0229] Example 6: Demethylation function of DNA-vector particles

[0230] 1. Lentiviral transfection of HCT116 and RKO cells

[0231] The cDNAs of hSTELLA and mSTELLA were cloned into the pLenti-III-EF1alpha vector (Applied Biological Materials#LV043) containing a puromycin resistance selection marker. The vector plasmid and packaging plasmids (psPAX2 and pMD2.G) were then co-transfected into HEK293T cells using Lipofectamine 3000 (Thermo Fisher Scientific). Lentiviral particles were harvested at 24, 48, and 72 hours after transfection and filtered through a 0.45 μm filter. The lentiviral particles were then concentrated using PEG 8000 and stored at -80°C.

[0232] HCT116 and RKO cells were transfected with lentiviral particles expressing hSTELLA and mSTELLA prepared using the above method. Simultaneously, an empty lentiviral vector (EV) was transfected as a control group. Three days after transfection, cells were treated with 1-2 μg / mL puromycin for 48 hours to screen for successfully transfected cells expressing the target proteins. Nine days after transfection, the selected HCT116-EV, HCT116-mSTELLA, HCT116-hSTELLA, RKO-EV, RKO-mSTELLA, and RKO-hSTELLA cells were collected for subsequent experiments.

[0233] 2. Verification of the demethylation function of mSTELLA expressed by lentiviral transfection

[0234] HCT116 and RKO cells transfected with the collected lentiviruses were used for DNA methylation analysis. Genomic DNA was extracted from the cells using the Promega Wizard Genomic DNA Purification Kit according to the manufacturer's instructions. The extracted genomic DNA underwent bisulfite conversion, deaminated unmethylated cytosine (C) to uracil (U), while methylated cytosine remained unchanged. The methylation status of whole-genome DNA was determined using the Illumina Infinium MethylationEPIC BeadChip methylation site detection chip. The raw data were preprocessed and normalized using R and the Bioconductor minfi package. Probes with p-values ​​> 0.01 were excluded from subsequent analyses, and methylation β-values ​​for the probes were defined. Probes were mapped to the human genome GRCh37 / hg19 version and annotated using the IlluminaHumanMethylationEPICanno.ilm10b2.hg19 package, and correlated with promoters, CpG islands, and cancer-specific methylation patterns.

[0235] Experimental results are as follows Figure 12 As shown in Figure A, the methylation of cancer-specific promoters in HCT116-mSTELLA cells and RKO-mSTELLA cells was significantly decreased, while there was no significant difference in the degree of cancer-specific promoter methylation between HCT116-hSTELLA cells and RKO-hSTELLA cells and empty vector transfected cells. This indicates that mSTELLA has a demethylation function, while hSTELLA does not. Figure 12 Figure B shows the relationship between gene upregulation and promoter DNA demethylation. The x-axis represents relative gene expression changes (log2 fold changes in hSTELLA and mSTELLA relative to EV), with orange vertical lines representing log2 fold differences of -1 and 1, respectively. The y-axis represents promoter DNA methylation changes, defined by the average difference (Δβ) of promoter probe values ​​for each promoter relative to EV; orange horizontal lines indicate genes with a promoter Δβ value of -0.2. Promoter methylation was decreased in HCT116-mSTELLA and RKO-mSTELLA cells, positively correlated with gene upregulation, indicating the reactivation of numerous potential tumor suppressor genes (TSGs) associated with abnormally high promoter methylation. Conversely, promoter methylation showed no significant changes in HCT116-hSTELLA and RKO-hSTELLA cells, and no significant gene upregulation was observed.

[0236] 3. Mouse model of xenograft lentivirus transfection expressing mSTELLA cells

[0237] Experimental mice: 5-6 week old NOD-Prkdcem26Cd52Il2rgem26Cd22 / Gpt(NCG) mice

[0238] Experimental Methods: NCG mice were housed under standardized conditions in a specific pathogen-free (SPF) grade animal facility. Xenograft mouse models were constructed using HCT116-EV cells, HCT116-mSTELLA cells, HCT116-hSTELLA cells, RKO-EV cells, RKO-mSTELLA cells, and RKO-hSTELLA cells transfected with the aforementioned lentiviruses. 1×10⁻⁶ cells were used to construct xenograft mouse models. 6 Cells were mixed with Matrigel (1:1) in 0.1 mL of PBS and subcutaneously injected into one side of the abdomen of each mouse. Tumor volume and mouse survival time were continuously observed and recorded. From day 5 onwards, tumor volume was measured every other day and calculated using the formula 0.5 × (length × width × height). The tumor volume was determined when it reached 2000 mm². 3 At that time, the mice were euthanized.

[0239] The results are as follows Figure 13 As shown in the figure, the data are expressed as mean ± standard error (n=5), ****P<0.0001, ***P<0.001, **P<0.01, and ns indicates no significance. Figures A, C, and E show the results of xenograft lentivirus-transfected HCT116 cell mice, and Figures B, D, and F show the results of xenograft lentivirus-transfected RKO cell mice. Figures A and B show the change in xenograft tumor volume in mice over time. "×" indicates that at least one mouse had a tumor volume exceeding 2,000 mmHg. 3 At that point, the mice were euthanized, and the experiment was stopped. In the groups transfected with HCT116-EV and HCT116-hSTELLA, mice showed tumor volumes reaching 2000 mmHg by day 25. 3 The experiment was terminated. In the HCT116-mSTELLA group, no mice reached a tumor volume of 2,000 mm² until the 37th day. 3 The RKO cell model showed the same trend, with tumors in mice in both the RKO-EV and RKO-hSTELLA groups reaching 2000 mmHg on day 19. 3 This led to the termination of the experiment, while in the RKO-mSTELLA group, it wasn't until the 37th day that mice reached a tumor volume of 2,000 mmHg. 3Figures C and D compare the tumor volume of HCT116 mouse xenografts on day 25 and RKO mouse xenografts on day 19. Lentivirally transfected mSTELLA significantly inhibited the in vivo proliferation of HCT116 and RKO tumor cells. Figures E and F show the survival curves of mouse xenografts. In the HCT116-EV group, mice reached ethical limits on day 31, in the HCT116-hSTELLA group, and on day 47, in the HCT116-mSTELLA group, and were euthanized, with the survival rate dropping to 0%. In the RKO-EV group, mice reached ethical limits on day 25, in the RKO-hSTELLA group, and on day 43, in the RKO-mSTELLA group, and were euthanized, with the survival rate dropping to 0%. Lentivirally transfected mSTELLA significantly prolonged the survival time of mice with xenografted HCT116 and RKO tumor cells.

[0240] As can be seen, the DNA-vector particles constructed by encapsulating mSTELLADNA with lentivirus in this invention exhibit significant demethylation effects and upregulate the expression of related genes at the cellular level, and significantly inhibit tumor growth and prolong the survival time of mice at the animal level.

[0241] The preferred embodiments of the present invention have been described in detail above. However, the detailed description of the specific embodiments is intended to illustrate, rather than limit, the scope of the invention, which is defined by the scope of the appended claims. Furthermore, different embodiments of the invention can be combined in any way without departing from the core spirit of the invention, and such combinations should also be considered part of the invention. To avoid repetition, all possible combinations will not be described in detail here.

[0242] While the invention has been described with reference to specific embodiments, those skilled in the art will understand that various modifications can be made and equivalents can be used without departing from the spirit and scope of the invention. Various modifications can be made to suit specific circumstances, materials, compositions, methods, and steps to conform to the purpose and spirit of the invention. All such modifications are intended to be within the scope of the claims.

Claims

1. An mRNA encoding the mouse developmental pluripotency-associated protein 3mSTELLA, characterized in that, The mRNA encodes any one of the following proteins: (1) Wild-type mSTELLA protein, the amino acid sequence of which is shown in SEQ ID NO.1; (2) A mutant protein that has more than 80% identity with the wild-type mSTELLA protein shown in SEQ ID NO.1 and has a similar DNA demethylation function; (3) A recombinant protein containing the amino acid sequence from position 80 to position 119 of the wild-type mSTELLA protein shown in SEQ ID NO.1 and having a DNA demethylation function; The amino acid sequence from position 80 to position 119 is: QSAFPKRRVRTLLSVLKDPIAKMRRLVRIEQRQKRLEGNE, where Q is located at the N-terminus and E is located at the C-terminus; (4) A recombinant protein containing more than 80% identity with the amino acid sequence from position 80 to position 119 of the wild-type mSTELLA protein shown in SEQ ID NO.1 and having a similar DNA demethylation function.

2. The mRNA encoding mouse developmental pluripotency-associated protein 3mSTELLA according to claim 1, characterized in that, The mRNA contains an open reading frame (ORF), and the nucleic acid sequence of the ORF includes any of the following: (1) The sequence shown in SEQ ID NO.2; (2) A degenerate or complementary sequence of the sequence shown in SEQ ID NO.2; (3) It has more than 80% identity with the sequence shown in SEQ ID NO.2 and encodes a protein with a similar DNA demethylation function; (4) A sequence comprising the sequence shown in SEQ ID NO.3 and encoding a recombinant protein having a DNA demethylation-like function; (5) A sequence that has more than 80% identity with the sequence shown in SEQ ID NO.3 and encodes a recombinant protein with a similar DNA demethylation function.

3. The mRNA encoding mouse developmental pluripotency-associated protein 3mSTELLA according to claim 2, characterized in that, The 5' end of the mRNA also includes a cap structure and / or a 5' UTR, and the 3' end also includes a 3' UTR and / or a polyA tail; Preferably, the open reading frame (ORF) further includes encoding a polypeptide or protein fused with the target protein for expression; the polypeptide or protein is used for the expression, transport, detection, or tracing of the target protein. Preferably, the polypeptide or protein includes a tag protein and / or a protein localization sequence; Preferably, the tag protein is selected from any one or a combination of at least two of the following: Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, or SUMO tag; Preferably, the protein localization sequence is a nuclear localization sequence (NLS). Preferably, the nuclear localization sequence is selected from any one or a combination of at least two of the following: NLS of SV40 large T antigen, NLS of histones H1, H2A, H2B, H3 or H4, NLS of cyclin A, B or D, NLS of ribonucleic acid polymerase I or II, NLS of nuclear factor κB, NLS of bovine serum albumin, NLS of proliferating cell nuclear antigen, NLS of adenovirus E1A, cAMP response element binding protein, NLS of HIV-1 viral protein, NLS1 or NLS2 of yeast nuclear localization sequence, MDMKKKDP containing the "MDM" motif, or NLS of KIKKAV not rich in arginine. Preferably, the 5'UTR is selected from the 5'UTR of the following genes or their homologs, fragments or variants: β-globin gene, heat shock protein 70 gene, axonoderm heavy chain 2 gene or 17β-hydroxysteroid dehydrogenase 4 gene. Preferably, the 5'UTR further comprises a KOZAK sequence; Preferably, the 5'UTR comprises a 5'UTR derived from the 17β-hydroxysteroid dehydrogenase 4 gene or a homolog, fragment or variant thereof, and / or a KOZAK sequence; Preferably, the 5'UTR comprises the nucleotide sequence shown in SEQ ID NO.4 and / or SEQ ID NO.5; Preferably, the 3'UTR is selected from the 3'UTR of the following genes or their homologs, fragments or variants: albumin gene, α-globin gene, β-globin gene, tyrosine hydroxylase gene, heat shock protein 70 gene, lipoxygenase gene or collagen α gene; Preferably, the 3'UTR is a 3'UTR derived from the albumin gene or a homology, fragment, or mutant thereof; Preferably, the 3'UTR comprises a nucleotide sequence as shown in SEQ ID NO. 6; Preferably, the polyA tail is 100-200 nucleotides in length; Preferably, the mRNA includes a chemically modified nucleoside; Preferably, the chemically modified nucleoside is selected from: pseudouridine, 5-methylcytidine, 5-methyluridine, 2-thiouridine, and N6-methyladenosine, wherein the artificially synthesized nucleotide analogue is selected from: N1-methylpseudouridine, 5-ethynyluridine, α-thioadenosine triphosphate, α-thiocytidine triphosphate, α-thioguanosine triphosphate, α-thiouridine triphosphate, α-thiopseudouridine triphosphate, or any one or a combination of at least two of α-thiopseudouridine N1-methylpseudouridine.

4. A DNA, characterized in that, The DNA encodes the mRNA of the mouse developmental pluripotency-associated protein 3mSTELLA as described in any one of claims 1-3.

5. A recombinant plasmid, characterized in that, The recombinant plasmid comprises the DNA sequence of claim 4; Preferably, the backbone of the recombinant plasmid is pCDNA3.1 plasmid; Preferably, the recombinant plasmid further comprises a start sequence, a T7 promoter, a 5'UTR, and a 3'UTR; Preferably, the starting sequence is pUC Ori, and the sequence of pUC Ori includes the nucleotide sequence shown in SEQ ID NO.15; Preferably, the sequence of the T7 promoter comprises the nucleotide sequence shown in SEQ ID NO.16; Preferably, the 5'UTR is selected from the 5'UTR of the following genes or their homologs, fragments or variants: β-globin gene, heat shock protein 70 gene, axonoderm heavy chain 2 gene or 17β-hydroxysteroid dehydrogenase 4 gene. Preferably, the 5'UTR further comprises a KOZAK sequence; Preferably, the 5'UTR comprises a 5'UTR derived from the 17β-hydroxysteroid dehydrogenase 4 gene or a homolog, fragment or variant thereof, and / or a KOZAK sequence; Preferably, the 5'UTR comprises the nucleotide sequence shown in SEQ ID NO.4 and / or SEQ ID NO.5; Preferably, the 3'UTR is selected from the 3'UTR of the following genes or their homologs, fragments or variants: albumin gene, α-globin gene, β-globin gene, tyrosine hydroxylase gene, heat shock protein 70 gene, lipoxygenase gene or collagen α gene. Preferably, the 3'UTR is a 3'UTR derived from the albumin gene or a homology, fragment, or mutant thereof; Preferably, the 3'UTR comprises a nucleotide sequence as shown in SEQ ID NO. 6; Preferably, the recombinant plasmid further comprises polyA, a resistance gene promoter, and a resistance gene; Preferably, the polyA has a length of 100-200 nucleotides; Preferably, the resistance gene promoter is an ampicillin resistance gene promoter; Preferably, the resistance gene is an ampicillin resistance gene or a neomycin resistance gene; Preferably, the recombinant plasmid comprises a nucleotide sequence as shown in SEQ ID NO.

17.

6. The use of the mRNA encoding mouse developmental pluripotency-associated protein 3mSTELLA according to any one of claims 1-3 in the preparation of mouse developmental pluripotency-associated protein 3mSTELLA.

7. A protein-carrier particle, characterized in that, The protein-carrier particle includes a protein and a carrier material that encapsulates the protein; The protein comprises any one or a combination of at least two of the proteins derived from the mRNA encoding the mouse developmental pluripotency-associated protein 3mSTELLA as described in any one of claims 1-3.

8. An mRNA-vector particle, characterized in that, The mRNA-carrier particle comprises the mRNA encoding the mouse developmental pluripotency-associated protein 3mSTELLA as described in any one of claims 1-3 and the carrier material encapsulating the mRNA.

9. A DNA-carrier particle, characterized in that, The DNA-carrier particle comprises the DNA of claim 4 and a carrier material encapsulating the DNA.

10. A tumor treatment drug, characterized in that, The tumor treatment drug comprises at least one of the following: the mRNA encoding mouse developmental pluripotency-associated protein 3mSTELLA as described in any one of claims 1-3; the DNA as described in claim 4; the recombinant plasmid as described in claim 5; the protein-carrier particle as described in claim 7; the mRNA-carrier particle as described in claim 8; or the DNA-carrier particle as described in claim 9. Preferably, the tumor treatment drug further includes an immune checkpoint inhibitor; Preferably, the immune checkpoint inhibitor includes at least one antibody selected from anti-PD1 antibody, anti-PD-L1 antibody, and anti-CTLA4 antibody.

11. The use of at least one of the following in the preparation of a medicament: the mRNA encoding mouse developmental pluripotency-associated protein 3mSTELLA as described in any one of claims 1-3; the DNA as described in claim 4; the recombinant plasmid as described in claim 5; the protein-carrier particle as described in claim 7; the mRNA-carrier particle as described in claim 8; or the DNA-carrier particle as described in claim 9. Preferably, the drug is an antitumor drug; Preferably, the tumor includes solid tumors and / or non-solid tumors; Preferably, the tumor includes any one of colorectal cancer, breast cancer, lung cancer, leukemia, or lymphoma; Preferably, the drug further contains a second active ingredient; Preferably, the second active ingredient is an immune checkpoint inhibitor; Preferably, the drug further contains pharmaceutically acceptable excipients.

12. The use of at least one of the following in the preparation of a medicament for the preparation of a treatment for diseases caused by DNA hypermethylation or related disease conditions: the mRNA encoding mouse developmental pluripotency-associated protein 3mSTELLA as described in any one of claims 1-3, the DNA as described in claim 4, the recombinant plasmid as described in claim 5, the protein-carrier particle as described in claim 7, the mRNA-carrier particle as described in claim 8, or the DNA-carrier particle as described in claim 9; Preferably, diseases caused by DNA hypermethylation include autoimmune diseases, neurological diseases, mental illnesses, reproductive diseases, cardiovascular diseases, metabolic diseases, blood diseases, and chronic inflammatory diseases; Preferably, autoimmune diseases caused by DNA hypermethylation include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and type 1 diabetes. Preferably, neurological diseases caused by DNA hypermethylation include Alzheimer's disease; Preferably, mental illnesses caused by DNA hypermethylation include depression, bipolar disorder, and autism spectrum disorder; Preferably, reproductive diseases caused by DNA hypermethylation include infertility; Preferably, cardiovascular diseases caused by DNA hypermethylation include atherosclerosis; Preferably, metabolic diseases caused by DNA hypermethylation include type 2 diabetes and obesity; Preferably, blood disorders caused by DNA hypermethylation include thalassemia and myelodysplastic syndrome; Preferably, chronic inflammatory diseases caused by DNA hypermethylation include inflammatory bowel disease; Preferably, disease-related conditions caused by DNA hypermethylation include reproductive disorders, recurrent miscarriages, abnormal embryonic development, birth defects, abnormal growth and development, neurodevelopmental disorders, and obesity.

13. The use of at least one of the following in cell reprogramming: the mRNA encoding mouse developmental pluripotency-associated protein 3mSTELLA as described in any one of claims 1-3, the DNA as described in claim 4, the recombinant plasmid as described in claim 5, the protein-carrier particle as described in claim 7, the mRNA-carrier particle as described in claim 8, or the DNA-carrier particle as described in claim 9; Preferably, the cell reprogramming includes inducing pluripotent stem cells, regulating gene expression, altering cell fate, and improving reprogramming efficiency; Preferably, the cell reprogramming application promotes tissue repair and regeneration.

14. The use of at least one of the following in the preparation of engineered immune cells: the mRNA encoding mouse developmental pluripotency-associated protein 3mSTELLA as described in any one of claims 1-3, the DNA as described in claim 4, the recombinant plasmid as described in claim 5, the protein-carrier particle as described in claim 7, the mRNA-carrier particle as described in claim 8, or the DNA-carrier particle as described in claim 9; Preferably, the immune cells are T cells or NK cells; Preferably, the engineered immune cells are used for cell immunotherapy.

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