COMORBIDITIES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial No.

62/406,517, filed October 11, 2016, which is incorporated by reference herein.

SEQUENCE LISTING

This application contains a Sequence Listing electronically submitted via EFS-Web to the United States Patent and Trademark Office as an ASCII text file entitled "Seq-List-

02700201_ST25.txt" having a size of 29 kilobytes and created on October 10, 2017. The information contained in the Sequence Listing is incorporated by reference herein.

BACKGROUND

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). 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 (Mann et al., 2007, Am Psychol 62:220-233; Pontzer et al., 2016, Curr Biol 26:410-417; Mark, 2008, Hypertension 51: 1426-1434; discussion 1434). It remains difficult to establish new therapies for these metabolic diseases.

SUMMARY

The difficulty in establishing therapies for these metabolic diseases stems primarily from the complex pathophysiology of obesity, of which excess energy intake and adipose chronic inflammation are two major components (Zhang et al., 2014Nutrients 6: 5153-5183; Monteiro and Azevedo, 2010, Mediators Inflamm 2010; Hill et al., 2012, Circulation 126: 126-132).

Normal eating behavior in both humans and rodents is tightly controlled by the gut-brain axis, in which glucagon-like peptide-1 (GLP-1) plays a dominant role (Hoist, 2007, Physiol Rev 87: 1409-1439). Responding to food intake, GLP-1 is produced by L-cells in the intestine and released into circulation, working on a variety of organs to regulate energy processing (Hoist, 2007, Physiol Rev 87: 1409-1439). 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. Reduction in GLP-1 release has been shown in obese patients, resulting in excess energy intake and imbalance of metabolic metabolism (Hoist, 2007, Physiol Rev 87: 1409-1439; Prasad-Reddy and Isaacs, 2015, Drugs Context 4: 212283; Garber, 2011, Diabetes Care 34 Suppl 2: S279-284). In addition, chronic inflammation is also crucial in producing obesity- related metabolic dysregulations (Monteiro and Azevedo, 2010, Mediators Inflamm 2010; Xu et al., 2003, J Clin Invest 112: 1821-1830; Weisberg et al., 2003, J Clin Invest 112: 1796-1808). Without chronic inflammation, a healthy weight gain would not lead to glucose intolerance and hepatic fat aggregation (Hotamisligil et al., 1993, Science 259: 87-91; Kim et al., 2007, J Clin Invest 117: 2621-2637; Kusminski et al., 2012, Nat Med 18: 1539-1549). The inventors hypothesized that simultaneously repressing energy intake and relieving inflammation would reduce body weight and restore metabolic homeostasis.

Described herein are recombinant proteins for use in the pharmacological treatment of obesity and related metabolic disorders. The therapeutic protein contains the sequence of a GLP- 1 receptor agonist and the sequence of an anti-inflammatory protein. In one embodiment, 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). This embodiment of the recombinant protein is also referred abbreviated herein as EATby taking the critical letters from Ex4 and hAAT. Using the approaches of gene transfer and protein administration, the therapeutic effects of EAT on weight loss, glucose homeostasis and hepatic steatosis were assessed in both high-fat diet-induced obese mice and genetically modified ob/ob mice. The evidence presented herein supports the feasibility of this new therapy for treatment of obesity-related metabolic disorders.

Provided herein is a recombinant protein including an agonist domain and an antiinflammatory domain. In one embodiment, the agonist domain has a 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.

Also provided is 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. In one embodiment, the polynucleotide further includes a vector, such as a viral vector.

In one embodiment, 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.

In one embodiment, the agonist domain includes, or has structural similarity with, a glucagon-like peptide 1, an exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, taspoglutide, or semaglutide. In one embodiment, the agonist domain includes, or has structural similarity with, the amino acid sequence of SEQ ID NO:3, 4, or 5. In one embodiment, the anti- inflammatory domain includes, or has structural similarity with, alpha-1 antitrypsin, alpha-1 antichymotrypsin, or alpha-1 antiproteinase. In one embodiment, the anti-inflammatory domain includes, or has structural similarity with, the amino acid sequence of SEQ ID NO: 6 or a truncation thereof. In one embodiment, the protein includes, or has structural similarity with, the amino acid sequence of SEQ ID NO:2. Further provided is a composition including a recombinant protein described herein or a recombinant polynucleotide described herein. In one embodiment, the composition includes a pharmaceutically acceptable carrier.

Also provided is a 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.

Provided herein are methods. In one embodiment, 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, and the agonist domain is located amino terminal to the anti-inflammatory domain. In one embodiment, the cell is ex vivo. In another embodiment, the cell is in vivo and the contacting the cell includes administering the

recombinant polynucleotide to a subject. The cell can be a mammalian cell, such as a human cell.

In one embodiment, 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. In one embodiment, the subject has a condition or is at risk of having a condition that can be treated by the composition. Examples of conditions include, but are not limited to, obesity, type 2 diabetes, type 1 diabetes, alpha- 1 antitrypsin deficiency, nonalcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis-cirrhosis, lung fibrosis, or a combination thereof In one embodiment, the concentration of the protein in the blood of the subject is at least 1 picograms/milliliter to no greater than 10 mg/ml.

As used herein, the term "protein" refers broadly to a polymer of two or more amino acids joined together by peptide bonds. The term "protein" also includes molecules which contain more than one protein joined by disulfide bonds, ionic bonds, or hydrophobic

interactions, or complexes of polypeptides that are joined together, covalently or noncovalently, as multimers (e.g., dimers, tetramers). Thus, the terms 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.

As used herein, the term "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.

While the polynucleotide 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.

As used herein, an "isolated" polypeptide or polynucleotide refers to a molecule that has been removed from a cell. For instance, 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.

Likewise, 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.

The term "and/or" means one or all of the listed elements or a combination of any two or more of the listed elements.

The words "preferred" and "preferably" refer to embodiments of the disclosure that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

It is understood that wherever embodiments are described herein with the language "include," "includes," or "including," and the like, otherwise analogous embodiments described in terms of "consisting of and/or "consisting essentially of are also provided.

Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

Conditions that are "suitable" for an event to occur, such as introduction of a

polynucleotide into a cell, or "suitable" conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and/or are

conducive to the event.

Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

In the description herein particular embodiments may be described in isolation for clarity. Unless otherwise expressly specified that the features of a particular embodiment are

incompatible with the features of another embodiment, certain embodiments can include a combination of compatible features described herein in connection with one or more

embodiments.

Reference throughout this specification to "one embodiment," "an embodiment," "certain embodiments," or "some embodiments," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 A-B shows a schematic presentation of plasmid constructs and their influence on high-fat diet-induced weight gain. (FIG. 1 A) Schematic presentation of plasmid constructs. SP=signal peptide, Pl=protein 1, P2=protein 2. (FIG. IB) Influence of these constructs on prevention of HFD-induced body weight gain. Male C57BL/6 mice were injected with plasmid DNA (20 μg per mouse) by hydrodynamic tail injection. The injected animals were kept on HFD (60% energy from fat) for 9 weeks. Values in (FIG. IB) represent average ± SD (n=5).

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 μg 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.

Treated animals were kept on HFD for additional 6 weeks. Values in (FIG. 2B) represent average ± SD (n=5).

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. Values in FIG. 3 A-D represent average ± SD (n=5). * P < 0.05 compared with the control, ** P < 0.01 compared with the control.

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)

Transcriptional levels of key genes responsible for chronic inflammation in EWAT. Values in FIG. 4A and FIG. 4C represent average ± SD (n=5). * P < 0.05 compared with the control, ** P < 0.01 compared with the control.

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. 5B) Areas under the curve (AUC) of glucose tolerance tests in FIG. 5 A. Values in FIG. 5 A and FIG. 5B represent average ± SD (n=5). ** P < 0.01 compared with the control. 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. 6D) Transcriptional levels of key genes responsible for lipogenesis in the liver. Values in FIG. 6A, FIG. 6B, and FIG. 6D represent average ± SD (n=5). * P < 0.05 compared with the control, ** P < 0.01 compared with the control.

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 μπι 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. 8D) Effect of plasmid transfer on body weight of CD-I mice with high-fat diet-induced obesity. Values in (FIG. 8D) represent average ± SD (n = 4). ** ⁵ < 0.01 compared with the control; ^## P < 0.01 compared with mice injected with pEx4. SP=signal peptide; Ex4=exendin 4; hAAT=human al antitrypsin; mAAT=mouse al antitrypsin.

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 μg 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. 91) Representative images of the liver and liver tissue sections stained by H&E and Oil red O. Bar=l cm. (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. Values in FIG. 9A, FIG. 9C, FIG. 9D, FIG. 9F-H, FIG. 9J, and FIG. 9K represent average ± SD (n = 5). * P < 0.05 compared with the control; ** P < 0.01 compared with the control.

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. (FIG. IOC) Profiles of glucose concentration (percentage of initial value) as a function of time upon intraperitoneal injection of insulin. (FIG. 10D) mRNA levels of Glut4 in WAT and BAT. (E) Representative images of H&E staining of the pancreatic islets. Values in FIG. 10A-D represent average ± SD (n = 5). ** P < 0.01 compared with the control.

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. 12 A)

Body weight change. (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. 12J) Representative images of H&E staining of the pancreatic islets. Values in FIG. 12A, C, D and F-I represent average ± SD (n = 5). * P < 0.05 compared with the control; ** P < 0.01 compared with the control.

FIG. 13A-E shows ^Jgene transfer blocks fatty liver development in ob/ob mice. (FIG.

13 A) Representative images of the liver and liver sections stained by H&E, Oil red O, or Nile red. Bar = 1 cm. (FIG. 13B) Liver weight. (FIG. 13 C) Liver triglyceride. (FIG. 13D) Blood concentrations of AST and ALT. (FIG. 13E) mRNA levels of critical genes controlling lipid and glucose metabolism in the liver. Values in FIG. 13B-E represent average ± SD (n = 5). * P < 0.05 compared with the control; ** P < 0.01 compared with the control.

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. 14E) Representative images of H&E staining of pivotal organs including the heart, liver, spleen, lungs, kidneys, pancreas, WAT and BAT. Values in FIG. 14A-D represent average ± SD (n = 5). * P < 0.05 compared with the control; ** P < 0.01 compared with the control.

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 (μ§:μ§). (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 μg of purified recombinant EAT protein, respectively. (FIG. 15D) Western blotting to verify the purified EAT protein using an anti-hAAT antibody - based detection system. M=protein marker.

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. Data were collected from 3 independent experiments. (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. 16D) Areas under the curve of glucose tolerance tests in (C). Values in FIG. 16A-) represent average ± SD (n=5). ** P < 0.01 compared with the control.

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. Values in FIG. 17B-D and FIG. 17F-I represent average ± SD (n = 5). * P < 0.05 compared with the control; ** P < 0.01 compared with the control.

FIG. 18A-C shows EAT protein treatment attenuates obesity-associated fatty liver in

C57BL/6 obese mice. (FIG. 18 A) Representative images of H&E staining and Oil red O staining of liver sections. (FIG. 18B) Liver triglyceride content. (FIG. 18C) Expression levels of genes encoding key components responsible for lipid production, lipid droplet formation, and chronic inflammation in the liver. Values in FIG. 18B-C represent average ± SD (n = 5). * P < 0.05 compared with the control; ** P < 0.01 compared with the control.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Provided herein are recombinant proteins having an agonist domain and anti- inflammatory domain. The order of the domains in the recombinant protein, from amino terminus to carboxy terminus, is the agonist domain followed by the anti-inflammatory domain. In one embodiment, the two domains are joined by an optional protein linker. A recombinant protein can be isolated or purified. In one embodiment, 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. Thus, 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. The protein being tested for GLP-1 agonist activity can be a separate protein or part of a recombinant protein described herein. Methods for evaluating whether a protein has GLP-1 agonist activity are described herein and include determining whether the protein causes a high-fat diet-induced obese mouse to have reduced body weight, reduced food consumption, or a combination thereof (see Example 1). In one embodiment, 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. In one embodiment, 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.

Examples of proteins that can be used as an agonist domain include, but are not limited to GLP-1 agonists. Examples of 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),

H(A/G)EGTFTSD VS S YLEGQ AAKEFIAWLVKG (SEQ ID NO:4), and

HGEGTF T SDL SKQMEEE A VRLFIE WLKNGGP S S GAPP SKKKKKK (SEQ ID NO:5). In one embodiment, the agonist domain depicted at SEQ ID NO:4 further includes an arginine at the C- terminal end, or a conservative substitution thereof.

Other examples of 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. In one embodiment, 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). From structure function information available, the skilled person has the predictable and reasonable expectation that certain regions of a GLP-1 agonist (e.g., non-conserved regions, individual amino acids, or a combination thereof) can be varied and that activity will not be affected. Likewise, the skilled person also has the predictable and reasonable expectation that varying certain regions of a GLP- 1 agonist (e.g., conserved regions, individual amino acids, or a combination thereof) will result in loss of activity.

The anti-inflammatory domain has anti-inflammatory activity. Thus, 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

10; 110(37): 15007-12). Other methods are also known and available to the skilled person (Cell Metab. 2013 Apr 2; 17(4):534-48, Arch Immunol Ther Exp (Warsz). 2012 Apr;60(2):81-97). In a preferred embodiment, 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

Francisco, CA). 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.

Examples of proteins that can be used as an anti-inflammatory domain include, but are not limited to, proteins that have anti-inflammatory activity. Examples of proteins having antiinflammatory activity include, but are not limited to, members of the serpin superfamily (Law et al., 2006, Genome Biology, 7:216). Specific examples of 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).

The amino acid sequence of proteins that can be used as an anti-inflammatory domain are known and readily available to the skilled person. One example of an anti-inflammatory protein is alpha-1 antitrypsin AAT:

EDPQGDAAQKTDTSHHDQDHPTF KITP LAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGL F LTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDT AL VNYIFFKGKWERPFEVKDTEEEDFHVDQ VTT VK WMMKRLGMFNIQHCKKL S S WVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLE EDRRSASLHLPKLSITGTY DLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAI PMSIPPEVKF KPFVFLMIEQNTKSPLFMGKVV PTQK (SEQ ID NO:6). Other examples of anti-inflammatory proteins useful as an anti-inflammatory domain include those having deletions of amino terminal amino acids of members of the serpin superfamily, such as serpinAl, serpinA3, and serpinA12. In one embodiment, 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., 2013, Theoretical Biol. Med Modelling, 10:36).

Other examples of 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

(see, for instance, Huntington, 2011, J Thromb Haemost 9(Suppl. l):6-34; Potempa et al., 1994, J. Biol. Chem., 269(23): 15957-15960; and Law et al., 2006, Genome Biology 7:216). From structure function information available, the skilled person has the predictable and reasonable expectation that certain regions of an anti-inflammatory protein (e.g., non-conserved regions, individual amino acids, or a combination thereof) can be varied and that activity will not be affected. Likewise, the skilled person also has the predictable and reasonable expectation that varying certain regions of an anti-inflammatory protein (e.g., conserved regions, individual amino acids, or a combination thereof) will result in loss of activity.

In one embodiment, 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. In one embodiment, a linker can be an amino acid sequence. For instance, 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).

In another embodiment, a linker can include an organic group. In one embodiment, 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. In another embodiment, 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.

In one embodiment, 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 PTQK. Another example of such a recombinant protein is SEQ ID NO:2:

HGEGTFT SDLSKQMEEEAVRLFIEWLKNGGP S SGAPPP SXEDPQGD AAQKTDT SHHDQD HPTF KITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGL F LTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHS EAFTVNFGDTEEAKKQINDYWKGTQGKIVDLVKELDRDTWALWYIFFKGKWERPFE VKDTEEEDFHVDQ VTT VK WMMKRLGMFNIQHCKKL S S WVLLMK YLGNAT AIFFLPDE GKLQHLENELTHDIITKFLENEDRRS ASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADL SGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQN TKSPLFMGKVVNPTQK, where X is a linker. In one embodiment, the linker is GGGGS (SEQ ID NO:7).

Also provided are recombinant proteins structurally similar to a recombinant protein described herein. As used 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. Thus, 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. In one embodiment, 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. In another embodiment, 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. For instance, 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. Likewise, 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. In one embodiment, a reference protein may be a recombinant protein described herein such as SEQ ID NO: 1 or SEQ ID NO:2. In another embodiment, 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.

Unless modified as otherwise described herein, 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). Alternatively, 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. The default values for all BLAST 2 search parameters may be used, including matrix =

BLOSUM62; open gap penalty = 11, extension gap penalty = 1, gap x dropoff = 50, expect = 10, wordsize = 3, and filter on.

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. For example, 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. Likewise, 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.

Thus, as used herein, 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.

Alternatively, as used herein, 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. For instance, a recombinant protein can include a tag, such as, but not limited to, a polyhistidine-tag (His-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. Optionally, the His-tag can then be cleaved. Other suitable affinity purification tags (e.g., maltose-binding protein) and methods of purification of proteins with those tags are known in the art.

In one embodiment, a recombinant protein described herein can include a signal peptide.

A signal peptide, often a continuous stretch of amino acids, 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.

Also provided are 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. In one embodiment, 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). Optionally, 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. In another embodiment, for instance when producing a recombinant protein having an organic group joining a GLP-1 agonist domain and an antiinflammatory domain, a polynucleotide can have a sequence encoding a GLP-1 agonist domain and a polynucleotide can have a sequence encoding an anti-inflammatory domain. The skilled person will appreciate that once the amino acid sequence of a protein is known, a 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

polynucleotide, i.e., an expression vector. The term vector includes, but is not limited to, plasmid vectors, viral vectors, cosmid vectors, and artificial chromosome vectors. Examples of viral vectors include, for instance, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, and herpes virus vectors. In one embodiment, a vector includes a

DNA transposon, and a polynucleotide encoding the recombinant protein is present between the inverted repeats of the DNA transposon. Typically, a vector is capable of replication in a host cell.

Selection of a vector depends upon a variety of desired characteristics in the resulting construct, such as a selection marker, vector replication rate, and the like. In some aspects, 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. In other aspects, 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. For example, the inclusion of 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.

In one embodiment, a vector can be useful in gene therapy. For instance, 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. As used herein, "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). As used herein, "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. In one embodiment, 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.

Also provided are compositions that include a recombinant protein described herein or a polynucleotide encoding a recombinant protein. Such compositions typically include a pharmaceutically acceptable carrier. As used herein "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. In general, a composition can be formulated to be compatible with its intended route of

administration. Administration may be systemic or local. Examples of 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. For parenteral administration, suitable carriers include physiological saline, bacteriostatic water, phosphate buffered saline (PBS), and the like. 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. 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. In many cases, it will be preferable to include 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.,

recombinant protein described herein or a polynucleotide encoding a recombinant protein) in the required amount in an appropriate solvent with one or a combination of ingredients routinely used in pharmaceutical compositions, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and any other appropriate ingredients. In the case of 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. For the purpose of oral therapeutic administration, 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. For administration by inhalation, 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. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such 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

suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

In those embodiments where a polynucleotide encoding a recombinant protein is administered, 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. 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. For a compound used in the methods described herein, 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). Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured using routine methods.

The compositions can be administered one or more times per day to one or more times per week, including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, 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.

Also provided are methods of using a protein and/or a polynucleotide described herein. In one embodiment, 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. In one embodiment, 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.

In one embodiment, 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. As used herein, the term "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. As used herein, the term "sign" 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. Typically, whether a subject has a condition, and whether a subject is responding to treatment, may be determined by evaluation of signs associated with the condition. In one embodiment, 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. In one embodiment, 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, 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.

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 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. In one embodiment, 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, whether by administration of the recombinant protein or administration of a polynucleotide encoding the recombinant protein, can result in the recombinant protein being present in blood, plasma, or serum. In one embodiment, 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. In one embodiment, the concentration of recombinant protein is no greater than 1000

milligrams/milliliter (mg/ml), no greater than 100 mg/ml, or no greater than 10 mg/ml. The 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 hematopoietic (e.g., blood or lymph) cells. Additional examples include stem cells, such as pluripotent or multipotent progenitor cells that develop or

differentiate into 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.

The subject can have signs of a condition. As used herein, 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.

Provided herein is a kit for treating a subject. The kit 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. Optionally, other reagents such as buffers and solutions needed to practice the disclosure are also included in separate containers. For instance, 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. As used herein, the phrase "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. In addition, the packaging material contains instructions indicating how the materials within the kit are employed to treat the subject. As used herein, 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.

Embodiments of the present disclosure are illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the disclosure as set forth herein.

Example 1

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. Overexpression of the EAT gene via a hydrodynamics-based procedure in mice prevented high-fat diet-induced obesity and insulin resistance and fatty liver. E^Jgene transfer to diet-induced obese animals induced weight loss, improved glucose homeostasis, and attenuated hepatic steatosis. In ob/ob mice, EAT gene transfer suppressed body weight gain and maintained metabolic homeostasis. The same set of metabolic benefits can also be achieved via EAT protein injection. Mechanistic studies reveal that the metabolic benefits obtained result from reduced food take and down-regulation of transcription of pivotal genes responsible for lipogenesis and lipid droplet formation in the liver and chronic inflammation in visceral fat. These results validate the feasibility of EA T therapy through gene transfer or protein administration in preventing and restoring metabolic homeostasis under diverse pathologic conditions and provide evidence in support of a new strategy to control obesity, fatty liver diseases and other metabolic disorders. Introduction

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.

This difficulty stems primarily from the complex pathophysiology of obesity, of which excess energy intake and adipose chronic inflammation are two major components [6-8]. Normal eating behavior in both humans and rodents is tightly controlled by the gut-brain axis, in which glucagon-like peptide-1 (GLP-1) plays a dominant role [9]. Responding to food intake, GLP-1 is produced by L-cells in the intestine and released into circulation, working on a variety of organs to regulate energy processing [9]. 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. Reduction in GLP-1 release has been shown in obese patients, resulting in excess energy intake and imbalance of metabolic metabolism [9-11]. In addition, chronic inflammation is also crucial in producing obesity-related metabolic dysregulations [7, 12, 13]. Without chronic inflammation, a healthy weight gain would not lead to glucose intolerance and hepatic fat aggregation [14-16].

In this study, we created a novel fusion gene for pharmacological intervention in obesity and related metabolic disorders. 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]. We abbreviated the fusion gene as EAT by taking the critical letters from Ex4 and A AT. Using the approaches of hydrodynamic gene transfer, we assessed the preventive and therapeutic effects of EAT on obesity, glucose homeostasis and hepatic steatosis in both high-fat diet-induced obese mice and genetically modified ob/ob mice. In this report, we provide direct evidence in support of the feasibility of a new fusion gene/protein-based strategy to manage obesity and related metabolic disorders.

Materials and Methods

Plasmid construction and preparation

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. For in vivo transgene overexpression, we used 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. A similar strategy and approach was used to clone other fusion genes into the same plasmid vector. Plasmid containing Ext4 and hAAT genes were similarly cloned to pLIVE vector. For preparation of EAT recombinant protein, 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

centrifugation. 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.

Preparation of EAT protein

We prepared 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). The transfection was performed with PEI (Mw=25,000 Da, branched) and conditions were optimized for weight ratio and the amount of DNA per plate. The optimized conditions were used for EAT overexpression in T-175 flasks. For transfection, 40 μg of plasmid DNA were mixed with 120 μg 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 μg 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).

Protease inhibition assay

The fluorescence-based elastase inhibition assay kit (#K782) was purchased from

BioVision (San Francisco, CA). The enzymatic assay was carried out following the protocol provided by manufacturer. In brief, purified EAT proteins were diluted at different

concentrations in the range of 0.0128-1000 μg/ml. The total reaction volume was 100 μΐ, and the excitation and emission wavelengths were 400 nm and 505 nm, respectively. The mixtures were incubated with substrate solution at 37 °C for 30 minutes before fluorescence intensity measurement using a Fluoro-Max 4 fluorimeter (Horiba Jobin Yvon).

Animals and treatments

All procedures performed on animals were approved by the Institutional Animal Care and Use Committee at the University of Georgia, Athens, Georgia (protocol number, #A2014-07- 008-Y1-A0). The high-fat diet (60% energy from fat) used in this study was purchased from Bio- Serv (#F3282, Frenchtown, NJ). Male C57BL/6 mice and CD-I mice were purchased from Charles River Laboratories (Wilmington, MA) and fed an HFD for 12-16 weeks to induce obesity. Leptin deficient ob/ob mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and kept on standard chow. Animals were housed under standard conditions with a 12 h light-dark cycle.

For the prevention study, animals were injected with plasmid DNA through a standard hydrodynamic tail vein injection and subsequently kept on HFD for 9 weeks. For repeated injection with normal CD1 mice, we hydrodynamically injected these mice 3 times, with an interval of 21 days, during the first 63 days. 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. For protein therapy, 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).

Histological examinations and liver triglyceride determination

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 μπι 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 μπι in thicknesses and fixed using 10% NBF. Liver triglyceride content was determined following a previously reported method with some modifications [42]. In brief, freshly collected 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).

Evaluation of glucose tolerance and insulin sensitivity

For the glucose tolerance test (GTT), mice were injected intraperitoneally with glucose at

2 g/kg body weight after 6 h fasting. Blood samples (10 μΐ each) were taken from the tail vein at varying time points and glucose concentrations were determined using glucose test strips and a TRUEtrack glucose meter purchased from Nipro Diagnostics Inc. (Fort Lauderdale, FL). For the insulin tolerance test (ITT), 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.

Determination of blood concentrations of insulin, TNFa, IL6, AST, and ALT

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.

Gene expression analysis

Total RNA was isolated using the TRIZOL reagent (Life Technologies, Grand Island, NY) for liver samples or RNeasy Lipid Tissue Mini Kit (QIAGEN, Valencia, CA) for adipose tissue samples according to the manufacturers' protocols. One microgram of total RNA was used for the first strand cDNA synthesis using a First-strand cDNA Synthesis kit purchased from OriGene (Rockville, MD). 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

Gene name Forward primer sequence (F) Reverse primer sequence (R)

Acc F: ATGGGCGGAATGGTCTCTTTC R: TGGGGACCTTGTCTTCATCAT

(SEQ ID NO: 10) (SEQ ID NO:52)

Acox F: CCGCCACCTTCAATCCAGAG R: CAA GTTCTCGATTTCTCGACGG

(SEQ ID NO: l l) (SEQ ID NO:53)

Adrp F: GACCTTGTGTCCTCCGCTTAT R: CAACCGCAATTTGTGGCTC

(SEQ ID NO: 12) (SEQ ID NO:54)

Atgl F: GGATGGCGGCATTTCAGACA R: CAAAGGGTTGGGTTGGTTCAG

(SEQ ID NO: 13) (SEQ ID NO:55)

Cdllb F: ATGGACGCTGATGGCAATACC R: TCCCCATTCACGTCTCCCA

(SEQ ID NO: 14) (SEQ ID NO:56)

Cdllc F: CTGGATAGCCTTTCTTCTGCTG R: GCACACTGTGTCCGAACTCA

(SEQ ID NO: 15) (SEQ ID NO:57)

Cd36 F: ATGGGCTGTGATCGGAACTG R: GTCTTCCCAATAAGCATGTCTCC

(SEQ ID NO: 16) (SEQ ID NO:58)

Chrebp F: AGATGGAGAACCGACGTATCA R: ACTGAGCGTGCTGACAAGTC

(SEQ ID NO: 17) (SEQ ID NO:59)

Cidea F: TGACATTCATGGGATTGCAGAC R: GGCCAGTTGTGATGACTAAGAC

(SEQ ID NO: 18) (SEQ ID NO:60)

Cptla F: CTCCGCCTGAGCCATGAAG R: CACCAGTGATGATGCCATTCT

(SEQ ID NO: 19) (SEQ ID NO:61)

Dgatl F: TCCGTCCAGGGTGGTAGTG R: TGAACAAAGAATCTTGCAGACG

(SEQ ID NO:20) (SEQ ID NO:62)

Dgat2 F: GCGCTACTTCCGAGACTACTT R: GGGCCTTATGCCAGGAAACT

(SEQ ID NO:21) (SEQ ID NO:63)

Dio2 F: AATTATGCCTCGGAGAAGACCG R: GGCAGTTGCCTAGTGAAAGGT

(SEQ ID NO:22) (SEQ ID NO:64)

Elov F: TTCTCACGCGGGTTAAAAATGG R: GAGCAACAGATAGACGACCAC

(SEQ ID NO:23) (SEQ ID NO:65)

F4/80 F: TGACTCACCTTGTGGTCCTAA R: CTTCCCAGAATCCAGTCTTTCC

(SEQ ID NO:24) (SEQ ID NO:66)

Fabp4 F: AAGGTGAAGAGCATCATAACCC R: TCACGCCTTTCATAACACATTCC

(SEQ ID NO:25) (SEQ ID NO:67)

Fas F: GGAGGTGGTG ATAGCCGGTAT R: TGGGTAATCCATAGAGCCCAG

(SEQ ID NO:26) (SEQ ID NO:68)

Fsp27 F: ATGGACTACGCCATGAAGTCT R: CGGTGCTAACACGACAGGG

(SEQ ID NO:27) (SEQ ID NO:69)

Fxr F: GCTTGATGTGCTACAAAAGCTG R: CGTGGTGATGGTTGAATGTCC

(SEQ ID NO:28) (SEQ ID NO:70)

G6p F: CGACTCGCTATCTCCAAGTGA R: GTTGAACCAGTCTCCGACCA

(SEQ ID NO:29) (SEQ ID NO:71)

Gapdh F: AGGTCGGTGTGAACGGATTTG R: TGTAGACCATGTAGTTGAGGTCA

(SEQ ID NO:30) (SEQ ID NO:72)

Hsl F: CCAGCCTGAGGGCTTACTG R: CTCCATTGACTGTGACATCTCG

(SEQ ID NO:31) (SEQ ID NO:73)

116 F: TAGTCCTTCCTACCCCAATTTCC R: TTGGTCCTTAGCCACTCCTTC

(SEQ ID NO:32) (SEQ ID NO:74)

Lead F: TCTTTTCCTCGGAGCATGACA R: GACCTCTCTACTCACTTCTCCAG

(SEQ ID NO:33) (SEQ ID NO:75)

Lpl F: GGGAGTTTGGCTCCAGAGTTT R: TGTGTCTTCAGGGGTCCTTAG

(SEQ ID NO:34) (SEQ ID NO:76)

Lxr F: CTCAATGCCTGATGTTTCTCCT R: TCCAACCCTATCCCTAAAGCAA

(SEQ ID NO:35) (SEQ ID NO:77)

Mead F: GGGTTTAGTTTTGAGTTGACGG R: CCCCGCTTTTGTCATATTCCG (SEQ ID NO:36) (SEQ ID NO:78)

Mcpl F: TTAAAAACCTGGATCGGAACCA R: GCATTAGCTTCAGATTTACGGGT

(SEQ ID NO:37) (SEQ ID NO:79)

Mgatl F: TGGTGCCAGTTTGGTTCCAG R: TGCTCTGAGGTCGGGTTCA

(SEQ ID NO:38) (SEQ ID NO:80)

Mgat2 F: TGGGAGCGCAGGTTACAGA R: CAGGTGGCATACAGGACAGA

(SEQ ID NO:39) (SEQ ID N0:81)

Oxpat F: TGTCCAGTGCTTACAACTCGG R: CAGGGCACAGGTAGTCACAC

(SEQ ID NO:40) (SEQ ID NO:82)

Pepck F: CTGCATAACGGTCTGGACTTC R: CAGCAACTGCCCGTACTCC

(SEQ ID NO:41) (SEQ ID NO:83)

Pgcla F: TATGGAGTGACATAGAGTGTGC R: CCACTTCA ATCCACCCAGAAAG

(SEQ ID NO:42) (SEQ ID NO:84)

Plinl F: GGGACCTGTGAGTGCTTCC R: GTATTGAAGAGCCGGGATCTTTT

(SEQ ID NO:43) (SEQ ID NO:85)

Ppara F: AGAGCCCCATCTGTCCTCTC R: ACTGGTAGTCTGCAAAACCAAA

(SEQ ID NO:44) (SEQ ID NO:86)

Pparyl F: GGAAGACCACTCGCATTCCTT R: GTAATCAGCAACCATTGGGTCA

(SEQ ID NO:45) (SEQ ID NO:87)

Ppary2 F: TCGCTGATGCACTGCCTATG R: GAGAGGTCCACAGAGCTGATT

(SEQ ID NO:46) (SEQ ID NO:88)

Ppard F: TCCATCGTCAACAAAGACGGG R: ACTTGGGCTCAATGATGTCAC

(SEQ ID NO:47) (SEQ ID NO:89)

Scd F: TTCTTGCGATACACTCTGGTGC R: CGGGATTGAATGTTCTTGTCGT

(SEQ ID NO:48) (SEQ ID NO:90)

Srebplc F: GCAGCCACCATCTAGCCTG R: CAGCAGTGAGTCTGCCTTGAT

(SEQ ID NO:49) (SEQ ID N0:91)

Tnfa F: CCCTCACACTCAGATCATCTTC R: GCTACGACGTGGGCTACAG

(SEQ ID NO:50) (SEQ ID NO:92)

Ucpl F: AGGCTTCCAGTACCATTAGGT R: CTGAGTGAGGCAAAGCTGATTT

(SEQ ID N0:51) (SEQ ID NO:93)

Determination of blood concentration of EAT protein

Standard ELISA protocol was followed to determine EAT protein concentration [39]. In brief, 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. After washing, 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. After washing, 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 μΐ/well) and washing buffer (400 μΐ/well), the sample volume used was 100 μΐ/well.

Statistics

Statistical analysis was performed using the Student's t test and one-way ANOVA. A P value below 0.05 (P < 0.05) was considered significantly different. All results were expressed as the mean ± SD.

Results

Plasmid constructs of fusion genes and in vivo assessment of their activity in suppressing high fat diet induced obesity

We constructed 6 plasmids coding for different fusion proteins including (1) hAAT- linker-S961 (fusion gene of human al antitrypsin with insulin receptor antagonist S961), (2) SCI-linker-hAAT (fusion gene of single chain insulin analog with human al antitrypsin), (3)

FGF21-linker-hAAT (fusion gene of fibroblast growth factor 21 with human al antitrypsin), (4) Ex4-linker-FGF21 (fusion gene of exendin 4 and fibroblast growth factor 21), (5) Ex4-linker- SOD3 (fusion gene of exendin 4 with superoxide dismutase 3), and (6) 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). 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.

The impacts of these constructs on diet-induced weight gain were assessed in mice. Each of the 6 plasmids (20 μg/mouse) was hydrodynamically injected into the tail vein of male C57BL/6 mice (n=5 each) that were kept on a high fat diet (EfFD) (60% energy from fat) for 9 weeks. Figure IB shows that, despite being constructed through the same strategy, these constructs showed different activity in suppressing diet-induced obesity. Four plasmids including phAAT-linker-S961, pSCI-linker-hAAT, pFGF21-linker-hAAT, and pEx4-linker-SOD3, showed no activity in suppressing FIFD-induced obesity. Animals injected with pEx4-linker-FGF21 exhibited a slight decrease in weight gain, while those treated with pEx4-linker-hAAT were blocked from FIFD-induced weight gain. During this 9-week FIFD feeding, the control animals injected with empty pLIVE gained 15.9 g compared to 2.1 g for animals injected with pEx4- linker-hAAT plasmids.

For the 5 constructs showing no potent activity, we performed a second injection of the same plasmid and examined whether they were able to reduce weight loss from the animals that were already obese. No weight reduction was seen in any of these animals with second plasmid injection (Figure 2). After 4 months FIFD feeding, all of the animals injected with either empty plasmid (control) or construct containing a fusion gene had a body weight around 50 g. These results suggest that, despite constructed through the same strategy, among all of 6 plasmids pEx4-linker-hAAT (pEAT) is the only one capable of blocking HFD-induced obesity. We therefore focused on this ϋ_4 fusion gene for further studies.

E^Jgene transfer blocks high-fat diet-induced weight gain, hyper adiposity, insulin resistance, fatty liver, and the expression of relevant genes

The impacts of EA T gene transfer on HFD-induced weight gain, glucose homeostasis, and fatty liver development were examined with 4 groups of C57/BL6 mice. Animals were hydrodynamically injected with 20 μg/mouse of pLIVE empty plasmid (control), or pEAT plasmid at 0.2 μg, 2.0 μg or 20.0 μg/mouse and fed an HFD for 9 weeks. Results in Figure 3A show that, at the end of the experiment, the blood concentration of EAT protein is -138 μg/ml in animals injected with 20.0 μg/mouse, compared to -50 μg/ml and -4 μg/ml with injection of 2.0 or 0.2 μg, respectively. The average food intake of animals (Figure 3B) 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 μg, 1.89 ± 0.13 with 2.0 μg, and 1.85 ± 0.17 g with 20.0 μg 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 μg (Figure 3C). There was no weight difference between animals injected with 2.0 and 20.0 μg pEAT plasmid. MRI analysis (Figure 3D) shows that the average lean mass of all animals is similar, but the fat mass is different, with control animals exhibiting the highest amount at -18.2 g, followed by that (-10.1 g) of animals injected with 0.2 μ . Approximately 4.9 g and 3.4 g of fat were seen in animals injected with 2.0 or 20.0 μg of pEAT plasmid, respectively.

White adipose tissue (WAT)were collected from each animal and their weight was measured. Results in Figure 4A show the highest amount of WAT in control mice, followed by animals with 0.2 μg of pEAT plasmid and the animals with the injection dose of 2.0 μg or 20.0 μg 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. The presence of macrophages in WAT, as indicated by CLS was confirmed by q-PCR analysis of macrophage specific marker genes. Figure 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).

E^Jgene transfer repressed diet induced liver enlargement (Figure 6A). The impact of EAT gene transfer on liver damage was assessed by the serum concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Concentrations of liver specific enzymes (Figure 6B) were higher in control mice (62 ±10 U/L, AST; 33 ± 6 U/L, ALT) compared to those of animals injected with pEAT plasmid (42 ± 7 U/L, AST; 24 ± 5 U/L, ALT). FIFD-induced hepatic fat accumulation was also blocked by EAT gene transfer as shown in Figure 6C by both H&E and Oil red O staining. Vacuole type structures seen in liver sections of control mice were not evidenced in mice receiving EAT gene transfer. No obvious fat accumulation was detected by Red oil O staining in pEAT treated animals compared to the controls. Results from q-PCR analysis of total RNAs extracted from the livers (Figure 6D) showed a decrease in the mRNA level of genes responsible for lipogenesis including Ppary2, Fsp27, and Cidea in animals injected with pEAT plasmid. These results demonstrate that a dose of 2.0 μg or greater of pEAT plasmid is sufficient to block FIFD-induced weight gain and obesity-associated macrophage activation, glucose resistance, and fatty liver development.

Further tissue section and H&E staining suggest that gene transfer did not produce any detectable histological change in the important organs including the heart, spleen, lungs, kidneys, and pancreas (Figure 7). These results suggest that ^Jgene transfer in animals attenuated HFD- induced adiposity, improved glucose intolerance and alleviated hepatic fat deposition, partly through suppressing food intake and relieving adipose chronic inflammation.

EAT gene transfer generated superior weight loss effect to either exendin 4 or hAAT alone in obese CD1 mice established via HFD feeding

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). To verify EA T expression and its activity, we hydrodynamically injected pEAT plasmids into obese mice and collected plasma samples 24 h after gene transfer. Western blotting confirmed EAT protein in mouse serum (Figure 8C), with apparent molecular weights of 56.5 kDa, compared to 52.0 and 51.2 kDa for hAAT and mouse AAT (mAAT), respectively. ^Jgene transfer produced a much stronger weight loss in CD-I mice with high-fat diet-induced obesity (Figure 8D) compared to that of pAAT and pEx4 at equal doses of 20 μg of plasmid DNA per mouse.

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

hydrodynamically injected pEAT plasmids into C57BL/6 mice with high-fat diet-induced obesity, a well-validated and widely used mouse model for obesity and related metabolic studies. Figure 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 (Figure 9C) 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). Consistently, 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).

E^Jgene transfer improves glucose homeostasis in obese mice

We next assessed the impact of EA T gene transfer on glucose metabolism in HFD- induced obese mice. Figure 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. Figure 10D shows that E^Jgene transfer increased Glut4 expression in WAT and BAT, by 1.9-fold and 2.7- fold, respectively. In alignment with the improved insulin sensitivity, E^Jgene transfer markedly reduced islet hypertrophy (Figure 10E). Importantly, 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

We next determined the activity of EAT gene transfer in the context of leptin deficiency in ob/ob mice. Overexpression of the EAT gene suppressed weight gain by -8.5 g (Figures 12A and 12B) during a feeding period of 8 weeks. The suppression in weight gain was primarily contributed by the lower increase in fat mass (Figure 12C), which was correlated with a decrease in food intake (Figure 12D). Histological examination of visceral fat revealed that E^Jgene transfer suppressed adipocyte hypertrophy and blocked development of CLS in WAT (Figure 12E). Further image quantification demonstrated that the average size of adipocytes in mice with E^Jgene transfer was -36.5% smaller than that of the controls (Figure 12F). Transcription levels of key inflammatory genes including F4/80, Cdllb, Mcp], T fa, and 116 were significantly lower in mice with ^Jgene transfer compared to the control (Figure 12G).

Consistent with the repressed weight gain and attenuated adipose chronic inflammation, tolerance tests revealed that ob/ob mice with EA T gene transfer had better glucose tolerance and insulin sensitivity (Figures 12H and 121). Histological examination using H&E staining demonstrated that ^Jgene transfer suppressed islet hypertrophy by -17.9% (Figure 12J).

EAT gene transfer blocks fatty liver in ob/ob mice

To study the pathological changes in the liver of ob/ob mice with or without EAT gene transfer, we performed a series of histological examinations. 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. To identify the factors potentially involved, we determined transcription levels of a variety of key genes responsible for lipid and glucose metabolism in the liver. 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).

Assessment of potential adverse effects of EA T gene transfer

Repeated injection of different amounts of pEAT plasmid was performed on CD-I mice fed standard chow and its impacts on animals were examined. Three doses of plasmid at 0.2, 2.0, and 20.0 μg/mouse were employed. Three injections of the same dose were performed on days 0, 22, and 44, and blood samples were collected after each injection. Results in Figure 14A show that blood concentrations of EAT protein are dose dependent. The peak level of EAT protein was -385 μg/ml in animals receiving 20.0 μg plasmid DNA, compared to -52 μg/ml in animals injected with 2.0 μg, and -5 μg/ml with 0.2 μg pEAT plasmid, respectively. EAT protein level declined slightly and regained peak levels with the 2 ^nd and 3 ^rd injection. Animals grow more slowly with higher doses of plasmid injection, but growth is evident at a later time, up to 6 months (Figure 14B). Body composition analysis shows the same amount of lean mass but different levels of fat mass depending on the amount of pEAT plasmid injected (Figure 14C). At the end of 6 months, animals were euthanized, and the internal organs were collected and weighed. 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). These data suggest that repeated injection of pEAT at different doses generated no detectable adverse effects nor immunogenicity. After 180 days, all animals injected with control plasmid or pEAT had comparable lean mass, while those treated with medium or high doses of pEAT plasmid showed lower fat mass.

Preparation, characterization, and verification of recombinant EAT protein

The above studies employing the gene transfer approach prove that pEAT is effective in blocking HFD-induced weight gain, insulin resistance, hyperglycosemia, and fatty liver development. And it also induces weight loss and improvement of glucose homeostasis and liver steatosis. To provide additional evidence in support of EAT activity, we prepared recombinant EAT protein and assessed its effects on C57BL/6 obese mice. FIEK293T cells were used to produce EAT protein and pcDNA3.1-EAT plasmids with His tag sequence and CMV promoter were used. Cells were transfected with polyethylenimine (PEI) in various conditions to optimize transfection efficiency (Figure 15 A). We typically use a PEI-to-DNA weight ratio of 3 : 1 for transfection. Two-step chromatography with Ni-column was employed to capture and polish EAT protein. Figure 15B shows that EAT protein was efficiently purified. In the range of 2-8 μg 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).

Verification of elastase inhibition activity and glucose-lowering activity of recombinant EAT protein

We validated the AAT activity of EAT protein through a fluorescence-based elastase inhibition assay. Purified EAT recombinant protein and hAAT were diluted at the same concentrations and added into a mixture of elastase and assay buffer with substrates. Florescence-based proteinase assay shows that purified EAT protein has the same activity as that of pure hAAT protein in inhibiting elastase activity (Figure 16 A). The exendin 4 peptide does not show any inhibition activity at equal molar level (Figures 16B). A glucose tolerance test was employed to assess the activity of exendin 4 in EAT. Compared to control animals and animals pre-injected with hAAT, animals pre-injected with either exendin 4 peptide or EAT protein induced a much faster decrease of blood glucose concentration (Figures 16C) with -40% decrease revealed by AUC (Figure 16D), suggesting a full preservation of exendin 4 activity in EAT. EAT protein therapy decreases body weight and promotes glucose tolerance in obese mice

We treated C57BL/6 mice with high-fat diet-induced obesity with a daily injection of EAT protein (Figure 17 A). Treatment with EAT recombinant protein significantly reduced body weight (-8.6%) and decreased average energy intake (Figures 17B and 17C). The weight loss was primarily contributed by the reduction in fat mass (Figure 17D). Protein therapy decreased the size of adipocytes in WAT and increased matrix density in BAT (Figure 17E). EAT protein injection down-regulated expression of genes encoding key proteins responsible for chronic inflammation in visceral white adipose tissue, including F4/80, Cdllb, Cdllc, Mcpl, Tnfa, and 116 (Figure 17F). Subsequent glucose intolerance tests revealed that EAT protein therapy significantly improved glucose homeostasis in obese mice, as evidenced by the increased glucose tolerance and improved insulin sensitivity (Figures 17G and 17H). Consistent with the attenuated insulin resistance, serum insulin levels of EAT protein-treated mice were significantly lower than those of the control animals (Figure 171).

EAT protein treatment attenuates fatty liver in obese mice

To examine the effect of EAT protein therapy on the liver, we performed H&E and Oil red O staining of the liver sections of treated obese mice. Figure 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).

Discussion

Obesity is a complex disease with severe comorbidities [21]. Targeting key components in obesity pathophysiology may prevent and reverse obesity and restore metabolic homeostasis. In the current study, we created a total of 6 fusion genes and found that the construct with 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. We demonstrated that both AAT and exendin 4 activities are preserved (Figure 16) in the EAT fusion protein. EAT gene transfer significantly reduced food intake in regular (Figure 3B), diet-induced and genetically obese mice (Figures 9D, 12D) and reduced adipose inflammation (Figures 4B, 9E, 12E). At the molecular level, 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). In addition, 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. In agreement with our findings, 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].

Beneficial effects of 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 μg of pEAT plasmid, although significant levels of EAT protein were also obtained at 0.2 μg/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]. Fusion of exendin 4 to hAAT creates larger protein molecules, which minimizes renal elimination. Physiological benefits seen in the animals are likely due to persistent exendin4 activity in EAT in suppressing food intake and regulating glucose homeostasis. Chronic inflammation in the adipose tissue is an integral component of obesity-associated metabolic dysregulations, particularly of insulin resistance [7]. Excess energy storage in fat causes adipocyte hypertrophy and apoptosis, leading to macrophage infiltration to remove the damaged adipocytes. Impairment of this process gives rise to the development of crown-like structures and causes sustained low-grade inflammation, resulting in compromised metabolic flexibility and insulin resistance [12, 13, 24]. Emerging evidence suggests that an imbalance between AAT and elastase substantially contributes to this pathological change [25]. Both obese men and mice had reduced levels of serum AAT, and importantly, AAT transgenic mice were protected from HFD-induced inflammation and insulin resistance [25]. Furthermore, AAT has also been tested recently in clinic for the treatment of diabetes, owing to its anti-inflammatory and immune-modulatory activities [26-28]. In alignment with these previous studies, we demonstrated in this study that EAT treatment suppressed adipocyte hypertrophy, blocked the formation of crown-like structures (Figures 6D, 9K, 13E), and thereby attenuated adipose inflammation and alleviated insulin resistance. However, because of the intricate connection between glucose metabolism and body weight, we cannot exclude the possibility that the improvement of insulin sensitivity is secondary to the reduction of body weight. It should be noted that insulin resistance is not always positively correlated with body weight. In fact, without adipose inflammation, some mouse strains are extremely obese while showing no sign of insulin resistance [14-16]. Conversely, when affected by adipose metabolic dysfunction and

inflammation, non-obese subjects in both humans and mice can exhibit insulin resistance [29, 30].

The hepatic fat deposition is controlled by a dynamic balance between liver lipid production, secretion, and communication with other organs [31]. In our study, 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. Though the underlying mechanism remains incompletely understood, recent studies demonstrate the existence of a GLP-1 receptor on hepatocytes, which directly implicates the GLP-1 pathway in hepatic fat regulation [32]. Extending these prior studies, we found in the current study that 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. In addition, the blocked hepatic steatosis may also be partly accounted for by improved liver-adipose tissue metabolic communication. It was reported earlier that the inter- organ crosstalk significantly affects lipid partitioning and storage in individual organs [36-38]. With the evidence presented herein, it is conceivable that EAT restores adipose dysfunction and promotes adipose storage capacity, thereby repressing lipid spillover and improving inter-organ crosstalk, resulting in a blockade of fatty liver development.

An important aspect of gene-based prevention and therapy is potential adverse effects.

Data shown in Figure 14 provide direct evidence in support that EAT over-expression is safe and does not result in undesirable consequence 6 months after EAT gene transfer with three repeated injections and doses.

In conclusion, by focusing on certain components of obesity pathophysiology we created a new fusion protein named EAT for the pharmacological management of obesity and its associated metabolic comorbidities. We found that in regular mice fed a HFD or standard chow, or in diet-induced C57BL/6 obese and ob/ob mice, that 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.

Flegal, KM, Carroll, MD, Kit, BK, and Ogden, CL (2012). Prevalence of obesity and trends in the distribution of body mass index among US adults, 1999-2010. JAMA 307: 491-497.

Pedersen, SD (2013). Metabolic complications of obesity. Best Pract Res Clin

Endocrinol Metab 27: 179-193.

Mann, T, Tomiyama, AJ, Westling, E, Lew, AM, Samuels, B, and Chatman, J (2007). Medicare's search for effective obesity treatments: diets are not the answer. Am Psychol 62: 220-233.

Pontzer, H, Durazo-Arvizu, R, Dugas, LR, Plange-Rhule, J, Bovet, P, Forrester, TE, et al. (2016). Constrained Total Energy Expenditure and Metabolic Adaptation to Physical Activity in Adult Humans. Curr Biol 26: 410-417.

Mark, AL (2008). Dietary therapy for obesity: an emperor with no clothes. Hypertension 51 : 1426-1434; discussion 1434.

Zhang, Y, Liu, J, Yao, J, Ji, G, Qian, L, Wang, J, et al. (2014). Obesity: pathophysiology and intervention. Nutrients 6: 5153-5183.

Monteiro, R, and Azevedo, I (2010). Chronic inflammation in obesity and the metabolic syndrome. Mediators Inflamm 2010.

Hill, JO, Wyatt, HR, and Peters, JC (2012). Energy balance and obesity. Circulation 126: 126-132.

Hoist, JJ (2007). The physiology of glucagon-like peptide I . Physiol Rev 87: 1409-1439.

Prasad-Reddy, L, and Isaacs, D (2015). A clinical review of GLP-1 receptor agonists: efficacy and safety in diabetes and beyond. Drugs Context 4: 212283.

Garber, AJ (2011). Long-acting glucagon-like peptide 1 receptor agonists: a review of their efficacy and tolerability. Diabetes Care 34 Suppl 2: S279-284.

Xu, H, Barnes, GT, Yang, Q, Tan, G, Yang, D, Chou, CJ, et al. (2003). Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance. J Clin Invest 112: 1821-1830. 13. Weisberg, SP, McCann, D, Desai, M, Rosenbaum, M, Leibel, RL, and Ferrante, AW, Jr. (2003). Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 112: 1796-1808.

14. Hotamisligil, GS, Shargill, NS, and Spiegelman, BM (1993). Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 259:

87-91.

15. Kim, JY, van de Wall, E, Laplante, M, Azzara, A, Trujillo, ME, Hofmann, SM, et al.

(2007). Obesity-associated improvements in metabolic profile through expansion of adipose tissue. J Clin Invest 111: 2621-2637.

16. Kusminski, CM, Holland, WL, Sun, K, Park, J, Spurgin, SB, Lin, Y, et al. (2012).

Mito EET-driven alterations in adipocyte mitochondrial activity reveal a crucial adaptive process that preserves insulin sensitivity in obesity. Nat Med 18: 1539-1549.

17. Janciauskiene, SM, Bals, R, Koczulla, R, Vogelmeier, C, Kohnlein, T, and Welte, T

(2011). The discovery of alphal -antitrypsin and its role in health and disease. RespirMed 105: 1129-1139.

18. Bergin, DA, Hurley, K, McElvaney, NG, and Reeves, EP (2012). Alpha- 1 antitrypsin: a potent anti-inflammatory and potential novel therapeutic agent. Arch Immunol Ther Exp (Warsz) 60: 81-97.

19. Koulmanda, M, Bhasin, M, Fan, Z, Hanidziar, D, Goel, N, Putheti, P, et al. (2012). Alpha 1 -antitrypsin reduces inflammation and enhances mouse pancreatic islet transplant survival. Proc Natl AcadSci USA 109: 15443-15448.

0. Kelley, LA, Mezulis, S, Yates, CM, Wass, MN, and Sternberg, MJ (2015). The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10: 845-858.

1. Guh, DP, Zhang, W, Bansback, N, Amarsi, Z, Birmingham, CL, and Anis, AH (2009).

The incidence of co-morbidities related to obesity and overweight: a systematic review and meta-analysis. BMC Public Health 9: 88.

2. Kooijman, S, Wang, Y, Parlevliet, ET, Boon, MR, Edelschaap, D, Snaterse, G, et al.

(2015). Central GLP-1 receptor signalling accelerates plasma clearance of triacylglycerol and glucose by activating brown adipose tissue in mice. Diabetologia 58: 2637-2646. 23. Wooddell, CI, Reppen, T, Wolff, JA, and Herweijer, H (2008). Sustained liver-specific transgene expression from the albumin promoter in mice following hydrodynamic plasmid DNA delivery. J Gene Med 10: 551-563.

24. Bu, L, Gao, M, Qu, S, and Liu, D (2013). Intraperitoneal injection of clodronate

liposomes eliminates visceral adipose macrophages and blocks high-fat diet-induced weight gain and development of insulin resistance. AAPSJ 15: 1001-1011.

25. Mansuy-Aubert, V, Zhou, QL, Xie, X, Gong, Z, Huang, JY, Khan, AR, et al. (2013).

Imbalance between neutrophil elastase and its inhibitor alphal -antitrypsin in obesity alters insulin sensitivity, inflammation, and energy expenditure. CellMetab 17: 534-548. 26. Fleixo-Lima, G, Ventura, H, Medini, M, Bar, L, Strauss, P, and Lewis, EC (2014).

Mechanistic evidence in support of alphal -antitrypsin as a therapeutic approach for type 1 diabetes. J Diabetes Sci Technol 8: 1193-1203.

27. Rachmiel, M, Strauss, P, Dror, N, Benzaquen, H, Horesh, O, Tov, N, et al. (2016).

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.

28. Jonigk, D, Al-Omari, M, Maegel, L, Muller, M, Izykowski, N, Hong, 1 et al. (2013).

Anti-inflammatory and immunomodulatory properties of alphal -antitrypsin without inhibition of elastase. Proc Natl Acad Sci USA 110: 15007-15012.

29. Wu, HT, Chang, CK, Tsao, CW, Wen, YJ, Ling, SM, Cheng, KC, et al. (2009). Insulin resistance without obesity induced by cotton pellet granuloma in mice. Lab Invest 89:

362-369.

30. Hammarstedt, A, Graham, TE, and Kahn, BB (2012). Adipose tissue dysregulation and reduced insulin sensitivity in non-obese individuals with enlarged abdominal adipose cells. Diabetol Metab Syndr 4: 42.

31. Tiniakos, DG, Vos, MB, and Brunt, EM (2010). Nonalcoholic fatty liver disease:

pathology and pathogenesis. Annu Rev Pathol 5: 145-171.

32. Gupta, NA, Mells, J, Dunham, RM, Grakoui, A, Handy, J, Saxena, NK, et al. (2010).

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, Gavrilova, 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.

34. Yu, S, Matsusue, K, Kashireddy, P, Cao, WQ, Yeldandi, V, Yeldandi, AV, et al (2003).

Adipocyte-specific gene expression and adipogenic steatosis in the mouse liver due to peroxisome proliferator-activated receptor gammal (PPARgammal) overexpression. J Biol Chem 278: 498-505.

35. Gao, M, Ma, Y, Alsaggar, M, and Liu, D (2016). Dual Outcomes of Rosiglitazone

Treatment on Fatty Liver. AAPS J 18 : 1023 - 1031.

36. Nov, O, Shapiro, H, Ovadia, H, Tarnovscki, T, Dvir, I, Shemesh, E, et al (2013).

Interleukin-lbeta regulates fat-liver crosstalk in obesity by auto-paracrine modulation of adipose tissue inflammation and expandability. PLoS One 8: e53626.

37. Gao, M, Bu, L, Ma, Y, and Liu, D (2013). Concurrent activation of liver X receptor and peroxisome proliferator-activated receptor alpha exacerbates hepatic steatosis in high fat diet-induced obese mice. PLoS One 8: e65641.

38. Moschen, AR, Wieser, V, and Tilg, H (2012). Adiponectin: key player in the adipose tissue-liver crosstalk. CurrMed Chem 19: 5467-5473.

39. Zhang, G, Song, YK, and Liu, D (2000). Long-term expression of human alphal- antitrypsin gene in mouse liver achieved by intravenous administration of plasmid DNA using a hydrodynamics-based procedure. Gene Ther 7: 1344-1349.

40. Liu, F, Song, Y, and Liu, D (1999). Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther 6: 1258-1266.

41. Gao, M, Ma, Y, Cui, R, and Liu, D (2014). Hydrodynamic delivery of FGF21 gene

alleviates obesity and fatty liver in mice fed a high-fat diet. J Control Release 185: 1-11. 42. Hara, A, and Radin, NS (1978). Lipid extraction of tissues with a low-toxicity solvent.

AnalBiochem 90: 420-426.

43. Livak, KJ, and Schmittgen, TD (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402-408. The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. Supplementary materials referenced in publications (such as supplementary tables, supplementary figures, supplementary materials and methods, and/or supplementary experimental data) are likewise incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The disclosure is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the disclosure defined by the claims.

Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.