Treatment of lymphatic edema with empapelin

Abstract

Lymphedema is a chronic disease caused by accumulation of liquid and fat in limbs due to lymphatic dysfunction. Herein, the inventor finds that the bioactive peptide empapin is a good candidate drug for restoring lymphatic flow in lymphedema. They find that the expression of empapin in female lymphedema arms is significantly reduced compared to normal arms. They use an empalin knockout mouse model, demonstrating the key effect of empalin because lymphedema lasts for more than 4 weeks after surgery and is associated with hypodermic lymphatic vessel production and increased dermis reflux. The inventor proves that the limb swelling is obviously relieved by intradermal injection of the empapin-lentiviral vector. This is associated with reduced dermal fibrosis and increased lymphatic density. Importantly, empapin stimulates eNOS-mediated lymphatic pumping by phosphorylation of Akt and eNOS in lymphatic endothelial cells (LEC), and the empapin can stimulate eNOS-mediated lymphatic pumping by phosphorylation of Akt and eNOS. This is related to the significant increase of E2F8-targeted gene expression in LEC by direct binding of E2F8 to a CCBE1 promoter. In a word, the result shows that the empapelin plays a key role in lymphedema, so that the empapelin is a new partner of the VEGF-C for preventing limb swelling and tissue fibrosis in the lymphedema.

Description

Field of the Invention

[0001] The present invention belongs to the field of medicine, in particular to the treatment of lymphedema. Background of the Invention

[0003] Lymphedema is a multifactorial disease that significantly impacts patients' quality of life (Greene, Grant et al. 2012, Hoffner, Peterson et al. 2018). Lymphedema is characterized by lymphatic dysfunction that results from genetic mutations (primary lymphedema) or cancer treatments (surgery, radiotherapy, chemotherapy, etc.), resulting in the inability of lymph fluid to return to the bloodstream (Mortimer and Rockson 2014).

[0004] Lymphatic stasis triggers changes in surrounding tissues, leading to adipose tissue accumulation and severe fibrosis. Adipose tissue deposition has been attributed to stimulation of lipogenesis in the affected limb (Aschen, Zampell et al. 2012, Zampell, Aschen et al. 2012). Recent evidence highlights a new concept, demonstrating an imbalance between de novo adipose tissue production and lipolysis, suggesting significant changes in adipokine synthesis (Koc, Wald et al. 2021, Sano, Hirakawa et al. 2022). Furthermore, a major hallmark of lymphedema associated with lymphostasis is the development of fibrosis in the skin and adipose tissue (Mortimer and Rockson 2014). Lymphostatic fibrosis defines the progression of lymphedema from the reversible stage to the elephantiasis stage. To date, there is no cure for lymphedema other than massage and compression bandages. However, once fibrosis develops, the tissue becomes denser, leading to lymphatic obstruction and worsening lymphedema. Importantly, fibrosis also affects collecting lymphatic pumping and increases limb swelling (Baik, Park et al. 2022)(Kataru, Wiser et al. 2019).

[0005] Numerous cytokines and peptides selectively participate in adipocyte metabolism, endothelial function, or tissue fibrosis. However, the bioactive peptide apelin combines beneficial effects across the entire limb tissue. Apelin is an endogenous ligand for the G-protein-coupled receptor APJ, expressed in multiple organs (Pope, Roberts et al. 2012). The first evidence linking apelin to the lymphatic vasculature came from the finding that apelin stimulates angiogenesis and lymphangiogenesis in the tumor setting (Berta, Hoda et al. 2014). Furthermore, our group previously described beneficial effects of apelin on cardiac lymphatic vasculature after cardiac ischemia (Tatin, Renaud-Gabardos et al. 2017). We found that apelin restored lymphatic morphology in precollecting vessels within the infarcted area, suggesting that apelin may be a promising candidate for restoring lymphatic morphology in damaged tissue (Tatin, Renaud-Gabardos et al. 2017). However, epazolin was originally described as an adipokine synthesized by white adipose tissue and other organs, such as the heart, kidney, and central nervous system (Castan-Laurell, Boucher et al. 2005) (Dai, Smith et al. 2013). The production and secretion of epazolin by adipocytes is controlled by insulin, and its beneficial effects on adipose tissue have made it a promising target for obesity and diabetes (Castan-Laurell, Boucher et al. 2005). epazolin is also a key player in fibroprotection in many organs, including the heart, lungs, and kidneys (Huang, Chen et al. 2016). epazolin inhibits the development of myocardial fibrosis after ischemia and atrial fibrillation by blocking the Ang2 pathway. In the heart, it is involved in maintaining normal lymphatic vessel shape after ischemia (Tatin, Renaud-Gabardos et al. 2017). It reduces renal and skin fibrosis by inhibiting TGFβ signaling (Yokoyama, Sekiguchi et al. 2018). Surprisingly, the protective effects of epazolin on white adipose tissue fibrosis are poorly studied. Specifically, the beneficial effects of epazolin on diet-induced obesity have been attributed to its ability to improve lymphatic and vascular integrity (Sawane, Kajiya et al. 2013). Epazolin stimulates nitric oxide production via PI3K / Akt signaling in blood endothelial cells (Busch, Strohbach et al. 2015). Summary of the Invention

[0006] The present invention is defined by the following claims. Specifically, the present invention relates to the use of epazolin for treating lymphedema. Detailed Description of the Invention

[0008] Lymphedema is a chronic disease caused by lymphatic dysfunction, leading to the accumulation of fluid and fat in the limbs. It is characterized by severe inflammation and fibrosis that affect mobility and increase the risk of skin infections. Here, the inventors discovered that the bioactive peptide epagliflozin is a good candidate for restoring lymphatic flow in lymphedema. They found that epagliflozin expression in women with lymphedema arms was significantly reduced compared to their normal arms. Using an epagliflozin knockout mouse model, they confirmed the key role of epagliflozin, as lymphedema persisted for more than 4 weeks after surgery and was associated with insufficient dermal lymphangiogenesis and increased dermal return. The inventors showed that intradermal injection of an epagliflozin-lentiviral vector significantly reduced limb swelling. This was associated with a reduction in dermal fibrosis and an increase in lymphatic density. Importantly, epagliflozin stimulated eNOS-mediated lymphatic pumping in lymphatic endothelial cells (LECs) through Akt and eNOS phosphorylation. This was associated with a significant increase in the expression of E2F8-targeted genes through direct E2F8 binding to the CCBE1 promoter in LECs. Together, our results suggest that epasitin plays a key role in lymphedema and is therefore a novel partner of VEGF-C in preventing limb swelling and tissue fibrosis in lymphedema.

[0009] Therefore, 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) an epasiin polypeptide or ii) a polynucleotide encoding an epasiin polypeptide.

[0010] The term "lymphedema" as used herein has its general meaning in the art and refers to a disease characterized by intense tissue swelling due to increased fluid retention in the tissues, localized accumulation of fatty tissue, and impaired immune function due to reduced lymphatic drainage. The term includes "primary lymphedema" and "secondary lymphedema." Primary lymphedema is a malformation of the lymphatic system characterized by swelling of the limbs that may be associated with other lymphatic accumulations. This disease is caused by an underlying developmental abnormality of the lymphatic system (abnormal lymphangiogenesis). It can be hereditary or non-hereditary, congenital or late-onset. In some embodiments, lymphedema is a secondary disease that can be caused by lymph node removal or lymphatic vessel damage. Such secondary lymphedema may also follow, for example, lymph node removal and injury associated with surgery, radiation therapy, neoplastic disease and its treatment, musculoskeletal injuries such as fractures, tendon releases and joint replacements, neurological disorders such as muscle paresis, vascular injury / surgery, epidermal injury, coagulation disorders such as deep vein thrombosis, scar tissue formation, tamoxifen treatment, filariasis, infection, lipedema or cellulitis.

[0011] The term "treatment" as used herein refers to both prophylactic or preventative treatment and curative or disease-modifying treatment, including treatment of patients with a risk of infectious disease or suspected infectious disease and patients who are ill or have been diagnosed with a disease or medical condition, and includes suppressing clinical relapse. Treatment can be applied to patients with a medical disease or who may eventually develop a disease, to prevent a disease or a recurrent disease, to cure a disease or a recurrent disease, to delay the onset of a disease or a recurrent disease, to alleviate the severity of a disease or a recurrent disease, or to improve one or more symptoms of a disease or a recurrent disease, or to prolong the patient's survival time so that it exceeds the expected survival time in the absence of such treatment." therapeutic regimen" refers to the treatment mode of a disease, such as the dosage mode used during treatment. A therapeutic regimen can include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or part of a therapeutic regimen) for the initial treatment of a disease. The overall goal of an induction regimen is to provide a high level of drug to the patient in the initial stages of a therapeutic regimen. An induction regimen may employ (part or all of) a "loading regimen," which may include administering a higher dose of the drug than the physician administered during the maintenance regimen, administering the drug more frequently than the physician administered during the maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or portion of a treatment regimen) used to maintain a patient during treatment for a disease, e.g., to keep the patient in remission for a long period of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering the drug at fixed intervals, such as weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupting treatment, intermittent treatment, treating upon relapse, or treating upon reaching specific predetermined criteria [e.g., disease manifestations, etc.]).

[0012] In particular, the methods of the present invention are particularly suitable for increasing the plasticity, contractility and / or dilatation of lymphatic vessels.

[0013] As used herein, the term "apelin" has its ordinary meaning in the art and refers to a 77-residue preprotein (NCBI reference sequence: NP-0059109.3, encoded by NCBI reference sequence NM-017413.3), which is processed into biologically active forms of apelin peptides, such as apelin-36, apelin-17, apelin-16, apelin-13, and apelin-12. The full-length mature peptide is called "apelin-36" and contains 36 amino acids, but the most effective isoform is a pyroglutamylated form of 13-mer apelin (apelin-13), called "Pyr-1-apelin-13 or Pyr1-apelin-13". Different apelin forms are described, for example, in U.S. Patent No. 6,492,324B1. An exemplary amino acid sequence of apelin is represented by SEQ ID NO: 1.

[0014] SEQ ID NO:1>sp|Q9ULZ1|APEL_HUMAN Apelin OS=Homo sapiens OX=9606 GN=APLN PE=2 SV=1

[0015] MNLRLCVQALLLLWLSLTAVCGGSLMPLPDGNGLEDGNVRHLVQPRGSRNGPGPWQGGRRKFRRQRPRLSHKGPMPF

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

[0017] As used herein, the term "apelin polypeptide" refers to a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 1.

[0018] As used herein, "percent identity" between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps and the length of each gap that need to be introduced to optimally align the two sequences. Mathematical algorithms can be used to compare sequences and determine the percent identity between two sequences, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443–53.). Algorithms such as EMBOSS Needle (pairwise alignment; available at www.ebi.ac.uk) can also be used to determine the percent identity between two nucleotide or amino acid sequences. For example, EMBOSS Needle can be used with the BLOSUM62 matrix, with a "gap opening penalty" of 10, a "gap extension penalty" of 0.5, a pseudo "end gap penalty", a "end gap opening penalty" of 10 and a "end gap extension penalty" of 0.5. Typically, "percent identity" is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For example, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, the identity is 60%. % identity is typically determined over the entire length of the query sequence being analyzed. Two molecules having the same primary amino acid sequence 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.

[0019] As used herein, the term "polynucleotide" refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. The term refers to the primary structure of the molecule. Thus, the term includes triple-stranded, double-stranded, and single-stranded deoxyribonucleic acids ("DNA"), as well as triple-stranded, double-stranded, and single-stranded ribonucleic acids ("RNA"). It also includes modified forms (e.g., by alkylation and / or capping) and unmodified forms of the polynucleotides. More specifically, the term "polynucleotide" includes the following: polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA and mRNA (whether spliced ​​or unspliced), any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing non-nucleotide backbones, such as polyamides (e.g., peptide nucleic acids "PNA") and polymorpholino polymers, as well as other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleobases in a configuration that allows base pairing and base stacking, such as those found in DNA and RNA. In some embodiments, the polynucleotide includes mRNA. On the other hand, the mRNA is a synthetic mRNA. In some embodiments, the synthetic mRNA includes at least one non-natural nucleobase. In some embodiments, all nucleobases of a certain class have been replaced by non-natural nucleobases (e.g., all uridines in the polynucleotides disclosed herein can be replaced by non-natural nucleobases such as 5-methoxyuridine). In some embodiments, a polynucleotide (e.g., synthetic RNA or synthetic DNA) comprises only natural nucleobases, ie, A, C, T, and G in the case of synthetic DNA, or A, C, T, and U in the case of synthetic RNA.

[0020] In some embodiments, the polynucleotides of the invention are messenger RNA (mRNA).

[0021] In some embodiments, the polynucleotide is inserted into a vector, such as a viral vector.

[0022] The term "viral vector" as used herein refers to a virion or viral particle that functions as a nucleic acid delivery vehicle, comprising a vector genome packaged within the virion or viral particle. Typically, the vector is a viral vector that is an adeno-associated virus (AAV), a retroviral vector, a bovine papilloma virus, an adenoviral vector, a vaccinia virus, or a polyoma virus.

[0023] In some embodiments, the viral vector is an AAV vector.

[0024] 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 mutant forms thereof. The AAV vector may have a complete or partial deletion of one or more AAV wild-type genes, preferably the rep and / or cap genes, but retain functional flanking ITR sequences.

[0025] In some embodiments, the viral vector is a retroviral vector.

[0026] As used herein, the term "retroviral vector" refers to a vector that contains structural and functional genetic elements primarily derived from retroviruses.

[0027] In some embodiments, the retroviral vector of the invention is derived from a retrovirus selected from the group consisting of an alpha retrovirus (e.g., avian leukosis virus), a beta retrovirus (e.g., mouse mammary tumor virus), a gamma retrovirus (e.g., murine leukemia virus), a delta retrovirus (e.g., bovine leukemia virus), an epsilon retrovirus (e.g., Walley cutaneous sarcoma virus), a lentivirus (e.g., HIV-1, HIV-2), and a foamy virus (e.g., human foamy virus).

[0028] In some embodiments, the retroviral vectors of the present invention are replication-defective retroviral particles that can transfer exogenously introduced genomic RNA rather than retroviral mRNA.

[0029] In some embodiments, the retroviral vector of the invention is a lentiviral vector.

[0030] As used herein, the term "lentiviral vector" refers to a vector that contains structural and functional genetic elements 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.

[0031] The structure and composition of the vector genome for preparing the retroviral vector of the present invention are consistent with those described in the art. In particular, minimal retroviral gene delivery vectors can be prepared from vector genomes, and in addition to the recombinant nucleic acid molecule of the present invention, they only comprise the sequence of the genomic non-coding region of the retroviral genome, which is necessary for providing recognition signals for DNA or RNA synthesis and processing. In some embodiments, the retroviral vector genome comprises all elements necessary for nucleic acid introduction and correct expression of target polynucleotides (i.e., transgenes). Examples of elements that can be inserted into the retroviral genome of the retroviral vector of the present invention are at least one (preferably two) long terminal repeats (LTRs), such as LTR5' and LTR3', psi sequences involved in retroviral genome encapsidation, and optionally at least one DNA flap structure (DNA flap) comprising cPPT and CTS domains. In some embodiments of the present invention, the LTR of the promoter and enhancer of U3 is deleted, preferably LTR3', and replaced with a minimal promoter that allows transcription during vector production, while adding an internal promoter to allow transgenic expression. In particular, the vector is a self-inactivating (SIN) vector containing a non-functional or modified 3' long terminal repeat (LTR) sequence. This sequence is copied to the 5' end of the vector genome during integration, resulting in the two LTRs inactivating promoter activity. Therefore, the vector genome can be a replacement vector in which all viral coding sequences between the two long terminal repeats (LTRs) have been replaced by the recombinant nucleic acid molecules of the present invention.

[0032] In some embodiments, the retroviral vector genome lacks functional gag, pol, and / or env retroviral genes. "Functional" refers to genes that are correctly transcribed and / or correctly expressed. Thus, in this embodiment, the retroviral vector genome of the invention comprises at least one of the gag, pol, and env genes that is not transcribed or incompletely transcribed; the expression "incompletely transcribed" refers to an alteration in the transcripts gag, gag-pro, or gag-pro-pol, where one or more are not transcribed. In some embodiments, the retroviral genome lacks gag, pol, and / or env retroviral genes.

[0033] 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 all or functional genes thereof.

[0034] Typically, the retroviral vectors of the present invention are non-replicative, i.e., the vector and retroviral vector genome are incapable of budding from infected host cells to form new particles. This can be achieved by lacking the gag, pol, or env genes in the retroviral genome, as described above; this can also be achieved by deleting other viral coding sequences and / or cis-acting genetic elements required for particle formation.

[0035] Therefore, the present invention covers the use of virus-like particles. The term "virus-like particle" or "VLP" as used herein refers to a structure that is similar to a virus particle but does not contain a viral genome, cannot replicate and is non-pathogenic. The particle typically contains at least one structural protein from a virus. Preferably, there is only one structural protein. Most preferably, there are no other non-structural components of the virus. Therefore, virus-like particles can be spontaneously self-assembled by viral structural proteins in vitro under appropriate conditions, while excluding genetic material and potential replication possibilities. Virus-like particles with a diameter of about 20 to 150 nm also have the characteristics of nanomaterials, such as a large surface area, surface accessible amino acids with reactive portions (such as lysine and glutamic acid residues), a regular (inerratic) spatial structure and good biocompatibility. Therefore, the assembled virus-like particles have great potential as a delivery system specifically for carrying various cargoes. In some embodiments, one or more zinc finger motifs of the Gag protein are replaced by one or more RNA binding domains. In some embodiments, the RNA binding domain is the Coat protein of MS2 phage, PP7 phage or Q3 phage, the prophage HK022 Nun protein, the U1A protein or the hPum protein. More preferably, the RNA binding domain is the Coat protein of MS2 phage or PP7 phage. Even more preferably, the RNA binding domain is the Coat protein of MS2 phage. These embodiments are particularly suitable for packaging the mRNA encoding the epazolin polypeptide into VLPs. Therefore, in some embodiments, the mRNA encoding the epazolin polypeptide encapsulated in the viral particles of the present invention contains at least one encapsidation sequence. "Encapsidation sequence" refers to the RNA motif (sequence and three-dimensional structure) specifically recognized by the RNA binding domain as described above. Preferably, the encapsidation sequence is a stem-loop motif. Even more preferably, the encapsidation sequence of the retroviral particle is the stem-loop motif of the RNA of MS2 phage or PP7 phage. The stem-loop motif, more specifically the stem-loop motif of the RNA of MS2 phage or the RNA of PP7 phage, can be used alone or repeated multiple times, preferably repeated 2 to 25 times, more preferably repeated 2 to 18 times, for example repeated 6 to 18 times. In some embodiments, the present invention relates to the use of non-integrating lentiviral particles constructed using phage capsid proteins and their homologous 19-nt stem-loops. Technology is used to replace the natural lentiviral Psi packaging sequence to achieve packaging of active mRNA into lentiviral particles (Prel A, Caval V, Gayon R, Ravassard P, Duthoit C, Payen E, Maouche-Chretien L, Creneguy A, Nguyen TH, Martin N, Piver E, Sevrain R, Lamouroux L, Leboulch P, Deschaseaux F, Bouillé P, Sensébé L, Pagès JC. Highly efficient invitro and in vivo delivery of functional RNAs using new versatile MS2-chimeric retrovirus-like particles. Mol Ther Methods Clin Dev. 2015 Oct 21; 2: 15039. doi: 10.1038 / mtm.2015.39. PMID: 26528487; PMCID: PMC4613645).

[0036] The retroviral vector of the present invention can be produced by any method known in the art, including transient transfection, in stable cell lines and / or by helper virus. Stable cell lines can also be preferably used for the production of vectors (Greene, MRet al.Transduction of Human CD34+Repopulating Cells with a Self-Inactivating Lentiviral Vector for SCID-X1Produced at Clinical Scale by aStable Cell Line.Hum.Gene Ther.Methods 23,297–308 (2012).). For example, the retroviral vector of the present invention can be obtained by a trans-complementation system (vector / packaging system), by using a plasmid containing the retroviral vector genome of the present invention and at least one other plasmid (with trans-providing coding polypeptide GAG, POL and envelope protein (or providing these polypeptides enough to make the part of retroviral particles formed) gag, pol and env sequence) in vitro transfection allows cells (such as 293T cells). For example, permissive cells are transfected with: a) a transcomplementing plasmid lacking the packaging signal psi and optionally deleting the accessory genes vif, nef, vpu and / or vpr, b) a second plasmid (envelope expression plasmid or pseudotyped env plasmid) comprising a gene encoding an envelope protein, and c) a plasmid vector comprising a recombinant retroviral genome, optionally deleting the promoter region of the 3'LTR or the U3 enhancer sequence of the 3'LTR, comprising a psi encapsidation sequence, a nuclear export element (preferably an HIV RRE element or other retroviral equivalent) between the 5' and 3' retroviral LTR sequences, comprising a nucleic acid molecule of the present invention and optionally an RNA promoter and / or a nuclear import sequence (cPPT sequence, e.g., CTS). Advantageously, the three plasmids used do not contain homologous sequences sufficient for recombination. Nucleic acids encoding gag, pol and env cDNAs can be advantageously prepared from viral gene sequences available in the prior art and databases according to conventional techniques. The transcomplementing plasmid provides nucleic acids encoding the retroviral gag and pol proteins. These proteins are derived from a lentivirus, most preferably from HIV-1. The plasmid lacks encapsidation sequences, sequences encoding the envelope, accessory genes, and advantageously also lacks the retroviral LTR. Thus, the sequences encoding the gag and pol proteins are advantageously placed under the control of a heterologous promoter (e.g., cellular, viral, etc.), which can be constitutive or regulated, weak or strong. Preferred is a plasmid comprising the transcomplementing sequence Δpsi-CMV-gag-pol-PolyA.In one embodiment, the plasmid is used for the expression of the empty virion. This plasmid allows expression of all proteins required for forming empty virions, except envelope glycoprotein. Plasmid trans-complementation can advantageously include TAT and REV genes. Plasmid trans-complementation advantageously does not contain vif, vpr, vpu and / or nef accessory genes. It is understood that gag and pol genes and TAT and REV genes can also be carried by different plasmids, and these plasmids can be separated. In this case, several plasmids of trans-complementation are used, and one or more of the proteins described in each plasmid encoding are used. The promoter used in plasmid trans-complementation, envelope plasmid and plasmid vector for promoting the expression of gag and pol, the mRNA of the vector genome and the transgenic is the same or different promoter, advantageously selected from ubiquitin promoter (ubiquitous promoter), or particularly selected from the promoter of the helper virus (i.e. adenovirus, baculovirus, herpes virus) of for example viral promoter CMV, TK, RSVLTR promoter and RNA polymerase III promoter such as U6 or H1 or encoding env, gag and pol. In order to produce the retroviral vector of the present invention, the above-mentioned plasmid can be introduced into competent cells and the virus produced in the harvest. The cells used can be any competent cells, particularly eukaryotic cells, particularly mammalian cells, such as human or animal cells. They can be somatic cells or embryonic stem cells or differentiated. Typically, cells include 293T cells, fibroblasts, hepatocytes, myocytes (skeletal, cardiac, smooth muscle, blood vessels, etc.), nerve cells (neurons, glial cells, astrocytes) or epithelial cells, kidney cells, eye cells, etc. It can also include insect cells, plant cells, yeast cells or prokaryotic cells. It can also be cells transformed by SV40 T antigen. The gag, pol and env genes encoded in the plasmid or helper virus can be introduced into the cells by any method known in the art that is suitable for the cell type under consideration. Usually, the cells and the vector system are contacted in a suitable device (plate, dish, tube, bag, etc.) for a period of time sufficient to allow the vector system or plasmid to be transferred into the cells. Typically, vector system or plasmid are introduced into cells by calcium phosphate precipitation, electroporation, transduction or use a kind of compound (such as lipid, polymer, liposome and peptide etc.) that promotes transfection.Preferably calcium phosphate precipitation.Cell is cultivated in any suitable culture medium, such as RPMI, DMEM, culture special culture medium without fetal bovine serum etc.Once transfection, retroviral vector of the present invention can be purified from the supernatant of cell.Retroviral vector purification can be completed by any suitable method to improve concentration, such as by density gradient purification (such as, cesium chloride (CsCl)) or by chromatographic technique (such as, column chromatography or batch chromatography).For example, carrier of the present invention can carry out two or three CsCl density gradient purification steps.Desirably, the vector is purified from infected cells using a method comprising lysing cells infected with the adenovirus, applying the lysate to a chromatography resin, eluting the adenovirus from the chromatography resin, and collecting fractions containing the retroviral vector of the invention.

[0037] In some embodiments, the vectors of the present invention include "control sequences," which is a collective term for promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which together provide for replication, transcription, and translation of the coding sequence in the recipient cell. All of these control sequences need not always be present, as long as the selected coding sequence is capable of replication, transcription, and translation in the appropriate host cell. Another nucleic acid sequence is a "promoter" sequence, which is used herein in its usual sense to refer to a region of nucleotides comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence. Transcriptional promoters can include "inducible promoters" (wherein the expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (wherein the 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 combined 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 are formulated using one or more lipid-based structures, including but not limited to liposomes, lipid complexes (lipoplex) 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, which are mainly composed of lipid bilayers and can be used as delivery vehicles for drug formulations. Liposomes can have different sizes, such as, but not limited to, multilamellar vesicles (MLVs), which can be hundreds of nanometers in diameter and can include a series of concentric bilayers separated by narrow aqueous compartments; small unicellular vesicles (SUVs), which can be less than 50 nanometers in diameter; and large unilamellar vesicles (LUVs), which can be between 50 and 500 nanometers in diameter. Liposome design can include but is not limited to opsonin (opsonin) or ligand, to improve the attachment of liposomes to unhealthy tissues, or activation events, such as but not limited to endocytosis.Liposomes can include low or high pH values ​​to improve the delivery of pharmaceutical preparations. As a non-limiting example, liposomes, such as synthetic membrane vesicles, are prepared by the methods, equipment and devices described in U.S. Patent Publication Nos. US20130177638, US20130177637, US20130177636, US20130177635, US20130177634, US20130177633, US20130183375, US20130183373 and US20130183372. 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 (as described in US20100324120), as well as liposomes that can deliver small molecule drugs, such as, but not limited to, liposomes from Janssen Biotech, Inc. (Horsham, Pa.). The polypeptides or polynucleotides of the present invention may be encapsulated by liposomes and / or may be contained in an aqueous core, which may then be encapsulated by liposomes (see International Publication Nos. WO2012031046, WO2012031043, WO2012030901, and WO2012006378 and U.S. Patent Publication Nos. US20130189351, US20130195969, and US20130202684). In some embodiments, the polynucleotides of the invention are formulated with stabilized plasmid-lipid particles (SPLPs) or stabilized nucleic acid lipid particles (SNALPs), which have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (see 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 Biol. 2010 33:177-178). et al. J Clin Invest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132; U.S. Patent Publication No. US20130122104).

[0039] In some embodiments, a polypeptide or polynucleotide of the present invention is administered in combination or in conjunction with ii) a VEGF-C polypeptide or ii) a polynucleotide encoding a VEGF-C polypeptide.

[0040] The term "VEGF-C" as used herein has the general meaning in the art and refers to vascular endothelial growth factor C encoded by the VEGF-C gene. An exemplary amino acid sequence of VEGF-C is represented by SEQ ID NO: 2.

[0041] SEQ ID NO:2>sp|P49767|VEGFC_HUMAN Vascular endothelial growth factorCOS=Homo sapiens OX=9606GN=VEGFC PE=1SV=1

[0042] MHLLGFFSVACSLLAAALLPGPREAPAAAAAFESGLDLSDAEPDAGEATAYASKDLEEQLRSVSSVDELMTVLYPEYWKMYKCQLRKGGWQHNREQANLNSRTE ETIKFAAAHYNTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSC RCMSKLDVYRQVHSIIRRSLPATLPQCQAANKTCPTNYMWNNHICRCLAQEDFMFSSDAGDDSTDGFHDICGPNKELDEETCQCVCRAGLRPASCGPHKELDRNS CQCVCKNKLFPSQCGANREFDENTCQCVCKRTCPRNQPLNPGKCACECTESPQKCLLKGKKFHHQTCSCYRRPCTNRQKACEPGFSYSEEVCRCVPSYWKRPQMS

[0043] In some embodiments, two polypeptides or polynucleotides can be administered to a patient separately or in the same composition. For example, a bicistronic polynucleotide encoding an epazolin polypeptide and a VEGF-C polypeptide can be used. In some embodiments, the polynucleotides encoding the polypeptides can be inserted into / contained in the same vector (e.g., a viral vector, a virus-like particle, etc.).

[0044] As used herein, the expression "therapeutically effective amount" refers to an amount of the active ingredient sufficient to treat or alleviate symptoms, with a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. For any particular subject, the specific therapeutically effective dosage level will depend on a variety of factors, including the disease being treated and the severity of the disease; the activity of the specific compound used; the specific composition used, the age, weight, general health, sex and diet of the subject; the time of administration, 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, one skilled in the art can start the dose of the compound at a dose lower than that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0045] Typically, the active ingredient of the present invention (i.e., polypeptide or polynucleotide) is combined with a pharmaceutically acceptable excipient and an optional sustained-release matrix (e.g., a biodegradable polymer) to form a pharmaceutical composition. Suitably, the term "pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other adverse reactions when administered to mammals, particularly humans. A pharmaceutically acceptable carrier or excipient refers to any type of non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation aid.

[0046] The present invention will be further described by the following figures and examples. However, these examples and figures should not be interpreted as limiting the scope of the present invention in any way. Attached photos

[0048] Figure 1 : Decreased expression of epasparginine in human lymphedema

[0049] A. Quantification of lymphatic vessel diameter (*p < 0.05). B. Quantification of dermal lymphatic density (p < 0.05). C. Quantitative RT-PCR analysis of genes associated with fibrosis and VEGFC maturation in transcutaneous lipectomy samples from patients with lymphedema (*p < 0.05) (n = 3). D. Quantitative RT-PCR analysis of adipokines in transcutaneous lipectomy samples from patients with lymphedema (*p < 0.05) (n = 3).

[0050] Figure 2. Etoplastin prevents secondary lymphedema

[0051] A. Schematic diagram of the experimental design of the secondary lymphedema model in mice injected with an Epstein-Barr lentiviral vector (LV-APL). Quantification of proximal leg swelling was performed 7 and 14 days after surgery in control limbs, lymphedema limbs (n=17), or limbs treated with Epstein-Barr lentiviral vectors for lymphedema (n=20) (*p<0.05). B. EIA dose of circulating Epstein-Barr in plasma of control (n=5) or Epstein-Barr-treated mice (n=5). C. Lymphangiography shows pathological remodeling of lymphatic vessels and dermal reflux in lymphedema, which is reversed by LV-APL (n=10). D. Masson trichrome staining of skin from lymphedema mice treated with or without Epstein-Barr (scale bar: 50 μm). E. Quantification of dermal thickness (*p<0.05). F. EIA of circulating VEGF-C in the plasma of control (n=5) or epazolin-treated mice (n=5). G. SHG signal from the deep collagen-rich layers of the dermis. H. Lyve-1 immunodetection of cutaneous lymphangiogenesis in epazolin-treated mice (Scale bar: 50 μm). I. Quantification of lymphangiogenesis in epazolin-treated mice (*p<0.05, **p<0.01). J. Quantification of lymphangiogenesis in epazolin-treated mice (**p<0.01).

[0052] Figure 3. Etoplastin and VEGFC exhibit complementary effects on lymphatic endothelial cells

[0053] AE. Comparison of bulk RNA sequencing in HDLECs treated with apache-, VEGF-C-, or apache + VEGF-C-conditioned medium. A. Heatmap of the top 30 significantly upregulated genes by apache and VEGF-C. B. Schematic representation of the number of genes upregulated by both apache and VEGF-C (43). C. Heatmap of the top 30 significantly upregulated genes by apache and apache + VEGF-C treatment. D. Schematic representation of the number of genes upregulated by apache and apache + VEGF-C (31). E. Schematic representation of the number of genes upregulated by apache, VEGF-C, and apache + VEGF-C (19).

[0054] Figure 4. Apoptosis-VEGF-C mRNA delivery: a new treatment option for lymphedema

[0055] A: Encapsulation of epazolin and VEGF-C mRNA molecules for in vivo delivery Schematic diagram of the vector. B. EIA dose of circulating epazolin in the plasma of control (n=5) or epazolin-treated mice (n=5). C. EIA dose of circulating VEGF-C in the plasma of control (n=5) or epazolin-treated mice (n=5). D. EIA dose of circulating VEGF-C in the plasma of control limbs, lymphedema limbs, or VEGF-C-treated mice. Quantification of proximal limb swelling in vehicle-treated lymphedema (n=10) 7 and 14 days after surgery (*p<0.05). E. Control limb, lymphedema limb, or VEGF-C treated limb. Quantification of proximal limb swelling in vehicle-treated lymphedema (n=10) 7 and 14 days after surgery (*p<0.05). F. Control limb, lymphedema limb, or limb treated with epazolin-VEGF-C. Quantification of proximal leg swelling in vehicle-treated lymphedema (n=10) 7 and 14 days after surgery. G. Quantification of proximal leg swelling in vehicle-treated lymphedema (n=10) 7 and 14 days after surgery. Quantification of proximal limb swelling in vehicle-treated lymphedema limbs (n=10) at 7 and 14 days after surgery (*p<0.05). H. VEGF-C-, Epstein-Barr-, or Epstein-VEGF-C- Representative images of lymphangiography in vehicle-treated mice. I. Quantification of lymphatic vessel dilation in mice treated with APLN-VEGF-C (**p<0.01). J. Quantification of lymphatic vessel dilation in mice treated with APLN-VEGF-C (**p<0.01). Quantification of proximal limb swelling in vehicle-treated mice after development of lymphedema (10 days post-surgery) (n=8) (*p<0.05). Example

[0056] method:

[0057] human tissue samples

[0058] Samples were obtained from 16 archived paraffin blocks of dermatolipectomy specimens obtained from patients with secondary lymphedema treated at Toulouse University Hospital, France, between 2015 and 2016. According to the International Society of Lymphology (ISL) classification, patients with a history of lymphedema between 1 and 12 years presented with stage 2 lymphedema. Eligible patients had a history of unilateral, non-metastatic breast cancer without recurrence for more than 5 years. The main clinical parameter used to diagnose significant lymphedema was clinical arm edema, with a volume difference of 10% or 200 milliliters (mL) between the affected and contralateral arm. Samples were selected as numbered specimens according to a protocol approved by the INSERM Institutional Review Board (DC-2008-452), the Ministry of State for Research (Ministère de la Recherche, ARS, CPP2, Authorization AC-2008-452), and the Ethics Committee. Where available, some control arm tissue samples removed for cosmetic purposes from the same patients were studied.

[0059] Lymphofluoroscopy

[0060] Near-infrared fluorescence lymphatic imaging was used to visualize the initial and conducting lymphatic vessels. 100 μg of indocyanine green was added. Dilute in 0.5 mL of pure water and inject intradermally into the first interdigital space. ) is fixed 15 cm above the examination area to observe fluorescent imaging of lymphatic flow. Indocyanine green lymphatic angiography results can be divided into two patterns: a normal linear pattern and an abnormal dermal return pattern.

[0061] Lymphoscintigraphy

[0062] Lymphoscintigraphy is used to diagnose the severity of lymphedema. This low-radiation test is contraindicated during pregnancy and breastfeeding. It is injected bilaterally subcutaneously between the first and second fingers. Large-sized nanoparticles of colloidal albumin radiolabeled with 99m-technetium are selectively captured by lymphatic capillaries and then drained by the lymphatic system. It enables comparative functional bilateral assessment of both upper limbs, including axillary lymph node uptake, lymphatic stasis, dermal reflux, and redirection into the deep lymphatic system to access the medial superior malleolar lymph nodes.

[0063] Mouse Model of Lymphedema

[0064] Mouse procedures were performed in accordance with EU and national regulations. C57Bl / 6 mice were provided by Envigo. All experiments have been approved by the local branch of the Ethics Committee of the Midi-Pyrénées, Inserm Rangueil-Purpan. Secondary lymphedema was established as previously described (Morfoisse et al. 2018). Briefly, lymphedema was established in the left upper limb of 6-week-old C57Bl / 6 female mice. Partial mastectomy of the second mammary gland was performed together with axillary and brachial lymph node dissection. Limb dimensions in the axillary region were measured over time using calipers. Mice remained edematous for 2 weeks. On the day of surgery, PBS or epazolin lentiviral vector (4 μL, 3 injections) were injected intradermally in the lymphedema limb. For Carrier, vehicle, epazolin, VEGFC and epazolin-VEGFC The vector was injected intradermally into the lymphedema limb (200 ng p24 divided into 3 injections, 2 μL each). G L-NAME was resuspended in water (1 mg / ml) and mice were allowed to drink water freely for 7 days.

[0065] Lymphangiography

[0066] Two weeks after surgery, mice were anesthetized with an intraperitoneal injection of ketamine (100 mg / kg) (Zoletil 100, Virbac) and xylazine (10 mg / kg) (Rompun 2%, Bayer). FITC-dextran (70,000 kDa, 2 mg / mL, Sigma) was injected into the footpads of the lymphedema limb and the control limb. The fluorescent molecule is taken up by lymphatic vessels and excreted from the blood vessels. Five minutes later, the skin was analyzed under a modular stereo microscope, Discovery V12 (Zeiss).

[0067] Histology

[0068] Skin from the lymphedema limb and control limb was collected 2 weeks after surgery and fixed O / N in 10% neutral buffered formalin at 4°C. The tissue was then embedded in paraffin and sliced ​​on a microtome. 5 μm sections were cut and placed on Superfrost Plus slides. The tissue was dewaxed, rehydrated, and microwaved in a microwave for 5 minutes three times with pH 9 Tris solution (H-3301, Vector Laboratories) to achieve antigen exposure. After cooling, the slides were washed with PBS and then blocked with 5% BSA solution at room temperature in a humid chamber. The sections were incubated O / N with the primary antibody at 4°C (goat anti-mouse Lyve1, R&DAF2125; rabbit anti-CD31, abcam Ab28364) and washed three times in PBS. The sections were incubated with the corresponding secondary antibody conjugated with Alexa-488 or Alexa-594 at room temperature at a dilution of au 1 / 400 for 1 hour. DNA was stained with DAPI. Slides were mounted with Dako fluorescent mounting medium (S3023). Images were acquired using an inverted fluorescence microscope (Leica, DMi8). Images were analyzed using Fiji software.

[0069] Assessment of fibrosis

[0070] Dermal fibrosis was assessed using Masson's trichrome staining (MST-100T, Cliniscience). Skin sections were dewaxed and the tissue was stained according to the manufacturer's recommendations. Images were acquired using a nanozoomer slide scanner. Dermal dimensions were quantified by measuring the length between the epidermis and hypodermis at least 10 times per field of view.

[0071] SHG imaging

[0072] Lymphedema skin and control tissues of mouse limbs were embedded in paraffin and sliced ​​on a microtome. 30 μm slices were cut and placed on Superfrost Plus slides. Tissues were dewaxed as described in the "Histology" section and used for SHG analysis. Bruker (Billerica, Massachusetts, USA) 2P Plus two-photon microscope was used for collection. The microscope was equipped with a Coherent (Santa Clara, California, USA) Chameleon Discovery laser and an Olympus (Shinjuku, Tokyo, Japan) 20x NA:1 objective lens. We used a laser wavelength of 900 nm and collected second harmonic generation (SHG) emission of 450 nm. Z stacks were acquired with a step size of 1 μm. Collagen fiber quantification was performed using at least 5 measurements of each skin section. This analysis was performed on at least 6 different mice in each case.

[0073] Collecting vessel contraction measurement

[0074] The contraction of the afferent collecting lymphatic vessels leading to the popliteal lymph nodes (PLN) was measured according to a previously described method (Liao S. et al.). Briefly, mice were anesthetized with an intraperitoneal injection of ketamine (100 mg / kg) and xylazine (10 mg / kg). 6 μL of FITC-dextran was injected into the footpad of the right lower limb. The skin was carefully removed to expose the afferent collecting lymphatic vessels leading to the PLN. The mice were then placed in a culture dish and placed on the stage of an inverted microscope (Leica, DMi8). Four 90-second videos were acquired for each mouse, and the number of contractions and expansions (the difference between the maximum and minimum diameters) was analyzed using Fiji software. To evaluate the effect of epazote, a lentiviral vector was injected into the dermis of the right lower limb 7 days before the experiment. For the L-NAME study, mice were allowed free access to water for 7 days.

[0075] Construction, production, purification, and quantification by p24 ELISA assay

[0076] Four plasmids were used to generate recombinant Particles: (i) pLVGagPol plasmid encoding viral gag and pol genes, which has been modified to contain PP7-capsid protein (PCP) within the gag gene, termed pLF-GagPolΔZF2_PCP (Mianne et al, 2022); (ii) pVSVG plasmid encoding VSV-G glycoprotein; and (iii) two plasmids encoding each RNA cargo, flanked by PP7 phage aptamers that enable RNA mobilization into lentiviral particles by interacting with the PP7 capsid protein cloned in the Gag sequence. All newly generated constructs were verified by restriction enzyme digestion and sequencing. Production was performed in 10-layer CellSTACK chambers (6360 cm2, Corning) after transfection of the four plasmids into HEK293T cells using a standard calcium phosphate procedure. Particles. 24 hours after transfection, the supernatant was discarded and replaced with fresh culture medium, and the cells were cultured at 37°C in a humidified environment of 5% CO2 in air. After replacing the culture medium, the supernatant was collected, clarified by centrifugation at 3000g for 5 minutes, and microfiltered through a 0.45μm pore size sterile filtration unit (Stericup, Millipore). The supernatant was harvested multiple times and all samples (crude harvest) were finally pooled. The crude harvest was concentrated and purified by ultrafiltration and diafiltration. For quantitative analysis, the HIV-1p24 ELISA kit (Perkin Elmer) was used according to the supplier's instructions to directly detect the p24 core antigen in the viral supernatant. The viral titer (expressed as the number of physical particles per milliliter) was calculated based on the amount of p24 (it is known that 1pg p24 is equivalent to 10E+4 physical particles).

[0077] Enzyme immunoassay (EIA)

[0078] The concentrations of epazolin in culture medium and mouse plasma were determined using an epazolin EIA kit (RAB0018, Sigma-Aldrich) according to the manufacturer's recommendations. The VEGFC ELISA kit was from R&D Systems.

[0079] Cell culture and treatment

[0080] Human dermal lymphatic endothelial cells (HDLEC) (single donor, adolescent foreskin, Promocell, C-12216, >95% of cells are CD31 positive and podoplanin positive) were cultured in endothelial cell culture medium MV2 (EGM-MV2, Promocell, C-22121). NIH3T3 were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma, D6429) supplemented with 10% fetal bovine serum (FBS, Gibco, 10270-06) and 1% penicillin-streptomycin. Endothelial cells were used at passages 3-6 and human fibroblasts were used at passages 4-7. Cells were cultured at 37°C in a 5% CO2 incubator. The culture medium was changed three times a week and the cells were passaged 1 / 3. To collect the conditioned medium, NIH3T3 were grown in 10 cm culture dishes, and after reaching confluence, the culture medium was removed and the cells were washed once with PBS. NIH3T3 cells were cultured overnight in 5 mL of low-serum medium (OptiMEM (Gibco)), and the culture medium was collected and used for experiments. HDLECs were treated with 50% conditioned medium / 50% MV2-05% FBS.

[0081] RNA extraction, reverse transcriptase, and qPCR

[0082] Total RNA was prepared using the RNeasy kit (Qiagen 74106) according to the supplier's instructions. 1 μg of RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, 4368813) containing Multi-Scribe reverse transcriptase according to the supplier's instructions. Quantitative real-time PCR was performed on a StepOne real-time PCR system (Thermofisher Scientific) using OneGreen FAST qPCR premix (Ozyme, OZYA008). All samples were analyzed in duplicate. Data were normalized to HPRT mRNA levels.

[0083] Immunoblotting

[0084] The cells were scraped and lysed in RIPA buffer (RIPA 2X, Biotech RB4476) supplemented with phosphatase inhibitors (PhosSTOP Easypack, Roche 0490687001) and protease inhibitors (Protease Inhibitor Cocktail, Sigma Aldrich). The lysate was centrifuged at 13500g for 10 minutes at 4°C. The supernatant was then collected and mixed with Laemmli buffer containing dithiothreitol (1mM DTT). Proteins were separated on a 4-15% SDS-PAGE gel and transferred to a nitrocellulose membrane (Trans-Blot Turbo RTA Transfer Kit, #1704271, Biorad). The membrane was blocked in 5% BSA-TBS-T (TBS-0.1% Tween 20) for 1 hour at room temperature and probed with primary antibodies overnight at 4°C. The antibodies used are as follows: phosphorylated AKT: AKT-p ser473 (CS#4060S), AKT: santa cruz H136 (S8312), phosphorylated ERK: ERK1 / 2-p(MAPKp42 / 44)(Thr202 / Thr204)(cell signalling#9106), ERK ERK1 / 2-(MAPKp42 / 44)(Thr202 / Thr204)(cell signalling#9102), phosphorylated eNOS (cell signalling#9571S), eNOS (cell signalling#5880S), E2F8 (Abcam, AB109596), VEGFR3 (R1D systemAF349), phosphorylated VEGFR3 (Affinity AF3676), CCBE1 (Sigma SAB1402017).

[0085] After three washes in TBS-T, the membrane was probed with HRP-conjugated secondary antibody diluted 1 / 10000. Signals were visualized using chemiluminescent detection reagents (Sigma) on a Chemidoc (Biorad) digital acquisition system.

[0086] Bulk RNA-Sequencing

[0087] RNA sequencing of primary human lymphatic endothelial cells

[0088] Total RNA from HDLECs transduced with or without the Epstein-Barr lentiviral vector was prepared using the RNeasy mini kit (Qiagen 74106). The total RNA was then subjected to a ribosomal RNA-depleted RNA sequencing (RNA-Seq) protocol by Genewiz using the PE 2x150 configuration of the Illumina HiSeq. Sequence reads were trimmed using Trimmomatic v.0.36 to remove possible aptamer sequences and poor-quality nucleotides. The trimmed reads were mapped to the human (Homo sapiens) GRCh38 reference genome available on ENSEMBL using the STAR aligner v.2.5.2b. The STAR aligner is a splicing aligner that detects splice junctions and merges them to help align the entire read sequence. This step generates a BAM file. The number of unique gene hits was calculated using feature Counts from the Subread package v.1.5.2. The number of hits was summarized and reported using the gene_id feature in the annotation file. Only unique reads falling within the exon region were counted. The distribution of read counts in the libraries was examined before and after normalization. The raw read counts were normalized to adjust for various factors, such as variations in sequencing yield between samples. These normalized read counts were used to accurately identify differentially expressed genes. A data quality assessment was performed to detect any samples that were not representative of their group and therefore could affect the quality of the analysis. The overall similarity between samples was assessed using the Euclidean distance between samples. This method is used to examine which samples are similar / different to each other and whether they meet the expectations of the experimental design. The shorter the distance, the more closely related the samples are. This distance was then used to cluster the samples. A principal component analysis was also performed to reveal similarities between samples based on a distance matrix.

[0089] Differential gene expression analysis

[0090] After extracting the gene hit counts, the gene hit count table was used for downstream differential expression analysis. Using DESeq2, gene expression was compared between epazolin-transduced LECs and non-transduced LECs. P-values ​​and log2 fold changes were generated using the Wald test. Genes with log2FC > 0.5 or log2FC < -0.5 and adjusted p-values ​​< 0.05 were defined as differentially expressed genes and used for downstream analysis. A volcano plot was used to visualize the overall transcriptional changes between the two groups being compared. Each data point in the volcano plot represents a gene. The log2 fold change of each gene is represented on the x-axis, and the log10 of its adjusted p-value is represented on the y-axis. Genes with adjusted p-values ​​less than 0.05 and log2 fold changes greater than 0.5 are represented by red dots. These represent upregulated genes. Genes with adjusted p-values ​​less than 0.05 and log2 fold changes less than 0.5 are represented by blue dots. These represent downregulated genes.

[0091] Gene Ontology (GO) analysis

[0092] Gene ontology analysis was performed on statistically significantly upregulated and downregulated gene sets using PANTHER software (version 16.0, http: / / pantherdb.org / ). Significantly differentially expressed gene sets were clustered according to the biological process or pathway of the genes using the Homo sapiens reference list, and overrepresentation of gene ontology terms was tested using Fisher's exact test. All GO terms with a false discovery rate (FDR) below 0.05 were considered significant and are listed in the supplementary data.

[0093] Chromatin immunoprecipitation (ChIP)

[0094] Human dermal lymphatic endothelial cells (HDLECs) that were not transduced or transduced with the Epstein-Barr lentiviral vector were directly cross-linked in culture medium using 1% formaldehyde for 15 minutes. 0.125M glycine was then added for 5 minutes. After washing twice with cold PBS, the cells were scraped and frozen at -80°C. The cells were lysed into the ChIP-IT Express Magnetic Chromatin Immunoprecipitation Kit (Active Motif 53008). The optimal ultrasonic treatment conditions were previously determined to obtain DNA fragments of approximately 500bp. The cells were ultrasonicated using a Diagenode Bioruptor sonicator (7 cycles, 30 seconds ON, 30 seconds OFF in a water bath) in a final volume of 350μl of shearing buffer specifically for the kit. The DNA concentration was determined using Nanodrop, and 25μg of chromatin was used in the reaction. The experiment was then performed according to the manufacturer's protocol. 4μg of E2F8 antibody (Abcam, AB109596) was used in the ChIP reaction. 10μl of each sample was retained as input. The reaction was incubated overnight at 4°C. Mock samples without antibody were similarly processed. Prior to qPCR, DNA was purified using the Active Motif Chromatin IP DNA Purification Kit (58002) and eluted in 50 μl of DNase / RNase-free water. 2 μl of purified chromatin was used for qPCR. In some experiments, ChIP reactions were supplemented with 10 ng of Drosophila melanogaster chromatin (spike-in chromatin, Active motif, 08221011) and 1 μg of an antibody recognizing H2Av (a Drosophila-specific histone variant) (spike-in antibody, Active motif, 61686) as an internal control for ChIP normalization.

[0095] Cell transduction

[0096] HDLECs were seeded at 100,000 cells / well in 6-well plates. After 24 hours, the cells were transduced with 1 mL of the Epstein-Barr lentiviral vector (final concentration: 5 μg / mL) diluted in 1 mL of OptiMEM medium containing protamine sulfate. Control cells (NT) not transduced with the Epstein-Barr lentiviral vector were treated at the same time and according to the same protocol; in this case, 2 mL of OptiMEM and 5 μg / mL protamine sulfate were added to the cells. The medium was changed after 24 hours. The cells were grown until confluence was reached, then passaged and expanded for further experiments. Epstein-Barr transduction was verified by RT-qPCR.

[0097] Statistical analysis

[0098] All results presented in this study represent at least three independent experiments. In all figures, "n" represents the number of biological replicates. Data are presented as mean ± standard error of the mean (sem). Statistical significance was determined using Prism ver.9.0 (GraphPad) by two-tailed Student's t-test, one-way ANOVA or two-way ANOVA test and Bonferroni post hoc test. When P value < 0.05, the difference was considered to be statistically significant. The symbols used were: ns> 0.05, * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.0001.

[0099] result:

[0100] Secondary lymphedema is characterized by increased diameter of lymphatic capillaries and poor collection and drainage.

[0101] Secondary lymphedema occurs months (and sometimes years) after cancer treatment, suggesting that this pathology is not simply a side effect of surgery. Lymphoscintigraphy is the primary imaging modality used to assess lymphatic system dysfunction. For decades, it has been considered the standard (Munn & Padera, 2014; Szuba et al, 2003). Lymphoscintigraphy in women who developed lymphedema after breast cancer showed a severe decrease in lymph collecting duct detection and axillary lymph node perfusion after injection of a radiotracer (data not shown). In addition, lymphofluorescence revealed a hypervascularized dermis with tortuous lymphatic capillaries (data not shown), which was associated with a strong desmoplastic reaction and dermal reflux (data not shown). This was confirmed using histological analysis, which showed an increase in cutaneous lymphatic vessel density ( Figure 1 A, 1B, and data not shown) and lymphangiogenesis and overload (data not shown). Surprisingly, no major differences were observed in genes involved in lymphangiogenic factor maturation, with the exception of CCBE1, which was significantly downregulated in lymphedema ( Figure 1 C). Because lymphedema is characterized by a large accumulation of fibrotic adipose tissue (AT) in the limbs, we also evaluated the expression of adipokines in lymphedema AT compared with normal arms ( Figure 1 D). We found that epaspatin expression was significantly reduced in lymphedema, whereas no differences were observed in adiponectin expression, leptin expression, or other adipokines ( Figure 1 D).

[0102] Lymphatic healing is impaired in epaspase knockout mice.

[0103] Our group previously described that epazolin improves normalization of lymphatic vessels in the heart after cardiac ischemia (Tatinet al. 2017). To investigate the role of epazolin in secondary lymphedema, we used a mouse model of lymphedema previously developed in our laboratory (Morfoisse et al. 2018). We performed a secondary mastectomy of the left upper limb and axillary and brachial lymph node dissection in epazolin-KO mice (data not shown). Using this model, reproducible lymphedema developed after 2 weeks and gradually returned to normal after 4 to 8 weeks. In epazolin-KO mice, lymphedema remained significantly maintained after 4 weeks, indicating an inability to restore lymphatic function (data not shown). Next, we examined lymphatic capillaries using lymphangiography after FITC-dextran injection into the footpad. We observed robust dermal reflux in epazolin-KO mice 4 weeks after surgery (data not shown). Lymphatic histological analysis showed that there was no difference in lymphatic basal density between WT and Epstein-Barr-KO mice (data not shown). In contrast, after lymphedema surgery, skin lymphangiogenesis was significantly reduced in Epstein-Barr-KO mice compared with WT mice (data not shown). This was associated with increased skin fibrosis in WT and Epstein-Barr-KO mice as shown using Masson's trichrome staining (data not shown). Since lymphedema leads to the accumulation of collagen fibers (one of the hallmarks of fibrosis development), we performed skin analysis by second harmonic generation (SHG) imaging (data not shown). Interestingly, the accumulation of collagen fibers in lymphedema was increased in Epstein-Barr-KO mice compared with WT mice (data not shown).

[0104] Epazopine has a regenerative function on lymphatic vessels in secondary lymphedema.

[0105] To evaluate the effect of epazolin on lymphatic healing, mice received intradermal injections of a lentiviral vector expressing epazolin (LV-epazolin) in the lymphedema limb. Significantly, lymphedema was significantly reduced in mice treated with epazolin ( Figure 2A Circulating epazolin levels were verified by mouse plasma ELISA, which showed that the plasma epazolin concentrations of mice treated with LV-epazolin were increased ( Figure 2B Next, lymphangiography was used to study lymphatic collection and drainage ( Figure 2C Consistently, we observed that secondary lymphedema resulted in pathological remodeling of the lymphatic vessels, with disorganized and abnormal lymphatic vessel morphology and an increased number of branch points in control limbs. Lymphatic leakage (dermal reflux) was also observed, suggesting a dysfunction of the superficial capillary network due to a lack of deeper collecting pumping ( Figure 3CIn contrast, mice treated with epazolin showed improved lymphatic shape, normalized morphology, and a reduction in the number of lymphatic vessel branches. Importantly, we did not observe dermal reflux in mice treated with epazolin, indicating improved lymphatic function ( Figure 3C Using Masson's trichrome staining, we observed increased dermal thickness in lymphedema limbs, consistent with the development of fibrosis ( Figure 2D We did not observe any dermal thickening in mice treated with etoposide ( Figures 2D and 2E ), which reflects the improvement of lymphedema pathology. Interestingly, we observed an increase in circulating VEGF-C (the major lymphangiogenic factor) after treatment with LV-epasthenia, suggesting that epasthenia may partially regulate VEGF-C protein synthesis ( Figure 2F A lentiviral vector expressing VEGF-C (LV-VEGFC) was used as a positive control ( Figure 2F The effect of etoposide on collagen deposition was also assessed using SHG (data not shown). We found that fibrosis was significantly reduced in mice treated with etoposide compared to controls ( Figure 2G ). The number of blood vessels was assessed using CD31 immunostaining. As expected, we did not find changes in the number of CD31-positive blood vessels in this lymphedema model (Morfoisse 2017) (data not shown). However, as previously described (Wysocka Marta B et al. 2018), treatment with the lentiviral vector for epazolidinone resulted in increased angiogenesis and vascular permeability (data not shown). In addition, lymphangiogenesis was assessed using Lyve1 immunostaining on skin sections ( Figures 2H, 2I, and 2J Consistent with the lymphangiographic results, an increase in the number of Lyve1-positive vessels was observed in the lymphedema limbs compared with the control limbs, whereas there was no significant difference when the control limbs were compared with the mice treated with LV-epamulin ( Figure 2I In contrast, we found that epazolin promoted significant dilation of lymphatic vessels ( Figure 2J Overall, our results suggest that epazolin has beneficial effects on secondary lymphedema by acting on lymphatic vessel plasticity and dilation.

[0106] Epamin controls LEC gene expression.

[0107] To investigate novel molecular mechanisms and signaling pathways regulated by apagin in LECs, we performed global transcriptome analysis of LECs stimulated for 24 hours with conditioned medium containing apagin or conditioned medium obtained from control NIH3T3 cells (data not shown). RNA sequencing sample quality and similarity assessment were verified (data not shown). Differential DESeq analysis revealed that 217 genes were dysregulated (p.adj < 0.05, Log2 fold change < -0.5 or > 0.5), of which 94 were upregulated and 123 were downregulated (data not shown). The top 30 downregulated or upregulated genes are shown in the heat map (data not shown), and the full list is provided in the data not shown. Gene ontology analysis of the downregulated genes indicated that no biological processes were significantly affected in HDLECs treated with apagin. In contrast, GO analysis of biological processes revealed that upregulated genes were enriched for terms related to extracellular matrix (ECM) remodeling and signaling (FDR < 0.05) (data not shown), including COL1A, FBN, ADATS2, and CCBE1 (data not shown). However, with the exception of collagen and calcium-binding EGF domain 1 (CCBE1), for which induction was strongly demonstrated (data not shown), induction of most of these genes was not confirmed by RT-qPCR in HDLECs. The CCBE1 protein is required for the activation of VEGF-C and the ADAMTS3 (thrombospondin motif-containing disintegrin and metalloproteinase-3) protease, enhancing ADAMTS3 cleavage activity and promoting VEGF-C maturation to its biologically active form. To investigate the effect of CCBE1 on VEGFR receptor activation, we knocked down CCBE1 in LECs using siRNA (data not shown). Cells were then stimulated with epazolin and subjected to Western blot analysis for P-VEGFR3 (data not shown). We found that knockdown of CCBE1 in LECs reduced the amount of VEGFR-3 protein. This was associated with a slight but significant decrease in VEGFR-3 phosphorylation in the presence of epazolin (data not shown). Interestingly, epazolin also stimulated the expression of the CCBE1 transcription factor E2F8 (data not shown). We then hypothesized that epazolin could participate in VEGF-C maturation by increasing E2F8 DNA binding to the CCBE1 promoter (data not shown). To answer this question, we performed chromatin immunoprecipitation (ChIP) experiments using E2F8 immunoprecipitation in epazolin-overexpressing HDLECs (data not shown). We found that epazolin significantly increased E2F8 binding to the CCBE1 promoter (data not shown). Interestingly, epazolin also induced E2F8 binding to the E2F1 transcription factor promoter, suggesting that it plays a role in other biological functions (data not shown) (Wells, Graveel et al. 2002).However, no binding to FLT4 was observed (data not shown). Taken together, these data indicate that epaspatin regulates HDLEC gene expression.

[0108] Epatin stimulates LEC function through Akt / eNOS signaling.

[0109] Next, we investigated in vitro which molecular pathways are involved in the response to epazolin. Epazolin is known to activate Erk and Akt signaling in human dermal lymphatic endothelial cells (HDLECs) in vitro (Kim, Kang et al. 2014) (Berta, Hoda et al. 2014). Consistent with the vasodilatory phenotype (data not shown), we hypothesized that the beneficial effects of epazolin on lymphedema were mediated in part by the AKT / eNOS pathway. To this end, we stimulated HDLECs with conditioned medium obtained from NIH3T3 cells transduced with LV-epazolin (previously depleted of VEGF-C). Epazolin synthesis was verified by RT-qPCR (data not shown) and ELISA on NIH3T3 cells (data not shown). Conditioned medium stimulation of HDLECs was confirmed by assessing the AKT and ERK pathways over a 24-hour time course, using medium containing VEGF-C as a positive control (data not shown). In this case, LECs responded to VEGF-C after 30 minutes, as we observed robust activation of AKT and ERK (data not shown), whereas epasitin stimulated Akt phosphorylation after 1 hour without a major effect on ERK (data not shown). Importantly, eNOS phosphorylation was observed in HDLECs in response to epasitin and VEGF-C (data not shown). Consistent with Akt activation in HDLECs, we found activation of cell migration (data not shown), while no effects on cell junctions or cytoskeletal remodeling were observed (data not shown). Importantly, eNOS phosphorylation was observed in HDLECs in response to epasitin and / or VEGFC, suggesting that both epasitin and VEGF-C stimulate lymphangiectasia via the eNOS pathway (data not shown).

[0110] Etoplastin stimulates lymphatic pumping through eNOS activation.

[0111] We next explored whether epagliflozin could control lymphatic vessel dilation, specifically its effects in collecting lymphatic vessels. Epagliflozin has been described in several cellular contexts to activate eNOS phosphorylation to promote vasodilation (Dray, Knauf et al. 2008) (Wysocka, Pietraszek-Gremplewicz et al. 2018). We then investigated whether epagliflozin could stimulate dilation of collecting lymphatic vessels, thereby stimulating lymphatic pumping (data not shown). Lymphatic flow in collecting lymphatic vessels is driven in part by autonomous contractions of smooth muscle cells. To assess the effects of epagliflozin on collecting duct contraction, we used a previously described intravital imaging approach (Liao, Jones et al. 2014) (data not shown). The frequency of duct contractions and dilations was assessed. Interestingly, we found that epagliflozin stimulated lymphatic pumping by increasing collector dilation (data not shown) without any effect on contraction frequency (data not shown). This effect was completely reversed by the nitric oxide synthase (NOS) inhibitor L-NAME (data not shown). Then, to test the role of NOS activation in response to epagliflozin in the setting of lymphedema in vivo, mice treated with LV-epagliflozin were treated with L-NAME (data not shown). Limb diameter was measured to assess edema (data not shown). Interestingly, L-NAME reversed the beneficial effects of epagliflozin on lymphedema, confirming that lymphatic pumping is the primary cause of this pathology. We also observed a significant increase in edema two weeks after surgery in the presence of epagliflozin + L-NAME (data not shown). Lymphangiography showed that L-NAME treatment also reversed the effects of epagliflozin on the lymphatic network. In fact, in mice treated with epagliflozin + L-NAME, we observed pathological remodeling of the lymphatic vessels, accompanied by dermal reflux and abnormal lymphatic branching (data not shown). Capillaries on skin sections were also quantified using Lyve1 immunodetection (data not shown). Increased lymphangiogenesis was systematically observed in lymphedema limbs, but we did not observe any differences between the different conditions (data not shown). However, mice treated with epazolin showed an increase in duct area, an increase that was suppressed by L-NAME treatment, restoring a phenotype similar to that of the control group (data not shown). We also investigated fibrosis, and surprisingly, L-NAME had no effect on fibrosis (data not shown). In summary, our results suggest that epazolin prevents lymphedema by promoting collecting duct pumping and pathological remodeling of lymphatic vessels. This phenotype appears to be mediated in part by activation of eNOS in lymphatic endothelial cells.

[0112] Epaminergic and VEGFC exhibited synergistic effects in regulating the expression of genes associated with collecting duct maintenance.

[0113] Most studies aimed at regenerating the lymphatic system have focused on the molecule VEGF-C. Despite this, VEGFC alone does not appear to improve lymphatic function in a mouse model of vascular injury, suggesting that it must be combined with other molecules to fully restore lymphatic function. Here, we show that epazolin controls lymphedema fibrosis, lymphatic function, and contractility of the collecting ducts. Our initial approach aimed to combine VEGF-C with epazolin to obtain a synergistic effect for the treatment of secondary lymphedema by targeting the entire lymphatic network from capillaries to collectors. When comparing the gene expression profiles of HDLECs stimulated with epazolin, VEGF-C, or epazolin + VEGF-C, we observed a similar induction of the top 30 genes, most of which are related to extracellular matrix remodeling (data not shown). Most of the genes induced by VEGF-C were also induced by epazolin ( Figures 3A and 3B Half of the genes induced by the combination of epazolin + VEGF-C were induced by epazolin ( Figure 3C, 3D ). When comparing the induction of genes common to both molecules, the synergistic effects of epazolin and VEGF-C focused on genes related to maintenance of the lymphatic microenvironment (collagens 1A1 and 6A) and VEGF-C maturation (ADAMTS2, E2F8) (Figure 3E). In addition, 33 genes were specifically upregulated by the combination of epazolin and VEGF-C (Figure 3E). When comparing the untreated group, the VEGFC alone group, or the epazolin alone group with the epazolin + VEGFC group, we found an increase in genes essential for collecting duct function, including connexin 37 and 47 (GJA4, GJC2) and claudin 5 (CDN5), while angiogenic genes were downregulated (VEGFA, FLT1, KDR, KI67) (data not shown). Expression of genes related to VEGFC maturation (ADAMTS2, CCBE1) was improved in the VEGFC-, epazolin-, and epazolin VEGFC groups compared to the WT group (data not shown).

[0114] Epasin-VEGFC RNA delivery: a new treatment option for secondary lymphedema.

[0115] In Western countries, secondary lymphedema occurs after cancer treatment, which makes the delivery of angiogenic molecules to cancer survivors ethically problematic. For safety reasons, we decided to use a next-generation vector called (Lf), this vector can deliver mRNA transiently from non-integrating particles. To enable the delivery of several heterologous mRNA molecules, we generated A vector containing two different mRNAs encoding VEGF-C or epazolin, respectively, was injected into a lymphedema mouse model ( Figure 4A)Compared with lentiviral vectors that induce permanent transgene expression without any effect on platelet numbers and immune cell populations, The efficiency of VEGF-C injection is highly dependent on the stability of mRNA (data not shown). Therefore, we first confirmed the presence of measurable circulating VEGF-C ( Figure 4B ) and Epsom ( Figure 4C) As expected, mRNA delivery was not as effective as lentiviral vectors, as we only observed partial inhibition of limb swelling using single mRNA delivery of VEGF-C or epazolin ( picture 4D, 4E This is to be expected due to the limited time of expression of the molecules. However, VEGF-C / Epascin dual mRNA Completely eliminated limb swelling ( Figures 4F and 4G ), reducing dermal reflux ( Figure 4H ) and restored lymphatic perfusion to the lymphedema limb ( Figure 4I This is associated with an increase in lymphatic vessel diameter ( Figure 4I Finally, to investigate whether epazolin-VEGFC mRNA could cure lymphedema, we injected mice that developed lymphedema (10 days after surgery) ( picture 4J ).in this case, The vector reversed lymphedema swelling, returning to normal after 11 days. These data demonstrate the synergistic effect of epazomatidylcholine and VEGF-C and suggest that despite transient expression of both transgenes, this combination produces a significant therapeutic effect, offering the prospect of using non-integrating RNA delivery vectors to treat lymphedema in patients who develop lymphedema after cancer treatment. Therefore, epazomatidylcholine is a highly effective molecule that binds to VEGF-C when used with a "safe" RNA delivery vector to treat lymphedema in patients who develop lymphedema after cancer treatment.

[0116] discuss:

[0117] Despite tremendous advances in understanding the molecular mechanisms driving lymphatic function over the past few decades, lymphedema, the most prominent pathology associated with lymphatic dysfunction, remains a significant unmet medical need (Mercier, Pastor et al. 2019). It is a painful, chronic condition that affects millions of people worldwide. Numerous factors contribute to its etiology. Primary lymphedema is an inherited disorder caused by gene mutations, while secondary lymphedema occurs following cancer treatment or filarial infection (Mortimer and Rockson 2014, Rockson 2018). However, both result in similar clinical signs: accumulation of fluid and fat in the limbs and a fibrotic, hypervascular dermis characterized by tortuous and leaky capillaries and inadequate perfusion of deep collecting ducts. Lymphoscintigraphy reveals severely reduced lymph node perfusion, indicating that collecting ducts remain but are unable to properly collect and deliver lymph. These observations support therapeutic strategies aimed at combining molecules to 1) normalize capillary territories and 2) regenerate lymphatic pumping in deep adipose depots. Although VEGF-C, the major lymphangiogenic growth factor, is now well established as a promising candidate for restoring the lymphatic capillary network (Hartiala, Suominen et al. 2020), its role in collecting vessels remains less well-characterized. VEGF-C binds to its tyrosine kinase receptor, VEGFR-3, to promote its biological activity (Alitalo, Tammela et al. 2005). Collecting ducts develop within a coordinated adipose environment, which undergoes significant changes during lymphedema. In particular, adipose tissue synthesizes numerous adipokines that are involved in the integrity of both blood and lymphatic vessels. Therefore, it is tempting to speculate that changes in adipokines production may affect lymphatic collecting function. Among these, epazolin is considered a key factor in stimulating LEC function (Kim, Kang et al. 2014). Aptanigrin stimulates lymphangiogenesis in cancer and is involved in the restoration of pre-collecting lymphatic vessel shape after myocardial infarction (Tatin, Renaud-Gabardos et al. 2017). Aptanigrin is a bioactive peptide that induces signaling after binding to its G protein-coupled receptor, APJ, located on the surface of lymphocytes (LECs). In addition to its effects on the endothelial monolayer, aptanigrin is also a potent anti-fibrotic molecule (Huang, Chen et al. 2016).

[0118] Gene expression analysis of skin lipectomy in women who developed secondary lymphedema after breast cancer revealed a significant reduction in epatagonist expression in lymphedema. The crucial role of epatagonist in lymphedema was confirmed in epatagonist knockout mice, whose exacerbated lymphedema was reversed by a lentiviral vector expressing epatagonist. In this mouse model of lymphedema, we found that epatagonist ameliorates lymphedema by targeting two key pathological hallmarks: lymphatic function and tissue fibrosis. Importantly, we found that the effects of epatagonist on lymphatic collection and pumping are directly controlled by nitric oxide synthase (NOS). NO production participates in endothelial homeostasis by controlling the regulation of vascular tone as a response to flow (Dimmeler, Fleming et al. 1999). eNOS mediates key aspects of vascular remodeling by converting mechanical stimulation into enhanced NO production. Endothelial NOS (eNOS) also regulates lymphatic homeostasis. In a mouse model of fibrosarcoma, eNOS mediates VEGF-C-induced lymphangiogenesis and tumor lymphatic metastasis (Lahdenranta, Hagendoorn et al. 2009). Other studies have shown that eNOS affects lymphatic flow through collecting lymphatic vessels but does not affect capillary diameter (Hagendoorn, Padera et al. 2004). Furthermore, in lambs with chronically increased pulmonary blood and lymph flow, impaired NO bioavailability in pulmonary lymphatic vessels was found (Datar, Gong et al. 2016).

[0119] Here, we determined that the effects of epazolin on lymphatic collecting duct pumping are mediated by eNOS. Importantly, epazolin was previously described to regulate aortic vascular tone by increasing Akt and eNOS phosphorylation in diabetic mice (Zhong, Yu et al. 2007). Here, we confirmed that the beneficial effects of epazolin on lymphatic collecting ducts are mediated by this pathway, suggesting that epazolin may be the origin of NO-mediated lymphatic pumping in many organs. The extent of Akt phosphorylation was found to be lower than that induced by VEGF-C, but it appears to effectively mediate its biological effects. Interestingly, epazolin had no effect on endothelial monolayer integrity, suggesting that the effects of epazolin are limited to functional and dynamic effects.

[0120] RNA sequencing of epazolin-stimulated LECs revealed that epazolin controlled the expression of genes involved in extracellular matrix remodeling, consistent with its effects on tissue fibrosis. Interestingly, epazolin also strongly stimulated the expression of CCBE1, a protein involved in the proteolytic activation of VEGF-C by ADAMTS3 (Jha, Rauniyar et al., 2017). This may partially explain the increase in circulating VEGF-C concentrations observed after epazolin treatment. Importantly, in humans, mutations in CCBE1 have been found to cause Hennekam syndrome, a congenital disorder characterized by lymphatic malformations leading to primary lymphedema, lymphangiectasia, and heart defects (Alders, Mendola et al., 2013). Mechanistically, we found that CCBE1 gene expression is controlled by epazolin, which directly increases promoter activation of its transcription factor, E2F8. Together, these data further confirm that epazolin is a key player in restoring lymphatic function in lymphedema. We therefore plan to evaluate the effect of apatinib in combination with VEGF-C in a phase I clinical trial for the treatment of secondary lymphedema, which will be conducted at the Toulouse Hospital. This pilot study, called Theralymph, will focus on women who develop lymphedema after breast cancer. However, an important ethical concern for cancer survivors who receive treatment is that pro-lymphangiogenic therapy can reactivate the tumor, even after more than five years without any recurrence. Therefore, the use of lentiviral gene therapy to permanently integrate the transgene quickly became apparent as not being the optimal solution for therapeutic delivery. Another problem is the short half-life of apatinib in plasma, less than five minutes (Japp and Newby 2016). This could be compensated by serial injections in the limbs, but this would significantly increase the risk of infection and desmoplastic reactions, which are common in patients with lymphedema. Adeno-associated viruses were also ruled out due to the size limitations of the transgene, which made it impossible to deliver multiple therapies simultaneously. We then decided to use vector, a novel class of non-integrating lentiviral vectors that can transiently deliver multiple mRNA particles (Prel, Caval et al. 2015). The phage capsid protein and its homologous 19-nt stem-loop are used to construct the vector to replace the natural lentiviral Psi packaging sequence, thereby enabling the packaging of active mRNA into lentiviral particles. Compared with the integrating lentiviral vector, the single epazolin mRNA However, when combined with VEGF-C, dual mRNA delivery completely eliminated limb lymphedema and restored lymph flow to the limb, suggesting that the mRNA delivery strategy allowed synthesis of both proteins sufficient to observe beneficial effects.

[0121] Over the past two years, we have seen the emergence of a new class of mRNA vaccines, a highly effective and low-toxic delivery method. We believe that mRNA can treat many diseases in a different way than traditional medicine, including lymphedema, a condition for which there is currently no treatment. The plasticity of the antagonist allows for the transient delivery of two mRNA molecules, allowing for the synergistic stimulation of the G protein-coupled receptor APJ and the tyrosine kinase receptor VEGFR-3. Given that lymphedema remains a multifactorial pathology with lymphatic endothelial dysfunction, adipose tissue accumulation, and fibrosis, we believe that multi-therapy will be the solution to cure this detrimental disease. Therefore, we plan to use the aptamer-VEGF-C The vector is undergoing a Phase I / II gene therapy clinical trial, which will be initiated at our hospital next year.

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Claims

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

2. The method of claim 1, wherein the patient suffers from secondary lymphedema.

3. The method according to claim 1, wherein the epazolin polypeptide or the polynucleotide encoding the epazolin polypeptide is suitable for increasing the plasticity, contractility and / or dilatation of lymphatic vessels.

4. The method of claim 1, wherein the polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO:

1.

5. The method of claim 1, wherein the polynucleotide encodes an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO:

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

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

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

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

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

11. The method of claim 1, wherein the polypeptide or polynucleotide is encapsulated in a virus-like particle.

12. The method of claim 1, wherein the polypeptide or polynucleotide is combined or co-administered with ii) a VEGF-C polypeptide or ii) a polynucleotide encoding a VEGF-C polypeptide.

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