WO2021016474A1 - Ibd therapy by inhibition of drd3 in regulatory t cells - Google Patents

Abstract

A method for treating gut inflammation in a mammal by administering to the mammal a pharmaceutical composition having one or more agents that inhibit, attenuate, disrupt and/or block the biological activity of DRD3 in the mammal. Also, an agent that inhibits, attenuates, disrupts and/or blocks the biological activity of DRD3 specifically expressed in regulatory CD4+ T-Cells.

Description

IBD THERAPY BY INHIBITION OF I⁾RD3 IN REGULATORY T CELLS

CROSS REFERENCE TO RELATED APPLICATION

The present application claims priority under 35 IISC 1 19(e) to LIS. .Patent Application. No.

62/877720 filed 23 July 2019, the disclosure of which is incorporated by reference herein in its entirety.

FIELD 0E THE INVENTION

The present invention provides a method and compositions for suppress and/or inhibiting intestinal inflammation, particularly inflammatory Bowel Diseases (IBD). The methods and compositions covered for tins invention, comprise the therapeutic use of agents that inhibit the biological activity of the dopamine D3 receptor (DRD3) in the domain of IBD. The methods and compositions comprised for this invention, arc useful for suppress and/or inhibiting the DRD3 biological activity specifically expressed in CD4+ T-Cells, particularly in the subpopulatian of regulatory CD4 " T-eeUs (Treg), thus attenuating the development of IBD. Then, one of the objects of the present invention is to propose the use of agents that inhibit the biological activity of DRD3 as useful in the preparation of a pharmaceutical composition for the treatment of IBD, particularly Crohn's disease (CD) and ulcerative colitis (UC), and more particularly for UC. Further, the present invention will be an effective treatment for patient with IBD, as for example, CD and UC, This invention also comprises therapeutic kits, that comprise DRD3 inhibitors, useful for the IBD treatment, particularly CD and UC.

BACKGROU D OF THE INVENTION,

Inflammatory bowel diseases (1BDs) form a group of chronic remittent inflammatory aff ctions of the gastrointestinal tract, among which Crohn's disease (CD) and ulcerative colitis (UC) are the most common. The overall IBD prevalence approximates 500-900 eases per 100,000 individuals, and has shown a marked increase daring the last decades. Evidence from inflammatory colitis ouse models and from samples obtained from UC and Crohn’s disease (CD) patients has indicated that gut inflammation in IBD is driven mainly by the inflammatory effector CD4^" T-cell subsets T-hdper 1 (Thl ) and I^'M? (Olson ei at., 2011). On the other hand, regulatory€D4 ^: T-eslls (Treg). a suppressive subset of lymphocytes that plays a crucial role in mainlining intestinal homeostasis in healthy conditions, seems to he dysfunctional in IBD (Wu ei al., 2014). The transfer of exogenous Treg cells can suppress inflammation induced by Thl and Thl 7 lymphocytes in mouse models of inflammatory colitis (Haber et al., 2004); and the main suppressive mechanisms rely on IL- I O and TGF-b secretion by these cells, in humans, Treg cells have been shown to be increased in the inflamed lamina propria of CD asid UC patients in comparison to nan-inflamed mucosa and mucosa from healthy controls, and l after isolation they retain their ability to suppress effector T-odls in vitro (Maul et al, 2005), thus suggesting that suppressive activity of Treg could be attenuated just in situ b mediators produced by the inflamed gut mucosa.

Cells residing in the gut may encounter dopamine, which can be produced from different sources, itcludingfhe intrinsic enteric nervous system, the intestinal epithelial layer (Pacheco et al, 2014), some components of the gut mierobiota (Clark and Mach, 2016), and certain immune cells, including dendritic cells and Treg (Cosentino et ah, 2007; Prado et al, 2012). Interestingly, inflamed gut mucosa from CD and UC patien ts involves a marked decrease of dopamine levels (Magro et ai, 2002), which may affect fee function of immune cells expressing dopamine receptors (DRs), including Treg and effector T-ed!s. importan tly, reduced levels of intestinal dopamine have been also observed in inflamed gut mucosa using animal models of inflammatory colitis (Magro et a!. , 2004; Pacheco et. al., 2014).

Dopamine exerts its effects by stimulating DRs, termed DRD1 -DRD5; ail of them belonging to the supc amAy ofG-protein coupled receptors. All these receptors have been found in CD4 ^! T-cells from human and mouse origin. If is important to consider feat each DR displays different affinities for dopamine: DRD3 > DRD5 > DRD4 > DRD2 > DRDI (KifoM) = 27, 228, 450, 1705, 2340, respectively), thereby their functional relevance depends on dopamine levels (Pacheco et al., 2014). Regarding the role of DRs expressed in CD4 ^: T celi§ upon inflammation, our recen t study showed that /.⁾«l?~defseient naive€D4⁺ T-cefts display impaired Thl differentiation and reduced expansion of Th 7 cells and consequently an attenuated manifestation of inflammatory colitis (Contrera et al, 2016). Taking into account die reduction in intestinal dopamine levels («1000 u in healthy individuals; ¾10⁽⁾ o.M in CD and UC patients) (Asano et al, 201.2; Magro et al., 2002) and the fact that DRD3 may be selectively stimulated at low dopamine concentrations, these results suggest that low dopamine levels present in the inflamed gut mucosa favour the inflammatory potential of CD4⁺ T-cells, thus promoting chronic inflammation. In the same direction, our previous work has shown that ib¾/.?~defieieuey results in atenuated inflammatory colitis in mouse (Contreras et ai, 2016).

On the other hand, emerging e i ence indicates t t high dopamine levels exert a strong anti inflammatory effect in several animal models of inflammation by stimulating low-affinity dopamine receptors, including DRD1 and ORD2 (Pacheco, 2017). In this regard, high dopamine concentrations in the gut of healthy individuals would stimulate DRD i in macrophages, attenuating the activation of the flammasome NLRP3 and thereby abrogating the production of inflammatory cytokines. Moreover, high dopamine le vels would promote DM>2 -stimulation, favouring the production of the anti-inflammatory cytokine IL-10 by CD4 T-ce!ls and suppressing both increased motility and ulcer development (Pacheco et ah, 2014). Indeed, a genetic polymorphism of DRD2 gene, which results in decreased receptor expression, has been reported as a risk factor for I8D (Magro et a!., 2006). Accordingly, although ihe frequency of Trg cells was not changed is the gat. suppressor function of intestinal Treg cells was compromised in inflammatory colitis (Wu et ai._s 2014), a condition associated to decreased dopamine levels (Msgxo el ah, 2004). Interestingly, the impairment of suppressive Treg function. was abolished by tie administration of cabergohue, a DRD2 agonist (Wu et al., 2014).

Taken together these findings suggest that, whereas D.RD2~signalMng in Treg cells promotes suppressive function in a healthy gut mucosa containing high dopamine levels, the selective DRD3- signalling in Treg impairs the suppressive activity in the inflamed gut mucosa containing low dopamine levels.

Current treatments for IBB

Actually available treatments for 1BD -consisting mainly of CI> and BC-. are general ahti-inilammatory drugs, which inhibit the immune response in general, including azathioprine (Az&san, Imuran), mereaptopuriue (Purinethol, Purixan), cyclosporine (Gengraf, Neoral, Sandimmune) and methotrexate (Trexali).

There are also biological therapies, most of them focused on neutralizing the action of TNFux. such as infliximab (Remicade), adalimumab (Humira) and goHmumab (Simpom) Other biological therapies currently used inhibit the action of the « integral subunit fr aliaoniah, Tysabri), «4(37 integ in (vedolizumab, Entyvio), 1L-I2 and II, -23 preventing binding to its IL-l 2R(fl receptor (ustekkmmah; Stelara),

in general, the limitations of the drugs currently used for the treatment of intestinal inflammatory diseases is the non -spec ill city, which inhibits the pathological inflammatory response, but also inhibits the beneficial immune responses that defend us against infectious pathogens and against the development of tumors.

The biological therapies mentioned above also involve die problem of non-specificity In different degrees; in this regard, TNF-«, 1L- 12 and IL-23 are inflammatory cytokines Invol ved i a general way in the pathogenic inflammatory immune responses, but also in immune responses beneficial tor the organism. With respect to integrin-b!ocking antibodies, these have a greater degree of specificity since they affect the entry of inflammatory cells into specific tissues. With respect to this, the blockade of «4 by natali mah inhibits the infiltration of lymphocytes both in the central nervous system (blocking a4b! ) and in the intestinal mucosa (blocking a4b7). In the case of vedoliau ab, blockade of a4b7 allows a specific inhibition of tire recruitment of rnllammaiory cells in the intestinal mucosa. However, this dreg, besides inhibiting the entry of inflammatory T cells into the intestinal mucosa, also inhibits the infiltration of regulatory T cells (Tregs) in this tissue, which carry out an immunosuppressive function and therefore beneficial in the context of intestinal inflammatory diseases. On the other hand, blockade of 7 edo! smah prevents the entry of T cells into both the colonic mucosa and the mucosa of the small intestine, the sits of elimination of inflammatory cells.

Considering all these evidences, we developed realization examples for the present invention which looking for the role of DRD3".tignall ng hr the function of Treg cells using an animal model of inflammatory colitis. Particularly, obtained experimental results revealed that DRD3-sigoalling in Treg Ceils atenuates suppressive activity and strongly reduces the gut tropism of these ceils. Accordingly, .OriB-defident mice were refractory to the development of ftfiatnMatOry colitis and the inhibition of DRBS-sigaallmg in Treg cells induced a potent anti-inflammatory effect with therapeutic effect in gut inflammation.

In this context, to solve the technical problem comisting in the absence of effective IBD therapies, and also the non-specificity of actually available IBD treatments, the present invention discloses methods and compositions useful for an effective IBD therapy, with high degree of specificity, based on the inhibition the DRD3 biological activity specifically expressed in CD4+ T-Cells. particularly in the subpopu!atioa of Tregs, thus attenuating the development of IBD. Therefore, tbs present invention provides new solutions for the design of improved therapeutic and preventive strategies against IBD, with greater level of effectivity and specificity.

BRIEF DESCRIPTION O THE INVENTION

The present invention provides therapeutic methods and pharmaceutical compositions useful for the treatment of intestinal inflammation, particularly useful for the treatment of Inflammatory Bowel Diseases (IBD), and more particularly useful for t e treatment of Crohn disease (CD) and ulcerative colitis (UC), and even more particularly useful for ulcerative colitis.

Any of the methods and compositions comprised by this invention, and characterized in the embodiments indicated below, may be useful for the effective treatment of intestinal inflammation, particularly for the treatment of IBD, particularly for CD and DC, and even more particularly for UC. in a particular embodiment, the methods and compositions useful for the treatment of IBD comprised by this invention, are useful tor blocking or attenuating or inhibiting the biological activity triggered or mediated by DRD3 in regulatory CD4+ T-Cells (Treg),

Is a particular embodiment, the methods and compositions useful for the treatment of IBD comprised by this invention, are useful for blocking or attenuating or inhib ting DRD3-signalling in Treg cells.

In a particular embodiment, the methods and compositions useful for the treatment of IBD comprised by this invention, which are useful for blocking or atenuating or inhibiting DRD3 -signalling in Treg cells, induce an enhanced Treg suppressive activity. In a particular embodiment, fee methods and compositions useful for fee treatment of 1SD comprised by this invention, which are useful for blocking or attenuating or inhibiting DRD3 '-signalling in Treg cells, induce a strongly increase of the Treg got tropism.

in a particular embodiment, fee methods and compositions useful for foe treatment of IBD comprised by this invention, are for blocking or inhibiting DRD3 -signalling in Treg ceils to induced a potent antiinflammatory effect indaeing an enhanced Treg suppressive activity and a strongly increase of the Treg gut tropism.

In a particular embodiment, fee present invention comprises a method for increasing a migration of regulatory T cells into gut mucosa in a mammal, comprising the step of transducing a Treg cell of the mammal with a retroviral vector containing a nucleic acid encoding an U A product that reduces Drd3 transcription in the mammal.

in a particular embodiment, the present invention comprises a method for increasing a migration of regulatory T cells into gut mucosa in a mammal, comprising foe ste of transducing a Treg cell of the mammal with a retroviral vector containing a nucleic acid encoding an ENA product that reduces Prd3 transcription in regulatory CD4+ T cells of the mammal.

in a particular embodiment, the present invention comprises a method for increasing regulatory T-cells gut tropism, comprising fee step of reduces Drd3 transcription in regulatory CD4T T cells of the mammal Said reduction in the transcription of Drd3 in regulatory CD4 + T cells, although it can be achieved through any of foe biotechnological tools available today ( pharmacological, biological, molecular, chemical, physical, inter alia), preferably in this invention is useful the targeting DRD3- expression on Treg cells, preferably through the generation of retroviral vectors, and even more particularly through viral vectors encoding biomo!ecules (e.g. nucleic acids and/or proteins) able of reducing die transcription ofDrdS.

In a particular embodiment, foe present invention comprises a method for inducing an enhanced Treg suppressive activity, comprising fee step of reduces rd3 transcription in regulatory CD4+ T cells of the mammal. Said reduction in the transcription of Brd3 in regulatory CD4 + T cells, although it can be achieved through any of foe biotechnological tools available today (pharmacological, biological, molecular, chemical, physical, inter alia), preferably in this invention is useful the targeting DRD3- expression on Treg cells, preferably through the generation of retroviral vectors, and even more particularly through viral vectors encoding biomo!eeoles (e.g, nucleic acids and/or proteins) able of reducing the transcription of Drd3.

in a particular embodiment, the present invention comprises a method for increasing regulatory T -ceils gut tropism and/or inducing an enhanced Treg suppressive activity, comprising the step of reduces Drd3 transcription in regulatory CD4v T ceils of the mammal. Said reduction in the transcription of Drd3 in regulatory CD4 + T ee!ls, although it can be achieved through any of the biotechnological tools available today (pharmacological biological, molecular chemical., physical, inter alia), preferably in this invention is useful the targeting BRD3-expressioo on Treg cells, preferably through die generation of retroviral vectors, and even more particularly through viral vectors encoding biomolecules (e.g. nucleic acids and/or proteins) able of reducing the transcription ofDrcB.

In a particular embodiment, the present invention comprises a method fhr increasing regulatory T~ceils gut tropism and/or inducing an enhanced Treg suppressive activity, comprising the step of blocking or attenuating or inhibiting the biological acti vity triggered or mediated by DRIB in regulatory CB4+ T* Celts (Treg) of a mammal. Said reduction in the biological activity of Dtd3 in regulatory CD4+ T-ceils, although it can be achieved through any of the biotechnological tools available today (e.g. pharmacological [e.g. PG01037], biological [e.g. shRNAj, inter alia), preferably in this invention is useful the targeting DRIB -expression on Treg cells, preferably through the generation of retroviral vectors, and even more particularly through viral vectors encoding biomoleeules (e.g. nucleic acids and/or proteins) able of reducing the transcription of ¾B and/or reducing the biological activity mediated by DRD3.

In another particular embodiment, the present invention comprises a method for attenuating got inflammation in a mammal in need thereof comprising the stop of transducing a Treg coll of the mammal with a retroviral vector containing a nucleic acid encoding an RNA product that reduces Drd3 transcription in the mammal

In another particular embodiment, the present invention comprises a method for attenuating gut inflammation in a mammal in need thereof comprising the step of introducing DrdS-defkient Treg cells into the mammal to cause an increase in migration of regulatory T cells into gut mucosa of the mammal aad/or cause an increase in Treg suppressive activity.

In another particular embodiment, the present in vention comprises any of the methods and compos? lions mentioned above useful for the treatment of gut inflammation, wherein the agent that reduces I¾d3 transcription in the mamma! is a nucleic acid encoding an RNA product, where the RNA product is an shRNA or a functionally similar nucleic acid molecule.

In another particular embodiment the present invention comprises any of the methods and compositions mentioned above useful for the treatiuent of gut inflammation, wherein the Drd3-dei ient Treg cells are introduced into the mammal intravenously.

Also this invention comprises methods fur inhibiting intestinal inflammation (e.g. IBΌ, CD, UC) in a subject comprising administering to the subject a pharmaceutical composition comprising shRNA and/or any other inhibitor or disrupter agent for blocking or inhibiting DRDS-signailing in Treg cells to induce a potent anti-inflammatory effect_* eliciting an enhanced ^"Frag suppressive activity and a strongly increase of the Treg gut trop ism. In a particular embodiment, die inhibitor or disrupter agent tor blocking or inhibiting DRD3-aigaalling in Treg cells comprised for the pharmaceutical compositions useful for the treatment of intestinal inflammation (e.g. IBIX CD. UC), is a chemical molecule or a biological molecule, as for example, peptides, antibodies_* nanobodies, aptamers, small ol cule_* antagonist, oligonucleotides and any other chemical or biological molecule with de property of blocking or inhibiting DRD3 -signalling in Treg cells to induce a poten anti-inflammatory effect, and in this way eliciting an enhanced Treg suppressive activity and/or a strongly ncrease of the Treg gat fropism.

in a particular embodiment, the inhibitor or disrupter agent for blocking or inhibiting Dl 3-signa3img in Treg cells comprised for the pharmaceutical compositions useful for the treatment of intestinal inflammation (e.g. IBD, CD, UC), la a nucleic acid, particularly as R. A, and even more particularly aa shRNA. In preferred embodiment of this invention, these nucleic a d are selected from the group of nucleic acid comprised in the Examples of this document or from other sources, which could be normal or modified nucleic acid, where in this last ease, nucleic acid could be modified at terminus and/or Internal regions, with linkers, spacers, special nucleotides, modified bonds, inter alia.

In a particular embodiment of the invention, the nucleic acids for blocking or inhibiting DRD3- signalling in Treg cells comprised for the pharmaceutical compositions useful for the treatment of intestinal inflammation (e.g, IBD, CD, UC), will generally contain phosphodiesier bonds, although in some eases, oligonucleotides probe analogs are included that may have alternate inter nucleoside linkages, comprising, but not limited to, phosphorothioate, peptide nucleic acid or PNA, phosphoiunnde, phosphorodithioa o. Other nucleic acids analogs include such as, hut not limited to, morpholine, locked oligonucleotides, peptidic nucleic acids or PNA or 2-o-(2-methoxy) ethyl modified 5^* and 3' end oligonucleotides. The nucleic acids may contain any combination of deoxyribo- and ribonucleotides, and any combination of bases. Including uracil, adenine_* thymine, cytosine, guanine, inoslne, xathanine hypoxathanine, isocytosine, isoguanine, inter alia.

In a particular embodiment, the methods and compositions useful for the treatment of IBD comprised by this invention, which are for blocking or inhibiting RD3 -signalling in Treg cells to induced a potent anti-inflammatory effect, Inducing an enhanced Treg suppressive activity and a strongly Increase of die Treg gut tropism, could be combined with any of the standard therapies actually available for the treatment of IBD.

Other preferred embodiments arc pharmaceutical composition for the treatment of IBD, which are for blocking or inhibiting DRD3-signaiiiag in Treg cells to Induced & potent anti-inflammatory effect_* inducing an enhanced Treg suppressive activity and a_: strongly increase of the Treg gut tropism, where said pharmaceutical composition comprise an of the agents with do property of blocking or inhibiting DRD3-sigrtailmg in Treg cells, and a pharmaceutically acceptable earner_* diluent or excipient. The compositions of the present invention can be utilized for therapeutics, diagnostics, prophylaxis and as research reagents an kits. Further, the present invention will be m effective treatment for patient with 18 D, as for example, Crohn Disease and Ulcerative colitis.

DEFINITIONS

IB P; is as umbrella term used to describe disorders that involve chronic irtfiammation of ie digestive tract. Types of 1BD indude: Ulcerative colitis, which is a condition causes long-lasting inflammation and sores (ulcers) in the innermost lining of your large intestine (colon) sod rectum; and Crohn’s disease, which is a type of 18 P that is characterized by inflammation of the lining of your digest? ve tract which often spreads deep into affected tissues. Both ulcerative colitis and Crohn's disease usually involve severe diarrhea_* abdominal pais, fatigue and weight loss. 1BD can be debilitating and sometimes leads to life-threatening complications.

BRIEF DESCRIPTION OF FIGURES figure L RRD3~cMIeienf mice are completely unresponsive to BBS-induced inflammatory solids. Eight-to-ten weeks old Drd3-snfieient (Drd3^<v*) and Drd3-deficient (Drd3 · ) mice were exposed to 1% DSS in the drinking water for 8 d and then switched to normal water. (A) Body weight was periodically registered throughout the whole time and the percentage of body weight change relative to initial weight was quantified and expressed as mean i: SEM. (8) On day 11, mice were sacrificed and the colon length was determined. Left panel shows representative images of a colon obtained from each experimental group. Right panel shows the quantification. Values arc cm of the whole colon expressed as mean i SEM. (€) Water consumption was monitored during three days either before or during DSS intoxication. Values are the volume of water consumed per mice. Data is expressed as mean ± SEM, (A-€) Data from 7 mice per group is shown. Two independent experiments gave similar results. *,

Mann- Whitney U-test

Figure 2. rd3-tleffieiency outside the adaptive immune system does not affect the development of DSS-iaduced inflammatory colitis, EighHo-ten week old Drrtd-suflicient (black symbols) and DrdJ-deficient (red symbols) iiogi-knockout mice were exposed to 1 ,75% DSS in the drinking water for 7 days. Afterward, the DSS-containing water was replaced by normal drinking water. Body weigh was registered and percent change relative to initial weight was quantified. Values represent mean * SEM. Data from 9-10 mice per group is shown. No significant differences were detected.

Figure 3. Brd3~deficieucy in CB4^÷ T-eells results in attenuated Thl and TM7 responses. Wild- type Cddi. /’’ CS7BL/6 recipient mice received ike i.v. transfer of 10·' total CD45.2' CD45.1^' CDT OT-1I T-cells one day before being s.c. immunized with 100 pg OVA;_® - _T peptide in CPA. Ten days later, viable CD4' CD45.2' cells obtained from the draining inguinal lymph node were restimulated with FMA and ionomycin in the presence of brefe!din A for 4h and intracellular cytokinemmnnostalmng was performed and analysed by flow cytometry. (A) Representative dot plots showing the expression oflL-17 and IF -y in the transferred (0045.2 ') a d endogenous (C045.2 ) populations from alive (ZA f) CD4 gated cells, CJD45,2⁺ producing 1L-17 and IPN-y are framed in the marked ares. Numbers indicate the percentage of CD45.2 ' positive for IL-17 or IFH-y. (B) Quantification of the percentage of transferred (CD 5.2^*) alive CZAq ) CD4* T-cells expressing IL-17 (left panel) and IFN-y (right panel). Each symbol represent data obtained from an indi idual mouse. Lines and error bars represent mean * SEM Data from 8-9 mice per group is shown. **, p<0.0i by impaired Student's t-test.

Figure 4. Prd3~defkient Treg display higher anit-inflammatory effect in BBS-induced inflammatory colitis. (A) DnfS mRNA transcription in Treg. Naive CD4~ T-cclls (CD3 CD4^! CD62L^* CD25^“ CD44 ) were isolated from die spleen of wild-type C57BL/6 mice by cell sorting and then incubated under biased conditions to different ate into Treg cells (inducible Treg; iTreg; gray bars) for different times (0, 1 , 2, 3 and 4 d). In addition, Treg (CD3 ' CD4 €025^K8Ϊ!) generated in vivo (Treg; black bar) were isolated from lire spleen of wild-type C57SL/6 mice by cell sorting. Total UNA was extracted and subsequently, the transcription le vels of Drd3 mRNA were analysed by qRT-PCR, Gapdh was used as house-keeping. Values correspond to mean ± SD. Data from three independent experiments are shown fe p<0,05; **, p<0,01 by one-way ANQVA. followed by Tukey’s posthoe test, ns, not significant (B-C) Drrtd-defideuey in. Treg results in stronger attenuation of inflammatory colitis Eight- to-ten weeks old wild-type C57BL/6 mice received the i.v. transfer of ^j¾¾£?-suiicient (DrcB '", black symbols) or dd-defieknt (Drd3^'Q red symbols) Treg cells (CD3⁺ CD4' CD25 ' cells; 3x 1 (F eells/motse) and then were exposed to 1.75% DSS in the drinking water for 8 d. Afterward, DSS- eontaiaiug drinking water was switched to normal water and mice were monitored for 5 additional days. As controls, a group of mice did not receive Treg transfer and was treated only with DSS (white symbols), whilst another group did not receive neither Treg transfer nor DSS (blue symbols). (B) Body weight was periodically registered throughout the time course of disease development and die percentage of body weight change relati ve to initial weight was quantified. (C) CD4^" T-cells were isolated from the mesenteric lymph nodes 13 d after Treg transfer and the production of IFN-G and IL- 17 A was determined by intracellular cytokine staining and analysed by flow cytometry. Numbers represent fee percentage of viable (ZAq gate) CD4^" T-cells producing (he corresponding cytokine. (B- €) Data from two independent experiments are shown t> ~ 8/group). Values represent mean G SEM. *, p<0.05; p<0 01 by Mann- Whitney U-test Figure 5. Brd3~defi e»ey does not affeet Treg generation. (A) .DrdT and DrdP naive CD4’ T- ce!Is were activated (» vtm under iTreg-polaxizing conditions &r 4 d and then CD2S and Foxp3 expression was analysed. Representative dot-plots are shown (left). Numbers in quadrants indicate the frequency of€1325^" FoxpJ’ among CD4^" !-ce!is. Quantification from three independent experiments is shown (right). Values are mean ± SEM. (B) Foxpo and CD4 staining on splenoeytes derived from Drd3 and Drd3 ^: mice. Representative dot-plots are shown (left). Numbers in quadrants indicate the frequency of FoxpT among CD4’ T-ce!is. Quantification from three independent experiments is shown (right). Values represent mean ± SEM, No significant differences were found.

Figure 6. Brd3-d k ncy increases the su ress ve potential of Treg m vtw attenuating th development of OSS induced inflammatory colitis. Treg cells t^'CD4^' GFP^'} were isolated from Drd3^{"; "} Foxp3^gfp or Drd3 F xp3ⁱⁱ mice and i.v. transferred (3,5 x 10 ce ls/mousc) into wild-type C37BL/6 recipient mice. Subsequently, transferred mice were exposed to 1 % DSS in the drinking water for 8 d Afterward, DSS-contaimng drinking water was switched to normal water and mice were monitored for some additional days. As control, a group of mice did not receive Treg transfer and was treated only with DSS. Body weight was periodically registered throughout the time course of disease development and the percentage of body weight change relative to initial weight was quantified. Values represeat mean ± SEM Data from four mice per group is shown. Three independent experiments gave similar results *, p<0,^QS; comparing Dni3 ^v Treg ~> DSS versus Drd3^~ Treg -> DSS by two-tailed Student’s t-test.

Figure 7. Drd3~slgsaiR«g reduces the suppressive activity an fL-ίq prod notion in regulatory T- ceMs in nf , (A and B) naive Drd3 CD45.1 €045,2 CD4⁺ CD25 T-cells (Tnaive) were loaded with 5 mM CFSE and activated with DCs and anti-CD3 Ab in the presence of CD45.1 CD45.2^' Treg (at different Tnaive Treg ratios). 72h later, Tnaive proliferation was quantified as the dilution of CFSE- associated fluorescence in the CD45. I ^: CD45.2 CD4^r population by flow cytometry. As a control to determine the maximal Tnaive proliferation, Tnaive were activated in the absence of Treg (No Tregs). (A) Tnaive wore eo-eu!tured with freshly isolated r¾£T^{v !} or .Drd3"’ Treg. (B) Prior to the co-culture with Tnaive, Drp ’ Treg were activated with anti-CD3 and anti-CD28 Abs either in the absence or in the presence of the DrdS-seleciive agonist PD 12890? (50 nM) aud incubated for 48h. (A and B) In left panels representative histograms of CFSE dilution profiles are shown. Markers show the population of naive T cells displaying CFSE dilution. Numbers on the histograms represent the percentage of naive T cells displaying CFSE dilution. In right panels, the extent of suppression is quantified as the percentage of inhibition of naive Teells proliferation relative to maximal proliferation (No Treg), where the percentage of suppression is 0 (dotted line). Dam represent mean□ SEM from three independent experiments. *, p<0,0S by impaired Student’s /-test, (C-D) naive Drd3 ^r: CD45.! CD45.2 CD4^” CD23^‘ T-ed!s (Tnaive) were loaded with 5 mM CTV and activated with anti-CD3 and an i-CD28 Abs sad eo-cultured with CD45.1 €D4S.2^:: nTreg (ratio XregrTnaive ^::: 1:8) isolated foxmDrd ^* Fatxp or Drd3"" FaxpS^ mice in {he presence of 0_S 1 or 1000 oM dopamine. After 72h, the extent of Tnaive proliferation was determined as the dilution of CTV-associated fluorescence in the CD45.F€045.2 CD4 ^: population by flow cytometry, (C) Representative histograms of CTV dilution profiles are shown. Markers show the population of Tnaive cells displaying CTV dilution. Numbers on the histograms represent the percentage of Tnaive cells displaying CTV dilution. (D) In right panel , the extent of Tnaive proliferation is quantified. Values represent mean D SEM Data from a representative experiment in triplicate is shown. V p<0,0S by enpaired Student’s /-test. n„s., not significant differences were observed. (E> nTreg were isolated from tire mesenteric lymph nodes from Dxd3^{< :} Fox S^ or DrdS Foxp3 mice, and then activated with anfi-CD3 and anfi€D28- coated dynabeads (Treg:dynabead ratio ^::: 1 :2) in the presence of 0, 100 or 1000 nM dopamine and incubated for 48h. Foxp3 expression was determined by the mean intensity fluorescence (MFI) associated to GPP. Values represent mean D SEM. Data from a representative experiment in triplicate is shown. *, p<0.05; **, p<0.01 by one-way ANOVA followed by Tukey’s posthoc test. (F~G) Treg were isolated from Drd3 ^* or Drd3^! mice and then activated with anti-G>3- and anfi-CD28- coated dynabeads (Txcg:dynabcad ratio = 1 :2) for 4Sh (F) Trcg cells were restimulated with PMA and ionomycia in the presence of brefeldm A, and intracellular lL-10 was immuaostained and analysed by flow cytometry. XL- 10 production was quantified as the percentage of IL-KT CD4 cells in the ZAq^* gate, (6) IL--10 production was determined in the cell culture supernatant by ELISA, (F and G) Values represent mean n SEM. Data from two independent experiments is shown. *, p<0.05 by unpaired Student's t-test

Figure 8. DrdS-xignalling attenuates the recruitment of regulatory T-cdls Into the gut mucosa upon inflammation, (A) Treg cells (€D4⁺ GFP ) were isolated from mesenteric lymph nodes of Drd3^{"; "} Foxp.¥^!p or Drd3 F xpS^ mice and the expression of CCR9 and «4b7 was analysed in the alive (ZAq } CD4⁺ GFP population by flow cytometry. Representative dot plots of CCR9 and «Ab7 expression axe shown in the left panel. Numbers on quadrants indicate the percentage of cells in the respective quadrant la the right panel, CCR9 (left graphs) and «4B7 (right graphs) expression was quantified as the percentage of positive cells (top graphs) or the MFi (bottom graphs) in the alive (ZAq ) CD4^’ GFF gate. Each symbol represent data obtained from a single mouse (n ~ 6-10 per group). Lines on the graphs represent mean i SEM, **, p<0,0i by unpaired Student's l-test. n.s., no significant differences were found. (B) Treg cells (CD4^÷ GFP^:) were isolated from mesenteric lymph nodes of DrdS ^* Foxp3^®P or DrdS^'" Faxp3^s!p mice and activated with anti CD3- and anti-€D 8- coated dynabeads (Tregtdynabead ratio ^::: 1 :2) in the presence of RA and 1L-2 for 7d. The expression of CCR9 and 4b? was analysed every other day in the alive (2Aqj CD4^' GFP population by flow cytometry'. Quantification of the frequency and density of CCR9 expression are shown in the top-left and the bottom-left panels respectively. Quantification of the frequency and density of a4b7 expression are shown in the top-middle and fee bottom-middle panels respectively. Quantification of the frequency of cells expressing both together C€R9 and 4b7 is sho n in the top-right panel. Quantification of the density of Foxp3 expression is shown in fee bottom-right panel. Values represent mean□ SEM from triplicates. Data from a representative fro three independent experiments is shown. *, p<0 05; **, p<⁽Uli; ***, p<l.u^')⁽ll by unpaired Student’s t-test. (C-E) Treg cells (CD4⁺ GFP) were isolated from meaenkric lymph nodes t&DrdZ ^* Foxp3 (Cd45J* *} and Drd3 ^" FoxpS^ (Cd43. G^: Cd45.2^*1 } mice,mixed at ratio l ; i and then i.v. transferred {10" total eelis per mouse) into wild-type CS7BL/6 (Cd45 *‘^*) recipient mice 4 d after initiated fee treatment with 1.75% DSS in fee drinking water. 24h later, mice were sacrificed and the arrival of Drd3*^M (Ci>45 1 ^* CD45.2^') dlJr S ^" (CD45.1 ^* CD4S .2 ) Treg was analysed in the spleen, mesenteric lymph nodes (MLN) colonic lamina propria (eLP) and Foyer’s patches by flow cytometry. As a control to normalise fee recruitment of transferred Treg to different target tissues the ratio of Drd3^*! -Tmg-to-Z <£ VFxeg was also analysed before the i.v. adoptive transfer (input). (C) Representative contour-plots analysing the expression of CD45.1 versus CD4S.2 in fee TCRS CD4^’ gated population. Numbers on the contour-plots indicate the percentage of Drd3 ^{' : "}’(black) and Drd3 ^" (red) Treg present in fee tissue. (D) The extent of migration was determined as the percentage of Treg present in the tissue normalised wife fee percentage of Treg present in the input (for each genotype). Values represent mean□ SO from eight mice per group. *, p<G.05; **, pO.Ol by two-tailed paired Wtlcoxau test. (E) The homing index was calculated as indicated in fee top panel. Quantification of fee homing index for each target tissue analysed is shown in the bottom panel. Each symbol represents fee homing index obtained for a single mouse in the corresponding tissue. Lines and error bars represent mean□ SEM eight mice per group. *, p<0,GS by one-way ANGVA followed by Tukcy’s posthoc test.

Figure 9, Brd3~defi e«cy does not affect Fexjp3 expression in Treg. Treg cells (CD4 GFP^’) were isolated from mesenteric lymph nodes of Drd3 '^"" Foxp3 or iVriJ" Foxp3 ^> mice and the expression of Foxp3 (GFP) was analysed in the alive (ZA f)€1)4^" population by flow cytometry . Foxp3 expression was quantified as fee percentage of GFP cells (left panel) or the density of GFP expression (MF1; right panel) in the alive (ZAq~) CD4’ gate. Each symbol represent data obtained from a single moose ( ^:::: 10-14 per group). Lines on the graphs represent mean ± SEM. n.s.. no significant differences were found.

Figure 10. Drd3-deficfency results in increased CC 9 expression on ITreg. Naive Cl>4^' CD62L^' CD44 CD25 T-ceils were isolated if ore Drd3^'f FaxpS^ or Brd ^ Foxp mice and activated wife anti~CO3 and anti-CD28 Ahs and ncubated wife IL-2, RA and TGF-bI for 6 d. Afterward, differentiated Treg were restimulafod wife a l~CD3 D28~coared Dynabeads (ratio Tregs;Dynabeads ^!!K 1 :2) for 24 h and fee expression of CCR9 was analysed in the alive iZaq^') GFP^' CD4⁺ population by flow cytometry. (A) Representative dot-plots showing CCR9 expression verso SSC-A. Numbers on. dot-plote indicate die percentage of€CR9÷ cells (inside the framed area). (B) Quantification of the percentage of CCR9* Treg cells. Values represent mean ± SBM. Data from six independent determinations ****, p<0 0001 by impaired Student's Host

Figure 11. Dr 3-deS e«cy does affect Just poorl the acquisition of got tropisro by splenic Treg, Treg cells (CD4^” GFF^”) were isolated front the spleen of DrS3 ^÷'^"” Foxp3 ot Drn3 rnp3 mice and activate with s.nti-CT>3~ and anti-CD28~ coated dynabeads (Trcg:dynabead mho = 1 :2) in the presence of RA and IL-2 for 7d. The expression of CCR9 and a4b7 was analysed every single day in the alive (ZAq ) CD4^” GFF^” population by flow cytometry. Quimtifica.tion of the frequency (A and B) and density (€ and D) of CCR9 (A and€) and 4b7 (B and D) expression are shown. Values represent mean ± SEM from triplicates. Data from a representative from three independent experiments is shown *, p<O.OS; **, pfoi.Ol by unpaired Student's tfrest.

Figure It, Optimizing the protocol to transduce T g with retroviral vectors. (A) Naive and regulatory G34 T cells (Treg) were sorted from wild-type mice and then activated for two days in the presence of 1L-2 and aaii~CD3/€D28~coated Dynabeads (Dynabeadsfiregs ratio = 1 :1). Then, cells were washed and spinoculaied with retrovirus supernatant (RV-shDrd3, encoding sh£)ni3 sod Gfp) into a retronectfo-eoated plate. Nest; cells were cultured fo three additional days with or without anti- CD3,CD28-eoa†ed Dynabeads (1 : 1). (B) Treg were sorted from wiki-type mice, activated for two days and then spinoculated with RV-sfaDRD3 supernatant as in (A). Next, cells were coltared for three additional days with different ratios of DynabeadstTregs. OFF expression was assessed by flow cytometry on fresh cells. (A and B) Representative density plots are shown. Numbers on framed regions indicate the percentage of OFF^" cells among alive (ZAq ) CD4+ cells.

Figure 13 Treg cells retain their FoxplF phenotype alter transducing t e with retroviral vectors codifying for shR A for rd3, Treg cells were sorted from wiMfrype mice and then acti vated for 2 d in the presence of IL-2 and anti-CD3/CD28-coate Dynabeads (Dynabeads:Tregs ratio 2:1). Then, cells were washed, spinoculated with RV-shDrd3 supernatant into a retronectin-coated plate and incubated for three additional days with anti-CD3/CD28~eoated Dynabeads (ratio DynabeadstTregs ~ 2: 1). OFF expression was assessed by flow cytometry on fresh cells (upper panels), whilst GFP and Foxp3 expression were analysed in permeabilieed cells (middle and lower parrels, respectively). Numbers on framed regions indicate: (i) the percentage of GFP^’ cells among alive (ZAq ) CD4^” cells (upper and middle panels), (ii) the percentage of Foxp3 ^: cells among alive (ZAq ) non-infected€04^’ cells or among alive (ZAq ) infected CD4 OFF" cells (lower panel), (A and B) Representative density plots arc shown. Figure 14. Tie transference of Tregs transduced e vim with un shMNA for Drd3 inio !>8S-t ried «rice exerts a potest therapeutic effect attenuating the development of Inffanunator colitis. Wild- type C57 1/6 mice were treated w th DSS 1 75% is the drinking water. During the first day of treatment with DSS, mice received the i.v. transference of2xl.0^> Tregs. Quo group received Tregs transduced ex vivo with retroviral particles codifying for a shRNA that attenuates the expression of the Dni3 transcript and GFP as a reporter gene (RV-sfaDrd3 red symbols ), whilst the other group received the transference of Tregs transduced ex v/vo with retroviral particles codifying only for OFF (KV-Coutrol; black symbols). GFP^* Tregs were purified by cell sorting before transference into recipient mice. (A) Body weight was daily determined and represented as % of body weight change respect to the initial weight. Values are mean ± SD. (B) Disease activity index (DAI) was also daily determined, which considers loss of body weight, depositions consistence, and the presence of blood in depositions. Values are mean -t SD. (C and D) After eight days of treatment with DSS, animals were sacrificed and histological analyses were performed in the colon (C) and the length of colons was determined (D). **, p<0,i)i; ***, J O.OOI, ****, p 0.0001 by unpaired Student's /-test for each time point

DESCRIPTION OF THE INVENTION

The present invention, which provides methods and compositions useful for an effective IBD therapy, with high degree of specificity, based on the inhibition of the BRD3 biological activity specifically expressed in regulatory€D4÷ T-Cells (Treg), also comprises the following inventive features;

A method for treating gut inflammation in a mamma! in need comprising administering to the mammal a pharmaceutical composition comprising one or more agents that inhibit attenuate, disrupt and/or block the biological activity' of DRIB i the mammal.

A method for increasing a migration of regulatory T cells into gut mucosa in a mammal in need comprising administering to the mammal a pharmaceutical composition comprising one or more agents that inhibit, attenuate, disrupt and/or block the biological activity of DRD3 in the mammal.

A method for increasing regulatory CD4+ T-eells (Treg) suppressive activity into gut mucosa in a mammal in need comprising administering to the mamma! a pharmaceutical composition comprising one or more agents tost inhibit, attenuate, disrupt and/or block the biological activity of DRD3 ia the mam al.

Any of these methods, wherein the one or more agents inhibit, attenuate, disrupt and/or block the biological activity of DRD3 specifically expressed in CD4·^;- T-Cells; wherein the one or more agents inhibit, attenuate, disrupt and or block the biological activity of DRD3 specifically expressed in CD4+ T-Cclls, is particularly in the subpopolation of regulatory CD4 - T-ceila (Treg); wherein the one or more agents that inhibit, attenuate, disrupt and/or block the biological activity of DRIB in Treg, reduces rd3 transcription; wherein the one or more agents that inhibit, attenuate, disrupt and/or block the biological activity of DRI>3 in Treg, is an RNA product that reduces ORl>3 transcription; wherein the RNA product is an shRNA; wherein the mammal in need suffer of an inflammatory bowel disease; wherein said inflammatory bowel disease is Crohn Disease; wherein said inflammatory bowel disease is Ulcerative Colitis.

A me thod for treating got inflammation in a mamma! in need, comprising the s tep of transducing a Treg cell of the mammal with a retroviral vector containing a nucleic acid encoding an RNA product that reduces Drd3 transcription in the mammal,

A method for increasing a migration of regulators' T cells into gut mucosa in a mammal in need, comprising the step of transducing a Treg ceil of the mammal with a retroviral vector containing a nucleic acid encoding an RNA product that reduces Drd3 transcription in the mammal.

A method for increasing Treg suppressive activity into gut mucosa in a mamma! in need, comprising the step of transducing a Treg cell of the mammal with a retroviral vector containing a nucleic acid ««coding an .RNA product that reduces Drd3 transcription in the mammal.

Any of these methods, wherein the R A product is an shRNA; wherein the RNA product have the ability to hybridize under defined conditions to a nucleic acid sequence selected from the group consisting of SEQ ID No.1 (DRD3 mRNA mouse) and SEQ ID Ne.2 (DRIB mRNA human); wherein the RNA product is an shBNA (small hairpin RNA); wherein the shRNA product is defined by SEQ ID No.3.

A method for treating gut inflammation in a mammal in need thereof comprising the step of introducing Drd3~defieient Treg cells into the mammal to cause an increase in migration of regulatory T cells into gut mucosa of the mammal

A method for treating gut inflammation in a mammal in need thereof comprising the step of introducing Drd3-defieie:n! Treg cells into the mammal to cause an increase in Treg suppressive activity into gut mucosa of the mammal.

Any of these methods, wherein the Drd3-def ieni Treg cells are introduced into the mammal intravenously; wherein the mammal in need suffer of an inflammatory bowel disease; wherein said inflammatory bowel disease is Crohn Disease; wherein said inflammatory bowel disease is Ulcerative Colitis,

Any of these methods, wherein the method further comprises administering one or more additional therapies; wherein the one or more additional therapies comprise auli-iuRammatory bowel disease therapy, wherein the additional anti-inflammatory bowel disease therapy comprise therapeutics selected from the group consisting of neutralizers of anti-TNFaipha (infliximab (Remieade), adaimiumab (Humira}. golimumab (Simponi), Certol unab . alpha integrin s banil inhibitors (naializumab (Tysabrl)), alph b ts? integrin inhibitors_: (vedol umab (Entyvio)), IL~12Rheial blocker that avoid its union to ligands IL-l 2 and JL-23 (ustekinumab (Stelara)), anti-MAd-CAM-l monoclonal antibody (PF-00547659), anti-IL-23 monoclonal antibody (MEDI2070), sphmgosine- 1 -phosphate (SIP) receptor agonist (Ozammod) antisense oligodeoxyimeieotide complementary to mRNA of Smad? (Mongersen), aminosalicylates (S-aminosalicylie acid (5-ASA); 5~ASA derivatives (Stliasalaa-uie, Asacol HD, Xtekicol, Pentasa, Colazal, Dipcotum, Ltslda, Apriso, Mesalatanc), corticosteroids (hydrocortisone, me ylprednisolone, prednisone, Budesonide, Budesonide MMX, hydrocortisone), antibiotics (ciprofloxacin, metronidazole, rifaximin, ormdazo!e, imidazole, anti- tuberculosis therapy, maeroUdes, fluoroquinolones, S-nitroimidasoles, aatimycobacterials), and immonosnodalators (thioporines (Purineihol, Parixan), 6-mercaptopurine, azathksprine (A¾asan, Imuran). methotrexate (Trcsall), cyclosporine (Gengraf, hi coral, Sandimmune),

An agent that inhibit atenuate, disrupt and/or block the biological activity of DRD3 specifically expressed in regulatory CD4+ T-Cells; wherein is an RNA preduet that reduces DRD3 transcription; wherein the RNA product have the ability to hybridize wider defined conditions to a nucleic acid sequence selected from the group consisting of SEQ ID No.l (DRD3 mRNA mouse) and SEQ ID o.2 (DRD3 mRNA human); wherein the RNA product is an sliRNA (small hairpin RNA); wherein the shRNA product is defined by SEQ ID Ne.3.

A polynucleotide encoding, upon expression, any of the agents before mentioned; a genetic vector that comprises any of these polynucleotide; where the genetic vector is a viral vector; where die viral vector is a retroviral vector; a CD4-f· T-eells that comprises any of the before mentioned genetic vectors; wherein the CD4-S- T-cells is a Treg.

A pharmaceutical composition comprising any of die before mentioned agents, and a pharmaceutically acceptable carrier, diluent or excipient; these pharmaceutical compositions, for use in the treatment of inflammatory bowel diseases, particularly for use in the treatment of Crohn disease or Ulcerative Colitis

A kit comprising any of the before mentioned pharmaceutical compositions, and instructions for administering the pharmaceutical composition to treat gut inflammation in a mammal, particularly inflammatory bowel disease, and even more particularly for use in the treatment of Crohn Disease or Ulcerative Colitis.

A kit comprising any of the before mentioned pharmaceutical compositions, and instructions for administering the pharmaceutical composition to increase a migration of Treg into gut mucosa in a mammal in need. A kit: comprising any of tire before mentioned pharmaceutical compositions, and instructions for administering the pharmaceutical composition to increase Treg suppressive activity into gut mucosa in a mammal in need.

Use of any of the before mentioned agents that inhibit, attenuate, disrupt and/or block the biological activit efI>RD3 specifically expressed in regulatory Ci>i÷ T-Ceils, for preparation of a medicament for treatment of inflammatory bowel diseases, particularly for preparation of a medicament for treatment of Crohn disease or ulcerative colitis.

Use of any of the before mentioned pharmaceutical compositions, for preparation of a medicament for treatment of inflammatory bowel diseases, particularly for preparation of a medicament tor treatment of Crohn disease or ulcerative colitis.

The present invention was developed using the following materials and methods:

Alice

Wild-type C57BL/6 (Drd ^"'* ; C 45.2 ^{! "}) and Ragf mice were obtained from The Jackson Laboratory. C57BL/6 Dr 3^ mice were kindly donated by Dr. Mam Caros (Joseph et ai., 20⁽12). C57BL/6 Ft?xp3^m reporter mice were kindly donated by Alexander Radensky (Fon.ten.ot et al , 2005). Both OT-II and B6.SJL -Ppnf ( Cd45.t '^*) were kindly provided by Dr, Maria Rosa Bono. Drd OT-H mice, R gf Drd3^"' QMS. / " Cd4$.2^>1 mdFoxp3 ^!> DnAT were generated by crossing parental mouse strains. We confirmed these new strains to he transgenic and Drd3~defteient by (low cytometry analysis of blood cells and PC.R of genomic DNA, respectively. Mice from 6 to 10 wk were used in all experiments. Ail procedures performed in animals were approved by and complied with regulations of the Institutional Animal Care and Use Committee at Fundaeion Ciencia & Vida

Reagents

Monoclonal antibodies (mAbs) for flow cytometry: so.ti-Fos.F3 (clone FJK-16S) conjugated to Phycoeryfhrin (PE)-Cyanine ? (Cy7) and Alloph cocyauin (APC), and aoti-IFN-y (clone XM01.2) conjugated to FE Cy7, anti-adp" (clone BATK32) conjugated to PE and antriCCRP (clone C W.1 .2) conjugated to APC or to AFC~Cy7 were obtained from sBioseience (San Diego.. CA, USA). Anti-CD4 (clone OKI .5) conjugated to APC and APC~Cy7; anti-CD25 (done PC61) conjugated to Fluorescein isothiocyanate (FITC); anti-CD44 ( one ΪM7) conjugated to PE; anti-CD62L (clone MEL 14) conjugated to APC-Cy7; aoti-fL~ l ?A (clone TC11 181710.1) conjugated to APC; aoti-CD45.2 (clone 104) conjugated to PE-Cy7; anti-CD45 i (clone A20) conjugated to Brilliant Violet (Sv)421; anti- TCRVo.2 (clone B20.1) conjugated to PE aad TCRVPS (clone MR9-4) conjugated to APC were purchased from Biolegend (San Diego, CA, USA). mAbs for Cell Culture; the followings Abs low in endotoxins and azide free (LEAF) were purchased from Biolegend.: &nti-CD28 (clone 37,51), aoti-CDBs (clone 145-20 1 ) and suti-IEN-y (clone AN- 18). Carrier-Free cytokines TGF-fl! and 1L-2 were purchased from Bfolegeod. Zombie Aqua (ZAq) Fixable Viability dye detectable by flow cytometry was purchased ftom Biolegend. Phorbol I2~myristaie 13-aeetate (PMA), ionomycm ami retinoic acid (RA) were purchased from Sigma- Aldrich (San Lois, MO, USA). Cell Trace Carboxyfiuorescein succinhnidyl ester (CFSE), Brefcldin A and Fetal Bovine Serum (PBS) were obtained from Life Technologies (Carlsbad, C^'A, USA). The peptide derived from the chicken ovalbumin (OVA ^- rw_: OT- II peptide or pOT-lI) was purchased from Oenescript (Piseataway, NJ, USA), Freund’s Complete Adjuvant (CFA) and anti-CD3/anti-CD28 conjugated dynabeads were purchased ftom Thermo Scientific, Bovine Serum Albumin (BSA) was purchased to Rockland (Limerick, PA, USA), D$S was obtained Bom MF Biomedicals Drd3 agonist, RΌ .128907, was purchased from TOCRIS. Cell trace violet (CTV) was obtained from Invitregen (Carlsbad, CA, USA). Ail tissue culture reagents were bought from Life Technologies.

CB4⁺ T-ce!l isol tion, activation and differentiation in vifm

Total CD4^÷ T-eelis were obtained by negative selection of sp!enocytes according to manufacturer Instructions (Miltenyi). Further purification of Treg (CD4^' CD25^h¾?5) and naive CD4⁺ T-eells (CD4^! CD62IZ CD44 CD25 ) ways achieved by labeling enriched CDd* T-celts with the corresponding antibodies and subsequent cell sorting using a FACS Aria II (Bi>), obtaining purities over 98%. Purification of Treg cells from FoxpS** mice was carried out by isolating OFF CD4^* cells by cell sorting. All in vitro experiments were performed using complete RP I medium (supplemented with 10% FB5, 2 mM L-Glntemine, 100 U/mL Penicillin, 100 ug/snL Streptomycin and 50 mM ϋ~ mercaptoethanol). To assess proliferation, Treg or naive CD4 T-ccl s were st.aioed with 5 mM CFSE or 5 mM CTV and stimulated for 3 d with 50 ng/we0 of plate-bound anti~CD3 mAh and 2 pg/niL soluble anti-CD28 mAh on fiat-botom 96-well plates (Thermo Scientific). The extent of T-cdi proliferation was determined as the percentage of dilution of CFSE- or CTV-associated fluorescence by flow cytometry.

To force the differentiation to iTreg, freshly purified naive CD4 T-cells svete incubated in the conditions indicated above, in the presence of 5 ng/mL TGF-G 1, 10 ug/mL J.L-2 and 100 nM RA. At different incubation times, the cells were assessed for gene and protein expression.

Flow cytometry

For intracellular cytokine staining analysis, cells were restimalated with 1 pg/mL ionomycm and 50 ug/mL PMA for 4 b, in the presence of 5 pg/mL brcfeldin A. Cell surface staining was carried out in PBS with 2% FBS. Fo intracellular staining, cells we e Erst stained with Zombie Aqua (ZAq) Fixable Viability kit (Biolegend), followed by staining fo cell-surface markers and then resuspended in fixatiou/permeabilization solution (3% BSA and 0.5% saponin in PBS). Samples including the analysis of Foxp3 were resuspended in fixation/permeabilixation solution (Foxp3 Fixation/Feraseabiliration; eBioscienee) according to the manufacturer instructions. Data were collected with a Canto II (ED) and results were analysed with FACSDiva (BD) and FlowJo software (Tree Star, Ashlars, OR, USA).

Quantitative RT-PCR

Total .RNA extracted from cells using the Torn! RNA EZNA kit (Omega Bio-Tck) was DNase-digested using the TURBO DMA-free kit (Ambitm) and 1 gg of RNA was used to synthesize eD A utilizing M-MLV reverse transcriptase, according to manufacturer^ instructions (Life Technologies). Quantitative gene expression analysis was erformed using Brilliant II SYBR Green QPCR Master Mix (Agilent), according to manufacturer's recommendations. Primers were used at a concentration of 0 5 mM. We used 40 PC.R cycles as follows: denatination 30 s at 95°€, annealing 30 s at bCBC and extension 30s at ?27^'C. Expression of target genes was normalize to Gapdh. The sequences of the primers used are the following: BrcB, sense 5^,-GAA€TC€TTAAG€€CCACCAT-3^! and antisense 5"- GAAGGCCCCGAGCACAAT-3 ^! ; and Gapdh, sense S’-TCCGTOTTCCTACCCCCAATG-S’ and atttisense S^{^}-GAGTGGGAGTTGCTGTTGAAG-Sh fn vitm suppression assays

Treg (C D4^" CD25^feiS ) obtained from Cd45. G^' " mice were activated with 50 ag of plate-bound anti€D3 and 2 pg/mL soluble anti-CD28 in the absence or presence of 50 nM PD 128907 (TOCR1S). After 2 d, activated Treg were washed and serially diluted to incubate with 5 x 10" CFSE-stained naive CD45.2" CD4" T-cells, I t^}4 CDl le" ceils obtained by positive selection (Mi!tenyi} and .1 pg mL anti-€D3 in roi d-bottom 96-well plates (Thermo Scientific), 3 d later, the degree of proliferation was assessed in the CD4 ' CD45.2^* population by flow cytometry. When indicated, suppression assays were performed with GFP" CD4" Treg isolated from Fox S^ Cd45 ^; " mice. In those cases, CD45. U CD45.2· CD4" CD25^" naive T-eclls were loaded with 5 mM CTV and activated with plate-bound anti-CD3 mAh (50 ng) and soluble anii-€D2§ Ab (2 m-g/mL) and co-euSteed with Treg cither in the absence or in the presence of dopamine (100 :nM or 1000 uM; Sigma -AMrich). After 72h, the extent of naive T-ce!l proliferation was determined as the dilution of CFSE- or CTV- associated fluorescence in the€045.1 " €045.2: 004" population by flow cytometry.

Su press on of acute colitis induced by BSS

WT recipient mice were iv. injected with 3 x 1 " Treg (€04"€D25^b¾¾) .1 d before starting the administration of 1% or 1 ,75% DSS in the thinking water. BSS was given for a total period of 8 d and then replaced with normal drinking water until the end of the experiment Body weight was recorded throughout the time-course of disease development. The extent of less of the initial body weight as used as the main parameter to determine disease severity. In some experiments, disease activity index (DAI) was also determined as a second readout of disease severity. For tins purpose, the percentage of loss of body weight, stool consistence and gross bleeding or ocoa.lt blood in feces were evaluated periodically throughout the time-course of disease development and each of these three parameters were scored with a scale between 0-4, as described before (Chen et aL 2007). Thus DAI scored from 0 (healthy) to 12 (severe colitis). At the end of the experiment the mesenteric lymph node was obtained to re-stknaiate with PM A and ionomyein in foe presence of brefe!din A and assess inflammatory populations by flow cytometry. Transverse sections of fixed colon were cut to 5 pm with a cryostat, mounted on xyknteed slides, and H&E stained to assess intestinal inflammation by light microscopy, as previously described (Ostanin et ai., 2009),

Retroviral transdnetien of Treg and naive 014^" T cells

For silencing Drd3 expression, we used the retroviral vector pBullet (Wcijtens et al., 1998). We smelted a region coding

© U6 promoter an shRNA directed to drd3 transcript (5⁵-TOC CCT CTC ITT GGT TTC AAC ACA AC- ) and the Hi promoter, into pBullet vector via Neol and Sail restriction sites (Genscript, Pisco may, NJ). pBullet vector drives the expression of foe entire construct by the CMV promoter upstream the Neol site. This vector was transfected into ftoenix-AMPHO cells. GFF cells were subsequently purified by ceil sorting to generate a stable cell line producing retrovirus coding shRNA for DrdS (RY-shDrd3) in foe supernatant. As a nofi-sileftciag control, we generated Phoenix- AMPHO cells stably secreting in the supernatant retroviral particles codifying only tor gfp (RY- Control),€04^" CD25^n¾R Treg cells were isolated fromlbw mice, and then activated with anfo-CDw- and aab-CD28~ coated dynabeads (at Tregs:Dynabeads ratio = 1:1) for 48b. Then, ceils were washed and spinocnlated with RV-shDrd3-containing or RV-Controi-containing supernatants into a etroneciin- eoated plats (Takara Bio, Japan). Afterward, cells were incubated with anti-€D3- and anti-CD28- coaled dynabeads (Trcgs: Dynabeads ratio = 2:1) for 72 h. Tranductiou was confirmed by GFP expression by flow cytometry.

Statistical Analyses

AM values are expressed as the e n ± SEM. Statistical analysis wore performed with two-tailed Student's ( est, when comparing only two groups and with one-way ANOVA followed by Tukey's post-hoc test, when comparing more foan two groups (G phPad Software). P values < 0,05 were considered significant.

EXAMPLES

Experiments made to develop the present invention are shown in foe foliowing examples;

Exam le 1. Genetic i¾xfd-deficie«cy in results is attenuate gut inflammation in a mouse model of Inflammatory colitis. By s ng n animal mods! of inflammatory colitis induced by the transfer of naive CD4^' T-eel!s nto X- eel! deficient mice, a model of inflammation that epends exclusively of T-ceil response, our previous study show that £W3-defkieney in CD4⁺ T-cel!s resulted in a significant attenuation in disease manifestation. (Contreras et &L 201 (>). Despite in UC aud CD T-eeil response plays a central role driving gat-isflammatiou, cells of the innate immune system play also a relevant role contributing to the inflammatory process {Sainaihan et al., 2008). Since in human IBD both, the adaptive and the innate immune system contribute to the disease development, we sought to analyse die relevance of DRD3- signalling in a mouse model of inflammatory colitis induced by the administration of dextras sodium sulphate (DSS) in the drinking water, a model that involves both, the innate and adaptive immune response (Sainathau et ah, 2008). To analyse the global role of BRBS-signalling in the development of inilammatory colitis, we first compared the development of inflammatory colitis induced by DSS in /> J~sufficient aud i. ?¾fi~defficient mice. Strikingly, the results show that iJr O deficieney resulted in a complete abrogation of disease manifestation, including the attenuation in fee loss of body weight (Fig, 1 A), which corresponds the main parameter to determine disease severity. An inhibited shortening of colon length (Fig. IB) was also observed in .OuB-deficicnt mice when compared with />¾?- suffic ent mice. Because .⁽Mid-deficiency has been previously associated to behavioural alterations in. mice, including depression and anxiety (Moraga-A aro et ah, 2014), we tested whether differences in the loss of body weight observed between IVtO-suificieat and 2¾¾/3 defficient mice were due to an unequal consumption of drinking water upon the DSS-treahnent. he results show that water consumption was similar in i.h¾’d~suffieient and niMeffieient mice before as well as after starting the DSS-administration (Fig. ID), thus ruling out the possibility that differences in body weigh observed between both genotypes was due to an unequal water consumption. Because DRD3 has been shown to be expressed not only in fee adaptive immune system, but also in neutrophils, eosinophils and Natural Killer cells (McKenna ct al., 2002), we next attempted to dissociate the relevance of DRD3- signalling in the adaptive immune system and outside fee adaptive immune system in the development of inflammatory colitis. For this purpose, we crossed Drd3^{' '} mice with Ragi mice to generate Brd3- defficient mice devoid of adaptive immune system. The results show that Brd3" Ragi" as well as Brd3^*” Ragi ^{' '} mice were susceptible to develop DSS-lnduced inflammatory colitis and feat both genotypes presented loss of body weight with similar intensity (Fig, 2), thus indicating feat DRD3- signalling outside the adaptive immune system lacks relevance in fee development of gut inflammation. Together, these results indicate that DRD3-signal.!ing in fee adaptive immune system plays as important role favouring the development of inflammatory colitis. xample 2. BRD3~s*g«a!Ii»g i« Treg cells attenuates their antyuflanunaiory eff ct in inflammatory col tis,

Qor previous study shows that Drofhddickncy in naive C04 T-eells, a subset devoid of reg, results in a selective attenuation of TM -mediated immunity In a model of antigen-specific immune respo se induced by the immunization with the ovalbumin (OVA) derived peptide O^'T-II (pOTII, which corresponds to QVA ) (Contreras et ah, 2016), Using the same antigen specific immunization model induced by pOTH, hem we observed that DraU-deiiciency in total CD4 T-cells, which includes the subset ofTreg cells, results in impaired Th.l - and Thl 7~ mediated immunity (Fig.3). Thus, together these results suggest that DmU-deficieney in Treg cells plays a relevant role in the control of Thl 7- mediated immunity. Of note, ThlT-mediated immunity has been extensively involved in gut inflammation (Olsen et a!., 2011).

To analyse the role of DRD3-signalling iu Treg in the control of got inflammation, we next aimed to analyse DRD3 expression in Treg cells. For this purpose, the levels of Dm’3 transcripts were quantified in Treg differentiated in vitro from naive CD4^’ T-ce!ls (iTreg) and generated in vivo (hereinafter called just Treg), which were isolated from the spleen. The results show that both !Treg and Treg display significant expression of Drd3 transcripts, nevertheless the extent of.D«s/i-transeription was higher in Treg (Fig. 4A), To analyse whether DRD3-signalling was relevant in the generation of Treg, we next conducted experiments to determine the impact of D/¾ -deficieney in die in viiro differentiation of naive CD4 T-cells into iXregs and in the generation ofTreg in vivo isolated from the spleen. The results show a similar extent of iTreg generation when compared the in vitro differentiation of £hrl?~suffieient and Zlrdiwleffteieni naive CD4 ^: T-eells (Fig, 5A). Similarly, no differences were detected in the frequency of Treg in the spleen of n i-sufficient and Oro'i-deffkient mice (Fig, 5B). Thus, these results suggest that DRIB -signalling does not play a relevant role in the generation ofTreg cells.

To determine the relevance a fflTRlTB -signalling in the suppressive potential of Treg in inflammatory colitis, we next evaluated the extent of dssea.se manifestation in OSS-treated mice that receive the adoptive transfer of i>ifd-sufficieot or f d-deffieieut Treg cells. Since we expected to observe a higher therapeutic effect in mice receiving Dm'i-deffieiesii Treg cells, we transferre a suboptimal amount of Treg (3x10^' cells per mouse) according to the results obtained previously by Huber and colleagues (Huber et al, 2004). According to our hypothesis, whereas Ibtfi-sufficient Treg did not exert a significant therapeutic effect in the development of inflammatory colitis, mice receiving the transfer of ilr i-defficient Treg displayed a complete attenuation in the loss of body weight (Fig. 4B). To evaluate the effect of DRD3-stgoailing in Treg cells in the control of inflammatory effector T-eell subsets in the gut-associated lymphoid tissues (GALT), we analysed the frequency of relevant T-helper cells in the mesenteric lymph nodes (MLN) of DSS-treated mice. Interestingly the results show no significant differences in the frequency of Thl cells among all experimental groups (Fig. 4C), Conversely, mice receiving Treg transfer displayed a strong reduction in the frequency of Thl ? cells in MLN in comparison with those mice treated only with DSS but withou t transfer of Treg ceils. In addition, mice receiving TWJ-deffieieut Treg show a more significant redaction in. Thi 7 frequency than that observed in mice receiving DnS-suffiefetd Treg cells (Fig, 4C), Thus, these results together with those shown in figure 3 and in our previous study (Contreras et ah, 20.16) suggest (hat DR03- signalling in Treg plays a relevant role limiting their suppressive activity preferentially on TM7- mediated responses. Because the CD3⁺€D4 ^: CD25 ^: subset of T-cell might contain not only Treg but also some few activated effector T -ceils, we repeated (he experiments from figure 48, hut transferring GEF Q>4⁺ Treg isolated from Fax 3 ^? reporter mice (Fontenot et ah, 2005} instead using the CD3 ^' CD4^" CD25" subset ofT-ceils. The results show that. DSS-treated mice receiving a suboptimai amount of E f-dcffieicut GFF^: CD4^i- Treg presented a significant improvement of disease manifestation in comparison with those DSS-treated mice receiving OrrfE-suiiicieut GPP' CD4^: Treg or with those only treated with DSS bur without Treg transfer (Fig, 6). Thus, these results (Fig.6) recapitulate those results obtained in figure 4B and thereby reinforces the conclusion that DRD3-signalling i Treg cells plays a relevant pro-inflammatory role favouring the development of inflammatory colitis.

Example 3, The selective stimulation of DMB3 attenuates the suppressive activity »f Treg To address the question of whether DRD3-signalling regulates Treg function, w first compared the suppressive activity of fh¾fi-sufficient and i. </3-defficient Treg using} an in vitro suppressive assay. Unexpectedly, we did not find any difference in the suppressive activity in these Treg ceils (Fig, 7A), As mentioned above, Treg cells isolated from inflamed lamina propria of IBD patients show impaired suppressive activity, nevertheless after isolation they retain their suppressive activity in vitro (Maui et al., 2005), suggesting that suppressive activity of Treg could be impaired just in situ by mediators produced by the inflamed tissue. Accordingly, we reasoned that suppressive activity of E d-sufficient Treg should be affected in the presence of DRD3~agoaists. To test this possibility, we activated Drd3~ sufficient Treg in the presence of the selective DRIB agonist, PD128907, and then assessed their ability to suppress the proliferation of naive CITE T-eells in vitro. Results showed that selective stimulation of DRD3 significantly reduced the ability of Treg to suppress CD4 T-cel! proliferation (Fig. 7B), indicating that DRD3~signaliing limits Treg ftmetion.

To confirm test DR.D3-stimulation attenuates Treg suppressive activity using now tee natural DRD3 ligand, dopamine, we conducted suppressive in vitro assays co-eu!turing wild-type naive CD4 T-eells with D J-sulIicieni or DnfJ-defficlent Treg in the presence of low dopamine levels (100 nM, which stimulates selectively DRD3), such as the situation found is inflamed gut mucosa, or in the presence of high dopamine levels (! OOO siM, which stimulates low-affinity and high-affinity DRs), conditions similar to those found in healthy gut mucosa (Asano et ah, 2012; Magro et ah, 2002). The results show that in the presence of 1000 nM dopamine, both Drtel-s&flicteot or DrfeEdeil eieoi Treg exert similar suppressive activity attenuating the proliferation of naive CiM^: T-eells (Fig. 7C-D), Nevertheless, In tee presence of 1.00 nM dopamine, tee suppressive activity of .DrdJ-snffieient Treg cells was significantly decreased in comparison to f ti3-defficisnt Treg activity. Thus. these results confirm once again that fee selective DRD3 stimulati«n limits Treg suppressi ve Inaction.

To address the molecular mechanism underlaying the DRD3-mediated attenuation of Treg s uppressi ve activity, we next evaluated how dopamine affected the density of Foxp3 expression, as fee decreased expression density of this maste transcription factor has been associated to reduced Treg activity and fee development of autoimmune disorders (Wan and Flavell, 2007). Interestingly, we observed that only when exposed to 100 aM dopamine, but not in the presence of 1000 nM dopamine, the density of Foxp3 expression was significantly reduced (Fig, 7E), thus suggesting that the selective DRIB- stimulation induces a reduction on the density of Foxp3 expression in Treg cells. Because one of fee main mechanisms used by Treg to suppress the activity of effector T -cells in the production of IL-IO, we also tested the possibility that PET -sigoallfeg regulates lL-10 production by Treg cells. According to the higher suppressive activity observed in i.h¾fi deffieient Treg, results show that />rdJ~defficiency resulted in increased production of ILTO by Treg cells (Fig. 7F~G). Together, these results indicate that fee selective stimulation of DRDS, a situation given in the gut mucosa upon inflammation, limits fee suppressive activity of Treg cells.

Exam le 4, i rd3-defieiene favours the ac uisition of gut tropism by Treg ceils

The higher attenuation of colitis development exerted by 2 3 deffieient Treg can be due to two non- excluding possibilities: 1 , To a higher suppressive activity, or 2, To an enhanced recruitment of Treg into fee gut mucosa. To address the second possibility, we first evaluated fee expression of key molecules involved in got homing, the C-C chemokine receptor CCR9. and the integrin a4b7 (More et al., 2003). For this purpose, we isolated Treg cells from mesenteric lymph nodes (MLN) of Drd3~ sufficient and Dr<B~defficient mice aad analysed fee expression of CCR9 and «4b?, Importantly, the results show that Ot03~d&fieienc)^! resulted in a significant and selective increase of CCR9, both in frequency and density of expression in in Treg cells from fee MLN in steady -state (Fig, 8A), Of note, we observed no differences in die expression of Foxp3 between Oidd-suffici t and IMB-dejfieient Treg obtained from die ML in steady-state (Fig, 9). interestingly, fee increased expression of CCR9 induced by the deficiency of Drd3 was also observed in flTegs differentiated from -naive CD4^' T-cells (Fig.

Afterward, we wondered whether fee selective increased CCR9 expression observed in rdi-defficienl Tmg cells was abrogated, maintained or exacerbated after T-ceil activation. To address this issue, we next isolated Treg cells from the MLN of B i-sufficient and iWi-deffieicnt mice and they were activated with anti-CD3/anti-CD28 coated dynabeads in fee presence of RA and 1L~2 and the dynamic of CCS9 and a4b7 expression was assessed at different time points during 7 d. According to fee role of RA in imprinting the gut tropis on Treg cells (Iwata et al, 2004), fee expression of both CCR.9 and a4b7 was increasing along fee time (Fig, SB), Furthermore, IViO-deliicieoi Treg cells maintained a higher frequency of and enhanced density of CCR9 expression along the time after T-eell activation up to 7 d, Conversely, 4b7 expression wa not affected by i>#¾fJ-deficieney in Treg cells (Fig. SB), It is noteworthy that Foxp3 expression was also similar in I>?¾/T-suffricieut and iTnTT-deffioient Treg cells Isolated from the MLN, both in steady-state, or after T-cel activation (Fig, 8B). Next, we attempted to determine whether the selective increase of CCR9 expression induced by / d3-defieiency was a general aspect of Treg ceils from different locations or it was a particular feature of Treg obtained from MLN. Accordingly , we isolated Treg cells from the spleen of i>rri5 suffiicient and iWJ-defficient mice and then were activated in vitro in the presence of RA and IL-2 and the dynamic of CCR9 and a4f)7 expression was assessed at different time point during 7 d. Importantly, the results show that expression of CCR9 and a4b? was similar in DrriJl-sufficient and r¾s?i-defficient Treg along the time (Fig. IT), suggesting that the regulation of CCR9 expression exerted by DRD3 -signalling seems to be confined to GALT associated Treg cells. Together, these results indicate that I fd-dsficiency increases the acquisition of gut tropism in Treg cells found in the MLN.

Exa ple 5, Lack of D.RII3~sIgnslSng in Treg Increas s their recruitment into Ike gut mucosa upon Inflammation

Since Drd3~defieiency significantly modified gut-tropism in Treg ce.Us₅ we next sought to evaluate whether DRD3-signallmg affects Treg recruitment into the gut mucosa upon inflammatory colitis. To this end, we transferred a mixture of Zir J-sufftcteni and Dri/i-defficient Treg cells in mice undergoing DSS-induced inflammatory colitis and 34 h later, we traced the destination of Treg cells analysing different tissues by flow cytometry. To quantify the extent of migration for each genotype, the percentage of Treg present in the tissue was normalised with the percentage of Treg present in the initial mixture. The results show that the fraction of /Wi-defficient Treg was significantly higher than drat of/Jfv/.?-sufFicieni Treg in the cl.F as well as in Fever’s patches (Fig, hCT ). Conversely, there were no differences in the distribution of i>i/3-deffieient and / x0-saffideni Treg cells in the spleen and MLN (Fig. SC-1⁾), Furthermore. to reinforce the conclusion that /Irdd-deficiency lead to a differentia! distribution of Treg cells favouring the recruitment of these cells into the gut mucosa, we also analysed the Homing Index (see the formula in figure 8E, top panel) as described previously (Villablanca and Mom, 2011). Tills analysis indicated that £>r<sB-defieieney results in an increased recruitment of Treg cells into the eLP in comparison to the spleen and MLN (Fig. 8E, bottom panel) . Of no te, despite there is a marked trend of higher recruitment of Treg into Foyer s patches led by ri/i-deficieney, there were no significant differences in the recruitment of Zht/Ldefficieui Treg into Payer’s patches in comparison with the spleen and MLN, probably due to the high dispersion of data obtained from Peyer’s patches (Fig. 8E, bottom panel). Thus, taken together these results indicate that . rdi-delleieney leads to enhanced recruitment of Treg into gut mucosa under inflammatory conditions, especially into the colonic lamina propria. xample 6. Targeting the transcription of srdS in Treg cells as a therapeutic ap roach to attenuate the development of inflammatory colitis

Because we observed a strong decrease of DSS-induced colitis manifestation when />i0-defiscieni Treg were transferred_* we nex t attempted to test the therapeutic potential of targeting RD3 cxpression on Treg cells to attenuate gut inflammation. For this purpose, we generated retroviral vectors codifying for an sliRNA directed to reduce Drd3 transcription (RV shDrd3), as described previously (Contreras et al., 2016). Prior to test the therapeutic potential of the inhibition of Drd3 transcription is Treg cells, we optimised the protocol of Treg transduction (Fig. 12) and confirmed that F»xp3 expression is not changed in Treg cells slier transduction wit V-ahDrd3 (Fig. 13). Afterward, we tested the therapeutic effect of Treg cells transduced with RV~shDrd3 using optimal condition (activating Treg cells with a dynabeads;Tregs ratio ~ 2; l), which was compared with the effect of Treg ceils transduced with control retroviral vectors (RV-Conlrol; codifying just for the reporter gene GFP) in the development of DSS- induced inflammatory colitis. Strikingly, the results show that the i.v. transfer of .RV-&hDr 3-transdeced Treg exerted a potent therapeutic effect attenuating disease manifestation in comparison when RV- contm!-transduced Treg were transferred (F%, 14). Importantly, the therapeutic effect exerted by RV- shDrd3-transdn.eed Treg cells was observed in multiple parameters, including the attenuation in the loss of body weight (Fig, 14A), decreased disease activity index (DAI; Fig. I4B), reduced alterations in the architecture of gut mucosa (Fig, 14C), and a lesser extern of colon shortening (Fig. 14D). Tims, these results show that the reduction of DRD3 expression in Treg cells exerts a potent therapeutic effect dampening gut inflammation.

References

SEQUENCES

SEQ ID No, 1

DRD3 mRNA mouse

1 tcgaattcct ctgtctgggc catggcacct ctgagccaga taagcagcca catcaactcc

61 acctgtgggg cagaaaactc cactggtgtc aaccgggccc gtccacatgc ctactacgcc

121 ctgtcctact gtgcactcat cctggccatc atctttggca acggtctggt atgtgcagct

181 gtgctgaggg agcgagccct acagaccacc accaactacc tagtggtgag cctggctgtg

241 gcagacctgc tggtggccac tttggtgatg ccgtgggtgg tgtacttgga ggtgacaggt

301 ggagtctgga atttcagccg catttgctgt gatgtttttg tcaccctgga tgtcatg tg

361 tgtacagcca gcatcctgaa cctctgtgcc atcagcatag acaggtacac agcagtggtc

421 atgccagttc actatcagca tggcaccggg cagagctcct gtcgacgtgt ggcgctcatg

481 attacggctg tgtgggtgct ggcctttgct gtgtcctgcc ctctcctctt tggtttcaac

541 acaacagggg atcccagcat ctgctccatc tccaaccctg attttgtcat ttactcttcg

601 gtggtgtcct t tatgttcc ctttggggtg actgtcctgg tctatgccag gatctac tg

661 gtcctgaggc aaaggcgaag aaaacggatc ctcactcgac agaacagcca gtgtatcagc

721 atcagacctg gcttccctca gcagtcttcc tqtctgcggc tgcatcccat tcggca ttt

781 tcaataaggg ccaggtttct gtcagatgcc acgggacaaa tggagcacat agaagacaaa

841 ccatatcccc agaaatgcca ggaccetetc ttgtcacatc tacagcccct ctctcctggc

SOI cagacacatg gagagctgaa acgctactac agcatctgcc aagacactgc cttgaggcat

961 ccaaacttcg aaggaggggg agggatgagc caagtggaga ggactcggaa ctcctt agc

1021 cccaccatgg cacccaagct cagcttagag gttcgaaaac tcagcaatgg caggttatcc

1061 acatccctga agctggggcc cctacagcct cggggagtac cacttcgaga gaagaaggcc

1141 acccagatgg tggtcattgt gctcggggcc ttcattgtct gttggctgcc cttcttcttg

1201 actcacqttc ttaataccca ctgtcaggca tgccacgtgt ccccagagct ttacaqaqcc

1261 acgacatggc ttggctacgt gaacagtgcc ctgaaccctg tgatctacac caccttcaac

1321 atagagttcc gcaaagcctt cctcaagatt ctatcctgct gaaggaggag aagagacatt

1381 ccctttaccc acttcaagat gccaggcagt ttggaccccc aggagggtct ggttggaatg

1441 actgccctgg cctct

SEQ ID No, 2

DRD3 mRNA human ggtaaactcc tcggtctcca gaaatcagaa gaaaatttta ggaaagagaa caaataatta

61 aaactctgta agtcttaatg aggtgctaag gaggaacccc acgaatgttt caggaagact

121 gttattcagc actgagggat tgaacatcag caaagcagga caaatgtcat aactgatggg

181 gacctgacaa ctctctgttc ccctgccttt ttaaataggt tatgcactca ctctaagaga

241 tgccagaaca aactataaag accaagataa atcaatactt tgt aggca ttaaaagctc

301 attaaaatgt gaagcccctt ggcatcacgc acctccctct gggctatggc atctctgagc

361 cagctgagta gccacctgaa ctacacctgt qqqqcagaga actccacagg tgccagccag 421 gcccgcccac atgcctacta tgccctctcc tactgcgcgc tcatcctggc catcgtcttc

481 ggcaatggcc tggtgtgcat qqctg tgctg aaggagcggg ccctgcagac taccaccaac 541 tacttagtag tgagcctggc tgtggcagac ttgctggtgg ccaccttggt gatgccctgg 601 gtggtatscc tggaggtgac aggtggagtc tggaatttca gccacatttg ctgtgatgtt 661 tttgtcaccc tggatgtcat gatgtgtaca gccagcatcc ttaatctctg tgccatcagc

721 a tagacaggt acactgcagt ggtcatgccc gttcactacc agcatggcac gggacagagc 781 tcctgtcggc gcgtggccct catgatcacg gccgtctggg tactggcctt tgctgtgtcc

841 tgccctcttc tgtttggctt taataccaca qqqqacccca ctgtctgctc catctccaac 901 cc gattttg tcatctactc ttcag ggtg tccttctacc tgccctttgg agtgactgtc 961 cttgtctatg ccagaatcta tgaggtgctg aaacaaagga gacggaaaag gatcctcact

1021 cgacagaaca gtcagtgcaa cagtgtcagg cctggcttcc cccaac a c cctctctcct 1081 gacccggcac atctggagct gaagcgttac tacagcatct gccaggacac tgccttgggt

1141 ggaccaggct tccaagaaag aggaggagag 11gaaaagag aggagaagac tcggaa11cc

1201 ctgagtccca ccatagcgcc caagctcagc ttagaagttc gaaaactcag caatggcaga

1261 t: t tcgacat ctttgaagct gggg cccccg caacctcggg gagtgcca ct tcgggagaag

1321 aaggcaaccc aaatggtggc cattgtgctt ggggccttca ttgtctgctg gctgcccttc

1381 11c11gaccc a t:g 11ctcaa tacccactgc cagaca t:gcc acgtgtcccc agagc111ac

1441 agtgccacga catggctggg ctacgtgaat agcgccctca accctgtgat ctataccacc

1501 ttcaatatcg agttccggaa agccttcctc aagatcctgt cttqctgagg g ge

SEQ ID No, 3

small hairpin RNA (shRNA) against DRD3 in mouse

5 T GCCCT CT CCT CTTT GGTTT C AACAC AAC 3 ^'

Claims

1. A method for treating got inflammation in a mammal in need comprising administering to the mammal a pharmaceutical composition comprising one or more agents that inhibit, attenuate, disrupt and/or block the biological activity of DRD3 in the mammal.

2. A method for increasing a migration of regulatory' T cells into gut mucosa in a mammal in need comprising administering to the mammal a pharmaceutical composition comprising one or more agents that inhibit, atenuate, disrupt and/or block the biological activity of DRD3 in the mammal

3. A method for increasing regulatory CD4+ T-cells (Treg) suppressive activity into gut mucosa in a mammal in need comprising administering to the mammal a pharmaceutical composition comprising one or more agents that inhibit, attenuate, disrupt and/or block the biological activity of DRD3 in the mammal.

4. The method of any precedent claims 1 to 3, wherein the one or more agents inhibit, attenuate, disrupt and/or block the biological activity of^'DRD3 specifically expressed in CD4+ T-Cells.

5. The method of claim 4, wherein the one or more agents inhibit, attenuate, disrupt and/or block the biological activity of DRD3 specifically expressed in CD4+ T-Ceils, is particularly in the subpopulation of regulatory CD4+ T-cells (Treg).

6. The method of claim 5, wherein the one or more agents that inhibit, attenuate, disrupt and/or block the biological activity of DRD3 in Treg, reduces Drcl3 transcription.

7. The method of claim 6, wherein the one or more agents that inhibit, attenuate, disrupt and/or block the biological activity of DRD3 in Treg, is an RNA product that reduces DRD3 transcription.

8. The method according to claim 7, wherein the RNA product is an shRNA.

9. The method of claim 8, wherein the mammal in need suffer of an inflammatory bowel disease.

10. The method of claim 9, wherein said inflammatory bow el disease is Crohn Disease.

1 1. The method of claim 9, wherein said inflammatory bowel disease is Ulcerative Colitis.

12. A method for treating gut inflammation in a mammal in need, comprising the step of transducing a Treg cell of the mammal with a retroviral vector containing a nucleic acid encoding an RNA product that reduces Drd3 transcription in the mammal.

13. A method for increasing a migration of regulatory T cells into gut mucosa in a mammal in need, comprising the step of transducing a Treg cell of the mammal with a retroviral vector containing a nucleic acid encoding an RNA product that reduces Drd3 transcription in the mammal.

14. A method for increasing Treg suppressive activity into gut mucosa in a mammal in need, comprising the step of transducing a Treg cell of the mammal with a retroviral vector containing a nucleic acid encoding an RNA product that reduces Drd3 transcription in the mammal.

15. The method according to claim 12, 13 or 14, wherein the RNA product is an sliRNA.

16. A method for treating gut inflammation in a mammal in need thereof comprising the step of introducing Drd3-deficient Treg cells into the mammal to cause an increase in migration of regulatory T cells into gut mucosa of the mammal .

17. A method for treating gut inflammation in a mammal in need thereof comprising the step of introducin Drd3 -deficient Treg cells into the mammal to cause an increase in Treg suppressive activity into gut mucosa of the mammal.

18. The method according to claim 16 or 17, wherein the I>rd3 -deficient Treg cells are introduced into the mammal intravenously.

19. The method of claim 18, wherein the mammal in need suffer of an inflammatory bowel disease.

20. The method of claim 19, wherein said inflammatory bowel disease is Croh Disease..

21. The method of claim 19, wherein said inflammatory bowel disease is Ulcerative Colitis.

22. The method of any one of claims 1 , 2, 3, 12, 13, 14, 16 or 17, wherein the method further comprises administering one or more additional therapies.

23. The method of claim 22, wherein the one or more additional therapies comprise anti inflammatory" bowel disease therapy.

24. The method of claim 23, wherein the additional anti-inflammatory bowel disease therapy comprises therapeutics selected from the group consisting of neutralizers of anti-TNFalpha (infliximab (Remicade), adalimumab (Hu ira), go!imumab (Simponi), Certolizumab), afpha4 integrin subunit inhibitors (natalizumab (Tysabri)), alpha4beta7 integrin inhibitors (vedo!izumab (Entyvio)), IL-12Rbetal blocker that avoid its union to ligands IL-12 and IL-23 (ustekinurnab (Stelara)), anti-MAd-CAM-1 monoclonal antibody (PF-00547659), anti -IL-23 monoclonal antibody (MEDI2070), sphingosine-1 -phosphate (SIP) receptor agonist (Ozanimod), antisense oligodeoxynucleotide complementary to mRNA of Smad? (Mongersen), aminosalicylates (5-ammosalicylic acid (5-ASA); 5-ASA derivatives (Sulfasalazine, Asacol HD, Delzicol, Pentasa, Colazal, Dipentum, Lialda, Apriso, Mesalazine), corticosteroids (hydrocortisone, methylprednisolone, prednisone, Budesonide, Budesonide MMX, hydrocortisone), antibiotics (ciprofloxacin, metronidazole, rifaximin, omidazole, tinidazole, anti-tuberculosis therapy, macrolides, fluoroquinolones, 5- nitroimidazoles, antimycobacterials), and inimimomodulators (thiopurines (Purinethol, Purixan), 6-mercaptopurine, azathioprine (Azasan, Imuran), methotrexate (Trexall), cyclosporine (Gengraf, Neoral, Sandimmune).

25. An agent that inhibit, attenuate, disrupt and/or block the biological activity of DRD3 specifically expressed in regulatory CD4+ T-Cells.

26. The agent according to claim 25, wherein is an RNA product that reduces DRD3 transcription.

27. The agent of claim 26, wherein the RNA product have the ability to hybridize under defined conditions to a nucleic acid sequence selected from the group consisting of SEQ ID No.l and SEQ ID No.2.

28. The agent of claim 27, wherein the RNA product is an shRNA.

29. The agent of claim 27, wherein the shRNA product is defined by SEQ ID No.3.

30. A polynucleotide encoding, upon expression, the agent of claim 25, 26, 27, 28 or 29.

31 A genetic vector that comprises a polynucleotide of claim 30.

32. The genetic vector of claim 31, where the genetic vector is a viral vector.

33. The genetic vector of claim 32, where the viral vector is a retroviral vector.

34. A CD4+ T-cells that comprises a genetic vector of claims 31.

35. A CD4+ T-cells of claim 34, wherein the CD4+ T-cells is a Treg.

36. A pharmaceutical composition comprising at least one agent of claims 25, 26, 27, 28 or 29, and a pharmaceutically acceptable carrier, diluent or excipient.

37. The pharmaceutical composition of claim 36, for use in the treatment of inflammatory bowel diseases.

38. The pharmaceutical composition of claim 37, for use in the treatment of Crohn disease.

39. The pharmaceutical composition of claim 37, for use in the treatment of Ulcerative Colitis.

40. A kit comprising the pharmaceutical composition of claim 36, and instructions for administering the pharmaceutical composition to treat gut inflammation in a mammal.

41. A kit comprising the pharmaceutical compositions of claim 40, and instructions for administering the pharmaceutical composition to treat an inflammatory bowel disease.

42. A kit comprising the pharmaceutical compositions of claim 41, and instructions for administering the pharmaceutical composition to treat Crohn Disease or Ulcerative Colitis.

43. A kit comprising the pharmaceutical composition of claim 36, and instructions for administering the pharmaceutical composition to increase a migration of Treg into gut mucosa in a mammal in need.

44. A kit comprising the pharmaceutical composition of clai 36, and instructions for administering the pharmaceutical composition to increase Treg suppressive activity into gut mucosa in a mammal in need.

45. Use of the agent of claim 25, 26, 21, 28 or 29, for preparation of a medicament for treatment of inflammatory bowel diseases.

46. Use of the agent of claim 45 for preparation of a medicament for treatment of Crohn disease.

47. Use of the agent of claim 46 for preparation of a medicament for treatment of Ulcerative Colitis.

48. Use of the pharmaceutical composition of claim 36 for preparation of a medicament for treatment of inflammatory bowel diseases.

49. Use of the pharmaceutical composition of claim 48 for preparation of a medicament for treatment of Crohn disease.

50. Use of tire pharmaceutical composition of claim 48 for preparation of a medicament for treatment of Ulcerative Colitis.