WO2015169994A1

WO2015169994A1 - Composition for modulating the activity of sidt1 for the treatment of ifn-mediated infectious, inflammatory or autoimmune diseases

Info

Publication number: WO2015169994A1
Application number: PCT/ES2015/070369
Authority: WO
Inventors: Concepción MARAÑÓN LIZANA; Marta ALARCÓN RIQUELME; Casimiro CASTILLEJO LÓPEZ; Céline COPPARD; Nieves VARELA HERNÁNDEZ
Original assignee: Fundación Pública Andaluza Progreso Y Salud
Priority date: 2014-05-07
Filing date: 2015-05-07
Publication date: 2015-11-12

Abstract

The invention relates to a composition for modulating the activity of SIDT1 for the treatment of infectious, inflammatory or autoimmune diseases mediated by interferons (IFN), preferably type I and type III IFN, more specifically for the treatment of psoriasis and lupus. The invention also relates to a method for selecting drugs that can be used in the treatment of said diseases and to a method for collecting data that can be used in the diagnosis of said diseases.

Description

Composition capable of modulating the activity of SIDT1 for the treatment of infectious inflammatory or autoimmune diseases mediated by IFN.

Technical field

The present invention is within the field of molecular biology and medicine, and specifically refers to a composition capable of modulating the activity of SIDT1 for the treatment of infectious, inflammatory or interferon-mediated autoimmune diseases (IFN), preferably IFN type I and III, and more specifically for the treatment of psoriasis and lupus. It also refers to a method of selecting drugs useful in the treatment of said diseases and a method for the collection of data useful in the diagnosis of said diseases.

Background of the invention

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease, characterized by the production of autoantibodies and the induction of tissue damage mediated by the deposition of autoantibody-autoantigen immune complexes. This disease results in serious complications of renal, cardiovascular and neurological type. The pathogenesis of SLE involves complex interactions of multiple genes and environmental factors. To date, several genes involved in susceptibility to SLE have been described (Harley, IT and Kaufman, CD 2009. Nat Rev 10 (5): p. 285-90), however the understanding of how the identified gene variants affect the Risk and progression of SLE is still insufficient. The described genetic associations, as well as the study of animal models and biological samples of patients with SLE have allowed the identification of several biological routes involved in the induction of the pathological phenomena characteristic of SLE, such as apoptotic cell clearance, processing of immunocomplexes, the function of Toll type receptors (TLR), or the production of type I interferons (especially IFNa), as well as their response to them.

Dendritic cells (DC) are professional antigen presenting cells (APCs), located at the interface between the innate and adaptive immune response. They are the only APCs capable of stimulating naive T lymphocytes [Villadangos, JA and P. Schnorrer, 2007. Nat Rev Immunol 7 (7): p. 543-555.] And, therefore, have a central role in inducing the autoimmune response. These cells have developed exclusive and probably predominant cross-presentation pathways, which allow them to be restricted to class I molecules of exogenous antigens, after internalization by endocytosis of phagocytosis [Rock, KL, 2003. Nat Immunol 4 (10): p . 941-3]. These pathways are crucial for the stimulation of a specific response of antigens that are not produced in the cytosol of DC, as are those recognized in the case of autoimmune diseases [Segura, E. and JA 2009. Curr Opin Immunol 21 (1): p. 105-10]. DC can mediate very different effects, such as immunity or tolerance, depending on the subpopulation considered and the microenvironment in which they are located [Steinman, RM, et al., 2003. Annu Rev Immunol, 21: p. 685-711]. human DC can be divided into two main subpopulations: conventional myeloid-type (cDC) DCs secrete large amounts of IL-12 and IL-23 after being stimulated with LPS, double-stranded DNA or microbial lipopeptides, thereby inducing a robust Th1 and CD8 effector responses. Phagocytic and presenting cells are prominent, and can be differentiated in vitro from circulating monocytes (MoDC). On the other hand, DC plasmacytoids (pDC) have been classically considered as poor immune system stimulators. However, recent results have shown that peptide-pulsed, or virus-infected, pDCs can effectively stimulate an effector T response [Lui, G., et ai, 2009. PLoS One 4 (9): p. e71 11; Di Pucchio, T., et al., 2008. Nat Immunol 9 (5): p. 551-7.], But also activate the CD4 ⁺ or CD8 ⁺ regulatory T lymphocytes [Moseman, EA, et al., 2004. J Immunol 173 (7): p. 4433-42]. Although they were supposed to be unable to perform cross-presentation, it has been shown that human circulating pDCs ex vivo cross-presentation of HIV antigens after phagocytosis of infected cells [Hoeffel, G., et al., 2007. Immunity 27 ( 3): p. 481-92]. They produce large amounts of type I and III IFNs in response to viruses, DNA fragments rich in unmethylated CpG motifs, or TLR-7 ligands. The pDCs stimulate the differentiation of plasma cells through IFN type I and IL-6 [Jego, G., et al., 2003. Immunity 19 (2): p. 225-34]. Type I interferons possess other important immunoregulatory functions, including stimulation of T lymphocytes [Bracci, L., et al., 2008. Expert Rev Vaccines 7 (3): p. 373-81]. The ability of any type of DC to induce immunity or tolerance is a consequence of the displacement of the balance between inhibitory or stimulatory factors, determined by the signals of the microenvironment in which the processing and presentation of the antigen occurs. The final phenotype of DC is reflected by the expression of costimulation molecules and cytokine secretion, for which the balance between IL-23, IL-12 and IL-10 is decisive for the polarization of naive T lymphocytes towards a Th1 / Th2 / Th17 / Treg phenotype. Although the interaction between DC and B lymphocytes has been less studied, it has been observed that pDC, but not cDC, can promote differentiation of memory B cells to plasma cells, through an IFNa-dependent mechanism [Poeck , H., et al., 2004. Blood 103 (8): p. 3058-64.], And also from the CD70-CD27 interaction [Shaw, J., et al., 2010. Blood 115 (15): p. 3051-7].

The role of DC in SLE was postulated several years ago, since high levels of type I IFNs are detected in SLE patients, and pDCs produce large amounts of these cytokines. In fact, circulating pDC levels are high in patients with SLE [Jin, O., et al., 2008. Lupus 17 (7): p. 654-62], and pDC infiltrates have also been found in the lesions of such patients [Ko, CJ, et al., 201 1. J Cutan Pathol, 38 (1 1): p. 889-92]. Therefore, pDCs have a central role in the immunopathogenesis of SLE.

SIDT1 (SID1 transmembrane family, member 1) is a very poorly studied membrane integral protein. Most of the known data on SIDT1 refers to its counterpart in C. elegans, SID1, a membrane protein involved in the mechanism of intercellular iRNA transfer. SID1 is a protein with 11 transmembrane domains, probably involved in the transport of nucleic acids, specifically its overexpression increases the entry of molecules of small RNA interreferencing (siRNA) into human epithelial cells [21-22]. Preliminary results point to a location in the endoplasmic reticulum and in endosomal-lysosomal vesicles of SID1. At first glance it seems difficult to conceive how SID1 could be involved in the pathophysiology of SLE. However, the authors of the present invention have shown that SIDT1 plays an important role in the recognition of exogenous nucleic acids and in the initiation of the IFN response in pDCs.

Advances in the discovery of the molecular mechanisms involved in viral diseases, as well as in IFN-mediated inflammatory or autoimmune diseases, such as psoriasis or lupus, would make it possible to have useful therapeutic agents in the treatment of these diseases and methods for the correct diagnosis from the same.

DESCRIPTION OF THE INVENTION

MEDICAL USES OF THE COMPOSITIONS OF THE INVENTION

The authors of the present invention have demonstrated involvement of SIDT1 in the recognition of exogenous nucleic acids and in the initiation of the IFN response in the pDCs, and their inhibition decreases the production of type I and III IFNs.

Therefore, a first aspect of the invention relates to a composition, hereinafter composition of the invention, comprising an agent modulating the activity of SIDT1 for the treatment, improvement or prevention of infectious, inflammatory or autoimmune diseases mediated by IFN

The term "SIDT1" (SID1 transmembrane family, member 1, also called in the literature SID1; SID-1; B830021 E24Rik) is located on chromosome 3 (3q13.2). Unless otherwise indicated, when the term "SIDT1" or "SID1" is used herein, it refers to both the gene and the human SIDT1 protein. In the context of the present invention, SIDT1 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the SIDT1 protein, and which would comprise various variants from: a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1,

b) nucleic acid molecules whose chain complement it hybrid with the polynucleotide sequence of a),

c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code,

d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 1. in that the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the SIDT1 protein. Among these nucleic acid molecules is the collection in the GenBank (NCBI) sequence NM_017699.2 or SEQ ID NO: 2.

SEQ ID NO: 1

MRGCLRLALLCALPWLLLAASPGHPAKSPRQPPAPRRDPFDAARGADFDHVYSGWNLST

ENIYSFNYTSQPDQVTAVRVYVNSSSENLNYPVLVWRQQKEVLSWQVPLLFQGLYQRSY

NYQEVSRTLCPSEATNETGPLQQLIFVDVASMAPLGAQYKLLVTKLKHFQLRTNVAFHFTA

SPSQPQYFLYKFPKDVDSVIIKWSEMAYPCSVVSVQNIMCPVYDLDHNVEFNGVYQSMTK

KAAITLQKKDFPGEQFFWFVI KPEDYACGGSFFIQEKENQTWN LQRKKN LEVTIVPSI KESV

YVKSSLFSVFIFLSFYLGCLLVGFVHYLRFQRKSIDGSFGSNDGSGNMVASHPIAASTPEGS

NYGTIDESSSSPGRQMSSSDGGPPGQSDTDSSVEESDFDTMPDIESDKNIIRTKMFLYLSD

LSRKDRRIVSKKYKIYFWNIITIAVFYALPVIQLVITYQTWNVTGNQDICYYNFLCAHPLGVLS

AFNNILSNLGHVLLGFLFLLIVLRRDILHRRALEAKDIFAVEYGIPKHFGLFYAMGIALMMEGV

LSACYHVCPNYSNFQFDTSFMYMIAGLCMLKLYQTRHPDINASAYSAYASFAVVIMVTVLG

VVFGKNDVWFWVIFSAIHVLASLALSTQIYYMGRFKIDLGIFRRAAMVFYTDCIQQCSRPLY

MDRMVLLWGNLVNWSFALFGLIYRPRDFASYMLGIFICNLLLYLAFYIIMKLRSSEKVLPVPL

FCIVATAVMWAAALYFFFQNLSSWEGTPAESREKNRECILLDFFDDHDIWHFLSATALFFSF

LVLLTLDDDLDWRRDQI PVF

SEQ ID NO: 2 gagttctctgcaggtagcagcctgggaaagcgaactcgcgcaccctcttgggggacgggctcttttgctttctatttcttctcctgcag aataaagaaataaaacaggtgtctgaggagaggttctattagagaggggagaatagagagtaattatcgttcagacaggcaa ccagggtgcacacgtatttgaaacagaccgaatttcctcctcgatgtggcgtcaggttgacttttcgagactagcgggtatttcttttta atgactccaatgcctttttattattgctgttattgaggttgagggagaagagatcggtctaaattctggctgggtaagtggggggattct cggcgatgagaaacgggggacttagaagccggaggaaaatcagcagccccacatctccacttctccagtccgccctactctc cacccgtgacctccagtggagaccccaggcggcagcatcagtatttgatcggcccttcgtcagcacgctgccagccctggccg gctgggttcgccaggcatcacccgctcggctctgaagcggacgcctggccctgcaccgggctttggaaggaccctctctgcgct cgccccctccccagggtggctccgctttcgagcccgggcgcggtgcccaccatgcgcggctgcctgcggctcgcgctgctctgc gcgctgccctggctcctgctggcggcgtcgcccgggcacccggcgaaatcccccaggcagcccccggcaccgcgccgcga ccccttcgacgctgccaggggcgccgatttcgatcatgtctacagcggggtggtgaacctcagcaccgagaacatctactctttc aactacaccagccagcccgaccaggtgacagccgtgagggtgtatgtgaacagttcctctgagaatctcaactacccggtcctt gttgtggttcgccagcagaaagaggtgctgtcctggcaggttcctctgctcttccaaggactataccagaggagctacaactatca agaagtgagccgcaccttatgtccctcagaagcaaccaatgagacgggacccttgcagcaactgatatttgtagatgtcgcatc catggcacccctgggtgctcagtacaaactgctagttaccaagctgaagcacttccagctccggacaaatgttgcctttcactttac tgccagcccctctcaacctcagtattttctatacaagtttcccaaagacgtggactcagt tatcattaaagtggtgtctgaaatggctt atccatgttctgttgtctcagtccagaatatcatgtgcccggtgtatgatctcgaccacaatgtggaatttaatggtgtctatcagtccat gaccaagaaagctgccatcacgctacagaagaaggattttccaggcgagcagttcttcgtggtatttgtgataaagcctgaagat tatgcctgtggaggatctttcttcatccaggaaaaggaaaaccagacctggaatctacagcgaaaaaagaaccttgaagtgac cattgtcccttccattaaagaatctgtttatgtgaaatccagtcttttcagtgtcttcatcttcctgtccttctacttgggatgccttcttgttgg gtttgttcattatctgaggtttcagagaaaatccattgatggaagctttgggtccaatgatggctctggaaatatggtggcatctcatcc cattgctgccagcacacccgaagggagcaattatgggacaatagatgagtcaagctccagtcctggaaggcagatgtcctcct ccgatggtgggccaccgggccagtcagacacagacagctccgtggaggagagcgacttcgacaccatgccagacattgag agtgataaaaacatcatccggaccaagatgttcctttacctgtcagatttgtccaggaaggaccggagaattgtcagcaaaaaat ataaaatttatttttggaacatcatcaccattgctgtgttttacgcgctgcccgtgatccagctggtcattacctatcagacagtggtaa atgtcactggcaaccaggacatctgttactacaacttcctctgtgctcaccccttgggcgtcctgagtgccttcaacaacattctcag caatctgggccacgtgcttctgggcttcctcttcctgctgatagtcttgcgccgcgacatcctccatcggagagccctggaagccaa g gacatctttgctgtggagtacgggattcccaaacactttggtctcttctacgctatgggcattgcattgatgatggaaggggtgctc agtgcttgctaccatgtctgccctaattattccaacttccaattcgacacctccttcatgtacatgatcgctggcctgtgcatgctgaag ctctatcagacccgccacccagacatcaatgccagcgcctactctgcctatgcctcctttgctgtggtcatcatggtcaccgtccttg gagtggtgtttggaaaaaatgacgtatggttctgggtcatcttctctgcaatccacgttctggcctcgctagccctcagcacccagat atattatatgggtcgtttcaagatagatttgggaattttccggcgggctgccatggtgttctacacagactgtatccagcagtgtagcc gacctctatatatggatagaatggtgttgctggttgtggggaatctggttaactggtccttcgccctctttggattgatataccgcccca aaaggtcctcccagtcccgctcttctgcatcgtggccaccgctgtgatgtgggctgccgccctatattttttcttccagaatctcagca gggactttgcttcctacatgctgggcatcttcatctgtaaccttttgctgtacctggccttttacatcatcatgaagctccgcagctctga gctgggagggaactccggccgaatcccgggagaagaaccgcgagtgcattctgctggatttcttcgatgaccatgacatctggc acttcctctctgctactgctctgtttttctcattcttggttttgttaactttggatgatgaccttgatgtggttcggagagaccagatccctgtc ttctgaacctccaacattaagagaggggagggagcgatcaatcttggtgctgtttcacaaaaattacagtgaccacagcaaagt aaccactgccagat gctccactcaccctctgtagagccaactctgcattcacacaggaaggagaggggctgcgggagatttaa acctgcaagaaaggaggcagaaggggagccatgttttgaggacagacgcaaacctgaggagctgagaaacacttgctcctt ccatctgcagctttgggagtgcaacagggataggcactgcatccaagtcaactcaccatcttggggtccctcccaccctcacgg agacttgccagcaatggcagaatgctgctgcacacttccctccagttgtcaccctgcccagaaaggccagcagcttggacttcct gcccagaaactgtgttggcccccttcacacctctgcaacacctgctgctccagcaagaggatgtgattctttagaatatggcgggg aggtgaccccaggccctgccctactgggatagatgttttaatggcaccagctagtcacctcccagaagaaactctgtatatttccc ccaggtttctgatgccatcagaagggctcaggagtggggtttgtcacacattcctcttaacaagtaactgtcactgggaccgagtc ctgggtgcttacatattccttcgtgtcttcatctcactgacctgtgtggacctcatcactctgactctgccttcttggaaaggccctgtca ctccacagatgtctggccagcttcaaggcagaaggaaaaacaggaaaagctcttttaacagcagcaggaacaagagaaatg actaaccatactaaaagactggtaacagcagcagcagccagacaggcctcaccttaaggacttgggctgccagagcaaattc agcagagcttatttggcctcccattcacacagctcagttctgtgcccacatcacctttggggaagaaatcagcattctaatcaggg acactacttcaggagtcctccacagcgagtccgtcatctgtcactttatgtagatcagggttctagacttcttccctgaggttctcaga agcagctctcaggatgaacgtattgtcctcttcccctcttcttgcaaagtgcacagctaatctaatgttgtctctcggttgcacctgaca ttctctccccagtaaggtgttggcaagctcagcatctgggttccactctcacactgtctggcagctctgtgtctgagaagttctacattg accaggcccccttgttgcctggagtatgacgtaatcagaaaatagacgtataaatgtgcacatgcgtatgtatttgcttgtgaaatta aagtcacctcttgcctctgctttcctgatcattc gttagagaaatggatcaggcatttttttaaattattattctttctctaaactatttgcatt gtgttcaaaaacccattttagaagtttgaacagcaagcttttcctgattttaaaaacacaaagttgctttcaatgaaatattttgtgatttt tttaaagtccccaaatgtgtacttagccttctgttattccttattctttaagcagtgttggcttccattgaccatatgaaggccaccaatta aatggttgtgttaatccaacatgtaaaaaactttttggcagggcacagtggctcacgcctgtaatcccaacactttgggaggctga ggcaggaggatcacttgagcccaggagattgacgccgcagtgaactatgattgtgcccctgcactccagcctggatgacagag tgagaccccatctcttaaaaaataaaaaaaaataaaaatttgaacctgtttcaaaaaaaaaaaaaaaaaaaa

The term "modulating activity" as used herein, refers both to inhibiting (decreasing) or stimulating (increasing) the level of activity of the SIDT1 protein in a cell. The activity of SIDT1 can be modulated by the modification of the levels and / or activity of the SIDT1 protein, or by the modification of the levels to which the SIDT1 gene is transcribed such that the levels of activity of the SIDT1 protein in The cell is modulated. Modulating agents can also be agonists (substances that are capable of binding to a receptor and eliciting a response in the cell, specifically an increase in SIDT1 activity), as antagonists (substances that not only do not activate the receptor, but also actually blocks its activation by agonists). In the context of the present invention, inhibition is the preferred form of modulation.

In a preferred embodiment of this aspect of the invention, the modulating agents comprised in the composition of the invention are selected from a list comprising: a) an organic molecule, b) an RNA molecule, c) an antisense oligonucleotide, d) an antibody, or e) a ribozyme F) A nuclease capable of editing chromosomal DNA (ZFN, Talen, CRISPR).

One skilled in the art could prepare organic molecules that can specifically bind SIDT1 without binding to other polypeptides or proteins, the organic molecules will preferably have a weight of 100 to 20,000 daltons, more preferably 500 to 15,000 daltons, and more preferably 1000 to 10,000 daltons. Bookstores of organic molecules are commercially available. The route of administration may be, without limitation, intraperitoneal, intrathecal, intravenous, intramuscular, subcutaneous, intraventricular, oral, enteral, parenteral, intranasal or dermal. Recently, with the development of antisense technology, nucleotide sequences specifically complementary to a particular DNA or RNA sequence could form complexes and block transcription or translation. Thus, with the progress of post-transcriptional gene silencing, and in particular of interfering RNA (interfering RNA or RNAi), tools have been developed that allow specific inhibition of gene expression. The inhibition of the expression of the SIDT1 protein would therefore constitute the inhibition of its biological activity, and in particular, of the activity that is contributing to the aggravation of the inflammatory or autoimmune characteristics mediated by IFN.

By "antisense polynucleotides" are meant ribonucleotide or deoxyribonucleotide chains that can inhibit the production of the SIDT1 protein by one of these three mechanisms:

1- Interfering transcription by hybridizing in the structural gene or in a regulatory region of the gene encoding SIDT1. Since transcription or expression is effectively blocked by hybridization of the antisense oligonucleotide with DNA, the production of SIDT1 decreases. 2- The binding of the antisense oligonucleotide in the cytoplasm with the mRNA, interfering with the formation of the translation construct itself, inhibiting the translation of mRNA into the protein.

3- The formation of a duplex antisense mRNA that allows rapid degradation of the duplex mRNA by RNases (such as RNase H). This results in a lower production of SIDT1.

Antisense oligonucleotides capable of modulating the activity of SIDT1 can be easily synthesized from the known sequence SEQ ID NO: 2 by applying knowledge of the state of the art. For example, and without limiting ourselves, it could be a sequence of ribonucleotides or RNA that belongs to the so-called siRNA (small interfering RNA), small interfering RNA or silencing RNA, capable of inhibiting the genetic expression of the SIDT1 protein. In the context of the present specification, "siRNA" (small interfering RNA or small interfering RNA) is understood to be a class of double stranded RNA 19 to 25 nucleotides long, and more preferably between 21 and 23 nucleotides, which is involved in the route of RNA interference, where siRNA interferes with the expression of a specific gene. In the present invention, this specific gene is SIDT1. It could also be any siRNA capable of hybridizing a nucleic acid molecule encoding the human SIDT1 protein that is collected in SEQ ID NO: 1. They could also be an RNA construct that contains at least any one of the possible nucleotide sequences of siRNA capable of inhibiting the expression of SIDT1, and without prejudice to the addition of any of the RNA sequences and constructions of the invention described above that are subject to modifications, preferably chemical, leading to greater stability against the action of ribonucleases. and with it to greater efficiency. Without these modifications involving the alteration of its mechanism of action, which is the specific binding to the RISC complex (RNA-induced silencing complex), activating it and manifesting a helicase activity that separates the two strands leaving only the antisense strand associated with the complex. The resulting ribonucleoproteic complex binds to the target mRNA (messenger RNA of SIDT1, which is collected in SEQ ID NO: 2). If the complementarity is not perfect, RISC is associated with the messenger and the translation is attenuated. But if it is perfect, RISC acts as RNasa, cutting the messenger and being free to repeat the process. Additionally, it is apparent to one skilled in the art that a large number of mRNA polynucleotides can be translated into SIDT1 as a consequence, for example, that the genetic code is degenerated. Any siRNA capable of inhibiting the translation of these mRNAs is also part of the invention.

The preparation of the siRNA sequence of the invention or of the RNA construct of the invention would be apparent to one skilled in the art, and could be carried out by chemical synthesis, which also allows the incorporation of chemical modifications in both different nucleotides of the product such as the incorporation of other chemical compounds at any of the ends. On the other hand, the synthesis could also be carried out enzymatically using any of the available RNA polymerases. Enzymatic synthesis also allows some chemical modification of inhibitor products or RNAs.

The nucleotide sequence design of the siRNA of the invention would also be apparent to one skilled in the art. Thus, it could be done through a random design in which 19-25 bases of the target mRNA are selected without taking into account the sequence or positional information it has in the transcript. Another non-limiting alternative of the present invention would be the conventional design by simple parameters developed by the pioneers of the art (Calipel, A. et al., 2003. J Biol Chem. 278 (43): 42409-42418) completed with an analysis BLAST nucleotide. Another possibility could be a rational design, in which a computer procedure is used to identify the optimal siRNA targets in an mRNA. The target sequences are analyzed in groups of 19 nucleotides at a time and those with the best characteristics are identified based on an algorithm that incorporates a large number of thermodynamic and sequence parameters. A genetic DNA construct could also be part of the composition of the invention, which would direct the in vitro or intracellular transcription of the siRNA sequence or RNA construct of the invention, and comprising at least one of the following types of sequences: a) DNA nucleotide sequence, preferably double stranded, comprising at least the sequence encoding the siRNA of the invention or the RNA construct of the invention for transcription, or, b) nucleotide sequence of DNA, preferably double stranded, corresponding to a gene expression system or vector comprising the sequence coding for the RNA sequence of the invention operably linked with at least one promoter that directs the transcription of said nucleotide sequence of interest, and with other sequences necessary or appropriate for transcription and its appropriate regulation in time and place, for example, start signals and termination, cutting sites, polyadenylation signal, origin of replication, transcriptional activators (enhancers), transcriptional silencers (silencers), etc. for use in those pathological contexts in which IFN levels are altered, and preferably, type I and III IFNs.

SIDT1 is contributing to the worsening of inflammatory and / or autoimmune characteristics and to the onset of the disease. Multiple of these constructs, systems or expression vectors can be obtained by conventional methods known to those skilled in the art (Sambrook et al. 2001. Molecular Cloning: A laboratory Manual. Coid Spring Harbor laboratory Press, New York)

The compositions of the present invention allow the transfection of the siRNA of the invention into a cell, in vivo or in vitro. Transfection could be carried out, but not limited to, direct transfection or vectors that facilitate the access of siRNA into the cell. Thus, examples of these vectors are, without limitation, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, Herpes simplex viruses, non-viral DNA plasmids, cationic liposomes and molecular conjugates. Thus, for example, the siRNAs of the present invention, as well as RNA or DNA precursors of these siRNAs, can be conjugated with release peptides or other compounds to favor the transport of these siRNAs into the cell.

Nucleases designed to block or activate gene expression are also part of the invention.

The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen binding site that specifically binds (immunoreacts with) the protein. SIDT1. Examples of portions of immunologically active immunoglobulin molecules, include F (ab) and F (ab ') 2 fragments that can be generated by treating the antibody with an enzyme such as pepsin. It can be a monoclonal or polyclonal antibody.

Antibodies capable of binding to the SIDT1 protein can be used to inhibit the activity of said protein. Many such antibodies are commercially available. The antibodies, or fragments thereof, may be able to inhibit the activity of the SIDT1 protein that contributes to the acquisition of the characteristics of inflammatory and / or autoimmune diseases. The antibodies can be polyclonal (typically include different antibodies directed against different determinants or epitopes) or monoclonal (directed against a single determinant in the antigen). The monoclonal antibody can be biochemically altered, by genetic manipulation, or it can be synthetic, possibly lacking the antibody in whole or in parts, of portions that are not necessary for the recognition of SIDT1 and being replaced by others that communicate to the antibody additional advantageous properties. The antibody can also be recombinant, chimeric, humanized, synthetic or a combination of any of the foregoing.

A "recombinant antibody or polypeptide" (rAC) is one that has been produced in a host cell that has been transformed or transfected with the nucleic acid encoding the polypeptide, or produces the polypeptide as a result of homologous recombination.

These rAc can be expressed and directed to specific cellular sub-compartments when the appropriate sequences for intracellular traffic are incorporated. These antibodies are called intrabodies, and have proven effective not only to divert proteins from their usual compartment or block interactions between proteins involved in signaling pathways, but also to activate intracellular proteins.

Also part of the invention are genetic DNA constructs capable of transcribing to a peptide, antibody or antibody fragment, for use in the treatment, improvement or prevention of infectious, inflammatory or autoimmune diseases mediated by IFN. Said genetic construction of DNA would direct the in vitro or intracellular transcription of the antibody sequence or fragment thereof, and comprises at least one of the following types of sequences: a) DNA nucleotide sequence, preferably double stranded, which It comprises, at least, the coding sequence of the antibody of the invention or of the antibody fragment of the invention for in vitro, or intracellular, transcription, b) DNA nucleotide sequence, preferably double stranded, corresponding to a system or vector of gene expression comprising the sequence coding for the antibody sequence or antibody fragment of the invention operably linked with at least one promoter that directs the transcription of said nucleotide sequence of interest, and with other sequences necessary or appropriate for transcription and its proper regulation in time and place, for example, start and end signals, cut sites, polyadenylation signal, origin of replication, transcriptional activators (enhancers), transcriptional silencers (silencers), etc. for use in those pathological contexts that pass with IFN release, preferably type I and III. A "ribozyme" as understood in the present invention, refers to a catalytic polynucleotide (typically RNA), which can be constructed to specifically recognize, by hybridization, an mRNA and fragment it or eliminate its expression. Ribozymes can be introduced into the cell as catalytic RNA molecules or as genetic constructs that are expressed to RNA catalytic molecules. The term "autoimmune disease" refers to a condition in a subject characterized by a cellular, tissue and / or organ injury caused by an immunological reaction of the subject to its own cells, tissues and / or organs. Illustrative, non-limiting examples of autoimmune diseases include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, Addison autoimmune disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune ooforitis and orchitis, autoimmune thrombocytopenia, autoimmune disease Behcet, bullous pemphigoid, cardiomyopathy, celiac disease, dermatitis, chronic fatigue, immune dysfunction syndrome (CFS), chronic inflammatory demyelinating polyneuropathy, Churg - Strauss syndrome, scarring penfigoid, CREST syndrome, cryogglutinin syndrome, discoid lupus , essential mixed cryoglobulinemia, fibromyalgia-fibromiositis, glomerulonephritis, Graves disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, dise Meiere de Meniere, mixed connective tissue disease, multiple sclerosis, immunomediated or type 1 diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammyelitis , primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, sarcoidosis, scleroderma, progressive systemic sclerosis, Sjógren's syndrome, Goodpasture's syndrome, rigid man's syndrome, lupus erythematosus, lupus erythematosus , Takayasu arteritis, temporal arteristis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis such as vasculitis, herpetiform dermatitis, vitiligo, Wegener granulomatosis, Glomerular basement membrane disease, antiphospholipid syndrome, Autoimmune diseases of the Mediterranean System, fever family, the myasthenic syndrome of Lambert-Eaton, sympathetic ophthalmia, polyocrinopathies, psoriasis, etc. In a particular embodiment, said autoimmune disease is systemic lupus erythematosus (SLE).

The term "treatment" or "treat" in the context of this specification, means the administration of a compound or formulation according to the invention to prevent, ameliorate or eliminate the disease or one or more symptoms associated with said disease. "Treatment" also covers the prevention, improvement or elimination of the physiological sequel of the disease.

The treatment of autoimmune diseases is typically done with immunosuppressants. These are substances that decrease the immune response. They can be either exogenous, such as immunosuppressive drugs, or endogenous. Examples of immunosuppressive drugs are glucocorticoids, which decreases both the expansion of B cell clones and the synthesis of antibodies; cytostatics, which affects the proliferation of both T cells and B cells, examples of alkylating agents are cytostatic agents, antimetabolites (for example, folic acid analogs, purine analogs, pyrimidine analogs, protein synthesis inhibitors ); antibodies or drugs that act on immunophilins such as cyclosporine, tacrolimus or sirolimus. SLE is treated with immunosuppression, mainly with cyclophosphamide or corticosteroids.

In another preferred embodiment of this aspect, the composition of the invention is used for the treatment of an autoimmune disease. In a more preferred embodiment of this aspect of the invention, the autoimmune disease is selected from the list consisting of: alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, Addison autoimmune disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia , autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac disease, dermatitis, chronic fatigue, immune dysfunction syndrome (CFS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome , scarring pemphigoid, CREST syndrome, cryogglutinin disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromiositis, glomerulonephritis, Graves disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura , the neu IgA clothing, juvenile arthritis, lichen planus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, immunomediated or type 1 diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, sarcoidosis, scleroderma, progressive systemic sclerosis, Sjógren's syndrome, Goodpasture's syndrome, Goodpasture's syndrome rigid man syndrome, lupus systemic erythematosus, lupus erythematosus, Takayasu arteritis, temporal arteristis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis such as vasculitis dermatitis herpetiformis, vitiligo, Wegener granulomatosis, Glomeripulous basal membrane disease, Autophosphine syndrome Nervous system, familial Mediterranean fever, Lambert-Eaton myasthenic syndrome, sympathetic ophthalmia, polyocrinopathies and psoriasis.

In a particular embodiment of the invention, the autoimmune disease is systemic lupus erythematosus. In another particular embodiment of the invention, the autoimmune disease is psoriasis.

The composition provided by this invention may be provided by any route of administration, for which said composition will be formulated in the pharmaceutical form appropriate to the route of administration chosen. SIDT1 activity modulating agents of said compositions are in a therapeutically effective amount. In the sense used in this description, the term "therapeutically effective amount" refers to the amount of modulating agents (or genetic constructs that allow their intracellular expression) calculated to produce the desired effect and, in general, will be determined, among other causes. , due to the characteristics of said agents (and constructions) and the therapeutic effect to be achieved. Pharmaceutically acceptable adjuvants and vehicles that can be used in said compositions are the vehicles known to those skilled in the art.

SCREENING METHOD OF DRUGS OF THE INVENTION

The invention provides methods for identifying compounds that can be used for the treatment of diseases related to SIDT1, and in particular inflammatory diseases or autoimmune diseases mediated by IFN, and preferably mediated by IFN type I and III.

These methods allow the identification of candidates, compounds to be tested or agents (for example, peptides, peptidomimetics, organic molecules, antisense oligonucleotides or other molecules) that can bind SIDT1 and / or have an activating or inhibiting effect of the biological activity of SIDT1 or of its expression, and thus determine whether those compounds would have an effect on the diseases in which SIDT1 is involved, and in particular those inflammatory or autoimmune diseases mediated by IFN.

Assays to identify these molecules, compounds or agents that modulate the activity of SIDT1 may employ cells expressing SIDT1, or in assays with isolated SIDT1 (or with its variants, such as biologically active fragments or fusion proteins that include a portion or part of YES DT1). Thus, another aspect of the invention consists in a method of selecting therapeutic agents useful in the treatment of inflammatory or autoimmune diseases mediated by IFN, and more specifically for the treatment of psoriasis and lupus; which comprises: a) contacting the compound to be analyzed with the SIDT1 polypeptide, and b) detecting the binding of said compound to be analyzed with the SIDT1 polypeptide.

Compounds that bind to the SIDT1 polypeptide would be identified as potential therapeutic agents against inflammatory or autoimmune diseases mediated by IFN, and more specifically for the treatment of psoriasis and lupus.

As stated, these assays may involve the complete SIDT1 polypeptide, a biologically active fragment thereof, or a fusion protein that involves all or a portion of the SIDT1 polypeptide. Determining the ability of a compound to modulate the activity of SIDT1 can be performed, for example, by determining the ability of SIDT1 to bind or interact with a target molecule of said compound, directly or indirectly. They can also be activity tests, directly or indirectly measuring the activity of SIDT1. It can also be an expression assay, directly or indirectly determining the expression of the SIDT1 mRNA or SIDT1 protein. These tests can also be combined with an in vivo test by measuring the effect of a test compound on the symptoms of SIDT1-related diseases, and in particular those inflammatory or autoimmune diseases mediated by IFN, and preferably mediated by IFN type I and III (by example, but not limited, on animal models or other model systems known in the art).

The compounds to be tested used in the method of selection of therapeutic agents are not limited to low molecular weight organic molecules, proteins (including antibodies), peptides, oliogonucleotides, etc. They can be natural and / or synthetic. For example, antibodies capable of binding to an epitope of SIDT1, which can be used therapeutically, as discussed above, can also be used in immunohistochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunoprecipitation assays, or other immunohistochemical assays known in The state of the art. SIDT1 can be used to immunize an animal, to obtain polyclonal antibodies. Monoclonal antibodies can also be prepared by techniques that allow the production of antibodies by cultured cell lines, including, but not limited to, hybridomas, human B-cell hybridomas or collections of nanobodies. Techniques for producing chimeric, humanized or synthetic antibodies are known. The therapeutic agents identified by the selection method described herein can be used in an animal or other model to determine the mechanism of action of said agent. Moreover, the therapeutic agents selected by the method described herein would be used in the treatment of diseases that occur with the alteration of SIDT1 and, in particular, IFN-mediated inflammatory or autoimmune diseases, preferably by type I and III IFNs, and more specifically psoriasis and lupus.

In another aspect of the invention a method of selection of therapeutic agents useful in the treatment of inflammatory diseases or autoimmune diseases mediated by IFN, preferably by type I and III IFN, and more specifically for the treatment of psoriasis and lupus, is described. which comprises: a) determining the activity of SIDT1 at an established concentration of the compound to be analyzed or in the absence of said compound, b) determining the activity of SIDT1 at a concentration of the compound to be analyzed different from that of a). Compounds that give rise to a different activity of SIDT1 would be identified as potential therapeutic agents against inflammatory or autoimmune diseases mediated by IFN, preferably by type I and III IFN, and more specifically for psoriasis and lupus.

Another aspect of the invention consists in a method of selecting therapeutic agents useful in the treatment of inflammatory or autoimmune diseases mediated by IFN, preferably by type I and III IFN, and more specifically psoriasis and lupus, comprising: a ) contacting the compound to be analyzed with the SIDT1 polynucleotide, b) detecting the binding of said compound to be analyzed with the SIDT1 polynucleotide

Compounds that bind to the SIDT1 polynucleotide would be identified as potential therapeutic agents against inflammatory or autoimmune diseases mediated by IFN, preferably by type I and III IFN, and more specifically from psoriasis and lupus.

METHOD OF DIAGNOSIS OF THE INVENTION

Diseases in which the alteration of the activity of SIDT1 can be diagnostic, and in particular inflammatory diseases or autoimmune mediated by IFN, and more specifically psoriasis and lupus, can be detected by measuring the amount of nucleic acids (DNA and / or RNA and / or mRNA) encoding SIDT1, or the amount of SIDT1 protein that is expressed, compared to normal cells. Oligonucleotide detection it can be done by methods well known in the state of the art (such as, but not limited to, probes with labeled nucleotides, DNA-DNA or DNA-RNA hybridization, PCR amplification using labeled nucleotides, RT-PCR). Methods for detecting the expression of the SIDT1 protein are also well known in the state of the art, such as, but not limited to, poly or monoclonal antibodies, ELISA, radioimmunoassay (RIA), and FACS (fluorescence activated cell sorting).

Therefore, in another aspect of the invention a method is described for the collection of data useful in the diagnosis and / or prognosis of inflammatory or autoimmune diseases mediated by IFN, and more specifically psoriasis and lupus, comprising: a) determine the expression of SIDT1 in a sample taken from a mammal, b) compare the values of the expression of SIDT1 obtained in a) with the standard values in healthy or diseased mammals.

IFN-mediated inflammatory or autoimmune diseases include, but are not limited to, systemic lupus erythematosus, systemic sclerosis, psoriasis, Sjógren syndrome, rheumatoid arthritis, Accardi-Goutiéres syndrome, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, antiphospholipid syndrome Addison autoimmune disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac disease, dermatitis, chronic fatigue, dysfunction syndrome Immune (CFS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, scarring penfigoid, CREST syndrome, cryoagglutinin disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromiositis, glomerulonephritis, Graves disease, syndrome Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, immunomediated diabetes mellitus or type 1 Myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaiter's phenomenon, Raynaiter's phenomenon sarcoidosis, scleroderma, progressive systemic sclerosis, Goodpasture syndrome, rigid man syndrome, systemic lupus erythematosus, lupus erythematosus, Takayasu arteritis, temporal arteristis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis such as vasculitis dermatitis herpetiformis, vitiligo, W granulomatosis egener, Glomerular basement membrane disease, antiphospholipid syndrome, Autoimmune Diseases of the Nervous System, family Mediterranean fever, Lambert-Eaton myasthenic syndrome, sympathetic ophthalmia and polyocrinopathies. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein, referring to polymeric forms of nucleotides of any length, both ribonucleotides and deoxyribonucleotides.

The terms "peptide", "oligopeptide", "polypeptide" and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which may be coding or non-coding, chemically or biochemically modified.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Fig. 1. Expression of the SIDT1 gene in several cell lines. A) Measurement of the abundance of the SIDT1 mRNA in different cell lines using qRT-PCR: Hela and JEK-293, epithelial cells; K562: granulocytic / pre-erythrocytic series; THP-1: monocytes; HL-60: promyloblast; daudi, bouquets, RL, raji, namalwa: B lymphocyte; Jurkat: T lymphocyte; NKL: NK cells; Gen2.2: pDC. B) Overexpression of SIDT1 in the Gen2.2 cell line after stimulation with TLR9 (ODN2006) and TKR7 (imiquimod) ligands measured by qRT-PCR. The expression of the SIDT1 gene is indicated using as reference the expression of the HPRT1 gene.

Fig. 2. Negatively regulated genes in Gen2.2 cells silenced for SIDT1, after stimulation with ODN2006 for 6h. The expression of the different genes was carried out by means of expression arrays. The table shows the relative expression in the silenced cells versus the cells treated with a control shRNA, without target. The table shows the first 50 genes sorted by importance of effect.

Fig. 3. Production of pro-inflammatory cytokines in Gen2.2 shSIDTI cells after stimulation with TLR ligands) (ODN2006) and TLR7 (Imiquimod). The cells were stimulated for 24h with ODN2006 or imiquimod and the amount of TNFa, IL-6 and IL-8 in the supernatant was measured by ELISA. As a control, non-transduced cells and transduced cells with a control shRNA were included. Fig. 4. Production of IFN pathway molecules in silenced gen2.2 cells for stimulated SIDT1. The gen2.2 shSIDTI cells and their controls were stimulated as in Figure 3 and the production of interferons of type I (IFNal) and III (IFNI1) and of the IFN-dependent chemokine CCL3 was measured by ELISA. Fig. 5. Partial inhibition of oligonucleotide entry into the cell by silencing SIDT1. The gen2.2 shSIDTI cells were incubated with the TLR9 stimulating oligonucleotide labeled ODN2006-Cy5 and their entry into the cell was measured by flow cytometry. As a control, non-transduced cells and transduced cells with a control shRNA were included. Fig. 6. SIDT1 silencing blocks the production of type I IFN by pDC isolated ex vivo in response to ODN2006. A) Measurement of SIDT1 gene expression in circulating pDCs transduced with shSIDTI lentiviral vectors The pDCs were isolated from blood from healthy donors by immunomagnetic methods. Expression was measured by branch-DNA techniques, using as HMBS control RNA. B) Production of type I IFN in the silenced pDCs after stimulation with ODN2006 ex vivo. The concentration was determined by the use of indicator cells after 24h of stimulation.

Fig. 7: Effect of SIDT1 silencing on type I IFN secretion after stimulation of DNA and RNA endosomal and cytosolic receptors. Gen2.2 shSIDTI cells and their controls were stimulated with ODN2006 or poly l: C in the presence or absence of lipofectamine. Type I IFN activity was measured in the supernatant after 24h of stimulation by the use of indicator cells.

Fig. 8: Phosphorylation of TBK-1 and F2a after stimulation of TLR9. Gen2.2 shSIDTI cells and their controls were stimulated with ODN2006 and phosphorylation of TBK-1 and elF2a was measured by Western blot. Actin protein was used to estimate the amount of total proteins in each of the lanes.

DETAILED EXHIBITION OF REALIZATION MODES

The authors of the present invention have verified that the SIDT1 mRNA is expressed in lymphoid-type hematopoietic cell lines (Fig. 1A): T and B lymphocytes, NK cells and plasmacytoid dendritic cells (pDC). His study focuses on pDCs, which are key cells for the organization of the immune response, because they are antigen presenting cells with a high capacity for cytokine production in response to microbial stimuli. The pDCs detect infectious agents or dead cells through exogenous nucleic acid endosomal receptors, which recognize RNA sequences (Toll-like receptor 7, TLR7), or DNA with non-methylated CpG motifs (TLR9). The pDCs are known as the natural interferon-producing cells (IFN), due to their very high capacity to produce large amounts of type I (a and β) and III (ΙΡΝλ) IFN, fundamental cytokines for controlling infection, as well as for Modulate the activity of T and B lymphocytes. Given the low frequency of circulating pDC and the difficulties for its cultivation, most of the results obtained in our study have been obtained using the Gen2.2 cell line, obtained from a patient with a Neoplasm with pDC phenotype. This cell line is currently the main cell model for the study of the biology of the pDC. According to the results, the SIDT1 mRNA is induced after stimulation with TLR7 (imiquimod) and TLR9 (ODN2006) ligands (Figure 1B) suggesting a role of SIDT1 in the response mediated by these receptors.

Expression arrays were performed using Gen2.2 cells transduced with lentiviral vectors carrying shRNA sequences specific for SIDT1, using shRNA control without target. As can be seen in Figure 2, the repressed genes in shSIDTI cells after stimulation with ODN2006 are enriched in genes related to the immune system, specifically with the IFN pathway. These results indicate that SIDT1 would play a central role in the regulation of the interferon pathway in response to stimulation of TLR9.

To verify this point, the production of cytokines in silenced Gen2.2 cells and their controls was studied, after stimulation with TLR7 (imiquimod) and TLR9 (ODN2006) ligands, finding that proinflammatory cytokine secretion (TNFa, IL-6, IL-8), is not affected (Fig. 3), but strongly the production of type I IFN (IFNal) and III (IFN 1), and CCL3, an IFN-dependent chemokine (Fig. 4). Through flow cytometry studies, the input of the ODN2006 oligonucleotide coupled to the Cy5 fluorescent marker could be quantified (Fig. 5), and a partial inhibition of the internalization of the fluorescent ligand could be observed, which is not sufficient to justify the marked effect on the IFN secretion. Finally, experiments were carried out on circulating primary pDC, obtained ex vivo by immunomagnetic methods from blood samples from healthy donors. As shown in Figure 6, the results obtained with the model cell line are confirmed using primary cells, since in pDCs silenced with shSIDTI lentiviral vectors (Fig. 6A) and stimulated with ODN2006, production inhibition was observed. of type I IFN of three logarithms of magnitude (Fig. 6B).

Example of the invention

Materials and methods Expression of SIDT1 in cell lines. The HeLa, HEK 293T, K562, THP-1, HL-60, daudi, RL, Raji, Namalwa, Jurkat, NKL, Gen2.2 cell lines were grown in RMPI medium supplemented with 10% fetal bovine serum at 37 ° C and 7% of C0 ₂ . These cell lines were used for purification of total RNA using the High Puré RNA kit (Roche). 1 μg of RNA was used as a template for cDNA synthesis using the Roche Transcriptor First-Strand cDNA kit, and then quantitative PCR experiments (qPCR) were performed using taqman probes specific to SIDT1 (Hs00214475_m1) and HPRT1 (Hs01003268_g1). As illustrated in Figure 1A, SIDT1 is preferentially expressed in lymphoid line cell lines: T and B lymphocytes, NK cells and plasmacytoid dendritic cells (pDC). Given the central role of regulating the immune system of the pDCs, this cell type was chosen as a model to study the function of SIDT1. Therefore, Gen2.2 cells were stimulated with ligands of the endosomal sensors of TLR-7 (imiquimod 3 μg / ml) and TLR-9 nucleic acids (ODN2006, 1 mM) for 24 and 48h. The results of qPCR in Figure 1 B show a strong overexpression of SIDT1 after stimulation, indicating a role of SIDT1 in the immune response mediated by these receptors.

Differential expression of genes in silenced Gen2.2 cells for SIDT1.

In order to study the role of SIDT1 in the response mediated by TLR-7 and -9 in the pDC, transductions were performed with lentiviral vectors carrying three shRNA sequences specific for SIDT1 (shRNA # 1 AGCCGTGAGGGTGTATGTGAA, (SEQ ID NO: 3 ); shRNA # 2 CTACTTGGGATGCCTTCTTGT (SEQ ID NO: 4); shRNA # 3 GTGCATTCTGCTGGATTTCTT (SEQ ID NO: 5)), cloned into the vector pLenti-H1 (AMSBIO). As a control, a lentivirus was used carrying a shRNA sequence without a target (shCTL GTCTCCACGCGCAGTACATTT (SEQ ID NO: 6)). In all cases a multiplicity of infection (moi) of 1 was used. Both Gen2.2 cells and cells transduced with The three shRNAs specific for SIDT1 (shSIDTI) as their control shRNA (shCTL) were stimulated for 6h with 1 μΜ ODN2006. RNA was purified after stimulation and was used for studies of RNA expression arrays.

Sample mapping was performed using the Ambion kit, lllumina ® TotalPrep RNA (Life Technologies), to obtain a biotin-labeled cRNA. This cRNA hybridized to the array of the human transcriptome lllumine expression array 12v4, and was subsequently revealed using streptavidin-Cy3.

The raw data were generated using the GenomeStudio software (lllumina) and subsequently analyzed in the R and Bioconductor statistical environment. The expression values of each probe were determined after correction of the background signal using the negative controls and subsequently quantile normalization was applied to eliminate technical variations. Probes with detection values p <0.01 in at least 10% of the samples were removed from the analysis and the average value of the expression of each gene was computed by applying the median to the value of all probes encoding the same gene. Differential expression analysis was carried out using the Limma package. Genes that showed a corrected p-value less than 0.05 were considered statistically significant. Figure 50 shows the 50 most repressed genes in Gen2.2 shSIDTI cells stimulated in relation to shCTL.

Functional analysis of differentially expressed genes was performed using the Genecodis tool. The annotations with corrected p-values less than 1 10 ^"4 were considered statistically enriched in the lists of inhibited genes and used to understand the biological processes underlying the experimental model. The most represented pathways correspond to the so-called" interferon signature ", characterized by genes connected with the production of type I interferons (IFNa / β) (Rónblom and Eloranta, Clin Opin Rheumatol) These data indicate the role of SIDT1 in the induction of IFN-related genes in response to stimulation of TLR-7 and -9.

Response to stimulation of TLR-7 and -9 in Gen2.2 shSIDTI.

To corroborate the result of the expression array, ODN2006 and imiquimod stimulations were performed on Gen2.2 cells and their controls for 24 hours, and several cytokines and chemokines were measured in the supernatant by ELISA assays (eBioscience), following the recommended protocol by the manufacturer. As illustrated in Figure 3, the stimulation of the production of TNFa, IL-6 and IL-8 proinflammatory cytokines is not altered by the silencing of SIDT1. However, there is a dramatic decrease in interferon type I (IFNal) and III (IFN 1), as well as CCL3 chemokine, which is regulated by IFN (Figure 4). These results confirm the differential expression data in expression arrays, and indicate a role of SIDT1 in regulating the response to stimulation of TLR-7 and -9, acting specifically in the interferon production pathway.

Partial inhibition of ODN2006 entry in Gen2.2 shSIDTI cells. Since other authors have hypothesized that SIDT1 would be a nucleic acid internalization channel, we conducted internalization studies using flow cytometry. Gen2.2 shSIDTI cells and their controls were incubated with the TLR-9 ODN2006 ligand labeled with the Cy5 fluorescent molecule, and the input was monitored by flow cytometry on a Canto FACS cytometer (BD Biosciences). As shown in Figure 5, the silencing of SIDT1 induces a decrease in the ODN2006-Cy5 input of approximately 30%, which is not sufficient to justify the drastic regulation of type I and III IFN, with conservation of the induction of proinflammatory cytokines. This result suggests that SIDT1 could be influencing the IFN response by a different mechanism to nucleic acid transport. SIDT1 silencing blocks the production of type I IFN in ex vivo primary pDCs.

Circulating pDCs were isolated from blood units from healthy donors using the BDCA-4 Positive Selection Kit in an Automacs Pro separator (both from Miltenyi Biotec), following the manufacturer's instructions. In all cases, pDCs with more than 90% purity were obtained. The purified pDCs were transduced with the shSIDTI or shCTL lentiviruses at a moi of 0.4 and were stimulated with 1 M ODN2006 in RPMI medium supplemented with 5% autologous inactivated plasma for 24 h. SIDT1 expression was quantified using the Quantigene Assay Kit (Panomics / Affimetrix) and the Glomax Multidetector System (Promega) luminometer, following the manufacturers instructions. Under these conditions, a decrease in SIDT1 expression was observed in the silenced cells of 50% (Figure 6A). Type I IFN of the supernatant was quantified using IFNa / β HEK-Blue IFNa / β (Invivogen) indicating cells, following the manufacturer's recommendations. As seen in Figure 6B, the silencing of SIDT1 in the primary pDC induces a decrease in IFNa / β production of three orders of magnitude.

These results using ex vivo primary pDC confirm those obtained with the model cell line and demonstrate that SIDT1 is a fundamental mediator for the induction of an interferon response after stimulation of the endosomal receptors of TLR-7 and -9 nucleic acids. Determination of the role of SIDT1 in endosomal nucleic acid receptors and in the IFN response mediated by cytosolic receptors

To determine whether the role of SIDT1 is limited to endosomal nucleic acid receptors, or on the contrary is also involved in the IFN response mediated by cytosolic receptors, stimulations with ODN2006 and poly l: C encapsulated in liposomes were performed through use. of lipofectamine, to allow the access of the ligands to the cytosol. As shown in Figure 7, Gen2.2 cells silenced for SIDT1 also showed an altered response after stimulation of cytosolic DNA receptors with CpG + lipofectamine. However, the IFN response mediated by cytosolic RNA receptors (poly 1: C + lipofectamine) was not altered in shSIDTI cells, which SIDT1 is not a general mediator of the IFN response to exogenous nucleic acids, but is a selective mediator of certain recognition pathways in the pDC.

Several key molecules have been identified in signal transduction that lead to the induction of an IFN response after stimulation of TLR9. Two of them were selected for the study of the role of SIDT1 through Western blot studies: TBK-1 activates the phosphorylation of different members of the IRF family, allowing its translocation to the nucleus to activate IFN signature genes, and therefore It is essential for the development of a complete response mediated by TLR9. On the other hand, it has been shown that the translation initiation factor of mRNA elF2a is involved in the response to stimulation of TLR9 and TLR3. As reflected in Figure 8, stimulation with ODN2006 induces phosphorylation of TBK-1 and elF2a. However, shSIDI cells phosphorylation of TBK1 and elF2a molecules is poor. These results suggest that SIDT1 is essential for the establishment of an optimal signal transduction cascade for the induction of type I and III interferons after stimulation of TLR7, TLR9 and cytosolic DNA receptors.

Claims

1.- The use of a composition comprising an agent modulating the activity of SIDT1 in the preparation of a medicament for the treatment, improvement or prevention of infectious, inflammatory or autoimmune diseases mediated by IFN.

2. - The use of a composition according to claim 1, wherein the modulating agents are selected from the list consisting of: a) a small molecule, b) an RNA molecule, c) an antisense oligonucleotide, d) an antibody, e) a ribozyme, f) an endonuclease capable of gene editing, or any combination thereof.

3. - The use of a composition according to any of claims 1-2, wherein the modulating agents are selected from the list consisting of: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID

NO: 5, or any of its combinations.

4. - The use of a composition according to any of claims 1 -3, wherein the IFN-mediated inflammatory or autoimmune disease is selected from the list comprising: alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, Addison autoimmune disease, diseases Autoimmune of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac disease, dermatitis, chronic fatigue, immune dysfunction syndrome (CFS), Chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, scarring pemphigoid, CREST syndrome, cryogglutinin disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromiositis, glomerulonephritis, Graves disease, Guillain-Barreim syndrome, thyroiditis, thyroiditis idiopathic pulmonary fibrosis, purpur to idiopathic thrombocytopenic (PTI), IgA neuropathy, juvenile arthritis, rheumatoid arthritis, lichen planus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, immunomediated or type 1 diabetes, myasthenia gravis, pemphigus vulgaris, anemia pernicious, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, Primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, sarcoidosis, scleroderma, progressive systemic sclerosis, Sjógren's syndrome, Goodpasture's syndrome, rigid man's syndrome, systemic lupus erythematosus, lupus erythematosus, Takayasu arteritis, temporal arteristis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis such as vasculitis, herpetiform dermatitis, vitiligo, Wegener granulomatosis, Glomerular basement membrane disease, antiphospholipid syndrome, Autoimmune diseases of the Nervous System, Mediterranean fever , the Lambert-Eaton myasthenic syndrome, sympathetic ophthalmia, polyocrinopathies, psoriasis, or any combination thereof.

5. The use of a composition according to any of claims 1-4, wherein the inflammatory or autoimmune disease mediated by IFN is systemic lupus erythematosus (SLE).

6. The use of a composition according to any of claims 1-4, wherein the inflammatory or autoimmune disease mediated by IFN is psoriasis.

7. A method of selecting therapeutic agents useful in the treatment of infectious, inflammatory or autoimmune diseases mediated by IFN, comprising: a) contacting the compound to be analyzed with the SIDT1 polypeptide, and b) detecting the binding of said compound to be analyzed with the SIDT1 polypeptide.

8. A method of selecting therapeutic agents useful in the treatment of infectious, inflammatory or autoimmune diseases mediated by IFN, comprising: a) determining the activity of SIDT1 at a certain concentration of the compound to be analyzed or in the absence of said compound, b ) determine the activity of SIDT1 at a concentration of the compound to be analyzed different from that of a).

9. A method of selecting therapeutic agents useful in the treatment of viral, inflammatory or autoimmune diseases mediated by IFN, comprising: a) determining the activity of SIDT1 at a certain concentration of the compound to be analyzed, b) determining the activity of SIDT1 in the presence of a compound that is known to modulate the activity of SIDT1.

10. A method of selecting therapeutic agents useful in the treatment of infectious, inflammatory or autoimmune diseases mediated by IFN, comprising: a) contacting the compound to be analyzed with the SIDT1 polynucleotide, b) detecting the binding of said compound to be analyzed with the SIDT1 polynucleotide.

1 1. A method for obtaining useful data in the diagnosis and / or prognosis and / or response to the treatment of viral, inflammatory or autoimmune diseases mediated by IFN, comprising: a) determining the expression of SIDT1 in an extracted sample of a mammal, b) compare the values of SIDT1 expression obtained in a) with the standard values in healthy or diseased mammals.