Synthetic micrornas for use in the treatment of melanoma

Abstract

Synthetic miRNA (S-miR) sets for use in the treatment of melanoma, and in particular for resistance to targeted therapy, pharmaceutical compositions and dosing regimens thereof are disclosed.

Description

[0001] SYNTHETIC MICRORNAS FOR USE IN THE TREATMENT OF MELANOMA FIELD OF THE INVENTION

[0002] The present invention relates to the field of medicine and biotechnology as it describes synthetic microRNA (miRNA) (S-miR) for use in the treatment of melanoma and in particular for resistance to targeted therapy against the BRAF / MEK signaling pathway (MARK) as well as therapeutic compositions comprising them.

[0003] TECHNICAL BACKGROUND

[0004] Melanoma is a malignant skin tumour that results from the uncontrolled transformation and proliferation of melanocytes. Depending on the tissues of origin, melanoma is classified into four main subtypes. Cutaneous melanoma (CM), acral melanoma (AM), mucosal melanoma (MM) and uveal melanoma (UV) (2). CM is the most common and around 50% of patients have a mutation in the BRAF gene (3). This led to the development of targeted therapy with BRAF inhibitors (BRAFi) and MEK inhibitors (MEKi), generally inhibiting the mitogen-activated protein kinase (MARK) pathway. Targeted therapy improved patient survival and prognosis (4,5). However, despite substantial improvement, patients receiving targeted therapy develop resistance to therapy within 9-11 months (6). Multiple mechanisms contribute to resistance, including genetic, epigenetic and microenvironmental changes (7). The rapid development of resistance is also linked to the high plasticity of melanoma (8). 15-20% of tumours show primary resistance to current therapies and acquired resistance remains a critical challenge (9,10). To overcome this problem several approaches were used including the use of RNA interference (RNAi), such as siRNA or miRNA (11).

[0005] SiRNAs and miRNAs are small RNAs of about 21-23 nucleotides, with a similar structure but different functions, determined by their sequence. Both molecules are loaded into a riboprotein complex called RISC, which regulates gene expression at the post-transcriptional level by recognizing target sequences on the 3’UTR of the messenger RNAs. The specificity of the recognition is conferred precisely by the siRNA or miRNA. SiRNA acts on a single messenger RNA (mRNA), recognizing it in a complementary way for the entire length of 21 nucleotides and inducing its degradation. In contrast, the miRNA does not recognize its target completely, but is mainly based on a sequence of 6 nucleotides 5' of the miRNA, called "seed". Instead of degradingmRNA, miRNA causes its destabilization and inhibits translation.

[0006] SiRNA and miRNA are used as anticancer therapeutic approaches, to restore correct gene expression. SiRNAs can target genes otherwise considered “unbeatable” by classical inhibitors and are therefore a powerful therapeutic approach.

[0007] International Patent Application No. WO2021231771A2 discloses the use of siRNAs to combat melanoma and cancer in general.

[0008] Strategies involving miRNAs as therapeutic agents employ mimic miRNAs, i.e., synthesized miRNAs that mimic the sequence of natural miRNAs, to restore normal gene expression in transformed cells, antimiRs, single-stranded oligonucleotides with sequence complementary to an endogenous miRNA to inhibit its function. It was shown that the use of existing miRNAs, thanks to the pleiotropic effect on multiple targets, reduced the proliferation of glioblastoma cells (12).

[0009] The use of a synthetic miRNA, targeting multiple genes simultaneously, has been tested on lung cancer, however targeting only two transcripts and thus few genes (2). International Patent Application No. WO2019198115A1 discloses endogenous miRNAs differentially expressed in melanoma cells as diagnostic and therapeutic agents.

[0010] International Patent Application No. WO2019198115A1 describes combinations of mimetics that in resistant cells have led to a reduction in cell viability of around 60-80%, depending on the combination used.

[0011] International Patent Application No. W02012005572A1 discloses miRNAs used as therapeutic agents in melanoma; in particular endogenous, deregulated miRNAs in melanoma, as diagnostic and therapeutic agents.

[0012] Closest prior art

[0013] International Patent Applications No. WO 2012 / 005572, WO 2019 / 198115, WO 2014 / 072357 describe miRNAs for the treatment of melanoma.

[0014] International Patent Application No. WO 2013 / 173637, and U.S. Patents US 2006 / 099612 and US 2011 / 178283 disclose oligonucleotides.The aforementioned prior art documents exclusively describe endogenous microRNAs or non-functional oligonucleotides, while the present invention relates to synthetic, rationally designed microRNAs with artificial sequences not present in nature and specific functional and chemical characteristics aimed at modulating molecular targets involved in tumour progression. In particular, the claimed sequences do not exhibit significant homology with known human microRNAs (<52%).

[0015] SUMMARY OF THE INVENTION

[0016] Technical Problem

[0017] Melanoma is the deadliest skin cancer, patients have a high mortality rate, and despite the progress made with targeted therapy and immunotherapy, the majority of patients develop resistance to these drugs, which reduces the survival rate. Targeted therapy consists of inhibitors of B-Raf Proto-Oncogene, Serine / Threonine Kinase (BRAF) (BRAFi), such as Dabrafenib, and inhibitors of Mitogen-Activated Protein Kinase Kinase 1 (MEK) (MEKi), such as Trametinib.

[0018] Strategies known in the art are capable of modulating or restoring the gene expression of individual genes that are identified as crucial in melanoma. These approaches are based on the identification and modulation of single crucial genes within metabolic pathways or networks. Unfortunately, melanoma cells, when subjected to common anticancer therapies, are able to develop resistance to them. This happens because cells are able to find alternative metabolic pathways to circumvent the inhibition of a single gene, thus making them resistant to common therapies. This behaviour reduces the efficacy of, or renders ineffective, common therapeutic approaches.

[0019] The inventors of the present invention have developed a different approach that relies on the simultaneous inhibition of the expression of hundreds of genes to counteract the onset of cancer cell resistance, thereby inhibiting entire metabolic pathways rather than individual genes. This approach is multi-gene and contrasts with single-gene therapies known in the art.

[0020] Another distinctive and characterizing feature is that said approach does not use natural and / or endogenous miRNAs but synthetic miRNAs designed specifically for aselected set of genes. Such an approach would be impossible to implement with endogenous miRNAs since these have a physiological role that has not been selected to inhibit the metabolic pathways we identified, on the contrary synthetic miRNAs offer high specificity for the treatment of melanoma resistant to common therapies because they are specifically designed for these pathways.

[0021] The known methodologies of the art are capable of reducing the viability of melanoma cells by 20-70% depending on the method and the miRNA used, but their efficacy in resistant cells or in combination with BRAFi has not been assessed to evaluate the ability to inhibit the onset of resistance.

[0022] In contrast, the inventors of the present invention showed a reduction in viability of about 85% even in situations of established resistance.

[0023] A set of synthetic miRNAs (S-miRs), non-naturally occurring artificial sequences, that function like miRNAs, were then selected that are able to improve selectivity and reduce off-target effects (i.e. , unintentional changes or effects that occur in the genome during the use of gene editing technologies) compared to endogenous miRNAs.

[0024] The S-miRs were selected using an algorithm developed by the inventors themselves that allowed an intentional and proactive targeted selection to be made to solve the technical problem.

[0025] The steps carried out for the selection of the S-miRs that are the subject matter of the present invention are summarized below and are also highlighted in the flow chart of Figure 1.

[0026] The process begins by selecting a list of genes whose sequences are analyzed.

[0027] Based on this information, the biochemical characteristics of the miRNA-target interaction are calculated.

[0028] This allows the identification of a set of optimized miRNA-like sequences, able to target the largest number of genes with maximum efficiency.

[0029] Transcriptomic datasets (RNAseq) derived from cell lines or patient samples were analyzed using single-cell sequencing (scRNAseq) technology or samples from patients before and after developing resistance to targeted therapy.The analyzed datasets come from GEO DataSets (Home - GEO DataSets - NCBI), an international public repository that freely stores and distributes microarray data, nextgeneration sequencing, and other forms of high-throughput functional genomics data submitted by the research community:

[0030] GSE97681 (Gene expression signature of vemurafenib resistance in WM989 and WM983B melanoma cells - Gene expression signature of vemurafenib resistance in WM989 and WM983B melanoma cells, https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE97681)

[0031] GSE205251 (GEMM melanoma tumours, untreated or treated with vemurafenib [bulk RNA-seq] - GEMM melanoma tumours, untreated or treated with vemurafenib [bulk RNA-seq] https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE205251) GSE103630 (expression changes in melanoma cell lines before MAPKi treatment vs. post-MAPKi resistance (RNA-seq Cell Line. batch 2) - Expression changes in melanoma cell lines pre MAPKi treatment vs. post-MAPKi resistance (RNA-seq_CellLine.batch2)

[0032] https: / / www.ncbi. nlm.nih.gov / geo / query / acc.cgi?acc=GSE103630)

[0033] GSE103725 (Changes in melanoma expression before MAPKi treatment vs. on MAPKi treatment [Y1.7. FPKM. batchi .2] - Expression changes in Melanomas pre MAPKi treatment vs. on MAPKi treatment [Y1.7. FPKM. batchi .2], https: / / www.ncbi. nlm.nih.gov / geo / query / acc.cgi?acc=GSE103725)

[0034] GSE203546 (Divergent BRAF Inhibitor Resistance Mechanisms Revealed through Epigenetic Mapping, https: / / www.ncbi. nlm.nih.gov / geo / query / acc.cgi?acc=GSE203546)

[0035] GSE202118 (Analysis of M229-Par and M229-Res cells by Illumina HiSeq instrument (4000 or equivalent), sequencing analysis - Analysis of M229-Par and M229-Res cells by Illumina HiSeq instrument (4000 or equivalent) seq analysis, https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE202118)

[0036] GSE108382 (SAKE (Single-cell RNA-Seq Analysis and Klustering Evaluation) identifies markers of resistance to targeted BRAF inhibitors in melanoma cell populations [Bulk RNA-Seq] - SAKE (Single-cell RNA-Seq Analysis and Klustering Evaluation) Identifies Markers of Resistance to Targeted BRAF Inhibitors in Melanoma Cell Populations [Bulk RNA-Seq], https: / / www.ncbi. nlm.nih.gov / geo / query / acc.cgi?acc=GSE108382).

[0037] Biological processes (BP) and metabolic pathways have been identified for each cellline or patient sample.

[0038] Biological processes (BP) and metabolic pathways in common between the samples, i.e. the most representative and those commonly activated in resistant cells, were selected. The selection of the most relevant processes was carried out based on their occurrence in the individual analyses, favouring those that appeared with greater frequency, as shown in table 1

[0039] Table 1

[0040] representative count

[0041] regulation of cell migration 39 response to external stimuli 19 development of the circulatory system 15 Catecholamine transport 8 cellular metabolic process 8 Chromosome organization 3 leukocyte aggregation 2 actin filament-based process 1 Cell adhesion 1 Cellular Communication 1 Organization or biogenesis of cellular components 1 chemical homeostasis 1 developmental process 1 immune system process 1 mi RNA transcription 1 response to stimulus 1

[0042]

[0043] signalling 1

[0044] The most representative metabolic pathways identified were: organization of the extracellular matrix (Extracellular matrix organization), signaling mediated by G-protein coupled receptors (Signalling by GPCR) and O-linked glycosylation.

[0045] Metabolic pathways are indicated in Table 2.

[0046] Table 2ID representative count_2

[0047] R-HSA- 1474244 Organization of the extracellular matrix 95 R-HSA-372790 Signalling by GPCR 53 R-HSA- 5173105 O-linked glycosylation 34 R-HSA- 1280215 Cytokine Signalling in Immune System 30 R-HSA-109582 Hemostasis 24 R-HSA-112316 Neuronal System 23 R-HSA-422475 Axon guidance 22 R-HSA- 6806834 Signalling by MET 18 R-HSA-168898 Toll-like receptor cascades 17 R-HSA- 5683057 MAPK Family Signaling Cascades 14 R-HSA- 1257604 PIP3 Activates AKT Signalling 14 R-HSA- 9658195 Leishmania infection 13 R-HSA-168249 Innate immune system 10 R-HSA- 2173782 Binding and uptake of ligands by scavenger receptors 7 R-HSA-

[0048]

[0049] 9006934 Receptor tyrosine kinase signaling 7

[0050] The most representative biological processes (BP) identified were: regulation of cell migration and circulatory system development.

[0051] All genes involved in the aforementioned representative biological processes and metabolic pathways were processed by a specific algorithm developed by the inventors themselves.

[0052] By way of example, the genes analyzed are shown in Table 3.

[0053] Table 3

[0054] analysis_x_7 analysis_x_7 analysis_x_7 analysis_x_7 analysis_x_7 analysis_x_7 analysis_x_8 ADAM8 TAS2R46 CCKAR SALL4 SMYD4 PROP1 MAPK10 ADAM 10 TAS2R30 APLN BAD STX4 INHBA EGLN3

[0055]

[0056] LAMC3 TAS2R19 CCKBR PTEN SOX9 EMC10 COL4A4CRTAP TAS2R20 ARHGEF7 CBX8 SAT1 FOXN4 IL2RB FBLN5 TAS2R50 WNT3A GATAD2B SRF PI16 JAK2 P3H3 ARHGEF26 FGD3 MTA3 CDX4 OTULIN ROCK1 CAPN9 GPSM1 KALRN RPTOR SYNJ2BP PIM1 CASP3 CEACAM8 ABHD12 F2RL3 TRIB3 AMOT TBCCD1 GLUL MMP24 RGS22 UCN2 RRAGD NRARP C2CD3 PRKCG ADAMTS8 TIAM2 CCRL2 RAC2 RAPGEF3 HRG PRKCD ADAMTS5 GCG TAAR5 RBBP4 CD99 DLL4 DAB2 EMILIN1 0PN1MW ARHGEF1 RBBP7 PYCARD DVL2 DENND3 CAPN11 RGS17 DGKI Rest AMOTL1 WASL SH3GL1 CAPN10 GHRH LPAR2 TRIM27 XG MOSPD3 EDNRA CMAl GHRHR ARHGEF2 RHEB GJA4 PITX2 IL3RA COL1A1 GHSR GPR37L1 RING1 TIE1 HEY1 CREB5 COL1A2 GIP TAAR2 RNF2 SH3BP1 DAPK3 CDK3 COL2A1 GIPR GPR55 RPS6KB2 DLX3 TBX6 F2RL2 COL3A1 NBEA S1PR2 RRAGC SYNPO2 SIRT6 RAB12 COL4A1 PDE7B GLP2R MLST8 LDLR PTCD2 SLC36A4 COL4A2 HCAR1 OPN4 BMI1 ASB4 MMP28 PPAT COL4A3 OXGR1 GNG8 SNAI2 LGALS3 ACVRL1 VAV1 COL4A4 C5AR2 ARHGEF6 SNAI1 ELK3 ACVR1B PDGFRB COL4A5 ARHGEF16 ROCK2 STRN EPPK1 DUSP22 CREB1 COL4A6 P2RY1O CCL4L2 NR2E1 PGDN EPHB4 CFI COL5A1 GLP1R GABBR2 TP53 NOX4 ARL13B FOXO4 COL5A2 GNA11 RGS6 TRAF6 TBXT DPP4 FLT3 COL6A1 GNA12 GNA14 TSC2 TEAD2 STAB2 IDH1 COL6A2 GNA15 ARHGEF1O USP7 GPR173 TRIP6 C3 COL6A3 GNAI1 ECE2 MAPKAP1 FLCN NPPB CBL COL7A1 GNAI2 ADGRE5 PIP4K2C DNAAF3 SALL1 YAP1 COL8A1 GNAI3 ARHGEF11 PHC3 CARDIO IL34 DAB2IP COL8A2 GNAL ARHGEF17 PREX2 BRMS1L NCL HIF1A COL9A1 GNAQ PLPPR4 TNKS2 CSF1R MIIP RGCC COL9A2 GN AS P2RY14 RNF146 ERAP1 PKM XAF1 COL9A3 GN ATI CDC42 PIP5K1A IGF1R CD9 SMAD4

[0057] POC1B- COLlOAl GNAT2 GALNT4 PIP5K1B SPN TMEM231 A2M COL11A1 GN AZ MUC12 PIP4K2B RHOD ATP5F1B CEBPB COL11A2 GNB1 CHST4 CASP9 TP53INP1 PDLIM7 BCR COL12A1 GNB2 B3GNT3 MAF1 HOXB13 GLI3 CTNNA1 COL13A1 GNB3 SPON2 AKT1S1 EPAS1 NACA LYN COL15A1 GNG3 SPON1 CBX2 SGK3 SEMA3B PDGFB

[0058]

[0059] COL16A1 GNG4 POMTl CBX4 MED12 GATA3 PRKAR1AC0L17A1 GNG5 ST6GALNAC2 TNKS NR2F2 DAPK2 CD19 C0L19A1 GNG7 B3GNT2 EED EMCN CD300A PLN COMP GNG10 B4GAT1 HDAC3 TBX1 TNFAIP3 CACNA1B COL6A6 GNG11 ADAMTS13 IER3 KLF2 AGGF1 MAPK9 COL26A1 GNGT1 ADAMTS7 USP13 MATR3 SP1 ARRB2 HAPLN1 GNGT2 ADAMTS6 IL33 EGFL7 ROM1 BDKRB1 ADAMTS14 GNRH1 GALNT6 MTA1 PLPP3 BBS5 IFNGR1 VCAN GNRH2 GALNT5 IL1RL1 ADTRP TREX1 ABCA1 NCAN GNRHR GALNT13 MTA2 TMSB10 STK11 ARHGAP23 CAPN12 CCR1O GALNT15 CD19 NOTO GJA5 SMAD7 CTRB1 GPR4 MUCL1 CD28 ZMYND8 KLK3 CAD CTSB XCR1 LARGE2 CD80 YAP1 RABGEF1 HECW2 CTSD NPBWR1 MUC17 CD86 FGF21 SPINK5 NFIX CTSG NPBWR2 GCNT7 GAB2 DHX36 TCF7L2 ARHGAP28 CTSK CXCR3 MUC15 DLG5 EP300 ZMPSTE24 PTPRC CTSL PRLHR B3GLCT SINHCAF ARHGAP24 OBSL1 PTPRE CTSV UTS2R B3GNTL1 DLL1 HK2 ADAP2 SH3BP1 CTSS GPR15 B3GALNT2 RNH1 PDGFD CPE JDP2 DAG1 NPW SBSPON TNFRSF14 OLFM1 ABL1 CDS1 DCN GPR17 GALNTL5 HLA-G RAB11A DPEP1 HCK TMPRSS6 GPR18 ADAMTS15 MIF NSDHL RAPGEF1 STAT6 COL22A1 GPR2O ADAMTS17 TJP1 ALOX15B KRT16 SMAD1 ADAMTS16 LPAR4 ADAMTS19 DDT CHST2 BAX IFITM3 ADAMTS18 MCHR1 B3GNT6 PTPRC MEF2C NDRG4 GADD45A DMD GPR25 MUC20 ALOX5 CD99L2 MACIR PLAUR DMP1 GPR27 THSD7A GREM1 ANGPTL3 RYR1 METTL3 ACAN GPER1 POFUT2 SERPINF2 SEMA6D RELN KRT1 DSPP GPR31 ST6GALNAC3 AIF1 MEIS1 TRAF3IP2 CDK1 ELANE GPR32 GALNT1 PTPRK ZNF580 IL17F GFAP A2M GPR150 GALNT2 MAPK15 EPN1 CXCL14 RPS6KB1 ELN GPR35 GALNT3 FAM 83 H B4GALT1 CYP19A1 ACTB FBLN1 GPR37 GALNT8 GFUS TNFRSF12A SEMA3G CACNA1E FBLN2 MLNR GCNT1 AGER FAM HOC PAD 12 NOS1 FBN1 GPR39 ST6GALNAC4 TNF TIPARP TNNC1 RELA FBN2 FFAR1 MUC19 ASB2 APPL2 AQP1 ZNF804A EFEMP1 FFAR3 C1GALT1C1 NOTCH4 NTN1 NISCH DNM1 SCUBE3 FFAR2 POMT2 UTY PLEKHG3 ENPEP PAK2 FGA GRK5 ADAMTSL5 MYH11 SHB CCN1 MYO1E FGB GRK6 GALNT18 TMSB4Y SLIT3 CDH13 EPGN FGF2 GRB2 B3GNT8 SRCIN1 PTPRT MIA3 MAPK12

[0060]

[0061] FGG ABR MUC21 E2F2 ENG AFDN PTPRGNID2 GRM1 GALNTL6 HMGBl POGLUT1 MCAM AKAP12 ITGA11 GRM2 MUC1 CXADR LMO4 PTPN23 PPP2R2B C0LGALT2 GRM3 MUC3A CDX2 CD40 ELP6 KIT FMOD GRM4 MUC4 ACTN4 ADA TTBK2 SMN1 FN1 GRM5 MUC5AC FREM2 DOCK7 CARMILl TPO NCSTN GRM6 MUC6 MAML1 PIP5KL1 HMGA2 IL4R CAPN7 GRM7 MUC7 SPRY2 PRICKLE4 NPHP3 SOCS3 COL24A1 GRM8 GALNT9 KLF5 OSBPL8 FGFRL1 JAK3 COL6A5 CXCL1 A4GNT FLT1 IL1B RIPOR1 PPP3CC OPTC CXCL2 GALNT7 POU4F1 SEMA3C FIELD HIF3A PCOLCE2 CXCL3 CFP SPATA13 HES1 SIN3A PPP3CA BMP10 GRP SEMA5B MAPRE2 MAP2K4 NRP2 GFPT1 P4HA3 GRPR ADAMTSL4 ANGPT4 SORL1 ODAD4 STMN1 LAMAl GPSM2 GALNT10 TMEM102 SFRP2 CLDN4 DVL1 EFEMP2 GPR132 POMGNT1 INSMI NEBL PLXNA1 TUBA8 HSPG2 ANXA1 THSD1 NDUFV2 SMYDl NOX5 DUSP1 TNC HCRT MUC13 ISM1 DOCK1 PSKH1 PTPN22 IBSP HCRTR1 C1GALT1 ATP5IF1 PDLIM5 KIF2A CFH ICAM1 HCRTR2 ADAMTSL3 SCRT2 CHRNA7 PTPRB GPER1 ICAM2 ADGRE2 GALNT16 NCOA6 PLXNC1 KAT2A TP53 ICAM3 TAAR6 GALNT11 GCNT2 HAS1 GP1BA BCL2L11 ICAM4 HR AS GALNT17 LRCH1 FLRT2 AXIN2 PTPN7 COL28A1 HRH1 ST6GAL1 LIF SPHK1 TAB2 ADCY4 APP HRH2 ST3GAL1 JAG1 TRIM32 IGFBP3 MYC ITGA6 HTR1A ST3GAL2 EPHA1 CER1 ZFPMl HSPA8 ITGA1 HTR1B ST3GAL4 RUNX1 CYGB TPMl CP ITGA2 HTR1D ST3GAL3 PPP1R16B HS6ST1 LEMD2 ELK1 ITGA2B HTR1E THBS2 SMAD2 DRCI SRI CALM1 ITGA3 HTR1F MUC5B GCSAM ING2 TLR3 IRAKI ITGA4 HTR2A B3GNT4 RIPPLY3 TENM4 SORBS2 PTX3 ITGA5 HTR2B GALNT14 GATA4 ABHD2 KCNK2 CTNND1 ITGA7 HTR2C GALNT12 ADIPOR2 FUZ CARMIL2 CHGA ITGA9 HTR4 THSD4 TWIST2 FHL2 CO RO 1 A PRKACA ITGAD HTR5A ADAMTS20 SLC2A1O SENP2 TERT PRKCH ITGAE HTR6 THSD7B ECSCR SLC12A6 CRELD1 AP2B1 ITGAL HTR7 ADAMTS12 FERMT2 GPC3 ARHGAP32 NUMA1 ITGAM IAPP ADAMTS10 PKNOX1 NPRL3 WARS2 RIPK1 ITGAV TAS2R60 B3GNT5 CACNA1C NFATC3 SPAG9 MAP2K3 ITGAX HCAR2 POMK PTGIS IFT74 PAK4 PLCG2 ITGB1 FFAR4 B3GNT9 SIX1 GJC1 MEF2A MAP2K6

[0062]

[0063] ITGB2 RXFP4 POMGNT2 ZBTB14 SYNPO2L ABCC8 PTK6ITGB3 GNAT3 GALNT4 STRA6 HIF1A SOD2 BCL2 ITGB4 QRFP SEMA5A MNAT1 CCDC103 SGCB SLC24A3 ITGB5 RGSL1 LARGE1 RHOJ CPNE3 FGF12 PTPRB ITGB6 IHH GCNT3 SEMA7A DNAI1 DNAJA4 ARHGAP10 ITGB7 CXCL8 ADAMTSL1 RIPK3 ADIPOQ CEP43 MAPK3 ITGB8 CXCR1 B3GNT7 GAS6 FBXW8 ETV2 IQGAP1 AGRN CXCR2 B4GALT6 SOX18 VEGFC SCARB1 DDHD2 KDR CXCL10 B4GALT5 RBI RAP2B B9D1 MYD88 KLK2 INSL3 MUC16 SGCG EPHB3 JMJD8 RPS6KA1 KLKB1 ITPR1 ADAMTSL2 EFNB2 ATP7A HOXA5 STAT3 CAPN8 ITPR2 RANBP9 DZIP1 MGAT5 HOXA7 SYNJ1 CHURC1- LAMA2 ITPR3 FNTB PATZ1 E2F7 RHOH IL6ST LAM A3 KEL RASA4 NFATC2 NKX2-1 HOXA13 FLNA LAMA4 KISSI PSME3 CLEC14A SMPD3 TMIGD2 PDE1C LAMA5 KNG1 PSMD14 NDP OSGIN1 ANTXR1 RCAN1 LAMB1 KPNA2 CNKSR1 DLC1 DNAAF1 SEMA3D ADORA1 LAMB2 KRAS LYPLA1 RCC2 ZNF304 NAA15 CCR2 LAMB3 RHOA CDC42EP2 STK24 DEFB124 FLT4 ADRA2C LAMC1 NPSR1 CDC42EP3 SGCZ HSPB6 CTNNA2 NTRK1 LAMC2 RHOB IGF2BP1 EGR3 LYL1 LGALS8 CDK2 LOX CCL4L1 NRG3 STC1 LDB1 PLCG2 MDK LOXL1 RHOC FRS3 GATA5 ASXL1 TNFAIP6 LRRC4 LOXL2 ARHGEF37 FRS2 IRX4 ANGPTL4 MAPK14 SYP LRP4 LHB DUSP10 LBP LEP TCAF2 F2R LTBP1 LHCGR RASGRP4 ING1 TGFBR3 HOXA3 SYN1 LTBP2 ARRB1 PSMB11 GDF2 PDGFC ATP8A1 JAK1 LTBP3 ARRB2 PSMA8 AKAP6 NKX2-6 HIF3A PTGER2 LUM CCL3L3 CSF2 ODAD2 CRIP1 ANO6 UNC5A MATN1 MC1R CSF2RA RGCC RECK NOD2 CALM2 MATN3 MC2R CSF2RB ELP5 IMMP2L EPN2 PAK1 MFAP1 MC3R CSK SLC39A12 STK4 FUT3 PAX8 MFAP2 MC4R NRG4 RAP2A F3 KRIT1 PDPK1 MFAP3 MC5R CDC42EP5 JARID2 TCAF1 SEMA4C TAB1 MFAP4 MCF2 PAQR3 TAFA5 SPHK2 IL24 WNT5A MMP1 CXCL9 KLB GJB6 VASH1 MCC SLA MMP2 MLN DAB2IP UNC5D HSPA5 HAMP CYCS MMP3 RGS21 SPRED1 CDH2 UNC5C PFN1 PPP2R2C MMP7 ARHGEF35 DLG1 RBM24 PIFO MIXL1 SMAD5 MMP8 MTNR1A DLG2 SIK1 EPOR SLC8B1 HLF

[0064]

[0065] MMP9 MTNRIB DLG3 SOX4 PRKD1 EPHB1 GATA3MMP10 NMB DLG4 MYO18B HPGD DYNC2H1 CAMK2B MMP11 NMBR DUSP1 AIR DDX58 CCL26 VAV2 MMP12 NPY DUSP2 DOCKS LRIG2 CCL24 STAT1 MMP13 NPY1R DUSP4 NKX3-1 NOTCH 1 HEYL PDGFRA MMP14 NPY2R DUSP5 NRP1 ODAD3 ECM1 LFNG MMP15 NPY5R DUSP6 TAB1 TSC1 CCDC40 RALBP1 MMP16 NR AS DUSP7 MGAT3 ALPK3 GAA PTPN12 MMP17 NTS DUSP8 JPH2 LRRC1O RNF41 JAG1 MMP19 NTSR1 DUSP9 IFT52 FERMT1 CLDN19 EGF CTRB2 GPR143 AG03 LGMN KIF2OB FBXW7 INSR CAPN14 OPRD1 AG04 JCAD AHI1 DOCK4 TG MUSK OPRK1 EGF FOXCI SEMA6A CLASP2 LDHB CEACAM6 OPRL1 SPRED2 ISL1 DCTN5 IL23A PTGER4 NCAM1 OPRM1 ERBB2 TET1 APELA FBXO31 SORBS1 NIDI OXT ERBB3 DKK1 PODXL RIC8A CD3E DDR2 OXTR ERBB4 SLITRK5 RRAS LGALS9 MPO P4HA1 P2RY1 EREG CCAR1 RUFY3 PKD1 ARHGAP29 P4HB P2RY2 ETV4 NEDD9 EXT1 PLXND1 ARHGEF7 FURIN P2RY4 RASGEF1A MKKS SUDS3 SAP130 HECW1 COL5A3 P2RY6 FGF1 CCM2L TBC1D24 IGSF1O FOXO3 SERPINE1 P2RY11 FGF3 POFUT1 VEZF1 PAX8 LRRK2 FUR PAK1 FGF4 LYN LRP1 MICAL2 CASP8 SPOCK3 ARHGEF4 FGF5 ZIC3 SAP30 AKIRIN1 SYN2 PCOLCE ARHGEF3 FGF6 ONECUT2 HAND2 FOXP1 ASNS TRAPPC4 TAS2R3 FGF7 ALPK2 GTPBP4 SOX6 GNAI1 PDGFA TAS2R4 FGF8 DDRGK1 NCKAP1L SOCS3 SOCS1 PDGFB TAS2R16 FGF9 SETDB2 FGFBP1 WNK1 MEF2C PECAM1 TAS2R1 FGF1O PTPRM VSIR ANGPTL6 JUN PLEC TAS2R9 FGFR1 CNN2 JMJD6 NRCAM SH3RF1 PLG TAS2R8 FGFR3 ID1 DIPK2A DNAH11 CACNA2D3 PLOD1 TAS2R7 FGFR2 RIN3 FKBPL MAZ SF3B1 PLOD2 TAS2R13 FGFR4 HHEX LRG1 PDLIM3 ANXA3 PPIB TAS2R10 MR AS MYLK2 FLRT3 APOLD1 IL1RN ASPN TAS2R14 RASA3 FOXS1 EPB41L5 FAM89B SMAD3 P3H2 DHH CNKSR2 GATA6 ANGPT2 MYLK NTNG2 PRKCA HEBP1 FOXO1 SOX17 SRGAP2C SHOX2 PDLIM2 PRSS1 PDE11A FOXO3 OVOL2 ABI3BP FADD AKAP5 PRSS2 PRLH TNRC6B SULF1 CYBB GGNBP2 C3AR1 KLK7 GAL RGL1 ADPRHL1 SPRY1 ROBO1 ARHGAP30 HTRA1 RXFP3 PSME4 CRK MOSPD2 DNMT1 ETS2

[0066]

[0067] PSEN1 PDE1A FLT3 OXCT1 SEMA4D ANK2 RAC1ADAMTS9 PDE1C FLT3LG FKBP1A IFT2O SRGAP3 MMP3 COL20A1 PDE2A FNTA PRDM1 MYOCD SELENOK ACAN PTPRS WNT16 FNTB BCL2 PDCL3 HSPB11 MAP2K7 JAM2 PDE3A ICMT ARSB MKS1 FOXN1 IRS1 NTN4 PDE3B FYN AJUBA MEGF8 EPHB2 TFAP2B BGN PDE4A EPGN PCNA TMEFF2 DDIT3 FGA CEACAM1 PDE4B SHC2 MYH7 EPB41L4B ACVR1C EPS15 SDC1 PDE4C RASGRP3 N FATCI CXCL17 ACVR1 MMP12 BCAN PDE4D FGF2O BICC1 FOXH1 TBX20 HRH1 SDC2 PDE7A AGO1 PLXNA3 IL18 GTF2I DNAJC6 SDC4 PDE8A GDNF NIPBL BBS7 MST1 TUBA1A TNN PDE1B GFRA1 TMSB15B CSF1 MDK NFIL3 P3H1 ACKR4 GFRA2 MTDH G6PD SETD2 PRKCA MMP25 PDPK1 GFRA3 TFAP2B CAMSAP3 HDAC9 FLT1 BMP1 PDYN FGF22 SAV1 PLET1 TNFSF12 NTRK3 BMP2 GHRL LAT SLC1A1 HIF1AN FGR KIF5C BMP4 GNG13 AGO2 GJA1 ASCL1 MYBPC3 CTDSP2 BMP7 PENK TNRC6A YWHAZ PPARD BRCA1 GRK2 CAPN15 PF4 KSR2 PGF MSN PTGS2 FUS DST PIK3CA ANGPT1 NUS1 BST1 DDAH1 FBN1 SPARC PIK3CG LAMTOR2 NXN HIPK2 PLXNA4 MMP9 SPP1 PIK3R1 GRIN1 MYDGF NKD1 VGLL4 LCK BSG PIK3R2 GRIN2B AP2B1 ADGRG1 EIF2AK3 MAP3K7 ADAM 17 PLA2G4A GRIN2D HACE1 TSPAN12 RARB MATK TGFB1 PLCB2 HGF MAPK11 MESP2 ARHGDIB HBEGF TGFB2 PLCB3 NRG1 BVES ANPEP NOG AGTR1 TGFB3 PLCB4 HSPB1 IQSEC1 NEK8 CACNA1G FOXS1 THBS1 S1PR5 DNAJB1 SYDE1 ARPIN NKX6-1 TGFBR2 TIMP1 PLXNB1 IL2 CCDC134 ANXA2 GYSI BDKRB2 TIMP2 PMCH IL2RA HMOX1 TAFAZZIN BMPER IL31RA ICAM5 PNOC IL2RB KAT2B PLA2G7 ATM MAP2K4 TLL1 P2RY13 IL2RG SERPINB3 ADGRF5 NAXE TIMP3 TLL2 GPR84 IL3 ADARB1 SYNE2 SH2D2A CSMD1 TNR GNG2 IL3RA VEGFD MYL2 CBLL1 TGFBR1 TNXB WNT4 IL5 F7 ADGRG3 HDAC4 ATP2B2 TPSAB1 TAS2R5 IL5RA GRB7 MTERF4 P2RX4 LTBP1 CAPN5 POMC IL6 F10 TMSB4X ID2 ID1 TTR PPBP IL6R APC FOXJ1 PFN2 PDE1A C0L14A1 ARHGEF38 IL6ST PLXNB3 RXRG PTN LRIG1 VCAM1 PLPPR1 IRS1 APEX1 RDX NEB CLTC

[0068]

[0069] VTN PPP1CA ARAF TBX18 JUNB SEMA3E ZAP70VWF AVP JAK1 TREM2 NOTCH3 THY1 ABL1 DDR1 PPP2CA JAK2 EYA1 TCF25 ACACB ARHGAP9 PXDN PPP2CB JAK3 PLVAP ADGRB1 BMPR2 SLC16A3 C0LGALT1 ARHGEF1OL JUN ZFAND5 ACE PAX6 CDKN2A SCUBE1 PPP2R1A AREG ANG MMRN2 XIRP2 PTBP1 ADAM12 PPP2R1B USP17L2 ADGRG6 MAP3K3 WNT5B ERCC6 MFAP5 AVPR1A KIT CEMIP2 SP1OO GATA2 TUBA4A COL18A1 PPP2R5D SPRED3 SMIM22 OR10J5 LEFTY1 SYN3 COL21A1 AVPR1B ARL2 TNFAIP2 TBX3 CFLAR BAX MADCAM1 PPP3CA MARK3 NEXMIF PDLIM4 IFT122 ARHGAP6 GDF5 PPP3CB MET TMEM204 NODAL CTNNB1 RUNX2 CAPN1 PPP3CC KITLG STAR D 13 SMURF2 C1QBP DAG LA CAPN2 PPP3R1 MAP3K11 UBP1 TRADD TH PXDN CAPN3 PPY MOVIO CCN3 TBX19 PDPN BIRC3 CAPNS1 AVPR2 MYC FUT9 PHLDA2 ADGRB2 PLCG1 CAPN6 NPY4R NEFL DSG2 SWAP70 ULK4 DUSP8 CAST PRKACA NF1 BMP5 IL27RA NTNG1 MCL1 CASP3 PRKACB NRTN CNMD HDAC6 DUSP3 PPM1L JAM3 PRKACG PEBP1 DACH1 HGS SRGAP1 DRD1 EMILIN2 ARHGEF40 PAK2 IFT140 PLCD3 FOXJ2 APEX1 LOXL4 PRKAR1A PAK3 NF2 HEXIM1 FAP SMO LTBP4 PRKAR1B ABHD17B HYAL1 TDGF1 RBM15 EPHB2 CAPNS2 PRKAR2A ZDHHC9 ZDHHC16 PLXDC1 CD200R1 HTR7 FBN3 PRKAR2B GOLGA7 SMAD7 CREB3L1 STIL PTPRR COL25A1 PRKCB SPTBN5 MIEN1 BEX4 KRT5 BAK1 LOXL3 PRKCD PDE6D FKBP1O MYL3 TTN NTN1 PHYKPL PRKCE PDGFRA ANKS6 CCN2 SYPL2 TIMP1 ITGA10 PRKCG PDGFRB TGFBI TDGF1P3 FUT4 HLX ITGA8 PRKCH PHB SDCBP OGT CCDC39 RIN1 COL27A1 PRKCQ PIK3CB CASS4 FOSL1 TNFRSF1A GLS2 CASK MAPK1 SHC3 YJEFN3 ZNHIT1 GLMN MAPK1 ACTN1 MAPK3 APBB1IP NINJ1 ADGRB3 MYOC MTOR SERPINH1 GNG12 IL17RD SMAD4 ACVR2B CALD1 EGR1 ADAM 19 MAPK7 PPP1CB RARE MTUS1 HSPB7 GEMIN6 ADAM 15 PRKX PPP1CC MMP21 SELE FMNL3 EPB41 ADAM9 LTB4R2 PPP2R5A PARD6B TBX5 CAV3 THBS1 MATN4 CCL28 PPP2R5B PTK2B KANK1 PAXIP1 TAGLN2 P4HA2 PSAP PPP2R5C OXSR1 SMYD2 CERS2 FOXO1 PLOD3 SUCNR1 PPP2R5E TBC1D32 GUI SRPK2 CD36 EMILIN3 NMUR2 PPP5C WDR11 SPNS2 LRP5 BCL2L1

[0070]

[0071] COL23A1 PYY PRKG2 PTPRG BRMS1 CHRD EREGCAPN13 ACKR3 MAPK4 RIN2 LRRC15 HOXB3 DRD2 MMP20 CYSLTR2 MAPK6 ACTC1 SIRT1 ARID2 PRMTl NRXN1 TRPC7 MAP2K1 PLN CTSH PTPRJ LAT ADAMTS4 LPAR5 MAP2K2 THBS4 PRRX1 FERMT3 TNFRSF11A ADAMTS3 PTAFR PSPN RBM20 LBX1 PKP2 GSK3B ADAMTS2 TAS2R38 PSMA1 RB1CC1 CPLANE1 STI Ml DLL1 ADAMTS1 PTCHI PSMA2 FRMD5 SEMA6B IL12A ERBB2 CD44 PTGDR PSMA3 MSX1 MINK1 TNFSF18 TRAF6 CD47 PTGER1 PSMA4 ESMI CFC1B APOD PTGER3 SH3PXD2A PTGER2 PSMA5 SHTN1 CLDN1 CD81 TTL SDC3 PTGER3 PSMA6 MED1 PRMTl LMNA LEPR CD151 PTGER4 PSMA7 LDB2 ARHGAP4 SLC9A3R1 TUBA3E CDH1 PTGFR PSMB1 HEY2 SGCD SRGAP2 DAG LB AKT3 PTGIR PSMB2 PANK2 SMAD3 TIRAP SHC1 INSL5 ABHD6 PSMB3 HYAL2 PRKD2 VASH2 ACPI ARHGEF33 PTH PSMB4 TMIGD1 STAT3 DNM1L SNAI1 NPY4R2 PTHLH PSMB5 STK3 CACYBP AAMP DLG1 0PN1MW3 PLEKHG5 PSMB6 SMAD6 FOLR1 PPP2R3A TUBA1C RASGRP1 PTH1R PSMB7 POPDC3 PTPN14 IFNG HNRNPA1 LPAR6 PTH2R PSMB8 BAS Pl MECP2 VIL1 MAP3K5 CDK5 NLN PSMB9 SMAD5 DSP EOMES SRC CALCRL PREXI PSMB10 PCSK5 DMTN S100A14 ARHGEF15 RASGRP2 CXCL16 PSMC1 TNMD FES S100A1 LPAR5 RAMP2 RASGRF2 PSMC2 PLXNB2 CDH5 HEG1 ADAMTS4 RAMP1 HRH4 PSMC3 JUP TFDP2 RNF213 CALM3 RAMP3 GNB4 PSMC4 FHOD3 CLN3 SVBP IL1R1 NET1 RXFP1 PSMC5 ATG5 ZNF268 IGF1 TNFAIP3 RGS19 NPS PSMC6 TMSB15A GPNMB LGR6 MAPK14 NMUR1 RGR PSMD1 CTDP1 BMPR1A ROBO4 FOXO6 VAV3 RGS1 PSMD2 MYO1E SVEP1 KCNJ8 S1PR1 LINC02210- CRHR1 RGS2 PSMD3 AKAP12 FAT4 GPX1 ARHGAP5 CXCL13 RGS3 PSMD4 SLIT2 NTNG2 MITF INPP5D RGS14 RGS4 PSMD5 FLNA CCM2 S100A11 RHOA CAMKK2 RGS7 PSMD7 CLEC7A EGLN1 LUZP1 CACNA1C CXCR6 RGS10 RGL3 BCR PLK2 ETS1 IGF2 GNA13 RGS12 PSMD8 TCAP ZMIZ1 MDM4 MAPK8 GNB5 RGS13 PSMD9 ZFPM2 BCAS3 IGFBP5 EPN1 RRH RGS16 PSMD10 SYK CERT1 ROBO2 VEG FA ADCY1 RHO PSMD11 KLRK1 NTRK2 CASP8 GRM1

[0072]

[0073] AHCYL1 RLN2 PSMD12 HOPX CAMKID SLAMF1 CXCL1ADCY2 PROK2 PSMD13 MEOX2 POU4F2 NOTCH 2 CAMK2G CYSLTR1 ROCK1 PSME1 GRHL2 NOS3 NPPA DCC CCR9 OPN1SW PSME2 ABI3 RBP4 SEMA4A CACNA1A CGA RPS6KA1 PTK2 FAM107A SGK1 STK39 ADAMTS5 PDE10A RPS6KA2 TNRC6C MYLK3 PGK1 MEF2D GNG12 CCL27 RPS6KA3 SPTBN4 AP1B1 PLXNA2 SEMA4F CDK4 NMU BDKRB1 PTPN3 NFATC4 CRB2 EPHA4 MMP19 NPFFR2 BDKRB2 PTPN7 STK26 CITED1 MSTN OXTR PROKR1 SAA1 PTPN11 RAP2C DOCK8 ILIA NOS3 GPR83 SCT PTPRA PLP1 NSD2 TNNT2 AREG ADCY3 SCTR ABHD17C KLF4 PROXI TNNI1 NOTCH 2 UTS2 CCL1 RGL2 PRICKLEI ARF6 ATOH8 GPSM3 EEF1AKMT4- ECE2 CCL2 RAC1 CRKL DNAH5 MACF1 SNRNP70 ADCY5 CCL3 RAFI STK1O PTP4A1 DOCKIO ERRFI1 ADCY6 CCL3L1 RAG1 TOMM70 CNTRL STAT1 GEMIN4 FZD1O CCL4 RAG 2 DPYSL3 BMERB1 ADIPOR1 SH2B3 AKAP13 CCL5 RALGDS PLEASE LDB3 CHI3L1 PTPRF GPR176 CCL7 RAP1A ANKRD1 SELP RTN4 SMURF1 GPR45 CCL11 RAP1B SLURP1 MAGI2 TALI ARHGEF2 PTGDR2 CCL13 RASA1 ALDH1A2 TRPV4 ITGB1BP1 PPP2CA HRH3 CCL16 RASA2 TYMP LRP2 GBX2 IDH2 CHRM1 CCL17 RASGRF1 ROR2 CALR SCG2 PTPN21 CHRM2 CCL19 RET QKI BBS4 SERPINE2 FH ADCY7 CCL2O BCL2L1 PRKG1 BST2 NFE2L2 SKP2 PTH2 CCL21 ACTB SUFU RASIP1 SEMA6C SRGAP3 CHRM3 CCL22 RPS27A KCNQ1 PDCD10 TMIGD3 FGFR2 CHRM4 CCL23 MAPK12 NRAP NR4A3 MAP4K4 AMOTL2 CHRM5 CCL25 GFRA4 RPS6KB1 ONECUT1 CLASP1 GRB2 MGLL CXCL6 SHC1 SMG9 LGALS12 SRGAP2B GNAS ADCY8 CXCL11 RASAL3 RSPO3 PRKDC SARS1 SP1 UCN3 CXCL5 SPTA1 SLC12A2 HECTD1 DNAI3 TGFB1 ADCY9 XCL1 SPTAN1 LIMCH1 PAM VANGL2 NRG1 ARHGEF25 CX3CL1 SPTB HCN4 FLVCR1 IL10 CDKN1A ADCYAP1 CXCL12 SPTBN1 ARID4A SAS Hl TP73 RHOH ADCYAP1R1 GPSM3 SPTBN2 CLDN5 BAK1 PTPN22 DNM1L RLN3 NPFFR1 BRAF MAP2K5 RREB1 SLC8A1 AKT1 FGD4 CXCR5 BTC PPM1F CCN6 HIPK1 RTA RXFP2 RGS18 TEK FBXO5 LEF1 DHRS3 CASP9 GPHB5 ITSN1 TGFA CD200 MCU TACSTD2 WIPF1

[0074]

[0075] CCR1 SHH TLN1 TBX2 PRL MYSM1 TIMP2CCR3 PLPPR2 ACTG1 GPI RFFL GADD45A MYODI CCR4 P2RY12 TYK2 SAP30L ZNF703 S100A7 PPME1 CCR5 PLEKHG2 UBA52 CSRP3 PRSS56 TNFRSF18 OPTN CCR6 SMO UBB IRX3 PLCE1 ACADM SH3GL2 CCR7 SOS1 UBC SLC2A12 ADGRA2 CD160 GLS CCR8 SOS2 VCL MYH9 PDCD4 FAS LG PINK1 ACKR2 SRC XPO1 STAB1 PARVA ERRFI1 DEPTOR CMKLR1 SST YWHAB NTF3 RAC KI WASF2 PRKCQ LTB4R SSTR1 SEMI MBD2 BAG4 ANGPTL7 KARS1 PCP2 SSTR2 SHOC2 MB EGR2 NEXN F2RL1 CNR1 SSTR3 FGF23 NGFR KIF14 NPR1 S1PR3 CNR2 SSTR4 DUSP16 SH3RF2 PDCD6 HNRNPU PTPRD ARHGEF19 SSTR5 ABHD17A SH3PXD2B SMOC2 SKI COL18A1 PR0KR2 BRS3 NCOA3 SFRP1 HPSE ABL2 HIC1 NMS XCL2 BRAP HAND1 ARHGAP22 RYR2 PDE3A CORT TAC1 KBTBD7 CSPG4 ARID4B CLIC4 PIM1 ADM TACR2 WDR83 MYH6 NOXI ATP2B4 BIRC2 ADORA1 TAC3 RASAL1 TRIBI ZP3 NBL1

[0076] TAAR9 TACR1 CUL3 HAS2 FUT1 NCK1

[0077] TAAR1 TACR3 MAPKAPK5 APOH ACTA2 NFKBIA

[0078] ADORA2A TBXA2R CDC14B MIB1 RPGRIP1L PXN

[0079] ADORA2B BTK CDC14A WT1 CORO1B EGFR

[0080] CREB1 TIAM1 LAMTOR3 CCBE1 CFC1 GRIK2

[0081] DGKK C3 IRS2 RRAS2 ELP3 PTPN1

[0082] CRH C3AR1 PEA15 FOXC2 SMARCD3 CYLD

[0083] CRHBP TRH ACTN2 BCAR1 WDR62 GPR17

[0084] CRHR1 TRHR FGF18 NIBAN2 CC2D2A ROR1

[0085] CRHR2 TRIO FGF17 ATP5F1A ZC3H12A APOA1

[0086] ADORA3 TRPC3 FGF16 PLCG1 KDM6B MMP2

[0087] ADRA1D TRPC6 IQGAP1 TGFBR2 EMP2 SMURF2

[0088] PIK3R6 TSHB SYNGAP1 CELA1 SPINT1 HTR1D

[0089] ADRA1B TSHR KSR1 AHR MTHFD1 PPP3CB

[0090] AD RAIA C5 CCND3 MAP2K3 LYVE1 ARHGAP22 ADRA2A C5AR1 ARTN TWIST1 SMAD1 ATP2A2

[0091] ADRA2B 0PN1MW2 KL GREB1L C10orf99 CAMK2A

[0092] GPBAR1 CCR2 RASAL2 CITED2 WASHCI TUBA1B

[0093] ADRA2C UCN PSMF1 IFT57 FER TRAF2

[0094] CX3CR1 VAV1 NRG2 MSX2 CTH LPAR1

[0095] ADRB1 VAV2 RAPGEF2 CCN4 ID3 PTPN6

[0096] ADRB2 VIP CDK1 GLIPR2 DIAPH1 UNC5B

[0097]

[0098] ADRB3 VIPR1 PSMD6 SERPINB7 STAT5A PLCD4GRK2 VIPR2 SEPTIN7 TBX4 DAND5 NET1 GRK3 DAG LA FGF19 SRPX2 PKD2 SMAD6 CD55 WNT1 RBX1 TCF21 NUMB PRKAA2 DGKA WNT2 RCE1 STAP1 ANXA3 PRKAA1 DGKB WNT3 HDAC5 CDH11 SLAMF8 SF3B3

[0099] COMMD3- DGKG WNT5A BMI1 RIPOR2 SPI1 SCRIB DGKH WNT6 CDKN1A MEAK7 APOE NCSTN DGKQ WNT7A CDKN1B XBP1 MCTP1 FGB PLPPR5 WNT7B STU Bl PPARA MARVELD3 SNRPG 0XER1 WNT8A RRAGB SEMA3F CASP7 SNIP1 GPHA2 WNT8B LAMTOR5 PHLDB2 EFNA3 LLGL1 DRD1 WNT10B RRAGA APOB EFNA1 SF3A3 DRD2 WNT11 WWP2 GSX2 MYADM AIMP2 DRD3 WNT2B CHD3 BCOR DAW1 SNRPB DRD4 WNT9A CHD4 SOX11 GLI2 NFKB1 DRD5 WNT9B CHUK OSR1 RARRES2 LIMSI AGT XK THEM4 FOXL1 CD274 PSEN1 HBEGF CXCR4 PIK3AP1 PRR5L PDLIM2 SNRPA1 AGTR1 FZD5 ATF2 SSH2 HTATIP2 PIK3CA AGTR2 CALCA CSNK2A1 CREB3 MYL7 FGG APLNR CALCB CSNK2A2 GPLD1 TGFBR1 BUD13 GPR183 FZD3 CSNK2B LDLRAD4 POPDC2 ILK ECE1 ARHGEF5 SLC38A9 TMEM100 CD34 QARS1 ECT2 CALCR ATN1 YTHDF3 CHD7 LARS1 S1PR1 ADM2 PHC1 SUN2 AIMP1 SNRPD2 LPAR1 PLPPR3 PHC2 BORCS8 CLDN3 SNRPD3 S1PR3 CALM1 EGR1 DNAAF4 GCSAML SF3B6 EDN1 WNT10A ESRI SLC9A1 PPP1R13L CLTA EDN2 CALM 2 ESR2 sex SNX17 SNRPB2 EDN3 CALM3 MECOM CYP1B1 AMOTL2 PRKAG3 EDNRA TAS1R2 EZH2 APPL1 VEG FA CCNB1 EDNRB TAS1R1 SCMH1 FOXF1 CD74 PRKAB1 EGFR GPR68 KDM1A NKX2-5 IFT172 HTATSF1 ADCY4 CAMK4 PHLPP2 SCRT1 TMED2 GEMIN7 ADGRE1 CAMK2A RCOR1 SOX14 CYRIB CCNA2 AKT1 CAMK2B PHLPP1 TNNI3 RAB25 AP2M1 AKT2 CAMK2D OTUD3 NRXN3 SIN3B SF3B2 F2 CAMK2G PIP5K1C RNF2O VEGFB DDX46 F2R FZD1 CBX6 CBY1 CUL7 MARS1

[0100]

[0101] F2RL1 FZD4 SUZ12 PLAA CAV1 PARVAF2RL2 FZD6 MKRN1 NSMF GLUL AIMP1

[0102] OPN5 FZD7 FRK E2F8 WARSI SF3B4

[0103] FGD2 FZD8 MTOR EPHA2 YY1 PIK3R1

[0104] DAGLB FZD9 RICTOR MPPl LEPR AP2A2

[0105] GPRC6A TAAR8 GABI CLUAP1 SCN5A EEF1E1

[0106] FGD1 TAS1R3 GSK3A PARP2 TLR4 EPRS1

[0107] ARHGEF15 OBSCN GSK3B SCAI ZFP36L1 DARS1

[0108] ARHGEF9 QRFPR ICOS XDH EVL IARS1

[0109] PLCB1 PPP1R1B HDAC1 TNFRSF1B PHACTR1 RARS1

[0110] MCF2L CAMKK1 HDAC2 BTG1 GRN SNRPF

[0111] ARHGEF12 PROK1 NR4A1 NDNF DAB2 SNRPD1

[0112] ARHGEF18 MCHR2 XIAP PTPRR ZNF609 SF3A2

[0113] PIK3R5 CASR IL1RAP PTPRU PRCP SNRPA

[0114] LPAR3 KISS1R INS SEC24B TMEM65 SNRPC

[0115] FPR1 ADGRE3 INSR CEMIP ANP32B SF3A1

[0116] FPR2 GPR65 IRAKI NDST1 MESP1 PSENEN

[0117] FPR3 GALR3 LAMTOR4 ATP1B2 SEMA4G PRKAB2

[0118] OPN3 RGS5 RHOG CPLANE2 RBPJ PPP3R1

[0119] NTSR2 ARHGEF39 LCK RNF207 IL12B PRKAG1

[0120] FSHB PIK3R3 MDIVI2 KDM6A SEMA4B LIMS2

[0121] FSHR DGKZ FOXO4 NAV3 MSC GEMIN5

[0122] GAST DGKE MYD88 NCK1 UNC5B DDX20

[0123] ACKR1 DGKD NEDD4 IL4 NTRK3 APH1A

[0124] FZD2 RGS8 TRAT1 MY01C SGPL1 SF3B5

[0125] GABBR1 RGS20 IRAK4 TNFSF14 MFGE8 CLTB

[0126] NPB NPFF HDAC7 TRIP11 COROIC CLTCL1

[0127] UTS2B PDE8B PIK3CD SEMA3A IFITM1 RBMX2

[0128] NGEF PTCH2 PIP4K2A FUT7 SLK SNRPE

[0129] GALR1 S1PR4 MBD3 KRT1 CIB1 LIMS3

[0130] TAS2R39 RGS11 PML PTK7 ILK PRKAG2

[0131] TAS2R40 RGS9 PPARG MINAR1 PDLIM1 PHF5A

[0132] TAS2R41 GALR2 GATAD2A ENPP2 VSTM4 GEMIN2

[0133] TAS2R43 HCAR3 LAMTOR1 SERPINF1 ANKRD17 CCNE2

[0134] TAS2R31 CCK PRR5 WDPCP DCHS1 AP2S1

[0135]

[0136] RORA IL1R1 LIMS4

[0137] The result of the aforementioned processing is a selected set of S-miRs capable of selectively and simultaneously targeting several genes involved in said biological processes and metabolic pathways.

[0138] S-miRs were selected based on a coefficient that takes into account both the ratio ofthe number of predicted targets to the total number of genes analyzed, and the characteristics of their interaction. In particular, strong binding sites, such as 8mer and 7mer-m8, as well as sites with sequences immediately adjacent to the AU-rich recognition site and links to the 3’were privileged, thus guaranteeing high effectiveness in targeting target genes. The threshold value is the highest number between 0.1 and 0.

[0139] As can be seen from the following table 4.

[0140] Table 4

[0141] Numbe

[0142] r of miRNA real_coeff.

[0143] UCCCAGCUGGUUGGGAUG 0.19811283

[0144] 1 8 CUG 9 UCAGCCUCAUGGGUACUU 0.18350513

[0145] 2_8 GGG 6 UCCCAGCAGGAAGGGAAG 0.22184807

[0146] 3_8 GCG 4 UGCUGGGAGGUUAGGUGA 0.22398074

[0147] 4 8 GAG 2 UGAGGCAGUCCAAUCCUU 0.13439957

[0148] 5 8 GAA 2 UGGCCAGGUUCCUCUCAA 0.11828049

[0149] 6 8 CUU 2 UCACUGCAUCUGCCGCCC 0.13115756

[0150] 7 8 CCCCC 1 UGCUGCUGGGAAUGCGCU 0.04367332

[0151] 8 8 GCU 4 UGGGCUGGUCCACAGGGG 0.03225256

[0152] 9 8 GGG 8 UCCUGGGCGGAAGUGCCU 0.05463052

[0153] 10 8 CCU 8 UCAGCCUCGGAAGUAGCC 0.27664492

[0154] 1 7 UGG 8 UGAGGCAGGGUUAUCCAU 0.17611966

[0155] 2 7 GAC 9 UGGCCAGGGGAAUCUGGG 0.12751014

[0156] 3 7 GGG 5 UCCUGCCUGGAACUCCAC 0.20551387

[0157] 4 7 AGG 2 UCACUGCAUCCUCCGCCC 0.18031366

[0158] 5 7 CCC 5 UCUCCUGAGGAACGCGGG 0.03883064

[0159] 6 7 GUU 2 UGCUGCUGGGAACUGUUU

[0160] 7_7 UUUUU 0.04255176

[0161]

[0162] 8 7 UCUGCAGCUCCUAAUAAAA 0.09489151AG 1

[0163] UGUGUCUGGGAAUGUGUG 0.03987681

[0164] 9_7 UGUGU 2

[0165] UGUGUGUCGGAAUGUGUG 0.02830807

[0166]

[0167] 10_7 UGUGU 5

[0168] The following S-miRs of Table 4 are the object of the present invention:

[0169] Table 5

[0170] SEQ.ID NO. NAME miRNA ST26 STANDARD

[0171] 1

[0172] UCCCAGCUGGUUGG TCCCAGCTGGTTGGGATG

[0173] 1_8 GAUGCUG CTG

[0174] 2 UCAGCCUCAUGGGU TCAGCCTCATGGGTACTTG 2_8 ACUUGGG GG

[0175] 3

[0176] UCCCAGCAGGAAGG TCCCAGCAGGAAGGGAA

[0177] 3_8 GAAGGCG GGCG

[0178] 4

[0179] UGCUGGGAGGUUAG TGCTGGGAGGTTAGGTGA

[0180] 4_8 GUGAGAG GAG

[0181] 5

[0182] UGAGGCAGUCCAAU TG AG G C AGTC C AATC CTTG

[0183] 5_8 CCUUGAA AA

[0184] 6 UGGCCAGGUUCCUC TGGCCAGGTTCCTCTCAA 6_8 UCAACUU CTT

[0185] 7

[0186] UCACUGCAUCUGCC TCACTGCATCTGCCGCCC

[0187] 7_8 GCCCCCCCC CCCCC

[0188] 8

[0189] UGCUGCUGGGAAUG TGCTGCTGGGAATGCGCT

[0190] 8_8 CGCUGCU GOT

[0191] 9 UGGGCUGGUCCACA TGGGCTGGTCCACAGGG 9_8 GGGGGGG GGGG

[0192] 10 UCCUGGGCGGAAGU TCCTGGGCGGAAGTGCC 10_8 GCCUCCU TCCT

[0193] 11

[0194] UCAGCCUCGGAAGU TCAGCCTCGGAAGTAGCC

[0195] 1_7 AGCCUGG TGG

[0196] 12

[0197] UGAGGCAGGGUUAU TGAGGCAGGGTTATCCATG

[0198] 2_7 CCAUGAC AC

[0199] 13

[0200] UGGCCAGGGGAAUC TGGCCAGGGGAATCTGGG

[0201] 3_7 UGGGGGG GGG

[0202] 14 UCCUGCCUGGAACU TCCTGCCTGGAACTCCCC 4_7 CCACAGG AGG

[0203] 15 UCACUGCAUCCUCC TCACTGCATCCTCCGCCC

[0204]

[0205] 5 7 GCCCCCC CCC16UCUCCUGAGGAACG TCTCCTGAGGAACGCGGG 6_7 CGGGGUU GTT

[0206] 17UGCUGCUGGGAACU TCCCAGCTGGTTGGGATG 7_7 GUUUUUUUU CTG

[0207] 18UCUGCAGCUCCUAA TCAGCCTCATGGGTACTT 8_7 UAAAAAG GGG

[0208] 19UGUGUCUGGGAAUG TCCCAGCAGGAAGGGAA 9_7 UGUGUGUGU GGCG

[0209] 20UGUGUGUCGGAAUG TGCTGGGAGGTTAGGTGA

[0210]

[0211] 10_7 UGUGUGUGU GAG

[0212] The pharmaceutical compositions containing thereof and use in the treatment of melanoma are object of the present invention.

[0213] Further features of the present invention will be clear from the following detailed description with reference to the provided experimental examples and to the accompanying Figures.

[0214] Figure 1 shows in a flowchart the operations that led to the selection of the S-miRs. Figure 2 shows the network of genes activated in the predicted resistant and target lines of S-miR_8_7. Causal pathways linking the S-miR_8_7 target proteins to the phenotypes noted in SIGNOR, identified with the ProxPath algorithm. The basic network is obtained by merging maps of curated networks in SIGNOR, Nodes represent biological entities: circles correspond to proteins, rectangles to phenotypes, squares to small molecules, and octagons to protein complexes. Gray nodes indicate target proteins of S-miR_8_7. The arcs represent interactions between proteins (arrows indicate activations and T-shaped arcs indicate inhibitions).

[0215] Figure 3 shows the molecular analyses following ectopic expression of S-miR. The analyses are carried out by RT-qPCR. (A) and (B) dose-dependent graph showing the expression of S-miR_8_7 and S-miR_10_8, respectively, upon increasing the dose of S-miR, after ectopic expression. The Y-axis shows the logarithm of 2A-ACT normalized to miR_194, which functions as an endogenous calibrator. The log base 10 of the tested S-miR concentrations, specifically 1nM, 5nM, 15nM and 30nM, is represented on the X-axis. (C) and (D) Histograms of S-miR_8_7 and SmiR_10_8, respectively. On the Y-axis the relative Fold Change of the gene is shown, the colors represent the control and expression conditions of the S-miR. (p-values: ns > 0.05, * < 0.05, ** < 0.01, *** <0.001).

[0216] Figure 4 shows the PCA (A) conditions are colored in grayscale and replicates with different shapes. (B) and (C) show a Volcano plot of modulated genes after ectopic expression of S-miR_8_7. Display of the — Iog10 (FDR) and the change in Iog2 of the fold change for each gene in relation to the covariate chosen. Upregulated genes (FDR < 0.1 and Iog2 fold change > 0.7) are highlighted in light gray. Downregulated genes (FDR < 0.1 and Iog2 fold change < 0.7) are indicated in dark grey. The vertical dashed lines indicate the thresholds for up-regulated and down-regulated genes, while the horizontal dashed line represents the FDR threshold.

[0217] Figure 5 shows S-miR treatment on resistant cells. (A) Dose-dependent curve of cell line sensitivity to BRAFi. The sensitive A375-S line is shown in black and the resistant A375-R3 in light grey. (B) Crystal Violet stained cell culture plate visually illustrates the degree of cell proliferation, where darker areas indicate increased viability and cell number. (C) The barplot shows the quantification of relative cell viability, expressed as fold change compared to control.

[0218] Figure 6 shows treatment with S-miR to inhibit the onset of resistance. Crystal Violet stained cell culture plate visually illustrates the degree of cell proliferation, where darker areas indicate increased viability and cell number. On the right, the barplot shows the quantification of the relative cell viability, expressed as fold change with respect to the control.

[0219] Figure 7 shows the dose-dependent effect of S-miRs. The barplot shows the quantification of relative cell viability, expressed as fold change compared to the control at different doses of S-miRs.

[0220] Figure 8 shows the outcome of the Pull-Down assay.

[0221] Figure 9 shows the outcome of the in vivo assay.

[0222] Table 10 shows the results of the PLGA assays.

[0223] SEQUENCES (sequence listing)Table 6

[0224] SEQ.ID NO. 1 miRNA TCCCAGCTGGTTGGGATGCTG RNA SYNTHETIC

[0225] 1_8 CONSTRUCT

[0226] SEQ.ID NO.2 miRNA : TCAGCCTCATGGGTACTTGGG RNA SYNTHETIC

[0227] 2_8 CONSTRUCT

[0228] SEQ.ID NO. 3 miRNA ? TCCCAGCAGGAAGGGAAGGCG RNA

[0229] 3_8

[0230] SEQ.ID NO. 4 miRNA TGCTGGGAGGTTAGGTGAGAG RNA SYNTHETIC

[0231] 4_8 CONSTRUCT

[0232] SEQ.ID NO. 5 miRNA i TGAGGCAGTCCAATCCTTGAA RNA SYNTHETIC

[0233] 5_8 CONSTRUCT

[0234] SEQ.ID NO. 6 miRNA TGGCCAGGTTCCTCTCAACTT RNA SYNTHETIC

[0235] 6_8 CONSTRUCT

[0236] SEQ.ID NO. 7 miRNA ; TCACTGCATCTGCCGCCCCCCCC RNA SYNTHETIC

[0237] 7_8 CONSTRUCT

[0238] SEQ.ID NO. 8 miRNA ; TGCTGCTGGGAATGCGCTGCT RNA SYNTHETIC

[0239] 8_8 CONSTRUCT

[0240] SEQ.ID NO. 9 miRNA ; TGGGCTGGTCCACAGGGGGGG RNA SYNTHETIC

[0241] 9_8 CONSTRUCT

[0242] SEQ.ID NO. 10 miRNA TCCTGGGCGGAAGTGCCTCCT RNA SYNTHETIC

[0243] 10 8 CONSTRUCT

[0244] SEQ.ID NO. 11 miRNA ? TCAGCCTCGGAAGTAGCCTGG RNA SYNTHETIC

[0245] 1 7 CONSTRUCT

[0246] SEQ.ID NO. 12 miRNA ? TGAGGCAGGGTTATCCATGAC RNA SYNTHETIC

[0247] 2_7 CONSTRUCT

[0248] SEQ.ID NO. 13 miRNA i TGGCCAGGGGAATCTGGGGGG RNA SYNTHETIC

[0249] 3_7 CONSTRUCT

[0250] SEQ.ID NO. 14 miRNA ; TCCTGCCTGGAACTCCCCAGG RNA SYNTHETIC

[0251] 4_7 CONSTRUCT

[0252] SEQ.ID NO. 15 miRNA = TCACTGCATCCTCCGCCCCCC RNA SYNTHETIC

[0253] 5_7 CONSTRUCT

[0254] SEQ.ID NO. 16 miRNA TCTCCTGAGGAACGCGGGGTT RNA SYNTHETIC

[0255] 6_7 CONSTRUCT

[0256] SEQ.ID NO. 17 miRNA TCCCAGCTGGTTGGGATGCTG RNA SYNTHETIC

[0257] 7_7 CONSTRUCT

[0258] SEQ.ID NO. 18 miRNA TCAGCCTCATGGGTACTTGGG RNA SYNTHETIC

[0259] 8_7 CONSTRUCT

[0260] SEQ.ID NO. 19 miRNA ; TCCCAGCAGGAAGGGAAGGCG RNA SYNTHETIC

[0261] 9_7 CONSTRUCT

[0262] SEQ.ID NO. 20 miRNA ; TGCTGGGAGGTTAGGTGAGAG RNA SYNTHETIC

[0263]

[0264] 10_7 CONSTRUCT

[0265] DETAILED DESCRIPTION OF THE INVENTION

[0266] An object of the present invention is at least one S-miR selected from the group consisting of SEQ. ID NOS. 1 to 20.

[0267] Preferably the S-miRs are SEQ. ID. NO. 10 and SEQ ID NO.18

[0268] Also within the scope of the present invention are sequences having at least 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 98%, 99% or 100% identity or similarity to the sequences S. SEQ. ID NOS. 1 to 20.The S-miRs can be subject to chemical modifications for their stabilization such as those shown in Table 7.

[0269] Table 7

[0270] Modifications Position

[0271] 2’0-Me 3’- or 5’ -end or central portion

[0272] PS Phosphodiester skeleton

[0273] 2’ F 3’- or 5’ -end or central portion Conjugation with lipids 3’- or 5’ -end

[0274]

[0275] Preferably the sequences SEQ. ID NOS. 1 to 20 may comprise at least one chemical modification selected from the group consisting of:

[0276] adding a methyl group to the 2' hydroxyl of the ribose of a 5’ or 3’ nucleotide or in the central portion of the sequence;

[0277] replacing an oxygen atom of the phosphate group with a sulphur atom on the phosphodiester backbone;

[0278] replacing the hydroxyl group at the 2’ position of the ribose with a fluorine atom in a 5’ or 3’ nucleotide or in the central portion of the sequence;

[0279] conjugation with lipids, of a 5’ or 3’ nucleotide.

[0280] preferably the lipids are selected from the group consisting of: Cholesterol, sphingomyelin, pegylated lipids such as for example 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)] (DSPE-PEG), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)] (DMG-PEG), 1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)] (DPPE-PEG) , cholesterol conjugated with polyethylene glycol (Cholesterol-PEG), ceramide conjugated with polyethylene glycol (Ceram ide-PEG).The S-miRs may be contained in particles, preferably nanoparticles or microparticles, more preferably lipid nanoparticles.

[0281] When encapsulated, S-miRs may not require chemical modifications.

[0282] Further object of the present invention is the use of S-miRs or particles containing thereof for use as a medicament in therapy.

[0283] Further object of the present invention is the use of S-miRs or particles containing thereof for use in the treatment of melanoma at any stage and of any subtype.

[0284] Melanoma in any of the subtypes: cutaneous melanoma (CM), acral melanoma (AM), mucosal melanoma (MM), and uveal melanoma (UV), as well as cutaneous melanoma characterized by the BRAF gene mutation.

[0285] The stages of melanoma are:

[0286] Stage 0 (Melanoma in situ): Melanoma is confined to the outermost layer of the skin (epidermis) and has not spread deeper.

[0287] Stage I: The tumour is still small and can be divided into IA and IB, depending on the thickness and presence of ulcerations.

[0288] Stage II: Melanoma is thicker and can be divided into HA, IIB and IIC, based on the thickness and presence of ulcerations.

[0289] Stage III: Melanoma has spread to regional lymph nodes or surrounding tissues.

[0290] Stage IV: Melanoma has spread to other parts of the body, such as the lungs, liver, or brain.

[0291] Pharmaceutical compositions comprising at least one

[0292] S-miR selected from the group consisting of SEQ. ID NOS. 1 to 20,

[0293] and / or

[0294] a sequence having at least 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 98%, 99% or 100% identity or similarity to the sequences S. SEQ. ID NOS. 1 to 20, and / orSEQ. ID NOS. 1 to 20 comprise at least one chemical modification selected from the group consisting of:

[0295] adding a methyl group to the 2' hydroxyl of the ribose of a 5’ or 3’ nucleotide or in the central portion of the sequence;

[0296] replacing an oxygen atom of the phosphate group with a sulphur atom on the phosphodiester backbone;

[0297] replacing the hydroxyl group at the 2’ position of the ribose with a fluorine atom in a 5’ or 3’ nucleotide or in the central portion of the sequence;

[0298] conjugation with lipids, of a 5’ or 3’ nucleotide.

[0299] or particles, nanoparticles or microparticles containing thereof

[0300] at least one pharmaceutically acceptable carrier and / or vehicle and / or pharmacologically acceptable excipients.

[0301] pharmaceutically acceptable carriers and vehicles and pharmacologically acceptable excipients are well known to those of skill in the art and are commercially available. The pharmaceutical compositions may further comprise in combination with the S-miRs at least one BRAF inhibitor (BRAFi) and / or a MEK inhibitor (MEKi).

[0302] The S-miRs and / or the pharmaceutical composition of the invention may be administered in any suitable manner.

[0303] Methods for preparing compositions are known or will be apparent to those skilled in the art, and are described in more detail in, for example, Remington's Pharmaceutical Science (17th ed., Mack Publishing Company, Easton, PA, 1985).

[0304] The dose to be administered is an amount sufficient to achieve the desired effect, in particular the treatment of melanoma. The physician may determine a suitable amount depending on the severity of the disease, the patient's condition, and other suitable parameters according to general knowledge in the art.

[0305] The S-miRs and the pharmaceutical compositions comprising them can be administered locally or systemically.A further object of the present invention is a dosage regimen that provides for the local or systemic administration of at least one

[0306] S-miR selected from the group consisting of SEQ. ID NOS. 1 to 20,

[0307] and / or

[0308] a sequence having at least 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 98%, 99% or 100% identity or similarity to the sequences S. SEQ. ID NOS. 1 to 20, and / or

[0309] SEQ. ID NOS. 1 to 20 comprise at least one chemical modification selected from the group consisting of:

[0310] adding a methyl group to the 2' hydroxyl of the ribose of a 5’ or 3’ nucleotide or in the central portion of the sequence;

[0311] replacing an oxygen atom of the phosphate group with a sulphur atom on the phosphodiester backbone;

[0312] replacing the hydroxyl group at the 2’ position of the ribose with a fluorine atom in a 5’ or 3’ nucleotide or in the central portion of the sequence;

[0313] conjugation with lipids, of a 5’ or 3’ nucleotide.

[0314] or particles, nanoparticles or microparticles containing thereof

[0315] or a pharmaceutical composition comprising at least one

[0316] S-miR selected from the group consisting of SEQ. ID NOS. 1 to 20,

[0317] and / or

[0318] a sequence having at least 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 98%, 99% or 100% identity or similarity to the sequences S. SEQ. ID NOS. 1 to 20, and / or

[0319] SEQ. ID NOS. 1 to 20 comprise at least one chemical modification selected from the group consisting of:adding a methyl group to the 2' hydroxyl of the ribose of a 5’ or 3’ nucleotide or in the central portion of the sequence;

[0320] replacing an oxygen atom of the phosphate group with a sulphur atom on the phosphodiester backbone;

[0321] replacing the hydroxyl group at the 2’ position of the ribose with a fluorine atom in a 5’ or 3’ nucleotide or in the central portion of the sequence;

[0322] conjugation with lipids, of a 5’ or 3’ nucleotide.

[0323] or particles, nanoparticles or microparticles containing thereof,

[0324] in combination with targeted therapy, namely with the systemic administration of at least one BRAF inhibitor (BRAFi) and / or a MEK inhibitor (MEKi).

[0325] This dosage regimen is useful to avoid relapses and selection of therapy-resistant cells. A further specific dosage regimen for the treatment of stage III melanoma involves the administration of local therapy by intra-lesional infusion with Talimogene laherparepvec (T-VEC) and local, intra- or peri-tumouralal administration, or by systemic route of at least one

[0326] S-miR selected from the group consisting of SEQ. ID NOS. 1 to 20,

[0327] and / or

[0328] a sequence having at least 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 98%, 99% or 100% identity or similarity to the sequences S. SEQ. ID NOS. 1 to 20, and / or

[0329] SEQ. ID NOS. 1 to 20 comprise at least one chemical modification selected from the group consisting of:

[0330] adding a methyl group to the 2' hydroxyl of the ribose of a 5’ or 3’ nucleotide or in the central portion of the sequence;

[0331] replacing an oxygen atom of the phosphate group with a sulphur atom on the phosphodiester backbone;replacing the hydroxyl group at the 2’ position of the ribose with a fluorine atom in a 5’ or 3’ nucleotide or in the central portion of the sequence;

[0332] conjugation with lipids, of a 5’ or 3’ nucleotide.

[0333] or particles, nanoparticles or microparticles containing thereof

[0334] or a pharmaceutical composition comprising at least one

[0335] S-miR selected from the group consisting of SEQ. ID NOS. 1 to 20,

[0336] and / or

[0337] a sequence having at least 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 98%, 99% or 100% identity or similarity to the sequences S. SEQ. ID NOS. 1 to 20, and / or

[0338] SEQ. ID NOS. 1 to 20 comprise at least one chemical modification selected from the group consisting of:

[0339] adding a methyl group to the 2' hydroxyl of the ribose of a 5’ or 3’ nucleotide or in the central portion of the sequence;

[0340] replacing an oxygen atom of the phosphate group with a sulphur atom on the phosphodiester backbone;

[0341] replacing the hydroxyl group at the 2’ position of the ribose with a fluorine atom in a 5’ or 3’ nucleotide or in the central portion of the sequence;

[0342] conjugation with lipids, of a 5’ or 3’ nucleotide.

[0343] or particles, nanoparticles or microparticles containing thereof.

[0344] A further dosage regimen involves local or systemic administration of at least one S-miR selected from the group consisting of SEQ. ID NOS. 1 to 20,

[0345] and / or

[0346] a sequence having at least 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 98%, 99% or 100% identity or similarity to the sequences S. SEQ. ID NOS. 1 to 20,and / or

[0347] SEQ. ID NOS. 1 to 20 comprise at least one chemical modification selected from the group consisting of:

[0348] adding a methyl group to the 2' hydroxyl of the ribose of a 5’ or 3’ nucleotide or in the central portion of the sequence;

[0349] replacing an oxygen atom of the phosphate group with a sulphur atom on the phosphodiester backbone;

[0350] replacing the hydroxyl group at the 2’ position of the ribose with a fluorine atom in a 5’ or 3’ nucleotide or in the central portion of the sequence;

[0351] conjugation with lipids, of a 5’ or 3’ nucleotide.

[0352] or particles, nanoparticles or microparticles containing thereof

[0353] or a pharmaceutical composition comprising at least one

[0354] S-miR selected from the group consisting of SEQ. ID NOS. 1 to 20,

[0355] and / or

[0356] a sequence having at least 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 98%, 99% or 100% identity or similarity to the sequences S. SEQ. ID NOS. 1 to 20, and / or

[0357] SEQ. ID NOS. 1 to 20 comprise at least one chemical modification selected from the group consisting of:

[0358] adding a methyl group to the 2' hydroxyl of the ribose of a 5’ or 3’ nucleotide or in the central portion of the sequence;

[0359] replacing an oxygen atom of the phosphate group with a sulphur atom on the phosphodiester backbone;

[0360] replacing the hydroxyl group at the 2’ position of the ribose with a fluorine atom in a 5’ or 3’ nucleotide or in the central portion of the sequence;conjugation with lipids, of a 5’ or 3’ nucleotide.

[0361] or particles, nanoparticles or microparticles containing thereof,

[0362] in conjunction with the systemic administration of at least one BRAF inhibitor (BRAFi) and / or one MEK inhibitor (MEKi), to reduce the local tumour mass and inhibit the emergence of resistant cells.

[0363] A method of treatment of a patient suffering from melanoma is also described, comprising the administration of the aforementioned S-miRs and / or particles comprising them and / or the aforementioned pharmaceutical compositions or the aforementioned dosing protocols / regimens in combination with other drugs or therapies known in the treatment of melanoma.

[0364] EXAMPLES

[0365] Example 1: Selection of S-miR sequences

[0366] Seven transcriptom ic data sets (RNAseq) and single cell sequencing data (scRNAseq) were analyzed in which cell lines or patient samples were collected before and after developing resistance to targeted therapy. Biological processes (BP) and pathways (metabolic pathways) have been identified for each cell line or patient sample. After analysis, common BPs and pathways were identified to obtain those most representative and those commonly activated in resistant cells. The most representative pathways are "Extracellular matrix organization", "Signalling by GPCR", and "O-linked glycosylation", while the most common BPs are "regulation of cell migration" and "circulatory system development".

[0367] All selected BP and pathway genes were collected and used as input to obtain the S-miR sequence. An artificial sequence functioning as a miRNA(miRNA-like), specifically selected to target a selection of these genes, was identified by means of an algorithm developed. Two S-miRs are selected with this algorithm, based on the type of starting gene analysis, called S-miR_8_7 and S-miR_10_8.

[0368] For S-miR_10_8, a selection of BPs and pathways were used to construct the sequence, while for S-miR_8_7, a network of causal interactions was constructed connecting the targets of S-miR_8_7 to the phenotypes identified using the ProxPath platform1. The sequences of the S-miRs are shown in Table 1 , along with the chemicalmodifications used to stabilize the S-miRs and make them less susceptible to degradation.

[0369] Table 7: Sequence and chemical modifications of S-miRs. Illustration of the 5’ - 3’ sequence of the S-miRs. Two strands that are joined together and form the miRNA-like dsRNA are shown. Strand 2 corresponds to the sense strand, strand 1 to the antisense strand.

[0370] Table 8

[0371] Name Oligo Sequence 5’ 3’ Legend

[0372] S-miR_8_7 Strand_1 X- AGCU = 2'-0-Me CUUUUUAUUAGGAGCUGCAGa

[0373] S-miR_8_7 Strand_2 z AGCU = RNA UCUGCAGCUCCUAAUAAAAAG UA- y

[0374] S- Strand_1 X- acgt = DNA miR_10_8 AGGAGGCACUUCCGCCCAGGa

[0375] S- Strand_2 z X = SpC3 miR_10_8 UCCUGGGCGGAAGUGCCUCCU

[0376] UA- y

[0377] y = Biotin

[0378] z = 5' Phos

[0379]

[0380] The method of operation of these S-miRs is the same as endogenous miRNAs, exploiting the same biochemical characteristics, yet, the molecular targets are different in that they depend on the sequence of the miRNA. Figure 2 shows the distribution of predicted genes targeted by S-miR_8_7 in the identified network. The homogenous distribution of target genes in the network is the key to efficiently inhibiting the entire network, rather than identifying a single gene, which represents a crucial node, and inhibiting the expression or function of that single gene.Example 2: Molecular Effect of S-miRs

[0381] To test the efficacy of S-miRs as therapeutics against melanoma, the molecular response following exogenous administration of S-miRs to melanoma cells (A375) was characterized. The presence of S-miRs was validated by RT-qPCR, normalizing against an endogenous calibrator. Increasing doses of S-miR_8_7 and S-miR_10_8 were tested in melanoma cells, specifically 1nM, 5nM, 15nM and 30nM as final dose. Figures 3Aand 3B confirm that increasing the dose of S-miR administered corresponds to an increase in the amount of S-miR measured by RT-qPCR within the cells. Subsequently, a target was selected for each of the two S-miRs and its expression was validated following S-miR treatment. A down-regulation of the messenger already at low doses of S-miR, such as 1nM, was confirmed for both S-miRs (Figure 3C and 3D). S-miR and Dabrafenib (BRAFi) were administered together for 10 days on sensitive cells, cells treated at 5 nM and 30 nM were subjected to RNA sequencing (RNAseq) to characterize the overall response of the cells following the combination of the two treatments. The PCA in Figure 4A shows that the cells cluster mainly according to the S-miR treatment, with a small dose-dependent difference, showing that the main difference is the S-miR treatment. Volcano plots (Figure 4 B, C) show differentially expressed genes. For S-miR_8_7, the predicted target genes, significantly modulated, are down-regulated. By increasing the dose to 30 nM, some of these targets are distributed in the volcano plot, with most of the target genes remaining downregulated anyway.

[0382] Example 3: S-miR on Resistant Cells

[0383] To test the efficacy of S-miRs as a treatment for melanoma, the phenotypic effect of S-miRs on melanoma cells resistant to targeted therapy (BRAFi) was tested. A BRAFi resistant melanoma cell line called A375-R3 was derived by culturing the cells with a BRAFi dose above the IC50 for 3 months. Sensitivity to BRAFi was tested by measuring the IC50 of A375-R3 and sensitive A375 (A375-S), showing how A375-R3 has a markedly reduced sensitivity (Figure 5A). A375-R3 was treated with S-miR and BRAFi for 7 days and the number and viability of the cells were analyzed with the Crystal Violet assay. A marked reduction in cell viability was observed, especially for S-miR_10_8. In the A375-R3 line, a reduction in viability of about 85% was observed, as shown in Figure 5.Example 4: S-miR as an Inhibitor of Pathways Leading to Resistance Subsequently, the ability of S-miRs to inhibit pathways leading to resistance was tested. To test this, sensitive cells were cultured with BRAFi and the two S-miRs for two weeks; on the fourteenth day, viability was analyzed using Crystal Violet. Figure 6 shows a marked reduction in cell viability, especially for S-miR_10_8.

[0384] To understand which dose corresponds to a cytotoxic effect, different doses - 1nM, 5nM, 15nM and 30nM - were administered to sensitive melanoma cells in combination with Dabrafenib for 10 days. Figure 7 shows the cytotoxic effect of the S-miRs, the 15 and 30 nM concentrations show the strongest efficacy with a comparable effect between them.

[0385] Example 5. Functional Binding Validation — Pull-Down Assay

[0386] To confirm the specificity of our computational pipeline, a pull-down assay was performed with biotinylated miRNA. Biotinylated Syn-mel-10-8 was administered to the cells and streptavidin capture recovered specifically bound RNA molecules. The sample was then analyzed by RT-qPCR, with comparison to a control miRNA (Syn-CTRL) devoid of predicted targets.

[0387] Real-time PCR quantification of the captured RNA fraction showed significant enrichment of our primary target genes compared to Syn-CTRL pull-down (foldenrichment >3x, p < 0.05). This biochemical confirmation demonstrates that Syn-mel-10-8 physically binds to the predicted target messenger RNAs, validating the consistency of the bioinformatics pipeline with the actual molecular activity (Figure 8). Example 6. In Vivo Proof of Concept and Delivery System Optimization

[0388] Phase I — First Generation Delivery (Conjugated with Cholesterol)

[0389] To assess the therapeutic potential of Syn-mel-10-8 in a physiological setting, a preliminary in vivo assay was performed using a standard nucleic acid delivery scaffold. Formulation: Fully modified miRNAs (2'-0-Me and 2'-F nucleotides for chemical stability) conjugated to cholesterol to facilitate passive cellular internalization.

[0390] Model: Human melanoma cells with acquired resistance implanted subcutaneously inimmunocompromised mice (Mus musculus, NOD-SCID). Upon reaching a predefined volume threshold, four intra-tumoural injections of Syn-mel-10-8 or Syn-CTRL were administered. Tumours were monitored with serial volumetric caliper measurement. Results: Significant reduction in tumour volume during the treatment window, confirming the biological efficacy of the Syn-mel-10-8 sequence in vivo (Figure 9). Limitation: The effect was limited to the duration of treatment, highlighting the need for a sustained release delivery system (Figure 9).

[0391] Phase II — New Generation PLGA Delivery System (Co-development)

[0392] A nanoparticle delivery system PLGA (poly-lactic-co-glycolic acid) was developed specifically optimized for Syn-mel-10-8. PLGA is FDA approved, biodegradable and allows a sustained and controlled release of the miRNA over days or weeks from a single administration.

[0393] Said system showed:

[0394] • prolonged release: unlike the multiple-dose cholesterol system, a single administration of PLGA guarantees superior and long-lasting silencing;

[0395] • In vitro superiority: viability assays on both sensitive and resistant cell lines confirmed deep and long-lasting down-regulation of resistance networks (Figure 10).

[0396] • Clonogenicity: colony formation assay — zero colonies (Figure 10). This is the key differentiating result that validates the PLGA system.

[0397] MATERIALSAND METHODS

[0398] Bioinformatics analysis and selection of S-miR sequences

[0399] The following transcriptom ics data was obtained from the GEO Data Sets platform: GSE97681, GSE205251, GSE103630, GSE103725, GSE203546, GSE202118, GSE108382. Raw counts were reanalyzed using the R program. Pathways, BPs, and phenotypes were obtained by annotation from the Gene Ontology database. The BROAD Institute GSEA was used to assess enrichment against collections of Hallmark gene sets curated by the BROAD Molecular Signatures Database version 7.4.1. SIGNOR database was used to identify causal interactions. The function to identify sequences of S-miRs was developed on the R program.RNAseq

[0400] The final sequencing libraries are 150 base-pair paired-end reads run on an Illumina NovaSeq platform. The quality of the reads was assessed using the FastQC tool (version 0.11.2, Babraham Institute, UK) and trimmed using TrimGalore software to remove adapters and low-quality bases (Q < 20). Subsequently, the reads were mapped to the Ensembl GRCh38 human reference genome using STAR version 2.5.0a, employing the gene annotations corresponding to Ensembl release 99, used to construct a transcriptome index and provided to STAR during alignment. The gene annotations were used to quantify read counts at the gene level using the HTSeq-count script version 0.8.0. Subsequently, transcriptom ic data normalization and differential gene expression (DEG) analysis were performed using the Bioconductor R packages RUVSeq version 1.28 and edgeR version 3.36. To identify genes with differential expression (DEGs), data are filtered to exclude from analysis genes with less than 1 count per million in less than 6 out of 12 total samples for each comparison. Volcano plots are created using the Bioconductor R EnhancedVolcano package version 1.12.0. Heatmap and hierarchical clustering generated with R pheatmap package version 1.0.12.

[0401] Real-Time qPCR

[0402] The total extraction of the RNA from the cells is carried out using the QIAGEN miRNeasy Mini kit, following the manufacturer's instructions. Quantitative PCR (qPCR) is performed in triplicate using the Sensitive SYBR Green from Meridian Bioscience and the StepOnePlus system (Applied Biosystems) for messenger RNA. The Hypoxanthine Phosphoribosyltransferase 1 (HPRT) gene is selected as a reference gene for normalization. Expression of miRNAs is quantified using the MiRCURY LNA SYBR Green PCR Kit, with normalization to the endogenous miR_194 control. All kits and tools were used according to manufacturer's recommendations.

[0403] Cell Cultures

[0404] A375 human melanoma cell line is obtained from the American Type Culture Collection (ATCC, USA). A375 is grown in DMEM (Euroclone SpA) medium supplemented with Glutamax, Pen / Strep and FBS. Cells were incubated in a humidified incubator at 37°C in a 5% CO2 atmosphere. All cell lines were confirmed to be free of mycoplasma bytesting prior to their use in experiments. The A375-R3 resistant line was derived by culturing the cells with Dabrafenib (BRAFi) at a concentration greater than the IC50 every 2-3 days.

[0405] Treatment

[0406] Ectopic expression of S-miRs is mediated using Lipofectamine RNAiMAX (Invitrogen) following the manufacturer's instructions.

[0407] Vitality Assay

[0408] Cell viability is tested with Crystal Violet. The cells are washed with PBS to remove debris and dead cells, fixed with 4% formaldehyde, washed again with PBS and allowed to dry. 2% Crystal Violet in 2% EtOH was added to the cells, then the plate was washed with bidistilled water. Quantification was performed by adding 10% acetic acid and measuring absorbance at 595 nm.

[0409] Accordingly, the present invention relates to:

[0410] • Sequence S-miR from SEQ.ID. NO. 1 to SEQ.ID,NO. 20, as well as sequences with at least 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 98%, 99% or 100% identity or similarity to said S-miRs as well as said S-miRs comprising at least one chemical modification selected from the group consisting of: addition of a methyl group to the 2' hydroxyl of the ribose of a 5’ or 3’ nucleotide or in the central portion of the sequence; replacement of an oxygen atom of the phosphate group with a sulphur atom on the phosphodiester backbone; replacement of the hydroxyl group in the 2' position of the ribose with a fluorine atom in a 5’ or 3’ nucleotide or in the central portion of the sequence; conjugation with lipids, of a 5’ or 3’ nucleotide and particles, preferably nanoparticles, more preferably lipid nanoparticles containing said S-miRs.

[0411] • The pharmaceutical compositions comprising S-miR above.

[0412] • Pharmaceutical compositions further comprising in combination at least one BRAF inhibitor (BRAFi) and / or a MEK inhibitor (MEKi).

[0413] • S-miR, particles and pharmaceutical compositions for use as a medicament in therapy, preferably in the treatment of melanoma at any stage and of anysubtype.

[0414] • Dosage regimen comprising the systemic or local administration of at least one S-miR and / or particles and / or pharmaceutical composition in combination with targeted therapy comprising the systemic administration of at least one BRAF inhibitor (BRAFi) and / or MEK inhibitor (MEKi) for the treatment of relapsed melanoma and / or selection of therapy-resistant melanoma tumour cells.

[0415] • Dosage regimen that provides for the systemic or local administration, intra- or peri-tumoural, of at least one S-miR s and / or particles and / or pharmaceutical composition in combination with local therapy by intra-lesional infusion with Talimogene laherparepvec (T-VEC) in conjunction with the systemic administration of at least one BRAF inhibitor (BRAFi) and / or one MEK inhibitor (MEKi), for the treatment of stage III melanoma.

[0416] • A dosage regimen that provides for the systemic or local administration, intra- or peri-tumoural, of at least one S-miR and / or particles and / or pharmaceutical composition s in conjunction with the systemic administration of at least one BRAF inhibitor (BRAFi) and / or one MEK inhibitor (MEKi), to reduce the local tumour mass and inhibit the onset of resistant cells.

[0417] Bibliography

[0418] 1. A Resource for the Network Representation of Cell Perturbations Caused by SARS-CoV-2 Infection - PubMed. https: / / pubmed.ncbi.nlm.nih.gov / 33809949 / .

[0419] 2. Rabbie, R., Ferguson, P., Molina-Aguilar, C., Adams, D. J. & Robles-Espinoza, C. D. Melanoma subtypes: genomic profiles, prognostic molecular markers and therapeutic possibilities. J Pathol 247, 539-551 (2019).

[0420] 3. Akbani, R. et al. Genomic Classification of Cutaneous Melanoma. Cell 161, 1681-1696 (2015).

[0421] 4. Long, G. V. et al. Dabrafenib plus trametinib versus dabrafenib monotherapy in patients with metastatic BRAF V600E / K-mutant melanoma: long-term survival and safety analysis of a phase 3 study. Ann Oncol 28, 1631-1639 (2017).

[0422] 5. Ascierto, P. A. et al. Update on tolerability and overall survival in COLUMBUS:landmark analysis of a randomised phase 3 trial of encorafenib plus binimetinib vs vemurafenib or encorafenib in patients with BRAF V600-mutant melanoma. Eur J Cancer 126, 33-44 (2020).

[0423] 6. Flaherty, K. T. et al. Combined BRAF and MEK inhibition in melanoma with BRAF V600 mutations. N Engl J Med 367, 1694-1703 (2012).

[0424] 7. Zhong, J. et al. BRAF Inhibitor Resistance in Melanoma: Mechanisms and Alternative Therapeutic Strategies. Curr Treat Options Oncol 23, 1503-1521 (2022).

[0425] 8. Diazzi, S., Tartare-Deckert, S. & Deckert, M. The mechanical phenotypic plasticity of melanoma cell: an emerging driver of therapy cross-resistance. Oncogenesis 12, 1-7 (2023).

[0426] 9. Meads, M. B., Gatenby, R. A. & Dalton, W. S. Environment-mediated drug resistance: a major contributor to minimal residual disease. Nat Rev Cancer 9, 665-674 (2009).

[0427] 10. Straussman, R. et al. Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion. Nature 487, 500-504 (2012).

[0428] 11. Sun, C. et al. Reversible and adaptive resistance to BRAF(V600E) inhibition in melanoma. Nature 508, 118-122 (2014).

[0429] 12. Bassot, A. et al. Identification of a miRNA multi-targeting therapeutic strategy in glioblastoma. Cell Death Dis 14, 630 (2023).

[0430] 13. Swetter, S. M. et al. NCCN Guidelines® Insights: Melanoma: Cutaneous, Version 2.2024. J Natl Compr Cane Netw 22, 290-298 (2024).

[0431] 14. Michielin, O., Akkooi, A. C. J. van, Ascierto, P. A., Dummer, R. & Keilholz, U. Cutaneous melanoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up f- Annals of Oncology 30, 1884-1901 (2019).

Claims

Claims1. An artificial miRNA-like sequence of sequence SEQ. ID. NO. or SEQ.ID. NO.18.

2. Sequences with at least 95%, 98%, 99% or 100% identity or similarity to the artificial miRNA-like sequences of claim 1.

3. The artificial miRNA-like sequence of claims 1 or 2 comprising a chemical modification selected from the group consisting of:adding a methyl group to the 2' hydroxyl of the ribose of a 5’ or 3’ nucleotide or in the central portion of the sequence;replacing an oxygen atom of the phosphate group with a sulphur atom on the phosphodiester backbone;replacing the hydroxyl group at the 2’ position of the ribose with a fluorine atom in a 5’ or 3’ nucleotide or in the central portion of the sequence; conjugation with lipids, of a 5’ or 3’ nucleotide.

4. Particles, preferably nanoparticles, more preferably lipid nanoparticles, comprising at least one artificial miRNA-like sequence according to any one of claims 1-3.

5. A pharmaceutical composition comprising at least one artificial miRNA-like sequence according to any one of claims 1-3 and / or particles according to claim 4 and at least one pharmaceutically acceptable carrier and / or vehicle and / or excipients.

6. The pharmaceutical composition according to claim 5 optionally further comprising in combination at least one BRAF inhibitor (BRAFi) and / or a MEK inhibitor (MEKi).

7. Artificial miRNA-like sequence according to any one of claims 1-3, particles according to claim 4, pharmaceutical composition according to any one of claims 5- 6 for use as a medicament in therapy.

8. The artificial miRNA-like sequence according to any one of claims 1-3, the particles according to claim 4, the pharmaceutical composition according to any one of claims 5-6 for use in the treatment of melanoma at any stage and of any subtype.

9. Dosage regimen providing for the systemic or local administration of at least one artificial miRNA-like sequence according to any one of claims 1-3 and / or particles according to claim 4 and / or pharmaceutical composition according to any one of claims 5-6 in combination with targeted therapy providing for the systemic administration of at least one BRAF inhibitor (BRAFi) and / or one MEK inhibitor (MEKi) for the treatment of relapsed melanoma and / or to prevent the selection of therapy-resistant melanoma tumour cells.

10. Dosage regimen providing for the systemic or local administration, intra- or peri-tumourally, of at least one artificial miRNA-like sequence according to any one of claims 1-3 and / or particles according to claim 4 and / or pharmaceutical composition according to any one of claims 5-6 in combination with local therapy by intra-lesional infusion with Talimogene laherparepvec (T-VEC) in conjunction with the administration systemically of at least one BRAF inhibitor (BRAFi) and / or one MEK inhibitor (MEKi), for the treatment of stage III melanoma.

11. Dosage regimen providing for the systemic or local, intra- or peri-tumoural administration of at least one artificial miRNA-like sequence according to any one of claims 1-3 and / or particles according to claim 4 and / or pharmaceutical composition according to any one of claims 5-6 in conjunction with the systemic administration of at least one BRAF inhibitor (BRAFi) and / or a MEK inhibitor (MEKi), to reduce the local tumour mass and inhibit the onset of resistant cells.

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