RNA compounds for treating proliferative disorders

Abstract

The invention is based on in vitro transcribed or in vitro synthesized RNA compounds for a translation of tumor suppressor proteins in subjects suffering from a proliferative disease such as cancer. The invention provides the RNA compounds, as well as compositions for their delivery or administration to subjects. The invention in some embodiments provides novel avenues for treating chemotherapy resistant cancer diseases.

Description

[0001] RNA COMPOUNDS FOR TREATING PROLIFERATIVE DISORDERS

[0002] FIELD OF THE INVENTION

[0003] [1] The invention is based on in vitro transcribed or in vitro synthesized RNA compounds for a translation of tumor suppressor proteins in subjects suffering from a proliferative disease such as cancer. The invention provides the RNA compounds, as well as compositions for their delivery or administration to subjects. The invention in some embodiments provides novel avenues for treating chemotherapy resistant cancer diseases.

[0004] DESCRIPTION

[0005] [2] High-grade serous ovarian cancer (HGSOC) is the most lethal gynecologic malignancy and the fifth most common cause of cancer-related death in women. The estimated annual incidence of this disease worldwide is over 200,000 individuals, with approximately 125,000 deaths. These cancers grow very fast, metastasize extensively, and show an aggressive course of the disease. Ovarian cancer (OC) cells stay within the peritoneal cavity and only disseminate to the mesothelium-lined surface! The current standard of care encompasses cytoreductive surgery, chemotherapy (platinum compounds, paclitaxel, and cyclophosphamide), bevacizumab (Avastin), and PARP (poly-ADP-ribose polymerase) inhibitors. Still, around 70% of patients will suffer from a relapse within three years following surgery and platinum-based chemotherapy and succumb to disease progression.

[0006] [3] Death-associated protein kinase (DAPK) is a member of the family of calcium / calmodulin (CaM)-regulated serine / threonine protein kinases. The DAPK gene is conserved during evolution from invertebrates including C. elegans to chordates and mammals. In humans the DAPK gene maps to chromosome 9q21.23. DAPK1 is a key modulator of cell death and autophagy which plays an important role in the ER-stress induced cell death pathway. DAPK1 is a central signal transducer in different pro-apoptotic pathways connected with various cell death mechanisms, and is activated by various external and internal apoptotic stimuli. This pro-apoptotic serine / threonine protein kinase is involved in type I apoptotic (Caspase-dependent) and type II autophagic (caspase-independent) mechanisms leading to cell death. The analysis of DAPK1 in cancer has revealed that DAPK1 acts as a tumor suppressor gene, participates in tumor development and metastatic activity by regulating autophagy and apoptosis5.

[0007] [4] A canonical kinase domain is located at the N-terminal end of DAPK1 followed by a Ca2+ / CaM autoregulatory domain which is able to suppress the enzymatic activity by interacting with the catalytic cleft in the absence of CaM thereby blocking the interaction with exogenous substrates. Upon CaM binding the autoregulatory domain dissociates from the catalytic region and enables the phosphorylation of substrates. Autophosphorylation at pSer308 within the CaM autoregulatory domain decreases the affinity for CaM thereby blocking the enzymatic activity of DAPK1. Towards the carboxyterminal end of DAPK1, the Ca2+ / CaM autoregulatory domain is followed by ankyrin repeats, the Ras of complex protein (ROC) - C- terminal of ROC (COR) domain and the death domains. Ankyrin repeats facilitate proteinprotein communications and are involved in DAPK1 degradation. Moreover, the ROC-COR domain promotes GTP hydrolysis indicating that DAPK1 is a GTP-binding protein with intrinsic GTPase activity and it plays a role in cytoskeletal localization. Furthermore, this domain contributes to homodimerization by both its kinase domain and the ROC domain. The death domain at the carboxyterminal end of DAPK1 represents a protein-protein interaction domain that can be found in many apoptosis-promoting proteins. Thus, DAPK1 has been implicated in important pathways leading to apoptosis and autophagy.

[0008] [5] Recently, RNA-based vaccines have emerged as valuable tools for immunotherapy. Novel strategies based on mRNA therapeutics came into view as efficacious and safe approaches to fighting infectious diseases and cancer based on their advantages such as relatively simple large-scale production, low side effects, and high efficacy. Unlike immune checkpoint blockade setting free immunosuppression and CAR-T cells eliminating cancer cells directly, recently developed cancer vaccines bring about the anticancer immune response via antigen-presenting cells, especially dendritic cells. Endeavours for replacing missing or faulty proteins by mRNA therapeutics have become an attractive strategy in different clinical areas like lung diseases and muscle atrophy but approaches in cancer are still rare.

[0009] [6] Thus, it is an object of the invention to provide novel treatment strategies to tackles proliferative disorders, in particular ovarian cancer.

[0010] BRIEF DESCRIPTION OF THE INVENTION

[0011] [7] Generally, and by way of brief description, the main aspects of the present invention can be described as follows:

[0012] [8] In a first aspect, the invention pertains to a ribonucleic acid (RNA) compound for use in the treatment of a proliferative disease in a subject, wherein the RNA compound encodes an amino acid sequence of a human DAPK1 protein.

[0013] [9] In a second aspect, the invention pertains a pharmaceutical composition comprising the RNA compound which encodes an amino acid sequence of a human DAPK1 protein.

[0014]

[0010] In a third aspect, the invention pertains to a method of treating a proliferative disorder in a subject, the method comprising a step of administering to the subject an RNA compound which encodes an amino acid sequence of a human DAPK1 protein.

[0015]

[0011] In a fourth aspect, the invention pertains a method of manufacturing a medicament for use in the treatment of proliferative disorder in a subject, wherein the method comprises a step of formulating an RNA compound which encodes an amino acid sequence of a human DAPK1 protein as a medicament.

[0016] DETAILED DESCRIPTION OF THE INVENTION

[0017]

[0012] In the following, the elements of the invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine two or more of the explicitly described embodiments or which combine the one or more of the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0018]

[0013] In the first aspect, the invention pertains to a ribonucleic acid (RNA) compound for use in the treatment of a proliferative disease in a subject, wherein the RNA compound encodes an amino acid sequence of a human DAPK1 protein, or a variant thereof.

[0019]

[0014] Human Death-associated protein kinase 1 (DAPK1) is encoded by the DAK1 gene and its sequence and coding gene sequences can be derived via the UniProt database in the version of November 03 2023, under the accession No. DAPK1355. The canonical amino acid sequence of wild type DAPK1 is also included herein as SEQ ID NO: 3. The canonical coding DNA sequence is shown in SEQ ID NO: 1 , and an RNA sequence is shown in SEQ ID NO: 2.

[0020]

[0015] A preferred embodiment of the invention pertains therefore to an RNA compound which comprises, or consists essentially of an RNA sequence which encodes human isoform 1 DAPK1 protein as shown in any one of SEQ ID Nos: 3, 6, 9, 12, 15, 18, or 21 , wherein SEQ ID NO: 18 is preferred in some embodiments.

[0021]

[0016] The RNA compound according to the invention has a sequence having at least 80% sequence identity to a nucleic acid sequence encoding a human DAPK1 protein (SEQ ID NO: 3). In the context of the invention, the term “sequences having at least 80% sequence identity” comprises 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 %, most preferably 100 % sequence identity to the respective sequence. In all cases, the identity is the identity over the total length of the sequences.

[0022]

[0017] Preferably, an RNA compound of the invention comprises an RNA sequence selected of SEQ ID NO: 2, 5, 8, 11 , 14, 17 or 20, or a sequence that is at least 80%, more preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence selected from any one of SEQ ID NO: 2, 5, 8, 11 , 14, 17 or 20. In this embodiment, it may be preferred that the selected RNA sequence is SEQ ID NO: 17.

[0023]

[0018] Am RNA compound of the invention that is particularly preferred is an RNA construct encoding for a protein according to SEQ ID NO: 18, or a sequence that is at least 80%, more preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence of SEQ ID NO: 18.

[0024]

[0019] An RNA compound of the invention may in certain embodiments encode for a variant human DAPK1 protein that comprises at least the domains: KD, AR, and DD.

[0025]

[0020] In one aspect, the RNA compound according to the invention is a variant of the RNA compound having a sequence encoding a protein according to SEQ ID NO: 3, i.e. human DAPK1. The term “variant” herein refers to biologically active derivatives of the respective RNA. In general, the term “variant” refers to molecules having a native sequence and structure with one or more additions, substitutions (generally conservative in nature) and / or deletions, relative to the native molecule, so long as the modifications do not destroy biological activity, and which are “substantially homologous” to the reference molecule. In general, the sequences of such variants will have a high degree of sequence homology to the reference sequence, e.g., sequence homology of more than 50%, generally more than 60%-70%, even more particularly 80%-85% or more, such as at least 90%-95% or more, when the two sequences are aligned. In case of DAPK1, a variant thereof shows a similar capability as a tumor suppressor protein.

[0026]

[0021] In a further embodiment, the invention relates to an RNA compound obtainable by transcription of a DNA sequence having at least 80% sequence identity to a nucleic acid sequence encoding for an amino acid sequence of SEQ ID NO: 1, or variant thereof, for use as a medicament. In a preferred embodiment, the RNA compound is for the use in the treatment of cancer.

[0027]

[0022] The RNA compound may be provided as an RNA molecule, or as a DNA based expression construct encoding for the RNA compound.

[0028]

[0023] The term “obtainable by transcription of a DNA sequence” is herein understood as meaning that the RNA compound is the transcription product or transcript of the respective DNA. Thus, the term “RNA compound obtainable by transcription of a DNA sequence having at least 80% sequence identity to a sequence encoding amino acid sequence of any one of sequences selected from SEQ ID NO: 1, 4, 7, 10, 13, 16 or 19, refers to any RNA compound that can be transcribed from a DNA sequence having a sequence that encodes a protein with an amino acid sequence that is at least 80% identical to the sequence identity to any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, or 21 , or any functional variant thereof. Comprised are any RNA compounds, wherein the RNA compound according to the invention may have been processed by undergoing posttranscriptional modifications such as RNA methylation, folding, cleavage or wrapping into extracellular cargo vesicles such as exosomes.

[0029]

[0024] The term "obtainable by transcription of a DNA sequence” is herein understood not to be limited with regard to the source of the RNA molecule. The RNA molecules according to the invention, including the RNA molecules used in the pharmaceutical compositions according to the invention, can be in vivo transcribed RNA molecules purified from cells or in vitro transcribed RNA.

[0030]

[0025] The skilled person knows how RNA can be purified from cells or transcribed in vitro. For example, RNA purification can be performed by using TRIzol Reagent (Invitrogen, CA, catalogue no. 15596018). For this, samples are lysed and homogenized in the appropriate volume of TRIzol Reagent. Samples are incubated at room temperature for 5 minutes, then 1 / 5 of the volume of chloroform is added, incubated and mixed vigorously. To separate the phases samples are centrifuged for 15 minutes at 13 000 x g at 18°C. The aqueous phase containing the RNA is transferred to a new tube and RNA is precipitated using isopropanol. The resulting pellet is washed using ice cold 70% ethanol, air dried and dissolved in a suitable buffer.

[0031]

[0026] The RNA compounds according to the invention may be purified from any eukaryotic cell expressing DAPK1 artificially or naturally, or may be purified from a transcription in a cell free system. Likewise, RNA purification can be performed using commercially available kits such as RNeasy Mini Kit (Qiagen, Germany, catalogue no. 74106) or Dynabeads™ mRNA DIRECT™ Purification Kit (Invitrogen, CA, catalogue no. 61012).

[0032]

[0027] The RNA compound for use of the invention is preferred, wherein the RNA compound encoding the amino acid sequence comprises the nucleotide sequence of SEQ ID NO: 2 (RNA sequence), or a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 2.

[0033]

[0028] The RNA compound for use of the invention is preferred, wherein the RNA compound encoding the amino acid sequence comprises the nucleotide sequence of SEQ ID NO: 5 (RNA sequence), or a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 5.

[0034]

[0029] The RNA compound for use of the invention is preferred, wherein the RNA compound encoding the amino acid sequence comprises the nucleotide sequence of SEQ ID NO: 8 (RNA sequence), or a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 8.

[0035]

[0030] The RNA compound for use of the invention is preferred, wherein the RNA compound encoding the amino acid sequence comprises the nucleotide sequence of SEQ ID NO: 11 (RNA sequence), or a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 11.

[0036]

[0031] The RNA compound for use of the invention is preferred, wherein the RNA compound encoding the amino acid sequence comprises the nucleotide sequence of SEQ ID NO: 14 (RNA sequence), or a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 14.

[0037]

[0032] The RNA compound for use of the invention is preferred, wherein the RNA compound encoding the amino acid sequence comprises the nucleotide sequence of SEQ ID NO: 17 (RNA sequence), or a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 17.

[0038]

[0033] The RNA compound for use of the invention is preferred, wherein the RNA compound encoding the amino acid sequence comprises the nucleotide sequence of SEQ ID NO: 20 (RNA sequence), or a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 20.

[0039]

[0034] The RNA compound of the invention is preferably an in vitro transcribed (IVT) RNA or is a synthetically polymerized RNA.

[0040]

[0035] The RNA compound of the invention may comprise the classical sequence motifs of messenger RNAs. However. The RNA molecules of the invention may also include various additional modifications or mutations in order to increase translation, reduce immunological adverse effects, or increase RNA stability in general. Many such modifications are well known in the art and are not individually listed herein for sake of conciseness.

[0041]

[0036] For example, an RNA compound of the invention may comprise a poly-A sequence in operable linkage to the sequence encoding the amino acid sequence. Preferably, when used, a poly-A sequence comprises at least 100 nucleotides. A poly-A sequence is usually located 3’ of the sequence encoding the amino acid sequence.

[0042]

[0037] By a further embodiment, the at least one RNA of the composition disclosed herein preferably comprises at least one of the following structural elements: a 5'- and / or 3'- untranslated region element (UTR element), particularly a 5'-UTR element which comprises or consists of a nucleic acid sequence which is derived from the 5'-UTR of a DAPK1 gene or from a fragment, homolog or a variant thereof, or a 5'- and / or 3'-UTR element which may be derivable from a gene that provides a stable mRNA or from a homolog, fragment or variant thereof; 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.

[0038] In another embodiment the RNA compound comprises a 5’ cap structure. 5 cap structures are well known in the art, but preferred Caps are selected from a Cap-0. Cap-1 or Cap-2 structure, preferably which is m7G(5’)ppp(5’) (2’0MeA)pG.

[0043]

[0039] Also preferred is an embodiment, wherein the RNA compound comprises a 5’ UTR comprising an elF4G aptamer.

[0044]

[0040] Preferred RNA compounds of the invention comprise at least one nucleotide which is a modified nucleotide, such as pseudo uridine or 5-methylcytidine, for example wherein the modified nucleotide is a replacement of at least one uracil with pseudo uridine, preferably is a replacement of all uracil positions with pseudo uridine.

[0045]

[0041] An RNA compound in accordance with the invention in preferred embodiments is derived from a mammalian vector based expression of DAPK1 mRNA and induces apoptosis in cancer cells in accordance with the invention.

[0046]

[0042] The RNA compound for use of the invention is preferred, wherein the RNA compound is administered to the subject by administering a pharmaceutically acceptable liposomal formulation comprising the RNA-compound.

[0047]

[0043] A proliferative disease in context of the invention is a tumor disease. A tumor disease is a cancer, in particular an ovarian cancer, and most preferred is a High-grade serous ovarian cancer (HGSOC). A tumor of the invention may be a metastatic tumor and / or a recurrent (relapsed) tumor.

[0048]

[0044] In a preferred embodiment of the invention a treatment in accordance with the first aspect involves the administration of the RNA compound to the subject, DAPK1 translation via the RNA compound in tumor tissue or tissue adjacent to a tumor, and thereby induction of apoptosis in tumor cells specifically.

[0049]

[0045] A subject to be treated in accordance with the invention in some embodiments received a surgery.

[0050]

[0046] Certain embodiments specifically pertain to the disease HGSOC and wherein the subject received or receives (while administrating the RNA compound of the invention, e.g by rinsing) a tumor debulking therapy in the intra peritoneal cavity. Thus, in certain embodiments the RNA compound is administered to the subject intraperitoneal.

[0051]

[0047] In some embodiment, the subject received or receives at least one additional anticancer therapy. For example, the additional anticancer therapy is chemotherapy, such as a chemotherapy involving administration of one or more platin compound. A chemotherapy administered to a subject in context of the invention includes administration of paclitaxel in combination with cisplatin or cisplatin in combination with cyclophosphamide.

[0048] A preferred embodiment of the invention pertains to the RNA compound for use in treatment as disclosed, wherein the proliferative disease is HGSOC, the subject received or receives debulking therapy, and wherein the RNA compound is administered into the intraperitoneal cavity of the subject.

[0052]

[0049] Further provided in another aspect is a pharmaceutical composition, the composition comprising the RNA compound recited above, and at least one pharmaceutically acceptable excipient.

[0053]

[0050] The invention also relates to a pharmaceutical composition comprising as active ingredient an RNA molecule obtainable by transcription of a DNA sequence having at least 80% sequence identity to SEQ ID NO: 3. In one embodiment, the pharmaceutical composition comprises as active ingredient an RNA molecule having at least 80% sequence identity to a nucleic acid sequence encoding for a protein of SEQ ID NO: 1.

[0054]

[0051] It is herein understood that a pharmaceutical composition comprises a therapeutically effective amount of the active ingredient as well as pharmacological excipients. The person skilled in the art knows how to formulate a pharmaceutical composition comprising an RNA molecule. In particular, the person skilled in the art is aware of methods for stabilizing an RNA molecule in a pharmaceutical composition.

[0055]

[0052] In the pharmaceutical composition according to the invention, the RNA molecule may be encapsulated in lipid nanoparticles, cellular or synthetic exosome mimics or in virus-like particles.

[0056]

[0053] Herein, the term “lipid nanoparticles” is defined as molecules that are spherical in shape and comprise a solid lipid core stabilized by a surfactant. The core lipids can be steroids, fatty acids, acylglycerols, waxes, and combinations of them. Surfactants may be biological membrane lipids such as phospholipids, sphingomyelins and bile salts (e.g., sodium taurocholate). All of these may be utilized as stabilizers in the lipid nanoparticles used for pharmaceutical compositions of the invention.

[0057]

[0054] Herein, the term “cellular or synthetic exosome mimics” is defined as nano-sized vesicles (exosomes), derived or purified from cells with modifications (cellular exosome mimics) or generated by artificial methods like cell extrusion (synthetic exosome mimics) that serve as cargos to deliver proteins, nucleic acids or other cellular components to neighbouring or distant cells.

[0058]

[0055] Herein, the term “virus-like particles” is defined as multiprotein structures that closely resemble the organization and conformation of viruses but contain no viral genetic material.

[0059]

[0056] Also disclosed is a method of treating a proliferative disorder in a subject, the method comprising a step of administering to the subject an RNA compound herein.

[0057] Also disclosed is a method of manufacturing a medicament for use in the treatment of proliferative disorder in a subject, wherein the method comprises a step of formulating an RNA compound as recited herein as a medicament. The method comprises a step of in-vitro transcribing an RNA compound as defined herein, in some preferred embodiments.

[0060]

[0058] Further, the method may comprise a step of formulating the RNA compound as a liposomal composition, wherein the liposome encapsulates the RNA compound.

[0061]

[0059] The invention certain embodiments also pertains to an isolated truncated DAPK1 protein, preferably selected from a protein having, or consisting of or consisting essentially of, an amino acid sequence shown in any one of SEQ ID NO: 3, 6, 9, 12, 15, 18 or 21, preferably 18, or a sequence at least 80%, preferably 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NO: 3, 6, 9, 12, 15, 18 or 21 , preferably 18.

[0062]

[0060] The invention may also pertain in certain aspects to a nucleic acid construct (NAC) or a vector comprising such NAC, encoding for an amino acid sequence shown in any one of SEQ ID NO: 3, 6, 9, 12, 15, 18 or 21 , preferably 18, or a sequence at least 80%, preferably 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NO: 3, 6, 9, 12, 15, 18 or 21, preferably 18.

[0063]

[0061] Proteins of the invention may comprise additional sequences, such as tags, for example a 3x flag tag (N-terminal end located).

[0064]

[0062] The invention also is described by the following itemized embodiments, which shall be understood and interpreted in context of this complete disclosure, in particular in view of the various definitions and explanations and the examples provided herein.

[0065]

[0063] Item 1 :A ribonucleic acid (RNA) compound for use in the treatment of a proliferative disease in a subject, wherein the RNA compound encodes an amino acid sequence of a human DAPK1 protein.

[0066]

[0064] Item 2: The RNA compound for use of item 1, wherein the RNA compound encoding the amino acid sequence comprises the nucleotide sequence of SEQ ID NO: 2 (RNA sequence preferred), or a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 2.

[0067]

[0065] Item 3: The RNA compound for use of item 1 or 2, wherein the RNA compound is an in vitro transcribed (IVT) RNA or is a synthetically polymerized RNA.

[0068]

[0066] Item 4: The RNA compound for use of any one of items 1 to 3, wherein the RNA compound comprises a poly-A sequence in operable linkage to the sequence encoding the amino acid sequence.

[0067] Item 5: The RNA compound for use of item 4, wherein the poly-A sequence comprises at least 100 nucleotides.

[0069]

[0068] Item 6: The RNA compound for use of item 4 or 5, wherein the poly-A sequence is located 3’ of the sequence encoding the amino acid sequence.

[0070]

[0069] Item 7: The RNA compound for use of any one of items 1 to 6, wherein the RNA molecule comprises a 5’ cap structure, such as a Cap-0. Cap-1 or Cap-2 structure, preferably which is m7G(5’)ppp(5’) (2’OMeA)pG.

[0071]

[0070] Item 8: The RNA compound for use of any one of items 1 to 7, wherein the RNA compound comprises a 5’ untranslated region (UTR),

[0072]

[0071] Item 9: The RNA compound for use of item 8, wherein the 5’ UTR comprises an elF4G aptamer.

[0073]

[0072] Item 10: The RNA compound for use of any one of items 1 to 9, wherein at least one nucleotide is a modified nucleotide, such as pseudo uridine or 5-methylcytidine, for example wherein the modified nucleotide is a replacement of at least one uracil with pseudo uridine, preferably is a replacement of all uracil positions with pseudo uridine.

[0074]

[0073] Item 11: The RNA compound for use of any one of items 1 to 10, wherein the RNA compound is administered to the subject by administering a pharmaceutically acceptable liposomal formulation comprising the RNA-compound.

[0075]

[0074] Item 12: The RNA compound for use of any one of items 1 to 11 , wherein the proliferative disease is a tumor disease.

[0076]

[0075] Item 13: The RNA compound for use of item 12, wherein the tumor disease is ovarian cancer.

[0077]

[0076] Item 14: The RNA compound for use of item 13, wherein the ovarian cancer is

[0078] High-grade serous ovarian cancer (HGSOC).

[0079]

[0077] Item 15: The RNA compound for use of any one of items 12 to 14, wherein the tumor is a metastatic tumor.

[0080]

[0078] Item 16: The RNA compound for use of any one of items 12 to 15, wherein the tumor is a relapsed tumor.

[0081]

[0079] Item 17: The RNA compound for use of any one of items 1 to 16, wherein the subject received a surgery.

[0080] Item 18: The RNA compound for use of any one of items 1 to 17, wherein the disease is HGSOC and the subject received tumor debulking therapy in the intra peritoneal cavity.

[0082]

[0081] Item 19: The RNA compound for use of any one of items 1 to 18, wherein the RNA compound is administered to the subject intraperitoneal.

[0083]

[0082] Item 20: The RNA compound for use of any one of items 1 to 19, wherein the subject received or receives at least one additional anticancer therapy.

[0084]

[0083] Item 21 : The RNA compound for use of item 20, wherein the additional anticancer therapy is chemotherapy.

[0085]

[0084] Item 22: The RNA compound for use of item 21, wherein the chemotherapy involves administration of one or more platin compound.

[0086]

[0085] Item 23: The RNA compound for use of item 21 or 22, wherein the chemotherapy is an administration of paclitaxel in combination with cisplatin or cisplatin in combination with cyclophosphamide.

[0087]

[0086] Item 24: The RNA compound for use of any one of items 1 to 23, wherein the proliferative disease is HGSOC, the subject received debulking therapy, and wherein the RNA compound is administered into the intraperitoneal cavity.

[0088]

[0087] Item 25: A pharmaceutical composition, the composition comprising the RNA compound recited in any one of items 1 to 24, and at least one pharmaceutically acceptable excipient.

[0089]

[0088] Item 26: The pharmaceutical composition of item 25, which is a liposomal formulation of the RNA compound.

[0090]

[0089] Item 27: The pharmaceutical composition of item 25 or 26, for a use in therapy as recited in any one of items 1 to 24.

[0091]

[0090] Item 28: A method of treating a proliferative disorder in a subject, the method comprising a step of administering to the subject an RNA compound recited in any one of items 1 to 24.

[0092]

[0091] Item 29: The method of item 28, wherein treatment is a use as recited in any one of items 1 to 24.

[0092] Item 30: A method of manufacturing a medicament for use in the treatment of proliferative disorder in a subject, wherein the method comprises a step of formulating an RNA compound as recited in any one of items 1 to 24.

[0093]

[0093] Item 31 : The method of item 30, wherein the method comprises a step of in-vitro transcribe an RNA compound as defined in any one of the preceding items.

[0094]

[0094] Item 32: The method of item 30 or 31 , wherein the method comprises a step of formulating the RNA compound as a liposomal composition, wherein the liposome encapsulates the RNA compound.

[0095]

[0095] The terms “of the [present] invention”, “in accordance with the invention”, “according to the invention” and the like, as used herein are intended to refer to all aspects and embodiments of the invention described and / or claimed herein.

[0096]

[0096] As used herein, the term “comprising” is to be construed as encompassing both “including” and “consisting of”, both meanings being specifically intended, and hence individually disclosed embodiments in accordance with the present invention. Where used herein, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. In the context of the present invention, the terms “about” and “approximately” denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by ±20%, ±15%, ±10%, and for example ±5%. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect. Where an indefinite or definite article is used when referring to a singular noun, e.g. "a", "an" or "the", this includes a plural of that noun unless something else is specifically stated.

[0097]

[0097] It is to be understood that application of the teachings of the present invention to a specific problem or environment, and the inclusion of variations of the present invention or additional features thereto (such as further aspects and embodiments), will be within the capabilities of one having ordinary skill in the art in light of the teachings contained herein.

[0098]

[0098] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[0099]

[0099] All references, patents, and publications cited herein are hereby incorporated by reference in their entirety.

[0100] BRIEF DESCRIPTION OF THE FIGURESAND SEQUENCES

[0101]

[0100] The figures show:

[0102]

[0101] Figure 1. Expression of DAPK1 and critical regulators of the mammalian cell cycle in cancer cells.

[0103]

[0102] Western blot analysis of DAPK1 expression in a (A) panel of cancer cell lines and (B) ovarian cancer cell lines. Cell lysates were immunoblotted for DAPK1, p53, p21 , PLK1 , and p- Actin. For all panels, one image representative of three independent experiments is shown.

[0104]

[0103] Figure 2. Mammalian vector-based expression of DAPK1 induces apoptosis in the HGSOC cell lines.

[0105]

[0104] HGSOC cell lines and primary HGSOC cells were transfected with mock- or with 1 pg recombinant DAPK1 -expressing vector. (A) Cell lysates were immunoblotted for Flag-DAPK1, PARP, p53, p21, and p-Actin. For all panels, one image representative of three independent experiments is shown. (B) The Caspase-3 / 7 activities were determined in HGSOC cell lines and primary HGSOC cells using a Caspase-Gio 3 / 7 assay. **P < 0.01 , ***P < 0.001 ; Student’s t-test, unpaired and two-tailed. (C) Ovarian cancer cell lines were transfected with mock- or with 1 pg recombinant DAPK1 -expressing vector in a dose-dependent manner and immunoblotted for DAPK1, PARP, PLK1 , p53, p21, pERK1 ,2, ERK1 ,2, LC3A / B-I / -II and p-Actin. (D) The dosedependent Caspase-3 / 7 activities were determined in ovarian cancer cell lines using a Caspase-Gio 3 / 7 assay. **P < 0.01, ***P < 0.001; Student’s t-test, unpaired and two-tailed.

[0106]

[0105] Figure 3. Mammalian vector-based expression of DAPK1 in the HGSOC cells.

[0107]

[0106] OVCAR-3 cells were transfected with mock- or with 1 pg recombinant DAPK1- expressing vector. (A) Cell lysates were immunoblotted for Flag-DAPK1, PARP, p53, p21, pERK1 ,2, ERK1,2 and p-Actin. For all panels, one image representative of three independent experiments is shown. (B) Cell lysates were immunoprecipitated using p53 antibodies. The resulting precipitate was immunoblotted for MDM2, DAPK1 and p53. For all panels, one image representative of three independent experiments is shown. (C) Immunofluorescence images

[0108]

[0107] of mock- or recombinant DAPK1 -expressing vector - transfected cells stained with LC3A / B-antibodies and DAPI. (D) The Caspase-3 / 7 activities were determined in HGSOC cell lines and primary HGSOC cells using a Caspase-Gio 3 / 7 assay. ***P < 0.001 ; Student’s t-test, unpaired and two-tailed. (E) OVCAR-3 cells were transfected with mock- or with 1 pg recombinant DAPK1 -expressing vector. Cell lysates were separated in total, cellular and nuclear fractions, immunoblotted for Flag-DAPK1, p53, p21, pERK1 ,2, ERK1,2, LC3A / B-I / -II and - Actin. For all panels, one image representative of three independent experiments is shown. Lysates were immunoprecipitated for p53 and subsequently immunoblotted for p53.

[0109]

[0108] Figure 4. The transient mammalian vector-based expression of DAPK1 sensitizes OVCAR-3 cells to Paclitaxel treatment.

[0110]

[0109] (A) 2D cell-proliferation of Paclitaxel-treated OVCAR-3 cells was measured using a CellTiter-Blue Cell Viability, 24 h, 48 h, and 72 h post-treatment. (B) OVCAR-3 cells were transfected with mock- or with 1 pg recombinant\DAPK-expressing vector, and the 2D cellproliferation of Paclitaxel-treated cells was measured using a CellTiter-Blue Cell Viability, 72 h post-treatment. Western Blot analysis showing the expression of Flag-DAPK1 after 24 h. (C) The dose-dependent Caspase-3 / 7 activities of OVCAR-3 cells transfected with mock- or with 1 pg recombinant\DAPK-expressing vector treated with increasing concentrations of Paclitaxel were determined using a Caspase-Gio 3 / 7 assay. ***P < 0.001; Student’s t-test, unpaired and two-tailed.

[0111]

[0110] Figure 5. Selection of minimal efficient DAPK constructs for the design of DAPK1 mRNA.

[0112]

[0111] Subclones were generated by DNA synthesis verified by DNA sequencing that represent subdomains of entire open reading frame of DAPK (clone 1: full-length open reading frame of DAPK, subclones 2-8 represent subdomains of full-length DAPK. Starting with the aminoterminal end the following domains can be found in full-length DAPK: kinase domain (KD), the Ca2+ / CaM autoregulatory domain (CM), ankyrin repeats (AR), the Ras of complex protein (ROC) - C-terminal of ROC (COR) domain and the death domain (DD). (B) Clones 1-8 were expressed as FLAG-tagged proteins and separated in a WB experiment. Cell lysates were immunoblotted for Flag-DDAPK1 , and p-Actin. (C) The Caspase-3 / 7 activities were determined in OVCAR-3 cells expressing clones 1-8 using a Caspase-Gio 3 / 7 assay. **P < 0.01, ***P < 0.001; Student’s t-test, unpaired and two-tailed. (D) OVCAR-3 cells were transfected with Flag- □ DAPK-expressing vectors, and the 2D cell-proliferation was measured using a CellTiter-Blue Cell Viability, 24 h, 48 h, and 72 h post- treatment. A WB experiment was performed to control for equal expression (Flag-immunoblot).

[0113]

[0112] Figure 6. The transient mammalian vector-based expression of □DAPK1-clones sensitizes OVCAR-3 cells to Paclitaxel treatment.

[0114]

[0113] (A) OVCAR-3 cells were transfected with Flag-DDAPK-expressing vectors, and the 2D cell-proliferation of Paclitaxel-treated cells was measured using a CellTiter-Blue Cell Viability, 24 h post- treatment. (B) The Caspase-3 / 7 activities were determined in control OVCAR-3 cells expressing clones 1-8 or Paclitaxel-treated cells expressing clones 1-8 using a Caspase-Gio 3 / 7 assay. *P < 0.05, **P < 0.01 , ***P < 0.001 ; Student’s t-test, unpaired and two-tailed.

[0115]

[0114] Figure 7. The stable mammalian vector-based expression of DDAPK1 -clones sensitizes OVCAR-8 cells to Paclitaxel treatment.

[0116]

[0115] OVCAR-8 clones were generated that stably express Flag-DDAPK1 clone 6 or Flag- □ DAPK1 clone 7. Cell lysates were immunoblotted for full-length DAPK1, DAPK1-clone 6 or DAPK1-clone 7. In addition, a Flag-IP was performed, and the resulting precipitate was blotted for Plk1. (B) Caspase-3 / 7 activities were determined in Paclitaxel-treated OVCAR-3 cells (control, cells expressing clone 6 or clone 7) using a Caspase-Gio 3 / 7 assay. **P < 0.01, Student’s t-test, unpaired and two-tailed.

[0117]

[0116] Figure 8. The stable mammalian vector-based expression of DDAPK1 -clones sensitizes OVCAR-8 cells to Cisplatin treatment.

[0118]

[0117] (A) OVCAR-8 cells and stably expressing clone 6 (DAPK1) were treated with Cisplatin and the 2D cell-proliferation was measured using a CellTiter-Blue Cell Viability, 24 h, 48 h, 72 h and 96 h post-treatment. (B) OVCAR-8 cells and stably expressing clone 6 were treated with Cisplatin and the 2D cell-proliferation was measured using a CellTiter-Blue Cell Viability, 72 h and 96 h post-treatment. (C) OVCAR-8 cells and stably expressing clone 6 (DAPK1) were treated with Cisplatin for 24 h, lysed and immunoblotted for Flag-DAPK1 and PARP.

[0119]

[0118] Figure 9. Design and treatment of ovarian cancer cells with in vitro-transcribed DAPK mRNA.

[0120]

[0119] (A) Key structural components of in vitro-transcribed (IVT) mRNA used in this study. Modifications of mRNA including a synthetic cap analog at the 5' -end, a poly(A) tail at the 3' - end and incorporation of modified nucleosides (e.g., 5-methylcytidine or pseudouridine), improve the stability of synthetic mRNAs, increase their translational activity in cells, and reduce their immunogenicity. (B) Purified IVT-Flag-DAPK-mRNA was analyzed using agarose gel electrophoresis. (C) Cell lysates were subjected to WB for key mediators of cell cycle regulation and apoptosis using Flag-, PARP-, Caspase-3-, PLK1-, Cyclin AI-, Cyclin B1-, Aurora A, CDK1-, pMLC2 and B-Actin-antibodies. (D) The dose-dependent Caspase-3 / 7 activities were determined using a Caspase-Gio 3 / 7 assay. (E) (left) Representative images show the number of 3D colonies (dose-dependent) on day 7. (right) The dose-dependent distribution of colonies is represented as bar graph. (F) Caspase-3 / 7 activities were determined in different ovarian cancer cell lines and (G) in primary cells using a Caspase-Gio 3 / 7 assay. Cell lysates were subjected to WB using Flag-, DAPK-, PARP-, and B-Actin-antibodies.

[0121]

[0120] Figure 10. Transfection of primary HGSOC Organoids using DAPK1 mRNA (clone 6) induces apoptosis.

[0121] (A) The Caspase-3 / 7 activities were determined in primary HGSOC cells and normal counterparts, both transfected with DAPK- clone 6 mRNA using a Caspase-Gio 3 / 7 assay. (B) (left) Live / dead staining was performed in primary organoids of HGSOC cells transfected with mock and DAPK1 -clone 6 mRNA, and (middle) quantified, (right) The Caspase-3 / 7 activities were determined in mock control cells or in cells transfected with DAPK-clone 6 mRNA using a Caspase-Gio 3 / 7 assay. *P < 0.1; Student’s t-test, unpaired and two-tailed. Cell lysates were subjected to WB using Flag-, and B-Actin-antibodies.

[0122]

[0122] Figure 11. Intraperitoneal injection of OVCAR-8 cells followed by intraperitoneal treatment of mice with liposomal DAPK1 (clone 6)-mRNA prevents tumor formation and organ dissemination in a Xenograft mouse model.

[0123]

[0123] (A) (upper) Bioluminescence imaging of luciferase-expressing metastatic OVCAR-8 cellbearing mice upon treatment with liposomal DAPK1 (clone 6)-mRNA. 2 x 106 OVCAR-8 cells stably expressing luciferase were i.p. injected followed by treatment with liposomal DAPK1 (clone 6)-mRNA for three weeks (2 x 5 pg liposomal DAPK1 (clone 6)-mRNA per week), (right) The color scale represents p / sec / cm2 / steradian. (lower) Tumor burden of control and liposomal DAPK1 (clone 6)-mRN A- treated nude mice determined weekly by bioluminescence over a period of nine weeks (n=8). (B) Determination of the body weight over the entire observation period. (C) (upper) Inspection of peritoneal structures and organs for tumor dissemination following laparotomy. Metastases are depicted by arrows, (lower) Size of organs was determined. (D) Bioluminescence imaging of removed murine organs (control vs. DAPK1 (clone 6)-mRNA treatment).

[0124]

[0124] Figure 12. The transient mammalian vector-based expression of DAPK1 sensitizes OVCAR-4 and OVCAR-8 cells to Paclitaxel treatment.

[0125]

[0125] (A) OVCAR-4 cells were transfected with 1 pg recombinant\DAPK-expressing vector, and the 2D cell-proliferation of Paclitaxel-treated cells was measured using a CellTiter-Blue Cell Viability, 24 h, and 72 h post-treatment. (B) OVCAR-8 cells were transfected with mock- or with 1 pg recombinant\DAPK-expressing vector, and the 2D cell-proliferation of Paclitaxel-treated cells was measured using a CellTiter-Blue Cell Viability, 48 h post- treatment. Lysates were blotted for FLAG-DAPK1.

[0126]

[0126] Figure 13. The stable mammalian vector-based expression of DAPK1 -clones sensitizes OVCAR-5 and -8 cells to Paclitaxel treatment.

[0127]

[0127] OVCAR-5 and -8 clones were generated that stably express Flag-DAPK1. Cell lysates were immunoblotted for full-length DAPK1. (A) Lysates of Paclitaxel-treated OVCAR-5 cells were blotted for FLAG-DAPK1. Caspase-3 / 7 activities were determined in Paclitaxel-treated OVCAR-5 cells using a Caspase-Gio 3 / 7 assay. **P < 0.01 , ***P < 0.001 ; Student’s t-test, unpaired and two-tailed. (B) Lysates of Paclitaxel-treated OVCAR-8 cells were blotted for FLAG-DAPK1. Caspase-3 / 7 activities were determined in Paclitaxel-treated OVCAR-5 cells using a Caspase-Gio 3 / 7 assay. *P < 0.05, **P < 0.01; Student’s t-test, unpaired and two-tailed.

[0128]

[0128] The following sequences are included in the sequence listing that forms part of the present disclosure:

[0129] Table 1 : Sequences of various constructs of the invention as listed in the appended sequence protocol.

[0130] EXAMPLES

[0129] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the description, figures and tables set out herein. Such examples of the methods, uses and other aspects of the present invention are representative only, and should not be taken to limit the scope of the present invention to only such representative examples.

[0130] The examples show:

[0131]

[0131] Example 1: Expression profile of DAPK in cell lines and primary cells

[0132]

[0132] The screening of a panel of ovarian cancer cell lines and primary ovarian cancer tissues revealed downregulation of DAPK1 in many cell lines and primary tissues.

[0133]

[0133] Example 2: Mammalian vector-based expression of DAPK1 in HGSOC cells induces apoptosis and autophagy (Figure 2)

[0134]

[0134] Mammalian vector-based expression of full-length DAPK1 in cell lines and primary tumor tissues induces PARP cleavage and Caspase 3 / 7 activity in a dose-dependent manner. Activation of ERK1 / 2 generally promotes cell proliferation, and its deregulated activity is a hallmark of many cancers. Upon increasing expression of DAPK1 activation of ERK1,2 (pERK1 ,2) is downregulated. Both observations, increase of PARP cleavage and Caspase 3 / 7 accompanied by reduced ERK activity, suggest an upregulation of apoptosis. Decrease of PLK1 expression, a marker of cellular proliferation, confirms the descending proliferative activity of cells transfected with increasing amounts of a mammalian DAPK1 -expressing vector.

[0135]

[0135] Microtubule-associated protein 1A / 1 B-light chain 3(MAP1LC3), commonly shortened to LC3, is a soluble protein with a molecular weight of approximately 17 kDa. LC3 is distributed throughout mammalian tissues and cultured cells and is a key component of autophagy, the recycling system of the eukaryotic cell. It is incorporated into the inner and outer membrane of autophagosomes during autophagosome biogenesis. Thus, LC3 is a specific marker of autophagy and, in particular, of autophagosome formation. The processing of autophagosome marker LC3 and its stepwise conversion from a cytosolic (LC3-I) to a membrane-bound, lipidated (LC3-II) form is exploited for the experimental measurement of autophagy. In western blotting, this lipidation event causes the LC3-II band to migrate faster enabling discrimination of the membrane and non-membrane bound forms. Upon increasing expression of DAPK1 activation, the LC3-II band shows more intense staining suggesting an upregulation of autophagy. LC3-I / I I were found in the cytoplasm and in the nucleus.

[0136]

[0136] Whether this observation indicates that autophagy controls nucleolar activity remains to be investigated.

[0137]

[0137] Example 3: Mammalian vector-based expression of DAPK1 in OVCAR-3 cells induces apoptosis and autophagy (Figure 3)

[0138]

[0138] A more detailed analysis of DAPK1 -overexpression in OVCAR-3 cells supported the induction of apoptosis (increased PARP cleavage and Caspase 3 / 7 activity).

[0139]

[0139] Most interestingly, overexpression of DAPK1 induces Mdm2 (a negative regulator of p53) cleavage. To induce apoptotic cell death, DAPK1 may require the activation of the p53 apoptotic pathway. In this case, induction of p19ARF by DAPK1 allows inactivation of Mdm2 to stabilize and activate p53. The activity of p19ARF-bound Mdm2 is lower than that of free Mdm2, suggesting that p19ARF promotes the stabilization of p53 by inactivating Mdm2.

[0140]

[0140] As an alternative mechanism, activated Caspase-2 directly cleaves the E3 ubiquitin ligase Mdm2 at Asp 367, leading to loss of the C-terminal RING domain responsible for p53 ubiquitination. As a consequence, N-terminally truncated Mdm2 binds p53 and promotes its stability.

[0141]

[0141] Example 4. Mammalian vector-based expression of DAPK1 sensitizes OVCAR-3 cells to Paclitaxel -treatment (Figure 4)

[0142]

[0142] Expression of DAPK1 cells reduces the EC50 of Paclitaxel in OVCAR-3 cells treated for 72 h to 0.5 nM compared to mock-treated cells (EC50 =1 nM).

[0143]

[0143] Example 5: Design and functional analysis of a minimal efficient DAPK1 clones for subsequent use as in vitro-t ran scribed (IVT) mRNA (Figure 5)

[0144]

[0144] Full-length DAPK with 4290 bp (No. 1) was truncated in 7 steps to the smallest deletion DAPK1 deletion clones (No. 8) with 825 bp. In each deletion steps at least one of multiple functional domains with DAPK1 was excised. All clones are Flag-tagged (N-terminal)

[0145] • Functional domains of DAPK1:

[0146] • Kinase / catalytic domain (KD)

[0147] • CaM-autoregulatory domain (CM)

[0148] • Ankyrin-repeats (AR)

[0149] • ROC-domain (ROC): ROC-COR domains promote GTP hydrolysis indicating that DAPK1 is a GTP-binding protein with intrinsic GTPase activity.

[0150] • COR-domain (COR)

[0151] • Death Domain (DD)

[0152]

[0145] Previous observations demonstrated that the deletion of the CaM domain in DAPK1 leads to a kinase that is constitutively active. Thus, the DAPK-delta CaM mutant was more active than wild-type DAPK1. Clones 3, 5 and 6 that are missing the CaM domain induced more Caspase 3 / 7 activity than full-length DAPK1 (Fig. 5C).

[0153]

[0146] Example 6: Mammalian vector-based expression of deletion clones of DAPK1 sensitizes OVCAR-3 cells to Paclitaxel-treatment (Figure 6)

[0154]

[0147] After an incubation period of only 24 h full-length DAPK1 (clone 1) had only a small sensitizing effect for OVCAR-3 cells for Paclitaxel. In contract, the sensitizing effect of clone 6 was 10-fold, that of clone 7 approx. 3-fold. Except clones 4 and 8, clones 1, 2, 3, 5, 6 and 7 induced high apoptotic activity in OVCAR-3 cells as measured by Caspase 3 / 7 activation.

[0155]

[0148] Example 7: Stable genomic integration of DAPKI / clone 6 and its truncated clones sensitizes OVCAR-3 cells to Paclitaxel-treatment (Figure 7)

[0156]

[0149] Stable genomic integration of clone 6 cells sensitized OVCAR-3 cells significantly to Paclitaxel. Based on the favorable results induced by clone 6 in terms of inhibition of cellular proliferation and induction of apoptosis (Fig. 5-7), clone 6 was chosen for further analysis. One additional reason for using clone 6 for future experiment was its length 2096 bp compared to full-length DAPK with 4290 bp (clone 1). The reduced length of clone 6 might be an important prerequisite for efficient liposomal transfection and high translational efficacy.

[0157]

[0150] Example 8: Mammalian vector-based expression of DAPKI / clone 6 sensitizes OVCAR-8 cells to Cisplatin-treatment (Figure 8)

[0158]

[0151] Expression of DAPK1 -clone 6 reduced the EC50 of ovarian cancer cells after 72 h treatment from 5.1 iM in wild-type cells to 3.1 iM in DAPKI / clone 6-expressing cells and after 96 h treatment from 4.8 iM in wild-type cells to 2.4 iM in DAPKI / clone 6-expressing cells. The analysis of PARP cleavage revealed DAPKI / clone 6-expression as promoter of apoptosis in Cisplatin-treated cells.

[0159]

[0152] Example 9: Transfection with DAPKI / clone 6-IVT mRNA induces apoptosis and reduces colony-formation of OVCAR-8 cells in a concentration-dependent manner (Figure 9)

[0160]

[0153] Transfection of OVCAR-8 cells with increasing doses of DAPKI / clone 6-IVT mRNA induced

[0161] • an increasing phosphorylation of an endogenous substrate myosin light-chain supporting the idea that cellular effects depend on the kinase activity of DAPK and its truncated clones.

[0162] • reduced cell cycle activity (downregulation of PLK1, Cyclin A / B1) and reduced colony forming ability

[0163] • increasing apoptosis (PARP cleavage, Casp. 3 cleavage, Casp. 3 / 7 activity in different ovarian cancer cell lines)

[0164]

[0154] very low increase of apoptosis in benign human cells

[0165]

[0155] Example 10: Transfection with DAPKI / clone 6-IVT mRNA shows minimal effect of benign human cells compared to their malignant counterparts (Figure 10)

[0156] Cell death and Caspase 3 / 7 activity show small increase in normal epithelial ovarian organoids upon transfection with DAPK1 / clone 6-IVT mRNA compared to their tumor counterparts.

[0166]

[0157] Example 11 : Xenograft experiments in mice (Figure 11)

[0167]

[0158] The inventors injected 2x106OVCAR-8 / Luc cells i.p. and administered 4-6 h later DAPK / clone 6-mRNA via the intraperitoneal route. Mice were treated for 3 weeks with 2 i.p. injections / week with DAPK1-mRNA (0.16 mg / kg) or control. I.p. spread and growth of OVCAR-8 cells was hampered upon twice weekly application of liposomal DAPK1 / clone 6-IVT mRNA. Regular screening of animal weight did not provide signs of weight loss indicating minimal side effects.

[0168]

[0159] The study of OVCAR-4 cells supports previous observations in OVCAR-3 cells in regard to the induction of apoptosis and autophagy by DAPK1(Fig. 3).

[0169]

[0160] References

[0170] 1. Gade, P. et al. Regulation of the death-associated protein kinase 1 expression and autophagy via ATF6 requires apoptosis signal-regulating kinase 1. Molecular and cellular biology 34, 4033-4048 (2014).

[0171] 2. Celik, S. et al. Methylation analysis of the DAPK1 gene in imatinib-resistant chronic myeloid leukemia patients. Oncol Lett s, 399-404 (2015).

[0172] 3. Shohat, G. et al. The pro-apoptotic function of death-associated protein kinase is controlled by a unique inhibitory autophosphorylation-based mechanism. The Journal of biological chemistry 276, 47460-47467 (2001).

[0173] 4. Raab, M. et al. Quantitative chemical proteomics reveals a Plk1 inhibitor-compromised cell death pathway in human cells. Cell research 24, 1141-1145 (2014).

[0174] 5. Bialik, S. & Kimchi, A. The death-associated protein kinases: structure, function, and beyond. Annual review of biochemistry 75, 189-210 (2006).

[0175] 6. Bialik, S., Bresnick, A.R. & Kimchi, A. DAP-kinase-mediated morphological changes are localization dependent and involve myosin-ll phosphorylation. Cell death and differentiation 11, 631-644 (2004).

[0176] 7. McShane, L.M. et al. REporting recommendations for tumor MARKer prognostic studies (REMARK). Nat Clin Pract Urol 2, 416-422 (2005).

[0177] 8. Raab, M. et al. Toxicity modelling of Plk1 -targeted therapies in genetically engineered mice and cultured primary mammalian cells. Nature communications 2, 395 (2011).

[0178] 9. Mergener, S., Siveke, J.T. & Pena-Llopis, S. Monosomy 3 Is Linked to Resistance to MEK Inhibitors in Uveal Melanoma. Int J Mol Sci 22 (2021).

[0179] 10. Tseng, J.-C., Vasquez, K.O. & Peterson, J.D. (2015).

[0180] 11. Bialkowska, A.B., Ghaleb, A.M., Nandan, M.O. & Yang, V.W. Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses. J Vis Exp (2016).

[0181] 12. Spankuch-Schmitt, B., Bereiter-Hahn, J., Kaufmann, M. & Strebhardt, K. Effect of RNA silencing of polo-like kinase-1 (PLK1) on apoptosis and spindle formation in human cancer cells. J Natl Cancer Inst 94, 1863-1877 (2002).

Claims

CLAIMS1. A ribonucleic acid (RNA) compound for use in the treatment of cancer in a subject, wherein the RNA compound encodes an amino acid sequence of a human Death-associated protein kinase 1 (DAPK1), and wherein the cancer is for example ovarian cancer.

2. The RNA compound for use of claim 1 , wherein the RNA compound encodes an amino acid sequence selected from an sequence shown in any one of SEQ ID NO: 3, 6, 9, 12, 15, 18 and 21, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence selected from any one of SEQ ID NO: 3, 6, 9, 12, 15, 18 and 21.

3. The RNA compound for use of claim 1 or 2, wherein the RNA compound encodes a truncated DAPK1 protein, preferable from any one of amino acid sequences shown in SEQ ID NO: 6, 9, 12, 15, 18 and 21.

4. The RNA compound for use of claim 3, wherein the truncated DAPK1 protein is delta CaM domain mutant.

5. The RNA compound for use of any one of claims 1 to 4, having an RNA sequence shown in SEQ ID NO: 17, or an RNA sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 17.

6. The RNA compound for use of any one of claims 1 to 5, wherein the RNA compound is an in vitro transcribed (IVT) RNA or is a synthetically polymerized RNA.

7. The RNA compound for use of any one of claims 1 to 6, wherein the RNA compound is administered to the subject by administering a pharmaceutically acceptable liposomal formulation comprising the RNA-compound.

8. The RNA compound for use of any one of claims 1 to 7, wherein the cancer is metastatic and / or relapsed.

9. The RNA compound for use of any one of claims 1 to 8, wherein the subject received a surgery, such as a tumor debulking therapy in the intra peritoneal cavity.

10. The RNA compound for use of any one of claims 1 to 9, wherein the cancer is HGSOC, the subject received debulking therapy, and wherein the RNA compound is administered into the intraperitoneal cavity.

11. The RNA compound for use of any one of claims 1 to 10, wherein the treatment comprises a sensitization of cells associated with the HGSOC to a treatment with another anti-cancer treatment, such as a chemotherapy.

12. The RNA compound for use of claim 11, wherein the chemotherapy is a therapy with a taxol-like agent, preferably paclitaxel; or wherein the chemotherapy is a treatment with a platin agent, such as cisplatin.

13. The RNA compound for use of any one of the preceding claims, wherein the treatment is a treatment of a chemotherapy resistant ovarian cancer, such as chemotherapy resistant HGSOC.

14. A pharmaceutical composition, the composition comprising the RNA compound recited in any one of claims 1 to 13, and at least one pharmaceutically acceptable excipient.

15. The pharmaceutical composition of claim 14, which is a liposomal formulation of the RNA compound.

16. The pharmaceutical composition of claim 14 or 15, for a use in therapy as recited in any one of claims 1 to 13.

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