Use of Apelin to Treat Lymphedema

Abstract

Lymphedema is a chronic condition caused by lymphatic dysfunction, which leads to fluid and fat accumulation in the limbs. In this study, we identified the biologically active peptide apelin as a promising candidate for restoring lymphatic flow in lymphedema. We found that apelin expression was significantly reduced in lymphedematous arms of women compared with normal arms. Using an apelin knockout mouse model, we confirmed the important role of apelin, as lymphedema persisted for more than 4 weeks after surgery and was associated with a lack of dermal lymphangiogenesis and increased backflow into the dermis. We demonstrated that intradermal injection of an apelin lentivector significantly reduced limb swelling, which was associated with reduced dermal fibrosis and increased lymphatic vessel density. Importantly, apelin stimulates eNOS-mediated lymphatic pumping through phosphorylation of Akt and eNOS in lymphatic endothelial cells (LECs). This was associated with a significant increase in E2F8 target gene expression through direct binding of E2F8 to the CCBE1 promoter in LECs. Collectively, these results indicate that apelin plays an important role in lymphedema and thus represents a novel partner for VEGF-C to prevent limb swelling and tissue fibrosis in lymphedema.

Description

[Technical Field]

[0001] The present invention is an invention in the medical field, particularly in the treatment of lymphedema. [Background technology]

[0002] Lymphedema is a multifactorial condition that substantially impacts the patient's quality of life (Greene, Grant et al. 2012, Hoffner, Peterson et al. 2018). It is characterized by impaired lymphatic transport into the circulation due to lymphatic dysfunction that occurs after genetic mutations (primary lymphedema) or after cancer treatment (surgery, radiation, chemotherapy, etc.) (Mortimer and Rockson 2014).

[0003] Lymphatic congestion alters surrounding tissues, leading to the accumulation of adipose tissue and severe fibrosis. Adipose tissue deposition has been shown to stimulate adipogenesis in the affected limb (Aschen, Zampell, et al. 2012; Zampell, Aschen, et al. 2012). Recent evidence has highlighted a new concept suggesting that an imbalance between de novo adipose tissue synthesis and lipolysis leads to significant changes in adipokine synthesis (Koc, Wald, et al. 2021; Sano, Hirakawa, et al. 2022). Furthermore, a key feature of lymphedema associated with lymphatic congestion is the development of fibrosis in the skin and adipose tissue (Mortimer and Rockson, 2014). Lymphostatic fibrosis defines the stage of lymphedema, ranging from reversible to elephantiasis. Currently, there is no treatment for lymphedema other than massage and compression bandaging. However, once fibrosis develops, tissue density increases, causing lymphatic obstruction that exacerbates lymphedema. Importantly, fibrosis also affects the pumping action of collecting lymphatic vessels, increasing limb swelling (Baik, Park, et al., 2022) (Kataru, Wiser, et al., 2019).

[0004] Many cytokines and peptides are selectively involved in adipocyte metabolism, endothelial function, or tissue fibrosis. However, the biologically active peptide apelin has beneficial effects on limb tissue as a whole. Apelin is an endogenous ligand for the G protein-coupled receptor APJ, which is expressed in various organs (Pope, Roberts, et al., 2012). The first evidence of a relationship between apelin and the lymphatic network was identified in the tumor environment, where apelin stimulates both angiogenesis and lymphangiogenesis (Berta, Hoda, et al., 2014). Our group also previously described the beneficial effects of apelin on the cardiac lymphatic network after myocardial ischemia (Tatin, Renaud-Gabardos, et al., 2017). We found that apelin could restore the shape of pre-collecting lymphatic vessels within the obstructed area, suggesting that apelin may represent a promising candidate for lymphatic vessel restoration in injured tissues (Tatin, Renaud-Gabardos, et al., 2017). However, apelin was first 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 apelin by adipocytes is regulated by insulin, and its beneficial role in adipose tissue has made it a promising target in obesity and diabetes (Castan-Laurell, Boucher, et al., 2005). Apelin also plays an important role in fibrosis prevention in many organs, including the heart, lungs, and kidneys (Huang, Chen, et al., 2016). Apelin prevents the development of myocardial fibrosis after ischemia and atrial fibrillation by blocking the Ang2 pathway. In the heart, apelin is involved in maintaining proper lymphatic vessel geometry after ischemia (Tatin, Renaud-Gabardos, et al., 2017). Apelin reduces kidney and skin fibrosis by blocking TGFβ signaling (Yokoyama, Sekiguchi, et al., 2018).It is surprising that the protective effect of apelin against white adipose tissue fibrosis has not been fully investigated. In particular, the beneficial effects of apelin against diet-induced obesity have been attributed to its ability to improve lymphatic and vascular integrity (Sawane, Kajiya, et al., 2013). Apelin stimulates NO production via PI3K / Akt signaling in vascular endothelial cells (Busch, Strohbach, et al., 2015). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 6,492,324B1 [Patent Document 2] U.S. Patent Publication No. 20130177638 [Patent Document 3] U.S. Patent Publication No. 20130177637 [Patent Document 4] U.S. Patent Publication No. 20130177636 [Patent Document 5] U.S. Patent Publication No. 20130177635 [Patent Document 6] U.S. Patent Publication No. 20130177634 [Patent Document 7] U.S. Patent Publication No. 20130177633 [Patent Document 8] U.S. Patent Publication No. 20130183375 [Patent Document 9] U.S. Patent Publication No. 20130183373 [Patent Document 10] U.S. Patent Publication No. 20130183372 [Patent Document 11] U.S. Patent Publication No. 20100324120 [Patent Document 12] International Publication No. 2012031046 [Patent Document 13] International Publication No. O2012031043 [Patent Document 14] International Publication No. 2012030901

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Non-licensed literature

[0006] [Non-licensed document 1] 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): pp. 443~53 [Non-licensed document 2] Prel A, Caval V, Gayon R, Ravassard P, Duthoit C, Payen E, Mauche-Chretien L, Creneguy A, Nguyen TH, Martin N, Piver E, Sevrain R, Lamouroux L, Leboulch P, Deschaseaux F, Bouille P, Sensebe L, Pages JC. "Highly efficient in vitro and in vivo delivery of functional RNAs using new versatile MS2-chimeric retrovirus-like particles.” Mol Ther Methods Clin Dev. 2015 / 10 / 21;2:15039. doi: 10.1038 / mtm.2015.39. PMID: 26528487; PMCID: PMC4613645 [Non-patent document 3] Greene, MR et al., Transduction of Human CD34 + Repopulating Cells with a Self-Inactivating Lentiviral Vector for SCID-X1 Produced at Clinical Scale by a Stable Cell Line. Hum. Gene Ther. Methods 23, pp. 297-308 (2012) [Non-patent document 4] Paunovska, Kalina, David Loughrey, and James E. Dahlman. "Drug delivery systems for RNA therapeutics." Nature Reviews Genetics (2022): pp. 1-16 [Non-Patent Document 5] Wheeler et al., Gene Therapy. 1999 6:271-281 [Non-patent document 6] Zhang et al., Gene Therapy. 1999 6:1438-1447 [Non-Patent Document 7] Jeffs et al., Pharm Res. 2005 22:362-372 [Non-patent document 8] Morrissey et al., Nat Biotechnol. 2005 2:1002-1007 [Non-Patent Document 9] Zimmermann et al., Nature. 2006 441:111-114 [Non-Patent Document 10] Heyes et al., J Contr Rel. 2005 107:276-287 [Non-Patent Document 11] Semple et al., Nature Biotech. 2010 28:172-176 [Non-Patent Document 12] Judge et al., J Clin Invest. 2009 119:661-673 [Non-Patent Document 13] deFougerolles, Hum Gene Ther. 2008 19:125~132 Summary of the Invention [Problem to be solved by the invention]

[0007] The invention is defined by the claims. In particular, the invention relates to the use of apelin to treat lymphedema. [Means for solving the problem]

[0008] Lymphedema is a chronic condition caused by lymphatic dysfunction, which leads to fluid and fat accumulation in the limbs. It is characterized by severe inflammation and fibrosis, which affect mobility and increase the risk of skin infections. Here, we identified the biologically active peptide apelin as a promising candidate for restoring lymphatic flow in lymphedema. We found that apelin expression was significantly reduced in the lymphedematous arms of women compared with their normal arms. Using an apelin knockout mouse model, we confirmed the important role of apelin, as lymphedema persisted for more than 4 weeks after surgery and was associated with a lack of dermal lymphangiogenesis and increased backflow into the dermis. We showed that intradermal injection of an apelin-lentivector significantly reduced limb swelling, which was associated with reduced dermal fibrosis and increased lymphatic vessel density. Importantly, apelin stimulates eNOS-mediated lymphatic pumping through phosphorylation of Akt and eNOS in lymphatic endothelial cells (LECs). This was associated with a significant increase in E2F8 target gene expression through direct binding of E2F8 to the CCBE1 promoter in LECs. Collectively, these results indicate that apelin plays an important role in lymphedema and thus represents a novel partner for VEGF-C to prevent limb swelling and tissue fibrosis in lymphedema.

[0009] Accordingly, 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 apelin polypeptide, or ii) a polynucleotide encoding an apelin polypeptide.

[0010] As used herein, the term "lymphedema" has its general meaning in the art and refers to a disorder characterized by increased fluid retention in tissues, localized accumulation of adipose 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 (but which may be associated with other lymphatic exudations) due to underlying abnormal development of the lymphatic system (abnormal lymphangiogenesis). This may or may not be genetic and may be congenital or acquired. In some embodiments, lymphedema is found as a secondary disorder that may result from lymph node resection or lymphatic vessel damage. Such secondary lymphedema may also be associated with, for example, surgery, radiation therapy, lymph node removal and injury associated with oncological disease and its treatment, musculoskeletal injuries such as fractures, tendon dissection, and joint replacement, neurological conditions such as muscle paralysis, vascular injury / surgery, integumentary injuries, coagulation disorders such as deep vein thrombosis, scar tissue formation, treatment with tamoxifen, filariasis, infection, steatosis, or cellulitis.

[0011] As used herein, the term "treatment" or "treating" refers to both prophylactic or preventative treatment and curative or disease-modifying treatment, including treatment of patients at risk of or suspected of having a disease, as well as patients who are ill or have been diagnosed with a disease or medical condition, and includes the suppression of clinical recurrence. Treatment may be administered to a patient with a medical disorder or a patient who may ultimately develop the disease in order to prevent, cure, delay the onset of, reduce the severity of, or alleviate one or more symptoms of the disorder or recurrent disorder, or to extend the patient's survival beyond that expected in the absence of such treatment. A "therapeutic regimen" refers to a pattern of treating a disease, e.g., a dosing pattern 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 portion of a therapeutic regimen) used in the initial treatment of a disease. The general goal of an induction regimen is to provide patients with a high level of drug during the initial period of the treatment regimen. The induction regimen may employ (partially or entirely) a "loading regimen," which may involve administering a higher dose of drug than the dose employed by the physician in the maintenance regimen, administering the drug more frequently than the frequency administered by the physician in the maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or part of a treatment regimen) used to maintain a patient during disease treatment, for example, to keep the patient in remission for a long period of time (months or years). The maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, such as weekly, monthly, or yearly), or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment upon relapse, or treatment when certain predetermined criteria (e.g., disease manifestation, etc.) are met).

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

[0013] As used herein, the term "apelin" has its general meaning in the art and refers to a 77-residue precursor protein (encoded by NCBI Reference Sequence: NP-0059109.3 and NCBI Reference Sequence: NM-017413.3), which is processed to produce 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, while the most potent isoform is a pyroglutamylated form of the 13-mer apelin (apelin-13), called "Pyr-1-apelin-13" or "Pyr1-apelin-13." Different forms of apelin are described, for example, in U.S. Patent No. 6,492,324 B1. An exemplary amino acid sequence of apelin is represented by SEQ ID NO: 1. SEQ ID NO: 1>sp|Q9ULZ1|APEL_HUMAN Apelin OS=Homo sapiens OX=9606 GN=APLN PE=2 SV=1 MNLRLCVQALLLLWLSLTAVCGGSLMLPDGNGLEDGNVRHLVQPRGSRNGPGPWQGGRR KFRRQRPRLSHKGPMPF

[0014] As used herein, the term "polypeptide" has its common meaning in the art and refers to a polymer of amino acids of any length. The polymer may contain modified amino acids. The term also encompasses amino acid polymers, whether natural or modified by intervention (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component). The definition also includes, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art.

[0015] As used herein, the term "apelin polypeptide" refers to a polypeptide comprising an amino acid sequence that has at least 90% identity to the amino acid sequence set forth in SEQ ID NO:1.

[0016] As used herein, the "percent identity" between two sequences is a function of the number of identical positions shared between the sequences (i.e., % identity = number of identical positions / total number of positions × 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. Sequence comparison and determination of percent identity between two sequences can be achieved using the mathematical algorithm described below. Percent identity between 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). Percent identity between two nucleotide or amino acid sequences can also be determined using algorithms such as EMBOSS Needle (pairwise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle can use the BLOSUM62 matrix, a "gap open penalty" of 10, a "gap extend penalty" of 0.5, no "end gap penalty," an "end gap open penalty" of 10, and an "end gap extend penalty" of 0.5. Generally, "percent identity" is a function of the number of matched positions divided by the number of positions considered for comparison, multiplied by 100. For example, if, after alignment, 6 out of 10 sequence positions are identical between two compared sequences, the identity is 60%. Percent identity is generally determined over the entire length of the query sequence over which the analysis is performed. Two molecules are identical if they have the same primary amino acid or nucleic acid sequence, regardless of the presence or absence of chemical and / or biological modifications.According to the present invention, a first amino acid sequence having at least 90% identity to a second amino acid sequence means that the first sequence has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the second amino acid sequence.

[0017] As used herein, the term "polynucleotide" refers to a polymer of nucleotides of any length, comprising ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. The term refers to the primary structure of the molecule. Thus, the term includes triplex-, double-, and single-stranded deoxyribonucleic acid ("DNA"), as well as triplex-, double-, and single-stranded ribonucleic acid ("RNA"). The term also includes modified forms, for example, by alkylation and / or capping, as well as unmodified forms of polynucleotides. More specifically, the term "polynucleotide" includes polyribonucleotides (containing D-ribose), including polydeoxyribonucleotides (containing 2-deoxy-D-ribose), tRNA, rRNA, hRNA, siRNA, and mRNA (spliced or unspliced), other types of polynucleotides that are N- or C-glycosides of purine or pyrimidine bases, as well as polymers containing non-nucleotide backbones, such as polyamides (e.g., peptide nucleic acids, "PNAs") and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers, provided that the polymers contain nucleobases in a configuration that allows for base pairing and base stacking as found in DNA and RNA. In some embodiments, the polynucleotide comprises mRNA. In another aspect, the mRNA is synthetic mRNA. In some embodiments, the synthetic mRNA contains at least one non-naturally occurring nucleobase. In some embodiments, all nucleobases of a particular class are substituted with non-natural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be substituted with non-natural nucleobases, such as 5-methoxyuridine). In some embodiments, a polynucleotide (e.g., synthetic RNA or synthetic DNA) contains only natural nucleobases, i.e., A, C, T, and G in the case of synthetic DNA, or A, C, T, and U in the case of synthetic RNA.

[0018] In some embodiments, a polynucleotide of the invention is messenger RNA (mRNA).

[0019] In some embodiments, the polynucleotide is inserted into a vector, such as a viral vector.

[0020] As used herein, the term "viral vector" refers to a virion or virus particle that functions as a nucleic acid delivery vehicle and includes a vector genome packaged within the virion or virus particle. Typically, the vector is a viral vector, i.e., adeno-associated virus (AAV), retroviral vector, bovine papilloma virus, adenoviral vector, vaccinia virus, or polyoma virus.

[0021] In some embodiments, the viral vector is an AAV vector.

[0022] As used herein, the term "AAV vector" refers to a vector derived from an adeno-associated virus serotype, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and variants thereof. An AAV vector has one or more of the AAV wild-type genes deleted in whole or in part, preferably the rep and / or cap genes, but retains functional flanking ITR sequences.

[0023] In some embodiments, the viral vector is a retroviral vector.

[0024] As used herein, the term "retroviral vector" refers to a vector that contains structural and functional genetic elements derived primarily from a retrovirus.

[0025] In some embodiments, the retroviral vectors of the present invention are derived from a retrovirus selected from the group consisting of an alpharetrovirus (e.g., avian leukemia virus), a betaretrovirus (e.g., mouse mammary tumor virus), a gammaretrovirus (e.g., murine leukemia virus), a deltaretrovirus (e.g., bovine leukemia virus), an epsilonretrovirus (e.g., Walley cutaneous sarcoma virus), a lentivirus (e.g., HIV-1, HIV-2), and a spumavirus (e.g., human spumavirus).

[0026] In some embodiments, the retroviral vectors of the present invention are viral particles of replication-defective retroviruses, which are capable of transporting the RNA of an exogenously transferred gene in place of retroviral mRNA.

[0027] In some embodiments, the retroviral vector of the present invention is a lentiviral vector.

[0028] As used herein, the term "lentiviral vector" refers to a vector containing structural and functional genetic elements derived primarily 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.

[0029] The structure and composition of the vector genome used to prepare the retroviral vectors of the present invention conform to those described in the art. In particular, minimal retroviral gene delivery vectors can be prepared from vector genomes that, excluding the recombinant nucleic acid molecules of the present invention, contain only the sequences of the retroviral genome (non-coding regions of the genome) necessary to provide recognition signals for DNA or RNA synthesis and processing. In some embodiments, the retroviral vector genome contains all elements necessary for nuclear import and proper expression of a polynucleotide of interest (i.e., a transgene). Examples of elements that can be inserted into the retroviral genome of the retroviral vectors of the present invention include at least one (preferably two) long terminal repeats (LTRs), such as a 5' LTR and a 3' LTR, a Psi sequence involved in encapsidation of the retroviral genome, and optionally at least one DNA flap containing a cPPT domain and a CTS domain. In some embodiments of the present invention, the LTR, preferably the 3' LTR, is deleted for its promoter and U3 enhancer and replaced with a minimal promoter that allows transcription during vector production, while an internal promoter that allows transgene expression is added. In particular, the vector is a self-inactivating (SIN) vector and contains a non-functional or modified 3' long terminal repeat (LTR) sequence. This sequence is copied to the 5' end of the vector genome upon integration, causing inactivation of the promoter activity of both LTRs. 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 with a recombinant nucleic acid molecule of the present invention.

[0030] In some embodiments, the retroviral vector genome lacks functional gag, pol, and / or env retroviral genes. "Functional" means genes that are correctly transcribed and / or correctly expressed. Thus, in this embodiment, the retroviral vector genome of the present invention includes at least one of the gag, pol, and env genes, but the gene is not transcribed or is incompletely transcribed. "Incompletely transcribed" expression refers to changes in transcripts such as gag, gag-pro, or gag-pro-pol, where one or some of these transcripts are not transcribed. In some embodiments, the retroviral genome lacks gag, pol, and / or env retroviral genes.

[0031] In some embodiments, the retroviral vector genome also lacks the coding sequences for the Vif, Vpr, Vpu, and Nef accessory genes (in the case of HIV-1 retroviral vectors), or complete or functional genes thereof.

[0032] Generally, retroviral vectors of the present invention are non-replicating, i.e., the vector and retroviral vector genome are incapable of forming new particles budding from infected host cells. This replication incompetence can be achieved by eliminating the gag, pol, or env genes from the retroviral genome, as noted in the preceding paragraph. It can also be achieved by deleting other viral coding sequences and / or cis-acting genetic elements necessary for particle formation.

[0033] Thus, the present invention encompasses the use of virus-like particles. As used herein, the term "virus-like particle" or "VLP" refers to a structure similar to a virus particle but lacking the viral genome, replication incapable, and pathogenic. The particle typically contains at least one structural protein derived from a virus. Preferably, only one structural protein is present. Most preferably, other nonstructural components of the virus are absent. Thus, virus-like particles can spontaneously self-assemble in vitro under appropriate conditions from viral structural proteins while excluding genetic material and potential replication capabilities. Virus-like particles, with diameters of approximately 20 to 150 nm, also possess characteristics of nanometer-sized materials, such as a large surface area, surface-accessible amino acids with reactive moieties (e.g., lysine and glutamic acid residues), a regular spatial structure, and good biocompatibility. Thus, assembled virus-like particles have great potential as delivery systems for specifically transporting various cargoes. In some embodiments, one or more zinc finger motifs of the Gag protein are replaced with one or more RNA-binding domains. In some embodiments, the RNA-binding domain is the coat protein of MS2 bacteriophage, PP7 phage, or Q3 phage, or the Nun protein, U1A protein, or hPum protein of prophage HK022. More preferably, the RNA-binding domain is the coat protein of MS2 bacteriophage or PP7 phage. Even more preferably, the RNA-binding domain is the coat protein of MS2 bacteriophage. These embodiments are particularly suitable for packaging mRNA encoding an apelin polypeptide into a VLP. Accordingly, in some embodiments, the mRNA encoding an apelin polypeptide encapsulated in a viral particle of the present invention comprises at least one encapsidation sequence. "Encapsidation sequence" refers to an RNA motif (sequence and three-dimensional structure) that is 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 a stem-loop motif of RNA of MS2 bacteriophage or PP7 phage, etc. The stem-loop motif, more specifically, the stem-loop motif of MS2 bacteriophage RNA or PP7 phage RNA, may be used alone or may be repeated several times, preferably 2 to 25 times, more preferably 2 to 18 times, for example 6 to 18 times. In some embodiments, the invention encompasses LentiFlash® technology, which is based on non-integrating lentiviral particles constructed using a bacteriophage coat protein and its cognate 19-nucleotide stem-loop (replacing the native lentiviral psi-packaging sequence) to achieve active packaging of mRNA into lentiviral particles (Prel A, Caval V, Gayon R, Ravassard P, Duthoith C, Payen E, Maouche-Chretien L, Creneguy A, Nguyen TH, Martin N, Piver E, Sevrain R, Lamouroux L, Leboulch P, Deschaseaux F, Bouille P, Sensebe L, Pages JC. "Highly efficient in vitro 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).

[0034] The retroviral vectors of the present invention can be produced by any method known in the art, including by transient transfection in stable cell lines and / or by means of helper viruses. The use of stable cell lines is also considered preferable for vector production (Greene, MR et al., Transduction of Human CD34 + Repopulating Cells with a Self-Inactivating Lentiviral Vector for SCID-X1 Produced at Clinical Scale by a Stable Cell Line. Hum. Gene Ther. Methods 23, pp. 297-308 (2012)). For example, retroviral vectors of the present invention can be obtained by transcomplementing a trans-complementation system (vector / packaging system) by transfecting permissive cells (such as 293T cells) in vitro with a plasmid containing a retroviral vector genome of the present invention and at least one other plasmid providing in trans the gag, pol, and env genes encoding the polypeptides GAG, POL, and envelope proteins, or sufficient portions of these polypeptides to allow the formation of retroviral particles.As an example, permissive cells are transfected with (a) a transcomplementation plasmid lacking the packaging signal psi (the plasmid has the accessory genes vif, nef, vpu, and / or vpr deleted), (b) a second plasmid containing a gene encoding an envelope protein (envelope expression plasmid or pseudotyping env plasmid), and (c) a plasmid vector containing a recombinant retroviral genome, optionally with the promoter region of the 3' LTR or the U3 enhancer sequence of the 3' LTR deleted (between the 5' and 3' ends of the retroviral LTR sequence, the psicapsid formation sequence, a nuclear export element (preferably the RRE element of HIV or other retroviral equivalents), and a nucleic acid molecule of the invention, optionally a promoter for the RNA and / or its nuclear import sequence (cPPT sequence, e.g., CTS)). Advantageously, the three plasmids used do not contain sufficient homologous sequences for recombination. Nucleic acids encoding the gag, pol, and env cDNAs can be advantageously prepared according to conventional techniques from viral gene sequences available in the prior art and databases. The transcomplementation plasmid provides nucleic acids encoding retroviral gag and pol proteins. These proteins are derived from a lentivirus, most preferably HIV-1. The plasmid lacks encapsidation sequences, envelope-encoding sequences, and accessory genes, and advantageously also lacks retroviral long terminal repeats. Thus, the gag and pol protein-encoding sequences are advantageously placed under the control of a heterologous promoter (e.g., a cellular, viral promoter, etc.), which may be constitutive or regulatable, low-strength or high-strength. Plasmids containing the transcomplementary Δpsi-CMV-gag-pol-PolyA sequence are preferred. This plasmid allows the expression of all proteins necessary for the formation of empty virions, except for the envelope glycoproteins. The transcomplementation plasmid advantageously contains the TAT and REV genes. The transcomplementation plasmid advantageously lacks the vif, vpr, vpu, and / or nef accessory genes.The Gag and pol genes, and the TAT and REV genes, may also be carried by different plasmids, possibly separately. In this case, multiple plasmids are used in transcomplementation, each encoding one or more of the proteins. The promoters used in the plasmid transcomplementation, envelope plasmid, and plasmid vector (driving expression of the coat proteins gag and pol, the vector genome mRNA, and the transgene, respectively) may be the same or different promoters, and are preferably selected from ubiquitous promoters or promoters native to viruses (e.g., CMV, TK, RSV LTR promoters), RNA polymerase III promoters (e.g., U6 or H1), or promoters of helper viruses encoding env, gag, and pol (i.e., adenovirus, baculovirus, herpesvirus promoters). To generate retroviral vectors of the invention, the above-mentioned plasmids can be introduced into competent cells, and the generated viruses can be harvested. The cells used can be any competent cells, particularly eukaryotic cells, especially mammalian cells (e.g., human or animal cells). These cells can be somatic or embryonic stem cells, or differentiated cells. Typically, the cells include 293T cells, fibroblasts, hepatocytes, muscle cells (e.g., skeletal muscle, cardiac muscle, smooth muscle, vascular muscle, etc.), nerve cells (neurons, glial cells, astrocytes), epithelial cells, kidney cells, and ocular cells. Insect cells, plant cells, yeast cells, or prokaryotic cells may also be included. Cells transformed with SV40 T antigen may also be included. 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, appropriate for the cell type under consideration. Typically, the cells and vector system are kept in contact in a suitable device (plate, dish, tube, pouch, etc.) for a time sufficient to allow the vector system or plasmid to be transferred into the cells.Generally, vector systems or plasmids are introduced into cells by calcium phosphate precipitation, electroporation, transduction, or by using transfection-facilitating compounds such as lipids, polymers, liposomes, and peptides. Calcium phosphate precipitation is preferred. Cells are cultured in any suitable medium, such as RPMI or DMEM (a special medium for culturing cells without fetal bovine serum). After transfection, the retroviral vectors of the present invention can be purified from the cell supernatant. Purification of the retroviral vector to increase its concentration can be achieved by any suitable method, such as density gradient purification (e.g., cesium chloride (CsCl)) or chromatographic techniques (e.g., column chromatography or batch chromatography). For example, the vectors of the present invention can be subjected to two or three CsCl density gradient purification steps. The vector is preferably purified from the infected cells using a method comprising lysing the adenovirus-infected cells, applying the lysate to a chromatography resin, eluting the adenovirus from the chromatography resin, and collecting the fraction containing the retroviral vector of the invention.

[0035] In some embodiments, vectors of the invention comprise "control sequences," which collectively refer to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, etc., which collectively enable the replication, transcription, and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present, so long as a selected coding sequence is capable of replication, transcription, and translation in an appropriate host cell. Another nucleic acid sequence is a "promoter" sequence, which is used herein in its conventional sense to refer to a nucleotide region containing a DNA regulatory sequence derived from a gene capable of binding RNA polymerase and initiating transcription of a downstream (3') located coding sequence. Transcriptional promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters."

[0036] In some embodiments, the polypeptides or polynucleotides of the present invention can be conjugated to 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 polypeptides or polynucleotides of the present invention are formulated using one or more lipid-based structures, including, but not limited to, liposomes, lipoplexes, or lipid nanoparticles (Paunovska, Kalina, David Loughrey, and James E. Dahlman. "Drug delivery systems for RNA therapeutics." Nature Reviews Genetics (2022): pp. 1-16). Liposomes are artificially prepared vesicles primarily composed of a lipid bilayer and can be used as delivery vehicles for administering pharmaceutical formulations. Liposomes can be of different sizes, including, but not limited to, multilamellar vesicles (MLVs) (which may contain a series of concentric bilayers separated by narrow aqueous compartments) that can be hundreds of nanometers in diameter, small unilamellar vesicles (SUVs) that can be less than 50 nm in diameter, and large unilamellar vesicles (LUVs) that can be 50-500 nm in diameter. Liposome designs can include, but are not limited to, opsonins or ligands to improve liposome attachment to compromised tissue or to activate events such as, but not limited to, endocytosis. Liposomes can also have a low or high pH to improve delivery of pharmaceutical formulations. As a non-limiting example, liposomes such as synthetic membrane vesicles are prepared by the methods, apparatus, and devices described in U.S. Patent Publication Nos. 20130177638, 20130177637, 20130177636, 20130177635, 20130177634, 20130177633, 20130183375, 20130183373, and 20130183372.In some embodiments, liposomes are formed from 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and MC3 (described in U.S. Patent Publication No. 20100324120), as well as liposomes capable of delivering small molecule drugs (e.g., DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.)). The polypeptides or polynucleotides of the invention can be encapsulated by liposomes and / or can be contained within an aqueous core which is then encapsulated by liposomes (see WO 2012031046, WO 2012031043, WO 2012030901, and WO 2012006378, and U.S. Patent Publication Nos. 20130189351, 20130195969, and 20130202684).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 (Wheeler et al., Gene Therapy. 1999 6:271-281; Zhang et al., Gene Therapy. 1999 6:1438-1447; Jeffs et al., Pharm Res. 2005 22:362-372; Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al., Nature. 2006 441:111-114; Heyes et al., J Contr Rel. 2005 107:276-287; Semple et al., Nature Biotech. 2010 28:172-176; Judge et al., J Clin Invest. 2009 119:661-673; deFougerolles, Hum Gene Ther. 2008 19:125-132; U.S. Patent Publication No. 20130122104).

[0037] In some embodiments, a polypeptide or polynucleotide of the invention is administered in combination with or in association with a VEGF-C polypeptide, or ii) a polynucleotide encoding a VEGF-C polypeptide.

[0038] As used herein, the term "VEGF-C" has its general meaning in the art and refers to vascular endothelial growth factor C, which is encoded by the VEGF-C gene. An exemplary amino acid sequence of VEGF-C is represented by SEQ ID NO:2. SEQ ID NO:2 >sp|P49767|VEGFC_HUMAN Vascular endothelial growth factor C OS=Homo sapiens OX=9606 GN=VEGFC PE=1 SV=1

[0039] [ka]

[0040] In some embodiments, either the polypeptide or the polynucleotide may be administered to a patient separately or in the same composition. For example, a bicistronic polynucleotide encoding both an apelin polypeptide and a VEGF-C polypeptide may be used. In some embodiments, the polynucleotides encoding the polypeptides may be inserted into or contained within the same vector (e.g., a viral vector, virus-like particle).

[0041] As used herein, a "therapeutically effective amount" refers to a sufficient amount of the active ingredient to treat or alleviate symptoms at a reasonable benefit-to-risk ratio applicable to any medical treatment. It is understood that the total daily dosage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, weight, general health, sex, and diet of the subject; the timing, route of administration, and excretion rate of the specific compound employed; the duration of treatment; drugs used in combination with the active ingredient; and similar factors known in the medical field. For example, it is well within the skill of the art to start administering the compound at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0042] Generally, the active ingredient (i.e., polypeptide or polynucleotide) of the present invention is combined with a pharmaceutically acceptable excipient, optionally with a sustained-release matrix (e.g., a biodegradable polymer, etc.), to form a pharmaceutical composition. The terms "pharmaceutical" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not elicit adverse allergic or other undesirable reactions when administered to mammals, particularly humans (if applicable). A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type.

[0043] The present invention is further illustrated by the following figures and examples, which, however, should not be construed in any way as limiting the scope of the present invention. [Brief explanation of the drawings]

[0044] [Figure 1] Figure 1A shows decreased apelin expression in human lymphedema. Figure 1A. Quantification of lymphatic vessel diameter (*p<0.05). Figure 1B. Quantification of dermal lymphatic density (p<0.05). Figure 1C. Quantitative RT-PCR analysis of genes involved in fibrosis and VEGFC maturation in dermal lipectomy samples from patients with lymphedema (*p<0.05) (n=3). Figure 1D. Quantitative RT-PCR analysis of adipokines in dermal lipectomy samples from patients with lymphedema (*p<0.05) (n=3). [Figure 2A] Figure 1 shows that apelin prevents secondary lymphedema. Schematic diagram of the experimental design for the secondary lymphedema model in mice injected with apelin lentivector (LV-APL). Quantification of proximal limb swelling 7 and 14 days after surgery in control limbs, lymphedematous limbs (n=17), or lymphedematous limbs treated with apelin lentivector (n=20) (*p<0.05). [Figure 2B]Figure 10: Apelin prevents secondary lymphedema. EIA dosimetry of circulating apelin in plasma from control mice (n=5) or apelin-treated mice (n=5). [Figure 2C] Figure 1: Apelin prevents secondary lymphedema. Lymphography revealed pathological remodeling of lymphatic vessels and reflux into the dermis in lymphedema, which was reversed by LV-APL (n=10). [Figure 2D] Figure 1 shows that apelin prevents secondary lymphedema. Masson's trichrome staining of skin obtained from mice with lymphedema treated or not with apelin (scale bar: 50 μm). [Figure 2E] Figure 1 shows that apelin prevents secondary lymphedema. Quantification of dermal thickness (*p<0.05). [Figure 2F] Figure 10: Apelin prevents secondary lymphedema.Dose determination by EIA of circulating VEGF-C in plasma of control mice (n=5) or apelin-treated mice (n=5). [Figure 2G] Figure 1 shows that apelin prevents secondary lymphedema.SHG signal from the deep collagen-rich layer within the dermis. [Figure 2H] Figure 1 shows that apelin prevents secondary lymphedema.Lyve-1 immunodetection of dermal lymphangiogenesis in apelin-treated mice (scale bar: 50 μm). [Figure 2I] Figure 1 shows that apelin prevents secondary lymphedema. Quantification of lymphangiogenesis in apelin-treated mice (*p<0.05, **p<0.01). [Figure 2J] Figure 1 shows that apelin prevents secondary lymphedema. Quantification of lymphatic vessel dilation in apelin-treated mice (**p<0.01). [Figure 3]Apelin and VEGF-C exhibit complementary effects on lymphatic endothelial cells. Figures 3A-3E. Comparison of bulk RNA sequencing in HDLECs treated with apelin, VEGF-C, or apelin + VEGF-C conditioned medium. Figure 3A. Heat map of the top 30 significant genes upregulated by apelin and VEGF-C. Figure 3B. Schematic representation of the number of genes upregulated by both apelin and VEGF-C (43). Figure 3C. Heat map of the top 30 significant genes upregulated by apelin and apelin + VEGF-C treatment. Figure 3D. Schematic representation of the number of genes upregulated by both apelin and apelin + VEGF-C (31). Figure 3E. Schematic representation of the number of genes upregulated by both apelin, VEGF-C, and apelin + VEGF-C (19). [Figure 4A] Figure 1: Apelin-VEGF-C mRNA delivery: a novel treatment option for lymphedema. Schematic diagram of the LentiFlash® vector encapsulating apelin mRNA and VEGF-C mRNA molecules for in vivo delivery. [Figure 4B] Figure 1: Apelin-VEGF-C mRNA delivery: a novel treatment option for lymphedema. EIA dosimetry of circulating apelin in plasma of control mice (n=5) or apelin-treated mice (n=5). [Figure 4C] Figure 1. Apelin-VEGF-C mRNA delivery: a novel treatment option for lymphedema. Dosimetry by EIA of circulating VEGF-C in plasma of control mice (n=5) or apelin-treated mice (n=5). [Figure 4D] Figure 1. Apelin-VEGF-C mRNA delivery: a novel treatment option for lymphedema. Quantification of proximal limb swelling 7 and 14 days after surgery in control limbs, lymphedematous limbs, or lymphedematous limbs treated with the VEGF-C LentiFlash® vector (n=10) (*p<0.05). [Figure 4E]Figure 1. Apelin-VEGF-C mRNA delivery: a novel treatment option for lymphedema. Quantification of proximal limb swelling 7 and 14 days after surgery in control limbs, lymphedematous limbs, or lymphedematous limbs treated with the VEGF-C LentiFlash® vector (n=10) (*p<0.05). [Figure 4F] Figure 1: Apelin-VEGF-C mRNA delivery: a novel treatment option for lymphedema. Quantification of proximal leg swelling 7 and 14 days after surgery in control limbs, lymphedematous limbs, or lymphedematous limbs treated with the apelin-VEGF-C LentiFlash® vector (n=10). [Figure 4G] Figure 1. Apelin-VEGF-C mRNA delivery: a novel treatment option for lymphedema. Quantification of proximal limb swelling 7 and 14 days after surgery in lymphedematous limbs (n=10) treated with APLN, VEGFC, or APLN+VEGFC LentiFlash® vectors (*p<0.05). [Figure 4H] Figure 1: Apelin-VEGF-C mRNA delivery: a novel treatment option for lymphedema. Representative lymphangiography images obtained from mice treated with VEGF-CC-LentiFlash®, Apelin-C-LentiFlash®, or Apelin-VEGF-C-LentiFlash® vectors. [Figure 4I] Figure 1. Apelin-VEGF-C mRNA delivery: a novel treatment option for lymphedema. Quantification of lymphatic vessel dilation in apelin-VEGF-C treated mice (**p<0.01). [Figure 4J] Figure 1. Apelin-VEGF-C mRNA delivery: a novel treatment option for lymphedema. Quantification of proximal limb swelling in mice treated with the APLN-VEGF-C LentiFlash® vector after lymphedema onset (10 days after surgery) (n=8) (*p<0.05). DETAILED DESCRIPTION OF THE INVENTION [Example]

[0045] method: human tissue samples Samples were obtained from archival paraffin blocks of 16 lipodermal excision specimens obtained from patients with secondary lymphedema treated at the Toulouse University Hospital in France between 2015 and 2016. Patients with a 1-12 year history of lymphedema presented with stage 2 lymphedema according to the International Society of Lymphology (ISL) classification. Eligible patients had a history of unilateral nonmetastatic breast cancer without recurrence for more than 5 years. The main clinical parameters used to diagnose significant lymphedema included clinical upper limb edema (a volume difference of 10% or 200 milliliters (mL) between the affected and contralateral upper limbs). Samples were selected as coded samples under a protocol approved by the INSERM Institutional Review Board (DC-2008-452), the National Research Department (Ministère de la recherche, ARS, CPP2, authorization number AC-2008-452), and the ethics committee. When available, several control arm tissue samples cosmetically removed from the same patients were also studied.

[0046] Fluorescence lymphography Near-infrared fluorescent lymphatic imaging was used to visualize the initial and transport lymphatic vessels. 100 μg of indocyanine green (Pulsion®) was diluted in 0.5 mL of pure water and then injected intradermally into the first interdigital space. Fluorescent images of lymphatic flow were observed by placing a camera (Photo Dynamic Eye, Hamamatsu®) 15 cm above the investigation area. Indocyanine green lymphangiography findings can be classified into two patterns: a normal linear pattern and an abnormal dermal reflux pattern.

[0047] Lymphoscintigraphy Lymphoscintigraphy was used to diagnose the severity of lymphedema. This is a low-dose radiation test, but is contraindicated during pregnancy and lactation. Bilateral subcutaneous injections were performed between the first and second toes. Large-sized 99m-technetium radiolabeled nanocolloidal albumin was selectively captured by lymphatic capillaries and then drained via the lymphatic system. This allowed for comparative, functional, and bilateral evaluation of both upper limbs, including uptake in the axillary lymph nodes, lymphatic congestion, reflux into the dermis, and bypass to the epitrochlear lymph nodes via the deep lymphatic system.

[0048] Mouse model of lymphedema Mice were treated in accordance with EU and national regulations. C57Bl / 6 mice were provided by Envigo. All experiments were approved by Inserm Rangueil-Purpan, the local branch of the Midi-Pyrenees Ethics Committee. 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 in conjunction with axillary and brachial lymphadenectomy. Limb size was measured over time in the axillary region using calipers. Mice developed edema over a 2-week period. On the day of surgery, PBS or the apelin lentiviral vector was injected intradermally into the lymphedematous limb (three 4 μL injections). For the LentiFlash® vector, vehicle, apelin, VEGFC, and apelin-VEGFC LentiFlash® vectors were injected intradermally into the lymphedematous limb (200 ng of p24 divided into three 2 μL injections). G For the treatment with L-nitro-L-arginine methyl ester (Sigma), L-NAME was suspended in water (1 mg / mL) and mice were allowed to drink freely for 7 days.

[0049] Lymphangiography Two weeks after surgery, mice were anesthetized by intraperitoneal injection of ketamine (10 mg / kg) (Zoletil 100, Virbac) and xylazine (10 mg / kg) (2% Rompun, Bayer). FITC-dextran (70,000 kDa, 2 mg / mL, Sigma) was injected into the plantar region of the lymphedematous and control limbs. The fluorescent molecule was taken up by lymphatic vessels and eliminated from the blood vessels. Five minutes later, the skin was analyzed under a modular stereomicroscope, Discovery.V12 Stereo (Zeiss).

[0050] histology Skin from lymphedematous and control limbs was harvested 2 weeks after surgery and fixed overnight in 10% neutral-buffered formalin at 4°C. The tissues were then embedded in paraffin and sectioned on a microtome. 5-μm sections were cut and placed on Superfrost Plus slides. The tissues were deparaffinized, rehydrated, and antigen unmasking was achieved in a microwave oven using Tris solution, pH 9 (H-3301, Vector Laboratories), for 5 minutes, three times. After cooling, the slides were washed in PBS and then blocked with 5% BSA solution in a humidified chamber at room temperature. The sections were incubated overnight at 4°C with primary antibodies (goat anti-mouse Lyve1, R&D AF2125; rabbit anti-CD31, Abcam Ab28364) and washed three times in PBS. Sections were incubated with the corresponding secondary antibodies conjugated to Alexa-488 or Alexa-594 (at a dilution of 1 / 400) for 1 hour at room temperature. DNA was stained with DAPI. Slides were mounted using Dako Fluorescence Mounting Medium (S3023). Images were acquired using an inverted fluorescence microscope (Leica, DMi8) and analyzed using Fiji software.

[0051] Assessment of fibrosis Dermal fibrosis was assessed using Masson's trichrome staining method (MST-100T, Cliniscience). Skin sections were deparaffinized, and tissues were stained according to the manufacturer's recommendations. Images were acquired on a NanoZoomer slide scanner. Dermal size was quantified using at least 10 measurements of the length between the epidermis and subcutaneous tissue per field.

[0052] SHG imaging Skin (lymphoedematous and control tissues) obtained from mouse limbs was paraffin-embedded and sectioned on a microtome. 30 μm sections were cut and placed on Superfrost Plus slides. Tissues were deparaffinized and used for SHG analysis as described in the "Histology" section. Data acquisition was performed using a Bruker (Billerica, Massachusetts, USA) 2P Plus two-photon microscope. The microscope was equipped with a Coherent (Santa Clara, California, USA) Chameleon Discovery laser and an Olympus (Shinjuku, Tokyo, Japan) 20x NA:1 objective. A 900 nm laser wavelength was used, and second harmonic generation (SHG) emission was collected at 450 nm. Z-stack images were acquired with a 1 μm step size. Collagen fiber quantification was performed using at least five measurements per skin section. This analysis was performed on at least six mice per condition.

[0053] Collecting duct contraction measurement Luminal constriction measurements of afferent collecting lymphatic vessels toward the popliteal lymph node (PLN) were performed as previously described (Liao S. et al.). Briefly, mice were anesthetized by intraperitoneal injection of ketamine (100 mg / kg) and xylazine (10 mg / kg). 6 μL of FITC-dextran was injected into the plantar region of the right hind paw. The skin was carefully removed to expose the afferent collecting lymphatic vessels toward the PLN. Next, the mouse was placed in a Petri dish and placed on the stage of an inverted microscope (Leica, DMi8). Four 90-second videos were acquired per mouse, and the values of constriction and dilation (the difference between the maximum and minimum diameters) were analyzed using Fiji software. To evaluate the effect of apelin, a lentiviral vector was injected into the dermis of the right hind paw 7 days before the experiment. For the L-NAME test, mice were allowed free access to water for 7 days.

[0054] LentiFlash® Construction, Production, Purification, and Quantitation by p24 ELISA Assay Four plasmids were used to generate recombinant LentiFlash® particles in HEK293T cells: (i) the pLVGagPol plasmid (designated pLF-GagPol ΔZF2_PCP) encoding the viral gag and pol genes modified to retain the PP7 coat protein (PCP) within the gag gene (Mianne et al., 2022); (ii) the pVSVG plasmid encoding the VSV-G glycoprotein; and (iii) two plasmids encoding RNA cargo flanked by PP7 bacteriophage aptamers to enable RNA recruitment into lentiviral particles through interaction with the PP7 coat protein cloned within the Gag sequence. All newly generated constructs were verified by restriction enzyme digestion and sequencing. After transfecting the four plasmids into HEK293T cells using standard calcium phosphate procedures, LentiFlash® particles were generated in a 10-layer CellSTACK chamber (6360 cm², Corning). Twenty-four hours after transfection, the supernatant was discarded and replaced with fresh medium. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2 in air. After medium replacement, the supernatant was collected, clarified by centrifugation at 3000 g for 5 minutes, and microfiltered through a 0.45 μm pore-size sterile filter unit (Stericup, Millipore). The supernatant was collected several times, and all samples were finally pooled (referred to as crude harvest). The crude harvest was concentrated and purified by ultrafiltration and diafiltration. For quantification, p24 core antigen was detected directly in the viral supernatant using an HIV-1 p24 ELISA kit (Perkin Elmer) according to the supplier's specifications. The viral titer (expressed as the number of physical particles per mL) was calculated from the amount of p24, assuming that 1 pg of p24 corresponds to 10E+4 physical particles.

[0055] Enzyme immunoassay (EIA) Apelin concentrations in the medium and mouse plasma were determined using an apelin EIA kit (RAB0018, Sigma-Aldrich) according to the manufacturer's recommendations. A VEGFC ELISA kit was obtained from R&D Systems.

[0056] Cell culture and treatments Human dermal lymphatic endothelial cells (HDLECs) (single-donor, juvenile foreskin-derived, Promocell, C-12216; over 95% of cells were CD31- and podoplanin-positive) were cultured in endothelial cell medium MV2 (EGM-MV2, Promocell, C-22121). NIH3T3 cells 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 cells were passaged at one-third intervals. To collect conditioned medium, NIH3T3 cells were grown in 10 cm dishes. After reaching confluence, the 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). The medium was then collected and used for the experiment. HDLECs were treated with 50% conditioned medium / 50% MV2-0 5% FBS.

[0057] RNA extraction and reverse transcriptase and qPCR Total RNA was prepared using the RNeasy kit (Qiagen 74106) according to the supplier's instructions. 1 μg of RNA was reverse transcribed using the high-Capacity cDNA Reverse Transcription Kit with Multi-Scribe reverse transcriptase (Thermo Fisher Scientific, 4368813) according to the supplier's instructions. Quantitative real-time PCR was performed on a StepOne Real-time PCR System (Thermo Fisher Scientific) using OneGreen FAST qPCR premix (Ozyme, OZYA008). All samples were analyzed in duplicate. Data were normalized to HPRT mRNA levels.

[0058] Immunoblotting 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 13,500 xg for 10 minutes at 4°C. The supernatant was then collected and mixed with Laemmli buffer containing dithiothreitol (1 mM 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 were as follows: Phospho AKT: AKT-p ser473 (CS#4060S), AKT: Santa Cruz H136 (S8312), Phospho ERK: ERK1 / 2-p (MAPKp42 / 44) (Thr202 / Thr204) (Cell Signaling #9106), ERK: ERK1 / 2 (MAPKp42 / 44) (Thr202 / Thr204) (Cell Signaling #9102), Phospho eNOS (Cell Signaling #9571S), eNOS (Cell Signaling #5880S), E2F8 (Abcam, AB109596), VEGFR3 (R1D The antibodies were: Phospho-VEGFR3 (Affinity Systems AF349), Phospho-VEGFR3 (Affinity Systems AF3676), and CCBE1 (Sigma SAB1402017). After three washes in TBS-T, the cells were probed with HRP-conjugated secondary antibodies (1 / 10,000 dilution). Signals were visualized on a Chemidoc (Biorad) digital acquisition system using chemiluminescent detection reagents (Sigma).

[0059] Bulk RNA sequencing RNA sequencing on primary human lymphatic endothelial cellsTotal RNA from apelin lentivector-transduced or untransduced HDLECs 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 implemented by Genewiz using an Illumina HiSeq PE 2x150 configuration. Sequence reads were trimmed using Trimmomatic v.0.36 to remove potential adapter sequences and low-quality nucleotides. The trimmed reads were mapped to the Homo sapiens GRCh38 reference genome available on ENSEMBL using STAR aligner v.2.5.2b. STAR aligner is a splice aligner that detects and incorporates splice junctions to aid in the alignment of all reads. A BAM file was generated as a result of this process. Unique gene hit counts were calculated using feature counts in the Subread package v.1.5.2. Hit counts were aggregated and reported using the gene_id feature in the annotation file. Only unique reads falling within exon regions were counted. The distribution of read counts within the library was confirmed before and after normalization. The original read counts were normalized to adjust for various factors (e.g., variability in sequencing yield between samples). These normalized read counts were used to accurately identify differentially expressed genes. Data quality assessment was performed to thoroughly detect samples that were not representative of the group and therefore potentially affect the quality of the analysis. The overall similarity between samples was evaluated by the Euclidean distance between samples. This method was used to determine which samples were similar or different to each other and whether the predictions from the experimental design were met. The shorter the distance, the more related the samples. Samples were then clustered using the distances. Principal component analysis was also performed to reveal similarities between samples based on the distance matrix.

[0060] Differential gene expression analysis After extracting gene hit counts, the gene hit count table was used for downstream differential expression analysis. DESeq2 was used to compare gene expression between apelin-transduced and non-transduced LECs. Wald tests were used to determine p-values and log2 fold changes. Genes with a log2FC greater than 0.5 or a log2FC less than -0.5 and an adjusted p-value less than 0.05 were defined as differentially expressed genes and used for downstream analysis. The overall transcriptional changes between the two groups being compared were visualized using volcano plots. Each data point in the volcano plot represents one gene. The log2 fold change of each gene is shown on the x-axis, and the log10 of its adjusted p-value is shown on the y-axis. Genes with an adjusted p-value less than 0.05 and a log2 fold change greater than 0.5 are indicated by red dots. These correspond to up-regulated genes. Genes with adjusted p-values <0.05 and log2 fold changes <0.5 are indicated by blue dots and correspond to down-regulated genes.

[0061] Gene Ontology (GO) analysis Gene ontology analysis was performed separately on statistically significant sets of up- and down-regulated genes using PANTHER software (version 16.0, http: / / pantherdb.org / ). Using the Homo sapiens reference list, sets of significantly and differentially expressed genes were clustered based on their biological processes or pathways, and overrepresentation of gene ontology terms was tested using Fisher's exact test. All GO terms with a false discovery rate (FDR) of less than 0.05 were considered significant and are listed in the Supplementary Data.

[0062] Chromatin immunoprecipitation (ChIP) Human dermal lymphatic endothelial cells (HDLECs) transduced with or without apelin lentivector were directly crosslinked in culture medium using 1% formaldehyde for 15 minutes. Then, 0.125 M glycine was added for 5 minutes. After washing twice with cold PBS, cells were scraped and frozen at -80°C. The cells were lysed and applied to the ChIP-IT Express Magnetic Chromatin Immunoprecipitation kit (Active Motif 53008). Optimal sonication conditions were previously determined to obtain approximately 500 bp DNA fragments. Cells were sonicated in the kit's specific shearing buffer (350 μl final volume) using a Diagenode Bioruptor Sonicator (7 cycles: 30 seconds on, 30 seconds off, in a water bath). DNA concentration was measured using a Nanodrop™, and 25 μg of chromatin was used per reaction. Experiments were then performed according to the manufacturer's protocol. Four micrograms of E2F8 antibody (Abcam, AB109596) was used per ChIP reaction. 10 μl of each sample was kept as input. Reactions were incubated overnight at 4°C. A mock sample without antibody was treated identically. 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 the Drosophila-specific histone variant, H2Av (spike-in antibody, Active Motif, 61686) as an internal control to normalize ChIP.

[0063] Cell transduction HDLECs were seeded at 100,000 cells / well in 6-well plates. After 24 hours, the cells were transduced with 1 mL of the apelin lentivector diluted in 1 mL of OptiMEM medium in the presence of a final concentration of 5 μg / mL of protamine sulfate. Control cells (NT) that were not transduced with the apelin lentivector were also treated simultaneously according to the same protocol; in this case, 2 mL of OptiMEM and 5 μg / mL of protamine sulfate were added to the cells. The medium was replaced after 24 hours. The cells were grown to confluence and then passaged and expanded for further experiments. Apelin transduction was verified by RT-qPCR.

[0064] statistical analysis All results presented in this study are representative of at least three independent experiments. In all figures, "n" represents the number of biological replicates. Data are expressed as the mean ± standard error of the mean (sem). Statistical significance was determined by two-tailed Student's t-test, one-way analysis of variance, or two-way analysis of variance with Bonferroni post-hoc test using Prism ver. 9.0 (GraphPad). Differences were considered statistically significant when P value < 0.05. The following symbols were used: ns > 0.05, ... * ≦0.05, ** ≦0.01, *** ≦0.0001.

[0065] result: Secondary lymphedema manifests as increased lymphatic capillary diameter and poor collection and drainage. Secondary lymphedema develops months, sometimes even 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 dysfunction. It has been considered the reference standard for decades (Munn and Padera, 2014; Szuba et al., 2003). Lymphoscintigraphy in women who developed lymphedema after breast cancer reveals detection of lymphatic collecting vessels and markedly reduced axillary lymph node perfusion after injection of a radiotracer (data not shown). Fluorescence lymphangiography also reveals a hypervascularized dermis with tortuous lymphatic capillaries (data not shown), associated with intense fibrotic reaction and reflux into the dermis (data not shown). This was confirmed using histological analysis of hyperplastic and overloaded lymphatic vessels, indicating increased lymphatic vessel density within the skin (data not shown) (Figure 1A, Figure 1B, and data not shown). Surprisingly, we observed no significant differences in genes involved in the maturation of lymphangiogenic factors, except for CCBE1, which was significantly downregulated in lymphedema (Figure 1C). Because lymphedema is characterized by a significant accumulation of fibrotic adipose tissue (AT) within the limbs, we also evaluated the expression of adipokines in lymphedematous AT compared with normal controls (Figure 1D). We found that apelin expression was significantly reduced in lymphedema, whereas no differences were observed in adiponectin expression, leptin expression, or other adipokines (Figure 1D).

[0066] Impaired lymphatic repair in apelin knockout mice. Our group previously reported that apelin improves lymphatic vascular normalization in the heart after cardiac ischemia (Tatin et al., 2017). To investigate the role of apelin in secondary lymphedema, we used a mouse model of lymphedema previously developed in our laboratory (Morfoisse et al., 2018). Apelin knockout mice underwent a second mastectomy in the left upper limb in conjunction with axillary and brachial lymph node dissection (data not shown). Using this model, reproducible lymphedema developed after 2 weeks and gradually returned to normal after 4–8 weeks. In apelin knockout mice, lymphedema remained significantly elevated even after 4 weeks, suggesting an inability to restore lymphatic function (data not shown). Next, lymphatic capillaries were examined using lymphography after injection of FITC-dextran into the plantar region. In apelin knockout mice, strong dermal reflux was observed 4 weeks after surgery (data not shown). Histological analysis of lymphatic vessels revealed no difference in lymphatic vessel basal density between WT and apelin KO mice (data not shown). In contrast, after lymphedema surgery, dermal lymphangiogenesis was significantly reduced in apelin KO mice compared with WT mice (data not shown). This was associated with increased skin fibrosis in both WT and apelin KO mice, as demonstrated using Masson's trichrome staining (data not shown). Because lymphedema leads to the accumulation of collagen fibers, one of the hallmarks of fibrosis, we performed skin analysis using second harmonic generation (SHG) imaging (data not shown). Interestingly, the accumulation of collagen fibers was increased in apelin KO mice compared with WT mice during lymphedema (data not shown).

[0067] Apelin has a regenerative function for lymphatic vessels in secondary lymphedema. To evaluate the effect of apelin on lymphatic healing, mice were intradermally injected with an apelin-expressing lentivector (LV-apelin) into the lymphedematous limb. Notably, lymphedema was significantly reduced in apelin-treated mice (Figure 2A). Circulating apelin levels were measured in mouse plasma by ELISA titration, revealing elevated plasma apelin concentrations in LV-apelin-treated mice (Figure 2B). Next, lymphatic collecting drainage was examined using lymphography (Figure 2C). Consistently, secondary lymphedema induces pathological remodeling of lymphatic vessels, with disorganized and abnormal luminal morphology and an increased number of branching points, compared with control limbs. Lymphatic leakage (backflow into the dermis) was also observed, revealing dysfunction of the superficial capillary network due to the lack of pumping activity of deep collecting lymphatic vessels (Figure 3C). In contrast, apelin-treated mice showed improved lymphatic morphology, including normalized morphology and a reduced number of luminal branches. Importantly, no reflux into the dermis was observed in apelin-treated mice, suggesting improved lymphatic function (Figure 3C). Using Masson's trichrome staining, dermal thickening was observed in lymphedematous limbs, consistent with the development of fibrosis (Figure 2D). No dermal thickening was observed in apelin-treated mice (Figures 2D and 2E), reflecting amelioration of lymphedema pathology. Interestingly, after LV-apelin treatment, an increase in circulating VEGF-C, a key lymphangiogenic factor, was observed, suggesting that apelin may partially regulate VEGF-C protein synthesis (Figure 2F). A VEGF-C-expressing lentivector (LV-VEGFC) was used as a positive control (Figure 2F). The effect of apelin on collagen deposition was also assessed using SHG (data not shown). A significant reduction in fibrosis was observed in apelin-treated mice compared to controls (Figure 2G). Blood vessel counts were assessed using CD31 immunostaining. As expected, no changes in the number of CD31-positive blood vessels were observed in this lymphedema model (Morfoisse 2017) (data not shown).However, as previously described in the literature (Wysocka Marta B et al., 2018), treatment with apelin lentivectors increased angiogenesis and vascular permeability (data not shown). In parallel, lymphangiogenesis was assessed using Lyve1 immunostaining on skin sections (Figures 2H, 2I, and 2J). Consistent with the lymphangiography results, an increased number of Lyve1-positive blood vessels was observed in lymphedematous limbs compared with control limbs, with no significant difference observed when comparing control mice with LV-apelin-treated mice (Figure 2I). Conversely, apelin was found to promote significant lymphatic vessel dilation (Figure 2J). Collectively, these results suggest that apelin may play a beneficial role in secondary lymphedema by acting on lymphatic vessel plasticity and dilatability.

[0068] Apelin regulates LEC gene expression. To investigate novel molecular mechanisms and signaling pathways regulated by apelin in LECs, we performed global transcriptome analysis on LECs stimulated for 24 hours with conditioned medium containing apelin or conditioned medium obtained from control NIH3T3 cells (data not shown). We verified the quality and similarity assessment of RNA-sequencing samples (data not shown). Differential DESeq analysis revealed that 217 genes were deregulated (p.adj<0.05 and Log2 fold change<-0.5 or >0.5), of which 94 genes were upregulated and 123 genes were downregulated (data not shown). The top 30 downregulated or upregulated genes are displayed on a heatmap (data not shown); the complete list is presented in the unpublished data. Gene Ontology (GO) analysis of the downregulated genes revealed that no biological processes were significantly affected in apelin-treated HDLECs. In contrast, GO analysis of biological processes revealed that up-regulated genes were enriched (FDR < 0.05) for terms related to extracellular matrix (ECM) remodeling and signal transduction (data not shown), including COL1A, FBN, ADATS2, and CCBE1. However, with the exception of collagen- and calcium-binding EGF domain 1 (CCBE1), whose induction was clearly confirmed, the induction of most of these genes was not verified by RT-qPCR in HDLECs (data not shown). CCBE1 protein, together with the ADAMTS3 (a disintegrin and metalloproteinase with thrombospondin motif-3) protease, is required for VEGF-C activation by enhancing the cleavage activity of ADAMTS3 and promoting the maturation of VEGF-C into its biologically active form. To examine the effect of CCBE1 on VEGF-C receptor activation, we performed knockdown of CCBE1 in LECs using siRNA (data not shown). Next, the cells were stimulated with apelin, and Western blot analysis of P-VEGFR3 was performed (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 apelin (data not shown). Interestingly, apelin also stimulated the expression of E2F8, a transcription factor for CCBE1 (data not shown). Therefore, we hypothesized that apelin might be involved in VEGF-C maturation by increasing E2F8 DNA binding to the CCBE1 promoter. To address this question, we performed chromatin immunoprecipitation (ChIP) experiments using E2F8 immunoprecipitation on apelin-overexpressing HDLECs (data not shown). We found that apelin significantly increased E2F8 binding to the CCBE1 promoter (data not shown). Interestingly, apelin also induced E2F8 binding to the E2F1 transcription factor promoter, suggesting a role in other biological functions (data not shown) (Wells, Graveel, et al., 2002). However, no binding to FLT4 was observed (data not shown). Overall, these data indicate that apelin regulates gene expression in HDLECs.

[0069] Apelin stimulates LEC function through Akt / eNOS signaling. Next, we investigated which molecular pathways are involved in the response to apelin in vitro. Apelin 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 its vasodilatory phenotype (data not shown), we hypothesized that the beneficial effects of apelin on lymphedema are mediated in part by the AKT / eNOS pathway. To this end, HDLECs were stimulated with conditioned medium obtained from LV-apelin-transduced NIH3T3 cells pre-depleted of VEGF-C. Apelin synthesis was verified by RT-qPCR (data not shown) and ELISA titration (data not shown) on NIH3T3 cells. Stimulation of HDLECs by conditioned medium was confirmed by assessing the AKT and ERK pathways over a 24-hour time course, and medium containing VEGF-C was used as a positive control (data not shown). In this context, LECs responded to VEGF-C after 30 minutes, with strong activation of AKT and ERK (data not shown), whereas apelin stimulated Akt phosphorylation after 1 hour without significant effect on ERK (data not shown). Importantly, eNOS phosphorylation was observed in HDLECs in response to apelin and VEGF-C (data not shown). Consistent with Akt activation in HDLECs, activation of cell migration was observed (data not shown), whereas no effect on cell junctions or cytoskeletal remodeling was observed (data not shown). Importantly, eNOS phosphorylation was observed in HDLECs in response to apelin and / or VEGF-C, suggesting that both apelin and VEGF-C stimulate lymphatic vessel dilation through the eNOS pathway (data not shown).

[0070] Apelin stimulates lymphatic pumping through activation of eNOS. Next, we explored whether apelin could regulate luminal dilation, particularly in collecting lymphatic vessels. Apelin has been described to activate eNOS phosphorylation and promote vasodilation in several cellular contexts (Dray, Knauf, et al., 2008) (Wysocka, Pietraszek-Gremplewicz, et al., 2018). We next investigated whether apelin could stimulate lymphatic collecting vessel dilation and thus lymphatic pumping (data not shown). Lymph flow is driven in part by the autonomous contraction of smooth muscle cells in collecting lymphatic vessels. To assess the effect of apelin on collecting vessel contraction, we used a previously described in vivo imaging method (Liao, Jones, et al., 2014) (data not shown). We assessed the number of luminal contractions and dilations. Interestingly, apelin was found to stimulate lymphatic pumping by increasing collecting vessel dilation 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). Next, to test the role of eNOS activation in vivo in response to apelin in lymphedema, LV-apelin-treated mice were subjected to L-NAME treatment (data not shown). Limb diameter was measured to assess edema (data not shown). Interestingly, L-NAME reversed the beneficial effects of apelin on lymphedema, confirming that lymphatic pumping is the primary pathogenesis of this pathology. Furthermore, a significant increase in edema was observed 2 weeks after surgery in the presence of apelin + L-NAME (data not shown). Lymphography revealed that L-NAME treatment also reversed the effects of apelin on the lymphatic network. Indeed, pathological remodeling of lymphatic vessels, with reflux into the dermis and abnormal lymphatic branching, was observed in apelin + L-NAME-treated mice (data not shown). Capillary quantification was also performed on skin sections using Lyve1 immunodetection (data not shown).Increased lymphangiogenesis was systematically observed in lymphedematous limbs, but no differences were observed between conditions (data not shown). Nevertheless, apelin-treated mice showed increased dilation (which was inhibited by L-NAME treatment) in terms of luminal area, recapitulating a similar phenotype compared with the control group (data not shown). We also examined fibrosis, but surprisingly, L-NAME had no effect on fibrosis (data not shown). Taken together, our results suggest that apelin prevents lymphedema by promoting the pumping function of collecting ducts and pathological remodeling of lymphatic vessels. This phenotype is likely mediated in part by activation of eNOS in lymphatic endothelial cells.

[0071] Apelin and VEGFC exhibit synergistic effects on regulating gene expression associated with the maintenance of collecting ducts. Most studies aimed at lymphatic system regeneration have focused on VEGF-C. Nevertheless, VEGF-C alone was ineffective in improving lymphatic function in a mouse vascular injury model, suggesting that it must be combined with other molecules to fully restore lymphatic function. Here, we found that apelin regulates lymphedematous fibrosis, lymphatic function, and collecting duct contractility. Our original approach aims to achieve synergistic effects in the treatment of secondary lymphedema by combining VEGF-C with apelin to target the entire lymphatic network, from capillaries to collecting ducts. When comparing gene expression profiles of HDLECs stimulated with apelin, VEGF-C, or apelin + VEGF-C, similar induction of the top 30 genes primarily related to extracellular matrix remodeling was observed (data not shown). Most of the genes induced by VEGF-C were also induced by apelin (Figures 3A and 3B). Half of the genes induced by the combination of apelin and VEGF-C were also induced by apelin (Figures 3C and 3D). When comparing the induction of genes shared by the two molecules, the cooperative effects of apelin and VEGF-C were concentrated in genes related to lymphatic microenvironment maintenance (collagen 1A1 and 6A) and genes related to VEGF-C maturation (ADAMTS2, E2F8) (Figure 3E). Furthermore, the combination of apelin and VEGF-C specifically upregulated 33 genes (Figure 3E). When comparing the untreated, VEGF-C alone, or apelin alone groups with the apelin + VEGF-C combination group, genes required for collecting duct function (including connexin 37 and 47 (GJA4, GJC2) and claudin 5 (CDN5)) were found to be increased, whereas angiogenic genes (VEGFA, FLT1, KDR, KI67) were downregulated (data not shown). The expression of genes related to VEGFC maturation (ADAMTS2, CCBE1) was improved in the VEGFC group, the apelin group, and the apelin + VEGFC group compared to the wild type (WT) (data not shown).

[0072] Apelin-VEGFC RNA delivery: a novel treatment option for secondary lymphedema. In Western countries, the development of secondary lymphedema after cancer treatment poses ethical concerns when delivering angiogenic molecules to cancer survivors. For safety reasons, we decided to use LentiFlash® (Lf), a next-generation vector that enables transient delivery of mRNA from non-integrating particles. Based on the ability of LentiFlash® to deliver several heterologous mRNA molecules, we created LentiFlash® vectors containing two different mRNAs, encoding VEGF-C or apelin, respectively, and injected them into a mouse lymphedema model (Figure 4A). The efficacy of LentiFlash® is strongly dependent on mRNA stability compared to lentivectors, which induce constitutive expression of transgenes without any effect on immune cell populations or platelet counts (data not shown). Therefore, we first confirmed the expression of circulating VEGF-C (Figure 4B) and apelin (Figure 4C), which became measurable 48 hours after injection, by ELISA. As expected, mRNA delivery by LentiFlash® was less effective than lentivectors, and only partial inhibition of limb swelling was observed when VEGF-C or apelin was used alone for mRNA delivery (Figures 4D and 4E). This is expected due to the time-restricted expression of these molecules. However, LentiFlash® delivery of the VEGF-C / apelin dual mRNA completely resolved limb swelling (Figures 4F and 4G), reduced backflow into the dermis (Figure 4H), and restored lymphatic perfusion in the lymphedematous limb (Figure 4I). This was associated with an increase in lymphatic vessel diameter (Figure 4I). Finally, to investigate whether apelin-VEGF-C mRNA could be a curative treatment for lymphedema, we injected it into mice with lymphedema (10 days after surgery) (Figure 4J). In that context, the LentiFlash® vector was shown to cause lymphedematous swelling to return to normal after 11 days.These data demonstrate the synergistic effect of apelin and VEGF-C, and demonstrate that the combination provides significant therapeutic benefit despite the transient expression of the two transgenes, raising the prospect of treating lymphedema using non-integrating RNA delivery vectors in patients who develop lymphedema after cancer treatment. Thus, apelin represents a highly effective molecule in combination with VEGF-C for the treatment of lymphedema using "safe" RNA delivery vectors in patients who develop lymphedema after cancer treatment.

[0073] Consideration: Although significant progress has been made over the past few decades in understanding the molecular mechanisms driving lymphatic function, lymphedema, the most common pathology associated with lymphatic dysfunction, remains an unsolved medical challenge (Mercier, Pastor, et al., 2019). Lymphedema is a painful, chronic condition affecting millions of people worldwide. Many factors contribute to the disease's pathogenesis. Primary lymphedema is an inherited disease induced by genetic mutations, while secondary lymphedema occurs after cancer treatment or filarial infection (Mortimer and Rockson, 2014; Rockson, 2018). However, both types share similar clinical signs: fluid and fat accumulation in the extremities associated with a fibrotic and hypervascularized dermis (characterized by tortuous, leaky capillaries and reduced perfusion of deep collecting ducts). Lymphoscintigraphy demonstrates significant reductions in lymph node perfusion, indicating that lymphatic collecting ducts, although still present, are unable to properly collect and transport lymph. These observations support therapeutic strategies focused on combining molecules to 1) normalize capillary zones and 2) restore lymphatic pumping activity in deep adipose depots. Although it is now well established that VEGF-C, a key lymphangiogenic growth factor, is the best candidate for restoring lymphatic capillary networks (Hartiala, Suominen, et al., 2020), its role in lymphatic collection is less effective. VEGF-C binds to its tyrosine kinase receptor VEGFR-3, promoting its biological activity (Alitalo, Tammela, et al., 2005). Collecting ducts develop within an adipose tissue-embedded environment, which undergoes significant alterations during lymphedema. In particular, adipose tissue synthesizes many adipokines involved in the integrity of blood and lymphatic vessels. Therefore, it is tempting to speculate that altering adipokine production could affect lymphatic collecting duct function. Among adipokines, apelin has been described as an important factor for stimulating LEC function (Kim, Kang et al., 2014).Apelin stimulates lymphangiogenesis in cancer and is involved in the restoration of precollecting lymphatic vessel shape after myocardial infarction (Tatin, Renaud-Gabardos, et al., 2017). Apelin is a biologically active peptide that induces signal transduction after binding to its G protein-coupled receptor, APJ, located on the surface of LECs. In addition to its effects on endothelial monolayers, apelin is a molecule with potent antifibrotic properties (Huang, Chen, et al., 2016).

[0074] Gene expression analysis of dermal fat excisions obtained from women with secondary lymphedema after breast cancer revealed a significant decrease in apelin expression in lymphedema. The critical role of apelin in lymphedema was confirmed in apelin-KO mice, which exhibited exacerbated lymphedema (which could be repaired by an apelin-expressing lentivector). In a mouse lymphedema model, apelin improved lymphedema by affecting two key features of the pathology: lymphatic function and tissue fibrosis. Importantly, the effect of apelin on the pumping function of lymphatic collecting ducts was found to be directly regulated by NOS. NO production is involved in endothelial homeostasis by controlling vascular tone as a blood flow adaptation (Dimmeler, Fleming, et al., 1999). eNOS mediates a key aspect of vascular remodeling by converting mechanical stimuli into enhanced NO production. Endothelial NOS (eNOS) also regulates lymphatic homeostasis. In a mouse fibrosarcoma model, eNOS mediates VEGF-C-induced lymphangiogenesis and tumor lymph node metastasis (Lahdenranta, Hagendoorn, et al., 2009). Other studies have shown that eNOS influences lymphatic flow through collecting lymphatic vessels without affecting capillary diameter (Hagendoorn, Padera, et al., 2004). Furthermore, impaired NO bioavailability within pulmonary lymphatic vessels was found in lambs exhibiting chronically increased pulmonary blood and lymph flow (Datar, Gong, et al., 2016). In this study, we found that the effect of apelin on the pumping function of lymphatic collectors is mediated by eNOS. Importantly, apelin has previously been shown to regulate aortic vascular tone in diabetic mice by increasing the phosphorylation of Akt and eNOS (Zhong, Yu, et al., 2007). In this study, we found that the beneficial effects of apelin on lymphatic collecting vessels are mediated through this pathway, suggesting that apelin may be the origin of NO-mediated lymphatic pumping in many organs.The extent of Akt phosphorylation was lower than that induced by VEGF-C, but appears to be sufficient to mediate its biological effects. Interestingly, apelin was found to have no effect on the integrity of the endothelial monolayer, suggesting that its role is limited to functional and dynamic effects.

[0075] RNA sequencing of apelin-stimulated LECs revealed that apelin regulated the expression of genes involved in extracellular matrix remodeling, consistent with its effects on tissue fibrosis. Interestingly, apelin also strongly stimulated the expression of CCBE1, a protein involved in the protease activation of VEGF-C by ADAMTS3 (Jha, Rauniyar, et al., 2017). This may partially explain the increased circulating VEGF-C levels observed after apelin treatment. Importantly, mutations in CCBE1 have been found to cause Hennekam syndrome, a congenital disorder characterized by lymphatic malformations leading to primary lymphedema, lymphangiectasia, and cardiac defects in humans (Alders, Mendola, et al., 2013). Mechanistically, CCBE1 gene expression is regulated by apelin, and apelin was found to directly increase the immobilization of its transcription factor, E2F8, on its promoter. Overall, these data further reinforce the finding that apelin is a key factor in restoring lymphatic function in lymphedema. Therefore, we proposed to evaluate the effects of apelin in conjunction with VEGF-C in a phase I clinical trial for secondary lymphedema to be initiated at Toulouse Hospital. This pilot study, called Theralymph, will focus on women who have developed lymphedema after breast cancer. However, a major ethical issue for survivors of cancer treatment is the possibility of tumor reactivation with pro-lymphangiogenic therapy, even if no recurrence has been observed for more than 5 years. Therefore, it was not long before we came to the conclusion that the use of lentiviral gene therapy, with its permanent transgene integration, was not the optimal solution for delivering treatment. Another issue is the short plasma half-life of apelin, less than 5 minutes (Japp and Newby, 2016). This could be supplemented by serial injections into the limbs, however, this significantly increases the risk of infection and fibrotic reactions, which are common in patients with lymphedema.Adeno-associated virus (AAV) was also rejected due to its transgene size limitations and inability to simultaneously deliver multiple therapies. We next decided to use the LentiFlash® vector, a novel class of non-integrating lentivectors capable of transiently delivering multiple mRNA particles (Prel, Caval, et al., 2015). LentiFlash® was constructed by replacing the native lentiviral Psi packaging sequence with a bacteriophage coat protein and its cognate 19-nucleotide stem-loop to actively package mRNA into lentiviral particles. Apelin mRNA alone, LentiFlash®, was less effective in reducing lymphedema than integrating lentivectors. However, when combined with VEGF-C, dual mRNA delivery completely resolved lymphedema and restored lymphatic flow within the limbs, demonstrating the beneficial effects of this mRNA delivery strategy, allowing sufficient synthesis of both proteins.

[0076] Over the past two years, we have witnessed the emergence of a new class of mRNA vaccines, vectors with high efficacy and low toxicity. We believe that mRNA can treat many diseases, including lymphedema, a pathology for which no treatment currently exists, in a manner distinct from conventional medicine. The flexibility of LentiFlash® allows for the temporary delivery of two mRNA molecules, which in this study stimulate the synergistic effects of APJ (a G-protein-coupled receptor) and VEGFR-3 (a tyrosine kinase receptor). Given that lymphedema persists as a multifactorial pathology involving lymphatic endothelial dysfunction, adipose tissue accumulation, and fibrosis, we believe that a multimodal therapy may provide a solution to eradicate this harmful condition. Therefore, we propose using the apelin-VEGF-C LentiFlash® vector in a Phase I / II gene therapy clinical trial to be initiated at our institution next year.

[0077] References: Throughout this specification various references describe the state of the art to which this invention pertains, the disclosures of which are hereby incorporated by reference into the present disclosure. (References) TIFF2025527176000002.tif226159TIFF2025527176000003.tif233159TIFF2025527176000004.tif233159TIFF2025527176000005.tif145159

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