Gene delivery to cells of vectors comprising CRAT nucleotidic or aminoacidic sequences

Abstract

Compositions and methods for the delivering of vectors comprising a CRAT nucleotidic or aminoacidic sequences to cells in mammals, including human subjects and their use, and methods, for the treatment of pathologies related to cellular trans-differentiation, activation of fibrosis, activation of inflammation and neo-angiogenesis, cancer, pathologies based on elevated lactic acid and lactate plasma levels, are disclosed.

Description

[0001] GENE DELIVERY TO CELLS OF VECTORS COMPRISING CRAT NUCLEOTIDIC OR AMINOACIDIC SEQUENCES

[0002] The present invention refers to compositions and methods for the delivering of vectors comprising a CRAT nucleotidic or aminoacidic sequences to cells in mammals, including human subjects and their use, and methods, for the treatment of pathologies related to cellular trans-differentiation, activation of fibrosis, activation of inflammation and neoangiogenesis, cancer, pathologies based on elevated lactic acid and lactate plasma levels.

[0003] Background of the invention

[0004] The pathophysiology of organ fibrosis involves various cellular effectors (neutrophils, macrophages, fibroblasts), up-regulation of profibrotic mediators (cytokines, chemokines, and growth factors), and processes where epithelial and endothelial cells undergo mesenchymal transition. Activated fibroblasts and myofibroblasts are the central cellular effectors in organ fibrosis, serving as the main source of matrix proteins. Cellular trans-differentiation such epithelial-mesenchymal transition, mesothelial- mesenchymal transition is characterized by cell hyperpro I iteration and / or collagen hyperproduction. These processes may be associated with pulmonary fibrosis, liver fibrosis, peritoneal fibrosis, inflammatory bowel disease, renal fibrosis, myocardial fibrosis, endometriosis, encapsulating peritoneal sclerosis and / or cancer. Cancer cell may be derived from a carcinoma, a sarcoma, a breast cancer, a pancreatic cancer, a lung cancer, a leukemia, a lymphoma, a brain cancer, a melanoma, a liver cancer, a stomach cancer, a small intestine cancer, a large intestine cancer, a kidney cancer, peritoneal cancer, a uterine cancer, an ovarian cancer, a bladder cancer, or a bone cancer.

[0005] Bonomini et al. "How to Improve the Biocompatibility of Peritoneal Dialysis Solutions (without Jeopardizing the Patient's Health)", Int J Mol Sci. 2021 Jul 26;22(15):7955. doi: 10.3390 / ijms22157955 disclose solutions for peritoneal dialysis with reduced glucose content and comprising different osmotically active ingredients optimised to control oxidative metabolism within the peritoneal membrane, to improve the metabolic profile of patients, who maybe glucose-intolerant, insulin-resistant, or diabetic, and for whom daily exposure to high doses of glucose is contraindicated.

[0006] Bonomini, et al. "The osmo-metabolic approach: A novel and tantalizing glucose-sparing strategy in peritoneal dialysis". J. Nephrol. 2021, 34: 503-519, doi: 10.1007 / s40620-020- 00804-2 disclose solutions for peritoneal dialysis wherein glucose is replaced by other osmolytes efficient in removing fluids as glucose, but less impactful on patient metabolism, such as icodextrin and amino acids, taurine, polyglycerol, carnitine and xylitol).

[0007] US patent n. US10874637 discloses methods and pharmaceutical compositions for the treatment of neurodegeneration in with brain iron accumulation (NBIA). It reports studies on CRAT encoding the carnitine acetyltransferase and REPSI involved in endocytosis and vesicle transport, and NBIA genes, reporting that a common feature in NBIA is iron overload which is related to increased levels and abnormal recycling of transferrin receptor as a common feature in NBIA, due to impaired palmitoylation of the receptor as a common consequence of the various disease causing mutations; and also shows that Artesunate improved TfRl palmitoylation in NBIA fibroblasts.

[0008] Guoyu Yu et al., Organelle-Derived Acetyl-CoA Promotes Prostate Cancer Cell Survival, Migration, and Metastasis via Activation of Calmodulin Kinase II, Cancer Res, 2018 May 15;78(10):2490-2502. doi: 10.1158 / 0008-5472.CAN-17-2392. Epub 2018 Mar 13 identifies a cell metabolic pathway that promotes prostate cancer metastasis and suggests prostate cancer may be susceptible to p-oxidation inhibitors. Disclose that acetyl CoA-CaMKII pathway promotes prostate cancer tumorigenesis and deletion of CaMKII by CRISPR / Cas9 in tumour cells abrogated cell survival, anchorage-independent growth and cell migration, while in animal model of prostate cancer metastasis, genetic ablation of CaMKII reduces cell metastasis; knockdown of the acetyl-CoA transporter carnitine acetyltransferase abolished CaMKII activation. Lasheras-Otero I. et al, The Regulators of Peroxisomal Acyl-Carnitine Shuttle CROT and CRAT Promote Metastasis in Melanoma, J Invest Dermatol. 2023 Feb;143(2):305-316.e5. doi: 10.1016 / j.jid.2022.08.038. Epub 2022 Sep 1 discloses that detached melanoma cells and isolated melanoma circulating tumour cells rewire lipid metabolism by upregulating fatty acid (FA) transport and FA beta-oxidation-related genes and in patients with melanoma, high expression of FA transporters and FA beta-oxidation enzymes significantly correlates with reduced progression-free and overall survival; carnitine transferases carnitine O-octanoyltransferase and carnitine acetyltransferase control the shuttle of peroxisome-derived medium-chain FAs toward mitochondria to fuel mitochondrial FA beta-oxidation and knockdown of carnitine O-octanoyltransferase or carnitine acetyltransferase and short-term treatment with peroxisomal or mitochondrial FA beta-oxidation inhibitors thioridazine or ranolazine suppressed melanoma metastasis in mice.

[0009] These two scientific papers (Guoyu Yu et al., 2018 and Lasheras-Otero I. et al, 2022, previously cited) suggest CraT is involved in the development of neoplasms.

[0010] W02004 / 028479 discloses sequence GSN:ADN04428 (length: 2804), relates to polynucleotides and polypeptides for treating psoriasis and nucleic acid useful for preparing a composition for diagnosing or treating psoriasis in a mammal.

[0011] WO2017 / 191274 discloses sequence GSP:BFB25973 (length: 626) and relates to RNA encoding therapeutic proteins which can be used for preparing a pharmaceutical composition for use in gene therapy for the treatment and prophylaxis of inherited or acquired diseases, disorders or conditions, e.g. metabolic or endocrine disorders, cancer, infectious diseases or immunodeficiencies.

[0012] WO 02 / 22660 discloses sequence GSP:ABB97444 and discloses a protein used to stimulate cell growth, to regulate haematopoiesis e.g. to treat aplastic anaemia, to help tissue regrowth e.g. in burn treatment, to regulate the immune system e.g. to treat multiple sclerosis, to regulate activin or inhibin e.g. to treat infertility, to regulate haemostasis or thrombolysis e.g. to treat stroke and cancer, to screen for drugs, to treat inflammatory conditions e.g. rheumatoid arthritis, and to treat nervous system disorders e.g. Parkinson's disease.

[0013] It is known that the long-term exposure to the high glucose load of peritoneal dialysis (RD) fluids can induce peritoneal fibrosis causing loss of peritoneal ultrafiltration capacity.

[0014] TGF-beta is a master regulator factor in the onset of peritoneal fibrosis and recent findings suggest that production of it is dependent on a hyper-glycolytic state caused by glucose-based PD solutions.

[0015] As known in the art different solutions for peritoneal dialysis are under investigation and they are based on reducing the amount of glucose, and using osmotic-metabolic agents that may provide metabolic benefits able to mitigate local and systemic glucose over- exposure.

[0016] The inventors of the present invention investigated the role of carnitine acetyltransferase (CrAT), also in combination with other active ingredients, such as L- carnitine, on the modulation of TGF-beta pro-fibrotic effects.

[0017] They surprisingly and unexpectedly found that inducing CrAT-overexpression, in different types of cells, may be a useful tool in avoiding the development of fibrosis, inflammation as well as the production of Vascular Endothelial Growth Factor (VEGF), being a proangiogenic factor.

[0018] CRAT overexpression influences cellular trans-differentiation, activation of fibrosis and inflammation and neoangiogenesis, in different types of cells and therefore can be useful for the treatment of many pathologies.

[0019] The above technical problem is therefore solved by providing a vector comprising a CRAT nucleotidic sequence. Also providing said vector for use in a method for treating pathologies related to cellular trans-differentiation, activation of fibrosis, activation of inflammation and neoangiogenesis, cancer, pathologies based on elevated lactic acid and lactate plasma levels,

[0020] Said vector is administered by enteral routes of administration, parenteral routes of administration, Inhalation route of administration, transcutaneous routes of administration wherein said administration causing over-expression of CRAT gene in host cells.

[0021] It is also object of the present invention a pharmaceutical composition comprising a vector comprising a CRAT nucleotidic sequence, and optionally further active ingredients, for use for the treatment and prevention of treating pathologies related to cellular trans-differentiation, activation of fibrosis, activation of inflammation and neoangiogenesis, cancer, pathologies based on elevated lactic acid and lactate plasma levels.

[0022] Brief description of drawings

[0023] Figure 1 shows in graph the relative CRAT gene expression in several clones, wild type and CRAT overexpressing.

[0024] Figure 2 shows the results of protein western blot analysis.

[0025] Figure 3 shows in graph relative expression of mesenchymal markers alphaSMA and Vimentin in wild type and CRAT overexpressing cells treated with different L-carnitine concentrations.

[0026] Figure 4 shows morphological aspects of wild type and CRAT-overexpressing cell treated with TGF-beta.

[0027] Figure 5 shows in graph relative expression IL-6 and IL-1B in wild type and CRAT overexpressing cells treated with different L-carnitine concentrations. Figure 6 shows in graph relative expression VEGF in wild type and CRAT overexpressing cells treated with different L-carnitine concentrations.

[0028] Figure 7 shows in graphs expression of alpha-SMA, VIM, IL-6 and IL-lbeta in WT mesothelial cells treated with TGF-beta added or not with DCA (wherein*p<0.05; **p<0.001 vs Control Untreated, #p<0.05; # #p<0.001 vs TGFb Untreated).

[0029] Figure 8 shows partial least square-discriminant analysis of metabolic profiles of WT and CrAT overexpressing mesothelial cells.

[0030] Detailed description of the invention

[0031] Within the meaning of the present invention, CRAT nucleotidic sequence means DNA or RNA or cDNA sequence being or corresponding to the coding sequence of CRAT gene.

[0032] Within the meaning of the present invention, the CRAT gene means the gene encoding for the protein coding region of the CRAT CDNA ORF (with open reading frame).

[0033] Within the meaning of the present invention, CRAT aminoacidic sequence means aminoacidic sequence of the enzyme Carnitine O-Acetyltransferase.

[0034] Within the meaning of the present invention vector means any system capable of transporting exogenous nucleotidic or aminoacidic sequences within a host cell and allowing its integration into the host genome and / or its expression in the host cell.

[0035] Within the meaning of the present invention, the vector is any standard vector allowing transfection and / or expression of CRAT in a host cell. The vector is any vector suitable to infect mammalian cells and human cells. The vector can be a plasmid.

[0036] Within the meaning of the present invention, suitable vectors are disclosed in Shugang Qin et al., Review Article Open, mRNA-based therapeutics: powerful and versatile tools to combat diseases, Signal Transduction and Targeted Therapy (2022) 7:166; https: / / doi.org / 10.1038 / s41392-022-01007-w, herein incorporated by reference. When the vector comprises a nucleotidic sequences may also comprises at least one controlling sequence upstream or downstream or operably linked to CRAT nucleotide sequence allowing or helping its integration into the host genome and / or its expression in the host cell.

[0037] Within the meaning of the present invention, host cell means any type of cell transfected with a vector and expressing the CRAT gene.

[0038] Preferably host cell is selected from the group consisting of: mesothelial cells, epithelial cells, pneumocytes, fibroblasts, endothelial cells, inflammatory cells, such as T- lymphocyte and macrophages, cardiomyocytes.

[0039] Within the meaning of the present invention, pathologies related to cellular transdifferentiation, activation of fibrosis, activation of inflammation and neo-angiogenesis, means pulmonary fibrosis, liver fibrosis, peritoneal fibrosis, inflammatory bowel disease, renal fibrosis, polycystic kidney disease, myocardial fibrosis, endometriosis, encapsulating peritoneal sclerosis, cancer, pulmonary hypertension, sepsis, vascular dementia.

[0040] Within the meaning of the present invention, pathologies based on elevated lactic acid and lactate plasma levels means ischemic crisis, hypoxic crisis, stroke, cardiac arrest, heart attacks, diabetic ketoacidosis.

[0041] Within the meaning of the present invention, cancer means carcinoma, sarcoma, breast cancer, pancreatic cancer, lung cancer, leukemia, lymphoma, brain cancer, melanoma, liver cancer, stomach cancer, small intestine cancer, large intestine cancer, kidney cancer, peritoneal cancer, uterine cancer, ovarian cancer, bladder cancer, bone cancer.

[0042] Object of the present invention is a vector comprising a CRAT nucleotidic sequence selected from the group consisting of SEQ. ID NO 1, SEQ.ID.NO.4, SEQ.ID.NO.6, SEQ.ID.NO.8, SEQ.ID.NO.10, SEQ.ID.NO.12. Another object of the present invention is a vector comprising a CRAT nucleotidic sequence selected from the group consisting of SEQ. ID NO 1, SEQ.ID.NO. 2, SEQ.ID.NO.

[0043] 3, SEQ.ID.NO.4, SEQ.ID.NO.5, SEQ.ID.NO.6, SEQ.ID.NO.7, SEQ.ID.NO.8, SEQ.ID.NO.9, SEQ.ID.NO.10, SEQ.ID.NO.il, SEQ.ID.N0.12, SEQ.ID.N0.13 for use in a method for treating pathologies related to cellular trans-differentiation, activation of fibrosis, activation of inflammation and neo-angiogenesis selected from the group consisting of: pulmonary fibrosis, liver fibrosis, peritoneal fibrosis, inflammatory bowel disease, renal fibrosis, polycystic kidney disease, myocardial fibrosis, endometriosis, encapsulating peritoneal sclerosis, pulmonary hypertension, sepsis, vascular dementia, cancer, pathologies based on elevated lactic acid and lactate plasma levels selected from the group consisting of: ischemic crisis, hypoxic crisis, cardiac arrest, heart attacks, diabetic ketoacidosis wherein said vector is administered by enteral routes of administration, parenteral routes of administration, inhalation routes of administration, transcutaneous routes of administration wherein said administration causing over-expression of CRAT gene in host cells.

[0044] Another object of the present invention is a vector comprising a CRAT nucleotidic sequence selected from the group consisting of SEQ. ID NO 1, SEQ.ID.NO.4, SEQ.ID.NO.6, SEQ.ID.NO.8, SEQ.ID.NO.il, SEQ.ID.N0.13 for use in a method for treating pathologies related to cellular trans-differentiation, activation of fibrosis, activation of inflammation and neo-angiogenesis selected from the group consisting of: pulmonary fibrosis, liver fibrosis, peritoneal fibrosis, inflammatory bowel disease, renal fibrosis, polycystic kidney disease, myocardial fibrosis, endometriosis, encapsulating peritoneal sclerosis, pulmonary hypertension, sepsis, vascular dementia, cancer, maculopathy. pathologies based on elevated lactic acid and lactate plasma levels selected from the group consisting of: ischemic crisis, hypoxic crisis, stroke, cardiac arrest, heart attacks, diabetic ketoacidosis, wherein said vector is administered by enteral routes of administration, parenteral routes of administration, inhalation routes of administration, transcutaneous routes of administration wherein said administration causing over-expression of CRAT gene in host cells.

[0045] Preferably the vector is selected from the group consisting of: lipid nanoparticles, protamine-condensed mRNAs, exosomes, extracellular vesicles, (EVs), mesoporous silica, CaP, plasmids, lipid nanoparticles with immunostimulatory potency, polymetric nanoparticles, polycationic peptides, polycationic proteins, crown-compounds.

[0046] Preferably the vector is plasmid.

[0047] Preferably lipid nanoparticles are cationic lipid nanoparticles, ionizable lipid nanoparticles.

[0048] More preferably cationic lipid nanoparticles for mRNA-CrAT delivery are selected from the group consisting of: N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), l,2-dioleoyloxy-3-trimethylammonium propane chloride (DOTAP), l,2-stearoyl-3-trimethylammonium-propane (DSTAP), and l,2-dimyristoyl-3- trimethylammonium-propane (DMTAP), dimethyldioctadecylammonium (DDA), DOTAP, DMTAP, DSTAP, N-(4-carboxybenzyl)-N,Ndimethyl-2,3-bis (oleoyloxy) propan-1- aminium (DOBAQ) and 3R-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC- Chol) can be used in combination with l,2-dioleoyl-sn-3-phosphoethanolamine (DOPE).

[0049] More preferably ionizable lipid nanoparticles for mRNA-CrAT delivery are selected from the group consisting of: amino lipid Dlin-MC3-DMA (MC3) either alone or in combination with DSPC, cholesterol, DMG-PEG2000, and DSPE-PEG2000 (50:10.5:38:1.4:0.1 mol ratio). More preferably polycationic peptides or polycationic proteins for mRNA-CrAT delivery are selected from the group consisting of: protamine, spermin or spermidine, polylysine, poly-arginine, cationic polysaccharides, including chitosan, cationic polymers, including poly( ethyleneimine ), poly(pro-pyleneimine ), polybrene, polyallylamines, and polyvi-nylamine, in particular the polymer comprises a polyami-doamine (PAMAM) polymer.

[0050] When the vector is for aminoacidic sequence it can be a crown-compound of formula (I):

[0051] Chemical Name: l,4,6,9,12,15,18,21-Octaoxa-spiro[4.17]docosane-2,3-dicarboxylic acid diethyl ester; a schematic of the structure is presented in below.

[0052] Chemical Elemental Structure: C20H34O12

[0053] Molecular Weight: 466.48 g / mol

[0054] Preferably the host cell is selected from the group consisting of: mesothelial, epithelial, pneumocytes, fibroblasts, endothelial cells, inflammatory cells such as T-lymphocyte, macrophages, cardiomyocytes.

[0055] Preferably pathologies related to cellular trans-differentiation, activation of fibrosis, activation of inflammation and neo-angiogenesis are selected from the group consisting of: pulmonary fibrosis, liver fibrosis, peritoneal fibrosis, inflammatory bowel disease, renal fibrosis, polycystic kidney disease, myocardial fibrosis, endometriosis, encapsulating peritoneal sclerosis, cancer, pulmonary hypertension, sepsis, vascular dementia, maculopathy.

[0056] Preferably cancer is selected from the group consisting of carcinoma, sarcoma, breast cancer, pancreatic cancer, lung cancer, leukemia, lymphoma, brain cancer, melanoma, liver cancer, stomach cancer, small intestine cancer, large intestine cancer, kidney cancer, peritoneal cancer, uterine cancer, ovarian cancer, bladder cancer, bone cancer.

[0057] Within the meaning of the present invention, pathologies based on elevated lactic acid and lactate plasma levels means ischemic crisis, hypoxic crisis, stroke, cardiac arrest, heart attacks, diabetic ketoacidosis.

[0058] Preferably enteral route of administration is selected from the group consisting of: oral administration, sublingual administration, rectal administration.

[0059] Preferably parenteral route of administration is selected from the group consisting of: intravascular administration, intramuscular administration, subcutaneous administration, intradermal administration, intraperitoneal administration, intrapleural administration, intracolonic administration, intravesical administration, intrauterine administration, intraurethral administration.

[0060] The CRAT sequences are:

[0061] SEQ. ID. 1 CRAT ORF (in bold the Cloning site)

[0062] TTTTGTAATACGACTCACTATAGGGCGGCCGGGAATTCGTCGACTGGATCCGGTACCGAGGAGA TCTGCCGCCGCGATCGCCTGTTAGCCTTCGCTGCCAGGACCGTGGTGAAGCCTCTGGGCTTCCT GAAGCCCTTCTCCTTGATGAAGGCTTCCAGCCGCTTCAAGGCACACCAGGATGCACTGCCACGG CTGCCCGTGCCCCCTCTCCAGCAGTCCCTGGACCACTACCTGAAGGCGCTGCAGCCCATCGTGA GTGAGGAGGAGTGGGCCCACACCAAGCAGCTGGTGGATGAGTTTCAGGCCTCAGGAGGTGTA GGGGAGCGCCTGCAGAAGGGGCTGGAGCGTCGGGCCAGGAAGACGGAGAACTGGCTGTCTG AGTGGTGGCTCAAGACCGCCTACCTCCAGTACCGCCAGCCTGTGGTCATCTACTCGAGCCCAGG

[0063] CGTGATGCTACCCAAGCAGGACTTCGTGGACCTGCAGGGTCAGCTCCGATTTGCTGCCAAACTC

[0064] ATTGAGGGTGTGTTGGATTTCAAGGTCATGATTGACAACGAGACCCTGCCCGTGGAGTACCTGG

[0065] GGGGGAAGCCACTGTGCATGAACCAGTACTATCAGATCTTGTCCTCCTGCCGAGTGCCGGGCCC

[0066] CAAGCAGGACACAGTCAGCAACTTCAGCAAGACCAAGAAGCCTCCCACGCACATCACCGTGGT

[0067] ACACAACTACCAGTTTTTTGAGCTGGATGTGTACCACAGTGACGGGACACCCCTCACTGCGGAT

[0068] CAGATCTTTGTGCAGCTGGAGAAGATCTGGAACTCATCCCTACAGACCAACAAGGAGCCTGTGG

[0069] GCATCCTCACCTCCAACCACCGCAACTCCTGGGCCAAGGCATACAACACCCTCATCAAAGACAA

[0070] GGTGAACCGGGATTCCGTGCGCTCCATCCAGAAGAGCATCTTCACCGTGTGCCTAGATGCAACC

[0071] ATGCCCAGGGTCTCAGAAGACGTGTACCGCAGCCACGTGGCAGGCCAGATGCTGCATGGGGGC

[0072] GGCAGCAGGCTCAACAGCGGCAACCGCTGGTTCGACAAGACGCTGCAGTTCATCGTGGCAAA

[0073] AGATGGCTCCTGTGGGCTTGTGTACGAGCATGCTGCAGCGGAGGGGCCCCCTATTGTCACCCTT

[0074] CTGGACTATGTCATCGAGTACACGAAGAAACCCGAGCTTGTGCGGTCTCCCATGGTGCCCCTGC

[0075] CCATGCCCAAGAAGCTGCGGTTCAACATCACCCCCGAGATCAAGAGCGACATCGAGAAGGCCA

[0076] AGCAGAACCTCAGCATCATGATCCAGGACCTGGATATCACCGTGATGGTGTTCCACCATTTTGGA

[0077] AAAGACTTCCCCAAGTCGGAGAAGCTAAGCCCAGATGCCTTCATCCAGATGGCTTTGCAGCTGG

[0078] CCTACTACAGGATCTACGGACAGGCATGTGCCACCTATGAAAGTGCCTCCCTGCGCATGTTTCAC

[0079] CTGGGCCGCACCGACACCATCCGCTCGGCTTCCATGGACTCACTCACCTTTGTCAAGGCCATGG

[0080] ATGACTCCAGCGTCACGGAGCACCAGAAGGTGGAGCTGCTGCGGAAGGCCGTGCAGGCCCAC

[0081] CGAGGCTACACCGACCGGGCCATCCGCGGGGAGGCCTTTGATCGACACCTGCTGGGCCTGAAG

[0082] CTGCAGGCCATCGAGGACCTGGTGAGCATGCCCGACATCTTCATGGACACCTCCTACGCCATCG

[0083] CCATGCACTTCCACCTCTCCACCAGCCAGGTCCCTGCCAAGACAGACTGTGTCATGTTCTTCGGG

[0084] CCCGTGGTCCCCGACGGCTACGGTGTCTGCTATAACCCCATGGAGGCCCACATCAACTTCTCCCT

[0085] GTCGGCCTACAACAGCTGCGCGGAGACCAACGCCGCCCGCCTGGCGCATTACCTGGAGAAGGC

[0086] GCTCCTGGACATGCGTGCCCTGCTGCAGAGCCACCCCCGGGCCAAGCTCACGCGT

[0087] SEQ. ID. 2 ENST00000318080.7 CRAT-201 cdna:protein_coding GTCCCCGCCCCACAGCCAACCTCCCGGGCACCGTTGCCCGCCCGGGGCCCGCTACCGGCCCAG

[0088] GCCCCGCCTCGCGAGTCCTCCTCCCCGGGTGCCTTCCCGCAGCCCGCTCGGCCCAGAGGGTGG

[0089] GCGCGGGGCTGCCTCTACCGGCTGGCGGCTGTAACTCAGCGACCTTGGCCCGAAGGCTCTAGC

[0090] AAGGACCCACCGACCCCAGCCGCGGCGGCGGCGGCGCGGACTTTGCCCGGTGTGTGGGGCG GAGCGGACTGCGTGTCCGCGGACGGGCAGCGAAGATGTTAGCCTTCGCTGCCAGGACCGTGG

[0091] TGAAGCCTCTGGGCTTCCTGAAGCCCTTCTCCTTGATGAAGGCTTCCAGCCGCTTCAAGGCACA CCAGGATGCACTGCCACGGCTGCCCGTGCCCCCTCTCCAGCAGTCCCTGGACCACTACCTGAAG

[0092] GCGCTGCAGCCCATCGTGAGTGAGGAGGAGTGGGCCCACACCAAGCAGCTGGTGGATGAGTT TCAGGCCTCAGGAGGTGTAGGGGAGCGCCTGCAGAAGGGGCTGGAGCGTCGGGCCAGGAAG

[0093] ACGGAGAACTGGCTGTCTGAGTGGTGGCTCAAGACCGCCTACCTCCAGTACCGCCAGCCTGTG GTCATCTACTCGAGCCCAGGCGTGATGCTACCCAAGCAGGACTTCGTGGACCTGCAGGGTCAGC

[0094] TCCGATTTGCTGCCAAACTCATTGAGGGTGTGTTGGATTTCAAGGTCATGATTGACAACGAGAC CCTGCCCGTGGAGTACCTGGGGGGGAAGCCACTGTGCATGAACCAGTACTATCAGATCTTGTCC

[0095] TCCTGCCGAGTGCCGGGCCCCAAGCAGGACACAGTCAGCAACTTCAGCAAGACCAAGAAGCC TCCCACGCACATCACCGTGGTACACAACTACCAGTTTTTTGAGCTGGATGTGTACCACAGTGACG

[0096] GGACACCCCTCACTGCGGATCAGATCTTTGTGCAGCTGGAGAAGATCTGGAACTCATCCCTACA GACCAACAAGGAGCCTGTGGGCATCCTCACCTCCAACCACCGCAACTCCTGGGCCAAGGCATA

[0097] C AACACCCTC ATCAAAG AC AAG GTG AACCG GG ATTCCGTGCG CTCCATCCAG AAG AG CATCTTC ACCGTGTGCCTAGATGCAACCATGCCCAGGGTCTCAGAAGACGTGTACCGCAGCCACGTGGCA

[0098] GGCCAGATGCTGCATGGGGGCGGCAGCAGGCTCAACAGCGGCAACCGCTGGTTCGACAAGAC GCTGCAGTTCATCGTGGCAGAAGATGGCTCCTGTGGGCTTGTGTACGAGCATGCTGCAGCGGA

[0099] GGGGCCCCCTATTGTCACCCTTCTGGACTATGTCATCGAGTACACGAAGAAACCCGAGCTTGTG CG GTCTCCCCTG GTG CCCCTG CCCATG CCC AAG AAG CTG CG GTTC AACATC ACCCCCG AG ATCA

[0100] AGAGCGACATCGAGAAGGCCAAGCAGAACCTCAGCATCATGATCCAGGACCTGGATATCACCGT GATGGTGTTCCACCATTTTGGAAAAGACTTCCCCAAGTCGGAGAAGCTAAGCCCAGATGCCTTC

[0101] ATCCAGATGGCTTTGCAGCTGGCCTACTACAGGATCTACGGACAGGCATGTGCCACCTATGAAA

[0102] GTGCCTCCCTGCGCATGTTTCACCTGGGCCGCACCGACACCATCCGCTCGGCTTCCATGGACTCA CTCACCTTTGTCAAGGCCATGGATGACTCCAGCGTCACGGAGCACCAGAAGGTGGAGCTGCTG

[0103] CGGAAGGCCGTGCAGGCCCACCGAGGCTACACCGACCGGGCCATCCGCGGGGAGGCCTTTGA

[0104] TCGACACCTGCTGGGCCTGAAGCTGCAGGCCATCGAGGACCTGGTGAGCATGCCCGACATCTTC

[0105] ATGGACACCTCCTACGCCATCGCCATGCACTTCCACCTCTCCACCAGCCAGGTCCCTGCCAAGAC

[0106] AGACTGTGTCATGTTCTTCGGGCCCGTGGTCCCCGACGGCTACGGTGTCTGCTATAACCCCATGG

[0107] AGGCCCACATCAACTTCTCCCTGTCGGCCTACAACAGCTGCGCGGAGACCAACGCCGCCCGCCT

[0108] GGCGCATTACCTGGAGAAGGCGCTCCTGGACATGCGTGCCCTGCTGCAGAGCCACCCCCGGGC

[0109] CAAGCTCTGAGCCCCTAGGACTCAGGCCTGCCAATGCCACAGCCAAGCCCACCCTGGGATGGG

[0110] CCACCCACCAGGGCTCAGCTCCTTGGTTCCCTCTTCCTTGGTTCCCTCTTCCCTGGTCCCCCCAA

[0111] ATCTACTGAGCCACGGACCGCATCCTCCAGGGGGCCTGCAGGCCCCAGCCAAGTGCCTTCCGT

[0112] GGGTCATCCCAGCACCTGCCAGGGCCCGACCTGGGGCTGAGTGCAGAGGCTGAGCAGGACGT

[0113] TAGGCCCGGGCCCCTGGCACGTCCTCCACCGGTGCCTCCTCTGGGGAAGGGAACCAGCCCTCC

[0114] AGAGCAGGAGACTGGCAAGAGCTCTTTGTCTCACCAGCTCAGCCCCGGCCACTCCCTGCCAAC

[0115] TCCATGACCAGGCCACCATCTGTACCCTGCTTCCCAAACTCCCAGGACCTGGAGACAGGATTGTC

[0116] TGGGGCCGAGGGGCCAGGGTGTGAGGTTTCACCTCCGTTGCGGCTGTGCTCCTGTGGATAACA

[0117] TTGCTAGCGAGCCGCCTCTGGTTCCACTCAGCTTGGTTCCTGCCCCCGCCCTGCTGTATGATATTA

[0118] ATGTGGAAGGTCATCAATAAAGGGGAATTGTGTTGGTGAA

[0119] SEQ. ID. 4 ENST00000681627.1 CRAT-219 cdna:protein_coding

[0120] GGCCCAGGCCCCGCCTCGCGAGTCCTCCTCCCCGGGTGCCTTCCCGCAGCCCGCTCGGCCCAG

[0121] AGGGTGGGCGCGGGGCTGCCTCTACCGGCTGGCGGCTGTAACTCAGCGACCTTGGCCCGAAG

[0122] GCTCTAGCAAGGACCCACCGACCCCAGCCGCGGCGGCGGCGGCGCGGACTTTGCCCGGTGTG

[0123] TGGGGCGGAGCGGACTGCGTGTCCGCGGACGGGCAGCGAAGATGTTAGCCTTCGCTGCCAGG

[0124] ACCGTGGTGAAGCCTCTGGGCTTCCTGAAGCCCTTCTCCTTGATGAAGGCTTCCAGCCGCTTCA

[0125] AGGCACACCAGGATGCACTGCCACGGCTGCCCGTGCCCCCTCTCCAGCAGTCCCTGGACCACTA

[0126] CCTGAAGGCGCTGCAGCCCATCGTGAGTGAGGAGGAGTGGGCCCACACCAAGCAGCTGGTGG

[0127] ATGAGTTTCAGGCCTCAGGAGGTGTAGGGGAGCGCCTGCAGAAGGGGCTGGAGCGTCGGGC CAGGAAGACGGAGAACTGGCTGTCTGAGTGGTGGCTCAAGACCGCCTACCTCCAGTACCGCCA

[0128] GCCTGTGGTCATCTACTCGAGCCCAGGCGTGATGCTACCCAAGCAGGACTTCGTGGACCTGCAG

[0129] GGTCAGCTCCGATTTGCTGCCAAACTCATTGAGGGTGTGTTGGATTTCAAGGTCATGATTGACA ACGAGACCCTGCCCGTGGAGTACCTGGGGGGGAAGCCACTGTGCATGAACCAGTACTATCAGA TCTTGTCCTCCTGCCGAGTGCCGGGCCCCAAGCAGGACACAGTCAGCAACTTCAGCAAGACCA AGAAGCCTCCCACGCACATCACCGTGGTACACAACTACCAGTTTTTTGAGCTGGATGTGTACCAC AGTGACGGGACACCCCTCACTGCGGATCAGATCTTTGTGCAGCTGGAGAAGATCTGGAACTCAT CCCTACAGACCAACAAGGAGCCTGTGGGCATCCTCACCTCCAACCACCGCAACTCCTGGGCCAA GGCATACAACACCCTCATCAAAGACAAGGTGAACCGGGATTCCGTGCGCTCCATCCAGAAGAG CATCTTCACCGTGTGCCTAGATGCAACCATGCCCAGGGTCTCAGAAGACGTGTACCGCAGCCAC GTGGCAGGCCAGATGCTGCATGGGGGCGGCAGCAGGCTCAACAGCGGCAACCGCTGGTTCGA CAAGACGCTGCAGTTCATCGTGGCAGAAGATGGCTCCTGTGGGCTTGTGTACGAGCATGCTGCA GCGGAGGGGCCCCCTATTGTCACCCTTCTGGACTATGTCATCGAGTACACGAAGAAACCCGAGC TTGTG CGGTCTCCCCTG GTG CCCCTG CCC ATG CCC AAG AAG CTG CG GTTC AACATC ACCCCCG A GATCAAGAGCGACATCGAGAAGGCCAAGCAGAACCTCAGCATCATGATCCAGGACCTGGATATC ACCGTGATGGTGTTCCACCATTTTGGAAAAGACTTCCCCAAGTCGGAGAAGCTAAGCCCAGATG CCTTCATCCAGATGGCTTTGCAGCTGGCCTACTACAGGATCTACGGACAGGCATGTGCCACCTAT

[0130] GAAAGTGCCTCCCTGCGCATGTTTCACCTGGGCCGCACCGACACCATCCGCTCGGCTTCCATGG ACTCACTCACCTTTGTCAAGGCCATGGATGACTCCAGCGTCACGGAGCACCAGAAGGTGGAGC TGCTGCGGAAGGCCGTGCAGGCCCACCGAGGCTACACCGACCGGGCCATCCGCGGGGAGGCC TTTGATCGACACCTGCTGGGCCTGAAGCTGCAGGCCATCGAGGACCTGGTCCCTGCCAAGACA GACTGTGTCATGTTCTTCGGGCCCGTGGTCCCCGACGGCTACGGTGTCTGCTATAACCCCATGGA G GCCCAC ATC AACTTCTCCCTGTCG GCCTACAACAG CTG CG CG G AG ACCAACG CCGCCCGCCTG GCGCATTACCTGGAGAAGGCGCTCCTGGACATGCGTGCCCTGCTGCAGAGCCACCCCCGGGCC AAGCTCTGAGCCCCTAGGACTCAGGCCTGCCAATGCCACAGCCAAGCCCACCCTGGGATGGGC CACCCACCAGGGCTCAGCTCCTTGGTTCCCTCTTCCTTGGTTCCCTCTTCCCTGGTCCCCCCAAAT

[0131] CTACTGAGCCACGGACCGCATCCTCCAGGGGGCCTGCAGGCCCCAGCCAAGTGCCTTCCGTGG GTCATCCCAGCACCTGCCAGGGCCCGACCTGGGGCTGAGTGCAGAGGCTGAGCAGGACGTTA

[0132] GGCCCGGGCCCCTGGCACGTCCTCCACCGGTGCCTCCTCTGGGGAAGGGAACCAGCCCTCCAG

[0133] AGCAGGAGACTGGCAAGAGCTCTTTGTCTCACCAGCTCAGCCCCGGCCACTCCCTGCCAACTCC

[0134] ATGACCAGGCCACCATCTGTACCCTGCTTCCCAAACTCCCAGGACCTGGAGACAGGATTGTCTG

[0135] GGGCCGAGGGGCCAGGGTGTGAGGTTTCACCTCCGTTGCGGCTGTGCTCCTGTGGATAACATT

[0136] GCTAGCGAGCCGCCTCTGGTTCCACTCAGCTTGGTTCCTGCCCCCGCCCTGCTGTATGATATTAAT GTG G AAGGTCATCAATAAAG G

[0137] SEQ. ID. 6 ENST00000680117.1 CRAT-213 cdna:protein_coding

[0138] CCCCGCCCCACAGCCAACCTCCCGGGCACCGTTGCCCGCCCGGGGCCCGCTACCGGCCCAGGC

[0139] CCCGCCTCGCGAGTCCTCCTCCCCGGGTGCCTTCCCGCAGCCCGCTCGGCCCAGAGGGTGGGC

[0140] GCGGGGCTGCCTCTACCGGCTGGCGGCTGTAACTCAGCGACCTTGGCCCGAAGGCTCTAGCAA

[0141] GGACCCACCGACCCCAGCCGCGGCGGCGGCGGCGCGGACTTTGCCCGGTGTGTGGGGCGGA

[0142] GCGGACTGCGTGTCCGCGGACGGGCAGCGAAGATGTTAGCCTTCGCTGCCAGGACCGTGGTG

[0143] AAGCCTCTGGGCTTCCTGAAGCCCTTCTCCTTGATGAAGGCTTCCAGCCGCTTCAAGGCACACC

[0144] AGGATGCACTGCCACGGCTGCCCGTGCCCCCTCTCCAGCAGTCCCTGGACCACTACCTGAAGGC

[0145] GCTGCAGCCCATCGTGAGTGAGGAGGAGTGGGCCCACACCAAGCAGCTGGTGGATGAGTTTC

[0146] AGGCCTCAGGAGGTGTAGGGGAGCGCCTGCAGAAGGGGCTGGAGCGTCGGGCCAGGAAGAC

[0147] GGAGAACTGGCTGTCTGAGTGGTGGCTCAAGACCGCCTACCTCCAGTACCGCCAGCCTGTGGT

[0148] CATCTACTCGAGCCCAGGCGTGATGCTACCCAAGCAGGACTTCGTGGACCTGCAGGGTCAGCTC

[0149] CGATTTGCTGCCAAACTCATTGAGGGTGTGTTGGATTTCAAGGTCATGATTGACAACGAGACCC

[0150] TGCCCGTGGAGTACCTGGGGGGGAAGCCACTGTGCATGAACCAGTACTATCAGATCTTGTCCTC

[0151] CTGCCGAGTGCCGGGCCCCAAGCAGGACACAGTCAGCAACTTCAGCAAGACCAAGAAGCCTC

[0152] CCACGCACATCACCGTGGTACACAACTACCAGTTTTTTGAGCTGGATGTGTACCACAGTGACGG

[0153] GACACCCCTCACTGCGGATCAGATCTTTGTGCAGCTGGAGAAGATCTGGAACTCATCCCTACAG

[0154] ACCAACAAGGAGCCTGTGGGCATCCTCACCTCCAACCACCGCAACTCCTGGGCCAAGGCATACA

[0155] ACACCCTCATCAAAGACAAGGTGAACCGGGATTCCGTGCGCTCCATCCAGAAGAGCATCTTCAC CGTGTGCCTAGATGCAACCATGCCCAGGGTCTCAGAAGACGTGTACCGCAGCCACGTGGCAGG

[0156] CCAGATGCTGCATGGGGGCGGCAGCAGGCTCAACAGCGGCAACCGCTGGTTCGACAAGACGC

[0157] TGCAGTTCATCGTGGCAGAAGATGGCTCCTGTGGGCTTGTGTACGAGCATGCTGCAGCGGAGG

[0158] GGCCCCCTATTGTCACCCTTCTGGACTATGTCATCGAGTACACGAAGAAACCCGAGCTTGTGCGG

[0159] TCTCCCCTG GTG CCCCTGCCC ATG CCC AAG AAG CTG CG GTTC AACATC ACCCCCG AG ATCAAG A

[0160] GCGACATCGAGAAGGCCAAGCAGAACCTCAGCATCATGATCCAGGACCTGGATATCACCGTGAT

[0161] GGTGTTCCACCATTTTGGAAAAGACTTCCCCAAGTCGGAGAAGCTAAGCCCAGATGCCTTCATC

[0162] CAGATGGCTTTGCAGCTGGCCTACTACAGGATCTACGGACAGGCATGTGCCACCTATGAAAGTG

[0163] CCTCCCTGCGCATGTTTCACCTGGGCCGCACCGACACCATCCGCTCGGCTTCCATGGACTCACTC

[0164] ACCTTTGTCAAGGCCATGGATGACTCCAGCGTCACGGAGCACCAGAAGGTGGAGCTGCTGCGG

[0165] AAGGCCGTGCAGGCCCACCGAGGCTACACCGACCGGGCCATCCGCGGGGAGGCCTTTGATCG

[0166] ACACCTGCTGGGCCTGAAGCTGCAGGCCATCGAGGACCTGGTGAGCATGCCCGACATCTTCATG

[0167] GACACCTCCTACGCCATCGCCATGCACTTCCACCTCTCCACCAGCCAGCTGCGCGGAGACCAAC

[0168] GCCGCCCGCCTGGCGCATTACCTGGAGAAGGCGCTCCTGGACATGCGTGCCCTGCTGCAGAGC

[0169] CACCCCCGGGCCAAGCTCTGAGCCCCTAGGACTCAGGCCTGCCAATGCCACAGCCAAGCCCAC

[0170] CCTGGGATGGGCCACCCACCAGGGCTCAGCTCCTTGGTTCCCTCTTCCTTGGTTCCCTCTTCCCT

[0171] GGTCCCCCCAAATCTACTGAGCCACGGACCGCATCCTCCAGGGGGCCTGCAGGCCCCAGCCAA

[0172] GTGCCTTCCGTGGGTCATCCCAGCACCTGCCAGGGCCCGACCTGGGGCTGAGTGCAGAGGCTG

[0173] AGCAGGACGTTAGGCCCGGGCCCCTGGCACGTCCTCCACCGGTGCCTCCTCTGGGGAAGGGA

[0174] ACCAGCCCTCCAGAGCAGGAGACTGGCAAGAGCTCTTTGTCTCACCAGCTCAGCCCCGGCCAC

[0175] TCCCTGCCAACTCCATGACCAGGCCACCATCTGTACCCTGCTTCCCAAACTCCCAGGACCTGGAG

[0176] ACAGGATTGTCTGGGGCCGAGGGGCCAGGGTGTGAGGTTTCACCTCCGTTGCGGCTGTGCTCC

[0177] TGTGGATAACATTGCTAGCGAGCCGCCTCTGGTTCCACTCAGCTTGGTTCCTGCCCCCGCCCTGC

[0178] TGTATGATATTAATGTGGAA

[0179] SEQ. ID. 8 ENST00000681325.1 CRAT-217 cdna:protein_coding CCCCGCCCCACAGCCAACCTCCCGGGCACCGTTGCCCGCCCGGGGCCCGCTACCGGCCCAGGC

[0180] CCCGCCTCGCGAGTCCTCCTCCCCGGGTGCCTTCCCGCAGCCCGCTCGGCCCAGAGGGTGGGC

[0181] GCGGGGCTGCCTCTACCGGCTGGCGGCTGTAACTCAGCGACCTTGGCCCGAAGGCTCTAGCAA GGACCCACCGACCCCAGCCGCGGCGGCGGCGGCGCGGACTTTGCCCGGTGTGTGGGGCGGA GCGGACTGCGTGTCCGCGGACGGGCAGCGAAGATGTTAGCCTTCGCTGCCAGGACCGTGGTG AAGCCTCTGGGCTTCCTGAAGCCCTTCTCCTTGATGAAGGCTTCCAGCCGCTTCAAGGCACACC AGGATGCACTGCCACGGCTGCCCGTGCCCCCTCTCCAGCAGTCCCTGGACCACTACCTGAAGGC GCTGCAGCCCATCGTGAGTGAGGAGGAGTGGGCCCACACCAAGCAGCTGGTGGATGAGTTTC AGGCCTCAGGAGGTGTAGGGGAGCGCCTGCAGAAGGGGCTGGAGCGTCGGGCCAGGAAGAC GGAGAACTGGCTGTCTGAGTGGTGGCTCAAGACCGCCTACCTCCAGTACCGCCAGCCTGTGGT CATCTACTCGAGCCCAGGCGTGATGCTACCCAAGCAGGACTTCGTGGACCTGCAGGGTCAGCTC CGATTTGCTGCCAAACTCATTGAGGGTGTGTTGGATTTCAAGGTCATGATTGACAACGAGACCC TGCCCGTGGAGTACCTGGGGGGGAAGCCACTGTGCATGAACCAGTACTATCAGATCTTGTCCTC CTGCCGAGTGCCGGGCCCCAAGCAGGACACAGTCAGCAACTTCAGCAAGACCAAGAAGCCTC CCACGCACATCACCGTGGTACACAACTACCAGTTTTTTGAGCTGGATGTGTACCACAGTGACGG GACACCCCTCACTGCGGATCAGATCTTTGTGCAGCTGGAGAAGATCTGGAACTCATCCCTACAG ACC AAC AAGG AG CCTGTG GG CATCCTC ACCTCCAACC ACCG CAACTCCTG GG CC AAG GC ATAC A ACACCCTCATCAAAGACAAGGTGAACCGGGATTCCGTGCGCTCCATCCAGAAGAGCATCTTCAC

[0182] CGTGTGCCTAGATGCAACCATGCCCAGGGTCTCAGAAGACGTGTACCGCAGCCACGTGGCAGG CCAGATGCTGCATGGGGGCGGCAGCAGGCTCAACAGCGGCAACCGCTGGTTCGACAAGACGC TGCAGTTCATCGTGGCAGAAGATGGCTCCTGTGGGCTTGTGTACGAGCATGCTGCAGCGGAGG GGCCCCCTATTGTCACCCTTCTGGACTATGTCATCGAGTACACGAAGAAACCCGAGCTTGTGCGG TCTCCCCTG GTG CCCCTG CCC ATG CCC AAG AAG CTG CG GTTC AACATC ACCCCCG AG ATCAAG A GCGACATCGAGAAGGCCAAGCAGAACCTCAGCATCATGATCCAGGACCTGGATATCACCGTGAT GGTGTTCCACCATTTTGGAAAAGACTTCCCCAAGTCGGAGAAGCTAAGCCCAGATGCCTTCATC CAGATGGCTTTGCAGCTGGCCTTTGATCGACACCTGCTGGGCCTGAAGCTGCAGGCCATCGAG

[0183] GACCTGGTGAGCATGCCCGACATCTTCATGGACACCTCCTACGCCATCGCCATGCACTTCCACCT CTCCACCAGCCAGGTCCCTGCCAAGACAGACTGTGTCATGTTCTTCGGGCCCGTGGTCCCCGAC

[0184] G G CTACGGTGTCTGCTATAACCCC ATG G AG G CCCAC ATC AACTTCTCCCTGTCG GCCTACAACAG

[0185] CTGCGCGGAGACCAACGCCGCCCGCCTGGCGCATTACCTGGAGAAGGCGCTCCTGGACATGCG

[0186] TGCCCTGCTGCAGAGCCACCCCCGGGCCAAGCTCTGAGCCCCTAGGACTCAGGCCTGCCAATG

[0187] CCACAGCCAAGCCCACCCTGGGATGGGCCACCCACCAGGGCTCAGCTCCTTGGTTCCCTCTTCC

[0188] TTG GTTCCCTCTTCCCTG GTCCCCCCAAATCTACTG AGCCACG G ACCG CATCCTCCAGG GG GCCT

[0189] GCAGGCCCCAGCCAAGTGCCTTCCGTGGGTCATCCCAGCACCTGCCAGGGCCCGACCTGGGGC

[0190] TGAGTGCAGAGGCTGAGCAGGACGTTAGGCCCGGGCCCCTGGCACGTCCTCCACCGGTGCCTC

[0191] CTCTGGGGAAGGGAACCAGCCCTCCAGAGCAGGAGACTGGCAAGAGCTCTTTGTCTCACCAG

[0192] CTCAGCCCCGGCCACTCCCTGCCAACTCCATGACCAGGCCACCATCTGTACCCTGCTTCCCAAAC

[0193] TCCCAGGACCTGGAGACAGGATTGTCTGGGGCCGAGGGGCCAGGGTGTGAGGTTTCACCTCC

[0194] GTTGCGGCTGTGCTCCTGTGGATAACATTGCTAGCGAGCCGCCTCTGGTTCCACTCAGCTTGGTT

[0195] CCTGCCCCCGCCCTGCTGTATGATATTAATGTGGAAGGTCATCAATAAAGGGGAAT

[0196] SEQ. ID. 10 ENST00000455396.2 CRAT-205 cdna:protein_coding

[0197] GGCCCAGAGGGTGGGCGCGGGGCTGCCTCTACCGGCTGGCGGCTGTAACTCAGCGACCTTGG

[0198] CCCGAAGGCTCTAGCAAGGACCCACCGACCCCAGCCGCGGCGGCGGCGGCGCGGACTTTGCC

[0199] CGGTGTGTGGGGCGGAGCGGACTGCGTGTCCGCGGACGGGCAGCGAAGATGTTAGCCTTCGC

[0200] TGCCAGGACCGTGGTGAAGCCTCTGGGCTTCCTGAAGCCCTTCTCCTTGATGAAGGCTTCCAGC

[0201] CGCTTCAAGGCACACCAGGATGCACTGCCACGGCTGCCCGTGCCCCCTCTCCAGCAGTCCCTGG

[0202] ACCACTACCTGAAGGCGCTGCAGCCCATCCTGTCTGAGTGGTGGCTCAAGACCGCCTACCTCCA

[0203] GTACCGCCAGCCTGTGGTCATCTACTCGAGCCCAGGCGTGATGCTACCCAAGCAGGACTTCGTG

[0204] GACCTGCAGGGTCAGCTCCGATTTGCTGCCAAACTCATTGAGGGTGTGTTGGATTTCAAGGTCA

[0205] TGATTGACAACGAGACCCTGCCCGTGGAGTACCTGGGGGGGAAGCCACTGTGCATGAACCAGT

[0206] ACTATCAGATCTTGTCCTCCTGCCGAGTGCCGGGCCCCAAGCAGGACACAGTCAGCAACTTCAG

[0207] CAAGACCAAGAAGCCTCCCACGCACATCACCGTGGTACACAACTACCAGTTTTTTGAGCTGGAT

[0208] GTGTACCACAGTGACGGGACACCCCTCACTGCGGATCAGATCTTTGTGCAGCTGGAGAAGATCT GGAACTCATCCCTACAGACCAACAAGGAGCCTGTGGGCATCCTCACCTCCAACCACCGCAACTC

[0209] CTGGGCCAAGGCATACAACACCCTCATCAAAGACAAGGTGAACCGGGATTCCGTGCGCTCCATC

[0210] CAGAAGAGCATCTTCACCGTGTGCCTAGATGCAACCATGCCCAGGGTCTCAGAAGACGTGTACC

[0211] GCAGCCACGTGGCAGGCCAGATGCTGCATGGGGGCGGCAGCAGGCTCAACAGCGGCAACCGC

[0212] TGGTTCGACAAGACGCTGCAGTTCATCGTGGCAGAAGATGGCTCCTGTGGGCTTGTGTACGAG

[0213] CATGCTGCAGCGGAGGGGCCCCCTATTGTCACCCTTCTGGACTATGTCATCGAGTACACGAAGA

[0214] AACCCGAGCTTGTGCGGTCTCCCCTGGTGCCCCTGCCCATGCCCAAGAAGCTGCGGTTCAACAT

[0215] CACCCCCGAGATCAAGAGCGACATCGAGAAGGCCAAGCAGAACCTCAGCATCATGATCCAGGA

[0216] CCTGGATATCACCGTGATGGTGTTCCACCATTTTGGAAAAGACTTCCCCAAGTCGGAGAAGCTA

[0217] AGCCCAGATGCCTTCATCCAGATGGCTTTGCAGCTGGCCTACTACAGGATCTACGGACAGGCAT

[0218] GTGCCACCTATGAAAGTGCCTCCCTGCGCATGTTTCACCTGGGCCGCACCGACACCATCCGCTCG

[0219] GCTTCCATGGACTCACTCACCTTTGTCAAGGCCATGGATGACTCCAGCGTCACGGAGCACCAGA

[0220] AGGTGGAGCTGCTGCGGAAGGCCGTGCAGGCCCACCGAGGCTACACCGACCGGGCCATCCGC

[0221] GGGGAGGCCTTTGATCGACACCTGCTGGGCCTGAAGCTGCAGGCCATCGAGGACCTGGTGAG

[0222] CATGCCCGACATCTTCATGGACACCTCCTACGCCATCGCCATGCACTTCCACCTCTCCACCAGCCA

[0223] GGTCCCTGCCAAGACAGACTGTGTCATGTTCTTCGGGCCCGTGGTCCCCGACGGCTACGGTGTC

[0224] TGCTATAACCCCATGGAGGCCCACATCAACTTCTCCCTGTCGGCCTACAACAGCTGCGCGGAGA

[0225] CCAACGCCGCCCGCCTGGCGCATTACCTGGAGAAGGCGCTCCTGGACATGCGTGCCCTGCTGC

[0226] AGAGCCACCCCCGGGCCAAGCTCTGAGCCCCTAGGACTCAGGCCTGCCAATGCCACAGCCAAG

[0227] CCCACCCTGGGATGGGCCACCCACCAGGGCTCAGCTCCTTGGTTCCCTCTTCCTTGGTTCCCTCT

[0228] TCCCTGGTCCCCCCAAATCTACTGAGCCACGGACCGCATCCTCCAGGGGGCCTGCAGGCCCCAG

[0229] CCAAGTGCCTTCCGTGGGTCATCCCAGCACCTGCCAGGGCCCGACCTGGGGCTGAGTGCAGAG

[0230] GCTGAGCAGGACGTTAGGCCCGGGCCCCTGGCACGTCCTCCACCGGTGCCTCCTCTGGGGAAG

[0231] GGAACCAGCCCTCCAGAGCAGGAGACTGGCAAGAGCTCTTTGTCTCACCAGCTCAGCCCCGGC

[0232] CACTCCCTGCCAACTCCATGACCAGGCCACCATCTGTACCCTGCTTCCCAAACTCCCAGGACCTG

[0233] GAGACAGGATTGTCTGGGGCCGAGGGGCCAGGGTGTGAGGTTTCACCTCCGTTGCGGCTGTG CTCCTGTGGATAACATTGCTAGCGAGCCGCCTCTGGTTCCACTCAGCTTGGTTCCTGCCCCCGCC

[0234] CTGCTGTATGATATTAATGTGGAAGGTCATCAATAAAGGGGAAT

[0235] SEQ. ID. 12 ENST00000681725.1 CRAT-221 cdna:protein_coding

[0236] CCCCGCCCCACAGCCAACCTCCCGGGCACCGTTGCCCGCCCGGGGCCCGCTACCGGCCCAGGC

[0237] CCCGCCTCGCGAGTCCTCCTCCCCGGGTGCCTTCCCGCAGCCCGCTCGGCCCAGAGGGTGGGC

[0238] GCGGGGCTGCCTCTACCGGCTGGCGGCTGTAACTCAGCGACCTTGGCCCGAAGGCTCTAGCAA

[0239] GGACCCACCGACCCCAGCCGCGGCGGCGGCGGCGCGGACTTTGCCCGGTGTGTGGGGCGGA

[0240] GCGGACTGCGTGTCCGCGGACGGGCAGCGAAGATGTTAGCCTTCGCTGCCAGGACCGTGGTG

[0241] AAGCCTCTGGGCTTCCTGAAGCCCTTCTCCTTGATGAAGGCTTCCAGCCGCTTCAAGGCACACC

[0242] AGGATGCACTGCCACGGCTGCCCGTGCCCCCTCTCCAGCAGTCCCTGGACCACTACCTGAAGGC

[0243] GCTGCAGCCCATCGTGAGTGAGGAGGAGTGGGCCCACACCAAGCAGCTGGTGGATGAGTTTC

[0244] AGGCCTCAGGAGGTGTAGGGGAGCGCCTGCAGAAGGGGCTGGAGCGTCGGGCCAGGAAGAC

[0245] GGAGAACTGGCTGTCTGAGTGGTGGCTCAAGACCGCCTACCTCCAGTACCGCCAGCCTGTGGT

[0246] CATCTACTCGAGCCCAGGCGTGATGCTACCCAAGCAGGACTTCGTGGACCTGCAGGGTCAGCTC

[0247] CGATTTGCTGCCAAACTCATTGAGGGTGTGTTGGATTTCAAGGTCATGATTGACAACGAGACCC

[0248] TGCCCGTGGAGTACCTGGGGGGGAAGCCACTGTGCATGAACCAGTACTATCAGATCTTGTCCTC

[0249] CTGCCGAGTGCCGGGCCCCAAGCAGGACACAGTCAGCAACTTCAGCAAGACCAAGAAGCCTC

[0250] CCACGCACATCACCGTGGTACACAACTACCAGTTTTTTGAGCTGGATGTGTACCACAGTGACGG

[0251] GACACCCCTCACTGCGGATCAGATCTTTGTGCAGCTGGAGAAGATCTGGAACTCATCCCTACAG

[0252] ACCAACAAGGAGCCTGTGGGCATCCTCACCTCCAACCACCGCAACTCCTGGGCCAAGGCATACA

[0253] ACACCCTCATCAAAGACAAGGTGAACCGGGATTCCGTGCGCTCCATCCAGAAGAGCATCTTCAC

[0254] CGTGTGCCTAGATGCAACCATGCCCAGGGTCTCAGAAGACGTGTACCGCAGCCACGTGGCAGG

[0255] CCAGATGCTGCATGGGGGCGGCAGCAGGCTCAACAGCGGCAACCGCTGGTTCGACAAGACGC

[0256] TGCAGTTCATCGTGGCAGAAGATGGCTCCTGTGGGCTTGTGTACGAGCATGCTGCAGCGGAGG

[0257] GGCCCCCTATTGTCACCCTTCTGGACTATGTCATCGAGTACACGAAGAAACCCGAGCTTGTGCGG

[0258] TCTCCCCTG GTG CCCCTG CCC ATG CCC AAG AAG CTG CG GTTC AACATC ACCCCCG AG ATCAAG A GCGACATCGAGAAGGCCAAGCAGAACCTCAGCATCATGATCCAGGACCTGGATATCACCGTGAT

[0259] GGTGTTCCACCATTTTGGAAAAGACTTCCCCAAGTCGGAGAAGCTAAGCCCAGATGCCTTCATC

[0260] CAGATGGCTTTGCAGCTGGCCTACTACAGGATCTACGGACAGGCATGTGCCACCTATGAAAGTG

[0261] CCTCCCTGCGCATGTTTCACCTGGGCCGCACCGACACCATCCGCTCGGCTTCCATGGACTCACTC

[0262] ACCTTTGTCAAGGCCATGGATGACTCCAGCGTCACGGAGCACCAGAAGGTGGAGCTGCTGCGG

[0263] AAGGCCGTGCAGGCCCACCGAGGCTACACCGACCGGGCCATCCGCGGGGAGGCCTTTGATCG

[0264] ACACCTGCTGGGCCTGAAGCTGCAGGCCATCGAGGACCTGGTGAGCATGCCCGACATCTTCATG

[0265] GACACCTCCTACGCCATCGCCATGCACTTCCACCTCTCCACCAGCCAGGTGGGCCACTGCCCCA

[0266] GGTGCCCCGGGGGCTGGAGGCAGAGAACCCACTGGTAGTTTGATGTGGCCTACACAGGCCCC

[0267] ACTGACCCATTGCCAACACAGCTGAGTTCCAGAGGAGGAGCTGGGGGCAGGGGCCTCTCTGC

[0268] AGCCCAGGCAGGCTGAGAAG

[0269] Preferably the CRAT aminoacidic sequence is selected from the group consisting of:

[0270] SEQ. ID. 3 CRAT-201 peptide: ENSP00000315013 pep:protein_coding

[0271] MLAFAARTVVKPLGFLKPFSLMKASSRFKAHQDALPRLPVPPLQQSLDHYLKALQPIVSEEEWAHT

[0272] KQLVDEFQASGGVGERLQKGLERRARKTENWLSEWWLKTAYLQYRQPVVIYSSPGVMLPKQDFV

[0273] DLQGQLRFAAKLIEGVLDFKVMIDNETLPVEYLGGKPLCMNQYYQILSSCRVPGPKQDTVSNFSKTK

[0274] KPPTHITVVHNYQFFELDVYHSDGTPLTADQIFVQLEKIWNSSLQ.TNKEPVGILTSNHRNSWAKAYN

[0275] TLIKDKVNRDSVRSIQKSIFTVCLDATMPRVSEDVYRSHVAGQMLHGGGSRLNSGNRWFDKTLQFI

[0276] VAEDGSCGLVYEHAAAEGPPIVTLLDYVIEYTKKPELVRSPLVPLPMPKKLRFNITPEIKSDIEKAKQNL

[0277] SIMIQDLDITVMVFHHFGKDFPKSEKLSPDAFIQMALQLAYYRIYGQACATYESASLRMFHLGRTDT

[0278] IRSASMDSLTFVKAMDDSSVTEHQKVELLRKAVQAHRGYTDRAIRGEAFDRHLLGLKLQAIEDLVS

[0279] MPDIFMDTSYAIAMHFHLSTSQVPAKTDCVMFFGPVVPDGYGVCYNPMEAHINFSLSAYNSCAET

[0280] NAARLAHYLEKALLDMRALLQSHPRAKL

[0281] SEQ. ID. 5 CRAT-219 peptide: ENSP00000506267 pep:protein_coding MLAFAARTVVKPLGFLKPFSLMKASSRFKAHQDALPRLPVPPLQQSLDHYLKALQPIVSEEEWAHT

[0282] KQLVDEFQASGGVGERLQKGLERRARKTENWLSEWWLKTAYLQYRQPVVIYSSPGVMLPKQDFV

[0283] DLQGQLRFAAKLIEGVLDFKVMIDNETLPVEYLGGKPLCMNQYYQILSSCRVPGPKQDTVSNFSKTK

[0284] KPPTHITVVHNYQFFELDVYHSDGTPLTADQIFVQLEKIWNSSLQ.TNKEPVGILTSNHRNSWAKAYN

[0285] TLIKDKVNRDSVRSIQKSIFTVCLDATMPRVSEDVYRSHVAGQMLHGGGSRLNSGNRWFDKTLQFI

[0286] VAEDGSCGLVYEHAAAEGPPIVTLLDYVIEYTKKPELVRSPLVPLPMPKKLRFNITPEIKSDIEKAKQNL

[0287] SIMIQDLDITVMVFHHFGKDFPKSEKLSPDAFIQMALQLAYYRIYGQACATYESASLRMFHLGRTDT

[0288] IRSASMDSLTFVKAMDDSSVTEHQKVELLRKAVQAHRGYTDRAIRGEAFDRHLLGLKLQAIEDLVPA

[0289] KTDCVMFFGPVVPDGYGVCYNPMEAHINFSLSAYNSCAETNAARLAHYLEKALLDMRALLQSHPR AKL

[0290] SEQ. ID. 7 CRAT-213 peptide: ENSP00000505542 pep:protein_coding

[0291] MLAFAARTVVKPLGFLKPFSLMKASSRFKAHQDALPRLPVPPLQQSLDHYLKALQPIVSEEEWAHT

[0292] KQLVDEFQASGGVGERLQKGLERRARKTENWLSEWWLKTAYLQYRQPVVIYSSPGVMLPKQDFV

[0293] DLQGQLRFAAKLIEGVLDFKVMIDNETLPVEYLGGKPLCMNQYYQILSSCRVPGPKQDTVSNFSKTK

[0294] KPPTHITVVHNYQFFELDVYHSDGTPLTADQIFVQLEKIWNSSLQ.TNKEPVGILTSNHRNSWAKAYN

[0295] TLIKDKVNRDSVRSIQKSIFTVCLDATMPRVSEDVYRSHVAGQMLHGGGSRLNSGNRWFDKTLQFI

[0296] VAEDGSCGLVYEHAAAEGPPIVTLLDYVIEYTKKPELVRSPLVPLPMPKKLRFNITPEIKSDIEKAKQNL

[0297] SIMIQDLDITVMVFHHFGKDFPKSEKLSPDAFIQMALQLAYYRIYGQACATYESASLRMFHLGRTDT

[0298] IRSASMDSLTFVKAMDDSSVTEHQKVELLRKAVQAHRGYTDRAIRGEAFDRHLLGLKLQAIEDLVS

[0299] MPDIFMDTSYAIAMHFHLSTSQLRGDQRRPPGALPGEGAPGHACPAAEPPPGQALSP

[0300] SEQ. ID. 9 CRAT-217 peptide: ENSP00000506574 pep:protein_coding

[0301] MLAFAARTVVKPLGFLKPFSLMKASSRFKAHQDALPRLPVPPLQQSLDHYLKALQPIVSEEEWAHT

[0302] KQLVDEFQASGGVGERLQKGLERRARKTENWLSEWWLKTAYLQYRQPVVIYSSPGVMLPKQDFV

[0303] DLQGQLRFAAKLIEGVLDFKVMIDNETLPVEYLGGKPLCMNQYYQILSSCRVPGPKQDTVSNFSKTK

[0304] KPPTHITVVHNYQFFELDVYHSDGTPLTADQIFVQLEKIWNSSLQ.TNKEPVGILTSNHRNSWAKAYN TLIKDKVNRDSVRSIQKSIFTVCLDATMPRVSEDVYRSHVAGQMLHGGGSRLNSGNRWFDKTLQFI

[0305] VAEDGSCGLVYEHAAAEGPPIVTLLDYVIEYTKKPELVRSPLVPLPMPKKLRFNITPEIKSDIEKAKQNL

[0306] SIMIQDLDITVMVFHHFGKDFPKSEKLSPDAFIQMALQLAFDRHLLGLKLQAIEDLVSMPDIFMDTS

[0307] YAIAMHFHLSTSQVPAKTDCVMFFGPVVPDGYGVCYNPMEAHINFSLSAYNSCAETNAARLAHYL

[0308] EKALLDMRALLQSHPRAKL

[0309] SEQ. ID. 11 CRAT-205 peptide: ENSP00000395458 pep:protein_coding

[0310] MLAFAARTVVKPLGFLKPFSLMKASSRFKAHQDALPRLPVPPLQQSLDHYLKALQPILSEWWLKTA

[0311] YLQYRQPVVIYSSPGVMLPKQDFVDLQGQLRFAAKLIEGVLDFKVMIDNETLPVEYLGGKPLCMNQ

[0312] YYQILSSCRVPGPKQDTVSNFSKTKKPPTHITVVHNYQFFELDVYHSDGTPLTADQIFVQLEKIWNSS

[0313] LQTNKEPVGILTSNHRNSWAKAYNTLIKDKVNRDSVRSIQKSIFTVCLDATMPRVSEDVYRSHVAGQ

[0314] MLHGGGSRLNSGNRWFDKTLQFIVAEDGSCGLVYEHAAAEGPPIVTLLDYVIEYTKKPELVRSPLVP

[0315] LPMPKKLRFNITPEIKSDIEKAKQNLSIMIQDLDITVMVFHHFGKDFPKSEKLSPDAFIQMALQLAYY

[0316] RIYGQACATYESASLRMFHLGRTDTIRSASMDSLTFVKAMDDSSVTEHQKVELLRKAVQAHRGYTD

[0317] RAIRGEAFDRHLLGLKLQAIEDLVSMPDIFMDTSYAIAMHFHLSTSQVPAKTDCVMFFGPVVPDGY

[0318] GVCYNPMEAHINFSLSAYNSCAETNAARLAHYLEKALLDMRALLQSHPRAKL

[0319] SEQ. ID. 13 CRAT-221 peptide: ENSP00000505817 pep:protein_coding

[0320] MLAFAARTVVKPLGFLKPFSLMKASSRFKAHQDALPRLPVPPLQQSLDHYLKALQPIVSEEEWAHT

[0321] KQLVDEFQASGGVGERLQKGLERRARKTENWLSEWWLKTAYLQYRQPVVIYSSPGVMLPKQDFV

[0322] DLQGQLRFAAKLIEGVLDFKVMIDNETLPVEYLGGKPLCMNQYYQILSSCRVPGPKQDTVSNFSKTK

[0323] KPPTHITVVHNYQFFELDVYHSDGTPLTADQIFVQLEKIWNSSLQ.TNKEPVGILTSNHRNSWAKAYN

[0324] TLIKDKVNRDSVRSIQKSIFTVCLDATMPRVSEDVYRSHVAGQMLHGGGSRLNSGNRWFDKTLQFI

[0325] VAEDGSCGLVYEHAAAEGPPIVTLLDYVIEYTKKPELVRSPLVPLPMPKKLRFNITPEIKSDIEKAKQNL

[0326] SIMIQDLDITVMVFHHFGKDFPKSEKLSPDAFIQMALQLAYYRIYGQACATYESASLRMFHLGRTDT

[0327] IRSASMDSLTFVKAMDDSSVTEHQKVELLRKAVQAHRGYTDRAIRGEAFDRHLLGLKLQAIEDLVS

[0328] MPDIFMDTSYAIAMHFHLSTSQVGHCPRCPGGWRQRTHW In a preferred embodiment a vector comprising a CRAT gene for use in a method for treating fibrotic stress, fibrosis and inflammation, wherein said vector is administered intraperitoneally or intramuscularly or intravenously or intrauterine or intracolonic or intrapleural or intravesical or intraurethral wherein said administration causing over-expression of CRAT gene in mesothelial cells, prostate carcinoma cells, prostate adenocarcinoma cells, lung carcinoma cells, endometriotic epithelial cells, retinal pigment epithelium

[0329] The invention also concerns a pharmaceutical composition comprising a therapeutically efficacious amount of a vector comprising a CRAT nucleotidic or aminoacidic sequence for use in a method for treating pathologies related to cellular trans-differentiation, activation of fibrosis, activation of inflammation and neo-angiogenesis, wherein said vector is administered by enteral routes of administration, parenteral routes of administration, inhalation routes of administration, transcutaneous routes of administration wherein said administration causing over-expression of CRAT gene in host cells, together with pharmaceutically acceptable vehicle and / or excipients.

[0330] The pharmaceutical composition may comprise at least one additional active ingredient, in a therapeutically active amount, selected from the group consisting of: carnitine, Dichloroacetate, oxamic acid, Shikonin, Oleanolic acid, and 3-Bromopyruvate, anticancer active agents, anti-fibrotic active agents, citrate.

[0331] Preferably the active ingredient is L-carnitine.

[0332] When present L-carnitine is in an amount (therapeutically effective) comprised between 50microM and 2mM, more preferably 2mM. In a preferred embodiment the effective expression of genes in mesothelial cells can be achieved with a non-invasive technique, through peripheral administration of a vector comprising a CRAT gene.

[0333] The preservation of the mesothelial cells monolayer of peritoneal membrane is mandatory to avoid ultrafiltration failure.

[0334] It is also disclosed a method for the treatment of patients undergoing to peritoneal dialysis which are long-term exposed to the high glucose to mitigate local and systemic glucose over-exposure, and to prevent and treat fibrotic stress, fibrosis and inflammation by administering a therapeutically effective amount of a pharmaceutical composition comprising a vector comprising a CRAT gene and optionally L- carnitine.

[0335] Preferably the vector is a plasmid-coding CRAT ORF.

[0336] Most preferably is a plasmid-coding CRAT ORF of SEQ.ID.l.

[0337] Preferably the pharmaceutical composition is administered intraperitoneally and more preferably during peritoneal dialysis.

[0338] The following Examples further illustrate the invention.

[0339] Examples

[0340] Example 1

[0341] Materials and Methods

[0342] Cell Culture: MeT5a cells (ATCC® CRL-9444™), from an immortalized human mesothelial cell line, were maintained in M199 (5.37 mM glucose) with 10% fetal bovine serum (FBS), EGF (3.3 nM), insulin (860 nM), trace elements B, hydrocortisone (400 nM), and an antibiotic solution. Cells were maintained in a humidified environment containing 5% CO2 at 37 °C, and the culture medium was replaced every 2 days. Cells were permitted to attach for 24 h and to grow to 80% confluence.

[0343] Cells were also seeded and cultured on a polyester filter (0.4-lm pore size; Transwell, 12 well type, Millipore), using complete medium. The inner and outer chambers were filled with 0.5 and 1.5 mL of the culture medium, respectively, and the culture medium was replaced every 2 days.

[0344] Transfection of CRAT overexpressing plasmid: in order to stably obtain CRAT- overexpressing MeT5a cells, we used a plasmid-coding CRAT ORF purchased from OriGene (CRAT (NM_000755) Human Tagged ORF Clone CAT#: RC212196L2).

[0345] CRAT sequence is SEQ. ID.NO.l

[0346] Cells were seeded in six-well plates and when they reached 70%-80% of confluence, they were transfected with Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. Forty-eight hours after transfection, cells were sorted for GFP tag and plated as single cell. Single clones were isolated and analysed for CRAT expression. The ones with the highest overexpression rate were used in the subsequent experiments.

[0347] Gene Expression Analysis: total RNA was extracted using the Trizol reagent (Invitrogen, Waltham, MA, USA), according to the manufacturer's instructions. Yield and purity were checked using Nanodrop (EuroClone, Pero, Italy), and total RNA from each sample was reverse-transcribed into cDNA using Superscript II Reverse Transcriptase (Invitrogen). Real-time PCR was performed on an ABI-Prism 7500 using Power SYBR Green Master Mix 2X (Applied Biosystems, Waltham, MA, USA). The comparative Ct method (DDCt) was used to quantify gene expression, and the relative quantification was calculated as 2-DDCt. The presence of non-specific amplification products was excluded by melting curve analysis. The primers used are from SEQ ID NO 14 to SEQ ID NO 35, as indicated in the following tablel: Table 1

[0348] Western Blotting: for protein expression analysis cells were washed with PBS and then treated for 3 h in PD or control solution and then re-filled with medium for 24 h. Cells were lysed in 50 mM Tris-HCI, pH 5.0, 150 mM NaCI, 0.5% Triton X-100 with Complete Protease Inhibitor Mixture (Roche Applied Science, Penzberg, Germany). Briefly, equal amounts of proteins were treated in reducing sample buffer and denatured for 10 min at 100 °C. Protein samples were then resolved in 10% SDS-PAGE and electrotransferred to nitrocellulose membranes. Non-specific binding was blocked for 1 h at room temperature with non-fat milk (5%) in TBST buffer (50 mM Tris-HCI, pH 7.4, 150 mM NaCI and 0.1% Tween 20). Membranes were exposed to primary antibodies recognising CRAT and ACTIN (Proteintech 15170-1-AP; Santa Cruz Biotechnology sc-47778) overnight at 4 °C and incubated with a secondary peroxidase-conjugated antibody for 1 h at room temperature. The signal was detected with Luminata™ Forte Western HRP Substrate (Millipore) according to the manufacturer's instructions and the signal was acquired with Mini HD9 (UVItec, Cambridge, UK). Gene expression analysis revealed a significant CRAT overexpression in several clones, as shown in figure 1 and the data was confirmed also at protein level by western-blot, as shown in figure 2.

[0349] TGF-beta has been reported to be able to induce fibrosis, through the activation of the EMT program, and inflammation. It is also known that TGF-beta is able to up-regulate VEGF a factor that sustain neoangiogenesis.

[0350] The experimental results prove that CRAT overexpression and the use of L-carnitine can hinder this process.

[0351] In detail wt and CRAT-overexpressing cells was treated with or without 20ng / ml of TGF- beta in presence of 50microM L-Carnitine (physiological concentration) or 2mM L- Carnitine (therapeutically concentration).

[0352] Gene expression results proved that the treatment with TGF-beta significantly increased the expression of mesenchymal markers alphaSMA and Vimentin in WT mesothelial cells. The addiction of 2mM L-Carnitine maintained the expression of these two markers at basal level.

[0353] The treatment with TGF-beta in CRAT-overexpressing clones failed to up-regulate alphaSMA and Vimentin expression also at physiological L-Carnitine concentration (50microM), as shown in figure 3.

[0354] These data were confirmed also by the morphological aspect of WT and CRAT- overexpressing cell treated with TGF-beta. Only in WT cells treated with TGF-beta are visible groups of elongated and spindle shaped cells, as shown in figure 4.

[0355] Gene expression analysis proved also that TGF-beta increase the inflammatory phenotype of WT mesothelial cells. The treatment of WT cells with 2mM L-Carnitine significantly reduced the up-regulation of lnterleukin-6 (IL-6) and Interleukin-lbeta (I L- lb). Similarly, the treatment with TGF-beta in CRAT overexpressing cells did not induce any changes in the expression of these pro-inflammatory markers, as shown in figure 5.

[0356] Another effect of TGF-beta in mesothelial cells is the up-regualtion of VEGF a factor that is responsible of an increased angiogenesis in endothelial cells with the consequent reduction of ultrafiltration capacity. Here we proved that TGF-beta increased VEGF expression WT mesothelial cells at physiological L-carnitine concentration (50microM) but this effect was significantly reduced by the addiction of 2mM L-Carnitine and completely prevented in CRAT-overexpressing cells (figure 6).

[0357] Dichloroacetate (DCA) is a potent chemical that stimulates the activity of the enzyme pyruvate (PDH) dehydrogenase and inhibits its regulatory enzyme pyruvate dehydrogenase kinase 2 (PDHK2) DCA is then a drug capable to shift pyruvate metabolism from lactate to acetyl-CoA (tricarboxylic acid cycle).

[0358] Preliminary results proved that in WT mesothelial cells treated with TGF-beta the use of DCA can significantly reduce the up-regulation of fibrotic markers alpha-SMA and VIM as well as the expression of pro-inflammatory cytokines IL-6 and IL-lbeta (figure 7).

[0359] In summary, the experimental data herein presented showed that wild type and CrAT- overexpressing mesothelial cells displayed a different metabolic profile: they showed a different behavior in response to TGF-beta and the treatment with TGF-beta significantly increased the expression of fibrotic markers alpha-smooth muscle actin (alpha-SMA) and vimentin (VIM) as well as that of pro-inflammatory markers interleukine-6 (IL-6) and interleukine-ip (IL-ip) and pro-angiogenic factor vascular endothelial growth factor (VEGF) at a physiological L-carnitine concentration (50 M). Treatment with supraphysiological L-carnitine levels (2mM) significantly reduced the upregulation of the markers analysed. Similarly, TGF-b was unable to modulate the expression of alpha-SMA, VIM, I L-6, IL-ip and VEGF in CrAT overexpressing cells.

[0360] Metabolomics analyses were performed on WT and CrAT overexpressing mesothelial cells. The results show that WT and CrAT overexpressing mesothelial cells displayed a different metabolic profiles, as showed by partial least square-discriminant analysis of figure 8.

[0361] Example 2

[0362] DU145 cells (ATCC® HTB-81™) prostate carcinoma cell line with epithelial morphology and PC3 (ATCC® CRL-1435™) prostate adenocarcinoma grade IV cell line, were maintained in RPMI with 10% fetal bovine serum (FBS), and an antibiotic solution.

[0363] A549 (ATCC® CCL-185™) lung carcinoma cell line, MCF-7 (ATCC® HTB-22™) and MDA- MB-231 (ATCC® HTB-26™) breast adenocarcinoma cell, 12Z ectopic endometriotic epithelial cell line (abmgood) were maintained in DMEM with 10% fetal bovine serum (FBS), and an antibiotic solution.

[0364] HK-2 (ATCC® CRL-2190™) proximal tubule epithelial cell line and ARPE-19 (ATCC® CRL- 2302™) normal retinal epithelial cells were maintained in DMEM-F12 with 10% fetal bovine serum (FBS), and an antibiotic solution.

[0365] Cells were maintained in a humidified environment containing 5% CO2 at 37 °C, and the culture medium was replaced every 2 days. Cells were permitted to attach for 24 h and to grow to 80% confluence.

[0366] In order to obtain CRAT-overexpressing cells, we used a plasmid-coding CRAT ORF purchased from OriGene (CRAT (NM_000755) Human Tagged ORF Clone CAT#: RC212196L2)

[0367] Cells were seeded in six-well plates and when they reached 70%-80% of confluence, they were transfected with Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. Forty-eight hours after transfection cells were treated with or without TGF-beta.

[0368] Total RNA was extracted using the Trizol reagent (Invitrogen, Waltham, MA, USA), according to the manufacturer's instructions. Yield and purity were checked using Nanodrop (EuroClone, Pero, Italy), and total RNA from each sample was reverse- transcribed into cDNA using Superscript II Reverse Transcriptase (Invitrogen). Real-time PCR was performed on an ABI-Prism 7500 using Power SYBR Green Master Mix 2X (Applied Biosystems, Waltham, MA, USA). The comparative Ct method (DDCt) was used to quantify gene expression, and the relative quantification was calculated as 2-DDCt. The presence of non-specific amplification products was excluded by melting curve analysis.

[0369] Results showed that the two prostate cancer cell lines analyzed displayed different EMT markers: DU145 (the one more epithelial-like) express more E-cadherin and less a-SMA and SNAIL respect to PC3 (the more mesenchymal-like). Results showed a higher CRAT expression in DU145 cells. (Table2).

[0370] Table 2

[0371] DU145 were treated with or without TGF-beta (lOng / ml) in presence or absence of different concentration of L-carnitine, D-carnitine (the inactive enantiomer) and DCA (lOmicroM).

[0372] TGF-beta reduced the expression of E-cadherin and up-regulated the expression of a- SMA and SNAIL. This effect was reduced by 50microM L-carnitine and almost abrogated by 2mM L-carnitine (table 3). Table 3

[0373] PC3 were treated with or without TGF-beta (lOng / ml) in presence or absence of different concentration of L-carnitine, D-carnitine (the inactive enantiomer) and DCA (lOmicroM).

[0374] The effect of TGF-beta on the expression of E-cadherin was mild since this cell line already express very low levels of this protein; however it reduced its expression and this effect is antagonized by L-carnitine. On this cell line TGf-beta up-regulated the expression of a-SMA and SNAIL. This effect was reduced by 50microM L-carnitine and almost abrogated by 2mM L-carnitine. Interestingly the treatment with 2mM L-Carnitine alone is able to reduces the basal expression of a-SMA and SNAIL in PC3 cells. (Table 4).

[0375] Table 4

[0376] PC3 cells were also transfected with CRAT-overexpressing plasmid and the resultant cells displayed a different morphology. From SEM photos (not shown) WT PC3 cells displayed an EMT associated phenotype with spindle shaped cells and loss of cell-cell contact, whereas CRAT-overexpressing PC3 cells displayed a more epithelial phenotype: cells grown one attached to the others, they have a more cobblestone appearance with loss of elongated shape

[0377] A549 is a lung carcinoma cell line that in response to TGF-beta is known to undergo EMT.

[0378] Here we confirmed that TGF-beta reduces the expression of E-cadherin and up-regulates the expression of a-SMA and Vimentin. This effect was reduced by 50 pM L-carnitine and nearly eliminated by 2 mM L-carnitine. Interestingly, treatment with 2mM L- Carnitine alone can reduce the basal expression of a-SMA and VIM in A549 cells.

[0379] Table 5

[0380] CRAT overexpression was also induced in A549 and the transient transfection with the CRAT-overexpressing plasmid produced an increase of CRAT gene expression of 3215,7±140,33 fold increase respect to control cells. CRAT overexpression prevented the increase of mesenchymal markers a-SMA, VIM and FN induced by TGf-beta.

[0381] Table 6

[0382] Results showed that the two brest cancer cell lines analyzed displayed different EMT markers: MCF7 (the one more epithelial-like) express more E-cadherin and less a-SMA and SNAIL respect to MDA-MB-231 (the more mesenchymal-like). Results showed a higher CRAT expression in MCF7 cells. (Table 7). Table 7

[0383] CRAT overexpression was also induced in MCF-7 and MDA-BM-231 breast cancer cells, and transient transfection with the CRAT-overexpressing plasmid resulted in a 1715,87122,78 (for MCF-7) and in a 325, 44± 17,84 (for MDA_MB-231) fold increase in 5 CRAT gene expression compared to control cells.

[0384] CRAT overexpression prevented the upregulation of mesenchymal markers a-SMA, VIM, and FN induced by TGF-beta in both cell lines. Interestingly CRAT overexpression reduced VIM expression MCF7 cells and a-SMA and FN in MDA-MB-231 cells (Tables 8 and 9)

[0385] 10 Table 8 Table 9

[0386] It has been established that EMT contributes do progression of endometriosis and 12z, a human ectopic endometriotic epithelial cell line, when treated with TGF-b undergo

[0387] EMT. Here we confirmed that TGF-beta upregulates the expression of 3 mesenchymal markers and both the concentrations of L-carnitine tested were able to reduce the TGF- beta effect. ( Table 10) Table 10

[0388] Macular degeneration is a disease that affects the central part of the retina where EMT process take part to the progression of the disease.

[0389] CRAT overexpression was also induced in ARPE-19 cells and the transient transfection with the CRAT-overexpressing plasmid produced an increase of CRAT gene expression of 1268,35±26,79 fold increase respect to control cells.

[0390] Here we confirmed that ARPE-19 cells exposed to TGF-beta up-regulates the expression of a-SMA, VIM and FM. We proved that CRAT overexpression blocked the up-regulation of a-SMA and VIM end reduced the increase of FN produced by TGF-beta. Table 11

Claims

CLAIMS1) Vector comprising a CRAT nucleotidic sequence selected from the group consisting of SEQ. ID NO 1, SEQ.ID.NO.4, SEQ.ID.NO.6, SEQ.ID.NO.8, SEQ.ID.NO.IO, SEQ.ID.NO.12.2) Vector of claim 1 for use in a method for treating a diseases selected from the group consisting of: pulmonary fibrosis, liver fibrosis, peritoneal fibrosis, inflammatory bowel disease, renal fibrosis, polycystic kidney disease, myocardial fibrosis, endometriosis, encapsulating peritoneal sclerosis, pulmonary hypertension, sepsis, vascular dementia ischemic crisis, hypoxic crisis, cardiac arrest, heart attacks, diabetic ketoacidosis, stroke and cancer wherein said vector is administered by enteral routes of administration, parenteral routes of administration, inhalation routes of administration, transcutaneous routes of administration wherein said administration causing over-expression of CRAT gene in host cells.3) Vector comprising a CRAT nucleotidic or aminoacidic sequence selected from the group consisting of SEQ. ID NO 1, SEQ.ID.NO. 2, SEQ.ID.NO. 3, SEQ.ID.NO.4, SEQ.ID.NO.5, SEQ.ID.NO.6, SEQ.ID.NO.7, SEQ.ID.NO.8, SEQ.ID.NO.9, SEQ.ID.NO.IO, SEQ.ID.NO.il, SEQ.ID.NO.12, SEQ.ID.NO.13 for use in a method for treating a diseases selected from the group consisting of: pulmonary fibrosis, liver fibrosis, peritoneal fibrosis, inflammatory bowel disease, renal fibrosis, polycystic kidney disease, myocardial fibrosis, endometriosis, encapsulating peritoneal sclerosis, pulmonary hypertension, sepsis, vascular dementia ischemic crisis, hypoxic crisis, cardiac arrest, heart attacks, diabetic ketoacidosis, maculopathy wherein said vector is administered by enteral routes of administration, parenteral routes of administration, inhalation routes of administration, transcutaneous routes of administration wherein said administration causing over-expression of CRAT gene in host cells.4) Vector according to anyone of claim 1-3 wherein the vector is selected from the group consisting of: lipid nanoparticles, protamine-condensed mRNAs, exosomes, extracellular vesicles, (EVs), mesoporous silica, CaP, plasmids, lipid nanoparticleswith immunostimulatory potency, polymetric nanoparticles, polycationic peptides, polycationic proteins, crown-compounds.5) Vector according to claim 4 wherein the plasmid is a plasmid- coding CRAT ORF.6) Vector according to claim 4 being 1,4,6,9,12,15,18,21-Octaoxa- spiro[4.17]docosane-2,3-dicarboxylic acid diethyl ester.7) Vector according to claim 2 or 3 wherein host cells are selected from the group consisting of: mesothelial, epithelial, retinal pigment epithelium, pneumocytes, fibroblasts, endothelial cells, inflammatory cells., cardiomyocytes.8) Vector according to claim 2 wherein cancer is selected from the group consisting of carcinoma, sarcoma, breast cancer, pancreatic cancer, lung cancer, leukaemia, lymphoma, brain cancer, melanoma, liver cancer, stomach cancer, small intestine cancer, large intestine cancer, kidney cancer, peritoneal cancer, uterine cancer, ovarian cancer, bladder cancer, bone cancer.9) Pharmaceutical composition comprising a therapeutically efficacious amount of a vector according to anyone of the preceding claims and pharmaceutically acceptable vehicle and / or excipients.10) Pharmaceutical composition according to claim 9 further comprising at least one additional active ingredient, in a therapeutically active amount, selected from the group consisting of: L-carnitine, Dichloroacetate, oxamic acid, Shikonin, Oleanolic acid, and 3-Bromopyruvate, anti-cancer active agents, anti-fibrotic active agents, citrate.

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