G protein-coupled receptor (GPCR) agonists and use thereof

Abstract

G protein-coupled receptor (GPCR) agonist peptides and their use are described. Also described are vectors for expressing the GPCR agonist peptides and method for treating a disease or disorder with the GPCR agonist peptides.

Description

DOCKET NO: 1134-242 PCTTITLEG PROTEIN-COUPLED RECEPTOR (GPCR) AGONISTS AND USE THEREOF

[0001] This application claims priority of U.S. Provisional Application No. 63 / 679,350, filed on August 5, 2024, and U.S. Provisional Application No. 63 / 823,219, filed on June 13, 2025, all of which are incorporated by reference in their entirety.

[0002] REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0003] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on July 24, 2025, is named “1134-242 PCT. xml” and is 383,257 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.FIELD

[0004] This application relates to therapeutic peptides, in particular, GPCR agonists.BACKGROUND

[0005] Recent development and use of GPCR agonists (GPCRAs), such as glucagon-like peptide-1 (GLP-1) receptor (GLP1R) agonists (GLPIRAs), that mimic the actions of a natural hormone, called GLP-1, have been highly effective for weight management and improved cardiovascular health when administered long-term in both diabetic and non-diabetic patients. The naturally occurring GLP-1 peptide has an extremely short half-life in circulation, thereby limiting its efficacy when delivered as a therapeutic. Modifications to GLP-1 protein and other related GLPIRAs, through amino acid changes to prevent cleavage / inactivation and / or the addition of molecular adducts (such as fatty acids, etc.), have helped increase the circulating half-life and therapeutic efficacy of these peptide therapeutics. The current FDA-approved GLP- IRAs for glycemic control include: Dulaglutide, Exenatide, Liraglutide, Lixisenatide / insulin glargine and semaglutide. The current FDA- approved GLP-1 agonists for weight loss include: Tirzepatide (dual GIP / GLP-1 receptor agonist) and Liraglutide. However, these treatments still require regular injections (daily to weekly), which may limit to their long-term use and adherence. Further, the success and popularity of these drugs for off- label weight loss in non- diabetic patients, in combination with the difficulty in their manufacturing, have resulted in an inadequate supply which may last for years to come.

[0006] Thus, there is a need in the art for novel GLP1R agonists. This application satisfies this unmet need.SUMMARY

[0007] One aspect of the present application relates to a polynucleotide expression construct that comprises from 5’ end to 3’ end: (1) a sequence encoding a 5’ ribozyme, (2) a sequence encoding an internal ribosome entry site (IRES), (3) a sequence for expressing a fusion G protein-coupled receptor (GPCR) agonist peptide and (4) a sequence encoding a 3 ’ribozyme, wherein the sequence for expressing the fusion GPCR agonist peptide comprises a sequence encoding a signal peptide and a sequence encoding a GPCR agonist peptide.

[0008] Another aspect of the present application relates to a polynucleotide expression cassette that encodes a fusion GPCR agonist peptide. The expression cassette comprises (1) a sequence encoding a signal peptide having an amino acid sequence selected from the group consisting of SEQ ID NOS:22-27 and SEQ ID NOS:370-393; and (2) a sequence encoding a GPCR agonist peptide having an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-21, 171-179, 196-205, 223-230, 250-254, 266-323 and 342-347.

[0009] Another aspect of the present application relates to a method for generating a circular RNA molecule encoding a fusion GPCR agonist peptide. The method comprises the step of introducing into a cell or tissue a nucleic acid molecule that comprises (1) a sequence encoding a 5’ ribozyme, (2) a sequence encoding an IRES, (3) a sequence for expressing the fusion GPCR agonist peptide, and (4) a sequence encoding a 3 ’ribozyme, wherein the sequence for expressing the fusion GPCR agonist peptide comprises a sequence encoding a signal peptide and a sequence encoding a GPCR agonist peptide, and wherein self-cleavage of the 3 ’ribozyme generates a 3’ phosphate and selfcleavage of the 5 ’ribozyme generates a 5’ hydroxy group, allowing for scarless ligation of the 5’ hydroxy group and 3’ phosphate of the RNA molecule to generate a circular RNA molecule encoding the fusion GPCR agonist peptide.

[0010] Another aspect of the present application relates to a fusion GPCR agonist peptide that comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:356, 358, 365 and 366.

[0011] Another aspect of the present application relates to a GPCR agonist peptide that comprises an amino acid sequence selected from (1) the group consisting of SEQ ID NOS: 1-21, 171- 179, 196-205, 223-230, 250-254, 266-323 and 342-347; or (2) the group consisting of SEQ ID NOS:305, 306, 312 and 314.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present application can be better understood by reference to the following drawings, wherein like references numerals represent like elements. The drawings are merely exemplary to illustrate certain features that may be used singularly or in combination with other features and the present disclosure should not be limited to the embodiments shown.

[0013] FIG. 1 shows in vitro transcribed CirculR RNA (panels A-E) is robustly translated without requiring the additional processing (cap and poly(A) tail) required to make linear in vitro transcribed RNA suitable for translation (panels A’-E’ and B”).

[0014] FIGS. 2A-2C show that human GLP-1 is processed from the Pre-pro-glucagon protein encoded by the GCG gene. (FIG. 2A) The human GCG gene is encoded by 6 exons on human chromosome 2. The relative locations of the processed peptides GCG, GLP-1 and GLP-2 from exons 3, 4, and 5 are labeled. (FIG. 2B) Sequence of the human pre-pro-glucagon protein (SEQ ID NO:34), which encodes a signal peptide (green, SEQ ID NO:27) that is cleaved to leave the proglucagon protein, that encodes the GCG (blue, SEQ ID NO:329), GLP-1 (red, SEQ ID NO: 174) and GLP-2 (brown, SEQ ID NO:272). (FIG. 2C) Proglucagon is expressed in both the pancreas and Intestinal L- cells, where is differentially processed by PC2 (pancreas) and PC 1 / 3 (Intestinal L-cells) into multiple bioactive peptide hormones, including GLP-1.

[0015] FIGS. 3A-3D show post-translational processing of human GLP-1, related GLP1R agonists and sequence modification to promote stability. As shown in FIG. 3 A, human GLP-1 is processed from proglucagon (1-42) (SEQ ID NO:167) as GLP-1 (1-37) (SEQ ID NQ:400), which is subsequently processed into GLP-1 (7-37) (SEQ ID NO: 174) and GLP-1 (7-36) (SEQ ID NO:173), the bioactive forms of the peptide. Cleavage and removal of the two N-terminal residues by DPP-IV to generate GLP-1 (a.a. 9-36)(SEQ ID NO:180) inactivates the peptide. Modifications to GLP-1 sequence, such as an Alanine to Glycine at position 8 (A8G; SEQ ID NO:319) to inhibit DPP-IV cleavage, or mutation of internal Lysine residues to prevent Trypsin cleavage, increase GLP-1 durability and retain GLP1R activation. As shown in FIG. 3B, the naturally occurring exendin-4 peptide expressed in the venom of the Gila Monster (SEQ ID NO: 14) is related to GLP-1 in both sequence and structure but is more stable due to a natural Glycine at position 8 (human GLP-1 numbering scheme) and an extended C-terminal tail (SEQ ID NO: 10). Further modifications to remove trypsin cleavage sites (TSME1) further enhances its stability (SEQ ID NO:11). As shown in FIG. 3C, dual GIP and GLP1R agonists (such as Tirzepatide (TZP; SEQ ID NO:8) can be more effective than just GLP1R agonists in controlling blood glucose levels. Modification of position 8 proposed here may make these peptides more stable (SEQ ID NO: 9). As shown in FIG. 3D,modifications to human GLP-1, such as addition of the extendin-4 C-terminal tail provide additional metabolic stability (GLP-EX; SEQ ID NO: 13).

[0016] FIGS. 4A-4C: Assessment of GLP1R signaling activity using a cAMP- responsive cellbased luciferase assay reporter. FIG. 4A shows a diagram showing the principles of the luciferasebased reporter for assessing GLP1RA activity. Cells are co-transfected with an expression plasmid encoding GLP1R (or other GPCR) and a luciferase reporter containing 6 copies of the cAMP response element (6X cAMP -RE-Luc). Upon stimulation by an GLP1R agonist (or other GPCR agonist), increased cAMP levels and signaling result in activation of the luciferase reporter. GLP1R agonists can be expressed from the same cells expressing the reporter constructs by co-transfection of plasmids encoding GLPIRAs (FIG. 4B) or secreted into supernatants and subsequently added to reporter cells to induce activation (FIG. 4C). While expression of the full length GCG preproglucagon protein (GCG), which encodes a natural signal peptide for secretion, is sufficient to activate the reporter cells, a signal peptide from CD5 was included to allow for secretion of the mature GLP-1 peptide. In FIG. 4D and 4E, co-transfection of different GLPIRAs were assessed for their ability to signal through the GLP1R (FIG. 4D) and GIPR (FIG. 4E). Constructs encoding the MGLP-1 peptide showed the higher activity than GLP-1, while the H7 signal peptide in combination with MGLP-1 showed the greatest activity overall.

[0017] FIGS. 5A-5E show discovery of a putative mammalian GLP-1 paralog, GLP- IB, in bats. Human GLP-1 (SEQ ID NO:176) is conserved across vertebrate species and shares a number of identical residues among GLP-1 orthologs and FIG. 5 A shows its similarity to the other vertebrate GLP1R agonist, exendin-4 (SEQ ID NO:328). Exendin-4 represents a GLP-1 gene duplication event, first found in the venom / saliva of Gila Monster (Heloderma suspectum), a species of venomous lizard found in the Southwestern United States. Exendin-4 acts as an agonist of GLP1R but has an extended C-terminus which results in longer stability in the body and was thus the first GLP1R agonist developed to treat metabolic syndromes. Exendin-4-like genes have now been found to be present in other vertebrate species, but this gene was lost in the lineage that gave rise to mammals. FIG. 5B shows the consensus sequence (SEQ ID NO:401) for known GLP-1 analogues (source Patent publication US20100317057A1). As shown in FIG. 5C, GLP-1 also shares a number of similarities with differences with the other peptide hormones produced from the preGCG gene, GCG (SEQ ID NO: 266) and GLP-2 (SEQ ID NO:272), but these differences are critical for their specific activation of their cognizant closely related G protein coupled receptors GPCRs, GCGR, GLP1R and GLP2R. Vertebrate species, including humans and other mammals, additionally contain other glucagon-like hormones, such as glucose-dependent insulinotropic peptide (GIP, SEQ ID NO:330), encoded by the GIP gene, which also functions as an incretin. FIG. 5D shows a BLAST protein search (blastp, NCBI)using human GLP-1 sequence in Bat species (SEQ ID NOs:331-333), identified numerous GLP-1 (pro-glucagon) orthologs, including orthologues in the Greater mouse-eared bat (Myotis myotis), herein referred to as GLP-1 A (SEQ ID NO:202) and GLP-1B (also referred to as GLP1X1, SEQ ID NO:227). Interestingly, this search also revealed the presence of an additional related sequence in myotis, named “hypothetical protein mMyoMyol_012148.” This sequence comes from a publication on first reference-quality genomes of six bat species (Rhinolophus ferriimequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Vipistrellus kuhlii, and Molossus molossus) published in 2020 and combines both long read genomic and transcriptomic data (D. Jebb et al., Nature 583, 578-584 (2020))

[0018] The newly discovered gene from Myotis myotis appears to be a paralog of GLP-1 A, thus it is named here as GLP-1B (SEQ ID NO:227), and differs from the GLP-1 analogue consensus sequence in two identically conserved positions, “TFTSD” to “iFnSD.” Despite these changes, a reverse search of Bat GLP-1B in the human genome revealed that human GLP-1 as the most related protein, followed by human Glucagon (GCG). A tblastn (protein sequence search of translated nucleotide sequences) search revealed that other bat species, such as Myotis daubentoniid, also contain both GLP-1 A and GLP-1B protein coding potential. FIG. 5E shows assessment of human GLP-1, AL myotis GLP-1 ortholog (GLP-1A) and GLP-1 paralog (GLP-1B), and GIP on activation of human GLP1R, GIPR and GCGR. As predicted, GIP was the only hormone to activate GIPR. None of the hormones tested activated GCGR. Interestingly, despite the unique sequence differences found in Bat GLP-1B, it functioned as a robust and specific GLP1R agonist similar to related human GLP-1 and AL myotis GLP-1 A.

[0019] FIGS. 6A-6C: Characterization of GLP-1B protein sequence. (FIG. 6A) Predicted full- length sequences of the AL myotis preproglucacon (ppGCG) proteins ppGCG-A (SEQ ID NO:47) and ppGCG-B (SEQ ID NO:48). Similar to human ppGCG, each AL myotis protein contains three peptide hormones related to human GCG, GLP-1 and GLP-2. However, ppGCG-B contains and potential additional N-terminal sequence that contains a potential DD domain, which may function as a protein interaction domain commonly found in proteins involved in signaling. FIG. 6B depicts alignment of conserved GCG, GLP-1, and GLP-2 regions of human (SEQ ID NO: 34) and AL myotis (SEQ ID NOs: 47 and 48) ppGCG protein sequences. FIG. 6C shows a prediction of DPP-IV and trypsin cleavage locations in human GLP-1 (SEQ ID NO:183) and AL myotis GLP1-A and GLP1-B (SEQ ID NO:233 and SEQ ID NO:281, respectively). GLP-1A and GLP— IB from Bat species may be resistant to DPP-IV cleavage due to a Serine substitution at position 2 in the mature GLP-1 sequence.

[0020] FIG. 7: Design of a circulR vector encoding a signal peptide (SP)-GLPIRA fusion protein, under the control of an IRES, for generating RNA using in vitro transcription. The RNA,when complexed in a lipid nanoparticles (LNPs), exosomes, virus-like particles (VLP), or other packaging method, can be delivered in vivo through injection, oral drop, or other delivery method, for expression and secretion from cells into the body (blood stream, gut or other tissue or cell type). Expression and secretion of GLP-1 analogues from cells into the body will activate GLP1R signaling in the body. The circularized RNA vector may provide more consistent and durable expression over time and is redosable. Adherence to long term GLP1RA treatments have been shown to provide significant benefits for weight control and protection from cardiovascular disease, neurological disease, and cancer.

[0021] FIGS. 8A-8D show GCGX-derived paralog sequences are functional receptor-specific activation. (FIG. 8A) Agonist-triggered glucagon-related receptor activation results in cAMP- dependent signaling pathways which can be monitored using a luciferase reporter containing six copies of a cAMP -response element (RE) (6X cAMP RE-Luc). (FIG. 8B) Alternatively, P-arrestin recruitment in response to agonist-triggered receptor activation can be monitored using the PRESTOTANGO system. Here, P-arrestin recruitment triggers the release of a tetracycline-responsive transactivator (tTA) that can be monitored using a tetracycline-dependent luciferase reporter (TET RE- Luc). (FIG. 8C) Diagram depicting the construct design for expressing the mature glucagon-related peptide hormones, which are secreted using a heterologous signal peptide (SP). (FIG. 8D) We cloned GCG, GLP-1, GLP-2 and GIP peptide hormones from representative vertebrates and tested their specific activation on GCGR, GLP1R, GLP2R and GIPR receptors using the cAMP RE-Luc reporter. We also cloned and tested GLP1X, GLP2X and GIPX paralog sequences and determined their receptor-specific activation.

[0022] FIGs. 9A-9D show Myotis GLP1X paralogs require a longer C-terminus to achieve full receptor-activation. The impact of C-terminal amino acid deletion and extension on the activity of GLP-1 orthologs and GLP1X paralogs were assayed using the cell based cAMP-responsive luciferase reporter assay. The function of human GLP-1, which naturally occurs as the (7-36) and (7-37) forms, is neither diminished or enhanced by increasing C-terminal extensions (FIG. 9A; SEQ ID NOs:167- 180). The canonical GLP-1 ortholog from Myotis bats is similarly unaffected by longer C-terminal extensions, despite sharing many of the same C-terminal amino acid differences as GLP1X paralogs (FIG. 9B; SEQ ID NOs:193-206). In contrast, GLP1X paralogs from Myotis bats show either reduced or no GLPIR-mediated cAMP -reporter activation when expressed as (7-36) forms (FIG. 9C; SEQ ID NOs:218-231 and FIG. 9D; SEQ ID NOs:242-255). Interestingly, GLP1X paralogs with longer C- terminal extensions gain receptor activation. Deletion of the two N-terminal amino acids, mimicking DPP-4 cleavage, abolished activity for both GLP-1 orthologs and GLP1X paralogs.

[0023] FIGS. 10A-10E show C-terminal extensions on Myotis GLP-1 and GLP1X peptides provide resistance to DPP-4 inhibition. To measure the resistance of GLP-1 orthologs and GLP1X paralogs to inactivation by DPP -4, we utilized an assay where the peptide agonists co-expressed with or without DPP -4 in cell supernatants and then transferred to reporter cells co-expressing GLP1R and a cAMP-responsive luciferase reporter (FIG. 10 A). In the absence of DPP -4, the results of this supernatant transfer were similar to those obtained when agonists were directly co-expressed with GLP1R and a cAMP-luciferase reporter, such as those data presented in Figure 5, including human GLP-1 (FIG. 10B; SEQ ID NOS:169-179), the canonical Myotis GLP-1 M.m.GLP-1 (FIG. 10C; SEQ ID NOS:195-205), or Myotis GLP1X paralogs M.m.GLPIXl (FIG. 10D; SEQ ID NOS:222-230) and M.d.GLPlX4 (FIG. 10E; SEQ ID NOS:246-254). Consistent with the labile nature of human GLP-1, DPP-4 completely inactivated its activity, and the presence or absence of C-terminal deletions or extensions showed no effect. Interestingly, however, Myotis GLP-1 M.m.GLP-1 (FIG. 10C) and the Myotis GLP1X paralog M.m.GLPIXl (FIG. 10D) showed resistance to DPP -4 inactivation, but only with C-terminal extensions that were greater than required to activate GLP1R in the absence of DPP -4.

[0024] FIG. 11 A-l ID show GLP-1 agonist modifications, when co-expressed with (w / ) or without (w / o) DPP4 overexpression in the same cells as human GLP1R and the cAMP -responsible luciferase reporter (FIGS. 11 A and 1 IB), or when GLP-1 agonists were co-expressed with (w / ) or without (w / o) DPP4 overexpression in separate cells, from which agonist containing supernatants were transferred to reporter cells (FIGS. 11C and 1 ID).

[0025] FIGS. 12A-12C show that the A8W modification of GLP-1 (7-36) provided robust resistance to DPP -4 inhibition. Substitution of tryptophan (W) at the same relative position in GLP-2 and GIP, but not GCG, provides resistance to DPP-4 inhibition. In the case of GCG, the tryptophan modification abolished activity with or without the addition of DPP -4. These data suggest that positional replacement of tryptophan is a general strategy for preventing DPP-4 inhibition of peptide hormones, however not all hormones tolerate this replacement.

[0026] FIG. 13 is a schematic depicting the ribozyme-mediated scarless cis-splicing to generate a circular RNA which can be translated via IRES-mediated translation. The 5’ ribozyme sequence and 3’ ribozyme sequence are listed as SEQ ID NO:402 and SEQ ID NO:403, respectively.

[0027] FIGS. 14A and 14B show CirculR 2.0 encoding a GFP-blasticidin-2A-luciferase (GBL) fusion protein can be efficiently delivered and expressed in vivo in liver using RNA-lipid nanoparticles. (FIG. 14A) CirculR 2.0 GBL RNA-LNPs were formulated using a Spark NanoAssemblr, injected into C57BL / 6J mice and imaged for bioluminescence after 6 hours, using IVIS imaging. CirculR 2.0 GBL injected mice showed robust luciferase activity in liver, compared to saline injected mice, demonstrating in vivo delivery and expression. (FIG. 14B) CirculR 2.0 vectorsencoding GLP-1 sequences were generated to express either human GLP-1 fused to IgG4 fc (GLP-1 - IgG4 fc) or human GLP-1 with the A8W modification which prevents DPP -4 inhibition (GLP-1 A8W).DETAILED DESCRIPTION

[0010] Reference will be made in detail to certain aspects and exemplary embodiments of the application, illustrating examples in the accompanying structures and figures. The aspects of the application will be described in conjunction with the exemplary embodiments, including methods, materials and examples, such description is non-limiting and the scope of the application is intended to encompass all equivalents, alternatives, and modifications, either generally known, or incorporated here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. One of skill in the art will recognize many techniques and materials similar or equivalent to those described here, which could be used in the practice of the aspects and embodiments of the present application. The described aspects and embodiments of the application are not limited to the methods and materials described.

[0028] As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the content clearly dictates otherwise.

[0029] The present application provides compositions and methods for increasing GLP1R activity. The present application also provides compositions comprising a GLP1R agonist peptide or a nucleic acid molecule encoding a GLP1R agonist peptide. The nucleic acid molecule may be a DNA molecule or an RNA molecule. The nucleic acid molecule may be a linear or a circular DNA or RNA molecule.I. Definitions

[0030] As used herein, the term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0031] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by a regulatory sequence such as a promoter and / or an enhancer.

[0032] As used herein, the term "expression vector" refers to a composition of matter which comprises a nucleotide sequence encoding a protein and / or an RNA and which can be used to deliver the nucleic acid sequence to the interior of a cell and express the encoded protein and / or RNA inside the cell. An expression vector typically comprises a regulatory sequence for expression of the protein or RNA encoded by the nucleotide sequence, wherein the regulatory sequence is operably linked to the nucleotide sequence. Expression vectors include non-viral vectors, such as plasmids, phagemids, and cosmids, and viral vectors, such as adenovirus vectors, adeno-associated virus (AAV) vectors, and retrovirus vectors.

[0033] The term “expression cassette” is a distinct component of vector DNA consisting of a gene and regulatory sequence to be expressed by a transfected cell. In each successful transformation, the expression cassette directs the cell's machinery to make RNA and protein(s). Some expression cassettes are designed for modular cloning of protein-encoding sequences so that the same cassette can easily be altered to make different proteins.

[0034] The term “functional variant” refers to a polypeptide or a polynucleotide that maintains one or more of the biological functions of an original polypeptide or a polynucleotide (also referred to as wild-type polypeptide or a polynucleotide). A functional variant is structurally similar or substantially structurally similar to a parent or reference compound of the present application, but differs, in some contexts slightly, in composition (e.g., one base, atom or functional group is different, added, or removed; or one or more amino acids are mutated, inserted, or deleted), such that the polypeptide or encoded polypeptide is capable of performing at least one function of the encoded parent polypeptide with at least 50% efficiency, preferably at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or at least 100% level of activity of the parent polypeptide. In some embodiments, a functional variant of an original polypeptide or polynucleotide maintains at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of at least one biological function of the original polypeptide or polynucleotide.

[0035] The term a “functional portion” or “functional fragment” refers to a polypeptide or polynucleotide that comprises only a domain, motif, portion or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% activity associated with the domain, portion or fragment of the parent or reference compound, preferably at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.9%, or at least 100% level of activity of the parent polypeptide, or provides a biological benefit (e.g., effector function).

[0036] The term “G protein-coupled receptor (GPCR)”, as used herein, refers to a group of evolutionarily related proteins that are cell surface receptors that detect molecules outside the cell and activate cellular responses. They are coupled with G proteins. They pass through the cell membrane seven times in the form of six loops (three extracellular loops interacting with ligand molecules, three intracellular loops interacting with G proteins, an N-terminal extracellular region and a C-terminal intracellular region) of amino acid residues, which is why they are sometimes referred to as seven-transmembrane receptors. GPCRs are activated by agonists of GPCR (GPCRAs). A GPCRA can bind either to the extracellular N-terminus and loops (e.g. glutamate receptors) or to the binding site within transmembrane helices (rhodopsin-like family). GPCRs can be grouped into six classes based on sequence homology and functional similarity. These families are:Class A (or 1) (Rhodopsin-like)Class B (or 2) (Secretin receptor family)Class C (or 3) (Metabotropic glutamate / pheromone)Class D (or 4) (Fungal mating pheromone receptors)Class E (or 5) (Cyclic AMP receptors)Class F (or 6) (Frizzled / Smoothened)In some embodiments, the term GPCR refers to Class B GPCRs. Examples of Class B GPCRs include, but are not limited to, GCGR, GLP1R, GLP2R, and GIPR.

[0037] The terms "nucleobase complementarity" and "complementarity" refer to a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). When used in reference to an oligonucleotide or portion thereof, the term "fully complementary" means that each nucleobase of the oligonucleotide or portion thereof is capable of pairing with a nucleobase of a complementary nucleic acid or contiguous portion thereof. Thus, a fully complementary region comprises no mismatches or unhybridized nucleobases in either strand. The term "partially complementary" means that one or more nucleobase of the oligonucleotide or portion thereof is not capable of pairing with the nucleobase(s) at the corresponding position(s) of a complementary nucleic acid or contiguous portion thereof.

[0038] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in amanner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0039] The term "nucleotide sequence" or interchangeably “nucleic acid sequence” refers to a single- or double-stranded nucleic acid. It can be DNA or RNA. It can also be single-stranded or double-stranded DNA. It also includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0040] The term "operably linked" refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0041] As used herein, the term "regulatory sequence" means a nucleic acid sequence which is required for expression of a coding sequence (either for protein or RNA) operably linked to the promoter / regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter / regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner. The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. A "constitutive" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell. An "inducible" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell. A "tissue-specific" promoter is a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causesthe gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0042] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[0043] The terms “treat” or “treatment” of a state, disorder, disease, or condition include: (1) preventing, delaying, or reducing the incidence and / or likelihood of the appearance of at least one clinical or sub-clinical symptom of the state, disorder, disease, or condition developing in a subject that may be afflicted with or predisposed to the state, disorder, disease, or condition, but does not yet experience or display clinical or subclinical symptoms of the state, disorder, disease, or condition; or (2) inhibiting the state, disorder, disease, or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof or at least one clinical or sub-clinical symptom thereof; or (3) relieving the state, disorder, disease, or condition, i.e., causing regression of the state, disorder, disease, or condition or at least one of the clinical or sub-clinical symptoms of the state, disorder, disease, or condition. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[0044] The term “effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result, or to elicit a desired therapeutic response in at least a sub-population of subjects, for example, ameliorate the symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0045] The term "subject" or "patient" as used herein is intended to include animals including humans, apes, monkeys, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. Preferably the subject is a mammal or human.

[0046] The phrase “pharmaceutically acceptable”, as used in connection with compositions described herein, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., a human). Preferably, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

[0047] The term "pharmaceutically acceptable carrier or excipient” refers to a vehicle that does not produce a severe adverse, allergic or other untoward reaction when administered to a subject, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a nontoxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.II. Polypeptides of the present applications

[0048] One aspect of the present application relates to a G protein-coupled receptor (GPCR) agonist peptide and functional variants thereof. In some embodiments, the GPCR agonist peptide of the present application comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-21, SEQ ID NOS: 171-179, SEQ ID NOS:223-230, SEQ ID NOS:266-323, SEQ ID NOS:342-345, and SEQ ID NOS:346-347

[0049] In some embodiments, the GPCR agonist peptides of the present application comprise an amino acid sequence selected from the group consisting of SEQ ID NOS: 305, 306, 312, 314 and 342-347

[0050] In some embodiments, the GPCR agonist peptide of the present application is fused to another peptide to form a fusion GPCR agonist peptide. In some embodiments, the fusion GPCR agonist peptide comprises (1) a signal peptide and (2) a GPCR agonist peptide. In some embodiments, the signal peptide of the fusion peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:22-27 and SEQ ID NOS:370-393.

[0051] In some embodiments, the GPCR agonist peptide of the fusion GPCR agonist peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:1-21, 171-179, 196-205, 223-230, 250-254, 266-323 and 342-347.

[0052] In some embodiments, the GPCR agonist peptide of the fusion GPCR agonist peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 305, 306, 312, 314 and 342-347.

[0053] In some embodiments, the GPCR agonist peptide of the fusion GPCR agonist peptide comprises the amino acid sequence of SEQ ID NO:342.

[0054] In some embodiments, the GPCR agonist peptide of the fusion GPCR agonist peptide comprises the amino acid sequence of SEQ ID NO:343.

[0055] In some embodiments, the GPCR agonist peptide of the fusion GPCR agonist peptide comprises the amino acid sequence of SEQ ID NO:344.

[0056] In some embodiments, the GPCR agonist peptide of the fusion GPCR agonist peptide comprises the amino acid sequence of SEQ ID NO:345.

[0057] In some embodiments, the fusion GPCR agonist peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:28-52.

[0058] In some embodiments, the fusion GPCR agonist peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:334-341 and SEQ ID NOS:348-369.

[0059] In some embodiments, the fusion GPCR agonist peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:356, 358, 365 and 366.

[0060] In some embodiments, the fusion GPCR agonist peptide further comprises additional chemical groups, such a diacidic fatty acid, and / or additional amino acid sequence that improve the stability of the fusion peptide. Such chemical groups and / or amino acid sequences can be attached to lysine residues of the fusion GPCR agonist peptide, with or without a linker / spacer sequence. Examples of the chemical groups that improvess the stability of the fusion peptide include, but are not limited to, diacidic fatty acid. Examples of the amino acid sequence that improves the stability of the fusion peptide include, but are not limited to, IgG4Fc sequences, albumin, albumin-binding domains from bacteria, such as SEQ ID NO:394, serum albumin binding knob domains, such as SEQ ID NOS:395 and 396, serum immunoglobulin (e.g., IgG4Fc) and albumin binding single-domain antibodies, such as SEQ ID NOS:397-399.

[0061] In some embodiments, the polypeptide of the present application comprises an amino acid sequence that is substantially homologous to an amino acid sequence described herein. Exemplary GPCR agonist peptides include, but are not limited to, peptide comprising a sequence selected from the group consisting of SEQ ID NOS:1-21, 171-179, 196-205, 223-230, 250-254, 266-323 and 342- 347.

[0062] In some embodiments, the present application provides a functional variant of a polypeptide described herein. For example, in some embodiments, a functional variant of a polypeptide of the present application has a degree of identity with respect to the original amino acid sequence of the polypeptide of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 81%, of at least 82%, of at least 83%, of at least 84%, of at least 85%, of at least 86%, of at least 87%, of at least 88%, of at least 89%, of at least 90%, of at least 91%, of at least 92%, of atleast 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%.

[0063] In some embodiments, a functional variant of a polypeptide of the present application comprises an amino acid sequence that is a portion of the amino acid sequence of the polypeptide described herein. For example, in some embodiments, the functional variant has a length with respect to the original amino acid sequence of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 81%, of at least 82%, of at least 83%, of at least 84%, of at least 85%, of at least 86%, of at least 87%, of at least 88%, of at least 89%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%. In some embodiments, the functional variant comprises one, two, three, four, five or more amino acid substitutions, one or more of which substitutions is optionally a conservative substitution.

[0064] In some embodiments, a functional variant of a polypeptide of the present application comprises an amino acid sequence that is a portion of the amino acid sequence of the polypeptide described herein and is substantially homologous to an amino acid sequence described herein. For example, in some embodiments, the functional variant has a degree of identity with respect to the original amino acid sequence of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 81%, of at least 82%, of at least 83%, of at least 84%, of at least 85%, of at least 86%, of at least 87%, of at least 88%, of at least 89%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5% and / or has a length with respect to the original amino acid sequence of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 81%, of at least 82%, of at least 83%, of at least 84%, of at least 85%, of at least 86%, of at least 87%, of at least 88%, of at least 89%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%.

[0065] In some embodiments, the polypeptides of the present application are generated from the polynucleotides of the present applications (as described in more details below) using standard molecular biology and tissue culture methods known in the art. In some embodiments, the polypeptides of the present application are synthesized using protein synthesizing technologies known in the art.III. Polynucleotides of the present applications

[0066] Another aspect of the present application relates to polynucleotides encoding the polypeptide of the present application, functional variants thereof, expression cassettes thereof and expression vectors thereof.

[0067] In some embodiments, the present application provides a polynucleotide comprising a sequence for expressing a fusion G protein-coupled receptor (GPCR) agonist peptide, wherein the sequence for expressing the fusion GPCR agonist peptide comprises a sequence encoding a signal peptide and a sequence encoding a GPCR agonist peptide.

[0068] In some embodiments, part of the polynucleotide encodes an GPCR agonist peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-21, SEQ ID NOS: 171-179, SEQ ID NOS: 196-205, SEQ ID NOS:223-230, SEQ ID NOS:250-254, SEQ ID NOS:266-323, SEQ ID NOS:342-345 and SEQ ID NOS:346-347

[0069] In some embodiments, part of the polynucleotide encodes an GPCR agonist peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 305, 306, 312, 314 and 342-347

[0070] In some embodiments, the polynucleotide encodes a fusion GPCR agonist peptide comprising (1) a signal peptide and (2) a GPCR agonist peptide. In some embodiments, the signal peptide of the fusion peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:22-27 and 370-393. In some embodiments, the GPCR agonist peptide of fusion peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:1-21, 171-179, 196-205, 223-230, 250-254, 266-323 and 342-347. In some embodiments, the fusion GPCR agonist peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:356, 358, 365 and 366. In some embodiments, the fusion GPCR agonist peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:28-52. In some embodiments, the fusion GPCR agonist peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 334-341 and SEQ ID NOS:348-369.

[0071] In some embodiments, the polynucleotide encodes a fusion peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: SEQ ID NOS: 305, 306, 312, and 314.

[0072] In some embodiments, part of the polynucleotide encodes a GPCR agonist peptide comprising the amino acid sequence of SEQ ID NO:342.

[0073] In some embodiments, part of the polynucleotide encodes a GPCR agonist peptide comprising the amino acid sequence of SEQ ID NO:343.

[0074] In some embodiments, part of the polynucleotide encodes a GPCR agonist peptide comprising the amino acid sequence of SEQ ID NO:344.

[0075] In some embodiments, part of the polynucleotide encodes a GPCR agonist peptide comprising the amino acid sequence of SEQ ID NO:345.

[0076] In some embodiments, part of the polynucleotide encodes a fusion GPCR agonist peptide comprising the amino acid sequence selected from the group consisting of SEQ ID NOS:28- 52 and SEQ ID NOS:348-369.

[0077] In some embodiments, part of the polynucleotide encodes a GPCR agonist peptide comprising an amino acid sequence that is substantially homologous to an GPCR agonist peptide described herein. Exemplary GPCR agonist peptides include, but are not limited to, peptide comprising a sequence selected from the group consisting of SEQ ID NOS: 1-21, 171-179, 196-205, 223-230, 250-254, 266-323 and 342-347.

[0078] In some embodiments, the polynucleotide of the present application comprises a nucleic acid sequence that is substantially homologous to a nucleic acid sequence described herein. Exemplary nucleotide sequences encoding GLP1R agonist peptides include, but are not limited to, the nucleotide sequences as set forth in the Table as SEQ ID NOS: 53-74.

[0079] In some embodiments, the polynucleotides of the present application comprise one or more codon optimized polynucleotides encoding one or more polypeptides of the present application for expression in e.g., human, mammalian or primate cells, such as Hui 17, HEK293T or CHO cells. The polynucleotides encoding the proteins of the present application may be codon optimized to improve the activity, stability or expression in host cells without changing the encoded amino acid sequence. Codon optimization replaces codons present in a polynucleotide sequence with preferred codons encoding the same amino acid, for example, codons preferred for mammalian expression. Thus, the amino acid sequence is not altered during the process. Codon optimization can be performed using gene optimization software. The codon optimized nucleotide sequence is translated and aligned to the original protein sequence to ensure that no changes were made to the amino acid sequence. Methods of codon optimization are known in the art and are described, for example, in U.S. Application Publication No. 2008 / 0194511 and U.S. Pat. No. 6,114,148.

[0080] In some embodiments, a functional variant of a polynucleotide of the present application has a degree of identity with respect to the original polynucleotide sequence of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 81%, of at least 82%, of at least 83%, of at least 84%, of at least 85%, of at least 86%, of at least 87%, of at least 88%, of at least 89%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%.

[0081] In some embodiments, a functional variant of a polynucleotide of the present application comprises a nucleic acid sequence that is a portion of the original polynucleotide sequence described herein. For example, in some embodiments, the functional variant has a length with respect to the original polynucleotide of at least 60%, of at least 65%, of at least 70%, of at least 75%, of atleast 80%, of at least 81%, of at least 82%, of at least 83%, of at least 84%, of at least 85%, of at least 86%, of at least 87%, of at least 88%, of at least 89%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%.

[0082] In some embodiments, a functional variant of a polynucleotide of the present application comprises a nucleic acid sequence that is a portion of the polynucleotide described herein and is substantially homologous to the polynucleotide described herein described herein. For example, in some embodiments, the nucleic acid has a degree of identity or percent identity with respect to the original nucleic acid sequence of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 81%, of at least 82%, of at least 83%, of at least 84%, of at least 85%, of at least 86%, of at least 87%, of at least 88%, of at least 89%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%. and / or has a length with respect to the original nucleic acid sequence of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 81%, of at least 82%, of at least 83%, of at least 84%, of at least 85%, of at least 86%, of at least 87%, of at least 88%, of at least 89%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%.

[0083] The polynucleotides of the present application may comprise any type of nucleic acid, including, but not limited to DNA and RNA. For example, in one embodiment, the composition comprises an isolated DNA molecule, including for example, an isolated cDNA molecule, encoding a fusion protein of the application. In one embodiment, the composition comprises an isolated RNA molecule encoding a fusion protein of the application, or a functional fragment thereof.

[0084] The polynucleotide molecules of the present application can be modified to improve stability in serum or in growth medium for cell cultures. Modifications can be added to enhance stability, functionality, and / or specificity and to minimize immunostimulatory properties of the nucleic acid molecule of the application. For example, in order to enhance the stability, the 3’ - residues may be stabilized against degradation, e.g., they may be selected such that they consist of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine by 2’- deoxythymidine is tolerated and does not affect function of the molecule.

[0085] In some embodiments of the present application, the polynucleotide molecules of the present application may contain at least one modified nucleotide analogue. For example, the ends may be stabilized by incorporating modified nucleotide analogues.

[0086] Non-limiting examples of nucleotide analogues include sugar- and / or backbone- modified ribonucleotides (i.e., include modifications to the phosphate-sugar backbone). For example, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. In exemplary backbone-modified ribonucleotides the phosphoester group connecting to adjacent ribonucleotides is replaced by a modified group, e.g., of phosphothioate group. In exemplary sugar-modified ribonucleotides, the 2’ OH-group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or ON, wherein R is C1-C6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.

[0087] Other examples of modifications are nucleobase-modified ribonucleotides, i.e., ribonucleotides, containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Bases may be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine; adenosine and / or guanosines modified at the 8 position, e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O- and N- alkylated nucleotides, e.g., N6-methyl adenosine are suitable. It should be noted that the above modifications may be combined.

[0088] In some embodiments, the polynucleotide molecules of the present application comprise at least one of the following chemical modifications: 2’-H, 2’-O-methyl, or 2’-OH modification of one or more nucleotides. In certain embodiments, a nucleic acid molecule of the application can have enhanced resistance to nucleases. For increased nuclease resistance, a nucleic acid molecule, can include, for example, 2’ -modified ribose units and / or phosphorothioate linkages. For example, the 2’ hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents. For increased nuclease resistance the nucleic acid molecules of the application can include 2’-O-methyl, 2’-fluorine, 2’-O-methoxyethyl, 2’-O-aminopropyl, 2’-amino, and / or phosphorothioate linkages. Inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA), e.g., 2’ -4’ -ethylene-bridged nucleic acids, and certain nucleobase modifications such as 2- amino-A, 2-thio (e.g., 2-thio-U), G-clamp modifications, can also increase binding affinity to a target.

[0089] In some embodiments, the polynucleotide molecules of the present application include a 2’-modified nucleotide, e.g., a 2’-deoxy, 2 ’-deoxy-2’ -fluoro, 2’-O-methyl, 2’-O-methoxyethyl (2’-O- MOE), 2’-O- aminopropyl (2’-O-AP), 2’-O-dimethylaminoethyl (2’-0-DMA0E), 2’-O- dimethylaminopropyl (2’-O-DMAP), 2’-O-dimethylaminoethyloxyethyl (2’-O-DMAEOE), or 2’-O-N- methylacetamido (2’-0-NMA). In one embodiment, the nucleic acid molecule includes at least one 2’- O-methyl-modified nucleotide, and in some embodiments, all of the nucleotides of the nucleic acid molecule include a 2’-O-methyl modification.

[0090] In certain embodiments, a polynucleotide molecule of the present application includes otherwise unmodified RNA and DNA as well as RNA and DNA that have been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, or as occur naturally in the human body. The art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al. (Nucleic Acids Res., 1994, 22:2183-2196). Such rare or unusual RNAs, often termed modified RNAs, are typically the result of a post-transcriptional modification and are within the term unmodified RNA as used herein. Modified RNA, as used herein, refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature, or different from that which occurs in the human body. While they are referred to as “modified RNAs” they will of course, because of the modification, include molecules that are not, strictly speaking, RNAs. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to be presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone.

[0091] Modifications of the polynucleotides of the application may be present at one or more of, a phosphate group, a sugar group, backbone, N-terminus, C-terminus, or nucleobase.

[0092] Expression cassettes

[0093] Another aspect of the present application relates to polynucleotide expression cassette encoding one or more polypeptides of the present application. In some embodiments, the expression cassette comprises a nucleotide coding sequence encoding a polypeptide of the present application, and a regulatory sequence operably linked to the nucleotide coding sequence. In some embodiments, the nucleotide coding sequence encodes a fusion polypeptide of the present application.

[0094] In some embodiments, the expression cassette encodes a fusion GPCR agonist peptide comprising: (1) a sequence encoding a signal peptide having an amino acid sequence selected from the group consisting of SEQ ID NOS:22-27 and 370-393; and (2) a sequence encoding a G protein- coupled receptor (GPCR) agonist peptide having an amino acid sequence selected from the group consisting of SEQ ID NOS:1-21, 171-179, 196-205, 223-230, 250-254, 266-323 and 342-347

[0095] In some embodiments, the expression cassette encodes a fusion GPCR agonist peptide having an amino acid sequence selected from the group consisting of SEQ ID NOS:356, 358, 365 and 366.

[0096] In some embodiments, the expression cassette encodes a fusion GPCR agonist peptide having an amino acid sequence selected from the group consisting of SEQ ID NOS:28-52.

[0097] In some embodiments, the expression cassette encodes a fusion GPCR agonist peptide having an amino acid sequence selected from the group consisting of SEQ ID NOS: 334-341 and SEQ ID NOS:348-369.

[0098] In some embodiments, the expression cassette further comprises one or more polyadenylation (poly A) sequences.

[0099] Expression constructs that produce circular polynucleotide molecules

[0100] Another aspect of the present application relates to polynucleotide expression constructs capable of generating a circular polynucleotide molecule. In some embodiments, the polynucleotide expression construct comprises from 5’ end to 3’ end: (1) a sequence encoding a 5’ ribozyme; (2) a sequence encoding an internal ribosome entry site (IRES); (3) a sequence for expressing a fusion GPCR agonist peptide; and (4) a sequence encoding a 3 ’ribozyme, wherein the sequence for expressing the fusion GPCR agonist peptide comprises a sequence encoding a signal peptide and a sequence encoding a GPCR agonist peptide. Exemplary ribozymes that can be incorporated into the polynucleotide expression constructs include, but are not limited to, one or more of SEQ ID NO:111, SEQ ID NO:112, SEQ ID NOs: 114-166, or any combination thereof. In some embodiments, the circular polynucleotide molecule is an RNA molecule.

[0101] In some embodiments, the fusion GPCR agonist peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:356, 358, 365 and 366. In some embodiments, the fusion GPCR agonist peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:28-52. In some embodiments, the fusion GPCR agonist peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 334-341 and 348-369. In some embodiments, the GPCR agonist peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:1-21, 171-179, 196-205, 223-230, 250-254, 266- 323 and 342-347. In some embodiments, the GPCR agonist peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:305, 306, 312 and 314. In some embodiments, the signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:22-27 and SEQ ID NOS:370-393 In some embodiments, the sequence for expressing the fusion GPCR agonist further comprises a sequence that improves the stability of the GPCR agonist.

[0102] Another aspect of the present application relates to methods of generating a circular RNA molecule encoding a glucagon-like peptide-1 (GLP-1) receptor (GLP1R) agonist peptide. In one embodiment, the method comprises the step of administering a DNA molecule or an in vitro transcribed linear RNA molecule comprising a coding region encoding a GLP1R agonist, wherein the coding sequence is between an upstream 5’ ribozyme sequence and a downstream 3 ’ribozyme.

[0103] In some embodiments, the method comprises a method for in vivo generation of a circular RNA molecule, the method comprising the step of providing a linear RNA molecule comprising a 3’ ribozyme, a coding region encoding a protein of interest and a 5’ribozyme to a subject of interest. In some embodiments, the linear RNA molecule further comprises an IRES, a leader sequence or a combination thereof. In one embodiment, the linear RNA in an in vitro transcribed RNA molecule comprising a 3’ ribozyme, a coding region encoding a protein of interest and a 5’ribozyme. In some embodiments, the linear RNA molecule further comprises an IRES, a leader sequence or a combination thereof.

[0104] In some embodiments, the method comprises a method for in vitro generation of a circular RNA molecule, the method comprising contacting a linear RNA molecule comprising a 3’ ribozyme, a coding region encoding a protein of interest and a 5’ribozyme with a ligase for ligating the 3’P and 5 ’OH ends generated upon cleavage of the ribozymes. In some embodiments, the linear RNA molecule further comprises an IRES, a leader sequence or a combination thereof.

[0105] Expression vectors

[0106] Another aspect of the present application relates to expression vectors comprising the expression construct or the expression cassette of the present application. In some embodiments, the expression vector is a non-viral expression vector such as plasmid or cosmid. In some embodiments, the non-viral vector is a plasmid.

[0107] In some embodiments, the expression vector is a viral expression vector. Viral expression vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0108] In some embodiments, the vector is a lentiviral vector (see, e.g., U.S. Patent No. 748,529 to Fang et al.; Ura et al., “Developments in Viral Vector-Based Vaccines,” Vaccines 2: 624- 641 (2014); and Hu et al., “Immunization Delivered by Lentiviral Vectors for Cancer and Infection Diseases,” Immunol. Rev. 239: 45-61 (2011), which are hereby incorporated by reference in their entirety).

[0109] In some embodiments, the vector is a retroviral vector (see e.g., U.S. Patent No. 748,529 to Fang et al., and Ura et al., “Developments in Viral Vector-Based Vaccines,” Vaccines 2: 624-641 (2014), which are hereby incorporated by reference in their entirety), a vaccinia virus, areplication deficient adenovirus vector, and a gutless adenovirus vector (see e.g., U.S. Pat. No. 5,872,005, which is incorporated herein by reference in its entirety).

[0110] In some embodiments, non-integrative viral vectors, such as AAV, may be utilized. In one instance, non-integrative vectors do not cause any permanent genetic modification. The vectors may be targeted to adult tissues to avoid having the subjects under the effect of constitutive expression from early stages of development. In some instances, non-integrative vectors effectively incorporate a safety mechanism to avoid over-proliferation of the GPCR agonist polypeptide expressing cells. The cells may lose the vector (and, as a consequence, the protein expression) if they start proliferating quickly.[OHl] In other embodiments, the vector is an adeno-associated virus (AAV) vector (see, e.g., Krause et al., “Delivery of Antigens by Viral Vectors for Vaccination,” Ther. Deliv. 2(1): 51 -70 (2011); Ura et al., “Developments in Viral Vector-Based Vaccines,” Vaccines 2: 624-641 (2014); Buning et al, "Recent Developments in Adeno- associated Virus Vector Technology," J. Gene Med. 10:717-733 (2008), each of which is incorporated herein by reference in its entirety).

[0112] As used herein, "adeno-associated virus vector" means an adeno-associated virus (AAV) comprising a naturally occurring or non-naturally occurring AAV capsid encapsidating a vector genome. Adeno-associated virus vector may be abbreviated "AAV vector," and depending on context, may be referred to by synonymous terms, such as "recombinant AAV vector," "rAAV vector," "rAAV," or just "vector." AAV vectors comprise a vector genome encapsidated by an AAV capsid. In some embodiments, the AAV vector genome comprises at least one AAV inverted terminal repeat (ITR) and a heterologous nucleotide sequence with a desired function when present or expressed in a transduced target cell. In some embodiments, the heterologous nucleotide sequence originates from a different type of virus, or an entirely different type of organism, such as an animal, plant, protist, fungus, bacteria, archaea, or other type of organism. In some embodiments, the heterologous nucleotide sequence replaces some or all of the native AAV rep and / or cap genes so that the vector is incapable of expressing functional Rep or VP proteins in transduced target cells. In some embodiments, the entire sequence of the vector genome consists of heterologous nucleotide sequences except for AAV inverted terminal repeat sequences positioned at the ends of the genome.

[0113] Examples of adeno-associated virus-based non integrative vectors include vectors based on any AAV serotype, i.e., AAVI, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVIO, AAVII and pseudotyped AAV. Vectors of interest include those capable of transducing a broad range of tissues at high efficiency, with poor immunogenicity and an excellent safety profile. In some cases, the vectors transduce post-mitotic cells and can sustain long-term gene expression (up to several years) both in small and large animal models of the related disorders.IV. Pharmaceutical compositions

[0114] Another aspect of the present application relates to a pharmaceutical composition. The pharmaceutical composition comprises (1) a polynucleotide or a polypeptide of the present application and (2) a pharmaceutically acceptable carrier.

[0115] In some embodiments, the pharmaceutical composition comprises a circular RNA molecule of the present application and / or a polynucleotide expression vector of the present application.

[0116] The pharmaceutical compositions can be administered to a subject by any route that results in prevention or alleviation of symptoms associated with disease or condition. For example, nucleic acid molecules can be administered parenterally, intravenously (I. V.), intramuscularly (I.M.), subcutaneously (S.C.), intradermally (I D ), orally, intranasally, etc. Examples of intranasal administration can be by means of a spray, drops, powder or gel and also described in U.S. Pat. No. 6,489,306, US20180344816, US20060078558, US20080070858, US20180298057, and US20150313924, which are incorporated herein by reference in their entireties.

[0117] The polypeptides of the present invention or the encoding nucleic acid molecule can be administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight, and other factors known to medical practitioners.

[0118] In some embodiments, the pharmaceutical composition further comprises or is formulated into a delivery vehicle for delivery of a linear or circular RNA molecule of the present application to a subject. Exemplary delivery vehicles include, but are not limited to, liposome or lipid nanoparticles.

[0119] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another instance, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution.

[0120] In some embodiments, a lipid nanoparticle encapsulating the polynucleotide or the polypeptide of the present application can be used for the introduction of the pharmaceuticalcompositions into suitable host cells. The formulation and use of such delivery vehicles can be carried out using known and conventional techniques.V. Methods of treatment

[0121] Another aspect of the present application relates to methods of treating, reducing the symptoms of, and / or reducing the risk of developing a disease or disorder in a subject. For example, in one embodiment, methods of the application of treat, reduce the symptoms of, and / or reduce the risk of developing a condition or disease including, but not limited to, Type 1 Diabetes (Insulin-Dependent Diabetes Mellitus (JDDM)), Type 2 Diabetes, Obesity and Weight Loss, Sleep Disorders (including Obstructive Sleep Apnea (OSA), Cancer, Dyslipidemia, Hypertension, Metabolic Syndrome, Beta-cell Preservation, Appetite Suppression, Gastric Emptying, Energy Expenditure, Thermogenesis, C rdiovascular Health, Heart Failure, Kidney Protection, Non-Alcoholic Fatty Liver Disease (NAFLD) / Nonalcoholic Steatohepatitis (NASH), Inflammation, Neuroprotection and Neurodeg enerative Diseases, Stroke, Dementia, Polycystic Ovary Syndrome (PCOS), Addiction, Osteoarthritis, Atherosclerosis, Osteoporosis, Short Bowel Syndrome, Inflammatory Bowel Disease (IBD), Radiation Enteritis, Chemotherapy-Induced Mucositis, Intestinal Ischemia / Reperfusion Injury, Celiac Disease, Gut Microbiome Dysbiosis, Pulmonary Arterial Hypertension (PAH), Asthma, Chronic Obstructive Pulmonary Disease (COPD), COVID-19-related ARDS, Sepsis, Alzheimer’s Disease, Parkinson’s Disease, Multiple Sclerosis, Rheumatoid Arthritis, Inflammatory Bowel, Disease, Irritable Bowel Syndrome, Gastroparesis, Erectile Dysfunction, Psoriasis, Short stature, or any combination thereof.

[0122] Thus, in one embodiment, the disease or disorder may be treated, reduced, or the risk can be reduced using the compositions and methods of the present application. Thus, in one embodiment, the method comprises administering to the subject one or more polynucleotide, polypeptides or pharmaceutical composition of the present application.

[0123] In some embodiments, the present application provides methods of treating or preventing GPCR-related conditions or disorders comprising administering an effective amount of a pharmaceutical composition comprising the polynucleotide or polypeptide disclosed herein to a subject in need thereof.

[0124] In some embodiments, the present application provides methods of treating or preventing glucagon related hormone (GRH)-related conditions or disorders comprising administering an effective amount of a pharmaceutical composition comprising the polynucleotide or polypeptide disclosed herein to a subject in need thereof.

[0125] Another aspect of the present application relates to a method for treating a GPCR- related condition in a subject. In some embodiments, the method comprises the step of administeringto the subject an effective amount of the polynucleotide, polypeptide or pharmaceutical composition of the present application.

[0126] In some embodiments, the GPCR-related condition is a glucagon-related hormone (GRH) related condition. Examples of GRH-related conditions include, but are not limited to, Type 1 Diabetes (Insulin-Dependent Diabetes Mellitus ( IDDM )), Type 2 Diabetes, Obesity and Weight Loss, Sleep Disorders (including Obstructive Sleep Apnea (OSA), Cancer, Dyslipidemia, Hypertension, Metabolic Syndrome, Beta-cell Preservation, Appetite Suppression, Gastric Emptying, Energy Expenditure, Thermogenesis, Cardiovascul r Health, Heart Failure, Kidney Protection, Non-Alcoholic Fatty Liver Disease (NAFLD) / Nonalcoholic Steatohepatitis (NASH), Inflammation, Neuroprotection and N eurodegen erative Diseases, Stroke, Dementia, Polycystic Ovary Syndrome (PCOS), Addiction, Osteoarthritis, Atherosclerosis, Osteoporosis, Short Bowel Syndrome, Inflammatory Bowel Disease (IBD), Radiation Enteritis, Chemotherapy-Induced Mucositis, Intestinal Ischemia / Reperfusion Injury, Celiac Disease, Gut Microbiome Dysbiosis, Pulmonary Arterial Hypertension (PAH), Asthma, Chronic Obstructive Pulmonary Disease (COPD), COVID-19-related ARDS, Sepsis, Alzheimer’s Disease, Parkinson’s Disease, Multiple Sclerosis, Rheumatoid Arthritis, Inflammatory Bowel, Disease, Irritable Bowel Syndrome, Gastroparesis, Erectile Dysfunction, Psoriasis, Short stature, or any combination thereof.

[0127] Another aspect of the present application relates to a method for increasing the activity of a GPCR in a subject. The method comprises the step of administering to the subject, an effective amount of the circular RNA molecule of the present application, a polypeptide of the present application and / or the polynucleotide of the present application.

[0128] The application is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the application should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present application and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.EXAMPLESExample 1: Therapeutic Expression of GLP-1 Receptor Agonists from Circular RNA forDurable Metabolic Control and Improved Cardiovascular Health

[0129] Small proteins / micropeptides can be functionally encoded and translated from small open reading frames. Therefore, a small open reading frame encoding a signal peptide for secretion with the mature active form of human GLP-1 and related GLPIRas was designed. These small ORFs can be expressed as either mini -genes (DNA) for delivery and expression from plasmid or viral vectors or translated directly from an RNA transcript (ex. linear mRNA or circular RNA) for expression in cells. Expression of small peptide hormones (such as GLP-1) from genetically encodable open reading frames allow for an alternative approach to peptide- based therapeutics, which have several potential advantages including consistent, durable expression and simple and low-cost manufacturing that can be rapidly scaled to meet demand.

[0130] A novel approach for the scarless trans-ligation of RNA in cells using ribozymes has been developed. Ribozymes (Rzs) are small, catalytic RNA sequences which are capable of nucleotide-specific self-cleavage.

[0131] Ribozyme-mediated RNA cleavage generates unique 3’ phosphate and 5’ - hydroxy termini, which resemble substrates for ubiquitous RNA repair pathways present in all three kingdoms of life. Ribozyme-mediated cleavage can be harnessed for the scarless trans- ligation of two independent RNA transcripts in mammalian cells, an approach named stitchR (stitch RNA), or the circularization of a single RNA, an approach called circulR (Figure 1).

[0132] In vitro transcribed CirculR RNA, when delivered to cells, is automatically circularized and suitable for protein translation. In contrast, linear RNA requires post-processing to add a 5’ cap and 3’ poly(A) tail, which is an additional step that represents -50-60% of the total cost of RNA manufacturing.

[0133] FIGS. 2A-2C demonstrate that human GLP-1 is processed from the pre-pro-glucagon protein encoded by the GCG gene.

[0134] FIG3. 3A-3D demonstrate post-translational processing of human GLP-1, related GLP1R agonists and sequence modification to promote stability. Without wishing to be bound by theory, it is possible that a combination of the extending-4 C-terminal tail and sequence modifications to prevent cleavage may further enhance efficacy (MGLP-EX).

[0135] FIGS. 4A-4E depict an assessment of GLP1R signaling activity using a cAMP- responsive cell-based luciferase assay reporter.

[0136] FIGS. 5A-5E depict the discovery of a putative mammalian GLP-1 paralog, GLP-1B, in bats.

[0137] FIGS. 6A-6C shows the characterization of the GLP-1B protein sequence.

[0138] FIG. 7 shows the design of a circulR vector encoding a signal peptide (SP)- GLP1RA fusion protein, under the control of an IRES, for generating RNA using in vitro transcription. TheRNA, when complexed in a lipid nanoparticles (LNPs), exosomes, virus-like particles (VLP), or other packaging method, can be delivered in vivo through injection, oral drop, or other delivery method, for expression and secretion from cells into the body (blood stream, gut or other tissue or cell type). Expression and secretion of GLP-1 analogues from cells into the body will activate GLP1R signaling in the body. The circularized RNA vector may provide more consistent and durable expression over time and is redosable. Adherence to long term GLP1RA treatments have been shown to provide significant benefits for weight control and protection from cardiovascular disease, neurological disease, and cancer.GLP-1RA activity assay using a cAMP-RE Luciferase reporter

[0139] Human HEK293T cells were maintained in complete media consisting of DMEM supplemented with 10% Fetal Bovine Serum, Pen / Strep, and cultured in an atmosphere of 5% CO2 at 37°C. Cells were passaged every 2 to 3 days. For the co-transfection, cells were plated in complete media (no antibiotics) at a density of 400,000 cells per ml in a 24-well plate. After 24 hours, cells were co-transfected using Fugene6 using manufactures’ protocols. Cells were transfected with an equal ratio of plasmids encoding a 6X cAMP-response element (RE) luciferase reporter (Addgene plasmid # 194384), an expression vector encoding human GLP1R (Addgene plasmid # 213253), and a single GLP1RA expression plasmid driven by CMV promoter (see list of sequences). A plasmid encoding GIPR (Addgene plasmid # 213252) was used to demonstrate GLP1R receptor specificity. As a transfection control, a CMV-Lacz transgene was included in the transfection reaction. After 24 hours of transfection, the cell media was replaced with low serum medium (1% FBS in OptiMEM) and culture for 6 hours, upon which time cells were lysed in IX Passive Lysis Buffer (Promega) and analyzed for luciferase activity using Luciferase Assay Substrate (Promega) using Manufacturer’s protocols.Animals

[0140] Genetically obese and diabetic mice Leprdb(db / db) or wildtype diet-induced obese (DIO) C57BL / 6J mice are purchased from Jackson Labs. Leprdb(db / db) are fed normal chow and DIO mice are fed high-fat diet (D12331, Research Diets) which contains 58% kilocalories from fat. Body composition analysis to measure fat and lean tissue mass is measured using nuclear magnetic resonance technology (MRI).

[0141] Mice treated with GLP1RA encoding circular RNAs show decreased levels of fasted blood glucose and significant decreases in levels of fasted insulin, and improved insulin sensitivity relative to vehicle-treated controls, following glucose tolerance and insulin tolerance tests (GTT / ITT). Example 2: Methods Plasmids

[0142] Small open reading frames were designed to express and secrete mature hormones agonists by utilizing a heterologous signal peptide (e.g. CD5), followed by the human codon optimized open reading frame encoding the mature peptide. Constructs were cloned using synthetic gBlocks (IDT) and subcloned into a mammalian expression vector containing a human CMV promoter, WPRE translational enhancer sequence and a polyadenylation sequence from bovine growth hormone. Cloned inserts were sequenced using Sanger sequencing (IDT) and the final maxi prepped plasmids (Nucleobond) were fully sequenced using Oxford nanopore sequencing (Plasmidsaurus).Receptor-Agonist Structural Modeling and Docking Prediction

[0143] Alphafold2 Multimer structural docking predictions were accessed through the COSMIC2 cloud platform user portal (https: / / cosmic-cryoem.org). Predictions were made between each agonist and a human receptor, lacking signal peptides. Structures were visualized using PyMOL. GLP-1RA activity assay using a cAMP-RE Luciferase reporter

[0144] Human HEK293T cells were maintained in complete media consisting of DMEM supplemented with 10% Fetal Bovine Serum, penicillin / streptomycin, and cultured in an atmosphere of 5% CO2 at 37°C. Cells were passaged every 2 to 3 days. For the co-transfection, cells were plated in complete media (no antibiotics) at a density of 400,000 cells per ml in a 24-well plate. After 24 hours, cells were co-transfected using Fugene6 using manufactures’ protocols. Cells were transfected with an equal ratio of plasmids encoding the 6X cAMP -response element (RE) luciferase reporter (Addgene plasmid # 194384), and expression plasmids encoding a human receptor (e.g. GLP1R) and glucagon- related hormone (please see list of sequences). A plasmid encoding LacZ driven by the CMV promoter (CMV-LacZ) was included as a transfection control. After 24 hours of transfection, the cells were lysed in IX Passive Lysis Buffer (Promega) and analyzed for luciferase activity using Luciferase Assay Substrate (Promega) using Manufacturer’s protocols.

[0145] For the supernatant-transfer assay, reporter cells were transfected with plasmids encoding the 6X cAMP -response element (RE) luciferase reporter (Addgene plasmid # 194384), an expression plasmid encoding a human receptor (e.g. GLP1R) and the CMV-LacZ transgene. To produce agonist containing supernatants, separate cells were transfected with plasmids encoding a GPCR agonist, and either empty plasmid or a plasmid encoding DPP-4. After 6 hours of transfection, reporter cells were harvested and re-seeded into 24 well plates, while complete media was changed on agonist producing cells to remove transfection reagents and plasmids. The next day, supernatants from agonist producing cells were transferred to reporter cells, and after 6 hours of incubation, the reporter cells were processed for luciferase assays.Example 3: Discovery of GLP1X and other glucagon-related paralogs in Myotis Bats

[0146] Multiple sequence alignments were used to compare the mature peptide hormone sequences of GCG, GLP-1, GLP-2 and GIP across 100 vertebrate species, spanning primates to fish (USCS genome browser). All these peptide hormones have been highly conserved across vertebrate evolution and have retained both a unique peptide length and hormone-specific motif of identically conserved residues.

[0147] This alignment also revealed that some species of bats acquired amino acid changes within GLP-1 that could alter cleavage by DPP-4 or C-terminal proteolytic processing. To further investigate the extent of these differences, we used a BLAST search of human and bat GLP-1 sequences on bat genomes. Similar to other vertebrates, GLP-1 orthologs in Myotis bats are similarly co-expressed with GCG and GLP-2 peptides within a pre-pro-protein (M. myotis ppGCG). Interestingly, this search also revealed the presence of an additional GLP-1 related sequence encoded within the Myotis myotis bat genome (hypothetical protein mMyoMyol_012148). Similar to pre-pro- glucagon, this hypothetical protein prediction has the potential to encode two additional glucagon-like peptides. Using a BLAST search of the human proteome, these additional glucagon-like peptides were most related to human GIP and human GLP-2. Further BLAT searches using the UCSC genome browser revealed a widespread expansion of glucagon-related paralogs in the genomes of Myotis bats, including Myotis dcnibenlonii. Myotis lucifugus, and Myotis ricketti.Example 4: Mapping of glucagon-related paralog sequences in Myotis bat genomes

[0148] In vertebrates, including humans and bats, the position of the GCG gene locus is systemically conserved between the genes DPP4 and FAP. To determine the location of the newly discovered glucagon-related peptide sequences, their position was mapped in the AL myotis genome (2020 BatlK build) using a BLAT search from the UCSC Genome Browser (D. Jebb et aL, Nature 583, 578-584 (2020). Interestingly, these new peptide sequences mapped ~24 megabases away from the canonical GCG gene locus, located on the same chromosome but oriented on the opposite strand. This was termed the expanded GCG locus, or GCGX, which is flanked by the MGAT5 and NCKAP5 genes in a region ~0.5 MB wide. In humans, the region flanked by MGAT5 and NCKAP5 genes is of a similar size (~0.5Mb) and distance from the canonical GCG locus (~28Mb), yet appears devoid of any annotated transcripts.

[0149] Due to their occurrence within a separate gene locus (GCGX), the novel glucagon- related peptide sequences were named based on their relatedness their closest human glucagon-family paralog and distinguished them using the letter X (GLP1X, GLP2X, and GIPX). Additional BLAT searches using human and bat GCG- and GCGX-derived peptide sequences revealed additional glucagon-related paralogs outside of their canonical GCG loci within the genomes of other Myotis bat species, including Myotis daubentonii (primary hap 2023 refseq), Myotis lucifugus(GCF_OOO 147115.1), and Myotis ricketti (AB -2021 2024). As in Myotis myotis, the GCGXlocus in M. daubentonii similarly occurs ~24 Mb away and is flanked by the same MGAT5 and NCKAP5 genes, suggesting this duplication event preceded the speciation of these two Myotis bat species.

[0150] It was also found expansions of glucagon-related sequences at the canonical GCG gene locus in Myotis myotis, which were named GCGY paralogs. Further, both M. daubentonii and A7. ricketti have an expanded number of glucagon -related sequences in their GCGX loci, which were numbered based upon their 5’ to 3’ occurrence within the GCGXlocus (GLP1X1, GLP1X2, etc.).Example 5: Divergence of GCGX and GCGY-derived paralogs from canonical glucagon-related hormone sequences

[0151] In total, 12 GLP1X, 6 GLP2X, 4 GIPX, 1 GLP1Y, and 1 GLP2Y paralogs were identified in fourATyotz bat species. Interestingly, no paralogs of GCG were found in the expanded regions. Next, multiple sequence alignments were used to compare the relatedness of GCGX and GCGY paralogs to canonical glucagon-related hormones from vertebrate species. Almost all of the novel paralogs contained either one or multiple amino acid changes that break the consensus motifs of identically conserved residues specific to GLP-1, GLP-2 and GIP hormone. These amino acid changes were unique to the mammalian GCGX and GCGY paralogs and not found in the other vertebrate paralogs Exendin-4 or GCGL, which themselves each diverge from the vertebrate ortholog consensus motifs by a single amino acid.Example 6: Modeling GCGX paralog agonist-receptor interactions

[0152] Since the amino acid changes have the potential to disrupt paralog structure or receptorspecific interactions, protein docking predictions using Alphafold2 Multimer structure prediction (J. Jumper et al.,. Nature 596, 583-589 (2021); R. Evans et al., bioRxiv, 2021.2010.2004.463034 (2022)) were next performed.

[0153] The structure and docking interactions of the canonical glucagon-related peptides and the predicted GCGX- and GCGF-derived paralogs with their cognate receptors were determined. Similar to the canonical glucagon-related peptides from human and Myotis bats, GCGX- and GCGY- derived paralogs were similarly predicted to form an alpha helix and dock in the same pocket as the canonical hormones, whether they broke from vertebrate consensus motifs or not. However, not all peptides are predicted to dock with the receptors, for example, the divergent GLP3X1 hormone predicted to be encoded within the Myotis ricketti GCGX gene cluster does not form a single alpha helix and is not predicted to bind in the same ligand pocket. The structure and docking predictions suggest that despite diverging from vertebrate consensus motifs, the expanded glucagon-related paralogs have the potential to function as bona fide receptor agonists.Example 7: Receptor-specific activation of GCGX- and GCGY-derived peptide hormones

[0154] To assess the function of the newly identified glucagon-related paralogs, cell-based luciferase reporter assays responsive to cAMP-signaling and P-arrestin recruitment as readouts for GPCR activation (W. K. Kroeze et al., 22, 362-369 (2015); Y. Wu et al., Biosensors (Basel) 13, (2022)) were utilized (FIGS. 8A and 8B). For these assays, co-expression of a human receptor (GCGR, GLP1R, GLP2R, or GIPR) and a pathway-specific luciferase reporter allows for the sensitive and agonist-specific activation of GPCRs in human HEK293T cells. For agonist expression, minigenes encoding a small open reading frame (smORF) containing a heterologous N-terminal signal peptide upstream of the mature hormone sequence were utilized to enable peptide secretion and precise cleavage at the N-terminus of the hormone (FIG. 8C). Transient co-transfections with plasmids encoding human GCG, GLP-1, GLP-2 or GIP minigenes resulted in the predicted receptor-specific activation of human GCGR, GLP1R, GLP2R, and GIPR receptors for both cAMP- and P-arrestin- pathway luciferase reporter assays, respectively (FIG. 8D).

[0155] The function of canonical glucagon-related hormones from vertebrate and mammalian species, the newly identified expanded mammalian paralogs from Myotis bats, and the previously identified vertebrate paralogs exendin-4 and GCGL were next tested and compared. These studies revealed that most of the GCGA-derived paralogs were functional and activated their predicted cognate receptors at levels similar to the human peptides (Figure 8D). For GLP1R, 9 of the 12 newly described GLP1X paralogs resulted in specific activation of GLP1R. For the GLP2X paralogs, two of the five peptides showed GLP2R-specific activation. Activation of human GIPR was observed for 2 of the 4 GIPX paralogs (A7. / -.GIPX I and A7. / -.GIPX2). The two GCGT-derived peptides did not show activation of any of the human receptors tested here.Example 8: Extended C-terminus in some GLP1X paralogs required for full receptor activation

[0156] The canonical GLP-1 ortholog in Myotis bats showed comparable activation of human GLP1R, despite containing a number of sequence changes relative to other mammalian GLP-1 orthologs, when expressed as a 31 amino acid peptide hormone. The GLP1X paralogs with the greatest activity (A / . / .GLP I X I / 2, A / . / .GLP I X3, GLPlXl, A / .t / .GLP I X2 and A / . / .GLP I X5) were similar in sequence to human GLP-1. However, two GLP1X paralogs ( / i / .GLP I X I and A / .t / .GLP I X4) were more divergent and showed either reduced (-50%) or no activation of human GLP1R when expressed as 31 amino acid peptides, respectively. Since the C-terminal sequences for these two GLP1X peptides were predicted within a hypothetical pro-protein and contain sequence changes that may alter their C- terminal processing, we sought to determine the impact of C-terminal sequences by generating C- terminal truncations and extensions for human GLP-1 (FIG. 9 A; SEQ ID NOs: 167-180), Myotis GLP-1 (FIG. 9B; SEQ ID NOs: 218-231), and the two GLP1X agonists (M. m.GLPIXl (FIG. 9C; SEQ ID NOs:266-279) and GLP!X4 (FIG. 9D; SEQ ID NOs: 314-327).

[0157] Human GLP-1 is naturally processed into a 31 amino acid peptide (7-37) and 30 amino acid peptide (7-36) which are both sufficient for GLP1R activation. Removal of the two N-terminal residues by DPP -4 (GLP-1 (9-36)) however abolishes its function. These findings were recapitulated in the cell-based cAMP -responsive luciferase reporter assay, in which minigenes encoding GLP-1 (7- 37) and (7-36) showed robust activation, whereas as expression of a peptide lacking the two N- terminal residues showed no activity (FIG. 9A). Human GLP-1 minigenes shorter than 27 amino acids showed no receptor activation, whereas genes with C-terminal extensions beyond 7-35 showed no substantial differences in their ability to activate GLP1R. A similar finding was found for the Myotis bat GLP-1 ortholog. In contrast, both of the two GLP1X agonists required longer peptide hormone lengths to achieve the same degree of GLP1R activation. For M.m. GLP1X1, robust receptor activation occurred at (7-37), and for A / .t / .GLP I X4, robust activation of GLP1R only occurred when expressed as the (7-39) length. Both bat GLP-1 and GLP1X peptides remained dependent upon the presence of the two N-terminal residues for receptor activation, suggesting that they could also be subject to negative regulation by DPP -4.Example 9: Myotis GLP-1 and GLP1X agonists with C-terminal extensions are resistant to DPP- 4 inactivation

[0158] To determine if Myotis GLP-1 and GLP1X peptides were regulated by DPP-4, cotransfection and expression of the peptide agonists were utilized in the presence or absence of DPP-4 expression in cultured cells. Post-transfection, supernatants were harvested and transferred to reporter cells co-transfected with human GLP1R and the cAMP-responsive luciferase reporter (FIG. 10A). In the absence of DPP-4 expression, supernatants harvested from cells expressing human GLP-1 (7-37) resulted in robust reporter activity. However, consistent with the labile nature of human GLP-1, coexpression of DPP-4 completely abolished the activity of human GLP-1 (FIG. 10B; SEQ ID NOs: 169-179), when expressed at its natural length and versions with longer C-terminal extensions. In contrast, however, Myotis GLP-1 (FIG. 10C; SEQ ID NOs: 220-230) and GLP1X paralogs m.GLPIXl (FIG. 10D; SEQ ID NOs: 270-278) and t / .GLPl X4 (FIG. 10E; SEQ ID NOs: 318- 326) with longer C-terminal extensions showed robust resistance to DPP-4 inhibition.Example 10: GLP-1 (7-36) A8W modification preserves GLP1R activation and confers resistance to DPP4 inhibition

[0159] A heterologous (CD5) signal peptide upstream of a small open reading frame encoding the 30 amino acid human GLP-1 peptide (7-36) was used to specifically express and secrete the mature form of the GLP-1 hormone. Co-expression in HEK293T cells with a plasmid encoding the human GLP-1 Receptor (GLP1R) and a cAMP-responsive luciferase reporter resulted in GLP1R signal transduction and robust luciferase activity. A GLP-1 / GLP1R signaling reporter assay was utilized todetermine the effects of GLP-1 modifications on receptor activation and resistance to the natural GLP- 1 inhibitor, Dipeptidyl Peptidase-4 (DPP4). Luciferase activities are shown for GLP-1 agonist modifications, when co-expressed with (w / ) or without (w / o) DPP4 overexpression in the same cells as human GLP1R and the cAMP-responsible luciferase reporter (FIGS. 11 A and 1 IB), or when GLP-1 agonists were co-expressed with (w / ) or without (w / o) DPP4 overexpression in separate cells, from which agonist containing supernatants were transferred to reporter cells (FIGS. 11C and 1 ID).Example 11: Modification of glucagon-related hormones provide resistance to DPP-4 inhibition

[0160] Amino acid substitutions in GLP-1 which provided resistance to DPP-4 inhibition were screened. Among these, substitution of tryptophan (W) for the naturally occurring alanine at position 8, A8W, both preserved GLP-1 function and provided almost complete resistance to DPP4 inhibition. Tryptophan substitution in other glucagon-related hormones, notably GLP-1 and GIP, is shown here to preserve their function and provide resistance to DPP -4 inactivation.

[0161] Results demonstrated that the A8W modification of GLP-1 (7-36) provided robust resistance to DPP -4 inhibition. FIGS. 12A to 12C show that substitution of tryptophan (W) at the same relative position in GLP-2 and GIP, but not GCG, provides resistance to DPP-4 inhibition. In the case of GCG, the tryptophan modification abolished activity with or without the addition of DPP-4. These data suggest that positional replacement of tryptophan is a general strategy for preventing DPP -4 inhibition of peptide hormones, however not all hormones tolerate this replacement.Example 12: CirculR RNA-LNP delivery and expression of GLP-1 peptides in circulation in vivo

[0162] FIG. 13 is a schematic depicting the ribozyme-mediated scarless cis-splicing to generate a circular RNA which can be translated via IRES-mediated translation. FIGS. 14A-14B show a circular RNA construct of the present application (CirculR 2.0, which encodes a GFP-blasticidin-2A- luciferase (GBL) fusion protein) can be efficiently delivered and expressed in vivo in liver using RNA- lipid nanoparticles. FIG. 14A, CirculR 2.0 GBL RNA-LNPs were formulated using a Spark NanoAssemblr, injected into C57BL / 6J mice and imaged for bioluminescence after 6 hours, using IVIS imaging. CirculR 2.0 GBL injected mice showed robust luciferase activity in liver, compared to saline injected mice, demonstrating in vivo delivery and expression. FIG. 14B, CirculR 2.0 vectors encoding GLP-1 sequences were generated to express either human GLP-1 fused to IgG4 fc (GLP-1 - IgG4 fc) or human GLP-1 with the A8W modification which prevents DPP -4 inhibition (GLP-1 A8W). GLP-1 encoding CirculR 2.0 RNA-LNPs were generated and injected into mice, from which blood was isolated and analyzed for GLP-1 expression using a multispecies GLP-1 ELISA (Thermofisher, BMS2194). Mice injected with either GLP-1 encoding CirculR 2.0 RNA-LNPs showed robust expression of GLP-1, compared to saline or CirculR 2.0 GBL injected mice. Recombinant human GLP-1 (rHuman GLP-1) and Myotis bat GLP-1 (rMyotis GLP-1) syntheticpeptides were included. The multispecies ELISA kit was sufficient to detect human GLP-1, but not Myotis bat GLP-1, which contains several amino acid changes.

[0163] CirculR 2.0 plasmids were first linearized with Xbal, cleaned up on a column, then used as templates for in vitro RNA transcription using the HiScribe® T7 High Yield RNA Synthesis Kit ( NEB 1040). Reactions were incubated overnight at 37 °C, then DNA templates were degraded using Turbo DNAse addition for 20 minutes. RNAs were then cleaned up using column purification. RNA-LNPs were produced using a Precision Medicine Spark NanoAssemblr using lipids ALC-0315, DSPC, Cholesterol, and DMG-PEG. LNPs were then cleaned up using 3.5kD Dialysis bags overnight.Example 13: Exemplary Expression Systems

[0164] CD5 signal peptide - GLP-1 ORF. The protein coding portion of GLP-1 (or derivative amino acid sequence which offers enhanced receptor activation, etc.) is placed downstream of a signal peptide sequence, which may either be from ppGCG (SEQ ID NO: 27) or any number of typical secreted proteins, such as the signal peptide from CD5 (SEQ ID NO: 22).

[0165] CD5-GLP-1 mini-gene sequence. The CD5 signal peptide - GLP-1 ORF nucleotide sequence (SEQ ID NO:79) is then placed under the control of a promoter, polyadenylation sequence, and any number of other translational regulatory sequences, such as 5’UTR, 3’UTR or translational enhancers, such as the WPRE sequence: ITR-hCMV promoter-kozak-CD5-GLP-l ORF- WPRE- bGHpA-ITR (SEQ ID NO: 105).

[0166] CD5-GLP-1 linear mRNA transcript sequence. For encoding GLP-1 in an mRNA transcript, the small ORF (signal peptide + GLP-1 ORF) is placed downstream of an RNA Polymerase promoter (such as T7 or T7 version 2.0). Any number of other translational regulatory sequences can be utilized, such as 5’UTR, 3’UTR, synthetic polyA (either encoded in the vector or enzymatically added post-transcription), and 5’ cap: T7 version 2.0 promoter, 5’UTR-Kozak-CD5-GLP-1 ORF- 3’UTR, poly(A) - SacI restriction site for linearization. (SEQ ID NO: 106).

[0167] CD5-GLP-1 circulR RNA transcript sequence. For GLP-1 encoded in a circular RNA, the small ORF is placed downstream of an IRES sequence and flanked by small ribozymes to mediate circularization: T7promoter-Rz-CVB3 IRES- CD5-GLP-1 ORF-Rz-Xbal site for linearization (SEQ ID NO: 109) Discussion

[0168] Glucagon-like peptides have played a conserved and significant role in regulating the metabolism of vertebrates across millions of years of evolution. The addition of glucacon-related paralogs were thought to be unique to non-mammalian species, however it has been shown here that mammals, specifically Myotis bats, have acquired a duplication of the GCG gene and harbor an expanded array of glucagon-like peptide sequences. Despite the acquisition of consensus-breakingamino acid changes, these peptides function as paralogs of glucagon-related hormones and have retained robust and receptor-specific activation. The present findings surprisingly found that some of the most divergent paralog agonists required C-terminal extensions for full receptor activation, which additionally confer resistance to DPP-4 inhibition. These findings highlight both a potential evolutionary expansion in the regulation of mammalian metabolism in bats and also offer new therapeutic opportunities for treating human metabolic disease.The GCGX locus likely arose from a gene duplication event

[0169] Bats, like other mammals, harbor a canonical GCG gene that gives rise to a pre-pro- protein that is processed into GCG, GLP-1, and GLP-2 hormones. Bats similarly encode a canonical GIP gene that generates the GIP hormone. The additional glucagon-like peptide sequences here identified in Myotis bats (M. myotis, M. daubentonii, M. lucifugus, andM. ricketti) map to a distinct genomic locus apart from the canonical GCG or GIP loci. For AT. myotis and daubentonii, the GCGX locus is found ~24 megabases away from the canonical GCG gene. Since the GCGX locus encodes 3 or more glucagon-related peptide sequences, the GCGX locus may have arisen from a duplication of the canonical GCG locus. Glucagon-like paralogs were also found expanded at the GCG locus of Myotis myotis (GCGY), which encoded glucagon-like peptide sequences related to GLP-1 and GLP-2.

[0170] 23 paralogs of glucagon-like peptide sequences across four Myotis bat species were identified here. Surprisingly, while no paralogs related to GCG were identified, paralogs of GIP (GIPX), which is normally found at a separate locus, were found clustered with GLP-1 and GLP-2 paralogs (GLP1X, GLP2X). The GIPX paralogs shared many of the hallmarks of mammalian GIP, notably a conserved Tyrosine (Y) at its N-terminus and a coding sequence that spanned an intron.Biological significance of Myotis glucagon-related hormone paralogs

[0171] While the half-life of human GLP-1 is on the order of minutes, exendin paralogs from vertebrates are longer lived (~6 hours), which enabled the development of exenatide, a synthetic exendin-4 that served as a first-in-class treatment for diabetes (REF). The recent development and application of durable human GLP-1 agonists, e.g. semaglutide, have been highly effective for treating weight management and improved cardiovascular health when administered in both diabetic and nondiabetic patients. The discovery of mammalian paralogs of GLP-1 in Myotis bats, which are resistant to DPP -4 inactivation, may themselves function as durable GLPIR-agonists or provide new approaches for increasing the durability of human GLP-1.

[0172] Among mammals, bats are subjected to some of the most extreme seasonal and diet- induced metabolic challenges. Myotis bats in turn possess some of the most dynamic metabolic and physiological adaptations, which remain poorly understood. The expansion of glucagon-relatedparalogs in mammals may have enabled additional metabolic adaptations during the evolution of Myotis bats. The presence of this additional locus in Myotis bats suggests this is a relevantly recent addition, and the diversity in paralog sequences between Myotis species suggests this is currently undergoing natural selection.

[0173] The discovery here of glucagon-related paralogs was possible by the genomic sequencing of bat species and aided by the fact that these paralogs are encoded within single exons. As the genomes of additional bat and other mammalian species are sequenced (only 15% of mammals are currently sequenced and 0.2% of all known species on earth), it is likely that additional glucagon- related paralogs will be discovered. Future studies on the spatio-temporal expression and regulation of these hormones in these species will no doubt shed light on their natural biological roles and perhaps uncover new opportunities for translational human therapeutics.List of Sequences

[0174] While various embodiments have been described above, such disclosures have been presented by way of example only and are not limiting. Thus, the breadth and scope of the subject compositions and methods should not be limited by any of the above-described exemplary embodiments but should be defined only in accordance with the following claims and their equivalents.

[0175] The above description is for the purpose of teaching the person of ordinary skill in the art how to practice the present application, and it is not intended to detail all those obvious modifications and variations of it which will become apparent to the skilled worker upon reading the description. It is intended, however, that all such obvious modifications and variations be included within the scope of the present application, which is defined by thefollowing claims. The claims are intended to cover the components and steps in any sequence which is effective to meet the objectives they intended unless the context specifically indicates the contrary.

Claims

1. WHAT IS CLAIMED IS:

1. A polynucleotide expression construct, comprising from 5’ end to 3’ end:(1) a sequence encoding a 5’ ribozyme;(2) a sequence encoding an internal ribosome entry site (IRES);(3) a sequence for expressing a fusion G protein-coupled receptor (GPCR) agonist peptide; and(4) a sequence encoding a 3 ’ribozyme, wherein the sequence for expressing the fusion GPCR agonist peptide comprises a sequence encoding a signal peptide and a sequence encoding a GPCR agonist peptide.

2. The polynucleotide expression construct of claim 1, wherein the fusion GPCR agonist peptide comprises an amino acid sequence selected from either (1) the group consisting of SEQ ID NOS:356, 358, 365 and 366, or (2) the group consisting of SEQ ID NOS:28-52, 334- 341, 348-355, 357, 359-364, and 367-369.

3. The polynucleotide expression construct of claim 1, wherein the GPCR agonist peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-21, 171-179, 196-205, 223-230, 250-254, 266-323 and 342-347.

4. The polynucleotide expression construct of claim 1, wherein the GPCR agonist peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:305, 306, 312, and 314.

5. The polynucleotide expression construct of and one of claims 1, 3 and 4, wherein the signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:22-27 and SEQ ID NOS:370-393.

6. The polynucleotide expression construct of any one of claims 1 to 5, wherein the sequence for expressing the fusion GPCR agonist peptide further comprises a sequence that improves the stability of the GPCR agonist peptide.

7. A circular nucleic acid molecule formed from the polynucleotide expression construct of any one of claims 1 to 6.

8. The circular nucleic acid molecule of claim 7 being circular RNA molecule or circular single-stranded DNA molecule.

9. A polynucleotide expression cassette encoding a fusion G protein-coupled receptor (GPCR) agonist peptide, comprising:(1) a sequence encoding a signal peptide having an amino acid sequence selected from the group consisting of SEQ ID NOS:22-27 and SEQ ID NOS:370-393; and(2) a sequence encoding a G protein-coupled receptor (GPCR) agonist peptide having an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-21, 171-179, 196-205, 223-230, 250-254, 266-323 and 342-347.

10. The polynucleotide expression cassette of claim 9, wherein the fusion GPCR agonist peptide has an amino acid sequence selected from either (1) the group consisting of SEQ ID NOS:28-52, 334-341 and 348-369, or (2) the group consisting of SEQ ID NOS:356, 358, 365 and 366.

11. The polynucleotide expression cassette of claim 9 or 10, further comprising a regulatory sequence operably linked to (1) and / or (2).

12. An expression vector comprising the expression construct of any one of claims 1 to 6, or the expression cassette of any one of claims 9 to 11.

13. The expression vector of claim 12, wherein the expression vector is a plasmid.

14. The expression vector of claim 12, wherein the expression vector is a viral vector.

15. The expression vector of claim 14, wherein the viral vector is an adeno-associated virus(AAV) vector.

16. A pharmaceutical composition, comprising:(1) the circular RNA molecule of claim 7, and / or the polynucleotide expression vector of any one of claims 12-15; and(2) a pharmaceutically acceptable carrier.

17. A method for treating a GPCR-related condition in a subject, comprising the step of: administering to the subject an effective amount of the pharmaceutical composition of claim 16.

18. The method of claim 17, wherein the GPCR-related condition is selected from the group consisting of type 2 diabetes, obesity, heart disease, sleep disorder, cancer, dyslipidemia, and hypertension.

19. A method of increasing the activity of a G protein-coupled receptor (GPCR) in a subject, comprising the step of: administering to the subject, an effective amount of the circular RNA molecule of claim 8, and / or the polynucleotide expression vector of any one of claims 12-15.

20. A method for generating a circular RNA molecule encoding fusion G protein-coupled receptor (GPCR) agonist peptide, comprising the step of: introducing into a cell or tissue a nucleic acid molecule comprising(1) a sequence encoding a 5’ ribozyme;(2) a sequence encoding an internal ribosome entry site (IRES);(3) a sequence for expressing a GPCR agonist peptide; and(4) a sequence encoding a 3 ’ribozyme, wherein the sequence for expressing the fusion GPCR agonist peptide comprises a sequence encoding a signal peptide and a sequence encoding a GPCR agonist peptide, and wherein self-cleavage of the 3 ’ribozyme generates a 3’ phosphate and self-cleavage of the 5 ’ribozyme generates a 5’ hydroxy group, allowing for scarless ligation of the 5’ hydroxy group and 3’ phosphate of the RNA molecule to generate a circular RNA molecule encoding the fusion GPCR agonist peptide.

21. A fusion GPCR agonist peptide comprising an amino acid sequence selected from: the group consisting of SEQ ID NOS:356, 358, 365 and 366.

22. A GPCR agonist peptide comprising an amino acid sequence selected from:(1) the group consisting of SEQ ID NOS: 1-21, 171-179, 196-205, 223-230, 250-254, 266-323 and 342-347; or(2) the group consisting of SEQ ID NOS:305, 306, 312 and 314.

Images