Use of NPY / y5r inhibitor in treating liver metastasis

Abstract

The present invention generally relates to an inhibitor (NPY / Y5R inhibitor) of NPY-5 receptor (Y5R) activation by neuropeptide Y (NPY) for use in the prevention or reduction of metastasis to the liver in a subject. The NPY / Y5R inhibitor is selected from at least one short interference RNA (siRNA) against NPY, at least one Y5R inhibitor, at least one anti-NPY antibody, at least one anti-Y5R antibody, and at least one antisense oligonucleotide (ASO) against NPY.

Description

[0001] USE OF NPY / Y5R INHIBITOR IN TREATING LIVER METASTASIS

[0002] FIELD OF THE INVENTION

[0003] The present invention generally relates to an inhibitor (NPY / Y5R inhibitor) of NPY-5 receptor (Y5R) activation by neuropeptide Y (NPY) for use in the treatment of liver metastasis in a subject. The NPY / Y5R inhibitor is selected from at least one short interference RNA (siRNA) against NPY, at least one Y5R inhibitor, at least one anti-NPY antibody, at least one anti-Y5R antibody, and at least one antisense oligonucleotide (ASO) against NPY.

[0004] BACKGROUND OF THE INVENTION

[0005] Metastasis remains the major cause of cancer-related death, but its mechanisms are poorly understood (Ganesh etal., 2021; Lambert etal., 2017). In particular, metastasis to the liver is a common hallmark of most types of solid cancer including melanoma, colorectal cancer, pancreatic cancer and breast cancer (Tsilimigras et al., 2021). Interactions between cancer cells and the microenvironment are crucial for the engraftment of liver metastases, which includes enabling tissue infiltration, organotropism, evasion of immune defenses and adaption to the liver niche (Tsilimigras eta / ., 2021; Van den Eynden eta / ., 2013; Gao et al., 2019; Mielgo et al., 2020).

[0006] Niche-derived cytokines, like transforming growth factor 0 (TGF-0) mediating stroma activation and immune evasion, and few so far identified chemokines inducing G- protein-coupled-receptor activation on cancer cells (Van den Eynden eta / ., 2013) are considered as key players of chemotactic liver infiltration and colonization. CXCL12 is one of the few identified chemoattractive chemokines, which is expressed in the liver niche and promotes metastasis by cross-talk with CXC-receptor 4 on multiple types of disseminated cancer cells (Mortezaee etal, 2021). CXCL12 was also shown to interact with TGF-p in the liver microenvironment to drive colon cancer metastasis. Further identification of such niche-derived factors and interactions promoting liver colonization will enable specific therapeutic option, and is therefore desirable (Ganesh etal., 2021; Lambert etal., 2017; Tsilimigras etal., 2021; Mortezaee etal., 2021).

[0007] Neuropeptide Y (NPY) and its G-protein-coupled receptor subtypes represent a highly conserved, chemokine-like system, which is involved in major cancer-related hallmarks including aging, autophagy, nutrient sensing, caloric restriction and hematopoietic stem-cell properties (Hirsch et al., 2012; Cerda-Reverter et al., 2000; Dumont et al., 1992; Reichmann et a!., 2016; Tatemoto et a!., 1982). NPY has been described to promote inflammation-induced tumorigenesis by enhancing epithelial cell proliferation (Jeppsson eta / ., 2017) and to exert proangiogenic actions which can be altered by its direct effect on tumor cell proliferation and survival resulting in stimulation or inhibition of tumor growth, depending on tumor type (Tilan etal., 2010). / IZ / ’ gene methylation has been described as a universal, longitudinal plasma marker for evaluating the clinical benefit of Regorafenib treatment in metastatic colorectal cancer (Jensen etal.,

[0008] 2019). In view of such observations, a connection has been suggested between nervous system dysfunctions and a range of neurotransmitters (including NPY), neurotropins and their receptors in colorectal cancer liver metastasis, and studies show a high complexity of brain-tumor interactions (Kasprzak etal., 2020).

[0009] With respect to the liver, NPY has been identified as a crucial target in cirrhosis and its related complications including portal hypertension (Dietrich et al., 2013; Hartl etal., 2015) and the primary liver cancer hepatocellular carcinoma (HCC) (Dietrich et al.,

[0010] 2020). Cirrhosis is a risk factor for primary liver cancer (HCC). However and notably, several studies indicate that liver cirrhosis reduces cancer patients' risk of liver metastases (i.e. secondary liver cancers) from extrahepatic tumors See, e.g., the systematic reviews by Seymour etal., 1999 and Dahl etal., 2011. See also the more recent publication by Mahdi etal., 2021 confirming that metastases are uncommon in cirrhotic livers. Moreover, numerous studies suggest that the NPY-system affects nonalcoholic fatty liver disease (NAFLD) via hepatic stellate cell activation, promotion of the metabolic syndrome and obesity, adipocyte- macrophage cross-talk promoting liver inflammation, and induction of hepatocytic cholesterol synthesis by activation of SREBP2-HMGCR-signaling.

[0011] Since NAFLD is the emerging epidemic of our century affecting one quarter of the global population, fatty change of the liver microenvironment was suggested to influence liver metastasis in multiple experimental and clinical studies. However, the question whether steatosis promotes or even inhibits liver metastasis in diverse types of cancer including melanoma, colorectal cancer and breast cancer remains controversial, since an equal number of studies reveal opposing results and clinical data provide evidence that the net effect is non-significant. These studies suggest that indirect, NAFLD-associated effectors depending on diverse circumstances (including co-morbidities, degree of steatosis, inflammatory activity and presence of fibrosis) as well as different experimental models, rather than fat accumulation per se, drives liver metastasis. The described CXCL12-CXCR4-chemokine-axis was shown to be deregulated in NAFLD and might represent one of such indirect effectors.

[0012] In contrast, the potential impact of the steatosis-associated NPY-system on fatty change of the metastatic liver microenvironment and associated liver colonization is completely unknown.

[0013] Two recent studies suggested that NPY might affect bone metastasis in Ewing sarcoma, however its potential impact on metastasis to the liver remained completely elusive (Hong et al., 2015; Lu et al., 2022). Notably, the recently published review of Sigorski et a!., 2025, which comprehensively discusses the role of NPY in cancer pathobiology including a potential effect in bone metastasis, indicates that further studies are required to determine the role of the NPY system in metastasis to other niches.

[0014] SUMMARY OF THE INVENTION

[0015] As shown in the examples section herein, the inventors now surprisingly found that hepatocyte-derived NPY is a major chemoattractive ligand promoting metastatic liver colonization. Specifically, niche-derived NPY was found to drive liver metastasis independent from steatosis via Y5R-mediated cAMP inhibition, ERK activation and CHK2 inactivation. While ERK-activation and cAMP inhibition mediated by Y5R had been confirmed recently by the inventors to drive hepatocellular carcinoma progression (Dietrich et a!., 2020), the existence of a potential NPY-Y5R-ERK / cAMP axis in liver metastasis had been unknown. Also, inactivation of the tumor suppressor CHK2 mediated by NPY-Y5R signaling had not been described before. CHK2 represents a potent tumor suppressor affecting metastasis in diverse types of cancer including melanoma, colorectal, pancreatic and prostate cancer (Pan eta / ., 2019).

[0016] The inventors' studies show that metastasis to the liver is strongly reduced in NPY knockout mice, which is independent from steatosis. NPY is expressed by hepatocytes in close proximity of metastatic cancer cells and induced by TGF-0 in the perimetastatic microenvironment. In contrast to niche-associated upregulation of NPY, systemic NPY-levels are not altered in metastatic patients and mice. Metastatic cancer cells express NPY-receptors, and specifically NPY-5 receptor (Y5R) was found to regulate cAMP- and ERK-signaling after NPY binding, thereby inducing chemotactic migration and invasion. Moreover, Y5R-mediated dephosphorylation of checkpoint kinase 2 (CHK2) was identified to promote clonogenicity in metastatic cancer cells. In line with these findings, strong Y5R expression is associated with a metastatic phenotype in several types of cancers. In vivo, small molecule-mediated inhibition of Y5R as well as therapeutic RNA interference targeting NPY significantly reduced liver metastasis, as demonstrated in the examples herein.

[0017] Next to targeting the responsible receptor Y5R on metastatic cancer cells, RNAi appears as a particularly elegant strategy to target hepatocytes in the (pre-) metastatic liver niche, thereby reducing the chemotactic ligand NPY and counteracting potential resistance mechanisms when targeting Y5R (which might be induced by compensatory upregulation of NPY or by further NPY receptors). In pre-clinical mouse experiments, the inventors provided an LNP-mediated drug delivery via i.p. injection of drug-loaded LNPs (lipid nanoparticles) inducing stable gene knockdown in the liver. RNAi mediated targeting of NPY did not reveal liver-associated or systemic side effects, and both systemic levels of the metabolic regulator NPY as well as its expression in other tissues was unaffected.

[0018] Taken together, these findings reveal a novel and surprising cross-talk between hepatocytes and cancer cells in the metastatic liver niche, and show that inhibiting this cross-talk, for example by inhibition of Y5R or NPY, or by targeting niche-associated NPY by RNA interference (using siRNA) or an antisense oligonucleotide (ASO), is useful as a therapeutic approach to control liver metastasis.

[0019] Accordingly, the present invention relates to an inhibitor (herein referred to as 'NPY / Y5R inhibitor') of NPY-5 receptor activation by NPY, especially hepatocyte-derived NPY, for use in the treatment of liver metastasis, especially the prevention or reduction of metastases to the liver, in a subject.

[0020] The present invention also relates to a method of treating liver metastasis, especially the prevention or reduction of metastases to the liver, in a subject, wherein the method comprises administering to the subject an NPY / Y5R inhibitor.

[0021] The NPY / Y5R inhibitor used according to the present invention is selected from:

[0022] (i) one or more than one short interference RNA (siRNA) against NPY,

[0023] (ii) one or more than one Y5R inhibitor,

[0024] (iii) one or more than one anti-NPY antibody,

[0025] (iv) one or more than one anti-Y5R antibody, and

[0026] (v) one or more than one antisense oligonucleotide (ASO) against NPY.

[0027] DETAILED DESCRIPTION OF THE INVENTION

[0028] Definitions

[0029] Unless otherwise defined herein, the singular article 'a' or 'an' means 'one or more than one', i.e. is not meant as a limitation to 'one'.

[0030] Unless otherwise defined herein, 'at least one' means 'one or more than one'.

[0031] Unless otherwise defined herein, the term 'nucleotide' refers to a DNA nucleotide or an RNA nucleotide, which may comprise one or more of a phosphonate modification, a ribose modification, and a base modification. In the context of DNA / RNA analogs, such as phosphorodiamidate morpholino oligonucleotides (PMOs), the term 'nucleotide' is used to refer to the nucleotide analog unit which, in case of PMOs, has a 6-membered morpholino ring instead of the (deoxy-) ribose of a DNA or RNA nucleotide and a phosphorodiamidate instead of the phosphonate of a DNA or RNA nucleotide.

[0032] The term 'cancer' as used herein refers to malignant diseases characterized by the abnormal cell growth with the potential to spread and / or invade to other parts of the body. Said spreading / invasion of cancer cells from the primary cancer to a different site within the body is generally known as metastasis. In the context of the present invention, 'metastasis' in particular means metastasis to the liver Oliver metastasis'). Metastases are often found at the same time as the primary cancer, or later. In some subjects with metastases, a primary cancer is found only later or not at all, for example, because the (too small) size and / or the (unusual) location of the primary cancer prevented its diagnosis, or because the primary cancer regressed leaving behind its metastasis.

[0033] Cancers include solid tumors as well as non-solid cancers (e.g. leukemia). Particular non-limiting examples of primary cancers are biliary tract cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colorectal cancer (e.g., colon cancer), endometrial cancer, esophageal cancer, gastric cancer, intraepithelial neoplasm, leukemia, lymphoma, liver cancer, lung cancer, orolaryngeal cancer, malignant melanoma, uveal melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer and renal cancer. The primary cancer may be different from primary liver cancer; particularly different from primary liver cancer, Ewing sarcoma, neuroblastoma, pheochromocytoma, paraganglioma, breast cancer, and prostate cancer. In preferred embodiments, the primary cancer is selected from stomach cancer, colorectal cancer (e.g., colon cancer), breast cancer, pancreatic cancer, lung cancer, and malignant melanoma, particularly from stomach cancer, colorectal cancer, pancreatic cancer, lung cancer, and malignant melanoma; more particularly from colorectal cancer and malignant melanoma.

[0034] The terms 'treatment' and 'treating' as used herein with regard to liver metastasis refer to an intervention to prevent, reduce, and / or slow progression of liver metastasis in the treated subject by administering an NPY / Y5R inhibitor described herein to the subject. A treatment of liver metastasis as described herein can thus be a prophylactic treatment, i.e., a treatment of the subject to protect the subject against, or prevent the subject from suffering from, liver metastases. The treatment of liver metastasis can be the prevention or reduction of liver metastases in a subject who has or had a primary cancer, wherein said prevention or reduction can - at least in part - be achieved by one or more of the following activities of the NPY / Y5R inhibitor in the subject:

[0035] (a) inhibiting the migration and / or invasion of cancer cells from the primary cancer into the liver of the subject;

[0036] (b) inhibiting the growth of metastatic cancer cells of the primary cancer in the liver of the subject;

[0037] (c) reducing the colony formation capacity (clonogenicity) of metastatic cancer cells of the primary cancer in the liver of the subject.

[0038] Liver metastasis in a subject, or prevention or reduction of liver metastasis, can be determined, for example, by monitoring the number and / or the size of liver metastases over time using known techniques, e.g. MRT. Known tumor markers can be used to detect liver metastases and distinguish them from other tissues such as healthy liver tissues and primary liver cancer (primary liver tumors).

[0039] The NPY / Y5R inhibitor described herein can be administered to the subject (i) prior to treatment of the primary cancer (particularly as neoadjuvant treatment), (ii) during treatment of the primary cancer (particularly as peri-operative treatment), or (iii) after treatment of the primary cancer, or (iv) at two or all three of (i)-(iii). Administration of the NPY / Y5R inhibitor after treatment of the primary cancer can be as an adjuvant treatment or as a palliative treatment (e.g., years after removal of the primary cancer). For example, the NPY / Y5R inhibitor can be used in a combinatory treatment for synchronous or metachronous liver metastatic cancer.

[0040] The NPY / Y5R inhibitor can be used as a monotherapy, or can be combined with first- and / or later-line cancer treatments, for example with one or more of chemotherapy, immunotherapy, radiation and surgical cancer removal.

[0041] Unless specified otherwise herein, the terms 'subject' and 'patient' are used herein synonymously and refer to a human or a non-human animal, for example a companion animal. Non-limiting examples of subjects or patients which can be treated with an NPY / Y5R inhibitor as described herein are mammals such as humans, cats, dogs, and horses. Preferably, the subject or patient is a human. Typically, the subject is suspected or known to have a primary cancer or to have had a primary cancer in the past. The inventors' studies demonstrated that treatment with an NPY / Y5R inhibitor as described herein can strongly reduce liver metastasis independent from steatosis. The subject treated with an NPY / Y5R inhibitor as described herein can therefore be a subject that has steatosis or a subject that does not have steatosis.

[0042] The NPY / Y5R inhibitor described herein is expediently administered in an effective amount. The term 'effective amount' as used herein in the context of a medical use or method of treating liver metastasis refers to the amount of NPY / Y5R inhibitor, or composition thereof, that is capable of eliciting the beneficial biological or medical response in a subject that is sought by the treating individual, i.e. medical doctor or other clinician, in particular a prophylactic or inhibitory effect against the formation of liver metastases, a reduction of the number of liver metastases and / or a reduction of the size of liver metastases.

[0043] For a treatment as described herein, the NPY / Y5R inhibitor can be administered in the form of a pharmaceutical composition comprising the NPY / Y5R inhibitor, a pharmaceutically acceptable carrier and, optionally, further excipients. The pharmaceutical composition can be formulated in any way that is compatible with its therapeutic application, including intended route of administration, delivery format and desired dosage.

[0044] Suitable administration routes for the NPY / Y5R inhibitor, or pharmaceutical composition thereof, include parenteral administration, for examples by injection. A preferred route of administration is subcutaneous administration. The optimal dose of the NPY / Y5R inhibitor administered will, of course, depend on the subject to be treated, particularly on the subject's weight, the subject's age, the manner of administration, and the judgement of the prescribing physician.

[0045] The term 'pharmaceutically acceptable' is used herein to refer to those compounds or compositions which are within the full range of medical judgment suitable for being administered to a human or non-human animal by the chosen route of administration, and thereby meet reasonable benefit I risk ratios without undue toxicity, irritation or other adverse events.

[0046] Neuropeptide Y (NPY), NPY-5 receptor (Y5R), and their encoding genes are known in the art (Larhammar etal., 2004). For example, sequence information for the 36-amino acid human NPY, its precursor and encoding gene is found under NCBI accession no. P69101, Uniprot entry P01303 and NCBI gene ID 4852. NPY is a highly conserved protein, and known to act as a neuropeptide and tissue hormone expressed in the brain and sympathetic neurons of the peripheral nervous system. As described in the examples section herein, strong NPY expression has been found in peri metastatic liver tissues, especially in hepatocytes which are the major target of siRNA therapies. Sequence information for human Y5R is found under Uniprot entry Q15761 and NCBI gene ID 4889. As described in the examples section herein, strong Y5R expression has been found in cancer cells, in particular in metastatic cancer cells in the liver. An NPY / Y5R inhibitor as described herein in particular inhibits the activation of Y5R by NPY, and thereby the downstream signaling of NPY-Y5R crosstalk. Said downstream signaling is known to include activation of Gi proteins resulting in potent antagonism of cAMP signaling (Dietrich et al., 2020). An inhibitory effect on NPY-induced Y5R activation can thus, for instance, be measured by bioluminescence resonance energy transfer (BRET) analysis of forskolin-induced cAMP activation applying cancer cells in vitra, see cAMP BRET assay in the examples section herein. NPY-Y5R-induced ERK- induction in (samples of protein lysates prepared from) cell lines (for in vitro proof) or in peri-metastatic liver tissues (for in vivo proof), especially in hepatocytes, can be measured qualitatively and quantitatively by Western blot analysis, e.g. using an anti- phospho-ERK antibody; see examples section herein. Other measurable effects of (inhibition of) Y5R activation include (inhibition of) metastatic cancer cell migration and invasion as well as (inhibition of) time-dependent dephosphorylation of checkpoint kinase 2 (phospho-CHK2) at Thr 68, as described in the examples herein, which can also be measured qualitatively and quantitatively by Western blot analysis, e.g. using samples of protein lysates prepared from cell lines (for in vitro proof) or metastatic liver tissues (for in vivo proof), especially hepatocytes, and using an anti-phospo-Chk2 antibody (see examples section herein).

[0047] SiRNA

[0048] The terms 'siRNA' and 'si-RNA" used herein are synonymous abbreviations of 'small interfering RNA' and refer to a double stranded RNA molecule which is capable of RNA interference (RNAi) of a target gene, particularly in a target-specific manner. Generally, it is believed that, after entering a target cell, the siRNA is introduced into the RNA- induced silencing complex (RISC), one strand of the siRNA Csense' or 'passenger' strand) is removed, and the remaining strand Cantisense' or 'guide' strand) directed RISC towards sequence-specific binding to the target gene messenger RNA (mRNA). Said binding to the target gene mRNA results in cleavage and degradation of the mRNA, thus preventing synthesis of NPY protein (translation). See, e.g., Friedrich et al. (BioDrugs, 2022, 36, 549-571). The siRNA described herein is in particular synthetic siRNA.

[0049] The term 'siRNA against NPY' as used herein refers to an siRNA capable of RNAi of a gene encoding NPY, expediently the NPY gene of the subject to be treated. Administration of siRNA against NPY thus inhibits expression of NPY by RNAi, in particular NPY expression in hepatocytes.

[0050] RNAi therapeutics have already been approved and represent breakthrough discoveries in medicine (Strnad etal., 2022; Balwani etal., 2020). From 2018 to 2023, five siRNA drugs (Vutrisiran, Patisiran, Givosiran, Lumasiran, Inclisiran) were approved for the treatment of patients - all of them specifically target gene expression in hepatocytes (Adams et al., 2018; Ahn et al., 2023). The approved and known "inpipeline" RNAi therapeutics are designed to treat hereditary disorders and metabolic (liver) diseases including amyloidosis, hypercholesteremia, alpha-l-anti-trypsine deficiency, NASH and diabetes (Strnad etal., 2022; Ahn etal., 2023). The concept of RNAi for treating cancer has not been evaluated in clinical phase II / III studies yet, mostly because siRNAs specifically target hepatocytes. This specific targeting, however, provides a particular advantage for the herein-described use of siRNAs against NPY in the treatment of liver metastasis by targeting NPY-expression of hepatocytes.

[0051] Design principles, advantageous chemical modifications, preparation methods and delivery for therapeutic siRNAs, which are applicable to the siRNAs against NPY used in the context of the present invention, are well known in the art. See, for, example, reviews by Hu et al. (Signal Transduction and Targeted Therapy, 2020, 5, 101) and Friedrich et al. (BioDrugs, 2022, 36, 549-571). Detailed information on design and modifications of siRNA is also found in Shmushkovich et al. (2018).

[0052] It is desirable to design siRNA such that any risk of immunostimulation is minimized. To this end, the siRNA should not contain immunostimulatory motifs, such as UGUGU. This and other putative immunostimulatory motif which should be avoided to minimize unwanted immune activation are described in Judge etal., 2005.

[0053] Common features of known effective siRNAs (Patisiran, Givosiran, Inclisiran, Lumasiran, Vutrisiran, Fitusiran, Revusiran, Tivanisiran, Teprasiran and Cemdisiran) include an U at position 1 (associated with best silencing); no C at position 7 (because C at this position is associated with poor silencing and higher off-target levels); an A at position 10 (known to improve on-target silencing); no G at position 14; and a C at position 19 (associated with good silencing).

[0054] Each strand of an siRNA is typically 15-30 nucleotides in length. SiRNAs against NPY used in the context of the present invention are preferably 19-29 nucleotides in length, more preferably 19-25 nucleotides in length, and even move preferably 19-24 nucleotides in length. Further preferred properties of siRNAs include a GC content (of the entire siRNA or, preferably, of the antisense strand of the siRNA) of 30-60%, particularly 30-43%; asymmetry of the siRNA strands (i.e., the sense strand and the antisense strand of the siRNA have different lengths, in particular the sense strand is shorter than the antisense strand); a lack of palindromes, CCC and GGG sequences, and internal repeats; a lack of putative immunostimulatory motifs (e.g., as described in Judge et al., 2005), such as UGUGU; a lack of single nucleotide polymorphisms in the target region (for parallel targeting of all SNP variants); a delta-delta-G of >0; an U at position 1; no G at position 14, particularly an A, an U or a C at position 14; no C at position 7, particularly an U, a G or an A at position 7, more particularly an A at position 7; a C at position 19; an A at position 10. Accordingly, siRNAs against NPY used in the context of the present invention are preferably characterized by one, two or all of the afore- mentioned preferred properties.

[0055] Exemplary siRNAs against human NPY include siRNAs having an antisense strand which comprises or consists of the nucleotide sequence set forth in any of SEQ ID NOs: 1-232 and SEQ ID NOs:263-292. Exemplary siRNAs against human NPY also include siRNAs having an antisense strand which comprises or consists of the nucleotide sequence set forth in any of SEQ ID NOs: 1-232, except that one or both of the last two nucleotides (nucleotides 20 and 21) of this sequence, independently, is missing or substituted by a different nucleotide. Exemplary siRNAs against human NPY may thus have an antisense strand which comprises, or consists of, at least nucleotides 1 to 19 of the nucleotide sequence set forth in any of SEQ ID NOs: 1-232, for example nucleotides 1 to 19, nucleotides 1-20, or nucleotides 1-21 of the nucleotide sequence set forth in any of SEQ ID NOs: 1-232, in particular nucleotides 1 to 19, nucleotides 1-20, or nucleotides 1-21 of the nucleotide sequence set forth in any of SEQ ID NO: 2, 75, 14, 11, 154, 1, 8, 9, 5, 4, 10, 184, 78, 80, 180, 27, 21, 83, 159, 22, 24, or 23, more particularly nucleotides 1 to 19, nucleotides 1-20, or nucleotides 1-21 of the nucleotide sequence set forth in SEQ ID NO:2 or SEQ ID NO:83.

[0056] A particular exemplary siRNA against human NPY for use according to the present invention is an siRNA having a sense strand and an antisense strand, wherein the antisense strand comprises, or consists of, at least nucleotides 1 to 19 (for example, nucleotides 1-19, 1-20 or 1-21, in particular all of nucleotides 1-21) of the nucleotide sequence set forth in SEQ ID NO:2, wherein the nucleotides of the siRNA are unmodified (as in SEQ ID NO:2) or, preferably, wherein at least part (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and particularly 100%) of the nucleotides of the siRNA (particularly at least part of said at least nucleotides 1 to 19 of the sequence set forth in SEQ ID NO: 2) has been chemically modified as described herein.

[0057] A particular exemplary siRNA against human NPY for use according to the present invention is an siRNA having a sense strand and an antisense strand, wherein the antisense strand comprises, or consists of, at least nucleotides 1 to 19 (for example, nucleotides 1-19, 1-20 or 1-21, in particular all of nucleotides 1-21) of the nucleotide sequence set forth in SEQ ID NO:75, wherein the nucleotides of the siRNA are unmodified (as in SEQ ID NO:75) or, preferably, wherein at least part (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 89%, at least 90%, at least 94%, and particularly 100%) of the nucleotides of the siRNA (particularly at least part of said at least nucleotides 1 to 19 of the sequence set forth in SEQ ID NO: 75) has been chemically modified as described herein.

[0058] A particular exemplary siRNA against human NPY for use according to the present invention is an siRNA having a sense strand and an antisense strand, wherein the antisense strand comprises, or consists of, at least nucleotides 1 to 19 (for example, nucleotides 1-19, 1-20 or 1-21, in particular all of nucleotides 1-21) of the nucleotide sequence set forth in SEQ ID NO:14, wherein the nucleotides of the siRNA are unmodified (as in SEQ ID NO: 14) or, preferably, wherein at least part (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 89%, at least 90%, at least 94%, and particularly 100%) of the nucleotides of the siRNA (particularly at least part of said at least nucleotides 1 to 19 of the sequence set forth in SEQ ID NO: 14) has been chemically modified as described herein.

[0059] A particular exemplary siRNA against human NPY for use according to the present invention is an siRNA having a sense strand and an antisense strand, wherein the antisense strand comprises, or consists of, at least nucleotides 1 to 19 (for example, nucleotides 1-19, 1-20 or 1-21, in particular all of nucleotides 1-21) of the nucleotide sequence set forth in SEQ ID NO:11, wherein the nucleotides of the siRNA are unmodified (as in SEQ ID NO: 11) or, preferably, wherein at least part (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 89%, at least 90%, at least 94%, and particularly 100%) of the nucleotides of the siRNA (particularly at least part of said at least nucleotides 1 to 19 of the sequence set forth in SEQ ID NO: 11) has been chemically modified as described herein.

[0060] A particular exemplary siRNA against human NPY for use according to the present invention is an siRNA having a sense strand and an antisense strand, wherein the antisense strand comprises, or consists of, at least nucleotides 1 to 19 (for example, nucleotides 1-19, 1-20 or 1-21, in particular all of nucleotides 1-21) of the nucleotide sequence set forth in SEQ ID NO:154, wherein the nucleotides of the siRNA are unmodified (as in SEQ ID NO: 154) or, preferably, wherein at least part (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 89%, at least 90%, at least 94%, and particularly 100%) of the nucleotides of the siRNA (particularly at least part of said at least nucleotides 1 to 19 of the sequence set forth in SEQ ID NO: 154) has been chemically modified as described herein.

[0061] A particular exemplary siRNA against human NPY for use according to the present invention is an siRNA having a sense strand and an antisense strand, wherein the antisense strand comprises, or consists of, at least nucleotides 1 to 19 (for example, nucleotides 1-19, 1-20 or 1-21, in particular all of nucleotides 1-21) of the nucleotide sequence set forth in SEQ ID NO:1, wherein the nucleotides of the siRNA are unmodified (as in SEQ ID NO:1) or, preferably, wherein at least part (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 89%, at least 90%, at least 94%, and particularly 100%) of the nucleotides of the siRNA (particularly at least part of said at least nucleotides 1 to 19 of the sequence set forth in SEQ ID NO:1) has been chemically modified as described herein.

[0062] A particular exemplary siRNA against human NPY for use according to the present invention is an siRNA having a sense strand and an antisense strand, wherein the antisense strand comprises, or consists of, at least nucleotides 1 to 19 (for example, nucleotides 1-19, 1-20 or 1-21, in particular all of nucleotides 1-21) of the nucleotide sequence set forth in SEQ ID NO:8, wherein the nucleotides of the siRNA are unmodified (as in SEQ ID NO:8) or, preferably, wherein at least part (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 89%, at least 90%, at least 94%, and particularly 100%) of the nucleotides of the siRNA (particularly at least part of said at least nucleotides 1 to 19 of the sequence set forth in SEQ ID NO: 8) has been chemically modified as described herein.

[0063] A particular exemplary siRNA against human NPY for use according to the present invention is an siRNA having a sense strand and an antisense strand, wherein the antisense strand comprises, or consists of, at least nucleotides 1 to 19 (for example, nucleotides 1-19, 1-20 or 1-21, in particular all of nucleotides 1-21) of the nucleotide sequence set forth in SEQ ID NO:9, wherein the nucleotides of the siRNA are unmodified (as in SEQ ID NO:9) or, preferably, wherein at least part (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 89%, at least 90%, at least 94%, and particularly 100%) of the nucleotides of the siRNA (particularly at least part of said at least nucleotides 1 to 19 of the sequence set forth in SEQ ID NO: 9) has been chemically modified as described herein. A particular exemplary siRNA against human NPY for use according to the present invention is an siRNA having a sense strand and an antisense strand, wherein the antisense strand comprises, or consists of, at least nucleotides 1 to 19 (for example, nucleotides 1-19, 1-20 or 1-21, in particular all of nucleotides 1-21) of the nucleotide sequence set forth in SEQ ID NO:5, wherein the nucleotides of the siRNA are unmodified (as in SEQ ID NO:5) or, preferably, wherein at least part (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 89%, at least 90%, at least 94%, and particularly 100%) of the nucleotides of the siRNA (particularly at least part of said at least nucleotides 1 to 19 of the sequence set forth in SEQ ID NO: 5) has been chemically modified as described herein.

[0064] A particular exemplary siRNA against human NPY for use according to the present invention is an siRNA having a sense strand and an antisense strand, wherein the antisense strand comprises, or consists of, at least nucleotides 1 to 19 (for example, nucleotides 1-19, 1-20 or 1-21, in particular all of nucleotides 1-21) of the nucleotide sequence set forth in SEQ ID NO:4, wherein the nucleotides of the siRNA are unmodified (as in SEQ ID NO:4) or, preferably, wherein at least part (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 89%, at least 90%, at least 94%, and particularly 100%) of the nucleotides of the siRNA (particularly at least part of said at least nucleotides 1 to 19 of the sequence set forth in SEQ ID NO:4) has been chemically modified as described herein.

[0065] A particular exemplary siRNA against human NPY for use according to the present invention is an siRNA having a sense strand and an antisense strand, wherein the antisense strand comprises, or consists of, at least nucleotides 1 to 19 (for example, nucleotides 1-19, 1-20 or 1-21, in particular all of nucleotides 1-21) of the nucleotide sequence set forth in SEQ ID NO:10, wherein the nucleotides of the siRNA are unmodified (as in SEQ ID NO: 10) or, preferably, wherein at least part (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 89%, at least 90%, at least 94%, and particularly 100%) of the nucleotides of the siRNA (particularly at least part of said at least nucleotides 1 to 19 of the sequence set forth in SEQ ID NO: 10) has been chemically modified as described herein.

[0066] A particular exemplary siRNA against human NPY for use according to the present invention is an siRNA having a sense strand and an antisense strand, wherein the antisense strand comprises, or consists of, at least nucleotides 1 to 19 (for example, nucleotides 1-19, 1-20 or 1-21, in particular all of nucleotides 1-21) of the nucleotide sequence set forth in SEQ ID NO:184, wherein the nucleotides of the siRNA are unmodified (as in SEQ ID NO: 184) or, preferably, wherein at least part (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 89%, at least 90%, at least 94%, and particularly 100%) of the nucleotides of the siRNA (particularly at least part of said at least nucleotides 1 to 19 of the sequence set forth in SEQ ID NO: 184) has been chemically modified as described herein.

[0067] A particular exemplary siRNA against human NPY for use according to the present invention is an siRNA having a sense strand and an antisense strand, wherein the antisense strand comprises, or consists of, at least nucleotides 1 to 19 (for example, nucleotides 1-19, 1-20 or 1-21, in particular all of nucleotides 1-21) of the nucleotide sequence set forth in SEQ ID NO:78, wherein the nucleotides of the siRNA are unmodified (as in SEQ ID NO:78) or, preferably, wherein at least part (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 89%, at least 90%, at least 94%, and particularly 100%) of the nucleotides of the siRNA (particularly at least part of said at least nucleotides 1 to 19 of the sequence set forth in SEQ ID NO: 78) has been chemically modified as described herein.

[0068] A particular exemplary siRNA against human NPY for use according to the present invention is an siRNA having a sense strand and an antisense strand, wherein the antisense strand comprises, or consists of, at least nucleotides 1 to 19 (for example, nucleotides 1-19, 1-20 or 1-21, in particular all of nucleotides 1-21) of the nucleotide sequence set forth in SEQ ID NO:80, wherein the nucleotides of the siRNA are unmodified (as in SEQ ID NO:80) or, preferably, wherein at least part (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 89%, at least 90%, at least 94%, and particularly 100%) of the nucleotides of the siRNA (particularly at least part of said at least nucleotides 1 to 19 of the sequence set forth in SEQ ID NO: 80) has been chemically modified as described herein.

[0069] A particular exemplary siRNA against human NPY for use according to the present invention is an siRNA having a sense strand and an antisense strand, wherein the antisense strand comprises, or consists of, at least nucleotides 1 to 19 (for example, nucleotides 1-19, 1-20 or 1-21, in particular all of nucleotides 1-21) of the nucleotide sequence set forth in SEQ ID NO:180, wherein the nucleotides of the siRNA are unmodified (as in SEQ ID NO: 180) or, preferably, wherein at least part (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 89%, at least 90%, at least 94%, and particularly 100%) of the nucleotides of the siRNA (particularly at least part of said at least nucleotides 1 to 19 of the sequence set forth in SEQ ID NO: 180) has been chemically modified as described herein.

[0070] A particular exemplary siRNA against human NPY for use according to the present invention is an siRNA having a sense strand and an antisense strand, wherein the antisense strand comprises, or consists of, at least nucleotides 1 to 19 (for example, nucleotides 1-19, 1-20 or 1-21, in particular all of nucleotides 1-21) of the nucleotide sequence set forth in SEQ ID NO:27, wherein the nucleotides of the siRNA are unmodified (as in SEQ ID NO:27) or, preferably, wherein at least part (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 89%, at least 90%, at least 94%, and particularly 100%) of the nucleotides of the siRNA (particularly at least part of said at least nucleotides 1 to 19 of the sequence set forth in SEQ ID NO: 27) has been chemically modified as described herein.

[0071] A particular exemplary siRNA against human NPY for use according to the present invention is an siRNA having a sense strand and an antisense strand, wherein the antisense strand comprises, or consists of, at least nucleotides 1 to 19 (for example, nucleotides 1-19, 1-20 or 1-21, in particular all of nucleotides 1-21) of the nucleotide sequence set forth in SEQ ID NO:21, wherein the nucleotides of the siRNA are unmodified (as in SEQ ID NO:21) or, preferably, wherein at least part (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 89%, at least 90%, at least 94%, and particularly 100%) of the nucleotides of the siRNA (particularly at least part of said at least nucleotides 1 to 19 of the sequence set forth in SEQ ID NO: 21) has been chemically modified as described herein.

[0072] A particular exemplary siRNA against human NPY for use according to the present invention is an siRNA having a sense strand and an antisense strand, wherein the antisense strand comprises, or consists of, at least nucleotides 1 to 19 (for example, nucleotides 1-19, 1-20 or 1-21, in particular all of nucleotides 1-21) of the nucleotide sequence set forth in SEQ ID NO:83, wherein the nucleotides of the siRNA are unmodified (as in SEQ ID NO:83) or, preferably, wherein at least part (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 89%, at least 90%, at least 94%, and particularly 100%) of the nucleotides of the siRNA (particularly at least part of said at least nucleotides 1 to 19 of the sequence set forth in SEQ ID NO: 83) has been chemically modified as described herein.

[0073] A particular exemplary siRNA against human NPY for use according to the present invention is an siRNA having a sense strand and an antisense strand, wherein the antisense strand comprises, or consists of, at least nucleotides 1 to 19 (for example, nucleotides 1-19, 1-20 or 1-21, in particular all of nucleotides 1-21) of the nucleotide sequence set forth in SEQ ID NO:159, wherein the nucleotides of the siRNA are unmodified (as in SEQ ID NO:159) or, preferably, wherein at least part (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 89%, at least 90%, at least 94%, and particularly 100%) of the nucleotides of the siRNA (particularly at least part of said at least nucleotides 1 to 19 of the sequence set forth in SEQ ID NO: 159) has been chemically modified as described herein.

[0074] A particular exemplary siRNA against human NPY for use according to the present invention is an siRNA having a sense strand and an antisense strand, wherein the antisense strand comprises, or consists of, at least nucleotides 1 to 19 (for example, nucleotides 1-19, 1-20 or 1-21, in particular all of nucleotides 1-21) of the nucleotide sequence set forth in SEQ ID NO:22, wherein the nucleotides of the siRNA are unmodified (as in SEQ ID NO:22) or, preferably, wherein at least part (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 89%, at least 90%, at least 94%, and particularly 100%) of the nucleotides of the siRNA (particularly at least part of said at least nucleotides 1 to 19 of the sequence set forth in SEQ ID NO: 22) has been chemically modified as described herein.

[0075] A particular exemplary siRNA against human NPY for use according to the present invention is an siRNA having a sense strand and an antisense strand, wherein the antisense strand comprises, or consists of, at least nucleotides 1 to 19 (for example, nucleotides 1-19, 1-20 or 1-21, in particular all of nucleotides 1-21) of the nucleotide sequence set forth in SEQ ID NO:24, wherein the nucleotides of the siRNA are unmodified (as in SEQ ID NO:24) or, preferably, wherein at least part (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 89%, at least 90%, at least 94%, and particularly 100%) of the nucleotides of the siRNA (particularly at least part of said at least nucleotides 1 to 19 of the sequence set forth in SEQ ID NO: 24) has been chemically modified as described herein.

[0076] A particular exemplary siRNA against human NPY for use according to the present invention is an siRNA having a sense strand and an antisense strand, wherein the antisense strand comprises, or consists of, at least nucleotides 1 to 19 (for example, nucleotides 1-19, 1-20 or 1-21, in particular all of nucleotides 1-21) of the nucleotide sequence set forth in SEQ ID NO:23, wherein the nucleotides of the siRNA are unmodified (as in SEQ ID NO:23) or, preferably, wherein at least part (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 89%, at least 90%, at least 94%, and particularly 100%) of the nucleotides of the siRNA (particularly at least part of said at least nucleotides 1 to 19 of the sequence set forth in SEQ ID NO: 23) has been chemically modified as described herein.

[0077] Exemplary siRNAs against murine NPY are siRNAs having an antisense strand which comprises or consists of the nucleotide sequence set forth in any of SEQ ID NOs:233- 262. Exemplary siRNAs against murine NPY also include siRNAs having an antisense strand which comprises or consists of the nucleotide sequence set forth in any of SEQ ID NOs: 233-262, except that one or both of the last two nucleotides (nucleotides 20 and 21) of this sequence, independently, is missing or substituted by a different nucleotide. Exemplary siRNAs against murine NPY may thus have an antisense strand which comprises, or consists of, at least nucleotides 1 to 19 of the nucleotide sequence set forth in any of SEQ ID NOs:233-262, for example nucleotides 1 to 19, nucleotides 1-20, or nucleotides 1-21 of the nucleotide sequence set forth in any of SEQ ID NOs:233-262, wherein the nucleotides of the siRNA are unmodified (as in SEQ ID NO: 233-262) or, preferably, wherein at least part (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 89%, at least 90%, at least 94%, and particularly 100%) of the nucleotides of the siRNA (particularly at least part of said at least nucleotides 1 to 19 of the sequence set forth in any of SEQ ID NOs: 233-262) has been chemically modified as described herein.

[0078] Additionally, the properties of siRNAs, including those of the exemplary siRNAs described herein, can be improved by chemical modifications of siRNA nucleotides. For example, such modifications can suppress immunostimulatory siRNA-driven activation of the innate immune response of the treated subject, improve chemical stability and efficacy, and / or decrease off-target-induced toxicity. SiRNAs against NPY used in the context of the present invention are therefore preferably characterized by one or more of such chemical modifications. For example, at least 50%, at least 60%, at least 70%, at least 80%, at least 89%, at least 90%, at least 94%, and in particular 100% of the nucleotides of an siRNA against NPY used in the context of the present invention have been chemically modified as described herein.

[0079] According to the chemical structure of basic building blocks of natural RNA, said chemical modifications can be categorized as modifications in the ribose moiety Cribose modification'), the phosphate backbone Cphosphonate modification'), and the nucleobases Chase modification'). The phosphonate modification may be one or more than one of methylphosphonate (MP), phosphorodithioate (PS2), methoxypropyl phosphonate (MPO), 5'-phosphorothioate (5'-PS), (S)-5'-C-methyl with phosphate, peptide nucleic acid (PNA), phosphorothioate (PS) Rp isomer, phosphorothioate (PS) Sp isomer, 5'-(£)-vinylphosphonate (5'-(£)-VP), and 5'-methyl phosphonate (5'-MP). The ribose modification may be one or more than one of 2'-O- methyl (2'0Me), 2'-deoxy-2'-fluoro (2'F), 2'-methyl-4-pyridine (2'-O-CH2Py(4)), PMO, 2'-O-methoxyethyl (2'-O-MOE), tricyclo-DNA (tcDNA), Locked Nucleic Acid (LNA), 2'- ara bi no-fl uoro (2'-Ara-F), 2'-O-benzyl, Glycol Nucleic Acid (GNA), Unlocked Nucleic Acid (UNA), and (S)-cEt-BNA. Alternatively or additionally, the ribose modification may be 2'-deoxy. The base modification may be one or more than one of pseudouridine (QJ), 2'-thiouridine (s2U), N6'-methyladenosine (m6A), N-ethylpiperidine 7'-EAA triazole modified adenine, N-ethylpiperidine 6'-triazole modified adenine, 6'- phenylpyrrolocytosine (PhpC), 5'-methylcytosine (m5C), 5'-fluoro-2'-deoxyuridine, 2',4'-difluorotoluyl ribonucleoside (rF), and 5'-nitroindole. 5-methyluracil (thymine) is also a base modification useful in the context of the present invention. A modified nucleotide of the siRNA may have one, or a combination of two or more of: a phosphonate modification, a ribose modification and a base modification. For example, in one or more nucleotides of the siRNA, in particular nucleotides which, if unmodified, would be U, the base modification may be thymine and the ribose modification may be 2'-deoxy.

[0080] Known strategies for efficient delivery of siRNA to hepatocytes, which can be applied to the siRNAs against NPY used in the context of the present invention, include conjugation of the siRNA with N-acetyl galactosamine (GalNAc), and siRNA delivery in the form of siRNA-loaded lipid nanoparticles (LNPs). See, for, example, reviews by Hu et al. (Signal Transduction and Targeted Therapy, 2020, 5, 101) and Friedrich et al. (BioDrugs, 2022, 36, 549-571).

[0081] Conjugation with GalNac is a further chemical modification of the siRNA. Delivery of the siRNAs in the form of GalNAc-siRNA conjugates is particularly preferred. For example, in GalNAc-siRNA conjugates, trivalent or tetravalent GalNAc moieties are coupled to the 5'-end or 3'-end of the siRNA sense strand via a linker. A particular example of such GalNAc-siRNA conjugate is an siRNA wherein the 3'-end of the sense strand carries a GalNAc-L96 moiety (structure shown in Figure 17). GalNAc is a ligand of the asialoglycoprotein receptor (ASGPR, ASGR1), which is an endocytic receptor that is highly expressed on hepatocytes, and barely expressed by other cells. The binding to ASGPR facilitates the uptake of GAINAc conjugated siRNA into hepatocytes by endocytosis. The interaction between GalNAc and ASGPR is pH-sensitive, such that dissociation of the receptor and GalNAc-siRNA conjugate occurs during acidification of the endosome. The GalNAc moiety is subsequently subject to enzymatic degradation that liberates the siRNA.

[0082] GalNAc-siRNA conjugates are preferably administered subcutaneously.

[0083] Particular examples of chemically modified siRNAs against human NPY for use according to the present invention are siRNAs having a sense strand and an antisense strand, wherein the antisense strand comprises, or consists of, at least nucleotides 1 to 19 (for example, nucleotides 1-19, 1-20 or 1-21, in particular all of nucleotides 1- 21) of the nucleotide sequence set forth in SEQ ID NO: 23, wherein at least part of (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 89, at least 90%, at least 94%, and particularly 100%) of the nucleotides have been chemically modified as described herein, and wherein the 3'-end of the sense strand siRNA carries a GalNAc moiety, in particular a GalNAc-L96 moiety, as described herein.

[0084] A specific exemplary chemically modified siRNA against human NPY for use according to the present invention is the siRNA which has the sense strand set forth in SEQ ID NO:614 and the antisense strand set forth in SEQ ID NO:610, and is the siRNA #1.1 shown in Figure 17, respectively.

[0085] A specific particular exemplary modified siRNA against human NPY for use according to the present invention is the siRNA which has the sense strand set forth in SEQ ID NO:615 and the antisense strand set forth in SEQ ID NO:611, and is the siRNA #1.2 shown in Figure 17, respectively.

[0086] A specific particular exemplary modified siRNA against human NPY for use according to the present invention is the siRNA which has the sense strand set forth in SEQ ID NO:616 and the antisense strand set forth in SEQ ID NO:612, and is the siRNA #18.1 shown in Figure 17, respectively.

[0087] A specific particular exemplary modified siRNA against human NPY for use according to the present invention is the siRNA which has the sense strand set forth in SEQ ID NO:617 and the antisense strand set forth in SEQ ID NO:613, and is the siRNA #18.2 shown in Figure 17, respectively.

[0088] Several classes of LNPs are known in the art and can be used for the delivery of siRNAs in the context of the present invention, including ionizable LNPs, cationic LNPs and neutral LNPs (Akinc etal., 2019; Akinc etal., 2010; de Fougerolles, 2008; Jayaraman et al., 2012; Maestro et al., 2021; Witzigmann et al., 2020; Woitok et al., 2020). Cationic LNPs and in particular ionizable LNPs are thereby particularly advantageous for siRNA delivery to hepatocytes. Examples of lipids which can be useful for the formation of LNPs for delivery of siRNAs in the context of the present invention include, but are not limited to, DLin-MC3-DMA ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- tetraen-19-yl 4-(dimethylamino)butanoate, CAS no. 1224606-06-7), e.g. in combination with cholesterol; DSPC ((2A)-2,3-bis(octadecanoyloxy)propyl 2- (trimethylazaniumyl)ethyl phosphate, CAS no. 816-94-4 (R enantiomer), CAS no. 4539- 70-2 (racemate); PEG-DMG (l,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene; DLin-DMA (N,N-dimethyl-2,3- / s[(9Z,12Z)-9,12-octadecadien-l-yloxy]-l-propanamine, CAS no. 871258-12-7); and L-319 (9-[4-(dimethylamino)-l-oxobutoxy]- heptadecanedioic acid, l,17-di-(2Z)-2-nonen-l-yl ester, CAS no: 1351586-50-9).

[0089] A NPY / Y5R inhibitor used in the context of the present invention can comprise one siRNA against NPY (this includes more than one identical siRNA molecules), or a combination of two or more different siRNAs against NPY, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 different siRNAs against NPY.

[0090] Y5R inhibitors which are small molecules

[0091] The term 'small molecule' as used herein refers to a compound having a molecular weight of not more than 1 kDa, for example from 0.1 kDa to 1 kDa.

[0092] Y5R inhibitors (Balasubramaniam etai., 1997; Li etai., 2015) which are useful in the context of the present invention include the following small molecules known to inhibit Y5R, for example:

[0093] / VL[[ / ra / 7s-4-[[(4-amino-2-quinazolinyl)amino]methyl]cyclohexyl]methyl]-l- naphthalenesulfonamide (CGP71683or a pharmaceutically acceptable salt thereof, for example CGP71683 hydrochloride (CAS no. 192322-50-2);

[0094] Zrans-N-[l-(2-fluorophenyl)-lH-pyrazol-3-yl]-l'-oxo-spiro[cyclohexane-l,3'(l'H)- furo[3,4-c]pyridine]-4-carboxamide (MK-0557; CAS no. 328232-95-7);

[0095] / IA[4-methyl-9-(l-methylethyl)-9 / ^carbazol-3-yl]-4-morpholinecarboxamide (5RA972; CAS no. 439861-56-0);

[0096] Zra / 7s-4-(tert-butylsulfonylamino)-N-[5-(trifluoromethyl)pyridin-2-yl]cyclohexane-l- carboxamide (S-2367, Velneperit; CAS no. 342577-38-2).

[0097] Whether or not a compound is an Y5R inhibitor can be determined as described herein, in particular: An inhibitory effect on NPY-induced Y5R activation can, for instance, be measured by bioluminescence resonance energy transfer (BRET) analysis of forskolin- induced cAMP activation applying cancer cells in vitro, see cAMP BRET assay in the examples section herein. NPY-Y5R-induced ERK-induction in (samples of protein lysates prepared from) cell lines (for in vitro proof) or in peri-metastatic liver tissues (for in vivo proof), especially in hepatocytes, can be measured qualitatively and quantitatively by Western blot analysis, e.g. using an anti-phospho-ERK antibody; see examples section herein. Other measurable effects of (inhibition of) Y5R activation include (inhibition of) metastatic cancer cell migration and invasion as well as (inhibition of) time-dependent dephosphorylation of checkpoint kinase 2 (phospho- CHK2) at Thr 68, as described in the examples herein, which can also be measured qualitatively and quantitatively by Western blot analysis, e.g. using samples of protein lysates prepared from cell lines (for in vitro proof) or metastatic liver tissues (for in vivo proof), especially hepatocytes, and using an anti-phospo-Chk2 antibody (see examples section herein). Antibodies against NPY or Y5R

[0098] Unless stated otherwise, the term 'antibody' as used herein refers to a (whole) immunoglobulin or an antigen-binding immunoglobulin fragment. Immunoglobulins are well known in the art and include antibodies such as, for example, IgA, IgD, IgE, IgG, and IgM. Antigen-binding immunoglobulins fragments are likewise well known in the art and include antigen-binding antibody fragments such as, for example, Fab, Fab', F(ab')2, single domain antibodies (such as the variable domain of camelid heavy chain antibodies, VHH), and single chain Fv (scFv).

[0099] The term 'anti-NPY antibody' as used herein refers in particular to an antibody which is capable of specifically binding to NPY and, when bound to NPY, inhibit the activation of Y5R by NPY.

[0100] The term 'anti-Y5R antibody' as used herein refers in particular to an antibody which is capable of specifically binding to Y5R and, when bound to Y5R, inhibit the activation of Y5R by NPY.

[0101] Techniques for generating antibodies against a known target antigen are well known in the art, and can be applied to generate antibodies against NPY or Y5R. Techniques for generating the antigen material for such purpose are likewise well known in the art, and can be applied for generating antigen material for the identification of anti- NPY antibodies or anti-Y5R antibodies. Such techniques include, for example, recombinant expression and purification techniques for generating a known protein (such as NPY or Y5R) or an antigenic portion thereof, and techniques for generating cells presenting a target antigen which is a receptor protein (such as Y5R) at their surface.

[0102] Whether or not an anti-NPY antibody or an anti-Y5R antibody (by binding NPY or Y5R, respectively) inhibits the activation of Y5R by NPY can be determined as described herein, in particular: An inhibitory effect on NPY-induced Y5R activation can, for instance, be measured by bioluminescence resonance energy transfer (BRET) analysis of forskolin-induced cAMP activation applying cancer cells in vitro, see cAMP BRET assay in the examples section herein. NPY-Y5R-induced ERK-induction in (samples of protein lysates prepared from) cell lines (for in vitro proof) or in peri-metastatic liver tissues (for in vivo proof), especially in hepatocytes, can be measured qualitatively and quantitatively by Western blot analysis, e.g. using an anti-phospho-ERK antibody; see examples section herein. Other measurable effects of (inhibition of) Y5R activation include metastatic cancer cell migration and invasion as well as (inhibition of) timedependent dephosphorylation of checkpoint kinase 2 (phospho-CHK2) at Thr 68, as described in the examples herein, which can also be measured qualitatively and quantitatively by Western blot analysis, e.g. using samples of protein lysates prepared from cell lines (for in vitro proof) or metastatic liver tissues (for in vivo proof), especially hepatocytes, and using an anti-phospo-Chk2 antibody (see examples section herein).

[0103] Antisense oligonucleotide (ASO)

[0104] The term 'ASO' (plural: 'ASOs7) used herein is an abbreviation of 'antisense oligonucleotide' and refers to a small (13-30 nucleotides in length), synthetic, singlestranded, chemically modified nucleic acid polymer which can be employed to sequence-specifically inhibit the expression of a target gene (which, in the context of the present invention, is the NPY gene). Based on their mode of action, ASOs can be divided in the following two categories: (1) ASOs causing target RNA degradation and (2) steric block ASOs (for review see Roberts et al., 2020).

[0105] Steric block ASOs are designed to bind to target transcripts with high affinity. Steric block ASOs have modified nucleotides that avoid RNAse H recognition. They are therefore not RNase-H-competent and thus do not induce degradation of their target mRNA, but interfere with mRNA-RNA interactions and / or mRNA-protein interactions and frequently affect mRNA splicing, (see Roberts et al., 2020) Examples for FDA- approved steric block ASOs are Eteplirsen, Golodirsen and Nusinersen.

[0106] ASOs causing target RNA degradation normally depend on the activity of RNAseH and are therefore also called 'RNase-H-competent ASOs'. RNAseH is a ubiquitous nuclease cleaving (i.e., degrading) the RNA strand in DNA-RNA heteroduplexes, such as the DNA-RNA heteroduplexes which are formed when DNA-based ASOs bind to their cognate mRNA transcripts. The cleavage at the ASO binding site results in the destruction of the target mRNA, thereby silencing target gene expression. As RNAseH is present in the cytoplasm and the nucleus, this approach is suitable to target mRNAs and ncRNAs in both compartments. In particular, the presence in the nucleus allows for a co-transcriptional degradation of gene transcripts which in consequence includes intron sequences as possible target sequence (see Lai et al., 2020, and Roberts et al., 2020). To date, at least four RNase-H-competent ASOs have received regulatory approval: Fomivirsen, Mipomersen, Inotersen and Volanesorsen. RNAse-H-competent ASOs have a "gapmer" structure, i.e. a structure characterized by a central "gap" region consisting of DNA nucleotides that allows for RNase H binding and cleavage of the base-paired target RNA strand. Said central DNA sequence is flanked by modified nucleotides which do not allow RNase H cleavage at these positions but increase exonuclease stability and binding affinity of the ASO. Gapmer ASOs typically use the RNAse-H-competent phosphorothioate (PS) linkage for all positions to improve nuclease stability. Terminal 2'-O-methoxyethyl (2'-O-MOE) and / or 2'-O-methyl (2'0Me) modifications have been used in ASOs for increased binding. Several 2'-O-MOE-modified PS-ASOs are in advanced clinical trials (Egli et al., 2023). Alternatively, terminal locked nucleic acid (LNA) modifications have been used and offer a number of advantages (Kurreck et al., 2002), such as an increase in melting temperature of 1.5-4°C per LNA (depending on the positions of the modified residues), increased RNAse H cleavage activity correlating with higher ASO binding affinity, strongly increased stability against nucleases, effective unassisted uptake of "naked" ASOs in vivo in cell culture. Several clinical studies claim good tolerance for LNA- containing ASOs (Bianchini et al., 2013; Van der Ree et al., 2014).

[0107] ASOs against NPY used in the context of the present invention may have a length of 16-30 nucleotides, particularly 16-25 nucleotides, more particular 16-22 nucleotides. The ASOs against NPY can comprise at least one of the following chemical modifications (a-d):

[0108] (a) at least one phosphonate modification independently selected from phosphorothioate (PS) Rp isomer, and phosphorothioate (PS) Sp isomer;

[0109] (b) at least one ribose modification independently selected from 2'-O-methyl (2'OMe), 2'-deoxy-2'-fluoro (2'F), 2'-O-methoxyethyl (2'-O-MOE), and Locked Nucleic Acid (LNA);

[0110] (c) at least one phosphonate and ribose modification selected from phosphorodiamidate morpholino;

[0111] (d) at least one base modification independently selected from pseudouridine (QJ),

[0112] 2'-thiouridine (s2U), N6'-methyladenosine (m6A), N-ethylpiperidine 7'-EAA triazole modified adenine, N-ethylpiperidine 6'-triazole modified adenine, 6'- phenylpyrrolocytosine (PhpC), 5'-methylcytosine (m5C), 5'-fluoro-2'- deoxyuridine, 2',4'-difluorotoluyl ribonucleoside (rF), and 5'-nitroindole.

[0113] The ASOs against NPY used in the context of the present invention can have the design of gapmer ASOs (RNAase-H-competent ASOs causing target RNA degradation) or steric block ASOs, wherein gapmer ASOs are preferred.

[0114] A particular example of ASOs against NPY which are steric block ASOs are ASOs having a nucleotide sequence which is a phosphorodiamidate morpholino oligonucleotide (PMO) sequence, i.e. a nucleic acid analog wherein the DNA bases are attached to a backbone of methylenemorpholine rings linked through phosphorodiamidate groups (instead of the backbone of the deoxyribose rings linked through phosphonate groups found in DNA). ASOs against NPY which are PMOs may have a length of 20-30 nucleotides, particularly 25-30 nucleotides.

[0115] ASOs against NPY which have the design of gapmer ASOs have a nucleotide sequence consisting of a central sequence portion which at each side is flanked by a flanking sequence portion. The central sequence portion is a RNAse-H-competent nucleotide sequence, i.e. a nucleotide sequence which allows for RNase H binding and cleavage of the base-paired target RNA strand. Preferably, the central sequence portion consists of unmodified DNA nucleotides. The flanking sequence portions are not RNAse-H- competent, i.e. are nucleotide sequences which do not allow for cleavage of a basepaired target RNA strand (specifically the NPY mRNA strand bound by the ASO) by RNase H. To this end, the nucleotides of the flanking sequence portions are chemically modified nucleotides, preferably nucleotides having at least one ribose modification selected from 2'-O-methyl (2'OMe), 2'-O- methoxyethyl (2'-O-MOE), and Locked Nucleic Acid (LNA).

[0116] Additionally, it is preferred that the ASOs against NPY which have the design of gapmer ASOs have the phosphonate modification phosphorothioate (PS) at all nucleotides of the central sequence portion and both flanking sequence portions. Said phosphorothioates can be selected from phosphorothioate (PS) Rp isomers, phosphorothioate (PS) Sp isomers, and mixtures thereof.

[0117] In a preferred example, the ASO against NPY has a nucleotide sequence consisting of a central sequence portion of DNA nucleotides which at each side is flanked by a flanking sequence portion of ribose-modified nucleotides, wherein the ribose modification in each ribose-modified nucleotide is Locked Nucleic Acid (herein referred to as 'LNA-based gapmer ASO7). In an even more preferred example, the ASO against NPY has a nucleotide sequence consisting of a central sequence portion of DNA nucleotides which at each side is flanked by a flanking sequence portion of ribose- modified nucleotides, wherein the ribose modification in each ribose-modified nucleotide is 2'-O-methyl (2'OMe) or 2'-O-methoxyethyl (2'-O-MOE) (herein referred to as '2'OMe-based gapmer ASO' and '2'-O-MOE-based gapmer ASO7). Preferably, each of the nucleotides of the central sequence portion and both flanking sequence portions of the LNA-based gapmer ASO, the 2'OMe-based gapmer ASO or the 2'-O-MOE-based gapmer ASO have the phosphonate modification phosphorothioate (PS), which can be selected from phosphorothioate (PS) Rp isomers, phosphorothioate (PS) Sp isomers, and mixtures thereof. The 2'OMe-based gapmer ASO or the 2'-O-MOE-based gapmer ASO is preferably 18-30 nucleotides, more preferably 20-22 nucleotides, in length. Given their strongly increased binding affinity, LNA-based gapmer ASOs may be shorter, preferably 16-20 nucleotides, more preferably 16-18 nucleotides, such as 16 nucleotides, in length.

[0118] In ASOs against NPY which have the design of gapmer ASOs, the central sequence portion may be 7-10 nucleotides in length and each of the flanking sequence portions may be 3-6 nucleotides in length. In particular examples of a LNA-based gapmer ASO, the central sequence portion is 8 nucleotides in length and each of the flanking sequence portions is 4 nucleotides in length, or the central sequence portion is 10 nucleotides in length and each of the flanking sequence portions is 3 nucleotides in length. In particular examples of 2'OMe-based or 2'-O-MOE-based gapmer ASOs, the central sequence portion is 10 nucleotides in length and each of the flanking sequence portions is 5 nucleotides in length. However, these classical, symmetrical ASO designs do not represent strict design rules.

[0119] The design of ASOs is rather flexible. Considerations for optimal ASO design are predominantly the right level of binding affinity to its target RNA and the recruitment of RNAse H. Information on design rules for optimal LNA-based gapmer ASOs is found in multiple studies (e.g., Papargyri et al., 2020; Hagedorn et al., 2017) Important parameters for an optimal LNA-based gapmer ASO design are the length of the ASO, the size of central "gap" region (central sequence portion of non-RNase-H-competent nucleotides), and the number of flanking LNA nucleotides.

[0120] The ASOs against NPY have a nucleotide sequence (meaning in particular the sequence of the bases) which, optionally apart from one non-complementary nucleotide (mismatch), is complementary to a sequence portion (Target sequence) within the sequence of the NPY transcript, preferably the sequence of Homo sapiens NPY mRNA, in particular the sequence of NCBI reference NM_000905.4.

[0121] As ASOs function through direct base-pairing to their target RNA, the identification of suitable target sequences can be achieved by straightforward bioinformatics sequence analysis such as described in Kuespert et al., 2020. Target sequence identification typically involves: avoiding of single nucleotide polymorphisms (SNPs) in the target transcript, mismatch analysis to the human transcriptome, and cross-reactivity to orthologous genes in relevant species for drug development as rodent and non-human primates. Even though a single mismatch to the target sequence, caused for example by a single nucleotide polymorphism (SNP) in target gene, may have little effect of ASO efficiency, the abundance of useful target sequences normally allows for completely avoiding target sequences which overlap with SNPs.

[0122] Mismatch analysis aims at identifying and avoiding potential off-target effects on unintended genes which are correlated with toxicity. The strength of off-target effects primarily depends on the binding affinity to an off-target sequence but also on the nature and expression of the off-target gene. Furthermore, the off-target binding affinity depends on multiple parameters such as the length and chemical properties of the ASO, and the number and position of the mismatches (Hagedorn et al., 2017). As an orientation, all current FDA approved ASO with a length of 20 to 22 nucleotides have more than 3 mismatches with any exon sequence in the human genome. Recent studies suggest that the activity and potential toxicity of gapmer ASOs can be predicted with reasonable confidence, relying on binding affinity and trinucleotide motives as critical parameters (Papargyri et al., 2020).

[0123] Cross-reactivity to orthologous genes of different species normally requires a basically complete match (typically 0-1 mismatches) of the target sequences in the orthologous genes. Cross-reactivity is typically desired for species required for preclinical development of the ASO, such as rodents (e.g., mice, rats) and if required also nonhuman primates (e.g., macaques). Depending on the level of conservation of the target gene, the selection of target sequences which allow for ASO cross-reactivity to the orthologous (NPY) genes of different species of interest normally considerably reduces the choice of target sequences.

[0124] Exemplary ASOs against human NPY include ASOs having a nucleotide sequence which comprises, or consists of, the nucleotide sequence set forth in any of SEQ ID NOs:351- 609, or of a portion of the nucleotide sequence set forth in any of SEQ ID NOs:351- 609 which is at least 16 (consecutive) nucleotides in length, wherein nucleotides of the ASO have been chemically modified as described herein. Unless specified otherwise herein, a reference to a nucleotide sequence set forth in any of SEQ ID NOs:351-609 is meant to refer to said nucleotide sequence set forth in any of SEQ ID NOs:351-609, which nucleotides have been chemically modified as described herein. In particular, the nucleotides of said exemplary ASOs against human NPY have been modified so as to function as gapmer ASO (RNAase-H-competent ASOs causing target RNA degradation) or as steric block ASO, preferably as gapmer ASO.

[0125] ASOs comprising or consisting of a nucleotide sequence set forth in any of SEQ ID NOs: 351, 597, 599 to 601, 604 to 605 and 608 to 609, or of a portion of said nucleotide sequence which is at least 16 (consecutive) nucleotides in length, bind to the NPY transcripts of Homo sapiens (NCBI Reference Sequence NM_000905.5), Macaca fascicularis (NCBI Reference Sequence XM_005549975.3), Macaca mu / atta (NCBI Reference Sequence NM_001032814.1), Mus musculus (NCBI Reference Sequence NM_023456.3) and Rattus c> / e^ / cz / s (NM123614.2). ASOs comprising or consisting of a nucleotide sequence set forth in any of SEQ ID NOs: 352 to 358, 361 to 365, 596 and 598, or of a portion of said nucleotide sequence which is at least 16 (consecutive) nucleotides in length, bind to the NPY transcripts of Homo sapiens (NCBI Reference Sequence NM_000905.5), Macaca fascicu / aris (NCBI Reference Sequence XM_005549975.3), Macaca mu / atta (NCBI Reference Sequence NM_001032814.1), Cam's lupus familiaris (NCBI Reference Sequence XM_038442248.1), Equus cabaiius CB Reference Sequence XM_001498508.5) and Fells catus (NCBI Reference Sequence XM_019825605.3).

[0126] ASOs comprising or consisting of the nucleotide sequence set forth in SEQ ID NO: 359, or of a portion of said nucleotide sequence which is at least 16 (consecutive) nucleotides in length, bind to the NPY transcripts of Homo sapiens (NCBI Reference Sequence NM_000905.5), Cam's lupus familiaris (NCBI Reference Sequence XM_038442248.1), Macaca fascicu / aris (NCBI Reference Sequence XM_005549975.3) and Macaca muiatta (NCBI Reference Sequence NM_001032814.1).

[0127] ASOs comprising or consisting of the nucleotide sequence set forth in SEQ ID NO: 360, or of a portion of said nucleotide sequence which is at least 16 (consecutive) nucleotides in length, bind to the NPY transcripts of Homo sapiens (NCBI Reference Sequence NM_000905.5), Equus cabaiius (NCBI Reference Sequence XM_001498508.5), Fells catus (NCBI Reference Sequence XM_019825605.3), Macaca fascicularis (NCBI Reference Sequence XM_005549975.3) and Macaca muiatta (NCBI Reference Sequence NM_001032814.1).

[0128] ASOs comprising or consisting of a nucleotide sequence set forth in any of SEQ ID NOs: 366 to 367, or of a portion of said nucleotide sequence which is at least 16 (consecutive) nucleotides in length, bind to the sequences of the NPY transcripts of / / cvnc> Sa S77s(NCBI Reference Sequence NM_000905.5), Cam's lupus familiaris (NCB Reference Sequence XM_038442248.1), Macaca fascicularis (NCBI Reference Sequence XM_005549975.3), Macaca muiatta (NCBI Reference Sequence NM_001032814.1) and Fells catus (NCBI Reference Sequence XM_019825605.3).

[0129] ASOs comprising or consisting of a nucleotide sequence set forth in any of SEQ ID NOs: 368 to 370, or of a portion of said nucleotide sequence which is at least 16 (consecutive) nucleotides in length, bind to the NPY transcripts of Homo sapiens (NCBI Reference Sequence NM_000905.5), Canis lupus familiaris (NCBI Reference Sequence XM_038442248.1), Equus cabaiius (NCBI Reference Sequence XM_001498508.5), Macaca fascicularis (NCBI Reference Sequence XM_005549975.3) and Macaca muiatta (NCBI Reference Sequence NM_001032814.1). ASOs comprising or consisting of a nucleotide sequence set forth in any of SEQ ID NOs: 371 to 393, 402 to 404, 406 to 407, 477 to 480 and 482 to 485, or of a portion of said nucleotide sequence which is at least 16 (consecutive) nucleotides in length, bind to the NPY transcripts of Homo sapiens (NCBI Reference Sequence NM_000905.5), Macaca fascicuiaris (NCBI Reference Sequence XM_005549975.3) and Macaca mulatta (NCBI Reference Sequence NM_001032814.1).

[0130] ASOs comprising or consisting of a nucleotide sequence set forth in any of SEQ ID NOs: 394 to 401 and 405, or of a portion of said nucleotide sequence which is at least 16 (consecutive) nucleotides in length, bind to the NPY transcripts of Homo sapiens (NCBI Reference Sequence NM_000905.5), Macaca fascicuiaris (NCBI Reference Sequence XM_005549975.3), Macaca mulatta (NCBI Reference Sequence NM_001032814.1) and Equus cabaiius (NCBI Reference Sequence XM_001498508.5).

[0131] ASOs comprising or consisting of a nucleotide sequence set forth in any of SEQ ID NOs: 408 to 409, 441 to 448, 456 to 476, 489 to 592 and 595, or of a portion of said nucleotide sequence which is at least 16 (consecutive) nucleotides in length, bind to the NPY transcript of Homo sapiens (NCBI Reference Sequence NM_000905.5).

[0132] ASOs comprising the nucleotide sequence set forth in SEQ ID NO: 440, or of a portion of said nucleotide sequence which is at least 16 (consecutive) nucleotides in length, bind to the NPY transcripts of Homo sapiens NCBI Reference Sequence NM_000905.5) and Canis lupus fa miiiaris (NCBI Reference Sequence XM_038442248.1).

[0133] ASOs comprising or consisting of a nucleotide sequence set forth in any of SEQ ID NOs: 481 and 487, or of a portion of said nucleotide sequence which is at least 16 (consecutive) nucleotides in length, bind to the NPY transcripts of / / cvnc>Sa ? / 7s(NCBI Reference Sequence NM_000905.5) and Macaca mulatta (NCBI Reference Sequence NM-001032814.1).

[0134] ASOs comprising or consisting of a nucleotide sequence set forth in any of SEQ ID NOs: 417 to 421, 486, 488 and 593 to 594, or of a portion of said nucleotide sequence which is at least 16 (consecutive) nucleotides in length, bind to the NPY transcripts of Homo sapiens (NCBI Reference Sequence NM_000905.5) and Macaca fascicuiaris (NCBI Reference Sequence XM_005549975.3).

[0135] ASOs comprising or consisting of a nucleotide sequence set forth in any of SEQ ID NOs: 602 to 603 and 606 to 607, or of a portion of said nucleotide sequence which is at least 16 (consecutive) nucleotides in length, bind to the NPY transcripts of Homo sapiens (NCBI Reference Sequence NM_000905.5), Macaca fascicuiaris (NCBI Reference Sequence XM_005549975.3), Macaca mulatta (NCBI Reference Sequence NM_001032814.1), Equus caballus (NCBI Reference Sequence XM_001498508.5), Mus musculus (NCBI Reference Sequence NM_023456.3) and Mus musculus (NCBI Reference Sequence NM_023456.3).

[0136] Known strategies for efficient delivery of ASOs can be applied to the ASO(s) against NPY used in the context of the present invention. Said strategies include the administration of 'naked' ASO, i.e. ASO which is not incorporated into delivery vehicles such as lipid nanoparticles (LNPs).

[0137] ASOs are relatively large, heavily charged biomolecules which cannot freely cross plasma membranes by passive diffusion. However, unlike siRNAs, ASO have been traditionally applied as "naked" molecules for in vivo experiments, whereas in vitro experiments initially relied on lipid-based transfection. In vitro, delivery to cultured cells was discovered significantly later and termed "gymnosis". (See, e.g., Stein et al., 2010.) The usefulness of 'naked' ASOs for delivery can be explained in part by the fact that ASO include modified nucleotides, protecting them against endonuclease and exonuclease activity in serum even without delivery vehicles such as lipid nanoparticles (LNPs).

[0138] An abundance of PS linkages throughout an ASO molecule facilitates the association to proteins, e.g. serum proteins such as serum albumin. For example, 95% of a PS- modified ASO was found to be bound to serum proteins, the far most abundant being serum albumin (Crooke et al., 2020). Whilst there are many nucleotide modifications, most notably ribose backbone modifications as LNAs, that protect from nuclease degradation, PS is unique in its ability to promote serum protein association. This protein association facilitates ASO delivery in at least two ways: 1) It prevents rapidly clearing from the bloodstream by glomerular filtration in the kidneys allowing ASO molecules to stay in circulation for 1-2 hours, which is sufficient to reach the target tissue. 2) It assists endocytic uptake by target cells (Crooke et al., 2020). PS modifications thereby facilitate ASO biodistribution and unassisted uptake of ASO by cells in vivo.

[0139] The unassisted transport of ASOs into cells was shown to work best with PS-containing ASOs carrying terminal LNA nucleotides (Stein et al., 2010). ASO concentrations required for efficient target RNA degradation are typically in a low micromolar range and thus usually 100-1000-fold higher than required for efficient siRNA-based target silencing with lipofection reagents. The advantage of unassisted delivery, such as delivery of 'naked' ASO, in vivo in vitro are the reduced chemical complexity and the avoidance of potential side effects caused by a delivery vehicle. As described above, chemically modified ASOs are able to enter target cells in easily accessible tissues such as liver without the assistance of delivery vehicles. Lipid conjugations, as used to allow delivery of naked siRNA, are therefore uncommon in ASOs.

[0140] The efficiency of ASO delivery to hepatocytes can be increased by using GalNAc-ASO conjugates. For example, addition of a GalNAc moiety to the 5' end of ASO molecules had been found to increase delivery to hepatocytes in mice by 10-fold (Prakash et al., 2014). The ASO against NPY used the context of the present invention is preferably conjugated with N-acetyl galactosamine (GalNAc). For example, trivalent or tetravalent GalNAc moieties can be coupled to the 5'-end or 3'-end of the ASO via a linker. GalNAc is a high-affinity ligand (Kd ~2.5 nM) of the asialoglycoprotein receptor (ASGPR), which is an endocytic receptor that is highly expressed on hepatocytes, and barely expressed by other cells. The binding to ASGPR facilitates the uptake of GalNAc-conjugated ASOs into hepatocytes by endocytosis. The interaction between GalNAc and ASGPR is pH- sensitive, such that dissociation of the receptor and GalNAc-ASO conjugate occurs during acidification of the endosome. The GalNAc moiety is subsequently subject to enzymatic degradation that liberates the ASO. GalNAc-conjugated ASOs are preferentially delivered to hepatocytes in vivo, whereas unconjugated ASOs are primarily detected in non-parenchymal liver cells (Roberts et al., 2020).

[0141] ASOs against NPY, and in particular GalNAc conjugates thereof, are preferably administered subcutaneously.

[0142] An NPY / Y5R inhibitor used in the context of the present invention can comprise one ASO against NPY (this includes more than one identical ASO molecules), or a combination of two or more different ASOs against NPY, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 different ASOs against NPY.

[0143] FURTHER PARTICULAR EMBODIMENTS

[0144] Further particular embodiments of the invention include the following embodiments A.1-A.15 recited in patent application EP 24 155 023.5 and embodiments B.1-B.22 recited in patent application EP 24 174 864.9.

[0145] A.l An inhibitor of NPY-5 receptor (Y5R) activation by hepatocyte-derived neuropeptide Y (NPY) for use in the treatment of liver metastasis in a subject, wherein said inhibitor (NPY / Y5R inhibitor) is selected from:

[0146] (i) at least one short interference RNA (siRNA) against NPY, (ii) at least one Y5R inhibitor which is a small molecule,

[0147] (iii) at least one anti-NPY antibody, and

[0148] (iv) at least one anti-Y5R antibody.

[0149] A.2 The NPY / Y5R inhibitor for use according to A.l, wherein the NPY / Y5R inhibitor of (i) is one siRNA against NPY, or a combination of two or more different siRNAs against NPY, particularly 2-10 different siRNAs against NPY.

[0150] A.3 The NPY / Y5R inhibitor for use according to A.2, wherein the siRNA(s) comprise(s) at least one of the following chemical modifications:

[0151] (i.1.1) at least one phosphonate modification selected from methylphosphonate (MP), phosphorodithioate (PS2), methoxypropyl phosphonate (MPO), 5'-phosphorothioate (5'-PS), (S)- 5'-C-methyl with phosphate, peptide nucleic acid (PNA), phosphorothioate (PS) Rp isomer, phosphorothioate (PS) Sp isomer, 5'-(£)-vinylphosphonate (5'-(£)-VP), and 5'-methyl phosphonate (5'- MP);

[0152] (1.1.2) at least one ribose modification selected from 2'-O-methyl (2'OMe), 2'- deoxy-2'-fluoro (2'F), 2'-methyl-4-pyridine (2'-O-CH2Py(4)), PMO, 2'- 0- methoxyethyl (2'-O-MOE), tricyclo-DNA (tcDNA), Locked Nucleic Acid (LNA), 2'-arabino-fluoro (2'-Ara-F), 2'-O-benzyl, Glycol Nucleic Acid (GNA), Unlocked Nucleic Acid (UNA), and (S)-cEt-BNA;

[0153] (1.1.3) at least one base modification selected from pseudouridine (QJ), 2'- thiouridine (s2U), N6'-methyladenosine (m6A), N-ethylpiperidine 7'- EAA triazole modified adenine, N-ethylpiperidine 6'-triazole modified adenine, 6'-phenylpyrrolocytosine (PhpC), 5'-methylcytosine (m5C), 5'- fluoro-2'-deoxyuridine, 2',4'-difluorotoluyl ribonucleoside (rF), and 5'- nitroindole.

[0154] A.4 The NPY / Y5R inhibitor for use according A.2 or A.3, wherein the siRNA(s) is / are:

[0155] (1.2) conjugated with N-acetyl galactosamine (GalNAc); and / or

[0156] (1.3) comprised by a lipid nanoparticle (LNP), particularly an ionizable lipid nanoparticle.

[0157] A.5 The NPY / Y5R inhibitor for use according to any one of A.2-A.4, wherein the treatment comprises injecting the siRNA(s), which are optionally conjugated with GalNAc or comprised by a LNP, into the subject, particularly by subcutaneous injection. A.6 The NPY / Y5R inhibitor for use according to any of A.2-A.5, wherein the siRNA(s) is / are characterized by one or more of the following features:

[0158] (1.4.1) each strand of the siRNA(s) has a length of 15-30 nucleotides, particularly of 19-29 nucleotides, more particularly of 19-25 nucleotides, and even more particularly of 19-24 nucleotides;

[0159] (1.4.2) a GC content of 30-60%;

[0160] (i.4.3.) asymmetry of the siRNA strands;

[0161] (i.4.4) a lack of palindromes, CCC and GGG sequences, and internal repeats.

[0162] A.7 The NPY / Y5R inhibitor for use according to A.l, wherein the NPY / Y5R inhibitor is a Y5R inhibitor of (ii) selected from:

[0163] / VL[[ / ra / 7s-4-[[(4-amino-2-quinazolinyl)amino]methyl]cyclohexyl]methyl]-l- naphthalenesulfonamide (CGP71683) or a pharmaceutically acceptable salt thereof, particularly CGP71683 hydrochloride;

[0164] / ra / 7s-N-[l-(2-fluorophenyl)-lH-pyrazol-3-yl]-l'-oxo-spiro[cyclohexane- l,3'(l'H)-furo[3,4-c]pyridine]-4-carboxamide (MK-0557);

[0165] / IA[4-methyl-9-(l-methylethyl)-9 / ^carbazol-3-yl]-4-morpholinecarboxamide (5RA972); and

[0166] Zra / 7s-4-(tert-butylsulfonylamino)-N-[5-(trifluoromethyl)pyridin-2- yl]cyclohexane-l-carboxamide (S-2367, Velneperit).

[0167] A.8 The NPY / Y5R inhibitor for use according to any one of A.1-A.7, wherein the treatment comprises injecting the Y5R inhibitor, particularly subcutaneously, into the subject.

[0168] A.9 The NPY / Y5R inhibitor for use according to any of A.1-A.8, wherein the subject has or had a primary cancer, and treatment of liver metastasis is the prevention or reduction of liver metastasis by one or more of the following activities of the NPY / Y5R inhibitor in the subject:

[0169] (a) inhibiting the migration and / or invasion of cancer cells from the primary cancer into the liver of the subject;

[0170] (b) inhibiting the growth of metastatic cancer cells of the primary cancer in the liver of the subject;

[0171] (c) reducing the colony formation capacity (clonogenicity) of metastatic cancer cells of the primary cancer in the liver of the subject.

[0172] A.10 The NPY / Y5R inhibitor for use according to A.9, wherein the primary cancer is selected from stomach cancer, colorectal cancer, breast cancer, pancreatic cancer, lung cancer, and malignant melanoma.

[0173] A.ll The NPY / Y5R inhibitor for use according to any of A.1-A.9, wherein the primary cancer is different from primary liver cancer.

[0174] A.12 The NPY / Y5R inhibitor for use according to any of A.l-A.ll, wherein the subject does not have steatosis.

[0175] A.13 The NPY / Y5R inhibitor for use according to any of A.l-A.ll, wherein the subject has steatosis.

[0176] A.14 The NPY / Y5R inhibitor for use according to any of A.1-A.13, wherein the subject is a mammal, particularly a human subject.

[0177] A.15 The NPY / Y5R inhibitor for use according to any of A.1-A.14, wherein the treatment comprises administering the NPY / Y5R inhibitor to the subject at one, two or all of the following times:

[0178] (a) prior to treatment of a primary cancer,

[0179] (b) during treatment of a primary cancer,

[0180] (c) after treatment of a primary cancer.

[0181] B.l An inhibitor of NPY-5 receptor (Y5R) activation by hepatocyte-derived neuropeptide Y (NPY) for use in the treatment of liver metastasis in a subject, wherein said inhibitor (NPY / Y5R inhibitor) is selected from:

[0182] (i) at least one short interference RNA (siRNA) against NPY,

[0183] (ii) at least one Y5R inhibitor which is a small molecule,

[0184] (iii) at least one anti-NPY antibody,

[0185] (iv) at least one anti-Y5R antibody, and

[0186] (v) at least one antisense oligonucleotide (ASO) against NPY.

[0187] B.2 The NPY / Y5R inhibitor for use according to B.l, wherein the NPY / Y5R inhibitor of (i) is one siRNA against NPY, or a combination of two or more different siRNAs against NPY, particularly 2-10 different siRNAs against NPY.

[0188] B.3 The NPY / Y5R inhibitor for use according to B.l or B.2, wherein the siRNA(s) comprise(s) at least one of the following chemical modifications:

[0189] (i.1.1) at least one phosphonate modification selected from methylphosphonate (MP), phosphorodithioate (PS2), methoxypropyl phosphonate (MPO), 5'-phosphorothioate (5'-PS), (S)- 5'-C-methyl with phosphate, peptide nucleic acid (PNA), phosphorothioate (PS) Rp isomer, phosphorothioate (PS) Sp isomer, 5'-(£)-vinylphosphonate (5'-(£)-VP), and 5'-methyl phosphonate (5'- MP);

[0190] (1.1.2) at least one ribose modification selected from 2'-O-methyl (2'OMe), 2'- deoxy-2'-fluoro (2'F), 2'-methyl-4-pyridine (2'-O-CH2Py(4)), PMO, 2'- 0- methoxyethyl (2'-O-MOE), tricyclo-DNA (tcDNA), Locked Nucleic Acid (LNA), 2'-arabino-fluoro (2'-Ara-F), 2'-O-benzyl, Glycol Nucleic Acid (GN A), Unlocked Nucleic Acid (UNA), and (S)-cEt-BNA;

[0191] (1.1.3) at least one base modification selected from pseudouridine (QJ), 2'- thiouridine (s2U), N6'-methyladenosine (m6A), N-ethylpiperidine 7'- EAA triazole modified adenine, N-ethylpiperidine 6'-triazole modified adenine, 6'-phenylpyrrolocytosine (PhpC), 5'-methylcytosine (m5C), 5'- fluoro-2'-deoxyuridine, 2',4'-difluorotoluyl ribonucleoside (rF), and 5'- nitroindole.

[0192] B.4 The NPY / Y5R inhibitor for use according to any one of B.1-B.3, wherein the siRNA(s) is / are:

[0193] (1.2) conjugated with N-acetyl galactosamine (GalNAc); and / or

[0194] (1.3) comprised by a lipid nanoparticle (LNP), particularly an ionizable lipid nanoparticle.

[0195] B.5 The NPY / Y5R inhibitor for use according to any one of B.1-B.4, wherein the treatment comprises injecting the siRNA(s), which are optionally conjugated with GalNAc or comprised by a LNP, into the subject, particularly by subcutaneous injection.

[0196] B.6 The NPY / Y5R inhibitor for use according to any of B.1-B.5, wherein the siRNA(s) is / are characterized by one or more of the following features:

[0197] (1.4.1) each strand of the siRNA(s) has a length of 15-30 nucleotides, particularly of 19-29 nucleotides, more particularly of 19-25 nucleotides, and even more particularly of 19-24 nucleotides;

[0198] (1.4.2) a GC content of 30-60%;

[0199] (i.4.3.) asymmetry of the siRNA strands;

[0200] (i.4.4) a lack of palindromes, CCC and GGG sequences, and internal repeats.

[0201] B.7 The NPY / Y5R inhibitor for use according to B.l, wherein the NPY / Y5R inhibitor is a Y5R inhibitor of (ii) selected from:

[0202] / VL[[ / ra / 7s-4-[[(4-amino-2-quinazolinyl)amino]methyl]cyclohexyl]methyl]-l- naphthalenesulfonamide (CGP71683) or a pharmaceutically acceptable salt thereof, particularly CGP71683 hydrochloride;

[0203] / ra / 7s-N-[l-(2-fluorophenyl)-lH-pyrazol-3-yl]-l'-oxo-spiro[cyclohexane- l,3'(l'H)-furo[3,4-c]pyridine]-4-carboxamide (MK-0557);

[0204] / IA[4-methyl-9-(l-methylethyl)-9 / -carbazol-3-yl]-4-morpholinecarboxamide (5RA972); and

[0205] Zra / 7s-4-(tert-butylsulfonylamino)-N-[5-(trifluoromethyl)pyridin-2- yl]cyclohexane-l-carboxamide (S-2367, Velneperit).

[0206] B.8 The NPY / Y5R inhibitor for use according to B.l, wherein the NPY / Y5R inhibitor of (v) is one ASO against NPY, or a combination of two or more different ASOs against NPY, particularly 2-10 different ASOs against NPY.

[0207] B.9 The NPY / Y5R inhibitor for use according to B.l or B.8, wherein the ASO(s) comprise(s) at least one of the following chemical modifications:

[0208] (v.1.1) at least one phosphonate modification independently selected from phosphorothioate (PS) Rp isomer, and phosphorothioate (PS) Sp isomer;

[0209] (v.1.2) at least one ribose modification independently selected from 2'-O- methyl (2'OMe), 2'-deoxy-2'-fluoro (2'F), 2'-O-methoxyethyl (2'-O- MOE), and Locked Nucleic Acid (LNA);

[0210] (v.1.3) at least one phosphonate and ribose modification selected from phosphorodiamidate morpholino;

[0211] (v.1.4) at least one base modification independently selected from pseudouridine (QJ), 2'-thiouridine (s2U), N6'-methyladenosine (m6A), N-ethylpiperidine 7'-EAA triazole modified adenine, N-ethylpiperidine 6'-triazole modified adenine, 6'-phenylpyrrolocytosine (PhpC), 5'- methylcytosine (m5C), 5'-fluoro-2'-deoxyuridine, 2',4'-difluorotoluyl ribonucleoside (rF), and 5'-nitroindole.

[0212] B.10 The NPY / Y5R inhibitor for use according to any of B.l, B.8 and B.9, wherein the ASO(s) is / are conjugated with N-acetyl galactosamine (GalNAc).

[0213] B.ll The NPY / Y5R inhibitor for use according to any of B.l and B.8-B.10, wherein the ASO(s) has / have a length of 16-30 nucleotides, particularly of 16-25 nucleotides, more particular of 16-22 nucleotides. B.12 The NPY / Y5R inhibitor for use according to any one of B.l and B.8-B.11, wherein the ASO(s) has / have a nucleotide sequence wherein:

[0214] (v.2.1) the nucleotide sequence consists of a central sequence portion of DNA nucleotides, flanked by chemically modified nucleotides (flanking sequence portions) which do not allow for cleavage of a base-paired RNA strand by RNAse H; optionally wherein central sequence portion is 7-10 nucleotides in length and each of the two flanking sequence portions is 3-6 nucleotides in length; or

[0215] (v.2.2) the nucleotide sequence is a phosphorodia midate morpholino oligonucleotide (PMO) sequence, optionally wherein the PMO sequence has a length of 20-30 nucleotides, particularly 25-30 nucleotides.

[0216] B.13 The NPY / Y5R inhibitor for use according to B.12, wherein the ASO(s) has / have a nucleotide sequence according to feature (v.4.1) which is further characterized by one or both of the following features:

[0217] (v.2.1.1) all nucleotides of the nucleotide sequence have a phosphonate modification independently selected from phosphorothioate (PS) Rp isomer, and phosphorothioate (PS) Sp isomer;

[0218] (v.2.1.2) the chemically modified nucleotides which do not allow for cleavage of a base-paired RNA strand by RNAse H are nucleotides having at least one ribose modification independently selected from: 2'-O- methyl (2'OMe), 2'-O-methoxyethyl (2'-O-MOE), and Locked Nucleic Acid (LNA); optionally wherein: the chemically modified nucleotides which do not allow for cleavage of a base-paired RNA strand by RNAse H are nucleotides having at least one ribose modification selected from: 2'-O-methyl (2'OMe) and 2'-O-methoxyethyl (2'-O-MOE); and the ASO has a length of 18-30 nucleotides, particularly of 20-22 nucleotides; or the chemically modified nucleotides which do not allow for cleavage of a base-paired RNA strand by RNAse H are nucleotides having the ribose modification Locked Nucleic Acid (LNA); and the ASO has a length of 16-20 nucleotides, particularly 16-18 nucleotides.

[0219] B.14 The NPY / Y5R inhibitor for use according to any one of B.l and B.8-B.13, wherein the treatment comprises injecting the ASO(s), which is / are optionally conjugated with GalNAc, into the subject, particularly by subcutaneous injection. B.15 The NPY / Y5R inhibitor for use according to any one of B.1-B.14, wherein the treatment comprises injecting the Y5R inhibitor, particularly subcutaneously, into the subject.

[0220] B.16 The NPY / Y5R inhibitor for use according to any of B.1-B.15, wherein the subject has or had a primary cancer, and treatment of liver metastasis is the prevention or reduction of liver metastasis by one or more of the following activities of the NPY / Y5R inhibitor in the subject:

[0221] (a) inhibiting the migration and / or invasion of cancer cells from the primary cancer into the liver of the subject;

[0222] (b) inhibiting the growth of metastatic cancer cells of the primary cancer in the liver of the subject;

[0223] (c) reducing the colony formation capacity (clonogenicity) of metastatic cancer cells of the primary cancer in the liver of the subject.

[0224] B.17 The NPY / Y5R inhibitor for use according to B.16, wherein the primary cancer is selected from stomach cancer, colorectal cancer, breast cancer, pancreatic cancer, lung cancer, and malignant melanoma.

[0225] B.18 The NPY / Y5R inhibitor for use according to any of B.1-B.16, wherein the primary cancer is different from primary liver cancer.

[0226] B.19 The NPY / Y5R inhibitor for use according to any of B.1-B.18, wherein the subject does not have steatosis.

[0227] B.20 The NPY / Y5R inhibitor for use according to any of B.1-B.18, wherein the subject has steatosis.

[0228] B.21 The NPY / Y5R inhibitor for use according to any of B.1-B.20, wherein the subject is a mammal, particularly a human subject.

[0229] B.22 The NPY / Y5R inhibitor for use according to any of B.1-B.21, wherein the treatment comprises administering the NPY / Y5R inhibitor to the subject at one, two or all of the following times:

[0230] (a) prior to treatment of a primary cancer,

[0231] (b) during treatment of a primary cancer,

[0232] (c) after treatment of a primary cancer.

[0233] DESCRIPTION OF THE FIGURES

[0234] Figure 1 A-B. Boyden chamber analysis and representative images (10-fold original magnification) of chemotaxis of human melanoma cells (SkMel28) towards recombinant NPY (lower chamber) with or without application of Y5R specific inhibitor CGP71683 (Y5Ri) (upper chamber) compared to migration towards serum free medium (n = 9). Arrowheads indicate migrated cells.

[0235] Figure 2. Representative images of colony number and colony size of human melanoma cells (SkMel28) in clonogenicity assays with application of increasing concentrations of Y5R inhibitor (Y5Ri) (n = 3).

[0236] Figure 3. Schematic illustration of the experimental procedure: splenic injection of murine melanoma cells (B16) in C57BL / 6 mice on day 0; intraperitoneal injection of Y5R inhibitor CGP71683 (Y5Ri) (15 mg / kg body weight per injection) or solvent only (DMSO) as control on indicated days; end of experiment eight days after cell implantation.

[0237] Figure 4. Explanted metastases bearing spleens and livers of DMSO (control) and Y5Ri treated mice after splenic injection of B16 melanoma cells.

[0238] Figure 5. Macroscopic classification of livers from DMSO (n = 11) and Y5Ri treated mice (n = 9) into very low I low versus moderate I high liver metastasis.

[0239] Figure 6. A. CHK2 level in NPY / _mice compared to control WT mice measured by IHC analysis. B. CHK2 level in Y5Ri-treated mice compared to control DMSO-treated mice measured by IHC analysis.

[0240] Figure 7. Schematic illustration of the experimental procedure: pre-treatment of C57BL / 6 mice with lipid nanoparticles (LNP) bearing control siRNA pool or siNPY pool (pool of siRNAs targeting murine NPY) one day before surgery (day -1); splenic injection of murine melanoma cells (B16) on day 0; intraperitoneal injection of LNPs with control siRNA pool or siNPY pool as indicated; end of experiment six days after cell implantation.

[0241] Figure 8. A. Quantification of NPY mRNA expression levels in liver tissues from mice treated with control LNPs ("Control", LNPs bearing control siRNA pool, n = 11) and from mice treated with LNPs bearing siRNA pool targeting murine NPY ("LNP-siNPY", n = 11). B. Quantification of NPY protein levels in liver tissues from mice treated with control LNPs (n = 11) and LNP-siNPY treated mice (n = 11).

[0242] Figure 9 A-B. ELISA based protein expression analysis of absolute NPY levels in liver tissues and serum samples from LNP-siNPY treated mice (n = 10) compared to mice treated with control LNPs (LNPs bearing control siRNA pool, n = 11) after splenic melanoma cell injection.

[0243] Figure 10. Analysis of relative spleen (A) and liver (B) weight after splenic injection of B16 melanoma cells and i.p. treatment with LNP-siNPY (n = 11) compared to mice treated with control LNPs (LNPs bearing control siRNA pool, n = 11).

[0244] Figure 11. Representative images of livers from mice treated with control LNPs (LNPs bearing control siRNA pool, n = 11) and LNP-siNPY treated mice (n = 11).

[0245] Figure 12. A. Quantification of number of metastases in liver tissues from LNP-siNPY treated mice (n = 10) compared to mice treated with control LNPs (LNPs bearing control siRNA pool, n = 11). B. Quantification of metastases size in liver tissues from LNP-siNPY treated mice (n = 10) compared to mice treated with control LNPs (LNPs bearing control siRNA pool, n = 11).

[0246] Figure 13. Classification of livers from mice treated with control LNPs (LNPs bearing control siRNA pool, n = 11) and LNP-siNPY treated mice (n = 11) into very low I low versus moderate I high liver metastasis.

[0247] Figure 14. A. Quantification of pERK protein expression in metastatic liver tissue samples of LNP-siNPY treated mice (n = 6) compared to mice treated with control LNPs (LNPs bearing control siRNA pool, n = 6). B. Quantification of pCHK2 protein expression in metastatic liver tissue samples of LNP-siNPY treated mice (n = 6) compared to samples from control treatment (control siPool bearing LNPs) (n = 6).

[0248] Figure 15. NPY mRNA expression in primary human hepatocytes after transfection with a siRNA pool against human NPY ("siNPY") or a negative control siRNA pool ("siCtr") for 24 hours. (n=3 independent experiments; *: p<0.01).

[0249] Figure 16. qRT-PCR quantification of NPY mRNA expression in human neuroblastoma cells (SK-N-SH - HTB-11) 24 hours after transfection with 3 nM of individual siRNAs (siRNAs 1-22) against human NPY or with mock control, positive control siRNA pool targeting NPY (NPY-cl), negative controls (Negl20 and GAPDH-C19) or toxic control siRNA pool (ToxPos). NPY mRNA expression is relative to Beta-Actin, with mock transfection control set to 100%. Data are depicted as means + / - SD.

[0250] Figure 17. Schematic representation of sequences and modifications of siRNAs #1.1, #1.2, #18.1 and #18.2 (also referred to as SINPY). The sense strand (SS) and the antisense strand (AS) are chemically modified as depicted. The 3'-end of the sense strands carries a triple-GalNAc (N-acetylgalactosamine-L96, GalNAc-L96) moiety for liver targeting.

[0251] Figure 18 and Figure 19. qRT-PCR results showing mRNA NPY levels in primary murine hepatocytes treated with siRNAs #1.1, #1.2, #18.1, #18.2 or controls after 24 hours post-transfection (Figure 18) or 48 hours post-transfection (Figure 19). Significant reduction of NPY mRNA levels was achieved for all four siRNAs, both after 24 and 48 hours post transfection. Data are depicted as means + / - SD. NPY mRNA expression is depicted relative to 18S, while Control or LNP-Control, respectively, were set 1.

[0252] Figure 20. qRT-PCR quantification of mRNA NPY levels in hepatocytes isolated 14 days post-treatment from BL / 6 mice treated with SINPY (#1.1, #1.2, #18.1 or #18.2 administered at 5mg / kg bodyweight) or PBS placebo control. Treatment with SINPY resulted in significant reduction of NPY mRNA, achieving knockdown efficacy of >95% as compared to NPY mRNA levels in mice treated with the placebo control.

[0253] Figure 21. qRT-PCR quantification of mRNA NPY levels in hepatocytes isolated 7 days post-treatment from BL / 6 mice treated with SINPY #18.2 administered at 2, 5, or 10 mg / kg bodyweight or PBS placebo control, respectively. SINPY #18.2 demonstrated a dose-dependent NPY mRNA knockdown efficacy, achieving efficacy of >80% with 2 mg / kg dose and more than 98% efficacy with 5 and 10 mg / kg.

[0254] Figure 22. Tumor volumes (relative to placebo control) of the primary tumors (pancreatic orthotopic tumors) of placebo (n=26) and SINPY #18.2 (n=28) treated mice. Ns: non-significant. Data are presented as mean ± SEM. Statistical significance was determined by 2-tailed, unpaired t test. *P < 0.05.

[0255] Figure 23 and Figure 24. Macroscopic classification of liver metastatic burden (number of animals having or lacking macroscopic liver metastases; Figure 23) and number of liver metastases per animal (Figure 24). Placebo: n=26; SINPY #18.2: n=28. Data are presented as mean ± SEM. Statistical significance was determined by 2-sided Fisher's exact test (f) and by 2-tailed, unpaired t test (g).

[0256] Figure 25. Kaplan-Meier curves depicting metastasis-free survival (MFS) (% of animals, Placebo: n=28; SINPY: n=28) and related statistical tests (Chi-square, P-value and HR (Hazard ratio and 95% confidence interval).

[0257] EXAMPLES

[0258] A Experimental Procedures

[0259] A.l Individual siRNAs and siRNA pools

[0260] The examples use the following individual siRNAs and siRNA pools:

[0261] - control siRNA pool of 30 non-targeting siRNAs having the antisense strand sequences of SEQ ID NOs: 321-350

[0262] - siRNA pool targeting murine NPY: pool of 30 siRNAs having the antisense strand sequences of SEQ ID NOs: 233-262 - siRNA pool targeting human NPY: pool of 30 siRNAs having the antisense strand sequences of SEQ ID NOs: 263-292

[0263] - individual siRNAs #1 to #22 targeting human NPY having the antisense strand sequence of SEQ ID NO:2, 75, 14, 11, 154, 1, 8, 9, 5, 4, 10, 184, 78, 80, 180, 27, 21, 83, 159, 22, 24 or 23, and a sense strand being the reverse complement thereto (see also Table 5)

[0264] - siRNA #1.1, siRNA #1.2, siRNA #18.1 and siRNA #18.2 targeting human NPY, which are chemically modified siRNAs derived from siRNA #1 and #18 having the structure depicted in Figure 17

[0265] Based on the structural information in Figure 17, the sequence protocol entries for the sense strand and antisense strand of each of these siRNAs have been prepared. The siRNAs can thus be defined as: siRNA #1.1 having the sense strand set forth in SEQ ID NO:614 and the antisense strand set forth in of SEQ ID NO:610 siRNA #1.2 having the sense strand set forth in SEQ ID NO:615 and the antisense strand set forth in of SEQ ID NO:611 siRNA #18.1 having the sense strand set forth in SEQ ID NO:616 and the antisense strand set forth in of SEQ ID NO:612 siRNA #18.2 having the sense strand set forth in SEQ ID NO:617 and the antisense strand set forth in of SEQ ID NO:613

[0266] A.2 Lipid nanoparticle mediated siRNA pool delivery

[0267] Lipid nanoparticles (LNP) were designed as described in detail by Evers et al., 2022. LNPs are considered promising drug delivery vehicles for liver specific clinical siRNA delivery (Evers et al., 2022). LNPs were prepared by microfluidic mixing using the NanoAssemblr Benchtop (Precision Nanosystems, Vancouver, Canada). An ethanolic phase containing lipids was mixed with an acidic aqueous phase (100 mM sodium acetate, pH 4.0) containing siRNA pools leading to the formation of LNPs. LNPs were produced at a flow rate ratio (aqueous:organic) of 3:1 and a total flow rate of 4.0 mL / min. Lipids were dissolved in 100% Ethanol (Merck, Darmstadt, Germany) at a total lipid concentration of 10 mM. The LNPs were composed of DLin-MC3-DMA ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, CAS no. 1224606-06-7), Cholesterol (Sigma Aldrich, Saint Louis, MO, USA), DSPC ((2A)-2,3-bis(octadecanoyloxy)propyl 2-(trimethylazaniumyl)ethyl phosphate, CAS no. 816-94-4 (R enantiomer), CAS no. 4539-70-2 (racemate); Lipoid, Ludwigshafen am Rhein, Germany) and PEG-DMG (l,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene; NOF Corporation, Tokyo, JP) at a molar percentage of 50 / 38.5 / 10 / 1.5, respectively. siRNA pools were encapsulated at an N / P ratio (ionizable lipid / RNA) of 6:1. Immediately after production, LNPs were dialyzed against an excess of phosphate buffered saline using Slide-a-Lyzer™ dialysis cassettes G2 with a membrane cutoff of 20 kDa for 16-24 h. After dialysis, LNPs were sterilized using 0.45 pm PVDF membrane filters. Purified LNP was kept at 4°C and used within 40 days after production. LNPs containing a specifically designed si-RNA pool targeting murine NPY were applied in preliminary experiments comparing tail vein vs. subcutaneous vs. i.p. (intraperitoneal) injection. I.p. injection was sufficient to significantly reduce NPY expression in the liver, while no effects were observed in other tissues or in the blood.

[0268] A.3 Animal models

[0269] Animals were obtained from Charles River Laboratories and housed under specific pathogen-free and controlled conditions (22°C, 55% humidity, 12 h day / night rhythm, free access to food and water). Melanoma tissues from a xenograft tumor model were obtained and described in previous studies (Dietrich et ai., 2018). A murine spleenliver metastasis model was used as previously described (Koch et al., 2015). Briefly, this model was performed by intrasplenic injection of B16 melanoma or CT26 colorectal carcinoma cells, respectively (1.5 x 105in 5 pl serum-free medium).

[0270] The following conditions were applied for pharmacologic inhibition of the Y5R using the specific small-molecule Y5R inhibitor CGP71683 ("Y5Ri"): Starting on the first day after cell implantation, mice of the Y5Ri group received a single dose (15 mg / kg bodyweight) every 24 h by intraperitoneal (i.p.) injection, while mice of the control group received the vehicle (ethanol (50%), DMSO (50%)).

[0271] For in vivo analysis of the effects of NPY depletion on melanoma-derived liver metastasis, the spleen-liver metastasis model was conducted using 129S-NpytmlRpa / J mice after crossing with C57BL / 6 mice to archive immunotolerance to the B16 cell line (BL / 6 background). The experiment was terminated on day seven after cell implantation.

[0272] Applying a further model of metastasis, RNA interference-mediated, therapeutic NPY knockdown was established using i.p. injection of a complex consisting of lipid nanoparticles and a siRNA pool against murine NPY ("LNP-siNPY"). Mice of the control group received a complex of lipid nanoparticles and a control siRNA pool. I.p. injections were conducted on the day before and on the first, third and fifth day after cell implantation. The experiment was terminated six days after cell implantation. In a further series of experiments, induction of mild steatosis prior to metastasis formation was applied. Mice were fed a Western-type diet having a high content of cholesterol, fructose / surcease and saturated fatty acids (38% Fat, 15% Lard, 15% Tallow, 4% C16:0, 4% C18:0; Dorn et al., 2024) for 6 weeks. Subsequently, the spleen-liver metastasis model was performed as described herein.

[0273] In all experiments, mice were killed by heart puncture under deep ketamine / xylazine- induced anesthesia. Blood samples were collected and further processed to obtain plasma samples. Non-tumorous and tumorous spleen and liver tissues as well as lung and gut tissue samples were immediately snap-frozen and stored at -80°C or were formalin-fixed for immunohistochemical analysis. All experimental protocols were approved by the Committee for Animal Health and Care of the local government and conformed to international guidelines on the ethical use of animals.

[0274] An orthotopic, syngeneic pancreatic cancer transplantation model for in vivo SINPY testing was generated using KPC661 cells (Falcomata et ai., 2022; Ischenko et ai., 2021). Briefly, under isofluran-anesthesia C57BL / 6N mice received buprenorphine and carprofen analgesia (0.1 mg / kg and 5 mg / kg, respectively). With a 10 mm cut / laparotomy at the subcostal region the abdomen was opened and the pancreas placed on a cotton wool stick. Using a 30G syringe 100.000 cells in 40 pl were injected into the pancreas tail. Successful injection was monitored by appearance of a submesothelial Matrigel bubble that remained stable during repositioning of the pancreas and wound closure (silk suture DSP 5-0 and wound clips).

[0275] A.4 Human patient tissues

[0276] Snap-frozen samples were immediately stored at -80°C. A tissue microarray consisting of tissues from nevi, primary melanoma and melanoma metastases was established as described previously (Koch etai., 2015). Primary melanoma tissues from patients with or without liver metastases were obtained from the Biobank of the Department of Dermatology, Universitatsklinikum Erlangen. Tissues samples from primary tumors of melanoma, breast cancer and pancreatic cancer patients, as well as tissues from liver metastases of melanoma, breast cancer, pancreatic cancer, and colorectal cancer patients were obtained from obtained from the Department of Surgery within the Liqulmm study. Clinicopathological features of the mentioned tissues are listed in Tables 1-3.

[0277] Table 1. Patient characteristics. BC: Breast cancer. CRC: Colorectal cancer. IR: immunoreactivity (score). MET: Metastatic tissue. MM: Malignant melanoma. Nd: not determined / missing. Other: Further types of cancer (i.e. gastric cancer (n=l), renal cancer (n=3), cholangiocellular carcinoma, fibrosarcoma (n=3), fibrosarcoma (n=l)). PC: Pancreatic cancer. Peri-tumor: Tumor-surrounding liver tissue.

[0278]

[0279] Table 2. Clinicopathological characteristics of melanoma patients (systemic NPY levels). Nd: not determined / missing

[0280]

[0281] Table 3. Clinicopathological characteristics and immunoreactivity in primary tumor tissues (TMA). BC: Breast cancer. CRC: Colorectal carcinoma. IR: immunoreactivity (score). MM: Malignant melanoma. Nd: Not determined / missing. PC: Pancreatic cancer A.5 Organoid model

[0282] Murine APTAK organoids were used for in vitro and in vivo experiments. These APTAK tumor organoids are deficient in Ape, Tp53, Tgfbr2 and express constitutively activated Kras and activated Aktl. Organoids were generated and CRC models were conducted in a similar way as previously described (Heichler etai., 2020). In an orthotopic mouse model for CRC and CRC (liver) metastasis, single cell suspensions from genetically engineered APTAK organoids were injected into the submucosa of wildtype mice.

[0283] A.6 Statistical analysis

[0284] Results were expressed as mean ± SEM. In order to compare groups, 2-tailed Student's t-tests or 1-way ANOVAs were used, along with Dunnett's multiple comparison test when appropriate. Correlation analysis was conducted using Spearman's and Pearson's coefficients, respectively. To analyze tissue microarrays, Fisher's exact test and Qui- Square test, as well as Spearman correlation analysis, and uni- and multivariate analysis applying the SPSS ordinal regression procedure (Polytomous Universal Model [PLUM]; link function: logit) were used. In siiico survival analysis was performed by computationally applying log-rank testing and hazard ratio estimates. A P value less than 0.05 was considered significant. Figures display the significance level as *P < 0.05, **P < 0.01, ***P < 0.001, and ****p < 0.0001. The number of experiments is depicted in the figure legends. Calculations were performed using Prism software (GraphPad Software Inc.) and SPSS (SPSS Statistics 23, IBM Corp.).

[0285] A.7 Cell culture

[0286] Normal human epidermal melanocytes (NHEM), the human primary melanoma cell lines Mel Juso, Mel Ei, Mel Wei and metastatic human melanoma cell lines SkMel28 (ATCC CRL-1469™), Mel Im, Mel Ju, SkMel3 (ATCC HTB-69™) were described previously (Jacob et ai., 1998; Heimerl et al., 2007). Primary human hepatocytes (PHH) were isolated and cultured as described elsewhere (Dietrich et al., 2017; Lee et al., 2013). In addition, the human breast cancer cell line MCF-7 (ATCC HTB-22™), the human pancreatic cancer cell line Panc-1 (ATCC CRL-1469™) and the human colon cancer cell lines HT29 (ATCC HTB-38™), Caco-2 (ATCC HTB-37™) and LoVo (ATCC CCL-229™) were used. The murine melanoma cell line B16 (ATCC CRL-6475™) was used for in vitro and in vivo studies. Furthermore, the murine pancreatic cancer cell line KPC and the murine colon cancer cell lines MC38 and CT26 (ATCC CRL-2638™) were used. Cell lines and characteristics are listed in Table 4.

[0287] Table 4. Summary and additional information of human and murine cell lines. BC: Breast cancer. CRC: Colorectal cancer. Hetero: Heterozygous genotype. Homo: Homozygous genotype. MET: Metastatic melanoma cell line. MM: Malignant melanoma. Nd: not determined I missing. PC: Pancreatic cancer. PT: Primary tumor melanoma cell line. Peri-tumor: Tumor-surrounding liver tissue. IR: immunoreactivity

[0288] (score). A.8 Treatment with recombinant proteins, agonists, inhibitors, and antibodies

[0289] As described in the examples, figures and figure legends, recombinant proteins, small molecule agonists and antagonists were used in this study. TGF-0 was purchased from R&D Systems (Minneapolis, USA). The activation of Y5R was conducted using the synthetic high-affinity Y5R-specific-agonist BWX46 ("Y5R-Ago") (CAS-No. 172997-92- 1; Tocris, Wiesbaden, Germany) and recombinant neuropeptide Y ("NPY") (Merck, Darmstadt, Germany). To inhibit Y5R-signaling with high affinity and selectivity over other NPY-receptors, the selective high-affinity small molecule inhibitor CGP71683 ("Y5R-Inh") (CAS-No. 192322-50-2; Tocris) was used. For YlR-inhibition, the specific small molecule inhibitor BIBO 3304 (Tocris) ("YIR-Inh") was used. For Y2R-inhibition, the specific small molecule inhibitor BIIE 0246 (CAS-No. 246146-31-6, Tocris) ("Y2R- Inh") was used. Forskolin (Merck) was used to elevate intracellular cAMP levels(Dietrich et al., 2020). Cinobufagin (MedChem Express, New Jersey, USA) was applied for dose-dependent induction of phospho-CHK2 (Pan etak, 2019). The MEK inhibitor U0126 (Merck) was used to specifically inhibit ERK activation.

[0290] A.9 siRNA- mediated gene knockdown

[0291] Briefly, for in vitro transfection, in each well (six-well plates), 2 x 104cells were seeded. Lipofectamine RNAimax transfection reagent (Life Technologies, Darmstadt, Germany) was applied as described (Dietrich etai., 2017). In order to knock down the expression of Y5R, a functionally verified siRNA pool targeting murine NPY or a functionally verified siRNA pool targeting human NPY (siTOOLs Biotech GmbH, Planegg, Germany; see information in section A.l herein), or individual RNAs targeting NPY (see Example B.ll) were used. The siRNA pools comprised 30 target-specific single siRNAs, allowing for highly efficient gene silencing as compared with the non-targeting control siRNA pool, while off-target effects of individual siRNAs can be reduced to a minimum (Hannus et a!., 2014). RNA and protein were isolated and functional assays were conducted 24 or 48 after transfection.

[0292] A.10 Immunohistochemistry & Immunofluorescence

[0293] Immunohistochemistry (IHC) and immunofluorescence analyses were performed using tissue microarrays consisting of human cancer tissue samples and mouse paraffin- embedded tissue sections as described previously (Dietrich et al., 2017; Dietrich eta / ., 2020). The following primary antibodies were used: anti-alpha-SMA antibody (catalog no. ab5694, Abeam; 1 in 500 dilution), anti-CD3 antibody (catalog no. C7930, Sigma- Aldrich, Saint Louis, Missouri, USA; 1 in 1000 dilution), anti-CD31 antibody (catalog no. DIA-310, Dianova GmbH, Hamburg, Germany; 1 in 50 dilution), anti-CD45 antibody (catalog no. 14-0452, Affymetrix, Santa Clara, California, USA; 1 in 50 dilution), anti- CD4 antibody (catalog no. 14-9766, Affymetrix; 1 in 100 dilution), anti-CD8 antibody (catalog no. 14-0808, Affymetrix; 1 in 50 dilution), anti-cleaved-caspase antibody (Cell Signaling, Frankfurt am Main, Germany; 1 in 50 dilution), anti-phospho-ERK antibody (catalog no. #9101, Cell Signaling; 1 in 100 dilution), anti-Ki-67 / MIB-l antibody (catalog no. M7240, Dako GmbH, Hamburg, Germany; 1 in 50 dilution), anti-Y5R antibody (catalog no. abl33757, Abeam; 1 in 1,000 dilution), anti-NPY antibody (catalog no. ab6173, Abeam; 1 in 500 dilution), anti-NPY antibody (catalog no. HPA044572, Sigma-Aldrich; 1 in 200 dilution), anti-NPY antibody (catalog no. abl0341, Abeam; 1 in 1,000 dilution), anti-NPY antibody (catalog no. BML-NA1233, Enzo Life Sciences, Farmingdale, USA; 1 in 500 dilution), anti-TGFpi antibody (catalog no. DM1047, Acris Antibodies; Herford, Germany; 1 in 100 dilution), anti-phospho-Chk2 antibody (catalog no. #2661, Cell Signaling; 1 in 50 dilution). For each antibody, immunohistochemistry staining was evaluated semi-quantitatively and scores were calculated as described previously (Dietrich eta / ., 2017; Dietrich eta / ., 2020).

[0294] A.11 Western blotting

[0295] As described in detail in previous studies (Dietrich et al., 2017), Western blots and densitometric quantification were performed using the following primary antibodies: anti-p-actin antibody (catalog no. A1978, Sigma-Aldrich; 1 in 5,000 dilution), anti- phospho-ERK antibody (catalog no. #9101, Cell Signaling, Frankfurt am Main, Germany; 1 in 4,000 dilution), anti-ERK antibody (catalog no. #9102, Cell Signaling; 1 in 1,000 dilution), anti-phospho-AKT antibody (catalog no. #9271, Cell Signaling, Frankfurt am Main, Germany; 1 in 2000 dilution), anti-AKT (catalog no. #9272, Cell Signaling, Frankfurt am Main, Germany; 1 in 2000 dilution), anti-Y5R antibody (catalog no. abl33757, Abeam; 1 in 30,000 dilution), anti-phospho-Chk2 antibody (catalog no. #2661, Cell Signaling, Frankfurt am Main, Germany; 1 in 1000 dilution), anti-phospho- p53 antibody (catalog no. #9284, Cell Signaling, Frankfurt am Main, Germany; 1 in 1000 dilution).

[0296] A.12 RNA isolation and reverse transcription

[0297] Using the E.Z.N.A. Micro Elute Total RNA Kit (Omega, Norcross, GA, USA), RNA was isolated as described elsewhere (Dietrich eta / ., 2017). Using the Superscript II Reverse Transcriptase Kit, cDNAs of total RNA fractions were generated (Invitrogen, Groningen, Netherlands). A.13 Analysis of mRNA-expression by quantitative RT-PCR (q RT-PCR)

[0298] As described previously (Dietrich et al., 2017; Dietrich et al., 2020), quantitative RT- PCR was performed using the following primer pairs: 18S rRNA (5'-TCT GTG ATG CCC TTA GAT GTC C-3' and 5'-CCA TCC AAT CGG TAG TAG CG-3'; SEQ ID NOs:293 and 294), CyclinDl (5'-GCC TGT GAT GCT GGG CAC TTC ATC TG-3' and 5'-TTT GGT TCG GCA GCT TGC TAG GTG AC-3'; SEQ ID NOs:295 and 296), Npy (murine) (5'-TGG CCA GAT ACT ACT CCG CT-3' and 5'-GCA GAC TGG TTT CAG GGG AT-3'; SEQ ID NOs:297 and 298), NPY (human) (5'-GCT AGG TAA CAA GCG ACT GGG-3' and 5'-TGG GCT GGA TCG TTT TCC AT-3'; SEQ ID NOs: 299 and 300), Npylr (murine) (5'-CAC CTG CAA CCA CAA TCT GC-3' and 5'-GAC GTC TTG GAC ACA TCC GT-3'; SEQ ID NOs:301 and 302), NPY1R (human) (5'-AAT GCT TCC CCG AGG TG TG-3' and 5'-TGG GGA AGA TCC GCT GGT AT-3'; SEQ ID NOs:303 and 304), Npy2r (murine) (5'-GGA TCT GCA TCA GGC ACA CT-3' and 5'-CGC CTT AGG TAG CAA GCC AT-3'; SEQ ID NOs:305 and 306), NPY2R (human) (5'-CTC CCT CGC CAC CAA AAC TTC-3' and 5'-GTG AAG GTG GGA GGA TCA GA GA-3'; SEQ ID NOs:307 and 308), Npy5r (murine) (5'-GGT ACA GCA AGA AGA CGG CA-3' and 5'- TTC TCG TGA GGG AAC GCT TG-3'; SEQ ID NOs:309 and 310), NPY5R (human) (5'-AAT ACT GCT GCC ACT CGG AA-3' and 5'-GGC CAG ATT GCC TAT GAG GA-3'; SEQ ID NOs:311 and 312), TGF-01 (human) (5'-GAG ATG GCA GGG ACT CTG ATA ACA CC-3' and 5'-AAA GTG CTA GGA TTA CAG GCG TGA GC-3'; SEQ ID NOs:313 and 314), Tgf-01 (murine) (5'-AGG AGA CGG AAT ACA GG GC-3' and 5'-CCA CGT AGT AGA CGA TGG GC3'; SEQ ID NOs:315 and 316), gp70 (murine) (5'-AGC CCC TTG AGA CTA CGG GT-3' and 5'-CGT CAA TGG GAA CCT GTG CG-3'; SEQ ID NOs:317 and 318), aSMA (murine) (5'-CCA GCC ATC TTT CAT TGG GAT-3' and 5'-CCC CTG ACA GGA CGT TGT TA-3'; SEQ ID NOs:319 and 320).

[0299] A.14 Clonogenicity assays

[0300] As described previously (Dietrich et al., 2017), clonogenic assays were performed to analyze stem cell behavior, attachment dependent colony formation and growth of cancer cells.

[0301] Anchorage independent colony formation and growth of cancer cells were analyzed using soft agar colony formation assays as described previously (Borowicz et a / ., 2014). Treatment with specific agonists and antagonists started 24 h after tumor cell seeding. Treatment was repeated after five and nine days. Microscopic analysis of the obtained colonies was conducted 14 days after tumor cell seeding.

[0302] A.15 Analysis of cell migration and invasion

[0303] As described previously (Dietrich et a!., 2017), Boyden chamber assays were used in order to analyze cell migration. Boyden chamber filters were additionally coated with Matrigel to analyze cell invasion.

[0304] A.16 NPY-ELISA

[0305] NPY-ELISA kits (murine and human) (Merck) were applied as described (Dietrich eta / ., 2020), according to the manufacturer's instructions to quantify NPY both in cell lysates and supernatants, as well as in serum samples.

[0306] A.17 cAMP BRET assay

[0307] A bioluminescence resonance energy transfer (BRET) assay was used to analyze cAMP signaling (Dietrich et al., 2020). The biosensor CAMYEL (cAMP sensor using YFP-Epac- RLuc) served for detection of changes in intracellular cAMP concentration upon activation of Y5R30. Transfected cells were treated with 5 pM coelenterazine and intracellular cAMP level was artificially raised using 10 pM forskolin for efficient detection of the BRET signal after Y5R activation.

[0308] A.18 Phospho-Kinase Array

[0309] A commercially available human Phospho-Kinase Array Kit (R&D Systems) was used to simultaneously determine the relative phosphorylation levels of 37 different kinase phosphorylation sites. Human melanoma cells (SkMel28) were treated for 5, 30 or 60 min with 100 nM of the Y5R agonist BWX46 and 300 pg total protein was used. The Assay was performed according to the manufacturer's instructions.

[0310] A.19 Graphical illustration

[0311] The graphical illustrations were drawn applying the GraphPad Prism Software (GraphPad Software, Inc., San Diego, CA, USA) and SPSS (SPSS Statistics 23, IBM Corp., USA). Parts of the figures were drawn by using pictures derived from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https: / / creativecommons.Org / licenses / by / 3.0 / ).

[0312] A.20 Study approval

[0313] In accordance with international guidelines on the ethical use of animals, the animal studies were approved (RUF-55.2.2-2532-2-566-11; RUF-55.2.2-2532-2-2083) by the local government's Committee for Animal Health and Care. Before enrolling in the study, patients signed an informed consent in accordance with the Helsinki Declaration.

[0314] A.21 Single-cell analysis (Single-nuclei RNA-Seq)

[0315] Snap frozen tissue samples were dissociated for generating single nuclei gene expression libraries following customized the protocols from 10X Genomics. Nuclei isolation was performed using the Chromium Nuclei Isolation Kit with RNase Inhibitor (lOxGenomics, PN-1000494) according to the user guide CG000505, Chromium Nuclei Isolation Kit, UG, RevA.

[0316] In brief, 200 pl of lysis buffer was added to the sample and dissociated with pestles provided by the vendor. After adding additional 300 pl of lysis buffer, the tissue was homogenized by pipette- mixing and incubated on ice for lOmin. Afterward, 500 pl of the suspension was loaded onto Nuclei Isolation Columns and centrifuged for 20 s at 16,000 ref, 4°C, followed by vortexing the flow-through for 10s and centrifugation for 3 min at 500 ref, 4°C in a swinging bucket rotor. Supernatant was removed and the pellet was gently resuspended in 500 pl of Debris Removal Buffer before centrifugation for 10 min at 700 ref, 4°C. After removal of the supernatant, the pellet was washed twice with 1ml of Wash Buffer followed by centrifugation for 5 min at 500 ref, 4°C.

[0317] For multiplexing a customized was applied to pool 6 samples per single-nuclei sequencing reaction. For this, nuclei were subsequently incubated after the first wash with 0.2 pM unique CMO Anchor- Barcode and 0.2 pM Co-Anchor and thereafter washed twice with 1 ml of Wash Buffer by centrifugation for 5 min at 500 ref, 4°C. The final nuclei pellet was taken up in 50 pl Resuspension Buffer (lOx Genomics).

[0318] Nuclei were counted using Acridine Orange / Propidium Iodide Stain (Logos, F23001) and an automated dual fluorescence cell counter (Logos, LUNA-FL™). Equal numbers of nuclei were pooled per multiplexed reaction resulting in a total of 20,000 nuclei per individual lOx Genomics Chromium Next GEM Single Cell 3' Kit v3.1 (PN-1000268) reaction. The pooled nuclei were loaded on a Chromium Next GEM Chip G (PN- 1000120) and run on the Chromium iX instrument as instructed by the manufacturer. Single-cell RNA-seq libraries were generated according to the manufacturer's instructions aiming for a maximum cell recovery of 10,000 cells. Library concentrations were quantified with the Qubit 2.0 Fluorometric Quantitation system (Life Technologies, Carlsbad, CA, USA) and the size distribution was assessed using the 2100 Bioanalyzer instrument (Agilent, Santa Clara, CA, USA). Libraries were sequenced by the Biomedical Sequencing Facility at the CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences on a NovaSeq 6000 instrument (Illumina, San Diego, CA, USA).

[0319] After quality control, the raw sequencing reads were aligned to the mouse reference genome GRCm38, by application of CellRanger (10X genomics) in order to obtain feature-barcode matrices. Raw count matrices were analysed using R package Seurat v. 5.1.0 Cells with greater than 10% mitochondrial RNA content, less than 200 genes detected or greater than 5000 genes detected were excluded from analysis. Individual samples were normalized with the standard workflow (NormalizeData, FindVariableFeatures, ScaleData).

[0320] Following the calculation of principal components, samples were integrated with Harmony. Clustering was then performed with the Louvain algorithm (n = 30 PCs, resolution = 1.0). Differentially expressed genes between all clusters were identified using the function "FindAIIMarkers" with a LogFC threshold > 0.25. Annotation with canonical marker genes of major cell types as well as subclusters and further confirmation by Enrichr-based gene set enrichment analysis (Kuleshov et al., 2016) and comparison of the gene signatures with the well-annotated metastatic melanoma cell clusters in both mice and humans defined by the Marine group (Pozniak et al., 2024; Karras etaL, 2022) was performed, hereby melanoma cells and other cell types were clearly defined. Significant differentially expressed genes between the individual groups (control vs NPY-siRNA) were calculated for each annotated cell cluster using the "FindMarkers" function of Seurat and the following thresholds: p-adjust<0.1 and Log2-Fold-Change (L2FC) >0.25.

[0321] B Experiments

[0322] B.l EXAMPLE 1: Reduced liver metastasis in NPY / _mice is uncoupled from steatosis

[0323] Controversial data suggested that the degree of steatosis could impact melanoma and colorectal cancer (CRC) associated liver metastasis. Only mild steatosis was associated with increased liver metastasis, while the presence of NAFLD-related fibrosis was shown to reduce liver metastasis. The inventors hypothesized that NPY-associated metabolic effects might affect mild / moderate fatty change of the liver microenvironment and associated metastasis.

[0324] To test the hypothesis, a preclinical mouse model of hepatic metastases via hemispleen injection of syngeneic melanoma cells was applied. This model produces invasive and proliferative liver metastasis without evidence of significant immune cell infiltration, inflammation or apoptosis, which was confirmed by immunostaining for inflammation and apoptosis markers, such as Caspase-3, CD45. CD31, CD8 and CD4. This was crucial for the purpose of exploring specific steatosis-associated effects, avoiding e.g. cytokine-driven bias. To induce bland (i.e. non-inflammatory and non-fibrotic) steatosis, short-term application of a Western-type diet (WTD) was performed for time period of 6 weeks, using wildtype and NPY’ / _mice. Wildtype mice fed with a normal diet served as controls.

[0325] In wildtype mice, WTD induced weight gain and mild to moderate liver steatosis, without evidence of significant inflammatory activity or fibrosis. Unexpectedly, NPY’ / _mice revealed similar WTD-induced weight gain, steatosis, inflammatory activity and fibrosis, when compared to NPY+ / +mice. Equal effects on steatosis, inflammatory activity and fibrosis were observed in males and females. Moreover, hepatic NPY expression was not affected both by short-term (6 weeks) and chronic (6 months) WTD-induced steatosis. Likewise, in mice with high fat diet-induced steatosis and in humans with NAFLD, hepatic NPY expression was not altered compared with non- steatotic liver tissues in wildtype mice, based on comparison of immunostainings for NPY of aforementioned liver tissue samples. Furthermore, systemic NPY levels were not affected in wildtype mice after short-term WTD-treatment and were even reduced after chronic WTD-administration in mice as well as in humans with NAFLD. Together, the degree of steatosis was not altered in NPY / _mice and conversely, fatty change of the liver did also not affect hepatic NPY expression in mice and humans.

[0326] Consistent with numerous studies, the inventors found that (bland) steatosis per se did not significantly promote liver metastasis. However, WTD-associated liver metastasis was markedly reduced in NPY / _mice as compared with wildtype mice. Both macroscopic metastatic tumor burden as well as the number and size of liver metastasis were strongly reduced in NPY / _mice, as observed in a macroscopic classification of liver metastasis from WT mice fed with CD, WT mice fed with WTD and NPY- / - mice fed with WTD. These data suggested that the NPY-system represents a steatosis-independent driver of liver metastasis.

[0327] B.2 EXAMPLE 2: NPY is expressed by peri-tumorous hepatocytes in the metastatic liver niche

[0328] NPY expression in the metastatic liver niche was characterized in more detail as follows:

[0329] Glycoprotein 70 (gp70) mRNA had previously been shown to be expressed by several murine cancer cell lines including the melanoma cell line B16 and the colorectal cell line CT26 but is absent in normal mouse tissues, inflamed tissues, non-transformed cell lines, and pre-cancerous lesions. Therefore, gp70 is known to represent a biomarker for high-sensitivity quantification of murine tumor burden.

[0330] The inventors found that NPY expression levels and NPY / gp70-ratios, respectively, were specifically induced in metastatic liver tissue samples as compared with non- metastatic liver tissue samples, tumorous spleen tissue samples (i.e., the primary tumor site) and non-tumorous spleen tissue samples, respectively. However, in comprehensive immunofluorescence and immunohistochemical analyses, liver- metastatic melanoma cells were found to not express NPY both in mice and in human melanoma-derived liver metastasis. Also, as compared with human epidermal melanocytes and human nevi tissues, NPY expression was found to be low or absent in melanoma cell lines and tumor tissues, respectively. In contrast, NPY was strongly expressed by peri-metastatic / microenvironment-associated hepatocytes in melanoma- derived liver metastasis in both mice and humans. Likewise, isolated primary murine and human hepatocytes were found to strongly express and secrete NPY protein, while NPY secretion was low in hepatic stellate cells and melanoma cell lines.

[0331] Beyond melanoma, low / absent NPY expression by metastatic cancer cells and strong induction of NPY in peri-metastatic hepatocytes was confirmed applying additional mouse models of colorectal cancer (CRC) cell- and organoid-derived liver metastasis as well as in liver metastases derived from CRC patients and in further types of human cancers including pancreatic cancer (PC) and breast cancer (BC). Immunofluorescence imaging of these samples showed strong signal for NPY in peri-metastatic area, compared to reduced NPY signal in are of metastases. In summary, NPY was found to be strongly induced and exclusively expressed by hepatocytes in the metastatic liver microenvironment.

[0332] B.3 EXAMPLE 3: TGF-p induces peri-metastatic NPY expression

[0333] NPY expression in the liver microenvironment was specifically expressed by peri- tumorous hepatocytes in close proximity to the metastatic invasion front. As shown recently by the inventors, NPY expression can be induced by hepatic transforming growth factor-beta 1 (TGF01) (Dietrich et al., 2020). Therefore, metastasis-related microenvironment activation (including TGF0 induction) might trigger close-by NPY expression.

[0334] TGF0 mRNA expression was enhanced in metastatic as compared with non-metastatic liver tissue samples. Likewise, TGF0 and NPY mRNA expression correlated significantly in liver metastatic tissues. Consistent with microenvironment-activation and associated TGF0 induction, also metastasis-related aSMA induction (Tsilimigras etak, 2021; Van den Eynden etal., 2013) and a significant correlation of TGF0 and aSMA expression in metastatic liver tissue samples was confirmed in the inventors' mouse model. In humans, immunohistochemistry analysis applying a tissue micro array consisting of diverse types of liver metastasis including melanoma and CRC revealed that perimetastatic TGF0 expression patterns strongly correlated with NPY expression. Application of hepatoblast-derived liver organoids (Dietrich et al., 2020) as well as primary human hepatocytes (PHH) confirmed that recombinant TGF0 is sufficient to induce NPY expression. It can be concluded that an activated liver microenvironment and TGF0 promote peri-metastatic NPY expression.

[0335] B.4 EXAMPLE 4: NPY promotes liver colonization and chemotactic cancer cell migration

[0336] Next, the inventors aimed at exploring the functional effects of NPY on liver metastasis. The initial data suggested that NPY might drive metastatic liver colonization independent from fatty change of the liver microenvironment. To confirm this, the described model of liver metastasis was performed applying NPY / _and wildtype mice fed with a normal diet. Also, in these non-steatotic conditions, liver metastasis was strongly reduced in NPY / _mice.

[0337] In contrast to the observed induction of NPY in peri-metastatic liver tissues, systemic NPY levels were not altered in mice with liver metastasis as compared with non- metastatic controls. Likewise, patients with melanoma-derived liver metastases did not reveal enhanced systemic NPY levels compared with patients with "non-liver" metastasis and patients without distant metastasis or a control population, respectively. Strong NPY expression in peri-metastatic liver tissues and no concomitant induction of systemic NPY-levels in liver-metastatic mice and humans suggested a potential liver-directed chemogradient of NPY as described for the CXCL12-CXCR4 axis (Mortezaee, 2021). Mechanistically, recombinant NPY induced dose-dependent chemotactic migration of the inventors' metastatic melanoma mouse model cell line in vitro. Moreover, murine liver lysates revealed strong chemoattractive effects on these metastatic melanoma cells, while significantly reduced effects were observed applying liver lysates derived from NPY / _mice, which could be rescued by addition of recombinant NPY. In the following, comprehensive experiments were performed to confirm that NPY is sufficient to induce dose-dependent chemotactic migration and invasion applying diverse human and murine melanoma, colorectal cancer, pancreatic cancer and breast cancer cell lines. Taken together, hepatocyte-derived NPY was identified to represent a major chemoattractive ligand strongly promoting liver colonization, chemotactic migration and invasion in vivo in vitro.

[0338] B.5 EXAMPLE 5: Liver metastatic cancer cells strongly express NPY-5- receptor

[0339] Next, the inventors aimed at determining the responsible receptor mediating the observed metastasis-promoting effects of niche-derived NPY.

[0340] In the metastatic mouse model cell line (B16), the NPY-5-receptor (Y5R) was identified to be strongly upregulated as compared with the benign murine melanocyte cell line Melan-a. Likewise, Y5R expression was significantly upregulated in metastatic tissues compared with non-metastatic tissues in the liver of this mouse model. Immunohistochemistry and immunofluorescence analyses revealed that marked Y5R expression was preserved by liver-metastatic B16 cells in vivo. Strong Y5R expression in tumor tissues was confirmed applying a further melanoma mouse model (Tg(Grml)). Analysis of human melanoma cell lines and benign melanocytes (NHEM) confirmed strong expression of Y5R mRNA and protein levels, respectively, in cancer cells in vitro. In vivo, strong Y5R expression also remained preserved in human melanoma cells applying a xenograft model. In melanoma patient-derived samples, Y5R was upregulated in primary and metastatic tumor tissues as compared with nevi tissues. In metastatic liver tissue samples derived from melanoma patients, Y5R was strongly expressed in the majority of patients.

[0341] Next to melanoma, Y5R mRNA and protein expression, respectively, was detected in both human and murine colorectal (CRC), pancreatic cancer (PC) and breast cancer (BC) cell lines. Strong Y5R expression was also observed in CRC tissue samples as well as in two different models of CRC-derived liver metastasis (hemispleen injection model of CRC-derived liver metastasis; orthotopic CRC-organoid-derived liver metastasis model), which confirmed marked expression of Y5R by liver-metastatic cancer cells in vivo. Applying the tissue micro array comprising diverse patient-derived liver metastasis, strong expression of Y5R by liver metastatic cancer cells was detected in CRC as well as in further types of cancer including PC and BC.

[0342] In line with these data, analysis of a further tissue micro array containing primary tumor tissues revealed that the emergence of liver metastasis is correlated with stronger Y5R expression as compared with non-liver-metastatic melanoma patients. Moreover, uveal and mucosal melanoma tissues which represent aggressive, high-risk subtypes for the emergence of liver metastasis were associated with stronger Y5R expression. Likewise, the emergence of liver metastasis was associated with stronger Y5R expression in CRC patient tissues as compared with non-metastatic and non-liver- metastatic-cohorts, respectively. Similar results were confirmed analyzing the complete cohort (n=l,014) comprising of melanoma (n=252), CRC (n=351), PC (n=176) and BC (n=235) tissues. Moreover, high Y5R immunohistochemistry scores correlated with a poor overall survival of CRC patients. These data were confirmed applying an additional TCGA (The Cancer Genome Atlas) derived patient dataset. It was concluded that Y5R is strongly expressed by liver-metastatic cancer cells and appeared to be a top candidate NPY-receptor potentially mediating NPY-induced liver colonization.

[0343] B.6 EXAMPLE 6: Y5R drives NPY-induced liver colonization

[0344] Functionally, small-molecule mediated specific inhibition of Y5R (Y5Ri) antagonized NPY-induced chemotactic migration in human melanoma cells (Figure 1A and IB). Conversely, a specific Y5R-agonist (Dietrich et al., 2020) significantly induced chemotactic migration in the same assay. Moreover, chemotactic migration induced by either hepatocyte-derived, NPY-containing supernatants or by liver lysates from wildtype mice, respectively, was significantly reduced by concomitant Y5R-inhibition. Reduction of chemotactic migration was also confirmed applying si-RNA-mediated inhibition of Y5R. Similar effects demonstrating Y5R-mediated migration were confirmed applying multiple human and murine melanoma, CRC, PC and BC cell lines as well as CRC organoids derived from a liver metastasis model.

[0345] Next to migration and invasion, efficient liver metastasis requires sufficient colony formation capacities of cancer cells (Tsilimigras et al., 2021; Van den Eynden et al., 2013). Y5Ri strongly reduced clonogenicity of human melanoma cells applying two- and three-dimensional colony formation assays (Figure 2). These effects were confirmed applying CRC, PC and BC cell lines. In vivo, a therapeutic approach applying specific small molecule-mediated Y5Ri significantly reduced liver metastasis (Figures 3-5). Y5Ri did not affect liver transaminases in mice and only slightly, however not significantly, enhanced cleaved caspase 3 expression in liver metastatic cells. In contrast, Ki67 expression in liver metastasis was significantly reduced in Y5Ri-treated mice as compared with control-treated mice. Likewise, metastatic CRC organoids confirmed significantly reduced Ki67 expression after Y5Ri compared with controls. Immunohistochemical analysis of human liver metastatic tissues confirmed that high Y5R expression was correlated with enhanced Ki67 expression.

[0346] In contrast, immunohistochemical staining of CD3, CD4 and CD8 of liver metastatic tissues applying both Y5Ri-treated mice and NPY / _(as compared with the respective controls) revealed no evidence for NPY-Y5R-signaling associated effects on immune cell infiltration / inflammation. Taken together, Y5R was found to mediate NPY-induced chemotactic migration and invasion. Furthermore, Y5Ri strongly reduced colony formation, proliferation and liver colonization both in vitro and in vivo.

[0347] B.7 EXAMPLE 7: Y5R promotes migration and clonogenicity via cAMP inhibition, ERK-activation and CHK2-dephosphorylation

[0348] Next, downstream signaling of NPY-Y5R crosstalk was analyzed. NPY potently antagonizes cAMP signaling via activation of Gi-proteins (Dietrich et al., 2020). Bioluminescence resonance energy transfer (BRET) analysis revealed significant inhibition of forskolin-induced cAMP activation in melanoma cells by NPY and Y5R- agonist treatment. Functionally, NPY-induced enhanced chemotactic migration was restored by co-treatment applying the cAMP inductor forskolin. In line with the inventors' data, cAMP activation was confirmed to reduce migration in diverse types of (cancer) cells (Chen etal., 2008). Next to cAMP inactivation, ERK activation is a strong inducer of cell motility in cancer (Vial et al., 2003). Likewise, the inventors' mouse model of liver metastasis revealed marked phospho-ERK (pERK) expression with particularly prominent staining of the invading tumor edges in close proximity to perimetastatic, NPY-expressing hepatocytes. In contrast, pERK expression in liver metastasis was strongly reduced in NPY / _compared with wildtype mice both in steatotic and non-steatotic conditions. Mechanistically, treatment of murine metastatic melanoma cells that were used in the inventors' mouse model with both NPY as well as the specific Y5R agonist was sufficient to significantly induce ERK-activation. These effects were confirmed applying human melanoma cell lines as well as human CRC cell lines and organoids. Applying the tissue micro array comprising diverse types of patient-derived liver metastasis, pERK expression was found to significantly correlate with Y5R expression. Moreover, marked correlation of Y5R and pERK expression in cancer cells expression was confirmed applying the primary tumor tissue micro array comprising large cohorts of melanoma, CRC, PC and BC patients. Functionally, Y5R- agonist-induced enhanced chemotactic migration was restored by co-treatment applying small-molecule mediated ERK inhibition. Together, NPY-Y5R crosstalk appeared to promote chemotactic migration and invasion of metastatic cancer cells via cAMP inactivation and ERK induction.

[0349] Next to migration / invasion, NPY-Y5R was associated with expression / proliferation of Ki67 / Ki67-positive cells, as immunostainings showed reduction of Ki67-positive cells upon inhibition of Y5R. In line with this, ERK inhibition was found to reduce proliferation in liver metastatic cancer cells and pERK strongly correlated with Ki67 expression in liver metastasis in humans, while the cAMP inductor forskolin revealed no effects on proliferation.

[0350] Beyond migration / invasion and proliferation, NPY-Y5R crosstalk strongly promoted colony formation (Figure 2). However, cAMP induction did not affect clonogenicity in melanoma and CRC cells, and ERK inhibition only slightly reduced clonogenicity when applying high doses of the ERK inhibitor. The inventors hypothesized that NPY-Y5R- crosstalk might affect further, yet unknown downstream effector(s) promoting clonogenicity in liver metastatic cancer cells. Subsequent multi-phospho-kinase array analysis revealed that Y5R-agonist treatment induced time-dependent dephosphorylation of checkpoint kinase 2 (CHK2) at Thr68 in human metastatic melanoma cells, as was shown by reduced phosphorylated CHK2 levels in phospho- kinase-array-based expression analysis after stimulation with Y5R-Ago for 5, 30 and 60 minutes. Activated CHK2 (pCHK2) is part of the DNA damage checkpoint pathway and represents a potent tumor suppressor. In melanoma and CRC cells, CHK2 can induce G2 / M cell cycle arrest (Pan et al., 2019). In breast cancer, inactivation or loss of CHK2 was shown to promote a metastatic phenotype. Y5R-agonist treatment confirmed dose-dependent reduction of pCHK2 levels in metastatic melanoma cells in vitro. Conversely, Y5R-inhibition induced pCHK2 in melanoma cells. These data were confirmed applying CRC organoids. In human liver metastasis, the tumor suppressor pCHK2 was poorly expressed in the majority of samples, and pCHK2 levels were inversely correlated with Y5R immunohistochemistry scores. Moreover, in the mouse models, significant induction of the tumor suppressor pCHK2 was found in NPY’ / _compared with wildtype mice as well as in Y5Ri-treated mice compared with controls (Figure 6A and 6B), confirming that pCHK2 is a NPY-Y5R target in vivo. Functionally, chemical induction of pCHK2 applying Cinobufagin (Cino) strongly reduced clonogenicity in metastatic melanoma cells. Marked reduction of clonogenic capacities of melanoma cells by Cino-mediated pCHK2 induction had been confirmed in previous studies (Pan et al., 2019). It was found that co-treatment applying Cino and high doses of the Y5R agonist was sufficient to rescue Cino-mediated inhibition of clonogenicity in metastatic melanoma cells. Together, NPY-Y5R crosstalk modulates cAMP-, ERK- and CHK2-signaling to promote liver metastasis.

[0351] B.8 EXAMPLE 8: Role of further NPY-receptors in cancer cell migration and clonogenicity

[0352] Besides Y5R, NPY can activate NPY-1 receptor (Y1R) and NPY-2 receptor (Y2R). In contrast to Y5R, Y1R and Y2R expression levels were not significantly overexpressed in metastatic as compared with non-metastatic liver tissue samples in the inventors' mouse model, which was established by comparing Y1R and Y2R mRNA levels in liver and liver metastases. Likewise, Y1R was not overexpressed in human melanoma cell lines as compared with human epidermal melanocytes, while Y2R was only slightly upregulated in melanoma cells. In metastatic melanoma cells, NPY-mediated enhanced chemotactic migration was not altered by co-treatment with specific small molecule mediated Y1R inhibition (YIRi) or Y2R inhibition (Y2Ri). Furthermore, clonogenicity of metastatic human melanoma cells was not affected by YIRi and Y2Ri, as there was no reduction in number of clones with increasing concentration of Y1R or Y2R inhibitors in clonogenicity assay. Next to human and murine melanoma cells, Y1R and Y2R was found to be expressed also by human and murine CRC, PC and BC cell lines. According to the findings in melanoma, YIRi and Y2Ri did not affect NPY-mediated chemotactic migration and clonogenicity in CRC cells (Figures 7A and 7B). Moreover, both YIRi and Y2Ri did not affect clonogenicity in PC and BC cell lines. Likewise, NPY-mediated chemotactic migration was not affected by Y2Ri in PC and BC cell lines. However, YIRi was sufficient to reduce NPY-mediated chemotactic migration in PC and BC cell lines.

[0353] In their mouse model, the inventors found that Y5Ri did not affect Y2R and Y5R expression levels in liver metastatic tissues, as there was no reduction in Y2R and Y5R mRNAs levels. However, NPY and Y1R expression in liver metastatic tissues as well as systemic NPY levels were upregulated upon Y5Ri treatment, as the levels of NPY and Y1R significantly increased. Moreover, while low expression of Y1R was confirmed in most human liver metastatic tissues derived from CRC, some CRC-derived liver metastasis revealed both strong Y5R and Y1R expression or even high Y1R expression, while Y5R expression was low. Likewise, analysis of TCGA-derived data revealed that next to Y5R, also Y1R (but not Y2R and NPY) were upregulated in in high-risk as compared with low-risk (based on prognostic index) CRC patient groups which was associated with a poor disease specific survival. Taken together, these findings revealed that, in contrast to Y5R, Y1R and Y2R appear not as strong promoters of migration and clonogenicity in cancer cells.

[0354] B.9 EXAMPLE 9: RNA interference targeting the metastatic liver niche represents an effective therapeutic strategy

[0355] In contrast to Y5Ri-mediated induction of NPY and Y1R expression in metastatic liver tissue samples, the expression of NPY-receptors (Y1R, Y2R, Y5R) was not affected in absence of the ligand, i.e. in metastatic liver tissue samples of NPY / _mice. Since NPY is expressed predominantly by hepatocytes in close proximity of metastatic cancer cells, specific targeting of the metastatic niche rather than targeting Y5R (on cancer cells) appeared to be a particularly attractive therapeutic approach and could avoid potential resistance mechanisms. Lipid Nanoparticles (LNPs) are a promising drug delivery vehicle for clinical siRNA delivery (Evers etal., 2022). The inventors designed lipid nanoparticles (LNPs) covering an siRNA pool consisting of 30 different specific siRNAs targeting murine NPY (LNP-siNPY) for a hepatocyte-specific in vivo knockdown (Figure 7). This therapeutic approach was sufficient to significantly reduce NPY mRNA and protein levels in liver tissues (Figures 8A and 8B). In contrast, NPY expression in "non-liver" tissues as well as systemic NPY-levels were not affected. Regarding NPY- mediated chemotactic migration, specific and significant reduction of NPY expression in liver tissues and concomitantly unaffected NPY serum levels upon LNP-siNPY treatment (Figure 9A) resulted in a strong reduction of NPY-liver-serum-ratios (Figure 9B). In contrast, body weights, spleen weights (i.e. the primary tumor site) and toxicity markers (liver transaminases and lactate dehydrogenase) were not affected upon LNP- siNPY treatment (Figure 10).

[0356] However, as compared with control treatment, LNPs bearing siNPY strongly reduced liver weights and macroscopic liver metastasis (Figure 11). Furthermore, immunohistochemistry analysis revealed that both the number and size of liver metastasis as well as Ki67-expression levels were markedly reduced in LNP-siNPY- treated mice (Figure 12 and Figure 13). Regarding NPY-Y5R- mediated downstream effectors as identified in this study, significantly reduced pERK levels as well as enhanced pCHK2 expression in liver metastasis of LNP-siNPY-treated mice confirmed the inventors' data (Figure 14). Moreover, strongly resembling the inventors' findings in NPY / _mice, targeting the ligand NPY did not induce any "compensatory" upregulation of NPY-receptors in metastatic liver tissue samples, as no changes in mRNA levels of Y1R, Y2R and Y5R in LNP-siNPY-treated mice were detected compared to the control. In summary, these findings show a novel cross-talk between niche- derived NPY and disseminated cancer cells and support a usefulness of agents targeting this cross-talk, such as RNA interference targeting niche-associated hepatocytes, in medical treatments to control liver metastasis.

[0357] B.1O EXAMPLE 10: RNA interference targeting human NPY in primary human hepatocytes

[0358] NPY mRNA expression was analyzed in primary human hepatocytes after transfection with a siRNA pool against human NPY ("siNPY") or a negative control siRNA pool C'siCtr") for 24 hours. The results confirmed that RNA interference is sufficient to significantly reduce NPY expression levels also in human primary hepatocytes (Figure 15). Moreover, additional analysis of LDH (toxicity marker) in cell supernatants after transfection of the siRNA pool against NPY as well as microscopic morphology analysis did not reveal any signs of toxicity induced by siNPY compared with the control siCTr treatment (not shown).

[0359] B.ll EXAMPLE 11: In vitro testing and validation of 22 exemplary siRNAs targeting NPY

[0360] For the testing of individual siRNAs, 22 siRNAs were chosen among the 232 siRNAs targeting human NPY and having the antisense strand sequences of SEQ ID NOs:l- 232 (and a sense strand reverse complement thereto) based the following in siiico criteria: targeting of all NPY transcripts in the NCBI gene database (www.ncbi.nlm.nih.gov / gene); absence of mismatch to any human non-NPY transcript in the NCBI gene database;

[0361] • GC-content of not more than 42%;

[0362] • delta-delta G of at least -1;

[0363] • targeting of the CDS or the 3' UTR of the human NPY transcript;

[0364] • targeting of at least one mammalian model organism;

[0365] • no overlap with SNPs;

[0366] • no immunogenic sequence motifs and also the following additional criteria regarding features characteristic of siRNA effectiveness based on previous clinical precedents:

[0367] • preference for U at position 1 of sequence hybridized to target mRNA;

[0368] • preference against C at position 7;

[0369] • preference for A at position 10;

[0370] • preference against G at position 14;

[0371] • preference for C at position 19.

[0372] Minimal off-target transcript hybridization, as predicted by bioinformatics analysis, was applied as a further criterion. Specifically, siRNAs that perfectly matched off-target transcripts between antisense positions 2 and 17 were excluded from selection.

[0373] The sequences of 22 pre-selected siRNA are listed in Table 5, together with the SEQ ID NO setting forth the nucleotide sequence of their respective antisense strands.

[0374] As described below, these 22 siRNAs were then evaluated by in siiico and in vitro testing. For further evaluation and pre-clinical studies, two siRNAs targeting both human and mouse NPY mRNA were chosen to facilitate subsequent testing in a mouse model.

[0375] Table 5. Antisense sequence of 22 pre-selected siRNAs with corresponding SEQ ID Nos. The sequences are represented according to WIPO Standard ST. 26 format.

[0376] * This is the number identifying the respective siRNA having the here listed antisense strand in the Figures and the description.

[0377] For in vitro testing and for each of siRNA #1 to #22 individually, human neuroblastoma cells (SK-N-SH, ATCC#: HTB-11) were transfected with 1 nM or 3 nM of the respective siRNA (gel-purified siRNA) as described in Methods section A.9 above. NPY expression was measured 24h post-transfection by RNA isolation, reverse transcription and qRT- PCR as described in the Methods sections A.12 and A.13 above.

[0378] Mock-transfected cells were used as controls (depicted in Fig. 16 as 100% of NPY mRNA expression relative to Beta-Actin). Negative controls included the pool of 30 non-targeting siRNA described in section A.l above ('Negl20') and a siRNA targeting an unrelated gene ('GAPDH-C19'). The pool of 30 siRNAs targeting murine NPY described in section A.l above ('NPY-cl') served as a positive control. Additionally, a toxic control siRNA pool ('ToxPos') was included.

[0379] As shown in Figure 16, all siRNAs, except for siRNA #3 and #13, achieved a knockdown efficacy exceeding 50% of NPY mRNA expression. The siRNAs with the highest knockdown efficiency in this assay were #1, 2, 5, 16, 17, 18, 19, and 21. The inventors consider that an siRNA having a relatively low knockdown effect in this assay is not necessarily less effective in vivo because some siRNAs might take effect slower than others but have a longer duration of action. This may be ascertained by the skilled person using, for example, an in vitro assay as described herein, where NPY-expressing cells such as human neuroblastoma cells (SK-N-SH, ATCC#: HTB-11) or murine hepatocytes (which may be isolated using a modified 2-step EGTA / collagenase perfusion procedure as described in Steiling et al., 2004) are treated with the siRNA (see Methods section A.9 and Example B.13), RNA is isolated and mRNA expression determined using quantitative RT-PCT (see Methods sections A.12 and A.13).

[0380] Some siRNAs described previously (directed against another target) have been reported to induce the cellular interferon response, the major and most important toxic mechanism of siRNAs (Reynolds et al., 2006). 2'-5'-oligoadenylate synthetase 2 is an enzyme that in humans is encoded by the 0AS2 gene. 2'-5'-oligoadenylate synthetase 2 is a member of the 2-5A synthetase family, essential proteins involved in the innate immune response to viral infection. Transcription of the 0AS2 gene is induced by interferons. The siRNAs #1-22 were therefore tested in an in vitro assay, at transfection concentrations of 1 nM and 3 nM siRNA, and detecting 0AS2 mRNA levels using qRT-PCR. As a control in this assay, a toxic control siRNA pool ('ToxPos') was included which has been previously validated to strongly induce expression of the 0AS2 gene, making it a reliable positive control for interferon induction. The results of this assay (data not shown) demonstrate that the ToxPos siRNA pool strongly induced 0AS2 mRNA levels (normalized relative to Beta-Actin). In contrast, none of the tested siRNAs targeting NPY mRNA (#1-22) triggered 0AS2 expression at both transfection concentrations (1 nM and 3 nM siRNA). This result confirms that all of the tested siRNAs #1-22 targeting NPY mRNA do not induce a siRNA-mediated interferon response.

[0381] As the next step, the inventors evaluated the potential gene knockdown viability effects of the tested siRNAs using DepMap scores (https: / / depmap.org / portal / ). The DepMap score represents (next to interferon induction / 0AS2 expression) a further relative measure of cell viability. Lower DepMap scores indicate reduced viability compared to the "Random" reference. The "Random" reference represents the distribution of the entire DepMap dataset (Tsherniak et al., 2017). The inventors determined DepMap scores for the seed sequences of each siRNA in a number of hepatocyte-derived cell lines (>10). For each of the eight analyzed siRNAs (siRNAs #1, 2, 5, 16, 17, 18, 19, 21) no evidence of markedly reduced DepMap scores compared to the "Random" reference was detected. In particular siRNAs #1, #18, and #21 even demonstrated slightly higher DepMap scores than "Random," indicating particularly low toxicity for these siRNA sequences.

[0382] B.12 EXAMPLE 12: Single cell RNA seq analysis showed specificity of NPY RNAi targeting is specific to melanoma and hepatocyte cells

[0383] Next, the inventors assessed whether NPY targeting (either using LNP-siNPY or siNPY) would affect gene expression in different cell types using single-cell analysis as described in methods section A.21 above.

[0384] Specifically, single-cell RNA seq analysis of metastatic liver tissues revealed that, in the various analyzed cell types, NPYi (LNP-siNPY or siNPY) affected gene expression basically exclusively in melanoma cells (which are known to express NPY) and hepatocytes (i.e., the direct target cells of LNPs), while gene expression in the other cell types was unaffected (see Table 6).

[0385] In hepatocytes, LNP-mediated inhibition of NPY by siRNAs did not affect the expression of specific toxicity-associated genes. However, it appeared to negatively regulate cholesterol metabolism, as identified using Enrichr-based gene set enrichment analysis (Kuleshov et al., 2016) of the up- and downregulated differentially expressed genes (DEGs) between control- and siNPY-treated hepatocytes (239 DEGs in Table 6; results for the respective specific genes not shown). This suggests potential beneficial side effects, including upregulation of biotransformation-related pathways and glycolysis. These findings support the inventor's approach of targeting the metastatic liver niche rather than cancer cells, demonstrating its feasibility with minimal potential side effects. Notably, the inhibition of NPY expression by si NPY does not induce toxic effects in hepatocytes. Additionally, the observed downregulation of cholesterol metabolism indicates possible beneficial effects on hypercholesterolemia, aligning with findings reported by Chen et al., 2020.

[0386] Table 6. Results of single RNA-seq analysis showing significant differential gene expression upon NPY siRNA knockdown only in melanoma and hepatocyte cell lines. Differentially expressed genes (DEG) between control- and siNPY-treatment are showed for each cell type using the following thresholds: p-adjust<0.1 and Log2-Fold- Change (L2FC) >0.25.

[0387] B.13 Design and in vitro testing of modified siRNA targeting

[0388] For further analyses in vitro and in vivo, the inventors then designed and synthesized highly modified therapeutic siRNAs based on siRNAs #1 and #18, which had been chosen for further experiments due to their additional targeting of mouse NPY (necessary for the animal model) and in view of the preceding analyses. The siRNA sequences of #1 and #18 were used to produce four highly modified siRNA drug candidates. Two different modification patterns were applied for each of #1 and #18 resulting in the variants #1.1 and #1.2 (based on #1) as well as #18.1 and #18.2 (based on #18). These siRNAs are also referred herein as SINPY with corresponding number. The structures of siRNAs #1.1, #1.2, #18.1 and #18.2 are depicted in Figure 17. Based on the information in Figure 17, the sequence protocol entries for the sense and antisense strands of siRNA #1.1 (SEQ ID NOs:610 and 614), #1.2 (SEQ ID NOs:611 and 615), #18.1 (SEQ ID NOs:612 and 616) and #18.2 (SEQ ID NOs:613- 617) have been prepared.

[0389] The introduced siRNA modifications, including 2'-O-methyl (2'-O-Me), 2'-fluoro (2'-F), and DNA nucleotide incorporation, are widely known and well-established in the art of siRNA technology (Friedrich et al., 2022).

[0390] To enable targeted liver delivery, the 3'-end of the sense strand of each of siRNAs #1.1, #1.2, #18.1 and #18.2 carries the triantennary GalNAc asialoglycoprotein receptor (ASGPR) ligand N-acetylgalactosamine-L96 (GalNAc-L96). GalNAc-L96 binds to the receptor ASGPR, which is expressed on hepatocytes, facilitating rapid endocytosis (Springer et al., 2018). This approach, i.e. conjugation with a GalNAc ligand of ASGPR for liver targeting, is also well-established in the art (Springer et al., 2018).

[0391] Before conducting in vitro and in vivo tests, each single-stranded oligonucleotide underwent quality control to ensure that it met high-quality standards before being used for duplex formation. Additionally, ESI mass analysis was routinely performed on all duplexes to confirm duplex formation and verify sequence integrity.

[0392] Next, the inventors tested the modified siRNAs in isolated murine hepatocytes. Primary murine hepatocytes were isolated using a modified 2-step EGTA / collagenase perfusion procedure as described previously (Steiling et al., 2004). After treatment of cultivated hepatocytes applying Control (isotonic PBS buffer) or siNPY (0.01, 0.1 and 1 nmol in isotonic PBS buffer) or LNP containing a negative Control-si RNA-Pool or a siRNA-Pool targeting murine NPY, RNA was isolated after 24 hours (Figure 18) or 48 hours (Figure 19) and reverse transcribed as described in Methods section A.12 above. Subsequently, qRT-PCR analysis was performed as described in Methods section A.13 above.

[0393] As shown in Figures 18 and 19, all four modified siRNAs successfully and very effectively reduced NPY mRNA 48 hours post-transfection, with siRNA #18.2 exhibiting the most significant effect. siRNA #18.2 significantly decreased NPY mRNA even at the lowest concentration of 0.01 nmol already 24 hours after transfection. Consequently, siRNA #18.2 was chosen for further therapeutic preclinical proof-of-principle studies in mice. Nevertheless, the inventors consider that an siRNA having a relatively low knockdown effect in this assay is not necessarily less effective in vivo because some siRNAs might take effect slower than others but have a longer duration of action, which may provide relevant benefits for the therapeutic application (lower injection frequency). It is conceivable that it may take about 2 to 4 weeks after administration of the siRNA until maximum knockdown efficiency is reached.

[0394] B.14 In vivo testing of modified siRNA targeting NPY in mice

[0395] In in vivo experiments, BL / 6 mice were treated using a single subcutaneous injection of PBS-buffered siRNAs targeting NPY mRNA (#1.1, #1.2, #18.1, #18.2). Unless indicated otherwise, each siRNA was administered at 5 mg / kg of mice bodyweight. A single subcutaneous injection of PBS was used as placebo control. 14 days posttreatment, mice were sacrificed, and hepatocytes were isolated as described in Example B.13. The isolated hepatocytes were cultured for 24 hours, after which qRT- PCR analysis was performed to quantify NPY mRNA expression levels. As depicted in Figure 20, NPY mRNA levels were drastically reduced in mice treated with each individual modified siRNA (#1.1, #1.2, #18.1, #18.2), with knockdown efficacy of >95% as compared to NPY mRNA levels in mice treated with the placebo control. Thus, all four modified siRNA achieved a very remarkable and durable NPY knockdown efficacy in vivo.

[0396] Furthermore, siRNA #18.2 was selected for additional experiments using different doses of this siRNA. Specifically, BL / 6 mice were treated using a single subcutaneous injection of PBS-buffered siRNA #18.2 applying 2, 5 or 10 mg / kg bodyweight, respectively, while placebo control was a single subcutaneous injection of PBS. After 7 days, mice were sacrificed, and hepatocytes were isolated as described in Example B.13. After 24 hours of cultivation (after isolation), qRT-PCR analysis was performed as described above to quantify NPY mRNA expression levels. As shown in Figure 21, siRNA #18.2 demonstrated a dose-dependent and very strong NPY mRNA knockdown efficacy, achieving efficacy of >80% with 2 mg / kg dose and more than 98% efficacy with 5 and 10 mg / kg.

[0397] B.15 In vivo testing of modified siRNA targeting NPY in mouse model of liver metastasis

[0398] Based on the previous findings, the modified siRNA #18.2 (hereafter referred to as SINPY) was chosen as a drug candidate for further therapeutic preclinical proof-of- concept studies in a mouse model of metastasis of (primary) pancreas cancer.

[0399] An orthotopic, syngeneic pancreatic cancer transplantation model was applied using KPC661 cells with an epithelial phenotype and spontaneous metastasis to the liver and lungs (Pereira et al., 2024; Ischenko et al., 2021; Erstad et al., 2018). In vitro, these cells (KPC661) were shown to express Y5R and migrate towards NPY-induced chemogradient according to inventors' earlier experiments (data not shown). Thus, this model allows formation of a pancreatic cancer-specific tumor-microenvironment, in line with tumor formation and metastasis of the genetic KPC model properly recapitulating disease progression in patients (Falcomata et al., 2022; Ischenko et al., 2021).

[0400] After orthotopic implantation of pancreatic cancer cells, mice were randomized into a Placebo (receiving vehicle only) (n = 28) and a SINPY (n = 28) arm. Treatment was given as one single s.c. injection per week (5 mg / kg), starting one week after initiation of the model. Mice were sacrificed after 21 days and analyzed for primary tumor, liver and lung metastases.

[0401] Application of SINPY did not reveal any clinically identifiable side effects compared to placebo treatment. Body weights, lung weights, liver weights and behavior were equal among both groups.

[0402] Pancreas organs and the respective pancreatic primary tumors were not affected by SINPY treatment (Figure 22). However, macroscopic liver metastastic burden (number of animals having or lacking macroscopic metastases in the liver) was strongly reduced in the SINPY group (Figure 23). Additionally, the number of liver metastasis per animal was reduced (Figure 24) and metastasis-free survival (MFS) was significantly enhanced (Figure 25) in the SINPY group compared to Placebo.

[0403] Together, by applying a clinically relevant mouse model of liver metastasis, these results provide further pre-clinical evidence for the safety and efficacy of SINPY administered by s.c. injection. Further toxicity studies as recommended for the investigational new drug application (IND) and subsequent investigator-initiated trial (ITT) have been initiated by the inventors.

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Claims

Claims1. An inhibitor of NPY-5 receptor (Y5R) activation by hepatocyte-derived neuropeptide Y (NPY) for use in the prevention or reduction of metastasis to the liver in a subject, wherein said inhibitor (NPY / Y5R inhibitor) is selected from:(i) at least one short interference RNA (siRNA) against NPY,(ii) at least one Y5R inhibitor which is a small molecule,(iii) at least one anti-NPY antibody, and(iv) at least one anti-Y5R antibody, and(v) at least one antisense oligonucleotide (ASO) against NPY.

2. The NPY / Y5R inhibitor for use according to claim 1, wherein the NPY / Y5R inhibitor is at least one siRNA against NPY and is for use in inhibiting NPY expression in hepatocytes in the subject, thereby preventing or reducing metastasis to the liver of the subject.

3. The NPY / Y5R inhibitor for use according to any of claims 1-2, wherein the NPY / Y5R inhibitor of (i) is one siRNA against NPY, or a combination of two or more different siRNAs against NPY, particularly 2-10 different siRNAs against NPY.

4. The NPY / Y5R inhibitor for use according to any of claims 1-3, wherein the siRNA(s) comprise(s) at least one of the following chemical modifications:(i.1.1) at least one phosphonate modification selected from methylphosphonate (MP), phosphorodithioate (PS2), methoxypropyl phosphonate (MPO), 5'-phosphorothioate (5'-PS), (S)- 5'-C-methyl with phosphate, peptide nucleic acid (PNA), phosphorothioate (PS) Rp isomer, phosphorothioate (PS) Sp isomer, 5'-(£)-vinylphosphonate (5'-(£)-VP), and 5'-methyl phosphonate (5'- MP);(i.1.2) at least one ribose modification selected from 2'-O-methyl (2'OMe), 2'- deoxy-2'-fluoro (2'F), 2'-methyl-4-pyridine (2'-O-CH2Py(4)), PMO, 2'- 0- methoxyethyl (2'-O-MOE), tricyclo-DNA (tcDNA), Locked NucleicAcid (LNA), 2'-arabino-fluoro (2'-Ara-F), 2'-O-benzyl, Glycol NucleicAcid (GNA), Unlocked Nucleic Acid (UNA), 2'-deoxy, and (S)-cEt-BNA;(i.1.3) at least one base modification selected from pseudouridine (QJ), 2'- thiouridine (s2U), N6'-methyladenosine (m6A), N-ethylpiperidine 7'- EAA triazole modified adenine, N-ethylpiperidine 6'-triazole modified adenine, 6'-phenylpyrrolocytosine (PhpC), 5'-methylcytosine (m5C), 5'- fluoro-2'-deoxyuridine, 2',4'-difluorotoluyl ribonucleoside (rF), 5- methyluracil, and 5'-nitroindole.

5. The NPY / Y5R inhibitor for use according to any one of claims 1-4, wherein the siRNA(s) is / are:(1.2) conjugated with N-acetyl galactosamine (GalNAc); and / or(1.3) comprised by a lipid nanoparticle (LNP), particularly an ionizable lipid nanoparticle.

6. The NPY / Y5R inhibitor for use according to any one of claims 1-5, wherein the siRNA(s), which is / are optionally conjugated with GalNAc or comprised by a LNP, is / are for being injected into the subject, particularly by subcutaneous injection.

7. The NPY / Y5R inhibitor for use according to any of claims 1-6, wherein the siRNA(s) is / are characterized by one or more of the following features:(1.4.1) each strand of the siRNA(s) has a length of 15-30 nucleotides, particularly of 19-29 nucleotides, more particularly of 19-25 nucleotides, and even more particularly of 19-24 nucleotides;(1.4.2) a GC content of 30-60%;(i.4.3.) asymmetry of the siRNA strands;(i.4.4) a lack of palindromes, CCC and GGG sequences, and internal repeats.

8. The NPY / Y5R inhibitor for use according to any of claims 1-7, wherein the NPY / Y5R inhibitor (i) is at least one siRNA selected from:(a) siRNAs having an antisense strand comprising, or consisting of, at least nucleotides 1 to 19 of a nucleotide sequence independently selected from the sequences set forth in SEQ ID NO: 2, 75, 14, 11, 154, 1, 8, 9, 5, 4, 10, 184, 78, 80, 180, 27, 21, 83, 159, 22, 24 and 23, particularly SEQ ID NO: 2 and 83; or(b) modified siRNAs having an antisense strand comprising, or consisting of, at least nucleotides 1 to 19 of a nucleotide sequence independentlyselected from the sequences set forth in SEQ ID NO:2, 75, 14, 11, 154, 1, 8, 9, 5, 4, 10, 184, 78, 80, 180, 27, 21, 83, 159, 22, 24 and 23, particularly SEQ ID NO: 2 and 83, wherein said nucleotide sequence has been chemically modified so as to comprise at least one of the chemical modifications listed in claim 4 and / or the siRNA is conjugated with GalNAc; particularly, wherein the modified siRNA (b) is selected from:(b.l) the modified siRNA having the sense strand set forth in SEQ ID NO:614 and the antisense strand set forth in SEQ ID NO: 610,(b.2) the modified siRNA having the sense strand set forth in SEQ ID NO:615 and the antisense strand set forth in SEQ ID NO: 611,(b.3) the modified siRNA having the sense strand set forth in SEQ ID NO:616 and the antisense strand set forth in SEQ ID NO:612, and(b.4) the modified siRNA having the sense strand set forth in SEQ ID NO:617 and the antisense set forth in SEQ ID NO:613.

9. The NPY / Y5R inhibitor for use according to claim 1, wherein the NPY / Y5R inhibitor is a Y5R inhibitor of (ii) selected from: / VL[[ / ra / 7s-4-[[(4-amino-2-quinazolinyl)amino]methyl]cyclohexyl]methyl]-l- naphthalenesulfonamide (CGP71683) or a pharmaceutically acceptable salt thereof, particularly CGP71683 hydrochloride;£ / 7S-N-[l-(2-fluorophenyl)-lH-pyrazol-3-yl]-l'-oxo-spiro[cyclohexane- l,3'(l'H)-furo[3,4-c]pyridine]-4-carboxamide (MK-0557); / IA[4-methyl-9-(l-methylethyl)-9 / -carbazol-3-yl]-4-morpholinecarboxamide (5RA972); andZra / 7s-4-(tert-butylsulfonylamino)-N-[5-(trifluoromethyl)pyridin-2- yl]cyclohexane-l-carboxamide (S-2367, Velneperit).

10. The NPY / Y5R inhibitor for use according to any one of claims 1-9, wherein the Y5R inhibitor is for being injected, particularly subcutaneously, into the subject.

11. The NPY / Y5R inhibitor for use according to any of claims 1-10, wherein the subject has or had a primary cancer, and the NPY / Y5R inhibitor is for use in one or more of:(a) inhibiting the migration and / or invasion of cancer cells from the primary cancer into the liver of the subject,(b) inhibiting the growth of metastatic cancer cells of the primary cancer in the liver of the subject,(c) reducing the colony formation capacity (clonogenicity) of metastatic cancer cells of the primary cancer in the liver of the subject, thereby preventing or reducing metastasis to the liver of the subject.

12. The NPY / Y5R inhibitor for use according to claim 11, wherein the primary cancer:(1) is different from primary liver cancer; and optionally is further different from Ewing sarcoma, neuroblastoma, pheochromocytoma, paraganglioma, breast cancer, and prostate cancer; and / or(2) is selected from stomach cancer, colorectal cancer, breast cancer, pancreatic cancer, lung cancer, and malignant melanoma; in particular from stomach cancer, colorectal cancer, pancreatic cancer, lung cancer, malignant melanoma, and uveal melanoma; more particularly from colorectal cancer and malignant melanoma.

13. The NPY / Y5R inhibitor for use according to any of claims 1-12, wherein the subject: (a) does not have steatosis, or (b) has steatosis.

14. The NPY / Y5R inhibitor for use according to any of claims 1-13, wherein the subject is a mammal, particularly a human subject.

15. The NPY / Y5R inhibitor for use according to any of claims 1-14, wherein the NPY / Y5R inhibitor is for being administered to the subject at one, two or all of the following times:(a) prior to treatment of a primary cancer,(b) during treatment of a primary cancer,(c) after treatment of a primary cancer.

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