Chimeric adiponectin polypeptides, extracellular vesicles containing the same, and uses thereof

Abstract

The present invention relates to a chimeric polypeptide comprising adiponectin, a nucleic acid encoding said chimeric polypeptide, and an extracellular vesicle comprising said chimeric polypeptide. The present invention further relates to the use of said extracellular vesicles as a medicament, in particular for treating various diseases.

Description

[Technical field]

[0001] The present invention relates to a chimeric polypeptide comprising adiponectin, a nucleic acid encoding said chimeric polypeptide, and an extracellular vesicle comprising said chimeric polypeptide.Furthermore, the present invention relates to the use of said extracellular vesicle as a medicament, in particular for treating various diseases. [Background technology]

[0002] Adipose tissue is an endocrine organ that secretes a wide variety of bioactive proteins (adipokines) and lipids, of which adiponectin is one of the most abundant adipokines. Adipokines are 244 amino acid cytokines found primarily in oligomeric complexes. Adiponectin is known to exert beneficial effects on a variety of human and animal conditions, including insulin resistance, cardiovascular disease, inflammatory conditions, and cancer. Thus, adiponectin is a promising candidate for the development of new drugs for a variety of diseases.

[0003] Despite this promising aspect, there is currently no adiponectin therapy available for clinical trials.In fact, the large-scale production of the native functional form of adiponectin, including post-translational modifications and proper highly multimerized complex assembly, is a difficult task.Combined with the short half-life of adiponectin, there is no production strategy that provides high levels of bioactive adiponectin at a reasonable price.Therefore, another approach is awaited to provide multimeric native active adiponectin.

[0004] In the present invention, the inventors have found that adiponectin can be produced in large quantities and in its native highly polymerized form when immobilized in extracellular vesicles, and that these adiponectin-rich extracellular vesicles can be purified and stored in characterized and qualified batches. Such standardized materials pave the way for promising strategies to treat various human conditions that benefit from adiponectin, such as diabetes, obesity, and related metabolic diseases, insulin resistance, cardiovascular diseases, inflammatory conditions, and cancer. Summary of the Invention

[0005] The present invention provides a method for producing a method comprising the steps of: i) the amino acid sequence of adiponectin, preferably wild-type adiponectin; ii) the amino acid sequence of the transmembrane domain of the transmembrane protein, and iii) Optionally, an amino acid sequence of a peptide that interacts with the Endosomal Sorting Complexes Required for Transport (ESCRT) cellular machinery. The present invention relates to a chimeric polypeptide comprising:

[0006] In one embodiment, the chimeric polypeptide further comprises at least one linker between the amino acid sequence of adiponectin and the amino acid sequence of the transmembrane domain.

[0007] In one embodiment, the chimeric polypeptide comprises, from N-terminus to C-terminus, components iii), ii, and i).

[0008] In one embodiment, the chimeric polypeptide further comprises a submembrane targeting domain, preferably the submembrane targeting domain is linked to an anchor molecule, preferably the anchor molecule is a fatty acid.

[0009] In one embodiment, the transmembrane domain is selected from the group comprising the transmembrane domain of CD40L and the transmembrane domain of CD8, preferably having SEQ ID NO: 34 and SEQ ID NO: 36, respectively, more preferably having SEQ ID NO: 35 and SEQ ID NO: 37, respectively.

[0010] In one embodiment, the pilot peptide comprises at least one YxxL motif having SEQ ID NO:1 or at least one DYxxL motif having SEQ ID NO:4, and at least one PxxP motif having SEQ ID NO:8, where "x" represents any amino acid residue.

[0011] In one embodiment, the pilot peptide comprises an amino acid sequence having SEQ ID NO: 30 or a variant thereof, wherein the variant of SEQ ID NO: 30 retains at least three YxxL and / or DYxxL motifs having SEQ ID NO: 1 and SEQ ID NO: 4, respectively, and at least four PxxP motifs having SEQ ID NO: 8, wherein "x" represents any amino acid residue.

[0012] The present invention also relates to a nucleic acid encoding the chimeric polypeptide of the present invention.

[0013] The present invention also relates to extracellular vesicles comprising the chimeric polypeptides of the present invention, preferably wherein the transmembrane domain of the chimeric polypeptide is anchored in the lipid bilayer of the extracellular vesicle and the adiponectin of the chimeric polypeptide is exposed on the outer surface of the extracellular vesicle.

[0014] In one embodiment, the extracellular vesicles are exosomes, preferably having a diameter in the range of about 30 nm to about 120 nm.

[0015] The present invention also relates to a population of extracellular vesicles of the present invention, which optionally further comprises soluble adiponectin.

[0016] In one embodiment, the extracellular vesicles of the invention or a population of extracellular vesicles of the invention are purified, preferably ultra-purified.

[0017] The present invention also relates to an extracellular vesicle of the invention or a population of extracellular vesicles of the invention for use as a medicament.

[0018] The present invention also relates to an extracellular vesicle of the invention or a population of extracellular vesicles of the invention for use in the treatment of a disease, disorder, or condition selected from the group including diabetes, obesity, insulin resistance, diseases associated with insulin resistance or deficiency, hypertension, dyslipidemia, hyperuricemia, atherosclerosis (including coronary artery disease, stroke, and peripheral arterial disease), fibrosis, inflammatory lung disease, nephrotic disease, sleep apnea, dry eye disease, inflammatory eye disease, gastritis and gastroesophageal reflux disease, inflammatory bowel disease, pancreatitis, osteoporosis, and inflammatory bone and joint diseases.

[0019] The present invention also relates to an extracellular vesicle or population thereof having adiponectin exposed on its outer surface for use in the treatment of a disease, disorder, or condition selected from the group including diabetes, obesity, insulin resistance, diseases associated with insulin resistance or deficiency, hypertension, dyslipidemia, hyperuricemia, atherosclerosis (including coronary artery disease, stroke, and peripheral arterial disease), fibrosis, nephrotic disease, sleep apnea, dry eye disease, inflammatory eye disease, gastritis and gastroesophageal reflux disease, inflammatory bowel disease, pancreatitis, osteoporosis, and inflammatory bone and joint diseases.

[0020] In one embodiment, the adiponectin is recombinant adiponectin, optionally fused to lactadherin or its functional C1 and / or C2 domains.

[0021] The present invention also provides a method of diagnosing obesity, insulin resistance, or a disease associated with insulin resistance or deficiency in a subject, comprising the steps of: a) measuring the level or amount of adiponectin-associated small extracellular vesicles in a sample previously collected from a subject; b) comparing the level or amount determined in step a) with a reference level or amount of adiponectin-associated small extracellular vesicles, the reference level or amount having been previously determined in a sample from a reference subject known not to be affected by obesity or insulin resistance, or in a pool of samples from more than one reference subject, all of whom are not affected by obesity or insulin resistance; c) if the level or amount determined in step a) is lower than the reference level or amount, concluding that the subject suffers from obesity, insulin resistance, or a disease associated with insulin resistance or deficiency. The present invention relates to a method comprising the steps of:

[0022] definition In the present invention, the following terms have the following meanings.

[0023] The word "about" preceding a number means ±10% of the value of said number.

[0024] "Adiponectin" (also known as "30 kDa adipocyte complement-related protein", "adipocyte complement-related 30 kDa protein", or abbreviated "ACRP30") is a protein derived primarily from adipocytes. As used herein, the term "adiponectin" includes adiponectin derived from any species that produces adiponectin. In humans, adiponectin is encoded by the ADIPOQ gene (also called the ACDC, ACRP30, APM1, or GBP28 gene). This gene encodes an adiponectin precursor having SEQ ID NO: 33, which is processed in vivo to its mature form having SEQ ID NO: 31.

[0025] "CD8" refers to a transmembrane glycoprotein that acts as a co-receptor for the T cell receptor (TCR). In humans, the CD8 transmembrane domain comprises the amino acid sequence having SEQ ID NO:36.

[0026] "CD40 ligand" (also called "CD40L" or "CD154") refers to a transmembrane protein that is a member of the tumor necrosis factor (TNF) superfamily. In humans, the CD40L transmembrane domain comprises the amino acid sequence having SEQ ID NO:34.

[0027] "Chimera", when referring to a polypeptide, refers to a polypeptide that combines at least two types of multiple domains that differ in their function and / or their cellular distribution, and at least two of these domains are derived from separate proteins of the same or different species, or from the same protein of different species.

[0028] "Diabetes" refers to a metabolic disease characterized by a chronic excess of sugar (glucose) in the blood, also known as "diabetes mellitus." One of the criteria used to diagnose diabetes is a fasting blood glucose level higher than 1.26 g / L blood (or about 7 mmol / L blood). There are several types of diabetes: "Type 1 diabetes", also called "juvenile diabetes", is characterized by the loss of insulin-producing beta cells of the islets of Langerhans in the pancreas in children or young adults (an autoimmune disease); and "Type 2 diabetes", also called "non-insulin-dependent diabetes", is characterized by the gradual development of insulin resistance (i.e., when insulin-sensitive cells lose their response to insulin). Insulin resistance is manifested by reduced glucose uptake by adipose tissue and muscle, and reduced inhibition of glucose production in the liver. In more advanced stages, type 2 diabetes can result in insulin deficiency, i.e., failure to synthesize insulin by endocrine pancreatic cells.

[0029] "Diseases associated with insulin resistance or deficiency" or "diseases associated with impaired glucose tolerance" refers to several metabolic diseases, including type 2 diabetes, metabolic syndrome, as well as cardiovascular disease (Ford, 2005. Diabetes Care. 28(7):1769-78), nonalcoholic fatty liver disease (Bugianesi et al., 2010. Curr Pharm Des. 16(17):1941-51), polycystic ovarian syndrome (PCOS) (Diamanti-Kandarakis, 2006. Endocrine. 30(1):13-7), Alzheimer's disease (Watson & Craft, 2003. CNS Drugs. 17(1):27-45), and cancer (Arcidiacono et al., 2012. Exp Diabetes Res. 2012:789174).

[0030] "Domain," when referring to a protein or polypeptide, refers to a region having structural and / or functional properties with respect to said protein or polypeptide. As used herein, "transmembrane domain" refers to a functional region of a protein or polypeptide that spans the phospholipid bilayer of a biological membrane.

[0031] "ESCRT" or "endosomal sorting complexes required for transport" refers to a cellular machinery originally made of five multisubunit protein complexes that act in concert with specialized endosomes to facilitate the transfer of specific cargo to vesicles that bud into the lumen from the limiting membrane. This machinery has been hijacked by some enveloped viruses to bud out of cell membranes, including the plasma membrane.

[0032] "Exosomes" refer to extracellular vesicles produced in the endosomal compartment of eukaryotic cells (Thery et al., 2018. J Extracell Vesicles. 7(1):1535750; Yanez-Mo et al., 2015. J Extracell Vesicles. 4:27066; van Niel et al., 2018. Nat Rev Mol Cell Biol. 19(4):213-228). Exosomes typically carry the CD81, CD63, and CD9 markers on their surface.

[0033] An "expression vector" refers to a vector capable of directing the expression of a nucleic acid sequence of interest (such as a nucleic acid of the present invention) in an appropriate host cell, the vector comprising a promoter operably linked to a nucleic acid sequence of interest, which is itself operably linked to a termination sequence.

[0034] "Extracellular vesicles" refer to any vesicles composed of a lipid bilayer that are naturally released from a cell and contain the cytosolic fraction of said cell. In particular, this expression includes vesicles secreted into the extracellular space, i.e. "exosomes".

[0035] "Insulin resistance" refers to the inability of a known amount of exogenous or endogenous insulin to increase glucose uptake and utilization in a subject to the same extent as in the normal population.

[0036] "Isolated" and any inflection thereof, and "purified" and any inflection thereof, are used interchangeably and mean that the molecular entity (e.g., polypeptide, nucleic acid, extracellular vesicle, etc.) so designated is substantially free of other components (i.e., contaminants) found in the natural environment in which the molecular entity is normally found. Preferably, an isolated or purified molecular entity (e.g., an isolated or purified polypeptide, an isolated or purified nucleic acid, an isolated or purified extracellular vesicle, etc.) is substantially free of other molecular entities that associate in a cell. "Substantially free of" means that the isolated or purified molecular entity represents more than 50%, preferably more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, and even more preferably more than 98% or 99% of a heterogeneous composition (i.e., is at least 50% pure). Purity can be assessed by a variety of methods known to those of skill in the art, including but not limited to chromatography, gel electrophoresis, immunoassays, compositional analysis, biological assays, and the like.

[0037] "Obesity" (or overweight) refers to a chronic disease (recognized by the World Health Organization since 1997) defined as an abnormal or excessive accumulation of body fat that can be harmful to health. Further, according to the World Health Organization, the definition of obesity is based on the measurement of the body mass index (BMI = weight / (height) 2 ) and classifies the disease into three levels: "overweight" when 25 kg / m 2 <BMI < 30 kg / m 2 ; "obesity" when 30 kg / m 2 <BMI < 40 kg / m 2 ; and "morbid obesity" when 40 kg / m 2 <BMI.

[0038] "Pilot peptide" refers to a peptide that interacts with an ESCRT protein. The pilot peptide can be directed to membrane vesicles, particularly vesicles that form exosomes, or to cellular compartments involved in the formation of membrane vesicles, particularly exosome-forming vesicles in eukaryotic cells.

[0039] "Subject" refers to a mammal, preferably a human. In one embodiment, the subject may be a "patient", i.e. a warm-blooded animal, more preferably a human, who is awaiting or receiving medical care, or who has been / is / may be the subject of past / present / future medical care, or who is being monitored for the development of disease.

[0040] A "membrane sub-targeting domain" or "membrane targeting domain" or "membrane recruitment domain" are used interchangeably and refer to a domain that can anchor itself to the plasma membrane and / or vesicle membrane in a cell, particularly a eukaryotic cell (e.g., an exosome-producing cell), without being inserted into said membrane, said anchoring being achieved by one or more anchoring molecules and / or by an interaction (e.g., electrostatic interaction) between the membrane sub-targeting domain. In certain embodiments, the membrane sub-targeting domain is capable of binding to or interacting with the inner surface of the plasma membrane (i.e., the cytoplasmic side of the plasma membrane) and / or the inner surface of the vesicle membrane (i.e., the lumenal side of the vesicle membrane).

[0041] "Therapeutically effective amount" refers to a level or amount of a chimeric polypeptide, nucleic acid, extracellular vesicle, population of extracellular vesicles, composition, pharmaceutical composition, medicament, etc., intended to (1) delay or prevent the onset of a disease, disorder, or condition; (2) delay or halt the progression, progression, or worsening of one or more symptoms of a disease, disorder, or condition; (3) bring about amelioration of symptoms of a disease, disorder, or condition; (4) reduce the severity or frequency of a disease, disorder, or condition; or (5) cure a disease, disorder, or condition, without causing significant negative or harmful side effects to the target. A therapeutically effective amount may be administered prior to the onset of a disease, disorder, or condition for a prophylactic or preventative effect. Alternatively or additionally, a therapeutically effective amount may be administered after the onset of a disease, disorder, or condition for a therapeutic effect.

[0042] "Transmembrane protein" refers to a protein that contains at least one transmembrane domain that allows anchoring to the phospholipid bilayer of a biological membrane. The transmembrane domain is generally hydrophobic-helical and may contain several, in particular 2, 3, 4, 5, 6, 7, 8, 9, or 10, or even 20 or more hydrophobic α-helices. It may also be arranged in β-sheets, e.g., β-barrel structures that are usually composed of 8 to 22 β-strands. Transmembrane proteins can also be classified according to the location of their N- and C-termini on different sides of the lipid layer: type I, type II, type III, and type IV. Type I transmembrane proteins are anchored to the lipid membrane with a stop-transfer anchor sequence, and their N-terminal domain is targeted to the lumen of the endoplasmic reticulum (ER) during synthesis (and to the extracellular space if the mature form is located on the cell membrane). Types II and III are anchored by a signal-anchor sequence, with type II targeted to the ER lumen by its C-terminal domain, whereas type III is targeted to the ER lumen by its N-terminal domain. Type IV is further subdivided into IV-A, whose N-terminal domain targets the cytosol, and IV-B, whose N-terminal domain targets the lumen.

[0043] "Treating" or "treatment" or "alleviation" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or delay (alleviate) the targeted condition or disorder. Those in need of treatment include those who already have the disorder, and those who are prone to have the disorder, or those in whom the disorder is to be prevented. In one embodiment, the "treatment" of a subject's disease, disorder, or condition is successful if, after administration of a therapeutic amount of a chimeric polypeptide, nucleic acid, extracellular vesicle, population of extracellular vesicles, composition, pharmaceutical composition, medicament, etc., the subject exhibits at least one of the following: alleviation to some degree of one or more of the symptoms associated with the disease, disorder, or condition to be treated; reduction in morbidity and mortality; and improvement in quality of life issues. The above parameters for assessing the success of treatment and improvement in disease are easily measurable by routine techniques familiar to physicians.

[0044] A "vector" refers to a nucleic acid capable of transporting a nucleic acid of interest (such as a nucleic acid of the present invention) to which it has been linked. A vector capable of directing the expression of a nucleic acid of interest (such as a nucleic acid of the present invention) is called an "expression vector." Generally, expression vectors are in the form of a plasmid. As used herein, the terms "plasmid" and "vector" are used interchangeably. However, other forms of expression vectors which serve equivalent functions are also encompassed by the term vector. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] Detailed Description The present invention provides a method for producing a method comprising the steps of: The amino acid sequence of adiponectin the amino acid sequence of the transmembrane domain of the transmembrane protein, and Optionally, an amino acid sequence of a peptide that interacts with the endosomal sorting and transport complex (ESCRT) cellular machinery. The present invention relates to a chimeric polypeptide comprising:

[0046] In one embodiment, components i), ii) and iii) are assembled N-terminally to C-terminally or C-terminally to N-terminally in the chimeric polypeptide.

[0047] According to the present invention, the chimeric polypeptide comprises the amino acid sequence of adiponectin.

[0048] In one embodiment, the amino acid sequence of adiponectin comprises or consists of the amino acid sequence of wild-type adiponectin.

[0049] In one embodiment, the amino acid sequence of adiponectin comprises or consists of the amino acid sequence of a variant adiponectin.

[0050] In one embodiment, the amino acid sequence of adiponectin comprises or consists of the amino acid sequence of wild-type human adiponectin. In one embodiment, the wild-type human adiponectin comprises or consists of the amino acid sequence having SEQ ID NO: 33 or a variant thereof. SEQ ID NO:33 MLLLGAVLLLLALPGHDQETTTQGPGVLLPLPKGACTGWMAGIPGHPGHNGAPGRDGRDGTPGEKGEKGDPGLIGPKGDIGETGVPGAEGPRGFPGIQGRKGEPGEGAYVYRSAFSVGLETY VTIPNMPIRFTKIFYNQQNHYDGSTGKFHCNIPGLYYFAYHITVYMKDVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQVYGEGERNGLYADNDNDSTFTGFLLYHDTN

[0051] The wild-type human adiponectin contains a peptide signal having the amino acid sequence of SEQ ID NO: 32, which is typically cleaved in vivo to form the mature form of wild-type human adiponectin. SEQ ID NO:32 MLLLGAVLLLLALPGHDQ

[0052] Thus, in one embodiment, the amino acid sequence of adiponectin comprises or consists of the amino acid sequence of the mature form of wild-type human adiponectin having SEQ ID NO: 31, i.e. the peptide signal having SEQ ID NO: 32 is cleaved from wild-type human adiponectin having SEQ ID NO: 33. SEQ ID NO:31 ETTTQGPGVLLPLPKGACTGWMAGIPGHPGHNGAPGRDGRGTPGEKGEKGDPGLIGPKGDIGETGVPGAEGPRGFPGIQGRKGEPGEGAYVYRSAFSVGLETYVTIPNMPIR FTKIFYNQQNHYDGSTGKFHCNIPGLYYFAYHITVYMKDVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQVYGEGERNGLYADNDNDSTFTGFLLYHDTN

[0053] In one embodiment, the amino acid sequence of adiponectin comprises or consists of an amino acid sequence having SEQ ID NO: 31, SEQ ID NO: 33, or a variant thereof.

[0054] In one embodiment, a variant of the amino acid sequence having SEQ ID NO:31 or SEQ ID NO:33 comprises an amino acid sequence sharing at least 70% overall sequence identity with the amino acid sequence of SEQ ID NO:31 or 33, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more overall sequence identity.

[0055] Additionally or alternatively, variants of the amino acid sequence having SEQ ID NO:31 or SEQ ID NO:33 comprise at least 70% local sequence identity to the amino acid sequence of SEQ ID NO:31 or SEQ ID NO:33, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more local sequence identity.

[0056] According to the invention, the chimeric polypeptide comprises the amino acid sequence of a transmembrane domain of a transmembrane protein.

[0057] In one embodiment, the transmembrane domain is a domain of a transmembrane protein derived from any organism, including mammals, viruses, and bacteria.

[0058] In one embodiment, the transmembrane domain is a domain of a transmembrane protein selected from the group including or consisting of a human protein, a protein of a non-human animal, a protein of a pathogenic organism or agent (particularly a viral protein, a bacterial protein, a parasitic protein), or a tumor cell protein.

[0059] In one embodiment, the transmembrane domain is a domain of a type I transmembrane protein, which may be used in a chimeric polypeptide as described herein above, with components i), ii) and iii), if present, being assembled N-terminally to C-terminally in the chimeric polypeptide (e.g., construct 1 in FIG. 1 ).

[0060] In one embodiment, the transmembrane domain is a domain of a retroviral transmembrane glycoprotein selected from the group including or consisting of bovine leukemia virus (BLV), human immunodeficiency virus (HIV) (e.g., but not limited to, HIV-1 or HIV-2), human T-cell leukemia virus (HTLV) (e.g., but not limited to, HTLV-1 or HTLV-2), and Mason-Pfizer Monkey Virus (MPMV).

[0061] In one embodiment, the transmembrane domain is a domain of the hemagglutinin transmembrane protein of an influenza virus.

[0062] In one embodiment, the transmembrane domain is that of CD8.

[0063] In one embodiment, the transmembrane domain is a type II transmembrane protein domain, which may be used in the chimeric polypeptide as described herein above, with components i), ii) and iii) being assembled, if present, from C-terminus to N-terminus in the chimeric polypeptide (e.g., constructs 2, 3, 4, 5 in FIG. 1).

[0064] In one embodiment, the transmembrane domain is a domain of the neuraminidase transmembrane protein of an influenza virus.

[0065] In one embodiment, the transmembrane domain is a domain of CD40 ligand (CD40L).

[0066] In one embodiment, the transmembrane domain is a domain of CD40 ligand (CD40L) or CD8.

[0067] In one embodiment, the transmembrane domain of CD40L comprises or consists of an amino acid sequence having SEQ ID NO: 34, or a variant thereof. In one embodiment, the transmembrane domain comprises or consists of an amino acid sequence having SEQ ID NO: 35, or a variant thereof. SEQ ID NO:34 IFMYLLTVFLITQMIGSALFAVYLH SEQ ID NO:35 MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDK

[0068] In one embodiment, the transmembrane domain of CD8 comprises or consists of an amino acid sequence having SEQ ID NO: 36, or a variant thereof. In one embodiment, the transmembrane domain comprises or consists of an amino acid sequence having SEQ ID NO: 37, or a variant thereof. SEQ ID NO:36 IYIWAPLAGICVALLLSLIITLICYH SEQ ID NO:37 DIYIWAPLAGICVALLLSLIITLICYHR

[0069] In one embodiment, a variant of an amino acid sequence having SEQ ID NO: 34, 35, 36, or 37 comprises an amino acid sequence sharing at least 70% overall sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more overall sequence identity with the amino acid sequence of SEQ ID NO: 34, 35, 36, or 37.

[0070] Additionally or alternatively, variants of the amino acid sequence having SEQ ID NO: 34, 35, 36, or 37 include amino acid sequences sharing at least 70% local sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more local sequence identity with the amino acid sequence of SEQ ID NO: 34, 35, 36, or 37.

[0071] In one embodiment, the chimeric polypeptide further comprises the amino acid sequence of a peptide that interacts with the endosomal sorting and transport complex (ESCRT) cellular machinery, otherwise known as a "pilot peptide."

[0072] In one embodiment, said pilot peptide is capable of targeting membrane vesicles, in particular exosome-forming vesicles, or cellular compartments involved in the formation of membrane vesicles, in particular exosome-forming vesicles in eukaryotic cells.

[0073] When incorporated into a chimeric polypeptide, such as the chimeric polypeptide of the present invention, the pilot peptide makes it possible to target said chimeric polypeptide to membrane vesicles and / or to the location of their formation, in particular to the membrane of a membrane vesicle so that said polypeptide can be secreted by the cell in association with the membrane vesicle (in particular as an exosome).

[0074] Pilot peptides that interact with ESCRT proteins are described in granted European Patent No. 2 268 816 and US Pat. No. 9,546,371, the relevant contents of which are incorporated herein by reference.

[0075] In one embodiment, the pilot peptide comprises at least one YxxL motif (SEQ ID NO:1), where "x" represents any amino acid residue. In particular, it may comprise one, two, or three YxxL motifs having SEQ ID NO:1.

[0076] In one embodiment, the YxxL motif or one of the YxxL motifs of the pilot peptide can be, for example, YINL (SEQ ID NO: 2) or YSHL (SEQ ID NO: 3).

[0077] In one embodiment, the pilot peptide contains a DYxxL motif (SEQ ID NO: 4), where "x" represents any amino acid residue.

[0078] In one embodiment, the DYxxL motif may be, for example, DYINL (SEQ ID NO:5).

[0079] Alternatively, or in addition, the pilot peptide contains at least one motif equivalent to a YxxL motif (SEQ ID NO:1), such as a YxxF motif (SEQ ID NO:6), where "x" represents any amino acid residue.

[0080] Alternatively, or in addition, the pilot peptide contains at least one motif that is equivalent to a DYxxL motif (SEQ ID NO:4), such as a DYxxF motif (SEQ ID NO:7), where "x" represents any amino acid residue.

[0081] In one embodiment, the pilot peptide further comprises at least one PxxP motif (SEQ ID NO: 8), where "x" represents any amino acid residue. In particular, it may comprise 1, 2, 3, or 4 PxxP motifs having SEQ ID NO: 8.

[0082] In one embodiment, the PxxP motif or one of the PxxP motifs of the pilot peptide is PSAP (SEQ ID NO: 9) or PTAP (SEQ ID NO: 10).

[0083] In one embodiment, the pilot peptide comprises at least one YxxL motif having SEQ ID NO:1 or at least one DYxxL motif having SEQ ID NO:4, and at least one PxxP motif having SEQ ID NO:8.

[0084] In one embodiment, the pilot peptide consists of an amino acid sequence having one, two, or three YxxL and / or DYxxL motifs having SEQ ID NO:1 or SEQ ID NO:4, respectively, and one, two, three, or four PxxP motifs having SEQ ID NO:8.

[0085] In one embodiment, the pilot peptide consists of an amino acid sequence having three YxxL and / or DYxxL motifs having SEQ ID NO:1 or SEQ ID NO:4, respectively, and four PxxP motifs having SEQ ID NO:8.

[0086] In one embodiment, the YxxL motif having SEQ ID NO:1 or at least one of the YxxL motifs having SEQ ID NO:1, if more than one, is located downstream, i.e., C-terminal to one or more PxxP motifs having SEQ ID NO:8.

[0087] Proteins having pilot peptides containing at least one YxxL motif having SEQ ID NO:1 include cellular proteins and viral proteins, in particular proteins of enveloped viruses, such as transmembrane glycoproteins of enveloped viruses, or proteins of herpes viruses, such as the LMP2-A protein of Epstein-Barr virus, which contains at least two YxxL motifs having SEQ ID NO:1.

[0088] In one embodiment, the pilot peptide is a peptide of a transmembrane glycoprotein of a retrovirus, hi one embodiment, the pilot peptide can be a peptide of a transmembrane glycoprotein of a retrovirus selected from the group including or consisting of bovine leukemia virus (BLV), human immunodeficiency virus (HIV) (e.g., but not limited to, HIV-1 or HIV-2), human T-cell leukemia virus (HTLV) (e.g., but not limited to, HTLV-1 or HTLV-2), and Mason-Pfizer monkey virus (MPMV).

[0089] In one embodiment, the pilot peptide has the following amino acid sequence: SEQ ID NO:11: PxxPxxxxPxxPxSxYxxLxPxxPExYxxLxPxxPDYxxL; SEQ ID NO:12: PxxPx n PxxPx n SxYxxLx n PxxPEx n YxxLx n PxxPDYxxL; SEQ ID NO: 13: PxxPxxxxPxxPxSxYxxLxPxxPExYxxLxPxxPDYxxLxxxx; and SEQ ID NO: 14: PxxPx n PxxPx n SxYxxLx n PxxPEx n YxxLx n PxxPDYxxLxxxx; (Wherein, "x" and "x n " represents any amino acid residue and any one or several amino acid residues, respectively) Contains one of the following:

[0090] In one embodiment, the pilot peptide has the following amino acid sequence: SEQ ID NO:15: PxxPxxxxxxxxxxxxYxxL; SEQ ID NO:16: PxxPxxxxxxxxxxxDYxxL; SEQ ID NO:17: PxxPxxYxxxxxxxxxYxxL; SEQ ID NO:18: PxxPxxYxxxxxxxxDYxxL; SEQ ID NO: 19: PxxPExYxxLxPxxPDYxxL; SEQ ID NO:20: PxxPx n YxxL; SEQ ID NO: 21: PxxPx n DYxxL; SEQ ID NO: 22: PxxPx n Yx n YxxL; SEQ ID NO: 23: PxxPx n Yx n DYxxL; SEQ ID NO:24: PxxPEx n YxxLx n PxxPDYxxL; SEQ ID NO:25: PxxPxxxxPxxPxxxYxxLxPxxPExYxxLxPxxPDYxxL; SEQ ID NO: 26: PxxPx n PxxPx n YxxLx n PxxPEx n YxxLx n PxxPDYxxL; SEQ ID NO: 27: PxxPxxxxPxxPxxxYxxLxPxxPExYxxLxPxxPDYxxLxxxx; and SEQ ID NO: 28: PxxPx n PxxPx n YxxLx n PxxPEx n YxxLx n PxxPDYxxLxxxx (Wherein, "x" and "x n " represents any amino acid residue and any one or several amino acid residues, respectively) Contains one of the following:

[0091] In particular, "n" may be greater than or equal to 1 and less than 50. "n" may in particular have any value from 1 to 20.

[0092] In one embodiment, the pilot peptide comprises between 6 and 100 amino acid residues, in particular between 20 and 80, 30 and 70, or 40 and 60 amino acid residues, such as 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid residues.

[0093] In one embodiment, the pilot peptide comprises or consists of an amino acid sequence having SEQ ID NO: 30, or a variant thereof. An exemplary nucleic acid sequence encoding a pilot peptide having SEQ ID NO: 30 comprises or consists of SEQ ID NO: 29, or a variant thereof. SEQ ID NO:29 GCGCCCCACTTCCCTGAAATCTCCTTCCCCCCTAAACCCGATTCTGATTATCAGGCCTTGCTACCATCCGCGCCAGAGATCTACTCTCACCTCTCCCCCACCAAACCCGATTACATCAACCTTCGACCGGCGCCCTAA SEQ ID NO:30 APHFPEISFPPKPDSDYQALLPSAPEIYSHLSPTKPDYINLRPAP

[0094] In one embodiment, a variant of the amino acid sequence having SEQ ID NO:30 comprises an amino acid sequence sharing at least 70% overall sequence identity with the amino acid sequence of SEQ ID NO:30, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more overall sequence identity.

[0095] Additionally or alternatively, variants of an amino acid sequence having SEQ ID NO:30 include amino acid sequences sharing at least 70% local sequence identity with the amino acid sequence of SEQ ID NO:30, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more local sequence identity.

[0096] In one embodiment, a variant of the amino acid sequence having SEQ ID NO:30 retains at least one, two, or three YxxL or DyxxL motifs having SEQ ID NO:1 or SEQ ID NO:4, respectively; and one, two, three, or four PxxP motifs having SEQ ID NO:8.

[0097] In one embodiment, a variant of SEQ ID NO:30 retains at least three YxxL and / or DYxxL motifs, having SEQ ID NO:1 and SEQ ID NO:4, respectively; and at least four PxxP motifs, having SEQ ID NO:8, where "x" represents any amino acid residue.

[0098] In one embodiment, a variant of the amino acid sequence having SEQ ID NO: 30 retains three YxxL and / or DYxxL motifs having SEQ ID NO: 1 or SEQ ID NO: 4, respectively, and four PxxP motifs having SEQ ID NO: 8.

[0099] In one embodiment, the chimeric polypeptide further comprises at least one linker.

[0100] In one embodiment, at least one linker connects the amino acid sequence of adiponectin and the amino acid sequence of the transmembrane domain.

[0101] In one embodiment, at least one linker is not cleavable. In one embodiment, at least one linker is cleavable.

[0102] In one embodiment, at least one linker is a Gly-Ser linker. Examples of Gly-Ser linkers include, but are not limited to, GS linkers, G2S linkers, G3S linkers, G4S linkers, including repeats and combinations thereof.

[0103] In one embodiment, at least one linker is (GGGS) nIt comprises or consists of the sequence (SEQ ID NO: 38), where n is a positive integer ranging from 1 to 10, preferably from 1 to 5.

[0104] In one embodiment, at least one linker is (GGGSGGGGS) n It comprises or consists of the sequence (SEQ ID NO: 39), where n is a positive integer ranging from 1 to 10, preferably from 1 to 5.

[0105] In one embodiment, at least one linker connecting the amino acid sequence of adiponectin and the amino acid sequence of the transmembrane domain comprises the sequence SGGGSGGGGSGGGSGGGGSGGGSGGGGSGGGGS (SEQ ID NO: 53). Said linker may be used in the chimeric polypeptide described herein above, in which components i), ii) and, if present, iii) are constructed from the N-terminus to the C-terminus in the chimeric polypeptide (e.g., construct 1 in FIG. 1).

[0106] In one embodiment, at least one linker connecting the amino acid sequence of adiponectin and the amino acid sequence of the transmembrane domain comprises the sequence GGGSGGGGSGGGSGGGGSGGGSGGGGSGGGSG (SEQ ID NO: 54). Said linker may be used in the chimeric polypeptides described herein above, in which components i), ii) and, if present, iii) are constructed from the C-terminus to the N-terminus in the chimeric polypeptide (e.g., constructs 2, 3, 4, 5 in FIG. 1).

[0107] In one embodiment, when the chimeric polypeptide comprises more than one linker, the two or more linkers can be the same or different.

[0108] In one embodiment, the chimeric polypeptide further comprises the amino acid sequence of a submembrane targeting domain.

[0109] In one embodiment, the submembrane targeting domain is added to a chimeric polypeptide as described herein above, where components i), ii), and, if present, iii) are assembled C-terminally to N-terminally in the chimeric polypeptide (e.g., constructs 2, 3, 4, 5 in Figure 1).

[0110] In one embodiment, the submembrane targeting domain is sufficient to allow the chimeric polypeptide to be anchored to the lipid bilayer of a cell or vesicle membrane, preferably via one or more anchor molecules and / or via interactions such as electrostatic interactions.

[0111] Thus, by its presence in the chimeric polypeptide, the submembrane targeting domain, when expressed in a cell, enables the chimeric polypeptide to be anchored to (or anchored within) the membrane of a cell or vesicle without inserting the chimeric polypeptide into said membrane.

[0112] In one embodiment, the submembrane targeting domain confers on the chimeric polypeptide the property of binding to the inner surface of a cell membrane (i.e., the cytoplasmic side of the cell membrane) and / or the inner surface of a vesicle membrane (i.e., the lumenal side of the vesicle membrane).

[0113] In one embodiment, the chimeric polypeptide further comprises a submembrane targeting domain, wherein the submembrane targeting domain is linked to the anchor molecule.

[0114] "Anchor molecule" refers to any molecule that can be inserted into at least one layer of the lipid bilayer of a cell or vesicle membrane. In particular, the anchor molecule is a lipid or lipid-containing molecule. Thus, the submembrane targeting domain is also said to be "lipid-anchored".

[0115] In one embodiment, the anchor molecule comprises or consists of one or more lipids or lipid-containing molecules, said lipids comprising hydrophobic carbon chains that enable them to be encapsulated in the lipid bilayer of a cell or vesicle membrane.

[0116] In one embodiment, the lipids are fatty acids, including but not limited to myristic acid, palmitic acid, and isoprenoids (eg, geranyl-geranyl and farnesyl).

[0117] In one embodiment, the anchor molecule is covalently linked to the submembrane targeting domain.

[0118] In one embodiment, the anchor molecule is linked to the submembrane targeting domain via a glycine (in the case of myristic acid), cysteine, or serine amino acid residue of the submembrane targeting domain, which may be via an amide or thioester bond.

[0119] In one embodiment, the just membrane targeting domain is a domain of an exogenous membrane protein, or a variant of the just membrane targeting domain of an exogenous membrane protein.

[0120] In one embodiment, the submembrane targeting domain comprises or consists of a consensus sequence that allows for attachment (e.g., by acylation or prenylation) of a fatty acid, in particular myristic acid, palmitic acid, or an isoprenoid (such as, for example, geranyl-geranyl and farnesyl).

[0121] In one embodiment, the submembrane targeting domain is as follows: (M) G-X1-X2-X3-S / C (SEQ ID NO: 45) (In the formula, X1, X2, and X3 independently represent any amino acid residue; (M) indicates an initiator methionine that, when located at the N-terminus of the chimeric polypeptide, can be removed in vivo by post-translational modification. It comprises or consists of the consensus sequence having SEQ ID NO:45.

[0122] In one embodiment, X1 is selected from the group consisting of: C, S, and L; and / or X2 is selected from the group including or consisting of S, I, V, M, and L; and / or X3 is selected from the group including or consisting of K, Q, H, F, C, and S.

[0123] In one embodiment, when a chimeric polypeptide described herein comprises a submembrane targeting domain linked to an anchor molecule, this is preferably located at the N-terminus of the chimeric polypeptide.

[0124] In one embodiment, the submembrane targeting domain may further comprise several basic amino acid residues, in particular selected from those comprising or consisting of K, R, and H. "Several" means at least two, preferably at least three, or more. These basic amino acid residues may be involved in the interaction with lipids of cell or vesicle membranes, in particular choline and its derivatives (e.g. phosphatidylcholine), thus allowing to increase the affinity of the submembrane targeting domain for these membranes.

[0125] In one embodiment, the basic amino acid residues may be located at the consensus sequence having SEQ ID NO:45 and / or outside this consensus sequence.

[0126] Thus, in one embodiment, the submembrane targeting domain comprises: (M) GXXKS (SEQ ID NO: 46), CKXK (SEQ ID NO: 47), and CKXKXXXXRRR (SEQ ID NO:49) (In the formula, X represents any amino acid residue, (M) indicates an initiator methionine that, when located at the N-terminus of the chimeric polypeptide, can be removed in vivo by post-translational modification. or comprising or consisting of an amino acid sequence selected from A variant of any of these sequences, said variant retaining the ability to anchor the submembrane targeting domain in the lipid bilayer of a cell or vesicle membrane.

[0127] In one embodiment, the submembrane targeting domain is derived from a protein of the SrC family of proteins. Examples of such proteins include, but are not limited to, Src, Yes, Lyn, Fyn, Lck, Blk, Fgr, Hck and Yrk proteins (Resh, 1994. Cell. 76(3):411-413), particularly the N-terminal part of one of these proteins, such as the 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 N-terminal amino acid residues of one of these proteins.

[0128] In one embodiment, the submembrane targeting domain is derived from a c-Src or v-Src protein, preferably c-Src.

[0129] Alternatively, the submembrane targeting domain can be derived from other acylated proteins, such as viral capsid proteins, including, but not limited to, the human immunodeficiency virus (HIV) MA protein or filovirus proteins.

[0130] In one embodiment, the submembrane targeting domain is derived from a Src protein.

[0131] In one embodiment, the submembrane targeting domain is derived from the Src protein and has the following amino acid sequence: (M) GSSKSKPKDPSQRRR (SEQ ID NO: 50), (M) GSSKSKPKDPSQRRRKSR (SEQ ID NO: 51) (M) GSSKSKPKDPSQRRRKSRGPGG (SEQ ID NO: 52), or A variant of either of these two sequences, said variant retaining the ability to anchor the submembrane targeting domain in the lipid bilayer of a cell or vesicle membrane. (wherein (M) represents an initiator methionine that, when located at the N-terminus of the chimeric polypeptide, can be removed in vivo by post-translational processing). It comprises or consists of one of:

[0132] In one embodiment, the submembrane targeting domain is derived from the Src protein and comprises or consists of an amino acid sequence having SEQ ID NO: 51, or a variant thereof.

[0133] In one embodiment, a variant of an amino acid sequence having SEQ ID NO:50, 51, or 52 comprises an amino acid sequence sharing at least 70% overall sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more overall sequence identity with the amino acid sequence of SEQ ID NO:50, 51, or 52.

[0134] Additionally or alternatively, variants of the amino acid sequence having SEQ ID NO:50, 51, or 52 include amino acid sequences sharing at least 70% local sequence identity with the amino acid sequence of SEQ ID NO:50, 51, or 52, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more local sequence identity.

[0135] In one embodiment, the submembrane targeting domain is derived from the Src protein and comprises or consists of an amino acid sequence having SEQ ID NO: 50, 51 or 52 or a variant thereof, preferably SEQ ID NO: 51, and further comprises one or more anchor molecules as defined above, in particular myristic acid (in the form of a myristyl moiety) linked to the glycine residue at position 2.

[0136] In one embodiment, when a chimeric polypeptide comprises a submembrane targeting domain, the submembrane targeting domain can be linked to the remainder of the chimeric polypeptide via at least one linker.

[0137] In one embodiment, the chimeric polypeptide comprises, from the N-terminus to the C-terminus: an amino acid sequence of adiponectin as defined herein above or a variant thereof; the amino acid sequence of a transmembrane domain of a transmembrane protein as defined herein above; The amino acid sequence of the pilot peptide as defined hereinabove It comprises or consists of:

[0138] Optionally, one or more linkers can be added between the components of the chimeric polypeptides of the invention.

[0139] In one embodiment, the chimeric polypeptide comprises, from the N-terminus to the C-terminus: an amino acid sequence of adiponectin as defined herein above or a variant thereof; optionally a linker; the amino acid sequence of a transmembrane domain of a transmembrane protein as defined herein above; optionally a linker; and The amino acid sequence of the pilot peptide as defined hereinabove It comprises or consists of:

[0140] An example of such a construct is shown in FIG. 1 as "Construct 1."

[0141] In one embodiment, the chimeric polypeptide comprises, from the N-terminus to the C-terminus: The amino acid sequence of human wild-type adiponectin or a variant thereof as defined herein above, Optionally, a linker, the amino acid sequence of the transmembrane domain of CD8 as defined herein; Optionally, a linker, and The amino acid sequence of the pilot peptide as defined herein above (preferably, the pilot peptide comprises at least one YxxL motif and / or DYxxL motif having SEQ ID NO: 1 or SEQ ID NO: 4, respectively, and at least one PxxP motif having SEQ ID NO: 8). It comprises or consists of:

[0142] In one embodiment, the chimeric polypeptide comprises, from the N-terminus to the C-terminus: The amino acid sequence of human wild-type adiponectin or a variant thereof as defined herein above, Optionally, a linker, the amino acid sequence of the transmembrane domain of CD8 as defined herein; Optionally, a linker, and The amino acid sequence of the pilot peptide as defined herein above (preferably, the pilot peptide comprises three YxxL and / or DYxxL motifs having SEQ ID NO: 1 or SEQ ID NO: 4, respectively, and four PxxP motifs having SEQ ID NO: 8). It comprises or consists of:

[0143] In one embodiment, the chimeric polypeptide comprises, from the N-terminus to the C-terminus: The amino acid sequence of human wild-type adiponectin having SEQ ID NO: 33 or a variant thereof; Optionally, a linker, The amino acid sequence of the transmembrane domain of CD8 having SEQ ID NO: 37, or a variant thereof; Optionally, a linker, and The amino acid sequence of the pilot peptide having SEQ ID NO: 30 or a variant thereof It comprises or consists of:

[0144] In one embodiment, the chimeric polypeptide comprises or consists of an amino acid sequence having SEQ ID NO: 40, or a variant thereof. SEQ ID NO:40 MLLLGAVLLLLALPGHDQETTTQGPGVLLPLPKGACTGWMAGIPGHPGHNGAPGRDGRGTPGEKGEKGDPGLIGPKGDIGETGVPGAEGPRGFPGIQGRKGEPGEGAYVYRSAFSVGLETYVTIPNMPIRFTKIFYNQQNHYDGSTGKFHCNIPGLYYFAYHITVYMKDVKVSLF KKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQVYGEGERNGLYADNDNDSTFTGFLLYHDTNSGGGSGGGGSGGGSGGGGSGGGGSGGGGSDIYIWAPLAGICVALLLSLIITLICYHRSRGAPHFPEISFPPKPDSDYQALLPSAPEIYSHLSPTKPDYINLRPAP

[0145] In one embodiment, a variant of the amino acid sequence having SEQ ID NO:40 comprises an amino acid sequence sharing at least 70% overall sequence identity with the amino acid sequence of SEQ ID NO:40, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more overall sequence identity.

[0146] Additionally or alternatively, variants of the amino acid sequence having SEQ ID NO:40 include amino acid sequences sharing at least 70% local sequence identity with the amino acid sequence of SEQ ID NO:40, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more local sequence identity.

[0147] In one embodiment, the chimeric polypeptide comprises, from the N-terminus to the C-terminus: Optionally, the amino acid sequence of a pilot peptide as defined herein above, the amino acid sequence of the transmembrane domain of a transmembrane protein as defined herein above, The amino acid sequence of adiponectin as defined herein above or a variant thereof It comprises or consists of:

[0148] Optionally, one or more linkers can be added between the components of the chimeric polypeptides of the invention.

[0149] In one embodiment, the chimeric polypeptide comprises, from the N-terminus to the C-terminus: Optionally, the amino acid sequence of a pilot peptide as defined herein above, Optionally, a linker, the amino acid sequence of the transmembrane domain of a transmembrane protein as defined herein above, Optionally, a linker, and The amino acid sequence of adiponectin as defined herein above or a variant thereof It comprises or consists of:

[0150] Examples of such constructs are shown in FIG. 1 as "Construct 2" and "Construct 3."

[0151] In one embodiment, the chimeric polypeptide comprises, from the N-terminus to the C-terminus: Optionally, the amino acid sequence of a pilot peptide as defined herein above; (preferably, the pilot peptide comprises at least one YxxL motif and / or DYxxL motif having SEQ ID NO:1 or SEQ ID NO:4, respectively, and at least one PxxP motif having SEQ ID NO:8), Optionally, a linker, the amino acid sequence of the transmembrane domain of CD40L as defined herein above, Optionally, a linker, and The amino acid sequence of human wild-type adiponectin or a variant thereof as defined herein above. It comprises or consists of:

[0152] In one embodiment, the chimeric polypeptide comprises, from the N-terminus to the C-terminus: the amino acid sequence of a pilot peptide as defined herein above; (preferably, the pilot peptide comprises at least one YxxL and / or DYxxL motif having SEQ ID NO:1 or SEQ ID NO:4, respectively, and at least one PxxP motif having SEQ ID NO:8), the amino acid sequence of the transmembrane domain of CD40L as defined herein above, The amino acid sequence of human wild-type adiponectin or a variant thereof as defined herein above. It comprises or consists of:

[0153] Optionally, one or more linkers can be added between the components of the chimeric polypeptides of the invention.

[0154] In one embodiment, the chimeric polypeptide comprises, from the N-terminus to the C-terminus: Optionally, the amino acid sequence of a pilot peptide as defined herein above (preferably, the pilot peptide comprises three YxxL and / or DyxxL motifs having SEQ ID NO: 1 or 4, respectively, and four PxxP motifs having SEQ ID NO: 8), Optionally, a linker, the amino acid sequence of the transmembrane domain of CD40L as defined herein above, Optionally, a linker, and The amino acid sequence of human wild-type adiponectin or a variant thereof as defined herein above. It comprises or consists of:

[0155] In one embodiment, the chimeric polypeptide comprises, from the N-terminus to the C-terminus: the amino acid sequence of a pilot peptide as defined herein above; (preferably, the pilot peptide comprises three YxxL and / or DyxxL motifs having SEQ ID NO: 1 or 4, respectively, and four PxxP motifs having SEQ ID NO: 8), the amino acid sequence of the transmembrane domain of CD40L as defined herein above, The amino acid sequence of human wild-type adiponectin or a variant thereof as defined herein above. It comprises or consists of:

[0156] Optionally, one or several linkers can be added between the components of the chimeric polypeptide of the invention.

[0157] In one embodiment, the chimeric polypeptide comprises, from the N-terminus to the C-terminus: The amino acid sequence of the pilot peptide having SEQ ID NO: 30 or a variant thereof; the amino acid sequence of the transmembrane domain of CD40L as defined herein above, The amino acid sequence of human wild-type adiponectin or a variant thereof as defined herein above. It comprises or consists of:

[0158] Optionally, one or several linkers can be added between the components of the chimeric polypeptide of the invention.

[0159] In one embodiment, the chimeric polypeptide comprises, from the N-terminus to the C-terminus: Optionally, the amino acid sequence of the pilot peptide having SEQ ID NO: 30, or a variant thereof; Optionally, a linker, The amino acid sequence of the transmembrane domain of CD40L having SEQ ID NO: 35 or a variant thereof; Optionally, a linker, and The amino acid sequence of human wild-type adiponectin having SEQ ID NO: 31 or a variant thereof It comprises or consists of:

[0160] In one embodiment, the chimeric polypeptide comprises, from the N-terminus to the C-terminus: The amino acid sequence of the pilot peptide having SEQ ID NO: 30 or a variant thereof; The amino acid sequence of the transmembrane domain of CD40L having SEQ ID NO: 35 or a variant thereof; The amino acid sequence of human wild-type adiponectin having SEQ ID NO: 31 or a variant thereof It comprises or consists of:

[0161] Optionally, one or several linkers can be added between the components of the chimeric polypeptide of the invention.

[0162] In one embodiment, the chimeric polypeptide comprises or consists of an amino acid sequence having SEQ ID NO: 41, or a variant thereof. SEQ ID NO:41 MSRGSMIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKGGGSGGGGGSGGGSGGGGSGGGGGGSGGGSGETTTQGPGVLLPLPKGACTGWMAGIPGHPGHNGAPGRDGRGTPGEKGEKGDPGLIGPKGDIGETGVP GAEGPRGFPGIQGRKGEPGEGAYVYRSAFSVGLETYVTIPNMPIRFTKIFYNQQNHYDGSTGKFHCNIPGLYYFAYHITVYMKDVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQVYGEGERNGLYADNDNDSTFTGFLLYHDTN

[0163] In one embodiment, where the pilot peptide is not optional in the above embodiments, the chimeric polypeptide comprises or consists of an amino acid sequence having SEQ ID NO: 42, or a variant thereof. SEQ ID NO:42 MSRGGGAPHFPEISFPPKPDSDYQALLPSAPEIYSHLSPTKPDYINLRPAPGGSRGSMIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKGGGSGGGGSGGGSGGGGSGGGSGGGSGGGSGETTTQGPGVLLPLPKGACTGWMAGIPGHPGHNGAPGRDGRDG TPGEKGEKGDPGLIGPKGDIGETGVPGAEGPRGFPGIQGRKGEPGEGAYVYRSAFSVGLETYVTIPNMPIRFTKIFYNQQNHYDGSTGKFHCNIPGLYYFAYHITVYMKDVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQVYGEGERNGLYADNDNDSTFTGFLLYHDTN

[0164] In one embodiment, a variant of the amino acid sequence having SEQ ID NO:41 or SEQ ID NO:42 comprises an amino acid sequence sharing at least 70% overall sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more overall sequence identity with the amino acid sequence of SEQ ID NO:41 or 42, respectively.

[0165] Additionally or alternatively, variants of the amino acid sequence having SEQ ID NO:41 or SEQ ID NO:42 include amino acid sequences sharing at least 70% local sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more local sequence identity with the amino acid sequence of SEQ ID NO:41 or 42, respectively.

[0166] In one embodiment, the chimeric polypeptide comprises, from the N-terminus to the C-terminus: the amino acid sequence of a just membrane targeting domain as defined herein above (preferably said just membrane targeting domain is linked to an anchor molecule), Optionally, the amino acid sequence of a pilot peptide as defined herein above, the amino acid sequence of the transmembrane domain of a transmembrane protein as defined herein above, The amino acid sequence of adiponectin as defined herein above or a variant thereof It comprises or consists of:

[0167] Optionally, one or several linkers can be added between the components of the chimeric polypeptide of the invention.

[0168] In one embodiment, the chimeric polypeptide comprises, from the N-terminus to the C-terminus: the amino acid sequence of a just membrane targeting domain as defined herein above; (preferably, said just membrane targeting domain is linked to an anchor molecule), Optionally, a linker, Optionally, the amino acid sequence of a pilot peptide as defined herein above, Optionally, a linker, the amino acid sequence of the transmembrane domain of a transmembrane protein as defined herein above, Optionally, a linker, and The amino acid sequence of adiponectin as defined herein above or a variant thereof It comprises or consists of:

[0169] Examples of such constructs are shown in FIG. 1 as "Construct 4" and "Construct 5."

[0170] In one embodiment, the chimeric polypeptide comprises, from the N-terminus to the C-terminus: the amino acid sequence of a just membrane targeting domain having the sequence (M)G-X1-X2-X3-S / C (SEQ ID NO: 45) as defined herein above (preferably, said just membrane targeting domain is linked to a fatty acid); Optionally, a linker, Optionally, the amino acid sequence of a pilot peptide as defined herein above; (preferably, the pilot peptide comprises at least one YxxL motif and / or DYxxL motif having SEQ ID NO:1 or SEQ ID NO:4, respectively, and at least one PxxP motif having SEQ ID NO:8), Optionally, a linker, the amino acid sequence of the transmembrane domain of CD40L as defined herein above, Optionally, a linker, and The amino acid sequence of human wild-type adiponectin or a variant thereof as defined herein above. It comprises or consists of:

[0171] In one embodiment, the chimeric polypeptide comprises, from the N-terminus to the C-terminus: an amino acid sequence of a just membrane targeting domain having the sequence (M)G-X1-X2-X3-S / C (SEQ ID NO: 45) as defined herein above, preferably said just membrane targeting domain being linked to a fatty acid, Optionally, a linker, Optionally, the amino acid sequence of a pilot peptide as defined herein above; (preferably, the pilot peptide comprises three YxxL and / or DyxxL motifs having SEQ ID NO: 1 or 4, respectively, and four PxxP motifs having SEQ ID NO: 8), Optionally, a linker, the amino acid sequence of the transmembrane domain of CD40L as defined herein above, Optionally, a linker, and The amino acid sequence of human wild-type adiponectin or a variant thereof as defined herein above. It comprises or consists of:

[0172] In one embodiment, the chimeric polypeptide comprises, from the N-terminus to the C-terminus: The amino acid sequence of the just membrane targeting domain having SEQ ID NO: 51 (preferably, said just membrane targeting domain is linked to a fatty acid, preferably myristic acid) Optionally, a linker, Optionally, the amino acid sequence of the pilot peptide having SEQ ID NO: 30, or a variant thereof; Optionally, a linker, The amino acid sequence of the transmembrane domain of CD40L having SEQ ID NO: 35 or a variant thereof; Optionally, a linker, and The amino acid sequence of human wild-type adiponectin having SEQ ID NO: 31 or a variant thereof It comprises or consists of:

[0173] In one embodiment, the chimeric polypeptide comprises or consists of an amino acid sequence having SEQ ID NO: 43, or a variant thereof. SEQ ID NO:43 MGSSKSKPKDPSQRRRKSRGPGGGSGGGSGGGSGGGSSRGSMIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKGGGSGGGGSGGGSGGGGSGGGSGGGSGGGSGETTTQGPGVLLPLPKGACTGWMAGIPGHPGHNGAPGRDGRDGTPGEKGEK GDPGLIGPKGDIGETGVPGAEGPRGFPGIQGRKGEPGEGAYVYRSAFSVGLETYVTIPNMPIRFTKIFYNQQNHYDGSTGKFHCNIPGLYYFAYHITVYMKDVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQVYGEGERNGLYADNDNDSTFTGFLLYHDTN

[0174] In one embodiment, where the pilot peptide is not optional in the above embodiments, the chimeric polypeptide comprises or consists of an amino acid sequence having SEQ ID NO: 44, or a variant thereof. SEQ ID NO:44 MGSSKSKPKDPSQRRRKSRGPGGGSGGGSGGGSGGGSSRGGGAPHFPEISFPPKPDDSDYQALLPSAPEIYSHLSPTKPDYINLRPAPGGSRGSMIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKGGGSGGGGSGGGSGGGGSGGGSGGGGGSGGGSGETTTQGPGVLLPLPKGACTGWMAG IPGHPGHNGAPGRDGRDGTPGEKGEKGDPGLIGPKGDIGETGVPGAEGPRGFPGIQGRKGEPGEGAYVYRSAFSVGLETYVTIPNMPIRFTKIFYNQQNHY DGSTGKFHCNIPGLYYFAYHITVYMKDVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQVYGEGERNGLYADNDNDSTFTGFLLYHDTN

[0175] In one embodiment, a variant of the amino acid sequence having SEQ ID NO:43 or SEQ ID NO:44 comprises an amino acid sequence sharing at least 70% overall sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more overall sequence identity with the amino acid sequence of SEQ ID NO:43 or SEQ ID NO:44, respectively.

[0176] Additionally or alternatively, variants of the amino acid sequence having SEQ ID NO:43 or SEQ ID NO:44 include amino acid sequences sharing at least 70% local sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more local sequence identity with the amino acid sequence of SEQ ID NO:43 or SEQ ID NO:44, respectively.

[0177] The present invention further relates to a nucleic acid encoding a chimeric polypeptide as defined herein above.

[0178] In one embodiment, the nucleic acid is comprised in a nucleic acid vector, such as a nucleic acid expression vector.

[0179] In one embodiment, the nucleic acid is included in a nucleic acid vector, such as a nucleic acid expression vector, and is operably linked to a regulatory element.

[0180] Examples of regulatory elements include, but are not limited to, promoters, Kozak consensus initiation sequences, polyadenylation signals, termination sequences (i.e., stop codons), etc. In particular, the regulatory elements are suitable for expression of nucleic acids in cells, such as bacteria, yeast, insect cells, mammalian cells, or human cells.

[0181] The present invention further relates to an extracellular vesicle (EV) comprising a chimeric polypeptide as defined herein above.

[0182] In one embodiment, the extracellular vesicle has adiponectin contained in the chimeric polypeptide on its outer surface. In one embodiment, the transmembrane domain of the chimeric polypeptide is anchored in the lipid bilayer of the extracellular vesicle.

[0183] As used herein, the expression "carried on its outer surface" means that the adiponectin contained in the chimeric polypeptide is partially or completely exposed to the outside of the extracellular vesicle. This configuration allows the adiponectin contained in the chimeric polypeptide to form oligomers with either other adiponectins of adjacent chimeric polypeptides or soluble adiponectins in the same extracellular vesicle.

[0184] In one embodiment, the extracellular vesicles are small molecule extracellular vesicles.

[0185] In one embodiment, the extracellular vesicles are exosomes.

[0186] In one embodiment, the exosomes have a diameter in the range of about 30 nm to about 150 nm, preferably about 30 nm to about 120 nm, more preferably about 40 to about 80 nm. In one embodiment, the exosomes have a diameter in the range of about 30 nm to about 120 nm.

[0187] A further object of the present invention is a population of extracellular vesicles as defined herein above.

[0188] In one embodiment, the population of extracellular vesicles is monodispersed in an aqueous solution, preferably a 0.9% aqueous NaCl solution and / or PBS.

[0189] "Monodisperse" means that the size of the extracellular vesicles in a population of extracellular vesicles is substantially uniform. "Substantially uniform" means that the extracellular vesicles have a narrow size distribution of approximately the average size. In one embodiment, the extracellular vesicles in 0.9% aqueous NaCl solution and / or PBS have a size that exhibits a standard deviation of less than 100%, for example, less than 75%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% with respect to the average size.

[0190] In one embodiment, the population of extracellular vesicles further comprises soluble adiponectin, i.e., adiponectin protein in free form, in other words, not contained in the chimeric polypeptide of the present invention.

[0191] In one embodiment, the soluble adiponectin is wild-type adiponectin. In one embodiment, the soluble adiponectin is a mutant adiponectin.

[0192] In one embodiment, the soluble adiponectin is wild-type human adiponectin. In one embodiment, the wild-type human adiponectin comprises or consists of the amino acid sequence having SEQ ID NO: 33 or a variant thereof. In one embodiment, the wild-type human adiponectin comprises or consists of the amino acid sequence having SEQ ID NO: 31 or a variant thereof.

[0193] A further object of the present invention is a method for obtaining an extracellular vesicle or a population of extracellular vesicles comprising a chimeric polypeptide as defined hereinabove.

[0194] General means and methods for obtaining extracellular vesicles or populations of extracellular vesicles are well known in the art. See, for example, Whitford & Guterstam, 2019. Future Med Chem. 11(10):1225-1236; Taylor & Shah, 2015. Methods. 87:3-10; Desplantes et al., 2017. Sci Rep. 7(1):1032.

[0195] In one embodiment, the method for obtaining an extracellular vesicle or a population of extracellular vesicles comprises the step of producing an extracellular vesicle or a population of extracellular vesicles as defined hereinabove.

[0196] In one embodiment, the step of producing the extracellular vesicle or population of extracellular vesicles comprises transfecting a cell with a nucleic acid encoding a chimeric polypeptide as defined herein above.

[0197] In one embodiment, the cell is a cell from a HEK293T cell or a derived cell line. In one embodiment, the cell is an adipocyte. In one embodiment, the cell is an immune cell, including but not limited to a mast cell, a lymphocyte (such as a T cell or a B cell), and a dendritic cell. In one embodiment, the cell is a stem cell, including but not limited to an embryonic stem cell, an adult stem cell (such as a hematopoietic stem cell, a mammary stem cell, an intestinal stem cell, a mesenchymal stem cell, an endothelial stem cell, a neural stem cell, an olfactory adult stem cell, or a neural crest stem cell), a cancer stem cell, an induced pluripotent stem cell (iPSC), and an induced stem cell (iSC).

[0198] In one embodiment, the method for obtaining extracellular vesicles or a population of extracellular vesicles further comprises culturing the transfected cells, preferably in a medium lacking extracellular vesicles (i.e., serum-free medium, medium supplemented with extracellular vesicle-depleted serum, or medium supplemented with extracellular vesicle-depleted platelet lysate) for a sufficient time to allow production of extracellular vesicles.

[0199] In one embodiment, the method for obtaining an extracellular vesicle or a population of extracellular vesicles further comprises purifying said extracellular vesicle or population of extracellular vesicles.

[0200] In one embodiment, the step of purifying the extracellular vesicles or population of extracellular vesicles comprises clarification of the transfected cell culture supernatant (e.g., by centrifugation or depth filtration), filtration, ultrafiltration, diafiltration, molecular sieve purification and / or ion exchange chromatography. Other methods for purifying extracellular vesicles or populations of extracellular vesicles include, but are not limited to, ultracentrifugation, tangential flow filtration (TFF) and BE-SEC chromatography.

[0201] In one embodiment, the extracellular vesicles or populations of extracellular vesicles of the invention are purified. Thus, the invention also relates to purified extracellular vesicles or populations of extracellular vesicles.

[0202] Methods for purification are well known to those of skill in the art and include, but are not limited to, the purification methods described herein above.

[0203] In one embodiment, the extracellular vesicles or populations of extracellular vesicles of the present invention are purified by ultracentrifugation to obtain semi-purified extracellular vesicles or populations of extracellular vesicles. Thus, the present invention also relates to semi-purified extracellular vesicles or populations of extracellular vesicles.

[0204] As used herein, semi-purified extracellular vesicles or populations of extracellular vesicles include the extracellular vesicles or populations of extracellular vesicles, proteins that are anchored to the membrane of the extracellular vesicles or populations of extracellular vesicles or tightly associated with the extracellular vesicles or populations of extracellular vesicles, and the crown of proteins associated with the extracellular vesicles or populations of extracellular vesicles (see FIG. 2B).

[0205] In one embodiment, the extracellular vesicles or populations of extracellular vesicles of the present invention are purified by tangential flow filtration and chromatography, in particular SEC and BE-SEC chromatography, to obtain ultra-purified extracellular vesicles or populations of extracellular vesicles.Thus, the present invention also relates to ultra-purified extracellular vesicles or populations of extracellular vesicles.

[0206] As used herein, ultra-purified extracellular vesicles or populations of extracellular vesicles include extracellular vesicles or populations of extracellular vesicles and proteins that are anchored to the membrane of the extracellular vesicles or populations of extracellular vesicles or tightly associated with the ultra-purified extracellular vesicles or populations of extracellular vesicles (see FIG. 2C).

[0207] An example of the production of extracellular vesicles in HEK293T cells is provided in the Examples section below.

[0208] The present invention further relates to a composition comprising, consisting essentially of, or consisting of a chimeric polypeptide, a nucleic acid, an extracellular vesicle, or a population of extracellular vesicles as described herein above.

[0209] As used herein, "consisting essentially of" with respect to a composition means that the chimeric polypeptide, nucleic acid, extracellular vesicle, or population of extracellular vesicles is the only biologically active therapeutic or agent in the composition.

[0210] In one embodiment, the composition further comprises soluble adiponectin, ie adiponectin in its free form as already defined above.

[0211] The present invention further relates to a pharmaceutical composition comprising, consisting essentially of, or consisting of a chimeric polypeptide, a nucleic acid, an extracellular vesicle, or a population of extracellular vesicles as defined herein above, and at least one pharma- ceutically acceptable excipient.

[0212] The term "pharmaceutical acceptable excipient" includes any solvent, diluent, dispersion medium, coating agent, antibacterial and antifungal agent, isotonic and absorption delaying agent, etc. The excipient does not produce any adverse, allergic or other undesirable reaction when administered to animals, preferably humans. For human administration, preparations must meet the sterility, pyrogenicity and general safety and purity standards required by regulatory agencies, such as the FDA or EMA.

[0213] Pharmaceutically acceptable excipients that may be used in these pharmaceutical compositions include, but are not limited to, ion exchange agents, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of vegetable oil saturated fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances (e.g., carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene polyoxypropylene block polymers, polyethylene glycol, and wool fat.

[0214] In one embodiment, the pharmaceutical composition further comprises soluble adiponectin, ie adiponectin in free form as already defined above.

[0215] The present invention further relates to a medicament comprising, consisting essentially of, or consisting of a chimeric polypeptide, a nucleic acid, an extracellular vesicle, or a population of extracellular vesicles as defined herein above.

[0216] In one embodiment, the medicament further comprises soluble adiponectin, ie adiponectin in free form as already defined above.

[0217] In one embodiment, the composition, pharmaceutical composition or medicament comprises a purified extracellular vesicle or a population of extracellular vesicles as defined herein above.

[0218] The present invention further relates to a kit of parts, comprising in a first part an extracellular vesicle or a population of extracellular vesicles as defined herein above, and in a second part a soluble adiponectin, i.e. adiponectin in free form as already defined above.

[0219] In one embodiment the two parts of the kit-of-parts are intended for simultaneous use or for sequential use in any order.

[0220] The present invention further relates to a chimeric polypeptide, a nucleic acid, an extracellular vesicle, a population of extracellular vesicles, a composition, a pharmaceutical composition, a medicament, or a kit-of-parts as defined herein above for use as a drug or medicament.

[0221] The present invention further relates to a chimeric polypeptide, a nucleic acid, an extracellular vesicle, a population of extracellular vesicles, a composition, a pharmaceutical composition, a medicament, or a kit-of-parts as defined herein above for use in the treatment of a disease, disorder, or condition in a subject in need thereof.

[0222] The present invention further relates to the chimeric polypeptide, nucleic acid, extracellular vesicle, population of extracellular vesicles, composition, pharmaceutical composition, medicament, or kit-of-parts as defined herein above in the manufacture of a medicament for treating a disease, disorder, or condition in a subject in need thereof.

[0223] The present invention further relates to a method for treating a disease, disorder or condition in a subject in need thereof, comprising or consisting of administering to said subject a chimeric polypeptide, a nucleic acid, an extracellular vesicle, a population of extracellular vesicles, a composition, a pharmaceutical composition, a medicament or a kit-of-parts as defined herein above.

[0224] In one embodiment, the disease, disorder, or condition is selected from the group comprising or consisting of obesity, insulin resistance, diseases associated with insulin resistance or deficiency, hypertension, dyslipidemia, hyperuricemia, atherosclerosis (including coronary artery disease, stroke, and peripheral arterial disease), fibrosis, inflammatory lung disease, nephrotic disease, sleep apnea, dry eye disease, inflammatory eye disease, gastritis and gastroesophageal reflux disease, inflammatory bowel disease, pancreatitis, osteoporosis, and inflammatory bone and joint disease. In one embodiment, the disease, disorder, or condition is diabetes.

[0225] Examples of diseases associated with insulin resistance or deficiency include, but are not limited to, type 2 diabetes, metabolic syndrome, cardiovascular disease, non-alcoholic fatty liver disease, polycystic ovarian syndrome, Alzheimer's disease, and cancer (particularly endometrial cancer, menopausal breast cancer, leukemia, colon cancer, gastric cancer, and prostate cancer). In one embodiment, the disease is type 2 diabetes.

[0226] Examples of inflammatory lung diseases include, but are not limited to, asthma, allergic asthma, emphysema, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome, bronchitis, pneumonia, cystic fibrosis, pulmonary fibrosis, and pulmonary sarcoidosis.

[0227] Examples of dry eye disease include, but are not limited to, aqueous tear deficiency, xerophthalmia, Sjogren's syndrome dry eye, non-Sjogren's syndrome dry eye, keratoconjunctivitis sicca, aqueous tear deficiency dry eye, evaporative dry eye, Stevens-Johnson syndrome, borderline ocular pemphigoid blepharitis, dry eye associated with allergic conjunctivitis, dry eye associated with post-viral conjunctivitis, dry eye associated with post-cataract surgery, dry eye associated with VDT work, and dry eye associated with contact lens wear.

[0228] Examples of inflammatory eye diseases include, but are not limited to, uveitis, scleritis, inflammation after ocular surgery, corneal transplants, corneal wound healing, conjunctivitis, retinal diseases, glaucoma, and ocular hypertension.

[0229] Examples of inflammatory bone and joint diseases include, but are not limited to, osteitis fibrosa cystica, osteomyelitis, sesamoiditis, Brodie abscess, periostitis, costochondritis, and polychondritis.

[0230] In one embodiment, the disease, disorder, or condition is or is associated with hypoadiponectinemia.

[0231] As used herein, "hypoadiponectinemia" refers to a reduced level of adiponectin in the bloodstream compared to standard levels. Methods for measuring levels of adiponectin in the bloodstream are well known to those of skill in the art and include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), AlphaLISA immunoassay, and immunoturbidimetry (such as latex particle enhanced immunoturbidimetry).

[0232] In one embodiment, the disease, disorder or condition is obesity.In one embodiment, the disease, disorder or condition is insulin resistance.In one embodiment, the disease, disorder or condition is insulin resistance or deficiency related disease, such as type 2 diabetes, metabolic syndrome, cardiovascular disease, non-alcoholic fatty liver disease, polycystic ovarian syndrome, Alzheimer's disease, and cancer; in particular type 2 diabetes and metabolic syndrome.In one embodiment, the disease, disorder or condition is type 2 diabetes.

[0233] The present invention also relates to an extracellular vesicle, or a population thereof, having adiponectin exposed on its outer surface for use in treating a disease, disorder, or condition selected from the group including obesity, insulin resistance, diseases associated with insulin resistance or deficiency, hypertension, dyslipidemia, hyperuricemia, atherosclerosis (including coronary artery disease, stroke, and peripheral arterial disease), fibrosis, inflammatory lung disease, nephrotic disease, sleep apnea, dry eye disease, inflammatory eye disease, gastritis and gastroesophageal reflux disease, inflammatory bowel disease, pancreatitis, osteoporosis, and inflammatory bone and joint disease. In one embodiment, the disease, disorder, or condition is diabetes.

[0234] In one embodiment, the extracellular vesicles are partially or completely coated with recombinant adiponectin.

[0235] "Recombinant adiponectin" refers to exogenous adiponectin that is not endogenously produced by cells. Extracellular vesicles partially or completely coated with recombinant adiponectin can be obtained by contacting extracellular vesicles with adiponectin in cellulo (e.g., by transfecting an extracellular vesicle-producing cell with a nucleic acid encoding adiponectin, thereby causing the cell to produce exogenous adiponectin) or ex cellulo (e.g., by providing adiponectin in a protein form that has been previously produced and further purified in a suitable recombinant expression system).

[0236] By "coated" it is meant that the adiponectin is exposed on the outer surface of the extracellular vesicle, to which the adiponectin is bound via any suitable type of interaction with an external component of the extracellular vesicle (such as, but not limited to, electrostatic interactions, protein-protein interactions, protein-lipid interactions, etc.).

[0237] In one embodiment, the extracellular vesicles are partially or completely coated with lactadherin, in particular recombinant adiponectin fused to functional C1 and / or C2 domains of lactadherin, as described in International Patent Publication No. 2003016522 (the relevant contents of which are incorporated herein by reference).

[0238] In one embodiment, the chimeric polypeptide, nucleic acid, extracellular vesicle, population of extracellular vesicles, composition, pharmaceutical composition, medicament, or kit-of-parts component defined herein above is formulated for administration to a subject in need thereof.

[0239] In one embodiment, administration to a subject can be parenterally, by inhalation spray, rectally, nasally, or via an implanted reservoir. The term "administering" specifically includes subcutaneous, intravenous, intramuscular, intraarterial, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques.

[0240] In one embodiment, the chimeric polypeptide, nucleic acid, extracellular vesicle, population of extracellular vesicles, composition, pharmaceutical composition, medicament, or kit-of-parts components defined herein above are administered to a subject in need thereof in a therapeutically effective amount.

[0241] However, it will be understood that the total daily usage of the chimeric polypeptides, nucleic acids, extracellular vesicles, populations of extracellular vesicles, compositions, pharmaceutical compositions, medicaments, or components of the kit-of-parts defined hereinabove will be decided by the attending physician within the scope of sound medical judgment.

[0242] In particular, the specific therapeutically effective dose level for any particular patient depends on a variety of factors, including the disease and severity of the disease being treated; the activity of the chimeric polypeptide, nucleic acid, extracellular vesicle, population of extracellular vesicles, composition, pharmaceutical composition, medicament, or kit-of-parts components used as defined hereinabove; the age, weight, general health, sex, and diet of the subject; the administration time, administration route, and excretion rate of the chimeric polypeptide, nucleic acid, extracellular vesicle, population of extracellular vesicles, composition, pharmaceutical composition, medicament, or kit-of-parts components used as defined hereinabove; the duration of treatment; drugs used in combination or simultaneously with the chimeric polypeptide, nucleic acid, extracellular vesicle, population of extracellular vesicles, composition, pharmaceutical composition, medicament, or kit-of-parts components used as defined hereinabove; and similar factors well known in the medical field. The total dose required for each treatment may be administered in multiple doses or in a single dose.

[0243] The present invention also provides a method of diagnosing obesity, insulin resistance, or a disease associated with insulin resistance or deficiency in a subject, comprising the steps of: a) measuring the level or amount of adiponectin-associated small extracellular vesicles in a sample previously collected from a subject; b) comparing the level or amount determined in step a) with a reference level or amount of adiponectin-associated small extracellular vesicles, a reference level or amount is predetermined in a sample from a reference subject known not to be affected by obesity or insulin resistance, or in a pool of samples from more than one reference subject, all of whom are not affected by obesity or insulin resistance; c) if the level or amount determined in step a) is lower than the reference level or amount, concluding that the subject suffers from obesity, insulin resistance, or a disease associated with insulin resistance or deficiency. The present invention relates to a method comprising the steps of:

[0244] The present invention also relates to the in vitro or in vivo use of the chimeric polypeptides, nucleic acids, extracellular vesicles, and populations of extracellular vesicles of the invention in non-therapeutic methods.

[0245] In one embodiment, the chimeric polypeptides, nucleic acids, extracellular vesicles, and populations of extracellular vesicles of the present invention are used in vitro or in vivo to assess the function or biological activity of adiponectin in various biological processes.

[0246] In one embodiment, the chimeric polypeptides, nucleic acids, extracellular vesicles, and populations of extracellular vesicles of the present invention are used in vitro or in vivo to determine the effect of adiponectin on a given biological process. [Brief description of the drawings]

[0247] [Figure 1] FIG. 1 shows five exemplary constructs of chimeric polypeptides according to the invention. [Diagram 2] Figure 2 is a schematic representation of EVs and their environment in culture medium (Figure 2A), semi-purified EVs (Figure 2B), and ultra-purified EVs (Figure 2C). Figure 2A: EVs in culture medium are associated with proteins and contaminants. Figure 2: EVs semi-purified by ultracentrifugation are associated with a crown of associated proteins. Figure 2C: EVs ultra-purified by TFF (tangential flow filtration) and chromatography are associated with membrane-anchored or tightly membrane-associated proteins. [Diagram 3] Figure 3 is an immunoblot showing that the chimeric polypeptide of sequence SEQ ID NO: 40 is expressed in cell extracts and EVs. It shows immunoblot analysis of extracts of cells and EVs expressing the chimeric polypeptide (3, 5) and control cells and EVs not expressing the chimeric polypeptide (2, 4), where expression of the chimeric polypeptide is detected by antibodies targeting pilot peptides (2, 3, 4, 5). (1) represents a molecular weight marker. [Figure 4]Figure 4 is an immunoblot showing that adiponectin contained in chimeric polypeptides in EVs is multimeric. It shows immunoblot analysis of EV extracts expressing the chimeric polypeptide of sequence SEQ ID NO: 40 (3, 4) or a control EV extract not expressing the chimeric polypeptide (2), where the chimeric polypeptide is revealed by an antibody targeting the pilot peptide. The experiments with extracts 2, 3 are carried out under reducing conditions (DTT), whereas the experiment with extract 4 is carried out under non-reducing conditions that preserve the multimeric structure. (1) represents the molecular weight marker. [Diagram 5] Figure 5 is a combination of three graphs showing the presence of EV markers and adiponectin on the surface of EVs expressing the chimeric polypeptide of sequence SEQ ID NO: 40. The EVs expressing the chimeric polypeptide were subjected to ELISA testing with antibodies targeting two EV markers, CD81 and CD63, and with an antibody targeting adiponectin. The anti-adiponectin antibody does not react with control EVs that do not express the chimeric polypeptide. [Figure 6]Figure 6 is a combination of diagram, immunoblot, and table showing the production of adiponectin in EVs semi-purified from culture medium. Figure 6A is a schematic diagram of semi-purified EVs. Figure 6B is a Western blot of semi-purified EVs in reducing conditions. Lane 0 corresponds to cells transiently transfected with a DNA construct containing an empty vector with only the pilot peptide (0). Lane "wt" corresponds to cells transiently transfected with DNA corresponding to wild-type adiponectin. The other lanes correspond to cells transiently transfected with DNA encoding chimeric adiponectin corresponding to construct 1 (SEQ ID NO: 40, lane 1), construct 2 (SEQ ID NO: 42, lane 2), construct 3 (SEQ ID NO: 41, lane 3), construct 4 (SEQ ID NO: 43, lane 4), construct 5 (SEQ ID NO: 44, lane 5). Figure 6C is a schematic diagram of wild type adiponectin (wt) and chimeric adiponectin corresponding to chimeric adiponectin differently anchored to the membrane and having constructs 1 through 5. Figure 6D is a table showing the sizes (kDa) of the proteins identified by the asterisks (*) in Figure 6B. [Figure 7] Figure 7 is a combination of a schematic and immunoblot showing the production of multimeric adiponectin in EVs semi-purified from culture medium. Figure 7A is a schematic of semi-purified EVs. Figure 7B is a Western blot of semi-purified EVs in non-reducing conditions. Lane 0 corresponds to cells transiently transfected with a DNA construct containing an empty vector with only the pilot peptide (0). Lane "wt" corresponds to the analysis of cells transiently transfected with DNA corresponding to wild-type adiponectin. The other lanes correspond to cells transiently transfected with DNA encoding chimeric adiponectin corresponding to construct 1 (SEQ ID NO: 40, lane 1), construct 2 (SEQ ID NO: 42, lane 2), construct 3 (SEQ ID NO: 41, lane 3), construct 5 (SEQ ID NO: 44, lane 5). [Figure 8]Figure 8 is a combination of a schematic and immunoblot showing the production of adiponectin in EVs ultra-purified from culture medium. Figure 8A is a schematic of ultra-purified EVs. Figure 8B shows a Western blot in reducing conditions of cell extracts and ultra-purified EVs. Lane (wt) corresponds to wild-type adiponectin. Lane 0 represents control cells, lane 2 represents membrane-anchored adiponectin with construct 2, and lane (2+wt) represents a mixture of adiponectin (wt) and membrane-anchored adiponectin. Figure 8C shows a Western blot in non-reducing conditions of ultra-purified EVs. Lane (wt) corresponds to wild-type adiponectin. Lane 0 represents control cells, lane 2 represents membrane-anchored adiponectin with construct 2, and lane (2+wt) represents a mixture of adiponectin (wt) and membrane-anchored adiponectin. [Figure 9] Figure 9 is a combination of immunoblots, histograms and graphs showing the characteristics of ultra-purified EVs. Figure 9A shows Western blots in reducing / non-reducing conditions of cell extracts and ultra-purified EVs with Alix marker as control (left panel: cell extracts in reducing conditions, middle panel: EVs in reducing conditions, right panel: EVs in non-reducing conditions). Lane (wt) corresponds to cells expressing wild-type adiponectin, lane 0 represents adiponectin of control cells, lane 2 represents membrane-anchored adiponectin with construct 2. Figure 9B is a combination of two graphs showing the detection by ELISA of CD81 EV marker (left graph) and adiponectin on EVs (right graph). Figure 9C is a histogram showing quantification of adiponectin by ELISA in EVs from control cells (left column), cells expressing wild-type adiponectin (middle column), and cells expressing chimeric adiponectin with construct 2 (right column). [Figure 10]Figure 10 is a combination of two histograms showing the characteristics of ultra-purified EVs in terms of EV concentration (Figure 10A) and size (Figure 10B). Column (EV "0") represents EVs of control cells, column (EV "wt") represents EVs of cells stably expressing wild type adiponectin, and (EV "2") represents EVs of cells expressing DNA encoding chimeric adiponectin with construct 2 (SEQ ID NO: 42). [Figure 11] FIG. 11 is a combination of six graphs showing the ELISA characterization of ultra-purified EVs showing adiponectin at time of production (T0 4°C) and 3 months after production stored in different conditions (4°C or -80°C). It represents the ELISA characterization of adiponectin (Adpn) and CD81 preservation of ultra-purified EVs at time of production (T0 4°C) and 3 months after production under storage at +4°C and -80°C. EV "0" represents the EVs of control cells. EV "wt" represents the EVs of cells stably expressing wild-type adiponectin and EV "2" represents the EVs of cells expressing DNA encoding chimeric adiponectin with construct 2 (SEQ ID NO: 42). [Figure 12] FIG. 12 is a histogram showing the quantification of adiponectin in ultra-purified EVs showing adiponectin stored in different conditions (4° C. or −80° C.) at time of production 0 (T0 4° C.) and 3 months after production. It represents the results of a quantitative ELISA analysis characterizing the preservation of adiponectin (Adpn) in ultra-purified EVs (EV “wt” and EV “2”) starting from culture medium of cells stably expressing adiponectin at time of production 0 (T0 4° C.) and 3 months after production under storage at +4° C. and −80° C. EV “0” represents the EV of control cells. EV “wt” represents the EV of cells stably expressing wild-type adiponectin and EV “2” represents the EV of cells expressing DNA encoding chimeric adiponectin with construct 2 (SEQ ID NO: 42). [Figure 13]FIG. 13 is a combination of two histograms showing the analysis of size and concentration of ultra-purified EVs presenting adiponectin stored under different conditions (4° C. or −80° C.) at time of production 0 (T0 4° C.) and 3 months after production. These histograms represent the size (nm) (left panel) and particle concentration (p / ml, particles / milliliter) (right panel) of ultra-purified EVs from culture medium of control cells (EV “0”) and cells stably expressing adiponectin (EV “wt” and EV “2”) at time of production 0 (T0 4° C.) and 3 months after production under storage at +4° C. and −80° C. EV “0” represents the EVs of control cells. EV “wt” represents the EVs of cells stably expressing wild-type adiponectin and EV “2” represents the EVs of cells expressing DNA encoding chimeric adiponectin with construct 2 (SEQ ID NO: 42). EXAMPLES

[0248] Adiponectin is one of the most important adipocytokines secreted by adipocytes and is known to exert beneficial effects in various human conditions, including diabetes, obesity, insulin resistance, cardiovascular disease, inflammatory conditions, and cancer. Although adiponectin appears to be a promising candidate for the development of new drugs to treat various diseases, there are currently no adiponectin therapies available for clinical trials.

[0249] Indeed, large-scale production of functional adiponectin is difficult due to its complexity. Adiponectin is a 244 amino acid cytokine that contains post-translational modifications and exists in three oligomeric complexes: low molecular weight (LMW), intermediate molecular weight (MMW), and high molecular weight (HMW) forms, meaning that functional adiponectin requires the presence of post-translational modifications and proper high-level multimerization. Although bacterial systems lack mammalian protein synthesis machinery and cannot produce functionally active adiponectin, the use of mammalian culture systems for mass production is not a scalable process. Furthermore, adiponectin has a short half-life in the circulation, making exogenous administration of recombinant adiponectin an unfeasible approach.

[0250] Interestingly, it was found that highly active HMW adiponectin is mainly present in the exosomal fraction in serum (Phoonsawat et al., Adiponectin is partially associated with exosomes in mouse serum, BBRC Vol. 448, Issue 3, 2014, Pages 261-266), and that adiponectin is mainly distributed on the outer surface of extracellular vesicles as a result of non-specific absorption of soluble adiponectin (Blandin et al., Extracellular vesicles are stable carriers of adiponectin with insulin-sensitive properties, http: / / dx.doi.org / 10.2139 / ssrn.4036824). Furthermore, adiponectin-associated extracellular vesicles have been shown to mediate insulin-sensitizing effects in target cells in vitro, and their injection into mice fed a high-fat diet prevents the animals from developing insulin resistance (Blandin et al., Extracellular vesicles are stable carriers of adiponectin with insulin-sensitive properties, http: / / dx.doi.org / 10.2139 / ssrn.4036824).

[0251] Although these results are encouraging because they indicate that administration of adiponectin-associated extracellular vesicles may be a valuable approach to treat diseases associated with alterations in adiponectin, such adiponectin-associated extracellular vesicles are not suitable for therapeutic applications. In particular, as shown in the examples herein below, ultra-purification of such adiponectin-associated extracellular vesicles induces the removal of surface adiponectin, resulting in ultra-purified extracellular vesicles that are almost devoid of adiponectin. Thus, there remains a need to provide a means to enable large-scale production of functional adiponectin, i.e., functional adiponectin on extracellular vesicles that are HMW and suitable for therapeutic applications.

[0252] Herein, the inventors have developed a novel chimeric adiponectin that allows the production, on an industrial scale, of extracellular vesicles that are loaded with functional adiponectin, are stable for several months, and may be useful for therapeutic applications, particularly for the treatment of insulin resistance and diabetes, as well as other diseases.

[0253] The invention is further illustrated by the following examples.

[0254] Example 1 Production of semi-purified extracellular vesicles containing chimeric adiponectin polypeptides material and method Production of adiponectin and adiponectin-bearing extracellular vesicles in mammalian cells

[0255] Extracellular vesicles were produced in HEK293T cells obtained from American Type Culture Collection (ATCC). Cells were cultured in DMEN supplemented with 5% heat-inactivated fetal bovine serum (iFBS), 2 mM GlutaMAX, and 5 μg / mL gentamicin at 37° C. in a humidified incubator with 5% CO2. HEK293T cells were routinely tested and found to be negative by MycoAlert™ Mycoplasma Detection Kit (Lonza Nottingham, Ltd.).

[0256] The nucleic acid sequences encoding wild-type adiponectin (SEQ ID NO: 31 or 33) and the nucleic acid sequences encoding chimeric adiponectin polypeptides targeted to exosomes (SEQ ID NO: 40-44) were inserted into eukaryotic expression vectors under the control of a CMV / HTLV chimeric promoter. If necessary, a zeocin-encoding resistance gene was added downstream of the CMW IRES sequence in tandem with the nucleic acid encoding adiponectin, allowing for the co-expression of zeocin resistance and the establishment of stable transfected cell lines. These nucleic acid sequences were transfected into HEK293T cells using PEI. If necessary, selection of stable transfected cell lines was obtained in the presence of 500 μg / mL zeocin for 15 days.

[0257] To obtain large-scale exosome production, HEK293T transfected cells were plated into 10 trays of cell chambers in 1 L of complete medium. After 24 hours, the cultures were fed with medium supplemented with extracellular vesicle-free iFBS and incubated for an additional 48 hours.

[0258] Adiponectin-extracellular vesicles and purification of extracellular vesicles Cell culture medium was harvested from transfected HEK293T cells and adiponectin-extracellular vesicle isolation was performed as previously described (Taylor & Shah, 2015. Methods. 87:3-10; Desplantes et al., 2017. Sci Rep. 7(1):1032; Corso G. et al. 2017. Scientific Reports. 7: 11561. DOI:10.1038 / s41598-017-10646-x). Briefly, cell culture supernatants were clarified by two successive centrifugations: 1300 rpm for 10 min and 4000 rpm for 15 min (both at 4° C.) followed by filtration through a 0.22 μm membrane filter. The supernatant was then concentrated by ultracentrifugation and diafiltration and loaded onto a size-exclusion chromatography (SEC) or BE-SEC column (CL2 B or Sephacryl S1000 or Captocore, GE Healthcare). Fractions containing extracellular vesicle biomarkers (CD81 and CD63) were identified by ELISA. Extracellular vesicle fractions containing adiponectin identified by Western blot were pooled, concentrated as necessary, and used for analysis and injection.

[0259] SDS-PAGE, Western blot, and antibodies The protein concentration of adiponectin-extracellular vesicles was measured using a BCA assay (Pierce BCA Protein Assay Kit, ThermoFisher Scientific). Adiponectin-extracellular vesicle preparations were lysed and separated by SDS-PAGE on 4-15% acrylamide gels (4-15% Mini-PROTEAN® TGX Stain-Free™ Gel kit, Bio Rad) and then transferred to PVDF membranes. For Western blotting under non-reducing conditions, a loading buffer without DTT was used.

[0260] Immunodetection of adiponectin was performed by primary antibodies against adiponectin (anti-adiponectin monoclonal antibody, clone ABM52A3, Abeomics Ref. #10-7597; or anti-adiponectin rabbit polyclonal antibody, Invitrogen Ref. #PA1 054), or anti-Ciloa pilot peptide (PP) (in-house antibody raised in rabbit).

[0261] Immunodetection of specific extracellular vesicle markers was performed with primary antibodies against either CD81 (Genetex Ref. #GTX101766), CD63 (Genetex Ref. #GTX132953), Alix (Proteintech #12422-1-AP), or syntenin (Fisher Scientific Ref. #11326573).

[0262] The membranes were then incubated with the corresponding HRP-conjugated secondary antibodies (donkey anti-mouse or anti-rabbit or anti-goat HRP, Jackson ImmunoResearch, Refs. #715-035-150, #711-035-152, or #715-038-147).

[0263] Signals were detected using an enhanced chemiluminescence detection kit (Super Signal West Pico Plus; ThermoFischer Scientific; Ref. 34580), and membranes were imaged by a ChemiDoc Imaging System (Bio Rad).

[0264] In addition, adiponectin on the surface of adiponectin-extracellular vesicles was detected by ELISA using these primary antibodies plus GeneTex (GTX112777) anti-adiponectin polyclonal antibody, as well as the respective secondary antibodies.

[0265] IgG ELISA specific for adiponectin and extracellular vesicle markers The surface content of extracellular vesicles in adiponectin, as well as surface markers specific for CD81 and CD63, was determined by ELISA using some of the antibodies mentioned above as well as anti-CD81 (Ancell; Ref. #ANC 302 020) or anti-CD63 (Agro-Bio; Ref. #S12086) antibodies.

[0266] Briefly, MaxiSorp ELISA plates (Nunc) were coated overnight at 4° C. with serial 1 / 2 dilutions (starting at 1 μg) of adiponectin-extracellular vesicles in 100 μL per well of 50 μM sodium carbonate / sodium bicarbonate (pH 9.6) buffer. The coated plates were washed three times with 200 μL of 1×PBS and saturated with 200 μL of 1×PBS containing 3% BSA per well for 1 h at 37° C. The plates were washed three times with 1×PBS and then incubated for 2 h at 37° C. with primary antibody dilutions (1:500 for adiponectin or 1:10000 for extracellular vesicle-specific markers) in 3% BSA and 5% FBS. This was followed by washing three times with 200 μL per well of 1×PBS and incubation with 100 μl per well of the corresponding secondary HRP-conjugated antibody (specified in the Western blot above) diluted 1:10000 in 1×PBS containing 3% BSA. After incubation with the secondary antibody, the plate was washed five times with 200 μL per well of 1×PBS and developed with 100 μL per well of TMB (Bio-Rad; Ref. #R8 / R9) for 30 minutes. The reaction was stopped by adding 50 μL per well of stop solution (2N sulfuric acid).

[0267] Absorbance was read at 450 nm using a ClarioStar Plus plate reader (BMG Labtech). The reciprocal endpoint titer was defined as the dilution with an OD3 at 450 nm three times higher than background.

[0268] result We demonstrated for the first time the production of semi-purified EVs containing chimeric adiponectin with adiponectin at either the N- or C-terminus (Figs. 1 and 2B).

[0269] Production of chimeric adiponectin with adiponectin at the N-terminus FIG. 3 shows that a chimeric polypeptide having SEQ ID NO: 40, comprising from the N-terminus to C-terminus adiponectin with its signal peptide (SEQ ID NO: 33), a (GGGSGGGGS)3 linker (having SEQ ID NO: 39), a CD8 transmembrane domain (having SEQ ID NO: 37), and a peptide pilot (having SEQ ID NO: 30), is expressed in cells and secreted into extracellular vesicles.

[0270] Interestingly, this chimeric polypeptide construct allowed the oligomerization of adiponectin in extracellular vesicles, as detected by immunoblot analysis (FIG. 4).

[0271] To confirm that adiponectin is expressed on the surface of extracellular vesicles, we performed an ELISA assay to label adiponectin and extracellular vesicle-specific markers in extracellular vesicles expressing a chimeric polypeptide having SEQ ID NO: 40 or control extracellular vesicles not expressing a chimeric polypeptide. As shown in Figure 5, two extracellular vesicle-specific markers (CD63 and CD81), as well as adiponectin, were detected in extracellular vesicles expressing the chimeric polypeptide. The lack of detection of adiponectin in the control extracellular vesicles confirms the specificity of adiponectin detection in extracellular vesicles expressing the chimeric polypeptide.

[0272] Production of chimeric adiponectin bearing adiponectin at both the N- and C-termini The constructs tested are provided in Figure 6C, and the sizes of the proteins revealed by anti-adiponectin antibodies are provided in Figure 6D. The sizes of adiponectin from cells transiently transfected with wild-type adiponectin (wt), DNA corresponding to chimeric adiponectin having construct 1 (SEQ ID NO: 40), DNA corresponding to chimeric adiponectin having construct 2 (SEQ ID NO: 42), DNA corresponding to chimeric adiponectin having construct 3 (SEQ ID NO: 41), DNA corresponding to chimeric adiponectin having construct 4 (SEQ ID NO: 43), and DNA corresponding to chimeric adiponectin having construct 5 (SEQ ID NO: 44) are in the range of 26,37 kDa, 36,92 kDa, 38,50 kDa, 32,98 kDa, 36,23 kDa, and 41,75 kDa, respectively.

[0273] These results further demonstrate that constructs 1, 2, 3, and 5, as well as wt adiponectin, express adiponectin in semi-purified EVs. Unexpectedly, construct 4 does not result in expression of adiponectin on EVs. Of note, semi-purified EVs carrying wt adiponectin or chimeric adiponectin with construct 2 (SEQ ID NO: 42) are the EVs that contain the highest amounts of adiponectin when analyzed under reducing conditions (Figures 6A and 6B).

[0274] Interestingly, when analyzed under non-reducing conditions (Figures 7A, 7B), the results show that semi-purified EVs carrying chimeric adiponectin corresponding to Construct 2 (SEQ ID NO: 42) contain higher amounts of highly oligomerized (HMW) adiponectin than EVs carrying wt adiponectin or EVs carrying chimeric adiponectin corresponding to Construct 3 (SEQ ID NO: 41) or Construct 5 (SEQ ID NO: 44).

[0275] Example 2 Production of ultra-purified extracellular vesicles containing chimeric adiponectin polypeptides material and method Production of adiponectin and adiponectin-bearing extracellular vesicles in mammalian cells Extracellular vesicles carrying wild-type or chimeric adiponectin polypeptides (SEQ ID NO: 42) were produced as described herein above.

[0276] Production of adiponectin in EVs ultra-purified from culture medium Culture media from cells stably expressing different DNA constructs: wild-type adiponectin (wt), membrane-anchored adiponectin with construct 2 (SEQ ID NO: 42) (2), both (2+wt), or control cells (9) were concentrated and purified using TFF and BE-SEC chromatography. Ultra-purified EVs and extracts from producing cells were subjected to SDS-PAGE separation under reducing or non-reducing conditions as described above herein and analyzed by Western blot, revealed with adiponectin primary antibody followed by HRP-conjugated secondary antibody.

[0277] Characterization of ultra-purified EVs Culture media from cells stably expressing different DNA constructs: wild-type adiponectin (wt), membrane-anchored adiponectin (2), or control cells (0) were concentrated and purified using TFF and BE-SEC chromatography. Ultrapurified EVs and extracts from producing cells were subjected to SDS-PAGE separation under reducing or non-reducing conditions and analyzed by Western blot, revealed with anti-Alix (EV marker) and anti-adiponectin primary antibodies followed by HRP-conjugated secondary antibodies.

[0278] The presence of EV markers (CD81) and adiponectin (Adpn) on the surface of EVs was detected by ELISA. Different types of EVs were immobilized on ELISA plates at dilutions ranging from 1 to 1 / 123 (1 = 50 μl of pure EVs) and detected with anti-CD81 or anti-adiponectin antibodies followed by a secondary anti-HRP antibody.

[0279] The amount of adiponectin was measured in the EV preparations by quantitative ELISA: EVs were lysed to detect the total amount of immobilized or associated adiponectin, and the concentration of adiponectin (ng / ml) was measured using a commercially available ELISA sandwich kit for quantification of human adiponectin.

[0280] result We also demonstrated the production of ultra-purified EVs containing chimeric adiponectin with adiponectin at the C-terminus (Figs. 1 and 2C).

[0281] The results of the production of ultra-purified EVs and the characterization of ultra-purified EVs are shown in Figure 8 and Figures 9 and 10.

[0282] Figure 8B (left panel) shows that WT adiponectin (lanes wt and wt+2) is better expressed in cells than immobilized (lanes 2 and wt+2). However, when EVs are ultrapurified, immobilized adiponectin (lanes 2 and wt+2) is much more stably targeted in EVs (resistant to ultrapurification) than WT adiponectin (lanes wt and wt+2). This is true when the corresponding constructs are alone (lanes wt and 2) or coexpressed (wt+2) (Figure 8B, right panel). In EVs ultrapurified and analyzed under non-reducing conditions (Figure 8C), construct 2 alone (lane 2) or coexpressed with WT adiponectin (lane 2+wt) are highly oligomerized. Only construct 2 allows stable targeting of large amounts of highly oligomerized (HMW) adiponectin on EVs. In contrast, even though the amount of WT adiponectin is high in cells, it is less present in semi-purified EVs and completely negligible in ultra-purified EVs. These results clearly demonstrate that the use of chimeric adiponectin construct 2 allows the production of ultra-purified EVs carrying large amounts of highly oligomerized adiponectin on their surface, and that ultra-purified EVs obtained from cells transfected with wt adiponectin are almost devoid of adiponectin.

[0283] Figures 9 and 10 present further characterization of ultra-purified EVs. Two EV markers, Alix and CD81, as well as adiponectin, are detected in EVs from cells stably expressing wt adiponectin or Construct 2 (SEQ ID NO: 42) (Figure 9A, B). Quantification of adiponectin by ELISA shows that ultra-purified EVs from cells expressing Construct 2 carry more adiponectin than ultra-purified EVs from cells expressing wt adiponectin (Figure 9C). These results are further confirmed by analysis with NanoAnalyzer, revealing that ultra-purified EVs from cells expressing Construct 2, carrying more adiponectin, are detected in greater quantities than ultra-purified EVs from cells expressing wt adiponectin (Figure 10A). As a control, the size of EVs is similar in both conditions (Figure 10B).

[0284] Taken together, these results show that chimeric adiponectin carrying adiponectin at its C-terminus, especially construct 2, allows obtaining ultra-purified EVs carrying large amounts of highly oligomerized (HMW) adiponectin on their surface, which is not the case for wt adiponectin. In conclusion, these results clearly demonstrate the advantages of the constructs of the present invention, especially construct 2, compared to wt adiponectin.

[0285] Example 3 Storage of ultra-purified EVs material and method Characterization by ELISA of ultra-purified EVs exhibiting adiponectin stored under different conditions (4°C or -80°C) 3 months after production The presence of EV markers (CD81) and adiponectin (Adpn) on the surface of EVs is detected by ELISA immediately after production and purification (T0) or after 3 months of storage at 4°C or -80°C (3 months at 4°C and 3 months at -80°C). Different types of EVs ultrapurified from culture medium of cells stably expressing adiponectin (wt and 2) or control cells (0) are immobilized on ELISA plates at dilutions ranging from 1 to 1 / 128 (1 = 50 μl pure EVs) and detected with anti-CD81 (upper panel) or anti-adiponectin (lower panel) antibodies followed by a secondary anti-HRP antibody.

[0286] Quantification of adiponectin in ultra-purified EVs after storage under different conditions (4°C or -80°C) 3 months after production. The amount of adiponectin is measured in EV preparations by quantitative ELISA immediately after production and purification (T0) or after 3 months of storage at 4° C. or -80° C. (3 months at 4° C. and 3 months at -80° C.) Different types of EVs ultrapurified from culture medium of cells stably expressing adiponectin (wt and 2) or control cells (0) are lysed to detect the total amount of immobilized or associated adiponectin, and the concentration of adiponectin (ng / ml) is measured using a commercially available ELISA sandwich kit for quantification of human adiponectin.

[0287] For quantification by ELISA, the concentration of adiponectin was measured using a sandwich ELISA kit (Human Adiponectin / Acrp30 DuoSet ELISA R&D Systems #DY1065-05) according to the manufacturer's protocol. To measure the total amount of adiponectin, including pre-experimental and fixed, EVs were lysed. Briefly, one volume of EVs was incubated with four volumes of lysis buffer (NP-40 1%, TNE 1X: Tris 0,1M, EDTA 1mM, and PMSF 0.25 mM final) for 30 min on ice. After the incubation step, EVs were diluted using the kit's reagent diluent, and 100 μl of lysed EVs were used for the ELISA. Two different dilutions were applied to each EV sample, and measurements were performed in technical replicates for each.

[0288] Analysis of size and concentration of ultra-purified EVs expressing adiponectin stored under different conditions (4°C or -80°C) 3 months after production. The size (nm) and concentration (particles per ml: p / ml) of EVs in the different preparations are measured using a NanoAnalyzer instrument (NanoFCM).

[0289] Batches of ultra-purified EVs were analyzed for their size (nm) and concentration of particles in particles per milliliter (p / ml) using a NanoAnalyzer instrument (nanoFCM). The instrument was calibrated with quality control beads (250 nm SiNPs) and size standard beads (S16M-Exo) prior to analysis, as recommended. Samples were diluted with 10 mL of 10% ethanol, as recommended. 8 Working sample concentrations of particles / ml were diluted in 1x PBS and acquired for 1 min at a maximum speed of 12.000 particles / min.

[0290] result We further demonstrated that ultra-purified EVs maintained large amounts of adiponectin when stored at 4 °C or -80 °C.

[0291] The results are shown in Figures 11, 12, and 13. These figures show that ultra-purified EVs from cells expressing either Construct 2 or wt adiponectin still retain adiponectin when the EVs are stored at 4°C or -80°C for 3 months (Figure 11), and that ultra-purified EVs from cells expressing Construct 2 still contain higher amounts of adiponectin than EVs from cells expressing wt adiponectin after storage at 4°C or -80°C for 3 months (Figure 12). These results are confirmed by analysis with a NanoAnalyzer, which reveals that only negligible variations in EV concentration are observed when adiponectin is immobilized on the EV membrane with Construct 2 (Figure 13, right panel). With respect to the size of the EVs, the variations in size measurements are not significant (Figure 13, left panel).

Claims

1. In any order, i) the amino acid sequence of adiponectin, and ii) the amino acid sequence of the transmembrane domain of the transmembrane protein A chimeric polypeptide comprising:

2. A chimeric polypeptide as described in claim 1, wherein the amino acid sequence of the adiponectin includes the amino acid sequence of wild-type adiponectin.

3. The chimeric polypeptide of claim 1, further comprising iii) the amino acid sequence of a pilot peptide, wherein the pilot peptide interacts with the Endosomal Sorting Complexes Required for Transport (ESCRT) cellular machinery.

4. The chimeric polypeptide of claim 1 , further comprising at least one linker between the amino acid sequence of the adiponectin and the amino acid sequence of the transmembrane domain.

5. The chimeric polypeptide of claim 3, comprising, from N-terminus to C-terminus, components iii), ii), and i).

6. The chimeric polypeptide of claim 1, further comprising a submembrane targeting domain.

7. The chimeric polypeptide of claim 1 , wherein the transmembrane domain is selected from the group consisting of the transmembrane domain of CD40L and the transmembrane domain of CD8.

8. A chimeric polypeptide described in claim 7, wherein the transmembrane domain of CD40L comprises the amino acid sequence of SEQ ID NO: 35 and the transmembrane domain of CD8 comprises the amino acid sequence of SEQ ID NO:

37.

9. The pilot peptide comprises at least one YxxL motif having SEQ ID NO: 1 or at least one DyxxL motif having SEQ ID NO: 4, and at least one PxxP motif having SEQ ID NO: 8, wherein "x" represents any amino acid residue. The chimeric polypeptide of claim 3.

10. the pilot peptide comprises an amino acid sequence having SEQ ID NO: 30 or a variant thereof; said variant of SEQ ID NO: 30 retains at least three YxxL and / or DyxxL motifs, having SEQ ID NO: 1 and SEQ ID NO: 4, respectively, and at least four PxxP motifs, having SEQ ID NO: 8, wherein "x" represents any amino acid residue; The chimeric polypeptide of claim 3.

11. A nucleic acid encoding the chimeric polypeptide of claim 1.

12. An extracellular vesicle comprising the chimeric polypeptide of claim 1.

13. An extracellular vesicle as described in claim 12, wherein the membrane-spanning domain of the chimeric polypeptide is fixed to the lipid bilayer of the extracellular vesicle, and the adiponectin of the chimeric polypeptide is exposed on the outer surface of the extracellular vesicle.

14. The extracellular vesicle of claim 12, wherein the extracellular vesicle is an exosome.

15. A population of extracellular vesicles described in claim 12.

16. A population of extracellular vesicles described in claim 15, further containing soluble adiponectin.

17. A purified extracellular vesicle according to claim 12 or a population of extracellular vesicles according to claim 15.

18. 16. An extracellular vesicle according to claim 12, or a population of extracellular vesicles according to claim 15, for use as a medicament.

19. 16. A pharmaceutical composition comprising the extracellular vesicles of claim 12 or a population of extracellular vesicles of claim 15 for use in treating a disease, disorder, or condition selected from the group consisting of diabetes, obesity, insulin resistance, diseases associated with insulin resistance or deficiency, hypertension, dyslipidemia, hyperuricemia, atherosclerosis (including coronary artery disease, stroke, and peripheral arterial disease), fibrosis, inflammatory lung disease, nephrotic disease, sleep apnea, dry eye disease, inflammatory eye disease, gastritis and gastroesophageal reflux disease, inflammatory bowel disease, pancreatitis, osteoporosis, and inflammatory bone and joint diseases.

20. The pharmaceutical composition for use according to claim 19, wherein the extracellular vesicles are partially or completely coated with recombinant adiponectin.

21. A pharmaceutical composition for use as described in claim 19, wherein the extracellular vesicles are partially or completely coated with recombinant adiponectin fused to lactadherin or its functional C1 and / or C2 domains.

Images