WO2018071528A1 - Protéines et méthode de traitement de l'obésité et de comorbidités associées - Google Patents

Protéines et méthode de traitement de l'obésité et de comorbidités associées Download PDF

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Publication number
WO2018071528A1
WO2018071528A1 PCT/US2017/056120 US2017056120W WO2018071528A1 WO 2018071528 A1 WO2018071528 A1 WO 2018071528A1 US 2017056120 W US2017056120 W US 2017056120W WO 2018071528 A1 WO2018071528 A1 WO 2018071528A1
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Prior art keywords
protein
domain
polynucleotide
agonist
inflammatory
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PCT/US2017/056120
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English (en)
Inventor
Dexi Liu
Mingming GAO
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University Of Georgia Research Foundation, Inc.
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Publication of WO2018071528A1 publication Critical patent/WO2018071528A1/fr

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    • 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
    • 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
    • C07K14/605—Glucagons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8125—Alpha-1-antitrypsin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Obesity (body mass index > 30) has become a major public health problem in recent years. The prevalence of obesity in the US is -35.5% and—35.8% among adult men and women, respectively (Flegal et al., 2012, JAMA 307: 491-497). Obesity is closely linked to a number of severe metabolic comorbidities such as diabetes and nonalcoholic fatty liver diseases (NAFLD and NASH) (Flegal et al., 2012, JAMA 307: 491-497; Pedersen, 2013, Best Pract Res Clin Endocrinol Metab 27: 179-193).
  • NAFLD and NASH nonalcoholic fatty liver diseases
  • GLP-1 glucagon-like peptide-1
  • the therapeutic protein contains the sequence of a GLP- 1 receptor agonist and the sequence of an anti-inflammatory protein.
  • the therapeutic protein includes Exendin-4 (Ex4), a potent agonist of the GLP-1 receptor, placed at the amino terminal end of the anti-inflammatory protein human alpha-1 antitrypsin (hAAT).
  • Ex4 Exendin-4
  • hAAT human alpha-1 antitrypsin
  • a recombinant protein including an agonist domain and an antiinflammatory domain.
  • the agonist domain has a GLP-1 receptor agonist activity
  • the anti -inflammatory domain has anti -inflammatory activity
  • the agonist domain is located amino terminal to the anti-inflammatory domain.
  • a recombinant polynucleotide including a coding region encoding a recombinant protein.
  • the recombinant protein includes an agonist domain and an antiinflammatory domain, where the agonist domain has GLP-1 receptor agonist activity, the anti- inflammatory domain has anti -inflammatory activity, and the agonist domain is located amino terminal to the anti-inflammatory domain.
  • the polynucleotide further includes a vector, such as a viral vector.
  • the agonist domain and the anti-inflammatory domain are joined by a linker.
  • the linker can include at least 1 amino acid and in one embodiment includes no greater than 10 amino acids. In one embodiment, the linker includes an organic group.
  • the agonist domain includes, or has structural similarity with, a glucagon-like peptide 1, an exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, taspoglutide, or semaglutide.
  • the agonist domain includes, or has structural similarity with, the amino acid sequence of SEQ ID NO:3, 4, or 5.
  • the anti- inflammatory domain includes, or has structural similarity with, alpha-1 antitrypsin, alpha-1 antichymotrypsin, or alpha-1 antiproteinase.
  • the anti-inflammatory domain includes, or has structural similarity with, the amino acid sequence of SEQ ID NO: 6 or a truncation thereof.
  • the protein includes, or has structural similarity with, the amino acid sequence of SEQ ID NO:2.
  • a composition including a recombinant protein described herein or a recombinant polynucleotide described herein.
  • the composition includes a pharmaceutically acceptable carrier.
  • the host cell that includes a recombinant polynucleotide described herein.
  • the host cell can be a mammalian cell, such as a human cell.
  • the host cell can be ex vivo or in vivo.
  • the method is for delivering a polynucleotide into a host cell, and includes contacting a cell with a recombinant polynucleotide under conditions suitable for introduction of the polynucleotide into the cell.
  • the polynucleotide includes a coding region encoding a recombinant protein including an agonist domain and an anti-inflammatory domain.
  • the agonist domain has GLP-1 receptor agonist activity
  • the antiinflammatory domain has anti-inflammatory activity
  • the agonist domain is located amino terminal to the anti-inflammatory domain.
  • the cell is ex vivo.
  • the cell is in vivo and the contacting the cell includes administering the
  • the cell can be a mammalian cell, such as a human cell.
  • the method is for treating a condition, and includes administering to a subject a composition including an effective amount of a recombinant protein described herein or a recombinant polynucleotide described herein.
  • the subject has a condition or is at risk of having a condition that can be treated by the composition.
  • the concentration of the protein in the blood of the subject is at least 1 picograms/milliliter to no greater than 10 mg/ml.
  • protein refers broadly to a polymer of two or more amino acids joined together by peptide bonds.
  • protein also includes molecules which contain more than one protein joined by disulfide bonds, ionic bonds, or hydrophobic
  • peptide, oligopeptide, and polypeptide are all included within the definition of protein and these terms are used interchangeably. It should be understood that these terms do not connote a specific length of a polymer of amino acids, nor are they intended to imply or distinguish whether the protein is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring.
  • polynucleotide refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and single- stranded DNA and RNA.
  • a polynucleotide may include nucleotide sequences having different functions, including for instance coding sequences, and non-coding sequences such as regulatory sequences. Coding sequence, non-coding sequence, and regulatory sequence are defined below.
  • a polynucleotide can be obtained directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques.
  • a polynucleotide can be linear or circular in topology.
  • a polynucleotide can be, for example, a portion of a vector, such as an expression or cloning vector, or a fragment.
  • DNA sequences described herein are listed as DNA sequences, it is understood that the complements, reverse sequences, and reverse complements of the DNA sequences can be easily determined by the skilled person. It is also understood that the sequences disclosed herein as DNA sequences can be converted from a DNA sequence to an RNA sequence by replacing each thymidine nucleotide with a uridine nucleotide.
  • an "isolated" polypeptide or polynucleotide refers to a molecule that has been removed from a cell.
  • an isolated polypeptide is a polypeptide that has been removed from the cytoplasm or from the membrane of a cell, and many of the polypeptides, nucleic acids, and other cellular material of its natural environment are no longer present.
  • an isolated polynucleotide is a polynucleotide that has been removed from the cytoplasm of a cell, and many of the polypeptides, nucleic acids, and other cellular material of its natural environment are no longer present.
  • a "purified" polypeptide or polynucleotide is one that is at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components of a cell.
  • Polypeptides and polynucleotides that are produced outside of a cell, e.g., through chemical or recombinant means, are considered to be isolated and purified by definition, since they were never present in a cell.
  • suitable conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and/or are
  • certain embodiments can include a combination of compatible features described herein in connection with one or more
  • FIG. 1 A-B shows a schematic presentation of plasmid constructs and their influence on high-fat diet-induced weight gain.
  • FIG. 2 shows impacts of transfer of different plasmid constructs on HFD-induced obese mice. Injections (indicated by the arrow) were performed on week 10 on the same groups of animals in FIG. IB. Plasmid DNA (20 ⁇ g per mouse) was injected through an adjusted hydrodynamic tail vein injection with a volume equal to -8% of the lean mass over 5-8 s.
  • FIG. 3A-D shows EAT gene transfer represses food intake and blocks HFD-induced adiposity.
  • FIG. 3A Blood levels of EAT protein 9 weeks after pEAT injection.
  • FIG. 3B EAT gene transfer reduced food intake of HFD.
  • FIG. 3C ⁇ Jgene transfer repressed HFD-induced weight gain.
  • FIG. 3D EAT gene transfer reduced fat mass while showing no significant impact on lean mass.
  • FIG. 4A-C shows EAT gene transfer blocks HFD-induced adipose hypertrophy and macrophage activation.
  • FIG. 4A EAT gene transfer reduced the weights of white fat depots.
  • FIG. 4B Representative images of H&E staining of EWAT and BAT.
  • FIG. 4C shows EAT gene transfer blocks HFD-induced adipose hypertrophy and macrophage activation.
  • FIG. 5 A-B shows EA T gene transfer improves glucose homeostasis.
  • FIG. 5 A Profiles of blood glucose concentration as function of time upon intraperitoneal injection of glucose.
  • FIG. 6A-D shows EAT gene transfer blocks HFD-induced fatty liver.
  • FIG. 6 A Liver weight at the end of the 9-week HFD feeding.
  • FIG. 6B Blood levels of AST and ALT.
  • FIG. 6C Representative images of H&E staining and Oil red O staining of the liver.
  • FIG. 7 shows absence of adverse effects of EA T gene transfer on major internal organs.
  • Animal were injected with 3 doses of pEAT plasmid DNA or empty plasmid. The injected animals were kept on HFD for 9 weeks.
  • Major internal organs including the heart, spleen, kidneys, lungs, and pancreas were collected and fixed using neutral buffer formalin. Tissue samples were embedded in paraffin and sectioned at 6 ⁇ in thickness. Histological examination was carried out via H&E staining of these tissue sections.
  • FIG. 8A-D shows schematic presentation of plasmid constructs and the effects of plasmid transfer on obese mice.
  • FIG. 8A Schematic presentation of plasmid constructs.
  • FIG. 8B Predicted structure of EAT protein based on PHYRE2 computer software.
  • FIG. 8C Western blotting of mouse plasma 24 h after hydrodynamic plasmid transfer.
  • FIG. 9A-K shows EAT gene transfer reduces body weight and improves fatty liver in diet-induced C57BL/6 obese mice.
  • Obese mice were kept on HFD and hydrodynamically injected with 20 ⁇ g of either pEAT or pLIVE empty plasmid (control). Animal body weight was monitored continuously for 21 days, at which time animals were sacrificed for tissue collection and histological and biochemical analysis.
  • FIG. 9A Effect of gene transfer on body weight.
  • FIG. 9B Representative images of mice at the end of experiment.
  • FIG. 9C Comparative body composition of animals with pEAT or control plasmid.
  • FIG. 9D Average food intake.
  • FIG. 9E Representative images of H&E staining of WAT and BAT.
  • FIG. 9F mRNA levels of key genes responsible for chronic inflammation in WAT.
  • FIG. 9G Circulating levels of TNFa and IL6 protein.
  • FIG. 9H Expression levels of genes controlling adaptive thermogenesis in brown fat.
  • FIG. 9J Relative level of triglyceride in the livers.
  • FIG. 9K Transcription levels of pivotal genes responsible for lipogenesis, lipid droplet formation, and inflammation in the liver.
  • FIG. 10A-E shows EAT gene transfer improves glucose tolerance and alleviates insulin resistance in diet-induced C57BL/6 obese mice.
  • Obese C57BL/6 mice were hydrodynamically transferred with pEAT or control plasmids. Animals were fasted for 6 h on day 15 and intraperitoneal injection of glucose solution was performed. Animals were fasted for 4 h before intraperitoneal injection of insulin solution on day 18. Blood samples were collected after insulin or glucose injection from mouse tails at different times and blood glucose concentrations were determined.
  • FIG. 10A Profiles of blood glucose concentration as function of time upon intraperitoneal injection of glucose.
  • FIG. 10B Serum concentration of insulin at the end of 21- day experiment.
  • IOC insulin-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide, mRNA levels of Glut4 in WAT and BAT.
  • FIG. 11 shows representative images of H&E staining of major organs from obese mice treated with or without EAT gene transfer. Samples were collected from C57BL/6 obese mice 3 weeks post plasmid DNA transfer and tissue sections were created and stained with H&E.
  • FIG. 12A-J shows effects of EAT gene transfer on transgenic ob/ob mice.
  • FIG. 12B Representative images of mice at end of the experiment.
  • FIG. 12C Body composition.
  • FIG. 12D Food intake.
  • FIG. 12E Representative images of H&E staining of WAT. Arrows point to crown-like structures.
  • FIG. 12F Adipocyte size.
  • FIG. 12G Expression of key genes responsible for chronic inflammation in WAT.
  • FIG. 12H Profiles of blood glucose concentration as function of time upon intraperitoneal injection of glucose.
  • FIG. 121 Profiles of glucose concentration (percentage of initial value) as a function of time upon intraperitoneal injection of insulin.
  • FIG. 13A-E shows ⁇ Jgene transfer blocks fatty liver development in ob/ob mice.
  • FIG. 13B Liver weight.
  • FIG. 13 C Liver triglyceride.
  • FIG. 13D Blood concentrations of AST and ALT.
  • FIG. 14A-E shows long-term effects of repeated injection of EAT gene constructs.
  • FIG. 14A Blood levels of EAT protein determined by ELISA.
  • FIG. 14B Weight gain of CD-I mice kept on standard chow.
  • FIG. 14C Body composition at 180 days after EAT gene transfer.
  • FIG. 14D Weights of internal organs.
  • FIG. 15A-D shows preparation and characterization of recombinant EAT protein.
  • FIG. 15A Representative images of HEK293T cells after PEI-based transfection with GFP reporter construct at different PEI to DNA ratios ( ⁇ : ⁇ ).
  • FIG. 15B SDS-PAGE characterization of samples collected from transfected 293 cells. Lane 1, total protein loaded on a nickel column; lane 2, flow through from the nickel column; lane 3, flow though of washing buffer; lane 4, elute by imidazole from the nickel column.
  • FIG. 15C SDS-PAGE determination of the purified EAT protein. Lane 1-3 were loaded 2, 4, and 8 ⁇ g of purified recombinant EAT protein, respectively.
  • FIG. 16A-D shows validation of elastase inhibition activity and exendin 4 activity of recombinant EAT.
  • FIG. 16A Inhibition of elastase enzyme activity by human AAT and recombinant EAT protein. Purified proteins were diluted at different concentrations, added to the reaction mixture, and incubated for 30 min. The excitation and emission wavelength used was 400 and 505 nm, respectively.
  • FIG. 16B Comparison of elastase inhibition activity of different components in EAT. Proteins and exendin 4 peptides were diluted using assay buffer to a final concentration of 20 nmol/ml. A fluorescence-based enzymatic assay was performed following the protocol provided with the kit.
  • FIG. 16C Effect of components in EAT on glucose clearance in glucose tolerance test.
  • HFD-induced obese mice 50 g were pretreated with a single intraperitoneal injection of saline, exendin 4, hAAT or EAT at 20 nmol/kg.
  • a standard IPGTT was carried out 30 min after the injection.
  • Blood glucose levels were measured at 0, 30, 60 and 120 min after glucose injection.
  • FIG. 17A-I shows EAT protein therapy reduces adiposity and improves glucose homeostasis in C57BL/6 obese mice.
  • FIG. 17A Schematic illustration of the treatment schedule. Daily injection (i.p.) of saline (control) or EAT protein (5 mg/Kg) was performed.
  • FIG. 17B Body weight change.
  • FIG. 17C Food intake.
  • FIG. 17D Body composition.
  • FIG. 17E Representative images of H&E staining of WAT and BAT sections.
  • FIG. 17F mRNA levels of vital inflammatory genes in WAT.
  • FIG. 17G Profiles of blood glucose concentration as function of time upon intraperitoneal injection of glucose.
  • FIG. 17H Profiles of glucose concentration (percentage of initial value) as a function of time upon intraperitoneal injection of insulin.
  • FIG. 171 Blood levels of insulin.
  • FIG. 18A-C shows EAT protein treatment attenuates obesity-associated fatty liver in
  • FIG. 18 A Representative images of H&E staining and Oil red O staining of liver sections.
  • FIG. 18B Liver triglyceride content.
  • recombinant proteins having an agonist domain and anti- inflammatory domain.
  • the two domains are joined by an optional protein linker.
  • a recombinant protein can be isolated or purified.
  • a recombinant protein includes more than one agonist domain, more than one anti-inflammatory domain, or a combination thereof.
  • the agonist domain has glucagon-like peptide-1 (GLP-1) agonist activity.
  • GLP-1 agonist activity glucagon-like peptide-1
  • a recombinant protein described herein that has both agonist and anti-inflammatory domains has GLP-1 agonist activity.
  • An agonist domain having GLP-1 agonist activity confers an appetite- controlling response in a well-validated mouse model for obesity under suitable conditions.
  • a protein having GLP-1 agonist activity causes a mouse exposed to a high-fat diet to have a weight reduction of at least at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the same type of mouse that did not receive the protein having GLP-1 agonist activity.
  • a protein having GLP-1 agonist activity causes a mouse exposed to a high-fat diet to have food consumption that is reduced by at least at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the same type of mouse that did not receive the protein having GLP-1 agonist activity.
  • proteins that can be used as an agonist domain include, but are not limited to GLP-1 agonists.
  • GLP-1 agonists also referred to in the art as GLP-1 receptor agonists or incretin mimetics, include but are not limited to glucagon-like peptide 1, exenatide (such as exendin-4), liraglutide, lixisenatide, albiglutide, dulaglutide, taspoglutide, and semaglutide. Many of these are approved for use in humans.
  • the amino acid sequence of each of these proteins is known and readily available to the skilled person. Specific examples of amino acid sequences include HGEGTF T SDL SK QMEEE A VRLF IE WLKNGGP S S GAPPP S (SEQ ID NO:3, also available at Genbank accession number AAB51130.1),
  • HGEGTF T SDL SKQMEEE A VRLFIE WLKNGGP S S GAPP SKKKKKK (SEQ ID NO:5).
  • the agonist domain depicted at SEQ ID NO:4 further includes an arginine at the C- terminal end, or a conservative substitution thereof.
  • GLP-1 agonists useful as an agonist domain include those having structural similarity with the amino acid sequence of a GLP-1 agonist.
  • An agonist domain having structural similarity with the amino acid sequence of a GLP-1 agonist has GLP-1 agonist activity.
  • the structure function relationship of proteins having GLP-1 agonist activity is known, and proteins having GLP-1 agonist activity have conserved amino acids and conserved domains.
  • GLP-1 agonists include the following conserved sequence at the amino- terminal end: HXEGTFTSDXSXXXEXXXXXXFIXWLXXGX (SEQ ID NO:8) (see, for instance, Moon, 2012, Frontiers in Endocrinol., doi: 10.3389/fendo.2012.00141).
  • GLP-1 agonist e.g., non-conserved regions, individual amino acids, or a combination thereof
  • varying certain regions of a GLP- 1 agonist e.g., conserved regions, individual amino acids, or a combination thereof
  • the anti-inflammatory domain has anti-inflammatory activity.
  • a recombinant protein described herein that has both agonist and anti-inflammatory domains has antiinflammatory activity.
  • An anti-inflammatory domain having anti-inflammatory activity alleviates adipose chronic inflammation, reduces adipose TNF-a expression, or a combination thereof, under suitable conditions.
  • Whether a protein has anti-inflammatory activity can be determined by in vitro and in vivo assays. In one assay, reduction of TNF-a release from bone marrow-derived mouse neutrophils in vitro can be evaluated (Proc Natl Acad Sci U S A. 2013 Sep.
  • anti-inflammatory activity can be determined by measuring the ability of an anti-inflammatory domain to inhibit neutrophil elastase. Methods for measuring the ability of a protein to inhibit neutrophil elastase are commercially available (e.g., BioVision, San
  • the protein being tested for anti-inflammatory activity can be a separate protein or part of a recombinant protein described herein (e.g., an anti-inflammatory domain).
  • a protein having anti -inflammatory activity reduces neutrophil elastase activity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) compared to the activity of neutrophil elastase in the absence of the protein having antiinflammatory activity.
  • proteins that can be used as an anti-inflammatory domain include, but are not limited to, proteins that have anti-inflammatory activity.
  • proteins having antiinflammatory activity include, but are not limited to, members of the serpin superfamily (Law et al., 2006, Genome Biology, 7:216).
  • members of the serpin superfamily include, but are not limited to, serpinAl (alpha- 1 antitrypsin, also referred to as AAT), serpinA3 (alpha 1-antichymotrypsin, also referred to as AACT), and serpinA12 (alpha 1-antiproteinase).
  • amino acid sequence of proteins that can be used as an anti-inflammatory domain are known and readily available to the skilled person.
  • an anti-inflammatory protein is alpha-1 antitrypsin AAT:
  • an anti-inflammatory domain includes the amino acid sequence SEQ ID NO: 6 with a deletion of the first 46 amino terminal amino acids or a subset thereof, e.g., a deletion of amino acids 1-46, 1-45, 1-44, 1-43, 1-42, 1-41, 1-40, 1-39, 1- 38, 1-37, 1-36, 1-35, 1-34, 1-33, 1-32, 1-31, 1-30, 1-29, 1-28, 1-27, 1-26, -25, 1-24, 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or the first amino acid (Pirooznia et al.
  • anti-inflammatory proteins useful as an anti-inflammatory domain include those having structural similarity with the amino acid sequence of an anti-inflammatory protein.
  • An anti-inflammatory domain having structural similarity with the amino acid sequence of an anti-inflammatory protein has anti-inflammatory activity.
  • the structure function relationship of proteins having anti-inflammatory activity is known, and proteins having antiinflammatory activity have conserved amino acids and conserved domains
  • a recombinant protein described herein includes a linker.
  • a linker is a compound that is incorporated within an amino acid sequence by covalent bonds and joins the protein domains in a recombinant protein.
  • a linker can be flexible or rigid, and in one embodiment is flexible.
  • a linker can be an amino acid sequence.
  • a linker can be at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 amino acids in length. It is expected that there is no upper limit on the number of amino acids in a linker used in a recombinant protein described herein; however, in one embodiment, a linker can be no greater than 10, no greater than 9, no greater than 8, or no greater than 7 amino acids in length.
  • Many linkers are known to a skilled person (see Chen et al. 2013, Adv, Drug Deliv. Rev., 65(10): 1357-1369).
  • An example of an amino acid linker includes GGGGS (SEQ ID NO:7).
  • a linker can include an organic group.
  • a linker that includes an organic group has the formula FfcN-R ⁇ CChH, wherein R 1 includes a substituted or unsubstituted, branched or straight chain Ci to C20 alkyl group, alkenyl group, or alkynyl group; a substituted or unsubstituted C3 to Cs cycloalkyl group; a substituted or unsubstituted C 6 to C20 aryl group; or substituted or unsubstituted C 4 to C20 heteroaryl group.
  • R 1 can be represented by the formula (CH2)n, where n is from 1 to 10.
  • the organic group can form bonds with an amino acid, e.g., it includes at least one amino group and at least one carboxyl group, where the carboxyl group can be a carboxylic acid or the ester or salt thereof.
  • a recombinant protein described herein includes an agonist domain having sequence similarity to an exendin-4 protein, a linker, and an anti-inflammatory domain having sequence similarity to an AAT protein.
  • An example of such a recombinant protein is SEQ ID NCv l :
  • HGEGTFT SDLSKQMEEEAVRLFIEWLKNGGP S SGAPPP SEDPQGD AAQKTDT SHHDQDH PTF KITP L AEF AF SLYRQLAHQ SNSTNIFF SP VSIATAF AMLSLGTKADTHDEILEGLNF LTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEA FTVOTGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVK DTEEEDFHVOQ VTT VK WMMKRLGMFNIQHCKKL S S WVLLMK YLGNAT AIFFLPDEGK LQHLE ELTHDIITKFLE EDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSG VTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF KPFVFLMIEQNTK SPLFMGKVV
  • HGEGTFT SDLSKQMEEEAVRLFIEWLKNGGP S SGAPPP SXEDPQGD AAQKTDT SHHDQD HPTF KITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGL F LTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHS EAFTVNFGDTEEAKKQINDYWKGTQGKIVDLVKELDRDTWALWYIFFKGKWERPFE VKDTEEEDFHVDQ VTT VK WMMKRLGMFNIQHCKKL S S WVLLMK YLGNAT AIFFLPDE GKLQHLENELTHDIITKFLENEDRRS ASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADL SGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQN TKSPLFMGKV
  • recombinant proteins structurally similar to a recombinant protein described herein.
  • a protein is "structurally similar" to a reference protein if the amino acid sequence of the protein possesses a specified amount of sequence similarity and/or sequence identity compared to the reference protein.
  • a protein may be "structurally similar" to a reference protein if, compared to the reference protein, it possesses a sufficient level of amino acid sequence identity, amino acid sequence similarity, or a combination thereof.
  • a recombinant protein described herein can have an amino acid sequence that is structurally similar to SEQ ID NO: 1 or SEQ ID NO:2.
  • a recombinant protein described herein includes an amino-terminal domain that has structural similarity with a protein having GLP-1 agonist activity, and a carboxy -terminal domain that has structural similarity with a protein having anti-inflammatory activity.
  • An agonist domain and an anti-inflammatory domain can include one or more additional amino acids at the amino-terminal end, the carboxy terminal end, or both ends without an appreciable reduction in activity.
  • An agonist domain and an anti-inflammatory domain can include one or more deleted amino acids at the amino-terminal end, the carboxy terminal end, or both ends without an appreciable reduction in activity.
  • a recombinant protein disclosed herein can include an agonist domain having one or more additional amino acids, or one or more deleted amino acids, at the amino-terminal end, the carboxy terminal end, or both ends.
  • a recombinant protein disclosed herein can include an anti-inflammatory domain having one or more additional amino acids, or one or more deleted amino acids, at the amino- terminal end, the carboxy terminal end, or both ends.
  • Structural similarity of two proteins can be determined by aligning the residues of the two proteins (for example, a candidate protein and any appropriate reference protein described herein) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order.
  • a reference protein may be a recombinant protein described herein such as SEQ ID NO: 1 or SEQ ID NO:2.
  • a reference protein may be a domain of a recombinant protein, such as a GPL-1 agonist domain (e.g., SEQ ID NO:3, 4, or 5) or an anti-inflammatory domain (e.g., SEQ ID NO:6 or a truncation thereof).
  • a candidate protein is the protein being compared to the reference protein.
  • a candidate protein can be produced using recombinant techniques, or chemically or enzymatically synthesized.
  • a pair-wise comparison analysis of amino acid sequences can be carried out using the BESTFIT algorithm in the GCG package (version 10.2, Madison WI).
  • proteins may be compared using the Blastp program of the BLAST 2 search algorithm, as described by Tatiana et al. (FEMS Microbiol Lett, 174:247-250 (1999)), and available on the National Center for Biotechnology Information (NCBI) website.
  • amino acid sequence similarity In the comparison of two amino acid sequences, structural similarity may be referred to by percent “identity” or may be referred to by percent “similarity.” “Identity” refers to the presence of identical amino acids. “Similarity” refers to the presence of not only identical amino acids but also the presence of conservative substitutions. A conservative substitution for an amino acid in a protein may be selected from other members of the class to which the amino acid belongs. For example, it is well-known in the art of protein biochemistry that an amino acid belonging to a grouping of amino acids having a particular size or characteristic (such as charge, hydrophobicity, or hydrophilicity) can be substituted for another amino acid without altering the activity of a protein, particularly in regions of the protein that are not directly associated with activity.
  • a particular size or characteristic such as charge, hydrophobicity, or hydrophilicity
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine.
  • Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • Conservative substitutions include, for example, Lys for Arg and vice versa to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr so that a free -OH is maintained; and Gin for Asn to maintain a free - H2.
  • active analogs of a protein containing deletions or additions of one or more contiguous or noncontiguous amino acids that do not eliminate a functional activity of a domain e.g., agonist activity or anti-inflammatory activity, are also contemplated.
  • reference to a protein as described herein reference to the amino acid sequence of a recombinant protein described herein (e.g., SEQ ID NO: 1), or reference to an agonist domain (e.g., SEQ ID NO:3, 4, or 5) or an anti-inflammatory domain (e.g., SEQ ID NO:6 or a truncation thereof ) can include a protein with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%), at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence similarity to the reference amino acid sequence.
  • reference to a protein as described herein reference to the amino acid sequence of a recombinant protein described herein (e.g., SEQ ID NO: l), or reference to an agonist domain (e.g., SEQ ID NO: 3, 4, or 5) or an anti-inflammatory domain (e.g., SEQ ID NO: 6 or a truncation thereof ) can include a protein with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the reference amino acid sequence.
  • a recombinant protein described herein can include additional domains.
  • a recombinant protein can include a tag, such as, but not limited to, a polyhistidine-tag (His-tag).
  • His-tag a polyhistidine-tag
  • Addition of a His-tag can be achieved by the in-frame addition of a nucleotide sequence encoding the His-tag directly to either the 5' or 3' end of a coding region that encodes a recombinant protein.
  • Incorporation of a His-tag into a protein permits the easy isolation of the recombinant protein by use of a nickel or cobalt affinity column.
  • the His-tag can then be cleaved.
  • Other suitable affinity purification tags e.g., maltose-binding protein
  • methods of purification of proteins with those tags are known in the art.
  • a recombinant protein described herein can include a signal peptide.
  • a signal peptide is typically located at the amino terminal end of a protein.
  • An amino terminal signal peptide can direct the polypeptide to which it is attached to the extracellular space.
  • An amino terminal signal peptide is removed from the protein by a specific cleavage event prior to secretion.
  • Protein sequences of many signal peptides that target a protein to the extracellular space are well known to the art.
  • An example of a signal peptide includes, but is not limited to, MPSSVSWGILLLAGLCCLVPVSLA (SEQ ID NO:9).
  • a signal peptide is useful in methods described herein where a polynucleotide encoding a recombinant protein is introduced into a cell.
  • polynucleotides encoding a recombinant protein described herein.
  • a polynucleotide encoding a recombinant protein having an agonist domain and an antiinflammatory domain is referred to herein as a recombinant polynucleotide.
  • a recombinant polynucleotide can have a nucleotide sequence encoding the amino acid sequence of a GLP-1 agonist domain followed by an anti-inflammatory domain, with an optional amino acid linker located between the two domains (e.g., SEQ ID NO:2).
  • a recombinant polynucleotide can also include nucleotides encoding a signal peptide, linked so that the nucleotides encoding a signal peptide and the nucleotides encoding a recombinant protein are contiguous and in the same reading frame.
  • a polynucleotide can have a sequence encoding a GLP-1 agonist domain and a polynucleotide can have a sequence encoding an anti-inflammatory domain.
  • nucleotide sequence encoding the protein can be readily determined by reference to the standard genetic code, wherein different nucleotide triplets (codons) are known to encode the same amino acid.
  • the class of nucleotide sequences encoding a selected protein sequence is large but finite, and the nucleotide sequence of each member of the class is readily determined. Further, when expression of a nucleotide sequence is desired in a specific cell type, reference to the codon bias of that cell can be used by the skilled person to optimize expression of the nucleotide sequence in the cell.
  • a nucleotide sequence encoding a GLP-1 agonist, an anti-inflammatory protein, or a recombinant protein may be produced using recombinant techniques, or chemically or enzymatically synthesized using routine methods, or can be isolated from a mammalian cell, including a primate cell such as a human cell.
  • a polynucleotide encoding a recombinant protein described herein can be present in a vector.
  • a vector is a replicating polynucleotide, such as a plasmid, virus, or cosmid, to which another polynucleotide may be attached so as to bring about the replication of the attached polynucleotide. Construction of vectors containing a polynucleotide of the disclosure employs standard ligation techniques known in the art. A vector may provide for further cloning
  • amplification of the polynucleotide i.e., a cloning vector, or for expression of the
  • a vector includes, but is not limited to, plasmid vectors, viral vectors, cosmid vectors, and artificial chromosome vectors.
  • viral vectors include, for instance, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, and herpes virus vectors.
  • a vector includes a
  • DNA transposon and a polynucleotide encoding the recombinant protein is present between the inverted repeats of the DNA transposon.
  • a vector is capable of replication in a host cell.
  • suitable host cells for cloning or expressing the vectors herein include eukaryotic cells.
  • Suitable eukaryotic cells include mammalian cells, such as a primate cell including a human cell, and fungi, such as S. cerevisiae and P. pastoris.
  • Examples of mammalian cells include, but are not limited to, Human Embryonic Kidney (HEK) 293T cells, Chinese hamster ovary (CHO) cells, and baby hamster kidney (BHK) cells.
  • suitable host cells for cloning or expressing the vectors herein include prokaryotic cells.
  • Suitable prokaryotic cells include E. coli.
  • Vectors may be introduced into a host cell using methods that are known and used routinely by the skilled person. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral -mediated nucleic acid transfer are common methods for introducing nucleic acids into host cells.
  • An expression vector optionally includes regulatory sequences operably linked to the coding region. The disclosure is not limited by the use of any particular promoter, and a wide variety of promoters are known. Promoters act as regulatory signals that bind RNA polymerase in a cell to initiate transcription of a downstream (3' direction) coding region.
  • the promoter used may be a constitutive or an inducible promoter. It may be, but need not be, heterologous with respect to the host cell.
  • a vector introduced into a host cell optionally includes one or more marker sequences, which typically encode a molecule that inactivates or otherwise detects or is detected by a compound in the growth medium.
  • a marker sequence may render the transformed cell resistant to an antibiotic, or it may confer compound-specific metabolism on the transformed cell.
  • Examples of a marker sequence are sequences that confer resistance to kanamycin, ampicillin, chloramphenicol, tetracycline, and neomycin.
  • a vector can be useful in gene therapy.
  • viral vectors such as adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, and herpes virus vectors can penetrate cells and introduce a polynucleotide into a cell so that the polynucleotide can be stably maintained.
  • Viral vectors can include any viral strain or serotype.
  • a viral vector that includes a polynucleotide encoding a recombinant protein described herein can be packaged, referred to herein as a "particle.”
  • a vector can be introduced into an ex vivo cell or an in vivo cell.
  • ex vivo refers to a cell that has been removed from the body of an animal.
  • Ex vivo cells include, for instance, primary cells (e.g., cells that have recently been removed from a subject and are capable of limited growth in tissue culture medium), and cultured cells (e.g., cells that are capable of long term culture in tissue culture medium).
  • primary cells e.g., cells that have recently been removed from a subject and are capable of limited growth in tissue culture medium
  • cultured cells e.g., cells that are capable of long term culture in tissue culture medium.
  • in vivo refers to a cell that is within the body of a subject.
  • a recombinant protein useful in the present disclosure may be produced using recombinant DNA techniques, such as an expression vector present in a cell. Such methods are routine and known in the art.
  • the polypeptides and fragments thereof may also be synthesized in vitro, e.g., by solid phase peptide synthetic methods.
  • the solid phase peptide synthetic methods are routine and known in the art.
  • the domains of a recombinant protein can be generated separately and then joined by an organic group using routine methods.
  • a polypeptide produced using recombinant techniques or by solid phase peptide synthetic methods may be further purified by routine methods, such as fractionation on immunoaffinity or ion- exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on an ani on-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration using, for example, Sephadex G-75, or ligand affinity.
  • routine methods such as fractionation on immunoaffinity or ion- exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on an ani on-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration using, for example, Sephadex G-75, or ligand affinity.
  • compositions that include a recombinant protein described herein or a polynucleotide encoding a recombinant protein.
  • Such compositions typically include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Additional active compounds can also be incorporated into the compositions.
  • a composition may be prepared by methods well known in the art of pharmaceutics.
  • a composition can be formulated to be compatible with its intended route of
  • Administration may be systemic or local.
  • routes of administration include parenteral (e.g., intravenous, intradermal, subcutaneous, intraperitoneal, intramuscular), enteral (e.g., oral), and topical (e.g., epicutaneous, inhalational, transmucosal) administration.
  • Appropriate dosage forms for enteral administration of the compound of the present disclosure may include tablets, capsules or liquids.
  • Appropriate dosage forms for parenteral administration may include intravenous administration.
  • Solutions or suspensions can include the following components: a sterile diluent such as water for administration, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; electrolytes, such as sodium ion, chloride ion, potassium ion, calcium ion, and magnesium ion, and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • a composition can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile solutions or dispersions.
  • suitable carriers include physiological saline, bacteriostatic water, phosphate buffered saline (PBS), and the like.
  • PBS phosphate buffered saline
  • a composition is typically sterile and, when suitable for injectable use, should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • polyol for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile solutions can be prepared by incorporating the active compound (e.g.,
  • dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and any other appropriate ingredients.
  • sterile powders for the preparation of sterile injectable solutions preferred methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterilized solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
  • Oral compositions can also be prepared using a fluid carrier. Pharmaceutically compatible binding agents and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • the active compounds are delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • any method suitable for administration of polynucleotide agents can be used, such as gene guns, bio injectors, and skin patches as well as needle-free methods such as micro- particle DNA vaccine technologies (Johnston et al., U.S. Pat. No. 6,194,389).
  • the active compounds may be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants.
  • a controlled release formulation including implants.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • Such formulations can be prepared using standard techniques.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.
  • Toxicity and therapeutic efficacy of the active compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Recombinant proteins exhibiting high therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from animal models.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of signs of disease, such as obesity).
  • IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of signs of disease, such as obesity.
  • Such information can be used to more accurately determine useful doses in humans.
  • Levels in plasma may be measured using routine methods.
  • compositions can be administered one or more times per day to one or more times per week, including once every other day.
  • treatment of a subject with an effective amount of an active compound can include a single treatment or, preferably, can include a series of treatments. For instance, in those embodiments where a polynucleotide is delivered, a skilled person can determine whether a single administration of a vector is sufficient or whether multiple doses of a vector are helpful to the subject.
  • the method includes delivering a polynucleotide into a host cell.
  • the host cell can be ex vivo or in vivo, and can be dividing or non-dividing.
  • the host cell can be mammalian, including a member of the family Muridae (a cell from a murine animal such as rat or mouse), or a primate cell, such as a human cell.
  • the method includes administration to a subject, and the subject may be in need thereof, such as for treatment of a condition.
  • Introduction of the polynucleotide into a cell can result in expression of a protein described herein. Production of the protein encoded by the polynucleotide may impart a therapeutic effect.
  • the method includes treating a condition in a subject.
  • the subject may be a mammal, including a member of the family Muridae, or a primate, such as a human.
  • condition refers to any deviation from or interruption of the normal structure or function of a part, organ, or system, or combination thereof, of a subject that is manifested by a characteristic sign or set of signs.
  • signal refers to objective evidence of a condition present in a subject. Signs associated with conditions referred to herein and the evaluation of such signs is routine and known in the art.
  • Conditions include, but are not limited to, obesity, type 2 diabetes, type 1 diabetes, alpha- 1 antitrypsin deficiency, lung fibrosis, and liver diseases resulting from fat accumulation and/or inflammation including, but not limited to, nonalcoholic fatty liver disease, non-alcoholic steatohepatitis, and liver fibrosis- cirrhosis.
  • a subject may be determined by evaluation of signs associated with the condition.
  • introduction into obese mice of a recombinant protein by gene transfer resulted in weight loss that was greater with the recombinant protein than the sum of weight loss observed when each domain of the recombinant protein when administered separately (see Figure 8D of the Examples), indicating that there was an unexpected synergy when the two domains were present in the same protein.
  • the stability of the recombinant protein was greater than the stability of each domain when evaluated separately.
  • Treatment of a condition can be prophylactic (also referred to as preventative) or, alternatively, can be initiated after the development of a condition. Treatment that is
  • prophylactic for instance, initiated before a subject manifests signs of a condition
  • treatment of a subject that is "at risk" of developing a condition is referred to herein as treatment of a subject that is "at risk" of developing a condition.
  • An example of a subject that is at risk of developing a condition is a person having a risk factor. Risk factors for the conditions described herein are known to the skilled person.
  • Treatment can be performed before, during, or after the occurrence of a condition described herein. Treatment initiated after the development of a condition may result in decreasing the severity of the signs of the condition, or completely removing the signs.
  • the method includes administering to the subject having a condition or at risk of developing a condition a composition including an effective amount of a recombinant protein described herein. In one embodiment, the method includes administering to the subject having a condition or at risk of developing a condition a composition including an effective amount of a polynucleotide encoding a recombinant protein described herein.
  • the polynucleotide includes a vector, such as a viral vector. The introduced polynucleotide may or may not be integrated into genomic DNA of the recipient cell, and may exist in the recipient cell only transiently.
  • the administration can result in the recombinant protein being present in blood, plasma, or serum.
  • the concentration of recombinant protein is at least 1 picograms/milliliter (pg/ml), at least 10 pg/ml, at least 100 pg/ml, at least 1 nanogram/milliliter (ng/ml), at least 10 ng/ml, at least 100 ng/ml, at least 1 microgram/milliliter (ug/ml), at least 10 ug/ml, or at least 100 ug/ml.
  • the concentration of recombinant protein is no greater than 1000
  • milligrams/milliliter no greater than 100 mg/ml, or no greater than 10 mg/ml.
  • concentration of the recombinant protein can be measured after the administration, for instance 1 week, 3 weeks, 5 weeks, 7 weeks, 9 weeks, or 11 weeks after the administration.
  • Cells that may be transduced include a cell of any tissue or organ type, of any origin (e.g., mesoderm, ectoderm or endoderm).
  • Non-limiting examples of cells include liver (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., beta islet cells), lung, central or peripheral nervous system, such as brain (e.g., neural, glial or ependymal cells) or spine, kidney, eye (e.g., retinal, cell components), spleen, skin, thymus, testes, lung, diaphragm, heart (cardiac), muscle, or gut (e.g., endocrine), adipose tissue (white, brown or beige), muscle (e.g., fibroblasts), synoviocytes, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells, inner ear nervous cells or hem
  • liver e.g., hepatocytes, sinusoidal endothelial cells
  • pancreas e.g., beta islet cells
  • lung central or peripheral nervous system, such as brain (e.g., neural, glial or ependymal cells) or spine, kidney, eye (retinal, cell components), spleen, skin, thymus, testes, lung, diaphragm, heart (cardiac), muscle or psoas, or gut (e.g., endocrine), adipose tissue (white, brown or beige), muscle (e.g., fibroblasts), synoviocytes, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells, or hematopoietic (e.g., blood or lymph) cells.
  • brain e.g., neural, glial or ependymal cells
  • spine kidney
  • eye retina
  • spleen skin
  • an "effective amount” is an amount effective to result in a change in at least one sign of the condition in the subject.
  • a recombinant protein or a polynucleotide encoding a recombinant protein can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect.
  • Other therapeutic compounds useful for the treatment of the conditions described herein are known and used routinely, and may be used as a second, supplemental agent, to complement the activity of a recombinant protein described herein.
  • kits for treating a subject includes a recombinant protein described herein or a polynucleotide encoding the recombinant protein in a suitable packaging material in an amount sufficient for at least one administration.
  • suitable packaging material in an amount sufficient for at least one administration.
  • other reagents such as buffers and solutions needed to practice the disclosure are also included in separate containers.
  • a kit may also include a solvent within which the active compound can be dissolved or suspended.
  • Instructions for use of the packaged proteins are also typically included in separate containers.
  • packaging material refers to one or more physical structures used to house the contents of the kit.
  • the packaging material is constructed by known methods, generally to provide a sterile, contaminant-free environment.
  • the packaging material may have a label which indicates that the active compound can be used for treating a subject.
  • the packaging material contains instructions indicating how the materials within the kit are employed to treat the subject.
  • the term "package” refers to a container such as glass, plastic, paper, foil, and the like, capable of holding within fixed limits the active compound, and other reagents "Instructions for use” typically include a tangible expression describing the active compound concentration or at least one method parameter, such as the amount to administer to a subject.
  • Obesity and its associated metabolic comorbidities represent a growing public health problem. Demonstrated herein is the use of a fusion gene of exendin-4 and a 1 -antitrypsin to control obesity and obesity-associated insulin resistance, fatty liver and hyperglycemia.
  • the fusion gene encodes a protein with exendin-4 peptide placed at the N-terminus of human a-1 antitrypsin with a linker and is designated EAT.
  • Obesity (body mass index > 30) has become a major public health problem in recent years. The prevalence of obesity in the US is -35.5% and—35.8% among adult men and women, respectively [1]. Obesity is closely linked to a number of severe metabolic comorbidities such as diabetes and nonalcoholic fatty liver disease (NAFLD) [1, 2]. Though behavior interventions such as exercise and energy restriction are believed to be effective in reducing body weight, overwhelming evidence suggests that these interventions are essential but not sufficient to maintain a healthy weight, particularly in individuals predisposed to obesity [3-5]. It remains difficult to establish a new therapy for these metabolic diseases.
  • NAFLD nonalcoholic fatty liver disease
  • GLP-1 glucagon-like peptide-1
  • GLP-1 is known to increase pancreatic insulin secretion, insulin sensitivities of both alpha and beta cells, and inhibits acid secretion and gastric emptying in the stomach.
  • the fusion gene contains the sequence of exendin-4 (Ex4), a potent agonist of the GLP-1 receptor, placed at the 5' end of the human a-1 antitrypsin (hAAT) gene, a coding sequence with a well-known function in suppressing inflammation [17-19].
  • Ex4 exendin-4
  • hAAT human a-1 antitrypsin
  • EAT a coding sequence with a well-known function in suppressing inflammation
  • the extendin 4 (Ext4) gene was designed according to its amino acid sequence and constructed using primers synthesized at Sigma- Aldrich (St. Louis, MO).
  • the hAAT gene was cloned from the plasmid used in a previous study [39].
  • the EAT fusion gene was created by placing an Ex4 sequence at the 5' end of the hAAT gene with a linker sequence encoding the GGGGS linker peptide.
  • a pLIVE plasmid vector purchased from Minis Bio (Madison, WI).
  • the EAT gene was cloned into the 3' end of the albumin promoter at the multi-cloning sites of pLIVE using restriction enzyme-mediated ligation.
  • Plasmid containing Ext4 and hAAT genes were similarly cloned to pLIVE vector.
  • EAT gene was cloned to pcDNA vector with a CMV promoter for peak level expression in FIEK293T cells.
  • Each of the plasmid constructs containing different genes was verified by restriction enzyme digestion and DNA sequencing. Plasmid DNA was prepared by means of cesium chloride-ethidium bromide gradient
  • the purity of the plasmid preparations was confirmed by OD260/280 ratio and by 1% agarose gel electrophoresis.
  • the purified plasmids were dissolved in saline and stored at -80 °C until use.
  • EAT recombinant protein using transient overexpression in FIEK293T cells purchased from ATCC (#CRL-3216).
  • the cells were cultured using Dulbecco's Modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS).
  • DMEM Dulbecco's Modified Eagle's medium
  • FBS fetal bovine serum
  • 40 ⁇ g of plasmid DNA were mixed with 120 ⁇ g of PEI and the complexes were kept at room temperature for 10-20 min before being added to a T-175 flask.
  • the DMEM medium with 1% FBS was changed every 2 days for a total of 8 days and all culture medium was collected for EAT protein purification using two-step chromatography, including medium passing through a Nickel Affinity Column (#25215, Thermo-Scientific) and then an ion- exchange column (#17-0510-10, GE Healthcare Life Sciences) for further polishing.
  • the purity of the final product was determined using 5-10% SDS-PAGE loaded with 2-8 ⁇ g recombinant protein per lane and stained with a kit (#1610803) purchased from Bio-Rad.
  • the purified EAT protein was further verified using Western blotting with an antibody recognizing AAT (#A0409, Sigma- Aldrich).
  • the fluorescence-based elastase inhibition assay kit (#K782) was purchased from
  • mice were injected with plasmid DNA through a standard hydrodynamic tail vein injection and subsequently kept on HFD for 9 weeks.
  • the injected mice were kept on standard chow for a total of 180 days.
  • Hydrodynamic gene transfer in obese mice was performed according to an established procedure with some modification [40, 41]. Briefly, appropriate volume of saline solution (equivalent to 8% lean mass of obese mice) containing different amounts of plasmid DNA were injected into the tail vein of mice over 5-8 s.
  • EAT protein was administered daily to the intraperitoneal cavity at 5 mg/kg.
  • Food consumption was determined by measuring the difference between the amount provided and the amount left twice weekly. Daily food intake per mouse was calculated based on the amount consumed divided by time and the number of mice per cage. Body composition was analyzed using an EchoMRI-100 (Echo Medical Systems, Houston, TX).
  • Liver samples were collected and fixed in 10% neutral buffered formalin ( BF). After dehydration using gradient ethanol, the samples were processed twice using xylene and embedded in paraffin. Tissue sections (6 ⁇ in thickness) were made, spread on a slide and baked at 37 °C for 2 h. The slides were stained with H&E, mounted with Permount medium (Fisher Scientific), and examined under an optical microscope (ECLIPSE Ti, Nikon). Image quantification was carried out using NIS-Elements imaging platform from Nikon Instruments Inc. (Melville, NY). For Oil red O and Nile red staining, frozen liver samples were cut at 8 ⁇ in thicknesses and fixed using 10% NBF. Liver triglyceride content was determined following a previously reported method with some modifications [42].
  • liver tissues (100-200 mg) were homogenized in a mixture of chloroform and methanol (2: 1) and tissue homogenates were incubated overnight at 4 °C. The mixture was centrifuged at 12,000 rpm for 20 min and supernatant collected. The collected fractions were dried, and lipids were re- dissolved in 1%) Triton X-100. The triglyceride concentration was determined using a commercial kit (#TR22203) from Thermo-Scientific (Waltham, MA).
  • mice were injected intraperitoneally with glucose at
  • mice were fasted for 4 h and blood glucose levels were measured after an intraperitoneal injection of insulin (0.75 U/kg) purchased from Eli Lilly (Indianapolis, IN) using blood samples collected from the tail vein at different time points.
  • Plasma samples were prepared by centrifuging freshly collected blood in a heparin- coated tube at 4,000 rpm for 5 min, and were then kept frozen at -80 °C until use. Insulin concentrations were determined using an insulin ELISA kit (#10-1247-01) purchased from Mercodia Developing Diagnostics (Winston-Salem, NC). Blood levels of TNFa and IL6 were determined using ELISA kits from eBioscience (San Diego, CA). AST and ALT levels were determined using biochemical kits purchased from Thermo-Scientific (Waltham, MA). All measurements were performed following the protocols provided by the manufacturer.
  • Real time PCR was performed on an ABI StepOne Plus Real Time PCR system (Foster City, CA) using PerfeCTa® SYBR® Green FastMix (Quanta Biosciences, Gaithersburg, MD) as the indicator. Primers were synthesized at Sigma (St. Louis, MO). Melting curve analysis of all real-time PCR products was conducted and showed a single DNA duplex. All primer sequences employed are summarized in Table 1. The data were analyzed using the AACt method [43]. Table 1. PCR Primer Sequences
  • Acox F CCGCCACCTTCAATCCAGAG R: CAA GTTCTCGATTTCTCGACGG
  • Adrp F GACCTTGTGTCCTCCGCTTAT R: CAACCGCAATTTGTGGCTC
  • Atgl F GGATGGCGGCATTTCAGACA R: CAAAGGGTTGGGTTGGTTCAG
  • Cdllb F ATGGACGCTGATGGCAATACC
  • R TCCCCATTCACGTCTCCCA
  • Cdllc F CTGGATAGCCTTTCTTCTGCTG
  • R GCACACTGTGTCCGAACTCA
  • Cd36 F ATGGGCTGTGATCGGAACTG R: GTCTTCCCAATAAGCATGTCTCC
  • Chrebp F AGATGGAGAACCGACGTATCA
  • R ACTGAGCGTGCTGACAAGTC
  • Cidea F TGACATTCATGGGATTGCAGAC
  • R GGCCAGTTGTGATGACTAAGAC
  • Cptla F CTCCGCCTGAGCCATGAAG R: CACCAGTGATGATGCCATTCT
  • Dgat2 F GCGCTACTTCCGAGACTACTT R: GGGCCTTATGCCAGGAAACT
  • Dio2 F AATTATGCCTCGGAGAAGACCG
  • R GGCAGTTGCCTAGTGAAAGGT
  • Elov F TTCTCACGCGGGTTAAAAATGG
  • R GAGCAACAGATAGACGACCAC
  • F4/80 F: TGACTCACCTTGTGGTCCTAA R: CTTCCCAGAATCCAGTCTTTCC
  • Fabp4 F AAGGTGAAGAGCATCATAACCC
  • R TCACGCCTTTCATAACACATTCC
  • Fsp27 F ATGGACTACGCCATGAAGTCT R: CGGTGCTAACACGACAGGG
  • Fxr F GCTTGATGTGCTACAAAAGCTG
  • R CGTGGTGATGGTTGAATGTCC
  • G6p F CGACTCGCTATCTCCAAGTGA
  • R GTTGAACCAGTCTCCGACCA
  • Gapdh F AGGTCGGTGTGAACGGATTTG
  • R TGTAGACCATGTAGTTGAGGTCA
  • Hsl F CCAGCCTGAGGGCTTACTG
  • R CTCCATTGACTGTGACATCTCG
  • Lpl F GGGAGTTTGGCTCCAGAGTTT R: TGTGTCTTCAGGGGTCCTTAG
  • Lxr F CTCAATGCCTGATGTTTCTCCT
  • R TCCAACCCTATCCCTAAAGCAA
  • Mead F GGGTTTAGTTTTGAGTTGACGG
  • R CCCCGCTTTTGTCATATTCCG (SEQ ID NO:36) (SEQ ID NO:78)
  • Mcpl F TTAAAAACCTGGATCGGAACCA
  • R GCATTAGCTTCAGATTTACGGGT
  • Oxpat F TGTCCAGTGCTTACAACTCGG
  • R CAGGGCACAGGTAGTCACAC
  • Pepck F CTGCATAACGGTCTGGACTTC R: CAGCAACTGCCCGTACTCC
  • Pgcla F TATGGAGTGACATAGAGTGTGC R: CCACTTCA ATCCACCCAGAAAG
  • Ppara F AGAGCCCCATCTGTCCTCTC R: ACTGGTAGTCTGCAAAACCAAA
  • Pparyl F GGAAGACCACTCGCATTCCTT R: GTAATCAGCAACCATTGGGTCA
  • Ppary2 F TCGCTGATGCACTGCCTATG
  • R GAGAGGTCCACAGAGCTGATT
  • Srebplc F GCAGCCACCATCTAGCCTG
  • R CAGCAGTGAGTCTGCCTTGAT
  • Tnfa F CCCTCACACTCAGATCATCTTC R: GCTACGACGTGGGCTACAG
  • Ucpl F AGGCTTCCAGTACCATTAGGT R: CTGAGTGAGGCAAAGCTGATTT
  • Standard ELISA protocol was followed to determine EAT protein concentration [39].
  • a rabbit anti-human AAT antibody was used to coat the ELISA plate overnight and blocked for 1 hr with blocking buffer (4% BSA in PBS-Tween buffer).
  • Serum prepared from animals was diluted serially with 1% BSA in PBS-Tween buffer and added to each well of the ELISA plate and incubated for 1 hr.
  • biotinylated goat anti-human AAT polyclonal antibody (1 : 1000 dilution in 1% BSA Tween buffer) was added and incubated for 1 hr at room temperature.
  • streptavidin-horseradish peroxidase conjugate (1 :50,000 dilution) was added, and incubated for 1 hr.
  • the substrate solution (3,3', 5,5'- tetramethylbenzidine) was added and the plate was read at 450 nm in an ELISA reader.
  • the AAT concentration was calculated based on a standard curve established in each plate using a known amount of pure hAAT. With the exception of blocking buffer (200 ⁇ /well) and washing buffer (400 ⁇ /well), the sample volume used was 100 ⁇ /well.
  • hAAT- linker-S961 fusion gene of human al antitrypsin with insulin receptor antagonist S961
  • SCI-linker-hAAT fusion gene of single chain insulin analog with human al antitrypsin
  • FGF21-linker-hAAT fusion gene of fibroblast growth factor 21 with human al antitrypsin
  • Ex4-linker-FGF21 fusion gene of exendin 4 and fibroblast growth factor 21
  • Ex4-linker- SOD3 fusion gene of exendin 4 with superoxide dismutase 3
  • Ex4-linker-hAAT fusion gene of exendin 4 with human al antitrypsin.
  • the fusion proteins encoded by the fusion genes are linked through a sequence encoding a Glycine-Glycine-Glycine-Glycine-Serine peptide linker.
  • the fusion genes were constructed using molecular biological techniques and cloned into the pLIVE vector ( Figure 1 A).
  • hAAT For 3 of these 6 fusion proteins, we placed hAAT at the C terminus while putting single chain insulin, FGF21, or exendin 4 at the N terminus. We also created proteins with exendin 4 kept at the N terminus while having FGF21, SOD3, or hAAT at the C terminus. At the same time, we included a fusion protein with hAAT at the N terminus and S961 peptide at the C terminus.
  • EfFD high fat diet
  • Figure IB shows that, despite being constructed through the same strategy, these constructs showed different activity in suppressing diet-induced obesity.
  • E ⁇ Jgene transfer blocks high-fat diet-induced weight gain, hyper adiposity, insulin resistance, fatty liver, and the expression of relevant genes
  • the average food intake of animals decreased as the amount of pEAT plasmid increased.
  • Control animals consumed 2.46 ⁇ 0.13 of HFD per day per animal compared to 2.12 ⁇ 0.11 with 0.2 ⁇ g, 1.89 ⁇ 0.13 with 2.0 ⁇ g, and 1.85 ⁇ 0.17 g with 20.0 ⁇ g of hydrodynamic injection of pEAT plasmid.
  • the average body weight of control mice is 45.0 g compared to 34.1 g at a dose of 0.2 ⁇ g ( Figure 3C). There was no weight difference between animals injected with 2.0 and 20.0 ⁇ g pEAT plasmid.
  • WAT White adipose tissue
  • Results in Figure 4A show the highest amount of WAT in control mice, followed by animals with 0.2 ⁇ g of pEAT plasmid and the animals with the injection dose of 2.0 ⁇ g or 20.0 ⁇ g per mouse showed the lowest and similar WAT.
  • the H&E staining of adipose tissues revealed the presence of crown-like structure (CLS) in the white adipose tissue of control animals ( Figure 4B), but not in animals with EAT gene transfer. More fat content was also seen in the brown adipose tissue (BAT) of control animals compared to that of pEAT treated animals.
  • FIG. 4C shows lower mRNA levels of F40/80, Cdllb, Cdllc and Mcpl in pEAT treated animals compared to those of the controls. Results of glucose tolerance tests showed improved glucose homeostasis in pEAT treated mice ( Figure 5).
  • Figure 8A shows the detailed design of plasmid vectors employed in the study, including plasmids containing a promoter, signal peptide for protein secretion, sequence of Ex4 hAAT, or EAT, a linker, and poly A signal.
  • a computer-based program [20] predicted that the EAT fusion protein has a globular structure with the secondary structure of each unit conserved ( Figure 8B).
  • EAT gene transfer reduces adiposity and improves fatty liver in high-fat diet-induced obese mice To systematically study the therapeutic effects of EAT gene transfer, we
  • FIG. 9A shows that transfer of the EAT gene progressively reduced body weight in these mice.
  • One single injection of the pEAT plasmid resulted in a weight loss of -24.7% within 3 weeks ( Figures 9A and 9B).
  • Body composition analysis revealed that the difference in body weight primarily resulted from reduction in fat mass (by -43.1%) and, to a lesser degree from lean mass (-12.8%). Gene transfer substantially repressed average food intake by -60.2% ( Figure 9D).
  • EAT gene transfer greatly reduced adipocyte hypertrophy in WAT and decreased fat deposition in brown adipose tissue (BAT) (Figure 9E).
  • E ⁇ Jgene transfer also down-regulated transcription of pivotal genes involved in WAT chronic inflammation, including F4/80, CDllb, CD 11c, MCP1, Tnfa and 116 (Figure 9F).
  • protein levels of TNFa and IL6 in blood were decreased by -41.7% and -53.2%, respectively, in treated mice compared to control (Figure 9G).
  • Thermogenic genes in BAT were up-regulated by EAT gene transfer (Figure 9H).
  • the treatment cured fatty liver ( Figure 91) and reduced hepatic fat content by -63.5% (Figure 9J).
  • the reduced hepatic fat deposition was associated with a decrease in mRNA levels of the genes responsible for lipid synthesis, lipid droplet formation and inflammation (Figure 9K).
  • FIG. 10A shows that EA T gene transfer markedly promoted glucose tolerance in these obese mice.
  • the peak glucose levels for mice with or without EA T gene transfer were -335.2 and -548.4 mg/dL, respectively, and the area under the curve was significantly reduced (-69.9%) in mice with E ⁇ Jgene transfer.
  • E ⁇ Jgene transfer also reduced blood insulin levels (Figure 10B), indicating improved insulin sensitivity.
  • This notion was further supported by the results of insulin tolerance tests, which showed that the mice with EAT gene transfer had a much stronger response to insulin injection (Figure IOC).
  • To study the underlying mechanism we measured mRNA levels of Glut4 in adipose tissues.
  • FIG 10D shows that E ⁇ Jgene transfer increased Glut4 expression in WAT and BAT, by 1.9-fold and 2.7- fold, respectively.
  • E ⁇ Jgene transfer markedly reduced islet hypertrophy (Figure 10E).
  • EAT gene transfer did not produce undesirable histological change in any of the major organs examined including the heart, spleen, lungs, and kidneys ( Figure 11)
  • E ⁇ Jgene transfer suppresses adiposity and improves glucose metabolism in leptin-deficient ob/ob mice
  • EAT gene transfer blocks fatty liver in ob/ob mice
  • Figure 13 A shows that EAT gene transfer blocked the enlargement of liver and remarkably repressed hepatic fat deposition in the context of leptin deficiency.
  • the average liver weight was -43.5% lower in the treated mice compared to that of the controls ( Figure 13B).
  • EAT gene transfer repressed hepatic triglyceride content by -63.1% ( Figure 13C).
  • the mice with ⁇ Jgene transfer showed much lower concentrations of serum AST and ALT compared to the controls ( Figure 13D), suggesting improved liver function.
  • EAT gene transfer decreased expression levels of pivotal genes for hepatic lipid production and lipid droplet formation while elevating transcription levels of key factors responsible for fatty acid oxidation (Figure 13E).
  • Figure 14D Results summarized in Figure 14D show similar weights of each of the internal organs including the heart, liver, spleen, lungs and the kidneys. No abnormal structures were identified by H&E staining of the tissue sections from the selected organs, or the WAT and BAT ( Figure 14E).
  • FIG. 15B shows that EAT protein was efficiently purified. In the range of 2-8 ⁇ g loaded proteins per lane, the purified EAT protein showed a single band, and no impurity was detected (Figure 15C). Immunoblotting confirmed the result, validating the identity of the purified EAT protein ( Figure 15D).
  • FIG 18A shows that EAT protein treatment greatly decreased the amount of lipid droplets in the liver. Further biochemical determination demonstrated that EAT protein treatment reduced hepatic fat content by -71.8% (Figure 18B). Examination of mRNA levels in the liver revealed that this protein treatment down-regulated transcription of genes responsible for hepatic lipogenesis and lipid droplet formation ( Figure 18C). At the same time, this treatment also reduced expression levels of several pro-inflammatory genes, including Mcpl, Tnfa, and 116 in the liver ( Figure 18C).
  • Obesity is a complex disease with severe comorbidities [21]. Targeting key components in obesity pathophysiology may prevent and reverse obesity and restore metabolic homeostasis.
  • exendin 4 a potent agonist of the GLP-1 receptor placed at the N-terminus of AAT is the only one with anti-obesity, anti-insulin resistance and anti-fatty liver activity.
  • both AAT and exendin 4 activities are preserved (Figure 16) in the EAT fusion protein.
  • EA T gene transfer decreased transcription of pivotal genes responsible for lipogenesis, lipid droplet formation and chronic inflammation in the liver ( Figures 6D, 9K, 13E).
  • EAT gene transfer also elevated expression of thermogenic genes in brown fat ( Figure 9H), indicating that this mechanism may also contribute to the weight loss observed in this study.
  • a recent study by Kooijman et al. provided strong evidence supporting exendin 4's ability to activate brown adipose tissue, thereby leading to a series of metabolic benefits in HFD-induced obese mice [22].
  • EAT gene transfer on animals depend on sufficient level of EAT protein in the blood. Hydrodynamic gene delivery was used in this study because of its high efficiency in gene delivery to the liver by a tail vein injection. The dose response curves show that sufficient levels of EAT protein were achieved at 2.0 ⁇ g of pEAT plasmid, although significant levels of EAT protein were also obtained at 0.2 ⁇ g/mouse. Persistent EA T gene expression in animals is largely due to the use of an albumin promoter in the pLIVE vector [23]. Increase in the circulation half-life of EAT for exendin 4 is another important factor for our design because the exendin 4 peptide is short lived in blood circulation [9].
  • the hepatic fat deposition is controlled by a dynamic balance between liver lipid production, secretion, and communication with other organs [31].
  • EAT treatment reversed fatty liver in diet-induced obese mice and blocked hepatic steatosis in ob/ob mice. This effect can be explained, at least partially, by decreased hepatic lipogenesis, reduced lipid droplet formation, and facilitated liver-adipose tissue crosstalk.
  • recent studies demonstrate the existence of a GLP-1 receptor on hepatocytes, which directly implicates the GLP-1 pathway in hepatic fat regulation [32].
  • EAT treatment reduced expression of Srebplc and its downstream targets, including Acc, Fas, and Scd responsible for lipogenesis in the liver.
  • the PPARy pathway appears to play a central role in controlling hepatic lipid droplet development by regulating CD36, FABP4, MGAT1 and lipid droplet surrounding proteins [33-35].
  • EAT treatment reduced transcription of all these crucial proteins, indicating down-regulation of the PPARy pathway as a potential mechanism of EAT therapy in improving fatty liver.
  • the blocked hepatic steatosis may also be partly accounted for by improved liver-adipose tissue metabolic communication.
  • EAT a new fusion protein named EAT for the pharmacological management of obesity and its associated metabolic comorbidities.
  • EAT therapy was able to generate various metabolic benefits, including the prevention of HFD-induced weight gain, insulin resistance and fatty liver, as well as therapeutic benefits of weight loss and improvement of metabolic homeostasis.
  • the primary mechanisms involved are sustained suppression of food intake by exendin 4 activity in EAT, although additional experiments are needed to examine the anti-inflammation effects of AAT in vivo.
  • Our findings open a new avenue for EAT-based treatment for obesity, diabetes, NAFLD, and related conditions.
  • liposomes eliminates visceral adipose macrophages and blocks high-fat diet-induced weight gain and development of insulin resistance.
  • Alpha- 1 antitrypsin therapy is safe and well tolerated in children and adolescents with recent onset type 1 diabetes mellitus. Pediatr Diabetes 17: 351-359.
  • Glucagon-like peptide- 1 receptor is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway .
  • Hepatology 51 1584-1592. 33. Matsusue, K, Haluzik, M, Lambert, G, Yim, SH, Gethosova, O, Ward, JM, et al (2003). Liver-specific disruption of PPARgamma in leptin-deficient mice improves fatty liver but aggravates diabetic phenotypes. J Clin Invest 111 : 737-747.
  • Interleukin-lbeta regulates fat-liver crosstalk in obesity by auto-paracrine modulation of adipose tissue inflammation and expandability.

Abstract

L'invention concerne une protéine recombinée comprenant un domaine agoniste et un domaine anti-inflammatoire. Dans un mode de réalisation, le domaine agoniste présente une activité agoniste au récepteur du peptide qui ressemble au glucagon (GLP-1), le domaine anti-inflammatoire présente une activité anti-inflammatoire, et le domaine agoniste est situé à l'extrémité amino terminale par rapport au domaine anti-inflammatoire. L'invention concerne également un polynucléotide recombiné comprenant une région codante codant pour la protéine recombinée. L'invention concerne en outre des méthodes, dont des méthodes de libération du polynucléotide recombiné dans une cellule hôte, et des méthodes de traitement d'une affection telle que l'obésité, le diabète de type 2, le diabète de type 1, la déficience en alpha-1 antitrypsine, la stéatose hépatique non alcoolique, la stéatohépatite non alcoolique, la fibrose-cirrhose du foie, la fibrose pulmonaire, ou une combinaison de ceux-ci.
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