RNA compounds for treating proliferative disorders

Abstract

This invention is based on in vitro transcription RNA compounds or in vitro synthetic RNA compounds for the translation of tumor suppressor proteins in subjects suffering from proliferative diseases such as cancer. The invention provides RNA compounds and compositions for their delivery or administration to subjects.

Description

Technical Field

[0001] The present invention is based on in vitro transcribed RNA compounds or in vitro synthesized RNA compounds for the translation of tumor suppressor proteins in a subject suffering from a proliferative disease such as cancer. The present invention provides RNA compounds and compositions for their delivery or administration to a subject.

Background Art

[0002] High-grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy and is the cause of the 5th leading cancer-related death in women. The estimated annual incidence of this disease exceeds 200,000 worldwide, and approximately 125,000 people have died. These cancers grow very rapidly, metastasize widely, and show a rapid course of the disease. Ovarian cancer (OC) cells remain in the peritoneal cavity and spread only on the surface covered with mesothelium 1. Current standard treatments include cytoreductive surgery, chemotherapy (platinum compounds, paclitaxel and cyclophosphamide), bevacizumab (Avastin), and PARP (poly ADP ribose polymerase) inhibitors. Nevertheless, about 70% of patients relapse within 3 years after surgery and platinum-based chemotherapy and succumb to the progression of the disease.

[0003] The TP53 gene plays a crucial role in human cancers and is the most frequently mutated human tumor suppressor gene, primarily through point mutations. Amino acid substitutions lead to disruptions in the binding of the tumor protein p53 to DNA. p53 protects the human genome by resisting cellular stress and DNA damage. Therefore, cells with mutant TP53 can develop an unstable genome, evade apoptosis, and ultimately develop into carcinogenesis. Multiple reports have shown that tumor cells with functionally inactive p53 are resistant to chemotherapy agents because the mechanism of efficacy of chemotherapy agents involves the activation of wild-type (wt) p53 due to DNA damage. The Cancer Genome Atlas Research Network has revealed that HGSOC is characterized by TP53 mutations in up to 96% of all cases, resulting in inactive or truncated p53 proteins. Based on the International Agency for Research on Cancer (IARC) TP53 database (http: / / www-p53.iarc.fr / ), most TP53 mutations in OC are missense mutations (>87.56%), which are mainly concentrated in hotspots (codons 175, 248, and 273) within the DNA-binding domain of p53. While a low prevalence (2–6%) of recurrently mutated genes such as BRCA1, BRCA2, RB1, and NF, and a long list of rarely mutated genes have been found in this malignancy, TP53 is actually the only gene that frequently mutates at the somatic level in HGSOC.10 Mutations in TP53 are likely an early event of tumorigenesis in the precursor lesions of OC, supporting the importance of mutated TP53 as a driving factor in this malignancy.

[0004] In recent years, RNA-based vaccines have emerged as a valuable tool for immunotherapy. Novel strategies based on mRNA therapeutics have been recognized as an effective and safe approach to combat infectious diseases and cancer, based on advantages such as relatively simple large-scale production, low side effects, and high efficacy. Unlike immune checkpoint inhibitors that relieve immunosuppression and CAR-T cells that directly eliminate cancer cells, recently developed cancer vaccines induce an anti-cancer immune response via antigen-presenting cells, particularly dendritic cells. While efforts to replace deficient or defective proteins with mRNA therapeutics have been an attractive strategy in various clinical areas such as lung diseases and muscle atrophy, approaches in cancer remain rare. Whether p53 rescue has therapeutic value depends heavily on the indication for the tumor and / or the patient.

[0005] Therefore, the object of the present invention is to provide novel therapeutic strategies for addressing proliferative diseases, particularly ovarian cancer. [Overview of the Initiative]

[0006] In general, the main aspects of the present invention can be described as follows.

[0007] In a first aspect, the present invention relates to a ribonucleic acid (RNA) compound for the treatment of proliferative disorders in a subject, the RNA compound encoding the amino acid sequence of the human p53 protein.

[0008] In a second aspect, the present invention relates to a pharmaceutical composition comprising an RNA compound encoding the amino acid sequence of the human p53 protein.

[0009] In a third aspect, the present invention relates to a method for treating a proliferative disorder in a subject, comprising the step of administering to the subject an RNA compound encoding the amino acid sequence of the human p53 protein.

[0010] In a fourth aspect, the present invention relates to a method for producing a pharmaceutical for the treatment of a proliferative disorder in a subject, comprising the step of formulating an RNA compound encoding the amino acid sequence of the human p53 protein as a pharmaceutical. [Modes for carrying out the invention]

[0011] Detailed description of the invention The elements of the present invention are described below. While these elements are listed in conjunction with specific embodiments, it should be understood that they can be combined in any way and in any number to create additional embodiments. The various examples and preferred embodiments described should not be construed as limiting the invention to only the explicitly described embodiments. This description should be understood as supporting and encompassing embodiments that combine two or more of the explicitly described embodiments, or embodiments that combine one or more of the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any substitution and combination of all elements described in this application should be considered disclosed by this description unless otherwise indicated in the context.

[0012] In a first aspect, the present invention relates to a ribonucleic acid (RNA) compound for the treatment of proliferative disorders in a subject, the RNA compound encoding the amino acid sequence of the human p53 protein or a variant thereof.

[0013] The cellular tumor antigen p53 is encoded by the TP53 gene, whose sequence and coding gene sequence can be derived from the UniProt database as of June 20, 2023, under accession number P04637. The amino acid sequence of wild-type p53 is also included herein as Sequence ID No. 1.

[0014] Therefore, preferred embodiments of the present invention relate to RNA compounds that contain or are essentially derived from p53 mRNA.

[0015] The RNA compound of the present invention has a sequence having at least 80% sequence identity with the nucleic acid sequence encoding the human p53 protein (SEQ ID NO: 1). In the context of the present invention, the term "sequence having at least 80% sequence identity" includes sequences having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, and most preferably 100% sequence identity with respect to each respective sequence. In all cases, identity is identity across the entire length of the sequence.

[0016] Preferably, the RNA compound of the present invention contains the RNA sequence of SEQ ID NO: 3, or a sequence that is at least 80%, more preferably 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 3.

[0017] In one embodiment, the RNA compound according to the present invention is a variant of an RNA compound having a sequence encoding the protein, i.e., human p53, according to Sequence ID No. 1. The term “variant” as used herein refers to a biologically active derivative of each RNA. Generally, the term “variant” refers to a molecule that has a native sequence and structure with one or more additions, substitutions (generally essentially conserved) and / or deletions compared to the native molecule, and is “substantially homologous” to the reference molecule, provided that the modifications do not disrupt its biological activity. Generally, the sequence of such a variant has a high degree of sequence homology to the reference sequence, e.g., more than 50%, generally more than 60%–70%, and more specifically more than 80%–85%, e.g., at least 90%–95% when the two sequences are aligned. In the case of p53, its variant exhibits similar capabilities to a tumor suppressor protein.

[0018] In further embodiments, the present invention relates to RNA compounds that can be obtained by transcribing a DNA sequence having at least 80% sequence identity with a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1 or a variant thereof, for use as a pharmaceutical. In preferred embodiments, the RNA compound is for use in the treatment of cancer.

[0019] The phrase "can be obtained by transcription of a DNA sequence" is understood herein to mean that the RNA compound is a transcript or copy of the respective DNA. Therefore, the phrase "RNA compound obtained by transcription of a DNA sequence having at least 80% sequence identity with the sequence encoding the amino acid sequence of SEQ ID NO: 1" refers to any RNA compound that can be transcribed from a DNA sequence having a sequence encoding a protein having an amino acid sequence that is at least 80% sequence-identical to SEQ ID NO: 1, or any functional variant thereof. The RNA compounds according to the present invention include any RNA compounds that may be processed by post-transcriptional modifications such as RNA methylation, folding, cleavage, or wrapping into extracellular cargo vesicles such as exosomes.

[0020] The phrase "can be obtained by transcription of a DNA sequence" is understood herein to be not limited to the source of the RNA molecule. The RNA molecule according to the present invention, including the RNA molecule used in the pharmaceutical composition according to the present invention, may be an in vivo transcribed RNA molecule or in vitro transcribed RNA purified from cells.

[0021] Those skilled in the art know how RNA can be purified from cells or transcribed in vitro. For example, RNA purification can be performed using TRIzol reagent (Invitrogen, CA, catalog no. 15596018). To do this, the sample is dissolved and homogenized in an appropriate volume of TRIzol reagent. The sample is incubated at room temperature for 5 minutes, then 1 / 5 volume of chloroform is added, incubated, and mixed vigorously. To separate the phases, the sample is centrifuged at 18°C ​​and 13000 × g for 15 minutes. The aqueous phase containing RNA is transferred to a new tube, and the RNA is precipitated using isopropanol. The resulting pellet is washed with ice-cold 70% ethanol, air-dried, and dissolved in a suitable buffer.

[0022] The RNA compound according to the present invention may be purified from any eukaryotic cell that artificially or naturally expresses p53, or may be purified from transcription in a cell-free system. Similarly, RNA purification can be performed using commercially available kits such as the RNeasy Mini Kit (Qiagen, Germany, catalog number 74106) or the Dynabeads(™) mRNA DIRECT(™) Purification Kit (Invitrogen, CA, catalog number 61012).

[0023] The RNA compound of the present invention is preferably an RNA compound encoding an amino acid sequence that comprises the nucleotide sequence of SEQ ID NO: 2 (preferred RNA sequence), or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2.

[0024] The RNA compound of the present invention is preferably in vitro transcribed (IVT) RNA or artificially polymerized RNA.

[0025] The RNA compound of the present invention may contain the classical sequence motifs of messenger RNA. However, the RNA molecules of the present invention may also contain various additional modifications or mutations in order to generally increase translation, reduce immunological adverse effects, or increase RNA stability. Many such modifications are well known in the art and are not individually listed herein for the sake of brevity.

[0026] For example, the RNA compound of the present invention may contain a polyA sequence operably linked to a sequence encoding an amino acid sequence. Preferably, when used, the polyA sequence comprises at least 100 nucleotides. The polyA sequence is usually located 3' of the sequence encoding the amino acid sequence.

[0027] In further embodiments, at least one RNA of the compositions disclosed herein preferably comprises at least one of the following structural elements: 5'- and / or 3'-untranslated region elements (UTR elements), particularly 5'-UTR elements comprising or consisting solely of a nucleic acid sequence derived from the 5'-UTR of the p53 gene or a fragment, homolog or variant thereof, or 5'- and / or 3'-UTR elements that may be derived from a gene or its homolog, fragment or variant thereof that provides stable mRNA; a histone stem-loop structure, preferably a histone stem-loop in its 3' untranslated region; a 5'-cap structure; a poly(A) tail (poly(A) sequence); or a poly(C) sequence.

[0028] In another embodiment, the RNA compound includes a 5' cap structure. While 5' cap structures are well known in the art, the preferred cap is selected from Cap-0, Cap-1, or Cap-2 structures, and is preferably m7G(5')ppp(5')(2'OMeA)pG.

[0029] Embodiments in which the RNA compound includes a 5'UTR containing an eIF4G aptamer are also preferred.

[0030] The preferred RNA compound of the present invention comprises at least one nucleotide, which is a modified nucleotide, for example, pseudouridine or 5-methylcytidine, where the modified nucleotide is, for example, a substitution of at least one uracil with pseudouridine, preferably a substitution of all uracil positions with pseudouridine.

[0031] In preferred embodiments, the RNA compound according to the present invention is derived from the expression of p53 mRNA based on a mammalian vector and induces apoptosis in cancer cells according to the present invention.

[0032] The RNA compound according to any one of claims 1 to 10, which is administered to a subject by administering a pharmaceutically acceptable liposomal formulation containing the RNA compound.

[0033] In relation to the present invention, proliferative diseases are neoplastic diseases. Neoplastic diseases are particularly ovarian cancer, most preferably high-grade serous ovarian cancer (HGSOC). The tumors of the present invention may be metastatic tumors and / or recurrent tumors.

[0034] In preferred embodiments of the present invention, the treatment according to the first aspect comprises administering an RNA compound to a subject, mediated p53 translation of the RNA compound in tumor tissue or tissue adjacent to the tumor, and thereby specifically inducing apoptosis in tumor cells.

[0035] In some embodiments, subjects treated according to the present invention underwent surgery.

[0036] In certain embodiments, specifically relating to the disease HGSOC, the subject has undergone or is undergoing intraperitoneal tumor debulking therapy (for example, by rinsing while administering the RNA compound of the present invention). Therefore, in certain embodiments, the RNA compound is administered intraperitoneally to the subject.

[0037] In some embodiments, the subject has received or is receiving at least one additional anticancer therapy. For example, the additional anticancer therapy is chemotherapy, such as chemotherapy involving the administration of one or more platin compounds. In connection with the present invention, the chemotherapy administered to the subject includes the administration of paclitaxel in combination with cisplatin or cisplatin in combination with cyclophosphamide.

[0038] A preferred embodiment of the present invention relates to a therapeutic RNA compound disclosed wherein the proliferative disorder is HGSOC, the subject has received or is receiving detoxification therapy, and the RNA compound is administered intraperitoneally to the subject.

[0039] In another embodiment, a pharmaceutical composition comprising the above-mentioned RNA compound and at least one pharmaceutically acceptable excipient is further provided.

[0040] The present invention also relates to a pharmaceutical composition comprising an RNA molecule obtained by transcription of a DNA sequence having at least 80% sequence identity with SEQ ID NO: 3 as an active ingredient. In one embodiment, the pharmaceutical composition comprises an RNA molecule having at least 80% sequence identity with the nucleic acid sequence encoding the protein of SEQ ID NO: 1 as an active ingredient.

[0041] It is understood herein that a pharmaceutical composition contains a therapeutically effective amount of active ingredient and pharmacological excipients. Those skilled in the art know how to formulate pharmaceutical compositions containing RNA molecules. In particular, those skilled in the art know how to stabilize RNA molecules in pharmaceutical compositions.

[0042] In the pharmaceutical composition according to the present invention, RNA molecules can be encapsulated in lipid nanoparticles, cellular or synthetic exosome mimics, or virus-like particles.

[0043] In this specification, the term “lipid nanoparticles” is defined as molecules having a spherical shape and containing a solid lipid core stabilized by a surfactant. The core lipid may be a steroid, fatty acid, acylglycerol, wax, or a combination thereof. The surfactant may be a phospholipid, sphingomyelin, or a biological membrane lipid such as bile salt (e.g., sodium taurocholate). All of these may be used as stabilizers in lipid nanoparticles used in the pharmaceutical compositions of the present invention.

[0044] In this specification, the term “cellular or synthetic exosome mimetic” is defined as a nano-sized vesicle (exosome) derived or purified from modified cells (cellular exosome mimetic), or produced by artificial methods such as cell extrusion (synthetic exosome mimetic) that function as cargo for delivering proteins, nucleic acids, or other cellular components to adjacent or distant cells.

[0045] In this specification, the term "virus-like particle" is defined as a multiprotein structure that closely resembles the composition and conformation of a virus but does not contain viral genetic material.

[0046] A method for treating a proliferative disorder in a subject is also disclosed, comprising the step of administering an RNA compound of this specification to the subject.

[0047] A method for producing a pharmaceutical for the treatment of a proliferative disorder in a subject is also disclosed, comprising the step of formulating an RNA compound described herein as a pharmaceutical. In some preferred embodiments, this method includes the step of in vitro transcription of the RNA compound as defined herein.

[0048] Furthermore, this method may include a step of formulating the RNA compound as a liposome composition, where the liposome encapsulates the RNA compound.

[0049] The present invention is also described by the following itemized embodiments, which shall be understood and interpreted in the context of this complete disclosure, in particular in light of the various definitions and descriptions and examples provided herein.

[0050] Item 1. A ribonucleic acid (RNA) compound for the treatment of proliferative disorders in a subject, wherein the RNA compound encodes the amino acid sequence of the human p53 protein.

[0051] Item 2. The RNA compound according to Item 1, wherein the RNA compound encoding an amino acid sequence comprises the nucleotide sequence of SEQ ID NO: 1 (preferred RNA sequence), or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of SEQ ID NO: 1.

[0052] Item 3. RNA compounds described in Item 1 or 2, which are either in vitro transcription (IVT) RNA or artificially polymerized RNA.

[0053] Item 4. An RNA compound according to any one of items 1 to 3, comprising a poly(A) sequence operably ligated to a sequence encoding an amino acid sequence.

[0054] Item 5. An RNA compound as described in Item 4, wherein the poly(A) sequence contains at least 100 nucleotides.

[0055] Item 6. An RNA compound as described in Item 4 or 5, wherein the poly(A) sequence is located at 3' of the sequence encoding the amino acid sequence.

[0056] Item 7. An RNA compound according to any one of items 1 to 6, wherein the RNA molecule comprises a 5' cap structure, for example, Cap-0, Cap-1, or Cap-2 structure, and is preferably m7G(5')ppp(5')(2'OMeA)pG.

[0057] An RNA compound listed in any one of items 1 through 7, including item 8.5' untranslated region (UTR).

[0058] RNA compounds listed in item 8, wherein item 9.5'UTR contains the eIF4G aptamer.

[0059] Item 10. An RNA compound according to any one of items 1 to 9, wherein at least one nucleotide is a modified nucleotide such as pseudouridine or 5-methylcytidine, for example, the modified nucleotide is a substitution of at least one uracil with pseudouridine, preferably all uracil positions are substituted with pseudouridine.

[0060] Item 11. An RNA compound described in any one of items 1 through 10, administered to a subject by administering a pharmaceutically acceptable liposomal formulation containing the RNA compound.

[0061] Item 12. RNA compounds listed in any one of items 1 through 11, whose proliferative disorder is a neoplastic disorder.

[0062] Item 13. The tumor disease is ovarian cancer, and the RNA compound is one of those listed in Item 12.

[0063] Item 14. The RNA compound described in Item 13, which indicates that the ovarian cancer is high-grade serous ovarian cancer (HGSOC).

[0064] Item 15. The tumor is a metastatic tumor, and the RNA compound is one of the items listed in items 12 through 14.

[0065] Item 16. The tumor is a recurrent tumor, and the RNA compound is one of the items listed in items 12 through 15.

[0066] Item 17. An RNA compound listed in any one of items 1 through 16, from which the subject underwent surgery.

[0067] Item 18. An RNA compound listed in any one of items 1 through 17, wherein the disease is HGSOC and the subject has received intraperitoneal tumor debulking therapy.

[0068] Item 19. An RNA compound described in any one of items 1 through 18, which is administered intraperitoneally to the subject.

[0069] Item 20. RNA compounds described in any one of items 1 through 19, in which the subject has received or is receiving at least one additional anti-cancer therapy.

[0070] Item 21. RNA compounds listed in Item 20, for which additional anti-cancer therapy is chemotherapy.

[0071] Item 22. RNA compounds as described in Item 21, wherein chemotherapy includes the administration of one or more platinum compounds.

[0072] Item 23. RNA compounds as described in Item 21 or 22, wherein the chemotherapy is administration of paclitaxel in combination with cisplatin or cisplatin in combination with cyclophosphamide.

[0073] Item 24. An RNA compound described in any one of items 1 through 23, wherein the proliferative disorder is HGSOC, the subject is undergoing detoxification therapy, and the RNA compound is administered intraperitoneally.

[0074] Item 25. A pharmaceutical composition comprising an RNA compound described in any one of items 1 to 24 and at least one pharmaceutically acceptable excipient.

[0075] Item 26. A pharmaceutical composition as described in Item 25, which is a liposome formulation of an RNA compound.

[0076] Item 27. A pharmaceutical composition according to item 25 or 26, for use in the treatment described in any one of items 1 through 24.

[0077] Item 28. A method for treating a proliferative disorder in a subject, comprising the step of administering an RNA compound described in any one of items 1 to 24 to the subject.

[0078] Item 29. The method of Item 28, wherein the treatment is for any of the uses described in Item 1 to 24.

[0079] Item 30. A method for producing a pharmaceutical product for the treatment of a proliferative disorder in a subject, comprising the step of formulating an RNA compound as described in any one of items 1 to 24.

[0080] Item 31. The method according to Item 30, comprising the step of transcribing an RNA compound defined in any one of the preceding items in vitro.

[0081] Item 32. The method according to item 30 or 31, comprising the step of formulating an RNA compound as a liposome composition, wherein the liposome encapsulates the RNA compound.

[0082] As used herein, terms such as "[of this] invention," "in accordance with the invention," and "according to the invention" are intended to refer to all aspects and embodiments of the invention described and / or claimed herein.

[0083] As used herein, the term “comprising” should be interpreted as encompassing both “including” and “consisting of,” both meanings being specifically intended and thus the individually disclosed embodiments of the present invention. As used herein, “and / or” should be interpreted as a specific disclosure of each of two designated features or components, the other of which may or may not be present. For example, “A and / or B” should be interpreted as (i) A, (ii) B, and (iii) A and B, each of which is a specific disclosure, as if each were individually described herein. In the context of the present invention, the terms “about” and “approximately” indicate intervals of precision that a person skilled in the art would understand to still guarantee the technical effect of the feature being discussed. These terms typically indicate deviations of ±20%, ±15%, ±10%, and, for example, ±5% from the given numerical value. As a person skilled in the art will understand, such specific deviations from the numerical value of a given technical effect depend on the nature of the technical effect. For example, natural or biological technical effects can generally have greater deviations than artificial or engineered technical effects. As those skilled in the art will understand, such specific deviations from the numerical value of a given technical effect depend on the nature of the technical effect. For example, natural or biological technical effects can generally have greater deviations than artificial or engineered technical effects. When an indefinite or definite article, e.g., "a," "an," or "the," is used when referring to a singular noun, this includes the plural of that noun unless otherwise specifically stated.

[0084] It should be understood that the application of the teachings of the present invention to specific problems or environments, and the inclusion of variations of the present invention or additional features therein (such as further aspects and embodiments) are within the capabilities of those skilled in the art in light of the teachings contained herein.

[0085] Unless otherwise indicated by the context, the above descriptions and definitions of features are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described herein.

[0086] All references, patents, and publications cited herein are incorporated herein by reference in their entirety. [Brief explanation of the drawing]

[0087] The drawing is shown below:

[0088] [Figure 1]This figure shows that liposome transfection with in vitro transcribed wt p53 mRNA induces cell cycle arrest and apoptosis in the HGSOC cell line OVCAR-8. a. OVCAR-8 cells were transfected with increased amounts of liposome wt and in vitro transcribed (IVT) Flag-tagged p53 mRNA. Cell lysates were subjected to Western blotting using Flag antibody and β-actin antibody. b. The dose-dependent cell cycle distribution of OVCAR-8 cells 24 hours after liposome transfection is shown as a bar graph. c. (Top) Cell lysates were subjected to Western blotting for important mediators of cell cycle regulation using Flag antibody, p53(pS20) antibody, p53 antibody, p21 antibody, PLK1 antibody, cyclin A1 antibody, cyclin B1 antibody, cyclin E1 antibody, Aurora A antibody, CDK1 antibody, p27 antibody, p16 antibody and β-actin antibody. (Below) The same cell lysates were subjected to WB for apoptosis regulators using PARP antibody, Caspase-3 antibody, Puma antibody, Noxa antibody, and Fas antibody. d. Cells were transfected with mock transfect or 0.5 μg p53 mRNA, and proliferation was measured 24 hours after treatment using the CellTiter-Blue cell viability assay. **P<0.01, ***P<0.001, Student's t-test, unpaired and two-sided. e. Dose-dependent caspase 3 / 7 activity was determined using the Caspase-Glo 3 / 7 assay. ***P<0.001, Student's t-test, unpaired and two-sided. f. Graph of annexin V-positive (%) cells. Demonstrates FACS analysis of gated transfected cells for annexin V-positive (early apoptosis) and annexin V / 7AAD-positive (late apoptosis) populations. [n=3 for each dose; ***=<0.001]. g. (Top) The dose-dependent cell cycle distribution of nocodazole-synchronous cells is shown as a bar graph. (n=3) (Bottom) Cell lysates were subjected to Western blotting using Flag antibody, cyclin E1 antibody, cyclin B1 antibody, PLK1 antibody, and β-actin antibody. h. (Left) A representative image shows the number of 3D colonies (dose-dependent) on day 10.(Right) The dose-dependent distribution of colonies is shown as a bar graph. (n=3) *P<0.05, ***P<0.001, Student's t-test, unpaired and two-tailed tests.

[0089] [Figure 2] This study demonstrates that restoring p53 function reduces numerical chromosomal instability (nCIN) in OVCAR-8 cells. a. Treatment schedule. OVCAR-8 cells were cultured for 28 days and treated with 200 ng of p53 mRNA every 48 hours. At the end of incubation, metaphase diffusion of untreated control and treated OVCAR-8 cells was prepared. Chromosomes were stained with Hoechst. b. Western blot using Flag antibody and β-actin antibody. c. (Top) Frequency distribution of chromosome number distribution in control and cells treated with 200 ng of p53 mRNA. (Bottom) Quantification of chromosome number in p53 mRNA-treated / untreated cells (mean ± SD). Results were statistically analyzed. ****p<0.001. d. Representative image of metaphase chromosome diffusion. Scale bar 20 μm.

[0090] [Figure 3]This figure shows that liposomal transfection with p53 mRNA results in higher p53 expression in primary HGSOC cells compared to normal counterparts. a. Primary human cells (tumor, normal) and OVCAR-8 cells from different origins were transfected with p53 mRNA. (Left) Cell lysates were subjected to WB using Flag antibody and β-actin antibody. (Right) Normalized levels of p53 expression are shown as bar graphs. b. (Top) Primary human HGSOC cells (tumor, normal) from patients P3-8 were transfected with 0.5 μg of p53 mRNA. Cell lysates were subjected to WB using Flag antibody and β-actin antibody. (Bottom left) Expression efficacy is shown as a bar graph. (Bottom right) Caspase 3 / 7 activity was determined using the Caspase-Glo 3 / 7 assay. **P<0.01, ***P<0.001, Student's t-test, unpaired and two-tailed test. c. (Left) Lysates of mock or p53 mRNA-transfected normal ovarian or tumor HGSOC (Patient 2) cells were subjected to WB using Flag antibody, p53 antibody, p21 antibody, CDK1 antibody, p27 antibody, p16 antibody, and β-actin antibody. (Right) Normalized levels of p53 expression are shown as a bar graph. d. (Left) The same cell lysates were subjected to WB using PARP antibody, Puma antibody, and Noxa antibody. (Right) Caspase 3 / 7 activity of mock or p53 mRNA-transfected normal and tumor HGSOC cells was determined using the Caspase-Glo 3 / 7 assay. (n=3) e. (Left) Representative image shows 3D colonies of transfected cells on day 10. (Right) Number of colonies is shown as a bar graph. (n=3) f. (Left) Representative image shows organoids on day 4. (Center) Organoid size was determined using ImageJ Software (n=8). *** P<0.001, Student's t-test, unpaired, two-tailed test. (Right) Caspase 3 / 7 activity of p53 mRNA transfected with HGSOC organoid mock or 2 μg of p53 mRNA was determined using the Caspase-Glo 3 / 7 assay. (n=3), *P<0.05, Student's t-test, unpaired and two-tailed test.(Below) Lysates of HGSOC organoids (patients 1-3), either mocks or transfected with p53 mRNA, were subjected to Western blotting using Flag antibody and β-actin antibody.

[0091] [Figure 4] This figure shows the identification of differentially expressed genes in p53 mRNA-transfected OVCAR-8 cells compared to mock-transfected counterparts. a. Heatmap of normalized gene expression represented by z-scores of mock (EGFP mRNA) or p53 mRNA-treated OVCAR-8 cells from three independent RNA-seq experiments (Student's t-test or Welch test, corrected for false-positive detection rate [FDR] q-adjusted p-value < 0.05 and absolute magnification change [FC] > 1.5 depending on variance). b. Volcano plot showing differentially expressed genes (DEGs) in RNA-seq between EGFP and p53 mRNA-treated OVCAR-8 cells (red dots = upcontrol, blue dots = downcontrol). c. Overrepresented Ingenuity pathways selected from pathway enrichment analysis of DEGs with FDR-adjusted p-value < 0.05 and FC > 1.5. Pathways with positive z-scores are shown in orange. Pathways with negative z-scores are shown in blue, and pathways without a specific pattern are shown in gray. d. Heatmap of all genes corresponding to the "p53 signaling pathway" from Ingenuity Pathway Analysis (IPA), showing P-values, FDR-adjusted P-values, and magnification changes (FC) for comparison between EGFP and p53 mRNA. Genes with WB validation are highlighted in red. e. Cell lysates were subjected to WB for the p53 signaling pathway using antibodies against p21, MDM2, Puma, p73, Fas, CDK1, and β-actin.

[0092] [Figure 5]This figure shows the analysis of differentially expressed genes in primary HGSOC cells. a. Primary HGSOC cells from patient 1 were transfected with p53 mRNA or EGFP-mRNA. Cell lysates were subjected to Western blotting using EGFP antibody, Flag antibody, and β-actin antibody. b-c. Heatmap (b) and volcano plot (c) showing differentially expressed genes (DEGs) in RNA-seq between primary HGSOC cells treated with EGFP versus p53 mRNA (P-value < 0.01, and absolute magnification change (FC) > 1.5) (red dots = up control, blue dots = down control). d. Transcriptome analysis of DEGs common to OVCAR-8 and primary HGSOC cells.

[0093] [Figure 6] This figure shows the upregulation of ovarian cancer signaling pathways when OVCAR-8 cells are treated with IVT p53 mRNA. a. Heatmap of all genes corresponding to the OC signaling pathway from Ingenuity Pathway Analysis (IPA), showing P-values, FDR-adjusted P-values, and magnification changes (FC) for comparison of EGFP and p53 mRNA in OVCAR-8 cells. Ingenuity Pathway Analysis of significant genes with FDR-adjusted P-values ​​<0.05 and FC >1.5 between EGFP and p53 mRNA treatment in OVCAR-8 cells. Downregulated genes are shown in blue / green (predicted / measured), and upregulated genes are shown in orange / red (predicted / measured). b. WB analysis using Flag antibody, EGFP antibody, GSK3B antibody, p-GSK3B antibody, β-catenin antibody, p-β-catenin antibody, AKT3 antibody, cyclin D antibody, CDK4 antibody, BRCA2 antibody, K-Ras antibody, PLK1 antibody, and β-actin antibody. c. (Top) TCF / LEF luciferase reporter response in cells to Wnt-3a stimulation. (Bottom) TCF / LEF luciferase reporter response in cells transfected with gradually increasing doses of p53 mRNA.

[0094] [Figure 7]This figure shows that application of liposome p53 mRNA to OVCAR-8 cells prevents intracystic growth and seeding in an orthotopic mouse model. a. Schematic diagram of the orthotopic mouse model. b. Bioluminescence imaging. (Left) On day 0, Luc-expressing OVCAR-8 cells were transfected (mock, p53 mRNA: 100 ng, 500 ng). 1 × 10⁶ cells / mouse were injected into the cyst (right ovary) of nude mice on day 1 and imaged using the IVIS Lumina system after D-luciferin application. Representative images (1 / week) of mice at baseline 8 weeks after orthotopic application of OVCAR-8 cells (mock or p53 mRNA treated) are shown. Photon flux is shown in a pseudo-colorized heatmap. (Right) BLI was performed at the indicated time points, and the data are shown in a graph. Photon flux was normalized to the baseline value obtained from mice on day 1. *P<0.05, **P<0.01, Wilcoxon test, unpaired and two-sided. c. (Left) Representative photographs of removed ovaries 8 weeks after intracystic application of mock or p53 mRNA-transfected (100ng, 500ng) OVCAR-8 cells in mice (n=8 in each group). (Center) Analysis of excised ovaries. Dose-dependent tumor volume and (right) BLI data are shown as bar graphs. *P<0.05, **P<0.01, Wilcoxon test, unpaired and two-sided test. d. BLI of removed organs 8 weeks after intracystic application of mock (n=8) or p53 mRNA-transfected (100ng, 500ng) OVCAR-8 cells (n=8 mice in each group) is shown as a bar graph. **P<0.01, ***P<0.001, Wilcoxon test, unpaired and two-sided test. e. Hematoxylin / eosin-stained sections of ovaries injected with p53 mRNA-transfected (100 ng, 500 ng) cells (n=8) or mock (n=8) OVCAR-8 cells at the end of the experiment (8 weeks). Scale bar, 300 μm. f. Hematoxylin / eosin-stained sections (8 weeks later) of metastases in the small intestine and colon after intracystic injection of mock or p53 mRNA-transfected (100 ng, 500 ng) OVCAR-8 cells, eight representative examples each. Scale bar, 300 μm.

[0095] [Figure 8] This figure shows that intravenous administration of liposomal p53 mRNA to mice after intravenous injection of OVCAR-8 cells prevents tumor formation and organ dissemination in an orthotopic mouse model. a. (Top) Bioluminescence imaging of mice containing luciferase-expressing metastatic OVCAR-8 cells treated with liposomal p53 mRNA. After intravenous injection of 2 × 10⁶ OVCAR-8 cells stably expressing luciferase, treatment with liposomal p53 mRNA for 3 weeks (2 × 5 μg liposomal p53 mRNA / week). (Right) Color scale represents p / sec / cm² / steradian. (Bottom) Tumor load of control and liposomal p53 mRNA-treated nude mice was measured weekly by bioluminescence over 9 weeks. *P<0.05, **P<0.01, ***P<0.001, Wilcoxon test, unpaired and two-sided. b. Determination of body weight throughout the observation period. c. Examination of peritoneal structures and organs for tumor dissemination after laparotomy. d. Bioluminescence imaging of removed mouse organs (control vs. p53 mRNA treatment).

[0096] The array is as follows:

[0097] Sequence ID 1 shows the amino acid sequence of the standard human p53 isoform:

[0098] MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIR VEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKKSKKGQSTSRHKKLMFKTEGPDSD

[0099] Sequence ID 2 shows the amino acid sequence of the flagged-tagged p53 human used in the example:

[0100] MDYKDHDGDYKDHDIDYKDDDDKMEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPT PAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDS DGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKKSKKGQSTSRHKKLMFKTEGPDSD.

[0101] Sequence ID 3 shows the mRNA sequence of the p53 expression construct of the present invention.

[0102] [Examples]

[0103] Herein, specific aspects and embodiments of the present invention will be described as examples with reference to the description, figures, and tables set forth herein. Such examples of the methods, uses, and other aspects of the present invention are representative only and should not be construed as limiting the scope of the present invention to such representative examples.

[0104] Examples are shown below.

[0105] Example 1: Mammalian vector-based expression of p53 induces apoptosis in HGSOC cells.

[0106] Since no drug sufficiently specific to rescue functional wt p53 in cancer cells harboring loss-of-function p53 mutants is yet available, we investigated reactivating p53 using different experimental strategies in HGSOC cell lines and primary OC cells. First, we tested five known OC cell lines for p53 expression. p53 expression was not detected in SKOV3 and OVCAR5, consistent with previous observations that they were classified as p53 deficient. In contrast, OVCAR-3, -4, and 8 cells showed high p53 protein expression due to mutations at R248Q, L130V, or a single nucleotide mutation (c.376-1G>A) at the splice junction, respectively. For further analysis, we selected different HGSOC cell lines, including OVCAR-8, with different genomic alterations confirmed by sequencing. In mutant TP53-carrying OVCAR-8 cells, exogenous expression of Flag-tagged wt p53, driven for 48 hours by a mammalian vector system, significantly reduced cell proliferation compared to controls. Evidence of reactivation of p53 activity is the induction of G0 / G1 cell cycle arrest. Therefore, we examined cells transfected with increased levels of wt p53 expression vector by flow cytometry 24 hours after transfection and observed an increase in the percentage of apoptotic cells in the sub-G1 phase and a moderate increase in the G0 / G1 population accompanied by significantly increased levels of caspase 3 / 7 activity. Annexin V-positive and PI-negative (Annexin V-FITC) + / PI - ) early apoptotic cells and annexin V / PI double-positive (annexin V-FITC + / PI +By determining late (terminal) apoptotic cells, the increased level of apoptosis in OVCAR-8 cells expressing increased levels of wt p53 was revealed. Western blot (WB) experiments for the detection of cleaved caspase-3 and PARP confirmed the increase in apoptosis. To further evaluate cell cycle progression in OVCAR-8 cells, the inventors performed WB tests on key regulators of cell cycle progression. CDK inhibitors (p16, p27) and p21, one of the best-studied p53 target genes, were upregulated with increased p53 expression by the vector. PLK1 and CDK1, two key elements in cell cycle regulation, were downregulated. Next, the inventors demonstrated the cytotoxicity of wt p53 in a dose-dependent manner by in vitro assays evaluating colony-forming ability compared to controls. Similar experiments in additional HGSOC cell lines OVCAR-3 and OVCAR-5 confirmed growth inhibition, cell cycle arrest, induction of apoptosis, and reduced colony formation ability upon wt p53 expression. Taken together, these data demonstrate that rescue of p53 function by vector-based re-expression of wt p53 induces cell cycle misregistration and death in HGSOC cells in a dose-dependent manner.

[0107] Example 2: Design, generation, and characterization of IVT mRNA in HGSOC cell lines.

[0108] Recently, the therapeutic use of mRNA as a vaccine has offered great hope in combating a range of infectious diseases. However, it remains unclear whether this class of biological agents can represent a new generation of "miracle cures" for cancer-targeted precision medicine, as hypothesized by Paul Ehrlich more than 100 years ago. To address this unanswered question, we used an IVT system based on a bacterial vector containing a T7 RNA polymerase promoter for mRNA transcription (human p53, firefly luciferase (Luc), enhanced green fluorescent protein (EGFP)), taking into account aspects of mRNA translation efficacy and decay rate. After IVT, thorough purification of the mRNA involved the use of DNase for degradation of the DNA template and the use of a spin column (silica membrane) to remove the digested template, excess NTPs, salts, and unused capping analogs. From this, IVT-mRNAs such as IVT-Flag tagged p53 mRNA, called p53 mRNA, were analyzed by agarose gel electrophoresis for quality control.

[0109] Based on the clinical advantages of liposomes as carriers of chemotherapeutic agents over conventional chemotherapy due to reduced therapeutic side effects and increased anticancer activity, the inventors utilized a liposome system to test its support quality for the delivery of IVT-mRNA (EGFP-mRNA, luc-mRNA) to HGSOC cells. For this purpose, the inventors first investigated the transfection efficiency in different HGSOC cell lines using control EGFP-mRNA encapsulated in positively charged lipid molecules (liposomes). To quantitatively test uptake, EGFP expression was measured by flow cytometry as a surrogate marker of transfection efficiency. A dose-dependent increase in EGFP expression was detected 24 hours after transfection with EGFP-mRNA (0.1-2 μg). 0.25 μg of EGFP-mRNA was sufficient for expression in 90% of cells, while doses of ≥0.5 μg resulted in EGFP fluorescence in 97-100% of cells, supporting superior transfection efficiency. This observation was confirmed by Western blotting (WB) showing high levels of EGFP at doses of ≥0.25 μg of EGFP-mRNA. In temporomandibular system analysis using 1 μg of EGFP-mRNA, a high percentage of EGFP-expressing cells (92.2–99.7%) persisted for 10 days post-transfection (d), decreasing to baseline levels (≤5.5%) between days 15 and 20. EGFP expression can be supported by WB for 7–8 days. As a second control IVT-mRNA, Luc-mRNA transfection was monitored by WB and by activity assays showing high enzymatic activity in cells transfected with 0.25–2 μg of Luc mRNA.

[0110] To further validate the usefulness of our approach for the expression of selected proteins driven by IVT-mRNA in OC cells, we tested additional cell lines (OVCAR-3, -4, -5, -8) by immunofluorescence, flow cytometry, and WB. Strong expression (≧70%) was detected in OVCAR-5 cells transfected with 1 μg of EGFP-mRNA, and very strong expression (≧95%) was detected in OVCAR-3, -4, and 8 cells. In summary, different experiments demonstrated high efficiency of liposome-based IVT-mRNA transfection and expression, regardless of OC cell line or mRNA type.

[0111] Example 3: Characterization of mRNA expression in primary normal ovarian cells and primary HGSOC cells.

[0112] Next, the inventors investigated the time dynamics of EGFP and Luc-mRNA expression in primary HGSOC cells. EGFP expression using 1 μg of mRNA was detectable by FACS testing, with the percentage of positive cells decreasing from 85% on day 1 to 74% on day 11. Western blotting (WB) validation showed EGFP expression for at least 7 days. Upon Luc-mRNA transfection, the corresponding enzyme activity was detectable for 7 days, and the WB signal was detectable for 4 days. Next, tumor organoids, a promising model in cancer research, were prepared from primary HGSOC tissue, metastatic tissue, and normal ovarian tissue. Organoids from primary tumor and metastatic sites showed efficient Luc activity or EGFP expression for up to 5 days. Luc activity in normal ovarian cells was detectable for only 1-2 days. To monitor the duration of Luc activity in mice by in vivo bioluminescence imaging (BLI), we transfected OVCAR-8 cells with Luc-mRNA for subsequent application to mice: (i) 1 × 10⁶ cells transfected with 2 μg of Luc-mRNA 6 (ii) Orthotopic transplantation of OVCAR-8 cells, (ii) 2 × 10⁶ transfected with 4 μg of Luc-mRNA 6(iii) IP injection of individual OVCAR-8 cells or (iii) IP injection of 10 μg of liposomal Luc-mRNA. Luciferase activity was detectable by BLI for 3 days in all three experimental settings. In summary, liposomes were well-suited for efficient and long-term IVT-mRNA expression in primary human cells (2D-, 3D) and mice.

[0113] Example 4: mRNA-based rescue of p53 function in HGSOC cell lines induces cell cycle arrest and cell death.

[0114] TP53 mutations are very common in HGSOC, but there are currently no approved clinical drugs to rescue wt p53 function. Therefore, we aimed to rescue p53 function in different preclinical models by transfecting HGSOC cell lines and primary cells (2D cultures, organoids) with IVT-mRNA encoding p53. Low doses of p53 mRNA (10-250 ng) induced strong p53 protein expression in OVCAR-8 cells (Figure 1a). As an indicator of p53 activity reactivation, compared to controls, a significant increase of 22.45% in G0 / G1 cells with a 57.14% decrease in S phase was observed with 0.1 μg of p53 mRNA 24 hours after transfection (Figure 1b), supporting our results using vector-based re-expression of p53. Based on the higher p53 expression levels using p53 mRNA compared to vector-based experiments, potent induction of cell cycle inhibitors p21, p16, and p27 was readily observed with low doses of p53 mRNA (≧0.1 μg), in parallel with downregulation of key cell cycle regulators such as PLK1, Aurora A, CDK1, and cyclin A / B (Figure 1c, top). These observations correlated with a significant decrease in cell viability of approximately twofold after 48 hours at a dose of 0.5 μg, accompanied by elevated levels of different criteria for cell death: Puma, Noxa, and Fas (Figure 1c, bottom), caspase-3 and PARP (Figure 1c, bottom), caspase 3 / 7 activity (Figure 1e), and annexin staining (Figure 1f) (Figure 1d). A dose-dependent increase in G1 was clearly observed in synchronized p53 mRNA-transfected cells (Figure 1g). As the dose of p53 mRNA increased, the loss of colony-forming ability in OVCAR-8 cells increased (Figure 1h). Furthermore, we tested p53-deficient HGSOC cells, such as OVCAR-5, for their sensitivity to wt p53 reexpression. Within 24 hours after p53 mRNA transfection, we were able to observe significant signs of cell death, including strong PARP cleavage, high levels of cells in sub-G0, and high levels of caspase 3 / 7 activity.In summary, our observations indicate that rescuing p53 function through transfection with wt p53 mRNA induces cell death in different HGSOC cell lines.

[0115] Example 5: Restoration of p53 reduces numerical chromosomal instability (nCIN) in the HGSOC cell line OVCAR-8.

[0116] Chromosomal instability (CIN) or karyotype instability is a fundamental characteristic of occlusive cell cultures (OCs), and most human OC cells exhibit aneuploidy. The OVCAR-8 cell line is no exception, as it is classified as a highly diploid cell line with high numerical complexity and a modal number of chromosomes ranging from 50 to 59. Our study aimed to determine whether the restoration of functional wt p53 could counteract nCIN and reduce the number of chromosomes in the cells to a near-diploid state. For this purpose, OVCAR-8 cells were treated with low doses of p53 mRNA every two days for 28 days, and chromosome distribution was assessed at the end of the incubation period using chromosome spreading (Figure 2, d). p53 expression significantly reduced the total number of chromosomes in cells treated with p53 mRNA compared to controls (Figure 2c, top). Re-expression of wt p53 reduced the average chromosome number from 53 to 47, providing further evidence of the important role p53 plays in maintaining genomic integrity in HGSOC cells (Figure 2c, bottom).

[0117] Example 6: mRNA-based rescue of p53 function in primary HGSOC cells induces cell cycle arrest and cell death.

[0118] To further investigate the rescue of p53 in preclinical scenarios, the inventors compared the effects of p53 mRNA on primary human cells (normal and cancerous) (Figure 3a). p53 expression driven by transfected p53 mRNA could be detected in different primary samples, except keratinocytes (Figure 3a). Initial comparisons of HGSOCs from the same patient with corresponding normal ovarian tissue revealed higher p53 expression in tumor cells despite equivalent amounts of transfected IVT-mRNA (Figure 3a). Further tissue analysis demonstrated that p53 expression (Figure 3b top, bottom left) and caspase 3 / 7 activity (Figure 3b, bottom right) in all tumor samples were significantly higher than the corresponding levels in normal ovarian cells. In contrast, similar experiments using IVT-GFP-mRNA resulted in nearly identical levels of GFP expression upon transfection with equivalent amounts of IVT-mRNA. When the transfection efficiency of fluorescent mRNA was tested in normal cells and HGSOC cells, no difference was detected, suggesting that the difference in transfection efficacy did not appear to be due to different p53 levels (tumor vs. normal). WB testing revealed that activation of cell cycle inhibitors p21, p16, and p27 (Figure 3c) and apoptosis (Figure 3d) were stronger in p53 mRNA-transfected primary HGSOC cells compared to normal counterparts, and correlated with a more significant decrease in HGSOC colony count (Figure 3e). Analysis of HGSOC organoids derived from patients P1-3 also demonstrated a significant increase in caspase 3 / 7 activity upon p53 mRNA treatment under 3D cell culture conditions (Figure 3f). In summary, p53 expression reduced the viability of primary cells of both types (normal vs. HGSOC), but the effect was significantly more pronounced in primary HGSOC cells compared to normal cells, which may be attributable to elevated levels of p53 in tumor cells (normal ≈ 21x vs. malignant ≈ 150x).

[0119] Example 7: Transcriptome changes in OVCAR-8 cells during rescue of wild-type p53 function.

[0120] Our observations of decreased proliferative activity and increased cell death in HGSOC-derived cell lines and primary cells supported a model of rescued p53 function upon re-expression of p53 by mRNA transfection. To deepen our understanding of the underlying biological processes and to address this aspect in a relatively unbiased manner, we transfected OVCAR-8 cells with 1 μg of p53 mRNA or a mock control (EGFP mRNA) in three independent experiments. A total of 25,540 human genes were profiled by mRNA sequencing 24 hours after transfection. The two groups were compared by unpaired t-tests considering group variances. All calculated p-values ​​were corrected using the Benjamini-Hochberg false-positive detection rate (FDR) method, and false positives were discarded by performing multiple tests. Differentially expressed genes (DEGs) between the two groups were selected according to the criteria of FDR q < 0.05 and absolute magnification change > 1.5. Transcriptome analysis revealed 3,061 upregulated transcripts and 1,662 downregulated transcripts (Figure 4a, b). These significant genes were further analyzed using Ingenuity Pathway Analysis (IPA) software to identify the overrepresented standard pathway associated with p53 mRNA transfection in OVCAR-8 cells (Figure 4c).

[0121] Consistent with the pro-apoptotic function of p53 as a tumor suppressor involved in cell cycle arrest and apoptosis induction, our Ingenuity pathway analysis revealed overexpression of several related pathways, such as "p53 signaling," "apoptotic signaling," and several "cell cycle" pathways, upon p53 mRNA transfection (Figure 4c). To confirm these findings, we investigated all genes involved in the IPA "p53 signaling" pathway and found that most of them (51 / 81, 63%) were differentially expressed with FDR q < 0.05 and absolute digit change > 1.5 (Figure 4d). Notably, most of the known downstream targets of p53 signaling (96%) were cooperatively upregulated by p53 mRNA transfection. The inventors further validated these results by performing Western blotting (WB) on selected proteins from the “p53 signaling pathway,” including p21(CDKN1A), MDM2, Puma(BBC3), p73, FAS(CD95), and CDK1 (Figure 4d). Their expression in OVCAR-8 cells was substantially upregulated or downregulated by p53 mRNA, consistent with transcriptome analysis. Regarding the “apoptotic signaling” pathway, most of the genes in this pathway (42 / 65, 65%) were significantly deregulated by p53 mRNA transfection. To demonstrate that p53 mRNA transfection induces apoptotic signaling in OVCAR-8 cells, the inventors extensively examined 13 of these proteins by WB.

[0122] Several "cell cycle" pathways, including "cell cycle regulation of chromosome replication," "cell cycle: G2 / M DNA damage checkpoint regulation," and "cyclin and cell cycle regulation," were significantly overexpressed and showed overall downregulation (negative z-score), similar to the "mitotic role of polo-like kinases" (Figure 4c). Most of the genes from these pathways were differentially expressed and validated by WB, confirming the downregulation of PLK1's cell cycle and mitotic roles by p53 mRNA transfection. Notably, the cellular senescence pathway was also upregulated by p53 mRNA transfection, potentially contributing to the observed cell death in OVCAR-8 cells, along with the induction of apoptosis.

[0123] Next, the inventors performed transcriptome analysis between p53 mRNA-transfected primary HGSOC cells from patient 1 and mock-transfected primary HGSOC cells (Figure 5a). High variability between samples resulted in low significance after FDR correction for several tests. Therefore, the inventors examined the significance of transcripts from primary HGSOC cells with P<0.01 and FC>1.5. A total of 156 transcripts were upregulated and 115 were downregulated (Figures 5b, c). Several genes from primary HGSOC cells were significant and shared pathways identified in OVCAR-8 cells (Figure 5d), supporting the relevance of both datasets.

[0124] Consistent with the critical function of p53 in OC development, our Ingenuity pathway analysis revealed multiple transcriptional changes via the “ovarian cancer signaling” pathway. Many genes in this pathway (55 / 76, 72%) were significantly deregulated (Figure 6a, b). As an example, we found that many genes in Wnt / β-catenin signaling were deregulated. This pathway promotes chemotherapy resistance, metastasis, and cancer stem cell regeneration in OC through abnormal activation of Wnt / β-catenin signaling, leading to hyperactivity of β-catenin. p53 mRNA treatment resulted in upregulation of AXIN1, which functions as a scaffold protein for components of the β-catenin disruption complex, including APC, CK1PP2A, and GSK3β. Furthermore, deregulation was observed for members of the frizzled receptor family, Wnt family, and TCF / LEF family (Figure 6a, b). In summary, these transcriptional changes resulted in a moderate decrease in β-catenin in p53 mRNA-treated cells (Figure 6b) and a decrease in transfection activity due to an increase in p53 mRNA, as measured by the TCF / LEF reporter kit for Wnt signaling.

[0125] Example 8: Orthotopic application of p53 mRNA-treated OVCAR-8 cells results in a dose-dependent reduction in tumor growth in an HGSOC xenograft model.

[0126] To investigate the viability of p53 mRNA-treated OVCAR-8 cells in the physiological microenvironment, we tested an orthotopic disease model. First, we tested whether OVCAR-8 cells stably expressing luciferase (Luc) (OVCAR-8 / Luc) showed an altered response to p53 reactivation compared to wt cells. Treatment with 100 ng of p53 mRNA correlated only with moderate cell death, as evidenced by low expression of apoptosis markers / activation, low caspase 3 / 7 activity, and moderate inhibition of cell growth, as evidenced by colony formation. However, transfection with 500 ng of p53 mRNA induced a strong apoptotic response and a strong decrease in colony formation efficacy. Therefore, stable expression of Luc in OVCAR-8 cells did not alter the response to p53 reactivation by p53 mRNA compared to wt OVCAR-8 cells. One day prior to xenotransplantation experiments, OVCAR-8 / Luc cells were transfected with mock or p53 mRNA (100 ng, 500 ng) (Figure 7a). To establish an orthotopic model, a Matrigel / OVCAR-8 cell suspension was injected into the right ovary of each mouse. Determination of tumor volume at 1 × / week over a 10-week observation period by BLI demonstrated delayed growth in tumors derived from cells treated with 100 ng of p53 mRNA and complete suppression of tumor growth derived from cells treated with 500 ng of p53 mRNA (Figure 7b). To validate in vivo measurements of Luc activity, mice were sacrificed at the end of the study and subjected to anatomical / histological examination of all organs by two independent veterinary pathologists. Examination of the removed ovaries based on volume determination (Figure 7c, left, center) and BLI measurements (Figure 7c, right) confirmed a dose-dependent reduction in tumor size by p53 mRNA compared to in vivo BLI measurements (Figure 7b). Histological examination revealed a large tumor mass and significantly altered morphology resulting from a massive carcinoma tissue in the right ovary injected with mock-treated OVCAR-8 / Luc cells (Figure 7e: Panels a, b). In the 100 ng p53 mRNA treatment group, the right ovary showed either tumor tissue (Figure 7e: Panels d, e) or normal histology.In the 500ng p53 mRNA-treated group, the right ovary and all other organs showed normal histology (Figure 7e: panels g, h). In all treatment groups (mock, 100ng, 500ng p53 mRNA), the left ovary that did not receive OVCAR-8 / Luc cells showed normal histology with follicular and corpus luteum structures and stromal tissue (Figure 7e: panels c, f, i). In mice injected with mock-treated cells, metastasis could be detected in the intestine, but metastatic activity was low in the 100ng group and absent in the 500ng group. In summary, the animal data revealed dose-dependent growth retardation of p53 mRNA-treated OVCAR-8 / Luc cells under orthotopic conditions.

[0127] Example 9: IP treatment of OVCAR-8 cells, followed by IP treatment with p53 mRNA, inhibits tumor cell dissemination.

[0128] In the advanced stages of occlusive disease (OC), intraperitoneal diffusion of tumor cells is common. Therefore, the goal of debulking surgery is the complete resection of all visible tumor cells. 37 Nevertheless, a high percentage of all OC patients succumb to the disease, indicating that the remaining cancer cells have a fatal impact on clinical outcomes. To further investigate the clinical relevance of p53 mRNA to HGSOC, we tested whether scattered tumor cells in the peritoneum have access to liposomal p53 mRNA. IP administration of drugs for OC has been shown to be more effective than systemic administration and is therefore recommended by the National Cancer Institute. Therefore, we investigated 2 × 10⁻⁶ 6 OVCAR-8 / Luc cells were injected via IP (intraperitoneal injection), and p53 mRNA was administered via the intraperitoneal route. Mice were treated for 3 weeks with either two IP injections per week of p53 mRNA (33 mg / kg) or a control. OVCAR-8 / Luc cells treated with the control grew exponentially, while IP treatment with liposomal p53 mRNA completely inhibited tumor cell growth (Figure 8a). Body weight developed normally in both groups throughout the observation period (Figure 8b).

[0129] In control animals treated with OVCAR-8 / Luc cell infiltration (IP) and mock-treated IP, macroscopic anatomical analysis revealed tumor masses on the peritoneal surface (Figure 8c, d). Most of these metastases were only weakly attached to organs. Large tumor masses were also detected in adipose tissue such as the omentum, ovarian fat body, and mesentery (Figure 8c, d). In control animals, organ enlargement was observed, such as the spleen, kidney, and uterus. Particularly large tumor masses attached to the small and large intestines resulted in adhesions between intestinal structures, suggesting intestinal stenosis. IVIS measurements of the omentum and intestines revealed the most prominent signals (Figure 8d). In mice treated with p53 mRNA, no intraperitoneal tumor masses were revealed by macroscopic anatomical analysis, and all organs appeared normal upon visual inspection. IVIS measurements of these organs showed no signal (Figure 8d).

Claims

1. An RNA compound for the treatment of high-grade serous ovarian cancer (HGSOC) in a subject, wherein the RNA compound is an RNA compound that encodes the amino acid sequence of the human p53 protein.

2. The RNA compound according to claim 1, wherein the RNA compound encoding the amino acid sequence comprises the nucleotide sequence of SEQ ID NO: 1 (preferred RNA sequence), or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence of SEQ ID NO:

1.

3. The RNA compound according to claim 1 or 2, wherein the RNA compound is in vitro transcription (IVT) RNA or artificially polymerized RNA.

4. The RNA compound according to any one of claims 1 to 3, wherein at least one nucleotide is a modified nucleotide such as pseudouridine or 5-methylcytidine, for example, the modified nucleotide is a substitution of at least one uracil with pseudouridine, preferably all uracil positions are substituted with pseudouridine.

5. The RNA compound according to any one of claims 1 to 4, wherein the RNA compound is administered to the subject by administering a pharmaceutically acceptable liposome formulation containing the RNA compound.

6. The RNA compound according to any one of claims 1 to 5, wherein the cancer is metastatic.

7. The RNA compound according to any one of claims 1 to 6, wherein the cancer is recurrent.

8. The RNA compound according to any one of claims 1 to 7, wherein the subject has undergone surgery.

9. The RNA compound according to any one of claims 1 to 8, wherein the subject has undergone tumor debulking therapy in the abdominal cavity.

10. The RNA compound according to any one of claims 1 to 9, wherein the RNA compound is administered intraperitoneally to the subject.

11. The RNA compound according to any one of claims 1 to 10, wherein the subject has received or is receiving at least one additional anti-cancer therapy.

12. The RNA compound according to any one of claims 1 to 11, wherein the proliferative disorder is HGSOC, the subject undergoes weight-loss therapy, and the RNA compound is administered intraperitoneally.

13. A pharmaceutical composition comprising an RNA compound according to any one of claims 1 to 12 and at least one pharmaceutically acceptable excipient.

14. The pharmaceutical composition according to claim 13, which is a liposome formulation of the RNA compound.

15. A pharmaceutical composition according to claim 13 or 14, used for the treatment described in any one of claims 1 to 11.

Images