Composition for preventing or treating bladder cancer

Abstract

The present invention relates to a composition for preventing or treating bladder cancer, in particular, to a composition for preventing or treating cancer, comprising a nucleic acid encoding a proenkephalin (PENK) protein, which is a cancer inhibitory protein that is inhibited by methylation in cancer cells, in particular, a PENK protein that is inhibited by methylation in bladder cancer cells. Thus, the composition inhibits proliferation of bladder cancer cells.

Description

Technical Field

[0001] This invention relates to compositions for the prevention or treatment of bladder cancer, and more specifically, to compositions comprising nucleic acids encoding a tumor suppressor protein—PENK (proenkephalin)—for the prevention or treatment of bladder cancer, wherein the tumor suppressor protein is inhibited by methylation in cancer cells, particularly PENK protein in bladder cancer cells, thereby inhibiting the proliferation of bladder cancer cells. Background Technology

[0002] Gene therapy has evolved into a therapeutic agent that treats or prevents diseases by adding new information to existing genes, modifying genes with that information, or deleting genes, and delivering the resulting gene to patient cells in the form of plasmid DNA (pDNA) or mRNA (messenger RNA) (Dunbar, C. E et al., Science 2018 1:12).

[0003] Gene therapy aims to cure diseases by correcting genetic defects in patients with hereditary or rare diseases. Recent technological innovations and investments in research and development have driven the global gene therapy market to grow at an average annual rate of 10%, and gene therapy is becoming a new blue-chip stock in the pharmaceutical market. Among them, mRNA is emerging as a promising treatment in the fields of vaccine development and protein replacement therapy. Gene therapy is based on the principle of introducing genes into cells in the form of pDNA or mRNA using viruses, liposomes, antisense technologies, etc., so that the genes can act as therapeutic agents (Dunbar, C. E et al., Science 2018 1:12).

[0004] In terms of stability, efficiency, and productivity, mRNA therapeutics are advantageously superior to DNA or viral therapeutics. First, mRNA is located in the cytoplasm and therefore has a low probability of mutation due to infection or insertion into genomic DNA. Second, mRNA can be modified in various forms in the cytoplasm, and thus intracellular stability can be improved by controlling its half-life or increasing the amount of translated protein. Third, mRNA is suitable for in vitro experiments, thus enabling rapid development and GMP production, and is advantageously suited for large-scale production (Wang, Y., Su et al. Molecular therapy 2013 358-367).

[0005] mRNA is RNA that transmits genetic information from DNA to ribosomes and induces protein expression through mRNA translation. Recombinant mRNAs used to develop therapeutics or vaccines are produced from linear DNA using promoters such as T7 or SP6 (but not limited to) and RNA polymerase, followed by 5' capping and poly-A adenylation (poly-A tailing) via in vitro transcription. The resulting mRNA has a structure similar to mature mRNA in the cytoplasm and consists of a 5' cap, a 5' UTR (untranslated region), a 3' UTR, poly-A, and the target gene to be expressed. The untranslated region (5' or 3' untranslated region) influences mRNA stability and translational activation depending on its sequence, thereby increasing half-life and expression levels. Additionally, poly-A prevents mRNA degradation in the cytoplasm, further increasing stability and expression levels. Therefore, finding the optimal untranslated region sequence and poly-A length is crucial for the effective and stable expression of proteins by mRNA therapeutics in vivo (Pardi, N et al., Nature reviews 2018 261-279).

[0006] Meanwhile, extensive research has been conducted on cancer treatment, which accounts for the highest proportion of human deaths. Commonly known cancer treatments include surgery, radiotherapy, chemotherapy, immunotherapy, and gene therapy. Chemotherapy has severe side effects, while radiotherapy involves excessive costs and time consumption, and exposes patients to risks associated with radioactive isotopes. Furthermore, immunotherapy has the drawback of relatively low treatment efficiency. Therefore, in the research of anticancer therapies, gene therapy, a novel treatment method that selectively introduces and expresses genes or proteins used for cancer treatment into cancer cells and cancerous tissue cells, is emerging as a promising alternative treatment. The principle of gene therapy involves using vectors such as viruses, liposomes, or antisense technologies to deliver genes in the form of plasmid DNA (pDNA), PCR products, or mRNA into cells, thereby allowing the gene to function as a therapeutic agent (Dunbar, CE et al., Science, 2018, 1:12). Among these, mRNA therapeutic agents offer advantages over DNA- or virus-based therapeutic agents in terms of stability, efficiency, and productivity.

[0007] Epigenetics refers to the phenomenon of functional changes, such as alterations in gene expression, occurring without changes in the DNA base sequence, through mechanisms including DNA methylation, covalent modification of histones, and ATP-dependent chromatin remodeling. A representative example is DNA methylation, which involves a methyl group attaching to a cytosine residue of a CpG dinucleotide, thereby affecting the expression of various genes. Increases or decreases in DNA methylation are primarily observed in cancer cells or tumor tissues, and extensive research is underway into the DNA methylation of specific genes in specific cancers. Therefore, elucidating the mechanisms by which gene expression is regulated through DNA methylation, including the regulation of tumor suppressor genes and oncogenes, is expected to be useful not only for early cancer diagnosis but also for prognostic and personalized treatment strategies (Kiselev IS et al., Acta Naturae, 2021).

[0008] Against this technical background, the inventors have discovered that the 5' regulatory region of the PENK gene is specifically methylated in bladder cancer cells, thereby inhibiting the expression of this gene. Furthermore, the inventors have found that when nucleic acids encoding the PENK protein are introduced into cell lines to induce overexpression, the proliferation of bladder cancer cell lines is inhibited. Based on these findings, this invention was completed. Summary of the Invention

[0009] One object of the present invention is to provide a composition, method or use for the prevention or treatment of bladder cancer.

[0010] According to one aspect of the invention, the above and other objectives can be achieved by providing a composition comprising a nucleic acid encoding PENK (enkephalinogenin) protein for the prevention or treatment of bladder cancer.

[0011] According to another approach, a method for preventing or treating bladder cancer is provided, which includes administering a nucleic acid encoding PENK (enkephalinogen 1) protein to a subject.

[0012] According to another aspect, the use of nucleic acids encoding PENK (pro-PENK) protein in the preparation of compositions for the prevention or treatment of bladder cancer is provided.

[0013] According to another aspect, a composition comprising a nucleic acid encoding PENK (enkephalinogen) protein and a carrier is provided for the prevention or treatment of bladder cancer.

[0014] According to another approach, a method for preventing or treating bladder cancer is provided, which includes administering a nucleic acid and a carrier encoding PENK (enkephalinogen 1) protein to a subject.

[0015] According to another aspect, the use of nucleic acids and carriers encoding PENK (pro-PENK) protein in the preparation of compositions for the prevention or treatment of bladder cancer is provided. Attached Figure Description

[0016] Figure 1 The methylation status of the promoter region of the gene encoding proenkephalin (PENK) and the expression of PENK mRNA and protein are shown ((A): methylation status of the promoter region of the PENK gene in normal cell lines and bladder cancer cell lines measured using pyrosequencing; (B): PENK mRNA expression in bladder cancer cell line (T24) analyzed; and (C): PENK protein expression in bladder cancer cell line (T24) analyzed.

[0017] Figure 2 The process of generating template DNA for expressing PENK gene mRNA using a PCR method is shown.

[0018] Figure 3 The mRNA expression of the control group and the PENK gene is shown as confirmed by in vitro transcription (IVT).

[0019] Figure 4 It shows the use of Figure 3 After transfecting PENK gene mRNA generated in the bladder cancer cell line (T24), the expression of PENK protein and apoptosis-related factors was confirmed by Western blot analysis ((A): PENK protein expression analysis; and (B): analysis of apoptosis-inducing factors in bladder cancer cells).

[0020] Figure 5 It shows the use of Figure 3 The effect of transfection of PENK gene mRNA produced in the process on the proliferation of bladder cancer cells (T24) ((A) Observation of the inhibition of cell proliferation by PENK mRNA under a microscope; and (B) Quantitative analysis of images presented in graphical form).

[0021] Figure 6 It shows the use of Figure 3 The effect of transfection of PENK gene mRNA produced in the bladder cancer cell line (T24) on bladder cancer cell colony formation ((A) Observation of the inhibition of colony formation by PENK mRNA using photographic images; and (B) Quantitative analysis of images presented as graphs).

[0022] Figure 7 It shows the use of Figure 3 The effect of transfection of PENK gene mRNA produced in the bladder cancer cell line (T24) on the wound healing ability of bladder cancer cells ((A) Observation of the inhibition of wound healing by PENK mRNA using microscopy; and (B) Quantitative analysis of images presented in graphical form).

[0023] Figure 8 It shows the use of Figure 3 The effect of transfecting the PENK gene mRNA produced in the bladder cancer cell line (T24) on the invasive ability of bladder cancer cells ((A) Observation of the inhibition of cell invasion by PENK mRNA using microscopy; and (B) Quantitative analysis of images presented in graphical form). Detailed Implementation

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well-known and commonly used in the art.

[0025] The inventors previously reported in previous studies that PENK Methylation of genes in bladder cancer (Oh et al., BMC Cancer, 2022; Oh et al., Journal of Molecular Diagnostics, 2023). Furthermore, the inventors have demonstrated... PENK Methylation, as a biomarker for the early diagnosis of bladder cancer using urine samples, further demonstrates its effectiveness even in patients with hematuria suspected of having bladder cancer. PENK Methylation can also be used to diagnose bladder cancer in its early stages.

[0026] In a specific embodiment of the present invention, the inventors discovered PENK The 5' regulatory region of the gene is specifically methylated in bladder cancer cells, thereby inhibiting the expression of the gene. Furthermore, the inventors demonstrated that when a PENK mRNA-based complex was administered to bladder cancer cell lines to induce overexpression, the proliferation of the bladder cancer cell lines was suppressed.

[0027] Based on these findings, in one aspect, the present invention relates to compositions for the prevention or treatment of bladder cancer, comprising nucleic acids encoding PENK (pro-enkephalin) protein. In another aspect, the present invention relates to a method for the prevention or treatment of bladder cancer, comprising administering to a subject nucleic acid encoding PENK (pro-enkephalin) protein. In yet another aspect, the present invention relates to the use of nucleic acids encoding PENK (pro-enkephalin) protein in the preparation of compositions for the prevention or treatment of bladder cancer.

[0028] Any nucleic acid can be used without restriction, as long as it contains nucleotides as basic structural units, and nucleic acids can include, for example, RNA or DNA.

[0029] RNA is synthesized from ribonucleotides, but in samples containing RNA that has been modified with some nucleosides introduced during synthesis, RNA that is essentially free of errors or damage can be a gene that exhibits the desired function in an organism.

[0030] The term "DNA" refers to a polynucleotide containing a sugar called deoxyribose and composed of purine or pyrimidine bases of adenine, thymine, cytosine, or guanine.

[0031] The term "RNA" refers to a polynucleotide containing a sugar called ribose and, usually, uracil rather than thymine, as one of the pyrimidine bases. RNA can consist of a single-stranded molecule transcribed from DNA and has a linear sequence of nucleotide bases complementary to the transcribed DNA strand.

[0032] As used herein, the term "nucleotide" refers to a glycoside containing a moiety, a base, and a covalently linked internucleotide bond such as a phosphate ester or a thiophosphate ester. The term "nucleotide" includes both naturally occurring nucleotides such as DNA or RNA, and non-naturally occurring nucleotides that may optionally contain modified moiety and / or base.

[0033] The term "ribonucleotide" or "deoxyribonucleotide" includes all naturally occurring and synthetic, unmodified and modified ribonucleotides or deoxyribonucleotides. Such modifications may include alterations to the sugar groups, bases, and / or bonds between ribonucleotides or deoxyribonucleotides within an oligonucleotide.

[0034] In the context of nucleic acids, the terms “polynucleotide,” “nucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably. These terms can refer to polymeric forms of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogues thereof. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Polynucleotides may include one or more modified nucleotides such as methylated nucleotides and nucleotide analogues. Modification of the nucleotide structure can occur before or after polymer assembly.

[0035] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides and / or ribonucleotides, or analogues thereof. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. The structure of a polynucleotide can be considered with reference to the 5' or 3' end, which indicates the directionality of the polynucleotide. In a single-stranded polynucleotide, adjacent nucleotides are typically linked by a phosphodiester bond between the 3' and 5' carbons. However, other internucleotide bonds can also be used, such as those involving methylene or phosphoramidite bonds. Each of the 5' and 3' carbons can be exposed at opposite ends of the polynucleotide, which can be referred to as the 5' and 3' ends or simply the ends. Due to the chemical groups attached to the respective ends, the 5' and 3' ends can also be referred to as the phosphoryl (PO4) end and the hydroxyl (OH) end, respectively.

[0036] The term "polynucleotide" is used interchangeably with the term "nucleic acid" and can include both double-stranded and single-stranded molecules. Examples of polynucleotides include, but are not limited to, genes or gene fragments (e.g., probes, primers, expression sequence tags (ESTs) or gene expression serialization analysis (SAGE) tags), genomic DNA, fragments of genomic DNA, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, complementary DNA (cDNA), recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers, or amplified copies of any of the foregoing. Polynucleotides can include modified nucleotides, such as methylated nucleotides and nucleotide analogs, including nucleotides with non-natural bases, or nucleotides with modified natural bases, such as aza- or denitro-purines.

[0037] Polynucleotides can be composed of a specific sequence of four nucleotide bases called adenine (A), cytosine (C), guanine (G), and thymine (T). Uracil (U) can exist as a natural substitute for thymine, for example, when the polynucleotide is RNA. Uracil can also be used in DNA. Therefore, the term "sequence" refers to the letter representation of a polynucleotide or any nucleic acid molecule containing natural and / or non-natural bases.

[0038] Nucleic acids may include the sequence shown in SEQ ID NO: 1 or a sequence having at least 90% homology with it.

[0039] [SEQ ID NO: 1] As used herein, the term "homology" refers to sequences exhibiting at least 90% homology, more preferably at least 95% homology, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology, as determined by optimally aligning the sequences of this invention with any other sequences and analyzing the aligned sequences using algorithms commonly used in the art. Alignment methods used for sequence comparison are well known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is accessible through NCBI and other means and can be used via the Internet in conjunction with sequence analysis programs such as BLASTP, BLASTN, BLASTX, TBLASTN, and TBLASTX. BLAST is available at www.ncbi.nlm.nih.gov / BLAST / . Methods for comparing sequence homology using this program can be found at www.ncbi.nlm.nih.gov / BLAST / blast_help.html.

[0040] Based on this, the nucleic acid sequence according to the present invention may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology with the sequences disclosed herein or the whole thereof.

[0041] The nucleic acid of this invention can be mRNA. The mRNA according to this invention can include the sequence of SEQ ID NO: 13.

[0042] [SEQ ID NO: 13] PENK mRNA sequence mRNA can be synthesized according to any of the various methods known in the art. For example, the mRNA according to the invention can be synthesized by in vitro transcription (IVT). IVT is typically performed using a linear or circular DNA template containing a promoter, a ribonucleotide triphosphate pool, a buffer system that may contain DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase) and deoxyribonuclease I (DNase I), pyrophosphatase, and / or ribonuclease (RNase) inhibitors. Conditions may vary depending on the application.

[0043] The mRNA according to the present invention can be purified to be free of reagents or impurities.

[0044] In some embodiments, the present invention can be used to purify mRNA containing one or more modifications that generally enhance stability. In some embodiments, the one or more modifications are selected from modified nucleotides, modified sugar-phosphate backbones, and 5' and / or 3' untranslated regions. In some embodiments, the present invention can be used to purify unmodified in vitro synthesized mRNA.

[0045] mRNA can be modified to enhance stability. Modifications to mRNA can include, for example, modifications to the nucleotides of RNA. Therefore, the modified mRNA according to the present invention can include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, the mRNA encoding the antibody (e.g., mRNA encoding the heavy and light chains) can include purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and modified nucleotide analogs or derivatives of purines and pyrimidines, such as, for example, 1-methyladenine, 2-methyladenine, 2-methylthio-N6-isopentenyladenine, N6- Methyladenine, N6-isopentenyladenine, 2-thiocytosine, 3-methylcytosine, 4-acetylcytosine, 5-methylcytosine, 2,6-diaminopurine, 1-methylguanine, 2-methylguanine, 2,2-dimethylguanine, 7-methylguanine, inosine, 1-methylinosine, pseudouracil (5-uracil), dihydrouracil, 2-thiouracil, 4-thiouracil, 5-carboxymethylamino 5-methyl-2-thiouracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thiouracil, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, piracetamidine, β-D-mannosyl-piracetamidine, wybutoxosine, phosphoramide ester, thiophosphate ester, peptide nucleotide, methylphosphonate, 7-dezoguanosine, 5-methylcytosine, and inosine, but not limited to these, and can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides).

[0046] In one embodiment, the nucleic acid includes a 5' untranslated region (UTR) upstream of the nucleic acid encoding the PENK protein and a 3' untranslated region (UTR) downstream of the coding region, wherein the 5' UTR may be included in a gene construct including a coronavirus leader sequence.

[0047] The SARS-CoV-2 leader sequence and an intergenic sequence that can serve as the 5' untranslated region (UTR) can be linked, followed by the coding sequence of the gene to be expressed, i.e., the nucleic acid encoding the PENK protein, and further linked to the SARS-CoV-2 3' UTR sequence and the poly(A) sequence, and the resulting construct can be introduced into cells.

[0048] In this case, in order to express the mRNA encoding the desired PENK protein in vitro, a promoter sequence (e.g., T7, SP6, etc.) can be linked to the 5' end of the sequence, and the mRNA encoding the PENK protein can be generated by in vitro transcription (IVT).

[0049] In particular, the present invention provides a gene construct comprising: a coding region for expressing mRNA encoding a PENK protein; a 5' untranslated region (UTR) located upstream of the coding region; and a 3' untranslated region (UTR) located downstream of the coding region, wherein the 5' UTR includes a coronavirus leader sequence.

[0050] Regarding the leader sequence, in coronaviruses, the genomic RNA and the transcribed subgenomic mRNA typically share a leader sequence of approximately 72 to 77 bp at the 5' end. This is a unique characteristic of coronaviruses and represents one of the most abundant viral targets present in infected cells. This is because all coronavirus subgenomic RNAs exhibit leader sequence ligation, where an approximately 72 bp leader sequence originating from the 5' end of the genomic RNA is attached to the 5' end of each subgenomic RNA. Consequently, the leader sequence has the highest copy number in the viral genome within the cell, followed by the subgenomic RNA encoding the N protein, which has the second highest copy number.

[0051] RNA includes, for example, mRNA. RNA is an abbreviation for ribonucleic acid. RNA is a nucleic acid molecule, a polymer composed of nucleotides. Nucleotides are typically monomers of adenosine monophosphate, uridine monophosphate, guanosine monophosphate, and cytidine monophosphate linked together by a so-called "backbone." This backbone is formed by phosphodiester bonds between sugars (i.e., ribose) and the phosphate moles of adjacent monomers. The specific continuous sequence of the monomers is called the "RNA sequence." RNA is typically acquired within cells through transcription of, for example, DNA sequences. In eukaryotic cells, transcription usually takes place in the nucleus or mitochondria. In vivo, mRNA (messenger RNA) is transcribed from DNA. For example, RNA processing in eukaryotic cells involves a variety of other post-transcriptional modifications such as splicing, 5'-capping, polyadenylation, and export from the nucleus or mitochondria. Messenger RNA typically provides a nucleotide sequence that can be translated into an amino acid sequence of a specific peptide or protein. Typically, mRNA includes a 5'-cap, a 5'-UTR, an open reading frame, a 3'-UTR, and a poly(A) sequence. In addition to messenger RNA, there are several other non-coding RNAs that can participate in regulating transcription and / or translation.

[0052] The 5'-UTR is located at the 5' end (i.e., "upstream") of the open reading frame. The 5'-UTR begins at the transcription start site and ends at the nucleotide preceding the start codon in the open reading frame. The 5'-UTR may contain elements that regulate gene expression, such as ribosome binding sites. The 5'-UTR can be modified posttranscribedly, for example, by adding a 5'-cap. The 5'-UTR corresponds to the sequence of the mature mRNA located between the 5'-cap and the start codon.

[0053] In particular, the 5' untranslated region (UTR) may include the sequence of SEQ ID NO.2.

[0054] [SEQ ID NO: 2] The 3'UTR is typically a portion of the mRNA located between the protein-coding region (i.e., the open reading frame) and the poly(A) sequence. The 3'UTR of mRNA is not translated into an amino acid sequence. The 3'UTR sequence is usually encoded by the gene that is transcribed into mRNA during gene expression. The genome sequence is first transcribed into mRNA containing optional introns. The mRNA then undergoes steps such as 5' capping, splicing, and 3' end modifications (such as 3' end polyadenylation), as well as optional endonuclease or exonuclease digestion. The 3'UTR is immediately adjacent to the 3' stop codon in the protein-coding region and includes nucleotides immediately adjacent to the 5' poly(A) sequence.

[0055] The 3' untranslated region (UTR) may include the 3' UTR sequence of SARS-CoV-2 and may include the sequence of SEQ ID NO:3.

[0056] [SEQ ID NO: 3] The 3' untranslated region (UTR) typically has a sequence of three nucleotide triplets that can be translated into a peptide or protein. The open reading frame (ORF) preferably includes a start codon, i.e., a combination of three subsequent nucleotides, typically encoding the amino acid methionine (ATG or AUG) at its 5' end, and a subsequent region typically a multiple of 3 nucleotides in length. The ORF is preferably terminated by a stop codon (e.g., TAA, TAG, TGA). This is the only stop codon in the ORF. Therefore, in the context of this invention, the ORF preferably begins with a start codon (e.g., ATG or AUG) and preferably terminates with a stop codon (e.g., TAA, TGA, or TAG, or UAA, UAG, UGA, respectively), and is a nucleotide sequence comprising three nucleotides. The ORF can be isolated or incorporated into longer nucleic acid sequences, such as vectors or mRNA. The ORF can also be referred to as a "protein-coding region." In particular, according to the invention, the nucleic acid encoding the PENK protein includes the sequence of SEQ ID NO: 1.

[0057] According to the present invention, the gene construct may further include a promoter and / or a poly(A) sequence.

[0058] The promoter may be located upstream of the 5' untranslated region (UTR). The promoter may include elements necessary for transcription, such as an RNA polymerase promoter. The promoter may include bacteriophage RNA polymerase promoters such as SP6 or T7, preferably a T7 promoter encoding an mRNA sequence.

[0059] The length of the poly(A) sequence can vary. For example, the poly(A) sequence can be about 20 to about 300 adenine nucleotides, preferably about 40 to about 200 adenine nucleotides, such as 60, 70, 80, 90 or 100 adenine nucleotides, more preferably about 50 to about 100 adenine nucleotides, or about 20 to about 400 adenine nucleotides.

[0060] In particular, in embodiments of the invention, the poly(A) sequence may have a length of 65 nucleotides.

[0061] The poly(A) sequence can be located downstream of the 3' untranslated region (UTR). For example, the poly(A) sequence can be directly linked or linked via a linker, such as via a linker of 1 to 50 nucleotides, preferably 1 to 20 nucleotides, or via a stretcher of 2, 4, 6, 8, 10, and 20 nucleotides.

[0062] In certain embodiments, the present invention may include a genetic construct containing the sequence shown in SEQ ID NO: 4.

[0063] [SEQ ID NO: 4] The composition may further include a delivery vector, particularly an acceptable vector, for delivering nucleic acids, such as mRNA expressed or synthesized from nucleic acids.

[0064] Based on this, in another aspect, the present invention relates to compositions for the prevention or treatment of bladder cancer, comprising nucleic acids and a carrier encoding PENK (enkephalinogenin) protein.

[0065] In another aspect, the present invention relates to a method for preventing or treating bladder cancer, comprising administering to a subject a nucleic acid and a carrier encoding PENK (enkephalinogen 1) protein.

[0066] In another aspect, the present invention relates to the use of nucleic acids and carriers encoding PENK (pro-PENK) protein in the preparation of compositions for the prevention or treatment of bladder cancer.

[0067] mRNA can be delivered via nanoparticles. For example, mRNA can be delivered via gold nanoparticles.

[0068] The surface of gold nanoparticles can be modified. Such modifications can be illustrated in detail in Acc Chem Res. 2019 June 18; 52(6): 1496-1506 and Pharmaceutics 2021, 13, 900, etc., which are incorporated herein by reference.

[0069] Gold nanoparticles can be linked to mRNA to form complexes with cationic endosomal disruptive polymers, and these complexes can be delivered into cells (Nature Biomedical Engineering, volume 1, pages 889-901 (2017)). Cationic endosomal disruptive polymers are, for example, polyethyleneimine, poly(arginine), poly(lysine), poly(histidine), poly-[2-{(2-aminoethyl)amino}-ethyl-asparagine](pAsp(DET)), block copolymers of poly(ethylene glycol)(PEG) and poly(arginine), block copolymers of PEG and poly(lysine), or block copolymers of PEG and poly{N-(N-(2-aminoethyl)-2-aminoethyl]asparagine} (PEG-pAsp(DET)).

[0070] In some cases, gold particles with arginine-modified surfaces can be used.

[0071] Arginine-modified gold particles can be assembled with nucleases or polynucleotides encoding them and / or cleavage factors or polynucleotides encoding them. As a result, the product is fused to the membrane of the target cell and then enters the cytoplasm (ACS Nano. 2017, 101:2452-2458).

[0072] The expressed mRNA can be delivered via liposomes, lipid nanoparticles (LNPs), or various nanoparticles. Liposomes or LNPs contain cationic lipids, non-cationic lipids, or neutral lipids, and may also contain other lipids such as PEG (polyethylene glycol) or cholesterol. These mRNA delivery systems are described in detail in U.S. Patent Publications 2018 / 0311176, 2019 / 0032051, and 2021 / 0046192, and International Patent Publications WO 2018 / 081480, WO 2020 / 097540, WO 2020 / 097548, and WO 2021 / 007278, which are incorporated herein by reference.

[0073] Cationic lipids, for example, are lipofectamine, a reagent used for liposome transfection.

[0074] Cationic lipids are exemplified in detail in documents such as US Patent Publications 2018 / 0311176 and 2019 / 0032051, including: N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), N,N-distearate-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleoyloxypropylamine (DODMA), 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-di... Linoleoxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleocarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleoyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleoyloxy-3-morpholinylpropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleothio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleoyloxy-3-trimethylaminopropane chloride DLin-TMA·Cl, 1,2-dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP·Cl), 1,2-dilinoleoyloxy-3-(N-methylpiperazinyl)propane (DLin-MPZ), or 3-(N,N-dilinoleoamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleoamino)-1,2-propanediol (DOAP), 1,2-dilinoleooxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleoyl-4-dimethylaminomethyl-[1 [3]-Dioxolane (DLin-K-DMA) or its analogues, (3aR,5s,6aS)-N,N-dimethyl-2,2-bis((9Z,12Z)-octadec-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxolane-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)bisdodecyl-2-ol (Tech G1), 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane, β-L-arginyl-2,3-L-diaminopropionic acid-N-palmitoyl-N-oleamide trihydrochloride, N',N'-bisoctadecyl-N-4,8-diaza-10-aminodecanoylglycine amide

[71] , 1,2-dilinoleoxy-3-dimethylaminopropane, DLin-KC2-DMA, aminolipid 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA, 1) 1,2-Distearate-N,N-dimethylaminopropane (DSDMA), dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), DLin-D-DMA, C12-200, 98N12-5, (20Z,23Z)-N,N-dimethylnonadecano-20,23-diene-10-amine, (17Z,20Z)-N,N-dimethylhexadecano-17,20-diene-9-amine, (1Z,19Z)-N5N-dimethylpentadecano-16,19-diene-8-amine, (13Z, (16Z)-N,N-dimethyldocosahexadec-13,16-diene-5-amine, (12Z,15Z)-N,N-dimethyldocosahexadec-12,15-diene-4-amine, (14Z,17Z)-N,N-dimethyldocosahexadec-14,17-diene-6-amine, (15Z,18Z)-N,N-dimethyldocosahexadec-15,18-diene-7-amine, (18Z,21Z)-N,N-dimethyldocosahexadec-18,21-diene-10-amine, (15Z,18Z)-N,N-dimethyldocosahexadec-15,18-diene -5-amine, (14Z,17Z)-N,N-dimethyltridecano-14,17-diene-4-amine, (19Z,22Z)-N,N-dimethyloctadecano-19,22-diene-9-amine, (18Z,21Z)-N,N-dimethylheptadecano-18,21-diene-8-amine, (17Z,20Z)-N,N-dimethylhexadecano-17,20-diene-7-amine, (16Z,19Z)-N,N-dimethylpentadecano-16,19-diene-6-amine, (22Z,25Z)-N,N-dimethyltridecano -22,25-diene-10-amine, (21Z,24Z)-N,N-dimethyltridecane-21,24-diene-9-amine, (18Z)-N,N-dimethylheptadecane-18-en-10-amine, (17Z)-N,N-dimethylhexadecane-17-en-9-amine, (19Z,22Z)-N,N-dimethylhexadecane-19,22-diene-7-amine, N,N-dimethylheptadecane-10-amine, (20Z,23Z)-N-ethyl-N-methylhexadecane-20,23-diene-10-amine, 1-[(11Z,1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethylheptadec-20-en-10-amine, (15Z)-N,N-dimethylheptadec-15-en-10-amine, (14Z)-N,N-dimethylheptadec-14-en-10-amine, (17Z)-N,N-dimethylheptadec-17-en-10-amine, (24Z)-N,N-dimethyltridec-24-en-10-amine, (20Z)-N,N-dimethylheptadec-20-en- 10-amine, (22Z)-N,N-dimethyl-3-carbo-22-en-10-amine, (16Z)-N,N-dimethyl-5-carbo-16-en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonyl-6-carbo-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonyl-6-carbo-13,16-dien-1-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadec-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethyl-6-nonadecan-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]6-nonadecan-10-amine, N, N-Dimethyl-21-[(1S,2R)-2-octylcyclopropyl]teicosto-10-amine, N,N-Dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecosto-10-amine, N,N-Dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadec-8-amine, N,N-Dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradec-5-amine, N,N-Dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodec-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadec-9-amine, 1-[(1S,2R)- [2-decylcyclopropyl]-N,N-dimethylpentadecanth-6-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecanth-8-amine, RN,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-(octyloxy)prop-2-amine, SN,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-(octyloxy)prop-2-amine, 1-{2-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-1-octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]prop-2-amine, 1-{2-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azacyclobutane, (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-yloxy]prop-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-2-amine, N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadec-9,12-dien-1- [Octyloxy]prop-2-amine, N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)prop-2-amine, (2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadec-6,9,12-trien-1-yloxy]-3-(octyloxy)prop-2-amine, (2S)-1-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)prop-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]-N,N-dimethylprop-2-amine, 1-[(11Z,14Z] -eicos(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)prop-2-amine, 1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)prop-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylprop-2-amine, (2S)-1-[(13Z)-docosa-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylprop-2-amine, 1-[(13Z)-docosa-13-en-1-yloxy]-N,N-dimethyl-3-( Octyloxy)propan-2-amine, 1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(1-methyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine ((2R)-N,N-dimethyl-H(1-metoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,[12Z)-octadec-9,12-dien-1-yloxy]prop-2-amine, N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]-methyl}cyclopropyl]octyl}oxy)prop-2-amine, N,N-dimethyl-1-{[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)prop-2-amine, with and (11E,20Z,23Z)-N,N-dimethyl-9-neco-11,20,2-trien-10-amine, 5-carboxyarginylglycine dioleamide (DOGS), dipalmitoylphosphatidylethanolamine-5-carboxyarginamide (DPPES), 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE), DMRIE-HP, Lipofectamine (DOSPA), 3b-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (“DC-Choi”), N-(1,2-dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (“DMRIE”), 1,2-dioleoyl-3-dimethylaminopropane (DODAP), DMDMA, cationic lipid-based transfection reagent TransIT-TKO, LIPOFECTIN, lipid transfection amines, OLIGOFECTAMINE or DHARMAFECT, DSDMA, DODMA, DL DLinDMA, γ-DLenDMA, DLin-K-DMA, DLin-K-C2-DMA (also known as DLin-C2K-DMA, XTC2 and C2K), DLin-K-C3-DMA, DLin-K-C4-DMA, DLen-C2K-DMA, y-DLen-C2K-DMA, DLin-M-C2-DMA (also known as MC2), DLin-M-C3-DMA (also known as MC3) or (DLin-MP-DMA) (also known as 1-B11), or mixtures thereof, but not limited thereto.

[0075] The non-cationic lipids are illustrated in detail in U.S. Patent Publications 2018 / 0311176 and 2019 / 0032051, and are, for example, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, or lysyl phosphatidylglycerol. In some cases, non-cationic lipids can be, for example, distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(-maleimidemethyl)-cyclohexane-1-carboxylic acid ester (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristylphosphatidylphosphate ethanolamine (DMPE), distearylphosphatidyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE (18-1-trans... PE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoylphosphatidylethanolamines, N-succinylphosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, or palmitoyloleoylphosphatidylglycerol (POPG), but not limited to these.

[0076] The neutral lipids are specifically illustrated in U.S. Patent Publications 2018 / 0311176, 2019 / 0032051, etc., and include, for example, but not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cerebroside, or cerebroside.

[0077] The PEG lipid may be included to prevent particle aggregation during mRNA delivery. Specific examples of such PEG lipids are illustrated in U.S. Patent Publications 2018 / 0311176 and 2019 / 0032051, and are, for example, PEG-diacylglycerol (DAG), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), or mixtures thereof. As a non-limiting example, PLGA may be terminated with a PEG conjugation of a lipid forming PLGA-DSPE-PEG, the PEG lipid being selected from PEG-c-DOMG, 1,2-dimyristoyl-sn-glycerol, methoxy polyethylene glycol (PEG-DMG), 1,2-distearyl-sn-glycerol, methoxy polyethylene glycol (PEG-DSG), PEG-c-DOMG, 1,2-distearyl-sn-glycerol, methoxy polyethylene glycol (PEG-DSG), 1,2-dipalmitoyl-sn- Glycerin, methoxy polyethylene glycol (PEG-DPG), PEG-lipid conjugates, such as, for example, PEG coupled to dialkoxypropyl groups (e.g., PEG-DAA conjugates), PEG coupled to diacylglycerol groups (e.g., PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamine groups, PEG coupled to ceramide groups, cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates, polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof. PEG can be PEG-dilauoxypropyl (C12), PEG-dimyristoxypropyl (C14), PEG-dispalmitoxypropyl (C16), PEG-distearateoxypropyl (C18), PEG-c-DOMG, PEG-DMG, or mixtures thereof, but is not limited thereto.

[0078] Peptides can be used for mRNA delivery. A peptide must have a cation that electrostatically interacts with the anionic phosphate group of a nucleic acid and may contain a positively charged amino acid that electrostatically interacts with the phosphate group. Details of peptides that can be used for mRNA delivery are described in AIMS Biophysics, 7(4): 323-338, which is incorporated herein by reference.

[0079] Peptides that can be used for mRNA delivery may include protamine. Protamine is a nuclear protein rich in cationic arginine that contributes to DNA stability during spermatogenesis in the testes, and can stabilize mRNA molecules and enable efficient delivery. The protamine-mRNA complex is described in detail in U.S. Patent No. 9,352,028, which is incorporated herein by reference.

[0080] Cell-penetrating peptides (CPPs) can also be promising cationic molecules for mRNA delivery. Amphiphilic CPPs such as arginine-rich RALA peptides (WEARLARALARARARHLARALARALRACEA), RALA, LAH4 (KKALLALALHHLAHLALHLALALKKA), and LAH4-L1 (KKALLAHALHLLALLALHLAHALKKA) can be used to deliver mRNA molecules.

[0081] In some cases, peptides, in addition to liposomes or LNPs (lipid nanoparticles), can also be used for mRNA delivery. The function of peptides is to package nucleic acids and prevent DNA or RNA from being degraded intracellularly or extracellularly. Examples of such peptides are described in detail in U.S. Patent Publication No. 2021 / 0170046, which is incorporated herein by reference, but is not limited thereto.

[0082] The composition may also contain at least one pharmaceutically acceptable support. A pharmaceutically acceptable support must be compatible with the active ingredient of the invention and may be an aqueous saline solution, sterile water, Ringer's solution, buffered saline solution, glucose solution, maltodextrin solution, glycerol, ethanol, or mixtures thereof, and may additionally contain other conventional additives, such as antioxidants, buffers, and antibacterial agents, as needed. Furthermore, the composition can be prepared into injectable formulations, such as aqueous solutions, suspensions, or emulsions, by adding diluents, dispersants, surfactants, binders, and lubricants. In particular, the composition is preferably formulated as a lyophilized product. The lyophilized product can be prepared using methods known in the art to which this invention pertains, and optionally using stabilizers for lyophilization. Moreover, the composition is preferably formulated according to each disease or ingredient using methods suitable in the art or methods disclosed in Remington's Pharmaceutical Science (Mack Publishing company, Easton, PA).

[0083] Those skilled in the art can determine the content of the active ingredient in the composition of the present invention and the method of administration based on the patient's symptoms and the severity of the disease. Furthermore, the composition can be formulated into various forms, such as powders, tablets, capsules, solutions, injections, ointments, and syrups, and can be provided in single-dose or multi-dose containers, such as sealed ampoules and bottles.

[0084] The compositions of the present invention can be administered orally or parenterally. Routes of administration of the compositions according to the present invention include, but are not limited to, for example, bronchial, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, percutaneous, subcutaneous, intraperitoneal, enteric, sublingual, or local administration. The dosage of the compositions according to the present invention varies depending on the patient's weight, age, sex, health condition and diet, timing of administration, method of administration, excretion rate, or severity of disease, and can be readily determined by those skilled in the art. Furthermore, the compositions of the present invention can be formulated into clinically suitable dosage forms using known techniques.

[0085] Subjects can be individuals with bladder cancer. Additionally, subjects can be mammals, with humans being preferred.

[0086] As used herein, the term "treatment" refers to any indication of success of any subjective or objective parameter, such as relief; reduction; the occurrence of symptoms or lesions that are tolerable to the patient, a decrease in pathology or condition; slowing of deterioration or worsening; the occurrence of a final point of regression that reduces the degree of debilitating effect on the patient; and the treatment or improvement of lesions, pathology, or condition, including improvement of the patient's physical or mental health. Treatment or relief of symptoms may be based on objective or subjective parameters, including physical examination, neuropsychiatric examination, and / or psychomedical evaluation.

[0087] The term "effective amount" generally refers to an amount sufficient to reduce the severity or frequency of symptoms, eliminate symptoms and underlying causes, prevent the occurrence of symptoms or underlying causes, or improve or correct damage caused by or related to a disease condition. In some embodiments, the effective amount is a therapeutically effective amount or a preventatively effective amount. A "therapeuticly effective amount" is an amount sufficient to correct a disease condition or symptom (particularly a condition or symptom related to the disease condition), or sufficient to prevent, halt, delay, or reverse the progression of a disease condition or any other adverse symptom in any way related to the disease. A preventatively effective amount refers to an amount in which a pharmaceutical composition has the intended preventative effect, for example, when administered to a subject, preventing or delaying the onset of a disease condition, or reducing the likelihood of the onset (or recurrence) of a disease condition or related symptoms.

[0088] The present invention will now be described in more detail with reference to the embodiments. However, it will be apparent to those skilled in the art that these embodiments are merely illustrative of the invention and should not be construed as limiting the scope of the invention.

[0089] Example 1. PENK expression in bladder cancer cell lines based on methylation. In order to determine the target PENKThe correlation between protein expression and DNA methylation was first investigated by analyzing the methylation level of the bladder cancer cell line T24 using pyrosequencing. The results showed that higher levels of methylation were observed in T24 cells compared to normal cells. Figure 1 (A)). Subsequently, mRNA and protein expression were analyzed by RT-PCR and Western blotting, respectively. The results showed that PENK expression was not detected. Figure 1 (B) and Figure 1 (C)). Therefore, this study preliminarily confirmed that hypermethylation inhibits the expression of mRNA and protein.

[0090] Example 2. For use by methylation inhibition PENK protein expression PENK Preparation of gene mRNA constructs 2-1. Used for PENK Preparation of IVT template for mRNA expression Based on the method for constructing mRNA constructs previously developed by the inventors (International Patent Application Publication No. WO2023 / 063769), a method for preparing mRNA constructs was developed. PENK protein expression PENK mRNA constructs. IVT template sequences previously constructed using this method are identified and shown in the table below.

[0091] [Table 1] Primer sequence In addition, in order to build for PENK The IVT template for mRNA delivery was used to append the PENK gene coding sequence to the 5' UTR of the SARS-CoV-2 N gene via PCR, followed by the SARS-CoV-2 3' UTR sequence and the polyA sequence (65 nucleotides). Figure 2 ).

[0092] To synthesize the 5' UTR, 1 ng of the plasmid DNA constructed above for control RNA expression was used as a template, along with 10 pmol of forward primer (SEQ ID NO: 5), 10 pmol of reverse primer (SEQ ID NO: 6), and 10 μl of 2X pfu premix (Master Mix) (Biofact, Daejeon, South Korea). To synthesize the 3' UTR, 10 pmol of forward primer (SEQ ID NO: 9), 10 pmol of reverse primer (SEQ ID NO: 8), and 10 μl of 2X pfu premix (Biofact, Daejeon, South Korea) were added. PCR (Applied Biosystem) reaction conditions were as follows: 95°C, 2 min (1 cycle); 95°C, 20 s, 60°C, 40 s, and 72°C, 1 min (30 cycles); 72°C, 5 min. The constructed PCR products were confirmed by electrophoresis on an agarose gel (2%). 1 ng of plasmid containing... PENK pCMV6- gene PENK Used as a template for amplification PENK Genes. A 10 pmol forward primer (SEQ ID NO: 10) was added, in which the 3' end sequence of the 5' UTR was linked to the 5' end sequence of the PENK gene; and a 10 pmol reverse primer (SEQ ID NO: 11) was added, in which the 3' end sequence of the PENK gene was linked to the 5' end sequence of the 3' UTR; and 10 μl of 2X pfu premix (Biofact, Daejeon Metropolitan City, South Korea) was added. PCR (Applied Biosystems) reaction conditions were as follows: 95℃, 2 min (1 cycle); 95℃, 20 s, 60℃, 40 s, and 72℃, 2 min (30 cycles); 72℃ (5 min). The constructed PCR products were confirmed by electrophoresis on an agarose gel (2%). The T7 promoter sequence, 5' UTR sequence, and... were ligated using PCR. PENKGene sequence, 3'UTR sequence, and 65-nucleotide poly(A) sequence. 100 pg of each of the above-constructed PCR products were mixed in equal volumes and used as templates. 10 pmol of forward T7 promoter sequence primer (SEQ ID NO: 5), 10 pmol of reverse primer (SEQ ID NO: 8), and 10 μl of 2X pfu premix (Biofact, Daejeon, South Korea) were added. The reverse primer consisted of the 3' terminal 20 nucleotides of the 3'UTR sequence linked to the 65-nucleotide poly(A) sequence. PCR (Applied Biosystems) reaction conditions were as follows: 95 °C, 2 min (1 cycle); 95 °C, 20 s, 60 °C, 40 s, and 72 °C, 2 min (30 cycles); 72 °C (5 min). PCR products were confirmed by agarose gel electrophoresis (2%). The PCR product was ligated into the pTOP Blunt V2 vector (Enzynomics, Daejeon, South Korea) and transformed into DH5α (Enzynomics, Daejeon, South Korea). After transformation, the product was plated on solid medium (Bioloard, Daejeon, South Korea) containing ampicillin (50 μg / mL, Sigma Aldrich, USA) and incubated at 37°C for 16 hours. Plasmid DNA was extracted using the HiGene™ Plasmid Mini Prep Kit (version 2.0) (Biofact, Daejeon, South Korea). The constructed IVT template sequence was confirmed by Sanger sequencing (SEQ ID NO: 4).

[0093] [Table 2] [SEQ ID NO: 4] 2-2. Control mRNA and PENK mRNA structure verification Based on Example 2-1, PCR reactions were performed in a total volume of 20 μL using 10 pmol of a forward primer corresponding to the forward sequence of the T7 promoter (SEQ ID NO: 12), 10 pmol of a reverse primer including the 3'UTR linked to nucleotide polyA 65 (SEQ ID NO: 8), and 10 μl of 2X pfu premix (Biofact, Daejeon, South Korea) to produce each mRNA. The PCR reaction conditions were as follows: 95°C, 2 min (1 cycle); 95°C, 20 s, 60°C, 40 s, and 72°C, 2 min 30 s (30 cycles); 72°C, 5 min. The amplified PCR products were purified by electrophoresis on a 2% agarose gel using a Qiaquick® gel extraction kit (QIAGEN, Hilden, Germany) and used as templates for in vivo tyrosine spectroscopy (IVT). IVT was performed using the Hiscribe T7 ARCA mRNA kit (NEB) according to the manufacturer's instructions. Using 1 μg of the above PCR product as a template, 10 μl of 2X ARCA / NTP MIX and 2 μl of T7 polymerase were added, and the mixture was incubated at 37°C for 16 hours to allow mRNA synthesis and the addition of an anti-reverse cap analog (ARCA) at the 5' end. After the reaction, 2 µl of deoxyribonuclease I was added, and the mixture was incubated at 37°C for 15 min to remove the DNA template. After the IVT reaction, the reaction solution was purified using the Monarch RNACleanup kit (NEB, Massachusetts, USA) and eluted in 50 μl of ribonuclease-free distilled water. The purified RNA was quantified using NanoDrop, and the mRNA product was confirmed by electrophoresis on an agarose gel (1%, 0.5X TBE). Figure 3 After electrophoresis, the size matched the expected size.

[0094] Example 3. In human cell lines PENK Confirmation of protein expression (control vs.). PENK ) 3-1. Transfection and Cell Harvesting Bladder cancer cell line T24 was cultured at 0.8 × 10⁻⁶ per well. 6Cells were seeded at a density of 1,000 cells / day in 60 mm culture plates (SPL, Pocheon, South Korea) and incubated at 37°C and 5% CO2 for 24 hours. mRNA synthesized via in vitro transcription (IVT) was mixed with Lipofectamine MessengerMAX at a ratio of 1:1.5 (w:v) and transfected into the T24 cell line. After incubation at 37°C and 5% CO2 for 24 hours, cells were harvested. Protein extraction was performed on the harvested cells using Pro-Prep (iNtRON Biotechnology, Seongnam, South Korea) according to the manufacturer's instructions.

[0095] 3-2. Confirmed by Western blotting PENK Protein expression and PENK Apoptosis function The extracted proteins were separated by SDS-PAGE using a Mini-PROTEAN® tetra vertical electrophoresis pool (Bio-Rad, California, USA) and transferred to a nitrocellulose membrane (Bio-Rad, California, USA). The membrane was blocked with 5% skim milk (Bio-Rad, Seoul, South Korea) for 1 hour, followed by contact with a solution diluted in 5% skim milk. PENK Polyclonal antibody (Invitrogen, Massachusetts, USA) was incubated at 4°C for 16 hours. The membrane was washed three times with 1x TBST for 10 minutes each time and incubated with mouse anti-rabbit IgG HRP secondary antibody diluted in 5% skim milk (Santa Cruz Biotechnology, Texas, USA) at room temperature for 2 hours, followed by three additional washes with 1x TBST. The membrane was treated with a 1:1 mixture of luminol / enhancer solution and peroxide solution (Cyanagen, Bologna, Italy) at room temperature for 1 minute, and protein expression was visualized using a fusion solo X imaging system (Vilber, Eberharzl, Germany). Figure 4 (A)).

[0096] In order to determine PENKThe apoptosis function was investigated, and the protein expression levels of apoptosis-related factors PARP and caspase-3 were also analyzed. Extracted proteins were SDS-PAGEed using a Mini-PROTEAN Tetra vertical electrophoresis pool (Bio-rad, California, USA) and transferred to nitrocellulose membranes, which were blocked with 5% skim milk for 1 hour. The membranes were incubated at 4°C for 16 hours with anti-PARP (Cell Signaling Technology, Massachusetts, USA) and anti-caspase-3 antibodies (Cell Signaling Technology, Massachusetts, USA), each diluted in 5% skim milk. The membranes were washed three times with 1 × TBST and incubated with mouse anti-rabbit IgG HRP secondary antibody at room temperature for 2 hours, followed by further washing. Detection was performed using a Fusion Solo X imaging system as described above. Figure 4 (B)). This indicates that PENK The gene induced apoptosis more effectively than the control gene, thereby effectively inhibiting the proliferation of bladder cancer cells, suggesting its potential use as a therapeutic agent for bladder cancer.

[0097] Example 4. Evaluation PENK Inhibitory effect on cell proliferation and colony formation 4-1. Cell growth inhibition test Bladder cancer cell line T24 was cultured at 0.05 × 10⁻⁶ per well. 6 T24 cells were seeded at a density of 1,000 cells / well in 24-well plates (SPL, Pocheon, South Korea) and incubated at 37°C and 5% CO2 for 24 hours. mRNA synthesized via IVT was mixed with Lipofectamine Messenger MAX at a ratio of 1:1.5 (w:v) and transfected into T24 cells, followed by incubation at 37°C and 5% CO2 for 24 hours. Cells were treated with Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer's instructions and incubated in a CO2 incubator for 1 hour. The absorbance of live cells at 450 nm was then measured using a Mobi microplate reader (Microdigital, Seoul, South Korea). The results showed that cell proliferation was reduced by approximately 37% in cells transfected with PENK mRNA compared to cells transfected with control mRNA. Figure 5 These results indicate that the PENK gene effectively inhibits the proliferation of bladder cancer cells, thus demonstrating its potential applicability as a therapeutic agent for bladder cancer.

[0098] 4-2. Evaluation of the inhibitory effect on colony formation (colony formation assay) Bladder cancer cell line T24 was cultured at 0.8 × 10⁻⁶ per well. 6T24 cells were seeded at a density of 1000 mcg / well in 60 mm culture plates (SPL, Pocheon, South Korea) and incubated at 37°C and 5% CO2 for 24 hours. mRNA synthesized via in vitro transcription (IVT) was mixed with Lipofectamine MessengerMAX at a ratio of 1:1.5 (w:v) and transfected into T24 cells. After incubation at 37°C and 5% CO2 for 24 hours, the cells were harvested. The harvested cells were then seeded at a density of 3 x 1000 mcg / well. 3 Cells were seeded at a density of [number] cells per well in 6-well plates and incubated at 37°C and 5% CO2 for 7 days. After colony formation, the culture medium was removed from each well, and the cells were fixed with 100% methanol at room temperature for approximately 20 minutes. After fixation, the cells were washed with distilled water, stained with 0.5% crystal violet, washed again with distilled water, and colony formation was evaluated. The results showed that, compared with cells transfected with the control gene, colony formation was reduced by approximately 37% in cells transfected with the PENK gene. Figure 6 These results indicate that the PENK gene effectively inhibits the colony formation of bladder cancer cells and therefore has potential use as a therapeutic agent for bladder cancer.

[0099] Example 5. Evaluation PENK Inhibitory effect on cell migration and invasion 5-1. Wound Healing Assay Bladder cancer cell line T24 was cultured at 0.05 × 10⁻⁶ per well. 6 T24 cells were seeded at a density of 1000 mRNA / v in 24-well plates (SPL, Pocheon, South Korea) and incubated at 37°C and 5% CO2 for 24 hours. MRNA synthesized via IVT was mixed with Lipofectamine MessengerMAX at a ratio of 1:1.5 (w:v) and transfected into T24 cells, followed by incubation at 37°C and 5% CO2 for 24 hours. Uniform wounds were then created using a Scar™ Scratcher (SPL, Pocheon, South Korea), and the cells were washed with PBS. Cells were incubated for another 24 hours, and images before and after incubation were analyzed using ImageJ software to measure cell migration distance within the wound area. Results showed that, compared to cells transfected with the control gene, cells transfected with the control gene... PENK The wound healing ability of gene-transfected cells decreased by approximately 40% after 24 hours. Figure 7 These results indicate that PENK The gene significantly reduces cell migration and wound healing ability, thereby effectively inhibiting the proliferation of bladder cancer cells, and has potential applicability as a therapeutic agent for bladder cancer.

[0100] 5-2. Transwell Invasion Measurement Bladder cancer cell line T24 was cultured at 0.05 × 10⁻⁶ per well. 6 T24 cells were seeded at a density of 1 × 10⁶ cells / well in 24-well plates (SPL, Pocheon, South Korea) and cultured at 37°C and 5% CO₂ for 24 hours. mRNA synthesized via in vitro transcription (IVT) was mixed with Lipofectamine MessengerMAX at a ratio of 1:1.5 (w:v) and transfected into T24 cells. After incubation at 37°C and 5% CO₂ for 24 hours, cells were harvested. The harvested cells were then incubated in serum-free RPMI medium supplemented with 0.5% BSA at a density of 1 × 10⁶ cells / well. 4 Cells were seeded at a density of [number] cells per cell type into the upper chamber of a matrix gel-coated polycarbonate filter (Corning, New York, USA), while serum-containing RPMI medium was added to the lower chamber. The cells were then incubated at 37°C and 5% CO2 for 24 hours. After incubation, the cells were fixed and stained using the Diff-Quik kit (Sysmex, Kobe, Japan), and invading cells were counted under a microscope. The results showed that, compared to cells transfected with the control gene, cells transfected with the PENK gene exhibited approximately 50% less chamber invasion. Figure 8 These results indicate that PENK Genes that regulate the motility of bladder cancer cells, thereby significantly reducing cell migration and invasion, have potential applicability as a therapeutic agent for bladder cancer.

[0101] Industrial applicability The present invention provides a composition for the prevention or treatment of bladder cancer, which exhibits the ability to inhibit the growth of bladder cancer cells, induce apoptosis, and inhibit cancer cell migration and invasion.

[0102] Although specific configurations of the invention have been described in detail, those skilled in the art will understand that this detailed description is provided for illustrative purposes as a preferred embodiment and should not be construed as limiting the scope of the invention. Therefore, the essential scope of the invention is defined by the appended claims and their equivalents.

[0103] Sequence List Free Text Electronic documents are attached.

Claims

1. A composition for the prevention or treatment of bladder cancer, comprising a nucleic acid encoding PENK (enkephalinogen) protein.

2. The composition according to claim 1, wherein, The nucleic acid includes the sequence represented by SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity with it.

3. The composition according to claim 1, wherein, The nucleic acid is DNA or RNA, wherein when the nucleic acid is RNA, the thymidine (T) residue in the nucleotide sequence is replaced by uracil (U).

4. The composition according to claim 1, wherein, The nucleic acids include: The 5' untranslated region (UTR) is located upstream of the coding region encoding the PENK protein; and 3' Untranslated Region (UTR), located downstream of the encoded region, The 5' untranslated region (UTR) is present in the gene construct that includes the coronavirus leader sequence.

5. The composition according to claim 4, wherein, The 5' untranslated region (UTR) comprises the nucleotide sequence represented by SEQ ID NO:

2.

6. The composition according to claim 4, wherein, The 3' untranslated region (UTR) comprises the nucleotide sequence represented by SEQ ID NO:

3.

7. The composition according to claim 4, wherein, The gene construct also includes a promoter and / or a poly(A) sequence.

8. The composition according to claim 1, wherein, The composition comprises a gene construct containing the nucleotide sequence represented by SEQ ID NO:

4.

9. A composition for the prevention or treatment of bladder cancer, comprising: Nucleic acid encoding PENK (pro-enkephalin) protein; And pharmaceutically acceptable carriers.

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