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

Abstract

The invention relates to delivery vehicles comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof, and their use in the treatment and / or prevention of various disease associated with EMT, MMT and / or EndoMT, and their inclusion in pharmaceutical compositions.

Description

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

[0002] Background

[0003] In 1926, Otto Heinrich Warburg first described how cancer cells metabolize glucose, converting it to lactate, even in the presence of oxygen (Warburg et al., J. Gen. Physiol. 8(6), 519- 530, 1927, doi:10.1085 / jgp.8.6.519). This process became known as "aerobic glycolysis" or the "Warburg effect", see Pavlova et a!., Cell Metabolism, 34(3), 355-377, 2022, doi:10.1016 / j.cmet.2022.01.007 for a review in cancer.

[0004] Initially thought to stem from mitochondrial damage, further research revealed that aerobic glycolysis is a metabolic reprogramming which occurs early in carcinogenesis, independent of oxygen levels (Warburg, Science, 123(3191), 309-314, 1956, doi: 10.1126 / science.123.3191.309). This process results in the loss of tumor suppressor function, altered signaling pathways, and oncogene activation, leading to cell proliferation and malignancy. While producing less ATP than the tricarboxylic acid (TCA or Krebs) cycle, aerobic glycolysis compensates through increased glucose consumption and lactate production, supporting tumor progression and creating a favorable tumor microenvironment (TME). Additionally, lactate production aids in regenerating NADH, regulating the cell's redox state, and generating intermediates for macromolecule synthesis (Angulo-Elizari et al., Drugs Drug Candidates, 2(3), 728-769, 2023, doi:https: / / doi.org / 10.3390 / ddc2030037). The pentose phosphate pathway (PPP) and glutaminolysis also enhance these metabolic benefits.

[0005] Another key cellular process associated with the Warburg effect or hyperglycolytic phenotype is the epithelial-mesenchymal transition (EMT), a type of cellular differentiation that can be easily observed during developmental processes (Montest et al., J. Clin. Invest., 129(1), 24-33, 2019, doi:10.1172 / JCI122132). Epithelial cells lose their cell polarity and cell-cell adhesion, becoming spindle shaped and acquiring migratory and invasive properties. Generally, epithelial cells express high levels of E-cadherin (E-CADH), whereas mesenchymal cells express high levels of N-cadherin (N- CADH), fibronectin (FN) and vimentin (VIM). For this reason, E / N-CADH, FN and VIM are considered markers of EMT. Through the regulation of EMT, systems, organs, and tissues are formed, and their function, growth, and regeneration are maintained (Kang et al., Int. J. Mol. Sci., 20(8), 2042, 2019, doi:10.3390 / ijms20082042). However, dysregulated EMT can cause numerous diseases, including maldevelopment, fibrosis, and neoplastic progression (see Fedele et al., Int. J. Mol. Sci., 23(2), 800; 2022, doi:10.3390 / ijms23020800 and Luo et al., Pharmacological Research, 202, 107144, 2024, doi: 10.1016 / j.phrs.2024.107144).

[0006] In the past two decades, there has been an upsurge in efforts to exploit the addiction of cancer cells to glucose and the Warburg effect for both cancer and fibrosis treatment (see Barba et al., Int. J. Mol. Sci., 25(6), 3142, 2024, doi:https: / / doi.org / 10.3390 / ijms25063142; Pan et al., Front. Biosci. (Landmark Ed), 29(9), 321, 2024, doi:10.31083 / j.fbl2909321; Wang et al., Front. Endocrinol. (Sec. Molecular and Structural Endocrinology), 15, 2024, doi: 10.3389 / fendo.2024.1379521 and Lv et al., Mol. Biol. Rep., 51, 389, 2024, doi:10.1007 / sll033-024-09307-w). Several enzymes in the glycolytic pathway have been targeted, some showing tumoricidal effects in vitro and in vivo, such as inhibitors of glucose transporters (GLUTW), hexokinase 2 (HK2), phosphofructokinase (PFK), 6- phosphofructo-2-kinase / fructose-2,6-biphosphatase 3 (PFKFB3), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phosphoglycerate mutase (PGAM1), pyruvate kinase (PK), lactate dehydrogenase A (LDHA) and monocarboxylate transporters (MCT).

[0007] Unfortunately, there has been little clinical success, given that glycolysis is crucial to normal cells' glucose metabolism.

[0008] Carnitine acetyl transferase (CRAT) is an enzyme which catalyses the reversible transesterification of the acetyl moiety from Acetyl-Coenzyme A (A-CoA) to carnitine, producing acetyl carnitine. Generally, carnitine acetyltransferases have molecular weights of about 70 kDa, and contain approximately 600 residues.

[0009] US10,874,637B2 (INSERM etal.) discloses methods and pharmaceutical compositions for the treatment of neurodegeneration in with brain iron accumulation (NBIA). It identifies a homozygous mutation c962G>A in CRAT, which results in a highly conserved arginine mutation (Arg321His, predicted to be deleterious) in a patient suffering from cerebellar atrophy and posterior leukodystophry. CRAT was not detected in the patient's fibroblasts by Western blot analysis. The inventors propose using artesunate, its prodrugs and artemisinin for the treatment of NBIA.

[0010] Yu et al., Cancer Res., 78(10), 2490-2502, 2018, doi:10.1158 / 0008-5472.CAN-17-2392 investigates the role of calcium / calmodulin-dependent kinase II (CaMKII) in prostate cancer (PCa) tumorigenesis. The authors examine whether the acetyl CoA-CaMKII pathway promotes PCa tumorigenesis and conclude that activated CaMKII promotes PCa cell survival, migration, and metastasis, providing a mechanism by which aberrant metabolism increases p-oxidation which in turn promotes PCa survival and metastasis through the modulation of a kinase. They propose the possibility of targeting this metabolic pathway for cancer treatment such as carnitine acetyltransferase (CRAT), an enzyme involved in transferring the acetate moiety to carnitine. Indeed, silencing CRAT led to reduced cell proliferation, colony formation, and cell migration.

[0011] Lasheras-Otero et al., J. Invest. Dermatol., 143(2), 305-316(e5), 2023, doi:10.1016 / j.jid.2022.08.038 investigated mechanisms relevant for melanoma circulating tumour cell survival by performing RNA sequencing. The authors show that knockdown of carnitine O- octanoyltransferase (CROT) and / or CRAT, and short-term treatment with peroxisomal or mitochondrial FA beta-oxidation inhibitors, thioridazine, or ranolazine, suppressed melanoma metastasis in mice.

[0012] Aromatase inhibitors (Al) are first-line therapy for postmenopausal women with ER+ breast cancer (BC). Al therapy reduces recurrence and prolongs survival by causing long-term estrogen deprivation (LTED) through inhibiting aromatase, which converts androgens to estrogens. However, up to 50% of ER+ BC recurs as Al-resistant metastatic disease within 10 years. Al-resistant BC upregulates androgen receptors (AR) and mitochondrial oxidative phosphorylation (OXPHOS), needing OXPHOS and faty acid oxidation (FAO). The liver and lung, common metastasis sites, have high levels of palmitate (PA). Sessions DT et al. used mutant ER-expressing MCF7 and T47D BC cell lines to assess AR antagonism via enzalutamide and shRNA knockdown, and found that AR supports growth, OXPHOS, FAO, and PA lipotoxicity resistance. AR positively regulates CRAT, suggesting that silencing or inhibiting CRAT may potentiate antiandrogen therapy in ER-mutant BC, particularly in the setting of Al resistance (SESSIONS, Dane T., et al. Endocrinology, 2025, bqafl68, doi.org / 10.1210 / endocr / bqafl68).

[0013] Summary

[0014] Unexpectedly, the inventors have found that overexpressing CRAT, an enzyme far from the traditional approach to targeting the Warburg hyperglycolytic phenotype, can counteract or reverse the epithelial-mesenchymal transition (EMT) process. This goes against the findings of Yu et al., and Lasheras-Otero et al., supra which instead suggest that inhibition of the CRAT enzyme may provide therapeutic utility.

[0015] Thus, the invention aims to provide a novel mechanism for the prevention or reversal of the EMT process.

[0016] As shown in the examples below, the inventors surprisingly and unexpectedly found that transfection of CRAT in different types of cells, resulting in higher-than-natural expression levels (overexpression) may be a useful tool in reducing or preventing the development of fibrosis, and inflammation, as well as reducing the detrimental effects of a number of signalling pathways which can result in EMT, such as TGF-p. They also found that CRAT expression can reduce vimentin (VIM) and fibronectin (FN), markers of mesenchymal cells, suggesting that CRAT may reverse the EMT process.

[0017] Without being bound by theory, it is expected that, whist data is provided for the CRAT gene therapy interventions to prevent or reverse the epithelial-mesenchymal transition (EMT) (see Example 2) as well as, the mesothelial-mesenchymal transition (MMT) (see Example 1), it is expected that diseases or disorders associated with the endothelial-mesenchymal transition (EndoMT) could equally be treated with the delivery vehicles expressing CRAT as described herein. Because each of the epithelial, mesothelial and endothelial cell phenotypes share a common embryonic origin and are all coming from the superfamily of epithelial cells, sharing common markers and causes for the mesenchymal transition, it is reasonable to extrapolate the data from the epithelial and mesothelial cells to endothelial cells (also see Figure 5 of Lu et al., infra). Thus, the invention aims to provide a treatment for diseases associated with each or EMT, MMT and EndoMT.

[0018] The data provided shows that CRAT gene therapy interventions, which result in long-term and stable CRAT expression in target cells (e.g. tissues and organs) may be useful for the treatment of many pathologies. These treatments may provide several benefits to patients, such as reduced dosing and fewer side effects than many treatments currently used to treat these pathologies.

[0019] The above technical problems may be solved by providing a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyltransferase or a functional fragment thereof as described elsewhere herein.

[0020] Any part of the disclosure (including the general description and concepts) may be combined with other parts of the disclosure, unless they do not make technical sense. It is not intended that this section or any headed section (which heading are used purely to aid understanding) is self- contained.

[0021] In a first configuration, there is provided a method of treating or preventing a disease or condition in a subject in need thereof, said method comprising administering a therapeutically or prophylactically effective amount of a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof, and whereby said disease or condition is treated or prevented.

[0022] In a second configuration, there is provided a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof for use in a method of treating or preventing a disease or condition.

[0023] In a third configuration, there is provided the use of a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof for the manufacture of a medicament for treating or preventing a disease or condition.

[0024] In a fourth configuration, there is provided a pharmaceutical composition comprising a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof and one or more pharmaceutically-acceptable excipients, diluents or carriers for use in a method of treating or preventing a disease or condition.

[0025] In a fifth configuration, there is provided a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof for use in therapy.

[0026] In a sixth configuration, there is provided a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof.

[0027] In a seventh configuration, there is provided a pharmaceutical composition comprising a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof and one or more pharmaceutically-acceptable excipients, diluents or carriers.

[0028] In an eighth configuration, there is provided a kit comprising: i. a pharmaceutical composition comprising a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof and one or more pharmaceutically-acceptable excipients, diluents or carriers; and ii. a label or instructions for use.

[0029] In a ninth configuration, there is provided a method of producing a pharmaceutical composition comprising a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof, said method comprising the steps of: a. providing said delivery vehicle, and b. formulating said delivery vehicle with one or more pharmaceutically acceptable excipients, diluents or carriers.

[0030] In a tenth configuration, there is provided a method of preparing a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof, said method comprising engineering said one or more nucleic acids into the delivery vehicle and operably linking said one or more nucleic acids to one or more promoters which are capable of expressing said one or more nucleic acids in a host cell, and introducing said one or more nucleic acids and one or more promoters into said delivery vehicle.

[0031] In an eleventh configuration, there is provided a method of increasing expression of carnitine acetyl transferase in a host cell, said method comprising contacting the host cell with a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof.

[0032] In a twelfth configuration, there is provided a host cell comprising one or more exogenous nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof.

[0033] Brief description of drawings

[0034] Figure 1: shows the relative expression of carnitine acetyl transferase (CRAT) in several MeT5o cell clones transfected with CRAT (clones 1 to 8) compared to wild type (WT) MeT5o cells.

[0035] Figure 2: shows the results of protein western blot analysis for MeT5o cell clones transfected with CRAT (3 clones) compared to wild type (WT) MeT5o cells. Actin is included as a positive control (expressed in all cells)

[0036] Figure 3: shows the relative expression of the mesenchymal markers o-SMA and vimentin in wild type and CRAT overexpressing MeT5o cells activated with TFG-p and treated with different L- carnitine concentrations.

[0037] Figure 4: shows morphological aspects of wild type and CRAT-overexpressing MeT5o cells treated with TGF-p. Figure 5: shows the relative expression of IL-6 and IL-ip in wild type and CRAT overexpressing MeT5o cells activated with TFG-p and treated with different L-carnitine concentrations.

[0038] Figure 6: shows the relative expression of VEGF in wild type and CRAT overexpressing MeT5o cells activated with TFG-p and treated with different L-carnitine concentrations.

[0039] Figure 7: The images are of PC3 cells (prostate adenocarcinoma grade IV cell line, which is an epithelial carcinoma). Left image (WT): Highly metastatic cell line of epithelial origin but characterized by EMT morphology: spindle shaped cells and loss of cell-cell contacts. Right image (CRAT overexpressing): P3 cells overexpressing CRAT display a more epithelial morphology: cells grow one attached to the other (with tight junctions), and have a more cobblestone appearance with a rounded shape, having lost the elongated shape of the WT cells.

[0040] Figure 8: shows a schematic representation of the effects of CRAT overexpression on an individual metabolism, including its impact on glycolysis, the synthesis of extracellular matrix components, lactic fermentation, and the Krebs cycle. Upward and downward arrows placed next to the pathways and molecular determinants (main enzymes and metabolites impacted by CRAT expression) mentioned indicate, respectively, an increase or a decrease in pathway rate, enzyme expression, or metabolite concentration.

[0041] Detailed description

[0042] Definitions

[0043] "Anti-fibrotic active agents", are well known to those skilled in the art, and in particular includes drugs which inhibit pyruvate dehydrogenase kinase (PDHK), such as leelamine, thymoquinone (TQ), AZD7545, anthraquinone 8c, JX06, PS10, Pfz3, VER2466608. The compounds described in Wu et al, Drug Des. Devel. Ther., 18, 4661-4679, 2024, PMID: 39440140, in particular compounds 9, 31 and 32 may be useful PDHK inhibitors. Other PDHK inhibitors include those fluorene derivatives described in Inoue etal., Bioorg. Med. Chem. Lett., 109, 129839, 2024, PMID: 38844173. Also see Li etal, Bioorg. Chem., 144, 107160, 2024 for a review.

[0044] Other anti-fibrotic active agents include, but are not limited to, active agents targeting TGF-p (e.g. pirfenidone, hydronidone, Luspatercept, AVID-200), active agents targeting receptor tyrosine kinase (e.g. nintedanib, ZSP 1603 or WXFL-152), active agents targeting Connective Tissue Growth Factor (e.g. pamrevlumab), active agents targeting Phosphoinositide 3-Kinase (e.g. parsaclisib, omipalisib, HEC-68498, buparlisib, umbralisib), active agents targeting Janus Kinase (e.g. ruxolitinib, fedratinib, momelotinib, pacritinib, jaktinib, itacitinib, ilginatinib), active agents targeting WNT / p- catenin (e.g. SM04646, PRI-724), active agents targeting Apoptosis Signal Regulating Kinase and Mitogen-Activated Protein Kinase (e.g. selonsertib, CC-90001, MG-S-2525), active agents targeting Lysyl Oxidase Homolog (e.g. epeleuton, tipelukast, PAT-1251), active agents targeting Peroxisome Proliferator-Activated Receptor (e.g. elafibranor, saroglitazar, lanifibranor, pemafibrate, ZSP0678), active agents targeting Farnesoid X Receptor (e.g. obeticholic acid, cilofexor, nidufexor, TERN-101, vonafexor, EDP-305, tropifexor, JKB-121, JKB-122), active agents targeting Glucagon-like peptide-1 and / or Gastric Inhibitory Polypeptide (e.g. semaglutide, tirzepatide, cotadutide, HM-15211), active agents targeting Cystic Fibrosis Transmembrane conductance Regulator, elexacaftor, ivacaftor, resmetirom, belapectin, azemiglitazone potassium, aramchol, imetelstat, KRT-232, panobinostat, HEC- 585, PLN-74809, BG00011, sotatercept, BI089-100, efruxifermin, pegbelfermin, Aldafermin, MK-3655, IDL-2965, TRK-250, PXS-5382A, FDL176, posenacaftor, GLPG2451, PU-H71, IQNIS-ENaCRx, AZD5634, BI 443651, Idelalisib, CHF 6333, ribociclib, ARO-HSD, CB4211, Interferon gamma, BBT- 877, ZL-2102, CB-280, N-6022, PRT-543, RXC007, tezacaftor, VX-121, LYT-100, CSL312, BI 1015550, rencofilstat, ZED1227, LASN01, metformin, treprostinil, HCC 45, HTD1801, duplimab, BAX602, allopurinol, ASC42, sacroglitizar magnesium, CNP-104, setanaxib, EP547, apibaxan, OCE-205 and efzofitimod and combinations thereof, see for example Table 1 of Zhao eta!., Signal Transduction and Targeted Therapy, 7, 206, 2022, doi: 10.1038 / s41392-022-01070-3

[0045] An "antioxidant" includes, but is not limited to compounds that protect the stability and integrity of a pharmaceutical composition by inhibiting reactions with oxygen. These compounds can act as radical scavengers, terminating oxidation reactions and reducing oxidative stress. They may play a crucial role in maintaining product quality and safety. For example, the antioxidant may be (but is not limited to) ascorbic acid or cysteine.

[0046] A "buffer" includes, but is not limited to, a solution that can resist pH change upon the addition of an acidic or basic components. For example, the buffer may be (but is not limited to) a citrate buffer, a phosphate buffer and / or an acetate buffer.

[0047] A "chelator" includes, but is not limited to, a compound that can bind metal ions, such as ethylenediaminetetraacetic Acid (EDTA).

[0048] "Constitutive promoter" refers to a promoter that is capable of facilitating continuous transcription of a coding sequence or gene under its control and / or to which it is operably linked in any growth conditions. Constitutive promoters and variants are well known in the art and are described elsewhere herein.

[0049] "Cancer active agent" includes small molecule drugs, chemotherapy, hormone therapy, immunotherapy, photodynamic therapy, stem cell therapy, and antibody therapy. For example, the cancer active agent may target a tumour associated antigen, which includes, but is not limited to CEA (anticarcinoembryonic antigen); Her2 / Neu; CD22 (sialicacid binding Ig-like lectin 2, SIGLEC2, SIGLEC- 2, B-lymphocyte cell adhesion molecule, BL-CAM, Leu-14); EPCAM (epithelial cell adhesion molecule, tumour-associated calcium signal transducer 1, TACSTD1, gastrointestinal tumour-associated protein 2, GA733-2, epithelial glycoprotein 2, EGP-2, epithelial cell adhesion molecule, Ep-CAM, KSA, KS1 / 4 antigen, M4S, tumour antigen 17-1A, EpCAM, CD326); EGFR (epidermal growth factor receptor, receptor tyrosine-protein kinase erbB-1, ERBB1, HER1, HER-1, ERBB); PMSA; CTLA-4 (cytotoxic T lymphocyte-associated antigen 4, CTLA4, CD152) CD30; CD20; CD33 (sialic acid binding Ig-like lectin 3, SIGLEC3, SIGLEC-3, gpG7, p67); CD80 (B7-1, CD28LG1); CD86 (B7-2, CD28LG2); CD2; CA125; Carbonic Anhydrase IX; CD70 (tumour necrosis factor superfamily member 7, TNFSF7, CD27LG, CD27L); CD74 (major histocompatibility class II invariant chain, MH2); CD56; CD40 (tumour necrosis factor receptor superfamily member 5, TNFRSF5, p50); CD19; c-met / HGFR; TRAIL-R1; DR5; PD-1; PDL1; IGF-1R; VEGF; VEGF-R2; Prostate stem cell antigen (PSCA); MUC1 sialylated carbohydrate, tumour-associated (CA242, cancer antigen 242); CanAg; Mesothelin; P-cadherin; Myostatin (GDF8); Cripto (TDGF1); ACVRL1 / ALK1; MUC5AC; CEACAM ((carcinoembryonic antigen-related cell adhesion molecules); CD137; CXCR4; Neuropilin; Glypicans; HER3 / EGFR; PDGFRa (platelet-derived growth factor receptor alpha subunit, PDGFR2, CD140a); EphA2; CD138.

[0050] "Chemotherapy" refers to a therapeutic agent whose primary purpose is to destroy cancer cells, typically by interfering with the tumour cell's ability to grow or multiply. There are many different types of chemotherapeutic agents, with more than 50 approved chemotherapy drugs available. Chemotherapeutic drugs can be classified based on how they work. Alkylating drugs kill cancer cells by directly attacking DNA, the genetic material of the genes. Cyclophosphamide is an alkylating drug. Antimetabolites interfere with the production of DNA and keep cells from growing and multiplying. An example of an antimetabolite is 5-fluorouracil (5-FU). Anti-tumour antibiotics are made from natural substances such as fungi in the soil. They interfere with important cell functions, including production of DNA and cell proteins. Doxorubicin and bleomycin belong to this group of chemotherapy drugs. Plant alkaloids prevent cells from dividing normally. Vinblastine and vincristine are plant alkaloids obtained from the periwinkle plant. Steroid hormones slow the growth of some cancers that depend on hormones. For example, tamoxifen is used to treat breast cancers that depend on the hormone estrogen for growth. DNA damage response (DDR) inhibitors, such as PARP inhibitors, block DNA repair mechanisms following single or double stranded breaks.

[0051] Examples of chemotherapeutic agents include platinum therapies (such as oxaliplatin Carboplatin and cisplatin) Adriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside (Ara-C), Cyclophosphamide, Thiotepa, Taxotere (docetaxel), Busulfan, Cytoxin, Taxol, Methotrexate, Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine, Vinorelbine, Teniposide, Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycins, Esperamicins (see, U.S. Patent No. 4,675,187), Melphalan, and other related nitrogen mustards.. Another example of chemotherapeutic agents is the class of antibody-conjugated toxins, including, but not limited to pyrrolobenzodiazepiness, maytansanoids, calicheamicin, etc. In an example, the chemotherapy is a standard of care cytotoxic chemotherapy for the cancer being treated.

[0052] "CRAT" and "carnitine acetyltransferase" refer to any carnitine O-acetyl transferase (CRAT) enzyme, derivative, orthologue, or homolog which is capable of catalyzing the conversion of carnitine to acetylcarnitine. Orthologues of CRAT can be found in various databases, for example see NCBI: https: / / www.ncbi. nlm.nih.gov / gene / 1384 / ortholog / ?scope=7776. The CRAT may be a naturally occurring, full length CRAT, or a truncated or engineered CRAT, providing that the enzymatic function is present. In some examples, the engineered CRAT may comprise one or more amino acid deletions, substitutions or additions, provided that the enzymatic function is still present. The CRAT may be fused to other domains or moieties, for example half-life extension moieties. The CRAT may be of any eukaryotic, e.g. mammalian, origin. In some examples, the origin of the CRAT is the same as the host cell or subject into which the CRAT is introduced. In a particular example, the CRAT is a human CRAT. In particular examples, the CRAT comprises the amino acid sequences described herein (e.g. any of SEQ ID Nos: 3, 5, 7, 9, 11, 13 or 14 to 20) or is encoded by one or more nucleotide sequences as described herein (e.g. any of SEQ ID Nos: l, 2, 4, 6, 8, 10 or 11).

[0053] A "delivery vehicle" as used herein refers to any means which can be transferred to a host cell. In a particular example, the delivery vehicle is a viral vector, as described elsewhere herein. In another example, the delivery vehicle is a non-viral delivery vehicle, as described elsewhere herein. In some embodiments, a delivery vehicle may include other mobile genetic elements, such as transposons and the like. In a particular embodiment, the delivery vehicle is not a plasmid.

[0054] "Drug-induced nephrotoxicity" also sometimes referred to as drug-induced renal failure, refers to injury of the kidney which is caused directly or indirectly by medication, including contrast agents. Approximately 20% of nephrotoxicity is induced by medication, and is more apparent in the older population, due to increased medication and lifespan. Mechanisms for drug-induced nephrotoxicity include changes in glomerular hemodynamics, tubular cell toxicity, inflammation, crystal nephropathy, rhabdomyolysis, and thrombotic microangiopathy. Various biomarkers have been identified for the assessment of nephrotoxicity. For a review of mechanisms and biomarkers, see Kim & Moon, Biomol. Ther., 20(3), 268-272, 2012, doi: 10.4062 / biomolther.2012.20.3.268.

[0055] "Endothelial to mesenchymal transition (endoMT)" refers to a process that changes endothelial cells into mesenchymal cells. The endothelial cells undergoing endoMT lose the expression of endothelial cell-specific proteins such as CD31 / platelet-endothelial cell adhesion molecule, von Willebrand factor, and vascular-endothelial cadherin. Instead, the cells acquire mesenchymal characteristics, becoming spindle shaped. As well as other growth factors described herein, EndoMT can be triggered by environmental stressors such as hyperglycaemia, Perez etal., Cytokine & Growth Factor Reviews, 33, 41-54, 2017, doi:10.1016 / j.cytogfr.2016.09.002. EndoMT has been associated with cardiovascular diseases (see for example, Kovacic et a!., J. Am. Coll. Cardiol., 73(2), 190-209, 2019, doi:10.1016 / j.jacc.2018.09.089), fibrotic diseases, atherosclerosis, pulmonary arterial hypertension, diabetes mellitus and cancers (see for example, Table 1 of Piera-Velazquez & Jimenez, Physiol. Rev., 99(2), 1281-1324, 2019, doi: 10.1152 / physrev.00021.2018; Thuan et a!., Front. Immunol., 9, 1985, 2018, doi: 10.3389 / fimmu.2018.01985; and Jin Choi et al., Experimental & Molecular Medicine, 52, 781-792, 2020, doi: 10.1038 / sl2276-020-0439-4 in cancer).

[0056] "Epithelial to mesenchymal transition (EMT)" refers to a process that changes epithelial cells into mesenchymal cells, giving them the ability to migrate and invade. During EMT, epithelial cells lose their cell polarity and cell-to-cell adhesion, and gain the properties of mesenchymal cells. Whilst important for embryonic development and cardiac repair, aberrant EMT is associated with numerous diseases, including various lung diseases, such as asthma, COPD, bronchiolitis obliterans syndrome and lung fibrosis (see for example, Bartis et al., Thorax, 69, 769-769, 2014, doi:10.1136 / thoraxjnl- 2013-204608). Without being bound by theory, preventing or reversing this process through the expression of CRAT, as described by the inventors herein, may open up new therapies for many new diseases.

[0057] "Mesothelial to mesenchymal transition (MMT)" refers to a process that causes mesothelial cells to lose their epithelial-like characteristics (e.g. cell polarity and cell-cell adhesion), and they acquire mesenchymal characteristics, becoming spindle shaped and acquiring migratory and invasive properties. As well as other growth factors described herein, MMT can be triggered by expression of SNAI1. MMT has been associated with long term use of peritoneal dialysis for the treatment of endstage kidney disease, see for example, Lopez-Cabrera, Advances in Medicine, 73134, 2014, doi: 10.1155 / 2014 / 473134.

[0058] Herein, any "disease or condition mediated or induced by an epithelial and / or mesothelial and / or endothelial to mesenchymal transition (EMT and / or MMT and / or endoMT)" is understood as a pathology characterized by a process in which epithelial and / or mesothelial and / or endothelial cells lose their polarity and cell-cell adhesion, acquiring mesenchymal traits such as enhanced migratory and invasive capabilities. During EMT, these cells alter their enzymatic profile by increasing the production of enzymes responsible for extracellular matrix degradation, thereby facilitating their movement and invasion into surrounding tissues. Exemplary pathologies include: fibrotic diseases (e.g. pulmonary fibrosis, liver fibrosis, renal fibrosis, myocardial fibrosis, peritoneal fibrosis, ischemic-induced fibrosis and hypoxic-induced fibrosis), diseases or conditions associated with fibrosis (e.g. inflammatory bowel disease, polycystic kidney disease, endometriosis, encapsulating peritoneal sclerosis and pulmonary hypertension); vascular dementia; eye related diseases (e.g. angiogenic eye disorders, ocular diseases in the anterior segment, ocular diseases in the posterior segment, neovascular related ophthalmic posterior segment diseases, retinal diseases, macular degeneration (including neovascular age- related macular degeneration (AMD) , such as neovascular AMD (also known as wet AMD)), diabetic retinopathies, diabetic macular oedema (DMO), choroidal neovascularisation (CNV), central retinal vein occlusion (CRVO), corneal neovascularization, retinal neovascularization, fibrovascular intraocular diseases, pathological scarring and drug-induced nephrotoxicity); cancer (e.g. 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 and bone cancer; or skin cancer, mesothelioma, peritoneal mesothelioma, prostate cancer and vascular tumours); inflammatory diseases or conditions; chronic obstructive pulmonary disease (COPD), epatithis and Crohn's disease; end-stage kidney disease, and the subject is receiving or has received peritoneal dialysis.

[0059] "Exogenous" or "heterologous" are used interchangeably and refer to a nucleotide sequence that is not normally found in a given cell in nature. The given cell is usually the host cell in the subject to be treated. Thus, an exogenous or heterologous nucleic acid may be one which is not naturally found in the host cell, i.e. the nucleic acid is exogenous to the host cell. As used herein, a heterologous sequence encompasses a nucleic acid sequence (or amino acid sequence) that is exogenously introduced into a given cell. A heterologous gene includes a native gene, or fragment thereof, that has been introduced into the cell. For example, a heterologous gene may include a native coding sequence that is a portion of a chimeric gene to include a native coding sequence that is a portion of a chimeric gene to include non-native regulatory regions that is reintroduced into the cell. A heterologous gene may also include a native gene, or fragment thereof, introduced into a non-native cell. Thus, a heterologous gene may be foreign or native to the host cell; a nucleic acid sequence that is naturally found in a given cell but expresses an unnatural amount of the nucleic acid and / or the polypeptide which it encodes; and / or two or more nucleic acid sequences that are not found in the same relationship to each other in nature.

[0060] As used herein, the term "fibrotic disease" or "fibrosis" relates to any kind of fibrosis, defined by the excessive accumulation of fibrous connective tissue (components of the extracellular matrix (ECM) such as collagen and fibronectin) in and around inflamed or damaged tissue, which can lead to permanent scarring, organ malfunction and, ultimately, death. As would be generally understood by the skilled person, fibrosis generally refers to tissue which is scarred, or to the accumulation of excess extracellular matrix components in a tissue. The fibrosis may be sufficiently established to impair the normal function of the tissue. Fibrosis includes pulmonary (or lung) fibrosis, liver fibrosis, in particular selected from non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty liver disease, heart fibrosis, mediastinal fibrosis, in particular lymph node fibrosis, retroperitoneal cavity fibrosis, in particular kidney fibrosis and aorta fibrosis, bone marrow fibrosis, skin fibrosis, as well as scleroderma or systemic sclerosis. In another example, the fibrotic disease is a fibrotic disease of the eye.

[0061] A "fibrosis associated disease" refers to a disease or condition which has a fibrotic component to the symptoms or pathology, but which disease or condition is not primarily caused by fibrosis. For example, most chronic inflammatory diseases have a certain fibrotic pathology, including, but not limited to scleroderma, rheumatoid arthritis, Crohn's disease, ulcerative colitis (UC), myelofibrosis, systemic lupus erythematosus (SLE) and chronic graft rejection. See Wynn & Ramalingam, Nat. Med., 18(7), 1028-1040, 2012, doi:10.1038 / nm.2807 for a discussion of fibrosis and fibrosis associated diseases, causes and pathology.

[0062] "Gene therapy" as used herein refers to the use of one or more nucleotide sequences, delivered to the cells of a subject, for overexpressing the encoded protein (in this case, carnitine acetyltransferase or a functional fragment thereof). The expression may be transient (and thus may require more than one administration to the subject) or stable, which may reduce the number and / or frequency of dosing, with resulting improved compliance and patient outcomes.

[0063] "Increasing" or an "increase", and other similar terms, as used herein in relation to transcription or expression of a gene or protein refers to an increase in gene / protein transcription or expression, such as at least a 10% increase in transcription or expression. In one example, the increase is at least a 20%, or at least a 30% increase. In one example, the increase is at least a 40%, at least a 50%, at least an 60% increase. In one example, the increase is at least an 70% increase. In one example, the increase is at least a 80% increase. In one example, the increase is at least a 90% increase. In one example, the increase is at least a two-fold increase. In one example, the increase is at least a three-fold increase. In one example, the increase is at least a four-fold increase. In one example, the increase is at least a 5-fold increase. In another example, the increase is statistically significant.

[0064] An "inducible promoter" refers to a promoter that transcribes a coding sequence or gene under its control and / or to which it is operably linked in the presence of an inducer of said promoter. The inducer may be one or more environmental condition(s) and / or one or more inducing molecule(s).

[0065] An "isotonic agent" includes, but is not limited to, a solution that has the same concentration of solutes as another solution (e.g. as the blood). For example, the isotonic agent may be (but is not limited to) sodium chloride (e.g. saline solution or Ringer solution).

[0066] "Peptide", "polypeptide" and "protein" are used interchangeably herein.

[0067] "Overexpression of CRAT" and similar phrases herein refers to an increase (as defined elsewhere herein) in expression of CRAT in a particular cell which has been transfected or otherwise modified to express more CRAT as compared to the original cell which has not been transfected or otherwise modified. Some original cells do not express CRAT at all.

[0068] "Overexpression of connective tissue growth factors (CTGF)" and similar phrases herein refers to an increase (as defined elsewhere herein) in expression of CTGF above that which is normally seen or expected by one skilled in the art for any given cell type. With respect to the present invention, an increase in CTGF is causative of EMT, MMT and EndoMT, see for example, Jiang et al., Cytokine, 61(1), 173-180, 2013, doi:10.1016 / j.cyto.2012.09.013 and Thuan etal., supra. CTGF has been shown to play a crucial roles in promoting lung fibrosis through various mechanisms, including EMT, as well as cooperation with TGF-p, see Isshiki et al., Pharmacol. Ther., 253,108578, 2024, doi: 10.1016 / j.pharmthera.2023.108578.

[0069] "Overexpression of epidermal growth factors (EGF)" and similar phrases herein refers to an increase (as defined elsewhere herein) in expression of EGF above that which is normally seen or expected by one skilled in the art for any given cell type. With respect to the present invention, an increase in EGF is thought to be causative of EMT, MMT and EndoMT, see for example, Koistinen et al., Matrix Biology, 63, 38-54, 2017, doi: 10.1016 / j. matbio.2016.12.007. EGF has been associated with EMT, as observed by increased expression of SNAIL, VIM and FN, and inhibited e-cadherin expression, in breast cancer cells, see Kim et al., Oncotarget., 7(51), 85021-85032, 2016, doi: 10.18632 / oncotarget.13116.

[0070] "Overexpression of fibroblast growth factors (FGF)" and similar phrases herein refers to an increase (as defined elsewhere herein) in expression of FGF above that which is normally seen or expected by one skilled in the art for any given cell type. With respect to the present invention, an increase in FGF is thought to be causative of EMT, MMT and EndoMT, see for example Pomella etal., Cells, 13(15), 1294, 2024, doi:10.3390 / cellsl3151294, and Lu etal., Pharmacol. Ther., 108757, 2024, doi: 10.1016 / j.pharmthera.2024.108757.

[0071] "Overexpression of platelet-derived growth factors (PDGF)" and similar phrases herein refers to an increase (as defined elsewhere herein) in expression of PDGF above that which is normally seen or expected by one skilled in the art for any given cell type. With respect to the present invention, an increase in PDGF is thought to be causative of EMT, MMT and EndoMT, see for example, Wu et al., Cancer Treat. Rev., 39(6), 640-646, 2012, doi: 10.1016 / j.ctrv.2012.11.006 and Thuan et al., supra.

[0072] "Overexpression of transforming growth factor-p (TGF- )" and similar phrases herein refers to an increase (as defined elsewhere herein) in expression of TGF-p above that which is normally seen or expected by one skilled in the art for any given cell type. With respect to the present invention, an increase in TGF-p is causative of EMT, MMT and EndoMT, see for example, Kim et al., Scientific Reports, 10, 10597, 2020, doi: 10.1038 / s41598-020-67325-7; Cooley etal., Sci. Transl. Med., 6(227), 227ra34, 2014, doi:10.1126 / scitranslmed.3006927; Wilson etal., Int. J. Mol. Sci., 21(11), 4158, 2020, doi:10.3390 / ijms21114158; and Thuan etal., supra.

[0073] "Overexpression of vascular endothelial growth factors (VEGF)" and similar phrases herein refers to an increase (as defined elsewhere herein) in expression of VEGF above that which is normally seen or expected by one skilled in the art for any given cell type. With respect to the present invention, an increase in VEGF is thought to be causative of EMT, MMT and EndoMT, see for example Kovacic et al., supra, Datlibagi, et al., Int. J. Mol. Sci., 24, 4509, 2023, doi: 10.3390 / ijms24054509, and Pomella et al., supra.

[0074] As used herein, with respect to treatment methods, "prevention" includes a reducing of the risk of contracting the disease. The "treatment or prevention" may be complete or partial treatment or prevention, i.e. a reduction, but not complete reduction of the disease / condition or symptoms thereof; or a reducing of the risk but not total prevention of the disease / condition or a symptom thereof. Similarly, the methods treat or prevent (i.e. reduces the risk of) an undesirable symptom of the disease or condition or the therapy.

[0075] Any percentage identity herein may be at least (about) 70%. For example, any percentage identity herein may be at least (about) 80%. For example, and in particular, any percentage identity herein may be at least (about) 90%. For example, and in particular, any percentage identity herein may be at least (about) 95%. For example, any percentage identity herein may be at least (about) 96%. For example, any percentage identity herein may be at least (about) 97%. For example, any percentage identity may be at least (about) 98%. For example, any percentage identity herein may be at least (about) 99%. With respect to CRAT, identity analysis has shown that sequences can have as low as 30-40% identity to each other, but form functional active enzyme. Thus, for CRAT sequences, the % identity may be at least (about) 30%, at least (about) 40%, at least (about) 50% or at least (about) 60%.

[0076] Any percent identity may be at least (about) 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to any given sequence.

[0077] Percent identity for amino acid sequences are determined using the blastP algorithm with the following parameters:-

[0078] The default parameters are adjusted for short input sequences, the expect threshold is set at 0.05 and the length of the seed sequence that initiates an alignment is set at 6. Regions of low compositional complexity are masked. The employed scoring matrix is 'BLOSUM62', with scoring costs to create and extend a gap being 11 and 1 respectively. Conditional compositional score matrix adjustment is employed to compensate for amino acid composition of compared sequences.

[0079] Percent identity for nucleotide sequences are determined using the blastn algorithm with the following parameters:-

[0080] The default parameters are automatically adjusted for short input sequences, the expect threshold is set at 0.05 and the length of the seed sequence that initiates an alignment is set at 28. Regions of low compositional complexity are masked. The query sequence is masked while producing seed sequences used to scan databases, but not masked for extensions. Matches are scored as +1 and mismatches are scored as -2.

[0081] For example, an amino acid sequence described herein is identical to the reference SEQ ID No, except for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes, in particular 1 to 5, for example 1 to 3, such as 1 or 2, e.g. 1 amino acid change. For example, a nucleic acid sequence described herein is identical to the reference SEQ ID No, except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotide changes.

[0082] For example, an amino acid sequence described herein is identical to the reference SEQ ID No except for a total number of amino acid changes wherein the total number is no more than (about) 5, 10, 15, 20, 25 or 30% of the number of amino acids in the reference sequence. For example, a nucleotide sequence described herein is identical to the reference SEQ ID No except for a total number of nucleotide changes wherein the total number is no more than (about) 5, 10, 15, 20, 25 or 30% of the number of nucleotides in the reference sequence.

[0083] "Reducing" or "decreasing", and other similar terms, as used herein in relation to transcription or expression of a gene or protein refers to a reduction or decrease in gene / protein transcription or expression, such as at least a 30% reduction or decrease of transcription or expression. In one embodiment, the reduction or decrease is at least a 40%, or at least a 50% reduction or decrease. In one embodiment, the reduction or decrease is at least a 60%, at least a 70%, at least an 80% reduction or decrease. In one embodiment, the reduction or decrease is at least an 85% reduction or decrease. In one embodiment, the reduction or decrease is at least a 90% reduction or decrease. In one embodiment, the reduction or decrease is at least a 95% reduction or decrease. In one embodiment, the reduction or decrease is at least a 97% reduction or decrease. In one embodiment, the reduction or decrease is at least a 99% reduction. In one embodiment, the reduction is a 100% reduction or decrease. In another example, the decrease is statistically significant,

[0084] As used herein, the terms "treat," "treatment," or "treating," refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a disease is reduced or halted. That is, "treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and / or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment). For treatment to be effective, a complete cure is not contemplated. The method can, in certain aspects, include cure as well.

[0085] Carnitine acetyl transferases and functional fragments thereof

[0086] The carnitine O-acetyl transferase (CRAT) enzyme catalyses the reversible reaction of carnitine to acetyl carnitine, using acetyl coenzyme A (CoA), as better detailed below.

[0087] Generally, carnitine acetyltransferases have molecular weights of about 70 kDa, and contain approximately 600 residues. CRAT contains two domains, an N domain and a C domain, and is composed of 20 o-helices and 16-p strands. The N domain consists of an eight-stranded p-sheet flanked on both sides by eight o-helices. The C domain consists of a six-stranded mixed p-sheet and eleven o-helices. The catalytic domain (in at least human and mouse CRAT) comprises His343located in the interface of the two domains, and a highly conserved Ser554is present to stabilise the intermediates in the transition state, see Hsiao et al., Protein Structure & Folding, 281(38), 28480- 28487, 2006, which is incorporated herein by reference in its entirety.

[0088] As described below, unexpectedly, overexpression of CRAT in cells has been shown to prevent or reverse EMT, opening significant opportunities for the treatment of a wide range of diseases. Preferably, the overexpression of CRAT in cells prevents or reverses EMT caused by the Warburg effect. The Warburg effect has been confirmed in numerous studies, Fantin etal., Cancer Cell, 9(6), 425-434, 2006, doi: 10.1016 / j.ccr.2006.04.023, observed that inhibiting lactate dehydrogenase, which prevents the conversion of pyruvate to lactate, reduced tumorigenicity. These data were interpreted as tumorigenicity dependent on high energy levels derived from glycolysis. Another study by Schulz et al., J Biol Chem, 281(2), 977-981, 2006, doi:10.1074 / jbc.M511064200, suggests that, rather than an increase in glycolysis being the leading cause of malignant tumor growth, the efficiency of mitochondrial energy conversion is the key metabolic factor.

[0089] Glycolysis consists of 10 enzymatic steps, with three key enzymes— hexokinase (HK), phosphofructokinase-1 (PFK1), and pyruvate kinase (PK)— catalyzing irreversible reactions. Other important enzymes include glucose transporter (GLUT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate dehydrogenase (PDH), and lactate dehydrogenase (LDH). Figure 8 shows a summary scheme of glycolysis in which some of the enzymes and metabolites involved are named. Overexpression of these enzymes can enhance tumor cell metabolism, promoting proliferation and survival.

[0090] Hexokinase (HK) converts glucose to glucose-6-phosphate, with HK2 linked to tumor aggressiveness and metastasis (Li et al., Am J Chin Med, 51(7), 1905-1925, 2023, doi: 10.1142 / S0192415X23500842). PKM2 converts phosphoenolpyruvate to pyruvate, existing in active tetramer and inactive dimer forms. In its dimer form, PKM2 exhibits low catalytic activity. This enables cells to accumulate glycolytic intermediates that can both enter the glycolytic pathway and be transformed into substrates for various metabolic pathways. Consequently, this process supplies cells with essential biosynthetic precursors and energy needed for the rapid proliferation and growth of tumor cells (Amin et al., Biochim Biophys Acta Rev Cancer, 1871(2), 331-341, 2019, doi:10.1016 / j.bbcan.2019.02.003). PFK1, the primary regulatory enzyme for glycolysis, is often overexpressed in tumor cells, enhancing glycolytic activity (Shen etal., Cell Oncol (Dordr), 43(4), 617- 629, 2020, doi:10.1007 / sl3402-020-00508-6). In addition, overexpressing it contributes to proliferation and survival (Wang etal., BMC Urol, 24(1), 111, 2024, doi: 10.1186 / sl2894-024-01457- 0). LDH reduces pyruvate to lactate.

[0091] Tumor malignancy and metastasis, particularly in pancreatic and lung cancers, are often associated with high levels of its isoform LDHA (Henderson et al., J Cell Mol Med, 24(23), 14026- 14038, 2020, doi: 10.1111 / jcmm.16013). Elevated LDHA impacts cancer metabolism and promotes a tumor-promoting microenvironment, making its inhibition a potential strategy to reduce tumor growth and enhance anti-tumor immunity (Verma et al., J Clin Invest, 134(17), 2024, doi: 10.1172 / JCI177606).

[0092] Numerous studies indicate that key enzymes are regulated by transcription, metabolite feedback, and cell signaling pathways. Most glycolytic enzymes, including HK2, PFK, PDH, PKM2, and LDHA, are upregulated by transcription factors c-MYC and HIF-1. These factors drive the metabolic shift from oxidation to glycolysis, enhancing glucose retention for lactate production and contributing to the Warburg effect in cancer and fibrotic diseases (Dang etal., Nat Rev Cancer, 8(1), 51-56, 2008, doi:10.1038 / nrc2274, Ung et at., Int J Biochem Cell Biol, 139, 106073, 2021, doi: 10.1016 / j.biocel.2021.106073). When c-MYC levels drop, the expression of HK2, PFKM, PKM2, and LDHA decreases, inhibiting glycolysis and reducing ATP, leading to endoplasmic reticulum stress and immunogenic cell death in colorectal cancer cells (Lei, J., eta!., Int Immunopharmacol, 121, 110350, 2023, doi: 10.3389 / fimmu.2024.1506426)

[0093] Inhibiting rate-limiting enzymes in glycolysis presents a potential strategy for cancer treatment by reducing glycolysis and ATP production, which can induce apoptosis in tumor cells. Researchers have developed inhibitors targeting key enzymes like PKM2, frequently overexpressed in cancers, which can slow tumor growth and enhance chemotherapy sensitivity (Wang, Y., et a!., Metabolites, 15(3), 2025). Clinical trials have investigated various glycolysis inhibitors, such as LDHA and PD kinase inhibitors, showing promising results in cancers like non-small cell lung cancer and hepatocellular carcinoma (Park, W., etal., Int J Mol Sci, 25, 807, 2024, doi: 10.3390 / ijms25020807; Yang, Y., etal., Front Biosci (Landmark Ed), 29, 178, 2024, doi:10.31083 / j.fbl2905178). However, glycolytic inhibitors often face challenges due to cytotoxic effects and potential complications like liver damage and immune suppression (Jia, K.G., et al., J Biochem, 167, 365-370, 2020, doi:10.1093 / jb / mvz099; Masola, V., etal., Nutrients, 13, 2282, 2021, doi:10.3390 / nul3072282).

[0094] As referred previously, fibrosis and glycolysis are related, and several glycolytic inhibitors have proven effective in preventing TGF-p-induced fibrotic marker expression. In TGF-p-treated dermal fibroblasts, 2-Deoxy-D-glucose (2-DG), significantly reduces collagen expression (Henderson, J., et al., J Cell Mol Med, 24, 14026-14038, 2020 doi: 10.1016 / j.cmet.16013) and shows notable efficacy in alleviating renal fibrosis in a unilateral ureteral obstruction (UUO) mouse model (Ding, H., et al., Am J Physiol Renal Physiol, 313, 561, 2017, doi:10.1152 / ajprenal.00036.2017). However, from a therapeutic perspective, complete glycolysis inhibition could lead to negative effects due to its critical role in energy metabolism. Therefore, a strategy that reduces glycolysis without entirely halting it is preferable. This can be accomplished with the glycolytic flux inhibitor 3-(3-pyridinyl)-l-(4-pyridinyl)- 2-propen-l-one (3PO), which limits cells' ability to quickly increase glycolysis instead of directly suppressing it (Ding, H., et al., Am J Physiol Renal Physiol, 313, 561, 2017, doi: 10.1152 / ajprenal.00036.2017).

[0095] In addition, PFKFB3 targeted elimination also mitigated fibrosis. Recent research has shown elevated PFKFB3 in the activation of hepatic stellate cells into proliferative, fibrogenic myofibroblasts, a process which is considered the central driver of hepatic fibrosis in human liver injury and which can be inhibited by 3PO. (Mejias, M., et a / ., Gastroenterology, 159, 273, 2020, doi: 10.1053 / j.gastro.2020.03.008, Tsuchida, T., et. al. Nat Rev Gastroenterol Hepatol 14, 397, 2017, doi:10.1038 / nrgastro.2017.38). This effect was observed in mouse and human cells, with in vivo inhibitory effects in two models also reducing fibrosis. Unexpectedly, the inventors found that CRAT overexpression favors glucose oxidation in diseases driven by cellular transitions, including EMT, mesothelial-to-mesenchymal (MMT), and endothelial-to-mesenchymal (EndoMT) transitions. Indeed, potentiation of CrAT activity is expected to indirectly enhance PDH activity by lowering intramitochondrial acetyl-CoA levels. In this scenario, a PDH activation could im proveg I ucose utilization, increase the overall glycolytic flux by coupling glycolysis with the Krebs cycle, while preventing mitochondrial overload of reducing equivalents. This would compete with glycolytic sidepathways , such as the lactic fermentation (i.e. the Warburg effect), nucleotide synthesis, extracellular matrix components synthesis (e.g. precursors collagen synthesis and hexosamine pathway (See Figure 8), lipid synthesis and glutaminolysis, which are thought to be favored in pathological environments.

[0096] Prevention or reversion of the epithelial-mesenchymal transition (EMT) can also be observed in vitro when cells are over-exposed to supra-physiologica concentrations of L-carnitine (e.g., 2 mM). However, the underlying mechanism of this evidence remains uncertain. In addition, the administration of exogenous L-carnitine to a human patient does not constitute a viable therapeutic strategy, particularly in individuals affected by diseases driven by cellular transitions, including epithelial-mesenchymal transition (EMT), mesothelial-to-mesenchymal transition (MMT), and endothelial-to-mesenchymal transition (EndoMT). This limitation mainly arises from the very poor bioavailibity restricted L-carnitine: when administered orally or intravenously, any plasma concentration above physiological levels (25-50 pM, Console et al., Front. Cell Dev. Biol. 8, 2020, doi:doi.org / 10.3389 / fcell.2020.583850 and Evans et ai., Clin Pharmacokinet, 42, 941-967, 2003, doi: 10.2165 / 00003088-2003421 10-00002) is rapidly excreted, making it impossible to achieve supra- physiological intracellular concentrations.

[0097] In contrast, CRAT may be expressed intracellularly in target cells via gene delivery approaches. This strategy has the advantage that CRAT enzyme is not eliminated from the organism like smallmolecule metabolites. Moreover, the unexpected findings of the inventors are the opposite of the antitumoral effects of CRAT inhibition reported above in the Background section regarding cancers of epithelial origin (ER-positive breast cancer and prostate cancer), since overexpression of CRAT restores an epithelial phenotype by reversing the EMT transition in several cancer cells of epithelial origin (Yu et al., Cancer Res., 78(10), 2490-2502, 2018, doi:10.1158 / 0008-5472.CAN-17-2392).

[0098] Considering the above, a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof is provided.

[0099] In a particular example, the carnitine acetyl transferase or a functional fragment thereof is capable of catalysing the conversion of carnitine (e.g. L-carnitine) to acetyl carnitine (e.g. L-acetyl carnitine).

[0100] Analysis of CRAT from various species shows significant sequence identity (35% to 100%). Thus, it is envisaged that a wide range of % identities of the CRAT amino acid sequence may still be able to provide the functional characteristics needed for the conversion of carnitine to acetyl carnitine. A skilled person, in light of the knowledge of the structure and function of the various CRAT proteins can envisage a number of substitutions, additions and / or deletions to the amino acid sequence of any given CRAT enzyme (for example conservative substitutions, additions and / or deletions), and will be able to predict and test functionality. Predictions of folding and function could be achieved using software such as Alphafold.

[0101] Functionality of any given potential CRAT enzyme may be tested by those skilled in the art, for example, using an assay such as a spectrophotometric assay or tandem mass spectrometry-based assay (see, for example, Kratochvil et al., Arch. Biochem. Biophys., 691, 108507, 2020, doi: 10.1016 / j. abb.2020.108507).

[0102] In a particular example, the CRAT expressed by the delivery vehicles described herein is of the same origin as the subject or host cell in which expression is desired. Thus, in one example, the subject is a human, and the CRAT encoded by the one or more nucleic acids is a human CRAT or functional fragment thereof. In another example, for example for carrying out preclinical studies, the subject is a mouse, rat or primate, and the CRAT encoded by the one or more nucleic acids is a mouse, rat or primate CRAT respectively.

[0103] In a particular example, the one or more nucleic acids encodes a carnitine acetyl transferase or functional fragment thereof comprises an amino acid sequence selected from SEQ ID No: 3, SEQ ID No:5, SEQ ID No:7, SEQ ID No:9, SEQ ID No:ll or SEQ ID No:13 or an amino acid having at least (about) 70% identity, or at least (about) 80% identity, in particular at least (about) 90% identity thereto. The % identity may be any % identity described herein.

[0104] In an example, the carnitine acetyl transferase or functional fragment thereof comprises an amino acid sequence of SEQ ID No:3. The carnitine acetyl transferase or functional fragment thereof may comprise an amino acid sequence of SEQ ID No:5. The carnitine acetyl transferase or functional fragment thereof may comprise an amino acid sequence of SEQ ID No:7. The carnitine acetyl transferase or functional fragment thereof may comprise an amino acid sequence of SEQ ID No:9. the carnitine acetyl transferase or functional fragment thereof may comprise an amino acid sequence of SEQ ID No: 11. The carnitine acetyl transferase or functional fragment thereof may comprise an amino acid sequence of SEQ ID No: 13. The amino acid sequence may have at least (about) 70% identity, or at least (about) 80% identity, in particular at least (about) 90% identity to the sequence above.

[0105] In a particular example, the carnitine acetyl transferase or functional fragment thereof is encoded by one or more nucleic acids comprising a nucleotide sequence selected from SEQ ID No: l, SEQ ID No:2, SEQ ID No:4, SEQ ID No:6, SEQ ID No:8, SEQ ID No: 10 or SEQ ID No: 11 or a nucleotide sequence having at least (about) 70% identity, or at least (about) 80% identity, in particular at least (about) 90% identity thereto. The % identity may be any % identity described herein.

[0106] In an example, the carnitine acetyl transferase or functional fragment thereof is encoded by one or more nucleic acids comprising a nucleotide sequence of SEQ ID No:l. The carnitine acetyl transferase or functional fragment thereof may be encoded by one or more nucleic acids comprising a nucleotide sequence of SEQ ID No: 2. The carnitine acetyl transferase or functional fragment thereof may be encoded by one or more nucleic acids comprising a nucleotide sequence of SEQ ID No:4. The carnitine acetyl transferase or functional fragment thereof may be encoded by one or more nucleic acids comprising a nucleotide sequence of SEQ ID No:6. The carnitine acetyl transferase or functional fragment thereof may be encoded by one or more nucleic acids comprising a nucleotide sequence of SEQ ID No:8. The carnitine acetyl transferase or functional fragment thereof may be encoded by one or more nucleic acids comprising a nucleotide sequence of SEQ ID No: 10. The carnitine acetyl transferase or functional fragment thereof may be encoded by one or more nucleic acids comprising a nucleotide sequence of SEQ ID No: 11. The nucleotide sequence may have at least (about) 70% identity, or at least (about) 80% identity, in particular at least (about) 90% identity to the sequence above.

[0107] In an example, the one or more nucleic acids comprises or consists of a DNA sequence. In another example, the one or more nucleic acids comprises or consists of an mRNA sequence, optionally wherein the mRNA sequence does not comprise the sequence of SEQ ID No:22.

[0108] In a particular example, the carnitine acetyl transferase or functional fragment thereof is human.

[0109] In other examples, the one or more nucleic acids encodes a carnitine acetyl transferase or functional fragment thereof comprises an amino acid sequence selected from SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19 or SEQ ID No:20 or an amino acid having at least (about) 70% identity, or at least (about) 80% identity, in particular at least (about) 90% identity thereto. The % identity may be any % identity described herein.

[0110] In any example herein, the carnitine acetyl transferase or functional fragment thereof does not comprise an amino acid sequence of SEQ ID No:21. In any example herein, the carnitine acetyl transferase or functional fragment thereof does not comprise an amino acid sequence of SEQ ID No:23. In any example herein, the carnitine acetyl transferase or functional fragment thereof is not encoded by the nucleotide sequence of SEQ ID No:22.

[0111] In any method or use herein, the one or more nucleic acids may be expressed in host cells. In any method or use herein, the one or more nucleic acids may be expressed in host cells in the subject. Particularly, the one or more nucleic acids may be integrated into the chromosome of the host cell (e.g. the host cell of the subject).

[0112] The subject and / or host cell may be any subject and / or host cell described herein.

[0113] In an example, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of reverting the fibrosis and / or cancer by decreasing o-smooth muscle actin (o-SMA) relative expression as compared to control host cells which do not comprise the one or more nucleic acids. In another example, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of decreasing o-SMA relative expression as compared to control host cells which do not comprise the one or more nucleic acids. In particular, the decreasing is a decrease of at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%.

[0114] Additionally or alternatively, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of preventing the induction of fibrosis by maintaining comparable (e.g. within a statistically significant level) expression of o-SMA, as compared to control host cells which have not been induced for fibrosis. The carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, may be capable of maintaining comparable (e.g. within a statistically significant level) expression of o-SMA, as compared to control host cells which have not been induced for fibrosis and / or for cancer. The carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, may be capable of preventing an increase (e.g. a statistically significant increase) in expression of o-SMA, as compared to control host cells which have not been induced for fibrosis and / or for cancer.

[0115] In another example, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancers in host cells, wherein the fibrosis and cancers are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of reverting the fibrosis by decreasing vimentin (VIM) relative expression as compared to control host cells which do not comprise the one or more nucleic acids. In another example, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancers in host cells, wherein the fibrosis and cancers are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of decreasing VIM relative expression as compared to control host cells which do not comprise the one or more nucleic acids. In particular, the decreasing is a decrease of at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%.

[0116] Additionally or alternatively, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancers in host cells, wherein the fibrosis and cancers are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of preventing the induction of fibrosis by maintaining comparable (e.g. within a statistically significant level) expression of VIM, as compared to control host cells which have not been induced for fibrosis. The carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, may be capable of maintaining comparable (e.g. within a statistically significant level) expression of VIM, as compared to control host cells which have not been induced for fibrosis and / or for cancer. The carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, may be capable of preventing an increase (e.g. a statistically significant increase) in expression of VIM, as compared to control host cells which have not been induced for fibrosis and / or for cancer.

[0117] In an example, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of reverting the fibrosis and / or cancer by decreasing interleukin-6 (IL-6) relative expression as compared to control host cells which do not comprise the one or more nucleic acids. In another example, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of decreasing IL-6 relative expression as compared to control host cells which do not comprise the one or more nucleic acids. In particular, the decreasing is a decrease of at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%.

[0118] Additionally or alternatively, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of preventing the induction of fibrosis and / or of cancer by maintaining comparable or reducing (e.g. within a statistically significant level) expression of IL-6, as compared to control host cells which have not been induced for fibrosis. The carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, may be capable of maintaining comparable or reducing (e.g. within a statistically significant level) expression of IL-6, as compared to control host cells which have not been induced for fibrosis and / or for cancer. The carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in an in vitro fibrosis model in host cells may be capable of preventing an increase (e.g. a statistically significant increase) in expression of IL-6, as compared to control host cells which have not been induced for fibrosis.

[0119] In another example, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro fibrosis models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of reverting the fibrosis and / or cancer by decreasing interleukin-ip (IL- 1P) relative expression as compared to control host cells which do not comprise the one or more nucleic acids. In another example, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of decreasing IL-ip relative expression as compared to control host cells which do not comprise the one or more nucleic acids. In particular, the decreasing is a decrease of at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%.

[0120] Additionally or alternatively, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of preventing the induction of fibrosis and / or of cancer by maintaining comparable (e.g. within a statistically significant level) expression of IL-ip, as compared to control host cells which have not been induced for fibrosis and / or for cancer. The carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, may be capable of maintaining comparable (e.g. within a statistically significant level) expression of IL- ip, as compared to control host cells which have not been induced for fibrosis and / or for cancer. The carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in an in vitro fibrosis model in host cells may be capable of preventing an increase (e.g. a statistically significant increase) in expression of IL- ip, as compared to control host cells which have not been induced for fibrosis and / or for cancer.

[0121] In another example, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of reverting the fibrosis and / or cancer by decreasing vascular endothelial growth factor (VEGF) relative expression as compared to control host cells which do not comprise the one or more nucleic acids. In another example, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of decreasing VEGF relative expression as compared to control host cells which do not comprise the one or more nucleic acids. In particular, the decreasing is a decrease of at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%.

[0122] Additionally or alternatively, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of preventing the induction of fibrosis and / or of cancer by maintaining comparable (e.g. within a statistically significant level) expression of VEGF, as compared to control host cells which have not been induced for fibrosis and / or for cancer. The carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, may be capable of maintaining comparable (e.g. within a statistically significant level) expression of VEGF, as compared to control host cells which have not been induced for fibrosis and / or for cancer. The carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, may be capable of preventing an increase (e.g. a statistically significant increase) in expression of VEGF, as compared to control host cells which have not been induced for fibrosis and / or for cancer.

[0123] In another example, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of reverting the fibrosis and / or cancer by decreasing zinc finger protein SNAI1 (SNAIL) relative expression as compared to control host cells which do not comprise the one or more nucleic acids. In another example, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of decreasing SNAIL relative expression as compared to control host cells which do not comprise the one or more nucleic acids. In particular, the decreasing is a decrease of at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%.

[0124] Additionally or alternatively, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of preventing the induction of fibrosis and / or cancer by maintaining comparable (e.g. within a statistically significant level) expression of SNAIL, as compared to control host cells which have not been induced for fibrosis and / or for cancer. The carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, may be capable of maintaining comparable (e.g. within a statistically significant level) expression of SNAIL, as compared to control host cells which have not been induced for fibrosis and / or for cancer. The carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in an in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, may be capable of preventing an increase (e.g. a statistically significant increase) in expression of SNAIL, as compared to control host cells which have not been induced for fibrosis and / or for cancer.

[0125] In another example, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of reverting the fibrosis and / or cancer by increasing E-cadherin relative expression as compared to control host cells which do not comprise the one or more nucleic acids. In another example, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of increasing E-cadherin relative expression as compared to control host cells which do not comprise the one or more nucleic acids. In particular, the increasing is an increase of at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%.

[0126] Additionally or alternatively, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of preventing the induction of fibrosis and / or of cancer by maintaining comparable (e.g. within a statistically significant level) expression of E-cadherin, as compared to control host cells which have not been induced for fibrosis. The carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, may be capable of maintaining comparable (e.g. within a statistically significant level) expression of E-cadherin, as compared to control host cells which have not been induced for fibrosis and / or cancer. The carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, may be capable of preventing a decrease (e.g. a statistically significant increase) in expression of E-cadherin, as compared to control host cells which have not been induced for fibrosis and / or for cancer.

[0127] In another example, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of reverting the fibrosis and / or cancer by decreasing fibronectin (FN) relative expression as compared to control host cells which do not comprise the one or more nucleic acids. In another example, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of decreasing FN relative expression as compared to control host cells which do not comprise the one or more nucleic acids. In particular, the decreasing is a decrease of at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%.

[0128] Additionally, or alternatively, the carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, is capable of preventing the induction of fibrosis and / or cancer by maintaining comparable (e.g. within a statistically significant level) expression of FN as compared to control host cells which have not been induced for fibrosis and / or for cancer. The carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, may be capable of maintaining comparable (e.g. within a statistically significant level) expression of FN, as compared to control host cells which have not been induced for fibrosis and / or for cancer. The carnitine acetyl transferase or functional fragment thereof encoded by the one or more nucleic acids, when expressed in an in vitro fibrosis model in host cells may be capable of preventing an increase (e.g. a statistically significant increase) in expression of FN, as compared to control host cells which have not been induced for fibrosis and / or for cancer.

[0129] In a particular example (relating to any example of measuring expression of o-SMA, VIM, IL- 6, IL-ip, VEGF, SNAIL, E-cadherin and / or FN), the fibrosis in the in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, is induced by the addition of TGF-p. In any example relating to any example of measuring expression of o-SMA, VIM, IL-6, IL- ip, VEGF, SNAIL, E-cadherin and / or FN, the in vitro model may be carried out in the presence of a physiologicalconcentration of L-carnitine (e.g. 50pM). Additionally or alternatively, the in vitro model may be carried out in the presence of or a therapeutically relevant concentration of L-carnitine (e.g. 2mM).

[0130] In any example relating to any example of measuring expression of o-SMA, VIM, IL-6, IL- ip, VEGF, SNAIL, E-cadherin and / or FN, the host cells may not be cells selected from MeT5o cells, DU 145 cells, A549 cells, MCF-7 cells, MDA-MB-231 cells, 12Z cells and / or ARPE-19 cells.

[0131] In any example relating to any example of measuring expression of o-SMA, VIM, IL-6, IL- ip, VEGF, SNAIL, E-cadherin and / or FN, the in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, may comprise the steps of: a. Culturing control host cells and host cells expressing the one or more nucleic acids in the presence of TGF-p (e.g. 20 ng / mL or 10 ng / mL) and physiological levels of L carnitine (e.g. 50pM) or therapeutically relevant levels of L-carnitine (e.g. 2mM), b. Measuring the expression of one or markers selected from o-SMA, VIM, IL-6, IL-ip, VEGF, SNAIL, E-cadherin and FN and control gene actin by gene expression analysis (e.g. by extracting total RNA, reverse transcribing the RNA and performing real-time PCR); and c. Comparing the level of expression of the one or more markers and control gene actin in the control cells with the level of expression in the host cells expressing the one or more nucleic acids and normalising (e.g. using the comparative Ct methods (DDCt). Gene expression data are presented in arbitrary units (a.u.).

[0132] In any example relating to any example of measuring expression of o-SMA, VIM, IL-6, IL- ip, VEGF, SNAIL, E-cadherin and / or FN, the in vitro models of fibrosis and / or cancer in host cells, wherein the fibrosis and cancer are preferably induced by EMT and / or MMT and / or EndoMT transitions, may comprise inducing the host cells as described in Example 1.3.1 and analysing gene expression as described in Example 1.1.3.

[0133] Delivery vehicles

[0134] Delivery vehicles allow the transfer of nucleotide sequences (DNA, RNA) to a target (host) cell. In some examples, the nucleotide sequences are comprised by an episome in the host cell, and the nucleotide sequence will not be transferred to all progeny cells upon mitosis. In other examples, the nucleotide sequences are stably integrated into the chromosome of the cell, and can be passed through several generations of daughter cells.

[0135] Host cells are as described elsewhere herein.

[0136] In a particular example, the delivery vehicle is a viral vector. The viral vector may be an adeno-associated virus (AAV) vector, an adenoviral vector, a lentiviral vector, an alphaviral vector, a picornaviral vector, a flaviviral vector, a vescicolar stomatitis viral vector or a retroviral vector.

[0137] The viral vector may be an adeno-associated virus (AAV) vector, an adenoviral vector, a herpes simplex viral vector, a retroviral vector, or a lentiviral vector.

[0138] The AAV vector may be an AAV vector particle. In some examples, the AAV vector particle comprises AAV3B capsid proteins, LK03 capsid proteins, or AAV9 capsid proteins. In a particular example, the AAV vector particle comprises AAV3B capsid proteins. Methods of preparing and modifying viral vectors and viral vector particles, such as those derived from AAV, are well known in the art. Suitable methods are described in Ayuso etal., Current gene therapy, 10(6), 423-436, 2010; Merten et al., Molecular Therapy-Methods & Clinical Development, 3, 16017, 2016; and Nadeau& Kamen, Biotechnology advances, 20(7-8), 475-489, 2003.

[0139] AAVs may be referred to in terms of their serotype. A serotype corresponds to a variant subspecies of AAV which, owing to its profile of expression of capsid surface antigens, has a distinctive reactivity which can be used to distinguish it from other variant subspecies. Typically, an AAV vector particle having a particular AAV serotype does not efficiently cross-react with neutralising antibodies specific for any other AAV serotype. AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10 and AAV11. The AAV vector of the invention may be an AAV3B, LK03, AAV9, or AAV8 serotype.

[0140] Typically, the AAV genome comprises at least one inverted terminal repeat sequence (ITR). An ITR sequence acts in cis to provide a functional origin of replication and allows for integration and excision of the vector from the genome of a cell. ITRs may be the only sequences required in cis next to the one or more nucleic acids. Suitably, one or more ITR sequences flank the nucleotide sequence encoding a complement protein (e.g. an inhibitor of the complement system).

[0141] The adenovirus is a double-stranded, linear DNA virus that does not go through an RNA intermediate. There are over 50 different human serotypes of adenovirus divided into 6 subgroups based on the genetic sequence homology. The natural targets of adenovirus are the respiratory and gastrointestinal epithelia, generally giving rise to only mild symptoms. Serotypes 2 and 5 (with 95% sequence homology) are most commonly used in adenoviral vector systems and are normally associated with upper respiratory tract infections in the young.

[0142] Adenoviruses have been used as vectors for gene therapy and for expression of heterologous genes. The large (36 kb) genome can accommodate up to 8 kb of foreign insert DNA and is able to replicate efficiently in complementing cell lines to produce very high titres of up to 1012. Adenovirus is thus one of the best systems to study the expression of genes in primary non-replicative cells.

[0143] Herpes simplex virus (HSV) is a neurotropic DNA virus with favourable properties as a gene delivery vector. HSV is highly infectious, so HSV vectors are efficient vehicles for the delivery of exogenous genetic material to cells. Viral replication is readily disrupted by null mutations in immediate early genes that in vitro can be complemented in trans, enabling straightforward production of high- titre pure preparations of non-pathogenic vector. The genome is large (152 Kb) and many of the viral genes are dispensable for replication in vitro, allowing their replacement with large or multiple transgenes. Latent infection with wild-type virus results in episomal viral persistence in sensory neuronal nuclei for the duration of the host lifetime. The vectors are non-pathogenic, unable to reactivate and persist long-term. The latency active promoter complex can be exploited in vector design to achieve long-term stable transgene expression in the nervous system. HSV vectors transduce a broad range of tissues because of the wide expression pattern of the cellular receptors recognized by the virus. Increasing understanding of the processes involved in cellular entry has allowed targeting the tropism of HSV vectors.

[0144] A retroviral vector may be derived from or may be derivable from any suitable retrovirus. A large number of different retroviruses have been identified. Examples include murine leukaemia virus (MLV), human T-cell leukaemia virus (HTLV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukaemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukaemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29) and avian erythroblastosis virus (AEV).

[0145] Lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include but are not limited to human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS); and simian immunodeficiency virus (SIV). Examples of non-primate lentiviruses include the prototype "slow virus" visna / maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV), and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).

[0146] The lentivirus family differs from retroviruses in that lentiviruses have the capability to infect both dividing and non-dividing cells. In contrast, other retroviruses, such as MLV, are unable to infect non-dividing or slowly dividing cells such as those that make up, for example, muscle, brain, lung and liver tissue.

[0147] Alpharviral vectors are described in more detail in Quetglas etal., Virus Res., 153(2), 179-96, 2010, doi:10.1016 / j.virusres.2010.07.027.

[0148] Vesicular stomatitis virus vectors are described in more detail in Fathi et al., Hum. Vaccin. Immunother., 15(10), 2269-2285, 2019, doi: 10.1080 / 21645515.2019.1649532.

[0149] The vector of the present invention may be a vaccinia viral vector. The vector of the present invention may be a vaccinia viral vector particle.

[0150] Vaccinia virus is a large enveloped virus that has an approximately 190 kb linear, doublestranded DNA genome. Vaccinia virus can accommodate up to approximately 25 kb of foreign DNA, which also makes it useful for the delivery of large genes. A number of attenuated vaccinia virus strains are known in the art that are suitable for gene therapy applications, for example the MVA and NYVAC strains.

[0151] Particular viruses include herpes simplex viruses (HSV) and adeno-associated viruses (AAV). For a review of delivery technology, see Howarth et al., Cell Biol. Toxicol., 26(1), 1-20, 2010, doi:10.1007 / sl0565-009-9139-5, which is incorporated herein by reference in its entirety.

[0152] In an example, the delivery vehicle is an engineered phage or a phagemid. For example, the delivery vehicle may be an M13-based engineered page and phagemid (see Ranjibar et al., Iran J. Basic Med. Sci., 21(9), 884-888, 2018, doi:10.22038 / IJBMS.2018.26191.6432, which is incorporated herein by reference in its entirety) and other engineered phage (see Horst etal., PNAS, 72(9), 3531- 3535, 1975, doi:10.1073 / pnas.72.9.3531, which is incorporated herein by reference in its entirety).

[0153] Other delivery vehicles that can be used in mammalian cells include conjugative plasmids (see for example, Water, Nat. Genet., 29(4), 375-376, 2001, doi:10.1038 / ng779, which is incorporated herein by reference in its entirety).

[0154] In other embodiments, the delivery vehicle is a non-viral delivery vehicle.

[0155] In an example, the delivery vehicle is a gold nanoparticle (see for example, Ding et al., Mol. Ther., 22(6), 1075-1083, 2014, doi: 10.1038 / mt.2014.30). The delivery vehicle may be a MXENES particle (see for example Rabiee et al., ACS Applied Bio. Materials, 4(6), 5106-5121, 2021, doi: 10.1021 / acsabm.lc00332 and Wang et al., Analytical chemistry, 91(13), 8622-8629, 2019). The delivery vehicle may be a transposon (see for example, Ivies & Izsvak, Curr. Gene. Ther., 6(5), 593- 607, 2006, doi: 10.2174 / 156652306778520647).

[0156] In another example, the delivery vehicle may be a cationic liposome. The delivery vehicle may be a modified dendrimer nanoparticle. The delivery vehicle may be a cationic polymer. The delivery vehicle may be a cationic polymer liposome. The delivery vehicle may be a polymer-lipid hybrid nanoparticle. The delivery vehicle may be a cationic nanoemulsion. The delivery vehicle may be a protamin-based core shell particle. The delivery vehicle may be a polymeric micelle. The delivery vehicle may be a cationic polysaccharide particle. The delivery vehicle may be a synthetic lipoprotein.

[0157] In an example, the delivery vehicle is selected from a lipid nanoparticle (e.g. lipid nanoparticles with immunostimulatory potency, see for example, Rohner etal., Nat. Biotechnol., 40(11), 1586-1600, 2022 doi: 10.1038 / s41587-022-01491-z and Kiaie et at., J. Nanobiotechnology, 20(1), 276, 2022, doi:10.1186 / sl2951-022-01478-7), a protamine-condensed mRNA, an exosome, an extracellular vesicle (EV), mesoporous silica, a CaP, a polymetric nanoparticle, a polycationic peptide, a polycationic protein and a crown-compound.

[0158] Lipid nanoparticles may be cationic lipid nanoparticles or ionizable lipid nanoparticles.

[0159] Examples of cationic lipid nanoparticles for nucleic acids which are mRNA encoding CRAT include N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-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-l-aminium (DOBAQ) and 3B-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) can be used in combination with l,2-dioleoyl-sn-3-phosphoethanolamine (DOPE).

[0160] Examples of ionizable lipid nanoparticles for nucleic acids which are mRNA encoding CRAT include 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).

[0161] Examples of polycationic peptides or polycationic proteins for nucleic acids which are mRNA encoding CRAT include protamine, spermin or spermidine, poly-lysine, 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.

[0162] A particular crown-compound is a compound of formula (I):

[0163] Chemical Name: 1,4,6,9,12, 15, 18, 21-Octaoxa-spiro[4.17]docosane-2,3-dicarboxylic acid diethyl ester; a schematic of the structure is presented in below.

[0164] Chemical Elemental Structure: C20H3 O12

[0165] Molecular Weight: 466.48 g / mol

[0166] Other suitable delivery vehicles 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.

[0167] When the one or more nucleotide sequences comprise mRNA, there may be additional challenges relating to immunogenicity, lack of stability, and inadequate translation efficiency. For a review of mRNA delivery technology, see Weng et al., Biotechnol. Adv., 40, 107534, 2020, doi:10.1016 / j.biotechadv.2020.107534; Qin et al, Signal Transduct. Target. Ther., 7(1), 166, 2022, doi:10.1038 / s41392-022-01007-w; Nitika et al., Life (Basel), 12(8), 1254, 2022, doi:10.3390 / lifel2081254; and Paunovska et al., Nat. Rev. Genet., 23, 265-280, 2022, doi: 10.1038 / S41576-021-00439-4. In an example, the delivery vehicle is not a pLenti7.3 expression vector. In an example, the delivery vehicle is not an isolated plasmid.

[0168] In another example, the delivery vehicle is not introduced into a host cell via electroporation.

[0169] There is provided a method of preparing a delivery vehicle as described herein, said method comprising operably linking said one or more nucleic acids to one or more promoters which are capable of expressing said one or more nucleic acids in a host cell, and introducing said one or more nucleic acids and one or more promoters into said delivery vehicle.

[0170] Promoters

[0171] The one or more nucleotide sequences encoding a carnitine acetyltransferase or a functional fragment thereof are controlled by one or more (in particular one) promoters. The one or more nucleotide sequences encoding a carnitine acetyltransferase or a functional fragment thereof may be operable linked to one or more (in particular one) promoters.

[0172] In a particular example, the promoter is a constitutive promoter.

[0173] In an example, the promoter is functional for expression of the one or more nucleic acids in a host cell. The host cell may be as described elsewhere herein, for example a host cell in vivo.

[0174] In a particular example, the promoter is not naturally associated with the expression of a carnitine acetyl transferase in the host cell.

[0175] The promoter may be a promoter which is naturally occurring in (i.e. endogenous to) the host cell. Alternatively, the promoter may be one which is naturally occurring in (i.e. endogenous to) any delivery vehicle (e.g. viral vector, conjugative plasmid, phage or phagemid) comprising any of the one or more nucleotide sequences described herein.

[0176] In other embodiments, the promoter is exogenous (i.e. heterologous) to the host cell or delivery vehicle, for example a promoter which is engineered into the delivery vehicle (e.g. viral vector, conjugative plasmid, phage or phagemid). The promoter may be one which is not naturally operably connected to the sequence in nature. The promoter may be one which is not naturally associated with the expression of a carnitine acetyl transferase, e.g. in the host cell.

[0177] In an example, the promoter is a eukaryotic promoter. In another example, the promoter is a virus (e.g. phage, AAV or lentivirus) promoter. In another example, the promoter is an animal promoter (optionally a mammalian or human promoter). In another example, the promoter is a plant promoter. In another example, the promoter is a fungus promoter (optionally a yeast promoter). In another example, the promoter is an insect promoter. In another example, the promoter is a synthetic promoter. In another example, the promoter is a viral, AAV or lentiviral promoter.

[0178] Constitutive promoters may be particularly useful for the expression of the one or more nucleotide sequences described herein, providing sustained and high level production of the carnitine acetyl transferase or functional fragment thereof. Thus, in one embodiment, each nucleotide sequence of the one or more (e.g. one) nucleotide sequences is under the control of a constitutive promoter (which may be the same or different).

[0179] In one example, the promoter is a promoter which is based on the sequence of a tac promoter. Tac promoters are based on a combination of promoters from the trp and lac operons, see de Boer, eta!., PNAS, 80(1), 21-25, 1983. doi:10.1073 / pnas.80.1.21, which is incorporated herein by reference in its entirety. Several tac-based promoters have been reported in the art, see e.g. Zhang et al., Microb. Cell Fact, 16:84, 2017, doi: 10.1186 / sl2934-017-0700-2, which is incorporated herein by reference in its entirety. In one example, the promoter is a tac promoter. In one example, the promoter is a Pc-tga promoter.

[0180] Constitutive promoters and variants are well known in the art and include, but are not limited to, BBa_J23100, a constitutive E. colics promoter (e.g. an osmY promoter (International Genetically Engineered Machine (iGEM) Registry of Standard Biological Parts Name BBa_J45992; BBa_J45993)), a constitutive E. colic2promoter (e.g. htpG heat shock promoter (BBa_J45504)), a constitutive E. coli a70promoter (e.g. lacq promoter (BBa_J54200; BBa_J56015), E. coli CreABCD phosphate sensing operon promoter (BBa_J64951), GlnRS promoter (BBa_K088007), lacZ promoter (BBa_K119000; BBa_K119001), M13K07 gene I promoter (BBa_M13101), M13K07 gene II promoter (BBa_M13102), M13K07 gene III promoter (BBa_M13103), M13K07 gene IV promoter (BBa_M13104), M13K07 gene V promoter (BBa_M 13105), M13K07 gene VI promoter (BBa_M 13106), M13K07 gene VIII promoter (BBa_M13108), M13110 (BBa_M13110)), a constitutive Bacillus subtilis c promoter (e.g. promoter veg (BBa_K143013), promoter 43 (BBa_K143013), PiiaG(BBa_K823000), PiepA (BBa_K823002), Pveg(BBa_K823003)), a constitutive Bacillus subtilis oBpromoter (e.g. promoter etc (BBa_K143010) or promoter gsiB (BBa_K143011)), a Salmonella promoter (e.g. Pspv2 from Salmonella (BBa_Kl 12706), Pspv from Salmonella (BBa_Kl 12707)), a bacteriophage T7 promoter (e.g. T7 promoter (BBa_I712074; BBa_I719005; BBa_J34814; BBa_J64997; BBa_K113010; BBa_K113011;

[0181] BBa_K113012; BBa_R0085; BBa_R0180; BBa_R0181; BBa_R0182; BBa_R0183; BBa_Z0251; BBa_Z0252; BBa_Z0253)), and a bacteriophage SP6 promoter (e.g. SP6 promoter (BBa_J64998)).

[0182] One way for measuring the strength of activity is by measuring the Anderson score of any given promoter. The activity of the reporters is measured by the relative fluorescence of the promoter when used in the control plasmid EX-Ptet-S-rbsRFP-P "RFP reporter" (see http: / / parts.igem.Org / Part:BBa_J61002) in strain TGI grown in LB media to saturation. BBa_J23119 is the "consensus" promoter sequence and the strongest member of the family. The Nhel and Avril restriction sites present within these promoter parts make them a scaffold for further modification. For more information, see http: / / parts.igem.Org / Part:BBa_J23114.

[0183] Thus, in one embodiment, the constitutive promoter is a strong constitutive promoter (for example a promoter having an Anderson Score (AS) of AS >0.4, such as >0.5). In another embodiment, the promoter has an Anderson score of between 0.1 and 0.4 or between 0.1 and 0.5.

[0184] Table 13: Anderson Promoter Collection a: also shown in the Anderson Catalog, see http: / / parts.igem.org / Promoters / Catalog / Anderson b: Strength is the Anderson Score (AS), e.g. a strength of 1 is a AS of 1. Reported activities of the promoters are given as the relative fluorescence of plasmids in strain TGI grown in LB media to saturation. A suitable plasmid is EX-Ptet-S-rbsRFP-P "RFP reporter" as described at http: / / parts.igem.Org / Part:BBa_J61002; insertion of a promoter element between Xbal and Spel sites results in a RFP reporter.

[0185] In some scenarios, where a constant supply of the carnitine acetyl transferase or functional fragment thereof is not required, an inducible promoter may be used. Thus, as an alternative to a constitutive promoter, an inducible promoter may be used, for example for the expression of the one or more nucleotide sequences encoding a carnitine acetyl transferase or functional fragment thereof. The inducible promoter may be active only under certain environmental conditions. For example, the inducible promoter may be active under environmental conditions which are specific to a particular tissue or organ of a subject.

[0186] In another embodiment, the inducible promoter is a temperature sensitive promoter, such as one which is active under physiological temperatures (e.g. approximately 35 to 39 °C, for example approximately 36 to 38 °C, such as approximately 37 °C). For a discussion on the use of this type of promoter (in this example, in a kill switch setting), see https: / / wyss.harvard.edu / news / kill-switches- for-engineered-microbes-gone-rogue / and the "cryodeath" system which is described in more detail in Stirling eta!., Mol. Cell, 68, 686-697. e683, 2017, which is incorporated herein in its entirety.

[0187] In an example, the inducible promoter is selected from an aTc (anhydrotetracycline)-inducible Tet promoter, an IPTG or lactose-inducible Lac promoter, a benzoic acid-inducible XylS / Pm promoter, an arabinose-inducible Ara promoter, a rhamnose-inducible Rha promoter, a bile acid-inducible BetA promoter, temperature controlled promoters and porphyrin-inducible promoters, and synthetic derivatives thereof.

[0188] Temperature controlled promoters are described, for example, in Villaverde et al., "Fine regulation of cI857-controlled gene expression in continuous culture of recombinant Escherichia coli by temperature!' , Appl. Environ. Microbiol., 59(10), 1993, 3485-3487, doi: 10.1128 / aem.59.10.3485- 3487.1993, which is incorporated herein by reference in its entirety.

[0189] Porphyrin-inducible promoters are described for example in W02020 / 252370A1 (Novome Biotechnologies, Inc.), which is incorporated herein by reference in its entirety.

[0190] In another embodiment, the promoter is active only in the presence of certain molecules present in the local physiological environment (such as molecules present only in a specific organ or tissue). These inducible promoters may therefore turn on and off production of the carnitine acetyltransferase or functional fragment thereof when in the desired location.

[0191] Host cells

[0192] A host cell is any type of cell to which the delivery vehicle has delivered the one or more nucleotide sequences and is capable of expressing the carnitine acetyl transferase or functional fragment thereof.

[0193] Thus, there is provided a host cell comprising one or more exogenous nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof (as described elsewhere herein).

[0194] In an example, the host cell comprises an episome comprising the one or more exogenous nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof.

[0195] However, it may be particularly useful to provide host cells which are able to pass the one or more exogenous nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof to subsequent generations of cells (e.g. daughter, granddaughter and great-granddaughter cells). If the one or more exogenous nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof are comprised by an episome, only one of the daughter cells will comprise the episome, and after many generations, very little CRAT (or functional fragment) will be expressed by any cell. Thus, in a particularly useful example, the one or more exogenous nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof are comprised by the chromosome of the host cell. For therapeutic uses, it is important that the one or more exogenous nucleic acids integrate into a "desert" region of the host cell, i.e. a location in the genome which does not significantly affect cell viability or function. In any method or use herein, the one or more nucleic acids may be expressed from the chromosome of host cells.

[0196] Particularly, the host cells are diseased cells. The host cells may be comprised by a diseased tissue or organ of the subject. In a specific example, the host cells are cancer cells.

[0197] The host cells are typically of the same origin as the subject when the delivery vehicle is being used is a method of therapy.

[0198] In a particular example, the host cells are mesenchymal cells. Without being bound by theory, it is envisaged that when the host cells are mesenchymal cells, the expression of CRAT may be able to revert the cells to either epithelial cells or endothelial cells.

[0199] In another particular example, the host cells are stem cells.

[0200] In a particular example, the host cells are selected from mesothelial cells, epithelial cells (such as retinal pigment epithelial cells or pneumocytes), endothelial cells, fibroblasts, inflammatory cells (such as T-lymphocyte and macrophages) and mesenchymal cells (e.g. cardiomyocytes).

[0201] In another particular example, the host cells are selected from mesothelial cells, prostate carcinoma cells, prostate adenocarcinoma cells, lung carcinoma cells, endometriotic epithelial cells and retinal pigment epithelial cells.

[0202] In an alternative example, the host cells are not cells selected from MeT5o cells, DU 145 cells, A549 cells, MCF-7 cells, MDA-MB-231 cells, 12Z cells and / or ARPE-19 cells. Additionally or alternatively, the host cells are not selected from mesothelial cells, epithelial cells (such as retinal pigment epithelial cells or pneumocytes), endothelial cells, fibroblasts, inflammatory cells (such as T-lymphocyte and macrophages). Additionally or alternatively, the host cells are not selected from mesothelial cells, prostate carcinoma cells, prostate adenocarcinoma cells, lung carcinoma cells, endometriotic epithelial cells and retinal pigment epithelial cells.

[0203] Pharmaceutical compositions and routes of administration

[0204] There is provided a pharmaceutical composition comprising a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof and one or more pharmaceutically acceptable excipients, diluents or carriers.

[0205] The delivery vehicles and one or more nucleic acids are as described elsewhere herein. The one or more pharmaceutically acceptable excipients, diluents or carriers may be a buffer. The composition may be buffered to a pH of from 3 to 9. The one or more pharmaceutically acceptable excipients, diluents or carriers may be a sugar. The one or more pharmaceutically acceptable excipients, diluents or carriers may be an isotonic agent. The one or more pharmaceutically acceptable excipients, diluents or carriers may be an antioxidant. The one or more pharmaceutically acceptable excipients, diluents or carriers may be an amino acid. The one or more pharmaceutically acceptable excipients, diluents or carriers may be a chelator. The one or more pharmaceutically acceptable excipients, diluents or carriers may be a surfactant. The one or more pharmaceutically acceptable excipients, diluents or carriers may be an emulsifier. The one or more pharmaceutically acceptable excipients, diluents or carriers may be saline. The one or more pharmaceutically acceptable excipients, diluents or carriers may be a combination of any of the foregoing.

[0206] The composition may be sterile. The composition may be pyrogen free.

[0207] In an example the pharmaceutically acceptable excipient, diluent or carrier is selected from citric acid, dextrose, acetic acid, glutamic acid, glycine, L-histidine, L-histidine monohydrochloride monohydrate, L-lysine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-threonine, mannitol, monosodium glutamate, pentetic acid, polysorbate 20, polysorbate 80, sodium acetate trihydrate, sodium chloride, sodium citrate, sorbitol, sucrose, trehalose, maltose, ethylenediaminetetraacetic acid (EDTA), Tween, glycerol, a glycol, ethanol, fructose, glycerine, glucose and / or sucralose.

[0208] Other acceptable carriers, excipients, or stabilizers are non-toxic to subjects at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatine, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; and metal complexes (e.g. Zn-protein complexes). In limited circumstances, due to stability of the cells (e.g. donor cells), target cells or transmissible elements (e.g. vectors, plasmids, phage or packaged phage particles), the formulation may include preservatives (such as octadecyldimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol). A skilled formulator is aware of agents which are compatible with the different modes of delivery of the delivery vehicle.

[0209] Particularly, the pharmaceutically acceptable excipient, diluent or carrier enhances solubility and / or stability of the delivery vehicle. The pharmaceutically acceptable excipient, diluent or carrier may act as a cryoprotectant or lyoprotectant. Methods of determining stability or solubility of the delivery vehicle in a pharmaceutical composition are routine and well known in the art. Examples include thermal shift assays, circular dichroism, differential scanning calorimetry. The pharmaceutical compositions may comprise any suitable binder, lubricant, suspending agent, coating agent or solubilising agent.

[0210] The pharmaceutical composition may further comprise one or more further therapeutically active molecules selected from carnitine, dichloroacetate (DCA), oxamic acid, shikonin, oleanolic acid, 3-bromopyruvate, anti-cancer active agents, anti-fibrotic active agents and citrate. The composition or delivery vehicle may be administered separately, sequentially or simultaneously of the one or more further therapeutically active molecules (e.g. selected from carnitine, dichloroacetate (DCA), oxamic acid, shikonin, oleanolic acid, 3-bromopyruvate, anti-cancer active agents, anti-fibrotic active agents and citrate).

[0211] The pharmaceutical composition (or delivery vehicle as described elsewhere herein) may further comprise one or more other active ingredients as described elsewhere herein. The delivery vehicle or pharmaceutical composition may be administered in combination with at least one further therapeutically active molecule. The at least one further therapeutically active molecule may be selected from small molecule drugs, chemotherapy, hormone therapy, immunotherapy, photodynamic therapy, stem cell therapy, and antibody therapy. In particular, the at least one further therapeutically active molecule is for use in treating cancer and is selected from small molecule drugs, chemotherapy, hormone therapy, immunotherapy, photodynamic therapy, stem cell therapy, and antibody therapy, and the disease or condition to be treated is cancer. In particular, the at least one further therapeutically active molecule is for use in treating or preventing fibrosis and is selected from small molecule drugs, chemotherapy, hormone therapy, immunotherapy, photodynamic therapy, stem cell therapy, and antibody therapy, and the disease or condition to be treated is fibrosis or a disease associated with fibrosis. Alternatively, the pharmaceutical composition or delivery vehicle may be administered sequentially, subsequently or separately to the at least one further therapeutically active agent.

[0212] Particularly, the active ingredient is L-carnitine. When present L-carnitine may be in a therapeutically effective amount. For example, a therapeutically effective amount of L-carnitine may be approximately 2 mM. A therapeutically effective amount of L-carnitine may be between 50 pM and 2 mM, in particular between 1 mM and 3mM (and optionally not 2 mM).

[0213] The composition or pharmaceutical composition may be an in vitro composition.

[0214] The pharmaceutical composition comprising the delivery vehicle may be freeze dried. In one example, the pharmaceutical composition comprising the delivery vehicle is a lyophilised formulation.

[0215] The composition may be formulated in a tincture, a capsule or a slow-release formulation. The formulation may be an oral tablet, comprised within a blister pack.

[0216] In one example, the pharmaceutical composition is formulated as a capsule or coated tablet.

[0217] Sustained-release preparations can also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, e.g. films, or microcapsule. Examples of sustained- release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L- glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.

[0218] The composition described herein may be comprised by a medical container. There is also provided a medical container comprising a pharmaceutical composition comprising a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof and one or more pharmaceutically acceptable excipients, diluents or carriers.

[0219] The medical device may be an ampoule, a syringe, or an inhaler. In an example, the medical container is selected from a sterile vial, a syringe, a bag and an injection device. In particular, the medical container is an intravenous (IV) bag. In an example, the bag, syringe or injection device is for intravenous, subcutaneous, intramuscular, intradermal and / or instraosseous administration. In particular, the bag, syringe or injection device is for intravenous or subcutaneous administration. The syringe may be a prefilled syringe. The injection device may be an auto injector, a jet injector or a pump device.

[0220] In an example, the composition is a solid composition comprised within an inhalation device or a sterile ampoule. Alternatively, the composition is a liquid composition comprised within an inhalation device or sterile ampoule.

[0221] In one example, the pharmaceutical composition comprising the delivery vehicle is formulated for rectal administration. In one example, the pharmaceutical composition is formulated for oral administration. In one example, the pharmaceutical composition is formulated for intravenous administration. In one example, the pharmaceutical composition is formulated for subcutaneous administration. In one example, the pharmaceutical composition is formulated for intramuscular administration. In one example, the pharmaceutical composition is formulated for intradermal administration. In one example, the pharmaceutical composition is formulated for instraosseous administration.

[0222] In one example, the pharmaceutical composition is formulated enteral routes of administration, parenteral routes of administration, inhalation routes of administration, or transcutaneous routes of administration. In an example, the delivery vehicle or pharmaceutical composition is administered by a route of administration selected from enteral, parenteral, inhaled and transcutaneous. In an example, the delivery vehicle or pharmaceutical composition is administered intraperitoneally, intramuscularly or intravenously. In an example, the delivery vehicle or pharmaceutical composition is administered intraperitoneally, intramuscularly or intravenously and the disease or condition to be treated or prevented is not fibrotic stress, fibrosis or inflammation.

[0223] In a particular example, the delivery vehicle or pharmaceutical composition is administered intraocularly. In a particular example, the delivery vehicle or pharmaceutical composition is administered transderma I ly. In a particular example, the delivery vehicle or pharmaceutical composition is administered topically. In a particular example, the delivery vehicle or pharmaceutical composition is administered intranasally. In a particular example, the delivery vehicle or pharmaceutical composition is administered intravitreally. In a particular example, the delivery vehicle or pharmaceutical composition is administered via intra-tumoral administration.

[0224] In an example, the enteral route of administration is selected from oral administration, sublingual administration and rectal administration.

[0225] In particular, a parenteral route of administration is selected from intravascular administration, intramuscular administration, subcutaneous administration, intradermal administration, intraperitoneal administration, intrapleural administration, intracolonic administration, intravesical administration, intrauterine administration, intraurethral administration.

[0226] In a particular example, the composition is administered only once to a subject. In another example, the composition is administered after host cells are extracted and tested for CRAT expression, and the composition is administered if the levels of CRAT are low or not detectable. The composition may be administered once symptoms of the disease or disorder (as described elsewhere herein) return. The composition may be administered annual, bi-annually, or every 5 years. The composition may be administered daily, weekly, monthly, every 3 months or every 6 months. It may be administered multiple times per day (e.g. twice or three times per day).

[0227] In a particular example, the composition is packaged with a label or instructions for use (for example for the treatment or prevention of any of the diseases described herein). The medical container may further comprise a label or instructions for use (for example for the treatment or prevention of any of the diseases described herein). The label or instructions for use may comprise a marketing authorisation number (e.g., an FDA or EMA authorisation number).

[0228] There is also provided a method of producing a pharmaceutical composition comprising a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof, said method comprising the steps of: a. providing said delivery vehicle (as described elsewhere herein), and b. formulating said delivery vehicle with one or more pharmaceutically acceptable excipients, diluents or carriers (as described elsewhere herein) The method may further comprise the step of c. packaging said composition. Alternatively, the method may further comprise the step of c. packaging said composition in a medical container as described elsewhere herein.

[0229] The method may further comprise the step of d. including in or on said packaging a label or instructions for use (for example for the treatment or prevention of any of the diseases described herein). The label or instructions for use may comprise a marketing authorisation number (e.g., an FDA or EMA authorisation number).

[0230] There is also provided a kit comprising: i. a pharmaceutical composition (as described elsewhere herein); and ii. a label or instructions for use.

[0231] There is also provided a kit comprising: i. a medical container (as described elsewhere herein); and ii. a label or instructions for use.

[0232] The label or instructions for use may comprise a marketing authorisation number (e.g., an FDA or EMA authorisation number).

[0233] Methods and uses

[0234] As described elsewhere, the work described herein supports the use of CRAT expression in the treatment of a number of diseases associated with the detrimental effects of EMT, MMT and / or EndoMT. These various transitions are thought to be caused by the overexpression of various growth factors, thus diseases and conditions which are associated with the overexpression of these growth factors may also be treated or prevented by the expression of CRAT or a functional fragment thereof in host cells.

[0235] Thus, there is provided, at a general level, a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof for use in therapy.

[0236] There is provided, also at a general level, a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof for use in gene therapy.

[0237] There is also provided a method of increasing expression of carnitine acetyl transferase in a host cell, said method comprising contacting the host cell with a delivery vehicle or pharmaceutical composition as described herein. The contacting may be ex vivo.

[0238] There is provided a method of treating or preventing a disease or condition in a subject in need thereof, said method comprising administering a therapeutically or prophylactically effective amount of a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof, and whereby said disease or condition is treated or prevented. There is also provided a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof for use in a method of treating or preventing a disease or condition.

[0239] There is also provided the use of a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof in the manufacture of a medicament for treating or preventing a disease or condition.

[0240] There is also provided a pharmaceutical composition comprising a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof and one or more pharmaceutically acceptable excipients, diluents or carriers for use in a method of treating or preventing a disease or condition.

[0241] The delivery vehicles, pharmaceutical compositions, one or more nucleic acids, diseases and conditions, and the like are all as described elsewhere herein.

[0242] The pathogenesis of the disease or condition may be mediated by the overexpression of at least one growth factor. In a particular example, the growth factor is transforming growth factor-p (TGF-p). The growth factor may be connective tissue growth factor (CTGF). The growth factor may be platelet-derived growth factor (PDGF). The growth factor may be fibroblast growth factor (FGF). The growth factor may be epidermal growth factor (EGF). The growth factor may be vascular endothelial growth factor (VEGF). The disease or condition may be mediated by a combination of growth factors, for example, 2, 3, 4, 5 or all of the growth factors.

[0243] The pathogenesis of the disease or condition may be mediated by a cellular transition. In particular, the pathogenesis of the disease or condition is mediated by epithelial to mesenchymal transition (EMT). Additionally or alternatively, the pathogenesis of the disease or condition may be mediated by mesothelial to mesenchymal transition (MMT). Additionally or alternatively, the pathogenesis of the disease or condition may be mediated by endothelial to mesenchymal transition (EndoMT).

[0244] Markers of EMT, MMT and / or EndoMT

[0245] Cells of embryonal epithelial origins (e.g. epithelial, mesothelial and endothelial cells) are characterised by high expression of e-cadherin. Therefore, e-cadherin is a marker of epithelial cells.

[0246] Mesenchymal cells are characterised by higher expression of o-small muscle actin (o-SMA), vimentin (VIM), fibronectin (FN) and N-cadherin. These are therefore markers of mesenchymal cells. Similarly, SNAIL1 is an EMT / MMT / EndoMT-associated transcription factor and it is also considered as a marker of such transitioning process.

[0247] IL-ip and IL-6 are cytokines associated with inflammation that, in specific pathological settings, may be associated with EMT / MMT / EndoMT. In these cases, IL-ip and IL-6 are also used for the cellular phenotype evaluation, as in the examples below for MeT5o cells. Another effect of TGF-p in mesothelial cells is the up-regulation of VEGF, a factor that is responsible for increased angiogenesis, subsequently leading to fibrosis. Thus, without being bound by theory, increases of VEGF are expected to correlate with detrimental pathology, leading to angiogenesis and pathology.

[0248] All these markers are used to evaluate the cell phenotype and assess whether a cellular transition is occurring. The ability of CRAT to reduce one or more of the mesenchymal markers o- SMA, VIM and / or FN, and / or one or more of the inflammatory cytokines IL-6 and / or IL-ip, and / or the angiogenesis inducer VEGF, and / or the fibrotic marker SNAIL; and / or also to increase the expression of e-cadherin can be used to demonstrate the ability of CRAT to prevent or reverse the EMT / MMT / EndoMT, and thus its utility in the diseases discussed below.

[0249] Fibrosis and diseases or conditions associated with fibrosis

[0250] TGF-p is a master regulator factor in the onset of peritoneal fibrosis. The pathophysiology of organ fibrosis involves various cellular effectors (neutrophils, macrophages, fibroblasts), up-regulation of profibrotic mediators (cytokines e.g. 11-6 and IL-ip, chemokines, and growth factors, e.g. VEGF), and processes where epithelial and endothelial cells undergo mesenchymal transition. Cellular transdifferentiation such EMT and MMT is characterized by cell hyperproliferation and / or collagen hyperproduction, contributing to fibrosis. In fact, EMT (which may be induced by TGF-p) has been described as one of the key cellular mechanisms of fibrosis, see Liu etai., Burns Trauma, 10, tkacOll, 2022, doi:10.1093 / burnst / tkac011. With the widely-reported association of EMT / MMT / EndoMT (and increases in markers such as TGF-p, o-SMA, FN, VIM, IL-6, IL- ip) in fibrotic diseases, the the delivery vehicles expressing CRAT or a functional fragment thereof described herein are expected to be particularly useful in the treatment of fibrotic diseases, diseases which have a fibrotic component and disease associated with fibrosis.

[0251] Zhao et ai, Signal Transduct. Target Ther., 8(1), 431, 2023, doi:10.1038 / s41392-023-01652- 9 describes that cardiac hypoxia (e.g. following myocardial infarction) induces EndoMT, contributing to cardiac fibrosis.

[0252] Thuan et al., supra describes the role of EMT and EndoMT in systemic sclerosis, atherosclerosis, Fuchs endothelial corneal dystrophy, and diabetic nephropathy.

[0253] As described in the examples below, CRAT expression is able to prevent or reverse EMT, and to reduce the expression of the pro-fibrotic marker SNAI1. Thus, it is expected that the delivery vehicles expressing CRAT or a functional fragment thereof will be useful in the treatment of fibrotic diseases or conditions.

[0254] Thus, in a particular example, the disease or condition is a fibrotic disease. In an example the fibrotic disease is selected from pulmonary fibrosis, liver fibrosis, renal fibrosis, myocardial fibrosis, peritoneal fibrosis, ischemic-induced fibrosis and hypoxic-induced fibrosis. In an example, the disease or condition is ischemic-induced fibrosis. In an example, the disease or condition is hypoxic-induced fibrosis. In an example, the disease or condition is subretinal fibrosis. In an example, the disease or condition is a fibrotic disease of the eye.

[0255] In another example, the disease or condition is a disease or condition associated with fibrosis. In an example, the disease or condition associated with fibrosis is selected from inflammatory bowel disease, polycystic kidney disease, endometriosis, encapsulating peritoneal sclerosis and pulmonary hypertension. In an example, the disease or condition is scleroderma. In an example, the disease or condition is rheumatoid arthritis. In an example, the disease or condition is myelofibrosis. In an example, the disease or condition is systemic lupus erythematosus (SLE). In an example, the disease or condition is chronic graft rejection. In an example, the disease or condition is Crohn's disease. In an example, the disease or condition is ulcerative colitis (UC). In an example, the disease or condition is pathological scarring. In an example, the disease is systemic sclerosis. In an example, the disease or condition is macular degeneration, e.g. age-related macular degeneration. In an example, the disease or condition is amyotrophic lateral sclerosis. In an example, the disease or condition is multiple sclerosis. In an example, the disease or condition is Alzheimer's disease. In an example, the disease or condition is vascular dementia. In an example, the disease or condition is Fuchs endothelial corneal dystrophy. In an example, the disease or condition is diabetic nephropathy.

[0256] Other conditions, such as hypoxia, neo-angiogenesis and glutaminolysis are typically present in fibrotic and cancer processes and thus may additionally benefit from therapy with delivery vehicles expressing CRAT or a functional fragment thereof.

[0257] It is known that the long-term exposure to the high glucose load of peritoneal dialysis (PD) fluids can induce peritoneal fibrosis, causing loss of peritoneal ultrafiltration capacity. Glucose overexposure results in the stimulation of growth factors, resulting in EMT, MMT and / or EndoMT. This leads to peritoneal membrane fibrosis which results in the structural and functional alteration of the peritoneal membrane. If a subject is left untreated, this fibrosis can result in dialysis failure. Thus, long-term use of PD may be associated with peritoneal fibrosis.

[0258] In a particular example, therefore, the disease or condition is end-stage kidney disease, and the subject is receiving or has received peritoneal dialysis. The administration of the delivery vehicle or pharmaceutical composition, in an example, mitigates the effects of glucose over-exposure, particularly wherein the subject has been treated with glucose-based peritoneal dialysis solution for at least one year. In an example, the subject is treated with PD daily or weekly.

[0259] For the treatment of end-stage kidney disease and / or peritoneal fibrosis, in a particular example, the delivery vehicle or pharmaceutical composition is administered intraperitoneally. More particularly, the delivery vehicle or pharmaceutical composition is administered during peritoneal dialysis.

[0260] There is also provided a method for the treatment of subjects treated with glucose-based peritoneal dialysis solutions to mitigate local and systemic glucose over-exposure, and to prevent fibrosis (in particular peritoneal fibrosis) by administering a delivery vehicle or pharmaceutical composition described herein.

[0261] The pharmaceutical composition may further comprise any anti-fibrotic active agents or combination thereof described elsewhere herein. The method or use may comprise administering the delivery vehicle or pharmaceutical compositions described herein simultaneously or sequentially with any anti-fibrotic active agents or combination thereof described elsewhere herein. In an example, the anti-fibrotic agent or combination thereof is administered separately to the delivery vehicle described herein.

[0262] In particular, the pharmaceutical composition may further comprise at least one further therapeutically active molecule which is for use in treating or preventing fibrosis and is selected from small molecule drugs, chemotherapy, hormone therapy, immunotherapy, photodynamic therapy, stem cell therapy, and antibody therapy, and the disease or condition to be treated is fibrosis or a disease associated with fibrosis. In particular, the method or use may comprise administering the delivery vehicle or pharmaceutical compositions described herein simultaneously or sequentially with at least one further therapeutically active molecule which is for use in treating or preventing fibrosis and is selected from small molecule drugs, chemotherapy, hormone therapy, immunotherapy, photodynamic therapy, stem cell therapy, and antibody therapy, and the disease or condition to be treated is fibrosis or a disease associated with fibrosis.

[0263] Cancer and tumour development

[0264] Cancers are a major contributor to disease burden worldwide, and projections forecast that global cancer burden will continue to grow. Cancer is a generic term for a large group of diseases that can affect any part of the body. A defining feature of cancer is the rapid creation of unwanted cells that grow beyond their usual boundaries, and which can then invade adjoining parts of the body and spread to other organs.

[0265] Many studies have proposed that induction of EMT is the primary mechanism by which epithelial cancer cells acquire malignant phenotypes that promote metastasis, see for example Thiery et al., Nature Reviews Cancer, 2(6), 442-54., 2002, doi:10.1038 / nrc822. Thus, generally, the overexpression of CRAT in host cells may be useful in the treatment of cancers and tumours.

[0266] The Examples show the potential therapeutic effect of cells overexpressing a human CRAT in DU145 and PC3 (prostate cancer) cells, in A549 (lung cancer) cells, MCF-7 and MDA-BM-231 (breast cancer) cells, thus demonstrating the potential therapeutic effect of the invention in a wide range of cancers.

[0267] In an example, the cancer is a solid tumour. In another example, the cancer is a blood-borne cancer.

[0268] In a particular example, the disease or condition is a cancer selected from 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 and bone cancer.

[0269] In another particular example, the disease or condition is skin cancer. In another particular example, the disease or condition is mesothelioma. In another particular example, the disease or condition is peritoneal mesothelioma. In another particular example, the disease or condition is prostate cancer. In another particular example, the disease or condition is vascular tumours.

[0270] In an example, the disease or condition is oral squamous cell carcinoma.

[0271] The pharmaceutical composition may further comprise any cancer active agents or combination thereof described elsewhere herein. The method or use may comprise administering the delivery vehicle or pharmaceutical compositions described herein simultaneously or sequentially with any cancer active agents or combination thereof described elsewhere herein. In an example, the cancer active agent or combination thereof is administered separately to the delivery vehicle described herein.

[0272] The method or use may comprise administering the delivery vehicle or pharmaceutical compositions described herein simultaneously or sequentially with at least one further therapeutically active molecule is selected from a cancer active agent selected from small molecule drugs, chemotherapy, hormone therapy, immunotherapy, photodynamic therapy, stem cell therapy, and antibody therapy, and the disease or condition to be treated is cancer.

[0273] The method or use may comprise administering the delivery vehicle or pharmaceutical compositions described herein simultaneously or sequentially with radiotherapy (and optionally any cancer active agents or combination thereof described elsewhere herein).

[0274] Eye diseases and disorders

[0275] Shu et al., Int. J. Mol. Sci., 21(12):4271, 2020, doi: 10.3390 / ijms21124271 describes that in the eye, EMT and EndoMT play key roles in the pathogenesis of subretinal fibrosis, the end-stage of age-related macular degeneration (AMD) that leads to profound and permanent vision loss. Also see Higashijima et al., Front. Ophthalmol. (Lausanne), 2, 1060087, 2023, doi:10.3389 / fopht. 2022.1060087.

[0276] Further neovascular AMD is currently treated with intravitreal injections of vascular endothelial growth factor (VEGF) inhibitors, which help improve vision. Thus, agents which reduce the expression of VEGF, as shown with the delivery vehicles expressing CRAT or a functional fragment thereof described herein may be useful for treatment of many types of AMD.

[0277] The Examples show the potential therapeutic effect of cells overexpressing a human CRAT in normal retinal epithelial cells which have been induced with TGF-p, thus demonstrating the potential therapeutic effect of the invention in eye-related disorders associated with the EMT / MMT / EndoMT transition. Thus, many diseases and disorders of the eye, especially those involving the cornea, retina and vascular components of the eye, and in particular those having a fibrotic component, are thought to involve the EMT / MMT / EndoMT process are expected to be able to be treated with the delivery vehicles expressing CRAT or a functional fragment thereof described herein.

[0278] In an example, the disease or condition is selected from angiogenic eye disorders, ocular diseases in the anterior segment, ocular diseases in the posterior segment, neovascular related ophthalmic posterior segment diseases, retinal diseases, macular degeneration (including age-related macular degeneration (AMD), such as neovascular AMD (also known as wet AMD)), diabetic retinopathies, diabetic macular oedema (DMO), choroidal neovascularisation (CNV), central retinal vein occlusion (CRVO), corneal neovascularization, retinal neovascularization and fibrovascular intraocular diseases. In an example, the disease is proliferative vitreoretinopathy (PVR). In an example, the disease is epiretinal membrane (ERM). In an example, the disease is proliferative diabetic retinopathy (PDR) and the delivery vehicle is optionally for treating retinal fibrosis associated with PDR. In an example, the disease is retinopathy of prematurity (ROP).

[0279] The pharmaceutical composition may further comprise active agents to treat fibrovascular intraocular diseases, such as bevacizumab, ranibizumab, brolucizumab, faricimab, aflibercept, and complement system-targeted therapies, or any combination thereof. The method or use may comprise administering the delivery vehicle or pharmaceutical compositions described herein simultaneously or sequentially with active agents to treat fibrovascular intraocular diseases, such as bevacizumab, ranibizumab, brolucizumab, faricimab, aflibercept, and complement system-targeted therapies, or any combination thereof.

[0280] Endometriosis

[0281] EMT has been established to be associated with endometriosis, with upregulation of TGF-p, as well as inflammatory cytokines, such as IL-6, IL-1 and VEGF, and a reduction in e-cadherin, see for example Cela et a!., Biomedicines, 9(11), 1681, 2021, doi:10.3390 / biomedicines9111681. In peritoneal and ovarian endometriotic lesions, epithelial markers were found downregulated in the epithelial cells, while the mesenchymal markers upregulated, see Yang & Yang, Oncotarget, 8(25), 41679-41689, 2017, doi: 10.18632 / oncotarget.16472.

[0282] The Examples show the potential therapeutic effect of cells overexpressing a human CRAT in human ectopic endometriotic epithelial cell line, thus demonstrating the potential therapeutic effect of the invention in endometriosis.

[0283] In a particular embodiment, the disease or condition is endometriosis. In an example, the disease or condition is superficial endometriosis. In an example, the disease or condition is cystic ovarian endometriosis. In an example, the disease or condition is deep infiltrating endometriosis.

[0284] The pharmaceutical composition may further comprise one or more active agents to treat endometriosis. The method or use may comprise administering the delivery vehicle or pharmaceutical compositions described herein simultaneously or sequentially with one or more active agents to treat endometriosis. First-line active agents for treating endometriosis include, but are not limited to, the combined oral contraceptive pill (COCP), progestagens and nonsteroidal anti-inflammatory drugs. Second-line active agents for treating endometriosis include, but are not limited to, gonadotropinreleasing hormone agonists and antagonists, non-hormonal complementary and alternative medicines (e.g. melatonin, cannabidiol, ligands of the Peroxisome Proliferator-Activated Receptor, pyruvate dehydrogenase kinase inhibitors). See, for example, Alonso et a!., Curr. Opin. Obstet. Gynecol., 36, 353-361, 2024 doi: 10.1097 / GC0.0000000000000983.

[0285] Inflammatory diseases

[0286] EMT can be associated with the stimulation of proinflammatory factors (see Table 1 of Suarez- Carmona, et a!., infra), and it is thought that there is a link between EMT and chronic inflammation, Suarez-Carmona, et al., Mol. Oncol., 11(7), 805-823, 2017, doi: 10.1002 / 1878-0261.12095. Lopez- Novoa & Nieto, EMBO Mol. Med., 1(6-7), 303-314, 2009, doi: 10.1002 / emmm.200900043 suggest that EMT is the "convergence point between inflammation and the progression of degenerative fibrotic diseases and cancer" and that factors associated with the control of the inflammatory response and in the induction of tumour cell death such as TGF-pi and hypoxia can also act as potent inducers of EMT in an inflammatory microenvironment.

[0287] In an example, the disease or condition is an inflammatory disease or condition. In an example, the disease or condition is chronic obstructive pulmonary disease (COPD). In an example, the disease or condition is epatithis. In an example, the disease or condition is Crohn's disease. In an example, the disease or condition is ulcerative colitis (UC).

[0288] Other diseases and conditions

[0289] In an example, the disease or condition is vascular dementia. According to several reports, liver fibrosis and pulmonary fibrosis (e.g. IPF) are associated with the incidence and / or progression of dementia, see for example Parikh et a!., Eur. J. Neurol., 29(9), 2622-2630, 2022, doi: 10.1111 / ene.15437; and Bors et a!., Chron. Respir. Dis., 12(4), 365-72, 2015, doi: 10.1177 / 1479972315603552. D'Ambrosi et al., Front. Immunol., 11, 1394, 2020, doi: 10.3389 / fimmu.2020.01394 report that fibrotic scarring has been implicated in the pathology of a number of CNS diseases and disorders, including amyotrophic lateral sclerosis, multiple sclerosis and Alzheimer's disease, and that these disease are also mediated by agents such as TGF-p and IL-6.

[0290] In an example, the disease or condition is amyotrophic lateral sclerosis (ALS). In an example, the disease or condition is multiple sclerosis. In an example, the disease or condition is Alzheimer's disease. In an example, the disease or condition is atherosclerosis. In an example, the disease or condition is pulmonary arterial hypertension. In an example, the disease or condition is diabetes mellitus.

[0291] In an example, the disease or condition is drug-induced nephrotoxicity. For example, Yuan et al., Am. J. Transplant., 15(6), 1682-1691, 2015, doi:https: / / doi.org / 10.1111 / ajt.l3161 describes that nephrotoxicity associated with the use of cyclosporine is likely to be induced by EMT. In an example, the disease or condition is a disease or condition related to the activation of fibrosis or the activation of inflammation. In an example, the disease or condition is a neoangiogenesis.

[0292] Subjects

[0293] Particularly, the subject is a human, for example a human patient.

[0294] In another example, the subject in a mammal.

[0295] In one embodiment, the subject is an animal. The subject may be a livestock or companion pet animal (e.g. a cow, pig, goat, sheep, horse, dog, cat or rabbit). The subject may be a laboratory animal (e.g. a mouse, rat, pig, dog, sheep, or primate).

[0296] Optionally, any method herein is not any of the following:

[0297] (a) a process for cloning a human being;

[0298] (b) a process for modifying the germ line genetic identity of a human being;

[0299] (c) a use of a human embryo;

[0300] (d) a process for modifying the genetic identity of an animals which is likely to cause it suffering without any substantial medical benefit to man or animal.

[0301] Optionally, any method herein is not a method of treatment on the human or animal body. Optionally, any method herein is not a surgical method.

[0302] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine study, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims. All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications and all US equivalent patent applications and patents are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Reference is made to the publications mentioned herein and equivalent publications by the US Patent and Trademark Office (USPTO) or WIPO, the disclosures of which are incorporated herein by reference for providing disclosure that may be used in the present invention and / or to provide one or more features (e.g. of a vector) that may be included in one or more claims herein.

[0303] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and / or." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0304] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0305] The term "or combinations thereof or similar as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0306] Any part of this disclosure may be read in combination with any other part of the disclosure, unless otherwise apparent from the context.

[0307] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and / or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0308] The present invention is described in more detail in the following non-limiting Examples.

[0309] Examples

[0310] Example 1

[0311] 1.1 Materials and Methods

[0312] 1.1.1 Cell Culture:

[0313] MeT5o 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.

[0314] 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.

[0315] 1.1.2 Transfection of CRAT overexpressing plasmid:

[0316] In order to stably obtain CRAT-overexpressing MeT5o cells, we used a plasmid-coding CRAT ORF purchased from OriGene (CRAT (NM_000755) Human Tagged ORF Clone CAT#: RC212196L2).

[0317] The CRAT sequence used is SEQ ID No:l.

[0318] 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.

[0319] 1.1.3 Gene Expression Analysis:

[0320] 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. Gene expression data are presented in arbitrary units (a.u.). The presence of non-specific amplification products was excluded by melting curve analysis. The primers used are from SEQ ID No:44 to SEQ ID No:63, as indicated in the following Table 1:

[0321] Table 1

[0322] 1.1.4 Western Blotting:

[0323] 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).

[0324] 1.2 Results

[0325] 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.

[0326] 1.3 Investigating the effect of CRAT expression and carnitine in cells treated with TGFB

[0327] Transforming growth factor beta (TGF-p) has been reported to be able to induce fibrosis, through the activation of EMT, and cause inflammation. It is also known that TGF-p is able to up- regulate VEGF, a factor that sustains neoangiogenesis. Thus, the addition of TGF-p to cells growing in vitro is a recognised model to induce the fibrosis and investigate the detrimental effects in diseaserelevant cell lines.

[0328] The experimental results show that CRAT overexpression and the use of L-carnitine can hinder this process.

[0329] 1.3.1 Materials and methods

[0330] In detail, wild type and CRAT-overexpressing cells was treated with or without 20 ng / mL of TGF-p in the presence of 50 pM L-Carnitine (physiological concentration) or 2 mM L-Carnitine (therapeutically relevant concentration).

[0331] Gene expression analysis was carried out as described in section 1.1.3 above. 1.3.2 Results a-SMA and vimentin:

[0332] Gene expression results show that the treatment with TGF-p significantly increased the expression of the mesenchymal markers o-small muscle actin (o-SMA) and Vimentin (VIM) in WT mesothelial cells (MeT5o cells). The addition of 2 mM L-Carnitine maintained (prevented increase) the expression of these two markers at basal level.

[0333] The treatment with TGF-p in CRAT-overexpressing clones (over-CRATl and over-CRAT2) failed to up-regulate (prevented an increase in) o-SMA and Vimentin expression also at physiological L-Carnitine concentration (50 pM), as shown in Figure 3.

[0334] These data were confirmed also by the observing the morphological aspects of WT and CRAT- overexpressing cells (MeT5o) treated with TGF-p. Only in WT cells treated with TGF-p are visible groups of elongated and spindle shaped cells, as shown in Figure 4.

[0335] Interleukin-6 and interleukin-ip:

[0336] Gene expression analysis also showed that treatment TGF-p increases the inflammatory phenotype of WT mesothelial cells (MeT5o). The treatment of WT cells with 2 mM L-Carnitine significantly reduced the up-regulation (prevented an increase) of Interleukin-6 (IL-6) and Interleukin- 1P (IL-1P).

[0337] Similarly, the treatment with TGF-p in CRAT overexpressing cells did not induce any changes in the expression of these pro-inflammatory markers, as shown in Figure 5.

[0338] Vascular endothelial growth factor (VEGF):

[0339] Another effect of TGF-p in mesothelial cells is the up-regulation of VEGF, a factor that is responsible for increased angiogenesis (which often accompanies fibrosis) in endothelial cells. In patients undergoing peritoneal dialysis specifically, VEGF-induced angiogenesis and fibrosis results in structural and functional changes to the peritoneal membrane, ultimately leading to loss of ultrafiltration capacity and dialysis failure. Here we show that TGF-p increases VEGF expression in WT mesothelial (MeT5o) cells at physiological L-carnitine concentrations (50pM), but this effect was significantly reduced by the addiction of 2 mM L-Carnitine (therapeutically relevant concentration), and was completely prevented in CRAT-overexpressing cells (Figure 6).

[0340] 1.4 Summary

[0341] In summary, the experimental data presented in Example 1 showed that wild type and CRAT- overexpressing mesothelial cells (MeT5o) displayed a different metabolic profile: they showed a different behaviour in response to TGF-p. The treatment with TGF-p in WT cells 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 (therapeutically relevant) L-carnitine levels (2 mM) significantly reduced the up-regulation of the markers analysed. Similarly, and importantly, TGF-p was unable to modulate the expression of o-SMA, VIM, IL-6, IL-ip and VEGF in mesothelial cells transfected to overexpress CRAT, suggesting that gene therapies to express CRAT in cells may provide beneficial downstream effects in the signalling pathways associated with fibrosis and inflammation.

[0342] Example 2

[0343] 2.1 Materials and methods

[0344] 2.1.1 Cell lines and cultures:

[0345] 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.

[0346] 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.

[0347] ARPE-19 (ATCC® CRL-2302™) normal retinal epithelial cells were maintained in DMEM-F12 with 10% fetal bovine serum (FBS), and an antibiotic solution.

[0348] HK-2 (ATCC® CRL-2190™) proximal tubule epithelial cell line were maintained in DMEM-F12 with 10% fetal bovine serum (FBS), and an antibiotic solution.

[0349] Hep G2 [HEPG2] is a cell line exhibiting epithelial-like morphology that was isolated from a hepatocellular carcinoma ( ATCC HB-8065 ™) were maintained in DMEM with 10% fetal bovine serum (FBS), and an antibiotic solution.

[0350] 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.

[0351] 2.1.2 Transfection of CRAT overexpressing plasmid:

[0352] 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)

[0353] 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-p.

[0354] 2.1.3 Gene Expression Analysis:

[0355] 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. Gene expression data are presented in arbitrary units (a.u.). The presence of non-specific amplification products was excluded by melting curve analysis.

[0356] 2.2 Results

[0357] Wild Type (WT) DU145 and PC3 cell analysis:

[0358] Results showed that the two prostate cancer cell lines (DU145 and PC3) analysed displayed different EMT markers: DU145 (the one more epithelial-like) expresses more E-cadherin and less o- SMA and SNAIL compared to PC3 (the more mesenchymal-like). Results showed a higher CRAT expression in DU145 cells. (Table 2).

[0359] Table 2

[0360] DU145 cells were treated with or without TGF-p (10 ng / mL) in presence or absence of different concentration of L-carnitine (active enantiomer) and D-carnitine (inactive enantiomer).

[0361] TGFp-induced WT DU145 cells:

[0362] TGF-p alone reduced the expression of E-cadherin and up-regulated the expression of both o-SMA and SNAIL. This effect was reduced by 50 pM L-carnitine and almost abrogated by 2mM L- carnitine (Table 3).

[0363] Table 3

[0364] Moreover, DU145 cells were treated with or without TGF-p (lOng / mL) in presence or absence of overexpression of CRAT. Results showed that CRAT overexpression prevented the up-regulation of a-SMA, VIM and FN induced by TGF-p on the prostate cancer cell line DU145, supporting the utility of CRAT overexpression in the exemplified disease cells (Table 3bis).

[0365] Table 3bis

[0366] TGFp-induced WT PC3 cells:

[0367] PC3 were treated with or without TGF-p (lOng / mL) in presence or absence of different concentration of L-carnitine (active enantiomer) and D-carnitine (inactive enantiomer).

[0368] The effect of TGF-p on the expression of E-cadherin (a key epithelial marker) was mild, since this cell line already express very low levels of this protein; however TGF-p did reduce its expression (suggesting potentially transition away from the epithelial cell type) and this effect is reversed by the addition L-carnitine. In this cell line, TGF-p up-regulated the expression of o-SMA and SNAIL. This effect was reduced by 50 pM 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).

[0369] Table 4

[0370] Transfection of PC3 cells with CRAT:

[0371] PC3 cells were also transfected with CRAT-overexpressing plasmid and the resultant cells displayed a different morphology. From SEM photos (Figure 7), 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.

[0372] Furthermore, PC3 cells were treated with or without TGF-p (lOng / mL) in presence or absence of overexpression of CRAT.

[0373] Results showed that CRAT overexpression prevented the up-regulation of a-SMA, VIM and FN induced by TGF-b on the prostate cancer cell line PC3, supporting the utility of CRAT overexpression in the exemplified disease cells (Table 4bis).

[0374] Table 4bis

[0375] WT A549 cell line analysis:

[0376] A549 is a lung carcinoma cell line that, in response to TGF-p, is known to undergo EMT.

[0377] Here we confirmed that TGF-p reduces the expression of E-cadherin and up-regulates the expression of o-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.

[0378] Table 5

[0379] Transfection of A549 cells with CRAT & induction with TGF-p:

[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 o-SMA, VIM and FN induced by TGF-p (Table 6).

[0381] Table 6

[0382] WT Breast Cancer cell line analysis (MCF7, MDA-MB-231):

[0383] Results showed that the two breast cancer cell lines analyzed displayed different EMT markers:

[0384] MCF7 (the one more epithelial-like) express more E-cadherin and less o-SMA and SNAIL respect to

[0385] MDA-MB-231 (the more mesenchymal-like). Results showed a higher CRAT expression in MCF7 cells. (Table 7).

[0386] Table 7

[0387] Transfection of MCF-7 and MDA-BM-231 cells with CRAT & induction with TGF-p:

[0388] 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 1715,87±22,78 (for MCF- 7) and in a 325, 44± 17,84 (for MDA_MB-231) fold increase in CRAT gene expression compared to control cells.

[0389] CRAT overexpression prevented the upregulation of mesenchymal markers a-SMA, VIM, and FN induced by TGF-p 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) Table 8

[0390] Table 9

[0391] TGF-p induced WT 12Z cell line analysis:

[0392] As discussed above, it has been established that EMT contributes to progression of endometriosis and 12z, a human ectopic endometriotic epithelial cell line, when treated with TGF-p undergo EMT. Here we confirmed that TGF-p upregulates the expression of 3 mesenchymal markers and both the concentrations of L-carnitine tested were able to reduce the TGF-p effect. (Table 10)

[0393] Table 10

[0394] ARPE-19 transfection with CRAT and analysis:

[0395] Macular degeneration is a disease that affects the central part of the retina where EMT contributes to the progression of the disease, see Shu etaL, supra.

[0396] 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.

[0397] 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-p.

[0398] Table 11 Transfection of HepG2 cells with CRAT & induction with TGF-p:

[0399] HepG2 is a liver adenocarcinoma cell line that, in response to TGF-p, is known to undergo EMT.

[0400] Here we confirmed that TGF-p up-regulates the expression of o-SMA, VIM and FN. This effect was reduced by CRAT overexpression.

[0401] HepG2 cells were treated with or without TGF-p (lOng / mL) in presence or absence of overexpression of CRAT (Table 12).

[0402] Table 12

[0403] Transfection of HK2 cells with CRAT & induction with TGF-p:

[0404] TGF-beta is a master activator of EMT and fibrosis also in the kidney

[0405] HK2 is a proximal tubular epithelial cell line that, in response to TGF-p, is known to undergo EMT.

[0406] Here we confirmed that TGF-p up-regulates the expression of o-SMA, VIM and FN. This effect was reduced by CRAT overexpression.

[0407] HK2 cells were treated with or without TGF-p (lOng / mL) in presence or absence of overexpression of CRAT (Table 13).

[0408] Table 13

[0409] Example 3

[0410] 3.1 Materials and methods

[0411] The mitochondrial respiratory function of control and treated HMRSV5 cells were measured using a Mito stress test assay with an XF24 extracellular flux analyzer (Seahorse Bioscience,), according to the manufacturer's instructions. Cells were seeded at a density of 5 x 104cells / well with 500 pL of growth medium (M199 medium containing 10% of FBS) in XF24 cell culture plates and incubated for 24 h at 37 °C with 5% of CO2. Subsequently, growth medium was removed from the wells, leaving on them only 50 pL medium. Then, cells were washed twice with 500 pL of pre-warmed assay XF base medium (102353-100) supplemented with 10 mM glucose (103577-100), 1 mM glutamine (103579-100), and 1 mM sodium pyruvate (103578-100); pH 7.4); eventually, 450 pL of assay medium (500 pL final) were added. Cells were incubated at 37 °C for 1 h to allow preequilibrating with the assay medium. Mitochondrial functionality was evaluated by the sequential injection of four compounds affecting bioenergetics. The final concentrations of the injected reagents were 1 pM oligomycin, 2 pM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and 1 and 2.5 pM rotenone / antimycin A. Optimal concentrations of inhibitors and uncouplers, as well as the cells' seeding density, were previously determined. A minimum of five replicates per treatment were used in each experiment. Next, basal respiration, maximal respiration, spare respiratory capacity, and ATP production were quantified (see Figure 9).

[0412] 3.1.1 Bioenergetics

[0413] It is known that TGF-p reinforces this hyper-glycolytic state, establishing a deleterious feedback loop that drives epithelial-to-mesenchymal transition (EMT) and fibrosis.

[0414] To assess the contribution of CRAT on this metabolic route, WT cells and CRAT- overexpressing human mesothelial cells were stimulated with TGF-p.

[0415] As expected, Figure 9 shows that TGF-p markedly increased glycolysis while reducing oxidative respiration in WT mesothelial cells. Interestingly, both these events were prevented by CRAT- overexpression.

[0416] Numbered embodiments

[0417] Embodiment 1. A method of treating or preventing a disease or condition in a subject in need thereof, said method comprising administering a therapeutically or prophylactically effective amount of a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof, and whereby said disease or condition is treated or prevented, optionally wherein the delivery vehicle is as defined in any one of embodiments 19 to 36 or wherein the delivery vehicle is comprised by a pharmaceutical composition as defined in any one of embodiments 37 to 40, or 42 to 45.

[0418] Embodiment 2. A delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof for use in a method of treating or preventing a disease or condition, optionally wherein the delivery vehicle is as defined in any one of embodiments 19 to 36 or wherein the delivery vehicle is comprised by a pharmaceutical composition as defined in any one of embodiments 37 to 40, or 42 to 45. Embodiment 3. The use of a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof in the manufacture of a medicament for treating or preventing a disease or condition, optionally wherein the delivery vehicle is as defined in any one of embodiments 19 to 36 or wherein the delivery vehicle is comprised by a pharmaceutical composition as defined in any one of embodiments 37 to 40, or 42 to 45.

[0419] Embodiment 4. A pharmaceutical composition comprising a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof and one or more pharmaceutically acceptable excipients, diluents or carriers for use in a method of treating or preventing a disease or condition, optionally wherein the delivery vehicle is as defined in any one of embodiments 19 to 36 or wherein the delivery vehicle is comprised by a pharmaceutical composition as defined in any one of embodiments 37 to 40, or 42 to 45.

[0420] Embodiment 5. The method according to embodiment 1, a delivery vehicle for the use according to embodiment 2, the use according to embodiment 3, or a composition for the use according to embodiment 4, wherein the pathogenesis of the disease or condition is mediated by:

[0421] (i) overexpression of at least one growth factor selected from transforming growth factor-p (TGF-p), connective tissue growth factors (CTGF), platelet-derived growth factors (PDGF), fibroblast growth factors (FGF), epidermal growth factors (EGF) and vascular endothelial growth factors (VEGF), in particular TGF- ; or

[0422] (ii) an epithelial, mesothelial and / or endothelial to mesenchymal transition (EMT, MMT and / or EndoMT, in particular EMT).

[0423] Embodiment 6. The method, delivery vehicle, use or pharmaceutical composition according to any one of embodiments 1 to 5, wherein the disease or condition is selected from a fibrotic disease (e.g. pulmonary fibrosis, liver fibrosis, renal fibrosis, myocardial fibrosis, peritoneal fibrosis, ischemic-induced fibrosis and hypoxic-induced fibrosis), a disease or condition associated with fibrosis (e.g. inflammatory bowel disease, polycystic kidney disease, endometriosis, encapsulating peritoneal sclerosis, and pulmonary hypertension), cancer and vascular dementia.

[0424] Embodiment 7. The method, delivery vehicle, use or pharmaceutical composition according to any one of embodiments 1 to 5, wherein the disease or condition is selected from angiogenic eye disorders, ocular diseases in the anterior segment, ocular diseases in the posterior segment, neovascular related ophthalmic posterior segment diseases, retinal diseases, macular degeneration (including age-related macular degeneration (AMD), such as neovascular AMD (also known as wet AMD)), diabetic retinopathies, diabetic macular oedema (DMO), choroidal neovascularisation (CNV), central retinal vein occlusion (CRVO), corneal neovascularization, retinal neovascularization, fibrovascular intraocular diseases, pathological scarring and drug- induced nephrotoxicity.

[0425] Embodiment 8. The method, delivery vehicle, use or pharmaceutical composition according to any one of embodiments 1 to 6, wherein the disease or condition is a cancer selected from 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 and bone cancer.

[0426] Embodiment 9. The method, delivery vehicle, use or pharmaceutical composition according to any one of embodiments 1 to 6, wherein the disease or condition is a cancer selected from skin cancer, mesothelioma, peritoneal mesothelioma, prostate cancer and vascular tumours.

[0427] Embodiment 10. The method, delivery vehicle, use or pharmaceutical composition according to any one of embodiments 1 to 5, wherein the disease or condition is an inflammatory disease or condition, optionally wherein the inflammatory disease or condition is selected from chronic obstructive pulmonary disease (COPD), epatithis and Crohn's disease.

[0428] Embodiment 11. The method, delivery vehicle, use or pharmaceutical composition according to any one of embodiments 1 to 5, wherein the disease or condition is end-stage kidney disease, and the subject is receiving or has received peritoneal dialysis, optionally wherein the administration of the delivery vehicle or pharmaceutical composition mitigates the effects of glucose over-exposure, particularly wherein the subject has been treated with glucose-based peritoneal dialysis solution for at least one year.

[0429] Embodiment llbis. The method, delivery vehicle, use or pharmaceutical composition according to any one of embodiments 1 to 5, wherein the disease or condition is selected from one or more of the following diseases:

[0430] -prostate carcinoma;

[0431] -prostate adenocarcinoma;

[0432] -lung carcinoma;

[0433] -breast adenocarcinoma;

[0434] -endometriosis;

[0435] -macular degeneration -fibrosis;

[0436] -liver adenocarcinoma.

[0437] Embodiment lltris The method, delivery vehicle, use or pharmaceutical composition according to embodiments 1 Ibis, wherein the disease or condition in an in vitro model is selected from the following diseases:

[0438] -prostate carcinoma expressed in DU 145 cell line;

[0439] -prostate adenocarcinoma expressed in PC3 cell line;

[0440] -lung carcinoma expressed in A549 cell line;

[0441] -breast adenocarcinoma expressed in MCF-7and MDA-MB-231 cell lines;

[0442] -endometriosis expressed in 12Z cell line;

[0443] -macular degeneration expressed in ARPE-19 cell line;

[0444] -fibrosis expressed in HK-2 cell line;

[0445] -liver adenocarcinoma expressed in Hep G2 cell line.

[0446] Embodiment 12. The method, delivery vehicle, use or pharmaceutical composition according to any one of embodiments 1 to 11, wherein the delivery vehicle or pharmaceutical composition is administered by a route of administration selected from enteral, parenteral, inhaled and transcutaneous.

[0447] Embodiment 13. The method, delivery vehicle, use or pharmaceutical composition according to any one of embodiments 1 to 11, wherein the delivery vehicle or pharmaceutical composition is administered intraperitoneally, intramuscularly or intravenously, and optionally wherein the disease or condition is not fibrotic stress, fibrosis or inflammation.

[0448] Embodiment 14. The method, delivery vehicle, use or pharmaceutical composition according to any one of embodiments 1 to 11, wherein the delivery vehicle or pharmaceutical composition is administered intraocularly, transderma I ly, topically, intranasally, intravitreally or via intra-tumoral administration.

[0449] Embodiment 15. The method, delivery vehicle, use or pharmaceutical composition according to any one of embodiments 1 to 14, wherein the delivery vehicle or pharmaceutical composition is administered in combination with at least one further therapeutically active molecule (such as an anti-fibrotic active agent or a cancer active agent), or in combination with radiotherapy, and wherein the administration of the at least one further therapeutically active molecule is administered separately, and optionally wherein the at least one further therapeutically active molecule is selected from carnitine, dichloroacetate (DCA), oxamic acid, shikonin, oleanolic acid, 3-bromopyruvate, anticancer active agents, anti-fibrotic active agents and citrate.

[0450] Embodiment 16. The method, delivery vehicle, use or pharmaceutical composition according to any one of embodiments 1 to 15, wherein the delivery vehicle or pharmaceutical composition is administered in combination with at least one further therapeutically active molecule selected from small molecule drugs, chemotherapy, hormone therapy, immunotherapy, photodynamic therapy, stem cell therapy, and antibody therapy, in particular any of the aforementioned which are for use in treating cancer, and when the disease or condition is cancer, or in particular any of the aforementioned which are for use in treating or preventing fibrosis, and when the disease or condition is fibrosis or a disease associated with fibrosis.

[0451] Embodiment 17. The method, delivery vehicle, use or pharmaceutical composition according to any one of embodiments 1 to 16, wherein the one or more nucleic acids is expressed in host cells in the subject, optionally wherein the one or more nucleic acids are integrated into the chromosome of the host cell.

[0452] Embodiment 18. A delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof for use in therapy, in particular for use in gene therapy, optionally wherein the delivery vehicle is as defined in any one of embodiments 19 to 36 or wherein the delivery vehicle is comprised by a pharmaceutical composition as defined in any one of embodiments 37 to 40, or 42 to 45.

[0453] Embodiment 19. A delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof.

[0454] Embodiment 20. The delivery vehicle according to embodiment 19, wherein the delivery vehicle is selected from a lipid nanoparticle (e.g. lipid nanoparticles with immunostimulatory potency), a protamine-condensed mRNA, an exosome, an extracellular vesicle (EV), mesoporous silica, a CaP, a polymetric nanoparticle, a polycationic peptide, a polycationic protein and a crown-compound.

[0455] Embodiment 21. The delivery vehicle according to embodiment 19, wherein the delivery vehicle is selected from a cationic liposome, a modified dendrimer nanoparticle, a cationic polymer, a cationic polymer liposome, a polymer-lipid hybrid nanoparticle, a cationic nanoemulsion, a protamin-based core shell particle, a polymeric micelle, a cationic polysaccharide particle, and a synthetic lipoprotein.

[0456] Embodiment 22. The delivery vehicle according to any one of embodiments 19 to 21, wherein the delivery vehicle is not an isolated plasmid, or is not a pLenti7.3 expression vector.

[0457] Embodiment 23. The delivery vehicle according to any one of embodiments 19 to 22, wherein the delivery vehicle is not introduced into a host cell via electroporation.

[0458] Embodiment 24. The delivery vehicle according to any one of embodiments 19, 22 or 23, wherein the delivery vehicle is a viral vector (for example an adeno-associated virus (AAV) vector, an adenoviral vector, a lentiviral vector, an alphaviral vector, a picornaviral vector, a flaviviral vector, a vescicolar stomatitis viral vector, a retroviral vector, an engineered phage or a phagemid), or a gold nanoparticle, a MXENES particle or a transposon.

[0459] Embodiment 25. The delivery vehicle according to any one of embodiments 19 to 24, wherein the carnitine acetyl transferase or functional fragment thereof: a. comprises an amino acid sequence selected from SEQ ID No:3, SEQ ID No:5, SEQ ID No:7, SEQ ID No:9, SEQ ID No: 11 and SEQ ID No: 13 or an amino acid having at least (about) 70% identity, at least (about) 80% identity, or at least (about) 90% identity thereto; or b. is encoded by a nucleotide sequence selected from SEQ ID No:l, SEQ ID No:2, SEQ ID No:4, SEQ ID No:6, SEQ ID No:8, SEQ ID No: 10 and SEQ ID No: 11 or a nucleotide sequence having at least (about) 70% identity, at least (about) 80% identity, or at least (about) 90% identity thereto.

[0460] Embodiment 26. The delivery vehicle according to any one of embodiments 19 to 24, wherein the carnitine acetyl transferase or functional fragment thereof comprises an amino acid sequence selected from SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19 and SEQ ID No:20, or an amino acid having at least (about) 70% identity, at least (about) 80% identity, or at least (about) 90% identity thereto.

[0461] Embodiment 27. The delivery vehicle according to any one of embodiments 19 to 24, wherein the carnitine acetyl transferase or functional fragment thereof does not comprise an amino acid sequence selected from SEQ ID No:21 and SEQ ID No:23, or wherein the carnitine acetyl transferase or functional fragment thereof is not encoded by the nucleotide sequence of SEQ ID No:22. Embodiment 28. The delivery vehicle according to any one of embodiments 19 to 27, wherein the one or more nucleic acids is operably linked to a constitutive promoter, optionally wherein the promoter is functional for expression of the one or more nucleic acids in a host cell, for example a host cell in vivo, optionally wherein the promoter is not naturally associated with the expression of a carnitine acetyl transferase.

[0462] Embodiment 29. The delivery vehicle according to any one of embodiments 19 to 28, wherein the expression of the carnitine acetyl transferase in an in vitro fibrosis model in host cells (optionally wherein the fibrosis is induced by the addition of TGF-p), i. is capable of reverting the fibrosis by decreasing, or is capable of decreasing, o-smooth muscle actin (o-SMA) relative expression as compared to control host cells which do not comprise the one or more nucleic acids, in particular wherein the decreasing is a decrease of at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%; and / or ii. is capable of preventing the induction of fibrosis by maintaining comparable (e.g. within a statistically significant level) expression of o-SMA, or is capable of maintaining comparable (e.g. within a statistically significant level) expression of o-SMA, or is capable of preventing an increase (e.g. a statistically significant increase) in expression of o-SMA, as compared to control host cells which have not been induced for fibrosis (e.g. by TGF-p), and optionally wherein the in vitro model is carried out in the presence of a physiological concentration of L-carnitine (e.g. 50pM), or a therapeutically relevant concentration of L-carnitine (e.g. 2mM), and further optionally wherein the host cells are not cells selected from MeT5o cells, DU 145 cells, A549 cells, MCF-7 cells, MDA-MB-231 cells, 12Z cells, HK2 cells, HepG2 cells and / or ARPE-19 cells.

[0463] Embodiment 30. The delivery vehicle according to any one of embodiments 19 to 29, wherein the expression of the carnitine acetyl transferase in an in vitro fibrosis model in host cells (optionally wherein the fibrosis is induced by the addition of TGF-p), i. is capable of reverting the fibrosis by decreasing, or is capable of decreasing, vimentin (VIM) relative expression as compared to control host cells which do not comprise the one or more nucleic acids, in particular wherein the decreasing is a decrease of at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%; and / or ii. is capable of preventing the induction of fibrosis by maintaining comparable (e.g. within a statistically significant level) expression of VIM, or is capable of maintaining comparable (e.g. within a statistically significant level) expression of VIM, or is capable of preventing an increase (e.g. a statistically significant increase) in expression of VIM as compared to control host cells which have not been induced for fibrosis (e.g. by TGF-p), and optionally wherein the in vitro model is carried out in the presence of a physiological concentration of L-carnitine (e.g. 50pM), or a therapeutically relevant concentration of L-carnitine (e.g. 2mM), and further optionally wherein the host cells are not cells selected from MeT5o cells, DU 145 cells, A549 cells, MCF-7 cells, MDA-MB-231 cells, 12Z cells, HK2 cells, HepG2 cells and / or ARPE-19 cells.

[0464] Embodiment 31. The delivery vehicle according to any one of embodiments 19 to 30, wherein the expression of the carnitine acetyl transferase in an in vitro fibrosis model in host cells (optionally wherein the fibrosis is induced by the addition of TGF-p) i. is capable of reverting the fibrosis by decreasing, or is capable of decreasing interleukin-6 (IL-6) relative expression as compared to control host cells which do not comprise the one or more nucleic acids, in particular wherein the decreasing is a decrease by at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%; and / or ii. is capable of preventing the induction of fibrosis by maintaining comparable or reducing (e.g. within a statistically significant level) expression of IL-6, or is capable of maintaining comparable or reducing (e.g. within a statistically significant level) expression of IL-6, or is capable of preventing an increase (e.g. a statistically significant increase) in expression of IL-6 as compared to control host cells which have not been induced for fibrosis (e.g. by TGF- P), and optionally wherein the in vitro model is carried out in the presence of a physiological concentration of L-carnitine (e.g. 50pM), or a therapeutically relevant concentration of L-carnitine (e.g. 2mM), and further optionally wherein the host cells are not cells selected from MeT5o cells, DU 145 cells, A549 cells, MCF-7 cells, MDA-MB-231 cells, 12Z cells, HK2 cells, HepG2 cells and / or ARPE-19 cells.

[0465] Embodiment 32. The delivery vehicle according to any one of embodiments 19 to 31, wherein the expression of the carnitine acetyl transferase in an in vitro fibrosis model in host cells (optionally wherein the fibrosis is induced by the addition of TGF-p), i. is capable of reverting the fibrosis by decreasing, or is capable of decreasing, interleukin-ip (IL-ip) relative expression as compared to control host cells which do not comprise the one or more nucleic acids, in particular wherein the decreasing is a decrease by at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%; and / or ii. is capable of preventing the induction of fibrosis by maintaining comparable (e.g. within a statistically significant level) expression of IL-ip, or is capable of maintaining comparable (e.g. within a statistically significant level) expression of IL-ip, or is capable of preventing an increase (e.g. a statistically significant increase) in expression of IL- ip, as compared to control host cells which have not been induced for fibrosis (e.g. by TGF-p), and optionally wherein the in vitro model is carried out in the presence of a physiological concentration of L-carnitine (e.g. 50pM), or a therapeutically relevant concentration of L-carnitine (e.g. 2mM), and further optionally wherein the host cells are not cells selected from MeT5o cells, DU 145 cells, A549 cells, MCF-7 cells, MDA-MB-231 cells, 12Z cells, HK2 cells, HepG2 cells and / or ARPE-19 cells.

[0466] Embodiment 33. The delivery vehicle according to any one of embodiments 19 to 32, wherein the expression of the carnitine acetyl transferase in an in vitro fibrosis model in host cells (optionally wherein the fibrosis is induced by the addition of TGF-p), i. is capable of reverting the fibrosis by decreasing, or is capable of decreasing, vascular endothelial growth factor (VEGF) relative expression as compared to control host cells which do not comprise the one or more nucleic acids, in particular wherein the decreasing is a decrease by at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%; and / or ii. is capable of preventing the induction of fibrosis by maintaining comparable (e.g. within a statistically significant level) expression of VEGF, or is capable of maintaining comparable (e.g. within a statistically significant level) expression of VEGF, or is capable of preventing an increase (e.g. a statistically significant increase) in expression of VEGF, as compared to control host cells which have not been induced for fibrosis (e.g. by TGF-p), and optionally wherein the in vitro model is carried out in the presence of a physiological concentration of L-carnitine (e.g. 50pM), and further optionally wherein the host cells are not cells selected from MeT5o cells, DU 145 cells, A549 cells, MCF-7 cells, MDA-MB-231 cells, 12Z cells, HK2 cells, HepG2 cells and / or ARPE-19 cells.

[0467] Embodiment 34. The delivery vehicle according to any one of embodiments 19 to 33, wherein the expression of the carnitine acetyl transferase in an in vitro fibrosis model in host cells (optionally wherein the fibrosis is induced by the addition of TGF-p), i. is capable of reverting the fibrosis by decreasing, or is capable of decreasing, zinc finger protein SNAI1 (SNAIL) expression as compared to control host cells which do not comprise the one or more nucleic acids, in particular wherein the decreasing is a decrease by at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%; and / or ii. is capable of preventing the induction of fibrosis by maintaining comparable (e.g. within a statistically significant level) expression of SNAIL, or is capable of maintaining comparable (e.g. within a statistically significant level) expression of SNAIL, or is capable of preventing an increase (e.g. a statistically significant increase) in expression of SNAIL, as compared to control host cells which have not been induced for fibrosis (e.g. by TGF-p), and optionally wherein the in vitro model is carried out in the presence of a physiological concentration of L-carnitine (e.g. 50pM), or a therapeutically relevant concentration of L-carnitine (e.g. 2mM), and further optionally wherein the host cells are not cells selected from MeT5o cells, DU 145 cells, A549 cells, MCF-7 cells, MDA-MB-231 cells, 12Z cells, HK2 cells, HepG2 cells and / or ARPE-19 cells.

[0468] Embodiment 35. The delivery vehicle according to any one of embodiments 19 to 34, wherein the expression of the carnitine acetyl transferase in an in vitro fibrosis model in host cells (optionally wherein the fibrosis is induced by the addition of TGF-p), i. is capable of reverting the fibrosis by increasing, or is capable of increasing, E-cadherin expression, as compared to control host cells which do not comprise the one or more nucleic acids, in particular wherein the increasing is an increase by at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%; and / or ii. is capable of preventing the induction of fibrosis by maintaining comparable (e.g. within a statistically significant level) expression of e-cadherin, or is capable of maintaining comparable (e.g. within a statistically significant level) expression of e-cadherin, or is capable of preventing a decrease (e.g. a statistically significant decrease) in expression of E-cadherin, as compared to control host cells which have not been induced for fibrosis (e.g. by TGF-p), and optionally wherein the in vitro model is carried out in the presence of a physiological concentration of L-carnitine (e.g. 50pM), or a therapeutically relevant concentration of L-carnitine (e.g. 2mM), and further optionally wherein the host cells are not cells selected from MeT5o cells, DU 145 cells, A549 cells, MCF-7 cells, MDA-MB-231 cells, 12Z cells, HK2 cells, HepG2 cells and / or ARPE-19 cells.

[0469] Embodiment 36. The delivery vehicle according to any one of embodiments 19 to 35, wherein the expression of the carnitine acetyl transferase in an in vitro fibrosis model in host cells (optionally wherein the fibrosis is induced by the addition of TGF-p), i. is capable of reverting the fibrosis by decreasing, or is capable of decreasing, fibronectin (FN) expression as compared to control host cells which do not comprise the one or more nucleic acids, in particular wherein the decreasing is a decrease by at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%; and / or ii. is capable of preventing the induction of fibrosis by maintaining comparable or reducing (e.g. within a statistically significant level) expression of FN, or is capable of maintaining comparable or reducing (e.g. within a statistically significant level) expression of FN, or is capable of preventing an increase (e.g. a statistically significant increase)in expression of FN as compared to control host cells which have not been induced for fibrosis (e.g. by TGF-p), and optionally wherein the in vitro model is carried out in the presence of a physiological concentration of L-carnitine (e.g. 50pM), or a therapeutically relevant concentration of L-carnitine (e.g. 2mM), and further optionally wherein the host cells are not cells selected from MeT5o cells, DU 145 cells, A549 cells, MCF-7 cells, MDA-MB-231 cells, 12Z cells, HK2 cells, HepG2 cells and / or ARPE-19 cells.

[0470] Embodiment 37. A pharmaceutical composition comprising a delivery vehicle as defined in any one of embodiments 19 to 36 and one or more pharmaceutically acceptable excipients, diluents or carriers.

[0471] Embodiment 38. The pharmaceutical composition according to embodiment 37 further comprising one or more further active ingredients selected from carnitine, dichloroacetate (DCA), oxamic acid, shikonin, oleanolic acid, 3-bromopyruvate, anti-cancer active agents, anti-fibrotic active agents and citrate.

[0472] Embodiment 39. The pharmaceutical composition according to embodiment 37 or embodiment

[0473] 38, wherein the one or more pharmaceutically acceptable excipients, diluents or carriers is selected from a buffer, a sugar, an isotonic agent, an antioxidant, an amino acid, a chelator, a surfactant, an emulsifier and / or saline.

[0474] Embodiment 40. The pharmaceutical composition according to any one of embodiments 37 to

[0475] 39, wherein the composition is comprised by a medical container.

[0476] Embodiment 41. A medical container comprising a pharmaceutical composition as defined in any one of embodiments 37 to 40. Embodiment 42. The pharmaceutical composition according to embodiment 40, or the medical container according to embodiment 41, wherein the medical container is selected from a sterile vial, a syringe, a bag and an injection device (in particular wherein the bag is an intravenous (IV) bag), optionally wherein the bag, syringe or injection device is for intravenous, subcutaneous, intramuscular, intradermal and / or instraosseous administration, (in particular wherein the bag, syringe or injection device is for intravenous or subcutaneous administration).

[0477] Embodiment 43. The pharmaceutical composition according to embodiment 40, or the medical container according to embodiment 41, wherein the composition is a solid composition comprised within an inhalation device or a sterile ampoule.

[0478] Embodiment 44. The pharmaceutical composition according to embodiment 40, or the medical container according to embodiment 41, wherein the composition is a liquid composition comprised within an inhalation device or sterile ampoule.

[0479] Embodiment 45. The pharmaceutical composition according to any one of embodiments 37 to 44, or the medical container according to any one of embodiments 41 to 44, wherein the composition is packaged with a label or instructions for use, or wherein the medical container further comprises a label or instructions for use, optionally wherein the label or instructions for use comprise a marketing authorisation number (e.g., an FDA or EMA authorisation number).

[0480] Embodiment 46. A kit comprising: a. a pharmaceutical composition as defined in any one of embodiments 37 to 44, or a medical container as defined in any one of embodiments 41 to 44; and b. a label or instructions for use, optionally wherein the label or instructions for use comprise a marketing authorisation number (e.g., an FDA or EMA authorisation number).

[0481] Embodiment 47. A method of producing a pharmaceutical composition comprising a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof, said method comprising the steps of: a. providing said delivery vehicle (optionally wherein the delivery vehicle is as defined in any one of embodiments 19 to 36), and b. formulating said delivery vehicle with one or more pharmaceutically acceptable excipients, diluents or carriers (optionally wherein the one or more pharmaceutically acceptable excipients, diluents or carriers are as defined in embodiment 39). Embodiment 48. The method according to embodiment 47, further comprising the steps of: c. packaging said composition (optionally in a medical container as defined in any one of embodiments 42 to 44); and d. optionally including in or on said packaging a label or instructions for use.

[0482] Embodiment 49. A method of preparing a delivery vehicle as defined in any one of embodiments 19 to 36, said method comprising operably linking said one or more nucleic acids to one or more promoters which are capable of expressing said one or more nucleic acids in a host cell, and introducing said one or more nucleic acids and one or more promoters into said delivery vehicle.

[0483] Embodiment 50. A method of increasing expression of carnitine acetyl transferase in a host cell, said method comprising contacting the host cell with a delivery vehicle as defined in any one of embodiments 19 to 36, optionally wherein the contacting is ex vivo.

[0484] Embodiment 51. A host cell comprising one or more exogenous nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof.

[0485] Embodiment 52. The host cell according to embodiment 51, the delivery vehicle according to any one of embodiments 28 to 36, or the method according to any one of embodiments 17, 49 or 50, wherein the host cells are selected from mesothelial cells, epithelial cells (such as retinal pigment epithelial cells or pneumocytes), endothelial cells, fibroblasts, inflammatory cells (such as T-lymphocyte and macrophages) and mesenchymal cells (e.g. cardiomyocytes).

[0486] Embodiment 53. The host cell according to embodiment 51, the delivery vehicle according to any one of embodiments 28 to 36, or the method according to any one of embodiments 17, 49 or 50, wherein the host cells are selected from mesenchymal cells and stem cells.

[0487] Embodiment 54. The host cell according to any one of embodiments 51 to 53, the delivery vehicle according to any one of embodiments 28 to 36, 52 or 53, or the method according to any one of embodiments 17, 49, 50, 52 or 53, wherein the host cells are not cells selected from MeT5o cells, DU145 cells, A549 cells, MCF-7 cells, MDA-MB-231 cells, 12Z cells and / or ARPE-19 cells, HK2 cells, HepG2 cells, and optionally wherein the host cells are not cells selected from mesothelial cells, epithelial cells (e.g. retinal pigment epithelial cells or pneumocytes), endothelial cells, fibroblasts and inflammatory cells (such as T-lymphocyte and macrophages).

[0488] Embodiment 55. The cell according to any one of embodiments 51 to 54, the delivery vehicle according to any one of embodiments 28 to 36, 52, 53 or 54 or the method according to any one of embodiments 17, 49, 50, 52, 53 or 54, wherein the host cells are diseased cells, or are cells comprised by a diseased tissue or organ of the or a subject, in particular wherein the host cells are cancer cells.

[0489] Table 12 - Sequences

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

Claims

CLAIMS1. A delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof, wherein the delivery vehicle is selected from:A. a lipid nanoparticle (e.g. lipid nanoparticles with immunostimulatory potency), a protamine- condensed mRNA, an exosome, an extracellular vesicle (EV), mesoporous silica, a CaP, a polymetric nanoparticle, a polycationic peptide, a polycationic protein and a crown-compound, orB. a cationic liposome, a modified dendrimer nanoparticle, a cationic polymer, a cationic polymer liposome, a polymer-lipid hybrid nanoparticle, a cationic nanoemulsion, a protamin-based core shell particle, a polymeric micelle, a cationic polysaccharide particle, and a synthetic lipoprotein, orC. a viral vector (for example an adeno-associated virus (AAV) vector, an adenoviral vector, a lentiviral vector, an alphaviral vector, a picornaviral vector, a flaviviral vector, a vescicolar stomatitis viral vector, a retroviral vector, an engineered phage or a phagemid), or a gold nanoparticle, a MXENES particle or a transposon and optionally, wherein the delivery vehicle is not an isolated plasmid, or is not a pLenti7.3 expression vector, and / or is not introduced into a host cell via electroporation.

2. The delivery vehicle according to claim 1, wherein the carnitine acetyl transferase or functional fragment thereof: a. comprises an amino acid sequence selected from SEQ ID No:3, SEQ ID No:5, SEQ ID No:7, SEQ ID No:9, SEQ ID No: 11 and SEQ ID No: 13 or an amino acid having at least (about) 70% identity, at least (about) 80% identity, or at least (about) 90% identity thereto; or b. is encoded by a nucleotide sequence selected from SEQ ID No:1, SEQ ID No:2, SEQ ID No:4, SEQ ID No:6, SEQ ID No:8, SEQ ID No: 10 and SEQ ID No: 11 or a nucleotide sequence having at least (about) 70% identity, at least (about) 80% identity, or at least (about) 90% identity thereto; or c. comprises an amino acid sequence selected from SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19 and SEQ ID No:20, or an amino acid having at least (about) 70% identity, at least (about) 80% identity, or at least (about) 90% identity thereto, and optionally wherein the carnitine acetyl transferase or functional fragment thereof does not comprise an amino acid sequence selected from SEQ ID No:21 and SEQ ID No:23, or wherein the carnitine acetyl transferase or functional fragment thereof is not encoded by the nucleotide sequence of SEQ ID No: 22.

3. The delivery vehicle according to claim 1 or claim 2, wherein the one or more nucleic acids is operably linked to a constitutive promoter, optionally wherein the promoter is functional for expression of the one or more nucleic acids in a host cell, for example a host cell in vivo, optionally wherein the promoter is not naturally associated with the expression of a carnitine acetyl transferase.

4. The delivery vehicle according to any one of claims 1 to 3, wherein the expression of the carnitine acetyl transferase in an in vitro fibrosis or cancer model in host cells, optionally wherein the fibrosis or cancer is induced by the addition of TGF-p and / or mediated by an epithelial, mesothelial and / or endothelial to mesenchymal transition (EMT, MMT and / or endoMT, in particular EMT):A: i. is capable of reverting the fibrosis or cancer by decreasing, or is capable of decreasing, o- smooth muscle actin (o-SMA) relative expression as compared to control host cells which do not comprise the one or more nucleic acids, in particular wherein the decreasing is a decrease of at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%; and / or ii. is capable of preventing the induction of fibrosis or cancer by maintaining comparable (e.g. within a statistically significant level) expression of o-SMA, or is capable of maintaining comparable (e.g. within a statistically significant level) expression of o-SMA, or is capable of preventing an increase (e.g. a statistically significant increase) in expression of o-SMA, as compared to control host cells which have not been induced for fibrosis (e.g. by TGF-p); and / orB: i. is capable of reverting the fibrosis or cancer by decreasing, or is capable of decreasing, vimentin (VIM) relative expression as compared to control host cells which do not comprise the one or more nucleic acids, in particular wherein the decreasing is a decrease of at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%; and / or ii. is capable of preventing the induction of fibrosis or cancer by maintaining comparable (e.g. within a statistically significant level) expression of VIM, or is capable of maintaining comparable (e.g. within a statistically significant level) expression of VIM, or is capable of preventing an increase (e.g. a statistically significant increase) in expression of VIM as compared to control host cells which have not been induced for fibrosis or cancer (e.g. by TGF-p); and / orC:i. is capable of reverting the fibrosis or cancer by decreasing, or is capable of decreasing interleukin-6 (IL-6) relative expression as compared to control host cells which do not comprise the one or more nucleic acids, in particular wherein the decreasing is a decrease by at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%; and / or ii. is capable of preventing the induction of fibrosis or cancer by maintaining comparable or reducing (e.g. within a statistically significant level) expression of IL-6, or is capable of maintaining comparable or reducing (e.g. within a statistically significant level) expression of IL- 6, or is capable of preventing an increase (e.g. a statistically significant increase) in expression of IL-6 as compared to control host cells which have not been induced for fibrosis or cancer (e.g. by TGF-p); and / orD: i. is capable of reverting the fibrosis or cancer by decreasing, or is capable of decreasing, interleukin-ip (IL-ip) relative expression as compared to control host cells which do not comprise the one or more nucleic acids, in particular wherein the decreasing is a decrease by at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%; and / or ii. is capable of preventing the induction of fibrosis or cancer by maintaining comparable (e.g. within a statistically significant level) expression of IL-ip, or is capable of maintaining comparable (e.g. within a statistically significant level) expression of IL-ip, or is capable of preventing an increase (e.g. a statistically significant increase) in expression of IL-ip, as compared to control host cells which have not been induced for fibrosis or cancer (e.g. by TGF- P); and / orE: i. is capable of reverting the fibrosis or cancer by decreasing, or is capable of decreasing, vascular endothelial growth factor (VEGF) relative expression as compared to control host cells which do not comprise the one or more nucleic acids, in particular wherein the decreasing is a decrease by at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%; and / or ii. is capable of preventing the induction of fibrosis or cancer by maintaining comparable (e.g. within a statistically significant level) expression of VEGF, or is capable of maintaining comparable (e.g. within a statistically significant level) expression of VEGF, or is capable of preventing an increase (e.g. a statistically significant increase) in expression of VEGF, ascompared to control host cells which have not been induced for fibrosis or cancer (e.g. by TGF- P); and / orF: i. is capable of reverting the fibrosis or cancer by decreasing, or is capable of decreasing, zinc finger protein SNAI1 (SNAIL) expression as compared to control host cells which do not comprise the one or more nucleic acids, in particular wherein the decreasing is a decrease by at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%; and / or ii. is capable of preventing the induction of fibrosis or cancer by maintaining comparable (e.g. within a statistically significant level) expression of SNAIL, or is capable of maintaining comparable (e.g. within a statistically significant level) expression of SNAIL, or is capable of preventing an increase (e.g. a statistically significant increase) in expression of SNAIL, as compared to control host cells which have not been induced for fibrosis or cancer (e.g. by TGF- P); and / orG: i. is capable of reverting the fibrosis or cancer by increasing, or is capable of increasing, E-cadherin expression, as compared to control host cells which do not comprise the one or more nucleic acids, in particular wherein the increasing is an increase by at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%; and / or ii. is capable of preventing the induction of fibrosis or cancer by maintaining comparable (e.g. within a statistically significant level) expression of e-cadherin, or is capable of maintaining comparable (e.g. within a statistically significant level) expression of e-cadherin, or is capable of preventing a decrease (e.g. a statistically significant decrease) in expression of E-cadherin, as compared to control host cells which have not been induced for fibrosis or cancer (e.g. by TGF-p); and / orH: i. is capable of reverting the fibrosis or cancer by decreasing, or is capable of decreasing, fibronectin (FN) expression as compared to control host cells which do not comprise the one or more nucleic acids, in particular wherein the decreasing is a decrease by at least (about) 30%, at least (about) 40%, at least (about) 50%, at least (about) 60%, at least (about) 70%, at least (about) 75%, or at least (about) 80%, at least (about) 85%, or at least (about) 90%, in particular at least (about) 50%; and / or ii. is capable of preventing the induction of fibrosis or cancer by maintaining comparable or reducing (e.g. within a statistically significant level) expression of FN, or is capable ofmaintaining comparable or reducing (e.g. within a statistically significant level) expression of FN, or is capable of preventing an increase (e.g. a statistically significant increase)in expression of FN as compared to control host cells which have not been induced for fibrosis or cancer (e.g. by TGF-p); and optionally wherein the in vitro model is carried out in the presence of a physiological concentration of L-carnitine (e.g. 50pM).

5. A pharmaceutical composition comprising a delivery vehicle as defined in any one of claims 1 to 4 and one or more pharmaceutically acceptable excipients, diluents or carriers selected from a buffer, a sugar, an isotonic agent, an antioxidant, an amino acid, a chelator, a surfactant, an emulsifier and / or saline, and optionally wherein the composition is comprised by a medical container.

6. The pharmaceutical composition according to claim 5, further comprising one or more further active ingredients, optionally selected from L-carnitine, dichloroacetate (DCA), oxamic acid, shikonin, oleanolic acid, 3-bromopyruvate, anti-cancer active agents, anti-fibrotic active agents and citrate.

7. A medical container comprising a pharmaceutical composition as defined in claim 5 or claim 6.

8. The medical container according to claim 7, wherein the medical container is selected from a sterile vial, a syringe, a bag and an injection device (in particular wherein the bag is an intravenous (IV) bag), optionally wherein the bag, syringe or injection device is for intravenous, subcutaneous, intramuscular, intradermal and / or intraosseous administration, (in particular wherein the bag, syringe or injection device is for intravenous or subcutaneous administration), for example, wherein the pharmaceutical composition is a solid composition comprised within an inhalation device or a sterile ampoule, or wherein the composition is a liquid composition comprised within an inhalation device or sterile ampoule, and further optionally wherein the composition is packaged with a label or instructions for use, or wherein the medical container further comprises a label or instructions for use, optionally wherein the label or instructions for use comprise a marketing authorisation number (e.g., an FDA or EMA authorisation number).

9. A delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof for use in a method of treating or preventing a disease or condition,optionally wherein the delivery vehicle is as defined in any one of claims 1 to 4, or wherein the delivery vehicle is comprised by a pharmaceutical composition as defined in any one of claims 5 or 6.

10. The delivery vehicle for the use according to claim 9 wherein the pathogenesis of the disease or condition is mediated by:(i) overexpression of at least one growth factor selected from transforming growth factor-p (TGF- P), connective tissue growth factors (CTGF), platelet-derived growth factors (PDGF), fibroblast growth factors (FGF), epidermal growth factors (EGF) and vascular endothelial growth factors (VEGF); or(ii) an epithelial, mesothelial and / or endothelial to mesenchymal transition (EMT, MMT and / or EndoMT, in particular EMT).

11. The delivery vehicle for the use according to claim 9 or claim 10, wherein the disease or condition is selected fromA: a fibrotic disease (e.g. pulmonary fibrosis, liver fibrosis, renal fibrosis, myocardial fibrosis, peritoneal fibrosis, ischemic-induced fibrosis and hypoxic-induced fibrosis), a disease or condition associated with fibrosis (e.g. inflammatory bowel disease, polycystic kidney disease, endometriosis, encapsulating peritoneal sclerosis and pulmonary hypertension), cancer and vascular dementia; orB. angiogenic eye disorders, ocular diseases in the anterior segment, ocular diseases in the posterior segment, neovascular related ophthalmic posterior segment diseases, retinal diseases, macular degeneration (including neovascular age-related macular degeneration (AMD) , such as neovascular AMD (also known as wet AMD)), diabetic retinopathies, diabetic macular oedema (DMO), choroidal neovascularisation (CNV), central retinal vein occlusion (CRVO), corneal neovascularization, retinal neovascularization, fibrovascular intraocular diseases, pathological scarring and drug-induced nephrotoxicity; orC. 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 and bone cancer; orD. skin cancer, mesothelioma, peritoneal mesothelioma, prostate cancer and vascular tumours; orE. an inflammatory disease or condition; orF. chronic obstructive pulmonary disease (COPD), epatithis and Crohn's disease; orG. end-stage kidney disease, and the subject is receiving or has received peritoneal dialysis, optionally wherein the administration of the delivery vehicle or pharmaceutical composition mitigates the effects of glucose over-exposure, particularly wherein the subject has been treated with glucose-based peritoneal dialysis solution for at least one year.

12. The delivery vehicle for the use according to claim 11 wherein the disease or condition is mediated by an epithelial, mesothelial and / or endothelial to mesenchymal transition (EMT, MMT and / or EndoMT, in particular EMT). and selected from one or more of the following diseases: -prostate carcinoma;-prostate adenocarcinoma;-lung carcinoma;-breast adenocarcinoma;-endometriosis;-macula degeneration;-fibrosis;-liver adenocarcinoma.

13. The delivery vehicle for the use according to any one of claims 9 to 12, wherein the delivery vehicle possibly included in a pharmaceutical composition, optionally the pharmaceutical composition of claim 5 or 6, is administeredA. by a route of administration selected from enteral, parenteral, inhaled and transcutaneous; orB. intraperitoneally, intramuscularly or intravenously, and optionally wherein the disease or condition is not fibrotic stress, fibrosis or inflammation; orC. intraocularly, transdermally, topically, intranasally, intravitreally or via intra-tumoral administration.

14. The delivery vehicle for the use according to any one of claims 9 to 13, wherein the delivery vehicle possibly included in a pharmaceutical composition, optionally the pharmaceutical composition of claim 5 or 6, is administered in combination with at least one further therapeutically active molecule (such as an anti-fibrotic active agent or a cancer active agent), or in combination with radiotherapy, and wherein the administration of the at least one further therapeutically active molecule is administered separately, and optionally wherein the at least one further therapeutically active molecule is selected from L- carnitine, dichloroacetate (DCA), oxamic acid, shikonin, oleanolic acid, 3-bromopyruvate, anticancer active agents, anti-fibrotic active agents and citrate.

15. The delivery vehicle for the use according to any one of claims 9 to 14, wherein the delivery vehicle possibly included in a pharmaceutical composition, optionally the pharmaceutical composition of claim 5 or 6, is administered in combination with at least one further therapeutically active molecule selected from small molecule drugs, chemotherapy, hormone therapy, immunotherapy, photodynamic therapy, stem cell therapy, and antibody therapy, in particular any of the aforementioned which are for use in treating cancer, and when the disease or condition is cancer, or in particular any of the aforementioned which are for use in treating or preventing fibrosis, and when the disease or condition is fibrosis or a disease associated with fibrosis.

16. The delivery vehicle for the use according to any one of claims 9 to 15, wherein the one or more nucleic acids is expressed in host cells in the subject, optionally wherein the one or more nucleic acids are integrated into the chromosome of the host cell.

17. A delivery vehicle for use according to any one of claim 9 to 16, wherein the method of treating or preventing a disease or condition is gene therapy, and optionally wherein the delivery vehicle is as defined in any one of claims 1 to 4 or wherein the delivery vehicle is comprised by a pharmaceutical composition as defined in any one of claims 5 or 6.

18. A method of producing a pharmaceutical composition comprising a delivery vehicle comprising one or more nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof, said method comprising the steps of: a. providing said delivery vehicle (optionally wherein the delivery vehicle is as defined in any one of claims 1 to 4), and b. formulating said delivery vehicle with one or more pharmaceutically acceptable excipients, diluents or carriers (optionally wherein the one or more pharmaceutically acceptable excipients, diluents or carriers are as defined in claim 5). optionally further comprising the steps of: c. packaging said composition (optionally in a medical container as defined in claim 7 or 8); and d. optionally including in or on said packaging a label or instructions for use.

19. A method of increasing expression of carnitine acetyl transferase in a host cell, said method comprising contacting the host cell with a delivery vehicle as defined in any one of claims 1 to 4, optionally wherein the contacting is ex vivo.

20. A host cell comprising one or more exogenous nucleic acids encoding a carnitine acetyl transferase or a functional fragment thereof (optionally wherein the carnitine acetyl transferase is as defined in any one of claims 2 to 4), or the delivery vehicle according to claim 3 or claim 4, or the delivery vehicle for the use according to claim 9, wherein the host cells are selected from:A. diseased cells, or are cells comprised by a diseased tissue or organ of the or a subject, in particular wherein the host cells are cancer cells; orB. mesothelial cells, epithelial cells (such as retinal pigment epithelial cells or pneumocytes), endothelial cells, fibroblasts, inflammatory cells (such as T-lymphocyte and macrophages) and mesenchymal cells (e.g. cardiomyocytes); and / orC. wherein the host cells are selected from mesenchymal cells and stem cells.

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