Use of 15-lipoxygenase for the treatment of lymphedema

Abstract

Lymphedema is characterized by the accumulation of protein-rich interstitial fluid, lipids and prominent inflammatory cell infiltrates in the limbs. It is a common disabling disease that causes significant morbidity and affects over 250 million people worldwide, yet there is no therapeutic treatment for lymphedema. Herein, we found that dermal and fat resections from patients with lymphedema exhibited inflammatory gene expression profiles compared to normal arms of the same patients. After lipidomic analysis, we identified a severe reduction in arachidonic acid-derived lipid mediators generated by 15-lipoxygenase (15-LOX) in lymphedematous arms. Using a mouse model of lymphedema, we recapitulated the causes of the human pathology, including the loss of intrinsic pro-resolution lipid mediators that play an essential role in the resolution of inflammation. This was associated with a lack of recruitment of regulatory T cells (Tregs) in adipose tissue of the injured limb. Importantly, we identified that lymphatic endothelial 15-LOX was responsible for Treg chemoattraction and transendothelial migration. These results were confirmed by the exacerbation of lymphedema and deterioration of lymphatic network in an original transgenic mouse model in which the ALOX15 gene was selectively deleted in the lymphatic system. Importantly, this was reversed by injection of 15-LOX-expressing lentivector. These results provide evidence that lymph lipoxygenase may be a novel therapeutic target for lymphedema by acting as a mediator of Treg infiltration into lymphedema adipose tissue.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention is in the field of medicine, in particular the treatment of lymphedema. [Background technology]

[0002] 2. Background of the Invention The lymphatic system provides a conduit for the resorption of interstitial fluid that leaks from the arteriovenous circulation. Further important lymphatic functions include digestive lipid absorption and transport of immune cells. 1 In the face of inherited defects or acquired lymphatic insults, the resulting lymphatic dysfunction can lead to a loss of normal immune responses or to the development of lymphatic insufficiency known as lymphedema (LD). 2 LD is characterized by the accumulation of protein-rich interstitial fluid, prominent inflammatory cell infiltrates, and a dysregulated regional immune response. Subsequent deposition of adipose tissue and fibrosis at sites of lymphatic dysfunction promotes progressive anatomical distortion and loss of function and chronic inflammation. 2 .

[0003] In response to injury, acute inflammation is a timely orchestrated defensive program critical for tissue repair and restoration of homeostasis. 3 It is divided into two phases: initiation and resolution. The initiation phase is characterized by tissue edema due to increased blood flow and permeability of the microvasculature. Polymorphonuclear neutrophils (PMNs) migrate to the injury site in response to chemical signals including proinflammatory lipid mediators (e.g. leukotriene B4 [LTB4]) and chemokines. The resolution phase is already in place at this early time point with the arrest of PMN influx at injury-appropriate levels and their timely apoptosis. 4 Monocytes then infiltrate the tissue where they execute a convergent phase by differentiating into macrophages. Macrophages respond to damage-associated molecular signatures present in the injured area, allowing a return to homeostasis for tissue repair and regeneration. 4-8Regulatory T (Treg) cells are a critical arm of the inflammation-resolution response, as they improve the clearance of apoptotic cells (efferocytosis) and thus enhance resolution. 9 .

[0004] Defective clearance may lead to exacerbated inflammation that impedes tissue repair. In this context, adaptive immune cells play a critical role in the host response to inflammation resolution and tissue repair, leading to chronic inflammation. 10,11 , resulting in tissue damage, excessive fibrosis, and loss of function as observed in lymphedema.

[0005] Resolution of vascular inflammation is a key driver for remodeling of the vascular wall and functional recovery. Resolution-promoting lipid mediators derived from polyunsaturated fatty acids orchestrate key cellular processes favoring resolution and return to immune homeostasis. Thus, the discovery of their presence in lymphatic vessels has generated great interest in their role in LD-induced inflammation. Importantly, studies have demonstrated that T cells are key regulators of inflammation in LDs. 12 They found that recruitment of Tregs to inflamed LD tissues is a key factor in T H 1 / T H 2We have demonstrated that LOX suppresses immune responses, limits tissue fibrosis, and improves lymphatic function. Human lipoxygenase (LOX) catalyzes the stereoselective dioxygenation of polyunsaturated fatty acids (arachidonic acid (AA), DHA, and EPA). 13 In particular, the 15-LOX enzyme is constitutively expressed in immune and endothelial cells, where it contributes to immune modulation. Indeed, the resolution of inflammation is impaired in 15-LOX-deficient mice, which is accompanied by an impaired wound-healing response with increased post-inflammatory fibrosis. 14 15-LOX catalyzes the oxygenation of AA, thus generating the metabolite 15-hydroxyeicosatetraenoic acid (15-HETE). The 15-HETE mediator exhibits chemotactic activity and, among other things, affects the trafficking of neutrophils. 15 , stimulates VEGFA synthesis and thus indirectly affects endothelial activation 16This differs from 12-HETE, which is produced by 12-LOX and is said to promote circular chemorepulsion defects in lymphoid monolayers when synthesized by tumor cells. 17 .

[0006] Pro-resolving lipid mediators are primarily synthesized locally by immune cells, but they are also produced in vascular tissues, including endothelial cells. These lipids have direct effects on endothelial-leukocyte interactions and may play a protective role after injury. 18 Therefore, they are considered not only as potential vascular therapeutics but also as potential biomarkers in vascular diseases.

[0007] Lymphedema is a chronic pathological condition associated with inflammation. Exploratory studies support the utility of targeted anti-inflammatory therapy with ketoprofen in patients with lymphedema. 19 Conversely, mouse models of obesity promote adipose tissue inflammation, impair lymphatic function, and exacerbate lymphedema. Mechanistically, increased infiltration of T helper type 2 (Th2) cells impairs both lymphangiogenic capillary responses and collecting duct function, in part through production of the Th2 cytokines IL-4 and IL-13. 20 Furthermore, CD11b + Macrophages produce high levels of nitric oxide and disrupt NO signaling in lymphatic endothelium, leading to decreased contractility and function of collecting lymphatic vessels 21 Thus, some inflammatory and immune cell subsets are detrimental to normal LEC function, which contribute to obesity-associated lymphatic dysfunction. On the other hand, regulatory T cells (Tregs), which suppress inflammation, have been shown to promote lymphatic regeneration and repair in a mouse model of tail lymphedema. 22 . Summary of the Invention

[0008] The invention is defined by the claims. In particular, the invention relates to the use of 15-lipoxygenase (15-LOX) for the treatment of lymphedema.

[0009] Detailed description of the invention: Lymphedema is characterized by the accumulation of protein-rich interstitial fluid, lipids and prominent inflammatory cell infiltrates in the limbs. It is a common disabling disease that causes significant morbidity and affects over 250 million people worldwide, yet there is no therapeutic treatment for lymphedema. Herein, we found that dermal and fat resections from patients with lymphedema exhibited inflammatory gene expression profiles compared to normal arms of the same patients. After lipidomic analysis, we identified a severe reduction in arachidonic acid-derived lipid mediators generated by 15-lipoxygenase (15-LOX) in lymphedematous arms. Using a mouse model of lymphedema, we recapitulated the causes of the human pathology, including the loss of intrinsic pro-resolution lipid mediators that play an essential role in the resolution of inflammation. This was associated with a lack of recruitment of regulatory T cells (Tregs) in adipose tissue of the injured limb. Importantly, we identified that lymphatic endothelial 15-LOX was responsible for Treg chemoattraction and transendothelial migration. These results were consistent with the original transgenic mouse model in which the ALOX15 gene was selectively deleted in the lymphatic system (Prox1CreERT2;ALOX15 fl / fl ) was confirmed by the worsening of lymphedema and deterioration of the lymphatic network in mice. Importantly, this was reversed by injection of a lentivector expressing 15-LOX. These results provide evidence that lympholipoxygenase may be a novel therapeutic target for lymphedema by acting as a mediator of Treg infiltration into lymphedematous adipose tissue.

[0010] Thus, a first object of the present invention relates to a method for treating lymphedema in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of i) a 15-LOX polypeptide or ii) a polynucleotide encoding a 15-LOX polypeptide.

[0011] As used herein, the term "lymphedema" has its general meaning in the art and refers to a disorder characterized by increased fluid retention in tissues due to reduced lymphatic drainage, local accumulation of adipose tissue, and intense tissue swelling due to impaired immune function. The term includes "primary lymphedema" and "secondary lymphedema." Primary lymphedema is a lymphatic dysplasia characterized by swelling of the limbs that may be complicated by other lymphatic exudations due to an underlying abnormality in the development of the lymphatic system (dyslymphangiogenesis). It may be hereditary or non-hereditary, and may be congenital or late-onset. In some embodiments, lymphedema is found as a secondary disorder that may result from dissection of lymph nodes or damage to lymphatic vessels. Such secondary lymphedema may also be associated with, for example, surgery, radiation therapy, oncological disease and its treatment, lymph node dissection and injury associated with musculoskeletal disorders such as bone fractures, tenotomy and joint replacement, neurological conditions such as muscle paresis, vascular injury / surgery, integumentary injuries, coagulation disorders such as deep vein thrombosis, scar tissue formation, tamoxifen treatment, filariasis, infections, lipedema or cellulitis. The term "treating lymphedema" encompasses both prophylactic and therapeutic treatment of lymphedema.

[0012] In particular, the polypeptides or polynucleotides of the invention are particularly suitable for improving the infiltration of Treg cells into tissues and thus resolving inflammation.

[0013] As used herein, the term "regulatory T cells" or "Treg cells" refers to cells that suppress, inhibit or prevent T cell activity. Typically, Treg cells have the following resting phenotype: CD4+CD25+FoxP3+.

[0014] As used herein, the term "15-LOX" refers to 15-lipoxygenase, a polyunsaturated fatty acid lipoxygenase. An exemplary amino acid sequence for 15-LOX is represented by SEQ ID NO: 1. The lipoxygenase domain ranges from amino acid residue 115 to amino acid 662.

[0015] [ka]

[0016] As used herein, the term "polypeptide" has its general meaning in the art and refers to a polymer of amino acids of any length. The polymer may contain modified amino acids. These terms also encompass amino acid polymers that are modified, naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. For example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art, are also included in the definition.

[0017] As used herein, the term "15-LOX polypeptide" refers to a polypeptide that includes the lipoxygenase domain of 15-LOX.

[0018] In some embodiments, a 15-LOX polypeptide of the present invention comprises an amino acid sequence having at least 90% identity to the amino acid sequence ranging from amino acid residue 115 to amino acid residue 662 of SEQ ID NO:1.

[0019] As used herein, the "percent identity" between two sequences takes into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. It is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100). The following mathematical algorithm can be used to achieve sequence comparison and percent identity determination between two sequences. The Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443-53.) can be used to determine the percent identity between two amino acid sequences. The percent identity of two nucleotide or amino acid sequences can also be determined using algorithms such as EMBOSS Needle (pairwise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle can be used with a BLOSUM62 matrix with a "Gap Open Penalty" of 10, a "Gap Extension Penalty" of 0.5, an "End Gap Penalty" of false, an "End Gap Open Penalty" of 10, and an "End Gap Extension Penalty" of 0.5. In general, the "percent identity" is a function of the number of matched positions divided by the number of positions compared, multiplied by 100. For example, if 6 out of 10 sequence positions are identical after alignment between two compared sequences, the identity is 60%. The percent identity is typically determined over the entire length of the query sequence over which the analysis is performed. Two molecules that have the same primary amino acid or nucleic acid sequence are identical, regardless of any chemical and / or biological modifications.According to the present invention, a first amino acid sequence having at least 90% identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% identity with the second amino acid sequence.

[0020] As used herein, the term "polynucleotide" refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. The term refers to the primary structure of the molecule. Thus, the term includes triple-, double-, and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-, double-, and single-stranded ribonucleic acid ("RNA"). It also includes modified forms of polynucleotides, for example, by alkylation and / or capping, as well as unmodified forms. More specifically, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), whether spliced ​​or not, including tRNA, rRNA, hRNA, siRNA, and mRNA, any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, as well as other polymers containing normucleotidic backbones, such as polyamides (e.g., peptide nucleic acids "PNA") and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers, provided that the polymers contain nucleobases in a configuration that allows for base pairing and base stacking as found in DNA and RNA. In some embodiments, the polynucleotide comprises an mRNA. In other aspects, the mRNA is a synthetic mRNA. In some embodiments, the synthetic mRNA comprises at least one unnatural nucleobase. In some embodiments, all nucleobases of a particular class are substituted with a non-natural nucleobase (e.g., all uridines in a polynucleotide disclosed herein can be substituted with a non-natural nucleobase, e.g., 5-methoxyuridine). In some embodiments, a polynucleotide (e.g., synthetic RNA or DNA) contains only natural nucleobases, i.e., A, C, T, and G in the case of synthetic DNA, or A, C, T, and U in the case of synthetic RNA.

[0021] In some embodiments, a polynucleotide of the invention is messenger RNA (mRNA).

[0022] In some embodiments, the polynucleotide is inserted into a vector, such as a viral vector.

[0023] As used herein, the term "viral vector" refers to a virion or virus particle that functions as a nucleic acid delivery vehicle and contains a vector genome packaged within the virion or virus particle. Typically, the vector is a viral vector that is an adeno-associated virus (AAV), a retroviral vector, a bovine papilloma virus, an adenovirus vector, a vaccinia virus, or a polyoma virus.

[0024] In some embodiments, the viral vector is an AAV vector.

[0025] As used herein, the term "AAV vector" refers to a vector derived from an adeno-associated virus serotype, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and variants thereof. An AAV vector may be deleted in whole or in part of one or more AAV wild-type genes, preferably the rep and / or cap genes, but may retain functional flanking ITR sequences.

[0026] In some embodiments, the viral vector is a retroviral vector.

[0027] As used herein, the term "retroviral vector" refers to a vector that contains structural and functional genetic elements primarily derived from a retrovirus.

[0028] In some embodiments, the retroviral vectors of the invention are derived from a retrovirus selected from the group consisting of an alpharetrovirus (e.g., avian leukosis virus), a betaretrovirus (e.g., mouse mammary tumor virus), a gammaretrovirus (e.g., murine leukemia virus), a deltaretrovirus (e.g., bovine leukemia virus), an epsilonretrovirus (e.g., walleye cutaneous sarcoma virus), a lentivirus (e.g., HIV-1, HIV-2), and a spumavirus (e.g., human spumavirus).

[0029] In some embodiments, retroviral vectors of the invention are replication-defective retroviral virus particles capable of delivering a genetic foreign import RNA in place of the retroviral mRNA.

[0030] In some embodiments, a retroviral vector of the present invention is a lentiviral vector.

[0031] As used herein, the term "lentiviral vector" refers to a vector that contains structural and functional genetic elements that are primarily derived from a lentivirus. In some embodiments, the lentiviral vector of the present invention is selected from the group consisting of HIV-1, HIV-2, SIV, FIV, EIAV, BIV, VISNA and CAEV vectors. In some embodiments, the lentiviral vector is an HIV-1 vector.

[0032] The structure and composition of the vector genome used to prepare the retroviral vector of the present invention are in accordance with those described in the art. In particular, a minimal retroviral gene delivery vector can be prepared from a vector genome that contains only the sequences of the retroviral genome, the non-coding regions of the genome, necessary to provide recognition signals for DNA or RNA synthesis and processing, apart from the recombinant nucleic acid molecule of the present invention. In one embodiment, the retroviral vector genome contains all the elements necessary for nuclear import and correct expression of the polynucleotide of interest (i.e., the transgene). Examples of elements that can be inserted into the retroviral genome of the retroviral vector of the present invention include at least one (preferably two) long terminal repeats (LTRs), such as LTR5' and LTR3', a psi sequence involved in the encapsidation of the retroviral genome, and optionally at least one DNA flap containing cPPT and CTS domains. In some embodiments of the present invention, the LTR, preferably LTR3', has the promoter and enhancer of U3 deleted and replaced by a minimal promoter that allows transcription during vector production, while an internal promoter is added to allow expression of the transgene. In particular, the vector is a self-inactivating (SIN) vector containing a non-functional or modified 3' long terminal repeat (LTR) sequence, which is copied to the 5' end of the vector genome during integration, resulting in the inactivation of promoter activity by both LTRs. Thus, the vector genome can be a replacement vector in which all viral coding sequences between the two long terminal repeats (LTRs) are replaced by the recombinant nucleic acid molecule of the present invention.

[0033] In some embodiments, the retroviral vector genome lacks functional gag, pol and / or env retroviral genes. By "functional" is meant correctly transcribed and / or correctly expressed genes. Thus, the retroviral vector genome of the invention in this embodiment contains at least one of the gag, pol and env genes that is not transcribed or is incompletely transcribed; the term "incompletely transcribed" refers to changes in the transcripts gag, gag-pro or gag-pro-pol, one of which or some of which are not transcribed. In some embodiments, the retroviral genome lacks gag, pol and / or env retroviral genes.

[0034] In some embodiments, the retroviral vector genome also lacks the coding sequences for the Vif-, Vpr-, Vpu-, and Nef-accessory genes (for HIV-1 retroviral vectors), or complete or functional genes thereof.

[0035] Typically, the retroviral vectors of the invention are non-replicating, i.e., the vector and retroviral vector genome are incapable of forming new particles that bud from an infected host cell. This may be achieved by the absence of gag, pol or env genes in the retroviral genome as described in the section above, but can also be achieved by deleting other viral coding sequences and / or cis-acting genetic elements required for particle formation.

[0036] The retroviral vectors of the invention can be produced by any method known in the art, including in stable cell lines and / or by transient transfection with helper viruses. The use of stable cell lines may also be preferred for vector production (Greene, MR et al. Transduction of Human CD34 + Repopulating Cells with a Self-Inactivating Lentiviral Vector for SCID-X1 Produced at Clinical Scale by a Stable Cell Line. Hum. Gene Ther. Methods 23, 297-308 (2012).). For example, the retroviral vectors of the invention can be obtained by transfecting permissive cells (such as 293T cells) in vitro with a plasmid containing the retroviral vector genome of the invention and at least one other plasmid by a transcomplementation system (vector / packaging system), providing in trans the gag, pol and env sequences encoding the polypeptides GAG, POL and envelope proteins, or portions of these polypeptides sufficient to allow the formation of retroviral particles.As an example, a permissive cell is transfected with a) a transcomplementation plasmid lacking the packaging signal psi, optionally with a deletion of the accessory genes vif, nef, vpu and / or vpr, b) a second plasmid (envelope expression plasmid or pseudotyping env plasmid) comprising a gene encoding an envelope protein, and c) a plasmid vector comprising a recombinant retroviral genome, optionally with a deletion of the promoter region of the 3'LTR or the U3 enhancer sequence of the 3'LTR, comprising a psi encapsidation sequence comprising a nucleic acid molecule of the invention between the retroviral 5'LTR and 3'LTR sequences, a nuclear export element (preferably the RRE element of HIV or other retroviral equivalents) and optionally a promoter and / or nuclear import sequence of an RNA (cPPT sequence, e.g. CTS). Advantageously, the three plasmids used do not contain sufficient homologous sequences for recombination. Nucleic acids encoding gag, pol and env cDNAs can be advantageously prepared by conventional techniques from viral gene sequences available in the prior art and databases. The transcomplementation plasmid provides the nucleic acids encoding the retroviral gag and pol proteins. These proteins originate from a lentivirus, most preferably from HIV-1. The plasmid lacks encapsidation sequences, sequences encoding the envelope and accessory genes, and advantageously also lacks retroviral LTRs. The sequences encoding the gag and pol proteins are therefore advantageously placed under the control of heterologous promoters, e.g. cellular, viral, which may be constitutive or regulatable, weak or strong. This is preferably a plasmid containing the sequence transcomplementant Δpsi-CMV-gag-pol-PolyA. This plasmid allows the expression of all proteins necessary for the formation of empty virions, except for the envelope glycoproteins. The transcomplementation plasmid may advantageously contain the TAT and REV genes.Plasmid transcomplementation advantageously lacks the vif, vpr, vpu and / or nef accessory genes. It is understood that different plasmids can also carry the gag and pol genes and the genes TAT ​​and REV, possibly separately. In this case, several plasmids are used for transcomplementation, each encoding one or more of said proteins. The promoters used for plasmid transcomplementation, envelope plasmids and plasmid vectors to drive the expression of the coat proteins gag and pol, the mRNA of the vector genome and the transgene, respectively, are the same or different promoters, advantageously selected from ubiquitous promoters or from specific promoters, such as the viral promoters CMV, TK, RSV LTR promoter and the RNA polymerase III promoters, such as U6 or H1, or the promoters of helper viruses (i.e. adenovirus, baculovirus, herpesvirus) encoding env, gag and pol. For the production of the retroviral vector of the invention, the above-mentioned plasmids can be introduced into competent cells and the produced viruses are recovered. The cells used can be any competent cell, in particular eukaryotic cells, in particular mammalian, e.g. human or animal cells. They can be somatic or embryonic stem cells or differentiated cells. Typically, the cells include 293T cells, fibroblasts, hepatocytes, muscle cells (skeletal muscle, cardiac muscle, smooth muscle, blood vessels, etc.), epithelial cells, neural cells (neurons, glial cells, astrocytes), such as kidney, eye, etc. It can also include insect, plant cells, yeast or prokaryotic cells. It can also be cells transformed by SV40 T antigen. The genes gag, pol and env encoded by the plasmid or the helper virus can be introduced into the cells appropriate for the cell type considered by any method known in the art. Usually, the cells and the vector system are contacted in a suitable device (plate, dish, tube, pouch, etc.) for a time sufficient to transfer the vector system or the plasmid into the cells.Typically, the vector system or plasmid is introduced into the cell by calcium phosphate precipitation, electroporation, transduction, or by using one of the transfection-facilitating compounds, such as lipids, polymers, liposomes, and peptides. Calcium phosphate precipitation is preferred. The cells are cultured in any suitable medium, such as RPMI, DMEM, a specific medium for culturing in the absence of fetal bovine serum. After being transfected, the retroviral vector of the present invention can be purified from the cell supernatant. Purification of the retroviral vector to increase concentration can be achieved by any suitable method, for example, by density gradient purification (e.g., cesium chloride (CsCl)) or by chromatographic techniques (e.g., column or batch chromatography). For example, the vector of the present invention can be subjected to two or three CsCl density gradient purification steps. The vector is desirably purified from the infected cells using a method that includes lysing the cells infected with adenovirus, applying the lysate to a chromatography resin, eluting the adenovirus from the chromatography resin, and collecting the fraction containing the retroviral vector of the present invention.

[0037] In some embodiments, vectors of the invention include "control sequences," which collectively refer to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, etc., that collectively provide for the replication, transcription, and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present, so long as the selected coding sequence is capable of being replicated, transcribed, and translated in an appropriate host cell. Another nucleic acid sequence is a "promoter" sequence, which is used herein in its ordinary sense to refer to a nucleotide region that contains a DNA regulatory sequence, where the regulatory sequence is derived from a gene that is capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence. Transcriptional promoters can include "inducible promoters" (expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters."

[0038] In some embodiments, the polypeptide or polynucleotide of the present invention can be conjugated with at least one other molecule. Typically, the molecule is selected from the group consisting of polynucleotides, polypeptides, lipids, lectins, carbohydrates, vitamins, cofactors, and drugs. In some embodiments, the polypeptide or polynucleotide of the present invention is formulated using one or more lipid-based structures, including but not limited to liposomes, lipoplexes, or lipid nanoparticles (Paunovska, Kalina, David Loughrey, and James E. Dahlman. "Drug delivery systems for RNA therapeutics." Nature Reviews Genetics (2022): 1-16). Liposomes are artificially prepared vesicles that can be composed primarily of lipid bilayers and can be used as a delivery vehicle for administration of pharmaceutical formulations. Liposomes can be of different sizes, including but not limited to, multilamellar vesicles (MLVs), which can be hundreds of nanometers in diameter and contain a series of concentric bilayers separated by narrow aqueous compartments, small univesicular vesicles (SUVs), which can be less than 50 nm in diameter, and large univesicular vesicles (LUVs), which can be 50-500 nm in diameter. Liposome designs can include, but are not limited to, opsonins or ligands to improve attachment of liposomes to unhealthy tissues or to activate events such as, but not limited to, endocytosis. Liposomes can contain low or high pH to improve delivery of pharmaceutical formulations. As a non-limiting example, liposomes such as synthetic membrane vesicles are prepared by the methods, apparatus and devices described in U.S. Patent Application Publication Nos. 20130177638, 20130177637, 20130177636, 20130177635, 20130177634, 20130177633, 20130183375, 20130183373 and 20130183372.In some embodiments, liposomes are formed from 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and MC3 (described in US20100324120), as well as liposomes capable of delivering small molecule drugs, such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.). The polypeptides of the polynucleotides of the present invention can be encapsulated by liposomes and / or can be contained within an aqueous core, which can then be encapsulated in liposomes (see WO 2012031046, WO 2012031043, WO 2012030901, and WO 2012006378 and U.S. Patent Application Publication Nos. 20130189351, 20130195969, and 20130202684).In some embodiments, the polynucleotides of the invention are formulated using stabilized plasmid-lipid particles (SPLPs) or stabilized nucleic acid lipid particles (SNALPs), which have been previously described and shown to be suitable for in vitro and in vivo oligonucleotide delivery (Wheeler et al. Gene Therapy. 1999 6:271-281; ​​Zhang et al. Gene Therapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al., Nature. 2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287; Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J Clin Invest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132; see U.S. Patent Application Publication No. 20130122104).

[0039] By "therapeutically effective amount" is meant a sufficient amount of active ingredient to treat or reduce symptoms at a reasonable benefit / risk ratio applicable to any medical treatment. It is understood that the total daily usage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used, the age, weight, general health, sex and diet of the subject; the administration time, route of administration and excretion rate of the specific compound used; the duration of treatment; the drugs used in combination with the active ingredient; and similar factors well known in the medical field. For example, it is well within the skill of the art to start the dose of the compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0040] Together, we identify herein a critical role for lymphatic endothelial production of 15-LOX metabolite, 15-HETE, as a key regulator of lymphedema.

[0041] As used herein, the term "15-HETE" refers to 15-hydroxyeicosatetraenoic acid. 15-LOX catalyzes the oxygenation of AA, thus generating the 15-hydroxyeicosatetraenoic acid (15-HETE) metabolite. 15-HETE mediators exhibit chemotactic activity, affecting, among other things, the trafficking of neutrophils and stimulating the synthesis of VEGFA. An exemplary representation of the chemical structure of 15-HETE is shown below:

[0042] [ka]

[0043] The results presented in this patent application favor the hypothesis that lymphatic endothelial 15-HETE-initiated Treg recruitment is a central event in the resolution of inflammation in lymphedema. Importantly, 15-HETE does not cause lymphatic damage as observed with 12-HETE and may therefore be a better treatment for lymphedema.

[0044] Thus, in another aspect, the present invention also relates to a method of treating lymphedema in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a 15-LOX metabolite, wherein the 15-LOX metabolite is 15-HETE.

[0045] Typically, the active ingredient (i.e., polypeptide or polynucleotide) of the present invention is combined with a pharma- ceutically acceptable excipient, and optionally a sustained release matrix, such as a biodegradable polymer, to form a pharmaceutical composition. The term "pharmaceutical" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to a mammal, particularly a human, as appropriate. A pharma- ceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In some embodiments, the pharmaceutical composition comprises i) a 15-LOX polypeptide, ii) a polynucleotide encoding a 15-LOX polypeptide, iii) 15-HETE and / or iv) a 15-HETE derivative.

[0046] As used herein, the term "15-HETE derivative" refers to a compound derived from another after transformation of the other. By way of example, a 15-HETE derivative may differ from 15-HETE in one or more atoms or functional groups.

[0047] The present invention is further illustrated by the following figures and examples, which should not, however, be construed as limiting the scope of the invention in any way. [Brief description of the drawings]

[0048] [Figure 1] Lymphatic endothelial ALOX15 controls lymphedema in vivo. Quantification of limb diameter in LD from mice treated with intradermal injection of ALOX15 lentivector (***p<0.001). [Diagram 2] 15-HETE inhibits lymphedema. Quantification of limb diameter in LD from mice treated with intradermal injection of 300 ng of 15-HETE (*p<0.01). [Diagram 3]15-HETE inhibits cutaneous lymphatic reflux in lymphedema. Left panel, limb lymphogram from 15-HETE-treated mice. Right panel, quantification of lymphatic branching points in 15-HETE-treated mice (*p<0.05). Example 1

[0049] Main methods Human tissue specimens A total of 17 lipocutaneous excision specimens were collected. Samples were obtained from long-term archival paraffin blocks of lymphedema from patients treated at the Rangueil Hospital, Toulouse, France, in 2015–2016. Samples were selected as coded specimens under a protocol approved by the INSERM Institutional Review Board (DC-2008-452) and the Research State Department (Ministry of Research, ARS, CPP2, authorization AC-2008-452). When possible, some control arm tissue samples were collected for aesthetic purposes.

[0050] Adipocytes isolated from arm subcutaneous adipose tissue were obtained from healthy adult women undergoing elective surgical procedures for aesthetic fat removal at Langueil Hospital, Toulouse, France. Informed consent was sought for the use of AT samples for research purposes only, prior to plastic surgery. Briefly, after digestion with collagenase (Sigma) and dispase (Gibco), the upper phase containing mature adipocytes was collected and washed three times in ECBM medium (Promocell, Heidelberg, Germany) containing 0.5% free fatty acid bovine albumin (Sigma, France). Mature adipocytes were maintained in cell culture cassettes (Clinicell25, Laboratoires MABIO International, Tourcoing, France) completely filled with ECBM / BSA medium. After 24 h incubation at 37°C in a humidified 5% CO2 cell incubator and centrifugation at 50 g for 2 min, the lower phase was collected, filtered through a 0.22 μm filter, and aliquots were frozen until use.

[0051] Mouse model All studies were approved by the local ethical committee, followed the guidelines of the European Convention for the Protection of Vertebrate Animals Used for Experimentation, and were performed according to the INSERM IACUC (France) guidelines for laboratory animal husbandry. All animal experiments were approved by the Inserm Rangueil-Purpan, a regional branch of the Midi-Pyrenees Ethics Committee, France. Randomization was ensured by selecting animals from different cages of the same experimental group.

[0052] Mice were housed in individually ventilated cages in a temperature- and light-controlled room in an SPF facility and had free access to food and water. Female C57BL / 6J mice (6 weeks old) were obtained from Janvier (Le Genest Saint Isle, France). The mouse model of lymphedema was performed as previously described (Morfoisse ATVB 2008).

[0053] result: Human lymphedema tissue displays an inflammatory profile. To broadly identify gene expression signatures associated with secondary lymphedema, we performed bulk-RNA sequencing and differential expression analysis (DEseq) followed by protein-coding RNA profiling on dermal and adipose tissue sections from four patients (data not shown). Analysis of the data identified 211 mRNAs that were upregulated (182 genes) or downregulated (29 genes) in the lymphedema arm (LD) compared to the control arm (CTL) (data not shown).

[0054] Overall, hierarchical cluster analysis indicates that inflammation- and lipid metabolism-related genes are differentially regulated in LD (data not shown). In particular, we found that PTX3, which is expressed in response to inflamed endothelium, is specifically regulated in LD. 24 , Amphiregulin (AREG) stimulates the expression of VEGFC 25, we observed a strong upregulation of IL6, a major inflammatory cytokine (data not shown).

[0055] Surprisingly, most of the genes involved in lymphangiogenesis, including VEGFC and its receptor Flt4, were upregulated (data not shown). Perhaps this led to the increased density of disorganized, leaky and dysfunctional lymphatic vessels observed in lymphedematous limbs when performing lymphangiography on patients. We observed upregulation of genes involved in matrix remodeling, including a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and tenascin C (TNC) (data not shown). Conversely, we observed downregulation of genes related to heparan sulfate (HS2ST1, MAMDC2, FGFR2) (data not shown).

[0056] To confirm the inflammatory state in lymphedema, flow cytometry analysis of dermal fat excisions was performed on lymphedema arms compared to normal arms of the same patients. Among immune cell populations, we found increased lymphocyte recruitment compared to macrophages (data not shown). In particular, T cells (CD4 positive) were upregulated relative to B lymphocytes (data not shown).

[0057] Lymphedematous adipose tissue shows a decrease in inflammation-resolving lipid mediators. LD promotes the accumulation of adipose tissue in the limbs, which in part contributes to interstitial fluid stasis leading to the accumulation of inflammatory cells. To understand whether lipid composition may be associated with changes in immune cell recruitment, we performed lipidomic analysis of skin and fat excisions from lymphedematous arms compared to non-injured arms (not shown). Differential analysis of arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) was performed in skin and adipose tissue (data not shown). We observed an overall decrease in lipid-derived mediators in both tissues. In particular, we found that arachidonic acid (AA)-derived lipid mediators, especially the pro-resolving molecules generated by 15-lipoxygenase (15-LOX), were strongly reduced in lymphedema (data not shown). We observed a decrease in the main AA derivatives, 15-hydroxyeicosatetraenoic acid (15-HETE), lipoxins A4 and B4 (LXA4 and LXB4) (data not shown). In contrast, we did not find any differences in lipids generated by DHA metabolism (data not shown). This was not explained by the overall expression of 15-LOX mRNA or protein expression in lymphedema tissues (data not shown). This discrepancy may be related to the small amount of tissue analyzed (μg) compared to patient explants (up to 2.5 kg). However, the number of 15-LOX positive cells was decreased in lymphedema tissue samples (data not shown). More importantly, we observed for the first time that 15-LOX is expressed in lymphatic endothelium (data not shown).

[0058] 15-LOX activity is reduced in a mouse lymphedema model To understand the molecular mechanisms underlying the inflammatory process in lymphedema, we performed a time course analysis of adipose tissue in a mouse lymphedema model (not shown). In this model, based on mastectomy with brachial and axillary lymph node dissection, mice showed increased skin reflux with marked swelling of the limbs (data not shown). At the early stage of LD (2 weeks after surgery), no significant differences in lipid content were observed (data not shown). In contrast, in acquired lymphedema (8 weeks after surgery), we found a pattern similar to that observed in human tissue samples (data not shown). In particular, we observed a decrease in AA-derived lipid mediators (15-HETE) generated by 15-LOX, while no differences were found in lipids generated by DHA metabolism (17-HDOHE) (data not shown). After 8 weeks of LD, we observed that changes in proresolvin content were associated with a decrease in 15-LOX mRNA and protein expression (data not shown). Importantly, downregulation of 15-LOX was also observed in lymphatic endothelium (data not shown).

[0059] 15-LOX promotes Treg recruitment into lymphedema tissues To evaluate the role of 15-LOX in LD, we used the pharmacological inhibitor PD146176. In the presence of 15-LOX inhibitors, LD swelling was increased (data not shown), with strong dermal reflux (data not shown) followed by dermal fibrosis (data not shown). Since we previously showed that T cell populations were recruited during the development of LD, we next performed flow cytometry analysis. We observed that CD4+ T cells increased during LD in the presence of 15-LOX inhibitors, which was accompanied by a decrease in the recruitment of Tregs (data not shown). This was confirmed by a decrease in the immunodetection of Tregs (Foxp3 positive cells) in lymphedema tissues (data not shown).

[0060] 15-LOX regulates T cell adhesion to LECs On the other hand, to investigate whether lymphatic endothelial 15-LOX could regulate T cell trafficking, we knocked down 15-LOX in primary cultures of HDLEC using siRNA (data not shown). Despite the low knockdown (30%), we observed a significant downregulation of the chemoattractant cytokine CCL21 in LEC-ALOX15KD (data not shown). We did not find any regulation of SPHK1 and sphingosine-1-phosphate receptor 1 (S1PR1) (data not shown), whereas SPHK2, which maintains endothelial integrity, was upregulated (data not shown). 26 Knockdown of 15-LOX significantly reduced lymphotoxin beta receptor (LTBR), which plays a critical role in lymphatic transendothelial Treg migration (data not shown). 27 We also observed a reduction in the expression of the claudin-5 adhesion molecule, which was associated with an increased inflammatory state of the lymphatic endothelium, as indicated by overexpression of iNOS (data not shown).

[0061] To decipher the role of 15-Lox lipid-derived mediators, we studied the effect of 15-HETE on cultured LECs. We identified a direct effect of 15-HETE on lymphatic endothelium, which promoted adhesion of T cells to lymphatic monolayers (data not shown) and had no effect on LEC proliferation (data not shown), but stimulated their migration (data not shown).

[0062] Selective lymphatic endothelial 15-LOX initiates inflammation resolution in lymphedema. To selectively study the role of lymphatic endothelial 15-LOX, we used Prox1CreERT2;Alox15, a gene in which the ALOX15 gene is selectively disabled in the lymphatic system after tamoxifen induction. fl / flConditional knockout mice were generated (data not shown). We observed a significant exacerbation of lymphedema in homozygous and heterozygous mice (data not shown). This was not associated with a reduction in lymphangiogenesis in lymphedematous limbs (Figures 6c and d). To study the effect of 15-LOX as a potential treatment for lymphedema, we generated a lentivector overexpressing the enzyme. Three intradermal injections of 15-LOX lentivector were performed at the time of surgery. In this rescue experiment, the development of LD was completely abolished using the lentivector, confirming the crucial role of 15-LOX in LD (Figure 1).

[0063] Consideration: Secondary lymphedema is a major complication of cancer treatment. It is a multifactorial pathology characterized by lymphatic endothelial dysfunction, accumulation of fluid and adipose tissue, intense skin fibrosis and chronic inflammation in the arms or legs. It was surprising to observe such a reproducible gene expression profile when comparing the lymphedema arm with the control arm in each patient, despite the many comorbidities associated with lymphedema. Half of these genes were involved in inflammatory activation, which is consistent with previous studies showing a critical role of inflammation in the development of lymphedema. Since lymphedema occurs months and sometimes years after cancer treatment, it is obvious to speculate that unseen chronic inflammation is occurring for months before physical signs such as skin fibrosis occur. To date, it is widely accepted that anti-inflammatory drugs are the best candidates for treating some symptoms of lymphedema. Unresolved inflammation is central to the pathophysiology of common vascular diseases such as atherosclerosis, aneurysms, and deep vein thrombosis. 5 Resolution of vascular inflammation is a key driver of vessel wall remodeling and functional recovery in these clinical settings. In that context, resolution-promoting lipid mediators derived from omega-3 polyunsaturated fatty acids orchestrate key cellular processes that drive resolution and return to homeostasis. Thus, identification of their effects on lymphatic vessels raises great interest in their properties in lymphedema.

[0064] Recent studies have also demonstrated elevated leukotriene B4 (LTB4) synthesis in patients with lymphedema. 28 LTB4 was detrimental to lymphatic repair at concentrations observed in established disease, suggesting that LTB4 is a promising drug target for the treatment of acquired lymphedema. We confirmed this hypothesis with lipidomic analysis of lymphedema tissues, which showed significant downregulation of AA-derived lipid mediators, including LTB4, in lymphedema.

[0065] Pro-convergence lipid mediators are synthesized locally in vascular tissues. They have a direct effect on vascular cell-leukocyte interactions and they play a protective role in injury responses. They are therefore considered as candidate biomarkers in vascular diseases as well as potential vascular therapeutics. Herein, we investigated the molecular and cellular mechanisms of convergence in lymphatic vasculature to improve tools for clinical measurements and to better define the potential for "convergence therapeutics" in lymphedema patients. Among the different enzymes that generate lipid mediators, we identified that 15-LOX was expressed both on immune cells and on lymphatic endothelial cells, suggesting an autocrine role on lymphatic function. No adverse effects of 15-HETE on lymphatic endothelium are observed compared to 12-HETE, which punctures lymphatic monolayers as previously described by Krejaschki and coworkers. 17 In our study, we did not find any regulation of 12-HETE in lymphedema. However, Krejaschki's study was limited to the paracrine effect of 12-HETE produced by tumor cells during the metastatic process. Herein, we first identified the presence of 15-LOX in lymphatic endothelial cells. Importantly, the expression of 15-LOX correlated with reduced infiltration of Tregs, especially the Foxp3+CD4+ subset, into lymphedema adipose tissue.

[0066] Treg cells accumulate in a variety of non-lymphoid tissues and exert both anti-inflammatory and homeostatic functions 29 Recently, a novel subset of Tregs was identified in visceral adipose tissue. 30,31,32 They show distinct transcriptomes for lymphoid and non-lymphoid tissues. 29,32,33 , and their accumulation is dependent on IL-33 30 Herein, we found that 15-HETE induces the synthesis of CCL21 by lymphatic endothelial cells to stimulate the chemoattraction of Tregs. Importantly, ALOX15 LECKO Using mice, we identified a critical role for lymphatic endothelial production of 15-HETE as a key regulator in lymphedema.

[0067] Taken together, these complementary results favor the hypothesis that lymphatic endothelial 15-HETE-initiated Treg recruitment is a central event in the resolution of inflammation in lymphedema. Importantly, 15-HETE did not induce lymphatic damage as observed with 12-HETE. 17 , and may therefore be a potential treatment for lymphedema without promoting any metastatic processes. Example 2

[0068] 15-HETE protects against lymphedema To evaluate the effect of 15-HETE on lymphedema, mice were injected intradermally at 300 ng into the limbs at the time of surgery (Figure 2). Compared to control mice injected with 15-HETE's adjuvant (ethanol), mice treated with 15-HETE did not develop lymphedema. This was associated with a strong reduction in cutaneous lymphatic reflux, a specific feature of lymphedema (Figure 3).

[0069] References: Throughout this application, various references are referred to that describe the state of the art to which this invention pertains, the disclosures of which are incorporated herein by reference into this disclosure.

[0070]

Table 1

Claims

1. A method of treating lymphedema in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of i) a 15-LOX polypeptide or ii) a polynucleotide encoding a 15-LOX polypeptide.

2. 2. The method of claim 1, wherein the 15-LOX polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence ranging from amino acid residue 115 to amino acid residue 662 of SEQ ID NO:

1.

3. The method of claim 1 , wherein the polynucleotide is messenger RNA (mRNA).

4. The method of claim 1 , wherein the polynucleotide is inserted into a vector, such as a viral vector.

5. The method of claim 4, wherein the viral vector is an AAV vector.

6. The method of claim 4, wherein the viral vector is a retroviral vector.

7. The method of claim 6 , wherein the retroviral vector is a lentiviral vector.

8. A method of treating lymphedema in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a 15-LOX metabolite, wherein the 15-LOX metabolite is 15-HETE.

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