Use of inhibitor of gasdermind for treatment of RAC2 monogenic disorders

Abstract

A growing number of patients presenting severe combined immunodeficiencies associated with Rac2 mutations have been identified in newborns. These mutations are associated with high frequency of infection, leukopenia and more rarely autoinflammatory features. Here, the inventors show that the Rac2 activating mutations activates the NLRP3 inflammasome leading to the secretion of IL-1β from macrophages depending on their activation strength. Therefore, inhibiting GasderminD (GSDMD) pathway should be considered as a potential treatment for these patients. The present invention relates to a method of treating Rac2 monogenic disorders in a subject in need thereof comprising the administration of an inhibitor of GasderminD (GSDMD).

Description

[0001] USE OF INHIBITOR OF GASDERMIND FOR TREATMENT OF RAC2

[0002] MONOGENIC DISORDERS

[0003] FIELD OF THE INVENTION:

[0004] The invention is in the field of inflammatory disorders and monogenic disorders. More particularly, the invention relates to methods and compositions for treatment of Rac2 monogenic disorders.

[0005] BACKGROUND OF THE INVENTION:

[0006] Among the Ras-like superfamily, 22 Rho GTPases have been identified in humans and until now the best characterized are Rho, Rac and Cdc42 (Ridley, 2006). These Rho GTPases were firstly described for their function as actin cytoskeleton dynamic regulators and later they were found to be involved in critical cellular processes including cell cycle, cell growth, metabolism, as well as innate immune processes (Etienne-Manneville and Hall, 2002; Bokoch, 2005). Rho GTPases are also known to be one of the preferential targets of microbial virulence factors, probably because of their role in controlling innate immune responses (Boquet and Lemichez, 2003).

[0007] Among the Rho GTPases, 3 isoforms of Rac have been found in humans, Rael, Rac2 and Rac3. Whereas Rael is ubiquitously expressed in humans and Rac3 highly expressed in the brain, the Rac2 GTPase expression is mostly restricted to hematopoietic system (Burridge and Wennerberg, 2004). This restricted expression pattern suggests a specific immune function for this Rac GTPase isoform. Strikingly, human and mouse Rac2 proteins only differ in two amino acids (Kim and Dinauer, 2001). In vivo studies using Rac2- / - mice have demonstrated the Rac2 specific role in most cells of the hematopoietic system. (Lougaris et al., 2020).

[0008] Like other Rho GTPases, Rac2 oscillates between an active GTP -bound and an inactive GDP -bound stage which is regulated by the GTPase activating protein (GAP), guanine nucleotide exchange factor (GEF) and guanosine nucleotide dissociation inhibitor (GDI). Importantly, point mutations affecting the 2 Switch domains of the Ras-like proteins affect the GTP cycling conferring to those mutants either a dominant loss or gain of function and were found associated with human diseases such as cancer (Cherfils and Zeghouf, 2013). Rac2 is following the same scheme, single point mutations affecting the switch 1 or 2 domains have been described and somatic mutations found in patients with immune disorders (G12R and G15D or D57N located respectively in the conserved domains named G-boxes 1 and 3 and P29R or Q61R, E62K or and D63V respectively in the Switch 1 and Switch 2 domain) (Bourne et al., 1991) (Ambruso et al., 2000; Accetta et al., 2011; Kawazu et al., 2013; Caye et al., 2015; Alkhairy et al., 2015; Hsu et al., 2019; Sharapova et al., 2019; Lagresle-Peyrou et al., 2021; Stern et al., 2021; Lougaris et al., 2019; Zhang and Chen et al., 2022; Zhang and Lv et al., 2023). Clinically, a common variable with Rac2 mutation switch is an immune deficiency and recurrent susceptibility to infections (Arrington et al., 2020). The E62K mutation is a gain of function mutation localized in the Switch 2 domain that was shown to be associated with a myeloid deficit and an altered neutrophil’s function. The Rac2 D63V is also a gain of function Rac2 mutation, leading to a juvenile myelomonocytic leukemia while the Rac2 G12R, was discovered in a patient with a bone marrow hypoplasia characterized by a severe combined immunodeficiency syndrome of autosomal dominant inheritance. The G12R mutation was shown to have a major impact on hematopoietic cells causing the clinical severity of the patient (Lagresle-Peyrou et al., 2021). The G15D mutation causes recuring infections by both bacteria and viruses and is associated with a dysregulation of T lymphocyte and neutrophil phenotype. P29R mutation is associated with an immunodeficiency combined with recurrent respiratory infections and lymphopenia. Interestingly, only one mutation was found with an Rac2 inhibitory activity: D57N. Located in the G3 box of Rac2, this mutation causes a severe phagocyte immune deficiency. Morphologically, the macrophages inhibited membrane ruffling, inhibited the formation of macropinosomes and induced an elongated, spread morphology (Hsu et al., 2019; Stern et al., 2021).

[0009] Interestingly, among the patients with different forms of primary immunodeficiencies associated to Rac2 mutations, more than 75% (15 / 19) had a confirmed or potential gain of function mutations with either Rac2 G12R, G15D, P29R, A59S, Q61R, E62K or D63V (Stern et al., 2021; Zhang and Chen et al., 2022; Zhang and Lv et al., 2023).

[0010] The Rac2 activation by the CNF1 bacterial toxin was recently demonstrated to trigger NLRP3 inflammasome activation (Dufies et al., 2021; Dufies and Boyer, 2021). This sensing mechanism was found to be critical for the innate immune response during bacteremia in mice and the signaling pathway found to be conserved in human macrophages (Diabate et al., 2015; Dufies et al., 2021). Importantly the CNF1 deamidates the Rac GTPases by post-translation deamidation of the glutamine 61 of Rac in a glutamate. This modification results a change in the protein sequence of Rac2 into Q61E mutant. This modification of the switch 2 of the protein destroys the GTPase activity locking the Rac GTPases into an active form (Boquet and Lemichez, 2003). Stinkingly, this modification is closely related to the mutation found in patients with genetic gain of function mutation of Rac2 linked to primary immunodeficiencies.

[0011] Here the inventors investigated whether the Rac2 mutations found in patients with primary immunodeficiencies confers to Rac2 the potential to activate the NLRP3 inflammasome.

[0012] SUMMARY OF THE INVENTION:

[0013] The invention relates to methods and compositions for treatment of Rac2 monogenic disorders. In particular, the invention is defined by the claims.

[0014] DETAILED DESCRIPTION OF THE INVENTION:

[0015] A growing number of patients presenting severe combined immunodeficiencies associated with Rac2 mutations have been identified in newborns. These mutations are associated with high frequency of infection, leukopenia and more rarely autoinflammatory features. Here, the inventors show that RAC2 activating mutations induce the NLRP3 inflammasome leading to the secretion of IL-10 and IL-18 from macrophages. This induction depends on the RAC2 mutation and in particular their activation state. It has been shown that Caspase-1 triggered GasderminD (GSDMD) cleavage and the subsequent GSDMD pore formation have been shown to be important events for IL-10 secretion (Shao, 2021). Therefore, inhibiting the GasderminD (GSDMD) pathway should be considered as a potential treatment for these patients.

[0016] Method of treatment

[0017] The present invention relates to a method of treating Rac2 monogenic disorders in a subject in need thereof comprising the administration of an inhibitor of GasderminD (GSDMD).

[0018] As used herein, the term “subject” or “patient” refer to any mammals, such as a rodent, a feline, a canine, and a primate. In a particular embodiment, the subject is human. In a particular embodiment, the subject is a newborn or a child or teenager or an adult. Particularly, in the present invention, the subject has or is susceptible to have Rac2 mutations disorders. Particularly, in the present invention, the subject has or is susceptible to have a Rac2 gain of function mutations including Rac2 G12R, G15D, P29R, A59S, Q61R, E62K or D63V. As used herein, the term “monogenic disorder” has its general meaning in the art and refers to a variation in a single gene and is typically recognized by its striking familial inheritance pattern.

[0019] As used herein, the term “Rac” has its general meaning in the art and refers to a subfamily of the Rho family of GTPases, small (~21 kDa) signaling G proteins (more specifically a GTPase). Rac acts as a molecular switch, remaining inactive while bound to GDP and activated once GEFs remove GDP, permitting Rac to bind GTP. When bound to GTP, Rac is activated. In its activated state, Rac participates in the regulation of cell movement, through its involvement in structural changes to the actin cytoskeleton. By changing the cytoskeletal dynamics within the cell, Rac-GTPases are able to facilitate the recruitment of neutrophils to the infected tissues, and to regulate degranulation of azurophil and integrin-dependent phagocytosis. Activated Rac also regulates the effector functions of the target proteins involved in downstream signaling. As an essential subunit of N0X2 (NADPH oxidase enzyme complex), Rac is required for ROS (reactive oxygen species) production involved in the formation of NETs (neutrophil extracellular traps), thus, facilitating the pathogen and debris clearance by neutrophils, and the reduction of inflammation. Rac comprises Rael, Rac2, Rac3, and RhoG subgroups.

[0020] As used herein, the term “Rac2” has its general meaning in the art and refers to Ras- related C3 botulinum toxin substrate 2. Rac2 is a small (~21 kDa) signaling G protein (to be specific, a GTPase), and is a member of the Rac subfamily of the family Rho family of GTPases. It is encoded by the gene Rac2. Rac2 is having the following human Gene ID number: 5880 and the following human uniprot number: Pl 5153 An exemplary amino acid sequence for the human Rac2 is represented by SEQ ID NO: 1.

[0021] SEQ ID NO : 1 >sp | P15153 | Rac2 HUMAN Ras-related C3 botulinum toxin substrate 2 0S=Homo sapiens OX=9606 GN=Rac2 PE=1 SV=1 MQAIKCVWGDGAVGKTCLLI SYTTNAFPGEYI PTVFDNYSANVMVDSKPVNLGLWDTAG QEDYDRLRPLSYPQTDVFLICFSLVSPASYENVRAKWFPEVRHHCPSTPI ILVGTKLDLR DDKDTIEKLKEKKLAPITYPQGLALAKEIDSVKYLECSALTQRGLKTVFDEAIRAVLCPQ PTRQQKRACSLL

[0022] As used herein, the term “Rac2 monogenic disorder” refers to a disorder with a single mutation in the protein Rac2. The Rac2 monogenic disorders are associated with high neutrophil dysfunction, lymphopenia, immunodeficiency frequency, infection, leukopenia and more rarely autoinflammatory features. As used herein, the term “neutrophil dysfunction” refers to a group of conditions that affect the body’s ability to fight bacterial and fungal infections. Neutrophils are a type of white blood cell that clear bacteria and fungi from the body. They are also called granulocytes or pus cells. Neutrophils are produced in bone marrow and released into the blood. They control infection by travelling into body tissues to kill microorganisms, such as bacteria and fungi. Neutrophils destroy bacteria and fungi through a process called phagocytosis. This process involves the neutrophils engulfing infected cells and using granules (small particles) and chemicals to destroy the bacteria and fungi. Neutrophils also produce chemical signals that attract other neutrophils at sites of infection, and an accumulation of neutrophils forms pus.

[0023] In particular neutrophil disorders include but are not limited to autoimmune Neutropenia of Infancy, Chediak-Higashi Syndrome, Chronic Granulomatous Disease (CGD), Cyclic Neutropenia, Drug-Induced Neutropenia, Ethnic Neutropenia, Glycogen Storage Disease Type IB, Idiopathic Neutropenia, Leukocyte Adhesion Defects (LAD), Myelokathexis / WHIM Syndrome, Reticular Dysgenesis or Severe Congenital Neutropenia (SCN).

[0024] As used herein, the term “lymphopenia” is the condition of having an abnormally low level of lymphocytes in the blood. Lymphocytes are a white blood cell with important functions in the immune system. These conditions can be acquired or inherited. Inherited conditions that can lead to lymphopenia: Ataxia telangiectasia, Chromosome 22ql l.2 deletion syndrome (sometimes called DiGeorge syndrome), Common variable immunodeficiency, Severe combined immunodeficiency syndrome (SCID) or Wiskott-Aldrich syndrome. Acquired conditions that can lead to lymphopenia: Infections (such as HIV, viral hepatitis, influenza, SARS CoV-2 (the virus that causes COVID-19), tuberculosis, pneumonia, Sepsis , or malaria), autoimmune disorders (such as Sjogren’s syndrome, lupus, or rheumatoid arthritis), blood cancer and other blood diseases (such as Hodgkin's disease and aplastic anemia), some medical treatments like blood and bone marrow transplant, cancer treatment, steroid therapy, or major surgery.

[0025] As used herein, the term “immunodeficiency”, also known as immune-compromised, is a state in which the immune system's ability to fight infectious diseases and cancer is compromised or entirely absent. Most cases are acquired ("secondary") due to extrinsic factors that affect the patient's immune system. Examples of these extrinsic factors include HIV infection and environmental factors, such as nutrition. Immune-compromisedmay also be due to genetic diseases / flaws such as SCID.

[0026] As used herein, the term “infection” has its general meaning in the art and refers to a penetration and proliferation in the body of a microorganism invisible to the naked eye (bacteria, virus), likely to cause a health problem. An infection can be local or generalized (sepsis).

[0027] As used herein, the term “leukopenia” has its general meaning in the art and refers to a decrease in the number of leukocytes (WBC). Found in the blood, they are the white blood cells, and are the body's primary defense against an infection. Thus, the condition of leukopenia places individuals at increased risk of infection. Low white cell count may be due to acute viral infections, such as a cold or influenza. It has been associated with chemotherapy, radiation therapy, myelofibrosis, aplastic anemia (failure of white cell, red cell and platelet production), stem cell transplant, bone marrow transplant, HIV, AIDS, and steroid use. Other causes of low white blood cell count include systemic lupus erythematosus, Hodgkin's lymphoma, some types of cancer, typhoid, malaria, tuberculosis, dengue, rickettsial infections, enlargement of the spleen, folate deficiencies, psittacosis, sepsis, Sjogren syndrome and Lyme disease. It has also been shown to be caused by deficiency in certain minerals, such as copper and zinc.

[0028] As used herein, the term “autoinflammatory features” are characterized by recurrent episodes of inflammation, accompanied by a wide range of symptoms, including fever, abdominal pain, skin rash, arthralgias, and myalgias.

[0029] As used herein, the term "gene" has its general meaning in the art and refers to a DNA sequence that codes for or corresponds to a particular sequence of amino acids which comprise all or part of one or more proteins or enzymes and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed.

[0030] As used herein, the “allele” has its general meaning in the art and refers to an alternative form of a gene (one member of a pair) that is located at a specific position on a specific chromosome which, when translated, results in functional or dysfunctional (including nonexistent) gene products.

[0031] As used herein, the term “protein” has its general meaning in the art and refers to large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, responding to stimuli, providing structure to cells and organisms, and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in protein folding into a specific 3D structure that determines its activity. As used herein, the term “mutation” has its general meaning in the art and refers to a substitution, deletion or insertion. The term "substitution" means that a specific amino acid residue at a specific position is removed and another amino acid residue is inserted into the same position. The term "deletion" means that one or more specific amino acid residues are removed. The term "insertion" means that one or more amino acid residues are inserted before or after a specific amino acid residue, more specifically, that one or more, preferably one or several, amino acid residues are bound to an a.-carboxyl group or an a, -amino group of the specific amino acid residue.

[0032] In some embodiments, the amino residue (G) at position 12 is substituted. In some embodiments, the amino residue (G) at position 12 is substituted by an amino acid residue (R). In some embodiments, the amino residue (G) at position 12 is substituted by an amino acid residue (V).

[0033] In some embodiments, the amino residue (G) at position 15 is substituted. In some embodiments, the amino residue (G) at position 15 is substituted by an amino acid residue (D).

[0034] In some embodiments, the amino residue (P) at position 29 is substituted. In some embodiments, the amino residue (P) at position 29 is substituted by an amino acid residue (R).

[0035] In some embodiments, the amino residue (P) at position 34 is substituted. In some embodiments, the amino residue (P) at position 34 is substituted by an amino acid residue (H).

[0036] In some embodiments, the amino residue (D) at position 57 is substituted. In some embodiments, the amino residue (D) at position 57 is substituted by an amino acid residue (N).

[0037] In some embodiments, the amino residue (A) at position 59 is substituted. In some embodiments, the amino residue (A) at position 59 is substituted by an amino acid residue (S).

[0038] In some embodiments, the amino residue (Q) at position 61 is substituted. In some embodiments, the amino residue (Q) at position 61 is substituted by an amino acid residue (E). In some embodiments, the amino residue (Q) at position 61 is substituted by an amino acid residue (R).

[0039] In some embodiments, the amino residue (E) at position 62 is substituted. In some embodiments, the amino residue (E) at position 62 is substituted by an amino acid residue (K).

[0040] In some embodiments, the amino residue (D) at position 63 is substituted. In some embodiments, the amino residue (D) at position 63 is substituted by an amino acid residue (V).

[0041] In some embodiments, the amino residue (N) at position 92 is substituted. In some embodiments, the amino residue (N) at position 92 is substituted by an amino acid residue (T).

[0042] As used herein, the term “polypeptide” has its general meaning in the art and refers to a polymer of amino acids of any length. The polymer can comprise modified amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art.

[0043] As used herein, the term “polynucleotide” as used herein refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”). It also includes modified, for example by alkylation, and / or by capping, and unmodified forms of the polynucleotide. More particularly, the term “polynucleotide” includes polydeoxyribonucleotides (containing 2- deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-gly coside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids “PNAs”) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. In some embodiments, the polynucleotide comprises an mRNA. In other aspect, the mRNA is a synthetic mRNA. In some embodiments, the synthetic mRNA comprises at least one unnatural nucleobase. In some embodiments, all nucleobases of a certain class have been replaced with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be replaced with an unnatural nucleobase, e.g., 5- methoxyuridine). In some embodiments, the polynucleotide (e.g., a synthetic RNA or a synthetic DNA) comprises only natural nucleobases, i.e., A, C, T and G in the case of a synthetic DNA, or A, C, T, and U in the case of a synthetic RNA.

[0044] The inventors show that patients bearing Rac2 mutations have an abnormal inflammasome activation profile revealed by an abnormal IL-lbeta secretion. The inventors treated cells with inhibitors of GasderminD (GSDMD) in a way to block the IL-lbeta secretion.

[0045] As used herein the term "inflammasome” relate to cytosolic multiprotein oligomers of the innate immune system responsible for the activation of inflammatory responses. Activation and assembly of the inflammasome promotes proteolytic cleavage, maturation and secretion of pro-inflammatory cytokines interleukin Ibeta (IL-lbeta) and interleukin 18 (IL-18), as well as cleavage of Gasdermin-D. The N-terminal fragment resulting from this cleavage induces a pro- inflammatory form of programmed cell death distinct from apoptosis, referred to as pyroptosis, and is responsible for secretion of the mature cytokines, presumably through the formation of pores in the plasma membrane. Inflammasome activation is initiated by different kinds of cytosolic pattern recognition receptors (PRRs) that respond to either microbe-derived pathogen- associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) generated by the host cell. Pattern recognition receptors involved in inflammasomes comprise NLRs (nucleotide-binding oligomerization domain and leucine-rich repeat-containing receptors) as well as AIM2 (absent in melanoma 2), IFI16 (IFN-inducible protein 16 ) as well as pyrin. Through their caspase activation and recruitment domain (CARD) or pyrin domain (PYD), the inflammasome receptors interact with the adaptor protein ASC, which then recruits pro-caspase- 1 via its CARD domain and activates the effector caspase through proteolytic cleavage. The activated caspase-1 finally cleaves the immature pro-inflammatory cytokines pro-IL-ip and pro-IL-18, as well as Gasdermin-D, which are responsible for inflammatory signaling and pyroptotic cell death, respectively. In addition to these so-called canonical inflammasomes, different studies also described non-canonical inflammasome complexes that act independently of caspase-1. In mice, the non-canonical inflammasome is activated by direct sensing of cytosolic bacterial lipopolysaccharide (LPS) by caspase-11, which subsequently induces pyroptotic cell death. In human cells, the corresponding caspases of the non-canonical inflammasome are caspase 4 and caspase 5.

[0046] As used herein the term "IL-lbeta" has its general meaning in the art and refers to Interleukin-1 beta. IL-1 beta is a member of the Interleukin 1 cytokine family. This cytokine is produced as a proprotein, which is proteolytically processed to its active form by Caspase 1 (CASP1 / ICE). This cytokine is an important mediator of the inflammatory response, and is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis.

[0047] As used herein, the terms “treating” or “treatment” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0048] In some embodiment, the present invention relates to an inhibitor of GasderminD (GSDMD) for use in the treatment of Rac2 monogenic disorders in a subject in need thereof.

[0049] As used herein, the term "GasderminD" (GSDMD) has its general meaning in the art and refers to a protein that in humans is encoded by the GSDMD gene on chromosome 8. It belongs to the gasdermin family which is conserved among vertebrates and comprises six members in humans, GSDMA, GSDMB, GSDMC, GSDMD, GSDME (DFNA5) and DFNB59 (Pejvakin). Members of the gasdermin family are expressed in a variety of cell types including epithelial cells and immune cells. GSDMA, GSDMB, GSDMC, GSDMD and GSDME have been suggested to act as tumour suppressors. The structure of full-length GSDMD consists of two domains, the 31 kDa N-terminal (GSDMD-N) and 22 kDa C-terminal (GSDMD-C) domains, separated by a linker region. GSDMD-C can be divided into four subdomains and is composed of 10 a-helices and two P-strands, forming a compact globular fold. The linker helix contacts the two helix-repeats which consist of four-helix bundles. The middle domain comprises an antiparallel P-strand and a short a-helix. The first flexible loop of GSDMD-C, which is located between GSDMD-N and the linker helix, stretches out and inserts into the GSDMD-N pocket, stabilizing the conformation of the full-length protein. GSDMD-N forms large transmembrane pores composed of 31 to 34 subunits that allow the release of interleukin- 1 (IL-1) family cytokines and drive pyroptosis. Human GSDMD is having the following Gene ID: 79792and is having the following UniProt number: P57764. Gasdermin D (GSDMD)- mediated pyroptosis and downstream inflammation are important self-protection mechanisms against stimuli and infections.

[0050] As used herein, the term "inhibitor of GasderminD" refers to a compound that regulate GasderminD reactions by slowing down or blocking them from occurring.

[0051] In a particular embodiment, the inhibitors of GasderminD (GSDMD) is a peptide, peptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide. The term “peptidomimetic” refers to a small protein-like chain designed to mimic a peptide.

[0052] In a particular embodiment, the inhibitors of GSDMD is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.

[0053] In some embodiments, the inhibitors of GSDMD is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of metabolites involved in GSDMD metabolism.

[0054] In a particular embodiment, the inhibitors of GSDMD is a siRNA. A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound. Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide-long doublestranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway whereby the siRNA interferes with the expression of a specific gene.

[0055] In a particular embodiment, the inhibitor of inhibitors of GSDMD is an anti-sense oligonucleotides (ASO). Anti-sense oligonucleotides include anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Antisense oligonucleotides, siRNAs, shRNAs of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically mast cells. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

[0056] In some embodiments, inhibitors of GSDMD is an endonuclease. In the last few years, staggering advances in sequencing technologies have provided an unprecedentedly detailed overview of the multiple genetic aberrations in cancer. By considerably expanding the list of new potential oncogenes and tumor suppressor genes, these new data strongly emphasize the need of fast and reliable strategies to characterize the normal and pathological function of these genes and assess their role, in particular as driving factors during oncogenesis. As an alternative to more conventional approaches, such as cDNA overexpression or downregulation by RNA interference, the new technologies provide the means to recreate the actual mutations observed in cancer through direct manipulation of the genome. Indeed, natural and engineered nuclease enzymes have attracted considerable attention in the recent years. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the errorprone nonhomologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR).

[0057] In a particular embodiment, the endonuclease is CRISPR-cas. As used herein, the term “CRISPR-cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.

[0058] In some embodiment, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR / Cas9 system has been described in US 8697359 Bl and US 2014 / 0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffini, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et al., 2013, Science, Vol. 339 : 823-826), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), zebrafish (Hwang et al., 2013, PLoS One, Vol. 8:e68708.), C. elegans (Hai et al., 2014 Cell Res. doi: 10.1038 / cr.2014.11.), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), plants (Mali et al., 2013, Science, Vol. 339 : 823-826), Xenopus tropicalis (Guo et al., 2014, Development, Vol. 141 : 707-714.), yeast (DiCarlo et al., 2013, Nucleic Acids Res., Vol. 41 : 4336-4343.), Drosophila (Gratz et al., 2014 Genetics, doi: 10.1534 / genetics.113.160713), monkeys (Niu et al., 2014, Cell, Vol. 156 : 836- 843.), rabbits (Yang et al., 2014, J. Mol. Cell Biol., Vol. 6 : 97-99.), pigs (Hai et al., 2014, Cell Res. doi: 10.1038 / cr.2014.11.), rats (Ma et al., 2014, Cell Res., Vol. 24 : 122-125.) and mice (Mashiko et al., 2014, Dev. Growth Differ. Vol. 56 : 122-129.). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. A recent exciting development is the use of the dCas9 version of the CRISPR / Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genome loci.

[0059] In some embodiment, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

[0060] In some embodiments, inhibitors of GSDMD is an antibody. As used herein, the term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody -based constructs and fragments are well known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93 / 1 1 161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001 ; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments. In some embodiments, the antibody is a “chimeric” antibody as described in U.S. Pat. No. 4,816,567. In some embodiments, the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409. In some embodiments, the antibody is a human antibody. A “human antibody” such as described in US 6,075,181 and 6,150,584. In some embodiments, the antibody is a single domain antibody such as described in EP 0 368 684, WO 06 / 030220 and WO 06 / 003388.

[0061] In a particular embodiment, the inhibitors of GSDMD is a monoclonal antibody. Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique.

[0062] In particular embodiment, the inhibitors of GSDMD is an intrabody. As used herein, the term "intrabody" generally refer to an intracellular antibody or antibody fragment. Antibodies, in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking / localization peptide sequences, such as, e.g., the endoplasmic reticulum retention. In some embodiments, the intrabody is a single domain antibody. In some embodiments, the antibody according to the invention is a single domain antibody. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.

[0063] In a particular embodiment, the inhibitors of GSDMD is a small organic molecule. The term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

[0064] In a particular embodiment, the GSDMD inhibitors, include but are not limited to disulfiram, necrosulfonamide (NSA), and dimethyl fumarate (DMF).

[0065] As used herein, the term “disulfiram” is an acetaldehyde dehydrogenase inhibitor. Disulfiram is having the following CAS Number : 97-77-8 and the following chemical structure :

[0066] As used herein, the term “necrosulfonamide” (NSA) is a necroptosis inhibitor acting by selectively targeting the mixed lineage kinase domain-like protein (MLKL). NSA is having the following CAS Number : 1360614-48-7 and the following chemical structure : As used herein, the term “dimethyl fumarate” (DMF) refers to a methyl ester of fumaric acid and has been described a prodrug. DMF is having the following CAS Number : 624-49-7 and the following chemical structure :

[0067] In some embodiments, the inhibitor of GasderminD is an inhibitor of GasderminD expression.

[0068] As used herein, an "inhibitor of expression" refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. In a particular embodiment of the invention, the inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme. For example, anti- sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of GSDMD mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of GSDMD, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding GSDMD can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566, 135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Small inhibitory RNAs (siRNAs) can also function as inhibitors of expression for use in the present invention. GSDMD gene expression can be reduced by contacting a subject or cell with a small double stranded R A (dsPvNA), or a vector or construct causing the production of a small double stranded R A, such that GSDMD gene expression is specifically inhibited (i.e. RNA interference or RNAi). Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically cells expressing GSDMD. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art. In some embodiments, the inhibitor consists in a vector that comprises the CRISPR / cas 9 protein and the appropriate RNA guide for disrupting the expression level of the gene encoding for GSDMD. In some embodiments, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA- guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

[0069] In a particular embodiment, i) an inhibitor of GSDMD and ii) a classical treatment as a combined preparation according to the invention for simultaneous, separate or sequential use in the treatment of Rac2 monogenic disorders.

[0070] In a particular embodiment, the classical treatment refers to immunosuppressive corticosteroids or non-steroidal therapies; immunotherapy: recombinant human IL- IB receptor antagonist; neutralizing monoclonal anti-IL-ip antibody; immune checkpoint inhibitors.

[0071] In a particular embodiment the invention relates to i) an inhibitor of GSDMD and ii) a corticosteroid used as a combined preparation for treating Rac2 monogenic disorders in a subject.

[0072] In a particular embodiment, i) an inhibitor of GSDMD ii) a corticosteroid as a combined preparation according to the invention for simultaneous, separate or sequential use in the treatment of Rac2 monogenic disorders.

[0073] As used herein, the term “corticosteroid” is well known in the art and refers to class of steroid hormones that are produced in the adrenal cortex as well as the synthetic analogues of these hormones. Two types of classes of corticosteroid exist in the art: glucocorticoids and mineralocorticoids. The corticosteroid for use in the invention is selected from the group consisting of: Flugestone (flurogestone); Fluorometholone; Medrysone; Prebediolone acetate; chlormadinone acetate, cyproterone acetate, medrogestone, medroxyprogesterone acetate, megestrol acetate, and segesterone acetate; Chloroprednisone; Cloprednol; Difluprednate; Fludrocortisone; Fluocinolone; Fluperolone; Fluprednisolone; Loteprednol; Methylprednisolone; Prednicarbate; Prednisolone; Prednisone; Tixocortol; Triamcinolone; Alclometasone; Beclometasone; Betamethasone; Clobetasol; Clobetasone; Clocortolone; Desoximetasone; Dexamethasone; Diflorasone; Difluocortolone; Fluclorolone; Flumetasone; Fluocortin; Fluocortolone; Fluprednidene; Fluticasone; Fluticasone furoate; Halometasone; Meprednisone; Mometasone; Mometasone furoate; Paramethasone; Prednylidene; Rimexolone; Ulobetasol (halobetasol); Amcinonide; Budesonide; Ciclesonide; Deflazacort; Desonide; Formocortal (fluoroformylone); Fluclorolone acetonide (flucloronide); Fludroxycortide (flurandr enol one, flurandrenolide); Flunisolide; Fluocinolone acetonide; Fluocinonide; Halcinonide; Triamcinolone acetonide; Cortivazol; RU-28362.

[0074] In a particular embodiment the invention relates to i) an inhibitor of GSDMD and ii) a nonsteroidal drug used as a combined preparation for treating Rac2 monogenic disorders in a subject.

[0075] In a particular embodiment, i an inhibitor of GSDMD and ii) a nonsteroidal drug as a combined preparation according to the invention for simultaneous, separate or sequential use in the treatment of Rac2 monogenic disorders.

[0076] As used herein, the term “nonsteroidal drug” refers to a class of drugs which decrease inflammation. The nonsteroidal drug for use in the invention is selected from the group consisting of: Aspirin (acetylsalicylic acid); Diflunisal (Dolobid); Salicylic acid and other salicylates Salsalate (Disalcid); Ibuprofen; Dexibuprofen ; Naproxen ; Fenoprofen ; Ketoprofen ; Dexketoprofen ; Flurbiprofen ; Oxaprozin; Loxoprofen; Indomethacin; Tolmetin; Sulindac; Etodolac; Ketorolac; Diclofenac; Aceclofenac; Nabumetone; Piroxicam; Meloxicam; Tenoxicam; Droxicam; Lornoxicam; Phenylbutazone; Mefenamic acid; Meclofenamic acid; Flufenamic acid; Tolfenamic acid; Celecoxib; Clonixin.

[0077] As used herein, the term “immunotherapy” has its general meaning in the art and refers to the treatment that consists in administering an immunogenic agent i.e. an agent capable of inducing, enhancing, suppressing or otherwise modifying an immune response.

[0078] In another embodiment the invention relates to i an inhibitor of GSDMD and ii) a neutralizing monoclonal anti-IL-ip antibody used as a combined preparation for treating Rac2 monogenic disorders.

[0079] In a particular embodiment, i) an inhibitor of GSDMD and ii) a neutralizing monoclonal anti-IL-ip antibody as a combined preparation according to the invention for simultaneous, separate or sequential use in the treatment of Rac2 monogenic disorders.

[0080] As used herein, the term “a neutralizing monoclonal anti-IL-ip antibody” refers to an antibody that blocks or reduces at least one activity of a polypeptide comprising the epitope to which the antibody specifically binds. The neutralizing antibody reduces IL-ip biological activity in in cellulo and / or in vivo tests. In the context of the invention, the neutralizing monoclonal anti-IL-ip antibody is canakinumab (trade name Haris, developed by Novartis). In another embodiment the invention relates to i) an inhibitor of GSDMD and ii) a recombinant human IL-lbeta receptor antagonist used as a combined preparation for treating Rac2 monogenic disorders in a subject.

[0081] In a particular embodiment, i) an inhibitor of GSDMD and ii) a recombinant human IL- 1B receptor antagonist as a combined preparation according to the invention for simultaneous, separate or sequential use in the treatment of Rac2 monogenic disorders. In the context of the invention, the recombinant human IL-1B receptor antagonist is Anakinra (marketed as Kineret® by Swedish Orphan Biovitru).

[0082] In another embodiment the invention relates to i) an inhibitor of GSDMD and ii) an immune checkpoint inhibitor used as a combined preparation for treating Rac2 monogenic disorders in a subject.

[0083] In a particular embodiment, i) an inhibitor of GSDMD and ii) an immune checkpoint inhibitor as a combined preparation according to the invention for simultaneous, separate or sequential use in the treatment Rac2 monogenic disorders.

[0084] As used herein, the term "immune checkpoint inhibitor" has its general meaning in the art and refers to any compound inhibiting the function of an immune inhibitory checkpoint protein.

[0085] As used herein the term "immune checkpoint protein" has its general meaning in the art and refers to a molecule that is expressed by T cells in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules). Immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al. , 2011. Nature 480:480- 489). Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD- 1, LAG-3, TIM-3 and VISTA. Inhibition includes reduction of function and full blockade. Preferred immune checkpoint inhibitors are antibodies that specifically recognize immune checkpoint proteins. A number of immune checkpoint inhibitors are known and in analogy of these known immune checkpoint protein inhibitors, alternative immune checkpoint inhibitors may be developed in the (near) future. The immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules and small molecules. Examples of immune checkpoint inhibitor includes PD-1 antagonist, PD-L1 antagonist, PD-L2 antagonist CTLA-4 antagonist, VISTA antagonist, TIM-3 antagonist, LAG-3 antagonist, IDO antagonist, KIR2D antagonist, A2AR antagonist, B7-H3 antagonist, B7-H4 antagonist, and BTLA antagonist. In some embodiments, PD-1 (Programmed Death-1) axis antagonists include PD-1 antagonist (for example anti-PD-1 antibody), PD-L1 (Programmed Death Ligand-1) antagonist (for example anti-PD-Ll antibody) and PD-L2 (Programmed Death Ligand-2) antagonist (for example anti-PD-L2 antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of MDX-1106 (also known as Nivolumab, MDX-1106-04, ONO-4538, BMS-936558, and Opdivo®), Merck 3475 (also known as Pembrolizumab, MK-3475, Lambrolizumab, Keytruda®, and SCH-900475), and CT-011 (also known as Pidilizumab, hBAT, and hBAT-1). In some embodiments, the PD-1 binding antagonist is AMP -224 (also known as B7-DCIg). In some embodiments, the anti-PD-Ll antibody is selected from the group consisting of YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. MDX-1105, also known as BMS-936559, is an anti-PD-Ll antibody described in W02007 / 005874. Antibody YW243.55. S70 is an anti-PD-Ll described in WO 2010 / 077634 AL MEDI4736 is an anti-PD- Ll antibody described in WO2011 / 066389 and US2013 / 034559. MDX-1106, also known as MDX-1 106-04, ONO-4538 or BMS-936558, is an anti-PD-1 antibody described in U.S. Pat. No. 8,008,449 and W02006 / 121168. Merck 3745, also known as MK-3475 or SCH-900475, is an anti-PD-1 antibody described in U.S. Pat. No. 8,345,509 and W02009 / 114335. CT-011 (Pidizilumab), also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in W02009 / 101611. AMP -224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in W02010 / 027827 and WO2011 / 066342. Atezolimumab is an anti-PD-Ll antibody described in U.S. Pat. No. 8,217,149. Avelumab is an anti-PD-Ll antibody described in US 20140341917. CA-170 is a PD-1 antagonist described in W02015033301 & WO2015033299. Other anti-PD-1 antibodies are disclosed in U.S. Pat. No. 8,609,089, US 2010028330, and / or US 20120114649. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody chosen from Nivolumab, Pembrolizumab or Pidilizumab. In some embodiments, PD-L1 antagonist is selected from the group comprising of Avelumab, BMS-936559, CA-170, Durvalumab, MCLA-145, SP142, STI-A1011, STIA1012, STI-A1010, STI-A1014, Al 10, KY1003 and Atezolimumab and the preferred one is Avelumab, Durvalumab or Atezolimumab.

[0086] In some embodiments, CTLA-4 (Cytotoxic T-Lymphocyte Antigen-4) antagonists are selected from the group consisting of anti-CTLA-4 antibodies, human anti-CTLA-4 antibodies, mouse anti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, humanized anti-CTLA- 4 antibodies, monoclonal anti-CTLA-4 antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4 antibodies, MDX-010 (Ipilimumab), Tremelimumab, anti-CD28 antibodies, anti-CTLA-4 adnectins, anti-CTLA-4 domain antibodies, single chain anti-CTLA- 4 fragments, heavy chain anti-CTLA-4 fragments, light chain anti-CTLA-4 fragments, inhibitors of CTLA-4 that agonize the co-stimulatory pathway, the antibodies disclosed in PCT Publication No. WO 2001 / 014424, the antibodies disclosed in PCT Publication No. WO 2004 / 035607, the antibodies disclosed in U.S. Publication No. 2005 / 0201994, and the antibodies disclosed in granted European Patent No. EP 1212422 B. Additional CTLA-4 antibodies are described in U.S. Pat. Nos. 5,811,097; 5,855,887; 6,051,227; and 6,984,720; in PCT Publication Nos. WO 01 / 14424 and WO 00 / 37504; and in U.S. Publication Nos. 2002 / 0039581 and 2002 / 086014. Other anti-CTLA-4 antibodies that can be used in a method of the present invention include, for example, those disclosed in: WO 98 / 42752; U.S. Pat. Nos. 6,682,736 and 6,207,156; Hurwitz et al., Proc. Natl. Acad. Sci. USA, 95(17): 10067-10071 (1998); Camacho et al., J. Clin: Oncology, 22(145): Abstract No. 2505 (2004) (antibody CP- 675206); Mokyr et al., Cancer Res., 58:5301-5304 (1998), and U.S. Pat. Nos. 5,977,318, 6,682,736, 7,109,003, and 7,132,281. A preferred clinical CTLA-4 antibody is human monoclonal antibody (also referred to as MDX-010 and Ipilimumab with CAS No. 477202-00- 9 and available from Medarex, Inc., Bloomsbury, N.J.) is disclosed in WO 01 / 14424. With regard to CTLA-4 antagonist (antibodies), these are known and include Tremelimumab (CP- 675,206) and Ipilimumab.

[0087] In some embodiments, the immunotherapy consists in administering to the subject a combination of a CTLA-4 antagonist and a PD-1 antagonist.

[0088] Other immune-checkpoint inhibitors include lymphocyte activation gene-3 (LAG-3) inhibitors, such as IMP321, a soluble Ig fusion protein (Brignone et al., 2007, J. Immunol. 179:4202-4211). Other immune-checkpoint inhibitors include B7 inhibitors, such as B7-H3 and B7-H4 inhibitors. In particular, the anti-B7-H3 antibody MGA271 (Loo et al., 2012, Clin. Cancer Res. July 15 (18) 3834). Also included are TIM-3 (T-cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade et al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al., 2010, J. Exp. Med. 207:2187-94). As used herein, the term “TIM-3” has its general meaning in the art and refers to T cell immunoglobulin and mucin domain-containing molecule 3. The natural ligand of TIM-3 is galectin 9 (Gal9). Accordingly, the term “TIM-3 inhibitor” as used herein refers to a compound, substance or composition that can inhibit the function of TIM-3. For example, the inhibitor can inhibit the expression or activity of TIM-3, modulate or block the TIM-3 signaling pathway and / or block the binding of TIM-3 to galectin-9. Antibodies having specificity for TIM-3 are well known in the art and typically those described in WO201 1155607, W02013006490 and WO2010117057. In some embodiments, the immune checkpoint inhibitor is an IDO inhibitor. Examples of IDO inhibitors are described in WO 2014150677. Examples of IDO inhibitors include without limitation 1-methyl-tryptophan (IMT), P- (3-benzofuranyl)-alanine, P-(3- benzo(b)thienyl)-alanine), 6-nitro-tryptophan, 6- fluoro-tryptophan, 4-methyl-tryptophan, 5 - methyl tryptophan, 6-methyl-tryptophan, 5 -methoxy -tryptophan, 5 -hydroxy-tryptophan, indole 3-carbinol, 3,3'- diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3- diacetate, 9- vinylcarbazole, acemetacin, 5 -bromo-tryptophan, 5 -bromoindoxyl diacetate, 3- Amino-naphtoic acid, pyrrolidine dithiocarbamate, 4-phenylimidazole a brassinin derivative, a thiohydantoin derivative, a P-carboline derivative or a brassilexin derivative. Preferably the IDO inhibitor is selected from 1-methyl-tryptophan, P-(3- benzofuranyl)-alanine, 6-nitro-L- tryptophan, 3-Amino-naphtoic acid and P-[3- benzo(b)thienyl] -alanine or a derivative or prodrug thereof.

[0089] In some embodiment, the method of the treatment of the present invention which also comprises a Rac2 inhibitor.

[0090] In another embodiment the invention relates to i) an inhibitor of GSDMD and ii) a Rac2 inhibitor used as a combined preparation for treating Rac2 monogenic disorders.

[0091] In a particular embodiment, i) an inhibitor of GSDMD and ii) a Rac2 inhibitor as a combined preparation according to the invention for simultaneous, separate or sequential use in the treatment of Rac2 monogenic disorders.

[0092] As used herein the term “Rac2 inhibitor” refers to an agent that blocks the activity of any protein in the Rho GTPase signal transduction pathway.

[0093] In some embodiment, the Rac2 inhibitor is a famesyl protein transferase inhibitor.

[0094] A used herein the term “farnesyl protein transferase inhibitor” or “FPT inhibitor” or “FTI” refers to a compound that: (i) potently inhibits FPT (but generally not geranylgeranyl protein transferase I) and (ii) blocks intracellular famesylation of Ras. FPT catalyzes the addition of an isoprenyl lipid moiety onto a cysteine residue present near the carboxy-terminus of the Ras protein. This is the first step in a post-translational processing pathway that is essential for both Ras membrane-association and Ras-induced oncogenic transformation. A number of FPT inhibitors have been reported, including a variety of peptidomimetic inhibitors as well as other small molecule inhibitors.

[0095] Famesyl transferase inhibitors generally fall into two classes: analogs of farnesyl diphosphate; and protein substrates for famesyl transferase. Farnesyl transferase inhibitors have been described in U.S. Pat. No. 5,756,528, U.S. Pat. No. 5,141,851, U.S. Pat. No. 5,817,678, U.S. Pat. No. 5,830,868, U.S. Pat. No. 5,834,434, and U.S. Pat. No. 5,773,455, incorporated herein by reference. Among the famesyl transferase inhibitors shown to be effective for inhibiting the transfer of the famesyl moiety to Ras-related proteins are L-739,749 (a peptidomimetic analog of the C-A-A-X sequence), L-744,832 (a peptidomimetic analog of the C-A-A-X sequence), SCH 44342(l-(4-pyridylacetyl)-4-(8-chloro-5,6 dihydro-IIH benzo[5,6]cyclohepta[l,2-b]pyridin-l l-yhdene)piperidine), BZA-5B (a benzodiazepine peptidomimetic), FTI-276 (a C-A-A-X peptidomimetic), and Bl 086 (a C-A-A-X peptidomimetic)

[0096] In some embodiment, the Rac2 inhibitor is S-famesylthiosalicylic Acid (FTS) and its derivatives and analogs. In some embodiment, the Rac2 inhibitor is imidazole-containing benzodiazepines and analogs. In some embodiment, the Rac2 inhibitor is chosen among; EHT1864 (CAS No. 754240-09-0), MBQ-167 (CAS No. 2097938-73-1) or NSC 23766 (CAS No. 1177865-17-6).

[0097] In some embodiment, the method of the treatment of the present invention which also comprises an IL-lbeta inhibitor.

[0098] In another embodiment the invention relates to i) an inhibitor of GSDMD and ii) an IL- lbeta inhibitor used as a combined preparation for treating Rac2 monogenic disorders.

[0099] In a particular embodiment, i) an inhibitor of GSDMD and ii) an IL-lbeta inhibitor as a combined preparation according to the invention for simultaneous, separate or sequential use in the treatment of Rac2 monogenic disorders.

[0100] As used herein the term “IL-lbeta inhibitor” an agent that blocks the activity of IL- lbeta.

[0101] In some embodiment, the IL-lbeta inhibitor are chosen from anakinra (CAS No. 143090-92-0), canakinumab (CAS No. 914613-48-2), gevokizumab (CAS No. 1129435-60-4) and rilonacept (CAS No. 501081-76-1).

[0102] As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g. an inhibitor of GSDMD) into the subject, such as by oral, mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof. In a particular embodiment, a topical administration is performed to the subject. More particularly, the an inhibitor of GSDMD are formulated as a cream for a topical administration. In another embodiment, an oral administration is performed to the subject. In a further embodiment, intravenous administration is performed to the subject.

[0103] By a "therapeutically effective amount" is meant a sufficient amount of an inhibitor of GSDMD for use in a method for the treatment of Rac2 monogenic disorders at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic 20 adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0104] Typically the active ingredient of the present invention is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. The term "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0105] Combined preparation:

[0106] The inhibitor of GSDMD as described above is also combined with a classical treatment. In one embodiment, the invention relates to i) an inhibitor of GSDMD and ii) an inhibitor of PAK1 / 2 used as a combined preparation for treating Rac2 monogenic disorders in a subject.

[0107] In another embodiment, the invention relates to i) an inhibitor of GSDMD and ii) a Caspase-1 inhibitor used as a combined preparation for treating Rac2 monogenic disorders in a subject.

[0108] In another embodiment, the invention relates to i) an inhibitor of GSDMD and ii) an inhibitor of NLRP3 used as a combined preparation for treating Rac2 monogenic disorders in a subject.

[0109] In one embodiment, the invention relates to i) an inhibitor of GSDMD and ii) an IL- Ibeta inhibitor used as a combined preparation for treating Rac2 monogenic disorders in a subject.

[0110] In one embodiment, the invention relates to i) an inhibitor of GSDMD and ii) a Rac2 inhibitor used as a combined preparation for treating Rac2 monogenic disorders in a subject.

[0111] As used herein, the terms “combined treatment”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication. The combined therapy may be dual therapy or bi-therapy.

[0112] As used herein, the term “administration simultaneously” refers to administration of 2 active ingredients by the same route and at the same time or at substantially the same time. The term “administration separately” refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes. The term “administration sequentially” refers to an administration of 2 active ingredients at different times, the administration route being identical or different.

[0113] Pharmaceutical composition:

[0114] An inhibitor of GSDMD 1 for use according to the invention alone and / or combined with a classical treatment as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.

[0115] Accordingly, in a further aspect, the invention relates to a pharmaceutical composition comprising an inhibitor of GSDMD for treating Rac2 monogenic disorders.

[0116] In a particular embodiment, the pharmaceutical composition according the invention, wherein the inhibitor of GSDMD is disulfiram, necrosulfonamide (NSA), and dimethyl fumarate (DMF). In some embodiment, the pharmaceutical composition of the present invention comprises an IL-lbeta inhibitor.

[0117] In some embodiment, the pharmaceutical composition of the present invention comprises a Rac2 inhibitor.

[0118] As used herein, the terms "pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuumdrying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0119] Method for screening:

[0120] In a further aspect, the invention relates to a method of screening a drug suitable for the treating Rac2 monogenic disorders comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the expression or activity of GSDMD.

[0121] Any biological assay well known in the art could be suitable for determining the ability of the test compound to inhibit the activity or expression of GSDMD. In some embodiments, the assay first comprises determining the ability of the test compound to bind to GSDMD. In some embodiments, a population of cells then contacted and activated so as to determine the ability of the test compound to inhibit the activity or expression of GSDMD. In particular, the effect triggered by the test compound is determined relative to that of a population of immune cells incubated in parallel in the absence of the test compound or in the presence of a control agent either of which is analogous to a negative control condition. The term "control substance", "control agent", or "control compound" as used herein refers a molecule that is inert or has no activity relating to an ability to inhibit a biological activity or expression. It is to be understood that test compounds capable of inhibiting the activity or expression of GSDMD, as determined using in vitro methods described herein, are likely to exhibit similar modulatory capacity in applications in vivo. Typically, the test compound is selected from the group consisting of peptides, petptidomimetics, small organic molecules, antibodies (e.g. intraantibodies), aptamers or nucleic acids. For example the test compound according to the invention may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo.

[0122] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0123] FIGURES:

[0124] Figure 1: The RAC2 A59S human patient mutation activates the NLRP3 inflammasome. U937 Cas9 (Cas9), NLRP3 KO or GasderminD KO (GSDMD KO) cells were co-transduced for 72h with Vpx containing virus like particles and HA-tagged RAC2 A59S or E62K encoding lentiviral vectors. Empty HIVSFFV-HARAC2-IRES-GFP vector was used as control. LPS was added for 8h to induce the expression of pro-IL-lB. Supernatants were analyzed using enzyme- linked immunosorbent assay (ELISA) for IL-ip. Data are mean ± SEM (Data are representative for n=3).

[0125] EXAMPLE:

[0126] Material & Methods

[0127] Patients and human samples

[0128] The study was conducted in accordance with French legislation and the principles of the Declaration of Helsinki. Informed consent was obtained from the patients’ parents or legal guardians, and the study protocol was approved by the regional independent ethics committee and the French Ministry of Research (2015-01-05 MS2; DC-2020-3994), as well as the French Advisory Committee on Data Processing in Medical Research (Comite Consultatif sur le Traitement de Flnformation en matiere de Recherche dans le domaine de la Sante, Paris, France; 15.297bis). Human blood from healthy donors was obtained from the Etablissement Frangais du Sang (13-PP-l 1 / CCTIRS N°14.266).

[0129] Cells isolation, culture, and reagents

[0130] HEK293T cells were obtained from ATCC (CRL-3216) and maintained according to the ATCC instructions. Human peripheral blood was obtained from healthy donors or from patients with Rac2 mutation with informed consent following the Declaration of Helsinki according to recommendations of an independent scientific review board. Human blood from healthy donors was obtained by Etablissement Frangais du Sang. Human blood from patients with Rac2 mutation was obtained in collaboration with IMAGINE. PBMCs (Peripheral Blood Mononuclear Cells, including monocytes and lymphocytes) were isolated using Ficoll (Cytiva) and red cells were lysed by using lysing buffer (BD Biosciences). Then, monocytes were purified from PBMCs using anti-CD14 microbeads (Miltenyi) and autoMACS® Pro Separator (Miltenyi). Both PBMCs and monocytes were cultivated in RPMI 1640 medium with glutamax- I (Life Technologies) supplemented with 10% (vol / vol) fetal bovine serum (Life Technologies). Macrophage differentiation was induced by addition of CSF-1 (100 ng / mL, Miltenyi) into the culture medium during 6 days. Inhibitors and reagents used in this study are: lOpM CP -456773 or MCC950 (Sigma-Aldrich), 5pM IPA-3 (Tocris), IpM AZ13711265 (AGV Discovery), 5pM Emricasan (Sigma- Aldrich), 100 ng / mL ultrapure LPS (Invivogen), 5 pM Nigericin (Invivogen).

[0131] Plasmid constructs, cell transfection and transduction

[0132] Human Rac2 mutations were obtained using the QuickChange Site Directed Mutagenesis Kit (Stratagene Europe) on pCDNA3.1-HA3-Rac2 wild-type.

[0133] Human Rac2 (h-Rac2) was subcloned from pCDNA3.1 Rac2 in pHSFFV-TIG-GFP (a kind gift of Els Verhoy en) using InFusion HD Cloning Kit (Takara Bio). Briefly, h-Rac2 wild type and D57N, Q61E, E62K, and D63V derivative mutants were PCR amplified using primers oligo IF and oligo 2R. PCR products were further cloned into pHSFFV-TIG-GFP previously digested using BamHI / XhoI restriction enzymes. H-Rac2 G12R, G12V, P34H, A59S, Q61R and N92T mutants were obtained by site-directed mutagenesis (QuickChange Lightning Site- Directed Mutagenesis Kit, Agilent Technologies) using the primers listed in supplementary table. All plasmid constructs were verified by sequencing (Eurofin).

[0134] For HA-Rac2 lentiviral particles production, HEK293T cells were co-transfected using calcium phosphate precipitation with pTIG-GFP-HA-Rac2 WT or mutants, psPAX2 packaging plasmid that co-expresses Gag-Pol, Tat, Rev (Addgene), and phCMV-VSV-G (Addgene).

[0135] To produce the Vpx-VLps, a plasmid SIV3+ (Mangeot PE, Duperrier K, Negre D, Boson B, Rigal D, Cosset FL, Darlix JL. High levels of transduction of human dendritic cells with optimized SIV vectors. Mol Ther. 2002 Mar;5(3):283-90. doi: 10.1006 / mthe.2002.0541. PMID: 11863418) coding for simian immunodeficiency virus gagpropol and Vpx and a plasmid phCMV-VSV-G were co-transfected by calcium phosphate preicpitaiton in HEK293T cells.

[0136] Supernatants containing lentiviral particles were collected 48 h after transfection, filtered through a 0.45 pm pore-size filter before overnight centrifugation at 3,000 g, 4°C. The supernatant was carefully removed by aspiration to obtain 100-150x concentration. To determine the concentration of lentiviral particles, HEK293T cells were infected with various amounts of lentiviral particles during 72h and GFP expression was tittered by FACS.

[0137] To transduce macrophages, a transduction protocol described earlier was modified (Berger G, Durand S, Goujon C, Nguyen XN, Corded S, Darlix JL, Cimarelli A. A simple, versatile and efficient method to genetically modify human monocyte-derived dendritic cells with HIV-l-derived lentiviral vectors. Nat Protoc. 2011 Jun;6(6):806-16. doi: 10.1038 / nprot.2011.327. Epub 2011 May 19. PMID: 21637200): human monocytes were seeded on 24-well plates in complete RPMI medium and derived in macrophages by adding CSF-1 for 6 days. When indicated, inhibitors were added 24h before cell transduction. Cells were then co-infected during 72h with a multiplicity of infection (MOI) of 10 particles / cell for pTIG-GFP-HA-Rac2 and an equivalent quantity of Vpx-VLP particles. To induce IL- IB secretion, fresh medium containing LPS was added for 8 hours. Supernatants were collected for cytokines assays and expression of transduced proteins in the cell lysates was verified by immunoblotting.

[0138] Immunoblotting

[0139] Total protein extracts were prepared by lysing cells with LDS sample buffer (Pierce) supplemented with 50 mM DTT (Euromedex) then fractionated on poly- acrylamide gels using SDS-PAGE, transferred to nitrocellulose membranes (BioRad), incubated with specific antibodies. Immobilon Western Chemiluminescent HRP Substrate (Millipore) and a PXi4 GeneSys imaging system (Syngene) were used for detection. Densitometric data analysis was performed using Imaged software (NIH, USA).

[0140] The primary antibodies used for immunoblotting were mouse anti-caspase- 1 (clone Casper- 1, Adipogen), mouse anti-NLRP3 (clone Cryo-2, Adipogen), mouse anti-P-actin (AC- 74, Sigma-Aldrich), mouse anti-Flag (clone M2, Sigma-Aldrich), mouse anti-GFP (clone7.1, 13.1, Roche), mouse anti-HA (16B12, BioLegend), mouse anti-GST (clone 26H1, CST). The secondary antibody was a polyclonal goat anti mouse immunoglobulins conjugated with HRP (Agilent Technologies).

[0141] Activated Rac GTPase pull-down

[0142] HEK293T cells were transfected using Lipofectamine 2000 (Life Technologies) with pCDNA3.1-HA3 plasmids encoding Rac2 WT, the constitutively active mutants Rac2G12V and the one mimicking CNF 1 -induced deamidation Rac2Q61E or harboring human mutations G12R, P34H, D57N, A59S, Q61R, E62K, D63V, N92T for 16h. Cells were lysed at 4°C using a lysis buffer (Tris 25mM pH 7.5, NaCl 150mM, MgC12 5mM, TritonXIOO 0.5%, glycerol 4%) and Pull-down assays were performed on Img of proteins using 30 pg of GST-PAK70-106. Total and activated HA-Rac2 was revealed by immunoblotting anti-HA. Equal amounts of proteins engaged in the Pull-down assays were confirmed by immunoblotting anti-P-actin. Equal amounts of GST-PAK70-106 proteins engaged in the Pull-down assays were confirmed by immunoblotting anti-GST. Reconstituted NLRP3 inflammasome in HEK293T cell system

[0143] HEK293T cells were transfected using Lipofectamine 2000 with plasmids encoding the NLRP3 inflammasome components as previously described (Nature microbiology, 2021). Briefly, HEK293T cells were transfected for 16h with plasmids encoding myc-NLRP3, ASC- GFP, mpro-Caspasel and pro-IL-113-Flag and various HA-Rac2 mutants. The monitoring of IL- 113 cleavage was performed using supernatant immunoblotting. Equal expression of transfected proteins in the cell lysates was confirmed by immunoblotting.

[0144] Immunofluorescence staining

[0145] Cells were fixed in 4% paraformaldehyde for 15 min, PFA was neutralized with 50 mM NH4C1 for 15 min, cells were permeabilized with 0.5% Triton X-100 for 5 min and blocked with 2% TBS-BSA. Cells were incubated with mouse anti-NLRP3 (clone Cryo-2, Adipogen) and rabbit anti-ASC (AG-25B-0006, Adipogen) antibodies for Ih followed by incubation with the secondary antibodies TexasRed anti-mouse IgG (TL2000, Vector Laboratories), Alexa Fluor® 488 anti-rabbit IgG (Life Technologies), Phalloidin iFluor 647 conjugate (abl76759, Abeam) and Hoechst 33342 (H1399, Thermo Fisher Scientific) for 30 min. Cells were imaged using a Nikon AIR confocal microscope.

[0146] Cytokine assays

[0147] IL-lb secretion in differentiated macrophages was induced with 100 ng.ml-1 LPS for 8h. Human IL-lb and human total IL-18 cytokine concentrations in cell supernatants were determined by DuoSet ELISA (DY201 and DY318 respectively) according to the manufacturer’s instructions (R&D Systems). Briefly, after overnight coating of capture antibody on 96-well microplates, supernatants and standards were added for 2 hours, followed by 2 hours incubation with detection antibody. Streptavidin-HRP was then added for 20 minutes before incubation in Substate solution for another 20 minutes. Optical density was determined immediately after addition of stop solution, using a microplate reader set to 450nm and 540nm to correct optical imperfections in the plate. The concentrations of cytokine were then determine using the generated standard curve.

[0148] LDH release

[0149] The culture medium of LPS-stimulated U937 cell lines were collected and centrifuged at 300g for 5 min to remove cellular debris. LDH measurement was performed using the LDH Cytotoxicity Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Data were plotted as the percentage of cytotoxicity considering a Triton X-100 treated well as 100%.

[0150] Flow cytometry analysis

[0151] Whole blood or PBMCs isolated from healthy donors or from patients with RAC2 mutation were used. Caspase-1 activation was detected using F AM- YV AD -FLIC A probe according to the manufacturer’s instruction (ICT098, Bio-Rad). Cells were collected and analyzed by flow cytometry using a Cytek Aurora cytometer (Cytek). Cytometry data were analyzed using FlowJo v.10.8.1. Doublets were excluded using a side scatter SSC-A (area) and SSC-H (height) plot. For whole blood, cells were incubated for 10 min with antibodies (APC- Vio 770 anti-CDlO (130-114-505, Miltenyi Biotec), Vioblue anti-CD14 (130-110-524, Miltenyi Biotec), PE anti-CD16 (130-113-393, Miltenyi Biotec), APC anti-CD45 (130-110- 633, Miltenyi Biotec) and PE-Vio 770 anti-CD66b (130-119-768, Miltenyi Biotec) before red blood cells lysis. For PBMCs, Vioblue anti-CD14 (130-110-524, Miltenyi Biotec) antidoby was incubated for 10 min with cells. Cells were then analyzed for Caspase-1 activation and formation of specks by checking the FAM-FLICA probe signal area (Caspase-1 -A) and height (Caspase-1 -H). Cells positive for Capase-1 specks were defined with a high Caspase-1 - A / Caspase-l-H ratio as compared to control cells.

[0152] Bulk RNAseq and Single cell RNAseq analysis

[0153] Overnight cultured MOLM-13 cells were transduced during 7h in the presence of lentiboost (Sirion) and with the appropriated lentiviral supernatant at a multiplicity of infection of 30. Two days after the transduction step, cells were collected, and pellet processed for transcriptomic analysis. The lentiviral constructs contain a GFP reporter gene allowing to check that more than 92% of the cells were transduced. For single cell RNAseq, blood samples were processed for Chromium Single Cell Gene Expression Flex analysis according to manufacturer protocol. Whole blood cells from control (n= 8078 cells) and RAC2 A59S patient (n= 14524 cells) were analyzed. Raw sequencing data were processed using the 10x Chromium CellRanger "multi" analysis pipeline (version 7.0.0). Reads were aligned to the human reference genome (GROG 8 -3.0.0) (lOx Genomics). Rstudio (version 4.3.1) and Seurat (version 5.0) was used to merge, scale and normalize gene expression data, as well as for clustering, differential gene expression analysis and visualizations. We used scType Cell Marker Database for Cell-type annotation. Statistical analyses

[0154] Statistical analyses were performed using GraphPad Prism v.8.2.1.

[0155] Supplementary table

[0156] Results

[0157] Activating human RAC 2 mutations activate the NLRP3 inflammasome

[0158] We noticed that some of the activating RAC2 variants are interestingly located very close to the site modified by the CNF1 toxin that catalyzes the RAC2 post translational modification Q61E thereby activating the NLRP3 inflammasome (Boyer et al., 2011; Dufies and Boyer, 2021; Dufies et al., 2021). The most striking example is the RAC2 Q61R variant that affects the same residue (Stem et al., 2021). We thus investigated whether this mutant behaved similar to the mutant mimicking the CNF1 toxin induced Q61E modification using the GST-PAK-RBD pull down assay. We measured similar binding strength of the PAK kinase for both Q61E and Q61R variants, but not for the RAC2 D57N, a variant associated with a RAC2 inhibitory effect found in patients suffering from immunodeficiency (Data not shown). We thus extended our study to neighboring mutations affecting position 62 (E62K), 63 (D63V) (Data not shown) as well as mutations in position 12 (G12R and G12V) (Data not shown), a position known to be important for the GTPase activity of RAC2 (Illenberger et al., 2003). We measured for all these mutants a strong GST-PAK-RBD binding capacity, confirming that these mutations correspond to a gain of function and indicating their ability to bind constitutively PAK kinases (Data not shown). We next investigated whether the RAC2 mutants found in genetic disorders could activate the NLRP3 inflammasome. The deamidation of the glutamine 61 of RAC2 into a glutamate triggered by the CNF1 toxin confers to RAC2 the ability to bind and activate constitutively PAK1 leading to NLRP3 phosphorylation and inflammasome activation (Dufies et al., 2021). We thus compared the capacity of RAC2 Q61E mimicking the CNF 1 -triggered modification to the RAC2 mutations found in immune disorders using a NLRP3 inflammasome reconstitution system. In this system, the members of the NLRP3 inflammasome (NLRP3, ASC, Caspase-1) and the pro-IL-lb cytokine are transfected in a non-professional immune cell (HEK293T) lacking the NLRP3 inflammasome. We determined that the RAC2 Q61R variant was able to trigger pro-IL-lb cleavage and secretion of the mature IL-lb indicating its capacity to activate the NLRP3 inflammasome (Data not shown). Similar results were found for RAC2 E62K and D63 V whereas the RAC2 D57N variant, associated with a RAC2 inhibitory effect, was similar to the control (empty vector) condition and lower than the wild type (WT) form of RAC2 in terms of pro-IL-lb cleavage and secretion of IL-lb (Data not shown). We detected an increased IL-lb secretion triggered by the RAC2 G12R compared to both the empty vector and the RAC2 wild type controls (Data not shown). Similar results were found with the RAC2 G12V mutant used here as a positive control (Data not shown). Altogether these results indicated that the NLRP3 inflammasome activation depends on RAC2 activation and PAK binding rather than the position of the mutated residue.

[0159] Activating RAC 2 mutations trigger inflammasome activation in circulating myeloid cells of patients and human primary macrophages

[0160] As previously mentioned, RAC2 expression is mostly restricted to hematopoietic system (Burridge and Wennerberg, 2004). To further determine the consequences of RAC2 activating mutation on inflammasome activation, we directly monitored the Caspase- 1 activity in blood circulating leukocytes of a patient harboring a RAC2 E62K mutation. To this aim, we used the F AM- YV AD-FLIC A probe that binds to the active Caspase-1 and first analyzed the global Caspase-1 activity of circulating leukocytes by quantifying the FAM-YVAD-FLICA mean fluorescence intensity (MFI) of monocytes, granulocytes, and lymphocytes. We found in a RAC2 E62K patient an increased Caspase- 1 activity specifically in monocytes and granulocytes that was not observed in lymphocytes compared to healthy controls (Data not shown). To further demonstrate that this Caspase-1 activity was related to the inflammasome activation, we next set-up an assay to analyze the speck formation, known to be specific of inflammasome oligomerization (Data not shown). The speck quantification was primarily developed to monitor the number of cells with ASC specks by FACS using cells expressing ASC-EGFP or labeled with ASC antibodies (Sester et al., 2016). Here, we adapted this technic to further quantify by FACS directly in the blood the number of cells with FAM-FLICA specks. To this aim we analyzed the number of cells harboring specks of activated Caspase-1 by measuring the A / H ratio (Data not shown). We validated our approach by quantifying the increased number of FAM- YV AD-FLIC A specks triggered by Nigericin, a well-known trigger of the NLRP3 inflammasome, directly in circulating monocytes of control healthy donors (S Data not shown). This Nigericin-triggered caspase-1 speck formation was correlated with an increased F AM- YV AD-FLIC A MFI in Nigericin treated monocytes Data not shown). Next, we measured directly in the blood of a RAC2 E62K patient an increased number of specks in both monocytes and granulocytes compared to controls. Interestingly, the strongest signal was found in monocytes with up to 12% of monocytes presenting specks compared to a mean of 4% in the controls {Data not shown). To further confirm the NLRP3 inflammasome activation triggered by RAC2 gain of function variants, we next expressed the RAC2 variants in monocytes derived macrophages isolated from the blood of healthy donors. We expressed these mutants using a lentiviral expression system and measured the secreted level of IL-lb and IL- 18, cytokines related to inflammasome activation, in primary human macrophages primed by LPS. Using these settings, we found that the over-expression of all tested gain of function variants induced higher level of secreted IL-lb and IL-18 {Data not shown). Conversely, we observed a decreasing trend in cytokines secretion associated with the expression of the D57N inactive variant of RAC2 as compared to the RAC2 WT {Data not shown). Next, we overexpressed RAC2 variants in MOLM-13, a cell line derived from a patient with acute myeloid leukemia. To determine the transcriptional impact of this inflammasome activation, we introduced the activating mutant RAC2 E62K into MOLM-13 and compared it to MOLM-13 over-expressing RAC2 WT. For both conditions RNAseq analysis was performed and compared to MOLM-13 cells expressing the corresponding control lentivector. Consistent with our previous data, the RNAseq analysis of RAC2 E62K over-expressing cells showed an increased IL-lb cytokine related genes signature (IL-lb and IL-l-RN encoding the Interleukin- 1 receptor antagonist protein) which was not observed in cells over-expressing RAC2 WT {Data not shown).

[0161] Identification of a RAC2 A59S as a gain of function variant activating NLRP3.

[0162] During the course of our study samples from a patient harboring a RAC2 A59S variant were addressed to us. This variant was recently clinically described and classified as a gain of function (Donko et al., 2024). Due to the proximity to the position 61 targeted by the CNF1 toxin and our findings concerning the RAC2 E62K patient, we tested whether this variant behaved like a gain of function mutant capable of activating the NLRP3 inflammasome. Using a GST-PAK-RBD pull down assay, we first identified that the A59S mutant behaved like an active mutant, similar to the RAC2 E62K mutant used here as a positive control {Data not shown). Next, we measured its capacity to activate the NLRP3 inflammasome in our inflammasome reconstitution system Data not shown). We previously identified the threonine in position 659 of NLRP3 as a phosphorylation site for PAK1 that was critical for the CNF1- toxin triggered IL-lb secretion (Dufies et al., 2021). We thus tested whether this phosphorylation site was important in the context of RAC2 A59S or E62K variants. Inflammasome reconstituted with the NLRP3 T659A mutant had an impaired IL-lb secretion compared to wild type NLRP3 Data not shown). Next, since Caspase- 1 triggered GasderminD (GSDMD) cleavage and the subsequent GSDMD pore formation have been shown to be important events for IL-lb secretion (Shao, 2021), we investigated whether the RAC2 A59S or E62K variants triggered IL-lb secretion was GSDMD dependent. To address this point, we took advantage of the U937 monocytic cell line. Consistent with what we observed in primary macrophages, the expression of RAC2 A59S or E62K variants in control U937 cells stimulated with LPS was sufficient to trigger IL-lb secretion {Figure 1). Using U937 NLRP3 knock-out (KO) cells we could confirm that the RAC2 A59S or E62K variants triggered-IL-lb secretion was NLRP3 dependent. Additionally, we measured an impaired IL-lb secretion in GSDMD KO cells {Figure 1). We next investigated the involvement of pyroptotic cell death in this phenomenon by investigating the impact of Ninjurinl (NINJ1), a protein required for pyroptosis (Kayagaki et al., 2021; Fu et al., 2024). Using U937 NINJ1 KO cells we observed that the IL- lb secretion triggered by RAC2 gain of function variants was not affected {Data not shown). In addition, we did not measure a significant increase in cell death triggered by the expression of RAC2 A59S or E62K in U937 cells as well as in U937 cells KO for NLRP3, NINJ1 {Data not shown) or GSDMD {Data not shown). To investigate in depth the impact of the activating variant RAC2 A59S we performed a single cell RNAseq analysis of blood circulating cells {Data not shown). This analysis showed increased numbers of both classical and non classical monocytes as well as myeloid dendritic cells when compared to a healthy control {Data not shown). In addition, and supporting our hypothesis, NLRP3 and IL-lb expression levels were increased in both monocytes and myeloid dendritic cells, while their expression in lymphocytes was not affected in our analysis {Data not shown).

[0163] To further confirm our data, we compared monocytes isolated from PBMCs of patients harboring the RAC2 A59S with monocytes isolated from RAC2 E62K mutation {Data not shown). In both cases, the PBMCs were collected and frozen using the same protocol and PBMCs from patients and control healthy donors were processed in parallel. The RAC2 A59S and RAC2 E62K patient’s monocytes analysis showed respectively 11.4% and 14.3% ofFAM- YVAD-FLICA positive specks while control monocytes isolated from healthy donors showed only 4%. We could repeat these experiments twice for blood from the same patients taken at different time points and observed similar results with an increased number of specks of activated Caspase-1 in RAC2 mutant patients compared to controls (Data not shown). The Caspase-1 activation was also visualized by measuring the F AM- YV AD -FLIC A mean fluorescence intensity (Data not shown). Importantly, we were able to differentiate monocytes isolated from the blood of both RAC2 A59S and RAC2 E62K variant patients into macrophages. For both RAC2 variants, macrophages treated with LPS showed cells positive for ASC specks colocalizing with NLRP3 while no ASC speck were found in macrophages treated with LPS isolated from healthy donors used as controls (Data not shown). The analysis of the cytokines secreted from both RAC2 A59S or RAC2 E62K patient's monocytes derived macrophages treated with LPS showed an increased IL-lb secretion that was drastically reduced upon NLRP3 inhibitor (MCC950) treatment (Data not shown) in accordance with our data generated in the NLRP3 inflammasome reconstitution experiments (Data not shown).

[0164] Discussion:

[0165] The precise mechanism of the immune phenotype associated with RAC2 variants in patients is still under debate. The most intriguing point is that both loss of function mutants and gain of function variants show an immune phenotype (Hsu, 2023). Here we show that the RAC2 gain of function mutations trigger NLRP3 inflammasome activation in a human cell reconstitution system as well as in monocytes and macrophages isolated from RAC2 A59S or RAC2 E62K patients. We show that this activation correlates with the capacity of the mutant proteins to bind the RAC -binding domain of PAK. Furthermore, this RAC2-triggered activation of the NLRP3 inflammasome was blocked by addition of inhibitors of GasderminD (GSDMD).

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Claims

CLAIMS:

1. A method of treating Rac2 monogenic disorders in a subject in need thereof comprising the administration of an inhibitor of GasderminD (GSDMD).

2. The method according to claim 1 wherein the subject has or is susceptible to have aRac2 gain of function mutation including Rac2 G12R, G15D, P29R, A59S, Q61R, E62K or D63 V.

3. The method according to claim 1 wherein the inhibitor GSDMD is a siRNA.

4. The method according to claim 1 wherein the inhibitor of GSDMD is disulfiram, necrosulfonamide (NSA), and dimethyl fumarate (DMF).

5. The method according to claims 1 to 4 which comprises an IL-lbeta inhibitor.

6. The method according to claims 1 to 4 which comprises a Rac2 inhibitor.

7. A pharmaceutical composition comprising an inhibitor GasderminD (GSDMD) for treating Rac2 monogenic disorders.

8. The pharmaceutical composition according to claim 7 wherein the GSDMD is disulfiram, necrosulfonamide (NSA), and dimethyl fumarate (DMF).

9. A method of screening a drug suitable for the treating Rac2 monogenic disorders comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the expression or activity of GSDMD.

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