Plasmid DNA constructs for peptide hormone expression

Abstract

In various aspects and embodiments, the present disclosure provides DNA constructs and compositions thereof for expressing peptide hormones in muscle tissue, and particularly peptide hormones such as glucagon-like peptide-1 (GLP-1) receptor agonists, glucose- dependent insulinotropic polypeptide (GIP) receptor agonists, and / or glucagon (GCG) receptor agonists, as well as dual and triple agonists. The disclosure further provides uses of these constructs in methods for treating disease, including metabolic diseases or diseases involving hyperglycemia, insulin resistance, obesity, and / or metabolic syndrome.

Description

[0001]Attorney Docket No.: RBF-003PC / 111846-5003 PLASMID DNA CONSTRUCTS FOR PEPTIDE HORMONE EXPRESSION CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 597,078, filed on November 8, 2023, the entire contents of which are incorporated herein. FIELD OF THE DISCLOSURE The present disclosure relates to, in part, DNA compositions comprising expression constructs for in vivo production of peptide hormones, including but not limited to a glucagon-like peptide-1 (GLP-1) receptor agonists, glucose-dependent insulinotropic polypeptide (GIP) receptor agonists, glucagon receptor agonists (GCG), as well as dual and triple agonists of the GLP-1, GIP, and GCG receptors. The present disclosure further relates to the use of such DNA constructs and methods for treating or preventing disease. DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY The instant application contains a sequence listing, which has been submitted in XML format via EFS-Web. The contents of the XML copy named “RBF-003PC_111846- 5003_Sequence_Listing”, which was created on November 8, 2024 and is 45,056 bytes in size, the contents of which are incorporated herein by reference in their entirety. BACKGROUND While protein and peptide therapeutics are widely used for treatment and prevention of disease (including chronic disease), conventional protein drugs generally require repeated (often frequent) administrations. As a result, it is difficult to maintain conventional protein drugs within their therapeutic window for long periods of time, thereby leading to inconvenient dosing schedules, ineffective therapy, and adverse side effects. For example, several long-lasting incretins, such as GLP-1 receptor agonists, have been developed for use in individuals with type 2 diabetes, including dulaglutide (Trulicity®), exenatide (Byetta®, Bydureon®), liraglutide (Victoza®), lixisenatide (Lyxumia®) and semaglutide (Ozempic®). These GLP-1 receptor agonists were developed and formulated in most cases for injectable or oral administration to patients, often daily or 1 DB1 / 142158622.1 Attorney Docket No.: RBF-003PC / 111846-5003 weekly. However, patient adherence to these administration schedules can be poor which prohibits many patients from realizing a full and lasting therapeutic potential for this chronic disease. Accordingly, there remains a need for improved therapy with GLP-1 receptor agonists and related agents, and which can reduce the administration frequency while maintaining steady circulating levels. In the various aspects and embodiments, the present disclosure addresses these and other problems. SUMMARY In various aspects and embodiments, the present disclosure provides DNA constructs and compositions thereof for expressing peptide hormones in muscle tissue, and particularly peptide hormones such as glucagon-like peptide-1 (GLP-1) receptor agonists, glucose- dependent insulinotropic polypeptide (GIP) receptor agonists, and / or glucagon (GCG) receptor agonists, as well as dual and triple agonists of the GLP-1, GIP, and GCG receptors. The disclosure further provides uses of these constructs in methods for treating disease, including metabolic diseases or diseases involving hyperglycemia, insulin resistance, obesity, and / or metabolic syndrome. The present disclosure in various aspects and embodiments provides DNA constructs encoding peptide hormones for sustained and durable expression in a mammalian host. In an aspect, the present disclosure provides a plasmid construct (and compositions comprising the same) having at least one expression cassette. The expression cassette comprises in the following order from 5´ to 3´: a) a CMV IE enhancer sequence; b) a chicken beta-actin (CBA) promoter sequence; c) a CMV IE intron A sequence; d) an open reading frame encoding one or more peptide hormones, such as an agonist at one or more of glucagon-like peptide-1 (GLP-1) receptor, glucose-dependent insulinotropic polypeptide (GIP) receptor, and / or a glucagon (GCG) receptor; and e) a transcription termination sequence. In embodiments, the DNA composition encodes the peptide hormone as a polypeptide fusion with a carrier protein for PK enhancement, optionally through a linking DB1 / 142158622.1 2 Attorney Docket No.: RBF-003PC / 111846-5003 sequence (e.g., Gly Ser linker as described herein). In embodiments, the polypeptide fusion is with IgG Fc, albumin, or albumin binding domain. In some embodiments, the polypeptide fusion is Fc, and may be IgG1, IgG2, or IgG4 Fc, which allows for dimeric presentation of the agonist due to formation of disulfide bridges between Fc chains. In embodiments, the construct comprises or further comprises a fusion of Angiopep-2. In embodiments, the construct encodes a GLP-1 receptor agonist, such as GLP-1 or exenatide, or variations thereof. An exemplary amino acid sequence for a GLP-1-hIgG4 Fc fusion construct is provided herein as SEQ ID NO: 16. A DNA coding sequence for SEQ ID NO: 16 is provided as SEQ ID NO: 17. The fusion protein of SEQ ID NO: 16 includes a signal peptide for cell secretion, and an optional antigenic tag (e.g., HIS tag). Since the Fc includes the hinge region, these constructs form homodimeric polypeptides via a disulfide bridge. In embodiments, the open reading frame encodes GIP, or a derivative thereof. An exemplary amino acid sequence for a GIP-hIgG4 Fc fusion construct is provided herein as SEQ ID NO: 18. A DNA coding sequence for SEQ ID NO: 18 is provided as SEQ ID NO: 19. The fusion protein of SEQ ID NO: 18 includes a signal peptide for cell secretion, and an optional antigenic tag (e.g., HIS tag). Since the Fc includes the hinge region, these constructs form homodimeric polypeptides via a disulfide bridge. When expressed with GLP-1 constructs using expression constructs disclosed herein for expressing two proteins (either in one or two open reading frames), both homodimeric and heterodimeric polypeptides are formed in vivo. In embodiments, the open reading frame encodes a dual agonist for GLP-1 receptor and GIP receptor. For example, the open reading frame may encode the amino acid sequence of tirzepatide or a derivative thereof. An exemplary amino acid sequence for a GLP-1 receptor / GIP receptor dual agonist (tirzepatide) with hIgG4 Fc fusion is provided herein as SEQ ID NO: 20. A DNA coding sequence for SEQ ID NO: 20 is provided as SEQ ID NO: 21. The fusion protein of SEQ ID NO: 20 includes a signal peptide for cell secretion, and an optional antigenic tag (e.g., HIS tag). Since the Fc includes the hinge region, these constructs form homodimeric polypeptides via a disulfide bridge. DB1 / 142158622.1 3 Attorney Docket No.: RBF-003PC / 111846-5003 In embodiments, the open reading frame encodes a triple agonist for GLP-1 receptor, GIP receptor, and GCG receptor. For example, the agonist may comprise the amino acid sequence of retratrutide, or a derivative thereof. An exemplary amino acid sequence for a triple agonist (retratrutide) with hIgG4 Fc fusion is provided herein as SEQ ID NO: 22. A DNA coding sequence for SEQ ID NO: 22 is provided as SEQ ID NO: 23. The fusion protein of SEQ ID NO: 22 includes a signal peptide for cell secretion, and an optional antigenic tag (e.g., HIS tag). Since the Fc includes the hinge region, these constructs form homodimeric polypeptides via a disulfide bridge. In embodiments, the open reading frame encodes a dual agonist for GLP-1 receptor and GCG receptor. For example, the open reading frame may encode the amino acid sequence of SEQ ID NO: 14, or a derivative thereof. An exemplary amino acid sequence for a GLP-1 receptor / GCG receptor dual agonist with hIgG4 Fc fusion is provided herein as SEQ ID NO: 24. A DNA coding sequence for SEQ ID NO: 24 is provided as SEQ ID NO: 25. The fusion protein of SEQ ID NO: 24 includes a signal peptide for cell secretion, and an optional antigenic tag (e.g., HIS tag). Since the Fc includes the hinge region, these constructs form homodimeric polypeptides via a disulfide bridge. In embodiments, the plasmid construct comprises two of said expression cassettes expressing different agonist sequences. For example, a first expression cassette can comprise an open reading frame encoding a GLP-1 receptor agonist; and a second expression cassette can comprise an open reading frame encoding a GIP receptor agonist or and GCG receptor agonist. In embodiments, the first expression cassette has an open reading frame encoding a GLP-1 receptor agonist (e.g., GLP-1 or exenatide); and the second expression cassette has an open reading frame encoding a GIP receptor agonist (e.g., GIP). In each case, the agonist may be expressed as a fusion with a carrier protein (such as IgG Fc) as described. Other agents that can be expressed or co-expressed using the expression constructs in embodiments include agents such as Bimagrumab, Maridebart, and Cagrilintide. In embodiments, the open reading frame encodes two members of the group of a GLP-1 receptor agonist, a GIP receptor agonist, and a GCG receptor agonist, with a peptide linker therebetween that induces ribosomal skipping, thereby allowing for both polypeptides DB1 / 142158622.1 4 Attorney Docket No.: RBF-003PC / 111846-5003 to be produced without translational fusion. The result of ribosomal skipping is co- translational cleavage of the nascent polyprotein. In each case, the GLP-1 receptor agonist, the GIP receptor agonist, and / or the GCG receptor agonist may comprise a fusion to a polypeptide for PK enhancement as described (e.g., IgG Fc). In some embodiments, the GLP-1 receptor agonist is GLP-1 or exenatide, and which is expressed with a GIP receptor agonist (e.g., GIP) in a single open reading frame. Exemplary constructs for co-expressing two proteins in a single open reading frame are provided herein as SEQ ID NOS: 8 and 9. In embodiments, proteins encoded by the construct may further encode detectable tags (such as antigen tags), to allow expression to be monitored over time. An exemplary tag is a HIS tag. In some embodiments, an exemplary construct (without the open reading frame) comprises the expression cassette of SEQ ID NO: 6. SEQ ID NO: 6 comprises the following elements: (a) a CMV IE enhancer sequence; (b) a CBA promoter sequence; (c) a CMV IE intron A sequence; and (d) a BGHpA sequence. Therapeutic polypeptide open reading frames can be cloned into the cassette of SEQ ID NO: 6 at a cloning site downstream of the CMV IE intron A sequence, such that the elements are functional to direct expression of the therapeutic polypeptide. Together, such elements provide for high expression of a therapeutic protein in mammalian cells (e.g., muscle cells). In some embodiments, an exemplary construct comprises the expression cassette of SEQ ID NO: 7. SEQ ID NO: 7 has elements similar to SEQ ID NO: 6, but includes a shorter CMV IE intron A, and is therefore desirable for constructing dual expression cassettes, where two (or more) expression cassettes are included on the plasmid (e.g., in tandem). Dual expression cassettes may be used to express, for example, a GLP-1 receptor agonist and a GIP receptor agonist (including as Fc fusions as described herein, or as fusions with other carrier proteins) on a single plasmid. In embodiments, an exemplary construct comprises the expression cassette of SEQ ID NO: 8. SEQ ID NO: 8 comprises the following elements: (a) an CMV IE enhancer sequence; (b) a CBA promoter sequence; (c) a CMV IE intron A sequence; (d) a furin-P2A sequence, and (e) a BGHpA sequence, which together provide for high expression of two DB1 / 142158622.1 5 Attorney Docket No.: RBF-003PC / 111846-5003 proteins in mammalian cells (e.g., muscle cells), with each polypeptide coding sequence cloned in frame on either side of the furin-P2A sequence. In embodiments, an exemplary construct comprises the expression cassette of SEQ ID NO: 9. SEQ ID NO: 9 comprises the following elements: (a) an CMV IE enhancer sequence; (b) a CBA promoter sequence; (c) a CMV IE intron A sequence; (d) a furin-T2A sequence, and (e) a BGHpA sequence, which together provide for high expression of two therapeutic proteins in mammalian cells (e.g., muscle cells), with each polypeptide coding sequence cloned in frame on either side of the furin-T2A sequence. In other aspects, the present disclosure provides a use of the DNA constructs described herein for treating or preventing disease. Accordingly, the present disclosure provides a method for treating or preventing a disease in a subject, where the method comprises administering an effective amount of the composition described herein to a subject in need thereof. In various embodiments, the disease is a metabolic disease. In some embodiments, the compositions of the disclosure find use in the treatment or prevention of various diseases or disorders related to obesity or overweight, diabetes mellitus, dysglycemia, and metabolic syndrome. In various embodiments, the constructs and methods described herein provide for stable expression (e.g., in muscle cells) for at least about 4 months, at least about 6 months, at least about 1 year, or at least about 18 months, or at least about 2 years. The methods described herein can be used with or without significant caloric restriction. In various embodiments, the composition is administered parenterally, such as by intramuscular injection. In embodiments, administering further comprises electroporation. In various embodiments, the disclosure reduces the frequency of administration, for example, as compared to administering protein drugs. In embodiments, administration of the compositions described herein is no more than about monthly. In embodiments, administration is no more than about quarterly, or no more about twice per year. In embodiments, administering is no more than about annually, or is about annually. DB1 / 142158622.1 6 Attorney Docket No.: RBF-003PC / 111846-5003 In embodiments, the method can further comprise monitoring for the presence of the expressed protein in the circulation of the subject. Such monitoring can be conducted at least once between administrations. For example, monitoring can be conducted from one to 12 times per year, in embodiments. Monitoring can be conducted by immunoassay, for example, using antibodies that detect the agonist or an antigenic tag included on the construct. Other aspects and embodiments of the invention will be apparent from the following detailed description and working examples. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an image showing delivering of a therapeutic protein-encoding plasmid (pDNA) into a muscle cell, followed by expression and production of the antibody or therapeutic protein in the muscle cell, and uptake of the antibody or therapeutic protein in the circulation. FIG. 2 illustrates a hurdle facing protein therapeutics, as protein therapeutics encounter a significant dilutional effect as the blood volume increases from e.g., a rabbit to a human. FIG. 3 illustrates the lengthy time and cost consuming process for manufacturing proteins, antibodies, and drug-related products by current methods in the field. FIG.4A, FIG.4B, FIG. 4C, and FIG.4D are graphs demonstrating the application of the plasmid DNA constructs disclosed herein to a diverse set of disease indications in mouse disease models. FIG. 4A shows intramuscular delivery of a construct encoding an anti-HER2 antibody in a mouse model of breast cancer. FIG. 4B shows intramuscular delivery of a construct encoding an anti-TNFα antibody in a mouse model of arthritis. FIG. 4C shows intramuscular delivery of a construct encoding an anti-Influenza A antibody in a mouse model for treatment of Flu. FIG. 4D shows intramuscular delivery of a construct encoding EPO in a mouse model for treatment of anemia. DB1 / 142158622.1 7 Attorney Docket No.: RBF-003PC / 111846-5003 FIG. 5 illustrates plasmid DNA constructs according to embodiments of the disclosure. Constructs in FIG. 5 show constructs encoding antibody heavy (HC) and light (LC) chains, but it is understood that open reading frames can encode therapeutic peptides and Fc fusions disclosed herein. FIG.6 shows two graphs, labelled 1 and 2, demonstrating in vivo expression level of antibodies (both heavy and light chains) in mice using plasmid constructs according to the disclosure. FIG. 7 is a graph comparing CMV and CBA promoters in the plasmid constructs according to the present disclosure. FIG.8 is a graph showing the durability of protein expression (mouse anti-Influenza A antibody) over a long time course (i.e., at least 1 year). FIG.9 illustrates DNA constructs expressing a therapeutic protein (4D5 antibody). FIG.10 is a graph showing the serum antibody concentration levels over a period of 1 year for different DNA constructs encoding the herceptin 4D5 antibody. FIG.11 illustrates DNA constructs expressing a therapeutic protein (s139 antibody). FIG.12 is a graph showing the serum antibody concentration levels over a period of 1 year for different DNA constructs encoding s139 IgG. FIG.13 shows a series of DNA constructs for expressing a therapeutic protein and employing various promoter designs. FIG. 14A, FIG. 14B, FIG. 14C, and FIG. 14D are graphs showing the levels of a therapeutic protein (s139 IgG) expressed in mice from different DNA constructs of FIG.13. FIG. 15 illustrates the design of Fc fusion constructs according to the present disclosure. Constructs in FIG.15 are shown with murine Fc for pharmacokinetic studies. FIG. 16A, FIG. 16B, FIG. 16C, and FIG. 16D are graphs showing electroporation evaluation of plasmid constructs encoding GLP-1 Fc fusion (FIG. 16A), GLP-1 Fc fusion DB1 / 142158622.1 8 Attorney Docket No.: RBF-003PC / 111846-5003 with GIP Fc fusion (FIG. 16B), GLP-1 / GIP dual agonist Fc fusion (FIG. 16C), and GLP- 1 / GIP / GCG triple agonist Fc fusion (FIG.16D). FIG.17 is a series of graphs showing the effects (weight gain / loss) of the constructs encoding non-fused GLP-1 in a mouse diet-induced obesity model. FIG. 18 is a series of graphs showing the impact of the constructs encoding non- fused GLP-1 peptide on glycemia. FIG. 19 shows a series of graphs comparing the effect of GLP-1-mIgG1 protein administration with Semaglutide (subcutaneous injection). FIG. 20 shows a series of graphs showing the effects (weight gain / loss) upon administering a GLP-1 fusion construct including fusion of Angiopep-2. FIG.21 are graphs showing expression of Bimagrumab or Maridebart in mice upon delivery of encoding DNA constructs. FIG.22 are graphs showing expression of human IgG4 fusion constructs (GLP-1 and GLP-1 / GIP dual agonist) in mice. FIG. 23 are graphs showing the results (weight gain / loss and food intake) of constructs encoding GLP-1 or GLP-1 / GIP dual agonists fused to an albumin binding domain (ABD035). FIG. 24 are graphs showing the results (weight gain / loss, food intake, and expression) of constructs encoding GLP-1-hIgG4 and GLP-1 / GIP-hIgG4). FIG.25A, FIG.25B, FIG.25C, and FIG.25D are graphs showing the results upon co-expression of additional therapeutic agents - Maridebart, Bimagrumab, and Cagrilintide—, along with GLP-1-Angiopep-2 fusion construct. DETAILED DESCRIPTION In various aspects and embodiments, the present disclosure provides DNA constructs and compositions thereof for expressing peptide hormones in muscle tissue, and particularly DB1 / 142158622.1 9 Attorney Docket No.: RBF-003PC / 111846-5003 peptide hormones such as glucagon-like peptide-1 (GLP-1) receptor agonists, glucose- dependent insulinotropic polypeptide (GIP) receptor agonists, and / or glucagon (GCG) receptor agonists, as well as dual and triple agonists for such receptors. The disclosure further provides uses of these constructs in methods for treating disease, including metabolic diseases or diseases involving hyperglycemia, dysglycemia, insulin resistance, obesity or overweight, and / or metabolic syndrome. The present disclosure in various aspects and embodiments provides DNA constructs encoding peptide hormones for sustained and durable expression in a mammalian host. The plasmid DNA constructs disclosed herein allow for production of such agents inside a patient’s cell (e.g., a muscle cell, or myocyte), such as a skeletal muscle cell. Such a production process allows for a muscle cell to act as an in vivo bioreactor in the expression and production of the peptide hormone. The plasmid DNA constructs disclosed herein lack cold-chain storage requirements, and allow for the simultaneous expression and production of multiple polypeptides in vivo, and compared to conventional protein therapeutic delivery, the administration of the DNA constructs disclosed herein can result in a significant decrease in administration frequency and maintain the circulating levels within a therapeutic window for a long duration (e.g., at least 4 months, or at least 6 months, or at least 12 months, or at least 18 months, or at least 24 months). Accordingly, the plasmid DNA constructs disclosed herein allow for the treatment or prevention of chronic disease using the patient’s own cells for the steady production of the therapeutic agent over time. An overview of the plasmid DNA construct and delivery system, as disclosed herein, is illustrated in FIG. 1. This figure shows the intramuscular injection of a plasmid DNA construct having an expression cassette as described herein. The plasmid DNA construct is delivered to muscle cells, allowing for steady expression and production of the therapeutic agent by the muscle cell. The expressed polypeptide moves into peripheral circulation, similar to a conventional therapeutic protein administered intravenously or subcutaneously. The plasmid DNA constructs described herein overcome many of the roadblocks relevant to typical therapeutic protein manufacturing and development, which are illustrated in FIG.3. The plasmid DNA constructs significantly reduce the timeline for manufacturing, DB1 / 142158622.1 10 Attorney Docket No.: RBF-003PC / 111846-5003 because no lengthy scale-up process is needed, since the therapeutic protein is expressed and produced in the patient’s own muscle cell. The manufacturing timeline and production costs are significantly reduced, giving a significant advantage of the presently described invention over current methods of protein expression, production, and manufacturing. Further, the invention in certain embodiments allows for one or multiple proteins to be produced in the patient in a sustained and durable manner, to thereby avoid the large fluxes in circulating levels of therapeutic proteins often associated with conventional protein therapy, and / or to avoid frequent administrations of therapeutic compositions. In an aspect, the present disclosure provides a plasmid construct (and compositions thereof) having at least one expression cassette. The expression cassette comprises in the following order from 5´ to 3´: a) a CMV IE enhancer sequence; b) a chicken beta-actin (CBA) promoter sequence; c) a CMV IE intron A sequence; d) an open reading frame encoding a peptide hormone, such as an agonist at one or more of glucagon-like peptide-1 (GLP-1) receptor, glucose-dependent insulinotropic polypeptide (GIP) receptor, and / or a glucagon (GCG) receptor; and e) a transcription termination sequence. The nucleotide sequence of CMV IE enhancer can be as shown herein (SEQ ID NO: 1). The term CMV IE enhancer includes derivatives having at least 100 consecutive nucleotides, or at least 200 consecutive nucleotides, or at least 300 consecutive nucleotides of the CMV IE enhancer represented by SEQ ID NO: 1. In these or other embodiments, the CMV IE enhancer may have from 1 to 50, or from 1 to 25, or from 1 to 10 nucleotide modifications independently selected from nucleotide substitutions, deletions, and insertions, without impacting the ability of the enhancer to support expression from the expression cassette. In various embodiments, the cis-acting elements are retained and unmodified. Meier JL. Et al., Requirement of Multiple cis-Acting Elements in the Human Cytomegalovirus Major Immediate-Early Distal Enhancer for Viral Gene Expression and Replication. J. Virology Vol. 76, Issue 1 (2002). In some embodiments, the CMV IE enhancer comprises the nucleotide sequence of SEQ ID NO: 1. The nucleotide sequence of chicken beta-actin promoter (CBA) can be as shown herein (see SEQ ID NO: 2). The term chicken beta-actin promoter (CBA) includes DB1 / 142158622.1 11 Attorney Docket No.: RBF-003PC / 111846-5003 derivatives having at least 100 consecutive nucleotides, or at least 200 consecutive nucleotides, or at least 250 consecutive nucleotides of the CBA promoter represented by SEQ ID NO: 2. In these or other embodiments, the CBA promoter may have from 1 to 50, or from 1 to 25, or from 1 to 10 nucleotide modifications independently selected from nucleotide substitutions, deletions, and insertions, without impacting the ability of the promoter to support expression from the expression cassette. In various embodiments, the cis-acting elements are retained and unmodified. Seo HW, Evaluation of combinatorial cis- regulatory elements for stable gene expression in chicken cells. BMC Biotechnol.2010; 10: 69. In some embodiments, the CBA promoter comprises the nucleotide sequence of SEQ ID NO: 2. The nucleotide sequence of CMV IE intron A can be as described herein (SEQ ID NOS: 3 or 5, for example). The term CMV IE intron A includes derivatives having at least 100 consecutive nucleotides, or at least 200 consecutive nucleotides, or at least 300 consecutive nucleotides of the CMV IE intron A represented by SEQ ID NO: 3 or SEQ ID NO: 5. In these or other embodiments, the CMV IE intron A may have from 1 to 50, or from 1 to 25, or from 1 to 10 nucleotide modifications (relative to SEQ ID NO: 3 or 5) independently selected from nucleotide substitutions, deletions, and insertions, without impacting the ability of the intron to support expression from the expression cassette. In various embodiments, the cis-acting elements are retained and are unmodified. Chapman BS, et al., Effect of intron A from human cytomegalovirus (Towne) immediate-early gene on heterologous expression in mammalian cells. Nucleic Acids Res. 1991 Jul 25; 19(14): 3979–3986. In some embodiments, the CMV IE intron A comprises the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 5. In some embodiments, the termination sequence is an artificial transcription termination sequence, and may include segments of a bovine growth hormone polyadenylation signal (BGHpA), and may include multiple polyadenylation sequences in tandem. The artificial transcription termination sequence initiates the process of releasing a newly synthesized RNA. Terminator sequences are typically found directly after 3’ regulatory elements, such as the polyadenylation or poly(A) signal, which can contribute to a stable RNA transcript. In some embodiments, the artificial transcription terminator DB1 / 142158622.1 12 Attorney Docket No.: RBF-003PC / 111846-5003 sequence promotes RNA processing and enhances gene-expression. In some embodiments, the artificial transcription terminator sequence comprises sequence from the bovine growth hormone(bGH) gene, and may include two synthetic poly(A) sequences. An exemplary nucleotide sequence for a termination sequence is shown as SEQ ID NO: 4. The term BGHpA includes derivatives having at least 100 consecutive nucleotides, or at least 200 consecutive nucleotides, or at least 250 consecutive nucleotides of the termination sequence of SEQ ID NO: 4. In these or other embodiments, the BGHpA termination sequence may have from 1 to 50, or from 1 to 25, or from 1 to 10 nucleotide modifications independently selected from substitutions, deletions, and insertions, without impacting the ability of the termination sequence to support transcription termination and processing of expressed transcripts. In various embodiments, the cis-acting elements are retained and are unmodified. In some embodiments, the BGHpA comprises the nucleotide sequence of SEQ ID NO: 4. In embodiments, the DNA composition encodes the peptide hormone as a polypeptide fusion with a carrier protein for PK enhancement, optionally through a linking sequence (e.g., Gly Ser linker). In embodiments, the polypeptide fusion is with IgG Fc or albumin. In some embodiments, the polypeptide fusion is Fc, and may be IgG1, IgG2, or IgG4 Fc. An exemplary IgG4 Fc amino acid sequence is shown as SEQ ID NO: 15. Derivatives of this sequence (including derivatives having known mutations to enhance PK) can be used, and such derivative can have from 1 to 10, or from 1 to 5, or from 1 to 3 amino acid modifications selected from substitutions, deletions, or insertions. In various embodiments, the amino acid modifications are amino acid substitutions. Other carrier proteins such as transferrin and elastic-like-peptide (ELP) are known in the art, and may be employed. In various embodiments, the carrier protein is Fc, allowing for dimeric presentation of the agonist, due to formation of disulfide bridges between Fc chains (see FIG.15). Generally, the carrier protein is a half-life extending moiety, such as albumin, albumin binding domain (e.g., ABD035), transferrin, an Fc domain, or XTEN (see US 8,492,530 which is hereby incorporated by reference in its entirety), or elastin-like protein, or a variant thereof. See, e.g., US 9,458,218, which is hereby incorporated by reference in its entirety. In some embodiments, the peptide hormone is fused to an albumin amino acid DB1 / 142158622.1 13 Attorney Docket No.: RBF-003PC / 111846-5003 sequence or domain, i.e., a human albumin or a fragment or variant thereof. See, for example, WO 2015 / 066550 and US 9,221,896, which are hereby incorporated by reference in their entirety. Albumin can be joined to the peptide hormone, optionally with an interposed linker (as described herein), preferably at the C-terminus of the peptide. In various embodiments, the albumin amino acid sequence binds to the neonatal Fc receptor (FcRn), e.g., human FcRn. The albumin amino acid sequence may be a variant of wild-type HSA. Various modifications to the albumin sequence that enhance its ability to serve as a circulation half- life extending carrier are known, and such modifications can be employed with the present invention. Exemplary modifications to the albumin amino acid sequence are described in US 8,748,380, US 10,233,228, US 10,208,102, and US 10,501,524, which are each hereby incorporated by reference in their entireties. Exemplary modifications include one or more (or all) of E505Q, T527M, and K573P as described therein. Generally, carrier proteins may be fused to the therapeutic peptide sequence via a linker sequence. Exemplary linkers have from 4 to about 20 amino acids, or in some embodiments from about 10 to about 20 amino acids (e.g., about 15 amino acids). While any linker can be used, in some embodiments flexible linkers such as linkers composed of Gly and Ser (or predominately Gly Ser) are preferred. In some embodiments, linkers can be selected from flexible and rigid peptide linkers. In some embodiments, flexible linkers are predominately or entirely composed of small and / or polar residues such as Gly, Ser, and Thr. An exemplary flexible linker comprises (GlyxSer)n linkers, where x is from 1 to 10 (e.g., from 2 to 6), and n is from 1 to about 10, and in some embodiments, is from 2 to about 6. In exemplary embodiments, x is 4, and n is 3. Due to their flexibility, these linkers are substantially unstructured. More rigid linkers include polyproline or poly Pro-Ala motifs and α-helical linkers. Generally, linkers of varying rigidity can be predominately composed of amino acids selected from Gly, Ser, Thr, Ala, and Pro. In embodiments, the peptide agonist (e.g., GLP-1 receptor agonist) is not fused to a PK-enhancing moiety. In all embodiments, the expression construct can encode GLP-1 that is modified to reduce proteolytic clipping as known in the art (e.g., proteolytic clipping by DPP-4). For example, GLP-1 sequences can have the Glycine at position 2, relative to SEQ ID NO: 10, substituted with Alanine. DB1 / 142158622.1 14 Attorney Docket No.: RBF-003PC / 111846-5003 In some embodiments, the construct encodes a GLP-1 receptor agonist, such as GLP- 1 or exenatide, or variations thereof having from one to four amino acid modifications (e.g., 1, 2, 3, or 4) independently selected from amino acid substitutions, deletions, and insertions. A reference GLP-1 amino acid sequence for GLP-1 is provided herein as SEQ ID NO: 10. In some embodiments, such modifications are amino acid substitutions. GLP-1 is a 30 amino acid peptide hormone produced by intestinal L cells, by a group of neurons of the central and autonomous nervous systems, and in lesser quantity in the endocrine pancreas. GLP-1 intestinal production is boosted by nutrient ingestion and, via GLP-1 receptors, stimulates insulin secretion. As a result, insulin secretion induced by oral glucose is significantly higher than the one resulting from intravenous administration, with equivalent plasma levels. This result, named the incretin effect, is due to actions of GLP-1 and of GIP. Though both hormones are incretins, they show opposite effects on glucagon secretion, since GIP enhances, and GLP-1 inhibits glucagon secretion. However, these actions are only triggered in the presence of hyperglycemia, as occurs in the postprandial period, and therefore do not cause hypoglycemia. In embodiments, several copies of the peptide agonist (e.g., GLP-1 receptor agonist) are expressed in tandem, such as from 2 to 5 copies expressed in tandem, according to this disclosure. An exemplary amino acid sequence for a GLP-1-hIgG4 Fc fusion construct is provided herein as SEQ ID NO: 16. A DNA coding sequence for SEQ ID NO: 16 is provided as SEQ ID NO: 17. The fusion protein of SEQ ID NO: 16 includes a signal peptide for cell secretion, and an optional antigenic tag (e.g., HIS tag). Since the Fc includes the hinge region, these constructs form homodimeric polypeptides in vivo via a disulfide bridge. See FIG.15. In embodiments, the open reading frame encodes GIP, or a derivative thereof having from one to four amino acid modifications (e.g., 1, 2, 3, or 4) independently selected from substitutions, deletions, and insertions. In some embodiments, such modifications are amino acid substitutions. A reference GIP amino acid sequence is provided herein as SEQ ID NO: 11. Glucose-dependent insulinotropic polypeptide or GIP (known previously as gastric DB1 / 142158622.1 15 Attorney Docket No.: RBF-003PC / 111846-5003 inhibitory polypeptide), is a hormone of the secretin family of hormones. While it is a weak inhibitor of gastric acid secretion, its main role, being an incretin, is to stimulate insulin secretion. GIP, along with GLP-1, belongs to a class of molecules referred to as incretins, which stimulate insulin release on oral food intake. GIP is derived from a 153-amino acid proprotein encoded by the GIP gene and circulates as a biologically active 42-amino acid peptide. It is synthesized by K cells in the duodenum and the jejunum of the gastrointestinal tract. In addition to its role as an incretin, GIP inhibits apoptosis of the pancreatic beta cells and promotes their proliferation. GIP also stimulates glucagon secretion and fat accumulation. GIP receptors are expressed in many organs and tissues including the central nervous system enabling GIP to influence hippocampal memory formation and regulation of appetite and satiety. An exemplary amino acid sequence for a GIP-hIgG4 Fc fusion construct is provided herein as SEQ ID NO: 18. A DNA coding sequence for SEQ ID NO: 18 is provided as SEQ ID NO: 19. The fusion protein of SEQ ID NO: 18 includes a signal peptide for cell secretion, and an optional antigenic tag (e.g., HIS tag). Since the Fc includes the hinge region, these constructs form homodimeric polypeptides in vivo via a disulfide bridge. See FIG.15. When expressed with GLP-1 constructs (such as that of SEQ ID NO: 16) using expression constructs disclosed herein for expressing two proteins (either in one or two open reading frames), both homodimeric and heterodimeric polypeptides are formed in vivo. See FIG.15. In embodiments, the open reading frame encodes a dual agonist for GLP-1 receptor and GIP receptor, many of which are known in the art. For example, the open reading frame may encode the amino acid sequence of tirzepatide, or a derivative thereof having from one to four amino acid modifications (e.g., 1, 2, 3, or 4) independently selected from substitutions, deletions, and insertions. A reference amino acid sequence for tirzepatide is provided herein as SEQ ID NO: 12. An exemplary amino acid sequence for a GLP-1 receptor / GIP receptor dual agonist (tirzepatide) with hIgG4 Fc fusion is provided herein as SEQ ID NO: 20. A DNA coding sequence for SEQ ID NO: 20 is provided as SEQ ID NO: 21. The fusion protein of SEQ ID NO: 20 includes a signal peptide for cell secretion, and an optional antigenic tag (e.g., HIS DB1 / 142158622.1 16 Attorney Docket No.: RBF-003PC / 111846-5003 tag). Since the Fc includes the hinge region, these constructs form homodimeric polypeptides in vivo via a disulfide bridge. See FIG.15. In embodiments, the open reading frame encodes a triple agonist for GLP-1 receptor, GIP receptor, and GCG receptor. For example, the agonist may comprise the amino acid sequence of retratrutide, or a derivative thereof having from one to four amino acid modifications (e.g., 1, 2, 3, or 4) independently selected from substitutions, deletions, and insertions. A reference amino acid sequence for a retratrutide is provided herein as SEQ ID NO: 13. An exemplary amino acid sequence for a triple agonist (retratrutide) with hIgG4 Fc fusion is provided herein as SEQ ID NO: 22. A DNA coding sequence for SEQ ID NO: 22 is provided as SEQ ID NO: 23. The fusion protein of SEQ ID NO: 22 includes a signal peptide for cell secretion, and an optional antigenic tag (e.g., HIS tag). Since the Fc includes the hinge region, these constructs form homodimeric polypeptides in vivo via a disulfide bridge. See FIG.15. In embodiments, the open reading frame encodes a dual agonist for GLP-1 receptor and GCG receptor, many of which are known in the art. For example, the open reading frame may encode the amino acid sequence of SEQ ID NO: 14, or a derivative thereof having from one to four amino acid modifications (e.g., 1, 2, 3, or 4) independently selected from substitutions, deletions, and insertions. An exemplary amino acid sequence for a GLP-1 receptor / GCG receptor dual agonist with hIgG4 Fc fusion is provided herein as SEQ ID NO: 24. A DNA coding sequence for SEQ ID NO: 24 is provided as SEQ ID NO: 25. The fusion protein of SEQ ID NO: 24 includes a signal peptide for cell secretion, and an optional antigenic tag (e.g., HIS tag). Since the Fc includes the hinge region, these constructs form homodimeric polypeptides in vivo via a disulfide bridge. See FIG.15. In embodiments, the plasmid construct comprises two of said expression cassettes (e.g., such as those of SEQ ID NO: 6 or 7) expressing different agonist sequences. For example, a first expression cassette can comprise an open reading frame encoding a GLP-1 DB1 / 142158622.1 17 Attorney Docket No.: RBF-003PC / 111846-5003 receptor agonist; and a second expression cassette can comprise an open reading frame encoding a GIP receptor agonist or GCG receptor agonist. In embodiments, the first expression cassette has an open reading frame encoding a GLP-1 receptor agonist (e.g., GLP-1 or exenatide); and the second expression cassette has an open reading frame encoding a GIP receptor agonist (e.g., GIP). In each case, the agonist may be expressed as a fusion with a carrier protein (such as IgG Fc) as described. In embodiments, the open reading frame encodes two members of the group of a GLP-1 receptor agonist, a GIP receptor agonist, and a GCG receptor agonist, with a peptide linker therebetween that induces ribosomal skipping. In each case, the GLP-1 receptor agonist, the GIP receptor agonist, and / or the GCG receptor agonist may comprise a fusion to a polypeptide for PK enhancement as described (e.g., IgG Fc). In some embodiments, the GLP-1 receptor agonist is GLP-1 or exenatide, and which is expressed with a GIP receptor agonist (e.g., GIP) in a single open reading frame. Exemplary constructs for co-expressing two proteins in a single open reading frame are provided herein as SEQ ID NOS: 8 and 9. Other peptides that can be expressed or co-expressed using the constructs disclosed herein include Maridebart, Bimagrumab, and Cagrilintide, and derivatives thereof (e.g., having from one to five amino acid substitutions). Bimagrumab is a monoclonal antibody that targets the activin receptor type II, which is involved in muscle growth and metabolism. This mechanism positions Bimagrumab as a potential agent for promoting muscle mass and function. Maridebart is a monoclonal antibody that functions as GLP-1 / GIP dual agonist. Cagrilintide is a long-acting amylin analog currently under investigation for its potential in weight management and glycemic control, particularly in individuals with obesity and type 2 diabetes. It acts as an amylin receptor agonist, mimicking the effects of the naturally occurring hormone amylin, which is co-secreted with insulin from pancreatic beta cells. Amylin plays a crucial role in regulating appetite, slowing gastric emptying, and improving glucose homeostasis. In embodiments, the peptide agonist (such as a GLP-1 receptor agonist, or a dual or triple agonist as described herein) can comprise a fusion to Angiopep-2. Angiopep-2 is believed to enhance the penetration of therapeutic agents across the blood-brain barrier DB1 / 142158622.1 18 Attorney Docket No.: RBF-003PC / 111846-5003 (BBB), a critical challenge in delivering proteins and peptides to the central nervous system (CNS). It is believed that GLP-1 may engage multiple pathways, including interactions within the CNS. For example, GLP molecules may target receptors in the CNS, contributing to appetite suppression, alongside their effects on gastrointestinal targets. In embodiments, the fusion to Angiopep-2 is at the N-terminus or the C-terminus of the expressed polypeptide, and in certain embodiments is at the C-terminus of the expressed polypeptide. An exemplary Angiopep-2 sequence is provided herein as SEQ ID NO: 27. In embodiments, the expression construct expresses a GLP-1-hIgG-Fc-Angiopep-2 fusion, or a GLP-1-Angiopep-2 fusion. Other fusion constructs employing GLP-1 receptor agonists and Angiopep-2 will be apparent from the present disclosure. In embodiments, Angiopep-2 can be fused indirectly through a linking sequence, which is optionally a Glycine Serine linker as described herein, or other linker of which many are described herein and known in the art. In embodiments, proteins encoded by the construct may further encode detectable tags (such as antigen tags), to allow expression to be monitored over time. An exemplary tag is a HIS tag. In exemplary sequences shown herein, the HIS tag is considered optional, and may be replaced with a different antigenic tag, or the constructs may comprise no tag. For example, the polypeptides can be encoded by cDNA. In embodiments, the polypeptides are encoded with a peptide linker therebetween that induces ribosomal skipping, thereby allowing for both polypeptides to be produced without translational fusion. Ribosomal skipping is a translational process wherein a viral peptide disrupts or prevents the ribosome from covalently linking (e.g., via inhibition of a peptidyl transferase) a new amino acid during translation, thereby cleaving the nascent protein and allowing translation to proceed. In some embodiments, the result of ribosomal skipping is co-translational cleavage of the nascent polyprotein. Exemplary such peptides include P2A peptide (e.g., from porcine teschovirus 1) and T2A peptide (e.g., asigna virus capsid protein). Nucleotide sequences encoding these peptides are shown in SEQ ID NOS: 8 and 9. To remove remaining amino acids from the upstream polypeptide (remaining from the peptide inducing ribosomal skipping), a proteolytic cleavage site can be incorporated on the N-terminal side of the peptide that induces ribosomal skipping. In some embodiments, DB1 / 142158622.1 19 Attorney Docket No.: RBF-003PC / 111846-5003 the proteolytic cleavage site is a furin cleavage site, which can have the consensus sequence RXR / K-R. In some embodiments, the furin recognition site can be incorporated on the N- terminal side of the peptide that induces ribosomal skipping, optionally with a linker peptide therebetween, such as a linker of 2 to about 20 amino acids, or a linker of from about 2 to about 10 amino acids. While any linker can be used, in some embodiments flexible linkers such as linkers composed of Gly and Ser are preferred. An exemplary linker is Gly Ser Gly. In some embodiments, linkers can be selected from flexible and rigid peptide linkers. In some embodiments, flexible linkers are predominately or entirely composed of small and / or polar residues such as Gly, Ser, and Thr. An exemplary flexible linker comprises (GlyxSer)n linkers, where x is from 1 to 10 (e.g., from 2 to 6), and n is from 1 to about 10, and in some embodiments, is from 2 to about 6. In exemplary embodiments, x is from 2 to 4, and n is from 2 to 4. Due to their flexibility, these linkers are substantially unstructured. More rigid linkers include polyproline or poly Pro-Ala motifs and α-helical linkers. Generally, linkers of varying rigidity can be predominately composed of amino acids selected from Gly, Ser, Thr, Ala, and Pro. Exemplary linker sequences contain at least 5 amino acids, and may be in the range of 5 to 30 amino acids or in the range of 5 to 20 amino acids. In some embodiments, an exemplary construct (without the open reading frame) comprises the expression cassette of SEQ ID NO: 6. SEQ ID NO: 6 comprises the following elements: (a) a CMV IE enhancer sequence; (b) a CBA promoter sequence; (c) a CMV IE intron A sequence; and (d) a BGHpA sequence. Therapeutic polypeptide open reading frames can be cloned into the cassette of SEQ ID NO: 6 at a cloning site downstream of the CMV IE intron A sequence, such that the elements are functional to direct expression of the therapeutic polypeptide. Together, such elements provide for high expression of a therapeutic protein in mammalian cells (e.g., muscle cells). In some embodiments, the plasmid DNA construct (without the therapeutic protein open reading frame) comprises a nucleotide sequence that has at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with SEQ ID NO: 6, or has 100% sequence identity to SEQ ID NO: 6. DB1 / 142158622.1 20 Attorney Docket No.: RBF-003PC / 111846-5003 In some embodiments, an exemplary construct comprises the expression cassette of SEQ ID NO: 7. SEQ ID NO: 7 has elements similar to SEQ ID NO: 6, but includes a shorter CMV IE intron A, and is therefore desirable for constructing dual expression cassettes, where two (or more) expression cassettes are included on the plasmid (e.g., in tandem). Dual expression cassettes may be used to express, for example, a GLP-1 receptor agonist and a GIP receptor agonist (including as Fc fusions as described herein, or as fusions with other carrier proteins) on a single plasmid. In some embodiments, the plasmid DNA construct comprises a nucleotide sequence that has at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with SEQ ID NO: 7, or has 100% sequence identity to SEQ ID NO: 7. In embodiments, an exemplary construct comprises the expression cassette of SEQ ID NO: 8. SEQ ID NO: 8 comprises the following elements: (a) an CMV IE enhancer sequence; (b) a CBA promoter sequence; (c) a CMV IE intron A sequence; (d) a furin-P2A sequence, and (e) a BGHpA sequence, which together provide for high expression of two proteins in mammalian cells (e.g., muscle cells), with each polypeptide coding sequence cloned in frame on either side of the furin-P2A sequence. In some embodiments, the plasmid DNA construct (without the therapeutic proteins coding sequences) comprises a nucleotide sequence having at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity, or 100% sequence identity, to SEQ ID NO: 8. In embodiments, an exemplary construct comprises the expression cassette of SEQ ID NO: 9. SEQ ID NO: 9 comprises the following elements: (a) an CMV IE enhancer sequence; (b) a CBA promoter sequence; (c) a CMV IE intron A sequence; (d) a furin-T2A sequence, and (e) a BGHpA sequence, which together provide for high expression of two therapeutic proteins in mammalian cells (e.g., muscle cells), with each polypeptide coding sequence cloned in frame on either side of the furin-T2A sequence. In some embodiments, the plasmid DNA construct (without the therapeutic protein coding sequence) comprises a nucleotide sequence that has at least 50%, or at least 55%, or at least 60%, or at least 65%, DB1 / 142158622.1 21 Attorney Docket No.: RBF-003PC / 111846-5003 or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity, or 100% sequence identity, to SEQ ID NO: 9. Plasmid constructs can be formulated for administration, for example, by intramuscular injection. Pharmaceutical compositions include, without limitation, solutions, emulsions, aqueous suspensions, and liposome-containing formulations. Compositions and formulations can contain sterile aqueous solutions, which also can contain buffers, diluents, and other suitable additives (e.g., penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers). In various embodiments, constructs are administered by intramuscular injection, and further comprising electroporation to facilitate entry of the plasmid into muscle cells. An exemplary device and process for intramuscular injection is disclosed in PCT / US2023 / 066671, which is hereby incorporated by reference in its entirety. For example, a device for gene transfer may comprise a pulse generator, a handpiece, and an array of electrodes arranged to maximize expression of a plasmid DNA construct delivered therethrough (through an injection needle tip contained within the device) while minimizing applied voltage and total electrical dose. For example, in some embodiments, the electrode array comprises six electrodes and a single DNA injection port. In some embodiments, the electrodes are arranged in a hexagonal pattern, which can provide for more consistent delivery into muscle cells. In some embodiments, the pulse(s) comprise perpendicular pulses and / or parallel pulses relative to the orientation of a muscle fiber. In other aspects, the present disclosure provides a use of the DNA constructs described herein for treating disease. Accordingly, the present disclosure provides a method for treating or preventing a disease in a subject, where the method comprises administering an effective amount of the composition described herein to a subject in need thereof. In various embodiments, the disease is a metabolic disease. In some embodiments, the compositions of the disclosure find use in the treatment or prevention of various diseases or disorders related to obesity and diabetes mellitus. In some embodiments, the methods disclosed herein prevent an onset or progression of a DB1 / 142158622.1 22 Attorney Docket No.: RBF-003PC / 111846-5003 disease, such as obesity and diabetes mellitus. In various embodiments, the constructs and methods described herein provide for stable expression (e.g., in muscle cells) for at least about 4 months, at least about 6 months, at least about 1 year, or at least about 18 months, or at least about 2 years. In embodiments, the subject may receive dosing using a schedule that is no more frequent than about quarterly, or about twice per year, or about once per year, or about once every two years. Subjects are generally mammalian subjects, such as human subjects, but in other embodiments may be veterinary subjects (e.g., dog, cat, horse, or pig) or livestock (e.g., cow, pig, sheep, etc.). In embodiments, the disease includes hyperglycemia and / or glucose dysregulation (e.g., dysglycemia). In embodiments, the disease comprises insulin resistance. In some embodiments, the subject has diabetes or is at risk of diabetes mellitus. For example, the subject may have prediabetes, which includes individuals with higher than normal blood sugar levels, but who are not considered diabetic. In some embodiments, the diabetes is type 1 diabetes. In embodiments, the subject is undergoing therapy with insulin (e.g., a basal or prandial insulin). In embodiments, the subject has type 2 diabetes. In embodiments, the subject has or is at risk of obesity. For example, the subject may be overweight. Obesity is the abnormal or excessive accumulation of body fat that presents a risk to an individual’s overall health. Obesity is a leading risk factor in a large number of serious conditions, including type 2 diabetes and its associated co-morbidities, and cardiovascular diseases such as heart disease and stroke, which are the leading causes of death worldwide. Obesity is now recognized by the World Health Organization (WHO) as an issue that has grown to epidemic proportion, even in children. For instance, in 2016, 1.9 billion adults worldwide were reportedly obese, and in 2019, 38.3 million children under the age of five worldwide were reportedly obese. According to the WHO, 422 million people worldwide have diabetes and 1.6 million deaths are directly attributed to diabetes each year. In certain embodiments, the patient is overweight or obese, and is therefore in need of weight loss, prevention of weight gain, and weight control overall. For example, the patient in some embodiments has a body mass index of from 25 to 30, or a body mass index of at least 30. In some embodiments, the patient has a body mass index of at least 35. Body DB1 / 142158622.1 23 Attorney Docket No.: RBF-003PC / 111846-5003 mass index or BMI is a measure of body fat based on height and weight, and the determination of BMI is well known. In embodiments, the method can be employed with or without significant caloric restriction. For example, in embodiments, the therapy described herein does not require or induce a substantial reduction in food intake in the patient to control weight gain, induce weight loss, or prevent weight gain. For example, the patient may have a positive energy balance prior to treatment, and thus is likely to continue weight gain in the absence of treatment. In some embodiments, the subject has or is at risk of metabolic syndrome. Metabolic syndrome may be defined by the presence in the patient of at least two of: (1) triglycerides >150 mg / dL (1.7 mmol / L); (2) HDL cholesterol <40 mg / dL (1.03 mmol / L) for a male, <50 mg / dL (1.29 mmol / L) for a female; (3) systolic BP >130 or diastolic BP >85 mm Hg, or the patient is being treated for hypertension; (4) fasting plasma glucose (FPG) >100 mg / dL (5.6 mmol / L) or the patient is being treated for hyperglycemia; and (5) an elevated waist circumference equal to or greater than 40 inches (102 cm) for men, or equal to or greater than 35 inches (88 cm) for women. In accordance with some embodiments, the patient's caloric intake may not be significantly altered by the treatment. For example, the patient may be placed on (or continue with) a diet that is not restricted in caloric intake by more than 20%, or not by more than 10%, or not by more than 5%, of the patient's caloric intake at the start of treatment. The caloric intake in conjunction with the treatment is sufficient to achieve a neutral or negative energy balance for the patient.. In accordance with other embodiments, the treatment is used in connection with caloric restriction. In various embodiments, the composition is administered parenterally, such as by intramuscular injection. In embodiments, administering further comprises electroporation as described. DB1 / 142158622.1 24 Attorney Docket No.: RBF-003PC / 111846-5003 In various embodiments, the disclosure reduces the frequency of administration, for example, as compared to administering protein drugs. In embodiments, administration of the compositions described herein is no more than about monthly. In embodiments, administration is no more than about quarterly, or no more about twice per year. In embodiments, administering is no more than about annually, or is about annually. In embodiments, the method can further comprise monitoring for the presence of the expressed protein in the circulation of the subject. Such monitoring can be conducted at least once between administrations. For example, monitoring can be conducted from one to 12 times per year, in embodiments. Monitoring can be conducted by immunoassay on peripheral blood (or component thereof), for example, using antibodies that detect the agonist or an antigenic tag included on the construct. As used herein, unless the context requires otherwise, the term about means ± 10% of an associated numerical value. As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the materials, compositions, devices, and methods of this technology. Similarly, the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features. Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the disclosure, the present technology, or embodiments thereof, may alternatively be described using more limiting terms such as “consisting of” or “consisting essentially of” the recited ingredients. Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials, similar or equivalent to those described herein, can be used in the practice or testing of the present disclosure, the preferred methods and materials are described herein. All publications, patents, and patent publications cited are incorporated by reference herein in their entirety for all purposes. DB1 / 142158622.1 25 Attorney Docket No.: RBF-003PC / 111846-5003 This disclosure is further illustrated by the following non-limiting examples. EXAMPLES Example 1: Design And Application Of Plasmid DNA Constructs To Treat A Variety of Diseases In the experiments of this example, plasmid DNA constructs were designed, as shown in FIG.5. The plasmid DNA constructs (see the first four DNA constructs in FIG.5, from top to bottom) were designed to comprise a) an enhancer sequence (CMV IE enhancer), b) a promoter sequence (CBA promoter), c) an intron sequence (CMV IE intron A), and d) a transcription termination. An arrow in each construct of FIG.5 indicates the location of a cloning site where a therapeutic protein can be encoded. In FIG.5 the therapeutic protein is indicated as antibody heavy and light chains (HC and LC), while it is understood that these open reading frame may instead encode the therapeutic peptides and proteins disclosed herein. Two proteins may be expressed in a single reading frame, using furin T2A or furin P2A to allow for co-translational cleavage. Exemplary dual expression constructs are also shown in FIG.5. In the experiments of this example, an electroporation device for gene transfer was used to deliver the DNA constructs to mice. The electroporation device has a handpiece, an array of electrodes arranged at one end of the handpiece and configured to be positioned at a tissue of a subject (e.g., muscle); and a pulse generator configured to generate electric pulses that cause the array of electrodes to emit electric fields in the targeted tissue to maximize expression of a plasmid DNA construct delivered therethrough while minimizing applied voltage and total electrical dose. The device for intramuscular injection is disclosed in PCT / US2023 / 066671, which is hereby incorporated by reference in its entirety. In the experiments shown in FIG.6, mice were administered different plasmid DNA constructs encoding a murine version (heavy and light chains) of the monoclonal antibody trastuzumab (4D5), and the serum antibody concentrations of 4D5 were measured at weeks 1-4 post administration. When mice were administered a total of either 5 µg or 25 µg of two plasmids, one encoding the heavy chain (HC) and one encoding the light chain (LC) of 4D5 (panel 1), the higher DNA dose was required in order to achieve a maximum serum antibody DB1 / 142158622.1 26 Attorney Docket No.: RBF-003PC / 111846-5003 concentration. In contrast, when animals were administered the same doses of plasmid DNA in which both the HC and LC were encoded in a single plasmid (a single expression cassette with co-translational cleavage) (panel 2), the lower dose of 5 µg was sufficient to achieve nearly the same serum antibody concentration as the higher dose of 25 µg. The experiments shown in FIG.7 demonstrate testing of different promoters in the plasmid DNA constructs. Two groups of mice were administered 5 µg of a plasmid DNA construct encoding a murine version of the monoclonal antibody trastuzumab (4D5) at week 0 (i.e. intramuscular injection), and serum antibody concentrations were measured at the indicated timepoints following administration. The plasmids that both groups received were the same except for the promoter. One group of animals received a construct including the CMV promoter, and the other group of animals received s construct where the CMV promoter was replaced with a chicken beta-actin (CBA) promoter. The animals that received the plasmid with CBA promoter had significantly higher (and surprising) levels of serum 4D5 antibody for a period of at least 48 weeks. The experiments in FIG.8 demonstrate stable antibody expression and production in mice for a period more than one year. In these experiments, mice were administered 5 µg of a plasmid DNA construct encoding the murine antibody S139 with the electroporation device as described, and antibody levels were monitored in the mice over the time course of the experiments. The experiments in FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D demonstrate that plasmid DNA constructs encoding an antibody or therapeutic protein, are applicable for treating and / or preventing a variety of diseases. In FIG. 4A, mice were implanted with human breast cancer tumor cells expressing the tumor antigen HER2. At time zero (dotted line) animals were either left untreated (black line), or administered plasmid DNA encoding a murine version of the anti-HER2 monoclonal antibody, trastuzumab (Herceptin) (red line), delivered by intramuscular injection into a muscle. The delivery of trastuzumab using the plasmid DNA constructs disclosed herein significantly suppressed tumor growth, as well as decreased tumor volume, as compared to the control group. In FIG.4B, mice bearing a transgene for human TNF-alpha develop a progressive, inflammatory arthritis disease. These animals were either administered an empty vector DB1 / 142158622.1 27 Attorney Docket No.: RBF-003PC / 111846-5003 plasmid DNA (control) or a plasmid DNA construct encoding a murine version of the anti- TNF-alpha antibody certolizumab, delivered by intramuscular injection into a muscle cell, at week zero and again at week 12. The y-axis shows the clinical arthritis score. Animals that received murine certolizumab based on a plasmid DNA construct had significantly slower disease progression and milder disease overall, as compared to the control group. In FIG. 4C, mice were administered a plasmid DNA construct encoding a monoclonal antibody directed against influenza A virus, delivered by intramuscular injection, or left untreated (control). At day zero, the animals were infected with a mouse- adapted H1N1 influenza A virus and monitored for virus-induced mortality. Animals that received the anti-influenza antibody based on a plasmid DNA construct were completely protected from virus-associated mortality, in contrast to the control group. In FIG. 4D, mice were administered a plasmid DNA construct encoding the therapeutic protein erythropoietin (epoetin alfa), delivered by intramuscular injection, or left untreated (control) at day 0. Seven days later (arrow), all the animals were treated with cisplatin to induce acute anemia. In contrast to the control animals, the animals that received erythropoietin based on a plasmid DNA construct were completely protected from anemia, as indicated by their blood hemoglobin levels. Example 2: Evaluation of Alternative DNA Construct Designs In the experiments of this example, plasmid DNA constructs were evaluated for the optimal combination of the promoter, enhancer, and polyA signal to enhance the expression of a therapeutic protein in mammalian cells. While prior studies from Xu et al. (“Optimization of transcriptional regulatory elements for constructing plasmid vectors” Gene, 2001 Jul 11;272(1-2):149-56) have shown the CA part of the overall CAG promoter, as well as intron A and the BGH poly A can provide some expression in skeletal muscle cells, this promoter, enhancer, and polyA signal combination were not tested together to evaluate the expression of a therapeutic protein in mammalian cells over any duration of time. In the experiments of this example, it was hypothesized that the CBA promoter would be more resistant to silencing, and thus, the combination of CBA promoter and Intron A in a DNA construct, as disclosed herein, would result in stable, high-level gene expression in skeletal muscle. DB1 / 142158622.1 28 Attorney Docket No.: RBF-003PC / 111846-5003 Indeed, the experiments of this example found that the combination of the CA part of the overall CAG promoter, intron A (or the Intron A deletion mutant, intron pCON3 [InpCON3]), and the BGH poly A significantly increase therapeutic protein expression level in vitro and also in skeletal muscle cells in vivo, as compared to other combinations of elements. In the experiments of this example, the electroporation device for gene transfer was used to deliver the DNA constructs to the mice. FIG. 9 shows a series of DNA constructs for encoding a therapeutic protein. The DNA constructs shown in FIG.9 represent the regulatory element configurations that were used to generate the data in the experiments shown in FIG.10. In FIG.9, the genes of interest are the heavy and light chains of 4D5, a mouse monoclonal antibody that binds HER2 (from which Herceptin was derived), and is used herein as an illustrative example. FIG.10 is a graph showing the serum antibody concentration levels over a period of 1 year for different DNA constructs encoding the Herceptin 4D5 antibody. In these experiments, the heavy chain and light chain antibody sequences were on two separate DNA constructs and significantly increased serum antibody concentrations were achieved with the CBA promoter, Intron A sequence, and bGHA elements. FIG. 11 shows a series of DNA constructs encoding a therapeutic protein. In FIG. 11, a single cassette is one set of regulatory elements (e.g., an enhancer, promoter, intron, terminator / poly A). One advantage of a single cassette, over a dual cassette, is the ability to express a therapeutic protein, such as an antibody heavy chain and light chain, from a single plasmid by inserting a furin-2A site between the sequence encoding the antibody heavy chain and the sequence encoding light chain. FIG.12 is a graph showing the serum antibody concentration levels over a period of 1 year for different DNA constructs encoding a therapeutic protein (i.e., murine anti- influenza virus antibody). In these experiments, significantly increased serum antibody concentrations with the CBA promoter and Intron A sequence were shown to significantly increase serum antibody concentrations. DB1 / 142158622.1 29 Attorney Docket No.: RBF-003PC / 111846-5003 FIG. 13 shows a series of DNA constructs encoding muscle-specific proteins for evaluation. The DNA constructs in FIG. 14 were engineered to carry the DES (desmin) promoter, a naturally occurring promoter of the desmin gene, or the CMV promoter. The desmin protein is a muscle-specific cytoskeletal protein belonging to the intermediate filament family, and is known to have high expression levels in mammalian muscle cells. DNA constructs were also engineered to have sequences that encode the Muscle creatine kinase (MCK) protein, which is induced to high levels during skeletal muscle differentiation. As shown in the graphs of FIG.14A, FIG.14B, FIG.14C, and FIG.14D, the CBA InA (CMV_CBA_InA) construct outperforms all other muscle specific promoters evaluated. Example 3: Evaluation of GLP-1 Plasmid Constructs by Electroporation In the experiments of this example, several GLP-1 plasmid constructs were designed (FIG. 15) and evaluated for delivery and long term expression. Constructs include single, dual, and triple agonist peptides, each comprising an Fc fusion (murine) for PK enhancement and a HIS tag: GLP-1-mIgG1(Fc)-tag, GLP-1-mIgG1(Fc)-tag + GIP-mIgG1(Fc)-tag, GLP- 1 / GIP-mIgG1(Fc)-tag, and GLP-1 / GIP / GCG-mIgG1(Fc)-tag. The constructs were tested in an mouse model and delivered by electroporation. In these experiments, each construct was electroporated at two doses of 5 µg and 25 µg into mouse muscle cells, and the concentration of each product in circulation was measured over time. The results of these experiments show that durable protein expression lasts for a long time period after initial administration for each construct and dose. Together, these experiments demonstrate that, inter alia, the plasmid DNA constructs disclosed herein allow for the simultaneous expression and stable production of therapeutic proteins in vivo at high levels, and compared to delivery of protein therapeutics, will result in a significant decrease in administration frequency, and have a robust therapeutic duration. Accordingly, the plasmid DNA constructs disclosed herein allow for the treatment of various metabolic diseases, including obesity and diabetes mellitus, using the patient’s own cells for the expression and production of the antibody or therapeutic protein. Example 4: Diet-induced Obesity Model DB1 / 142158622.1 30 Attorney Docket No.: RBF-003PC / 111846-5003 This Example tests various expression constructs according to this disclosure in a mouse diet-induced obesity model. The experiments evaluated expression of constructs as well as the efficacy of constructs in inducing weight loss / reducing weight gain and improving glycemia. The experiments of this example employ three primary readouts: (1) the expression of the peptide over time, (2) weight gain / loss, and (3) insulin resistance as measured by glucose metabolism according to standard protocols. The experiments involved subjecting mice to a high-fat diet (HFD) (e.g., for eight weeks) to induce obesity, followed by 12 weeks of HFD or Standard Diet (SD), after which test mice receive 4 injections of a test DNA construct. The HFD or SD is continued post- administration. Mice were evaluated for construct expression, weight loss / weight gain, as well as insulin resistance.To measure insulin resistance, animals fasted to establish baseline serum glucose levels, followed by glucose injection and subsequent monitoring of glucose metabolism. The results in FIG. 17 involve mice that received constructs encoding non-fused GLP-1 peptides (with amino acid substitution to reduce proteolytic DPP-4 clipping). Results showed less weight gain for test mice over about 10 weeks. Mice that were maintained on a high-fat diet gained less weight compared to controls, while those on a standard diet maintain a lower weight overall or did not gain weight. The results show that constructs encoding a non-fused GLP-1 peptide demonstrate efficacy in weight management over a long duration with a single administration. The experiments in FIG. 18 show the results of the non-fused GLP-1 peptide constructs on glycemia over eight weeks. Mice maintained on HFD or SD show effects on glycemia for several weeks. The experiments in FIG. 19 show the effects on weight gain / loss, food intake, and glycemia upon administering GLP-1-mIgG1 fusion protein or Semaglutide, each injected subcutaneously. As shown, the proteins show similar potency. GLP-1-Fc constructs were prepared with an Angiopep-2 fusion at the C-terminus. Angiopep-2 is believed to enhance the penetration across the blood-brain barrier (BBB) The experiments in FIG.20 show that constructs encoding the fusion of Angiopep-2 exhibit good DB1 / 142158622.1 31 Attorney Docket No.: RBF-003PC / 111846-5003 durability. The results in FIG.20 show a duration of effect lasting approximately 15 weeks (or close to four months) from a single injection. FIG. 21 shows the results of administering constructs encoding Bimagrumab or Maridebart and the resulting expression levels in mice. Bimagrumab is a monoclonal antibody that targets the activin receptor type II, which is involved in muscle growth and metabolism. Maridebart is a monoclonal antibody and functions as GLP-1 receptor and GIP receptor dual agonist, and is being explored for its ability to reduce lean muscle loss. Both constructs showed expression in immune tolerized mice. The results show that Maridebart demonstrates particularly high expression. However, some individual animals exhibited a decline in serum levels, indicated by dashed lines on the graphs. This decline is attributed to the development of anti-drug antibodies (ADAs) in those animals. In these experiments, to avoid ADA responses, a standard protocol is employed where animals are pre-treated with a cocktail of anti-CD4 and anti-CD8 antibodies to transiently deplete T-cell populations.. FIG.22 shows the expression of human IgG4 fusion proteins: one containing GLP- 1 (GLP-1-hIgG4), and the other comprising a GLP-1 / GIP dual agonist (GLP-1 / GIP- hIgG4). The left side of the figure features the human IgG4 fusion construct with GLP-1 (semaglutide, while the right side displays results for constructs encoding the human IgG4 fusion proteins with a dual agonist ; referred to as GLP / GIP in FIG.22. The data in FIG.22 shows that both constructs are expressed over many weeks. FIG. 23 shows the results of two different constructs on weight gain / loss and food intake. In these experiments, the constructs include GLP-1 fused with ABD035, which is an albumin-binding domain (ABD), and a GLP / GIP dual agonist fused with ABD035. ABD035 enhances the half-life of therapeutic proteins by promoting their binding to serum albumin. In the analysis of the high-fat diet group (top panel), both constructs show effectiveness, with the GLP-1-ABD035 performing better at weeks three and four. In the groups switched to a standard diet (bottom panel), the GLP-1 with ABD035 construct outperforms the GLP / GIP dual agonist. The results indicate that both constructs exhibit efficacy in promoting weight loss. FIG.24 shows the results of two different constructs on weight loss and food intake. In these experiments, the constructs include GLP-1-hIgG4 and a GLP / GIP dual agonist fused DB1 / 142158622.1 32 Attorney Docket No.: RBF-003PC / 111846-5003 to hIgG4. In the analysis of the high-fat diet group (top panel), both constructs show effectiveness. In the groups switched to a standard diet (bottom panel), the GLP-1 construct outperforms the GLP / GIP dual agonist in this study. The results indicate that both constructs exhibit efficacy in promoting weight loss. FIG.25A, FIG.25B, and FIG.25C are graphs showing the results of expression of three therapeutic agents (Maridebart, Bimagrumab, and Cagrilintide) along with expression of the GLP-1-Angiopep-2 construct. Cagrilintide is a long-acting amylin analog currently under investigation for its potential in weight management and glycemic control, particularly in individuals with obesity and type 2 diabetes. It acts as an amylin receptor agonist, mimicking the effects of the naturally occurring hormone amylin, which is co-secreted with insulin from pancreatic beta cells. Amylin plays a crucial role in regulating appetite, slowing gastric emptying, and improving glucose homeostasis. The experiments in FIG.25A, FIG. 25B, and FIG.25C show that Maridebart, when expressed in both high-fat diet (HFD) and standard diet (SD) conditions, exhibits an additive effect on glycemic control. Similarly, Cagrilintide shows an additive effect on glycemia in the context of the high-fat diet. These experiments show that the combination of these agents with GLP-1-Angiopep-2 enhance glycemic management, particularly under conditions of dietary stress. The experimental results indicate the constructs disclosed herein improve glucose metabolism and reduce insulin resistance in the high-fat diet over a long duration of time. A significant advancement highlighted in this study is the potential for extended dosing intervals. Current GLP-1 receptor agonists require weekly injections, but the results shown in this Example demonstrate that the constructs of this disclosure allow for administration at comparatively low frequency. This reduction in dosing frequency would lead to substantial cost savings and improved patient compliance. From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims. DB1 / 142158622.1 33 Attorney Docket No.: RBF-003PC / 111846-5003 SEQUENCES SEQ ID NO: 1 – CMV IE enhancer ATTGGCTATTGGCCAACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATAT ATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGC CCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAA TGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACG CCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGG GACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGG SEQ ID NO: 2 – Chicken beta-actin promoter TCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGT ATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGC GGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGC GGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGC GCGCGGCGGGCG SEQ ID NO: 3 – CMV IE intron A CCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTA TAGACTCTATAGGCACACCCCTTTGGCTCTTATGCATGCTATACTGTTTTTGGCTTGGGGCCTATA CACCCCCGCTTCCTTATGCTATAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCAT TATTGACCACTCCCCTATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCAC AACTATCTCTATTGGCTATATGCCAATACTCTGTCCTTCAGAGACTGACACGGACTCTGTATTTTT ACAGGATGGGGTCCCATTTATTATTTACAAATTCACATATACAACAACGCCGTCCCCCGTGCCCGC AGTTTTTATTAAACATAGCGTGGGATCTCCACGCGAATCTCGGGTACGTGTTCCGGACATGGGCTC TTCTCCGGTAGCGGCGGAGCTTCCACATCCGAGCCCTGGTCCCATGCCTCCAGCGGCTCATGGTCG CTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCACAGCACAATGCCCACCACCACC AGTGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTCTGAAAATGAGCGTGGAGATTGGGCTCGC ACGGCTGACGCAGATGGAAGACTTAAGGCAGCGGCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTA TTCTGATAAGAGTCAGAGGTAACTCCCGTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGA GCAGTACTCGTTGCTGCCGCGCGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTT TCCATGGGTCTTTTCTGCAGTCACCGTC SEQ ID NO: 4 – Artificial transcription terminator sequence AGATCACTTCTGGCTAATAAAAGATCAGAGCTCTAGAGATCTGTGTGTTGGTTTTTTGTGGATCTG CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAG GTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTC ATTCTATTCTGGGGGGTGGGGTGGGGCAGCACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGC ATGCTGGGGATGCGGTGGGCTCTATGGGTACCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTC TCTCTCGGTACCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCGGTACCAGGTGCTG AAGAATTGACCCGGTTCCTCCTGGGCCAGAAAGAAGCAGGCACATCCCCTTCTCTGTGACACACCC TGTCCACGCCCCTGGTTCTTAGTTCCAGCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCG CCTTCAATCCCACCCGCTAAAGTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAACCAA ACCTAGCCTCCAAGAGTGGGAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAA TGCCTCCAACATGTGAGGAAGTAATGAGAGAAATCATAGAATT SEQ ID NO: 5 -- CMV IE intron A (short) DB1 / 142158622.1 34 Attorney Docket No.: RBF-003PC / 111846-5003 CCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTA TAGACTCTATAGGCACACCCCTTTGGCTCTTATGCATCTCGTTGCTGCCGCGCGCGCCACCAGACA TAATCGCTGACACACTGACAGACTGTTCCTTTCCTTTTTTTTTTTTTGCAGTCACCGTC SEQ ID NO: 6 – Construct 1 CMV IE enhancer Chicken beta-actin promoter CMV IE intron A Artificial transcription terminator sequence ATTGGCTATTGGCCAACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATAT ATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGC CCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAA TGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACG CCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGG GACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCC ACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTT TAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGG CGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAA GTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGCC GCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATA GACTCTATAGGCACACCCCTTTGGCTCTTATGCATGCTATACTGTTTTTGGCTTGGGGCCTATACA CCCCCGCTTCCTTATGCTATAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTA TTGACCACTCCCCTATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAA CTATCTCTATTGGCTATATGCCAATACTCTGTCCTTCAGAGACTGACACGGACTCTGTATTTTTAC AGGATGGGGTCCCATTTATTATTTACAAATTCACATATACAACAACGCCGTCCCCCGTGCCCGCAG TTTTTATTAAACATAGCGTGGGATCTCCACGCGAATCTCGGGTACGTGTTCCGGACATGGGCTCTT CTCCGGTAGCGGCGGAGCTTCCACATCCGAGCCCTGGTCCCATGCCTCCAGCGGCTCATGGTCGCT CGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCACAGCACAATGCCCACCACCACCAG TGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTCTGAAAATGAGCGTGGAGATTGGGCTCGCAC GGCTGACGCAGATGGAAGACTTAAGGCAGCGGCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTATT CTGATAAGAGTCAGAGGTAACTCCCGTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGC AGTACTCGTTGCTGCCGCGCGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTC CATGGGTCTTTTCTGCAGTCACCGTCGCTAGCCTCGAGAGATCACTTCTGGCTAATAAAAGATCAG AGCTCTAGAGATCTGTGTGTTGGTTTTTTGTGGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGT TGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATA AAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCA GCACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGG TACCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCGGTACCTCTCTCTCTCTCTCTC TCTCTCTCTCTCTCTCTCTCTCTCGGTACCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGCCA GAAAGAAGCAGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGGTTCTTAGTTCCAG CCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCAATCCCACCCGCTAAAGTACTTG GAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAACCAAACCTAGCCTCCAAGAGTGGGAAGAAATT AAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAATGCCTCCAACATGTGAGGAAGTAATGAG AGAAATCATAGAATT DB1 / 142158622.1 35 Attorney Docket No.: RBF-003PC / 111846-5003 SEQ ID NO: 7 – Construct 2 CMV IE enhancer Chicken beta-actin promoter CMV IE intron A Artificial transcription terminator sequence ATTGGCTATTGGCCAACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATAT ATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGC CCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAA TGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACG CCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGG GACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCC ACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTT TAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGG CGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAA GTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGCC GCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATA GACTCTATAGGCACACCCCTTTGGCTCTTATGCATCTCGTTGCTGCCGCGCGCGCCACCAGACATA ATCGCTGACACACTGACAGACTGTTCCTTTCCTTTTTTTTTTTTTGCAGTCACCGTCGCTAGCCTC GAGAGATCACTTCTGGCTAATAAAAGATCAGAGCTCTAGAGATCTGTGTGTTGGTTTTTTGTGGAT CTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGG AAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGT GTCATTCTATTCTGGGGGGTGGGGTGGGGCAGCACAGCAAGGGGGAGGATTGGGAAGACAATAGCA GGCATGCTGGGGATGCGGTGGGCTCTATGGGTACCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCT CTCTCTCTCGGTACCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCGGTACCAGGTG CTGAAGAATTGACCCGGTTCCTCCTGGGCCAGAAAGAAGCAGGCACATCCCCTTCTCTGTGACACA CCCTGTCCACGCCCCTGGTTCTTAGTTCCAGCCCCACTCATAGGACACTCATAGCTCAGGAGGGCT CCGCCTTCAATCCCACCCGCTAAAGTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAAC CAAACCTAGCCTCCAAGAGTGGGAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGA AAATGCCTCCAACATGTGAGGAAGTAATGAGAGAAATCATAGAATT SEQ ID NO: 8 – Construct 3 CMV IE enhancer Chicken beta-actin promoter CMV IE intron A Furin P2A Artificial transcription terminator sequence ATTGGCTATTGGCCAACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATAT ATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGC CCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAA TGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACG CCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGG GACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCC ACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTT TAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGG CGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAA GTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGCC DB1 / 142158622.1 36 Attorney Docket No.: RBF-003PC / 111846-5003 GCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATA GACTCTATAGGCACACCCCTTTGGCTCTTATGCATGCTATACTGTTTTTGGCTTGGGGCCTATACA CCCCCGCTTCCTTATGCTATAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTA TTGACCACTCCCCTATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAA CTATCTCTATTGGCTATATGCCAATACTCTGTCCTTCAGAGACTGACACGGACTCTGTATTTTTAC AGGATGGGGTCCCATTTATTATTTACAAATTCACATATACAACAACGCCGTCCCCCGTGCCCGCAG TTTTTATTAAACATAGCGTGGGATCTCCACGCGAATCTCGGGTACGTGTTCCGGACATGGGCTCTT CTCCGGTAGCGGCGGAGCTTCCACATCCGAGCCCTGGTCCCATGCCTCCAGCGGCTCATGGTCGCT CGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCACAGCACAATGCCCACCACCACCAG TGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTCTGAAAATGAGCGTGGAGATTGGGCTCGCAC GGCTGACGCAGATGGAAGACTTAAGGCAGCGGCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTATT CTGATAAGAGTCAGAGGTAACTCCCGTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGC AGTACTCGTTGCTGCCGCGCGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTC CATGGGTCTTTTCTGCAGTCACCGTCGCTAGCAGGAGAAAGAGAGGATCCAGCGGCGAAGGCAGAG GCAGCCTTCTTACATGTGGCGACGTGGAAGAGAACCCCGGACCTCTCGAGAGATCACTTCTGGCTA ATAAAAGATCAGAGCTCTAGAGATCTGTGTGTTGGTTTTTTGTGGATCTGCTGTGCCTTCTAGTTG CCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGT CCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGG TGGGGTGGGGCAGCACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGT GGGCTCTATGGGTACCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCGGTACCTCTC TCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCGGTACCAGGTGCTGAAGAATTGACCCGGTT CCTCCTGGGCCAGAAAGAAGCAGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGGT TCTTAGTTCCAGCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCAATCCCACCCG CTAAAGTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAACCAAACCTAGCCTCCAAGAG TGGGAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAATGCCTCCAACATGTGA GGAAGTAATGAGAGAAATCATAGAATT SEQ ID NO: 9 – Construct 4 CMV IE enhancer Chicken beta-actin promoter CMV IE intron A Furin T2A Artificial transcription terminator sequence ATTGGCTATTGGCCAACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATAT ATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGC CCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAA TGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACG CCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGG GACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCC ACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTT TAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGG CGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAA GTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGCC GCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATA GACTCTATAGGCACACCCCTTTGGCTCTTATGCATGCTATACTGTTTTTGGCTTGGGGCCTATACA CCCCCGCTTCCTTATGCTATAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTA TTGACCACTCCCCTATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAA CTATCTCTATTGGCTATATGCCAATACTCTGTCCTTCAGAGACTGACACGGACTCTGTATTTTTAC DB1 / 142158622.1 37 Attorney Docket No.: RBF-003PC / 111846-5003 AGGATGGGGTCCCATTTATTATTTACAAATTCACATATACAACAACGCCGTCCCCCGTGCCCGCAG TTTTTATTAAACATAGCGTGGGATCTCCACGCGAATCTCGGGTACGTGTTCCGGACATGGGCTCTT CTCCGGTAGCGGCGGAGCTTCCACATCCGAGCCCTGGTCCCATGCCTCCAGCGGCTCATGGTCGCT CGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCACAGCACAATGCCCACCACCACCAG TGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTCTGAAAATGAGCGTGGAGATTGGGCTCGCAC GGCTGACGCAGATGGAAGACTTAAGGCAGCGGCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTATT CTGATAAGAGTCAGAGGTAACTCCCGTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGC AGTACTCGTTGCTGCCGCGCGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTC CATGGGTCTTTTCTGCAGTCACCGTCGCTAGCAGGAGAAAGAGAGGATCCAGCGGCGCCACCAACT TCAGCCTGCTTAAACAGGCAGGCGACGTGGAAGAGAACCCCGGACCTCTCGAGAGATCACTTCTGG CTAATAAAAGATCAGAGCTCTAGAGATCTGTGTGTTGGTTTTTTGTGGATCTGCTGTGCCTTCTAG TTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCAC TGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGG GGGTGGGGTGGGGCAGCACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGC GGTGGGCTCTATGGGTACCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCGGTACCT CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCGGTACCAGGTGCTGAAGAATTGACCCG GTTCCTCCTGGGCCAGAAAGAAGCAGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCT GGTTCTTAGTTCCAGCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCAATCCCAC CCGCTAAAGTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAACCAAACCTAGCCTCCAA GAGTGGGAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAATGCCTCCAACATG TGAGGAAGTAATGAGAGAAATCATAGAATT SEQ ID NO: 10 – GLP-1 HGEGTFTSDVSSYLEGQAAKEFIAWLVRGR SEQ ID NO: 11 – GIP YGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ SEQ ID NO: 12 -- GLP-1 / GIP receptor dual agonist YGEGTFTSDYSIALDKIAQKAFVQWLIAGGPSSGAPPPS SEQ ID NO: 13 -- GLP-1 / GIP / GCG receptor triple agonist YAQGTFTSDYSILLDKKAQAAFIEYLLEGGPSSGSPPPS SEQ ID NO: 14 -- GLP-1 / GCG receptor dual agonist QGTFTSDKSEYLDSERARDFVAWLEAGG SEQ ID NO: 15 – hIgG4(FC) DB1 / 142158622.1 38 Attorney Docket No.: RBF-003PC / 111846-5003 AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS RWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGS SEQ ID NO: 16 -- GLP-1-hIgG4(Fc) (His-tagged) Signal peptide GLP-1 Linker Hinge hIgG4(Fc) (His-tagged) MGWSCIILFLVATATGVHSHGEGTFTSDVSSYLEGQAAKEFIAWLVRGRGGGGSGGGGSGGGGSAE SKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSHHHHHH SEQ ID NO: 17 -- DNA encoding SEQ ID NO: 16 ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCCACGGCGAG GGCACCTTCACCAGCGACGTGAGCAGCTACCTGGAGGGCCAGGCCGCCAAGGAGTTCATCGCCTGG CTGGTGAGGGGCAGGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGCCGAG AGCAAGTACGGCCCCCCCTGCCCCCCCTGCCCCGCCCCCGAGGCCGCCGGCGGCCCCAGCGTGTTC CTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTG GTGGACGTGAGCCAGGAGGACCCCGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAC AACGCCAAGACCAAGCCCAGGGAGGAGCAGTTCAACAGCACCTACAGGGTGGTGAGCGTGCTGACC GTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGGCCTGCCC AGCAGCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCTG CCCCCCAGCCAGGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTAC CCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCC CCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAGGCTGACCGTGGACAAGAGCAGGTGG CAGGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAG AGCCTGAGCCTGAGCCTGGGCAAGGGCGGCGGCGGCAGCCACCACCACCACCACCAC SEQ ID NO: 18 -- GIP-hIgG4(Fc) (His-tagged) Signal peptide GIP Linker Hinge hIgG4(Fc)(HIS tagged) MGWSCIILFLVATATGVHSYGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGGS GGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA DB1 / 142158622.1 39 Attorney Docket No.: RBF-003PC / 111846-5003 KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSHHHHHH SEQ ID NO: 19 -- DNA encoding SEQ ID NO: 18 ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCTACGGCGAG GGCACCTTCATCAGCGACTACAGCATCGCCATGGACAAGATCCACCAGCAGGACTTCGTGAACTGG CTGCTGGCCCAGAAGGGCAAGAAGAACGACTGGAAGCACAACATCACCCAGGGCGGCGGCGGCAGC GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGCCGAGAGCAAGTACGGCCCCCCCTGCCCCCCCTGC CCCGCCCCCGAGGCCGCCGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTG ATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAGGAGGACCCCGAGGTG CAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAG TTCAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAG GAGTACAAGTGCAAGGTGAGCAACAAGGGCCTGCCCAGCAGCATCGAGAAGACCATCAGCAAGGCC AAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCCAGGAGGAGATGACCAAGAAC CAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGC AACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTC CTGTACAGCAGGCTGACCGTGGACAAGAGCAGGTGGCAGGAGGGCAACGTGTTCAGCTGCAGCGTG ATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCTGGGCAAGGGCGGC GGCGGCAGCCACCACCACCACCACCAC SEQ ID NO: 20 -- GLP-1 / GIP-hIgG4(Fc) (His-tagged) Signal peptide GLP-1 / GIP receptor dual agonist Linker Hinge hIgG4 (Fc) (HIS-tagged) MGWSCIILFLVATATGVHSYGEGTFTSDYSIALDKIAQKAFVQWLIAGGPSSGAPPPSGGGGSGGG GSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSHHHHHH SEQ ID NO: 21 -- DNA encoding SEQ ID NO: 20 ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCTACGGCGAG GGCACCTTCACCAGCGACTACAGCATCGCCCTGGACAAGATCGCCCAGAAGGCCTTCGTGCAGTGG CTGATCGCCGGCGGCCCCAGCAGCGGCGCCCCCCCCCCCAGCGGCGGCGGCGGCAGCGGCGGCGGC GGCAGCGGCGGCGGCGGCAGCGCCGAGAGCAAGTACGGCCCCCCCTGCCCCCCCTGCCCCGCCCCC GAGGCCGCCGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGC AGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAGGAGGACCCCGAGGTGCAGTTCAAC TGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTTCAACAGC ACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAG TGCAAGGTGAGCAACAAGGGCCTGCCCAGCAGCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAG CCCAGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCCAGGAGGAGATGACCAAGAACCAGGTGAGC CTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAG DB1 / 142158622.1 40 Attorney Docket No.: RBF-003PC / 111846-5003 CCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGC AGGCTGACCGTGGACAAGAGCAGGTGGCAGGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAG GCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCTGGGCAAGGGCGGCGGCGGCAGC CACCACCACCACCACCAC SEQ ID NO: 22 -- GLP-1 / GIP / GCG-hIgG4(Fc) (His-tagged) Signal peptide GLP-1 / GIP / GCG triple agonist Linker Hinge hIgG4 (Fc) (HIS-tagged) MGWSCIILFLVATATGVHSYAQGTFTSDYSILLDKKAQAAFIEYLLEGGPSSGSPPPSGGGGSGGG GSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSHHHHHH SEQ ID NO: 23 -- DNA encoding SEQ ID NO: 22 ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCTACGCCCAG GGCACCTTCACCAGCGACTACAGCATCCTGCTGGACAAGAAGGCCCAGGCCGCCTTCATCGAGTAC CTGCTGGAGGGCGGCCCCAGCAGCGGCAGCCCCCCCCCCAGCGGCGGCGGCGGCAGCGGCGGCGGC GGCAGCGGCGGCGGCGGCAGCGCCGAGAGCAAGTACGGCCCCCCCTGCCCCCCCTGCCCCGCCCCC GAGGCCGCCGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGC AGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAGGAGGACCCCGAGGTGCAGTTCAAC TGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTTCAACAGC ACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAG TGCAAGGTGAGCAACAAGGGCCTGCCCAGCAGCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAG CCCAGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCCAGGAGGAGATGACCAAGAACCAGGTGAGC CTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAG CCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGC AGGCTGACCGTGGACAAGAGCAGGTGGCAGGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAG GCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCTGGGCAAGGGCGGCGGCGGCAGC CACCACCACCACCACCAC SEQ ID NO: 24 -- GLP-1 / GCG-hIgG4(Fc) (His-tagged) Signal peptide GLP-1 / GCG receptor dual agonist Linker Hinge hIgG4 (Fc) (HIS-tagged) MGWSCIILFLVATATGVHSQGTFTSDKSEYLDSERARDFVAWLEAGGGGGGSGGGGSGGGGSAESK YGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA DB1 / 142158622.1 41 Attorney Docket No.: RBF-003PC / 111846-5003 KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSHHHHHH SEQ ID NO: 25 -- DNA encoding SEQ ID NO: 24 ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCCAGGGCACC TTCACCAGCGACAAGAGCGAGTACCTGGACAGCGAGAGGGCCAGGGACTTCGTGGCCTGGCTGGAG GCCGGCGGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGCCGAGAGCAAG TACGGCCCCCCCTGCCCCCCCTGCCCCGCCCCCGAGGCCGCCGGCGGCCCCAGCGTGTTCCTGTTC CCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGAC GTGAGCCAGGAGGACCCCGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCC AAGACCAAGCCCAGGGAGGAGCAGTTCAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTG CACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGGCCTGCCCAGCAGC ATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCTGCCCCCC AGCCAGGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGC GACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTG CTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAGGCTGACCGTGGACAAGAGCAGGTGGCAGGAG GGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTG AGCCTGAGCCTGGGCAAGGGCGGCGGCGGCAGCCACCACCACCACCACCAC SEQ ID NO: 26 -- ABD035 AGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGAAAACTCACCC GGCCCGGACACTGTAAGGATTGACAGACTGAGAGCTCTTTCTTAATTCAGTGGGTGGTGG TGCATGGCCGTTCTTAGTTGGTGGAGCGATTTGTCTGGTTAATTCCGATAACGAACGAGA CTCTGGCCTATTAAATAGACGAGATATTACTCAGTATCTCGCGCTTCTTAGAGGGACAAG CGGCGTCTAGTCGCATGAAAAAGAGCAATAACAGGTCTGTGATGCCCTTAGATGTCCGGG GCTGCACGCGCGCTACACTGAAAGAATCAGTGGGCGTTTTACCTGGTTCGGAAGAACCGG GTAACCCAAGTAATTTCTTTCGTGCTTGGGATAGGGAATTGCAATTATTTCCCTTAAACG AGGAATTCCCAGTAAACGCGGGTCATAAGCTCGTGTTGATTACGTCCCTGCCCTTTGTAC ACACCGCCCGTCGCTACTACCGATTGGATGACTTAGTGA SEQ ID NO: 27 -- ANGIOPEP-2TFFYGGSRGKRNNFKTEEY DB1 / 142158622.1 42

Claims

Attorney Docket No.: RBF-003PC / 111846-5003 CLAIMS What is claimed is:

1. A DNA composition comprising a plasmid construct having at least one expression cassette, wherein the expression cassette comprises in the following order from 5´ to 3´: a) a CMV IE enhancer sequence; b) a chicken beta-actin promoter sequence; c) a CMV IE intron A sequence; d) an open reading frame encoding a peptide hormone, such as an agonist at one or more of glucagon-like peptide-1 (GLP-1) receptor, glucose-dependent insulinotropic polypeptide (GIP) receptor, and / or a glucagon (GCG) receptor; and e) a transcription termination sequence.

2. The DNA composition of claim 1, wherein the peptide hormone comprises a fusion to a polypeptide for PK enhancement.

3. The DNA composition of claim 2, wherein the polypeptide fusion is Fc or albumin.

4. The DNA composition of any one of claims 1 to 3, wherein the GLP-1 receptor agonist is GLP-1 or exenatide.

5. The DNA composition of any one of claims 1 to 3, wherein the open reading frame encodes a dual agonist for GLP-1 receptor and GIP receptor, and optionally comprises the amino acid sequence of tirzepatide.

6. The DNA composition of any one of claims 1 to 3, wherein the open reading frame encodes a triple agonist for GLP-1 receptor, GIP receptor, and GCG receptor, and optionally comprises the amino acid sequence of retratrutide.

7. The DNA composition of any one of claims 1 to 6, wherein the termination sequence is an artificial transcription termination sequence comprising segments of a bovine growth hormone polyadenylation signal (BGHpA). DB1 / 142158622.1 43Attorney Docket No.: RBF-003PC / 111846-5003 8. The DNA composition of any one of claims 1 to 7, wherein the plasmid construct comprises two of said expression cassettes, a first expression cassette having an open reading frame encoding a GLP-1 receptor agonist; and a second expression cassette having an open reading frame encoding a GIP receptor agonist or a GCG receptor agonist.

9. The DNA composition of claim 8, wherein the first expression cassette has an open reading frame encoding a GLP-1 receptor agonist; and the second expression cassette has an open reading frame encoding a GIP receptor agonist.

10. The DNA composition of claim 1, wherein the open reading frame encodes a GLP- 1 receptor agonist, and either a GIP receptor agonist or a GCG receptor agonist, with a peptide linker therebetween that induces ribosomal skipping, and which optionally comprises a P2A peptide or a T2A peptide.

11. The DNA composition of claim 10, wherein the GLP-1 receptor agonist, the GIP receptor agonist, and / or the GCG receptor agonist comprise a fusion to a polypeptide for PK enhancement.

12. The DNA composition of claim 11, wherein the polypeptide fusion is Fc, albumin, or albumin binding domain.

13. The DNA composition of any one of claims 1 to 12, further comprising fusion of Angiopep-2 amino acid sequence.

14. The DNA composition of any one of claims 10 to 13, wherein the GLP-1 receptor agonist is GLP-1 or exenatide, and which is expressed with a GIP receptor agonist.

15. The DNA composition of any one of claims 10 to 14, wherein the peptide linker comprises a P2A peptide.

16. The DNA composition of claim 15, wherein the peptide linker comprises a T2A peptide. DB1 / 142158622.1 44Attorney Docket No.: RBF-003PC / 111846-5003 17. The DNA composition of any one of claims 10 to 16, wherein the peptide linker further comprises a furin recognition site on the N-terminal side of the peptide that induces ribosomal skipping.

18. The DNA composition of claim 17, wherein the linker sequence comprises a linker between the furin recognition site and the peptide that induces ribosomal skipping.

19. The DNA composition of any one of claim 1 to 18, comprising an open reading frame encoding an agent selected from Bimagrumab, Maridebart, or Cagrilintide.

20. A method for treating or preventing a disease in a subject, comprising administering an effective amount of the composition of any one of claims 1 or 19 to a subject in need thereof.

21. The method of claim 20, wherein the disease is a metabolic disease.

22. The method of claim 20, wherein the subject has or is at risk of obesity.

23. The method of claim 20, wherein the subject has diabetes or is at risk of diabetes.

24. The method of claim 23, wherein the diabetes is type 1 diabetes.

25. The method of claim 23, wherein the diabetes is type 2 diabetes.

26. The method of claim 20, wherein the subject or is at risk of metabolic syndrome.

27. The method of any one of claims 20 to 26, wherein administering comprises intramuscular injection and electroporation.

28. The method of claim 27, wherein administering is no more than about monthly.

29. The method of claim 28, wherein administering is no more than about quarterly.

30. The method of claim 28, wherein administering is no more about twice per year.

31. The method of claim 28, wherein administering is no more than about annually. DB1 / 142158622.1 45Attorney Docket No.: RBF-003PC / 111846-5003 32. The method of any one of claims 28 to 31, further comprising monitoring for the presence of the expressed protein in the circulation at least once between administrations. DB1 / 142158622.1 46

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