WO2013013708A1

WO2013013708A1 - Treatment of acute rejection in renal transplant

Info

Publication number: WO2013013708A1
Application number: PCT/EP2011/062849
Authority: WO
Inventors: Josep Maria GRINYÓ I BOIRA; Oriol BESTARD MATAMOROS; Josep Maria Cruzado Garrit; Juan TORRES AMBROS; Jose Maria ̠ARAN PERRAMON
Original assignee: Fundació Institut D'investigació Biomèdica De Bellvitge
Priority date: 2011-07-26
Filing date: 2011-07-26
Publication date: 2013-01-31

Abstract

The invention is related to methods and compositions involving CD40 silencing by RNA interference and an immunosuppressive agent for preventing the rejection of kidney transplant in mammals and increasing tolerance in a recipient mammal to a kidney transplant.

Description

TREATMENT OF ACUTE REJECTION IN RENAL TRANSPLANT

TECHNICAL FIELD The present invention relates to the field of immunomodulation and, more in particular, to methods for preventing rejection of kidney transplant based on the silencing of CD40 using RNA interference strategies.

BACKGROUND ART

Tissue and organ transplants save many lives threatened by disease and cancer eachyear. Allogeneic grafts (or allografts) from human donor skin, kidney, liver, pancreas andheart are now commonplace, and xenogeneic grafts (or xenografts) from non-human mammalian donor organisms are also being studied for their potential use as a broadly available source of tissues and organs.

The long term success of allogeneic and xenogeneic grafts requires that the graft survives the host's immune surveillance of foreign antigens. It has long been recognized that the normally functioning immune system of the transplant recipient recognizes the transplanted organ as "non-self tissue and thereafter mounts an immune response to the presence of the transplanted organ. Left unchecked, the immune response will generate a plurality of cells and proteins that will ultimately result in the loss of biological functioning or the death of the transplanted organ. Therefore the long term success of allogeneic and xenogeneic transplants requires that immune responses mounted by the recipient against the donor graft be suppressed or prevented.

In order to prevent host immune rejection of tissue and organ grafts, transplant recipients are typically treated with one or more cytotoxic agents in an effort to suppress the transplant recipient's immune response against the transplanted organ or tissue. For example, cyclosporine (cyclosporin A), a cyclic polypeptide consisting of 11 amino acid residues and produced by the fungus species Tolypocladium inflatum Gams, is currently the drug of choice for administration to the recipients of allogeneic kidney, liver, pancreas and heart (i. e., wherein donor and recipient are of the same species of mammals) transplants.

However, administration of cyclosporin is not without drawbacks as the drug can cause kidney and liver toxicity as well as hypertension. Moreover, use of cyclosporin can lead to malignancies (such as lymphoma) and lead to opportunistic infection due to the "global" nature of the immunosuppression it induces in patients receiving long term treatment with the drug, i. e., the hosts normal protective immune response to pathogenic microorganisms is down-regulated thereby increasing the risk of infections caused by these agents. Other drugs for the prevention of graft rejection include: FK- 506 (which has a similar mode of action as cyclosporine and is thought to be as potent as cyclosporin in its immunosuppressive qualities while having fewer toxic side effects than cyclosporin); steroids, such as prednisone, methylprednisalone, and Azathioprine (an analog of 6-mercaptopurine) (which are non-specific immunosuppressive drugs used to prolong allograft survival in transplantation recipients); and monoclonal antibodies (such as OKT3 monoclonal antibodies which are directed against the CD3 antigen present on T-cells and which have also been employed as non-specific immunosuppressive therapeutic agents in allograft recipients). Another strategy used to suppress host immune rejection of graft cells in total lymphoid irradiation (TLI), another form of non-specific immunosuppressive therapy that has been used clinically and experimentally to prolong allograft survival. The radiation exposure and treatment schedule for TLI were developed for the treatment of Hodgkin's disease and were subsequently found to be immunosuppressive. TLI induces production of the "global" immunosuppression mentioned above and has the same limitations of other global immunosuppressive therapies.

A particularly severe drawback of the immunosuppressive drug therapies is that they must be administered indefinitely to suppress allogeneic graft rejection, and tolerance to the foreign tissue does not develop. Moreover, the general non-specific suppression of recipient allograft rejection by drug or irradiation treatment carries the risk of increased susceptibility to infection and malignancy. Accordingly, alternative means of preventing host rejection of allogeneic or xenogeneic grafts would be desirable The humoral branch of the immune response plays an important role in both acute and chronic allograft rejection. The appearance of alloantibodies after renal transplantation is a critical setback that results in allograft loss. Ligation of CD40 on B-cells is crucial for antibody production, which can be inhibited or delayed by the blockade of the CD40/CD40L pathway. The co-stimulatory molecule CD40,also known as tumor necrosis factor receptor superfamily member 5 (TNFRSF5), is a type-I trasmembrane receptor. CD40 is constitutively expressed on a wide variety of cells, such as B and T lymphocytes, antigen presenting cells (APC), dendritic cells (DC), macrophages and activated endothelial and renal tubular cells. Its ligand, CD40L, also known as CD 154, is expressed more broadly than initially believed. In renal transplantation, CD40 is over- expressed on tubular epithelial cells, macrophages and Tcells. CD40 expression is homogeneous in the tubulo-interstitium in all grades of acute rejection, while in grafts suffering acute vascular rejection, grades II and III, it is strongly expressed on endothelial cells. Vascular damage involves strong activation of the endothelium and it is well known that CD40 actively participates in these processes (Pluvinet R et al. Blood 2008; 112: 3624-3637).

The interference of the interaction CD40-CD40L by using anti-CD40 antibodies as antagonists has been attempted as therapeutic strategy against graft rejection. Potentially the CD40-CD40L interaction can be inhibited by targeting either CD40 or CD40L.

The use of anti-CD40L antibodies as therapeutic strategy against graft rejection has been described e.g. in U.S. Patent No. 5,876,718 (Noelle et al.), which discloses methods of inducing T cell non-responsiveness to transplanted tissues and of treating graft-versus-host disease with anti-CD40L monoclonal (mouse) antibodies. EP0742721B1 (Noelle et al.) discloses methods of inhibiting a humoral immune response to a thymus-dependent antigen that use anti-CD40L monoclonal (mouse) antibodies. U.S. Patent No. 6,375,950 describes methods for inducing T cell unresponsiveness to donor tissue or organs in a transplant recipient through use of anti- CD40L monoclonal (murine) antibodies. However, the use of anti-CD40L monoclonal antibodies results in thromboembolic complications derived from the activation and aggregation of platelets, which express CD40L (Kawai T et al. Nat. Med. 2000; 6: 114) On the other hand, human clinical studies using an anti-human CD 154 Mab have been halted because of thromboembolic events. As this anti-CD 154 Mab is constructed as a humanized IgGl, which binds extremely well to Fc-receptors, the anti-CD 154 Mab may cross-link CD154 to Fc- receptors resulting in the formation of blood clots. Recently, CD40 was reported to be constitutively expressed on platelets and found to be functionally important using soluble CD 154 as stimulus for platelet activation.

Therefore, it is necessary to develop alternative strategies for disrupting CD40 signalling capable of preventing graft rejection and that overcome the problems associated to the methods based on the use of anbti-CD40 antibodies.

SUMMARY OF THE INVENTION In one aspect, the invention relates to a method A method for increasing tolerance in a recipient mammal to a kidney transplant that comprises the administration to said subject of an interfering RNA that silences CD40 gene expression or a polynucleotide coding for said interfering RNA.

In another aspect, the invention relates to a method for the treatment or prevention of acute rejection of a renal transplant in a subject in need thereof that comprises the administration to said subject of an interfering RNA that silences CD40 gene expression or a polynucleotide coding for said interfering RNA.

In a further aspect, the invention relates to a pharmaceutical composition that comprises an interfering RNA that silences CD40 gene expression or a polynucleotide coding for said interfering RNA and an immunosuppressive agent.

Finally, the invention relates to a method for the prevention and/or the treatment of acute rejection in renal transplant in a subject in need thereof that comprises the administration of an interfering RNA that silences CD40 gene expression or a polynucleotide coding for said interfering RNA and an immunosuppressive agent.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the knock-down efficiency of siRNA sequences against rat CD40. The synthesized siRNAs were assayed in HEK293 cells over-expressing a luciferase-rat CD40 partial cDNA gene fusion. Results are given as mean values + SD from three independent experiments performed in triplicate. ANOVA, *p< 0.05 vssiRNA-C.

Figure 2 shows the cumulative survival of rats after CD40 siRNA administration. The siCD40 group doubled the mean survival time (MST) of NoTreat group. The combination of both treatments significantly prolonged MST. In both siRNA treated groups, seven rats survivedmore than the mean survival time. Log Rank test, P=0.0009. a vs NoTreat, b vs Rp.

Figure 3 shows representative photomicrographs of kidneys. A to D: Haematoxylin/Eosin, 400x; E to H: IL-7R immunostaining, 200x. A: Grafts treated with unspecific siRNA (NoTreat); B: Grafts treated with sub-therapeutic rapamycin (Rp); C: Grafts treated with rat anti-CD40 siRNA (siCD40); D: Grafts treated with the combination therapy (siCD40-Rp); E: NoTreat graft showing intense IL-7R expression in tubuli (asterisk) and vessels (arrow); F: Rp; G: siCD40; H: siCD40-Rp. Figure 4 shows apelin expression. 1) mRNA quantification of apelin expression in the kidney. Values are expressed as fold time respect to non transplanted kidney tissue. ANOVA, Scheffe's test P=0.0626, b vsRp, c vssiCD40. 2) Semi-quantification of apelin expression in tubular epithelial cells, glomeruli and vascular epithelial cells, graded from 0 to 4+. Kruskall- Wallis test, P<0.005 a vsNoTreat, b vsRp, c vs siCD40. 3) Representative photomicrographies of apelin immunostaining (200x). Apelin expression was detected in a variable degree in tubular epithelial cells (asterisk), glomeruli(arrow head) and vascular epithelial cells (arrow). Apelin was slightly positive in NoTreat groupwith severe acute vascular damage (A), Rp group (B) and siCD40-Rp group (D); siCD40 groupshowed an intense immunostaining in the vessels and glomeruli (C). Detail (400x) of NoTreat(E) and siCD40 (F) groups. Note the intense staining of the vessel (arrow) in the siCD40 group.

DETAILED DESCRIPTION OF THE INVENTION Methods for increasing tolerance in a recipient subject to a donor kidney and for the treatment or prevention of acute rejection of a renal transplant The authors of the present invention have found that the silencing of CD40 gene expression by RNA interference results in increased tolerance of a kidney when transplanted into a donor subject. This increased tolerance is observed as an increased survival of the transplanted subject (see example 2), in decreased degree of histological rejection findings in the transplanted organ (see example 3) as well as in a decreased expression of intra-graft mediators of the immune response (see example 5).

Thus, in a first aspect, the invention relates to a method (hereinafter first method of the invention) for increasing tolerance in a recipient mammal to a kidney transplant that comprises the administration to said subject of an interfering RNA that silences CD40 gene expression or a polynucleotide coding for said interfering RNA.

The term "tolerance" according to the invention refers to an immunological tolerance. "Immunonoligicaltolerance," as used herein, refers to a state wherein the immune system of a fully immunocompetent transplant recipient subject mammal is non- responsive to the transplantwithout the need for long-term immunosuppression, leading to a lack of rejection of the transplant.

The term "increasing tolerance", as used herein, refers to decreasing the severity of or eliminating one or more of the general characteristics of graft rejection. Such characteristics are the result of an immune response directed against the graft (foreign) tissue and include, for example, progressive infiltration of mononuclear cells, such as lymphocytes, into the foreign tissue, production of lymphocytotoxic antibodies, cytolysis, necrosis, vasculitis, hemorrhage and fibrosis. Another readily observable indication of improved tolerance will be prolonged survival of transplanted graft tissue in a recipient as compared to a non-immunosuppressed recipient (control). The increase in tolerance to a kidney transplant can be measured as a reduction in any of the signs mentioned above with respect to those appearing in a control immunocompetent recipient subject which has received a kidney transplant and which has not received any treatment aimed at increasing the tolerance to the transplant. Thus, the method according to the present invention allows a reduction in the one or more of the above signs of rejection of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% (i.e. complete abolishment of any sign of rejection).

In another aspect, the invention is related to a method for the prevention and/or the treatment of acute rejection in a renal transplant in a subject in need thereof that comprises the administration to said subject of an interfering RNA that silences CD40 gene expression or a polynucleotide coding for said interfering RNA.

Alternatively, the invention is related to an interfering RNA that silences CD40 gene expression or a polynucleotide coding for said interfering RNA for its use in the treatment and/or the prevention of acute rejection in a renal transplant in a subject in need thereof.

Alternatively, the invention is related to the use of an interfering RNA that silences CD40 gene expression or a polynucleotide coding for said interfering RNA to obtain a medicament for its use in the treatment and/or the prevention of acute rejection in a renal transplant in a subject in need thereof.

The term "prevention" is understood to mean the administration of an oligonucleotide according to the invention or of a medicament containing it in an initial or early stage of the disease, or also to avoid its appearance.

As used herein, the term "treatment" refers to both therapeutic measures and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the acute rejection after a renal transplant. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented. The term "acute rejection" refers to an immune reaction evoked by allografted organs. The acute rejection has its onset 2-60 days after transplantation, and it is characterized by interstitial vascular endothelial cell swelling, interstitial accumulation of lymphocytes, plasma cells, immunoblasts, macrophages, neutrophils; tubular separation with edema/necrosis of tubular epithelium; swelling and vacuolization of the endothelial cells, vascular edema, bleeding and inflammation, renal tubular necrosis, sclerosed glomeruli, tubular 'thyroidization'.

The term "renal transplant", also called kidney transplant, refers to the replacement of a diseased, damaged, or missing kidney with a donor kidney.

By "subject" or "individual" or "animal" or "patient" or "mammal," is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.

There are several types of immunological attacks made by the recipient against the donor organ which can lead to rejection of the allograft including cellular rejection (acute), humoral rejection, and chronic rejection (vasculopathy, chronic allograft nephropathy, bronchiolitis obliterans syndrome).

Humoral rejection (also referred herein to as antibody-mediate rejection or ABMR) includes hyperacute rejection (HAR) or accelerated humoral rejection (ACHR) and is a type of rejection characterized by acute allograft injury that is resistant to potent anti-T cell therapy, by the detection of circulating donor specific antibodies, and the deposition of complement components in the graft. ABMR with elevated circulating alloantibodies and complement activation that occurs in 20-30 percent of acute rejection cases has a poorer prognosis than cellular rejection. In a preferred embodiment, the methods according to the present invention act by decreasing humoral rejection of ABMR against the transplanted kidney.

In accordance with the present invention, the donor and/or the recipient of the graft may be mammal. In one embodiment, at least the recipient is human. The donor may be alive or deceased at the time the graft is removed. The graft may be an autograft, an allograft or a xenograft. In one embodiment, the graft is an allograft.

The expression "an interfering RNA that silences CD40 gene expression", as used herein, relates to a RNA molecule which is capable of causing degradation of CD40 the mRNA and an inhibition of translation by the process of RNA interference. Suitable means for determining whether a given interfering RNA is capable of silencing CD40 include any means for determining the levels of the CD40 mRNA in a sample, including RT-PCR, Northern blot and the like as well as any means for determining the levels of CD40 protein, including immunological methods such as ELISA, Western blot, immunohistochemistry. An interfering RNA is considered as capable of silencing CD40 when cells treated with the interfering RNA or which express the interfering RNA as a consequence of having been contacted with a polynucleotide encoding said interfering RNA when it results in a decrease of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% in the levels of the CD40 mRNA or CD40 protein with respect to the same cells which have not been contacted with the interfering RNA or the polynucleotide encoding said interfering RNA. The expression "RNA interference" or RNAi is a process of sequence-specific post- transcriptional gene repression which can occur in eukaryotic cells. In general, this process involves degradation of an mRNA of a particular sequence induced by double- stranded RNA (dsRNA) that is homologous to that sequence. This dsRNAis capable of causing the silencing of gene expression by means of converting said RNA into siRNA by means of an RNase type III (Dicer). One of the siRNA strands is incorporated into the ribonucleoprotein complex referred to as the RNA-induced silencing complex (RISC). The RISC complex uses this single strand of RNA to identify mRNA molecules that are at least partially complementary to the RNA strand of the siRNA incorporated in the RISC that are degraded or undergo an inhibition in their translation. Thus, the siRNA strand that is incorporated into the RISC is known as a guide strand or antisense strand. The other strand, which is known as a transient strand or sense strand, is eliminated from the siRNA and is partly homologous to the target mRNA. The degradation of a target mRNA by means of the RISC complex results in a reduction in the expression levels of said mRNA and of the corresponding protein encoded thereby. Furthermore, RISC can also cause the reduction in the expression by means of the inhibition of the translation of the target mRNA. The invention contemplates the use of interfering RNA specific for CD40 as such as well as the use of polynucleotides encoding for said interfering RNA.

As used herein, the term "specific for CD40" refers to small inhibitory RNA duplexes that, by means of showing a substantial degree of sequence complementarity with CD40 mRNA, induce the RNA interference (RNAi) pathway to negatively regulate gene expression of CD40.

As used herein, and unless otherwise indicated, the term "complementary," when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 degrees centigrade or 70 degrees centigrade for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

Specific interfering RNAs include RNAs that show base-pairing to the target polynucleotide over the entire length of the first and second nucleotide sequence. Such sequences can be referred to as "fully complementary" with respect to each other herein. However, where a first sequence is referred to as "substantially complementary" with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as "fully complementary." "Complementary" sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non- Watson-Crick base pairs includes, but not limited to, G:U Wobble or Hoogstein base pairing.

The terms "complementary", "fully complementary" and "substantially complementary" herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide which is "substantially complementary to at least part of a messenger RNA (mRNA) refers to a polynucleotide which is substantially complementary to a contiguous portion of the mRNA of interest (e.g., encoding target gene). For example, a polynucleotide is complementary to at least a part of a target gene mRNA if the sequence is substantially complementary to a non-interrupted portion of a mRNA encoding target gene. As used herein the term "oligonucleotide" embraces both single and double stranded polynucleotides. The double stranded oligonucleotides used to effect RNAi are preferably less than 50 base pairs in length and, more preferably, comprise about 25, 24, 23, 22, 21, 20, 19, 18 or 17 base pairs of ribonucleic acid. Optionally the dsRNA oligonucleotides of the invention may include 3' overhang ends. Exemplary 2-nucleotide 3' overhangs may be composed of ribonucleotide residues of any type and may even be composed of 2'- deoxythymidine residues, which lowers the cost of RNA synthesis and may enhance nuclease resistance of siRNAs in the cell culture medium and within transfected cells (see Elbashir et al, Nature 411 : 494-8, 2001). Exemplary concentrations of dsRNAs for effecting RNAi are about 0.05 nM, 0.1 nM, 0.5 nM, 1.0 nM, 1.5 nM, 25 nM or 100 nM, although other concentrations may be utilized depending upon the nature of the cells treated, the gene target and other factors readily discernable to the skilled artisan. Exemplary dsRNAs may be synthesized chemically or produced in vitro or in vivo using appropriate expression vectors. Exemplary synthetic RNAs include 21 nucleotide RNAs chemically synthesized using methods known in the art (e.g., Expedite RNA phosphoramidites and thymidine phosphoramidite (Proligo, Germany). Synthetic oligonucleotides are preferably deprotected and gel-purified using methods known in the art (see, e.g., Elbashir et al., Genes Dev. 15: 188-200, 2001). Longer RNAs may be transcribed from promoters, such as T7 RNA polymerase promoters, known in the art. A single RNA target, placed in both possible orientations downstream of an in vitro promoter, will transcribe both strands of the target to create a dsRNA oligonucleotide of the desired target sequence. Any of the above RNA species will be designed to include a portion of nucleic acid sequence represented in a target nucleic acid, such as, for example, a nucleic acid that hybridizes, under stringent and/or physiological conditions, to the polynucleotide encoding human CD40.

The specific sequence utilized in design of the interfering RNA for use according to the present invention may be any contiguous sequence of nucleotides contained within the expressed CD40 gene message. Programs and algorithms, known in the art, may be used to select appropriate target sequences. In addition, optimal sequences may be selected utilizing programs designed to predict the secondary structure of a specified single stranded nucleic acid sequence and allowing selection of those sequences likely to occur in exposed single stranded regions of a folded mRNA. Methods and compositions for designing appropriate oligonucleotides may be found, for example, in U.S. Pat. No. 6,251,588, Birmingham, A et al. 2007, Nature Protocols, 2:2068-2078, Ladunga, I. 2006, Nucleic Acids Res. 35:433-440 and Martineau, H., Pyrah, L, 2007, Toxicol. Pathol, 35 :327-336 and Pei and Tuschl, 2006, Nature Methods, 3 :670-676, the contents of which are incorporated herein by reference. Messenger RNA (mRNA) is generally thought of as a linear molecule which contains the information for directing protein synthesis within the sequence of ribonucleotides, however studies have revealed a number of secondary and tertiary structures that exist in most mRNAs. Secondary structure elements in RNA are formed largely by Watson-Crick type interactions between different regions of the same RNA molecule. Important secondary structural elements include intramolecular double stranded regions, hairpin loops, bulges in duplex RNA and internal loops. Tertiary structural elements are formed when secondary structural elements come in contact with each other or with single stranded regions to produce a more complex three dimensional structure. A number of researchers have measured the binding energies of a large number of RNA duplex structures and have derived a set of rules which can be used to predict the secondary structure of RNA (see, e.g., Jaeger et al, Proc. Natl. Acad. Sci. USA 86: 7706, 1989; and Turner et al, Annu. Rev. Biophys. Biophys.Chem. 17: 167, 1988). The rules are useful in identification of RNA structural elements and, in particular, for identifying single stranded RNA regions which may represent preferred segments of the mRNA to target for silencing RNAi, ribozyme or antisense technologies. Accordingly, preferred segments of the mRNA target can be identified for design of the RNAi mediating dsRNA oligonucleotides as well as for design of appropriate ribozyme and hammerhead ribozyme compositions of the invention. Different types of molecules, such as small interfering RNA and short hairpin RNA, have been used effectively in the RNAi technology.

Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of double-stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway where the siRNA interferes with the expression of a specific gene. In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related pathways, e.g., as an antiviral mechanism or in shaping the chromatin structure of a genome. Synthetic siRNAs have been shown to be able to induce RNAi in mammalian cells. This discovery led to a surge in the use of siRNA/RNAi for biomedical research and drug development. A siRNA can be chemically synthesised or can be obtained through in vitro transcription. siRNAs typically consist of a double RNA strand with a length between 15 and 40 nucleotides and can contain a 3' and/or 5' overhanging region with 1 to 6 nucleotides. The length of the overhanging region is independent of the total length of the siRNA molecule. siRNAs act by means of the degradation or the post-transcriptional silencing of the target messenger. The siRNAs of the invention are substantially homologous with a pre-selected region of the target CD40 mRNA. "Substantially homologous" is understood as that they have a sequence which is sufficiently complementary or similar to the target mRNA such that the siRNA is capable of causing the degradation thereof by RNA interference. The siRNAs suitable for causing said interference include siRNAs formed by RNA, as well as siRNAs containing different chemical modifications such as:

siRNAs in which the bonds between the nucleotides are different from those that occur in nature, such as phosphorothioate bonds,

conjugates of the siRNA strand with a moiety that promotes penetration of the siRNA into biological membranes. In a particular example, the siRNA may be modified by coupling to a cholesterol molecule. The cholesterol conjugate may be coupled to the 5' or to the 3' end of the siRNAwith a functional reagent, such as a fluorophore,

modifications of the ends of the siRNA strands, particularly the 3' end by means of the modification with different functional groups of the hydroxyl in position 2',

nucleotides with modified sugars such as O-alkylated moieties in position 2' such as 2'-0-methylribose p 2'-0-fluororibose,

nucleotides with modified bases like halogenated bases (for example 5- bromouracil and 5-iodouracil), alkylated bases (for example 7- methylguanosine).

In a preferred embodiment, the siRNAs for use according to the present invention comprise two overhanging nucleotides at the 3' end of each of the RNA strands, are stabilized with a partial phosphorothioatebackbone, contains 2'-0-methyl sugarmodificationon the sense and antisense strands and additionally has a cholesterol conjugate to the 3' end of the sense strand by means of a pyrrolidine linker. The siRNAs of the invention can be obtained using a series of techniques well-known to a person skilled in the art. For example, the siRNA can be chemically synthesised starting from ribonucleosides protected with phosphoramidite groups in a conventional DNA/RNA synthesizer. Short hairpin RNA (shRNA) is yet another type of RNA that may be used to effectRNAi. AnshRNA is a RNA molecule formed by two antiparallel strands connected by a hairpin region and wherein the sequence of one of the antiparallel strands is complementary to a pre-selected region in the target mRNA. The shRNAs are formed by a short antisense sequence (with 19 to 25 nucleotides), followed by a loop of 5 to 9 nucleotides followed by the sense strand. shRNAs can be chemically synthesized from ribonucleosides protected with phosphoramidite groups in a conventional DNA/RNA synthesizer or they can be obtained from a polynucleotide by means of in vitro transcription. shRNAs are processed inside the cell by the RNase Dicer that eliminates the hairpin region giving rise to siRNAs as has been previously described. shRNAs can also contain distinct chemical modifications as has been previously described in the case of siRNAs.

Currently, short-interfering RNAs (siRNAs) and short-hairpin RNAs (shRNAs) are being extensively used to silence various genes to silence functions carried out by the genes. It is becoming easier to harness RNAi to silence specific genes, owing to the development of libraries of ready-made shRNA and siRNA gene-silencing constructs by using a variety of sources. For example, RNAi Codex, which consists of a database of shRNA related information and an associated website, has been developed as a portal for publicly available shRNA resources and is accessible at http://codex.cshl.org. RNAi Codex currently holds data from the Hannon-ElledgeshRNA library and allows the use of biologist-friendly gene names to access information on shRNA constructs that can silence the gene of interest. It is designed to hold user-contributed annotations and publications for each construct, as and when such data become available. Olson et al. (Nucleic Acids Res. 34(Database issue): D153-D157, 2006, incorporated by reference) have provided detailed descriptions about features of RNAi Codex, and have explained the use of the tool. All these information may be used to help design the various siRNA or shRNA targeting AMPA receptor or other proteins of interest.

In another aspect, the invention contemplates the use of a polynucleotide which encodes for the interfering RNA specific for CD40.

A "polynucleotide", "nucleic acid," or "nucleic acid molecule" is a polymeric compound comprised of covalently linked subunits called nucleotides. Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single-stranded or double-stranded. DNA includes cDNA, genomic DNA, synthetic DNA, and semi- synthetic DNA. The "polynucleotide coding for an interfering RNA that silences CD40 gene expression" is a polynucleotide the transcription of which gives rise to the previously described siRNA or shRNA. This polynucleotide comprises a single promoter region regulating the transcription of a sequence comprising the sense and anti sense strands of the shRNAs and miRNAs connected by a hairpin or by a stem-loop region. In principle, any promoter can be used for the expression of the shRNAs and miRNAs provided that said promoters are compatible with the cells in which the siRNAs are to be expressed. Thus, the promoters suitable for carrying out this invention include those for the expression of genes whose expression is specific of renal cells. Gene promoters specific of renal cells include, but are not limited to, the uromodulin promoter, the Tamm- Horsfall protein promoter or the type 1 gamma-glutamyltranspeptidase promoter.

In addition, the polynucleotides encoding siRNAsmay comprise two transcriptional units, each formed by a promoter regulating the transcription of one of the strands formed in siRNA (sense and antisense). The polynucleotides encoding siRNAs can contain convergent or divergent transcriptional units. In the divergent transcription polynucleotides, the transcriptional units encoding each of the DNA strands forming the siRNA are located in tandem in the polynucleotide such that the transcription of each DNA strand depends on its own promoter, which can be the same or different (Wang, J. et al, 2003, Proc. Natl. Acad. Sci. USA, 100:5103-5106 and Lee, N.S., et al., 2002, Nat. BiotechnoL, 20:500-505). In the convergent transcription polynucleotides, the DNA regions giving rise to the siRNAs form the sense and antisense strands of a DNA region that is flanked by two inverted promoters. After the transcription of the sense and antisense RNA strands, they will form the hybrid corresponding to the functional siRNA.

The polynucleotides encoding for the siRNAs or for the shRNAs of the invention can be found isolated as such or forming part of vectors allowing the propagation of said polynucleotides in suitable host cells. Vectors suitable for the insertion of said polynucleotides are vectors derived from expression vectors in prokaryotes such as pUC18, pUC19, Bluescript and the derivatives thereof, mpl8, mpl9, pBR322, pMB9, ColEl, pCRl, RP4, phages and "shuttle" vectors such as pSA3 and pAT28, expression vectors in yeasts such as vectors of the type of 2 micron plasmids, integration plasmids, YEP vectors, centromere plasmids and the like, expression vectors in insect cells such as vectors of the pAC series and of the pVL, expression vectors in plants such as pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE series and the like, and expression vectors in eukaryotic cells, including baculovirus suitable for transfecting insect cells using any commercially available baculovirus system. The vectors for eukaryotic cells include preferably viral vectors (adenoviruses, viruses associated to adenoviruses such as retroviruses and, particularly, lentiviruses) as well as non-viral vectors such as pSilencer 4.1-CMV (Ambion), pcDNA3, pcDNA3.1/hyg, pHMCV/Zeo, pCR3.1, pEFI/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER- HCMV, pUB6/V5-His, pVAXl, pZeoSV2, pCI, pSVL and PKSV-10, pBPV-1, pML2d and pTDTl .

The vectors may also comprise a reporter or marker gene which allows identifying those cells that have been incorporated the vector after having been put in contact with it. Useful reporter genes in the context of the present invention include lacZ, luciferase, thymidine kinase, GFP and on the like. Useful marker genes in the context of this invention include, for example, the neomycin resistance gene, conferring resistance to the aminoglycoside G418; the hygromycinphosphotransferase gene, conferring resistance to hygromycin; the ODC gene, conferring resistance to the inhibitor of the ornithine decarboxylase (2-(difluoromethyl)-DL-ornithine (DFMO); the dihydrofolatereductase gene, conferring resistance to methotrexate; the puromycin-N- acetyl transferase gene, conferring resistance to puromycin; the ble gene, conferring resistance to zeocin; the adenosine deaminase gene, conferring resistance to 9-beta-D- xylofuranose adenine; the cytosine deaminase gene, allowing the cells to grow in the presence of N-(phosphonacetyl)-L-aspartate; thymidine kinase, allowing the cells to grow in the presence of aminopterin; the xanthine-guanine phosphoribosyltransferase gene, allowing the cells to grow in the presence of xanthine and the absence of guanine; the trpB gene of E. coli, allowing the cells to grow in the presence of indol instead of tryptophan; the hisD gene of E. coli, allowing the cells to use histidinol instead of histidine. The selection gene is incorporated into a plasmid that can additionally include a promoter suitable for the expression of said gene in eukaryotic cells (for example, the CMV or SV40 promoters), an optimized translation initiation site (for example, a site following the so-called Kozak's rules or an IRES), a polyadenylation site such as, for example, the SV40 polyadenylation or phosphoglycerate kinase site, introns such as, for example, the beta-globulin gene intron. Alternatively, it is possible to use a combination of both the reporter gene and the marker gene simultaneously in the same vector.

The interfering RNA for use in the methods of the present invention are targeted to CD40. The term "CD40" as used herein refers to a 45- to 50-kDa type I integral membrane glycoprotein also known as tumor necrosis factor receptor superfamily member 5 (TNFRSF5). This receptor has been found to be essential in mediating a broad variety of immune and inflammatoryresponses including T cell-dependent immunoglobulin classswitching, memory B cell development, and germinal centerformation.

Human CD40 gene is deposited in GenBank (version dated March 12^th 2011) with accession number NG 007279.1. Two transcripts are deposited in GenBank for the human CD40. mRNA transcript 1 (mRNAl) is the transcript variant of human CD40 that encodes the longer isoform of 1,616 bp or isoform 1. This mRNAl is deposited in GenBank with accession number NM_001250.4 mRNA transcript 2 (mRNA2), is a transcript variant of human CD40 of 1554 pb that lacks a coding segment, which leads to a translation frame shift, compared to variant mRNAl . The resulting isoform 2 contains a shorter and distinct C-terminus,compared to isoform l.The mRNA2 is deposited in GenBank with accession number M l 52854.2. Two human protein isoforms are deposited in GenBank: isoform 1 ( P 001241.1) of 277 aminoacids and isoform 2 ( P_690593.1) of 203 aminoacids.

The interfering RNAs according to the present invention may be targeted to any region of the CD40 mRNA provided that an effective silencing is achieved. Methods for determining the degree of silencing of the CD40 mRNA have been described above. In a preferred embodiment, the interfering RNAs are targeted to the regions in the CD40 mRNA corresponding to positions 173-193, 192-212, 479-499, 709-729, 62-82, 137- 157, 214-234, 242-262 or 188-214of the human CD40 mRNA wherein the numbering corresponds to the position with respect to the start codon in the CD40 cDNA as defined in NCBI accession X60592.1.

a preferred embodiment, the siRNAs are those shown in Table 1

Targeted region in

human CD40 Sequence SEQ ID NO

mRNA

5' -UGCCUUCCUUGCGGUGAAA-3' 1

173-193

5' -UUUCACCGCAAGGAAGGCA- 3 ' 2

5' -GCGAAUUCCUAGACACCUG-3' 3

192-212

5' -CAGGUGUCUAGGAAUUCGC-3' 4

5' -UGUCACCCUUGGACAAGCU-3' 5

479-499

5' -UGCUUGUCCAAGGGUGACA-3' 6

5' -UUUUCCCGACGAUCUUCCU-3' 7

709-729

5' -AGGAAGAUCGUCGGGAAAA- 3 ' 8

5' -CCACCCACUGCAUGCAGAG-3' 9

62-82

5' -CUCUGCAUGCAGUGGGUGG-3' 10

5' -CUGGUGAGUGACUGCACAG-3' 11

137-157

5' -CUGUGCAGUCACUCACCAG-3' 12

214-234 5' - CAGAGAGACACACUGCCAC- 3 ' 13 5 ' -GUGGCAGUGUGUCUCUCUG- 3 ' 14

5 ' -UACUGCGACCCCAACCUAG- 3 ' 15

242-262

5 ' -CUAGGUUGGGGUCGCCAGUA- 3 ' 16

5 ' - GAAAGC GAAUU C CUAGACAC CU GGAAC - 3 ' 17

188-214

5 ' -GUUCCAGGUGUCUAGGAAUUCGCUUUC- 3 ' 18

Table 1. Sequence composition and target localization within human CD40 mRNA of siRNAs designed to screen for efficient CD40 mRNA silencing. Numbering is provided from the ATG start codon in the CD40 mRNA as shown in NCBI accession number X60592 (version 1 of 14-NOV-1997)

Other illustrative, non-limitative, examples of interfering RNA specific for the sequence of CD40 include the mouse CD40 siRNA sc-29998, the mouse CD40 shRNA plasmid sc-29998-SH, the mouse CD40 shRNA lentiviral particles sc-29998-V, the human CD40 siRNA sc-29250, the human shRNA plasmid sc-29250-SH and the human CD40 shRNA lentiviral particles sc-29250-V, all of them from Santa Cruz Biotechnology and the human CD40 hairpin siRNA eukaryotic expression vectors as in Chen L. & Zheng XX, Chinese J Cell Mol. Immunol 2005; 21(2): 163-6.

Preferred interfering RNAs targeted to human CD40 gene are those targeted towards a stable internal loop within the secondary structure of the CD40 mRNA.

In a particular embodiment of the invention, the interfering RNA that silences CD40 gene expression is a short interfering RNA (siRNA). In certain instances, the interfering RNA may be modified by a non-ligand group in order to enhance the activity, cellular distribution or cellular uptake of the dsRNA. Procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties include lipid moieties, such as cholesterol (Letsinger et al, Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al, Bioorg. Med. Chem. Lett., 1994, 4: 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al, Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al, Bioorg. Med. Chem. Let., 1993, 3 :2765), a thiocholesterol (Oberhauser et al, Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al, EMBO J., 1991, 10: 111; Kabanov et al, FEBS Lett., 1990, 259:327; Svinarchuk et al, Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al, Tetrahedron Lett., 1995, 36:3651; Shea et al, Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al, Nucleosides and Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al, Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al, Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et ah, J. Pharmacol. Exp. Ther., 1996, 277:923). Typical conjugation protocols involve the synthesis of the interfering RNA bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the RNA still bound to the solid support or following cleavage of the RNA in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate. In some embodiments, an interfering RNA described herein is covalently bound to a lipophilic ligand. Exemplary lipophilic ligands include cholesterol; bile acids; and fatty acids {e.g., lithocholic-oleyl acid, lauroyl acid, docosnyl acid, stearoyl acid, palmitoyl acid, myristoyl acid, oleoyl acid, or linoleoyl acid)

The patient that is treated according to the methods of the present invention may receive a kidney from a different species. In this case, the kidney is known as xenograft. However, in a preferred embodiment, the patient treated according to the first method of the invention receives a kidney from the same species from which the kidney derives, in which case, the kidney will be known as allograft. In a preferred embodiment, the recipient is a human. In a still more preferred embodiment, the donor kidney is of human origin and the recipient is also human.

In a particular embodiment of the invention, the interfering RNA that silences CD40 gene expression or the polynucleotide coding said interfering RNA is administered to the kidney by intra-arterially infusion, as shown in Example 1. In another embodiment, the interfering RNA or the polynucleotide coding for said interfering RNAof the invention are administered by means of the so-called "hydrodynamic administration" in which the interfering RNA or the polynucleotide coding for said interfering RNAare introduced intravascularly into the organism at high speed and volume, which results in high transfection levels with a more diffuse distribution (Alino, S.F. et al. 2010. J Gene Med, 12:920-6). A modified version of this technique has made it possible to obtain positive results for silencing through the naked siRNAs of exogenous genes (Lewis et al, 2002, Nat. Gen., 32: 107-108; McCaffrey et al, 2002, Nature, 418:38-39) and endogenous genes (Song et al, 2003, Science, Nat. Med., 9:347-351) in multiple organs. It has been shown that the effectiveness of the intercellular access depends directly on the volume of the fluid administered and the speed of the injection (Liu et al., 1999, Science, 305: 1437-1441). In mice, the administration has been optimized at values of 1 ml/10 g of body weight in a period of 3-5 seconds (Hodges, et al, 2003, Exp. Opin. Biol. Then, 3 :91-918). The exact mechanism allowing in vivo cell transfection with siRNAs after their hydrodynamic administration is not fully known. In the case of mice, it is thought that administration through the tail vein takes place at a rate that exceeds the heart rate and that the administrated fluid accumulates in the superior vena cava. This fluid subsequently accesses the vessels in the organs, and after that, through fenestrations in said vessels, accesses the extravascular space. In this way, the siRNA comes into contact with the cells of the target organism before it is mixed with the blood, thus reducing the possibilities of degradation through nucleases.

The interfering RNA or the polynucleotide coding for said interfering RNA of the invention can be administered forming part of liposomes, conjugated to cholesterol or conjugated to compounds capable of causing the translocation through cell membranes such as the TAT peptide, derived from the HIV-1 TAT protein, the third helix of the homeodomain of the D. melanogasterA termapaedia protein, the VP22 protein of the herpes simplex virus, arginine oligomers and peptides such as those described in WO07069090 (Lindgren, A. et al, 2000, Trends Pharmacol. Sci., 21 : 99- 103; Schwarze, S.R. et al, 2000, Trends Pharmacol. Sci., 21 :45-48, Lundberg, M. et al, 2003, Mol. Therapy, 8: 143-150 and Snyder, E L. and Dowdy, S.F., 2004, Pharm. Res., 21 :389- 393). Alternatively, the interfering RNA or the polynucleotide coding for said interfering RNA of the invention may be administered forming part of polyplexes which are complexes of polymers with DNA. Most polyplexes consist of cationic polymers and their production is regulated by ionic interactions. One large difference between the methods of action of polyplexes and lipoplexes is that polyplexes cannot release their DNA load into the cytoplasm, so to this end, co-transfection with endosome-lytic agents (to lyse the endosome that is made during endocytosis, the process by which the polyplex enters the cell) such as inactivated adenovirus must occur. However, this is not always the case, polymers such as polyethylenimine have their own method of endosome disruption as does chitosan and trimethylchitosan.

Alternatively, the interfering RNA or the polynucleotide coding for said interfering RNAof the invention can be administered associated to dendrimers which are repeatedly branched, roughly spherical large molecules capable of delivering the oligonucleotides.

The amount of interfering RNA or the polynucleotide coding for said interfering RNA required for the therapeutic or prophylactic effect will naturally vary according to the elected interfering RNA or polynucleotide coding for said interfering RNA, the nature and the severity of the illness to be treated, and the patient.A specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the patient's age, body weight, general health, sex, and diet, and the time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. Judgment of such factors by medical caregivers is within the ordinary skill in the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

For prevention of kidney allograft rejection, it is preferred that the recipient patient is treated once he has received the transplanted organ. However, the allograft may be treated prior to transplantation, which includes perfusion of the allograft in the donor subject and ex vivo perfusion. Methods of organ perfusion are well known in the art. In general, harvested kidney are perfused with the compositions of the invention in a pharmacologically acceptable carrier such as, for example, lactated Ringer's solution, University of Wisconsin (UW) solution, Euro-Collins solution or Sachs solution. Simple flushing of the organ or pulsatile perfusion may be used. Perfusion time is generally dependent on the length of ex vivo viability of the organ being transplanted; these viability times vary from organ to organ and are known in the art. In particular, kidneys, for example, may be transplanted up to 48 hr or even 72 hr after harvesting. Dosage may range from 0.001 μg to 500 μg each of the interfering RNA or the polynucleotide encoding said interfering RNA.

Compositions of the invention

The authors of the present invention have unexpectedly found that the administration of interfering siRNAs specific for CD40 are capable of increasing the immunosuppressive effect of subtherapeutical combinations of an immunosuppressive agent (see e.g. examples 2 and 3 of the present invention). Thus, in another aspect, the invention relates to a pharmaceutical composition comprising

(i) An interfering RNA that silences CD40 gene expression or a polynucleotide coding for said interfering RNA and

(ii) an immunosuppressive agent.

As used in the present invention, the expression "pharmaceutical composition" relates to a formulation which has been adapted to administer a predetermined dose of one or several therapeutically useful agents to a cell, a group of cells, an organ, a tissue or an animal.

The terms "interfering RNA specific for CD40" and "polynucleotide encoding for a interfering RNA specific for CD40" have been described in detail above and are used with the same meaning in the context of the present invention.

In a preferred embodiment, the interfering RNA is targeted to a region selected from the group consisting of a region located at positions 173-193, 192-212, 479-499, 709-729, 62-82, 137-157, 214-234, 242-262 or 188-214 of the human CD40 mRNA wherein the numbering corresponds to the position with respect to the start codon in the CD40 cDNA as defined in NCBI accession X60592.1. In a still more preferred embodiment, the interfering RNA comprises a sequence as defined in Table 1. In another preferred embodiment, the interfering RNA is a short interfering RNA (siRNA).

The compositions of the invention may contain one or more interfering RNAs or polynucleotides coding for said interfering RNA according to the invention. In a preferred embodiment, the composition of the invention comprises several the interfering RNAs or polynucleotide coding for said interfering RNAs, being targeted to different regions of one and the same target mRNA.

The term "immunosuppresive agent", as used herein, refers to a substance that acts to inhibit, slow or reverse the activity of the immune system in a subject treated therewith. This would include substances that suppress cytokine production, down- regulate or suppress self-antigen expression, or mask the MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines (see US 4,665,077); nonsteroidal antiinflammatory drugs (NSAIDs); ganciclovir, tacrolimus, glucocorticoids such as Cortisol or aldosterone, anti-inflammatory agents such as a cyclooxygenase inhibitor, a 5 - lipoxygenase inhibitor, or a leukotriene receptor antagonist; purine antagonists such as azathioprine or mycophenolate mofetil (MMF); trocade (Ro32-355); a peripheral sigma receptor antagonist such as ISR-31747; alkylating agents such as cyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde (which masks the MHC antigens, as described in US 4, 120,649); anti -idiotypic antibodies for MHC antigens and MHC fragments; cyclosporin A; steroids such as corticosteroids or glucocorticosteroids or glucocorticoid analogs, e.g., prednisone, methylprednisolone, including SOLU- MEDROL^(R) methylprednisolone sodium succinate, rimexolone, and dexamethasone; dihydrofolate reductase inhibitors such as methotrexate (oral or subcutaneous); antimalarial agents such as chloroquine and hydroxychloroquine; sulfasalazine; leflunomide; cytokine release inhibitors such as SB-210396 and SB-217969, monoclonal antibodies and a MHC II antagonist such as ZD2315; a PGl receptor antagonist such as ZD4953; a VLA4 adhesion blocker such as ZD7349; anti-cytokine or anti-cytokine receptor antibodies including anti- interferon-alpha, -beta, or -gamma antibodies, anti- T F-alpha antibodies (infliximab (REMICADE(R)) or adalimumab), anti-T F-alpha immunoadhesin (etanercept), anti-T F- beta antibodies, interleukin-1 (IL-1) blockers such as recombinant HuIL-lRa and IL-1B inhibitor, anti-interleukin-2 (IL-2) antibodies and anti-IL-2 receptor antibodies; IL-2 fusion toxin; anti-L3T4 antibodies; leflunomide; heterologous anti-lymphocyte globulin; OPC- 14597; NISV (immune response modifier); an essential fatty acid such as gammalinolenic acid or eicosapentaenoic acid; CD-4 blockers and pan-T antibodies, preferably anti-CD3 or anti-CD4/CD4a antibodies; co-stimulatory modifier (e.g., CTLA4-Fc fusion, also known as ABATACEPT™); anti-interleukin-6 (IL-6) receptor antibodies and antagonists; anti- LFA-1 antibodies, including anti-CD 1 la and anti-CD 18 antibodies; soluble peptide containing a LFA-3-binding domain (WO 1990/08187); streptokinase; IL-10; transforming growth factor-beta (TGF-beta); streptodornase; RNA or DNA from the host; FK506; RS- 61443; enlimomab; CDP-855; P P inhibitor; CH-3298; GW353430; 4162W94, chlorambucil; deoxyspergualin; rapamycin; T-cell receptor (Cohen et al, US 5, 114,721); T-cell receptor fragments (Offner et al, Science, 251 : 430- 432 (1991); WO 1990/11294; Janeway, Nature, 341 : 482-483 (1989); and WO 91/01133); BAFF antagonists such as BAFF antibodies and BR3 antibodies; zTNF4 antagonists (Mackay and Mackay, Trends Immunol, 23 : 1 13-5 (2002)); biologic agents that interfere with T cell helper signals, such as anti-CD40 receptor or anti-CD40 ligand (CD 154), including blocking antibodies to CD40-CD40 ligand (e.g., Durie et al, Science, 261 : 1328-30 (1993); Mohan et al, J. Immunol, 154: 1470-80 (1995)) and CTLA4-Ig (Finck et al, Science, 265: 1225-7 (1994)); and T-cell receptor antibodies (EP 340, 109) such as T10B9.

In a particular embodiment of the invention, the immunosuppressive agent is an inhibitor of the mammalian target of rapamycin (mTOR), also known as mechanistic target of rapamycin or FK506 binding protein 12-rapamycin associated protein 1 (FRAPl). mTOR is a serine/threonine protein kinase that regulates cell growth, cell motility, cell survival, protein synthesis and transcription. mTOR belongs to the phosphatidilinositol 3-kinase-related kinase protein family. mTOR inhibitors include, without limitation, Pp242 (Torkinib), WYE-354, Ku-0063794, PI- 103, rapamycin (sirolimus) and its derivatives such as temsirolimus(Torisel, CCI-779), everolimus (RAD001, Afinitor), ridaforolimus (AP23576) and deforolimus (MK-8669).In a preferred embodiment of the invention, the inhibitor of mTOR is rapamycin, also known as sirolimus. Other analogs of rapamycin include: rapamycin oximes (U.S. Pat. No. 5,446,048); rapamycin aminoesters (U.S. Pat.No. 5,130,307); rapamycin dialdehydes (U.S. Pat.No. 6,680,330); rapamycin 29-enols (U.S. Pat.No. 6,677,357); O-alkylated rapamycin derivatives (U.S. Pat.No. 6,440,990); water soluble rapamycin esters (U.S. Pat. No. 5,955,457); alkylated rapamycin derivatives (U.S. Pat. No. 5,922,730); rapamycin amidino carbamates (U.S. Pat.No. 5,637,590); biotin esters of rapamycin (U.S. Pat.No. 5,504,091); carbamates of rapamycin (U.S. Pat.No. 5,567,709); rapamycin hydroxyesters (U.S. Pat. No. 5,362,718); rapamycin 42-sulfonates and 42-(N- carbalkoxy)sulfamates (U.S. Pat. No. 5,346,893); rapamycin oxepane isomers (U.S. Pat.No. 5,344,833); imidazolidyl rapamycin derivatives (U.S. Pat.No. 5,310,903); rapamycin alkoxyesters (U.S. Pat.No. 5,233,036); rapamycin pyrazoles (U.S. Pat.No. 5, 164,399); acyl derivatives of rapamycin (U.S. Pat.No. 4,316,885); reduction products of rapamycin (U.S. Pat.Nos. 5, 102,876 and 5,138,051); rapamycin amide esters (U.S. Pat.No. 5,118,677); rapamycin fluorinated esters (U.S. Pat.No. 5,100,883); rapamycin acetals (U.S. Pat.No. 5,151,413); oxorapamycins (U.S. Pat.No. 6,399,625); and rapamycin silyl ethers (U.S. Pat.No. 5,120,842), each of which is specifically incorporated by reference.

The pharmaceutical compositions according to the invention may further comprise a pharmaceutically acceptable excipient or carrier. "Pharmaceutically acceptable excipient" is understood to be an inactive substance therapeutically speaking, used to incorporate the active principle and which is acceptable for the patient from a pharmacological/toxicological viewpoint and for the pharmaceutical chemist that manufactured it from a physical/chemical standpoint with respect to the composition, formulation, stability, acceptance by the patient and bioavailability. The number and the nature of the pharmaceutically acceptable excipients depend on the desired administration form. The pharmaceutically acceptable excipients are known by the person skilled in the art (Fauli and Trillo C. (1993) "Tratado de FarmaciaGalenica", Luzan 5, S.A. Ediciones, Madrid). Said compositions may be prepared by the conventional methods known in the state of the art ("Remington: The Science and Practice of Pharmacy", 20th edition (2003) Genaro A.R., ed., Lippincott Williams & Wilkins, Philadelphia, US). Suitable pharmaceutically acceptable carriers including, e.g., ion exchangers, alumina, aluminium stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat.

The term "pharmaceutically effective amount" relates to a quantity capable of exercising a therapeutic effect, and which may be determined by the person skilled in the art using typically used means.

The invention contemplates pharmaceutical compositions especially prepared for the administration of the interfering RNA or the polynucleotide coding for said interfering RNA of the invention in naked form, i.e., in the absence of compounds that protect the interfering RNA or the polynucleotide coding for said interfering RNA from degradation by the nucleases of the organism, which entails the advantage of eliminating the toxicity associated with the reagents used for the transfection. Suitable routes of administration for the naked interfering RNA or the polynucleotide coding for said interfering RNA include the intravascular, intratumoral, intracranial, intraperitoneal, intrasplenic, intramuscular, subretinal, subcutaneous, mucous, topic or oral route (Templeton, 2002, DNA Cell Biol, 21 :857-867). The initial concerns in regard to the capacity of molecules such as siRNAs to induce an immune response when administered naked have been investigated by Heidel et al. (Nat. Biotechnol, 2004, 22: 1579-1582). By means of the determination of the plasma interleukin and interferon levels after the intraperitoneal and intravenous administration of siRNAs, no immune response was observed while, at the same time, it was observed that the systematic administration of the siRNA was well tolerated. The pharmaceutical composition of the invention comprises a pharmaceutically effective amount of an interfering siRNA that silences CD40 gene expression or a polynucleotide coding for said siRNA and a pharmaceutical effective amount of an immunosuppressive agent also comprises a pharmaceutically acceptable carrier. A pharmaceutically-acceptable carrier can be, for example, water, sodium phosphate buffer, phosphate-buffered saline, normal saline or Ringer's solution or other physiologically-buffered saline, or other solvent or vehicle such as a glycol, glycerol, an oil such as olive oil or an injectable organic ester. A pharmaceutically acceptable carrier can also contain physiologically acceptable compounds that act, for example, to stabilize or increase the absorption of the siRNA or the polynucleotide coding for said siRNA. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the composition. Therapeutic uses of the compositions of the present invention

The compositions according to the present invention can be used for increasing tolerance in a recipient mammal to a kidney transplant of for the treatment or prevention of acute rejection of a renal transplant in a subject in need thereof by the combined administration of the CD40-specific interfering RNA or the polynucleotide encoding said interfering RNAand the immunosuppressive agent(s).

Thus, in another aspect, the invention relates to a method for increasing tolerance in a recipient mammal to a kidney transplant comprising the administration to said subject a composition comprising

(ii) an immunosuppressive agent. In another aspect, the invention relates to a method for the treatment or prevention of acute rejection of a renal transplant in a subject in need thereof that comprises the administration to said subject of a composition comprising (i) An interfering RNA that silences CD40 gene expression or a polynucleotide coding for said interfering RNA and

(ii) an immunosuppressive agent. The terms "interfering RNA that silences CD40 gene expression", "polynucleotide coding for said interfering RNA" and immunosuppressive agent have been described above in detail and are used with the same meaning in the context of the therapeutic methods of the composition. In a preferred embodiment, the interfering RNA is targeted to a region selected from the group consisting of a region located at positions 173-193, 192-212, 479-499, 709-729, 62-82, 137-157, 214-234, 242-262 or 188-214 of the human CD40 mRNA wherein the numbering corresponds to the position with respect to the start codon in the CD40 cDNA as defined in NCBI accession X60592.1. In a still more preferred embodiment, the interfering RNA comprises a sequence as defined in Table 1.

In another preferred embodiment, the interfering RNA is a short interfering RNA (siRNA).

In a particular embodiment of the invention, the immunosuppressive agent is an inhibitor of the mammalian target of rapamycin (mTOR). In another preferred embodiment, the mTOR inhibitor is rapamycin or an analog thereof.

By "combined administration" it has to be understood that the CD40-specific interfering RNA or the polynucleotide encoding said interfering RNA can be administered together or separately, simultaneously, concurrently or sequentially with the immunosuppressive agent in any order, e.g. the administration of the CD40-specific interfering RNA or the polynucleotide encoding said interfering RNA can be made first, followed by the administration of one or more immunosuppressive agent(s); or the administration of CD40-specific interfering RNA or the polynucleotide encoding said interfering RNA can be made last, preceded by the administration of one or more immunosuppressive agent (s); or the administration of the CD40-specific interfering RNA or the polynucleotide encoding said interfering RNA can be made concomitantly with one or more immunosuppressive agent(s). The compositions of the invention are suitable for administration to any type of mammal, preferably a human being. Suitable routes for administering siRNA molecules/ft vivo include, but are not limited to, subcutaneous, intradermal, intramuscular, intraocular, intrathecal, intracerebellar, intranasal, intratracheal, hypodermic, intraperitoneal, intrahepatic, intratesticular, intratumoral, hypodermic injection and intravascular perfusion. Optimal dosages of the components of the composition to be administered may be readily determined by those skilled in the art, and will vary with the particular compound used, the strength of the preparation, the mode of administration, and the severity of the condition to be treated. In addition, factors associated with the particular patient being treated, including patient age, weight, diet and time of administration, will result in the need to adjust dosages and interval, in particular for providing a synergistic effect in lowering blood glucose level in a subject. The frequency and/or dose relative to the simultaneous or separate administrations can be adapted by one of ordinary skill in the art, in function of the patient, the pathology, the form of administration, etc. In a preferred embodiment, the immunosuppressive agent and, in particular, the rapamycin is administered a subtherapeutic dosis. As used herein, the phrase "subtherapeutic dose" refers to a dosage, which is less than that dosage which would produce a therapeutic result in the subject if administered in the absence of the other agent or agents.

A "subtherapeutic dose" can be defined in relative terms (i.e., as a percentage amount (less than 100 percent) of the amount of pharmacologically active agent conventionally administered). For example, a subtherapeutic dose amount can be about 1 percent to about 25 percent of the amount of pharmacologically active agent conventionally administered. In some embodiments, a subtherapeutic dose can be about 1 percent, 2 percent, 3 percent, 5 percent, 10 percent, 12 percent, 15 percent, 20 percent, or 25 percent of the amount of pharmacologically active agent conventionally administered. Alternatively, a "subtherapeutic dose" is one that results in blood levels of a pharmacological agent which is lower, either systemically or locally, than that obtained when an established therapeutic dose for that particular pharmacological agent is administered. Accordingly, a "subtherapeutic dose" can result from the administration of a pharmacological agent at a lower than established dosage, or via a route or dosing schedule different from an established therapeutic dosage or administration protocol, as discussed below.

In a preferred embodiment, the immunosuppressive agent is amTOR inhibitor. In a still more preferred embodiment, the mTOR inhibitor is rapamycin.In a preferred embodiment, rapamycin is administered at a subtherapeutic dosage. Suitable subtherapeutic rapamycin dosages include, for example, between 0.01-1 mg/kg, between 0.02-0.9 mg/kg, between 0.03-0.8 mg/kg, between 0.04-0.7 mg/kg,between 0.05-0.6 mg/kg,between 0.06-0.5 mg/kg,between 0.07-0.4 mg/kg,between 0.08-0.3 mg/kg, between 0.09-0.2 mg/kg,between 0.1-1.1 mg/kg orbetween 0.1-1 mg/kg.

The dose can be repeated as needed as determined by those of ordinary skill in the art. Thus, in some embodiments of the methods set forth herein, a single dose is contemplated. In other embodiments, two or more doses are contemplated. Where more than one dose is administered to a subject, the time interval between doses can be any time interval as determined by those of ordinary skill in the art. For example, the time interval between doses may be about 1 hour to about 2 hours, about 2 hours to about 6 hours, about 6 hours to about 10 hours, about 10 hours to about 24 hours, about 1 day to about 2 days, about 1 week to about 2 weeks, or longer, or any time interval derivable within any of these recited ranges.

The invention is described in detail below by means of the following examples which are to be construed as merely illustrative and not limitative of the scope of the invention.

EXAMPLES

MATERIALS AND METHODS siRNA design and screening

siRNA duplexes targeting the partial rat CD40 mRNA sequence (GenBankAcc.N⁰ AF241231)were designed according to the siRNA user guide (http://www.rockefeller.edu/labheads/tuschl/sirna.html). For the initial screening, six siRNAs were synthesized by in vitro transcription (Silencer™ siRNA construction kit, Ambion, Austin, TX), and three more (TNFRSF5-1, TNFRSF5-2 andTNFRSF5-3) were chemically synthesized (Cenix BioScience; Silencer pre-designed siRNAs,Ambion). A scrambled non-silencing siRNA was used as control for off-target effects. To assess the rat CD40 mRNA silencing efficiency of siRNA molecules, the GeneEraser™Luciferase Suppression-Test System was used (Stratagene, La Jolla, CA, USA). A 547pbCD40 partial coding sequence was cloned into the 3'UTR of the luciferase gene to form thetargeting vector pTarget-luc-CD40. In vitro transfection of siRNAs (ΙΟΟηΜ) was performed inHEK-293 cells using Oligofectamine (Invitrogen, Paisley, UK). Six hours later, the cells were transfected with pTarget-luc-CD40 (400ng DNA; Polyfect Transfection Reagent, Qiagen, Hilden,Germany). After 48h of incubation, the luciferase activity from lysates was determined(Luciferase Assay System, Promega, Madison, WI; Luminometer TD-20/20, Turner Designs, Sunnyvale, CA). For normalization of transfection efficiency, co-transfected pCMV-Pgal wasdetermined (up to 500ng DNA). siRNA transfer to renal tissue

To set up the siRNA transfer protocol, preliminary studies were performed to evaluate the siRNA dose, carrier vector and administration procedure. The protocol that offered optimal tissue transfer was as follows. Chemically synthesized siRNA duplexes (Qiagen Hilden, Germany) were dissolved in DEPC water. Following renal washing with 1 mL of EuroCollins solution at 4°C (EC), donor kidneys were intra-arterially infused with 30μg of either siCD40 or control unspecific siRNA duplex in a final lmL volume of isotonic saline solution. Immediately after siRNA infusion, kidneys underwent electroporation held with a tweezers-type electrode. The electroporation protocol (six pulses of 20 ms/each, 1 Hz frequency, at 100 V/cm) was applied twice per kidney to ensure that the whole tissue was covered by the tweezers-type electrode.

Animals and surgical transplantation technique For functional assessment of CD40 silencing, inbred male Wistar-Agouti rats (250 g BW) received an allogeneic kidney from Brown-Norway rats (250 g BW) (Charles River by Harlan UKLimited, Bicester, UK). Kidneys were preserved in EC while the recipient was prepared for transplantation. The surgical technique has been described previously in Herrero-Fresnedaet al. (Transplantation 2005; 79: 165-173). Recipient rats were bi-nephrectomized at the moment of transplantation. Animals did not receive any immunosuppressant and were maintained in accordance with the Guidelines of the Committee on Care and Use of Laboratory Animals and Good Laboratory Practice. Treatments and Groups. Follow-up

The groups were as follows: NoTreat group: unspecific siRNA as control (n= 16); Rp group: daily oral sub-therapeutic rapamycin (0.5mg/Kg, Sirolimus, Wyeth) for the first 15 days after transplantation (n = 14);siCD40 group: siCD40 (n= 21); and siCD40-Rp group: siCD40 and sub-therapeutic rapamycin (n = 21). Serum creatinine (sCr, μιηοΙ/L) was determined by Jaffe's reaction (Olympum Autoanalyzer, Hamburg, Germany) every 2 days beginning the day after surgery. For the survival study, rats were ideally followed for 100 days. The indefinite survival was established at this time point.Upon sacrifice, grafts were excised and processed for histological and molecular studies. Histological Studies

Coronal 1-2 mm-thick slices of graft kidneys were fixed in buffered formalin, dehydrated and embedded in paraffin. For light microscopy, 3-4 μπι-thick tissue sections were stained with hematoxylin-eosin and periodic acid-Schiff A pathologist blinded to the treatment groups assessed all sections for tubulitis, interstitial infiltration, vasculitis, glomerulitis, mesangiolysis, acute tubular necrosis, peritubular capillary infiltration, capillary thrombosis, intimal arteritis, fibrinoid necrosis, transmural infiltration, endothelial denudation and hemorrhage, following the Banff criteria for acute/active lesion scoring 42. Immunohi stochemi stry

Representative paraffin-embedded tissue sections were immunoperoxidase-stained for CD40 (1 :50; Research Diagnostics Inc, USA), NFKB p65 subunit (1 : 1000; Abeam PLC, UK), apelin(l :500; Phoenix Pharmaceuticals, USA), IL-7R (1 :50; Santa Cruz Biotechnology, USA) and C4d(l :30; Biomedica Gruppe, Austria), and immunofluorescence-stained for IgG (1 :300; Sigma,Spain). Negative controls were performed by immunostaining-matched serial sections without the primary antibodies. The immunoperoxidase-stained samples were revealed with diaminobenzidine (D-5637, Sigma, Spain) and counterstained with hematoxylin. For IgG, samples were directly observed under fluorescence light microscopy. C4d and IgG (assessed in peritubular capillaries), apelin and IL-7R were semi-quantitatively scored: 0 denoted negative staining, 1 positive staining in <25% of the sample, 2 positive staining in 25-50%, 3 positive staining in 50-75%, and 4 positive staining in 75-100%). NFKB p65immunostaining was considered positive when it was located inside the nuclei of different cells(TECs, VECs and interstitial infiltrate). CD40 immunostaining was only used to localize theprotein expression without further semi-quantification.

Quantification of gene expression in renal grafts

For molecular studies, the kidney was immediately frozen in liquid nitrogen and stored at -80°C. Total RNA was extracted and reverse-transcribed to cDNA as previously described 43. Negative controls for reverse transcription were carried out using distilled water. Tissue expression levelsfor CD40 and other immune-inflammatory mediators were quantified by TaqMan real-time PCR(ABI Prism® 7700, Applied Biosystems, Spain) using the comparative CT method (AppliedBiosystems, Spain).PCR reactions and amplification were performed as previously described 43. Pooled values of healthy non-transplanted kidneys were used as the reference value. Results were expressed as"many fold of the unknown sample" with respect to the reference value (arbitrary units).

Quantification of circulating donor specific antibodies

The presence of circulating donor specific antibodies (DSA) class-I was quantified on recipient serum samples incubated with donor spleen cells and measured by flow cytometry. Plasma samples were collected at the moment of sacrifice. Donor splenocytes were isolated from a Brown-Norway rat spleen by Ficoll® density gradient and freshly used. Different controls were added: serum from non-transplanted Wistar- Agouti rat as naive; serum from a transplanted WArat with high anti-HLA antibody titer as positive control, and splenocytes from Lewis rat as negative control. Briefly, 5x105 splenocytes were incubated with 25 μΐ of recipient serum for 30min at room temperature, washed in PBS, incubated in the dark (30min, 4°C) with a 1 :25 mix of anti- CD3(eBioscience, Ltd. Iceland, Ireland) and anti-IgG Fc portion (UK, Jackson Immuno Research,Baltimore USA), fixed with 1% paraformaldehyde and analyzed by flow cytometry. Fluorescence increase of 15% with respect to the negative control was considered as positive. Results were expressed as percentage of positive cells with respect to the total number of CD3+ spleen cells.

Quantification of the CD40- and CD45RA-positive population in the spleen

At sacrifice, the recipient's spleen was harvested in PBS. The splenocytes were isolated by Ficoll® density gradient and crio-preserved at -180°C.To quantify the percentage of CD40+ and CD45RA+ splenocytes, cells were thawed and recovered by standard methods. 5x105 splenocytes were incubated in the dark (20 min, 4°C) with antibodies (5μ1 CD40 and 2μ1 CD45RA, BD Pharmingen, Spain). After washing with PBS,7- amino-actinomycin D (1 : 10) was added to control cell viability. Cells were analyzed by flow cytometry. Results are expressed as mean percentage of positive cells to the total number of splenocytes.

Statistical analysis

Overall survival was analyzed using the Log Rank test. Serum creatinine differences at any time point, gene expression, plasma cytokine levels and DSA titters were analyzed by ANOVA followed by Scheffe's test. For histological parameters and DSA presence, Chi Square P value was calculated from the contingency table. Semi-quantitative immunostaining was analyzed through the non-parametric Kruskal-Wallis test. Values of p<0.05 were considered as statistically significant. Data are presented as mean ± SEM.

EXAMPLE 1

Selection and characterization of effective anti-rat CD40 siRNAs

Nine siRNAs were designed and generated to target different positions within the coding region of rat CD40 mRNA (Table 2). Table 2. Sequence composition and target localization of the siRNAs designed to screen for efficient rat CD40 mRNA silencing.

Targeted

siRNA anti- SEQID region in rat Sequence

rat CD40 NO

CD40 mRNA

5' -ACAGUACCUCCAAGGUGGCUU-3' 19 siRNA-2 124-145

5' -GCCACCUUGGAGGUACUGUUU-3' 20

5' -ACCGACUAGUUAGCCACUGUU-3' 21 siRNA-4 171-192

5' -CAGUGGCUAACUAGUCGGUUU-3' 22

5' -UGCCAACCGUGCGACUCAGUU-3' 23 siRNA-6 212-233

5' -CUGAGUCGCACGGUUGGCAUU-3' 24

5' -CUCAAUCAAGGGCUUCAGGUU-3' 25 siRNA-10 290-251

5' -CCUGAAGCCCUUGAUUGAGUU-3' 26

5' -GGGCUUCAGGUUAAGAAGGUU-3' 27 siRNA-12 299-320

5' -CCUUCUUAACCUGAAGCCCUU-3' 28

5' -GUGUCAUCCAUGGACAAGCUU-3' 29 siRNA-21 517-538

5' -GCUUGUCCAUGGAUGACACUU-3' 30 siRNA 5' -GCUCUUGAGAAGACCCAAUUU-3' 31

157-175

TNFRSF5-1 5' -AUUGGGUCUUCUCAAGAGCUG-3' 32 siRNA 5' -GGCGAAUUCUCAGCUCACUUU-3' 33

193-211

TNFRSF5-2 5' -AGUGAGCUGAGAAUUCGCCUG-3' 34 siRNA 5' -GUGUGUUACGUGCAGUGACUU-3' 35

66-84

TNFRSF5-3 5' -GUCACUGCACGUAACACACUG-3' 36

The RNAi efficacy of each of the nine synthesized siRNAs was tested in HEK-293 cells using the GeneEraserTM Luciferase Suppression-Test System. These siRNAswere co- transfected into HEK-293 cells along with a luciferase/CD40 fusion construct. Luciferase activity was determined at two days post-transfection (Figure 1). Five of the designed siRNAs had significant knockdown efficacy, although the most potent silencing efficiency (82-84%) was achieved with the siRNAs TNFRSF5-1 and TNFRSF5-2 (Figure 1). Thereby, the inventors chose siRNA TNFRSF5-2 (renamed as siCD40) for the subsequent in vivo study. EXAMPLE 2

Effect of CD40 silencing in Renal function and Survival

NoTreat animals developed severe renal insufficiency from the 5th day post- transplantation dying before 9 days. Treatment with 0.5 mg/Kg/day of rapamycin was unable neither to avoid renal insufficiency nor to increase survival (Figure 2). Seven long-surviving rats were found in both siCD40-treated groups. Five out of those (three in the siCD40 group and two in the siCD40-Rp group) displayed stable chronic renal insufficiency (sCr around 250-300 μιηοΙ/L), surviving for 39, 48, 53, 57 and 57 days. The remaining 2 rats (siCD40-Rp group) achieved indefinite survival with stable renal function throughout the study (sCrl00=69 and 130μmol/L)(Figure 2).

EXAMPLE 3

Effect of CD40 silencing in the Rejection Pattern

Conventional histology of NoTreat grafts showed a variable degree of tubule-interstitial damage, perivascular edema and hemorrhage, fibrinoid necrosis either in the vessel wall or in glomeruli, endothelial denudation and cell infiltration mainly composed by polymorphonuclear cells but few lymphocytes; all these features were indicative of acute vascular humoral rejection (Figure 3 A). Grafts treated with sub-therapeutic rapamycin displayed mixed features of both cellular and humoral rejection: there was notable cell infiltration in all renal parenchyma including the vessels, with the characteristic humoral signs of karyorrhexis and activated endothelium in vessel walls (Figure 3B). Only 12% of both siCD40-treated groups displayed features of acute vascular humoral rejection. Forty-nine per cent of grafts presented cellular rejection, occasionally affecting vessels and more frequently displaying slight tubulitis and interstitial infiltrate mainly composed by lymphocytes (17% grade III, 3% grade II A, 5% grade IA, and 24% borderline). Altogether, under light microscopy the siCD40- treated animals presented a more cellular and apparently chronic pattern of rejection (Figure 3C&D). Five out of the seven long-surviving rats, treated with siCD40, displayed only grade 1 tubulitis and interstitial infiltrate with some degree of fibrosis. The other two rats, which achieved indefinite survival, displayed a normal histology in one case, and only grade 2 tubulitis with interstitial infiltrate in the other. In the NoTreat group, most of the grafts were C4d+ and IgG+ in peritubular capillaries, while CD40 silencing reduced the positivity for these humoral parameters (Table 3). In addition, donor specific antibodies (DSA) were evident in 83% of NoTreat animals while serum from non-transplanted WA rats did not present pre-formed DSA against BN splenocytes(1.77±0.53% DSA+ cells). Treatment with either rapamycin or siCD40 partially reduced the donor specific antibodies (DSA). Importantly, the combination of both treatments reduced not only the presence of DSA but also the percentage of positive cells (Table 3). Some DSA-positive animals did not show histological features of humoral rejection. However, all grafts displaying a histological pattern of antibody- mediated acute humoral rejection were DSA-positive. Most of the DSA-negative animals presented cellular rej ection.

Table 3. Rejection pattern, "n" indicates the number of assessed samples for each parameter in each group. The percentages of DSA-positive cells, splenocytes expressing CD40, CD45Ra (B cells) and double staining with respect to the total number of a b C splenocytes were analyzed by ANOVA and Scheffe's test, χ² for the rest of the parameters. P<0.005. vs No Treat, vs Rp., vs siCD40.

EXAMPLE 4

Effect of CD40 silencing in local and systemic CD40 expression All renal structures expressed variable CD40+ immunostaining: TECs, vessel SMCs and glomeruli. When CD40 was silenced, protein expression disappeared in glomeruli and vessels, and TEC immunostaining diminished .CD40 gene expression in NoTreat grafts was over-expressed twenty fold compared with control non-transplanted kidneys. Rapamycin partially reduced CD40 expression. Intragraft gene silencing effectively reduced CD40 expression to syngeneic values (3.45±0.75). The addition ofsiCD40 plus rapamycin did not further reduce CD40 gene expression (Table 3). To evaluate the effect of intra-graft CD40 silencing on the systemic B-cell response, we usedFACS to quantify the percentage of CD45RA+ (B-cells), CD40+ and double labeled splenocytes at the moment of sacrifice. Results showed that both groups treated with siCD40had fewer B+ splenocytes than the NoTreat group. Furthermore, the siCD40-treated group expressed the lowest percentage of CD40+/CD45RA+ splenocytes, and supplementation with rapamycin did not reduce this cell population further (Table 3 and Figure 4). EXAMPLE 5

Effect of CD40 silencing in Intra-graft expression of mediators

Intra-graft gene expression of TLR3, TLR4 and the downstream intermediate MyD88 was reduced in both siCD40-treated groups, particularly in the siCD40-Rp group. In contrast, its expression was strongly activated in the NoTreat group. Complement regulators CFH and CFI were also down-regulated in all treated groups, especially in the combined therapy (Table 4).

Table 4. Gene expression of Immune-Inflammatory Mediators in Renal Grafts: At sacrifice, the grafts were immediately frozen in liquid nitrogen and stored at -80°C. Gene expression was quantified by TaqMan real-time PCR and expressed as fold time respect to non-transplanted kidney tissue. ANOVA, Scheffe's test P<0.005, ^]vsNoTreat, 2vsRp, ³vs siCD40, ⁴ vs siCD40Rp.

Therefore local injection of siRNA does not seem to activate innate immune responses. Pluvinet R. et al. (Blood 2008; 112:3624-3637)had previously shown a clear up- regulation of apelin in endothelial cells as a consequence of siRNA-mediated CD40 silencing in vitro. In the present in vivo study the inventors showed a down-regulation of apelin gene expression in NoTreat animals which, as shown, had severe acute vascular damage. Upon CD40 silencing, apelin expression returned to values similar to syngeneic rats, but this effect was not observed in rapamycin treatment (Figure 4). Analysis of the local pro-inflammatory status in NoTreat rats showed the expected cytokine over-expression (Table 4). The anti-inflammatory IL-11 was over-expressed by CD40 silencing, following a similar expression pattern to apelin in the treated groups. In contrast, IL-15expression was specifically down-regulated upon CD40 silencing; whereas IL-7R expression was reduced in all treated grafts (Table 5). Further staining of grafts for IL-7R expression showed preferential localization in tubular epithelial and vascular endothelial cells (Figure 3A). In Rp group there was a slight decrease in IL-7R expression (Figure 3B) while both siCD40-treated groups showed a clear reduction in tubuli and almost null expression in the vessels(Figures 3C and 3D). Finally, downstream of the inflammatory cascade, the gene expression of the NFkB was reduced by all treatments, particularly when CD40 silencing was combined with the subtherapeutic dose of rapamycin (Table 5). Accordingly, NFkB nuclear translocation was reduced in siCD40-treated grafts (Table 5). In contrast, it was translocated from cytoplasm to nuclei in the NoTreat and Rp groups, especially in interstitial infiltrate and vascular endothelial cells.

Table 5. Evaluation of IL-7R and KF-kB protein location in immunostaining.IL-7R was semi-quantitatively graded from 0 to 4+ and analyzed by Kruskall-Wallis test, P<0.005, 1 vs NoTreat, 2 vs Rp. NFkB was considered positive when nuclei were immunostained. Results are expressed as number of positive samples vs. number of assessed samples. Results were analyzed by Chi Square.

Claims

1. A method for increasing tolerance in a recipient mammal to a kidney transplant that comprises the administration to said subject of an interfering RNA that silences CD40 gene expression or a polynucleotide coding for said interfering

RNA.

A method for the treatment or prevention of acute rejection of a renal transplant in a subject in need thereof that comprises the administration to said subject of an interfering RNA that silences CD40 gene expression or a polynucleotide coding for said interfering RNA.

The method as defined in claims 1 or 2 wherein the interfering RNA is targeted to a region in the CD40 mRNA selected from the group consisting of a region located at positions 173-193, 192-212, 479-499, 709-729, 62-82, 137-157, 214- 234, 242-262 or 188-214 of the human CD40 mRNA wherein the numbering corresponds to the position with respect to the start codon in the CD40 cDNA as defined in NCBI accession X60592.1.

The method as defined in any of claims 1 to 3 wherein the interfering RNA comprises a sequence as defined in Table 1.

The method as in claims 1 to 4 wherein the interfering RNA is a short interfering RNA (siRNA).

The method as in any of claims 1 to 5 wherein the interfering RNA or the polynucleotide encoding said interfering RNA is administered by intravascular infusion.

The method according to claim6 wherein the intravascular infusion is carried out in the renal artery.

8. The method according to 7 wherein the kidney is an allograft.

9. The method as defined in any of claims 1 to 8 wherein the recipient mammal is a human.

A composition comprising

(i) An interfering RNA that silences CD40 gene expression

polynucleotide coding for said interfering RNA and

(ii) an immunosuppressive agent. 11. A composition as defined in claim 10 wherein the interfering RNA is targeted to a region selected from the group consisting of a region located at positions 173-193, 192-212, 479-499, 709-729, 62-82, 137-157, 214-234, 242-262 or 188-214of the human CD40 mRNA wherein the numbering corresponds to the position with respect to the start codon in the CD40 cDNA as defined in NCBI accession X60592.1.

12. A composition as defined in any of claims 10 or 11 wherein the interfering RNA comprises a sequence as defined in Table 1. 13. A composition according to claim 10 wherein component (i) is a short interfering RNA (siRNA).

14. The composition as in any of claims lOto 13 wherein the immunosuppressive agent is an mTOR inhibitor.

15. The composition as in claim 14 wherein the mTOR inhibitor is rapamycin or an analogue thereof.

A method for increasing tolerance in a recipient mammal to a kidney transplant comprising the administration to said subject a composition comprising

(ii) an immunosuppressive agent.

17. A method for the treatment or prevention of acute rejection of a renal transplant in a subject in need thereof that comprises the administration to said subject of a composition comprising

(ii) an immunosuppressive agent.

18. The method as in claims 16 or 17 wherein the interfering RNA is targeted to a region of the human CD40 gene.

19. The method as defined in claim 18 wherein the interfering RNA is targeted to a region selected from the group consisting of a region located at positions 173-193, 192-212, 479-499, 709-729, 62-82, 137-157, 214-234, 242-262 or 188-214of the human CD40 mRNA wherein the numbering corresponds to the position with respect to the start codon in the CD40 cDNA as defined in NCBI accession X60592.1.

20. The method as defined in any of claims 17 to 19 wherein the interfering RNA comprises a sequence as defined in Table 1.

21. The method as defined in any of claims 17 to 20 wherein the interfering RNA is a short interfering RNA (siRNA). 22. The method as defined in any of claims 17 to 21 wherein the interfering RNA is administered subcutaneously, intradermally, intramuscularly, intraocularly, intrathecally, intracerebellarly, intranasally, intratracheally, hypodermically, intraperitoneally, intrahepatically, intratesticularly, intratumorally, hypodermically, by injection or by intravascular perfusion.

23. The method asdefined inany of claims 17 to 22 wherein the immunosuppressive agent is an mTOR inhibitor.

24. The method as in claim 23 wherein the mTOR inhibitor is rapamycin or analogue thereof.

25. The method according to claim 24wherein rapamycin is administered subtherapeuticdosis.