Use of particles for the treatment of cardiac events associated with obesity

Abstract

The inventors showed that intravenous administration of the small extracellular vesicles (sEVs) loaded with a plasmid encoding a dominant-negative mutant of NLRP3 under the control of the SF1 promoter (SF1-NLRP3-DN) slightly reduced body weight but did not modify food intake in atherosclerosis-prone ApoE knockout mice fed a high-fat diet, but induced an increase in brow adipose tissue (BAT) thermogenesis. Glucose tolerance was not affected after treatment with SF1-NLRP3-DN-loaded EVs. High-fat diet feeding induced an increase in the number of arrhythmias, as well as an increase in the onset of atrial fibrillation (AF) induced by transoesophageal stimulation, but also spontaneous arrhythmias that was not modified by the treatment with non-loaded EVs. Interestingly, EVs loaded with SF1-NLRP3-DN almost completely prevented both induced and spontaneous arrhythmias (conduction block) and AF. Finally, echocardiographic analyses showed that SF1-NLRP3-DN-loaded EVs did not induce any change in the structural and functional capacity of the heart. Accordingly, the present invention relates to the use of particles for the treatment of cardiac events associated with obesity.

Description

[0001]USE OF PARTICLES FOR THE TREATMENT OF CARDIAC EVENTSASSOCIATED WITH OBESITY FIELD OF THE INVENTION: The present invention is in the field of medicine, in particular nanobiomedecine and cardiology. BACKGROUND OF THE INVENTION: Obesity is a rapidly growing problem that is reaching epidemic proportions worldwide and is associated with an increased risk of cardiovascular morbidity and mortality.1,2Obesity increases the risk of metabolic disorders leading to the development of atherosclerotic cardiovascular events in particular cardiac arrhythmias and atrial fibrillation (AF), the most frequent cardiac arrhythmia in clinical practice. Hypothalamus is the critical brain region responsible for regulating energy homeostasis, which is achieved by balancing the levels of food intake controlled by arcuate nucleus (ARC) and energy expenditure controlled by the ventromedial hypothalamus (VMH). Hypothalamicinflammation has been linked to a variety of metabolic disorders, and starts early when animalsare exposed to a hypercaloric environment; it is apparent even before the development of disease phenotypes, implying that hypothalamic inflammation is a crucial driver of metabolic diseases.3,4The hypothalamus has also been implicated through endocrine signaling systems, directly or as a consequence of underlying cardiac pathology, to contribute to the pathogenesisof AF (See https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC8208746 / ).The CANTOS study shows that targeting IL-1ß resulting from the NLRP3 (NOD-like receptor family, pyrin domain-containing protein 3) inflammasome pathway activation as an anti-inflammatory anti-inflammatory therapy led to a significant lowering rate of recurrentcardiovascular events (i.e. stroke, myocardial infection, heart failure) independently of lipid- level lowering.5The inventors have described that large (lEVs) and small (sEVs) extracellular vesicles (EVs),membrane-bound vesicles released by most cell types, account for specific vascular complications leading to atherosclerosis, in addition to represent predictive factors of cardiovascular diseases, in obese and diabetic patients.6,7Recently, the inventors developed a nanobiomedicine approach using neuronal-targeted EVs todeliver a plasmid encoding an AMPKα1 dominant negative mutant (AMPKα1-DN) for targeting specific SF1 neurons in the VMH following intravenous injections.8SF1-AMPKα1-DN-loaded sEVs significantly decreased body weight in obese mice. Notably, this effect wasfeeding-independent but involved sympathetic nerve activation and an increase in brow adipose tissue (BAT) thermogenesis. SUMMARY OF THE INVENTION:The present invention is defined by the claims. In particular, the present invention relates to theuse of particles for the treatment of cardiac events associated with obesity.DETAILED DESCRIPTION OF THE INVENTION: The inventors showed that intravenous administration of the small extracellular vesicles (sEVs) loaded with a plasmid encoding a dominant-negative mutant of NLRP3 under the control of theSF1 promoter (SF1-NLRP3-DN) slightly reduced body weight in atherosclerosis-prone ApoEknockout mice fed a high-fat diet. Notably, this effect was independent of body weight gain butwas associated with an increase in brow adipose tissue (BAT) thermogenesis. Glucose tolerancewas not affected after treatment with SF1-NLRP3-DN-loaded EVs. High-fat diet feeding induced an increase in the number of arrhythmias, as well as an increase in the onset of AFinduced by transesophageal stimulation, but also spontaneous arrhythmias that was notmodified by the treatment with non-loaded EVs. Interestingly, EVs loaded with SF1-NLRP3-DN almost completely prevented both arrhythmias (conduction block) and AF, both induced and spontaneous. Finally, echocardiographic analyses showed that SF1-NLRP3-DN-loadedEVs did not induce any change in the structural and functional capacity of the heart.Accordingly, the present invention relates to the use of particles for the treatment of cardiacevents associated with obesity. Main definitions: As used herein, the terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. Polypeptides when discussed in the context of gene therapy refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function of the intact protein. As used herein, the term “fusion protein” is referred herein as to a protein formed by at least two domains, wherein the at least two domains have been joined, one after the other, so that they are synthetized or translated as a single unit, and thus the two domains of the fusion protein are part of a single polypeptide. As used herein, the term “polynucleotide” as used herein refers to polymers of nucleotides ofany length, including ribonucleotides, deoxyribonucleotides, analogues thereof, or mixturesthereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-,double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- andsingle-stranded ribonucleic acid (“RNA”). It also includes modified, for example by alkylation, and / or by capping, and unmodified forms of the polynucleotide. More particularly, the term “polynucleotide” includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA and mRNA,whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycosideof a purine or pyrimidine base, and other polymers containing normucleotidic backbones, forexample, polyamide (e.g., peptide nucleic acids “PNAs”) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. In some embodiments, the polynucleotide comprises a mRNA. In other aspect, the mRNA is a synthetic mRNA. In some embodiments, the synthetic mRNA comprises at least one unnatural nucleobase. In some embodiments, all nucleobases of a certain class have been replaced with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be replaced with an unnatural nucleobase, e.g., 5-methoxyuridine). In some embodiments, the polynucleotide (e.g., a synthetic RNA or a synthetic DNA) comprises only natural nucleobases, i.e., A, C, T and G in the case of a synthetic DNA, or A, C, T, and U in the case of a synthetic RNA. As used herein, the term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as, for example, a gene, a cDNA, or a mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "polynucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.As used herein, the term “NLRP3” has its general meaning in the art and refers to the NLRfamily pyrin domain containing 3 protein (previously known as NACHT, LRR, and PYDdomains-containing protein 3 [NALP3] and cryopyrin) that is encoded by the NLRP3 gene(Gene ID: 114548). NLRP3 is a cytosolic protein with three domains: a leucine-rich repeat at the carboxy terminal, a central nucleotide-binding and oligomerization domain (NACHT),which possesses ATPase activity, and a pyrin domain (PYD) at the amino terminal. NLRP3activates an inflammasome complex that mediates the processing and secretion of pro- inflammatory cytokines such as interleukin-1 beta (IL-1β) and interleukin-18 (IL-18). NLRP3 is involved in the innate immune response to various infections and stress signals, and its dysregulation has been implicated in several inflammatory disorders, such as gout, type 2diabetes, Alzheimer's disease, and atherosclerosis. An exemplary amino acid sequence forNLRP3 is shown as SEQ ID NO:1 (human), SEQ ID NO:2 (human) or SEQ ID NO:3(mouse). SEQ ID NO:1 >sp|Q96P20|NLRP3_HUMAN NACHT, LRR and PYD domains- containing protein 3 OS=Homo sapiens OX=9606 GN=NLRP3 PE=1 SV=3 MKMASTRCKLARYLEDLEDVDLKKFKMHLEDYPPQKGCIPLPRGQTEKADHVDLATLMID FNGEEKAWAMAVWIFAAINRRDLYEKAKRDEPKWGSDNARVSNPTVICQEDSIEEEWMGL LEYLSRISICKMKKDYRKKYRKYVRSRFQCIEDRNARLGESVSLNKRYTRLRLIKEHRSQ QEREQELLAIGKTKTCESPVSPIKMELLFDPDDEHSEPVHTVVFQGAAGIGKTILARKMM LDWASGTLYQDRFDYLFYIHCREVSLVTQRSLGDLIMSCCPDPNPPIHKIVRKPSRILFL MDGFDELQGAFDEHIGPLCTDWQKAERGDILLSSLIRKKLLPEASLLITTRPVALEKLQH LLDHPRHVEILGFSEAKRKEYFFKYFSDEAQARAAFSLIQENEVLFTMCFIPLVCWIVCT GLKQQMESGKSLAQTSKTTTAVYVFFLSSLLQPRGGSQEHGLCAHLWGLCSLAADGIWNQ KILFEESDLRNHGLQKADVSAFLRMNLFQKEVDCEKFYSFIHMTFQEFFAAMYYLLEEEK EGRTNVPGSRLKLPSRDVTVLLENYGKFEKGYLIFVVRFLFGLVNQERTSYLEKKLSCKI SQQIRLELLKWIEVKAKAKKLQIQPSQLELFYCLYEMQEEDFVQRAMDYFPKIEINLSTR MDHMVSSFCIENCHRVESLSLGFLHNMPKEEEEEEKEGRHLDMVQCVLPSSSHAACSHGL VNSHLTSSFCRGLFSVLSTSQSLTELDLSDNSLGDPGMRVLCETLQHPGCNIRRLWLGRC GLSHECCFDISLVLSSNQKLVELDLSDNALGDFGIRLLCVGLKHLLCNLKKLWLVSCCLT SACCQDLASVLSTSHSLTRLYVGENALGDSGVAILCEKAKNPQCNLQKLGLVNSGLTSVC CSALSSVLSTNQNLTHLYLRGNTLGDKGIKLLCEGLLHPDCKLQVLELDNCNLTSHCCWD LSTLLTSSQSLRKLSLGNNDLGDLGVMMFCEVLKQQSCLLQNLGLSEMYFNYETKSALET LQEEKPELTVVFEPSW SEQ ID NO:2 >sp|Q96P20|NLRP3_HUMAN NACHT, LRR and PYD domains-containing protein 3 OS=Homo sapiens OX=9606 GN=NLRP3 PE=1 SV=2 MASTRCKLARYLEDLEDVDLKKFKMHLEDYPPQKGCIPLPRGQTEKADHVDLATLMIDFN GEEKAWAMAVWIFAAINRRDLYEKAKRDEPKWGSDNARVSNPTVICQEDSIEEEWMGLLE YLSRISICKMKKDYRKKYRKYVRSRFQCIEDRNARLGESVSLNKRYTRLRLIKEHRSQQE REQELLAIGKTKTCESPVSPIKMELLFDPDDEHSEPVHTVVFQGAAGIGKTILARKMMLD WASGTLYQDRFDYLFYIHCREVSLVTQRSLGDLIMSCCPDPNPPIHKIVRKPSRILFLMD GFDELQGAFDEHIGPLCTDWQKAERGDILLSSLIRKKLLPEASLLITTRPVALEKLQHLL DHPRHVEILGFSEAKRKEYFFKYFSDEAQARAAFSLIQENEVLFTMCFIPLVCWIVCTGL KQQMESGKSLAQTSKTTTAVYVFFLSSLLQPRGGSQEHGLCAHLWGLCSLAADGIWNQKI LFEESDLRNHGLQKADVSAFLRMNLFQKEVDCEKFYSFIHMTFQEFFAAMYYLLEEEKEG RTNVPGSRLKLPSRDVTVLLENYGKFEKGYLIFVVRFLFGLVNQERTSYLEKKLSCKISQ QIRLELLKWIEVKAKAKKLQIQPSQLELFYCLYEMQEEDFVQRAMDYFPKIEINLSTRMD HMVSSFCIENCHRVESLSLGFLHNMPKEEEEEEKEGRHLDMVQCVLPSSSHAACSHGLVN SHLTSSFCRGLFSVLSTSQSLTELDLSDNSLGDPGMRVLCETLQHPGCNIRRLWLGRCGL SHECCFDISLVLSSNQKLVELDLSDNALGDFGIRLLCVGLKHLLCNLKKLWLVSCCLTSA CCQDLASVLSTSHSLTRLYVGENALGDSGVAILCEKAKNPQCNLQKLGLVNSGLTSVCCS ALSSVLSTNQNLTHLYLRGNTLGDKGIKLLCEGLLHPDCKLQVLELDNCNLTSHCCWDLS TLLTSSQSLRKLSLGNNDLGDLGVMMFCEVLKQQSCLLQNLGLSEMYFNYETKSALETLQ EEKPELTVVFEPSW SEQ ID NO:3 >sp|Q8R4B8|NLRP3_MOUSE NACHT, LRR and PYD domains-containing protein 3 OS=Mus musculus OX=10090 GN=Nlrp3 PE=1 SV=1 MTSVRCKLAQYLEDLEDVDLKKFKMHLEDYPPEKGCIPVPRGQMEKADHLDLATLMIDFN GEEKAWAMAVWIFAAINRRDLWEKAKKDQPEWNDTCTSHSSMVCQEDSLEEEWMGLLGYL SRISICKKKKDYCKMYRRHVRSRFYSIKDRNARLGESVDLNSRYTQLQLVKEHPSKQERE HELLTIGRTKMRDSPMSSLKLELLFEPEDGHSEPVHTVVFQGAAGIGKTILARKIMLDWA LGKLFKDKFDYLFFIHCREVSLRTPRSLADLIVSCWPDPNPPVCKILRKPSRILFLMDGF DELQGAFDEHIGEVCTDWQKAVRGDILLSSLIRKKLLPKASLLITTRPVALEKLQHLLDH PRHVEILGFSEAKRKEYFFKYFSNELQAREAFRLIQENEVLFTMCFIPLVCWIVCTGLKQ QMETGKSLAQTSKTTTAVYVFFLSSLLQSRGGIEEHLFSDYLQGLCSLAADGIWNQKILF EECDLRKHGLQKTDVSAFLRMNVFQKEVDCERFYSFSHMTFQEFFAAMYYLLEEEAEGET VRKGPGGCSDLLNRDVKVLLENYGKFEKGYLIFVVRFLFGLVNQERTSYLEKKLSCKISQ QVRLELLKWIEVKAKAKKLQWQPSQLELFYCLYEMQEEDFVQSAMDHFPKIEINLSTRMD HVVSSFCIKNCHRVKTLSLGFFHNSPKEEEEERRGGRPLDQVQCVFPDTHVACSSRLVNC CLTSSFCRGLFSSLSTNRSLTELDLSDNTLGDPGMRVLCEALQHPGCNIQRLWLGRCGLS HQCCFDISSVLSSSQKLVELDLSDNALGDFGIRLLCVGLKHLLCNLQKLWLVSCCLTSAC CQDLALVLSSNHSLTRLYIGENALGDSGVQVLCEKMKDPQCNLQKLGLVNSGLTSICCSA LTSVLKTNQNFTHLYLRSNALGDTGLRLLCEGLLHPDCKLQMLELDNCSLTSHSCWNLST ILTHNHSLRKLNLGNNDLGDLCVVTLCEVLKQQGCLLQSLQLGEMYLNRETKRALEALQE EKPELTIVFEISW As used herein, the term “dominant negative” has its general meaning in the art and refers to a type of polypeptide that interferes with the normal function of another polypeptide of the same or similar sequence. A dominant negative polypeptide usually has a mutation that reduces or abolishes its activity, but retains the ability to bind to the same targets as the wild-type polypeptide. By doing so, it prevents the wild-type polypeptide from performing its function, resulting in a loss-of-function phenotype. As used herein, the expression “derived from” refers to a process whereby a first component (e.g., a first polypeptide), or information from that first component, is used to isolate, derive or make a different second component (e.g., a second polypeptide that is different from the first one). As used herein, the “percent identity” between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology.48 (3): 443–53). The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. In general, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and / or biological modification. According to the invention a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73;74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98;99 or 100% of identity with the second amino acid sequence. As used herein, the term “mutation” has its general meaning in the art and refers to a substitution, deletion or insertion. In particular, the term "substitution" means that a specific amino acid residue at a specific position is removed and another amino acid residue is insertedinto the same position. Within the specification, the mutations are references according to thestandard mutation nomenclature. As used herein, the term “particle” refers to a small object that behaves as a whole unit with respect to its transport and properties i.e. a discrete unit of matter, where the atoms or molecules from which it is formed essentially embody the particle. As used herein, the term "extracellular vesicle" or “EV” has its general meaning in the art and refers to a cell-derived vesicle comprising a membrane that encloses an internal space. Extracellular vesicles comprise all membrane-bound vesicles that have a smaller diameter than the cell from which they are derived. Generally, extracellular vesicles range in diameter from 30nm to 1000nm, and can comprise various macromolecular cargo either within the internal space, displayed on the external surface of the extracellular vesicle, and / or spanning the membrane.As used herein, the terms "isolated" "isolating” "purified" "purifying," "enriched," and"enriching," as used herein with respect to cells, means that the particles at some point in time were separated, purified, and capable of therapeutic use. "Highly purified," "highly enriched,"and "highly isolated," when used with respect to said particles, indicates that the particles areat least about 70%, about 75%, about 80%, about 85% about 90% or more of the particles, about 95%, at least 99% pure, at least 99.5% pure, or at least 99.9% pure or more of the particles, andcan typically be about 95% or more of the particles.As used herein, the term “load” refers to the introduction or insertion of a substance or objectinto or onto a particle of the present invention. As used herein, the term “loading” refers tointroducing or inserting a substance or object into or onto the particle of the present invention.As used herein, the term “functionalized” is used interchangeably with the terms “attached” and “bound”. As used herein, the term "subject", “host”, “individual” or “patient” refers to a mammal,typically a human being, male or female at any age that is in-need of a therapy. As used herein the term "obesity" refers to a condition characterized by an excess of body fat. The operational definition of obesity is based on the Body Mass Index (BMI), which is calculated as body weight per height in squared meter (kg / m²). Obesity refers to a condition whereby an otherwise healthy patient has a BMI greater than or equal to 30 kg / m², or a condition whereby a patient with at least one co-morbidity has a BMI greater than or equal to 27 kg / m². An "obese patient" is an otherwise patient with a BMI greater than or equal to 30 kg / m² or a patient with at least one co-morbidity with a BMI greater than or equal 27 kg / m². A "patient at risk of obesity" is an otherwise healthy patient with a BMI of 25 kg / m² to less than 30 kg / m² or a patient with at least one co-morbidity with a BMI of 25 kg / m² to less than 27 kg / m². Theincreased risks associated with obesity may occur at a lower BMI in people of Asian descent.In Asian and Asian-Pacific countries, including Japan, "obesity" refers to a condition whereby a patient with at least one obesity-induced or obesity-related co-morbidity that requires weight reduction or that would be improved by weight reduction, has a BMI greater than or equal to 25 kg / m². An "obese patient" in these countries refers to a patient with at least one obesity- induced or obesity-related co-morbidity that requires weight reduction or that would be improved by weight reduction, with a BMI greater than or equal to 25 kg / m². In these countries, a "patient at risk of obesity" is a person with a BMI greater than 23 kg / m2to less than 25 kg / m². As used herein, the term "obesity-related disease" encompasses disorders that are associated with, caused by, or result from obesity. As used herein, the term “atrial fibrillation” or “AF” has its general meaning in the art andrefers to a type of arrythmia that occurs more frequently in people who are overweight or obese.Atrial fibrillation is a condition where the upper chambers of the heart (the atria) beat faster and out of sync with the lower chambers of the heart (the ventricles). This can cause blood clots, stroke, heart failure, and other complications. Obesity is a risk factor for developing atrialfibrillation, as it can lead to inflammation, high blood pressure, sleep apnea, and metabolicchanges that affect the electrical activity of the heart. Studies have shown that losing weight can reduce the frequency and severity of atrial fibrillation episodes and improve the quality of life of patients.As used herein, the term "treatment" or "treat" refers to both prophylactic or preventivetreatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]). As used herein, the term “pharmaceutical composition” refers to a composition described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and / or excipients. The pharmaceutical compositions as provided herewith typically include a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Particles of the present invention:The first object of the present invention relates to an isolated particle loaded with i) a dominantnegative polypeptide of NLRP3 or ii) a polynucleotide encoding thereof. Polypeptides: In some embodiments, the particle of the present invention is loaded with a dominant negative polypeptide of NLRP3. In some embodiments, the dominant negative polypeptide of NLRP3 comprises or consists in an amino acid sequence having at least 90% of identity with the amino acid sequence as setforth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.In some embodiments, the dominant negative polypeptide of NLRP3 comprises or consists inan amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 whereinone or more amino acid residues at position 23, 24 or 27 are mutated in SEQ ID NO:1 orwherein one or more amino acid residues at position 21, 22 or 25 are mutated in SEQ ID NO:2or SEQ ID NO:3.In some embodiments, the dominant negative polypeptide of NLRP3 comprises or consists inan amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 whereinthe lysine (K) at position 23 in SEQ ID NO:1 or the lysine (K) at position 21 in SEQ ID NO:2or SEQ ID NO:3 is substituted by an amino acid residue having a negative charged side chain.In some embodiments, the dominant negative polypeptide of NLRP3 comprises or consists inan amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 whereinthe lysine (K) at position 23 in SEQ ID NO:1 is substituted by a glutamic acid residue (E) orwherein the lysine (K) at position 21 in SEQ ID NO:2 or SEQ ID NO:3 is substituted by aglutamic acid residue (E). In some embodiments, the dominant negative polypeptide of NLRP3 comprises or consists inan amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 whereinthe lysine (K) at position 24 in SEQ ID NO:1 is substituted by an amino acid residue having anegative charged side chain or wherein the lysine (K) at position 22 in SEQ ID NO:2 or SEQID NO:3 is substituted by an amino acid residue having a negative charged side chain. In someembodiments, the dominant negative polypeptide of NLRP3 comprises or consists in an aminoacid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 wherein the lysine(K) at position 24 in SEQ ID NO:1 is substituted by a glutamic acid residue (E) or wherein thelysine (K) at position 22 in SEQ ID NO:2 or SEQ ID NO:3 is substituted by a glutamic acidresidue (E). In some embodiments, the dominant negative polypeptide of NLRP3 comprises or consists inan amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 whereinthe methionine (M) at position 27 in SEQ ID NO:1 is substituted by an amino acid residuehaving a negative charged side chain or wherein the methionine (M) at position 25 in SEQ IDNO:2 or SEQ ID NO:3 is substituted by an amino acid residue having a negative charged sidechain. In some embodiments, the dominant negative polypeptide of NLRP3 comprises orconsists in an amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ IDNO:3 wherein the methionine (M) at position 27 in SEQ ID NO:1 is substituted by a glutamicacid residue (E) or wherein the methionine (M) at position 25 in SEQ ID NO:2 or SEQ ID NO:3is substituted by a glutamic acid residue (E). In some embodiments, the dominant negative polypeptide of NLRP3 comprises or consists inan amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 that ischaracterized by one or more substitutions selected from the group consisting of K23E, K24E,and M27E in SEQ ID NO:1 or that is characterized by one or more substitutions selected fromthe group consisting of K21E, K22E, and M25E in SEQ ID NO:2 or SEQ ID NO:3.In some embodiments, the dominant negative polypeptide of NLRP3 comprises or consists inan amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3comprising the K23E, K24E, and M27E substitutions in SEQ ID NO:1 or comprising the K21E,K22E, and M25E substitutions in SEQ ID NO:2 or SEQ ID NO:3.Polynucleotides: In some embodiments, the particle of the present invention is loaded with a polynucleotide that encodes for a dominant negative polypeptide of NLRP3. In some embodiments, the polynucleotide encodes for the dominant negative polypeptide ofNRLP3 that comprises or consists in an amino acid sequence having at least 90% of identitywith the amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.In some embodiments, the polynucleotide encodes for the dominant negative polypeptide ofNLRP3 that comprises or consists in an amino acid sequence as set forth in SEQ ID NO:1, SEQID NO:2 or SEQ ID NO:3 wherein one or more amino acid residues at position 23, 24 or 27 inSEQ ID NO:1 are mutated or wherein one or more amino acid residues at position 21, 22 or 25in SEQ ID NO:2 or SEQ ID NO:3 are mutated.In some embodiments, the polynucleotide encodes for the dominant negative polypeptide of NLRP3 that comprises or consists in an amino acid sequence as set forth in SEQ ID NO:1, SEQID NO:2 or SEQ ID NO:3 wherein the lysine (K) at position 23 in SEQ ID NO:1 is substitutedby an amino acid residue having a negative charged side chain or wherein the lysine (K) atposition 21 in SEQ ID NO:2 or SEQ ID NO:3 is substituted by an amino acid residue having anegative charged side chain. In some embodiments, the polynucleotide encodes for the dominant negative polypeptide of NLRP3 that comprises or consists in an amino acid sequenceas set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 wherein the lysine (K) at position23 in SEQ ID NO:1 is substituted by a glutamic acid residue (E) or wherein the lysine (K) atposition 21 in SEQ ID NO:2 or SEQ ID NO:3 is substituted by a glutamic acid residue (E).In some embodiments, the polynucleotide encodes for the dominant negative polypeptide ofNLRP3 that comprises or consists in an amino acid sequence as set forth in SEQ ID NO:1, SEQID NO:2 or SEQ ID NO:3 wherein the lysine (K) at position 24 in SEQ ID NO:1 is substitutedby an amino acid residue having a negative charged side chain or wherein the lysine (K) atposition 22 in SEQ ID NO:2 or SEQ ID NO:3 is substituted by an amino acid residue having anegative charged side chain. In some embodiments, the polynucleotide encodes for the dominant negative polypeptide of NLRP3 that comprises or consists in an amino acid sequenceas set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 wherein the lysine (K) at position24 in SEQ ID NO:1 is substituted by a glutamic acid residue (E) or wherein the lysine (K) atposition 22 in SEQ ID NO:2 or SEQ ID NO:3 is substituted by a glutamic acid residue (E).In some embodiments, the polynucleotide encodes for the dominant negative polypeptide ofNLRP3 that comprises or consists in an amino acid sequence as set forth in SEQ ID NO:1, SEQID NO:2 or SEQ ID NO:3 wherein the methionine (M) at position 27 in SEQ ID NO:1 issubstituted by an amino acid residue having a negative charged side chain or wherein themethionine (M) at position 25 in SEQ ID NO:2 or SEQ ID NO:3 is substituted by an aminoacid residue having a negative charged side chain. In some embodiments, the polynucleotide encodes for the dominant negative polypeptide of NLRP3 that comprises or consists in an aminoacid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 wherein themethionine (M) at position 27 in SEQ ID NO:1 is substituted by a glutamic acid residue (E) orwherein the methionine (M) at position 25 in SEQ ID NO:2 or SEQ ID NO:3 is substituted bya glutamic acid residue (E). In some embodiments, the polynucleotide encodes for the dominant negative polypeptide ofNLRP3 that comprises or consists in an amino acid sequence as set forth in SEQ ID NO:1, SEQID NO:2 or SEQ ID NO:3 that is characterized by one or more substitutions selected from thegroup consisting of K23E, K24E, and M27E in SEQ ID NO:1 or that is characterized by one ormore substitutions selected from the group consisting of K21E, K22E, and M25E in SEQ IDNO:2 or SEQ ID NO:3.In some embodiments, the polynucleotide encodes for the dominant negative polypeptide ofNLRP3 that comprises or consists in an amino acid sequence as set forth in SEQ ID NO:1, SEQID NO:2 or SEQ ID NO:3 comprising the K23E, K24E, and M27E substitutions in SEQ IDNO:1 or comprising the K21E, K22E, and M25E substitutions in SEQ ID NO:2 or SEQ IDNO:3. In some embodiments, the polynucleotide is an RNA or a DNA molecule. In some embodiments, the polynucleotide of the present invention is a DNA molecule, in particular a DNA plasmid. In some embodiments, the polynucleotide of the present invention is an RNA molecule, in particular a messenger RNA (mRNA). In some embodiments, the polynucleotide comprises a sequence that has been codon optimized for expression in a mammalian cell. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences. A variety of codon optimization methods is known in the art, and include, e.g., methods disclosed in at least U.S. Pat. Nos. 5,786,464 and 6,114,148. In order to confirm the presence of the polynucleotide in the donor cell, a variety of assays may be performed. Such assays include, for example, "molecular biological" assays well known such as Southern and Northern blotting, RT-PCR and quantitative PCR; or "biochemical" assays, such as detecting the presence or absence of a particular peptide. In some embodiments, the polynucleotide of the present invention is operatively linked to oneor more control sequences. As used herein, the term "control sequence'" refers collectively topromoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translatedin an appropriate host cell. In particular, the control sequence is a "promoter" sequence, whichis used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of bindingRNA polymerase and initiating transcription of a downstream (3'-direction) coding sequence.Transcription promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters”. In some embodiments, the polynucleotide of the present invention is operably linked and under the control of a steroidogenic factor 1 (SF1) promoter. As used herein, the term “nuclear receptor steroidogenic factor-1” or “SF1” refers to a transcription factor essential for terminaldifferentiation and maintenance of ventromedial nucleus neuronal (VMH) populations. Thepresence of the said promoter restricts the expression of the polynucleotide of the present invention into SF1 expressing cells or tissues. As used herein, the term “SF1-expressing cell or tissue” refers to cells or tissues that are able to express the SF1 transcription factor.Typically, the SF1 expressing cell is a SF1 expressing neuron located in the VMH region. The hypothalamus is organized in anatomically distinct interconnected hypothalamic nuclei including the arcuate nucleus (ARC), the ventromedial nucleus (VMH), the paraventricular nucleus (PVH), the lateral hypothalamic area (LHA) and the dorsomedial nucleus (DMH) that among others, participate in the regulation of energy metabolism. Thus, as used herein, the term“ventromedial nucleus of the hypothalamus” or “VMH” refers to a hypothalamic region.The VMH region is known for being involved in feeding behaviour and energy expenditure regulation via the brown adipose tissue (BAT) thermogenesis. The VMH is related to othernuclei through axonal projections. In some embodiments, the sequence of the SF1 promotercomprises or consists of the nucleic acid sequence as set forth in SEQ ID NO:4. SEQ ID NO 4: Steroidogenic factor 1 (SF1) polynucleotide sequence. AAAACAAAACAAAACAAAACAAAACAAAACAAAACAAACAAACAAACAAACAAACAAAAACCCTTCTTT CCTACCTGGTCCTAGTACCCACATAGTCCTACCTGAAGTCCCTGAAGCCACACCCTTAGCCCAGCAGTC TTGGCACAACCTCAGTTTCCCCAGCTACCAATGGACCATATCTGCAGCTCCCAGAGAAGCCACCAAAAA GGCCACACAAACCCCACCTGATGGGTTCCACCATGCCATTTCTCCACACTAGCCATTCTGACTCCTCAC TCAGATCTGGGACAAGCTGGACCACGCAGCCCAGGCAAGGACCCAGGGAGGAAGCCATTCAAGGGGAGA AACTCCCAGCCTGGTAAGGGAGCAGGCCATAAATCAGGTCCCACTCCCACCCAGTCGCTAACAAGCCGC TGCCTATCTGCCTACATGGGGTCCCTGCCTCAGGCTCCCTCATCAGCCTGGACAGCCAGCTGGCCAAGG TCTCTCCAGTGCCTTGGCCTCTGCCCCCACCCAGGGCCCCCATAAAGATAGGGATATTTTTTTTTCTTT TAGAAGAGTGAAAAAAGATATAGACCCAAATGAAGAGAAACACCAACAAAGGAGGAGAAAGGCCTGCAG AGTCACGTGGGGGCAGAGACCAATTGGGCCTCCGGTGGCCCCCCCACCCACGAGGGGAGGAGGAAAGGA CGATCGGACAGGGCCAGTTTCCAGTCCGCCGCTGCCCGCCCGCTGCTGGGT Particles: Any particles which have been described in the art for cargo delivery into cells may be used.Such nanoparticles include for example liposomes, micelles, extracellular vesicles,nanospheres, and nanoparticles. In addition to the above, biological particles can also be utilised as particles in accordance with the present invention. Examples of these include viral particles (which normally have a size of 20 nm to 300 nm), virus-like particles (e.g. particles that are composed of only the shell of a viral particle), HDL and LDL nanoparticles (which normally have a size of 5-30 nm), self- assembled nanoparticles, bacterial particles, and extracellular vesicles. In some embodiments, the nanoparticles comprises at least a core with one or more polymers, or their copolymer, such as, e.g., one or more of dextran, carboxymethyl dextran, chitosan, trimetylchitosan, polyvinylalcohol (PVA), polyanhydrides, polyacylates, polymethacrylates, polyacylamides, cellulose, hydromellose, starch, dendrimers, polyamino acids, polyethyleneglycols, polyethyleneglycol-co-propyleneglycol, aliphatic polyesters, including poly(lactic acid (PLA), poly(glycolic acid), and their copolymers including poly(lactic-co- glycolylic)acid (PLGA), or poly(^-caprolactone). Other suitable polymers may comprise polyamino acid selected from the group consisting of poly(g-glutamic acid), poly(a-aspartic acid), poly(e-lysine), poly(a-glutamic acid), poly(a-lysine), poly-asparagine, or derivativesthereof, and mixtures thereof. In general, the surface of the nanoparticles may also befunctionalised or coated to produce a desirable physical characteristic such as solubility, biocompatibility, and for facilitating chemical linkages with other biomolecules, such as the syncytin-1 fusion protein of the present invention. In some embodiments, the surface of the nanoparticles can be functionalized by incorporating one or more chemical linkers such as, without limitation: carboxyl groups, amine groups, carboxyl / amine, hydroxyl groups, polymers such as silane, dextran or PEG or their derivatives.In some embodiments, the particle is a virus particle or a virus-like particle. As it is readilyunderstood by the one skilled in the art, a virus particle that is used according to the invention may be selected in a group comprising Moloney murine leukemia virus-derived vector particles, Bovine immunodeficiency virus-derived particles, Simian immunodeficiency virus-derived vector particles, Feline immunodeficiency virus-derived vector particles, Human immunodeficiency virus-derived vector particles, Equine infection anemia virus-derived vector particles, Caprine arthritis encephalitis virus-derived vector particle, Baboon endogenous virus- derived vector particles, Rabies virus-derived vector particles, Influenza virus-derived vector particles, Norovirus-derived vector particles, Respiratory syncytial virus-derived vector particles, Hepatitis A virus-derived vector particles, Hepatitis B virus-derived vector particles, Hepatitis E virus-derived vector particles, Newcastle disease virus-derived vector particles, Norwalk virus-derived vector particles, Parvovirus-derived vector particles, Papillomavirus- derived vector particles, Yeast retrotransposon-derived vector particles, Measles virus-derivedvector particles, and bacteriophage-derived vector particles. In some embodiments, the virusparticle of the present invention is a retrovirus-derived particle. In some embodiments, the virus particle of the present invention is a lentivirus-derived particle.In some embodiments, the particle is an extracellular vesicle (EV). In some embodiments, theEVs are spherical or round-shaped. Further, the EVs may have a size of greater than 2 nm. TheEVs may have a size of greater than 5 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm,80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm or 160 nm. The EVs mayhave a range of size distribution, such as between 30 nm to 50 nm, 30 nm to 100 nm, typically 30 nm to 150 nm or 30 nm to 200 nm. The size distribution may be determined by various means. In principle, the size may be determined by size fractionation and filtration through amembrane with the relevant size cut-off. The EVs size may then be determined by trackingsegregation of component proteins with SDS-PAGE or by a biological assay. The size may also be determined by electron microscopy or by Nanoparticle tracking analysis (NTA). In some embodiments, the EVs comprise markers derived from the cells used to produced them (hereinafter referred to as “donor cell”). Donor cells include without limitation epithelial cells, circulating immune cells, hematopoietic cells, bone marrow cells, circulating vascular progenitor cells, cardiac cells, chondrocytes, bone cells, beta cells, hepatocytes, and neurons… Typically, the donor cells are mammalian cells. In some embodiments, the donor cells include purified primary cells and immortalized cell lines. In some embodiments, the donor cells are cells in suspension (e.g. circulating leukocytes (PBMC)), or adherent cells (e.g. endothelial cells). In some embodiments, the donor cells include pluripotent stem cells. As intended herein, the expression "pluripotent stem cells" relates to division-competent cells which are liable to differentiate in one or more cell types. Pluripotent stem cells encompass stem cells, in particular adult stem cells (e.g. mesenchymal stem cells (MSC)) and embryonic stem cells. The term also encompasses induced pluripotent stem cells (IPS). In some embodiments, the donor cell is a mesenchymal stem cell. As used, herein, the term "mesenchymal stem cell" or "MSC" has its general meaning in the art and refers to multipotent stromal cells that can differentiate into a variety of cell types, including: osteoblasts (bone cells), chondrocytes (cartilage cells),myocytes (muscle cells) and adipocytes (fat cells) (See for example Wang, Stem Cells 2004;22(7);1330-7; McElreavey; 1991 Biochem Soc Trans (1);29s; Takechi, Placenta 1993 March / April; 14 (2); 235-45; Takechi, 1993; Kobayashi; Early Human Development; 1998; Jul. 10; 51 (3); 223-33; Yen; Stem Cells; 2005; 23 (1) 3-9). In some embodiments, the donor cells are immune cells, more typically antigen presenting cells,even more typically dendritic cells. In some embodiments, the donor cell is a cell with lowimmunogenicity, typically an immature immune cell, typically an immature antigen presenting cell, most typically an immature dendritic cell or monocyte. By “immature” is referred herein as a cell that is not activated or biologically active, or that does not present activation markers or molecule in the surface. An immature dendritic cell has different morphological phenotype than the mature one. Immature dendritic cells have a round and smooth surface, while mature cells, such as mature dendritic cells, have a rough surface with multiple pseudopodia. Immature dendritic cells produce large quantities of EVs devoid of T-cell activators such as majorhistocompatibility complex II (MHC-II), cluster of differentiation 80 (CD80) and cluster ofdifferentiation 86 (CD86). Thus, in some embodiments, the donor cell, typically the immature dendritic cell, does not express activation markers, typically T-cell activator markers or molecules such as MHC-II, CD80 or CD86, or a combination thereof. In some embodiments,the donor cell is an immature immortalized cell, typically an immature immortalized dendriticcell or monocyte, most typically the JAWS II cell line from the American Type Culture Collection CRL-1194; ATCC; Manassas, VA, USA or a cell derived from said JAWS II cellline. In some embodiments, the donor cell is an immature antigen presenting cell characterizedby having a statistically significant reduced expression of at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of at least one T-cell activator molecule in comparison to the expression of said T-cell activator molecule in a mature antigen presenting cell. The percentage of reduction of said T-cell activator molecule can be measured by techniques known in the art, e.g. by flow cytometry or RT-PCR. Typically, the donor cells are grown in a conditioned media, and the EVs are produced and released to said media. In the context of the present invention, the term “conditioned media” is understood as the supernatant of cell cultures. Since the population of EVs is released into the extracellular media when the intracellular multivesicular bodies fuse with the plasma membrane of the donor cell, the EV outer membrane would be similar as the one of the donor cells. Thus, in some embodiments, the population of EVs are positive for cellular markers, typically cellular immature dendritic cell (iDC) markers. In some embodiments, the EVs comprise one or more of the specific exosome markers selected from the groups consisting of ALIX, TSG101, CD9 or CD81, or any combination thereof. In some embodiments, the EVs are characterized by lacking the MHC-II, CD80, CD86 or GRP94 markers, or any combination thereof. In some embodiments, the EVs have an immature phenotype, so that they are low immunogenic.In some embodiments, the EVs of the present invention are prepared from a donor that has beengenetically engineered to express the components of the EVs and optionally the cargo(s) ofinterest (e.g. the NRLP3 dominant negative polypeptide or the polynucleotide). Typically, thedonor cell is transduced in order to express one or more polynucleotides that encode for the different components of the EVs. It is contemplated that the polynucleotide construct can be introduced into the donor cells as naked DNA or in a suitable vector. Naked DNA generally refers to the DNA contained in a plasmid expression vector in the proper orientation for expression. Physical methods for introducing a polynucleotide construct into a donor cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Other means can be used including colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. In some embodiments, the polynucleotide construct is introduced into the donor cell by a viral vector that is an adeno-associated virus (AAV), a retrovirus, lentivirus, bovine papilloma virus, an adenovirus vector, a vaccinia virus, a polyoma virus, or an infective virus. In some embodiments, the EVs of the present invention are prepared by any method well knownin the art. In some embodiments, the EVs of the present invention are prepared by methods for3D culture that are well known in the art, and include, but are not limited to standard culture in 2D flasks, hanging drop culture, culturing on matrices, culturing on microcarriers, culturing on synthetic extracellular scaffolds, culturing on chitosan membranes, culturing under magnetic levitation, suspension culture in rotating bioreactors, or culturing under non-contact inhibition conditions. See, e.g., Haycock J W. (2011). “3D cell culture: a review of current approaches and techniques.”. Methods Mol Biol. 695: 1-15; Lee, J; Cuddihy M J, Kotov N A. (14 Mar. 2008). Three-dimensional cell culture matrices: state of the art. doi:10.1089 / teb.2007.0150; Pampaloni, Francesco (October 2007). “The third dimension bridges the gap between cell culture and live tissue”. Nature Reviews 8: 839-845; and Souza, Glauco (14 Mar. 2010). “Three-dimensional tissue culture based on magnetic cell levitation”. Nature Nanotechnology:291-296; the entire content of each is hereby incorporated by reference.In some embodiments, the EVs of the present invention are prepared by the method as described in the EXAMPLE.The techniques used to load EVs with various cargo include free-thaw cycles to fuse EVs andliposomes, sonication, extrusion, permeabilization with saponin, and electroporation. Severalcommercial kits are available for loading nucleic acids into EVs and the skilled person wouldbe familiar with them. Typically, the at least one polynucleotide, typically a plasmid, is mainlylocated in the core of the EVs.In some embodiments, the particle of the present invention, and in particular the EVs of thepresent invention, is functionalized with one or more glycoprotein(s) for allowing the (specific)fusion of the particles to the targeted cells of and / or allowing the crossing of barriers such asblood brain barrier. In particular, the glycoprotein gains entrance of the particle into neuronalcells and brain tissues. In some embodiments, the particle of the present invention, and inparticular the EVs of the present invention, is functionalized with one or more viralglycoprotein(s). In some embodiments, the viral glycoprotein is VSV-G or a fragment or variantthereof. In some embodiments, the viral glycoprotein is CNV-G, or a fragment or variantthereof, from neurotropic Chandipura vesiculovirus that shares many properties with VSV-G.In some embodiments the glycoprotein is selected from the viral glycoproteins such as those from Carajas virus (Cara-G), Cocal virus (Coca-G), Mar aba virus (Mara-G), Rabies virus (RabV-G), Ebola virus (MOKV-G), Chikungunya virus (E1E2E3), Nipah virus (NiF and NiG),SARS-CoV2 (Spike S), and MERS or SARS coronavirus (Spike MS). In some embodiments,the particle of the present invention, and in particular the EV of the present invention isfunctionalized with one or more endogenous human fusiogenic proteins such as Syncytin 1 (ERVW-1), Synctin 2 (ERVFRD-1) or HERV-K env (HERV-K-G) that share many structural elements with class I retroviral glycoproteins. In some embodiments, the particle of the presentinvention, and in particular the EVs of the present invention, is functionalized with a fusionprotein comprising or consisting of the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b (Lamp2b), wherein said fusion protein comprises, consists or consists essentially of an amino acid sequence with at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:5. SEQ ID NO 5: Fusion protein: Neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b (Lamp2b). Note: (Notunderlined: Lamp2b sequence)Underlined: RVG sequence) MCLSPVKGAKLILIFLFLGAVQSNALIVNLTDSKGTCLYARYTIWMPENPRPGTPCDIFTNSRGKRASN GSGGAEWEMNFTITYETTNQTNKTITIAVPDKATHDGSSCGDDRNSAKIMIQFGFAVSWAVNFTKEASH YSIHDIVLSYNTSDSTVFPGAVAKGVHTVKNPENFKVPLDVIFKCNSVLTYNLTPVVQKYWGIHLQAFV QNGTVSKNEQVCEEDQTPTTVAPIIHTTAPSTTTTLTPTSTPTPTPTPTPTVGNYSIRNGNTTCLLATM GLQLNITEEKVPFIFNINPATTNFTGSCQPQSAQLRLNNSQIKYLDFIFAVKNEKRFYLKEVNVYMYLA NGSAFNISNKNLSFWDAPLGSSYMCNKEQVLSVSRAFQINTFNLKVQPFNVTKGQYSTAQECSLDDDTI LIPIIVGAGLSGLIIVIVIAYLIGRRKTYAGYQTL Specific embodiments:In some embodiments, the particle of the present invention relates to an isolated EV that isloaded with a DNA polynucleotide that encodes the amino acid sequence as set forth in SEQID NO: 1, 2 or 3 that comprises that comprises K23E, K24E, and M27E substitutions in SEQID NO:1 or K21E, K22E, and M25E substitutions in SEQ ID NO:2 or SEQ ID NO:3 wherein said polynucleotide is operably linked and under the control of the SF-1 promoter that consistsof the nucleic acid sequence as set forth in SEQ ID NO:4 and the EV is functionalized with afusion protein that consists of the amino acid sequence as set forth in SEQ ID NO:5.Therapeutic uses: A further object of the present invention relates to the use of a particle as a drug. A further object of the present invention relates to a method of therapy in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a particle of the present invention. A further object of the present invention relates to a method of treating of an obesity related disease comprising administering to the patient a therapeutically effective amount of a particle of the present invention. In some embodiments, the patient can be human or any other animal (e.g., birds and mammals) (e.g., domestic animals such as cats and dogs; livestock and farm animals such as horses, cows, pigs, chickens, etc.). Typically said patient is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, and mouse) and a primate (e.g., a monkey, chimpanzee, and a human). In some embodiments, the patient is a non-human animal. In some embodiments, the patient is a farm animal or pet. In some embodiments, the patient isa human. More particularly, the patient is an obese patient. In some embodiments, the patientis an elderly patient. As used herein, the term "elderly patient" refers to an adult patient sixty- five years of age or older. Examples of obesity-related disorders include overeating and bulimia, diabetes, hypertension, elevated plasma insulin concentrations and insulin resistance, dyslipidemia, hyperlipidemia, abnormal heart rhythms and arrhythmias, myocardial infarction, congestive heart failure, coronary heart disease, angina pectoris, cerebral infarction, cerebral thrombosis and transient ischemic attack, and osteoarthritis. Other examples include pathological conditions showing reduced metabolic activity or a decrease in resting energy expenditure as a percentage of total fat-free mass. Further examples of obesity-related disorders include metabolic syndrome, also known as syndrome X, insulin resistance syndrome, type II diabetes, impaired fasting glucose, impaired glucose tolerance, inflammation, such as systemic inflammation of the vasculature, atherosclerosis, hypercholesterolemia, hyperuricaemia, as well as secondary outcomes of obesity such as left ventricular hypertrophy. Obesity-related disorders also include the liver abnormalities associated with obesity such as non-alcoholic fatty liver disease (NAFLD) a rising cause of cirrhosis associated to obesity and metabolic syndrome. coronary artery disease in humans.In some embodiment, the method of the present invention is particularly suitable for thetreatment of irregular heartbeat disorders associated with obesity that include, but are not limited to atrial and ventricular arrhythmia, atrial and ventricular fibrillation, atrial and ventricular tachyarrhythmia, atrial and ventricular tachycardia, catecholaminergic polymorphic ventricular tachycardia (CPVT), and exercise-induced variants thereof. In some embodiments, the method of the present invention is particularly suitable for thetreatment of atrial fibrillation associated with obesity.The particle of the invention may be administered to the patient at therapeutically effective doses to provide the therapeutic effects. These doses can be given once or repeatedly, such as daily, every other day, weekly, biweekly, or monthly. In some embodiments, the composition may be administered to a subject in one dose, or in two doses, or in three doses, or in four doses, or in five doses, or in six doses or more. The interval between dosages may be determined based the practitioner's determination that there is a need thereof. Administration of the particles to the patient can be by any means known in the art. Exemplary modes of administration include rectal, transmucosal, topical, transdermal, inhalation, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, and intraarticular) administration, and the like, as well as direct tissue or organ injection, alternatively, intrathecal, direct intramuscular, intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Compositions: A further object of the present invention relates to a composition that comprises an amount ofthe particles of the present invention. Compositions as described herein encompasspharmaceutical compositions that are used for the purpose of performing a method of therapyin a patient in need thereof. The particles may be formulated in a conventional manner usingone or more physiologically acceptable carriers or excipients. The particles may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The particle compositions may take such forms assuspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulation agents such as suspending, stabilizing and / or dispersing agents. Liquid preparations of theparticle compositions may be prepared by conventional means with pharmaceuticallyacceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts. Alternatively, the compositions may be in powder form for constitution with asuitable vehicle, e.g., sterile pyrogen-free water, before use. The compositions may, if desired,be presented in a pack or dispenser device that may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. In some embodiments, the composition may be in liquid or solid (e.g. lyophilized) form. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES: Figure 1. Effect of systemic treatment with SF1-NLRP3-DN-loaded sEVs on energybalance in C57BL / 6 mice fed with high-fat diet. A-E, Body weight change (g) (A, P=0.1848),percentage (%) (B), food intake (g) (C, P=0.5946), daily food intake (g) (D) and cumulative food intake (g) (E) of mice after intravenous injection with control (non-loaded; n = 12 mice) or SF1-NLRP3-DN-loaded (n = 12 mice) sEVs every 3 days for 28 days. F-H, Correlation analysis between body weight change (%) and daily food intake (g) (F), energy expenditure(kcal / h / kg) (G), and energy efficiency (g / kcal) (H). I-J, Blood glucose at day 0 (I) and at day28 (J). Data are expressed as mean ± SEM. Statistical significance was assessed by a Friedman test (A, C, I-J), Mann-Whitney test (B, D-E, G-H), and Pearson correlation coefficient (F). Figure 2. Effect of systemic treatment with SF1-NLRP3-DN-loaded sEVs on BATthermogenesis in C57BL / 6 mice fed with high-fat diet. A-C, Representative thermographicimages (scale bars, 2 cm) (A), quantification (°C) (B), and delta average (%) (C) of interscapular BAT temperature quantification of mice after intravenous injection with control (non-loaded; n = 12 mice) or SF1-NLRP3-DN-loaded (n = 12 mice) sEVs every 3 days for 28 days. D, Correlation analyses between body weight change (%) and average BAT temperature (°C). Data are expressed as mean ± SEM. Statistical significance was assessed by a Friedman test (B), Mann-Whitney test (C) and Pearson correlation coefficient (D). Figure 3. Effect of systemic treatment with SF1-NLRP3-DN-loaded sEVs on atrialfibrillation in C57BL / 6 mice fed with high-fat diet. A-B, Representative surfaceelectrocardiograms (ECG) (A) and percentage of pacing-induced arrhythmias (%) (B, P=0.5148) at day 28 of mice after intravenous injection with control (non-loaded; n = 7 mice) or SF1-NLRP3-DN-loaded (n = 7 mice) sEVs every 3 days for 28 days. C-E, P wave duration (ms) (C), PR interval (ms) (D) and QT duration (ms) (E) at day 28. Data are expressed as mean ± SEM. Statistical significance was assessed by a Chi-2 square test (B) and Mann-Whitney test (C-E).Figure 4. Effect of systemic treatment with SF1-NLRP3-DN-loaded sEVs on energybalance in ApoE KO mice fed with high-fat diet. A-E, Body weight change (g) (A,P=0.0612), percentage (%) (B), food intake (g) (C, P=0.8338), daily food intake (g) (D) and cumulative food intake (g) (E) of mice after intravenous injection with control (non-loaded; n = 29 mice) or SF1-NLRP3-DN-loaded (n = 30 mice) sEVs every 3 days for 28 days. F-H, Correlation analysis between body weight change (%) and daily food intake (g) (F), energy expenditure (kcal / h / kg) (G) and energy efficiency (g / kcal) (H) of mice after intravenous injection with control (non-loaded; n = 29 mice) or SF1-NLRP3-DN-loaded (n = 30 mice) sEVs every 3 days for 28 days. I-K, Blood glucose before (mg / dl) (I) and after treatment (mg / dl) (J), and area under the curve (K) after intravenous injection of mice with control (non-loaded; n = 18 mice) or SF1-NLRP3-DN-loaded (n = 18 mice) sEVs. Data are expressed as mean ± SEM. Statistical significance was assessed by a Friedman test (A, C, I-J), Mann-Whitney test (B, D- E, G-H, K), and Pearson correlation coefficient (F). Figure 5. Effect of systemic treatment with SF1-NLRP3-DN-loaded sEVs on BAT thermogenesis in ApoE KO mice fed with high-fat diet. A-C, Representative thermographic images (scale bars, 2 cm) (A), quantification (°C) (B), and delta average (%) (C) of interscapular BAT temperature quantification of mice after intravenous injection with control (non-loaded; n = 25 mice) or SF1-NLRP3-DN-loaded (n = 23 mice) sEVs every 3 days for 28 days. D, Correlation analyses between body weight change (%) and BAT temperature (°C) (D). Data are expressed as mean ± SEM. Statistical significance was assessed by a Friedman test (B), Mann-Whitney test (C) and Pearson correlation coefficient (D).Figure 6. Effect of systemic treatment with SF1-NLRP3-DN-loaded sEVs on atrialfibrillation in ApoE KO mice. A-B, Representative surface electrocardiograms (ECG) (A) and percentage of pacing-induced arrhythmias (%) (B, P = 0.0009) at day 28 of mice without any injection (n = 3 mice Standard Diet, SD) or after intravenous injection with control (non- loaded; n = 7 mice SD; n = 18 mice High-Fat Diet, HFD), or SF1-NLRP3-DN-loaded (n = 22mice HFD) sEVs every 3 days for 28 days. C-E, P wave duration (ms) (C), PR interval (ms)(D) and QT duration (ms) (E) at day 28 of mice without any injection (n = 4 mice SD) or after intravenous injection with control (non-loaded; n = 7 mice SD; n = 18 mice HFD), or SF1-NLRP3-DN-loaded (n = 21 mice HFD) sEVs every 3 days for 28 days. Data are expressed asmean ± SEM. Statistical significance was assessed by a Chi-2 square (B) and Kruskal-Wallis test (C-E). Figure 7. Effect of systemic treatment with SF1-NLRP3-DN-loaded sEVs on heart ratevariability during the activity phase in ApoE KO mice fed with a high-fat diet. A-B,Representative surface electrocardiograms recorded by telemetric approach (ECG) (A) and incidence of spontaneous arrhythmias (%) (B) at day 0 and at day 28 of mice after intravenous injection with control (non-loaded; n = 8 mice) or SF1-NLRP3-DN-loaded (n = 8 mice) sEVsevery 3 days for 28 days. C-F, Heart rate (bpm) (C), standard deviation of NN intervals (SDNN,ms) (D), root mean square of successive differences (RMSSD, ms) (E), percentage of differences of more than 6 ms between successive R-R intervals (pNN6, %) (F) during theactivity phase at day 0 and at day 28 of mice. G-I, Normalized Low Frequencies (LF) (n.u.)(G), normalized High Frequencies (HF) (n.u.) (H), LF / HF ratio (I) during the activity phase atday 0 and at day 28 of mice. J-M, Heart rate (bpm) (J), standard deviation of NN intervals(SDNN, ms) (K), root mean square of successive differences (RMSSD, ms) (L), percentage of differences of more than 6 ms between successive R-R intervals (pNN6, %) (M) during theresting phase at day 0 and at day 28. N-P, Normalized Low Frequencies (LF) (n.u.) (N),normalized High Frequencies (HF) (n.u.) (O), LF / HF ratio (P) during the resting phase at day 0 and at day 28. Data are expressed as mean ± SEM. Statistical significance was assessed by a Friedman test. Figure 8. Effect of systemic treatment with SF1-NLRP3-DN-loaded sEVs on bloodpressure in ApoE KO mice feed with a high-fat diet. A–C, Systolic blood pressure (SPB,mmHg) (A), diastolic blood pressure (DBP, mmHg) (B) and heart rate (bpm) (C) at day 28 of mice after intravenous injection with control (non-loaded; n = 4 mice, green) or SF1-NLRP3-DN-loaded (n = 7 mice, blue) sEVs every 3 days for 28 days. Data are expressed as mean ±SEM. Statistical significance was assessed by a Mann-Whitney test. Figure 9. Effect of systemic treatment with SF1-NLRP3-DN-loaded sEVs onechocardiographic analysis in ApoE KO mice with a high-fat diet. A-D, Heart rate (bpm)(A), diameter in systole (s, mm) (B), volume in systole (s, µl) (C) and left ventricle mass (LV mass, mg) (D) at day 28 of mice after intravenous injection with control (non-loaded; n = 7mice) or SF1-NLRP3-DN-loaded (n = 11 mice) sEVs every 3 days for 28 days. E-H, Strokevolume (µl) (E), ejection fraction (%) (F), fractional shortening (%) (G) and cardiac output (ml / min) (H) at day 28 of mice. I-L, Left ventricle anterior wall in systole (LVAW;s, mm) (I), in diastole (LVAW;d , mm) (J), left ventricle posterior wall in systole (LVPW;s, mm) (K) and in diastole (LVPW;d , mm) (L) at day 28. Data are expressed as mean ± SEM. Statistical significance was assessed by a Mann-Whitney test. Figure 10. Effect of systemic treatment with SF1-NLRP3-WT-loaded sEVs on energybalance in NLRP3 KO mice fed with high-fat diet. A-F, Body weight (g) (A), body weightchange (g) (B), percentage (%) (C), food intake (g) (D), daily food intake (g) (E) and cumulative food intake (g) (F) of mice after intravenous injection with control (non-loaded; n = 8 mice) or SF1-NLRP3-WT-loaded (n = 8 mice) sEVs every 3 days for 28 days. G-I, Correlation analysis between body weight change (%) and daily food intake (g) (G), energy expenditure (kcal / h / kg) (H) and energy efficiency (g / kcal) (I) after 28 days. Data are expressed as mean ± SEM. Statistical significance was assessed by a Friedman test (A-B, D), Mann-Whitney test (C, E-F, H-I), and Pearson correlation coefficient (G). Figure 11. Effect of systemic treatment with SF1-NLRP3-WT-loaded sEVs on BAT thermogenesis in NLRP3 KO mice fed with high-fat diet. A-C, Representative thermographic images (scale bars, 2 cm) (A), quantification (°C) (B), and delta average (%) (C) of BAT interscapular temperature quantification of mice after intravenous injection with control (non-loaded; n = 8 mice) or SF1-NLRP3-WT-loaded (n = 8 mice) sEVs every 3 days for 28 days. Data are expressed as mean ± SEM. Statistical significance was assessed by a Friedman test (B) and Mann-Whitney test (C). Figure 12. Effect of systemic treatment with SF1-NLRP3-WT-loaded sEVs on atrialfibrillation in NLRP3 KO mice. A-B, Representative surface electrocardiograms (ECG) (A)and percentage of induced arrhythmias (%) (B, P = 0.0009) at day 28 of mice non-injected (n = 6) and after intravenous injection with control (non-loaded; n = 8 mice) or SF1-NLRP3-WT- loaded (n = 9 mice HFD) sEVs every 3 day for 28 days. C-F, Sinus cycle length (ms) (C), P wave duration (ms) (D), PR duration (ms) (E) and QT duration (ms) (F) at day 28. G-I, Sinus node recovery time (SNRT) at 120ms (G), 100ms (H) and 80ms (I) at day 28. Data are expressed as mean ± SEM. Statistical significance was assessed by a Chi-2 square (B) and Kruskal-Wallis test (C-I). Figure 13. Effect of systemic treatment with SF1-NLRP3-WT-loaded sEVs on heart ratevariability in NLRP3 KO mice fed with a high-fat diet. A-B, Representative surfaceelectrocardiograms (ECG) (A) and incidence of spontaneous arrhythmias (%) (B) at day 0 and at day 28 of mice after intravenous injection with control (non-loaded; n = 7 mice) or SF1- NLRP3-WT-loaded (n = 7 mice) sEVs every 3 day for 28 days. C-E, Standard deviation of NN intervals (SDNN, ms) (C), root mean square of successive differences (RMSSD, ms) (D), percentage of differences of more than 6 ms between successive R-R intervals (pNN6, %) (E) during the activity phase at day 0 and at day 28 of mice after intravenous injection with control (non-loaded; n = 8 mice) or SF1-NLRP3-DN-loaded (n = 8 mice) sEVs every 3 day for 28 days. F-I, Normalized Low Frequencies (LF) (n.u.) (F), normalized High Frequencies (HF) (n.u.)(G), LF / HF ratio (H) and heart rate (bpm) (I) during the activity phase at day 0 and at day 28of mice. J-L, Standard deviation of NN intervals (SDNN, ms) (J), root mean square of successive differences (RMSSD, ms) (K), percentage of differences of more than 6 ms between successive R-R intervals (pNN6, %) (L) during the resting phase at day 0 and at day 28. M-P, Normalized Low Frequencies (LF) (n.u.) (M), normalized High Frequencies (HF) (n.u.) (N), LF / HF ratio (O) and heart rate (bpm) (P) during the resting phase at day 0 and at day 28. Data are expressed as mean ± SEM. Statistical significance was assessed by a Chi-2 square (B) and a Friedman test (C-P). EXAMPLE: Methods:Animals. Adult (8–10 weeks) male ApoE knockout (Charles River, Wilmington, MA), NLRP3knockout or C57 / Bl6 mice were housed at an ambient temperature of 23±2°C under a 12 hourslight / 12 hours dark cycle. Animals were allowed to free access to water and a standard chow diet or high-fat diet (HFD, 60% kcal from fat with 0.2% cholesterol (D12492; Research Diets, New Brunswick, NJ) for 16 weeks (i.e. 12 weeks HFD regimen and 4 weeks of smallextracellular vesicles (sEV) treatment). All procedures followed the European ParliamentDirective 2010 / 63 / EU on the protection of animals and were approved by the institutional Ethics Committee for Animal Experiments, Languedoc Roussillon, France (Project No. Apafis# 35413-2022020917375444v5 and Apafis# 38287-2022081215103789v4).sEV production. To convey neuronal targeting capacities to sEVs and mitigate host immuneresponses, we employed immature dendritic cells which were genetically modified to express a fusion protein of lysosome-associated membrane protein 2b (Lamp2b); a protein highly expressed in sEV membranes; fused to a specific glycoprotein derived from the neurotrophic rabies virus (RVG). This RVG facilitates blood-brain-barrier (BBB) penetration via its interaction with the nicotinic acetylcholine receptor (nAChR). sEVs were deeply characterized using transmission electron microscopy, nanoparticle tracking analysis (NTA), and western blot for membrane protein marker expression, as shown previously8. Additionally, the sEVs were loaded with a plasmid encoding a dominant-negative mutant of NLRP3 under the control of the SF1 promoter (SF1-NLRP3-DN), or with a plasmid encoding a wild-type of NLRP3 under decontrol of the SF1 promoter (SF1-NLRP3-WT), to restrict their action to SF1 cells in the VMH.sEV loading. sEVs (100 μg) were incubated with 10 μl of Exo-Fect solution (SystemBiosciences, Palo Alto, CA), 5 μg of plasmid (SF1-NLRP3-DN or SF1-NLRP3-WT) and 70 μlof PBS for 10 min at 37 °C. Then, after adding 30 μl of Exo-Quick solution (System Biosciences), the mixture was placed at 4 °C for 30 minutes. After, the samples werecentrifugated for 3 minutes at 21,000 g to pellet the sEVs before being resuspended in 0.9%NaCl.Systemic treatment with sEVs. One hundred μg of non-loaded, SF1-NLRP3-DN or SF1-NLRP3-WT loaded sEVs were injected in the tail vein of the mice after 12 weeks of diet, every 3 days for 28 days. Body weight and food intake were daily measured.Temperature measurements. Skin temperature surrounding BAT were recorded (days 0 and28) with an infrared camera (C3-X: compact infrared thermal-imaging camera, FLIR; West Malling, Kent, UK) and analyzed with a specific software package (FLIR-Tools Software, FLIR; West Malling). Glucose tolerance test (GTT). Glycemia was measured with a glucometer (Freestyle Optium Neo, Abbott, Oxon, UK), after an intraperitoneal injection of 2g / kg of glucose (B. Braun,Melsungen, Germany). Animals were fasted 6 hours before the procedure.Echocardiography (ECG). Cardiac function was evaluated in vivo by conventionalechocardiography under general anesthesia (2% isoflurane in O2, body temperature maintainedat 37°C, and 450–600 / minutes heart rate). Transthoracic echocardiography (Vevo3100;VisualSonics, Toronto, Canada) was performed. TM and 2D modes were used with long and short axis parasternal views. Image analysis was performed using VisualSonics Vevo3100 software.Telemetry. Mice were placed under general anesthesia (2% isoflurane in O2) for subcutaneousimplantation of a telemetric device (TA10ETA-F10, Data Sciences International, Saint Paul, MN) in the animal's back, with two subcutaneous electrodes (lead II ECG). Then, mice were placed in a room with regulated temperature (23±2°C) and under a 12 hours light / 12 hours darkcycle to conserve circadian rhythm during the 24 hours continuous record of ECG. ECG recordswere performed at day 0 and day 28. Analysis of heart rate variability (HRV) was performedon Ponemah® software (Ponemah system v6.0, Data Sciences International, Saint Paul, MN) andaccording to previous work (Thireau J, Zhang BL, Poisson D, Babuty D. Heart rate variability in mice: a theoretical and practical guide. Exp Physiol.2008 Jan;93(1):83-94.). Heart rate variability. Heart rate variability parameters were studied on different analysis domains. First, temporal domain was studied with SDNN (Standard Deviation of all Normal- to-Normal (N-N) Intervals), which corresponds to global variability, and RMSSD (Root MeanSquare of Successive N-N intervals Differences) and pNN6 (percentage of mean number ofsuccessive N-N intervals exceeding 6 ms), which are two short term variability parameters and reflected parasympathetic activity. Then, with fast Fourier transform, frequential domain was studied, with low frequencies analysis (LF range: 0.4–1.5 Hz) that reflect sympathetic and baroreflex activities; high frequencies (HF range: 1.5–5 Hz), that reflect parasympathetic activity; LF / HF ratio, which is a measurement of interaction between sympathetic nervous system (LF) and parasympathetic nervous system (HF), and that reflects sympatho-vagal balance9, 10.Atrial Electrophysiologic exploration. Electrophysiological study was performed by closedchest transesophageal approach on mice under inhalation anesthesia (2.5% isoflurane in O2), using a Powerlab acquisition system coupled to LabChart software (ADInstruments, Dunedlin, New Zealand), as previously described11. Briefly, surface ECGs were recorded using micro- needle electrode in a Lead-II Einthoven derivation. Normal sinus cycle length (RR, ms), P- wave duration, as the PR and QT intervals were measured. Programmed electrical stimulations using transesophageal atrial pacing were performed with a specialized octopolar catheters (EPR-800, 1.1 French, 8 circular electrodes, 1.0 mm inter-electrode distance) (Millar) with an STG2004 stimulator with the MC-Stimulus II controller (MultiChannel Systems, Reutlinger, Germany). Atrial fibrillation was defined by transesophageal ECG criteria including rapid andfragmented P waves, irregular AV-nodal conduction, and ventricular rhythm persisting for atleast one second. All along the protocol, body temperature was kept constant at 37 °C and spontaneous respiration was monitored.Blood pressure measurement. Non-invasive blood pressure (systolic, diastolic, and mean) andheart rate were measured using tail-cuff method with BP-2000 blood pressure analysis system (Bioseb, Vitrolles, France). Mice were trained once a day for 7 days to get used to the device prior the beginning of experimental protocol. Ten successive measurements were recorded and averaged.Sample processing. Mice were killed by cervical dislocation. After decapitation, blood wasrapidly recovered and processed to obtain serum / plasma, and tissues were immediately homogenized on ice. All samples were stored at −80 °C.Statistical analysis. Data are expressed as mean ± SEM; some data are expressed as apercentage of the appropriate controls. Statistical significance was determined by the testsindicated in the Figure Legends. P < 0.05 was considered significant. Data analysis wasperformed using Prism Software (GraphPad 9.5.1, Boston, MA).Results: The aim of this study is to develop a new strategy for the treatment or prevention of atherosclerotic cardiovascular events associated with obesity and resultant atrial fibrillation by reducing the activity of hypothalamic NLRP3 in SF1 neurons. To this end, we used sEVs ascarriers of plasmids encoding a dominant-negative NLRP3 mutant (NLRP3-DN) or wild-typeNLRP3 (NLRP3-WT). sEVs are derived from multivesicular bodies and contain proteins, lipids and genetic information capable of modifying the phenotype and function of target cells, enabling them to play a crucial role in physiology and pathophysiology. To confer specificity to the expression of this NLRP3-DN mutant or NLRP3-WT only in hypothalamic SF1- expressing neurons, the SF1 promoter was used to drive its expression. In particular, to avoid any invasive cranial surgery / procedure, the aim of this study was also to specifically target NLRP3 within hypothalamic SF1 neurons using systemic delivery routes, making it affordable for potential therapeutic use. The present invention thus provides systemically administered SF1-NLRP3-DN-loaded sEVs for use in the treatment or prevention of atherosclerotic cardiovascular events associated with obesity and resultant atrial fibrillation. Immature dendritic cells were used to generate large quantities of sEVs and to avoid any systemic inflammatory response following their administration. To confer neuronal targeting capabilities to the generated sEVs, immature dendritic cells were genetically engineered to transiently express a fusion protein consisting of (i) lysosome-associated membrane protein 2b (Lamp2b), a protein highly expressed in sEV membranes, fused to (ii) a specific rabies neurotrophic virus (RVG) glycoprotein, which enables blood-brain barrier (BBB) crossing by binding to the nicotinic acetylcholine receptor (nAChR).We used three experimental models of obesity, C57BL / 6 ApoE knockout and NLRP3 knockoutmice fed a high-fat diet. In C57BL / 6 mice, intravenous administration of SF1-NLRP3-DN-loaded sEVs did not significantly reduce body weight, food intake, glucose tolerance or BAT temperature (Figures1A,B,C,D,E,F,G,H,I,J and 2A,B,C,D), but it decreased P wave duration without affecting thepercentage of induced arrythmias (Figure 3A,B,C,D,E). Intravenous administration of SF1-NLRP3-DN-loaded sEVs slightly, but not significantly, reduced body weight in ApoE knockout mice fed a high-fat diet. Also, neither food intake nor energy expenditure were modified. Glucose tolerance was not affected after treatment with SF1-NLRP3-DN-loaded sEVs (Figure 4A,B,C,D,E,F,G,H,I,J,K). Interestingly, the treatment was associated with an increase in BAT thermogenesis (Figure 5A,B,C,D). ApoE knockout mice fed a high-fat diet and treated with unloaded sEVs showed an increase in the number of arrhythmias (conduction disturbances) as well as an increase in the incidence of AF induced by transesophageal stimulation (anesthetized approach) but also spontaneousarrhythmias (using telemetry methodology) (Figures 6A,B,C,D,E and 7A,B,C,D,E,F,G,H,I,J,K,L,M,N,O,P). Interestingly, sEVs loaded with SF1-NLRP3-DN prevented both atrio-ventricular conduction block and AF, both induced and spontaneous. In addition, sEVs loaded with SF1-NLRP3-DN prevented the increase in P-wave duration observed in ApoE knockout mice treated with a high-fat diet, which may explain the prevention of atrial arrhythmias. Note that there is no change in PR interval and QT duration (Figure 6A,B,C,D,E). In addition, sEVs loaded with SF1-NLRP3-DN treatment seem to modulate the activity of the autonomic nervous system (ANS). Indeed, HRV analyses showed that sympatho- vagal influences were modified by the injection of sEVs loaded with SF1-NLRP3-DN. Depending on the period (awake or resting phase of the mice), both temporal and frequency domain parameters indicated that the treatment increased the relative sympathetic / vagal ratio. In other words, treatment increased the relative contribution of the sympathetic component of the ANS (increase in LFnu) relative to the vagal contribution (decrease in HFnu, RMSSD or pNN6). The exact mechanism could not be determined and could be due to decreased vagal activity, increased sympathetic activity, or a combined effect of both. ANS interference with AF and neuromodulation therapies of AF are still under investigation or several results were equivocal (https: / / www.neuromodulationjournal.org / article / S1094-7159(22)00029-0 / fulltext and https: / / www.jacc.org / doi / 10.1016 / j.jacbts.2023.03.019). In the present study, the observed increased LF / HF ratio appears to correlate with improvement in cardiac phenotype, such as restoration of sinus rhythm (telemetry approach) or reduction in the rate of AF initiation (trans- esophageal pacing) (Figure 7A,B,C,D,E,F,G,H,I,J,K,L,M,N,O,P). Interestingly, the increase in sympathetic activity is associated with the increase in BAT, which reflects thermogenesis. sEVs loaded with SF1-NLRP3-DN treatment did not affect systolic and diastolic blood pressure or heart rate (Figure 8A,B,C). Finally, echocardiographic analyses showed that sEVs loaded with SF1-NLRP3-DN did not induce any changes in structural and contractile cardiac functions (Figure 9A,B,C,D,E,F,G,H,I,J,K,L). Intravenous administration of SF1-NLRP3-WT-loaded sEVs in NLRP3 knockout mice fed with high-fat diet did not result in any modifications in body weight, food intake, energy expenditure, energy efficiency or an increase in BAT temperature (Figure 10A,B,C,D,E,F,G,H,I and Figure 11A,B,C).In NLRP3 knockout mice, the treatment with SF1-NLRP3-WT-loaded sEVs induced anincrease in the number of arrhythmias (conduction disturbances) as well as an increase in the incidence of AF induced by transesophageal stimulation, whereas unloaded sEVs did not induce any changes. This effect was associated with an increase in P-wave duration which may explain the occurrence of atrial arrhythmias (Figure 12A,B,C,D,E,F,G,H,I,). Note that there is no change in PR interval and QT duration.NLRP3 knockout mice fed with high-fat diet treated with unloaded sEVs did not displaymodifications on the occurrence of AF nor conduction disturbances. Interestingly, sEVs loaded with SF1-NLRP3-WT increased spontaneous arrhythmias (using telemetry methodology)(Figure 13A,B,C,D,E,F,G,H,I,J,K,L,M,N,O,P). In addition, sEVs loaded with SF1-NLRP3-WT treatment seem to modulate the activity of the autonomic nervous system (ANS). Indeed,HRV analyses showed that sympatho-vagal influences were modified by the injection of sEVs loaded with SF1-NLRP3-WT. Depending on the period (awake or resting phase of the mice),both temporal and frequency domain parameters indicated that the treatment decreased therelative sympathetic / vagal ratio. In other words, treatment decreased the relative contributionof the sympathetic component of the ANS (decrease in LFnu) relative to the vagal contribution(increase in HFnu or pNN6). In the present study, the observed decreased LF / HF ratio appearsto correlate with worsened in cardiac phenotype, such as increase of sinus rhythm (telemetryapproach).These data demonstrated that the presence of NLRP3 in the VMH, as conveyed by sEVs loadedwith SF1-NLRP3-WT, plays a pivotal role in the induction of cardiac arrhythmias in obese mice.The present invention provides for the use of EVs as therapeutic vectors which are administeredsystemically to deliver a plasmid encoding a dominant negative isoform of NLRP3 for use in the treatment or prevention of cardiac arrhythmias and atrial fibrillation associated with obesity. REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. 1. Dietrich & Horvath. Nat Rev Drug Discov 11:675;2012. doi 2. Tschop et al. Cell Metab 24:51;2016. doi 3. Dragano et al. Neuroscience 437:215,2020. doi 4. Milbank et al. Pharmacol Ther 157:65;2016. doi 5. Ridker et al. N Engl J Med 377:1119;2017. doi 6. Martinez & Andriantsitohaina Circ Res. 120:1674;2017. doi 7. Malloci et al. Antioxid Redox Signal 30:813;2019. doi 8. Milbank et al. Nat Metab. 3:1415;2021. doi 9. Malik et al. Circulation 17:354;1996. doi 10. Thireau et al. Exp Physiol.93(1):83;2008. doi 11. Fossier et al. J Am Coll Cardiol.80(23):2205;2022 doi.

Claims

CLAIMS:

1. An isolated particle loaded with i) a dominant negative polypeptide of NLRP3 or ii) apolynucleotide encoding thereof.

2. The isolated particle according to claim 1 wherein the dominant negative polypeptideof NLRP3 comprises or consists in an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2 orSEQ ID NO:3.

3. The isolated particle according to claim 1 wherein the dominant negative polypeptideof NLRP3 comprises or consists in an amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 wherein one or more amino acid residues at position23, 24 or 27 of SEQ ID NO:1 are mutated, or wherein one or more amino acid residuesat position 21, 22 or 25 of SEQ ID NO:2 or SEQ ID NO:3 are mutated.

4. The isolated particle according to claim 3 wherein the dominant negative polypeptideof NLRP3 comprises or consists in an amino acid sequence as set forth in SEQ ID NO:1 wherein: -the lysine (K) at position 23 is substituted by an amino acid residue having anegative charged side chain, preferably by a glutamic acid residue (E), or -the lysine (K) at position 24 is substituted by an amino acid residue having anegative charged side chain, preferably by a glutamic acid residue (E), or -the methionine (M) at position 27 is substituted by an amino acid residue having anegative charged side chain, preferably by a glutamic acid residue (E); or comprises or consists in an amino acid sequence as set forth in SEQ ID NO:2 or SEQID NO:3 wherein:- the lysine (K) at position 21 is substituted by an amino acid residue having anegative charged side chain, preferably by a glutamic acid residue (E), or -the lysine (K) at position 22 is substituted by an amino acid residue having anegative charged side chain, preferably by a glutamic acid residue (E), or- the methionine (M) at position 25 is substituted by an amino acid residue having anegative charged side chain, preferably by a glutamic acid residue (E).

5. The isolated particle according to claim 3 wherein the dominant negative polypeptideof NLRP3 comprises or consists in an amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 that is characterized by one or more substitutionsselected from the group consisting of K23E, K24E, and M27E in SEQ ID NO:1 or thatis characterized by one or more substitutions selected from the group consisting of K21E, K22E, and M25E in SEQ ID NO:2 or SEQ ID NO:3.

6. The isolated particle according to claim 3 wherein the dominant negative polypeptideof NLRP3 comprises or consists in an amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 comprising the K23E, K24E, and M27E substitutionsin SEQ ID NO:1 or comprising the K21E, K22E, and M25E substitutions in SEQ IDNO:2 or SEQ ID NO:3.

7. The isolated particle according to any one of claims 1 to 6 wherein the polynucleotideis an RNA or a DNA molecule.

8. The isolated particle according to claim 7 wherein the polynucleotide is an RNAmolecule, in particular a messenger RNA (mRNA).

9. The isolated particle according to claim 8 wherein the polynucleotide is operably linkedand under the control of a steroidogenic factor 1 (SF1) promoter.

10. The isolated particle according to any one of claims 1 to 9 that is an extracellular vesicle(EV).

11. The isolated particle according to claim 10 wherein the EV comprises one or more ofthe specific exosome markers selected from the groups consisting of ALIX, TSG101, CD9 or CD81, or any combination thereof and / or is characterized by lacking the MHC-II, CD80, CD86 or GRP94 markers, or any combination thereof.

12. The isolated particle according to claim 10 wherein the EV is functionalized with afusion protein comprising or consisting of the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b (Lamp2b), wherein said fusionprotein comprises, consists or consists essentially of an amino acid sequence with at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:5.

13. The isolated particle according to any one of claims 1 to 12 that is an isolated EV thatis loaded with a DNA polynucleotide that encodes the amino acid sequence as set forth in SEQ ID NO:1 that comprises the K23E, K24E, and M27E substitutions or as set forthin SEQ ID NO:2 or SEQ ID NO:3 that comprises the K21E, K22E, and M25Esubstitutions wherein said polynucleotide is operably linked and under the control ofthe SF-1 promoter that consists of the nucleic acid sequence as set forth in SEQ ID NO:4 and the EV is functionalized with a fusion protein that consists of the amino acid sequence as set forth in SEQ ID NO 5.

14. A method of therapy in a patient in need thereof comprising administering to the patienta therapeutically effective amount of an isolated particle according to any one of claims 1 to 13.

15. The method according to claim 14 for treating of an obesity related disease, preferablyan atrial fibrillation associated with obesity.

16. A pharmaceutical composition comprising an isolated particle according to any one ofclaims 1 to 13.

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