Tardigrade DSUP protein for use in the therapeutic treatment of diabetes

Abstract

The invention relates to a new therapeutic agent for use in the prevention and / or treatment of diabetes, more particularly type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM). The new therapeutic agent is the "Damage suppressor" (DSUP) protein of a tardigrade organism of the genus Ramazzottius, preferably of the species Ramazzottius varieornatus. Also described are an isolated nucleic acid comprising a nucleotide sequence encoding the DSUP protein, an expression vector comprising said isolated nucleic acid, and a pharmaceutically acceptable vehicle containing internally said isolated nucleic acid or said expression vector, for use in the aforementioned therapeutic applications.

Description

[0001] Tardigrade DSUP protein for use in the therapeutic treatment of diabetes

[0002] The present invention falls within the field of therapeutic treatments of diabetic diseases, in particular type 1 diabetes mellitus and type 2 diabetes mellitus.

[0003] In recent decades, diabetes mellitus has taken on the characteristics and dimensions of a global health emergency due to its high prevalence. In 1980, the World Health Organization (WHO) estimated that there were approximately 108 million people affected by diabetes worldwide. This number has now quadrupled, reaching 451 million diabetic patients in 2017. According to reliable projections, in 2045 there will be about 700 million patients.

[0004] Type 1 diabetes (T1DM) is an autoimmune disease, characterized by a progressive destruction and dysfunction of pancreatic beta cells producing insulin, driven by inflammatory processes and by the direct action of autoreactive T lymphocytes (Nigi L, et al. Pancreatic Alpha-Cells Contribute Together With Beta-Cells to CXCL10 Expression in Type 1 Diabetes. Front Endocrinol (Lausanne). 2020 Sep 15; 11 : 630. doi: 10.3389 / fendo.2020.00630) resulting in chronic hyperglycemia.

[0005] Type 2 diabetes mellitus (T2DM) is defined as a chronic metabolic disease characterized by hyperglycemia, caused by insulin resistance and / or dysfunction and destruction of insulinsecreting beta cells, resulting in a reduction in beta-cell mass (American Diabetes Association (ADA): https: / / diabetes.org / about-diabetes / type-2) (Weir GC, Gaglia J, Bonner- Weir S. Inadequate P-cell mass is essential for the pathogenesis of type 2 diabetes. Lancet Diabetes Endocrinol. 2020 Mar; 8 (3): 249-256. doi: 10.1016 / S2213-8587 (20) 30022-X. Epub 2020 Jan 29. PMID: 32006519; PMCID: PMC7098467). (Cui D, Feng X, Lei S, Zhang H, Hu W, Yang S, Yu X, Su Z. Pancreatic P-cell failure, clinical implications, and therapeutic strategies in type 2 diabetes. Chin Med J (Engl). 2024 Apr 5; 137(7): 791-805. doi: 10.1097 / CM9.0000000000003034. Epub 2024 Mar 13. PMID: 38479993; PMCID: PMC 10997226).

[0006] In detail, beta-cell apoptosis in type 1 diabetes is mainly due to autoimmunity. In type 2 diabetes, beta-cell death occurs as a combined consequence of increased circulating glucose and saturated fatty acids, along with factors secreted by adipocytes (adipokines) and inflammatory phenomena due to chronic activation of the innate immune system. In both types of diabetes, inflammatory mediators within the pancreatic islets, resulting in chronic inflammation, trigger a common signaling pathway that causes beta-cell apoptosis. From an etiopathological point of view, in both T1DM and T2DM, beta cell apoptosis may be caused by cytokines secreted by cells of the immune system. In fact, cytokines, such as interferon gamma (IFN-y) and tumor necrosis factor alpha (TNF-a), are secreted by activated T cells and macrophages, causing direct damage to beta cells. In particular, IFN-y induces the production of nitric oxide and reactive oxygen species in beta cells, leading to oxidative stress and subsequent cell death (Kruger C, et al. The importance of aquaporin-8 for cytokine-mediated toxicity in insulin-producing cells. Free Radical Biology and Medicine 2021 Oct; 174: 135-143. doi: 10.1016 / j .freeradbiomed.2021.08.003.). TNF- a promotes inflammation and improves the expression of cell adhesion molecules, facilitating the recruitment of more immune cells in the pancreatic islets. The prolonged release of pro- inflammatory cytokines creates a vicious cycle, amplifying the immune response and causing further damage to beta cells (Pan X, et al. Chemokines in Type 1 Diabetes Mellitus. Front Immunol. 2022 Feb 15; 12:690082. doi: 10.3389 / fimmu.2021.690082.) (Donath, M.Y., Shoelson, S.E. Type 2 diabetes as an inflammatory disease. Nat Rev Immunol. 2011 Feb; 11(2):98-107. doi: 10.1038 / nri2925. Epub 2011 Jan 14. PMID: 21233852. ) (Eguchi K, Nagai R. Islet inflammation in type 2 diabetes and physiology. J Clin Invest. 2017 Jan 3; 127 (1): 14-23. doi: 10.1172 / JCI88877. Epub 2017 Jan 3. PMID: 28045399; PMCID: PMC5 199688). Over time, this immune-mediated destruction of beta cells results in a loss of insulin production, which leads to hyperglycemia and the clinical manifestations of diabetes.

[0007] Therefore, therapeutic approaches aimed at blocking beta-cell apoptosis caused by inflammatory phenomena could represent an important new development in the treatment of type 1 and 2 diabetes (Donath MY, Starling J, Maedler K, Mandrup-Poulsen T. Inflammatory mediators and islet beta-cell failure: a link between type 1 and type 2 diabetes. J Mol Med (Berl). 2003 Aug; 81 (8): 455-70. doi: 10.1007 / s00109-003-0450-y. Epub 2003 Jul 18. PMID: 12879149.) (Newsholme P, Keane K, de Bittencourt PUT Jr, Krause M. The Impact of Inflammation on Pancreatic P-Cell Metabolism, Function and Failure in T1DM and T2DM: Commonalities and Differences [Internet], Type 1 Diabetes. InTech; 2013. Available from: http: / / dx.doi.org / 10.5772 / 55349) (Eizirik, DL, Pasquali, L. & Cnop, M. Pancreatic P-cells in type 1 and type 2 diabetes mellitus: different pathways to failure. Nat Rev Endocrinol 16, 349-362 (2020). https: / / doi.org / 10.1038 / s41574-020-0355-7). This is even more relevant in light of the chronic complications common to both T1DM and T2DM. Long-term complications include diabetic nephropathy, angiopathy, and retinopathy. In addition, both forms of diabetes mellitus are associated with an increased risk of stroke, circulatory disorders and heart attack (Domingueti CP, Dusse LM, Carvalho Md, de Sousa LP, Gomes KB, Fernandes AP. Diabetes mellitus: The link between oxidative stress, inflammation, hypercoagulability and vascular complications. J Diabetes Complications. 2016 May-Jun; 30 (4): 738-45. doi: 10.1016 / j.jdiacomp.2015.12.018. Epub 2015 Dec 18. PMID: 26781070.).

[0008] Currently, conventional antidiabetic therapies are based on the use of hypoglycemic drugs, anti-hyperglycemic drugs (sulfonylurea, meglitinide), GLP-1 receptor agonists (tirzepatide) or dopamine-D2 agonists, various inhibitors (for dipeptidyl peptidase 4, sodium-glucose-2 cotransporter), or insulin and its analogues.

[0009] However, the beneficial effects of the aforementioned therapies are generally associated with serious side effects due to the prolonged use of the same drugs. In addition, insulin production is increasingly difficult, with repercussions on its availability and relative market price.

[0010] In fact, the new drugs for the treatment of type 1 diabetes mellitus (T1DM) consist of immunotherapies that aim to modulate the function of the cells of the immune system. For example, the FDA has recently approved treatment with the drug Teplizumab, an anti-CD3 drug that has been shown to delay the onset of type 1 diabetes by an average of 2 years, for subjects positive for autoantibodies who have not yet developed the disease.

[0011] However, recently, the scientific community has also begun to give attention to drugs aimed at preventing the death of pancreatic beta cells and preserving their function. In fact, it has recently been shown that Verapamil, a calcium antagonist already used for cardiovascular diseases, is able to partially preserve the stimulated secretion of peptide C up to 1 year after diagnosis compared to placebo, in children and adolescents with newly diagnosed T1DM (Forlenza GP, et al; Study Group. Effect of Verapamil on Pancreatic Beta Cell Function in Newly Diagnosed Pediatric Type 1 Diabetes: A Randomized Clinical Trial. JAMA. 2023 Mar 28; 329(12): 990-999. doi: 10.1001 / jama.2023.2064.).

[0012] Tardigrades, also known as "water bears", are microscopic metazoans with bilateral symmetry typically ranging in length from 0.1 mm to 1.2 mm. There are currently more than 1400 species of tardigrades (marine, limnic and terrestrial) (Degma P. & Guidetti R. (2023) Actual checklist of Tardigrada species), mainly distinguished in two evolutionary lines (Eutardigrada and Heterotardigrada) (Guidetti R. & Bertolani R. (2005) Tardigrade taxonomy: an updated check list of the taxa and a list of characters for their identification. Zootaxa, 845: 1-46) that have colonized very different habitats: they are found from urban areas to polar regions, from oceanic zones to the driest deserts and are mainly housed in the interstices of mosses and lichens, in humus and in grasslands. They are aquatic organisms, so even terrestrial species are active only if covered by at least a film of water and for this reason they are called limnoterrestrial (Guidetti R., et al. (2011) On dormancy strategies in tardigrades. Journal of Insect Physiology 57: 567-576.).

[0013] All tardigrade species have a head and four metameres, each with a pair of limbs; two or more claws are usually inserted at the end of each limb. These organisms are eutelic, that is, at birth each individual has the same number of cells that it will have as an adult, so growth occurs only by increasing volume, even if cell divisions are possible to replace lost cells. In tardigrades, gas exchanges occur passively through the epidermis and cuticle. Circulatory organs are also absent, since this function is achieved through the large pseudocoelomic body cavity filled with liquid and passively fluctuating cells that accumulate reserve substances. Tardigrades have a dorsal brain connected by nerve cords to four ventral ganglia, a pair of ocelli with photoreceptor function, somatic and visceral muscles, a complete digestive system with a piercing-sucking buccopharyngeal system and reproductive organs (Gross V., et al. (2018) Miniaturization of tardigrades (water bears): Morphological and genomic perspectives. Arthropod Structure & Development, 48: 12-19). These animals have evolved extraordinary tolerance to hostile environmental conditions and it has been this peculiar resilience that has allowed them to adapt to extreme environments. Tardi grades can enter anhydrobiosis, a metabolically inactive state induced by water deficiency (Welnicz W., et al. (2011) Anhydrobiosis in tardigrades - The last decade. Journal of Insect Physiology, 57: 577-583) in which they can remain for months or even years.

[0014] In the anhydrobiotic state, tardigrades are able to survive chemical and physical conditions that would be lethal to almost all other living beings. For example, they can withstand exposure to extreme temperatures (from -273 °C to 100°C), high hydrostatic pressures, organic solvents such as ethanol, ionizing and UV radiation (Welnicz W., et al (2011) Anhydrobiosis in tardigrades-The last decade. Journal of Insect Physiology, 57: 577-583). In addition, tardigrades are the first animals to have survived exposure to space vacuum.

[0015] The molecular mechanisms underlying these unique tolerance capabilities involve several components that work together as bioprotectors (Yoshida Y., and Tanaka S. (2022) Deciphering the Biological Enigma-Genomic Evolution Underlying Anhydrobiosis in the Phylum Tardigrada and the Chironomid Polypedilum vanderplanki. Insects, 13: 557). In particular, three new families of proteins, unique to the tardigrade phylum, have been identified, called "tardigrade disordered proteins" (TDPs): cytoplasmic proteins, known as "cytoplasmic abundant heat-soluble proteins" (CAHS); secreted proteins, known as "secretory abundant heat-soluble proteins" (SAHS); mitochondrial proteins, known as "mitochondrial abundant heat-soluble proteins" (MAHS) (Yamaguchi A, et al. Two novel heat-soluble protein families abundantly expressed in an anhydrobiotic tardigrade. PLoS One. 2012; 7(8): e44209. doi: 10.1371 / joumal.pone.0044209. Epub 2012 Aug 28. PMID: 22937162; PMCID: PMC3429414.; Tanaka S, et al. Novel mitochondria-targeted heatsoluble proteins identified in the anhydrobiotic Tardigrade improve the osmotic tolerance of human cells. PLoS One. 2015 Feb 12; 10 (2): eOl 18272. doi: 10.1371 / journal. pone.0118272. PMID: 25675104; PMCID: PMC4326354). These proteins are defined as "intrinsically disordered proteins" (IDPs), i.e. they lack a stable three- dimensional structure and their function is achieved precisely through this structural dynamism (Handa T, et al. Perspectives on the evolutionary and functional importance of intrinsically disordered proteins. Int J Biol Macromol. 2023 Jan 1; 224:243-255. doi: 10.1016 / j .ijbiomac.2022.10.120. Epub 2022 Oct 17. PMID: 36257361).

[0016] The anhydrobiotic state can cause damage to DNA and proteins, so tardigrade organisms have also evolved maintenance and repair mechanisms necessary to restore cellular functions after rehydration; in particular, tardigrades efficiently activate DNA repair systems that maintain genomic stability (Yoshida Y., and Tanaka S. (2022) Deciphering the Biological Enigma-Genomic Evolution Underlying Anhydrobiosis in the Phylum Tardigrada and the Chironomid Polypedilum vanderplanki. Insects, 13: 557).

[0017] Among the different species belonging to the phylum Tardigrada, the species Ramazzottius varieornatus is one of the most stress tolerant. Proteins involved in anhydrobiosis, such as CAHS and SAHS, are constitutively expressed at high levels in this organism (Hashimoto T, et al; Extremotol erant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein. Nat Commun. 2016 Sep 20;7: 12808. doi: 10.1038 / ncommsl2808) making it always ready to adapt quickly to water deprivation conditions.

[0018] In addition to water deprivation, the tardigrades of the species R. varieornatus are able to tolerate high doses of radiation (Horikawa DD, et al. Analysis of DNA repair and protection in the Tardigrade Ramazzottius varieornatus and Hypsibius dujardini after exposure to UVC radiation. PLoS One. 2013 Jun 6; 8 (6): e64793. doi: 10.1371 / journal. pone.0064793. PMID: 23762256; PMCID: PMC3675078). Since DNA molecules are the main target of radiation damage, the researchers Hashimoto T. et al hypothesized, during a study on the gene repertoire of R varieornatus, that this organism possessed a unique tool for protecting its DNA in the nucleus. The study thus identified a protein exclusive to this organism, called "Damage suppressor" (DSUP), which has the task of preserving the genetic material. To further investigate the protection mechanisms, directly or indirectly mediated by this protein, human cells were engineered to express DSUP, showing a 40% reduction in X-ray induced DNA fragmentation compared to the non-engineered counterpart and maintaining viability and proliferative capacity (Hashimoto T, et al. Extremotol erant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein. Nat Commun. 2016 Sep 20; 7: 12808. doi: 10.1038 / ncommsl2808). Radiations can affect DNA by causing direct damage and / or can cause indirect damage through the formation of reactive oxygen species (ROS), which in turn damage the genetic material. Therefore, the DSUP protein is able to preserve DNA from direct damage caused by ionizing radiation but also from indirect damage mediated by ROS (Hashimoto T, et al; Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein. Nat Commun. 2016 Sep 20; 7: 12808. doi: 10.1038 / ncommsl2808).

[0019] The DSUP protein specifically binds nucleosomes by participating in the complex of proteins associated with DNA (Chavez C, et al. The tardigrade damage suppressor protein binds to nucleosomes and protects DNA from hydroxyl radicals. Elife. 2019 Oct 1; 8: e47682. doi: 10.7554 / eLife.47682) and, as demonstrated by transfection in human cell cultures, incorporation into nucleosomes is not deleterious (Hashimoto T, et al. Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique proteins. Nat Commun. 2016 Sep 20; 7: 12808. doi: 10.1038 / ncommsl2808). Studies on protein sequence and functionality have revealed the presence of a largely unstructured portion and a C-terminal region responsible for nuclear localization and association with DNA (Hashimoto T, et al. Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein. Nat Commun. 2016 Sep 20; 7: 12808. doi: 10.1038 / ncommsl2808.; Hashimoto T, Kunieda T. DNA Protection Protein, a Novel Mechanism of Radiation Tolerance: Lessons from Tardigrades. Life (Basel). 2017 Jun 15; 7 (2): 26. doi: 10.3390 / life7020026. PMID: 28617314; PMCID: PMC5492148).

[0020] The amino acid sequence of the DSUP protein is rich in serine, alanine, glycine and lysine (S AGK) residues that favor the acquisition of a disordered conformation; it is therefore likely that DSUP is organized in a mass diffused on chromatin functioning as a molecular shield (Chavez C, et al. The tardigrade damage suppressor protein binds to nucleosomes and protects DNA from hydroxyl radicals. Elife. 2019 Oct 1; 8: e47682. doi: 10.7554 / eLife.47682). Other studies have shown that this protein also acts as a damage sensor, allowing the activation of repair systems. In human cells transfected with the DSUP protein and subjected to oxidative stress by H2O2, an increase in the expression of genes involved in DNA repair has been demonstrated, but more significantly of genes involved in antioxidant systems. In the case of exposure to UV radiation, on the other hand, human cells transfected with the DSUP protein and irradiated show a significant increase in the expression of genes involved in DNA repair.

[0021] Patent application W02022170056 discloses a recombinant CMV viral vector that comprises one or more exogenous genes, including the gene encoding the tardigrade DSUP protein. This patent application mentions the use of the recombinant CMV viral vector for the prevention and / or therapeutic treatment of diseases associated with aging, but provides support and experimental evidence only for a vector comprising the gene encoding the enzyme telomerase reverse transcriptase (TERT) or the gene encoding the peptide follistatin 344 (FS344).

[0022] Patent JP6583606 discloses the protective activity of the tardigrade DSUP protein against damage caused to DNA molecules by radiation or oxidative stress.

[0023] In this context, there is therefore a dramatic need for therapeutic strategies that are effective in the treatment of diabetic diseases, by contrasting their onset and progression, and that are suitable for obtaining a lasting clinical response while reducing at the same time possible adverse effects to a minimum.

[0024] This need has now been met by the present inventors, who have found that the DSUP protein from tardigrades of the genus Ramazzottius is able to exert a protective activity on pancreatic beta cells by promoting their survival and improving their functionality, thereby promoting insulin production and blood glucose regulation.

[0025] Accordingly, the DSUP protein from tardigrades of the genus Ramazzottius represents a new therapeutic agent for the prevention and / or treatment of diabetes.

[0026] The present invention is based on the results obtained by the inventors in the experimentation and research activities described in the experimental section that follows. Briefly, in vitro studies conducted by the present inventors have revealed that the expression of the DSUP protein in a murine pancreatic beta cell line stably transfected with the gene encoding said protein is able to exert a significant protective action on these cells by contrasting the apoptotic processes activated by cytokine-mediated inflammatory stress. Further studies have also verified that pancreatic beta cells expressing the DSUP protein do not show alterations in their functionality, maintaining the ability to produce and secrete the insulin hormone unchanged.

[0027] In light of the above, the DSUP protein from tardigrades of the genus Ramazzottius therefore represents an important innovative therapeutic tool in the field of diabetic pathologies. The protective action of this protein on stress factors and inflammatory processes that lead to pancreatic beta cell dysfunction and death allows for increased cell survival and, at the same time, promotes autogenous insulin production. Accordingly, the therapeutic approach based on the DSUP protein advantageously allows limiting, if not abolishing, the use of conventional anti-diabetic drugs which, as illustrated above, frequently cause the onset of significant adverse effects, or at least reducing the use of such drugs at significantly lower therapeutic doses.

[0028] Therefore, an object of the present invention is a "Damage suppressor" (DSUP) protein isolated from a tardigrade organism of the genus Ramazzottius, for use in the prevention and / or therapeutic treatment of diabetes.

[0029] The invention also relates to an isolated nucleic acid comprising or consisting of a nucleotide sequence coding for a DSUP protein, an expression vector comprising said isolated nucleic acid, a pharmaceutically acceptable vehicle containing internally said isolated nucleic acid and / or said expression vector of the invention, for the aforementioned therapeutic use.

[0030] Further features and advantages of the invention are identified in the appended claims and illustrated in detail in the following description.

[0031] The attached independent and dependent claims form an integral part of the present description.

[0032] Within the context of the present invention, a DSUP protein from tardigrades belonging to the species Ramazzottius varieornatus is most preferred.

[0033] The DSUP protein of the tardigrade Ramazzottius varieornatus is a 445 amino acid protein (UniProt Primary accession number: PODOW4), whose amino acid sequence is known per se and is reported in the sequence listing as SEQ ID NO. 1 :

[0034] MASTHQSSTEPSSTGKSEETKKDASQGSGQDSKNVTVTKGTGSSATSAAIVKTGGS QGKDSSTTAGSSSTQGQKFSTTPTDPKTFSSDQKEKSKSPAKEVPSGGDSKSQGDT KSQSDAKSSGQSQGQSKDSGKSSSDSSKSHSVIGAVKDVVAGAKDVAGKAVEDA PSIMHTAVDAVKNAATTVKDVASSAASTVAEKVVDAYHSVVGDKTDDKKEGEH SGDKKDDSKAGSGSGQGGDNKKSEGETSGQAESSSGNEGAAPAKGRGRGRPPAA AKGVAKGAAKGAAASKGAKSGAES SKGGEQS SGDIEMAD AS SKGGSDQRDS AA TVGEGGASGSEGGAKKGRGRGAGKKADAGDTSAEPPRRSSRLTSSGTGAGSAPA AAKGGAKRAAS S S STPSNAKKQ ATGGAGKAAATKAT AAKS AASKAPQNGAGAK KKGGKAGGRKRK.

[0035] This protein may, for example, be administered to a subject with diabetes in the form of a recombinant protein. Techniques for producing recombinant proteins are well-established, and the person skilled in the art is able to apply them without the need for any inventive activity.

[0036] Alternatively, the patient may be administered an isolated nucleic acid comprising or consisting of a nucleotide sequence coding for the DSUP protein capable of being translated into the protein.

[0037] Therefore, a further object of the invention is an isolated nucleic acid that comprises or consists of a nucleotide sequence coding for a tardigrade DSUP protein as defined above, for use in the prevention and / or therapeutic treatment of diabetes.

[0038] The nucleotide sequence coding for the DSUP protein of Ramazzottius varieornatus is known per se and is reported in the sequence listing as SEQ ID NO. 2: 5'ATGGCATCCACACACCAATCATCCACAGAACCCTCTTCCACAGGTAAATCTG AGGAAACGAAGAAAGATGCTTCGCAAGGGAGCGGGCAAGACTCCAAGAACGT AACCGTTACCAAAGGTACCGGTTCCTCCGCCACCTCAGCTGCCATTGTCAAGA CAGGAGGATCCCAAGGCAAAGATTCCTCTACTACAGCGGGCTCTTCTAGTACT CAGGGACAGAAGTTCAGTACTACACCTACCGACCCGAAAACTTTCAGCTCTGA CCAAAAGGAGAAATCCAAAAGCCCAGCCAAAGAAGTCCCGTCTGGTGGCGAT AGTAAGTCCCAAGGTGACACCAAGTCTCAAAGCGACGCCAAATCTTCTGGACA

[0039] AAGTCAGGGCCAGTCTAAAGACAGCGGCAAATCATCTTCCGACAGTAGCAAG AGTCACTCTGTCATCGGAGCTGTCAAAGACGTCGTTGCAGGCGCCAAAGATGT CGCAGGAAAAGCCGTCGAGGATGCTCCTAGCATCATGCATACTGCAGTCGATG

[0040] CTGTGAAGAACGCAGCCACGACTGTGAAGGATGTGGCATCGTCGGCTGCATCG ACTGTGGCGGAGAAGGTAGTCGATGCTTACCACAGTGTGGTGGGAGACAAGA CGGACGACAAGAAAGAGGGCGAGCACAGCGGCGACAAGAAGGACGACTCCA AAGCTGGAAGTGGCTCTGGACAAGGTGGTGACAACAAGAAGTCTGAAGGAGA

[0041] GACTTCTGGCCAAGCAGAATCCAGCTCTGGCAACGAAGGAGCTGCTCCAGCCA AAGGCCGTGGTCGTGGACGGCCTCCAGCAGCTGCTAAAGGAGTTGCTAAGGGT GCTGCAAAGGGCGCTGCCGCCTCCAAAGGAGCCAAGAGCGGTGCTGAATCCTC

[0042] CAAGGGAGGAGAACAGTCGTCAGGAGATATCGAGATGGCAGATGCTTCCTCC AAGGGAGGCTCGGACCAGAGGGATTCCGCGGCGACCGTTGGCGAAGGTGGTG CATCAGGCAGTGAGGGTGGAGCTAAGAAAGGCAGAGGGCGGGGCGCTGGTAA

[0043] GAAAGCGGATGCGGGTGATACGTCCGCTGAGCCGCCTCGGCGGTCGTCCCGCC TGACGTCTTCAGGTACAGGGGCGGGTTCCGCTCCAGCTGCAGCGAAAGGCGGA GCGAAGCGTGCTGCTTCTTCCTCCAGTACACCTTCCAACGCTAAGAAGCAAGC

[0044] GACTGGAGGTGCTGGCAAAGCTGCTGCCACCAAAGCAACTGCTGCCAAATCGG CAGCCTCTAAAGCTCCCCAGAATGGCGCAGGTGCCAAGAAGAAGGGAGGAAA GGCTGGAGGACGGAAGAGGAAGTAA3

[0045] According to an alternative embodiment of the present invention, the nucleotide sequence encoding the DSUP protein of Ramazzottius varieornatus comprises or consists of the sequence SEQ ID NO. 3:

[0046] 5'ATGGCCTCTACACACCAGAGCAGCACCGAGCCTAGCTCTACAGGCAAGAGCG AGGAGACCAAGAAGGACGCCTCTCAGGGAAGCGGACAGGACAGCAAGAACGT GACCGTGACCAAGGGCACAGGATCTAGCGCCACAAGCGCCGCTATCGTGAAG ACAGGAGGCAGCCAGGGCAAGGATAGCAGCACAACAGCCGGCTCTAGCAGCA CACAGGGCCAGAAGTTCAGCACCACCCCTACAGACCCCAAGACCTTCAGCAGC GACCAGAAGGAGAAGTCCAAGAGCCCCGCCAAGGAAGTGCCTAGCGGAGGCG ATTCTAAGTCTCAGGGCGACACCAAGAGCCAGAGCGACGCCAAGTCTAGCGG ACAGTCTCAGGGCCAGAGCAAGGATAGCGGCAAGAGCAGCAGCGACAGCTCT AAGAGCCACAGCGTGATCGGAGCCGTGAAAGACGTGGTGGCAGGAGCCAAAG ACGTGGCAGGAAAGGCAGTGGAGGACGCCCCTAGCATCATGCACACAGCAGT GGACGCCGTGAAGAACGCAGCCACAACAGTGAAGGACGTGGCCTCTAGCGCC GCTTCTACAGTGGCCGAGAAAGTGGTGGACGCATACCACAGCGTCGTGGGCGA TAAGACCGACGACAAGAAGGAGGGAGAGCACAGCGGCGACAAGAAGGACGA TAGCAAGGCCGGAAGCGGAAGCGGACAGGGCGGCGATAATAAGAAGTCCGAG GGAGAAACAAGCGGACAGGCCGAGTCTAGCAGCGGAAACGAAGGAGCCGCTC CAGCCAAAGGCAGAGGAAGAGGCAGACCTCCAGCCGCCGCTAAAGGAGTGGC TAAAGGAGCCGCCAAAGGAGCAGCCGCTTCTAAAGGAGCCAAGAGCGGAGCC GAGTCTTCTAAGGGAGGCGAGCAGTCTAGCGGCGATATCGAGATGGCCGACGC CTCTTCTAAAGGCGGAAGCGATCAGAGAGACAGCGCCGCTACAGTGGGAGAA GGAGGAGCTAGCGGAAGCGAAGGAGGAGCTAAGAAGGGCAGAGGCAGAGGA GCCGGCAAAAAAGCCGACGCAGGAGATACAAGCGCCGAGCCTCCCAGAAGAA GCAGCAGACTGACCTCCTCTGGAACAGGAGCAGGAAGCGCTCCAGCAGCCGC TAAAGGAGGAGCCAAAAGAGCCGCTTCTAGCAGCAGCACCCCTAGCAACGCC AAGAAGCAGGCTACAGGAGGAGCCGGAAAAGCCGCCGCTACAAAAGCCACAG CCGCCAAGTCTGCCGCTTCTAAAGCTCCTCAGAACGGAGCCGGCGCCAAGAAG AAGGGCGGCAAAGCCGGAGGCAGAAAGAGGAAGCACCACCACCACCACCACT AG3'.

[0047] Advantageously, thanks to a process of gene codon optimization, the nucleotide sequence SEQ ID NO. 3 is particularly suitable for the expression of the DSUP protein in a human cell, more particularly in a human pancreatic beta cell. In particular, codon optimization is a useful tool when expressing genes heterologously (in a different host organism), if problems occur during the cloning of a gene or when optimizing the level of gene expression.

[0048] Most amino acids can be translated by multiple codons, but codon bias reflects the preference for one codon over another and varies between species. This can lead to a reduction in the translation of a protein when a gene is inserted into a different host species. Optimization consists in exploiting the codon bias of the host organism to produce the same amino acid sequence with greater efficiency.

[0049] Preferably, the isolated nucleotide sequence encoding the DSUP protein according to the invention is selected from the deoxyribonucleic acid (DNA) sequences SEQ ID NO. 2 and 3. It is also understood that the present invention contemplates the use of ribonucleic acid (RNA) sequences corresponding to the aforementioned coding sequences SEQ ID NO. 2 and 3.

[0050] Yet another object of the present invention is an expression vector comprising an isolated nucleic acid as defined above, for use in the prevention and / or therapeutic treatment of diabetes.

[0051] Expression vectors suitable for use in gene therapy are known and described in the state of the art, so their choice and use fall within the ability of the person skilled in the art.

[0052] In one embodiment according to the invention, the expression vector is a plasmid vector, preferably a pEFl / V5-His vector containing the promoter for the alpha-subunit of the human elongation factor 1 ("human elongation factor 1 alpha-subunit", hEF-lalpha) which allows high levels of expression in a wide range of species and cell types; a C-terminal tag encoding the V5 epitope and a poly-histidine peptide.

[0053] In the context of the present invention, in the expression vector the nucleotide sequence encoding the DSUP protein may be operatively linked to one or more regulatory sequences, allowing its expression in a host cell, preferably in a pancreatic beta cell, more preferably in a human pancreatic beta cell. The selection of the most appropriate regulatory sequences to be used in the present invention is well within the skills of the those of ordinary skill in the art. By way of non-limiting example, promoters are cited, for example, inducible promoters, as well as enhancer elements, internal ribosome entry sites and / or transcription termination signals. For example, the nucleotide sequence encoding the DSUP protein may be operatively linked to a promoter such as the hEF-lalpha promoter. According to the invention, the isolated nucleic acid comprising or consisting of a nucleotide sequence coding for the DSUP protein and / or the expression vector containing it, can be encapsulated within a pharmaceutically acceptable vehicle.

[0054] Therefore, a pharmaceutically acceptable vehicle containing internally an isolated nucleic acid and / or an expression vector as defined above also fall within the scope of the present invention.

[0055] The expression "pharmaceutically acceptable vehicle", as used herein, means a medium suitable for transporting a therapeutic agent in an animal organism, in particular in the human organism, which is preferably capable of operating a localized release of the therapeutic agent, thereby allowing a more targeted therapeutic treatment. The term "pharmaceutically acceptable" refers to vehicles that may be administered to animal organisms without toxicity at concentrations in accordance with the efficacy of the therapeutic agent.

[0056] In one embodiment according to the invention, the pharmaceutically acceptable vehicle is in the form of a nanoparticle, more preferably a lipid nanoparticle (LNPs).

[0057] Lipid-based nanoparticles for use in the delivery of therapeutic agents are known and described in the state of the art, so the selection and use thereof fall within the skill of those of ordinary skill in the art. By way of non-limiting example, liposomes, micelle, lipid nanoemulsions (LNE), solid lipid nanoparticles (SLN), lipid nanoparticles and nanostructured lipid carriers (NLC) are mentioned.

[0058] Alternatively, the pharmaceutically acceptable vehicle according to the invention may be an extracellular vesicle (EV), preferably a human extracellular vesicle, even more preferably a human extracellular vesicle engineered to target exclusively pancreatic beta cells, to be used as a delivery strategy.

[0059] As is known in the art, extracellular vesicles (EVs) are a heterogeneous population of small particles enclosed by a lipid bilayer released by all types of living cells. Once secreted into the extracellular environment, EVs can induce a response in target cells in two different ways, namely by activating membrane receptors or by releasing their cargo into the cytoplasm, which may contain proteins, nucleic acids and / or lipids. Non-limiting examples of EVs include microvesicles, released by plasma membrane budding, and exosomes, derived from the endosomal compartment.

[0060] The person skilled in the art is able to select the most suitable extracellular vesicles to be used in the present invention.

[0061] As already reported for numerous drugs at different stages of the clinical phase or already on the market, lipid nanoparticles and extracellular vesicles represent particularly efficient and safe systems for drug delivery.

[0062] According to a further embodiment of the present invention, the pharmaceutically acceptable vehicle is a viral particle. Such a viral particle may be, for example, a lentivirus, a retrovirus, an adenovirus or an adeno-associated virus. Preferably, the viral particle is a recombinant adeno-associated virus (rAAV).

[0063] Advantageously, in the aforementioned embodiments of the invention, the use of a pharmaceutically acceptable vehicle allows for the release of the isolated nucleotide sequence and / or the vector according to the invention in a specific area of the animal or human organism, thereby avoiding potentially harmful effects on organs not directly involved.

[0064] As described above, the ability to exert a protective role on the apoptosis of pancreatic beta cells induced by the mobilization of reactive T cells directed against beta-cell antigens (immune-mediated damage) make the isolated DSUP protein, the isolated nucleic acid comprising or consisting of a nucleotide sequence encoding said protein, the expression vector comprising said nucleic acid and the pharmaceutically acceptable vehicle as defined above particularly suitable for use in applications aimed at promoting the survival of pancreatic beta cells in a subject affected by diabetes and improving the functionality of these cells. The expression "functionality of pancreatic beta cells", as used herein, primarily means the production and secretion of the insulin hypoglycemic hormone by these cells.

[0065] In one embodiment according to the invention, diabetes is type 1 diabetes mellitus.

[0066] In another embodiment according to the invention, diabetes is type 2 diabetes mellitus.

[0067] A further object of the present invention is a pharmaceutical composition comprising an isolated DSUP protein from tardigrades of the genus Ramazzottius, preferably of the species Ramazzottius varieornalus. an isolated nucleic acid, an expression vector and / or a pharmaceutically acceptable vehicle as defined above, in combination with pharmaceutically acceptable excipients and / or diluents.

[0068] According to the invention, the pharmaceutical composition is suitable for use in the therapeutic and preventive medical applications indicated above with reference to the DSUP protein, the isolated nucleic acid, the expression vector, and / or the pharmaceutically acceptable carrier for use according to the invention.

[0069] The pharmaceutical composition of the present invention can be formulated into any suitable form, for example for administration via the enteral route (oral or gastroenteric, rectal, sublingual), parenteral route (percutaneous, inhalation, ocular, intravenous, intra-arterial, intramuscular, intradermal, intranasal, subcutaneous, intraperitoneal), topical route (direct contact of the drug with the site of action and / or with the skin and / or mucous membranes). Of course, the selection of suitable carriers, excipients and / or diluents is carried out depending on the desired form of administration, and this selection is within the skills of those of ordinary skill in the art. The selection of the dose of active ingredient and the dosage regimen also falls within the skills of those of ordinary skill in the art, and the selection thereof may depend on various factors such as the age of the patient and the degree of disease progression.

[0070] In one embodiment, the therapeutic treatment with the tardigrade DSUP protein, the isolated nucleic acid, the expression vector, the pharmaceutically acceptable vehicle and / or the pharmaceutical composition as defined above, additionally comprises the administration of one or more hypoglycemic agents. Suitable hypoglycemic agents (for oral administration) include biguanides, sulfonylureas, glinides, glitazones, DPP -4 enzyme inhibitors, intestinal alpha-glucosidase inhibitors, and renal SGLT-2 glucose transporter inhibitors.

[0071] The experimental section that follows is provided for illustrative purposes only and does not limit the scope of the invention as defined in the appended claims. In the experimental section, reference is made to the accompanying drawings, wherein:

[0072] - Figure 1 shows a series of micrographs indicative of the localization of the DSUP protein expressed in murine MIN6 pancreatic beta cells. Nuclei labeling by DAPI (micrographs on the left in the three panels), localization of the DSUP protein by histidine tail (HIS) labeling (micrographs in the center in the three panels), merge (micrographs on the right in the three panels);

[0073] - Figure 2 shows three graphs illustrating the survival rate of MIN6 cells transfected with the DSUP plasmid (DSUP+), the empty plasmid (Empty), or not transfected (CTRL), A) after treatment with H 2 O 2 100 pM for 90 minutes or 4 hours and B) after treatment with H2O2 100 pM or 250 pM overnight (O / N);

[0074] - Figure 3 illustrates the count of pyknotic nuclei (graphs A-B) and nuclei positive for Propidium Iodide (PI+) staining (graphs C-D) of MIN6 cells treated with a cytokine mixture for 24 hours (24h) or 48 hours (48h) and transfected with DSUP plasmid (DSUP), control plasmid (PEMPTY), or not transfected (CTR). The data are reported as ratio between treated (CYT) and untreated (NT) cells (mean ± SD) of the percentage of positive picnotic nuclei out of the total nuclei. Statistical analysis performed using ANOVA analysis with Tuckey’s multiple comparison test;

[0075] - Figure 4 illustrates the extent of Glucose-Stimulated Insulin Secretion (GSIS) in MIN6 cells treated with cytokine mixture for 24 hours or 48 hours (gray bars in histograms) and transfected with DSUP plasmid or empty control plasmid (empty plasmid), or in nontransfected MIN6 cells (white bars in histograms). In the histograms, the data are reported as (A) Insulin Content in % (IC%) (Ratio of secreted insulin to total insulin * 100) and as (B) Stimulation Index (ratio of IC% detected with 20 mM glucose stimulus and basal stimulus at 2 mM glucose, normalized to total insulin content). Declaration pursuant to Article 170 bis, paragraphs 2, 3 and 4 of the Italian Industrial

[0076] Property Code

[0077] This invention has been made in accordance with the provisions of Article 170-bis, paragraphs 2, 3 and 4 of the Italian Industrial Property Code.

[0078] EXAMPLES

[0079] 1. MATERIALS AND METHODS

[0080] 1.1 Culture and transfection of MIN6 cells

[0081] For their experiments, the inventors used an in vitro model of immortalized murine beta cells (MIN6).

[0082] MIN6 cells were obtained from AddexBio (San Diego, CA, USA), cultured between passages 21-35 and maintained in culture in DMEM (cat. 5671, Sigma- Aldrich, St. Louis, MO, USA) supplemented with 1% L-Glutamine, 1% Antibiotic / Antifungal (A5955, Sigma- Aldrich, St. Louis, MO, USA), 15% FBS (cat. ECS0180L, Euroclone, Milan, Italy), 1% sodium pyruvate (cat. ECM0542D, Euroclone, Milan, Italy) and P-Mercaptoethanol 50 pM. The MIN6 cells were stably transfected with a plasmid containing the nucleotide sequence coding for the DSUP protein (hereinafter referred to as "DSUP plasmid") and an empty plasmid (donated by Dr. Jlenia Brunetti, University of Siena).

[0083] For the construction of the DSUP plasmid, a plasmid was used that inserts a histidine tail at the C-terminus of the protein to be used in the localization studies since no anti-DSUP antibodies are currently available. The insert encoding the DSUP protein was obtained by amplifying the DSUP gene from the pCXN2KS-DSUP construct (Addgene plasmid # 90019; http: / / n2t.net / addgene :90019), which contains the coding sequence of the DSUP gene of Ramazzottius varieornatus (SEQ ID NO. 2) (Hashimoto et al., 2016). For insert amplification, there were used primers that insert the restriction sites for the Spel and EcoRI enzymes at the 5' and 3' ends of the insert, respectively. The pEFl / V5-His plasmid (Invitrogen) and the insert were then digested at 37°C for 4 hours using the Spel and EcoRI restriction enzymes in the presence of the Cut Smart buffer (New England BioLabs). Subsequently, the digestion products were purified. The insert was purified directly using the GeneElute PCR Clean-up kit (Sigma- Aldrich), while the plasmid was purified from band (QIAquick gel extraction kit-Qiagen), after running on 2% agarose gel. Both purifications were quantified at the Nanodrop (ThermoScientific) which also returns the 260 / 280 nm ratio. The T4 DNA ligase enzyme was used in the insertion phase of the nucleotide insert coding for the DSUP protein in the pEFl / V5-His plasmid. The recombinant vector obtained was cloned in competent Escherichia coli cells, DH5alpha strain, extracted by miniprep (GeneJET Plasmid Miniprep kit, Thermo Scientific), quantified at Nanodrop and stored at - 20°C.

[0084] The expression vector thus obtained and the empty plasmid were transfected into MIN6 cells using the Lipofectamine® 2000 reagent (Life Technologies, Carlsbad, CA, USA) and the stably transfected cells were selected using the antibiotic G418 at a final concentration of 500 pg / mL (SERVA Electrophoresis GmbH, Heidelberg, Germany) for three weeks. The verification of transfection and transcription of theDSUP protein coding sequence was carried out by PCR. For this purpose, total RNA was extracted from the cell pellets using the SV total RNA isolation system (Promega) following the manufacturer's instructions and reverse transcribed into cDNA using the M-MuLV-RH cDNA synthesis kit (Experteam). The transcription of DSUP protein coding sequence was evaluated by endpoint PCR using the following primers: forward 5 TCCAC AGAACCCTCTTCCAC -3 ' (SEQ ID NO. 4) and reverse 5'- GACGATGCCACATCCTTCAC -3' (SEQ ID NO. 5) (annealing T: 55 °C, 35 cycles, amplicon length: 560 bp). The PCR products were visualized in a 2% agarose gel with ethidium bromide.

[0085] 1.2 Localization of the DSUP protein within the cells

[0086] The localization of the DSUP protein after expression in murine MIN6 pancreatic beta cells was evaluated by immunofluorescence assay using an anti-6X His tag monoclonal antibody and subsequently an anti-mouse Alexa Fluor 546. The nuclei were labeled with DAPI. The images were acquired using the Leica SP8 confocal microscope (Leica Microsystem) with 40X immersion lens.

[0087] 1.3 Cell viability

[0088] The MTT metabolic test (Vybrant® MTT Cell Proliferation Assay Kit, Molecular Probes) was used to quantify the cell viability of MIN6 cells after stable transfection with the DSUP plasmid. To evaluate the resistance induced by the expression of DSUP against free radicals, the cells transfected with both the DSUP plasmid and the empty plasmid were plated at a density of 340,000 cells / mL in a 96-well plate. After 24 hours of incubation, the control and transfected cells were treated with hydrogen peroxide (H2O2) at 100 pM for 90 minutes, 4 hours and overnight (O / N) and with 100 / 250 pM O / N in complete medium (10% FBS, + / - G418). After treatment, the cells were incubated at 37 °C for 4 hours with the MTT tetrazolium dye (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (yellow) which, in healthy cells, is converted by mitochondrial enzymes into an insoluble formazan compound (purple). Solubilization was carried out with DMSO (50 pL) at 37 °C for 10 minutes. Accordingly, the number of viable cells was determined by measuring the absorbance at 540 nm in a microplate reader (Tecan). Each experiment was performed in triplicate and was repeated at least three times.

[0089] 1.4 Induction of pro-inflammatory stress

[0090] Pro-inflammatory stress on MIN6 cells was induced with a cytokine mix composed of IL- ip (5 ng / mL, cat. 15271, Sigma-Aldrich, St. Louis, MO, USA), TNFa (30 ng / mL, cat. T7539) and ZFNy (10 ng / mL, cat. 485MI- R&D System, Minneapolis, MN, USA) for 24 h and 48 h.

[0091] 1.5 Apoptosis evaluation by counting pyknotic nuclei

[0092] The pyknotic nuclei present in the MIN6 cells under examination were counted using Hoechst 33342 (cat. 62249, Invitrogen, Waltham, MA, USA) diluted 1 : 100 and added to the MIN6 cells previously plated in 24-well plates (1 pL / well). Manual counting was used to determine the number of pyknotic nuclei (normalized with respect to the total number of nuclei) in five different areas / well. Furthermore, to verify late apoptosis, the Propidium Iodide (PI) reagent diluted 1 : 100 was used and added to the MIN6 cells previously plated in 24-well plates (1 pL / well). Manual counting was performed with an inverted fluorescence microscope (Leica DMI3000 B, Leica Microsystems, Wetzlar, Germany) with 40X magnification.

[0093] For statistical analysis the One way ANOVA test with the Tukey’s multiple comparison test was used Results with p < 0.05 were considered significant.

[0094] 1.6 Glucose-Stimulated Insulin Secretion Test (GSIS)

[0095] MIN6 cells, 24 hours after transfection with DSUP plasmid or control empty plasmid (CTR), were incubated with 300 pL of Krebs starvation solution composed of 610mM NaCl, lOOmM HEPES, 24mM KC1, 20mM NaHCO3, 6mM KH2PO4, 20mM NaCl, 6mM MgSO47H2O, 5 mM CaCh and 2 mM glucose (basal Krebs solution) for 30 minutes at 37°C. After washing in basal Krebs solution, the cells were incubated with 300 pL of basal Krebs solution (2 mM glucose) or stimulus (20 mM glucose) for 45 minutes at 37°C. Secreted insulin levels were measured using the Mercodia Mouse Ultrasensitive Insulin ELISA (#10- 1249-01) and the values were normalized to the total insulin content. The total contents were extracted with 300 pL of cold acid ethanol. Results with a p-value < 0.05 were considered significant.

[0096] 1.7 Culture and transfection of EndoC-BHl cells

[0097] The human beta cell line EndoC-PHl, obtained from UniverCell-Biosolutions (Toulouse- France), was used between passages 48-51. In detail, the EndoC-PHl cells were plated in flasks treated with coating medium [coating medium: DMEM with high glucose content (4500 g / L) (cat. 51441 C), 1% Penicillin / Streptomycin (cat. P0781), 1% ECM (cat. E1270) and 0.2% bovine plasma fibronectin (cat. Fl 141) - all from Sigma-Aldrich, St. Louis, MO, USA] and maintained in culture in low glucose DMEM (1000 g / L) [DMEM (cat. D6046) supplemented with 2% BSA fraction V (cat. 10775835001), P-mercaptoethanol 50 pM (cat. M7522), 1% L-glutamine (cat. G7513), 1% Penicillin / Streptomycin (cat. P0781), Nicotinamide lOmM (cat. N0636), Transferrin 5.5 pg / mL (cat. T8158) and Sodium selenite

[0098] 6.7 ng / mL (cat. S5261) - all produced by Sigma-Aldrich (St. Louis, MO, USA).

[0099] In order to modulate the expression of the DSUP protein in this human beta cell line, the EndoC-PHl cells were plated at a density of 1.37 x 105cells / cm in 24-well plates. After 24 hours, the EndoC-PHl cells were transiently transfected with 500 nM of synthetic mRNA DSUP (custom synthesized by Tebu-Bio through mRNA synthesis by in vitro plasmid transcription) using the RNAiMAX Lipofectamine transfection reagent (cat. 13778-150, Invitrogen, Waltham, MA, USA) for 24 hours, 48 hours and 72 hours in order to verify the timing with the greatest transfection efficiency to be able to perform survival, apoptosis and functionality tests.

[0100] 1.8 Verification of the presence of the DSUP transcript in human EndoC-BHl cells

[0101] To verify the successful transfection of the human EndoC-PHl cells, RNA was extracted from cell pellets at 24, 48 and 72 hours after transfection using the RNeasy Mini Kit (Qiagen). The RNA was quantified at the Nanodrop and, subsequently, 100 ng / ul for each sample were reverse transcribed into complementary DNA (cDNA) using the i Script™ cDNA Synthesis Kit (Biorad). The samples were then amplified by end point PCR in the Fast method (Roche) in a mixture containing 2.5 mM MgCh, 200 uM dNTPS, 10 uM primers and 1 U of Taq at an annealing temperature of 60°C. The PCR products were run in 2.5% agarose gel.

[0102] The sequence of primer pairs required to amplify the entire DSUP sequence is shown below: pair 1

[0103] F: CAAGAGCGAGGAGACCAAGA (SEQ ID NO. 6)

[0104] R: CTGAAGGTCTTGGGGTCTGT (SEQ ID NO. 7) Amplification product of 213 base pairs (bp); pair 2 F: CAGGACAGCAAGAACGTGAC (SEQ ID NO: 8)

[0105] R: CCTTTCCTGCCACGTCTTTG (SEQ ID NO. 9)

[0106] 454 bp amplification product pair 3

[0107] F: ACAGACCCCAAGACCTTCAG (SEQ ID NO: 10)

[0108] R: ACTTTCTCGGCCACTGTAGA (SEQ ID NO. 11)

[0109] 359 bp amplification product pair 4

[0110] F: CAAAGACGTGGCAGGAAAGG (SEQ ID NO: 12)

[0111] R: CTTCGTTTCCGCTGCTAGAC (SEQ ID NO. 13)

[0112] 308 bp amplification product pair 5

[0113] PF: GCTACAGTGGGAGAAGGAGG (SEQ ID NO: 14)

[0114] PR: CCTCCTGTAGCCTGCTTCTT (SEQ ID NO. 15) 242 bp amplification product

[0115] 2. RESULTS

[0116] 2.1 The DSUP protein localizes in MIN6 cells at the nuclear and cytoplasmic level

[0117] In literature, the DSUP protein is described as having nuclear activity since it is involved in DNA damage repair. The localization of the protein on the DNA occurs through its carboxyterminal domain. Since there are no anti-DSUP antibodies, the present inventors have constructed an expression vector that carries the coding sequence for the DSUP protein linked to a coding sequence for a histidine tail (HIS) that would allow the localization of the protein to be followed once expressed within the cell. As shown in Figure 1, the DSUP protein localizes in the control MIN6 cells (untreated) both at the cytoplasmic level and at the nuclear level.

[0118] 2.2. The expression of the DSUP protein protects pancreatic beta cells from oxidative stress In order to examine the effect of the DSUP protein on cell death induced by reactive oxygen species (ROS), the present inventors exposed the MIN6 cells to hydrogen peroxide (H2O2) at 100 pM for 90 minutes, 4 hours and overnight (O / N) and with 100 / 250 pM O / N. The percentage of cell survival (assessed by MTT) was higher in cells expressing the DSUP protein (DSUP+) for all treatments and for each relative duration (Figure 2A and Figure 2B). At all concentrations, cell survival was lower in the basal control condition and in cells transfected with the empty plasmid.

[0119] 2.3 The expression of the DSUP protein protects pancreatic beta cells from apoptosis induced by inflammatory stress

[0120] The present inventors then investigated whether the expression of the DSUP protein, induced by stable transfection in the murine MIN6 beta cell line, could protect these cells from apoptosis induced by inflammatory stress following treatment with a mixture of cytokines containing IL-ip, TNF-a and IFN-y.

[0121] As shown in Figure 3, the ratio of pyknotic nuclei detected in the cytokine-treated cell sample to pyknotic nuclei detected in the untreated cell sample was significantly reduced (p=0.04) 48 hours after treatment (Figure 3B) in MIN6 cells expressing the DSUP protein compared to MIN6 cells transfected with control plasmid and compared to non-transfected MIN6 cells.

[0122] Similarly, the ratio of Propidium Iodide (PI+) staining positive cells detected in the cytokine- treated sample and PI+ cells detected in the untreated sample was significantly reduced (p=0.04) 48 hours after treatment (Figure 3B) in MIN6 cells expressing the DSUP protein compared to MIN6 cells transfected with control plasmid and compared to non-transfected MIN6 cells. These data demonstrate that the expression of the DSUP protein induced by stable transfection, promotes a protection of the MIN6 cells 48 hours (Figure 3B and 3D), but not 24 hours (Figure 3 A and 3C) after treatment with a cytokine mixture.

[0123] 2.4 The expression of the DSUP protein does not alter the functionality of pancreatic beta cells After confirming the involvement of the DSUP protein in the protection from apoptosis induced by inflammatory stress, the inventors of the present invention conducted dedicated studies in order to verify whether this protein could influence, positively or negatively, the beta cell function in basal conditions or following inflammatory stress. Therefore, after expression of the DSUP protein, induced by stable transfection in the murine MIN6 beta cell line, these cells were subjected to stress with a mixture of cytokines containing IL-ip, TNF- a and IFN-y for 24 hours and 48 hours.

[0124] Subsequently, the stressed cells were treated with high concentrations of glucose (20 mM) compared to basal glucose concentrations (2 mM) to verify cell functionality. The Glucose- Stimulated Insulin Secretion (GSIS) test revealed that inflammatory stress does not produce a reduction in the functionality of non-transfected MIN6 cells, transfected with CTR plasmid as well as MIN6 cells expressing the DSUP protein. Cellular function was expressed as Insulin Content in % (IC%; Figure 4A) and as Stimulation Index (Stimulation index, understood as the ratio of insulin content in % between insulin secreted upon 20 mM glucose stimulus and insulin secreted at 2 mM glucose, normalized for total insulin content) (Figure 4B). Furthermore, no difference in functionality was observed between the MIN6 cells expressing the DSUP protein that were not subjected to inflammatory stress compared to the cells not transfected or transfected with CTR control empty plasmid (Figure 4A-4B).

[0125] The above results show that the expression of the DSUP protein in MIN6 cells does not cause alteration of the functionality (understood as the ability to synthesize and secrete insulin) of the beta cells, maintaining them functional and capable of secreting insulin like those not transfected.

[0126] 2.5 Transfection with DSUP mRNA is effective in human pancreatic beta cells

[0127] The results of the end point PCR reactions on human pancreatic beta cells (EndoC-PHl) transfected at 24, 48 and 72 hours with a synthetic mRNA coding for DSUP, showed that the transfection procedure was efficient at all times tested, since for all conditions, amplification of the sequence encoding the whole protein was observed.

Claims

CLAIMS1. An isolated "Damage suppressor" (DSUP) protein, from a tardigrade organism of the genus Ramazzottius, for use in the prevention and / or therapeutic treatment of diabetes.

2. The isolated DSUP protein for use according to claim 1, wherein the tardigrade organism belongs to the species Ramazzottius varieornatus.

3. The isolated DSUP protein for use according to claim 2, which consists of the amino acid sequence of SEQ ID NO. 1.

4. An isolated nucleic acid comprising a nucleotide sequence encoding a DSUP protein according to any one of claims 1 to 3, for use in the prevention and / or therapeutic treatment of diabetes.

5. The isolated nucleic acid for use according to claim 4, wherein the nucleotide sequence encoding the DSUP protein is selected from the group consisting of SEQ ID NO. 2, 3 and corresponding RNA sequences thereof.

6. An expression vector comprising an isolated nucleic acid according to claim 4 or 5, for use in the prevention and / or therapeutic treatment of diabetes.

7. The expression vector for use according to claim 6, wherein the nucleotide sequence encoding the DSUP protein is operatively linked to one or more regulatory sequences located on the vector.

8. A pharmaceutically acceptable vehicle containing internally an isolated nucleic acid according to claim 4 or 5 or an expression vector according to claim 6 or 7, for use in the prevention and / or therapeutic treatment of diabetes.

9. The pharmaceutically acceptable vehicle for use according to claim 8, wherein the pharmaceutically acceptable vehicle is selected from a lipid nanoparticle and an extracellularvesicle (EV).

10. The pharmaceutically acceptable vehicle for use according to claim 8, wherein the pharmaceutically acceptable vehicle is a viral particle.

11. The pharmaceutically acceptable vehicle for use according to claim 10, wherein the viral particle is selected from the group consisting of lentiviruses, retroviruses, adenoviruses or adeno-associated viruses.

12. The isolated DSUP protein for use according to any one of claims 1 to 3, the isolated nucleic acid for use according to claim 4 or 5, the expression vector for use according to claim 6 or 7, or the pharmaceutically acceptable vehicle for use according to any one of claims 8 to 11, wherein the treatment is a therapy that promotes the survival of pancreatic beta cells in a subject affected by diabetes.

13. The isolated DSUP protein for use according to any one of claims 1 to 3 or 12, the isolated nucleic acid for use according to any one of claims 4, 5 or 12, the expression vector for use according to any one of claims 6, 7 or 12, or the pharmaceutically acceptable vehicle for use according to any one of claims 8 to 12, wherein diabetes is selected from type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM).

14. A pharmaceutical composition comprising an isolated DSUP protein according to any one of claims 1 to 3, an isolated nucleic acid according to claim 4 or 5, an expression vector according to claim 6 or 7, and / or a pharmaceutically acceptable vehicle according to any one of claims 8 to 11, and a pharmaceutically acceptable excipient and / or diluent, for use in the prevention and / or therapeutic treatment of diabetes.

15. The pharmaceutical composition for use according to claim 14, wherein the treatment is a therapy that promotes the survival of pancreatic beta cells in a subject affected by diabetes.

16. The pharmaceutical composition for use according to claim 14 or 15, wherein diabetes is selected from type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM).

17. The pharmaceutical composition for use according to any one of claims 14 to 16, which is in a pharmaceutical form for enteral, parenteral or topical administration.

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