Methods for the diagnosis, treatment and characterization of NLRP3-associated autoinflammatory diseases

Abstract

Using NLRP3-deficient U937 cells reconstituted with doxycycline-inducible NLRP3 variants in response to NLRP3 induction, the present inventors have developed a novel functional cell-based assay to screen for NLRP3 variants that uncouples NLRP3 induction, priming and activation. The inventors studied and characterized 38 NLRP3 variants by assessing pyroptosis and IL-1β / 18 5 secretion in, priming and / or activation. The results were confirmed in primary monocytes from patients carrying different variants. The present invention pertains to a method for characterizing NLRP3 mutations that allows discriminating gain-of-function mutants from polymorphism without any impact on NLRP3 activity. The invention also relates to methods for the diagnosis of NLRP3- associated autoinflammatory diseases and for predicting a response to NLRP3 inhibitors based on 10 the detection of specific NLRP3 mutations in a sample obtained from a patient. The invention also relates to new NLRP3 inhibitors.

Description

METHODS FOR THE DIAGNOSIS, TREATMENT AND CHARACTERIZATION OF NLRP3- ASSOCIATED AUTOINFLAMMATORY DISEASESTechnical Field

[0001] The present invention pertains to novel methods for the characterization of NLRP3 mutations, and for the diagnosis and treatment of NLRP3-associated autoinflammatory diseases (NLRP3-AID). The present invention particularly pertains to a method for characterizing NLRP3 mutations. The present invention further relates to a method for the diagnosis of NLRP3-associated autoinflammatory diseases in a patient comprising detecting specific NLRP3 mutations in a sample obtained from said patient, as well as to a method for predicting a response to an NLRP3 inhibitor. The invention also relates to new NLRP3 inhibitors.Background Art

[0002] Cryopyrinopathies (Cryopyrin-associated periodic syndrome, CAPS), now referred to as NLRP3 mutation-associated autoinflammatory diseases (NLRP3-AID), are a group of autoinflammatory diseases caused by gain-of-function mutations of NLRP3. NLRP3 is a cytosolic stress sensor that upon activation assembles the inflammasome signaling complex controlling IL-1 p and IL-18 secretion and pyroptosis, a pro-inflammatory cell death coupled with the release of many alarmins. NLRP3 is activated by a two-step mechanism engaged by coordinated priming and activation signals.

[0003] NLRP3-AID were historically described as three dominant clinical entities: familial cold urticaria (FCAS), Muckle-Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease (NOMID, also referred to as chronic infantile neurological cutaneous and joint syndrome, CINCA - Hoffman et al., Nat Genet 29, 301-5 (2001); Booshehri, L. M. & Hoffman, J. Clin. Immunol. 39, 277-286 (2019)). All subtypes classically display cold-induced urticaria. FCAS was described as a benign form with mainly cutaneous features and arthralgia, whereas Muckle-Wells syndrome patients were described with urticaria, chronic inflammation and even recurrent fever, sensorineural deafness, ocular inflammation, headache, arthritis and could be complicated by inflammatory AA amyloidosis. NOMID begins at birth and is characterized by central nervous system inflammation such as chronic meningitis, skin involvement with a diffuse non-itchy urticarial rash, joint involvement including deforming arthropathy preferentially affecting the knees and facial dysmorphia characterized by the presence of frontal bumps and nasal saddle deformation. AA amyloidosis can complicate all forms of NLRP3-AID.

[0004] Most NLRP3-AID patients are highly responsive to IL-1 -targeted therapies, especially on cutaneous, ocular and articular features and prevention of inflammatory amyloidosis (Booshehri, L. M. & Hoffman (2019)). Nevertheless, the clinical response in patients with stable deafness, bone deformity, chronic renal failure is often poor, which is why early diagnosis is critical to prevent irrevocable damages.

[0005] Beside clinical presentation, NLRP3-AID diagnosis mostly relies on genetics. However, classical genetic approaches, including analysis of familial segregation and recurrent associationwith the disease, are poorly efficient to distinguish the gain-of-fu notion pathogenic NLRP3 mutations from non-pathogenic variants.

[0006] Indeed, NLRP3-AID is a rare disease which prevalence ranges between 1-3 per million. Despite some genotype-phenotype correlations, the symptoms and the severity may vary between patients bearing identical mutations, even within one family. In addition, few low penetrance variants of NLRP3 are associated with typical or atypical NLRP3-AID phenotypes, but also found in asymptomatic people. Finally, NLRP3-AID can be caused by somatic mosaicism, especially in NOMID often associated with de novo mutations (Louvrier, C. et al., J. Allergy Clin. Immunol. 145, 1254-1261 (2020)). Of the 204 amino-acid substitutions or deletions in NLRP3 described today, only 11 % have been fully determined to be pathogenic (n=22) or benign (n=1), while 49% are likely pathogenic (n=96) or likely benign (n=4) and 40% are variants of uncertain significance (n=51 , VUS), unsolved (n=6) or not classified (n=24) (Van Gijn, et al., J. Med. Genet. 55, 530-537 (2018)).

[0007] Classical genetic approaches can therefore not allow for the accurate characterization of NLRP3 mutations. The identification of gain-of-function NLRP3 variants requires functional approaches. In addition, the diversity of symptoms and their genotype-correlation suggest that gain- of-function mutants may be heterogeneous in their activity, and that clinical manifestations may depend, at least in part, on the grade of gain-of-function. Current functional characterizations of NLRP3 variants are largely based on the detection of IL- 1 p secretion in response to priming by LPS (Booshehri, L. M. & Hoffman (2019)). Nevertheless, this approach does not allow distinguishing between constitutively active and priming-activated mutants and does not allow detecting mutations that would bypass the priming requirement.

[0008] There is therefore a need for new assays that allow analyzing and characterizing NLRP3 mutations. The correct characterization of NLRP3 mutations is essential for the accurate and early diagnosis of NLRP3-AID, and would be helpful in identifying residues that are critical to NLRP3 activity. The identification of key residues could allow screening and developing new therapeutic compounds and allow predicting their efficacy in inhibiting NLRP3 activity.Summary of the Invention

[0009] The invention is defined by the claims.

[0010] The present inventors have developed a novel functional cell-based assay to screen for NLRP3 variants that uncouples NLRP3 induction, priming and activation. The inventors studied and characterized various NLRP3 variants by assessing pyroptosis and IL-1 p / 18 secretion in NLRP3- deficient U937 cells reconstituted with doxycycline-inducible NLRP3 variants in response to NLRP3 induction, priming and / or activation. The results were confirmed in primary monocytes from 15 patients carrying 9 different variants. These analyses efficiently discriminated gain-of-function mutants from polymorphism without any impact on NLRP3 activity. The assay disclosed herein allowed identifying a group of mutants responsive to the activation signal in the absence of prior priming that could not be detected in previous assays, leading to misdiagnosis of non-NLRP3-AID.

[0011] Therefore, according to a first embodiment, the present invention pertains to a method for characterizing a mutated NLRP3 protein, said method comprising the steps of: a. Expressing said mutated NLRP3 protein in cells; b. Treating a group of said cells so as to induce NLRP3 induction alone, another group of said cells so as to induce NLRP3 induction and priming, another group of said cells so as to induce NLRP3 induction and activation, and another group of said cells so as to induce NLRP3 induction, priming and activation; and c. Evaluating the response of said cells to the treatments and characterizing said mutated NLRP3 protein accordingly.

[0012] Step b. can advantageously be performed by treating said cells with a TLR ligand such as LPS to induce NLRP3 priming. Step b. can also advantageously be performed by treating said cells with nigericin to induce NLRP3 activation.

[0013] By evaluating the response of said cells to the treatment, typically by monitoring cell death such as pyroptosis, or by monitoring IL-1 p / 18 secretion, it is possible to characterize the mutated protein and particularly to determine whether the corresponding mutation is a gain-of-function mutation or a simple polymorphism that is not associated with NLRP3-AID. The method therefore allows determining whether the mutant is 1 / a constitutively active mutant (i.e. which induces cell death upon “simple” induction of NLRP3 expression), 2 / a mutant that requires indifferently priming or activation further to induction of NLRP3 expression, 3 / a mutant that requires priming further to induction of NLRP3 expression, 4 / a mutant that requires activation further to induction of NLRP3 expression or 5 / a mutant that does not correspond to a gain-of-function mutation.

[0014] The method according to the present invention allowed characterizing specific mutations and determining whether said mutations are associated with NLRP3-AID or not. The mutations characterized in the context of the present invention are A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, A352V, L353P, K375E, T436N, A439T, A439V, E525K, K565E, E567G, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M, L1016F, R918Q, R918X, Y859H and K829T. Therefore, it is possible to determine, based on the detection of these specific mutations, whether a patient suffers from an NLRP3-AID.

[0015] Therefore, according to a further embodiment, the present invention pertains to an in vitro method for the diagnosis of an NLRP3-AID in a patient, said method comprising a step of detecting, in a biological sample obtained from said patient, an NLRP3 mutation selected from the group consisting of K565E, E567G, K568N, Y570C, (S726G / S896P), S726G, A75V, K375E, G767S, S896P, T952M and L1016F in the amino acid sequence of the human NLRP3 protein as set forth in SEQ ID No. 1.

[0016] As shown herein, the presence of a mutation selected from the group consisting of R260W, D303H, D303N, A352V, E525K, K565E, E567G, K568N, Y570C, E311 K, I334V, T348M, T436N,A439T, E690K, V262G, G301 D, L353P, A439V, G569R, R168Q, V198M, M701T, Q703K, (S726G / S896P), R918Q, R918X, K829T, Y859H and S726G indicates that said patient suffers from an NLRP3-AID. On the other hand, the presence of a mutation selected from the group consisting of A75V, 1313V, K375E, G767S, S896P, T952M and L1016F, alone, indicates that said patient does not suffer from NLRP3-AID.

[0017] The method for the diagnosis of an NLRP3-AID according to the present invention can further comprise detecting at least one mutation selected from the group consisting of Y859C, R168Q, V198M, R260W, V262G, G301 D, D303N, D303H, E311 K, 1313V, I334V, T348M, A439T, A352V, L353P, A439V, T436N, E525K, G569R, E690K, M701T, Q703K and R918Q in the amino acid sequence of the human NLRP3 protein as set forth in SEQ ID No. 1 . These mutations indicate that the patient suffers from an NLRP3-AID.

[0018] The inventors have further showed that the presence of said mutations can allow predicting the response of a patient to inflammasome inhibitors such as NLRP3 inhibitors. The inventors have indeed tested various NLRP3 inhibitors on NLRP3-mutated cells and shown that response to these inhibitors varies based on the type of mutation. The present inventors have particularly shown that:

[0019] Mutants A75V, R168Q, V198M, R260W, V262G, D303N, E311 K, 1313V, I334V, T348M, A352V, K375E, T436N, A439T, A439V, K565E, E567G, K568N, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F are inhibited by MCC950-like inhibitors. But that mutants G301 D, D303H, L353P, E525K, G569R do not respond to such inhibitors.

[0020] All mutants A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, A352V, L353P, K375E, T436N, A439T, A439V, E525K, K565E, E567G, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F are inhibited by either inhibitor CY-09 or inhibitor G5.

[0021] Mutants A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, A352V, L353P, K375E, T436N, A439T, A439V, E525K, K565E, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F are inhibited by inhibitor CRT0066101 but not mutant E567G.

[0022] Therefore, according to another embodiment, the present invention pertains to an in vitro method for predicting a response to an NLRP3 inhibitor in a patient, said method comprising a step of detecting, in a biological sample obtained from said patient, an NLRP3 mutation selected from the group of 34 mutations consisting of A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, A352V, L353P, K375E, T436N, A439T, A439V, E525K, K565E, E567G, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F with respect to the amino acid sequence of the human NLRP3 protein as set forth in SEQ ID No. 1 .

[0023] In the context of the present invention the presence: of one of said 34 mutations indicates that the patient will respond to a treatment comprising a G5-like inhibitor;- of one of mutations A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, L353P, K375E, T436N, A439T, A439V, E525K, K565E, E567G, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F indicates that the patient will respond to a treatment comprising a CY-09-like inhibitor;- of one of mutations A75V, R168Q, V262G, 1313V, I334V, K375E, T436N, A439T, K565E, E567G, K568N, Y570C, E690K, M701 T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F indicates that the patient will respond to a treatment comprising an MCC950-like inhibitor; of one of mutations G301 D, D303H, E525K and G569R indicates that the patient will not respond to a treatment comprising an MCC950-like inhibitor;- of one of mutations A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, A352V, L353P, K375E, T436N, A439T, A439V, E525K, K565E, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F indicates that the patient will respond to a treatment comprising a CRT0066101-like inhibitor; and of mutant E567G indicates that the patient will not respond to a treatment comprising a CRT0066101-like inhibitor.

[0024] The present invention therefore also relates to:A G5-like inhibitor for use in the treatment of an NLRP3-AID in a patient who has been identified as displaying a mutation selected from the group consisting of A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, A352V, L353P, K375E, T436N, A439T, A439V, E525K, K565E, E567G, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F with respect to the amino acid sequence of the human NLRP3 protein as set forth in SEQ ID No. 1 ;A CY-09-like inhibitor for use in the treatment of an NLRP3-AID in a patient who has been identified as displaying a mutation selected from the group consisting of A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, L353P, K375E, T436N, A439T, A439V, E525K, K565E, E567G, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F with respect to the amino acid sequence of the human NLRP3 protein as set forth in SEQ ID No. 1 ; an MCC950-like inhibitor for use in the treatment of an NLRP3-AID in a patient who has been identified as displaying a mutation selected from the group consisting of A75V, R168Q, V262G, 1313V, I334V, K375E, T436N, A439T, K565E, E567G, K568N, Y570C, E690K, M701 T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F withrespect to the amino acid sequence of the human NLRP3 protein as set forth in SEQ ID No. 1 ; a CRT0066101-like inhibitor for use in the treatment of an NLRP3-AID in a patient who has been identified as displaying a mutation selected from the group consisting of CRT0066101 A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, A352V, L353P, K375E, T436N, A439T, A439V, E525K, K565E, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F with respect to the amino acid sequence of the human NLRP3 protein as set forth in SEQ ID No. 1 .

[0025] Having identified and characterized the specific mutations disclosed in the context of the present invention, it is possible to test and identify new NLRP3-inhibitors. Therefore, according to a further embodiment, the present invention also pertains to an in vitro method for screening a candidate NLRP3 inhibitor, said method comprising: i. Expressing, in a cell, a mutated NLRP3 protein having a mutation selected from the group consisting of A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, A352V, L353P, K375E, T436N, A439T, A439V, E525K, K565E, E567G, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, R918Q, T952M and L1016F with respect to the amino acid sequence of the human NLRP3 protein as set forth in SEQ ID No. 1 ; ii. Treating said cell with a candidate NLRP3 inhibitor;Hi. Inducing, in said cell, NLRP3 induction, and NLRP3 priming and / or activation depending on the mutation expressed; and iv. Selecting said compound if it significantly inhibits NLRP3 expression and / or activity in said cell treated with said candidate NLRP3 inhibitor.

[0026] The mutation expressed in step i. can also be selected from Y859H, R918X and K829T.

[0027] Finally, the characterization method according to the present invention also allowed identifying residues that are critical to NLRP3 activity. These residues are located within two specific domains of NLRP3 that can be targeted by using NLRP3 binding agents. These two domains are 1 / the interaction motif between the acidic loop (comprised between amino acids No.687-703) and, on the one side, the concave side of the LRR domain, and, on the other side, the C-terminal tip of the LRR domain of NLRP3 (amino acids 740-1034), ( i.e. comprising amino acids E690, M701 , Q703 and Y859), and 2 / the accessible surface of the HD2 domain (comprising amino acids K565, E567, K568, G569, Y570). The data gathered in the context of the present invention therefore shows that it is possible to modulate NLRP3 activity by using compounds that specifically target these zones.

[0028] Therefore, the present invention also pertains to a compound modulating NLRP3 activity, wherein said compound specifically binds to the accessible surface of the HD2 domain of NLRP3 orto the interaction motif between the acidic loop and the concave side and the C-terminal tip of the LRR domain of NLRP3.

[0029] Accordingly, the present invention also pertains to an in vitro method for modulating NLRP3 activity in a cell, wherein said method comprises treating said cell with an NLRP3 binding compound which specifically binds to the accessible surface of the HD2 domain of NLRP3 or to the interaction motif between the acidic loop and the concave side and the C-terminal tip of the LRR domain of NLRP3.

[0030] The present invention finally pertains to an NLRP3 binding compound for use in modulating NLRP3 activity in a cell, wherein said compound specifically binds to the accessible surface of the HD2 domain of NLRP3 or to the interaction motif between the acidic loop and the concave side and the C-terminal tip of the LRR domain of NLRP3.Brief Description of Drawings

[0031] Figure 1 : Assessment of the activity of NLRP3 variants upon expression, priming and / or activation signals. A / LRP3-deficient KO U937 reconstituted with doxycycline-inducible NLRP3 variants were treated with doxycycline, LPS priming signal and / or nigericin activation signal. NLRP3 activity was assessed by measuring pyroptosis and IL-10 / 18 secretion.

[0032] Figure 2: Relative cell death of U937 expressing NLRP3 variants as compared to U937 expressing WT NLRP3 in response to NLRP3 expression, priming and / or activation. A / LRP3-deficient U937 cells reconstituted with doxycycline-inducible NLRP3 variants were treated with doxycycline (1 |j.g / ml, 3h), LPS (40 ng / ml, 2h) and nigericin (15 |j.g / ml) before cell death was monitored by PI incorporation over time quantified by time-lapse high content microscopy. The figure shows Regression coefficient (RC) heatmap from the generalized linear mixed model for each variant as compared to the WT. Positive RC denotes increased cell death in the considered variant as compared to the WT, and conversely. RC corresponding to statistically significant increases (dark grey) or decreases (light grey) in cell death are color-coded (p<0.05). Not significant, p>0.05. Based on the results, variants are classified in 5 functional groups: constitutive active variants (group#1 , red), variants active upon either priming or activation signal (group#2, green), variants active upon priming signal (group#3, yellow), variants active upon activation signal (group#4, blue), and mutants active upon priming and activation signals (no gain-of-function, group#5, black).

[0033] Figure 3: Inhibition of cell death of U937 expressing each NLRP3 variants in response to NLRP3 expression, priming and / or activation in presence of NLRP3 inhibitors. Regression coefficient (RC) heatmap from the linear mixed model of PI incorporation AUC for each NLRP3 variant with a specific treatment for each inhibitor, as compared to vehicle only. Negative coefficient denoted inhibition of cell death, and conversely. Significant values: RC <-0.1. Not significant: p>0.05. Variant*treatment combinations that do not trigger cell death were not tested and are represented in black.

[0034] Figure 4: Localization of disease mutant sites in the structure of human NLRP3. A, Bar diagram showing the domain architecture of NLRP3 with the mutant sites of the five variant groupsindicated. B, Display of the inactive spherical NLRP3-ADP decamer (7pzc) (Coll et al., Nat. Med. 21 , 248-255 (2015)) and the active concentrical NLRP3-ATP decamer bound to NEK7 (in grey) (8ej4) (Xiao, L., Magupalli, V. G. & Wu, H. Nature 613, 595-600 (2023)). C, The conformational transition from the inactive to the active state in NLRP3 involves an 85° rotation of the FISNA-NBD-HD1 subdomains in the NACHT relative to the remainder. The missense mutations of the five variant groups are highlighted as spheres in the inactive conformation. D, Close-up of residues E690, M701 and Q703 in the loop section of the transition LRR and Y859 in the concave side of the LRR. E, Localization of the constitutively active group#1 mutant sites in the NACHT domain of NLRP3. F, Residues T436 and A439 are at the pivot point of the FISNA-NBD-HD1 rotation. The active state (8ej4) is shown in grey relative to the inactive state (7pzc). G, Variant group#4 residues R168 and V198 in the FISNA domain and S726 in the transition LRR are located in variable regions of NLRP3.Detailed description of the invention

[0035] The present invention pertains to methods for the characterization of NRLP3 mutations, and for the diagnosis and treatment of NLRP3-associated autoinflammatory diseases (NLRP3-AID). The present inventors have developed a cell-based assay that allow characterizing NLRP3 mutations. The present inventors have identified and characterized specific NLRP3 mutations that were shown to be correlated with NLRP3-AID, thereby allowing for the accurate diagnosis of NLRP3-AID. These specific mutations can also be used for predicting a response to NLRP3 inhibitors in a patient, and for screening candidate NLRP3 inhibitors. The cell-based assay according to the present invention allowed identifying key residues in the control of NLRP3, thereby offering the possibility of identifying efficient NLRP3 inhibitors specifically targeting said residues.

[0036] According to a first embodiment, the present invention pertains to a method for characterizing a mutated NLRP3 protein, said method comprising the steps of: a. Expressing said mutated NLRP3 protein in cells; b. Treating a group of said cells so as to induce NLRP3 induction, another group of said cells so as to induce NLRP3 induction and priming, another group of said cells so as to induce NLRP3 induction and activation, and another group of said cells so as to induce NLRP3 induction, priming and activation; and c. Evaluating the response of said cells to the treatments and characterizing said mutated NLRP3 protein accordingly.

[0037] “NLRP3” or “NLR family pyrin domain containing 3” is a cytosolic stress sensor that upon activation assembles the inflammasome signaling complex controlling IL-1 p and IL-18 secretion and pyroptosis. NLRP3 functions as a pattern recognition receptor (PRR) that recognizes pathogen- associated molecular patterns (PAMPs). NLRP3 is a tripartite protein that contains an amino-terminal pyrin domain (PYD), a central NACHT domain and a carboxy-terminal leucine-rich repeat (LRR) domain (Swanson and Ting., Nature Reviews Immunology 19.8 (2019): 477-489). The NACHT domain is an ATP / ADP fixation site that is essential for NLRP3 self-association and function. The LRR domain is thought to induce autoinhibition by folding back onto the NACHT domain (Swansonand Ting., 2019). The amino acid sequence of the human NLRP3 protein corresponds to the amino acid as set forth is SEQ ID No.1 :_

[0038] Table 1 : Amino acid sequence of the human NLRP3 protein.

[0039] In the context of the present invention, the positions of the different amino acids referred to within the NLRP3 protein are made in reference to the above sequence (SEQ ID No. 1). This nomenclature corresponds to that of the Infevers database European nomenclature (Milhavet, F. et al., Hum. Mutat. 29, (2008) 803-808).

[0040] NLRP3 is activated by a two-step mechanism engaged by coordinated priming and activation signals after NLRP3 induction (i.e. expression). Priming is usually provided by pattern recognition receptor (PRR) ligands, including the Toll-like receptor 4 (TLR4) agonist LPS, as well as cytokines. Priming was first described to increase the cellular level of key proteins in the pathway by transcriptional up-regulation of NLRP3 itself, the inducible inflammasome substrate pro- IL-1 p and many other inflammatory genes (Bauernfeind, F. G. et al., J Immunol 183, 787-91 (2009)). Nevertheless, priming is now recognized to also trigger an ensemble of NLRP3 post-translational modifications that renders NLRP3 competent for activation (Juliana et al., J Biol Chem 287, 36617- 22 (2012); McKee, C. M. & Coll, J. Leukoc. Biol. 108, 937-952 (2020) ; Niu et al., Nat. Commun. 12, 5862 (2021)). Activation signals correspond to diverse cell stresses, that target plasma membrane ion permeability (including the bacterial toxin nigericin), lysosomal rupture or mitochondrial functions for most of them. These signals converge towards a disruption of vesicular trafficking, which could be the common trigger for NLRP3 activation (Zhang et al., Nat. Immunol. 24, 30-41 (2023)). Then, NLRP3 assembles an inflammasome complex controlling caspase-1 activation. Caspase-1 cleaves pro-IL-1 p and pro-IL-18 into their mature forms, as well as Gasdermin D (GSDMD), which in turn forms pores in the plasma membrane, and ultimately mediates IL-1 p / 18 release and initiates pyroptosis.

[0041] The method according to the present invention aims at characterizing an NLRP3-mutated protein, i.e. an NLRP3 mutant which comprises a mutation as compared to the human wild-type NLRP3 amino acid sequence as shown in SEQ ID No. 1 .

[0042] A "mutation" refers to any detectable change in genetic material, e.g. DNA, RNA, cDNA, or in an amino acid sequence encoded by such a genetic material. This includes gene mutations, in which the structure (e.g. DNA sequence) of a gene is altered, as well as protein mutations, in which the amino-acid structure of the protein is altered. Generally, a mutation is identified in a subject by comparing the sequence of a nucleic acid or of a polypeptide expressed in said subject with the corresponding nucleic acid or polypeptide expressed in a control population. When the mutation to be detected is a somatic mutation, i.e. a mutation that occurs in a specific cell line other than the germline, it can also be detected by comparing the sequence of a nucleic acid or of a polypeptide expressed by said cell line with the corresponding nucleic acid or polypeptide expressed by another cell line. A mutation can be a deletion (i.e. removal of at least one nucleotide in the gene sequence), an insertion (i.e. addition of at least one nucleotide in the gene sequence) or a substitution (i.e. replacement of one nucleotide with another). In the context of the present invention, the mutation is located in the exons (i.e. in the coding parts of the gene) and has an effect on the encoded NLRP3 protein (i.e. the mutation is not a silent mutation). The mutation can be a missense mutation (which result in a codon that codes for a different amino acid) a nonsense mutations (which results in a premature stop codon or a nonsense codon in the transcribed mRNA and thus in a truncated protein) or a frameshift mutation (which results from the insertion or deletion of a number of nucleotides in a DNA sequence that is not divisible by three and induce a change the reading frame resulting in a completely different translation from the original). In the context of the present invention, the NLRP3 mutated protein is preferably the result of the expression of an NLRP3 gene comprising a missense mutation which results in a substitution (i.e. replacement) of at least one amino acid with another in the NLRP3 amino acid sequence. The nomenclature used for referring to mutations in the context of the present invention is consistent with that of Antonarakis, S.E. and the Nomenclature Working Group (“Recommendations for a nomenclature system for human gene mutations." Hum.Mut. 11 : 1- 3 (1998)). This nomenclature has been largely accepted and is the standard nomenclature for referring to mutations within DNA / RNA / Amino Acid sequences. For instance, in the context of the present invention the mutation “R260W’ denotes that amino acid 260 (Arginine, R) is changed to a (Tryptophan, W). The expression” (S726G / S896P)” indicates that the protein comprises both mutation S726G and S896P.

[0043] The method according to the present invention comprises a step of expressing a mutated NLRP3 protein to be characterized in cells.

[0044] The “cells” can be of any cell type which usually expresses NLRP3. Typically, the cells are selected from monocytes, macrophages, and dendritic cells (DCs). According to a specific embodiment, said cells are monocytes. Such cells / cell lines are easily available to the skilled person. The cell line U-937 available from the American Type Culture Collection catalogue can e.g. be used.

[0045] According to a specific embodiment, the cells can be modified so that they do not express an endogenous NLRP3 protein. By a cell that “does not express an endogenous NLPR3 protein”, it is herein meant that the expression of NLRP3 in said cells has been inactivated by genetic engineering. The A / LRP3 gene naturally / endogenously comprised in said cell can therefore not allow for the synthesis of an NLRP3 protein. The skilled person knows many techniques that allow inactivating the expression of a gene in a cell. Inactivation of a gene can be carried out by the various methods known by the skilled person, including knock out, particularly by directed mutagenesis or homologous recombination. A particularly preferred method consists in using the CRISP / Cas9 system so as to target, bind and cut the NLRP3 gene so as to turn is off. A method for inactivating NLRP3 expression by using the CRISPR / CAS9 system is disclosed in the experimental section of the present description.

[0046] The mutated NLRP3 protein is expressed in said cells by any means known to the skilled person. Expressing a gene / protein in a target cell is a routine procedure for the skilled person. The nucleotide sequence encoding the NLRP3 mutated protein is typically placed in an expression vector. A typical expression vector contains eukaryotic DNA elements, such as a transcription initiation sequence for the exogenous gene, for instance a promoter, and DNA elements which control the processing of the transcripts, such as termination / polyadenylation sequences. The gene is typically placed under the control of an inducible promoter, which will allow for the expression of a gene under specific conditions. One can particularly use the doxycycline inducible system to control the gene expression (De Boeck and Verfaillie, Molecular Biology of the Cell 32.17 (2021): 1517-1522). The doxycycline system, such as the Tet-One™ Inducible Expression System, allows inducing the expression of a target gene but only in the presence of doxycycline. It provides a simple and efficient method for controlling the expression of a target gene. According to a specific embodiment, the NLRP3 gene expressed in the context of the present invention is placed under the control of a doxycycline induction system. According to such an embodiment, NLRP3 induction is obtained by treating the cell with doxycycline. The method according to the present invention comprises treating the cells into which the mutated NLRP3 is expressed so as to induce NLRP3 induction, priming and / or activation. The cells are divided in subgroups which are treated so as to mimic different steps of NLRP3 activation.

[0047] This step aims at reproducing the conditions of NLRP3 induction, priming and / or activation to evaluate if and under which conditions the mutant is activated. This step is therefore carried out by inducing the signals that are required for NLRP3 induction / priming / activation in a cell.

[0048] According to the present invention, among the cells into which NLRP3 is expressed after step a., a part (i.e. group) of the cells is treated so as to induce NLRP3 induction alone, another part / group of the cells is treated so as to induce NLRP3 induction and priming, another part / group of the cells is treated so as to induce NLRP3 induction and activation, and another part / group of the cells is treated so as to induce NLRP3 induction, priming and activation. To do so, the cells are treated with compounds inducing NLRP3 induction, priming or activation.72

[0049] As mentioned above, NLRP3 induction corresponds to the expression of NLRP3 within the cell, i.e. to the NLRP3 gene transcription. Therefore, a compound “inducing NLRP3 induction” is a compound that allow for the expression of the NLRP3 mutant in said cell. As mentioned above, when NLRP3 is placed under the control of a doxycycline induction system, the compound inducing NLRP3 induction is doxycycline.

[0050] NLRP3 priming is provided by activation of cytokines or pathogen-associated molecular patterns (PAMPs) and renders NLRP3 susceptible to activation. Priming induces an upregulation in the expression of NLRP3 and multiple post-translational modifications of NLRP3 that render NLRP3 sensitive to activation signals. These events can be induced through the recognition of various pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) that engage pattern-recognition receptors (PRRs) such as Toll-like receptors (TLRs) or nucleotide-binding oligomerization domain-containing protein 2 (NOD2), or through cytokines such as tumor necrosis factor (TNF) and IL-1 p that lead to nuclear factor-KB (NF-KB) activation and gene transcription.

[0051] A compound “inducing NLRP3 priming” can therefore be selected from the group consisting of TLR (in particular TLR4 and TLR2) ligands. Such compounds include e.g. LPS, Pam3CSK4 heat kill bacteria, Poly(l:C), Flagellin, ssRNA, CpG oligonucleotides, Imiquimod, Resiquimod, IL-1 and TNF. In the context of the present invention, said compound inducing NLRP3 priming is preferably LPS.

[0052] NLRP3 activation corresponds to the final step allowing for inflammasome formation. A compound “inducing NLRP3 activation” can be selected from the group consisting of nigericin, ATP, Imiquimod, CL097, HLLOMe and particles (monosodium urate, silica, asbestos, amyloid). In the context of the present invention, said compound inducing NLRP3 activation is preferably nigericin.

[0053] Compounds inducing NLRP3 induction, priming and activation can be introduced simultaneously or subsequentially. Typically, when present, the compound inducing NLRP3 induction is introduced first, followed by the compound inducing NLRP3 priming, and finally by the compound inducing NLRP3 activation. The compound inducing NLRP3 priming is typically introduced within 2 hours, preferably 1 hour after that inducing NLRP3 induction. The compound inducing NLRP3 activation is typically introduced within 3 hours, preferably 2 hours afterthat inducing NLRP3 induction. The compound inducing NLRP3 activation is typically introduced within 4 hours, preferably 3 hours after that inducing NLRP3 induction.

[0054] According to a specific embodiment, step b. can further comprise treating the cell with at least one NLRP3 inhibitor so as to further characterize said mutation and its response to said inhibitor.

[0055] Many “NLRP3 inhibitors” have been developed and can be easily obtained (Zhang et al., International Journal of Molecular Medicine 51.4 (2023): 1-21). NLRP3 inhibitors act by preventing NLRP3 induction, priming and / or activation. They inhibit the activation of the NLRP3 inflammasome. NLRP3 inhibitors include 1 / inhibitors of TLR4 such as TAK-242, or resatorvid; 2 / inhibitors of NF-KB such as BAY 11-7082; 3 / NLRP3 inflammasome pathway inhibitors targeting the microenvironmentof NLRP3 inflammasome assembly such as the IP3R inhibitor 2-aminoethoxydiphenyl borate and the Ca2+ chelator BAPTA-AM; 4 / NLRP3 binding compounds such as MCC950-like inhibitors, oridonin, OLT1 177, INF39, tranilast, inhibitor CY-09, JC124, 3,4-Methylenedioxy-p-nitrostyrene (MNS), parthenolide and BOT-4-one; 5 / NLRP3 inflammasome pathway inhibitors targeting caspase- 1 such as belnacasan, Ac-YVAD-cmk, AI-44 and FC1 1 A-2; 6 / deubiquitinase inhibitors such as the G5 inhibitor; 7 / protein kinase D inhibitors such as inhibitor CRT0066101. According to a preferred embodiment, the NLRP3 inhibitor according to the present invention is selected from the inhibitor CY-09, the inhibitor G5, the inhibitor CRT0066101 and MCC950-like inhibitors.

[0056] Inhibitor “CY-09” is a selective and direct NLRP3 inhibitor. CY-09 Cas No. is CAS 1073612- 91-5. It binds to the ATP-binding motif of NLRP3 NACHT domain and inhibits NLRP3 ATPase activity, resulting in the suppression of NLRP3 inflammasome assembly and activation (Jiang et al. Journal of Experimental Medicine 214.11 (2017): 3219-3238). A “CY-09-like inhibitor” refers to compounds having the same chemical structure as CY-09. CY-09-like inhibitors are of formula 1 :wherein n is selected from 0, 1 , 2, 3, 4, 5 or 6, preferably 0 or 1 ; andR is a phenyl substituted with at least one group selected from a hydrogen, a fluor, a C1-C6 alkyl and a CF3. Preferably, R is a mono-substituted phenyl, more preferably a meta-monosubstituted phenyl.

[0057] Preferably, the CY-09-like inhibitor is of formula 2:Formula 2

[0058] More preferably of formula 3:Formula 3 wherein n is as defined above; andR' is a group selected from a hydrogen, a fluor, a C1-C6 alkyl and a CF3.

[0059] According to an even more preferred embodiment, the CY-09-like inhibitor is of formula 4:Formula 4 wherein n is as defined above.

[0060] CY-09-like inhibitors include compounds such as CY-09 and C172 (see Jiang et al. Journal of Experimental Medicine 214.11 (2017) : 3219-3238) .

[0061] Inhibitor “G5”, having as chemical name is 3,5-bis((4-Nitrophenyl)methylene)-1 ,1-dioxide, tetrahydro-4H-thiopyran-4-one, is a deubiquitinase inhibitor that maintains NLRP3 in an ubiquitinated inactive form (Py, B. F., Kim, M. S., Vakifahmetoglu-Norberg, H. & Yuan, J. Deubiquitination of NLRP3 by BRCC3 critically regulates inflammasome activity. Mol Cell 49, (2013) 331-8). Also referred to as “Ubiquitin Isopeptidase Inhibitor I, G5”, its Cas No. is CAS 108477-18-5. An “G5-like inhibitor” refers to compounds having the same mechanism of action as G5, i.e. that maintain NLRP3 in an ubiquitinated inactive form. G5-like inhibitors include G5 and compounds such as Thiolutin and Holomycin.

[0062] Inhibitor “CRT0066101” is a protein kinase D inhibitor which impairs NLRP3 phosphorylation at the Golgi during the activation process (Zhang et al. J Exp Med 214, (2017) 2671-2693). Its chemical name is 2-[4-[[(2R)-2-aminobutyl]amino]-2-pyrimidinyl]-4-(1 -methyl-1 H-pyrazol-4-yl)phenol dihydrochloride and its Cas No. is 1883545-60-5. An “CRT0066101-like inhibitor” refers to compounds having the same mechanism of action as CRT0066101 , i.e. protein kinase D inhibitors that impair NLRP3 phosphorylation at the Golgi during the activation process. CRT0066101-like inhibitors include CRT0066101 and compounds such as CID755673, 3-IN-PP1 , 1-NA-PP1 and SD- 208.

[0063] “MCC950-like inhibitors” include NLRP3 inhibitors that interact with NLRP3 into a cleft in between the NACHT and the LRR domains in proximity to the ATP binding site and locks NLRP3 in an inactive conformation (Li et al. European Journal of Pharmacology 928 (2022): 175091). They include MCC950 which is well-known potent and selective NLRP3 inhibitor (having the Cas No. CAS 210826-40-7), but also MCC950 derivatives which have the same mechanism of action as MCC950 but have been modified so as to prevent the hepatotoxicity observed upon administration MCC950. MCC950-like inhibitors include MCC950 N-cyano-sulfoximineurea derivatives (Agarwal et al., ACS Medicinal Chemistry Letters 11 .4 (2020): 414-418) and alkenyl sulfonylurea derivatives (Agarwal et al., Bioorganic & Medicinal Chemistry Letters 30.21 (2020): 127571), NP3-146 and NP3-301 (PMID: 34687713)

[0064] The method according to the present invention further comprises characterizing the tested NLRP3 mutant by evaluating the response of the NLRP3 mutant-containing cells to the treatment inducing induction / priming and / or activation.

[0065] The “response” of said cells is evaluated by measuring pyroptosis (i.e. cell death resulting from inflammasome activation) and IL-1 p / 18 secretion. The skilled person knows how the evaluate and monitor pyroptosis and IL-1 p / 18 secretion in a cell culture. Methods for monitoring pyroptosis and measuring IL-1 p / 18 secretion are e.g. disclosed in the experimental section of the present application. The response can also directly be evaluated by measuring cell death, e.g. by LDH assay which aims at measuring cell death by detecting the release of lactate dehydrogenase in the culture medium. The response is typically observed between 10 minutes to 2 hours after having introduced the last compound inducing either NLRP3 induction, priming or activation.

[0066] Based on said response, it is possible to characterize the studied mutant, i.e. to determine under which conditions it is activated and whether it is a gain-of-function mutant or a mutant resulting from a simple polymorphism that is not associated with NLRP3-AID. The mutant can therefore be classified as 1 / a constitutively active mutant (i.e. which induces cell death upon “simple” induction of NLRP3 expression), 2 / a mutant that requires either priming or activation further to induction of NLRP3 expression, 3 / a mutant that requires priming further to induction of NLRP3 expression, 4 / a mutant that requires activation further to induction of NLRP3 expression or 5 / a mutant that does not correspond to a gain-of-function mutation.

[0067] As explained above, the method according to the present invention allowed characterizing specific NLRP3 mutations. These specific mutations are disclosed in Table 2 below:77

[0068] Table 2: Mutations according to the present invention.

[0069] The above mutations can be used as a marker for diagnosing an NLRP3-AID.

[0070] Therefore, according to a further embodiment, the present invention therefore pertains to an in vitro method for the diagnosis of an NLRP3-AID in a patient, said method comprising a step of detecting, in a biological sample obtained from said patient, an NLRP3 mutation selected from the group consisting of K565E, E567G, K568N, Y570C, (S726G / S896P), S726G, A75V, K375E, G767S, S896P, T952M and L1016F in the amino acid sequence of the human NLRP3 protein as set forth in SEQ ID No. 1 , wherein, the presence of a mutation selected from the group consisting of K565E, E567G, K568N, Y570C, (S726G / S896P) and S726G indicates that said patient suffers from an NLRP3-AID; and the presence of a mutation selected from the group consisting of A75V, K375E, G767S, S896P, T952M and L1016F, alone, indicates that said patient does not suffer from NLRP3-AID.

[0071] According to another embodiment, the in vitro method according to the present invention can comprise detecting, in said biological sample obtained from said patient, an NLRP3 mutation selected from Y859H, R918X and K829T. The presence of a mutation selected from Y859H, R918X and K829T indicates that said patient suffers from an NLRP3-AID.

[0072] “NLRP3-AID” for “NLRP3-associated autoinflammatory diseases” previously referred to as “cryopyrin-associated periodic syndromes (CAPS)” are a group of autoinflammatory disorders caused by gain-of-fu notion mutations of NLRP3. NLRP3-AID encompass three dominant clinical entities: familial cold-associated syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal- onset multisystem inflammatory disease (NOMID), also known as chronic infantile neurologic cutaneous articular syndrome (CINCA).

[0073] In the context of the present invention, the “biological sample” can be any sample allowing for the detection of a mutation in the NLRP3 gene or protein. As explained in the experimental section of the present description, most of the mutations according to the present invention are germinal mutations and can therefore be detected in any biological sample obtained from the patient. Some mutations can be somatic mutations and can therefore be detected in biological samples comprising monocyte cell lines such as blood samples. Recent improvements in sequencing techniques, suchas the development of next generation sequencing (NGS), provide high performances which allow detecting mutations, including somatic mutations, in whole blood samples. Preferred biological samples are a cell or tissue sample. The biological sample can be whole blood or PBMC (peripheral blood mononuclear cells obtained from the patient. In a preferred embodiment according to the present invention, the biological sample is a blood sample obtained from the patient.

[0074] In the context of the present invention, an NLRP3-AID is diagnosed, i.e. detected in a patient by evaluating whether the patient displays a mutation among mutations K565E, E567G, K568N, Y570C, (S726G / S896P), S726G, A75V, K375E, G767S, S896P, T952M and L1016F, and according to a further embodiment among mutations Y859H, R918X and K829T. This step is performed by analyzing the sample for the presence at least one of these mutations. According to the present invention, the method for the diagnosis of an NLRP3-AID therefore comprises analyzing the sample for the presence of at least one (i.e. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15) of these mutations. The method can comprise analyzing the sample for the presence of at least 5, at least 10, at least 12 or of all 15 of these 15 mutations.

[0075] According to a further embodiment, the method for the diagnosis of an NLRP3-AID according to the present invention can further comprise a step of analyzing the sample for the presence of an NLRP3 mutation selected from the group consisting of R260W, D303H, D303N, A352V, E525K, E311 K, I334V, T348M, T436N, A439T, E690K, V262G, G301 D, L353P, A439V, G569R, R168Q, V198M, M701T, Q703K, Y859C, R918Q and 1313V. The presence of a mutation selected from the group consisting of R260W, D303H, D303N, A352V, E525K, E311 K, I334V, T348M, T436N, A439T, E690K, V262G, G301 D, L353P, A439V, G569R, R168Q, V198M, M701T, Q703K, Y859C and R918Q indicates that the patient suffers from an NLRP3-AID. On the other hand, the presence of mutation 1313V, alone, indicates that said patient does not suffer from NLRP3-AID.

[0076] The presence of a mutation in NLRP3 gene or NLRP3 protein can be detected by any means known by the skilled person’s common general knowledge. The detection of a mutation in a known gene / protein is a routine procedure that is within the skilled person’s common general knowledge. A mutation may be detected by analysing NLRP3 nucleic acid molecules. Nucleic acid molecules include mRNA, genomic DNA and cDNA derived from mRNA. DNA or RNA can be single stranded or double stranded. They may be utilized for detection by amplification and / or hybridization with a probe, for instance. The nucleotide sequence can be obtained from a genomic DNA sample isolated from the biological sample. In this case, any biological sample containing genomic DNA can be used. NLRP3 mutations may be detected in RNA or DNA samples, preferably after amplification. For instance, the isolated RNA may be subjected to coupled reverse transcription and amplification, such as reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that are specific for the mutated site or that enable amplification of the region containing the mutated site. Conditions for primer annealing may be chosen to ensure specific reverse transcription (where appropriate) and amplification; so that the appearance of an amplification product be a diagnostic of the presence of a mutation according to the invention. Otherwise, RNA may be reverse-transcribed and amplified, or DNA may be amplified, after whichthe mutated site may be detected in the amplified sequence by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art.

[0077] The mutation of NLRP3 may also be detected at the protein level by detecting the mutated form of the NLRP3 protein. In this context, the biological sample obtained from a patient may be analysed in presence of antibodies specific of the mutated form of the protein, i.e. antibodies that are capable of distinguishing between the mutated form and the wild-type protein (or any other protein), to determine the presence or absence of the mutation specified by the antibody.

[0078] The antibodies may be monoclonal or polyclonal antibodies, single chain or double chain, chimeric antibodies, humanized antibodies, or portions of an immunoglobulin molecule, including those portions known in the art as antigen binding fragments Fab, Fab', F(ab')2 and F(v). They can also be immunoconjugated, e.g. with a toxin, or labelled antibodies.

[0079] Whereas polyclonal antibodies may be used, monoclonal antibodies are preferred because they are more reproducible in the long run.

[0080] Polyclonal antibodies can be obtained from serum of an animal immunized against the appropriate antigen, which may be produced by genetic engineering for example according to standard methods well-known by one skilled in the art (see e.g. Harlow et al. (1988)).

[0081] Monoclonal antibodies refer to a population of antibody molecules that contains only one species of antibody combining site capable of immunoreacting with a particular epitope. A monoclonal antibody thus typically displays a single binding affinity for any epitope with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different epitope, e.g. a bispecific monoclonal antibody. Laboratory methods for preparing monoclonal antibodies are well known in the art (see, for example, Harlow et al., 1988).

[0082] Aptamers, which are a class of molecule that represents an alternative to antibodies in term of molecular recognition, can also be used for detecting the mutated form of NLRP3 in the context of the present invention. Aptamers are oligonucleotide or oligopeptide sequences that are capable of recognizing virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).

[0083] All the probes, primers, aptamers or antibodies used in this context may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or anyother labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal.

[0084] In the context of the present invention, the presence of a mutation selected from the group consisting of R260W, D303H, D303N, A352V, E525K, K565E, E567G, K568N, Y570C, E311 K, I334V, T348M, T436N, A439T, E690K, V262G, G301 D, L353P, A439V, G569R, R168Q, V198M, M701T, Q703K, (S726G / S896P), S726G, Y859C, Y859H, K829T, R918X and R918Q indicates that said patient suffers from an NLRP3-AID. This means that detecting a single of these mutations in the biological sample allows diagnosing the patient with NLRP3-AID.

[0085] In the context of the present invention, the presence of a mutation selected from the group consisting of A75V, 1313V, K375E, G767S, S896P, T952M and L1016F indicates that said patient does not suffer from NLRP3-AID. The presence of one of these mutations alone (i.e. in the absence of a mutation associated with NLFP3-AID) allows ruling out NLRP3-AID as a diagnosis.

[0086] The presence of said mutations can also allow predicting whether a patient will respond to NLRP3 inhibitors. Said mutations can therefore be used as biomarkers for selecting the most appropriate anti-NLRP3 treatment for each patient. The present inventors have particularly shown that:

[0087] Mutants A75V, R168Q, V198M, R260W, V262G, D303N, E311 K, 1313V, I334V, T348M, A352V, K375E, T436N, A439T, A439V, K565E, E567G, K568N, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F respond to MCC950-like inhibitors but not mutants G301 D, D303H, L353P, E525K and G569R.

[0088] All mutants A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, A352V, L353P, K375E, T436N, A439T, A439V, E525K, K565E, E567G, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F respond to CY-09-like or G5-like inhibitors.

[0089] Mutants A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, A352V, L353P, K375E, T436N, A439T, A439V, E525K, K565E, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F respond to CRT0066101-like inhibitors but not mutant E567G.

[0090] Therefore, according to another embodiment, the present invention pertains to an in vitro method for predicting a response to an NLRP3 inhibitor in a patient, said method comprising a step of detecting, in a biological sample obtained from said patient, an NLRP3 mutation selected from the group of 34 mutations consisting of A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, A352V, L353P, K375E, T436N, A439T, A439V, E525K, K565E, E567G, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F with respect to the amino acid sequence of the human NLRP3 protein as set forth in SEQ ID No. 1 .

[0091] In the context of the present invention, the presence:27 of one of said 34 mutations indicates that the patient will respond to a treatment comprising a G5-like inhibitor;- of one of mutations A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, L353P, K375E, T436N, A439T, A439V, E525K, K565E, E567G, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F indicates that the patient will respond to a treatment comprising a CY-09-like inhibitor- of one of mutations A75V, R168Q, V262G, 1313V, I334V, K375E, T436N, A439T, K565E, E567G, K568N, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F indicates that the patient will respond to a treatment comprising an MCC950-like inhibitor; of one of mutations G301 D, D303H, E525K and G569R indicates that the patient will not respond to a treatment comprising an MCC950-like inhibitor;- of one of mutations A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, A352V, L353P, K375E, T436N, A439T, A439V, E525K, K565E, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F indicates that the patient will respond to a treatment comprising a CRT0066101-like inhibitor; and of mutant E567G indicates that the patient will not respond to a treatment comprising a CRT0066101-like inhibitor.

[0092] The method according to the present invention allows predicting a patient’s response to a specific NLRP3 inhibitor, i.e. whether the patient will benefit (respond) or not from receiving the treatment. Patients who will benefit from receiving the treatment are also referred to as “responders”. Administering the treatment to these patients will allow treating their disease and / or improving their condition. Responders will achieve a response, i.e. the NLRP3-AID symptoms are reduced or stabilized after treatment. On the contrary, patients who will not benefit from receiving a known treatment are referred to “non-responders” or “poor-responders”. Administering the treatment to these patients will not allow treating their disease / reducing their symptoms, or at least less efficiently than in responders. For these patients, the selection of an alternative treatment strategy would thus be advantageous.

[0093] The method according to the present invention allows for the correct and efficient identification of responders to CY-09-like inhibitors, G5-like inhibitors, CRT0066101-like inhibitors and MCC950- like inhibitors.

[0094] The present invention therefore also relates to:A G5-like inhibitor for use in the treatment of an NLRP3-AID in a patient who has been identified as displaying a mutation selected from the group consisting of A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, A352V,L353P, K375E, T436N, A439T, A439V, E525K, K565E, E567G, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F with respect to the amino acid sequence of the human NLRP3 protein as set forth in SEQ ID No. 1 ;A CY-09-like inhibitor for use in the treatment of an NLRP3-AID in a patient who has been identified as displaying a mutation selected from the group consisting of A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, L353P, K375E, T436N, A439T, A439V, E525K, K565E, E567G, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F with respect to the amino acid sequence of the human NLRP3 protein as set forth in SEQ ID No. 1 ;An MCC950-like inhibitor for use in the treatment of an NLRP3-AID in a patient who has been identified as displaying a mutation selected from the group consisting of A75V, R168Q, V262G, 1313V, I334V, K375E, T436N, A439T, K565E, E567G, K568N, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F with respect to the amino acid sequence of the human NLRP3 protein as set forth in SEQ ID No. 1 ;A CRT0066101 -like inhibitor for use in the treatment of an NLRP3-AID in a patient who has been identified as displaying a mutation selected from the group consisting of CRT0066101 A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, A352V, L353P, K375E, T436N, A439T, A439V, E525K, K565E, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F with respect to the amino acid sequence of the human NLRP3 protein as set forth in SEQ ID No. 1 .

[0095] Having identified and characterized the 38 mutations disclosed in the context of the present invention, it is possible to test and identify new NLRP3-inhibitors.

[0096] According to a further embodiment, the present invention therefore also pertains to a method for screening a candidate NLRP3 inhibitor, said method comprising: i. Expressing, in a cell, a mutated NLRP3 protein having a mutation selected from the group consisting ofA75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, A352V, L353P, K375E, T436N, A439T, A439V, E525K, K565E, E567G, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, R918Q, T952M and L1016F with respect to the amino acid sequence of the human NLRP3 protein as set forth in SEQ ID No. 1 ; ii. Treating said cell with a candidate NLRP3 inhibitor;Hi. Inducing NLRP3 induction, priming and / or activation depending on the mutation expressed;iv. Selecting said compound if it inhibits NLRP3 expression and / activity in said cell treated with said candidate NRLP3 inhibitor.

[0097] The mutation expressed in step i. can also be selected from Y859H, R918X and K829T.

[0098] The cell is as defined above.

[0099] The candidate NLRP3 inhibitor may be of any type. It may be of natural origin or may have been produced by chemical synthesis. This may involve a library of structurally defined chemical compounds, uncharacterized compounds or substances, or a mixture of compounds.

[0100] Step Hi. comprises inducing NLRP3 induction, priming and / or activation “depending on the mutation expressed”. As explained above, the mutations according to the present invention correspond either 1 / a constitutively active mutant (i.e. which induces cell death upon “simple” induction of NLRP3 expression), 2 / a mutant that requires indifferently priming or activation further to induction of NLRP3 expression, 3 / a mutant that requires priming further to induction of NLRP3 expression, 4 / a mutant that requires activation further to induction of NLRP3 expression or 5 / a mutant that does not correspond to a gain-of-fu notion mutation. Step Hi. aims at reproducing the conditions under which said NLRP3 mutant is activated. As detailed in Table 2 and the experimental section, depending on the mutant, these conditions require NLRP3 induction alone or in combination with NLRP3 priming alone, NLRP3 activation alone, or NLRP3 priming and activation.

[0101] According to a specific embodiment, steps ii. And Hi. can be permuted, i.e NLRP3 induction, priming and / or activation can be induced prior to treating said cell with a candidate NLRP3 inhibitor.

[0102] In the context of the present invention, the compound is selected as being a promising NLRP3 inhibitor when it inhibits NLRP3 expression and / activity of at least 40%, preferably 50%, more preferably 75%, even more preferably 90% in said cell as compared to a control cell. Said control being a cell which has been obtained by performing steps i., ii. and Hi. but not iv.

[0103] “NLRP3 expression” corresponds to the expression of the NLRP3 gene. The skilled person can easily determine the expression level of NLRP3 by using several techniques that are routine to the skilled person.

[0104] The expression level of the NLRP3 protein can be evaluated by measuring the expression level of the NLRP3 gene. The expression level of a gene is typically determined by analyzing the mRNA(s) transcribed from that gene. These nucleic acid molecules can typically be extracted from a sample and analyzed by standard methods. One method commonly used is to subject the isolated mRNAs to reverse transcription ("RT") coupled with polymerase chain reaction ("PCR") amplifications using oligonucleotide primers specific to the genes of interest. Quantification of mRNAs can typically be performed using one of two real-time quantification technologies called SYBR Green® or TaqMan®.

[0105] The expression level of NLRP3 can also be directly evaluated by measuring the amount of the NLRP3 protein detected in the sample. Such methods typically involve contacting the sample to be analyzed with an agent capable of specifically binding the target protein. This agent is usually apolyclonal or monoclonal antibody. The presence of the protein is then typically detected by standard immunodetection methods after separation of the proteins by electrophoresis (technique also called "Western blotting") or by immunoassays by direct, indirect, competition or immunocapture methods (techniques also called "ELISA"). The formation of a complex between the protein of interest and the antibody(s) targeting said protein is usually detected and quantified by measuring an enzymatic reaction generating a colored, chemiluminescent or fluorescent product.

[0106] “NLRP3 activity” corresponds to the ability of NLRP3 to activate the inflammasome. NLRP3 activity can be directly monitored by assessing cell death such as pyroptosis, as well as IL-1 p / 18 secretion in the sample (as disclosed herein).

[0107] In addition to allowing for the characterization of the 38 specific mutations disclosed above, the characterization method according to the present invention also allowed identifying residues that are critical to NLRP3 activity. These residues are located within two specific domains of NLRP3 that can be targeted by using NLRP3 binding agents. These two domains are 1 / the interaction motif between the acidic loop (comprised between amino acids No.687-703) and, on the one side, the concave side of the LRR domain of NLRP3 and, on the other side, the C-terminal tip of the LRR domain of NLRP3 (amino acid residues 740-1034) on the other side, and 2 / the accessible surface of the HD2 domain (comprising amino acids K565, E567, K568, G569, Y570). The data gathered in the context of the present invention therefore shows that it is possible to modulate NLRP3 activity by using compounds that specifically target these zones.

[0108] Therefore, the present invention also pertains to a compound modulating NLRP3 activity, wherein said compound specifically binds to the accessible surface of the HD2 domain of NLRP3 or to the interaction motifs between the acidic loop and concave side of the LRR domain of NLRP3 on the one side, and the C-terminal tip of the LRR domain of NLRP3 on the other side.

[0109] According to the present invention “the accessible surface of the HD2 domain of NLRP3” corresponds to amino acids 565-570 of the NLRP3 sequence as set forth in SEQ ID No.1 .

[0110] The “interaction motifs between the acidic loop and concave side and the C-terminal tip of the LRR domain of NLRP3” correspond to a zone comprised between amino acids No.687- 703 and 740-1034 of the NLRP3 protein. The acidic loop acts as a glue which, under the NLRP3 inactive conformation, binds the two NLRP3 subunits (the concave side of the LRR domain of NLRP3 on the one side and the C-terminal tip of the LRR domain of NLRP3 on the other side). The LRR domain of NLRP3 corresponds to amino acids 740-1034 in Seq ID No. 1 . The acidic loop corresponds to amino acids H683-C704 in Seq ID No. 1. The concave side of the LRR domain of NLRP3 corresponds to amino acids R772-L780 + V799-N806 + K828-C835 + T856-N863 + K886-N891 + H914-R918 + V943-N948 + K971-L974 of SEQ ID No. 1. The C-terminal tip of the LRR domain of NLRP3 corresponds to amino acids Y1007-E1017 + S1033-W1034 of SEQ ID No.1 . The compound modulating NLRP3 activity according to the present invention therefore binds to the amino acids involved in the interaction between the acidic loop and concave side of the LRR domain of NLRP3, and / or in the interaction between the acidic loop the C-terminal tip of the LRR domain of NLRP3.

[0111] The compound specifically binds to the specified zones within the NLRP3 molecule. The compound according to the present invention is therefore an NLRP3 binding agent, i.e. a compound that has the ability to target and associate with these specific zones of the NLRP3 protein through molecular binding. The nature of the interaction between NLRP3 and the binding agent can be a non-covalent, reversible covalent or irreversible covalent binding. The skilled person knows which compounds can be qualified as NLRP3 binding agents and may therefore be useful in the context of the present invention..

[0112] Knowing the specific zones to be targeted within the NLRP3 molecule, the skilled person knows how to synthetize suitable binding agents targeting these zones. NLRP3 binding agents can e.g. be selected from polyclonal or monoclonal antibodies, antibody fragments, nucleic acids, peptides, aptamers or small molecules.

[0113] Antibody fragments such as scFv and single-domain antibodies (sdAb) can be of particular interest in this context. scFv are IgG fragments made of an N-terminal, variable domain of the H chain (VH) and an N-terminal, variable domain of the L chain (VL) joined by a short synthetic peptide linker. An sdAb is an antibody fragment consisting of a single monomeric variable antibody domain. sdAbs or “nanobodies®” have been derived from heavy-chain antibodies found in Camelidae species (such as camel, llama, dromedary, alpaca and guanaco) and are also referred to as VHH fragments. sdAb also include VNAR fragments derived from heavy chain antibodies found in cartilaginous fish, such as sharks. sdAbs have also been generated from a heavy chain / light chain of conventional IgGs. sdAbs have extremely interesting properties as they are generally quite stable and often resist extreme pH and temperature conditions that can be problematic for conventional antibodies and antibody fragments. sdAbs tend to be more resistant to the action of proteases and can be produced in vitro with high production yields.

[0114] According to a preferred embodiment, the compound modulating NLRP3 activity is selected from antibodies, aptamers, scFv and sdAb, preferably an sdAb.

[0115] The compound according to the present invention “modulates” NLRP3 activity, meaning that it allows either stimulating or inhibiting NLRP3 activity. Such compounds may be useful in various application wherein the inflammasome-induced inflammation needs to be either increased (such as in immunodepressed states) or decreased (such as during NLRP3-AID).

[0116] Inhibiting NLRP3 is a therapeutic strategy that is already used in the treatment of various disorders. The most obvious group of disorders that can be treated by inhibiting NLRP3 are NLRP3- AID. However, NLRP3 inhibition is also used in the treatment of various disorders (referred to as NLRP3-associated disorders) including atherosclerosis, ischemic stroke, Alzheimer’s disease, diabetes mellitus and inflammatory bowel diseases (Zhang et al., International Journal of Molecular Medicine 51 .4 (2023): 1-21).

[0117] The present invention therefore pertains to an NLRP3 binding compound for use in modulating NLRP3 activity in a cell, such as for treating NLRP3-AID or one of the NLRP3-associated disorder as detailed above, wherein said compound specifically binds to the accessible surface ofthe HD2 domain of NLRP3, or to the interaction motif between the acidic loop and concave side of the LRR domain of NLRP3 on the one side and the C-terminal tip of the LRR domain of NLRP3 on the other side.

[0118] The present invention also pertains to an in vitro method for modulating NLRP3 activity in a cell, wherein said method comprises treating said cell with an NLRP3 binding compound which specifically binds to the accessible surface of the HD2 domain of NLRP3 or to the interaction motif between the acidic loop and concave side of the LRR domain of NLRP3 on the one side and the C- terminal tip of the LRR domain of NLRP3 on the other side .

[0119] The invention will now be illustrated by means of the following examples.Examples

[0120] Reconstitution of / VL / ?P3-deficient U937 human monocytes with inducible NLRP3 variants associated with autoinflammation.

[0121] In this study 38 NLRP3 variants identified in patients suffering auto-inflammation suggestive of NLRP3-AID were analyzed (as disclosed in Table 1 above). 23 variants were selected among the most common variants associated with NLRP3-AID in France (Cuisset et al., Ann Rheum Dis 70, (2011) 495-9). 11 variants were included along the study upon the request of clinicians for diagnosis purpose. Variants Y859H, K829T, R918X and R918Q were further included. In order to compare the impact of NLRP3 variants on inflammasome activity in a standardized genetic background, U937 human monocytes knocked-out for NLRP3 by CRISPR / Cas9 and stably reconstituted them with doxycycline-inducible NLRP3 variants (Figure 1) were used (Lagrange et al., Nat. Commun. 9, (2018) 242). The expression levels of NLRP3 variants were similar or lower than the level of control WT NLRP3 in reconstituted U937 cell lines treated with doxycycline, excluding that their putative gain- of-function may be caused by higher expression levels. In addition, NLRP3 levels in doxycycline- treated reconstituted U937 monocytes were similar to endogenous NLRP3 level in U937 cells treated with LPS, indicating that the system was physiologically relevant.

[0122] Functional screen of NLRP3 variants using pyroptosis as readout.

[0123] The activities of NLRP3 variants were tested under (1) induction of their expression by doxycycline (Dox), (2) expression induction followed by LPS as a priming signal (Dox+LPS), (3) expression induction followed by nigericin as an activation signal (Dox+Nig) and (4) expression induction followed by both priming and activation signals (Dox+LPS+Nig) (Figure 1). NLRP3 activity was assessed by using pyroptosis as a readout. Propidium iodide incorporation was measured over time in Hoechst-counterstained cells using high content screening (HCS) microscopy. Cell death percentages over time for each variant and following each treatment were compared to those of U937 cells reconstituted with WT NLRP3 by fitting a linear mixed model (Figure 2). NLRP3 variants could be classified in 5 functional groups based on their activation upon induction, priming and / or activation signals. U937 cells expressing group#1 NLRP3 variants, as for example K568N, underwent cell death upon doxycycline-mediated induction of NLRP3 expression. Group#1 variants corresponded therefore to constitutively active mutants. U937 cells expressing group#2 NLRP3variants, as E311 K, underwent cell death upon NLRP3 expression and, either priming with LPS or activation with nigericin. U937 cells expressing group#3 NLRP3 variants, as A439V, underwent cell death upon NLRP3 expression and priming with LPS. U937 cells expressing group#4 NLRP3 variants, as R168Q, or Y859H, K829T, R918X and R918Q (data not shown), underwent cell death upon NLRP3 expression and activation with nigericin. Finally, U937 cells expressing group#5 NLRP3 variants, as T952M, underwent cell death upon NLRP3 expression, priming with LPS and activation with nigericin. Group#5 variants corresponded to variants with no gain-of-fu notion in reconstituted U937 cells. In the present experimental scheme, cell death monitoring was initiated when nigericin was added, i.e. 3h after doxycycline treatment. In order to investigate early cell death for group#1 NLRP3 variants, propidium iodide incorporation immediately following doxycycline treatment was imaged. Permeabilization of U937 expressing group#1 NLRP3 variants was detectable starting from 2.5h following doxycycline treatment, validating our experimental scheme.

[0124] Sensitivity of NLRP3 variants to NLRP3 chemical inhibitors.

[0125] The sensitivity of the NLRP3 variants to inflammasome inhibitors was next investigated, apart for Y859H, K829T, R918X and R918Q which were not tested. Direct NLRP3 inhibitors MCC950 and CY-09 were tested. MCC950 interacts with NLRP3 into a cleft in between the NACHT and the LRR domains in proximity to the ATP binding site and locks NLRP3 in an inactive conformation (Hochheiser et al., Nature (2022) doi:10.1038 / s41586-022-04467-w; Brinkschulte et al., Commun. Biol. 5, 1176 (2022); Tapia-Abelian et al., Nat. Chem. Biol. 15, 560-564 (2019); Dekker et al. J. Mol. Biol. 433, 167309 (2021)). CY-09 binding to NLRP3 depends on the integrity of NLRP3 ATP binding site and its inhibitory mechanism remains debated (Jiang et al. J. Exp. Med. 214, 3219-3238 (2017); Brinkschulte et al (2022)). Two indirect inhibitors were also tested: the deubiquitinase inhibitor G5, which impairs NLRP3 deubiquitination by BRCC3 (Py, B. F., Kim, M. S., Vakifahmetoglu-Norberg, H. & Yuan, Mol Cell 49, 331-8 (2013)), and the protein kinase D inhibitor CRT0066101 , which impairs NLRP3 phosphorylation at the Golgi during the activation process (Zhang et al., J Exp Med 214, 2671-2693 (2017)). The optimal dose of each inhibitorwas determined using A / LRP3-deficient U937 reconstituted with WT NLRP3 treated with doxycycline, LPS and nigericin to reach similar 80% inhibition for all tested inhibitors. Cell death was then measured following Dox (group#1), Dox+LPS (groups#1-3), Dox+Nig (groups#1 , 2, 4), or Dox+LPS+Nig (groups#1-5) in the presence of these inhibitors added before the last treatment for each condition. For each variant and for each treatment, we compared cell death in the presence of each of the inhibitors to vehicle only by fitting a linear mixed-effects model to AUC value databases (Figure 3). None of the group#5 variants showed reduced sensitivity to any of the inhibitors, confirming the absence of gain-of-function of these variants.

[0126] Most gain-of-function mutants are sensitive to MCC950, including all the mutants of group#4. Nevertheless, consistent with previous studies, several NLRP3 mutants were resistant. D303H and E525K (group#1) as well as G301 D, L353P and G569R (group#3) were resistant to MCC950 independently of the signals (priming or activation) provided. These mutations may affect NLRP3 ability to bind to MCC950, or impair the ability of MCC950 to lock NLRP3 in a closed conformation.I334V, A439T and E690K (group#2), and V262G, A439V and T348M (group#3) were sensitive to MCC950 when added before the priming signal, but were resistant to MCC950, when added on primed U937 before the activation signal. In this last condition, these mutants may switch to an active conformation upon priming before the addition of MCC950. Similarly, K565E, E567G, K568N and Y570C (group#1) were resistant to MCC950, when added before the activation signal on primed cells. Noteworthy, K568N and Y570C were also partly resistant to MCC950, when added before the activation signal on non-primed cells.

[0127] E31 1 K, I334V, A439T and E690K (group#2) were inhibited by MCC950 when added before priming or activation signal on non-primed cells, while T436N (group#2) was resistant to MCC950 when added before the priming signal, but sensitive to MCC950 when added before the activation signal. This suggested that while both priming and activation signals resulted in active T436N form, the structural switches underlying this activation process may differ. R260W (group#1) was inhibited by MCC950 when added before the LPS priming signal, or before the activation signal on primed cells. Although MCC950 was much less potent in this last condition, this may indicate that the LPS- induced structural switch in these mutants was reversible and that MCC950 may lock back NLRP3 in a closed conformation. D303N (group#1) was sensitive to MCC950 when added before its protein induction, but resistant if added later before the priming or the activation signal, suggesting that upon induction D303N may transiently adopt a closed conformation stabilized by MCC950, but quickly switch to an irreversible active conformation which may be stabilized by its oligomerization. In contrast to MCC950, no NLRP3 variants showed strong resistance to the direct inhibitor CY-09. E525K, K568N (group#1) and R168Q (group#4) showed partial resistance to CY-09 when added before the activation signal to non-primed cells. D303H (group#1) and T436N (group#2) showed partial resistance when CY-09 was added before the priming signal. A352V (group#1) showed partial resistance when CY-09 was added upon induction or before the activation signal.

[0128] Concerning the indirect inhibitors, no NLRP3 variants showed strong resistance to the deubiquitinase inhibitor G5. E525K (group#1) and R168Q (group#4) showed partial resistance when G5 was added before the activation signal to non-primed cells. A352V (group#1) was partially resistant when G5 was added before the induction or the activation signal. T436N (group#2) was partially resistant when G5 was added before the priming signal. CRT0066101 was inefficient in inhibiting any mutant of groups#1 , 2 or 4 when added before the activation signal in non-primed cells. In addition, E567G (group#1) and T436N (group#2) were resistant to CRT0066101 when added before priming, and A352V (group#1) was partially resistant to CRT0066101 when added upon induction. Noteworthy, observed resistance to all inhibitors of A352V (if added before induction or activation of unprimed cells), T436N (if added before priming) and E525K (if added before activation of unprimed cells) may be artifactual as these mutants showed the weakest gain-of-function of their groups in these conditions (Figure 2).

[0129] Activity of NLRP3 variants in patient primary monocytes.

[0130] In addition, a functional assay on primary monocytes was developed. It is adapted for small volume pediatric blood samples as it requires less than 0.2x106monocytes. Pyroptosis of primarymonocytes from 15 patients bearing NLRP3 variants was assessed in response to LPS and / or nigericin, and its sensitivity to MCC950 inhibition. Priming and activation signals have both been independently described to be dispensable for NLRP3 activation in human monocytes (Gaidt et al., Immunity 44, 833-846 (2016); Gritsenko et al., Front. Immunol. 11 , 565924 (2020)). In the experimental settings (dose, kinetics), LPS priming alone did not cause pyroptosis in monocytes from healthy donors. In the absence of priming, nigericin triggered pyroptosis in a MCC950-sensitive manner although with slower kinetics than in the presence of priming. In this context, additional priming signals from death of neighboring cells upon transportation or monocytes purification procedures could not be excluded.

[0131] NLRP3 induction and priming could not be discriminated in this experimental setting, and monocytes from patients bearing NLRP3 variants of group#1 (R260W, E525K+D646Y and K565E) and group#3 (T348M and A439V) showed increased pyroptosis in response to LPS as compared to their respective control monocytes from healthy donors. Monocytes from patients bearing NLRP3 variants of group#4 (S726G+S896P, Q703K and V198M) showed increased pyroptosis in response to nigericin. Monocytes from a patient bearing a NLRP3 variant of group#5 (T952M) showed no gain- of-function relative to their controls. MCC950 inhibited pyroptosis of all but monocytes from patient bearing E525K, consistent with inhibition assays on reconstituted U937 cells (Figure 3).

[0132] Discussion

[0133] NLRP3-AID clinical manifestations are diverse and most of them are poorly specific. Diagnosis relies largely on the identification of NLRP3 variants by genetic analysis, but only 11 % of known variants have been fully characterized as benign or pathogenic (Infevers 29 / 05 / 2023), while the functional link to the disease remains to be established for all the others (Van Gijn et al. J. Med. Genet. 55, 530-537 (2018)). In addition, gain-of-fu notions of NLRP3 variants have been assessed by monitoring NLRP3 activity in response to priming signal, which does not discriminate constitutive from priming-induced activity, and NLRP3 activity in response to activation signals in the absence of priming has not been considered (Rieber et al., Clin. Immunol. Orlando Fla 157, 56-64 (2015)). By reconstitution of A / LRP3-deficient U937 monocytes, the inventors functionally characterized 38 NLRP3 variants, including the most frequent in France, on an identical genetic background. For 9 variants, results were confirmed on primary monocytes from 15 patients. The inventors evidenced the functional diversity of NLRP3 variants which were classified in 5 groups. Identification of NLRP3 variants with no gain-of-function (group#5) will exclude NLRP3-AID diagnosis and guide decision toward broader genetic analysis for patients positive for these variants after gene panel approaches. Genotype-phenotype correlation are difficult to assess. Indeed, severities vary between patients bearing identical mutations, and in the anti-IL-1 therapy era, delayed access to treatment rather than the genotype determines symptoms severity. Nevertheless, the low genotype overlap between somatic and germline mutations suggest that somatic mutations, that may be responsible for 0.5%- 19% of NLRP3-AID cases, would be highly detrimental and incompatible with life if germline. Interestingly, the functional diversity identified in the present study strongly correlates with somatic vs germline mutation. All somatic mutations included in the study (n=5) correspond to constitutivelyactive mutants (group#1 , n=4) or mutants activated by priming or activation signal (group#2, n=1). 44% of group#1 mutants are associated with mosaicism (4 / 9), 20% of group#2 (1 / 5), but none of groups#3-4 (0 / 6, each).

[0134] Comparison of the sensitivity of NLRP3 mutants to two direct and two indirect inhibitors evidenced that direct inhibitor CY-09 was efficient on more variants than MCC950, and that the deubiquitinase inhibitor G5 was efficient on all mutants. This suggests that refining the modes of action of these compounds may be of particular interest for the development of potent inhibitors with therapeutic potential against most NLRP3-AID mutants.

[0135] Functional assays on primary patients’ monocytes are limited by the ethical issues related to blood drawn from pediatric patients, and by technical issues related to the low number of monocytes purified from pediatric samples, the cell stress caused by transportation, and the requirement for rapid delivery and processing of clinical samples. Reconstitution of NLRP3-deficient U937 lines with doxycycline-inducible NLRP3 variants enabled the comparison of NLRP3 variants on identical genetic background and discriminated NLRP3 constitutive activity vs priming-induced and / or activation-induced activity. Noteworthy, expression levels were similar for most of the NLRP3 variants, excluding any technical artifact caused by overexpression of some mutants. In contrast, the 2 mutants with the lowest expressions, E567G and D303N, were found constitutively active suggesting that their lower expressions likely resulted from their cytotoxic activity. Time-lapse assessment of pyroptosis at the single cell level proved to be a very sensitive and robust readout.

[0136] Here are described five functional groups of NLRP3 variants, classified according to their cellular response to induction, priming and / or activation signals. This led to the definition of a new group of variants, group#4, responding to the activation signal in the absence of priming. Our data revise previous studies that concluded to the absence of gain-of-function of some of these variants based on unresponsiveness to priming signals despite the higher prevalence in auto-inflammatory patients, and should be considered for atypical patient diagnosis toward NLRP3-AID (Rieber et al., Clin. Immunol. Orlando Fla 157, 56-64 (2015); Theodoropoulou et al., Front. Immunol. 11 , 877 (2020)). Gain-of-function of most group#4 variants in response to Dox+Nig were of low magnitude, and could be observed using pyroptosis (R168Q, V198M, M701T, Q703K, S726G, Y859C). Identification of constitutively active mutants (group#1) by treatment of reconstituted U937 with doxycycline indicated that other key proteins of the pathway (ASC, caspase-1 , GSDMD) pre-existed in functional forms in U937, and that priming and activation signals acted on NLRP3 itself.

[0137] Given the recent advances in NLRP3 structure determination, the identification of gain-of- function mutations provides insights into the key regulatory mechanisms of inflammasome assembly. A bar diagram depicts the localization of mutant sites according to the domain architecture of NLRP3 (Figure 4A). Based on our current knowledge, the transition from the inactive spherical decamer to the active concentrical decamer requires the release of the LRR domains by binding to NEK7 (Figure 4B). This conformational transition goes along with an 85° rotation of the NBD and HD1 subdomains relative to the WHD and HD2 subdomains within the NACHT, which is the central mechanism of STAND ATPase activation and accompanied by a nucleotide exchange from ADP to ATP (Figure4C) (Hochheiser et al., (2022) ;Xiao, L., Magupalli, V. G. & Wu, H. (2023)). Some disease mutations may destabilize the inactive spherical “cage” structure with the PYD effector domains buried inside, that relies mostly on interactions between the LRR domains (Hochheiser et al., (2022); Andreeva et al., bioRxiv2021 .09.12.459968 (2021) doi:10.1101 / 2021 .09.12.459968; Ohto et al., Proc. Natl. Acad. Sci. U. S. A. 119, e2121353119 (2022)). In human NLRP3, a loop section of 42 residues mediates the interlaced LRR dimer assembly with the acidic loop binding on one side into the concave side of its LRR structure and on the other side to the tip of the cognate LRR (Figures 4D). Y859 maps to the concave side of the LRR directly interacting with the acidic loop (687-700) comprising E690, suggesting that these two NLRP3-AID mutation sites may destabilize the inactive spherical structure (Hochheiser et al., (2022)). Residues M701 and Q703 from group#4 similarly reach out into the concave side of the canonical LRR and any mutation here might influence the intertwined LRR dimer assembly.

[0138] The group#1 variants of constitutively active mutants locate all in the NACHT domain in proximity to the nucleotide binding site. D303 is in the Walker B motif with the typical aaaaDE motif degenerated to aaaaDGxDE in NLRs (where “a” denotes an aliphatic residue) (Brinkschulte et al., (2022)). The two acidic residues catalyze the intrinsic ATP hydrolysis mechanism that is required for the relapse from an active to an inactive state, and it is well perceived that any mutation in this motif enriches the active state of NLRP3. A352 is following the sensor 1 motif (R349) on b-strand 4, interacting with D303 on b-strand 3 from the back (Figure 4E). Its mutation to a larger residue could also disrupt the inactivation mechanism. The R260 residue at the tip of b-strand 2 is buried in the structure of the inactive NACHT domain (7pzc) but on the surface close to the g-phosphate group in the active NACHT conformation (8ej4). Its mutation to a larger Trp-residue might prevent the formation of the tightly closed inactive state. The acidic E525 in WHD forms a salt bridge with the sensor 1 R349 following H-bonds with HD2. A charge reversal to Lys will dismiss these interactions. Finally, residues K565, E567 and K568 are on the accessible surface of the HD2 domain, both in the inactive and active state. Their disease related mutations point to an important surface patch of this side. The succeeding residue Y570 is again inside and interacts with the NBD, yet its mutation to Cys could indirectly influence the three preceding residues. This similarly holds for the G569R mutation of group#3, which might also affect the integrity of the KsesxEKGY stretch.

[0139] The group#3 G301 D mutant is proximate to D303 and part of the degenerated Walker B motif. The G301 E mutation has been shown to have an eight-fold reduced hydrolysis activity, enriching thus the active state. Residues T436 and A439 from groups #2-3 are located in the WHD near the pivot point switching between the inactive and active conformation (Figure 4F) (Hochheiser et al., (2022) ;Xiao, L., Magupalli, V. G. & Wu, H. (2023)). The new group#4 disease variants R168 and V198 are both located in the FISNA domain. R168 is next to T167 that interacts with the adenine base of the bound nucleotide (Figure 4G). V198M may stabilize the FISNA domain in the active conformation, as V198 is located in the activation loop comprising S196PVSP, whose priming- associated phosphorylation is likely to destabilizes the inactive cage complex (Hochheiser et al., (2022) ;Xiao, L„ Magupalli, V. G. & Wu, H. (2023)).

[0140] MCC950 binds NLRP3 in a cleft spanned by subdomains NBD, HD1 , WHD, HD2 and transition LRR, stabilizing the inactive conformation of the spherical decamer. (Hochheiser et al., (2022)). The present results on the inhibition of cell death upon NLRP3 WT or NLRP3-AID variant expressions confirmed the sensitivity to MCC950 of V198M, E311 K, T348M, the partial sensitivity of A352V, A439V, D303N, and the full resistance of L353P (Coll et al., (2015); Tapia-Abelian et al (2019); Vande Walle et al., PLoS Biol. 17, e3000354 (2019); Weber et al., Rheumatol. Oxf. Engl. 61 , e299-e313 (2022). D303H and E525K (group#1) and G301 D, L353P and G569R (group#3) were fully resistant to MCC950, either because they do not bind to MCC950, such as L353P (Vande Walle et al., (2019)), or MCC950 is not sufficientto stabilize the inactive conformation. The group#3 mutants were activated upon priming, indicating that the mechanism of NLRP3 priming may not rely solely, or at all, on an inactive cage structure destabilization. Indeed, mutants activated by the activation signal nigericin without priming (group#4) were highly sensitive to MCC950 to a similar extent as the WT NLRP3, suggesting that these mutants form inactive structures and that the activation signal is at least involved in destabilizing this inactive form. Alternatively, MCC950 may inhibit NLRP3 activity independently of stabilizing the cage perse, but ratherthe closed conformation of the NACHT domain, e.g. when bound to NEK7, or additional other mechanisms.

[0141] CY-09 is supposed to compete with nucleotide-binding to NLRP3; yet, a recent study has reported that CY-09 does not inhibit NLRP3 ATPase activity (Brinkschulte et al., (2022)). The present results partly confirmed NLRP3 A352V (group#1) sensitivity to CY-09 (Jiang et al., 2017). In contrast to MCC950, no gain-of-fu notion mutations showed full resistance to CY-09, consistent with CY-09 targeting residues indispensable for NLRP3 inflammasome assembly, suggesting that CY-09 and its derivatives may have broader activity than MCC950 for future clinical perspectives against NLRP3- AID.

[0142] G5 is a deubiquitinase inhibitor that maintains NLRP3 in an ubiquitinated inactive form (Py, B. F., Kim, M. S., Vakifahmetoglu-Norberg, H. & Yuan, J., 2013). Consistent with the lack of specificity of G5 targeting multiple deubiquitinases, and the reports of multiple ubiquitination sites on NLRP3, none of the NLRP3 gain-of-function mutants showed resistance to G5 (Py, B. F., Kim, M. S., Vakifahmetoglu-Norberg, H. & Yuan, J., 2013). Among all mutants, NLRP3 A352V (group#1) and T436N (group#2) showed lower sensitivity to G5 upon treatment with doxycycline only and LPS only, respectively. However, as the amplitude of the activation was low in these conditions, the low percentage of inhibition may therefore be artefactual. Noteworthy, constitutively active mutants were inhibited by G5, indicating that ubiquitination and deubiquitination may occur at least in part independently of priming and activation signals. The hypothesis that G5 may have an additional target downstream of NLRP3 in the pathway is ruled out by the specificity of G5 to inhibit the NLRP3 but neither the AIM2 nor the NLRC4 inflammasomes (Py, B. F., Kim, M. S., Vakifahmetoglu-Norberg, H. & Yuan, J., 2013).

[0143] CRT0066101 is a pan-PKD inhibitor targeting PKDs-mediated NLRP3 S293 phosphorylation upon activation signals that controls the release of NLRP3 from an intracellular membrane compartment to the cytosol for the assembly of the inflammasome (Zhang et al., 2017). None of thegroup#2 and #4 mutants were inhibited by CRT0066101 when activated by activation signal in the absence of priming (Figure 3, Dox+Nig only), indicating that in this context NLRP3 activation occurs independently of PKDs. Constitutive activity of most of the group#1 mutants were sensitive to CRT0066101 , indicating that basal PKD activity may be required.

[0144] Altogether, the present study constitutes the most comprehensive comparative analysis of NLRP3 mutations associated with auto-inflammation reported so far. The present results reveal the functional diversity of NLRP3 gain-of-function mutations that should be taken in consideration for atypical NLRP3-AID diagnosis and specific targeted treatments perspectives.

[0145] Methods

[0146] Study approval

[0147] The study was carried out in the setting of the ENFLAMAI protocol, which was previously approved by the French Comite de Protection des Personnes (CPP, #L16-189) and by the French Comite Consultatif sur le Traitement de I’lnformation en matiere de Recherche dans le domaine de la Sante (CCTIRS, #16.864). The experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services’ Belmont Report. Anonymous healthy donors’ blood was provided by the Etablissement Fran ais du Sang (EFS) in the framework of the convention #14-1820 between Inserm and EFS. Informed consent was received from participants prior to inclusion in the study.

[0148] NLRP3 variants nomenclature

[0149] All NLRP3 amino-acids are numbered according to the Infevers database European nomenclature (Milhavet et al., 2008). Their pathogenicity was classified by an International consortium of expert geneticists as previously described (Van Gijn et al., 2018).

[0150] Cell culture

[0151] Lenti-X™ 293T cells (Takara) were cultured at 0.5-1x106cells / ml in Dulbecco’s modified Eagle’s Medium (DMEM) GlutaMax™-! supplemented with 1X penicillin / streptomycin (PS) and 10% Fetal Bovine Serum (FBS) (Gibco). NLRP3-deficient U937 cells have been previously described. U937 and U937 reconstituted NLRP3-deficient cells were cultured at 0.25-1x106cells / ml in Roswell Park Memorial Institute (RPMI) 1640 GlutaMax™-! supplemented with 1X PS and 10% FBS (Gibco). Monocytes were purified from 10-20 ml of blood samples (drawn the day before in heparin tubes) using EasySep™ Direct Human Monocyte Isolation kit (StemCell) and cultured at 0.07-0.15x106cells / ml in RPMI-1640 GlutaMax™-! supplemented with 1X PS and 10% FBS (Gibco).

[0152] Plasmids

[0153] Human NLRP3 were cloned in pENTR™1A (Invitrogen) from Flag-NLRP3 encoding plasmids kindly shared by F. Martinon (Unil, Lausanne, Switzerland). Mutations were performed using QuickChange™ II kit (Agilent Technologies). cDNAs were transferred in plnducer21 using recombination Gateway LR clonase Enzyme mix kit (Thermofisher) (Meerbrey et al., Proc. Natl. Acad.Sci. U. S. A. 108, 3665-3670 (2011)). pMD2.G and pCMVR8.74 were gifts from D. Trono (Addgene plasmid #12259 and #22036).

[0154] Reagents

[0155] The following reagents were used: polyethylenimine (PEI, Sigma), polybrene (Santa-Cruz), phorbol myristate acetate (PMA, Invivogen), doxycycline (Sigma), LPS (O111 :B4, Sigma), nigericin (Invivogen), ATP (Sigma), MCC950 (Adipogen), CY-09 (tocris), G5 (Calbiochem), CRT0066101 (Tocris), propidium iodide (PI, Immunochemistry technologies), Hoechst (Immunochemistry technologies)

[0156] Lentivector preparation and cell transduction

[0157] 1 .6x106Lenti-X™ 293T cells were co-transfected with pMD2.G (10 mg), pCMVR8.74 (30 mg) and plnducer21-NLRP3 or its variants (40 mg) using PEI to produce lentivectors. Lentivectors were concentrated by ultracentrifugation (30,000 rpm, 1 h) and used to transduced 1.5x105NLRP3- deficient U937 cells in the presence of polybrene (8mg / ml) (Lagrange et al, 2018). 4 days later, transduced GFP-positive cells were sorted by cytometry as bulk.

[0158] Western-blot analysis

[0159] To assess NLRP3 protein levels, U937 cells were differentiated with PMA (50 ng / ml, 16h) and treated with LPS (40 ng / ml, 3h) or doxycycline (0.5-2 mg / ml, 3h). Cells were directly lysed in sample buffer 2X. Samples were analyzed by SDS-PAGE and transferred to PVDF membranes. The following antibodies were used: anti-NLRP3 (Cryo2, Adipogen), anti-IL-1 p (D3U3E, Cell Signaling), anti-IL-18 (D2F3B, Cell Signaling), anti-LaminB (B-10, Santa Cruz Biotechnology), anti-actin (C4, Sigma), HRP-anti-Mouse IgG (H+L) (Promega).

[0160] Cell death assay

[0161] To assess U937 permeabilization, 0.3X106cells / ml were platted in media without phenol red. The next day, cells were treated with doxycycline (1 pg / ml, 3h), LPS (40 ng / ml, 2h) and nigericin (15 pg / ml) before time-lapse imaging using CQ1 high content screening microscope (Yokogawa). In inhibition assay, MCC950 (1 pM), CY-09 (50 pM), G5 (1 pM), CRT0066101 (0.5 pM) or DMSO vehicle (1 % final in all conditions) were added 20 min before doxycycline, LPS or nigericin as indicated. To assess monocyte permeabilization, 0.07-0.15x106cells / ml were platted in media without phenol red and directly treated with MCC950 (1 pM, 3h15), LPS (40 ng / ml, 3h) and nigericin (5 pg / ml) before time-lapse imaging using CQ1 high content screening microscope (Yokogawa). PI (1 .25 pg / ml) and Hoechst (0.2 pg / ml) were added 2h before imaging. 2 images / well were taken every 15 min for 2h using 10X objectives (UPLSAPO 10X / 0.4). Images were quantified using the CQ1 software (Yokogawa).

[0162] Cytokine secretion assay

[0163] To assess IL-18, IL-i p and TNF secretion, 0.25X106reconstituted U937 cells / ml can be differentiated with PMA (50 ng / ml, 16h). The next day, cells are treated with doxycycline (1 pg / ml,4h), LPS (40 ng / ml, 3h) and nigericin (15 pg / ml, 1 h). Cell-free media were then analyzed using IL- 18, IL-1 p and TNF ELISA kits (RnD Systems).

[0164] Statistical analysis

[0165] The cell death was observed at 10 time points ranging from 0 to 135 minutes by 15 minutes interval for each of the 38 NLRP3 variants and NLRP3 WT undergoing 5 treatments ( / .e. untreated, Dox, Dox+LPS, Dox+Nig and Dox+LPS+Nig), and studied as a proportion of the whole cell population.

[0166] The database was divided into 50 data subsets (5 treatments * 10 time points). For each subset a generalized linear mixed model (glmm) was fitted, beta family with logit link producing 50 results for each variant. In our model, the NLRP3 variant type was set as the explanatory variable with the WT as the reference. The outcome variable in the model was the frequency of dead cells bounded in [0,1], glmm can similarly to Imm correct for the technical batch effect induced by the individual experiment. The p-values obtained were corrected by the Bonferroni approach to take into account results for the 5 treatments. The R statistical environment, version 4.1 .3, was used with the glmmTMB function from the glmmTMB package. Results for Dox, Dox+LPS and Dox+Nig treatments are shown. For visualisation purpose, cut-offs were fixed to regression coefficients 1.5 / -1.5 for Dox and Dox+LPS, and 0.5 / -0.5 for Dox+Nig, with p-value<0.05. As data examples, raw data of duplicates of representative experiments were analyzed by two-way ANOVA multiple comparison using NLRP3 variant and treatments as variable (Graphpad, Prism). Experiments were independently repeated 2- 8 times.

[0167] IL-1 p, IL-18 and TNF concentrations were measured for the 35NLRP3 variants and NLRP3 WT upon 3 treatments ( / .e. Dox, Dox+LPS, Dox+Nig). One dataset per treatment was created, and a linear mixed-effects model (Imm) was realized for each dataset to assess the dependence of the cytokine concentration on the variant. Each dataset comprised 564 observations. The outcome in the model was the log 1 p (log 1 p(X)=log(1 +X)) of the cytokine concentration given in pg / ml while the right side of the model was the NLRP3 variant. The NLRP3 WT was set as the reference. Imm was appropriate due to its ability to correct for the batch effect triggered by the individual experiment. We used the R statistical environment, version 4.1 .3, with the Ime function from the nlme package. For visualisation purpose, cut-offs were fixed to regression coefficients 1 / -1 with p-value<0.05. As data examples, raw data of duplicates of representative experiments were analyzed by two-way ANOVA multiple comparison using NLRP3 variant and treatments as variable (Graphpad, Prism). Experiments were independently repeated 3-18 times.

[0168] The effect of the NLRP3 inhibitors was tested by comparing AUC obtained in presence of each of the 4 inhibitors ( / .e. MCC950, CY-09, G5 and CRT0066101) to the AUC obtained without inhibitor (vehicle only, used as the reference state). A linear mixed-effects model (Imm) was fitted for each genotype undergoing a given treatment, in a dataset constructed by the extraction of genotype*treatment from all data. The model response variable was log10(AUC) and the explanatory variable was the inhibitors. As data diagnosis proved a lawful steady batch effect of the independent experiment, Imm modeling was used to correct for this batch effect technical bias in the data. The p-values obtained were corrected by the Bonferroni approach to take into account results for the 4 treatments (Dox, Dox+LPS, Dox+Nig and Dox+LPS+Nig). For visualisation purpose, cut-offs were fixed to regression coefficients <-0.1 with p-value<0.05. Regression coefficient=log10(Fold change). The R statistical environment, version 4.1 .3, was used with the Ime function from the nlme package. As data examples, AUC of duplicates of representative experiments were analyzed by two-way ANOVA multiple comparison using treatments and inhibitors as variable (Graphpad, Prism). Experiments were independently repeated twice.Table 3 : NLRP3 variants included in the study (Infevers database, personal observations and Louvrier et al.).

[0170] Frequencies based on exome sequencing of general population of all origins. *, patients with somatic mutation were asymptomatic.

[0171] PYD, pyrin domain; NACHT, NAIP, CIITA, HET-E and TEP1 domain; LRR, Leucin-rich repeats domain; trLRR, transition LRR; FISNA, Fish-specific NACHT associated domain; NBD, nucleotide binding domain; WHD, winged helix domain; HD1 / 2, helical domain; VUS, variant of uncertain significance.

[0172] NLRP3 variants Y859H, K829T, R918X and R918Q, located in the LRR domain, were further included in the study.

Claims

Claims

1. An in vitro method for characterizing a mutated NLRP3 protein, said method comprising the steps of: a. Expressing said mutated NLRP3 protein in cells; b. Treating a group of said cells so as to induce NLRP3 induction alone, another group of said cells so as to induce NLRP3 induction and priming, another group of said cells so as to induce NLRP3 induction and activation, and another group of said cells so as to induce NLRP3 induction, priming and activation; and c. Evaluating the response of said cells to the treatments and characterizing said mutant accordingly.

2. The method according to claim 1 , wherein NLRP3 priming is induced by treating the cells with a TLR ligand such as lipopolysaccharides (LPS), and / or NLRP3 activation is induced by treating said cell with nigericin.

3. The method according to claim 1 or 2, wherein the method further comprises a step of treating said cells with an NLRP3 inhibitor.

4. The method according to any one of claims 1 to 3, wherein the response in step c. is evaluated by monitoring pyroptosis and / or IL-1 p / 18 secretion in said cells.

5. The method according to any one of claims 1 to 4, wherein characterizing said mutated protein consists in indicating whether the mutation is a gain-of-function mutation or not.

6. An in vitro method for the diagnosis of an NLRP3-associated autoinflammatory disease (NLRP3-AID) in a patient, said method comprising a step of detecting, in a biological sample obtained from said patient, an NLRP3 mutation selected from the group consisting of K565E, E567G, K568N, Y570C, (S726G / S896P), S726G, A75V, K375E, G767S, S896P, T952M and L1016F in the amino acid sequence of the human NLRP3 protein as set forth in SEQ ID No. 1 , wherein, the presence of a mutation selected from the group consisting of K565E, E567G, K568N, Y570C, (S726G / S896P) and S726G indicates that said patient suffers from an NLRP3-AID; and the presence of a mutation selected from the group consisting of A75V, K375E, G767S, S896P, T952M and L1016F, alone, indicates that said patient does not suffer from NLRP3-AID.

7. The method according to claim 6, wherein said method comprises analyzing the sample for the presence of all mutations K565E, E567G, K568N, Y570C, (S726G / S896P), S726G, A75V, K375E, G767S, S896P, T952M and L1016F.

8. The method according to claim 5 or 6, wherein said method further comprises detecting the presence of an NLRP3 mutation selected from the group consisting of R260W, D303H, D303N, A352V, E525K, E311 K, I334V, T348M, T436N, A439T, E690K, V262G, G301 D, L353P, A439V, G569R, R168Q, V198M, M701T, Q703K, Y859C , R918Q and 1313V, in the amino acid sequenceas set forth in SEQ ID No. 1 , and wherein the presence of a mutation selected from the group consisting of R260W, D303H, D303N, A352V, E525K, E311 K, I334V, T348M, T436N, A439T, E690K, V262G, G301 D, L353P, A439V, G569R, R168Q, V198M, M701T, Q703K, Y859C and R918Q indicates that the patient suffers from an NLRP3-AID, and the presence of mutation 1313V, alone, indicates that said patient does not suffer from NLRP3-AID.

9. An in vitro method for predicting a response to an NLRP3 inhibitor in a patient, said method comprising a step of detecting, in a biological sample obtained from said patient, an NLRP3 mutation selected from the group of 34 mutations consisting of A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, A352V, L353P, K375E, T436N, A439T, A439V, E525K, K565E, E567G, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F with respect to the amino acid sequence of the human NLRP3 protein as set forth in SEQ ID No. 1 , wherein- the presence of one of said 34 mutations indicates that the patient will respond to a treatment comprising a G5-like inhibitor;- the presence of one of mutations A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, L353P, K375E, T436N, A439T, A439V, E525K, K565E, E567G, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F indicates that the patient will respond to a treatment comprising a CY-09-like inhibitor- the presence of one of mutations A75V, R168Q, V262G, 1313V, I334V, K375E, T436N, A439T, K565E, E567G, K568N, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F indicates that the patient will respond to a treatment comprising an MCC950-like inhibitor;- the presence of one of mutations G301 D, D303H, E525K and G569R indicates that the patient will not respond to a treatment comprising an MCC950-like inhibitor;- the presence of one of mutations A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, A352V, L353P, K375E, T436N, A439T, A439V, E525K, K565E, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F indicates that the patient will respond to a treatment comprising a CRT0066101 - like inhibitor;- the presence of mutant E567G indicates that the patient will not respond to a treatment comprising a CRT0066101-like inhibitor.

10. The method according to any one of claims 6 to 9, wherein said biological sample is a blood sample.

11. A G5-like inhibitor for use in the treatment of an NLRP3-AID in a patient who has been identified as displaying a mutation selected from the group consisting of A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, A352V, L353P, K375E,T436N, A439T, A439V, E525K, K565E, E567G, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F with respect to the amino acid sequence of the human NLRP3 protein as set forth in SEQ ID No. 1 ;

12. A CY-09-like inhibitor for use in the treatment of an NLRP3-AID in a patient who has been identified as displaying a mutation selected from the group consisting of A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, L353P, K375E, T436N, A439T, A439V, E525K, K565E, E567G, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F with respect to the amino acid sequence of the human NLRP3 protein as set forth in SEQ ID No. 1 .

13. An MCC950-like inhibitor for use in the treatment of an NLRP3-AID in a patient who has been identified as displaying a mutation selected from the group consisting of A75V, R168Q, V262G, 1313V, I334V, K375E, T436N, A439T, K565E, E567G, K568N, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F with respect to the amino acid sequence of the human NLRP3 protein as set forth in SEQ ID No. 1 .

14. A CRT0066101-like inhibitor for use in the treatment of an NLRP3-AID in a patient who has been identified as displaying a mutation selected from the group consisting of CRT0066101 A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, A352V, L353P, K375E, T436N, A439T, A439V, E525K, K565E, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, T952M and L1016F with respect to the amino acid sequence of the human NLRP3 protein as set forth in SEQ ID No. 1 .

15. An in vitro method for screening a candidate NLRP3 inhibitor, said method comprising: i. Expressing, in a cell, a mutated NLRP3 protein having a mutation selected from the group consisting of A75V, R168Q, V198M, R260W, V262G, G301 D, D303H, D303N, E311 K, 1313V, I334V, T348M, A352V, L353P, K375E, T436N, A439T, A439V, E525K, K565E, E567G, K568N, G569R, Y570C, E690K, M701T, Q703K, (S726G / S896P), S726G, G767S, Y859C, S896P, R918Q, T952M and L1016F with respect to the amino acid sequence of the human NLRP3 protein as set forth in SEQ ID No. 1 ; ii. Treating said cell with a candidate NRLP3 inhibitor;Hi. Inducing, in said cell, NLRP3 induction, and NLRP3 priming and / or activation, depending on the mutation expressed; and iv. Selecting said compound if it inhibits NLRP3 expression and / or activity in said cell treated with said candidate NRLP3 inhibitor.

16. A compound modulating NLRP3 activity, wherein said compound specifically binds to the accessible surface of the HD2 domain of NLRP3 or to the interaction motif between the acidic loop and concave side of the LRR domain of NLRP3 on the one side, and the C-terminal tip of the LRR domain of NLRP3 on the other side.

17. An in vitro method for modulating NLRP3 activity in a cell, wherein said method comprises treating said cell with an NLRP3 binding compound which specifically binds to the accessible surface of the HD2 domain of NLRP3 or to the interaction motif between the acidic loop and concave side of the LRR domain of NLRP3 on the one side, and the C-terminal tip of the LRR domain of NLRP3 on the other side .

18. An NLRP3 binding compound for use in modulating NLRP3 activity in a cell, wherein said compound specifically binds to the accessible surface of the HD2 domain of NLRP3 or to the interaction motif between the acidic loop and concave side of the LRR domain of NLRP3 on the one side, and the C-terminal tip of the LRR domain of NLRP3 on the other side.

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