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WO2025137510A1 - Compositions d'arnm d'adiponectine et leurs procédés d'utilisation - Google Patents

Compositions d'arnm d'adiponectine et leurs procédés d'utilisation Download PDF

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WO2025137510A1
WO2025137510A1 PCT/US2024/061376 US2024061376W WO2025137510A1 WO 2025137510 A1 WO2025137510 A1 WO 2025137510A1 US 2024061376 W US2024061376 W US 2024061376W WO 2025137510 A1 WO2025137510 A1 WO 2025137510A1
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apn
lnp
mrna
composition
cells
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Qisheng Tu
Jinkun Chen
Rady Eid EL-ARABY
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Tufts University
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Tufts University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/5759—Products of obesity genes, e.g. leptin, obese (OB), tub, fat

Definitions

  • T2D type 2 diabetes
  • Adipokines bioactive chemicals produced by adipocytes
  • APN adiponectin
  • compositions including adiponectin (APN) RNA and nanoparticles are disclosed herein.
  • a composition including an adiponectin mRNA and a nanoparticle is provided.
  • the APN mRNA includes SEQ ID NO: 1-2 or a sequence having at least 95% identity to SEQ ID NO: 1-2, or the APN mRNA encodes a polypeptide of SEQ ID NO: 3 or a polypeptide having at least 95% identity to SEQ ID NO: 3.
  • the nanoparticle may be a lipid nanoparticle (LNP).
  • a pharmaceutical composition in which the pharmaceutical composition includes APN-mRNA, a nanoparticle (optionally an LNP), and a pharmaceutically acceptable carrier.
  • a method of treating a condition that could benefit from administering APN is provided.
  • the condition to be treated is a metabolic disorder.
  • the condition to be treated is a bone disorder, such as a metabolic bone disorder.
  • the method can be used to increase weight loss.
  • FIG. 1A C2C12 myotubes viability was assessed using the CCK8 assay. After transfection with APN-mRNA-LNP, the half maximal inhibitory concentration (IC50) was determined after 24 hours.
  • FIG. 1B Relative APN Expression in differentiated Myotubes cell C2C12
  • FIG. 1C Relative APN Expression in Kidney mesangial cell SV40- MES13.
  • FIG.1D APN protein expression, three separate blot analyzes of the samples were conducted.
  • FIG. 1E Optical densitometry was used to semi-quantify the bands, and ImageJ (1.53t) digital imaging processing software was used for analysis.
  • FIG.1F IPGTT with AUC of IPGTT
  • FIG.1G ITT curve with AUC of ITT.
  • FIG.1H Blood glucose levels at different time points were evaluated by glucometer
  • FIG.1I Body weight at different time points. Values are mean ⁇ SD.
  • FIGS. 2A-2B APN in situ expression.
  • FIG. 3 Summary of the studied gene expression in the different studied tissues. The diagram was built based on the data from the Mouse Genome Database (MGD) and the Gene Expression Database (GXD). informatics.jax.org.
  • FIGS. 4A-4E Relative gene expression in different tissues.
  • FIG. 4A Glut-4 gene in differentiated myotubes cell C2C12, Kidney mesangial cell SV40-MES13, Skeletal muscles, Liver, Kidney, White fats, and Brown fats. For each tissue type the legend from top to bottom is organized in the graph from left to right.
  • FIG. 4A Glut-4 gene in differentiated myotubes cell C2C12, Kidney mesangial cell SV40-MES13, Skeletal muscles, Liver, Kidney, White fats, and Brown fats. For each tissue type the legend from top to bottom is organized in the graph from left to right.
  • DGKd gene in differentiated myotubes cell C2C12, Kidney mesangial cell SV40-MES13, Skeletal muscles, Liver, and Kidney are shown in the graphs from left to right. Within each tissue the legends from top to bottom show the organization of treatments from left to right.
  • FIG.4C PKCe gene in differentiated myotubes cell C2C12, Kidney mesangial cell SV40-MES13, Skeletal muscles, Liver, Kidney, White fats, and Brown fats are shown in the graphs from left to right. Within each tissue, the legends from top to bottom show the organization of treatments from left to right. Gene expression was calculated based on the Law of Fold-change, which is 2 ⁇ CT .
  • FIGS.5A-5E Relative insulin receptor (IR) expression in different studied tissues. The legend from top to bottom is shown from left to right in the graphs.
  • FIG. 5A Differentiated Myotubes cell C2C12;
  • FIG. 5B Kidney mesangial cell SV40-MES13;
  • FIG. 5A Differentiated Myotubes cell C2C12;
  • FIG. 5B Kidney mesangial cell SV40-MES13;
  • FIGS. 6A-6B Pancreas sections in the studied groups.
  • FIG.7A Relative EGFR Expression in different Differentiated Myotubes cell C2C12;
  • FIG.7B Kidney mesangial cell SV40-MES13;
  • FIG.7C kidney.
  • FIGS. 7D Overall correlation between APN and EGFR gene expression in kidney.
  • FIGS. 8A-8E Relative gene expression of the pro-inflammatory cytokines.
  • TNF ⁇ gene in differentiated myotubes cell C2C12, Kidney mesangial cell SV40-MES13, Skeletal muscles, Liver, Kidney, White fats, and Brown fats.
  • the legends from top to bottom represent the 5 treatment groups from left to right.
  • FIG. 8B IL-6 gene in differentiated myotubes cell C2C12, Kidney mesangial cell SV40-MES13, Skeletal muscles, Liver, Kidney, White fats, and Brown fats.
  • the legends from top to bottom represent the treatment groups from left to right.
  • FIGS.9A-9E Histopathology results in the studied groups.
  • PKC protein kinase C
  • IR insulin receptor
  • IRS-1 insulin receptor substrate 1
  • FIGS. 11A-11J Physiochemical properties, transfection evaluations, and intracellular uptake of the nanoparticles.
  • FIGS.11E-11J show characterization of physiochemical 7 properties, transfection evaluations, and intracellular uptake of a different nanoparticle.
  • FIG. 11E LNPs size distribution tested by DLS.
  • FIG. 11F LNPs transfection efficacy evaluated on Hek 293 cells by delivering mCherry encoded mRNA, the fluorescence images were taken after 2 days post transfection.
  • FIG. 11G TEM images of LNPs (scale bar, 100 nm).
  • FIG. 11H Physicochemical characterization of LNPs.
  • FIG. 11I Cytotoxicity evaluation of LNPs with different dose on Hek 293 cell line.
  • FIGS. 13A-13I AA3-DLin vaccines induced robust SARS-CoV-2 spike-specific antibody endpoint titers in the immunized mice.
  • FIG.12F IC50 titers of spike pseudovirus neutralizing antibody.
  • FIG. 12G PRNT50 titers of AA3-DLin vaccines in the immunized mouse sera. Data are displayed as means ⁇ SD. A one-way ANOVA with multiple comparison tests and unpaired t-test for comparison of two groups were used to analyze the statistical significance (ns, not significant; *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001).
  • FIGS. 13A-13I A one-way ANOVA with multiple comparison tests and unpaired t-test for comparison of two groups were used to analyze the statistical significance (ns, not significant; *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001).
  • FIG. 13A Schematic overview of the synthesis of APN-LNP. Evaluation of cytotoxicity of APN-LNP by CCK8 assay in preadipocytes and mature adipocytes transfected for 24 h. Preadipocytes (FIG. 13B) and mature adipocytes (FIG. 13C) were transfected with APN-LNP (0.25 ⁇ g/ml) for 24 h, with empty-LNP as the control.
  • FIG.15A MC3T3-E1 cells were transfected with APN-LNP (0.25 and 1.0 ⁇ g/ml) for 24 h in OPTI medium and further incubated in AMEM. Adiponectin mRNA expression was evaluated at 24 h, 72 h, 120 h and 240 h by qPCR. Osteogenesis related markers Bsp (FIG. 15B) and Ocn (FIG. 15C) expression in MC3T3-E1 were evaluated at 24 h.
  • FIGS.16A-16D Schematic representation of (FIG.16A) surgical procedure of 0.25 mm defect using RISystem Internal Fixation System and (FIG. 16B) surgery timeline of the femoral fracture model in male DIO mice.
  • FIG. 18A H&E staining of liver tissues.
  • FIG. 18B Gene expression of osteogenic markers Bsp and Runx2 in contralateral femur tissues.
  • FIG.19 Schematic overview of the synthesis and action of APN-LNP. Transfection with APN-LNP inhibited adipogenesis in 3T3-L1 adipocytes and Mmp9 expression in RANKL- stimulated RAW264.7 cells.
  • compositions may be administered to subjects via injection to treat conditions including metabolic syndrome, diabetes, type II diabetes, obesity, insulin resistance, hyperinsulinemia, hyperglycemia, adipokine dysregulation, bone regeneration, or bone repair.
  • the compositions may result in weight loss in the subjects administered the compositions and may allowed for glucose regulation to be restored.
  • compositions provided here results in at least one of Glut-4 reactivation, improvement in the Insulin Resistance, increased DGKd, decreased PKC ⁇ , insulin receptor activation, activation of insulin secretion through the reactivation of the islets of Langerhans, EGFR inhibition, reduction of inflammatory cytokines (TNF- ⁇ , IL-1 ⁇ , IL-6), decrease blood glucose levels, and reduction of hepatocellular steatosis as compared to a subject not administered the composition.
  • Lipid nanoparticles Lipid nanoparticles are able to deliver therapeutic agents and have been shown to be useful in a variety of systems.
  • LNPs can be used to deliver mRNA encoding antiviral proteins, such as interferons, or to delivery CRISPR/Cas9 systems for gene editing to target viral genomes to treat viral infections.
  • LNPs can be used to treat a variety of conditions, including viral, bacterial, or fugal infections, neurological disorders, cancer, or regenerative medicine.
  • the term “lipid nanoparticle” is a spherical vesicle made of lipids. The lipids can be cationic, ionizable, phospholipids, or cholesterol.
  • LNPs can be made up of various types of lipids, including but not limited to ionizable lipids, helper or neutral lipids, sterol lipids, cholesterol, and lipids attached to polyethylene glycol (PEG).
  • the lipid composition may comprise proteolipids (e.g., protamine), carrier proteins, and/or small molecules.
  • the lipid composition may comprise a single lipid group or multiple lipid groups.
  • Nonlimiting examples of lipid groups 10 include cationic lipids, anionic lipids, neutral lipids, polyethylene glycol (PEG)ylated lipids, ionizable lipids, helper lipids, stealth lipids, or cholesterols.
  • Nonlimiting examples of lipids include DOSPA 2,3-dioleyloxy-N-[2- (sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate, DOTMA 1,2- di- O-octadecenyl-3-trimethyl ammonium propane, DOTAP 1,2-Dioleoyl-3- trimethyalammoniumpropane, and DC-Cholesterol 313-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol.
  • Nonlimiting examples of ionizable lipids include SM-1029-Heptadecanyl 8-((2- hydroxyethyl)(6-oxo-6- (undecyloxy)hexyl)amino)octanoate, ALC-0315 4- hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) Dlin-MC3-DMA, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,2823icol23htraen-19-yl4-(dimethylamino) butanoate, and DODMA 1,2-Dioleyloxy-3-dimethylamino propane.
  • Nonlimiting examples of stealth lipids include PEGIG (R)-2,3- bis(myristoyloxy)propyl-1-(methoxy poly (ethylene glycol) 2000) carbamate and ALC-0159 2-[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide.
  • LNPs are made of AA3-Dlin, DOPE, Cholesterol, and DMG-PEG 2000.
  • the molar ratios of the lipids used in lipid nanoparticles may vary. In Example 1, the lipid nanoparticles used comprise a molar ratio of 40:40:25:0.5 (AA3-Dlin: DOPE: Cholesterol: DMG-PEG).
  • Lipid nanoparticles may range in size from approximately 20nm to 500nm, 30 nm to 250 nm, 50nm to 200 nm or any range between those contemplated here.
  • the average size of lipid nanoparticles may be approximately 100 nm or between 90nm and 110nm or between 80nm and 120nm.
  • Lipid nanoparticles can also be characterized by a polydispersity index (PDI), which is a measure of the distribution of the molecular mass in a given polymer sample.
  • PDI polydispersity index
  • Lipid nanoparticles can be further characterized by their zeta potential, which is defined as the electrical potential at its surface.
  • Example 1 the average size of the LNP is 96.5 nm, the PDI is 0.166, and the zeta potential is -3.5 mV.
  • Example 2 AA3-DLin LNPs are used, and are fabricated using microfluidic-chip device by mixing the organic phase containing ionizable lipid, DOPE, cholesterol, and DMG-PEG and the water phase of mRNA.
  • the average size of the LNP is 100 11 nm, with PDI of 1.56, and the zeta potential is -4.6 mV.
  • the lipid nanoparticle can be a self- assembly lipid-polymer nanoparticle. Nanoparticles of the present invention may be modified by any means known in the art.
  • the nanoparticle may be modified to decrease degradation or filtration or to increase evasion, function or targeting. Nanoparticles may also be modified to increase contact, binding or internalization by target cells. Nanoparticles may be modified with a variety of ligands such as small molecules, surfactants, dendrimers, polymers, and biomolecules. LNPs having any such modification are encompassed within the invention. Nanoparticles are not limited to lipid nanoparticles. Other nanoparticles include, but are not limited to, polymer-based nanoparticles, inorganic nanoparticles, and hybrid nanoparticles.
  • Polymer-based nanoparticles such as polymeric micelles, liposomes, and dendrimers, can be engineered for controlled drug released and improved stability.
  • Inorganic nanoparticles such as gold nanoparticles, iron oxide nanoparticles, and silica nanoparticles, can be used for various applications including imaging, drug delivery, and therapy.
  • Hybrid nanoparticles combine features of different types of nanoparticles, such as lipid-polymer nanoparticles or inorganic-polymer hybrid nanoparticles, offering unique properties and functionalities.
  • the nanoparticles may be further modified with various functional groups, such as targeting ligands (e.g., antibodies, peptides) to enhance cellular uptake and specificity, or with fluorescent dyes for imaging and tracking purposes.
  • LNPs may be used to deliver therapeutic agents like small molecules, nucleic acids, and proteins such as monoclonal antibodies. They can protect drugs from degradation, increase their solubility, and enable targeted delivery.
  • Lipid nanoparticles can encapsulate mRNA.
  • the LNPs are used to transport mRNA. There can be one or more types of mRNA present in an LNP.
  • the LNP can encapsulate a reporter gene, such as mCherry, and a therapeutic gene, such as Adiponectin.
  • LNPs containing a therapeutic gene can be referred to as therapeutic LNPs.
  • Lipid nanoparticles can be encapsulated into a lipid nanoparticle or a rapidly eliminating lipid nanoparticle and the lipid nanoparticles or a rapidly eliminating lipid nanoparticle can then be encapsulated into a polymer, hydrogel and/or surgical sealant described herein and/or known in the art.
  • the lipid nanoparticles can be formulated for sustained release (e.g., on the scale of hours, days, weeks, months, or years).
  • polynucleotide or “nucleic acid” are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or multi-stranded DNA or RNA, genomic DNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • cDNA complementary DNA
  • RNA e.g., messenger RNA (mRNA) or microRNA (miRNA)
  • mRNA messenger RNA
  • miRNA microRNA
  • the backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA) or modified or substituted sugar or phosphate groups.
  • the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidates and thus can be an oligodeoxynucleoside phosphoramidate (P--NH2) or a mixed phosphoramidate-phosphodiester oligomer.
  • P--NH2 oligodeoxynucleoside phosphoramidate
  • Other polynucleotide modifications include modified nucleosides such as pseudouridine, N1-methylpseudouridine, 5-methylcytidine, 5-methyluridine, 2’-O-methyluridine, 2-thiouridine, and N6-methyladenosine.
  • a double-stranded polynucleotide can be obtained from the single stranded polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer.
  • Polynucleotide sequences provided herein are provided as the cDNA encoding for the APN-mRNA of interest (SEQ ID NO: 1 (human) and SEQ ID NO: 4 (mice)).
  • APN is a large alternatively spliced protein so other isoforms or alleles found in the same species as the subject may be used in the compositions and methods disclosed here.
  • specific modifications include substituting cytidine residues with 5-Methylcylidine and uridine residues with pseudouridine. In some embodiments, specific modifications include at least one of a poly(A) tail and a 7-methylguanylate cap. In some embodiments, modifications include substituting cytidine, substituting uridine, a poly(A) tail, and a 7-methylguanylate cap in the same RNA strand. These modifications can decrease anti-RNA immune response and enhance RNA stability.
  • the term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined 13 sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • the cDNA sequence (SEQ ID NO: 1 and 4), mRNA sequence (SEQ ID NO: 2 and 5), and protein sequence for Adiponectin (SEQ ID NO: 3 and 6) in human and mice are provided.
  • an mRNA capable of encoding SEQ ID NO: 3 or SEQ ID NO: 6 are administered to a subject.
  • sequence identity refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • Nucleic acid and protein sequence identities can be evaluated by using any method known in the art. For example, the identities can be evaluated by using the Basic Local Alignment Search Tool (“BLAST”).
  • the BLAST programs identity homologous sequences by identifying similar segments between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from protein or nuclei acid sequence database.
  • the BLAST program can be used with the default parameters or with modified parameters provided by the user.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, GIy, VaI, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, GIu, Asn, GIn, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G
  • amino acid residue e.g., Ala, Pro, Ser, Thr, GIy, VaI, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp,
  • substantially identical'' of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 95% sequence identity to the polynucleotide encoding the polypeptide of interest described herein. Alternatively, percent identity can be any integer from 95% to 100%. In one embodiment, the sequence identity is at least 95%, alternatively at least 99%. More preferred embodiments include at least: 96%, 97%, 98%,99% or 100% compared to a reference sequence using the programs described herein; preferably BLAST using standard 14 parameters, as described.
  • the term "substantial identity" of amino acid sequences for purposes of this invention means polypeptide sequence identity of at least 95%, preferably 98%, most preferably 99% or 100%.
  • Preferred percent identity of polypeptides can be any integer from 95% to 100%. More preferred embodiments include at least 96%, 97%, 98%, 99%, or 100%.
  • the APN mRNAs in the compositions may be SEQ ID NO: 1-2 or a sequence having at least 95% identity to SEQ ID NO: 1-2 or maybe an mRNA encoding a polypeptide of SEQ ID NO: 3 or a polypeptide having at least 95% identity to SEQ ID NO: 3.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living microorganism is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • compositions refers to the combination of an active agent, compound, or ingredient with a pharmaceutically acceptable carrier or excipient, making the composition suitable for diagnostic, therapeutic, or preventive use in vitro, in vivo, or ex vivo.
  • pharmaceutically acceptable carrier or excipient refers to a carrier or excipient that is useful in preparing a pharmaceutical formulation that is generally safe, non-toxic, and is neither biologically or otherwise undesirable, and includes a carrier or excipient that is acceptable for veterinary use as well as human pharmaceutical use.
  • a “pharmaceutically acceptable carrier or excipient” as used in the specification and claims includes both one and more than one such carrier or excipient.
  • pharmaceutically acceptable salt refers to any acid or base addition salt whose counter-ions are non-toxic to the subject to which they are administered in pharmaceutical doses of the salts.
  • therapeutic agent e.g., a therapeutic polypeptide, nucleic acid, or transgene
  • a therapeutic agent may be used in a treatment as described herein.
  • the polynucleotide comprises an APN sequence.
  • treatment covers any treatment of a disease in a mammal and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease.
  • the therapeutic agent may be administered before, during or after the onset of disease or injury.
  • the treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest.
  • the subject therapy will desirably be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.
  • compositions including adiponectin (APN) mRNA and a lipid nanoparticle are described.
  • the APN-mRNA may be taken up by the lipid nanoparticle to increase stability and ability to administer the APN-mRNA to a subject.
  • composition of the APN mRNA may include SEQ ID NO: 1-2 or a sequence having at least 95% identify to SEQ ID NO: 1-2 or is capable of encoding SEQ ID NO: 3.
  • APN-mRNA may include at least one nucleotide modification.
  • the composition may further include a pharmaceutically acceptable carrier.
  • the composition may be administered with a required frequency such as one a week, every other week, once a month, once every other month, once a quarter or annually to tret the condition.
  • the administration amount and schedule may be different depending on the condition being treated in the subject.
  • a “therapeutically effective amount” refers to the amount or dose of the pharmaceutical composition that, upon single or multiple dose administration to the subject, provides the desired effect in the subject under diagnosis or treatment.
  • the disclosed methods may include administering an effective amount of the disclosed compounds to treat or alleviate symptoms of metabolic disorders. The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active agent(s) or to maintain the desired effect.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to determine a desirable concentration range and route of administration. “Therapeutically effective amount” further refers to an amount that does not induce excessive adverse side effects, such as effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of the present disclosure.
  • the pharmaceutical compositions of the present disclosure are formulated for administration by subcutaneous injection. In some embodiments, the pharmaceutical compositions of the present disclosure are formulated for administration by intravenous or intramuscular injection. Although not required, the compositions may optionally be supplied in unit dosage form suitable for administration of a precise amount. The administration of the compositions may increase the expression of APN protein after administration of the composition in a tissue selected from the group consisting of muscle, liver, pancreas, kidney and fat.
  • Methods of Treating Metabolic Bone Disorders and Inflammation “Metabolic disorder” refers to a disorder that negatively alters a subject’s metabolic processes.
  • metabolic disorders include by are not limited to diabetes, type II diabetes, obesity, hyperinsulinemia, hyperglycemia, adipokine dysregulation, and obesity.
  • Symptoms of metabolic disorders include weight loss or weight gain, lethargy, and excessive thirst.
  • Effects of metabolic disorders include but are not limited to dysregulation of insulin receptor activation, dysregulation of insulin secretion, insulin resistance, altered glucose uptake, diabetic nephropathy 17 symptoms, fat (hydropic) degradation in muscles, liver, and kidneys, and increased inflammatory cytokines.
  • Metabolic disorders can also cause chronic inflammation, decline in pancreatic ⁇ cell’s ability to produce insulin, and increase of oxidative stress in the kidneys.
  • the administration of the composition may result in at least one of Glut-4 reactivation, improvement in the Insulin Resistance, increased DGKd, decreased PKC ⁇ , insulin receptor activation, activation of insulin secretion through the reactivation of the islets of Langerhans, EGFR inhibition, reduction of inflammatory cytokines (TNF- ⁇ , IL-1 ⁇ , IL-6), decrease blood glucose levels, and reduction of hepatocellular steatosis as compared to a subject not administered the composition.
  • the subject is in need of a treatment for inflammation.
  • Inflammation refers to the release of pro-inflammatory cytokines from immune-related cells and the activation of the innate immune system.
  • Innate immune responses are capable of not only combating infectious microbes but also contributing to pathological situations, such as sepsis, obesity, atherosclerosis, autoimmunity, osteoarthritis, and cancer.
  • the subject has chronic inflammation.
  • Chronic inflammation refers to a slow, long-term inflammation lasting several months to years.
  • Chronic inflammation may be associated with a number of inflammation-mediated disorders, conditions, or diseases, such as diabetes, cardiovascular disease, artherosclerosis, arthritis and joint diseases, allergies, and chronic obstructive pulmonary disease.
  • Risk factors that contribute to chronic inflammation include, obesity, age, diet, smoking, low sex hormones, stress, and sleep disorders.
  • the subject has acute inflammation. Tissue damage due to trauma, microbial invasion, or noxious compounds can induce acute inflammation. Acute inflammation may start rapidly, become severe in a short time, and symptoms may last for hours, days, or a few weeks, e.g., 1-6 weeks.
  • the subject may have an infection, such as a viral or bacterial infection, or suffer from a condition such as sepsis, septic shock, or endotoxemia.
  • the subject has a bone disorder, which may be caused by metabolic disorders or a subject is in need of bone regeneration or bone healing after a fracture or other bone injury. Adiponectin has been shown to have beneficial effects on bone metabolism, potentially aiding in the treatment of osteoporosis.
  • BV/TV bone volume to total volume ratio
  • Tb.N trabecular number
  • a subject is in need of weight loss.
  • a subject may have or be in the process of developing a metabolic disorder at least partially caused by weight gain. Additionally or alternatively, the subject may have a metabolic disorder that causes weight gain.
  • Non-alcoholic fatty liver disease Adiponectin has been shown to improve insulin sensitivity and reduce hepatic steatosis in animal models of NAFLD.
  • Cardiovascular diseases Adiponectin has anti-inflammatory and anti-atherosclerotic properties, suggesting potential benefits in conditions like atherosclerosis, heart failure, and stroke.
  • Metabolic syndrome Adiponectin deficiency is associated with metabolic syndrome, a cluster of conditions that increase the risk of heart disease, stroke, and type 2 diabetes.
  • Neurodegenerative diseases some studies suggest a role for adiponectin in neuroprotection and cognitive function.
  • Periodontitis Adiponectin has anti-inflammatory properties and may help to reduce inflammation and promote tissue repair in periodontal disease. Miscellaneous Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which the invention pertains. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
  • the terms “a”, “an”, and “the” mean “one or more.”
  • a molecule should be interpreted to mean “one or more molecules.”
  • “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus ⁇ 10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.
  • Adiponectin mRNA conjugated with lipid nanoparticles targets pathogenesis of Type 2 diabetes APN, also known as Acrp30, AdipoQ, ApM1, and GBP28 [9], is a 30 kDa monomeric protein encoded by the ADIPOQ gene on chromosome 3q27, spanning approximately 15.8 kb. This protein is associated with a susceptibility locus for cardiovascular disease, type 2 diabetes, and metabolic syndrome [10]. Similar to leptin [11], monomeric APN is primarily produced and released in white adipose tissue. Glycosylation and hydroxylation play a critical role in controlling its activity and receptor binding [12].
  • adiponectin levels have an inverse relationship with adipose tissue.
  • adiponectin levels are typically between 2 and 30 ng/ml [13].
  • APN levels are negatively correlated with insulin resistance and BMI [8, 14].
  • Obesity-related adipose tissue growth triggers an inflammatory profile that lowers adiponectin secretion and levels [7].
  • Chronic inflammatory diseases such as T2D, obesity, and atherosclerosis are associated with decreased serum APN concentrations [8, 15, 16].
  • T2D T2D
  • atherosclerosis are associated with decreased serum APN concentrations [8, 15, 16].
  • adiponectin deficiency plays a role in T2D pathogenesis [17, 18].
  • T2D type 2 Diabetes
  • AdipoR1/AdipoR2 adiponectin receptors
  • APPL1Rab5 or APPL1-AMP-AMPK-mediated translocation of glucose transporter 4 (GLUT4) adiponectin receptors
  • GLUT4 glucose transporter 4
  • Insulin resistance is commonly characterized by decreased GLUT4-dependent glucose absorption in skeletal muscles and adipose tissue [38].
  • EGFR epidermal growth factor receptor
  • both C2C12 myotubes and SV40-MES13 kidney mesangial 22 cells showed significantly higher APN protein expression after 24, 48, and 72 h of transfection as compared with the PC and Empty-LNP groups (p ⁇ 0.001) (FIGS.1D-1E).
  • APN-mRNA-LNP Administration Leads to Reduction in Blood Glucose Levels and Attenuated Body Weight Gain in DIO Mice Mice were fed with a high-fat diet (HFD) and tested at 23 weeks of age using the insulin tolerance test (ITT) and the intraperitoneal glucose tolerance test (IPGTT). These tests aimed to assess glucose tolerance and insulin sensitivity before the administration of APN-mRNA-LNP.
  • APN-mRNA-LNP increased APN expression in situ Regarding gene expression
  • the evaluation of APN expression in the studied groups was performed after three days, one week, and two weeks of injection.
  • 23 APN expression significantly increased (p ⁇ 0.001) by 8.31-fold as compared with the PBS and Empty-LNP groups. This increase persisted after one week (6.93-fold) and further intensified after two weeks, reaching 24.18-fold changes in skeletal muscle tissue samples (FIG.2A).
  • liver tissue Similar trend was observed in liver tissue, where APN increased by 3.38-fold after three days, 6.36-fold after one week, and 24.75-fold changes after two weeks (p ⁇ 0.001) as compared with the PBS and Empty-LNP groups (FIG.2A). While APN expression was slightly higher in kidney and pancreas samples, there were 2.88- and 2.55-fold changes after three days, respectively. In addition, there were 2.59- and 3.11-fold changes after one week, and 3.27- and 3.13-fold changes after two weeks (FIG.2A), respectively, with a significance level of p ⁇ 0.01 when compared with the PBS and Empty-LNP groups. In adipose tissues, especially in white fat tissues (W.
  • APN showed remarkable overexpression after three days, reaching 71.21, 40.80 after one week and 26.11-fold changes after two weeks (p ⁇ 0.001) when compared with the PBS and Empty- LNP groups (FIG.2A).
  • APN increased gradually after three days to 4.68, 6.53 after one week, and reached 23.33 after two weeks (p value ⁇ 0.001) when compared with the PBS and Empty-LNP groups (FIG.2A).
  • the untreated groups PBS and Empty-LNP
  • the adiponectin protein was not expressed in muscle, liver, and kidney samples.
  • APN-mRNA-LNP skeletal muscles showed a significant increase in adiponectin expression (5, 70, and 40%), along with comparable intensity scores (1, 3, and 2).
  • adiponectin expression increased gradually (40, 50, and 70%) with intensity scores (2, 3, and 3), respectively.
  • adiponectin was expressed after two weeks of injection (60% with a score of 3 on the intensity scale).
  • the administration of APN-mRNA-LNP resulted in in situ adiponectin expression in the muscles, kidney, and liver of DIO mice (FIG.2B).
  • APN-mRNA-LNP treatment affects T2D pathways following increased APN expression
  • T2D Type 2 Diabetes
  • Glut-4 reactivation improvement in the Insulin Resistant Pathway (DGKd and PKC ⁇ )
  • IR activation activation of insulin secretion through the reactivation of the island of Langerhans EGFR Pathway Inhibition
  • Reduction of Inflammatory Cytokines TNF- ⁇ , IL-Ib, IL-6
  • Fatty Changes The expected gene expression in various studied tissues is summarized in FIG.3, utilizing information from the Mouse Genome Database (MGD) and the Gene Expression Database (GXD).
  • Glut-4 reactivation (glucose uptake improvement): In vitro, the results showed a significant increase in Glut4 gene expression after APN- mRNA-LNP transfection for 24, 48, and 72 h in C2C12 myotubes (p ⁇ 0.001 at all timepoints) and SV40-MES13 kidney mesangial cells (p ⁇ 0.001, 0.001, and 0.01, respectively), as compared with the PC and Empty-LNP groups. In vivo, Glut-4 gene expression showed significant improvement in skeletal muscles, liver, kidney, W. fat, and B.
  • Insulin receptor (IR) activation and the histological changes of pancreas In vitro The results showed a significant downregulation of IR gene expression in the untreated group (EmptyLNP) as compared with the NC group.
  • IR gene expression significantly increased in C2C12 myotubes in all timepoints (FIG. 5A), as well as in SV40MES13 kidney mesangial cells (FIG. 5B), when compared with the PC and Empty-LNP groups.
  • the injection of APN-mRNA-LNP results in IR activation in the diabetic mouse model.
  • pancreas specimens were collected and fixed in a 10% buffered formaldehyde solution for 24 h for subsequent histological examinations.
  • a comparison of the islet diameter before and after injection at various time points was conducted.
  • the results showed that pancreatic tissue sections from the PBS and Empty-LNP groups exhibited large-sized islets of Langerhans with average diameters of 283 and 258.3 ⁇ M, respectively.
  • the diameter slightly decreased in the APN-mRNA-LNP group after three days (136 ⁇ M) and one week (121.25 ⁇ M).
  • kidney specimens were collected, fixed in a 10% buffered formaldehyde solution for 24 h, and then cut into 3 ⁇ M-thick sections for PAS staining.
  • TNF- ⁇ , IL-6, and IL1 ⁇ the common proinflammatory cytokines directly related to T2D
  • results demonstrated that TNF- ⁇ , IL-6, and IL1b gene expression were significantly upregulated in the untreated group (PBS and Empty-LNP) compared to the NC group.
  • gene expression in SV40MES13 renal mesangial cells and C2C12 myotubes decreased significantly at all time points following APN-mRNA-LNP transfection.
  • Results showed that TNF- ⁇ gene expression was significantly downregulated in skeletal muscles, liver, W. fats, and B.
  • IL-1 ⁇ gene expression was significantly reduced in skeletal muscles, liver, kidney, W. fat, and B. fat tissues as compared with the PBS and Empty-LNP groups (FIGS.8A-8C).
  • the PC and Empty-LNP groups showed considerably higher levels (p ⁇ 0.001) than the NC group.
  • TNF- ⁇ and IL-6 protein expression in 28 the treated groups was significantly lower than that of the PC and Empty-LNP groups after 24, 48, and 72 h of transfection, similar to the normal negative control group in C2C12 myotubes (p ⁇ 0.001) at all timepoints, as well as in SV40-MES13 kidney mesangial cells (p ⁇ 0.001) at all timepoints (FIG. 8D-8E).
  • adipose tissue The invasion of adipose tissue is pathologically associated with obesity, insulin resistance, and diabetes through the generation of cytokines and chemokines by immune cells (B cells and T cells) and macrophages [55].
  • B cells and T cells immune cells
  • macrophages APN overexpression.
  • Skeletal muscles from the femur, liver, and kidney samples were used to evaluate this relationship.
  • liver tissue from the APN-mRNA-LNP groups after 3 days, 1 week, or 2 weeks showed a significant reduction in hepatocellular steatosis, affecting almost 10-20%, 10-20%, and 5-10% of cells in a descending order, respectively.
  • the proportion of hepatocellular hydropic degeneration appeared simultaneously with intervals of 80–90%, 60–80%, and 90–100%, respectively, following 3 days, 1 week, and 2 weeks of injection (FIG.8B, FIG.8D).
  • Light microscopy of kidney tissue sections from PBS and Empty-LNP mice showed almost identical features, with an average of 60–70% and 50–60%, respectively (black arrow).
  • T2D is characterized by a malfunction in the body's capacity to regulate and utilize glucose as an energy source. This chronic condition results in elevated blood sugar levels. Over time, high blood sugar can lead to complications affecting various systems and major organs including the liver, kidney, heart, and brain. There are two main issues associated with T2D: insufficient insulin production by the pancreas, and reduced sensitivity of cells to insulin, leading to poor sugar uptake.
  • APN adiponectin
  • APN-mRNA-LNP a protein closely involved in the insulin resistance pathways.
  • we successfully increased the direct production of insulin boosted the uptake of glucose into cells, and decreased inflammation associated with uncontrolled hyperglycemia.
  • APN-mRNA-LNP As a novel therapeutic agent to prevent and treat various diseases.
  • the present study evaluated the Glut-4 gene expression at both gene or protein level in the C2C12 myotubes and SV40-MES13 kidney mesangial cells, as well as in the skeletal muscles, liver, kidney, W. fat, and B. fat tissues. Significantly elevated levels of Glut-4 expression were observed in these tissues.
  • DAG As a precursor of triglycerides and phospholipids, DAG contributes to the metabolism of lipids, and functions as a second messenger in cellular signaling. A temporal increase in intracellular DAG mass is associated with glucose-induced insulin resistance in animals [39]. DAG is also phosphorylated to produce phosphatidic acid (PA), which is needed for it to act as a lipid second messenger and regulate signals related to metabolic and mitogenic responses [61].
  • PA phosphatidic acid
  • DGKd is a class of enzymes important for reducing DAG signaling and catalyzing the phosphorylation of DAG to PA [62].
  • Peripheral insulin resistance and moderate obesity are implicated in decreased DGKd protein expression.
  • Skeletal muscle DGKd is downregulated in those with poorly managed blood sugar levels [43].
  • a persistent upturn in intracellular DAG promotes aberrant signal transduction and intracellular lipid accumulation through the initiation of PKC isoforms and insulin resistance [40, 41].
  • DGKd showed significantly downregulated gene and/or protein expression in C2C12, SV40-MES13 cells, even in the skeletal muscles, liver, kidney, W. fat, and B.
  • the islets vary greatly in size; on average, they have a diameter of between 50 and 200 ⁇ M.
  • Insulin secretion is activated in DIO mice injected with APN-mRNA-LNP, based on the concept that island size and insulin production are correlated irreversibly [65].
  • IL-6 levels significantly decreased in the kidney, W. fat, and B. fat tissues after 1 and 2 weeks of APN-mRNA-LNP injection, while no significant changes were observed in skeletal muscles and liver IL-6 levels.
  • the expression of the IL-1 ⁇ gene significantly decreased in all examined organs, including skeletal muscles, the liver, kidney, W. fat, and B. fat tissues. Therefore, it can be said that APN mRNA-LNP treatment resulted in a reduction of proinflammatory cytokines (IL-1b, IL-6, and TNF- ⁇ ).
  • ATP- citrate lyase (ACL), fatty acid synthase (FAS), and stearoyl-CoA desaturase (SCD)-1 are genes that are activated by chronically elevated plasma glucose levels via two different mechanisms: directly, by increasing the citric acid (TCA) cycle activity and synthesis of Acyl CoA, which serves as a substrate for both gluconeogenesis and de novo lipogenesis (DNL) [71]; and indirectly, by activating the expression of carbohydrate response element binding protein (ChREBP) and liver X receptor ⁇ (LXR ⁇ ), which in turn promotes ACL, FAS, and SCD-1 gene transcription [72].
  • TCA citric acid
  • DNL de novo lipogenesis
  • ChREBP carbohydrate response element binding protein
  • LXR ⁇ liver X receptor ⁇
  • glucotoxicity increases lipotoxicity and activates ChREBP in the kidney, skeletal muscles, and pancreas, decreasing insulin production (pancreas) and aggravating IR in these tissues.
  • ChREBP expression is decreased in adipocytes by regulating the release of specific adipokines and lipid species, which may exacerbate the condition of IR [72].
  • APN-mRNA-LNP stimulates the formation of endogenous APN in muscles, liver, kidney, pancreas, and fat cells.
  • APN-mRNA-LNP after being administered, was proven to be safe for experimental animals because it quickly disappears from the body and does not integrate into the genome.
  • the myoblasts were seeded in Dulbecco’s modified Eagle medium (DMEM, No.2492921, Gibco) as growth medium (GM), which contained D-glucose (HG, 4.5 g/l) and L-Glutamine, supplemented with 10% fetal bovine serum (FBS) and 1% (100 U/ml) penicillin/ streptomycin (P/S) under humidified atmospheric conditions at 37 ⁇ C and 5% CO2. Subculturing was done by trypsinization, and cells were diluted to 2 x 10 5 cells/ml and seeded in plates.
  • DMEM Dulbecco’s modified Eagle medium
  • GM growth medium
  • FBS fetal bovine serum
  • P/S penicillin/ streptomycin
  • SV40 MES13 Mouse mesangial glomerulus SV40MES13 kidney cells were obtained from the American Type Culture Collection (ATCC CRL-1927) (Manassas, VA). SV40 MES13 were cultured in 3:1 mixture of ATCC-formulated Dulbecco’s Modified Eagle’s medium (DMEM, No.
  • F12 contained GlutaMAX-1 Ham (Nutrition Mixture, No. 2193045, GIBCO), supplemented with 5% FBS and 1% (100 U/ml) P/S under humidified atmospheric conditions at 37 ⁇ C and 5% CO 2 .
  • the complete medium was replaced every 48 h and subcultured at a ratio of 1:5 [48]. All the cell experiments were performed below passage number 10 (C2C12) or 7 (SV40 MES13) in a humidified environment at 37°C and 5% CO 2 .
  • High glucose (HG) stress The cells were grown to subconfluency until they reached the required cell number for the experimental setup.
  • T7 RNA polymerase in vitro transcription produced cmRNA encoding APN (IVT).
  • the full-length mRNA for APN (NM 009605) was produced (BioSynthesis, Inc., USA).
  • the mRNA APN was chemically altered by ribonucleotide substitution, in which uridine residues were replaced with pseudouridine and cytidine residues with 5-methylcylidine to increase RNA stability while reducing the anti-RNA immune response.
  • a poly(A) tail extending to 120 nucleotides
  • a 7- methylguanylate cap at the 3 and 5 ends, respectively
  • cmRNA APN The quantity and quality of cmRNA APN was assessed using the NanoDrop 2000C (Thermo Fisher Scientific, USA). The purity and size were confirmed by automated capillary electrophoresis using a Fragment Analyzer (Advanced Analytical, USA). High-performance liquid chromatography was used to analyze the nucleotides (BioSynthesis, Inc., USA). To generate APN mRNA contained in LNPs given by the Co-I Dr. Xu's lab, a reliable self-assembly approach was used in our experimentation [50, 51].
  • AA3-DLin LNPs with a molar ratio of 40:40:25:0.5 (AA3-DLin: DOPE: Cholesterol: DMG-PEG) were used to deliver APN mRNA for the following studies.
  • the preparation and characterization of AA3-DLin LNPs used in this study were well-established and evaluated in previous publications [51-53].
  • the mCherry encoded mRNA purchased from Trilink was encapsulated into AA3-DLin LNPs to transfect Hek 293 cells. The mCherry positive cells were then observed under a fluorescent microscope for intracellular uptake investigation.
  • the physicochemical properties, transfection evaluations, and intracellular uptake of the nanoparticles are shown in FIGS.
  • FIGS. 11E-11J show physiochemical properties, transfection evaluations, intracellular uptake, and cell survival of a different but highly similar nanoparticle.
  • APN-mRNA-LNP Cytotoxicity Cytotoxicity of APN-mRNA-LNP was assessed by cell viability assay in C2C12 differentiated myotubes. C2C12 cells were harvested after 6 days of differentiation and seeded in 96-well plates at a density of 7.0 ⁇ 10 4 . The myotube cells were treated with APN-mRNA-LNP at various concentrations (0-64) ⁇ g/ml for 24 h in blank medium, in parallel with blank wells, and in an EmptyLNP well.
  • Cell viability assays were determined using the manufacturer’s protocol of 36 Cell Counting Kit-8 (CCK8) (No. TS511, Dojindo, Japan). Briefly, 10 ml of WST-8 were added to each treated cell and incubated for 3 h at 37°C. Thereafter, absorbance was measured at 450 nm. The half maximal inhibitory concentration (IC50) for APN-mRNA-LNP was calculated depending on the readings of the CCK-8 test using the AAT Bioquest online tools.
  • IC50 half maximal inhibitory concentration
  • APN-mRNA-LNP administration In vitro: For APN-mRNA-LNP transfection, cells were cultured in high-glucose media “HG stress 10 g/L (55mM D-glucose)” for 72 h, then the media were replaced with blank media. One ⁇ g/ml APN-mRNA-LNP was added to three treated plates (24, 48, and 72 h), as well as to parallel plates containing PBS as a control, a negative control (seeded in normal media) and a positive control (PC) (seeded in HG media), and an Empty-LNP group (seeded in HG media). Cells were extracted after 24 h of transfection for gene and protein expression analysis.
  • mice All in vivo procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Tufts University.
  • DIO C57BL/6J
  • mice Strain #:380050, RRID: IMSRJAX: 380050
  • HFD high-fat diet
  • Mice were maintained at constant temperature (23 ⁇ 2 °C), humidity (45–55%), and a 12-h light/dark cycle with ad libitum access to water and food.
  • mice Blood glucose and body weight were assessed weekly to ensure that the rats had acclimated for at least 2 weeks prior to the start of the study.
  • the intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were carried out to assess insulin sensitivity and glucose tolerance, respectively, in order to prevent significant variations between groups. After 12 hours of fasting, the IPGTT was used to evaluate glucose tolerance (21:00–9:00).
  • a Clarity Plus Blood Glucose Meter was used to measure the blood sugar (Clarity Diagnosis; China).
  • APN-mRNA-LNP Injection Male C57BL/6J mice on a high-fat diet (HFD) received 0.3 mg/kg ( ⁇ 10 ug/mouse) of APN- mRNA-LNP intravenously once at 25 weeks of age. The control group received the same dose of phosphate buffered saline (PBS), while the second positive LNP group received Empty-LNP at the same time.
  • PBS phosphate buffered saline
  • the liver, skeletal muscles, kidney, pancreas, W. fat, and B. fat tissues were collected and directly placed in liquid nitrogen before being stored at -800C for mRNA analysis.
  • An anti-rabbit IgG (HRP-linked antibody) was used as a secondary antibody (#7074S, Cell Signaling). Three separate blot analysis of the samples were conducted. Optical densitometry was used to semiquantify the bands, and ImageJ digital imaging processing software was used for analysis (ImageJ 1.53t, National Institutes of Health, Bethesda, MD, USA). GAPDH was used as an endogenous control to normalize the expression of each protein under investigation. Histopathological studies The target tissues (liver, kidney, skeletal muscles, and pancreas) were obtained from the experimental animals after sacrifice and fixed in a 10% buffered formaldehyde solution for 24 h to perform histological examinations.
  • 11E-11J show physiochemical properties, transfection evaluations, intracellular uptake, and cell survival of a set of nanoparticles.
  • the developed AA3-DLin LNPs are fabricated using microfluidic-chip device by mixing the organic phase containing ionizable lipid, DOPE, cholesterol, and DMG-PEG and the water phase of mRNA.
  • the TEM image showed uniform spherical shape of LNPs around 100 nm size which is consistent with DLS results (FIG.11G).
  • FIG.11F shows the transfection efficacy of LNPs evaluated by delivering mCherry encoded mRNA on Hek 293 cells.
  • the fluorescence images (taken after 2 days post transfection) showed the LNPs have excellent transfection efficacy as well as remarkable cell viability compared to commercial lipofectamine 3000 (FIG. 11I).
  • the mRNA release profile was evaluated (FIG. 11J), the results showed the LNPs could release out 80% encapsulated mRNA after 2 days (FIG.11J). More importantly, the AA3-DLin LNPs successfully delivered the luciferase-encoded mRNA (mLuc-LNPs) in vivo.
  • the mLuc-LNPs were intramuscularly injected into BALB/c mice and generated strong luciferase expression at 2.35*10 8 total flux (p/s) at 6 h post-injection (FIG. 12A).
  • the comparison studies were performed with FDA-approved MC3 LNPs and ALC-0315 LNPs through intramuscular injection of luciferase mRNA under same preparation conditions. The total flux was recorded at 6 h post-injection and the results demonstrated that the AA3-DLin LNP formulations outperformed these commercial FDA-approved LNP formulations (FIG. 12B).
  • the full-length wild-type spike protein encoded mRNA was formulated into AA3- DLin LNPs to develop a new kind of AA3-DLin mRNA-LNP COVID-19 vaccines.
  • the AA3- DLin vaccines delivered spike mRNA efficiently in the Hek 293 cells with strong spike protein expression demonstrated by Western Blot and immunofluorescence analysis.
  • the AA3-DLin vaccine transfected lysates exhibited clear full-length spike protein bands at ⁇ 180 kDa with GAPDH as the loading control (FIG. 12C). Additionally, the expressed spike protein was also probed by fluorescence-labeled antibodies (FIG.12D).
  • a prime/boost manner of vaccination was applied to each group two weeks apart.
  • the spike-specific IgG antibody evaluation, pseudovirus neutralization, and real virus changeling studies were performed accordingly.
  • the results showed that the antibodies increased significantly after booster injections with a dose-dependent relationship exemplified by endpoint titers, IC50 titers, and PRNT50 titers (FIGS.12E-12G).
  • AA3-DLin LNPs successfully deliver the spike-encoded mRNA in vitro and in vivo, and strong immunogenicity was detected in the vaccinated mice groups (ACS Nano, 2022, 16, 11, 18936– 18950). Similar LNPs will be used with the mRNAs described herein for delivery.
  • APN mRNA-loaded lipid nanoparticle (LNP) preparation We employ a robust, self-assembly method to prepare nucleic acids (APN, EGFP and Luc mRNAs) encapsulated in LNPs as described above.
  • the AA3-DLin LNPs were fabricated using a microfluidic-chip device by mixing the organic phase containing ionizable lipid, DOPE, cholesterol, and DMG-PEG and the water phase of mRNA.
  • the LNP size and zeta potential were determined by using a ZetaPALS dynamic light-scattering detector.
  • the mRNA in the LNPs were analyzed by using the Quant-iT RiboGreen assay.
  • the LNPs were used fresh or kept at ⁇ 80°C to use later.
  • Example 3 Effects of modified messenger RNA of adiponectin delivered by lipid nanoparticles on adipogenesis and bone metabolism in vitro and in vivo Introduction 48
  • Obesity is a chronic disease characterized by excessive accumulation of body fat. It is a major risk factor for many diseases, among them type 2 diabetes, cardiovascular disease, and bone disorders [1–3].
  • the levels of pro-inflammatory cytokines are elevated, leading to chronic inflammation in the body [4–7].
  • the chronic inflammation inhibits osteogenesis and promotes osteoclastogenesis, which contributes to delayed bone healing during the bone remodeling process after bone injury [8].
  • mRNA messenger RNA
  • mRNA therapy is a new and promising approach to treating diseases [21–23]. It involves delivering mRNA into cells, where it can be translated into proteins. mRNA therapy has several advantages over traditional protein therapy, including higher efficiency, lower toxicity, and greater specificity.
  • LNPs preparation We employed a robust, self-assembly method to prepare APN mRNA encapsulated LNPs provided by the Co-I Dr. Xu’s lab (FIG.13A) [32, 33].
  • the LNP formulation, LNP preparation methods, LNP characterization and optimization studies, has been extensively characterized and optimized in our previous studies [32]. This formulation has demonstrated effective mRNA delivery in vitro and desirable properties such as for in vivo applications.
  • APN-LNP 3T3-L1 cell line from ATCC (CL-173) was cultured in Dulbecco’s modified eagle medium with 4.5 g/L D-glucose (DMEM, Gibco) supplemented with 10% newborn calf serum (Gibco) and 1% penicillin-streptomycin (Gibco). Passage 3-8 was used for experiment. Cells were seeded on 6 well plate at a density of 4 ⁇ 10 4 cells/ml and allowed to grow for 2 days to reach 100% cell confluent (D0).
  • D0 cell confluent
  • Adipogenic Induction medium containing 10 ⁇ g/ml insulin (Sigma-Aldrich), 1 ⁇ M dexamethasome (Sigma-Aldrich), and 0.5 mM isobutylmethylxanthine (IBMX) (Sigma-Aldrich) in complete DMEM medium for 72 hours (D1-D3).
  • ADM Adipogenic Differentiation Medium
  • D4-D5 10 ⁇ g/ml insulin for 48 hours
  • D6-D7 were maintained in DMEM medium for another 2 days (D6-D7). Adipocytes in different differentiation stages were transfected with APN-LNP.
  • MC3T3-E1 cell line was purchased from ATCC (CRL-2593) and maintained in Alpha MEM (AMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin.
  • Transfection was carried out using 0.25 ⁇ g/mL APN-LNP for 12 hours in Opti-MEM. After transfection, the medium was replaced with AMEM containing 100 ⁇ g/mL receptor activator of nuclear factor-kappa B ligand (RANKL) (Pepro Tech Inc) and incubated for 48 hours. Cells were collected at 24 and 72 hours post-transfection for further analysis. Cytotoxicity assays 3T3-L1 cells were induced in AIM and in ADM as described above. Transfection with APN-LNP in Opti medium was conducted on days 0, 3 and 7. The empty-LNP group was used as a control.
  • APN-LNP nuclear factor-kappa B ligand
  • Cytotoxicity was evaluated using the Cell Counting Kit-8 (CCK-8, Dojindo), with absorbance measured at 450 nm using a microplate reader. The cytotoxic effects of APN-LNP on MC3T3-E1 cells were assessed following the same protocol used for 3T3-L1 cells.
  • livers of all mice were collected and processed to histological analysis for H&E staining.
  • ⁇ CT Analysis The collected femurs were scanned using Bruker’s Skyscan 1172 (Bruker) at 9.0- ⁇ m- voxel resolution at Tufts Medical Center. A 0.5 mm aluminum (Al) X-ray filter was used, and the X-ray settings were 50 kV and 500 ⁇ A. Scans were performed in 0.3° rotation steps.
  • RNA isolation from organs and qPCR analysis Total RNA from organs excluding bone was extracted using TRIzol TM Reagent (Invitrogen) under the manufacturer’s instructions.
  • APN-LNP cytotoxicity of APN-LNP on 3T3-L1 at different concentrations was determined using CCK-8 assay. No obvious cytotoxicity was found for APN- LNP up to 4 ⁇ g/ml in preadipocytes and mature adipocytes (FIGS.13B-13C).
  • mRNA and protein expression of APN after transfection with APN-LNP We then evaluated the expression of APN after transfection by qPCR. The results showed significantly increased APN levels in either preadipocytes or mature adipocytes (FIGS. 13D-13E).
  • APN-LNP could promote osteogenesis and inhibit osteoclastogenesis in murine cell lines [19,20].
  • MC3T3-E1 Cell survival We determined the cytotoxicity of APN-LNP on MC3T3-E1 cells at different concentrations using CCK-8 assay. No obvious cytotoxicity was found for APN-LNP up to 2 ⁇ g/ml when transfection was performed (FIG.14). Compared with empty-LNP group, transfection with 4 ⁇ g/ml APN-LNP significantly decreased MC3T3-E1 cell numbers.
  • APN-LNP Transfection with APN-LNP promotes expression of APN and osteogenic markers in MC3T3- E1 cells
  • APN mRNA expression was significantly upregulated at 24, 72, and 120 hours post- transfection (FIG.15A).
  • the highest APN expression levels were found at 24 hours.
  • APN expression in the 1.0 ⁇ g/ml group increased more than 28,000- fold compared to the empty-LNP control, while in the 0.25 ⁇ g/ml group, the fold change exceeded 800-fold.
  • Osteogenic markers Bsp and Runx2 expression in contralateral femurs of the experimental mice After detecting the contralateral femur tissues, osteogenic markers Bsp and Runx2 mRNA expression were significantly higher in the APN-LNP group compared to the empty-LNP and control group (FIG.18B).
  • Inflammation related markers TNF ⁇ and IL-10 in white adipose tissues (WAT) Additionally, we assessed the influence of APN-LNP on the expression of inflammatory cytokines in WAT. We found that APN-LNP administration increased the mRNA levels of anti- inflammatory marker IL-10 (Figure.5C). Nevertheless, no differences were found regarding gene expression of pro-inflammatory marker TNF ⁇ (FIG.18C).
  • APN-LNP APN messenger RNA and packaged the mRNA with LNPs to form the APN-LNP.
  • APN is primarily secreted by adipose tissue.
  • APN-LNP could transfect 3T3-L1 adipocytes and upregulate APN gene and protein expression in this cell line.
  • increased APN protein levels were detected in the cellular extracts and cell culture supernatant in transfected mature adipocytes, which indicated that modified APN-LNP mRNA delivered by LNPs can be translated into protein transcripts in vitro as expected.
  • APN-LNP inhibited expression of adipogenesis-related markers such as Ppar ⁇ , Lpl and Hsl in two adipocyte cell stages at gene levels.
  • Ppar ⁇ is an important regulator in promoting adipogenesis and lipid accumulation [37–39].
  • APN-LNP transfection increased gene and protein expression of APN, further leading to decreased adipogenic genes expression, which may exert an anti-adipogenic effect in 3T3-L1 cells.
  • overexpression of APN suppressed adipogenesis in cultured bone marrow mesenchymal stem cells [40].
  • APN is shown to be expressed in adipocytes, human and murine osteoblasts, and myocytes [41,42].
  • MC3T3-E1 osteoblastic cell line and RAW264.7 cell line The latter could be differentiated into osteoclasts with RANKL stimulation and is usually used for osteoclastic research. They are two commonly used cell lines in studying bone metabolism in vitro.
  • Our results showed that transfection with APN-LNP stimulated MC3T3-E1 cells and upregulated expression of APN for a few days. As the concentration of APN-LNP increased, this effect even persisted 57 longer.
  • APN-LNP transfection in MC3T3-E1 osteoblastic cells upregulated the expression of Bsp and Ocn dose dependently, which suggests that APN-LNP may promote osteogenesis.
  • Other researchers have reported that APN treatment can increase cell proliferation and osteogenic markers expression in murine and human osteoblasts, which was consistent with our results [18–20].
  • various studies reported a negative role of APN on osteoclastogenesis in RAW264.7 cells [18–20].
  • Our results showed that transfection with APN-LNP significantly inhibited Mmp9 expression level in RANKL-stimulated RAW264.7 cells.
  • APN-LNP may inhibit osteoclastogenesis by inhibiting Mmp9 expression. Although many studies have revealed that APN can inhibit osteoclastogenesis, this is the first report of its inhibitory effect on Mmp9 production in RAW264.7 cells. And further studies will be performed to investigate the underlying mechanisms. In this study, we aimed to explore if APN-LNP could decrease fasting blood glucose and improve femoral fracture healing in male DIO mice. At timepoint T1, we found that the fasting blood glucose and body weight decreased not only in APN-LNP group, but also in empty-LNP and control group.
  • APN-LNP decreased fasting blood glucose and body weight significantly, which indicated a beneficial impact of APN-LNP on regulating metabolism.
  • APN-LNP could partially improve bone healing.
  • the micro-environment of body is very complex. Notably, bone healing process is complex and influenced by many factors, including the type of fracture, the patient's age and health status, as well as any underlying medical conditions.
  • APN-LNP may be effective in promoting bone healing in certain subsets of fracture healing, but not in others.
  • Our findings are consistent with our previous research, which demonstrated the efficacy of APN-LNP in improving insulin sensitivity and kidney function in diabetic mice. Specifically, both studies revealed the ability of APN-LNP to reduce inflammation, suggesting a common underlying mechanism of action. While the previous study focused on metabolic improvements, 58 the current investigation highlights the treatment of bone disorders. The consistent beneficial effects observed across these distinct disease models underscore the broad therapeutic potential of APN-LNP and warrant further exploration of its mechanisms of action. In conclusion, our results suggest that APN-LNP could inhibit adipogenesis and promote osteogenesis.
  • APN-LNP improved bone healing by promoting osteogenesis and reducing inflammation in a femoral fracture model of male DIO mice.
  • this study provides promising preclinical data on the potential of APN-LNP as a therapeutic agent for bone disorders in obesity (FIG.19).
  • References for Example 3 1. Nagareddy, P.R.; Kraakman, M.; Masters, S.L.; Stirzaker, R.A.; Gorman, D.J.; Grant, R.W.; Dragoljevic, D.; Hong, E.S.; Abdel-Latif, A.; Smyth, S.S.; et al.
  • Adipose Tissue Macrophages Promote Myelopoiesis and Monocytosis in Obesity. Cell Metabo lism 2014, 19, 821–835, doi:10.1016/j.cmet.2014.03.029.
  • Adiponectin A Promising Target for the Treatment of Diabetes and Its Complications. Life 2023, 13, 2213, doi:10.3390/life13112213. 13. Tilija Pun, N.; Park, P.-H. Adiponectin Inhibits Inflammatory Cytokines Production by Beclin-1 Phosphorylation and B-Cell Lymphoma 2 mRNA Destabilization: Role for Autophagy Induction.
  • MiRNA-218 Regulates Osteoclast Differentiation and Inflammation Response in Periodontitis Rats through Mmp9. Cell Microbiol 2019, 21, e12979, doi:10.1111/cmi.12979. 44. Gu, J.-H.; Tong, X.-S.; Chen, G.-H.; Liu, X.-Z.; Bian, J.-C.; Yuan, Y.; Liu, Z.-P. Regulation of Matrix Metalloproteinase-9 Protein Expression by 1 ⁇ ,25-(OH) 2 D 3 during Osteoclast Differentiation. J Vet Sci 2014, 15, 133, doi:10.4142/jvs.2014.15.1.133. 45.

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Abstract

La présente invention concerne des compositions comportant un ARNm d'adiponectine (APN) et une nanoparticule. La nanoparticule peut être une nanoparticule lipidique. Les compositions peuvent être incorporées dans des compositions pharmaceutiques. L'invention concerne des procédés d'utilisation des compositions de l'invention comprenant des méthodes de traitement du diabète, du diabète de type II, de l'obésité, de la résistance à l'insuline, de l'hyperinsulinémie, de l'hyperglycémie, de la dysrégulation de l'adipokine, de la parodontite, du syndrome métabolique, de l'ostéoporose, de la stéatose hépatique non alcoolique, de la perte osseuse ou d'une maladie neurodégénérative.
PCT/US2024/061376 2023-12-20 2024-12-20 Compositions d'arnm d'adiponectine et leurs procédés d'utilisation Pending WO2025137510A1 (fr)

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US20120129917A1 (en) * 2009-08-11 2012-05-24 Opko Curna, Llc Treatment of adiponectin (adipoq) related diseases by inhibition of natural antisense transcript to an adiponectin (adipoq)
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US20100016216A1 (en) * 2002-01-18 2010-01-21 Garth Cooper Adiponectin and uses thereof
US20070190539A1 (en) * 2005-08-05 2007-08-16 Bristol-Myers Squibb Company Human MLR single nucleotide polymorphisms associated with dose-dependent congestive heart failure and methods of use thereof
US20120129917A1 (en) * 2009-08-11 2012-05-24 Opko Curna, Llc Treatment of adiponectin (adipoq) related diseases by inhibition of natural antisense transcript to an adiponectin (adipoq)
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EL-ARABY RADY E., TU QISHENG, XIE YING, ABOUSHOUSHA TAREK, LI ZHONGYU, XU XIAOYANG, ZHU ZOE X., DONG LILY Q., CHEN JAKE: "Adiponectin mRNA Conjugated with Lipid Nanoparticles Specifically Targets the Pathogenesis of Type 2 Diabetes", AGING AND DISEASE, vol. 16, no. 2, 1 April 2025 (2025-04-01), US , pages 1059 - 1079, XP093332362, ISSN: 2152-5250, DOI: 10.14336/AD.2024.0162 *

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