Compositions and methods for expression of GLP-1 receptor agonists

Abstract

The present disclosure relates generally to nucleic acid molecules, e.g., self-replicating RNA (srRNA) expressing glucagon-like peptide 1 receptor (GLP-1R) agonists, as a monogenic or multigenic along with GPCR agonists, GIPR agonists, and GCGR agonists, pharmaceutical compositions and recombinant cells containing the same, as well as the use of such srRNA molecules, recombinant cells, and compositions for activating GLP-1R signaling in a subject. Also provided are methods for preventing and / or treating various health conditions associated with GLP-1R signaling.

Description

Attorney Docket No.: 058462-523001WOCOMPOSITIONS AND METHODS FOR EXPRESSION OF GLP-1 RECEPTOR AGONISTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 744,134, filed on January 10, 2025. The disclosure of the above-referenced application is herein expressly incorporated by reference it its entirety, including any drawings.INCORPORATION OF THE SEQUENCE LISTING

[0002] The material in the accompanying Sequence Listing is hereby incorporated by reference into this application. The accompanying Sequence Listing XML file, named 058462_523001WO_Sequence Listing. XML, was created on January 7, 2026, and is 124,525 bytes in size.FIELD

[0003] The present disclosure relates generally to the field of molecular virology and particularly relates to nucleic acid molecules, e.g., self-replicating RNA (srRNA) expressing agonist(s) of glucagon-like peptide 1 receptor (GLP-1R), glucose-dependent insulinotropic peptide receptor (GIPR), G-protein-coupled receptor (GPCR), and / or glucagon receptor (GCGR), pharmaceutical compositions, and recombinant cells containing the same, as well as the use of such srRNA molecules, recombinant cells, and compositions for eliciting a pharmacodynamic effect in a subject. Also provided are methods for preventing and / or treating various health conditions associated with the function of .BACKGROUND

[0004] G-protein-coupled receptors (GPCRs) constitute the largest family of cell surface receptor proteins. In recent years, glucagon-like peptide-1 receptor (GLP-1R) and its agonists have garnered widespread attention in the medical community. GLP-1R, a core member of the GPCR family, is widely present on the surfaces of various cells in the human body.

[0005] Upon activation, GPCRs couple to GTP -binding proteins that can be divided into four subclasses, Gas-, Gai / o-, Gaq and Gal2 / 13. Gs and Gi / o regulate adenylate cyclases leading to an increase (Gas) or a decrease (Gai / o) in cAMP production, or to an increase inAttorney Docket No.: 058462-523001WOintracellular calcium concentration (Gaq). Ga(12 / 13) activates Rho GEFs, which in turn activate Rho. GPCRs can also engage P-arrestins. Historically, P-arrestin-1 and P-arrestin-2 were believed to serve an exclusive role in GPCR desensitization. However, it has been shown that P-arrestins can also function to activate signaling cascades. Many human diseases are associated with the dysfunction of GPCRs. Thus, GPCRs represent some of the most attractive therapeutic or molecular targets in the pharmaceutical industry.

[0006] By specifically binding to the key hormone GLP-1, GLP-1R regulates blood glucose levels and lipid metabolism. This receptor and its agonists hold significant therapeutic potential, reshaping the treatment approaches for multiple diseases, including diabetes, cardiovascular disorders, and neurodegenerative diseases.

[0007] Recombinant GLP-1R agonists and derivatives, including GLP-1 and homologs, have been used as therapies to manage Type 2 diabetes and obesity. Due to their widespread use and the requirement of frequent dosing, the industry has struggled to keep up with manufacturing demands for the drugs. In addition to the inconvenience and cost to the patient, frequent dosing also carries the risk of patient non-compliance.

[0008] There is a need in the art to identify better and more robust methods and systems for expressing products of interest in srRNA-based expression platforms. There is also need in the art for new compositions and methods for treating diseases associated with insulin deficiency and high blood glucose. The present disclosure provides, inter alia, solutions to various problems existing with previous attempts to use GLP-1R agonists and offers improved methods for treatment of various health conditions.SUMMARY

[0009] The present disclosure relates generally to nucleic acid molecules, e.g., selfreplicating RNA (srRNA) expressing glucagon-like peptide 1 receptor (GLP-1R) agonists (GLP-lRAs) and GLP-1RA potentiators, pharmaceutical compositions and recombinant cells containing the same, as well as the use of such srRNA molecules, recombinant cells, and compositions for activating GLP-1R in a subject. Also provided are methods for preventing and / or treating various health conditions associated with GLP-1R. In particular, some embodiments of disclosure relate to nucleic acid constructs that include a nucleic acid sequence encoding a modified alphavirus genome or srRNA, wherein at least a portion of the nucleic acid sequence encoding the viral structural proteins of the modified alphavirus genome or srRNA has been replaced by a coding sequence for a polypeptide constructAttorney Docket No.: 058462-523001WOcomprising a GLP-1R agonist, either alone or with a GPCR agonist, a GIPR agonist, and / or a GCGR agonist. In some embodiments, the nucleic acid sequence further includes coding sequences for one or more heterologous signal peptides that directs secretion of one or more GLP-lRAs alone or with GPCR, GIPR, and GCGR agonists. In some embodiments, at least one of the one or more GLP-1R agonists includes a structural modification to increase halflife.

[0010] In one aspect of the disclosure, provided herein are nucleic acid constructs including a nucleic acid sequence encoding a modified alphavirus genome or self-replicating RNA (srRNA), wherein at least a portion of the nucleic acid sequence encoding the viral structural proteins of the modified alphavirus genome or srRNA has been replaced by a coding sequence for a polypeptide construct comprising one or more glucagon-like peptide-1 receptor (GLP-1R) agonists.

[0011] Non-limiting exemplary embodiments of the nucleic acid constructs of the disclosure can include one or more of the following features. In some embodiments, the nucleic acid sequence further includes coding sequences for one or more glucagon receptor (GCGR) agonists, glucose-dependent insulinotropic peptide (i.e., gastric inhibitory polypeptide; GIP), GIP receptor (GIPR) agonists, and / or G-protein-coupled receptor (GPCR) agonists. In some embodiments, the nucleic acid sequence further includes coding sequences for one or more heterologous signal peptides that directs secretion of the one or more GLP-1R agonists, alone or in combination with GCGR agonist, GIP, GIP receptor (GIPR) agonist, and / or GPCR agonist. In some embodiments, the one or more heterologous signal peptides is selected from signal peptides of human AACT, human FGF21, human FZD3, mouse IGKV3-10, human IGKV3-20, human INS, human INSR, human IPSP, human PORIM, human SAMP, and a combination of any thereof. In some embodiments, the one or more agonists of GLP-1R, GCGR, GPCR, GIPR, and GIP are operably linked to one another within a single open reading frame (i.e., in a polycistronic ORF). In some embodiments, the one or more agonists of GLP-1R, GCGR, GPCR, GIPR, and GIP are operably linked to one another by one or more linkers.

[0012] In some embodiments of the disclosure, at least one of the agonists of GLP-1R, GCGR, GPCR, GIPR, and GIP includes a structural modification to increase half-life. In some embodiments, the structural modification includes one or more mutations, N-extension, and / or C-terminal extensions. In some embodiments, the structural modification includes fusion to a Fc region of an immunoglobulin. In some embodiments, the modified alphavirusAttorney Docket No.: 058462-523001WOgenome or srRNA includes no nucleic acid sequence encoding viral structural proteins. In some embodiments, the nucleic acid sequence encoding the polypeptide construct is operably linked to a promoter sequence. In some embodiments, the promoter sequence is or includes a 26S subgenomic (sg) promoter.

[0013] In some embodiments of the disclosure, the srRNA is a capped srRNA including a 5’-cap. In some embodiments, the 5’-cap is a cap-0, cap-1, cap-2, or cap analogue. In some embodiments, the modified alphavirus genome or srRNA is of an alphavirus belonging to the VEEV / EEEV group, or the SFV group, or the SINV group. In some embodiments, the alphavirus is Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), Chikungunya virus (CHIKV), Madariaga virus (MADV), Western equine encephalitis virus (WEEV), or Sindbis virus (SINV).

[0014] In some embodiments, the polypeptide construct includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-25. In some embodiments, the coding sequence for the polypeptide construct includes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 26-51.

[0015] In one aspect, provided herein are recombinant cells including a nucleic acid construct as disclosed herein. In a related aspect, provided herein are cell cultures including at least one recombinant cell as described herein and a culture medium. Non-limiting exemplary embodiments of the recombinant cells of the disclosure can include one or more of the following features. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a vertebrate animal cell or an invertebrate animal cell. In some embodiments, the recombinant cell is an insect cell. In some embodiments, recombinant insect cell is a mosquito cell. In some embodiments, the recombinant cell is a mammalian cell. In some embodiments, the recombinant cell is selected from the group consisting of a monkey kidney CV1 cell transformed by SV40, a human embryonic kidney cell (HEK), a baby hamster kidney cell (BHK) or a derivative cell thereof, a mouse sertoli cell, a monkey kidney cell, a human cervical carcinoma cell, a canine kidney cell, a buffalo rat liver cell, a human lung cell, a human liver cell, a human fat cell or adipocyte, a mouse fat cell or adipocyte a mouseAttorney Docket No.: 058462-523001WOmammary tumor, a TRI cell, a FS4 cell, a Chinese hamster ovary cell (CHO), an African green monkey kidney cell, a human A549 cell, a human cervix cell, a human CHME5 cell, a human PER.C6 cell, aNSO murine myeloma cell, a human epidermoid larynx cell, a human fibroblast cell, a human HUH-7 cell, a human MRC-5 cell, a human muscle cell, a human endothelial cell, a human astrocyte cell, a human macrophage cell, a human RAW 264.7 cell, a mouse 3T3 cell, a mouse L929 cell, a mouse connective tissue cell, a mouse muscle cell, and a rabbit kidney cell.

[0016] In another aspect, provided herein are transgenic animals including: (a) a nucleic acid construct as disclosed herein; and / or (b) a recombinant cell as disclosed herein. In some embodiments, the transgenic animal is a vertebrate animal or an invertebrate animal. In some embodiments, the transgenic animal is an insect. In some embodiments, the transgenic animal is a non-human transgenic animal. In some embodiments, the transgenic animal is a mammalian. In some embodiments, the mammalian is a non-human mammalian.

[0017] In one aspect, provided herein are compositions, e.g., pharmaceutical compositions including a pharmaceutically acceptable excipient and one or more of the following: a nucleic acid construct as disclosed herein; and / or (b) a recombinant cell as disclosed herein.

[0018] Non-limiting exemplary embodiments of the pharmaceutical compositions of the disclosure can include one or more of the following features. In some embodiments, the pharmaceutical composition includes a nucleic acid construct as disclosed herein, and a pharmaceutically acceptable excipient. In some embodiments, the composition is formulated with a delivery vehicle into a delivery system, wherein the delivery system includes a liposome, a viral replicon particle (VRP), a lipid-based nanoparticle (LNP), a polymer nanoparticle, a physiologic buffer, a microsphere, an immune stimulating complex (ISCOM), a conjugate of bioactive ligand, or a combination of any thereof. In some embodiments, the LNP delivery system includes a cationic lipid, an ionizable cationic lipid, an anionic lipid, or a neutral lipid. In some embodiments, the lipid of the LNP delivery system is present in mass ratio of lipid to RNA from about 100:1 to about 4:1. In some embodiments, the LNP delivery system includes lipid-based nanoparticles having an average diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. In some embodiments, the LNP delivery system includes lipid-based nanoparticles having an average diameter of about 1-1000 nm, about 1-1000 nm, about 1-500 nm, about 1-250 nm, about 25-200 nm, about 25-100 nm, about 35-75 nm, or about 25-60 nm.

[0019] In some embodiments, the pharmaceutical composition is formulated for one orAttorney Docket No.: 058462-523001WOmore of intranasal administration, intrathecal administration, transdermal administration, intraperitoneal administration, intramuscular administration, intratracheal administration, intranodal administration, intratumoral administration, intraarticular administration, intravenous administration, subcutaneous administration, intravaginal administration, intraocular administration, rectal administration, and oral administration. In some embodiments, the pharmaceutical composition is formulated intramuscular administration.

[0020] In another aspect, provided herein are methods for activating GLP-1 receptor signaling in a subject, the method includes administering to the subject a composition including: (a) a nucleic acid construct as disclosed herein; (b) a recombinant cell as disclosed herein; and / or (c) a pharmaceutical composition as disclosed herein. In another aspect, provided herein are methods for eliciting a pharmacodynamic effect in a subject in need thereof, the method includes administering to the subject a composition including: (a) a nucleic acid construct as disclosed herein; (b) a recombinant cell as disclosed herein; and / or (c) a pharmaceutical composition as disclosed herein. Non-limiting exemplary embodiments of the methods disclosed herein can include one or more of the following features. In some embodiments, the pharmacodynamic effect includes one or more of the following: immunogenicity effect, a biomarker response, a therapeutic effect, a prophylactic effect, a desired effect, an undesired effect, an adverse effect, and effect in a disease model. In some embodiments, the pharmacodynamic effect includes eliciting an immune response in the subject.

[0021] In another aspect, provided herein are methods preventing and / or treating a health condition in a subject in need thereof, the method includes prophylactically or therapeutically administering to the subject a composition including: (a) a nucleic acid construct as disclosed herein; (b) a recombinant cell as disclosed herein; and / or (c) a pharmaceutical composition as disclosed herein. Non-limiting exemplary embodiments of the methods disclosed herein can include one or more of the following features. In some embodiments, the administered composition results in slowing of gastric emptying, reducing the release of glucagon, decreasing blood glucose, enhancing insulin sensitivity, and / or stimulating insulin production in the subject. In some embodiments, the subject is having or suspected of having a metabolic disorder associated with GLP-1R signaling. In some embodiments, the metabolic disorder associated with GLP-1R signaling is diabetes, obesity, hypertension, hyperlipidemia, a cardiovascular disease, or a musculoskeletal disease. In some embodiments, the composition is administered to the subject individually as a single therapy (monotherapy) or as a firstAttorney Docket No.: 058462-523001WOtherapy in combination with at least one additional therapies.

[0022] In yet another aspect, provided herein are kits for including: (a) a nucleic acid construct as disclosed herein; (b) a recombinant cell as disclosed herein; and / or (c) a pharmaceutical composition as disclosed herein, and instructions for performing a method as disclosed herein. In some embodiments, the kit is for a method of preventing and / or treating a health condition. In some embodiments, the health condition is the health condition is a metabolic disorder associated with GLP-1R signaling. In some embodiments, the metabolic disorder associated with GLP-1R signaling is diabetes, obesity, hypertension, hyperlipidemia, a cardiovascular disease, or a musculoskeletal disease.

[0023] Each of the aspects and embodiments described herein are capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect.

[0024] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 depicts non-limiting examples of monogenic and multigenic designs of therapeutic srRNAs in accordance with some embodiments of the disclosure, in which the nucleic acid sequence encoding viral structural proteins of the original virus have been completely deleted. The therapeutic srRNA designs described in this figure contain native 5’ UTR and 3’ UTR derived from an alphavirus, and further contain one or more heterologous gene of interest (GOI) that are incorporated in either a monogenic or a multigenic expression cassette placed under control of a 26S subgenomic promoter. The coding sequences for the non- structural proteins nsPl, nsP2, nsP3, and nsP4 are indicated (nSPl-4). GIP: glucosedependent insulinotropic polypeptide; GCG: glucagon; P2A: autoproteolytic peptide sequence from porcine teschovirus-1 2A; IRES: internal ribosomal entry site. GLP-1RA: GLP-1R agonist.

[0026] FIG. 2 summarizes the results of experiments performed to illustrate that GLP-1R agonists with heterologous signal peptides can be encoded into srRNA vectors capable of RNA replication. The Y-axis represents the frequency of BHK-21 cells that contain double stranded RNA (dsRNA), the replication intermediate of srRNA replication, after transfectionAttorney Docket No.: 058462-523001WOwith srRNA.

[0027] FIG. 3 summarizes the results of experiments performed to illustrate that the signal peptide identity in srRNA vector affects secretion of the expressed GLP-1R agonist. The Y-axis represents the concentration of GLP-1 in cell culture media after BHK-21 cells were transfected with the corresponding srRNA. In these experiments, it was observed that not all signal peptides result in detectable secreted GLP-1R agonist (GLP-1), as determined by a cell-based assay.

[0028] FIG. 4 summarizes the results of additional experiments performed to illustrate that the signal peptide identity in srRNA vector affects secretion of an exemplary GPCR agonist, glucagon (GCG). The Y-axis represents the concentration of GCG in cell culture media after BHK-21 cells were transfected with the corresponding srRNA. In these experiments, it was observed that not all signal peptides result in detectable secreted GCG, as determined by ELISA.

[0029] FIGS. 5A-5B summarize the results of experiments performed to illustrate that the signal peptide identity in srRNA vector affects expression of another exemplary GLP-1R agonist, Exenatide. The Y-axes represent the concentration of Exenatide in cell culture media after BHK-21 cells were transfected with the corresponding srRNA. In FIG. 5A, the functional concentration of Exenatide as determined by reporter cell line was used for quantifying GLP-1 receptor signaling. In FIG. 5B, concentration of Exenatide was determined by ELISA using an Exendin-4 reference.

[0030] FIGS. 6A-6B summarize the results of experiments performed to illustrate that the signal peptide identity in srRNA vector affects expression of another exemplary GLP-1R agonist, GLP-l-Fc. The Y-axes represent the concentration of GLP-l-Fc in cell culture media after BHK-21 cells were transfected with the corresponding srRNA. In FIG. 6A, the functional concentration of GLP-l-Fc as determined by reporter cell line was used for quantifying GLP-1 receptor signaling. In FIG. 6B, the concentration of GLP-l-Fc was determined by ELISA.

[0031] FIGS. 7A-7B summarize the results of experiments illustrating in vitro expression and secretion of GLP-l-Fc and Exenatide in different srRNA-based vectors. The Y-axis represent the concentration of GLP-1R agonists (GLP-l-Fc in FIG. 7A and Exenatide in FIG. 7B) quantified by ELISA in cell lysates or cell culture media after BHK-21 cells were transfected with the corresponding srRNA.

[0032] FIG. 8 schematically summarizes the results of experiments performed to evaluateAttorney Docket No.: 058462-523001WOGLP-l-Fc’s in vivo expression from a monogenic EEEV srRNA-based vector by multiple routes of administration.

[0033] FIGS. 9A-9C summarizes the results of experiments performed to evaluate GLP-1 -Fc’s expression and secretion from different multigenic srRNA-based vectors with different heterologous signal peptides. FIG. 9A: The srRNA designs described in this figure contain native 5’ UTR and 3’ UTR derived from an alphavirus, and further contain a multigenic expression cassette placed under control of a 26S subgenomic promoter. The coding sequences for the non-structural proteins nsPl, nsP2, nsP3, and nsP4 are indicated (nSPl-4). GIP: glucose-dependent insulinotropic polypeptide; GCG: glucagon; P2A: autoproteolytic peptide sequence from porcine teschovirus-1 2 A; IRES: internal ribosomal entry site. GLP-1 -Fc: GLP-1 polypeptide operably linked to the Fc region of an immunoglobulin. Description of the srRNA designs used in these experiments can also be found in Table 2. In FIGS. 9B-9C, the Y-axis represent the concentration of GLP-Fc expressed with a msIgGK signal peptide (FIG. 9B) or an Insulin signal peptide (FIG. 9C) quantified by ELISA in cell culture media after BHK-21 cells were transfected with the corresponding srRNA.

[0034] FIGS. 10A-10C summarizes the results of experiments performed to evaluate expression and secretion of GLP-1R agonist, GCG, and GIP from different multigenic srRNA-based vector with different heterologous signal peptides. FIG. 10A: The designs described in this figure contain native 5’ UTR and 3’ UTR derived from either EEEV or VEEV, and further contain a multigenic expression cassette placed under control of a 26S subgenomic promoter. The coding sequences for the non-structural proteins nsPl, nsP2, nsP3, and nsP4 are indicated (nSPl-4). GIP: glucose-dependent insulinotropic polypeptide; GCG: glucagon; P2A: autoproteolytic peptide sequence from porcine teschovirus-1 2 A; IRES: internal ribosomal entry site. GLP-l-Fc: GLP-1 polypeptide operably linked to the Fc region of an immunoglobulin. Description of the srRNA designs used in these experiments can also be found in Table 2. In FIGS. 10B-10C, the Y-axis represent the concentration of secreted glucagon (FIG. 10B) or secreted GIP (FIG. 10C) quantified by ELISA in cell culture media after BHK-21 cells were transfected with the corresponding srRNA. LLOD: Lower limit of detection.Attorney Docket No.: 058462-523001WODETAILED DESCRIPTION OF THE DISCLOSURE

[0035] Provided herein are, inter alia, viral expression systems including self-replicating RNAs (srRNAs) based on alphaviruses with superior expression potential which are suitable for expressing heterologous molecules such as, agonists of glucagon-like peptide- 1 receptor (GLP-1R), for the purposes of therapeutic treatment of human health conditions or diseases, diabetes, obesity, hypertension, hyperlipidemia, cardiovascular diseases, and musculoskeletal diseases. These srRNA-based expression systems address the requirement of frequent dosing of existing therapeutic agents containing agonists and derivatives. For example, some embodiments of the disclosure relate to nucleic acid constructs such as, e.g., expression constructs and vectors, containing a modified genome or srRNA of an alphavirus in which at least a portion of the nucleic acid sequence encoding the viral structural proteins of the modified alphavirus genome or srRNA has been replaced by coding sequences for a polypeptide construct containing a glucagon-like peptide- 1 receptor (GLP-1R) agonist, either alone or along with GPCR agonists, GIPR agonists, and / or GCGR agonists. Further provided are recombinant cells that are genetically engineered to include one or more of the nucleic acid constructs disclosed herein. Biomaterials and recombinant products derived from such recombinant cells are also within the scope of the application. Also provided are compositions and methods useful for (i) activating GLP-1 receptor, (ii) modulating a pharmacodynamic effect, and / or (iii) for preventing and / or treating a health condition associated with GLP-1R signaling in a subject in need thereof.

[0036] As discussed above, recombinant GLP-1R agonists and derivatives, including GLP1 and homologs, have been used as therapies to manage Type 2 diabetes and obesity. Due to their widespread use and the requirement of frequent dosing, the industry has struggled to keep up with manufacturing demands for the drugs. In addition to the inconvenience and cost to the patient, frequent dosing also carries the risk of patient non-compliance.

[0037] Current formulations typically contain a single GLP-1R agonist, which may be therapeutically limiting based on the interaction of the single molecule with the receptor.

[0038] Self-replicating RNAs can generate one or multiple GLP-1R agonists in situ at low doses for extended periods, which can address manufacturing bottlenecks, reduce dose frequency, and improve therapeutic efficacy. Unlike recombinant protein administration, the production of secreted proteins from an mRNA or srRNA vectors presents a challenge. The protein must be expressed from the vector and be released from the cell for it to function. N-terminal signal peptides can be used to instruct the protein to be secreted from the cell, butAttorney Docket No.: 058462-523001WOnot all signal peptides function equivalently. As described in greater detail below, the inventors have identified signal peptides that are functional and non-functional with regards to secretion of functional GLP-1R agonists expressed from srRNA vectors in vitro and in vivo.DEFINITIONS

[0039] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.

[0040] The singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A,” “B,” “A or B,” and “A and B .”

[0041] The terms “administration” and “administering,” as used herein, refer to the delivery of a bioactive composition or formulation by an administration route including, but not limited to, intranasal, transdermal, intravenous, intra-arterial, intramuscular, intranodal, intraperitoneal, subcutaneous, intramuscular, oral, intravaginal, and topical administration, or combinations thereof. The term includes, but is not limited to, administering by a medical professional and self-administering.

[0042] The term “agonist” of a receptor used in the context of the present disclosure refers to a chemical compound, molecule, or substance (ligand) that is capable of binding to the receptor and activating a downstream signaling pathway or cascade through the targeted receptor. For example, if a GLP-1 receptor is a target receptor, the agonist binds to the GLP-1 receptor and activates GLP-1 receptor-mediated silencing. As such, an exemplary GLP-1 receptor agonist can be a GLP-1 polypeptide or an analog thereof. The agonists described in various embodiments of the present disclosure are polypeptide agonists, e.g., those can be encoded by nucleic acid sequences.

[0043] The terms “cell,” “cell culture,” and “cell line” refer not only to the particularAttorney Docket No.: 058462-523001WOsubject cell, cell culture, or cell line but also to the progeny or potential progeny of such a cell, cell culture, or cell line, without regard to the number of transfers or passages in culture. It should be understood that not all progeny are exactly identical to the parental cell. This is because certain modifications may occur in succeeding generations due to either mutation (e.g., deliberate or inadvertent mutations) or environmental influences (e.g., methylation or other epigenetic modifications), such that progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein, so long as the progeny retain the same functionality as that of the original cell, cell culture, or cell line.

[0044] The term “effective amount,” “therapeutically effective amount,” or “pharmaceutically effective amount” of a composition of the disclosure, e.g., nucleic acid constructs (e.g., srRNA constructs), recombinant cells, and / or pharmaceutical compositions, generally refers to an amount sufficient for the composition to accomplish a stated purpose relative to the absence of the composition (e.g., achieve the effect for which it is administered, stimulate an immune response, prevent or treat a disease, or reduce one or more symptoms of a disease, disorder, infection, or health condition). An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). The exact amount of a composition including a “therapeutically effective amount” will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0045] The term “construct” refers to a recombinant molecule, e.g., recombinant nucleic acid or polypeptide, including one or more isolated nucleic acid sequences or amino acid sequences from heterologous sources. For example, polypeptide constructs can be chimeric polypeptide molecules in which two or more amino acid sequences of different origin are operably linked to one another in a single polypeptide construct. Similarly, nucleic acid constructs can be chimeric nucleic acid molecules in which two or more nucleic acid sequences of different origin are assembled into a single nucleic acid molecule. Thus, representative nucleic acid constructs include any constructs that contain (1) nucleic acidAttorney Docket No.: 058462-523001WOsequences, including regulatory and coding sequences that are not found adjoined to one another in nature (e.g., at least one of the nucleotide sequences is heterologous with respect to at least one of its other nucleotide sequences), or (2) sequences encoding parts of functional RNA molecules or proteins not naturally adjoined, or (3) parts of promoters that are not naturally adjoined. Representative nucleic acid constructs can include any recombinant nucleic acid molecules, linear or circular, single-stranded or double-stranded DNA or RNA nucleic acid molecules, derived from any source, such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, capable of genomic integration or autonomous replication, including a nucleic acid molecule where one or more nucleic acid sequences have been operably linked. In some embodiments, nucleic acid constructs of the present disclosure can include the necessary elements to direct expression of a nucleic acid sequence of interest that is also contained in the construct. Such elements may include control elements such as a promoter that is operably linked to (e.g., so as to direct transcription of) the nucleic acid sequence of interest, and optionally includes a polyadenylation sequence.

[0046] In some embodiments of the disclosure, the nucleic acid construct may be incorporated within a vector. The term “vector” is used herein to refer to a nucleic acid molecule or sequence capable of transferring or transporting another nucleic acid molecule. Thus, the term “vector” encompasses both DNA-based vectors and RNA-based vectors. The term “vector” includes cloning vectors and expression vectors, as well as viral vectors and integrating vectors. An “expression vector” is a vector that includes a regulatory region, thereby capable of expressing DNA sequences and fragments in intro, ex vivo, and / or in vivo. In some embodiments, a vector may include sequences that direct autonomous replication in a cell such as, for example a plasmid (DNA-based vector) or a self-replicating RNA vector. In some embodiments, a vector may include sequences sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. In some embodiments, the vector of the disclosure can be single-stranded vector (e.g., ssDNA or ssRNA). In some embodiments, the vector of the disclosure can be double-stranded vector (e.g., dsDNA or dsRNA). In some embodiments, a vector is a gene delivery vector. In some embodiments, a vector is used as a gene delivery vehicle to transfer a gene into a cell.

[0047] In addition to the components of the construct, the vector may include, for example, one or more selectable markers, one or more origins of replication, such as prokaryotic and eukaryotic origins, at least one multiple cloning site, and / or elements to facilitate stableAttorney Docket No.: 058462-523001WOintegration of the construct into the genome of a cell. Two or more constructs can be incorporated within a single nucleic acid molecule, such as a single vector, or can be incorporated within two or more separate nucleic acid molecules, such as two or more separate vectors. An “expression construct” generally includes at least a control sequence operably linked to a nucleotide sequence of interest. In this manner, for example, promoters in operable connection with the nucleotide sequences to be expressed are provided in expression constructs for expression in a cell. For the practice of the present disclosure, compositions and methods for preparing and using constructs and cells are known to one skilled in the art.

[0048] The term “operably linked,” as used herein, denotes a physical or functional linkage between two or more elements, e.g., polypeptide sequences or polynucleotide sequences, which permits them to operate in their intended fashion. For example, the term “operably linked” when used in context of the nucleic acid molecules described herein or the coding sequences and promoter sequences in a nucleic acid molecule means that the coding sequences and promoter sequences are in-frame and in proper spatial and distance away to permit the effects of the respective binding by transcription factors or RNA polymerase on transcription. It should be understood that operably linked elements may be contiguous or non-contiguous (e.g., linked to one another through a linker). Thus, operably linked segments, portions, regions, and domains of the polypeptides or nucleic acid molecules disclosed herein may be contiguous or non-contiguous e.g., linked to one another through a linker). In the context of polypeptide constructs, “operably linked” refers to a physical linkage (e.g., directly or indirectly linked) between amino acid sequences (e.g., different segments, portions, regions, or domains) to provide for a described activity of the constructs.

[0049] The term “recombinant” when used with reference to a cell, a nucleic acid, a protein, or a vector, indicates that the cell, nucleic acid, protein or vector has been altered or produced through human intervention such as, for example, has been modified by or is the result of laboratory methods. Thus, for example, recombinant proteins and nucleic acids include proteins and nucleic acids produced by laboratory methods. Recombinant proteins can include amino acid residues not found within the native (non-recombinant or wild-type) form of the protein or can be include amino acid residues that have been modified, e.g., labeled. The term can include any modifications to the peptide, protein, or nucleic acid sequence. Such modifications may include the following: any chemical modifications of the peptide, protein or nucleic acid sequence, including of one or more amino acids,Attorney Docket No.: 058462-523001WOdeoxyribonucleotides, or ribonucleotides; addition, deletion, and / or substitution of one or more of amino acids in the peptide or protein; creation of a fusion protein, e.g., a fusion protein comprising one or more polypeptide agonists; and addition, deletion, and / or substitution of one or more of nucleic acids in the nucleic acid sequence. The term ’’recombinant” when used in reference to a cell is not intended to include naturally-occurring cells but encompass cells that have been engineered / modified to include or express a polypeptide or nucleic acid that would not be present in the cell if it was not engineered / modified.

[0050] The term “percent sequence identity,” as used herein in the context of two or more nucleic acids or proteins, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (e.g., about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. See e.g., the NCBI website at ncbi.nlm.nih.gov / BLAST. Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the complement of a query sequence. This definition includes sequence comparison performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences. This definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. Sequence identity can be calculated over a region that is at least about 20 amino acids or nucleotides in length, or over a region that is 10-100 amino acids or nucleotides in length, or over the entire length of a given sequence. Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux etal., Nucleic Acids Res (1984) 12:387), BLASTP, BLASTN, FASTA (Atschul etal., J. Mol. Biol. (1990) 215:403). Sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with the default parameters thereof. Additional methodologies that can suitably be utilized to determine similarity or identity amino acid sequences include those relying on position-specific structure-scoring matrix (P3SM) thatAttorney Docket No.: 058462-523001WOincorporates structure-prediction scores from Rosetta, as well as those based on a length-normalized edit distance as described previously in, e.g., Setcliff et aL, Cell Host & Microbe 23(6), May 2018.

[0051] The term “pharmaceutically acceptable excipient” as used herein refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive, or diluent for administration of a compound(s) of interest to a subject. As such, “pharmaceutically acceptable excipient” can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable carrier” includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds (e.g., antibiotics and additional therapeutic agents) can also be incorporated into the compositions.

[0052] As used herein, a “subject” or an “individual” includes animals, such as human e.g., human individuals) and non-human animals. In some embodiments, a “subject” or “individual” is a patient under the care of a physician. Thus, the subject can be a human patient or an individual who has, is at risk of having, or is suspected of having a health condition of interest (e.g., diabetes or obesity) and / or one or more symptoms of the health condition. The subject can also be an individual who is diagnosed with a risk of the health condition of interest at the time of diagnosis or later. The term “non-human animals” includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, non-human primates, and other mammals, such as e.g., sheep, dogs, cats, cows, chickens, and non-mammals, such as amphibians, reptiles, etc.

[0053] It is understood that aspects and embodiments of the disclosure described herein include "comprising,” "consisting,” and "consisting essentially of' aspects and embodiments. As used herein, "comprising" is synonymous with "including,” "containing,” or "characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of' excludes any elements, steps, or ingredients not specified in the claimed composition or method. As used herein, "consisting essentially of' does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term "comprising,” particularly in a description of components of a composition or in a description of steps of a method, is understood to encompass those compositions and methods consistingAttorney Docket No.: 058462-523001WOessentially of and consisting of the recited components or steps.

[0054] Where a range of values is provided, it is understood by one having ordinary skill in the art that all ranges disclosed herein encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. In some embodiments, where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above.Furthermore, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0055] Certain ranges are presented herein with numerical values being preceded by the term “about” which, as used herein, has its ordinary meaning of approximate. The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. In some embodiments, the term “about” indicates the designated value ± up to 10%, up to ± 5%, or up to ± 1%.Attorney Docket No.: 058462-523001WO

[0056] Headings, e.g., (a), (b), (i) etc., are presented merely for ease of reading the specification and claims. The use of headings in the specification or claims does not require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.

[0057] It should be appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.GLUCAGON-LIKE PEPTIDE 1 RECEPTOR (GLP-1R) AGONISTS

[0058] The glucagon-like peptide-1 (GLP-1) receptor, known as GLP-1R, is a vital component of the G protein-coupled receptor (GPCR) family and is found primarily on the surfaces of various cell types within the human body. This receptor specifically interacts with GLP-1, a key hormone that plays an integral role in regulating blood glucose levels, lipid metabolism, and several other crucial biological functions. By specifically binding to GLP-1, it regulates blood glucose levels and lipid metabolism. This receptor and its agonists hold significant therapeutic potential, reshaping the treatment approaches for multiple diseases, including diabetes, cardiovascular disorders, and neurodegenerative diseases. GLP-1 is a peptide produced by the cleavage of proglucagon, mainly synthesized in the intestinal mucosal L-cells, pancreatic islet a-cells, and neurons in the nucleus of the solitary tract. GLP-IRAs mimic the action of endogenous GLP-1, activating GLP-1R, thereby enhancing insulin secretion, inhibiting glucagon release, delaying gastric emptying, and reducing food intake through central appetite suppression. These mechanisms make GLP-lRAs powerful tools for controlling blood glucose and improving metabolic syndrome. Furthermore, their multifaceted mechanisms of action suggest potential applications beyond traditional metabolic disorders. From the discovery of the GLP-1 fragment GLP-1 to the development of more stable and long-acting GLP-1 analogs, these milestones represent significantAttorney Docket No.: 058462-523001WObreakthroughs in the medical field. Additionally, multi -agonist approaches combining GLP-1R agonists with glucose-dependent insulinotropic peptide (GIP), and glucagon receptor agonists, enhance efficacy due to the additive anorectic and insulinotropic effects of GLP-1 and GIP with the increase in energy expenditure by glucagon.

[0059] Insulin regulates the concentration of blood sugar and blood lipid levels through the promotion of glucose and lipid uptake into cells. Type 2 Diabetes Mellitus (T2DM or T2D) is a complex metabolic disorder characterized by hyperglycemia arising from a combination of insufficient insulin secretion together with the development of insulin resistance. T2D and obesity are closely linked, with obesity accounting for 80-85% of the risk of developing T2D. Incretin-based therapies represent a promising class of agents for the treatment of T2D.In cretins including the glucagon -like peptide- 1 (GLP-1) and the glucose-dependent insulinotropic polypeptide (GIP) are endogenous peptide hormones secreted from the intestine in response to food intake. GLP-1 and GIP exert their action through G-protein coupled receptor, the GLP-1 and the GIP receptors, respectively. GLP-1R is expressed in pancreatic P-cells as well as various tissues including liver, smooth muscle, heart, kidney, gastrointestinal tract, lungs, pituitary, white adipose tissues, and the central nervous system. GLP-1 lowers postprandial glucose excursion by potentiating glucose-stimulated insulin secretion from pancreatic P-cells and has recently been shown to promote P-cell survival in rodents. In addition, GLP-1 exerts extra-pancreatic actions such as promoting gastric emptying, weight loss, intestinal growth and increasing insulin sensitivity in peripheral tissues.

[0060] In recent years, GLP-1 medications have become a focal point in the medical community due to their innovative treatment mechanisms, significant therapeutic efficacy, and broad development prospects. The ongoing development of new indications for GLP-1 drugs has offered promising prospects for further expanding therapeutic interventions, showcasing their significant potential in the medical field, including the extensive benefits of using GLP-lRAs in treating a broad spectrum of diseases, such as obesity, cardiovascular diseases, non-alcoholic fatty liver disease (NAFLD), neurodegenerative diseases, musculoskeletal inflammation, and various forms of cancer. Moreover, multi-agonist approaches combining GLP-1R agonists with glucose-dependent insulinotropic peptide (GIP), and glucagon receptor agonists, enhance efficacy due to the additive anorectic and insulinotropic effects of GLP-1 and GIP with the increase in energy expenditure by glucagon. Additional information regarding GLP-1R and GLP-lRAs can be found in, e.g., Zheng et al.,Attorney Docket No.: 058462-523001WOSignal Transduction and Targeted Therapy 9, No. 1 (2024): 234-234, which is incorporated by reference herein in its entirety.ALPHAVIRUS SELF-REPLICATING RNASAlphaviruses

[0061] In some embodiments of the disclosure, the replicon, e.g., srRNA, is derived from a virus belonging to the Alphavirus genus. The Alphavirus genus has been widely studied and the life cycle, mode of replication, etc., of these viruses are well characterized. Alphaviruses are small, enveloped RNA viruses with a single-stranded, positive-sense RNA genome. More information in this regard can be found in, e.g., Arrigo NC et al., supra 2010 and Corrin T. et al., Vector-Borne and Zoonotic Diseases, Vol. 21, No. 5, 2021. In addition, alphaviruses have been shown to replicate very efficiently in animal cells which makes them valuable as vectors for production of protein and nucleic acids in such cells. Transmission between species and individuals occurs mainly via mosquitoes making the alphaviruses a contributor to the collection of Arboviruses, also known as Arthropod-Borne Viruses.

[0062] The Alphavirus genus includes, inter alia, the Sindbis virus (SINV), the Semliki Forest virus (SFV), the Ross River virus (RRV), Venezuelan equine encephalitis virus (VEEV), and Eastern equine encephalitis virus (EEEV), which are all closely related and are able to infect various vertebrates such as mammalians, rodents, fish, avian species, and larger mammals such as humans and horses as well as invertebrates such as insects. Each of these alphaviruses has a single stranded RNA genome of positive polarity enclosed in a nucleocapsid surrounded by an envelope containing viral spike proteins. Alphavirus particles are enveloped, tend to be spherical (although slightly pleomorphic), and have an isometric nucleocapsid. Alphavirus genomes are single-stranded RNA of positive polarity of approximately 11-12 kb in length, flanked by 5’ and 3’ untranslated regions (UTRs) and are capped at the 5' end and polyadenylated at the 3' end, and comprise two open reading frames with a first frame (~7 kb) encoding the nonstructural proteins with enzymatic function and a second frame (~4 kb) encoding the viral structural proteins (e.g., the capsid protein CP, El glycoprotein, E2 glycoprotein, E3 protein and 6K protein). The non- structural polyprotein (nsP) is cleaved into four different proteins (nsPl, nsP2, nsP3, and nsP4) which are necessary for the transcription and translation of viral mRNA inside the cytoplasm of host cells.

[0063] The 5’ two-thirds of the alphavirus genome encodes a number of nonstructural proteins (nsPs) necessary for transcription and replication of viral RNA. These proteins areAttorney Docket No.: 058462-523001WOtranslated directly from the RNA and together with cellular proteins form the RNA-dependent RNA polymerase essential for viral genome replication and transcription of subgenomic RNA. Four nonstructural proteins (nsPl, nsP2, nsP3, nsP4) are produced as a single polyprotein constitute the virus’ replication machinery. The processing of the polyprotein occurs in a highly regulated manner, with cleavage at the P2 / 3 junction influencing RNA template use during genome replication. This site is located at the base of a narrow cleft and is not readily accessible. Once cleaved, nsP3 creates a ring structure that encircles nsP2. These two proteins have an extensive interface. Mutations in nsP2 that produce noncytopathic viruses or temperature sensitive phenotypes cluster at the P2 / P3 interface region. P3 mutations opposite the location of the nsP2 noncytopathic mutations prevent efficient cleavage of P2 / 3. This in turn can affect RNA infectivity altering viral RNA production levels.

[0064] The 3’ one-third of the genome comprises subgenomic RNA which serves as a template for translation of all the structural proteins required for forming viral particles: the core nucleocapsid protein C, and the envelope proteins P62 and El that associate as a heterodimer. The viral membrane-anchored surface glycoproteins are responsible for receptor recognition and entry into target cells through membrane fusion. The subgenomic RNA is transcribed from the p26S subgenomic promoter present at the 3’ end of the RNA sequence encoding the nsP4 protein. The proteolytic maturation of P62 into E2 and E3 causes a change in the viral surface. Together the El, E2, and sometimes E3, glycoprotein “spikes” form an E1ZE2 dimer or an E1 / E2ZE3 trimer, where E2 extends from the center to the vertices, El fills the space between the vertices, and E3, if present, is at the distal end of the spike. Upon exposure of the virus to the acidity of the endosome, El dissociates from E2 to form an El homotrimer, which is necessary for the fusion step to drive the cellular and viral membranes together. The alphaviral glycoprotein El is a class II viral fusion protein, which is structurally different from the class I fusion proteins found in influenza virus and HIV. The E2 glycoprotein functions to interact with the nucleocapsid through its cytoplasmic domain, while its ectodomain is responsible for binding a cellular receptor. Most alphaviruses lose the peripheral protein E3, while in Semliki viruses it remains associated with the viral surface.

[0065] Alphavirus replication has been reported to take place on membranous surfaces within the host cell. In the first step of the infectious cycle, the 5’ end of the genomic RNA is translated into a polyprotein (nsPl-4) with RNA polymerase activity that produces a negative strand complementary to the genomic RNA. In a second step, the negative strand is used as aAttorney Docket No.: 058462-523001WOtemplate for the production of two RNAs, respectively: (1) a positive genomic RNA corresponding to the genome of the secondary viruses producing, by translation, other nsP and acting as a genome for the virus; and (2) subgenomic RNA encoding the structural proteins of the virus forming the infectious particles. The positive genomic RNA / subgenomic RNA ratio is regulated by proteolytic autocleavage of the polyprotein to nsPl, nsP2, nsP3 and nsP4. In practice, the viral gene expression takes place in two phases. In a first phase, there is main synthesis of positive genomic strands and of negative strands. During the second phase, the synthesis of subgenomic RNA is virtually exclusive, thus resulting in the production of large amount of structural protein.Self-replicating RNA

[0066] As will be appreciated by the skilled artisan, the term “self-replicating RNA” (srRNA) refers to RNA molecule that contains all of the genetic information required for directing its own amplification or self-replication within a permissive cell. Therefore, srRNA is sometimes also referred to as “self-amplifying RNA” (saRNA). In some embodiments, the srRNA is a “replicon,” which can be a linear or circular section of DNA or RNA which replicates sequentially as a unit. Non-limiting examples of replicons include “replicon RNA” or “RNA replicon.” To direct its own replication, the srRNA generally (1) encodes polymerase, replicase, or other proteins which may interact with viral or host cell-derived proteins, nucleic acids or ribonucleoproteins to catalyze the RNA amplification process; and (2) contain cv.s-acting RNA sequences required for replication and transcription of the subgenomic RNA. These sequences may be bound during the process of replication to its self-encoded proteins, or non-self-encoded cell-derived proteins, nucleic acids or ribonucleoproteins, or complexes between any of these components.

[0067] In some embodiments of the disclosure, an alphavirus srRNA construct (e.g., srRNA, saRNA, or RNA replicon molecule) generally contains the following elements: 5' viral or defective-interfering RNA sequence(s) required in cis for replication, sequences coding for biologically active alphavirus non- structural proteins (e.g., nsPl, nsP2, nsP3, and nsP4), a subgenomic promoter (sg) for the subgenomic RNA (sgRNA), 3' viral sequences required in cis for replication, and optionally a polyadenylate tract (poly(A)). In some instances, a subgenomic promoter (sg) that directs expression of a heterologous sequence can be included in the srRNA construct of the disclosure.

[0068] Further, the term srRNA molecule (e.g., srRNA, saRNA, or RNA repliconAttorney Docket No.: 058462-523001WOmolecule) generally refers to a molecule of positive polarity, or “message” sense, and the srRNA may be of length different from that of any known, naturally-occurring alphavirus. In some embodiments of the present disclosure, the srRNA does not contain at least a portion (e.g., region, domain, fragment, or site) of the coding sequence for one or more of the alphavirus structural proteins; and / or sequences encoding structural genes can be substituted with heterologous sequences. In those instances, where the srRNA is to be packaged into a recombinant alphavirus particle, it can contain one or more sequences, so-called packaging signals, which serve to initiate interactions with alphavirus structural proteins that lead to particle formation.

[0069] The srRNA constructs of the disclosure generally have a length of at least about 2 kb. For example, the srRNA can have a length of at least about 2 kb, at least about 3 kb, at least about 4 kb, at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, at least about 11 kb, at least about 12 kb or more than 12 kb. In some embodiments, the srRNA can have a length of about 4 kb to about 20 kb, about 4 kb to about 18 kb, about 5 kb to about 16 kb, about 6 kb to about 14 kb, about 7 kb to about 12 kb, about 8 kb to about 16 kb, about 9 kb to about 14 kb, about 10 kb to about 18 kb, about 11 kb to about 16 kb, about 5 kb to about 18 kb, about 6 kb to about 20 kb, about 5 kb to about 10 kb, about 5 kb to about 8 kb, about 5 kb to about 7 kb, about 5 kb to about 6 kb, about 6 kb to about 12 kb, about 6 kb to about 11 kb, about 6 kb to about 10 kb, about 6 kb to about 9 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, about 7 kb to about 11 kb, about 7 kb to about 10 kb, about 7 kb to about 9 kb, about 7 kb to about 8 kb, about 8 kb to about 11 kb, about 8 kb to about 10 kb, about 8 kb to about 9 kb, about 9 kb to about 11 kb, about 9 kb to about 10 kb, or about 10 kb to about 11 kb. In some embodiments, the srRNA can have a length of about 6 kb to about 14 kb. In some embodiments, the srRNA can have a length of about 6 kb to about 16 kb.COMPOSITIONS OF THE DISCLOSURE

[0070] As described in greater detail below, one aspect of the present disclosure relates to nucleic acid constructs including a nucleic acid sequence encoding a modified viral genome or srRNA, wherein at least a portion of the nucleic acid sequence encoding the viral structural proteins of the modified alphavirus genome or srRNA has been replaced by a coding sequence for a polypeptide construct comprising one or more glucagon-like peptide-1 receptor (GLP-1R) agonists. Also provided are recombinant cells, cell cultures, andAttorney Docket No.: 058462-523001WOtransgenic animals that have been engineered to include (a) a nucleic acid construct and / or (b) a recombinant cell as disclosed herein. The present disclosure further provides compositions, e.g., pharmaceutical compositions, including one or more of the following: (a) a nucleic acid construct as described herein; and (b) a recombinant cell as described herein.A. Nucleic acid constructs

[0071] As described in greater detail below, one aspect of the present disclosure relates to novel nucleic acid constructs encoding an alphavirus genome or an srRNA as described herein. In some embodiments, the nucleic acid constructs include a nucleic acid sequence encoding a modified genome or srRNA of an alphavirus, wherein at least a portion of the nucleic acid sequence encoding the viral structural proteins of the modified alphavirus genome or srRNA has been replaced by a coding sequence for a polypeptide construct comprising one or more glucagon-like peptide-1 receptor (GLP-1R) agonists, or along with GCGR agonists, GIP, GIP receptor (GIPR) agonists, and / or GPCR agonists. In some embodiments, the agonists of the disclosure are polypeptide agonists which can be encoded by nucleic acid sequences. In some embodiments, a modified alphavirus genome or srRNA of the disclosure can include deletion(s), substitution(s), and / or insertion(s) in one or more of the genomic regions of the parent alphavirus genome. In some embodiments, the sequence encoding a srRNA of the disclosure can be operably linked, e.g., placed under the control of elements required for expression (e.g., promoter sequences), which allow expression of the srRNA in a host cell, in a subject, or in an ex-vivo cell-free expression system.

[0072] Non-limiting exemplary embodiments of the nucleic acid constructs, e.g., genomes and srRNA constructs, of the disclosure can include one or more of the following features. In some embodiments, the nucleic acid sequence further comprises coding sequences for one or more signal peptides that directs secretion of the one or more GLP-1R agonists, wherein the one or more signal peptides is heterologous relative to the one or more GLP-1R agonists, GPCR agonists, GIPR agonists, and / or GCGR agonists (e.g., the signal peptides are derived from heterologous sources).

[0073] Examples of GLP-1R agonists suitable for the compositions and methods of the disclosure include, but are not limited to, GLP-1 and its homologs, e.g., exendin-4, Exenatide, Liraglutide, Lixisenatide, Albiglutide, Dulaglutide, Semaglutide, Beinaglutide, Polyethylene glycol liraglutide, and derivatives / variants of any thereof. In some embodiments, the polypeptide construct includes coding sequences for at least two, at leastAttorney Docket No.: 058462-523001WOthree, at least four, at least five, at least six, or at least seven GLP-1R agonists or derivative thereof.

[0074] As discussed above, GLP-1R agonists exert central effects on appetite and food intake, while GPCR agonists drive increased energy expenditure in animal models and humans. The effects of GPCR agonist and GLP-1R agonists are believed to be synergistic in driving greater degrees of weight loss compared to a GLP-1R agonist alone. GPCR agonists also enhance lipolysis and suppress liver fat synthesis, providing an additional pathway for liver fat reduction and NASH resolution.

[0075] Examples of GPCR agonists suitable for the compositions and methods of the disclosure include, but are not limited to, GCG (1-29), GKP-1 (7-36 amid), GIP (1-42), N28D glucagon, IUB288, GCG CEX, GCG 104, G108, HM15136, GLP-l / GCG, and GLP- 1 / GIP / GCG. Additional information regarding suitable GPCR agonists can be found in, for example, Novikoff A. and Muller TD, Peptides, 2023, Jul; 165-171003, which is incorporated by reference herein in its entirety.

[0076] Non-limiting examples of GCGR agonists suitable for the compositions and methods of the disclosure include, but are not limited to those described in Coskun et al., Molecular Metabolism, Volume 18, December 2018, pp. 3-14 and Scheen AJ, Ann. Endocrinol. (Paris) 2023 Apr;84(2):316-321; both of which are incorporated by reference herein in their entireties.

[0077] In some embodiments, the polypeptide construct includes coding sequences for two, three, four, five, six, or seven GLP-1R agonists, GPCR agonists, GIPR agonists, and / or GCGR agonists, or a derivative of any thereof. In some embodiments, the polypeptide construct includes coding sequences for less than two e.g., one), less than three, less than four, less than five, less than six, or less than seven GLP-1R agonists, GPCR agonists, GIPR agonists, and / or GCGR agonists or a derivative of any thereof.

[0078] The polypeptide agonists have affinity for GLP-1R, GPCR, GIPR, and / or GCGR as may be determined using a cellular assay, wherein the polypeptides are conjugated to a nonionic glycolipid surfactant. The polypeptides may be selective for one receptor, or in certain embodiments have dual or tri-binding affinity properties for the respective receptors (e.g., GLP-1R, GPCR, GIPR, and / or GCGR).

[0079] Accordingly, dual and triple agonists of GLP-1R, GPCR , GIPR, and / or GCGR are also contemplated for use in the compositions and methods of the present disclosure. In some embodiments of the disclosure, the polypeptide construct includes coding sequences for two, three, four, five, six, or seven dual or triple agonists of GLP-1R agonists, GPCR agonists,Attorney Docket No.: 058462-523001WOGIPR agonists, and / or GCGR agonists, or a derivative of any thereof. In some embodiments, the polypeptide construct includes coding sequences for less than two (e.g., one), less than three, less than four, less than five, less than six, or less than seven dual or triple agonists of GLP-1R agonists, GPCR agonists, GIPR agonists, and / or GCGR agonists, or a derivative of any thereof.

[0080] Exemplary agonists of GLP-1R, GPCR, GIPR, and / or GCGR suitable for the compositions and methods of the disclosure include GGGtri-agonist, GIP / GLP co-agonist peptide, GIP / GLP co-agonist peptide II, C2816, a GLP-1 / cholecystokinin receptor-1 (CCK1) co-agonist, ZP3022, a GLP-1 / gastrin co-agonist, GLP-1 / xenin co-agonist, GIP / xenin co-agonist, GLP-1 / gastrin / xenin tri-agonist, NNC 9204-1177 (NN9277), LY3305677, JNJ-54728518, LY2944876 / TT-401, CPD86, LY3298176 (Tirzepatide), LY3437943, SAR438335, ZP-I-98, ZP-DI-70, HM15211, NN9423 / MAR423, PB-719, and DD01. More information regarding these agonists, including their amino acid sequences, can be found in PCT Publication No. WO2022178366A1 and WO2021168386A1, both of which are herein incorporated by reference.

[0081] As discussed above, the nucleic acid molecules and constructs of the disclosure can include one or more heterologous signal peptides that directs secretion of the one or more agonists of GLP-1R, GCGR, GIPR, and / or GPCR. In principle, there are no particular limitations to the signal peptides. Examples of heterologous signal peptides suitable for the compositions and methods of the disclosure include, but are not limited to signal peptides of human AACT, human FGF21, human FZD3, mouse IGKV3-10, human IGKV3-20, human INS, human INSR, human IPSP, human PORIM, and human SAMP.

[0082] In some embodiments, the one or more agonist polypeptides of GLP-1R, GCGR, GPCR, GIPR, and GIP are operably linked to one another within a single open reading frame (z.e., in a polycistronic ORF).

[0083] In some embodiments, the one or more agonist polypeptides of GLP-1R, GCGR, GPCR, GIPR, and GIP are operably linked to one another by one or more linkers.

[0084] In some embodiments, at least one of the one or more agonist polypeptides of GLP-1R, GCGR, GPCR, GIPR, and GIP comprises a structural modification to increase half-life. In some embodiments, the structural modification comprises one or more mutations, N-extension, and / or C-terminal extensions. In some embodiments, the structural modification comprises fusion to a Fc region of the IgG subclass of antibodies that lacks the IgG heavy chain variable region. Exemplary Fc regions can include a mutation that inhibits complementAttorney Docket No.: 058462-523001WOfixation and Fc receptor binding, or it may be lytic, i.e., able to bind complement or to lyse cells via another mechanism, such as antibody-dependent complement lysis.

[0085] The “Fc region” can be a naturally occurring or synthetic polypeptide that is homologous to the IgG C -terminal domain produced by digestion of IgG with papain. IgG Fc has a molecular weight of approximately 50 kDa. The agonist polypeptides can include the entire Fc region, or a smaller portion that retains the ability to extend the circulating half-life of a chimeric polypeptide of which it is a part. In addition, full-length or fragmented Fc regions can be variants of the wild-type molecule. That is, they can contain mutations that may or may not affect the function of the polypeptides; as described further below, native activity is not necessary or desired in all cases. In some embodiments, the agonist polypeptides as described herein include an IgGl, IgG2, IgG3, or IgG4 Fc region.

[0086] The Fc region can be “lytic” or “non-lytic,” but is typically non-lytic. A non-lytic Fc region typically lacks a high affinity Fc receptor binding site and a CTq binding site. The high affinity Fc receptor binding site of murine IgG Fc includes the Leu residue at position 235 of IgG Fc. Thus, the Fc receptor binding site can be destroyed by mutating or deleting Leu 235. For example, substitution of Glu for Leu 235 inhibits the ability of the Fc region to bind the high affinity Fc receptor. The murine CTq binding site can be functionally destroyed by mutating or deleting the Glu 318, Lys 320, and Lys 322 residues of IgG. For example, substitution of Ala residues for Glu 318, Lys 320, and Lys 322 renders IgGl Fc unable to direct antibody-dependent complement lysis. In contrast, a lytic IgG Fc region has a high affinity Fe receptor binding site and a C q binding site. The high affinity Fc receptor binding site includes the Leu residue at position 235 of IgG Fc, and the CT q binding site includes the Glu 318, Lys 320, and Lys 322 residues of IgGl. Lytic IgGFc has wild-type residues or conservative amino acid substitutions at these sites. Lytic IgG Fc can target cells for antibody dependent cellular cytotoxicity or complement directed cytolysis (CDC). Appropriate mutations for human IgG are also known (see, e.g., Morrison et al., The Immunologist 2: 119-124, 1994; and Brekke et al.. The Immunologist 2 '. 125, 1994).

[0087] Table 1 provides a list of exemplary nucleic acid constructs expressing a GLP-1R agonist with various heterologous signal peptides which were encoded into srRNA vectors capable of RNA replication in transfected BHK-21 cells in accordance with some embodiments of the disclosure. The amino acid sequences of these GLP-1R agonists, along with their corresponding coding sequences, are provided in the Sequence Listing.Attorney Docket No.: 058462-523001WOTABLE 1: Exemplary nucleic acid constructs expressing a GLP-1R agonist with various heterologous signal peptides. Fc: the fragment crystallizable region (Fc region) domain of immunoglobulin.

[0088] In some embodiments, the coding sequences for a polypeptide construct include the GLP-1R agonists and the configurations and / or ordinality described in Tables 1 and 2. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-25.

[0089] In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-25, wherein one, two, three, four, five, or more nucleotides of the aminoAttorney Docket No.: 058462-523001WOacid sequence may be substituted by a different amino acid.

[0090] As described above, in some embodiments of the disclosure, the nucleic acid constructs include a nucleic acid sequence encoding a modified genome or srRNA of an alphavirus, wherein at least a portion of the nucleic acid sequence encoding the viral structural proteins of the modified alphavirus genome or srRNA has been replaced by a coding sequence for a polypeptide construct comprising one or more GLP-1R agonists, alone or in combination with GCGR agonists, GIPR agonists, and / or GPCR agonists. In some embodiments, the modified alphaviral genome or srRNA is devoid of at least a portion of the nucleic acid sequence encoding one or more of the viral structural proteins CP, El, E2, E3, and 6K of the unmodified alphaviral genome or srRNA. In some embodiments, the modified viral genome or srRNA is devoid of a portion of or the entire sequence encoding CP. In some embodiments, the modified alphaviral genome or srRNA is devoid of a portion of or the entire sequence encoding El. In some embodiments, the modified alphaviral genome or srRNA is devoid of a portion of or the entire sequence encoding E2. In some embodiments, the modified alphaviral genome or srRNA is devoid of a portion of or the entire sequence encoding E3. In some embodiments, the modified alphaviral genome or srRNA is devoid of a portion of or the entire sequence encoding 6K. In some embodiments, the modified viral genome or srRNA is devoid of a portion of or the entire sequence encoding a combination of CP, El, E2, E3, and 6K. Accordingly, some embodiments of the disclosure provide a modified alphaviral genome or srRNA in which the coding sequence for non-structural proteins nsPl, nsP2, nsP3, and nsP4 of the unmodified alphaviral genome or srRNA is present, however at least a portion of or the entire sequence encoding one or more structural proteins (e.g., CP, El, E2, E3, and 6K) of the alphaviral genome or srRNA is absent.

[0091] In some embodiments, the modified viral genome or srRNA is devoid of a substantial portion of the nucleic acid sequence encoding one or more viral structural proteins. The skilled artisan will understand that a substantial portion of a nucleic acid sequence encoding a viral structural polypeptide can include enough of the nucleic acid sequence encoding the viral structural polypeptide to afford putative identification of that polypeptide, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as The Basic Local Alignment Search Tool (BLAST; publicly available at, for example, guides.lib.berkeley.edu / ncbi / blast and / / blast.ncbi. nlm.nih.gov / Blast.cgi). Accordingly, a substantial portion of a nucleotide sequence comprises enough of the sequence to affordAttorney Docket No.: 058462-523001WOspecific identification and / or isolation of a nucleic acid fragment comprising the sequence. For example, a substantial portion of a nucleic acid sequence can include at least about 20%, for example, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% of the full-length nucleic acid sequence. As described above, the present disclosure provides nucleic acid molecules and constructs which are devoid of partial or complete nucleic acid sequences encoding one or more viral structural proteins. The skilled artisan, having the benefit of the sequences as disclosed herein, can readily use all or a substantial portion of the disclosed sequences for the compositions and methods of the disclosure. Accordingly, the present application comprises the complete sequences as disclosed herein, e.g., those set forth in the accompanying Sequence Listing and Figures, as well as substantial portions of those sequences as defined above.

[0092] In some embodiments, modified viral genome or srRNA of the disclosure is devoid of at least a portion of the nucleic acid sequence encoding the viral capsid protein. In some embodiments, modified viral genome or srRNA of the disclosure is devoid of a substantial portion of the nucleic acid sequence encoding the viral capsid protein. In some embodiments, the modified viral genome or srRNA is devoid of the entire sequence encoding the viral capsid protein, e.g., the modified viral genome or srRNA includes no nucleic acid sequence encoding the viral capsid protein of the viral unmodified genome or srRNA.

[0093] In some embodiments, modified viral genome or srRNA of the disclosure is devoid of at least a portion of the nucleic acid sequence encoding one or more of the following viral structural proteins: El, E2, E3, and 6K. In some embodiments, modified viral genome or srRNA of the disclosure is devoid of a substantial portion of the nucleic acid sequence encoding one or more of the following viral structural proteins: El, E2, E3, and 6K. In some embodiments, the modified viral genome or srRNA is devoid of the entire sequence encoding one or more of the following viral structural proteins: El, E2, E3, and 6K, e.g., the modified viral genome or srRNA includes no nucleic acid sequence encoding one or more of the following viral structural proteins: El, E2, E3, and 6K of the viral unmodified genome or srRNA.

[0094] In some embodiments, the modified viral genome or srRNA is devoid of the entire sequence encoding viral structural proteins, e.g., the modified viral genome or srRNA includes no nucleic acid sequence encoding the structural proteins of the viral unmodified genome or srRNA.

[0095] In some embodiments, the nucleic acid constructs of the disclosure further includeAttorney Docket No.: 058462-523001WOone or more expression cassettes, e.g., for expression of a polypeptide construct of interest, e.g., a polypeptide construct comprising one or more agonist polypeptides (e.g., agonist polypeptides of GLP-1R, GCGR, GIP, and / or GPCR). In some embodiments, the polypeptide construct comprises one or more GLP-1R agonist polypeptides. In principle, the nucleic acid constructs disclosed herein can generally include any number of expression cassettes. In some embodiments, the nucleic acid constructs disclosed herein can include at least two, at least three, at least four, at least five, or at least six expression cassettes. The skilled artisan will understand that the term “expression cassette” refers to a construct of genetic material that contains coding sequences and enough regulatory information to direct proper transcription and / or translation of the coding sequences in a cell, in vivo and / or ex vivo. The expression cassette may be inserted into a vector for targeting to a desired host cell and / or into a subject. Accordingly, in some embodiments, the term expression cassette may be used interchangeably with the term “expression construct.” In some embodiments, the term "expression cassette" refers to a nucleic acid construct that includes a gene encoding a protein or functional RNA operably linked to regulatory elements such as, for example, a promoter and / or a termination signal, and optionally, any or a combination of other nucleic acid sequences that affect the transcription or translation of the gene.

[0096] In some embodiments, at least one of the expression cassettes includes a promoter operably linked to a heterologous nucleic acid sequence. In some embodiments, the heterologous nucleic acid sequence may encode a polypeptide construct of interest, e.g., a polypeptide construct comprising one or more agonist polypeptides (e.g., agonist polypeptides of GLP-1R, GCGR, GIPR, GIP, and / or GPCR). In some embodiments, the polypeptide construct comprises one or more GLP-1R agonist polypeptides. Accordingly, the nucleic acid constructs as provided herein can find use, for example, as an expression vector that, when including a regulatory element (e.g., a promoter) operably linked to a heterologous nucleic acid sequence, can affect expression of the heterologous nucleic acid sequence (e.g., heterologous nucleic acid sequence encoding a polypeptide construct comprising one or more agonist polypeptides, for example, agonist polypeptides of GLP-1R, GCGR, GIPR, GIP, and / or GPCR). Both naturally-occurring promoter sequences and synthetically-designed (and / or produced) promoter sequences are suitable. Accordingly, in some embodiments, the promoter is or comprises naturally occurring sequences isolated from or derived from a genomic sequence of a gene. In some embodiments, the promoter may be synthetically produced or designed by altering known DNA elements. In some embodiments, at least oneAttorney Docket No.: 058462-523001WOof the expression cassettes includes a subgenomic (sg) promoter operably linked to a heterologous nucleic acid sequence. In some embodiments, the sg promoter is a 26S subgenomic promoter.

[0097] In some embodiments, at least one nonstructural protein (nsP), or a portion thereof, of the modified alphaviral genome or srRNA is heterologous relative to the remainder of the modified alphaviral genome or srRNA. In some embodiments, the modified alphaviral genome or srRNA further includes a nucleic acid sequence encoding a heterologous nsP or a portion thereof. In some embodiments, the nucleic acid molecules of the disclosure further include one or more untranslated regions (UTRs). In some embodiments, at least one of the UTRs is a heterologous UTR relative to the remainder of the modified alphaviral genome or srRNA. In some embodiments, the nucleic acid molecules of the disclosure further include a heterologous 5’ UTR. In some embodiments, the nucleic acid molecules of the disclosure further include a heterologous 3’ UTR. In some embodiments, the nucleic acid molecules of the disclosure further include a native 5’ UTR. In some embodiments, the native or heterologous 5’ UTR is not modified (e.g., non-modified 5’ UTR). In some embodiments, the native or heterologous 5’ UTR has been further modified (e.g., modified 5’ UTR) for enhancing expression, translation and / or stability of a downstream polynucleotide sequence. Exemplary modifications suitable for the compositions and methods of the disclosure include nucleotide substitutions, deletions, and insertions. In some embodiments, the nucleic acid molecules of the disclosure further include a native 3’ UTR. In some embodiments, the native 3’ UTR is not modified (e.g., non-modified 3’ UTR). In some embodiments, the native 3’ UTR has been further modified (e.g., modified 5’ UTR) for enhancing expression, translation and / or stability of a polynucleotide sequence placed upstream of the 3’ UTR. For example, in some embodiments, the nucleic acid molecules of the disclosure can include a native 5’ UTR and a heterologous 3’ UTR. In some embodiments, the nucleic acid molecules of the disclosure can include a heterologous 5’ UTR and a native 3’ UTR. In some embodiments, both 5’ UTR and 3’ UTR are heterologous relative to the remainder of the modified alphaviral genome or srRNA.

[0098] In some embodiments, at least one of expression cassettes includes a coding sequences for a polypeptide construct of interest (PCI). In some embodiments, the coding sequence for the polypeptide construct of interest includes coding sequences for one or more genes of interest (GOI). In some embodiments, the polypeptide construct of interest includes a single agonist polypeptide sequence (e.g., single GLP-1R agonist polypeptide in aAttorney Docket No.: 058462-523001WOmonogenic PCI). In some embodiments, the coding sequence for the PCI includes coding sequences for a plurality of agonist polypeptides (e.g., agonists of GLP-1R, GCGR, GPCR, GIPR, and GIP in multigenic PCI (e.g., bigenic, trigenic, or tetragenic, etc.). In some embodiments, each of the coding sequences of the plurality of agonist polypeptides (e.g., agonists of GLP-1R, GCGR, GPCR, GIPR, and GIP) is operably linked to a separate promoter sequence. In some embodiments, the coding sequences of the plurality of agonist polypeptides e.g., agonists of GLP-1R, GCGR, GPCR, GIPR, and GIP) are operably linked to one another within a single open reading frame, e.g., in a polycistronic ORF (see, e.g., Examples 1 and 5, FIG. 1 and Table 2). In some embodiments, the coding sequences of the plurality of agonist polypeptides (e.g., agonists of GLP-1R, GCGR, GPCR, GIPR, and GIP) are configured into two or more ORFs that are connected to one another by a sequence for an internal ribosomal entry site (IRES) (see, e.g., Examples 1 and 5, FIG. 1 and Table 2, Rep-877, Rep-897, Rep-898, and Rep-899). In some embodiments, the coding sequence of the polycistronic ORF(s) is operably linked to a promoter sequence. In some embodiments, at least one of the promoter sequences is a subgenomic (sg) promoter. In some embodiments, the sg promoter is a 26S genomic promoter.

[0099] In some embodiments, the plurality of agonist polypeptides (e.g., agonists of GLP-1R, GCGR, GPCR, GIPR, and GIP) can be linked to one another directly or indirectly (e.g., via one or more connector sequences, for example via linkers). For example, in some embodiments, the plurality of GLP-1R agonist polypeptides can be directly linked to one another, e.g., adjacently to one another. In some embodiments, at least two (e.g., 2, 3, 4, or 5) of the plurality of GLP-1R agonist polypeptides are operably linked to one another by one or more connector sequences (e.g., linkers). In some embodiments, the length and amino acid composition of the connector sequences can be optimized to vary the orientation, flexibility, and / or proximity of the GLP-1R agonist polypeptides relative to one another to achieve a desired activity or property of the PCI. In some embodiments, a connector sequence of the plurality of connector sequences (e.g., linkers) includes one or more autoproteolytic peptide sequences. Non-limiting examples of autoproteolytic peptide sequences suitable for the methods and compositions of the disclosure include autoproteolytic cleavage sequences derived from calcium-dependent serine endoprotease (furin), porcine teschovirus-1 2 A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), Equine Rhinitis A Virus (ERAV) 2A (E2A), Thosea asigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), and Flacherie Virus 2A (BmIFV2A). In some embodiments, the polypeptide constructs of theAttorney Docket No.: 058462-523001WOdisclosure include one or more P2A autoproteolytic cleavage sequences. In some embodiments, at least two of the plurality of GLP-1R agonist polypeptides are operably linked to each other via a P2A autoproteolytic cleavage sequence.

[0100] In some embodiments, the coding sequences of the plurality of GLP-1R agonist polypeptides and their ordinality are configured as shown in FIG. 10.

[0101] In some embodiments, the coding sequences of the plurality of GLP-1R agonist polypeptides are operably linked to one another by one or more internal ribosomal entry sites (IRES). Non-limiting examples of IRES suitable for the methods and compositions of the disclosure include viral IRES sequences, cellular IRES sequences, and artificial IRES sequences. Examples of suitable IRES sequences include, but are not limited to, Kaposi’s sarcoma-associated herpesvirus (KSHV) IRES, hepatitis virus IRES, Pestivirus IRES, Cripavirus IRES, Rhopalosiphum padi virus IRES, fibroblast growth factor IRES, platelet-derived growth factor IRES, vascular endothelial growth factor IRES, insulin-like growth factor IRES, picomavirus IRES, encephalomyocarditis virus (EMCV) IRES, Pim-1 IRES, p53 IRES, Apaf-1 IRES, TDP2 IRES, L-myc IRES, and c-myc IRES. In some embodiments, the internal ribosomal entry site (IRES) is from encephalomyocarditis virus (EMCV).

[0102] In some embodiments of the methods described herein, the recombinant alphavirus srRNA is of a virus belonging to the Alphavirus genus of the Togaviridae family. In some embodiments of the disclosure, the modified alphavirus genome or srRNA is of an alphavirus belonging to the Venezuelan equine encephalitis virus / Eastem Equine Encephalitis virus (VEEV / EEEV) group, or the Semliki Forest virus (SFV) group, or the Sindbis virus (SINV) group. In some embodiments, the modified alphavirus genome or srRNA is of an alphavirus belonging to the BFV complex, EEEV complex, MIDV complex, NDUV, complex, SFV complex, VEEV complex, WEEV complex. In some embodiments, the alphavirus is Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Pixuna virus (PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), O’Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (B ABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu virus (NDUV), Madariaga virus (MADV), or Buggy Creek virus. In some embodiments, the alphavirus is VEEV, EEEV, CHIKV, or SINV. In some embodiments, theAttorney Docket No.: 058462-523001WOalphavirus is VEEV. In some embodiments, the alphavirus is EEEV. In some embodiments, the alphavirus is Western Equine Encephalitis virus (WEEV). In some embodiments, the alphavirus is CHIKV. In some embodiments, the alphavirus is SINV.

[0103] In some embodiments, the alphavirus is Chikungunya virus (CHIKV). Non-limiting examples of CHIKV strains suitable for the compositions and methods of the disclosure include CHIKV S27, CHIKV LR2006-OPY-1, CHIKV YO123223, CHIKV DRDE, CHIKV 37997, CHIKV 99653, CHIKV Ag41855, and Nagpur (India) 653496 strain. Virulent and avirulent CHIKV strains are both suitable. Additional examples of CHIKV strains suitable for the compositions and methods of the disclosure include but are not limited to those described in Afireen etal. Microbiol. Immunol. 2014, 58:688-696, Lanciotti and Lambert ASTMH 2016, 94(4):800-803 and Langsjoen et al. mBio. 2018, 9(2):e02449-17. In some embodiments, the modified CHIKV genome or replicon RNA (e.g., self-replicating RNA) is derived from CHIKV strain S27. In some embodiments, the modified CHIKV genome or replicon RNA is derived from CHIKV strain DRDE. In some embodiments, the modified CHIKV genome or replicon RNA is derived from CHIKV strain DRDE-06. In some embodiments, the modified CHIKV genome or replicon RNA is derived from CHIKV strain DRDE-07. In some embodiments, the modified CHIKV genome or replicon RNA is derived from CHIKV strain S27.

[0104] In some embodiments, the alphavirus is Eastern Equine Encephalitis virus (EEEV). Non-limiting examples of EEEV strains suitable for the compositions and methods of the disclosure include EEEV 792138, 783372, BeAn5122, BeAr300851, BeAr436087, C-49, FL91-4679, FL93-939, GML903836, MP-9, PE6, and V105-00210. Virulent and avirulent EEEV strains are both suitable. Additional suitable EEEV strains include, but are not limited to those described in the Virus Pathogen Resource website (ViPR; which is publicly available atwww.viprbrc.org / brc / vipr_genome_search. spg?method=SubmitForm&blockId=868&decorat or=toga). In some embodiments, the modified EEEV genome or replicon RNA (e.g., selfreplicating RNA) is derived from EEEV strain FL93-939.

[0105] In some embodiments, the alphavirus is Sindbis virus (SINV). In some embodiments, the modified genome or RNA replicon e.g., self-replicating RNA) is of a SINV strain. Non-limiting examples of SINV strains suitable for the compositions and methods of the disclosure include SINV strain AR339, AR86, and Girdwood. Examples of SINV strains suitable for the compositions and methods of the disclosure include, but are notAttorney Docket No.: 058462-523001WOlimited to those described in Sammels et al. J. Gen. Virol. 1999, 80(3):739-748, Lundstrbm and Pfeffer Vector Borne Zoonotic Dis. 2010, 10(9):889-907, Sigei et al. Arch, of Virol.2018, 163:2465-2469 and Ling et al. J. Virol. 2019, 93:e00620-19. Additional suitable SINV strains include, but are not limited to those described in the Virus Pathogen Resource website (ViPR; which is publicly available atwww.viprbrc.org / brc / vipr_genome_search. spg?method=SubmitForm&blockId=868&decorat or=toga). Virulent and avirulent SINV strains are both suitable. In some embodiments, the modified genome or RNA replicon is of a SINV strain Girdwood. In some embodiments, the modified genome or RNA replicon is of a SINV strain AR86. In some embodiments, the modified SINV genome or replicon RNA is derived from SINV strain Girdwood. In some embodiments, the modified SINV genome or replicon RNA is derived from SINV strain AR86. In some embodiments, the at least one heterologous nsP or portion thereof of the modified genome or RNA replicon is derived from a SINV strain AR86. In some embodiments, the at least one heterologous nsP or portion thereof is nsPl, nsP3, nsP4, or a portion of any thereof, or a combination of any of the foregoing. In some embodiments, the modified genome or RNA replicon is of a SINV strain AR86.

[0106] In some embodiments, the alphavirus is Western Equine Encephalitis virus (WEEV). Non-limiting examples of WEEV strains suitable for the compositions and methods of the disclosure include WEEV California, McMillan, IMP181, Imperial, Imperial 181, IMPR441, 71V-1658, AG80-646, BFS932, COA592, EP-6, E1416, BFS1703, BFS2005, BSF3060, BSF09997, CHLV53, KERN5547, 85452NM, Montana-64, S8-122, and TBT-235. Additional examples of WEEV strains suitable for the compositions and methods of the disclosure include 5614, 93A27, 93A30, 93A38, 93A79, B628(C1 15), CBA87, CNTR34, CO921356, Fleming, Lake43, PV012357A, PV02808A, PV72102, R02PV001807A, R02PV002957B, R02PV003422B, R05PV003422B, R0PV003814A and R0PV00384A. Virulent and avirulent WEEV strains are both suitable. Additional suitable WEEV strains include, but are not limited to those described in Bergren NA et al., J. Virol. 88(16): 9260-9267, August 2014, and in the Virus Pathogen Resource website (ViPR; which is publicly available athttps: / / www.viprbrc.org / brc / vipr_genome_search. spg?method=SubmitForm&blockId=57240 &decorator=toga). In some embodiments, the modified WEEV genome or srRNA is derived from WEEV strain Imperial. In some embodiments, the modified WEEV genome or srRNA is derived from WEEV strain McMillan.Attorney Docket No.: 058462-523001WO

[0107] In some embodiments, the alphavirus is Madariaga virus (MADV), formerly referred to as South American Eastern Equine Encephalitis virus (SA EEEV). Non-limiting examples of MADV strains suitable for the compositions and methods of the disclosure include ArgLL, ArgB, BeAn-5122, ArgM, 24443 (TR59), 25714 (BG60), BeAr 18205, 900188 (PA62), BeAr 81828, BeAr 126650, 68U231, 77U1104 (PE70), 75V1496, BeAr 300851, 75U40, and El Delirio (Arrigo NC etal., supra 2010). Additional examples of MADV strains suitable for the compositions and methods of the disclosure include 76V25343, 77U1 (BR77), BeAr348998, IVICPan57151, BeAn416361, 903836 (PA84), BeAr436087, 435731 (PA86), C49 (CO92), PE-0.0155-96 (0.0155), PE-3.0815-96 (3.0815), PE-16.0050-98 (16.0050), PE-18.0140-99 (18.0140), and PE-18.0172-99 (18.0172) (Arrigo NC et al., supra 2010). Additional suitable MADV strains include, but are not limited to those described in Arrigo NC et al., supra 2010, and in the Virus Pathogen Resource website (ViPR; which is publicly available atwww.viprbrc.org / brc / vipr_genome_search. spg?method=SubmitForm&blockId=868&decorat or=toga). In some embodiments, the modified MADV genome or srRNA is derived from MADV strain BeAr300851.TABLE 2: Exemplary srRNA-based vectors disclosed in the present disclosure. Signal peptides, number of agonists, and their ordinality in the respective expression cassettes are indicated. WEEV-McM: WEEV-McMillan. SINV-G: SINV-Girdwood. WEEV-I: WEEV-Imperial.Attorney Docket No.: 058462-523001WO

[0108] Table 2 above provides a list of exemplary nucleic acid constructs expressing a GLP-1R agonist (GLP-l-Fc), either alone or in combination with a GIPR agonist and / or a GCGR agonist in accordance with some embodiments of the disclosure. In each construct, the srRNA-based vector, the signal peptide, the number of agonists and their ordinality in the expression cassette are shown. While some constructs contain coding sequences for multiple polypeptides operably linked to one another within a single open reading frame (e.g., in a polycistronic ORF), others include coding sequences for multiple polypeptides configured into two ORFs that are connected to one another by a sequence for an internal ribosomal entry site (IRES). P2A: autoproteolytic peptide sequence from porcine teschovirus-1 2A. In Table 2, the heterologous signal peptides, the agonists, the autoproteolytic peptide P2A sequences, and internal ribosomal entry sites (IRES) are shown in N-terminus to C-terminus direction ( / .< ., in 5’ to 3’ direction) of the corresponding coding sequences. The amino acid sequences of these GLP-1R agonists, along with their corresponding coding sequences, are provided in the Sequence Listing.

[0109] In some embodiments, the coding sequences for multiple polypeptides are configured into two ORFs that are connected to one another by a sequence for an internal ribosomal entry site (IRES) (see, e.g., Rep-877, Rep-897, Rep-898, and Rep-899). In some embodiments, the first ORF includes, in 5’ to 3’ direction, coding sequences for the following: (msIgGK SP)-GIP-P2A-(PORIM SP)-GCG. In some embodiments, the second ORF includes, in 5’ to 3’ direction, coding sequences for the following: (msIgGK SP)-(GLP-Attorney Docket No.: 058462-523001WO1-Fc). In some embodiments, the second ORF includes, in 5’ to 3’ direction, coding sequences for the following: (INS SP)-(GLP-l-Fc).

[0110] In some embodiments, the coding sequences for multiple polypeptides are configured into two ORFs that are connected to one another by a sequence for an internal ribosomal entry site (IRES) wherein the first ORF includes, in 5’ to 3’ direction, coding sequences for the following: (msIgGK SP)-GIP-P2A-(PORIM SP)-GCG; and the second ORF includes, in 5’ to 3’ direction, coding sequences for the following: (msIgGK SP)-(GLP-1-Fc). (See, Rep-877 and Rep-898). In some embodiments, the coding sequences for multiple polypeptides are configured into two ORFs that are connected to one another by a sequence for an internal ribosomal entry site (IRES) wherein the first ORF encodes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24; and the second ORF comprises or is a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11.[OHl] In some embodiments, the coding sequences for multiple polypeptides are configured into two ORFs that are connected to one another by a sequence for an internal ribosomal entry site (IRES) wherein the first ORF includes, in 5’ to 3’ direction, coding sequences for the following: (msIgGK SP)-GIP-P2A-(PORIM SP)-GCG; and the second ORF includes, in 5’ to 3’ direction, coding sequences for the following: (INS SP)-(GLP-1-Fc). (See, Rep-897 and Rep-899). In some embodiments, the coding sequences for multiple polypeptides are configured into two ORFs that are connected to one another by a sequence for an internal ribosomal entry site (IRES) wherein the first ORF encodes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24; and the second ORF comprises or is a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 25.

[0112] In some embodiments, the srRNA constructs of the disclosure can include a 5’ cap. The term “5’ cap” as used herein refers to a structure found on the 5’ end of some eukaryotic RNAs, e.g., RNA transcripts, and generally includes a dinucleotide or a guanosine nucleotide connected to an RNA, e.g., mRNA, via a 5'- to 5'-triphosphate linkage (also referred to as Gppp or G(5')ppp(5')). In some embodiments, a guanosine nucleoside included in a 5' capAttorney Docket No.: 058462-523001WOmay be modified, for example, by methylation at one or more positions e.g., at the 7-position) on a base (guanine), and / or by methylation at one or more positions of a ribose. In some embodiments, a guanosine nucleoside included in a 5' cap includes a 2'0 methylation at a ribose (2'0MeG). In some embodiments, a guanosine nucleoside included in a 5' cap includes methylation at the 7-position of guanine (m7G). In some embodiments, a guanosine nucleoside included in a 5' cap includes methylation at the 7-position of guanine and a 2' O methylation at a ribose (m7(2'OMeG)).

[0113] Multiple distinct cap structures can be used to generate the 5' cap of in vitro transcribed srRNA. In some embodiments, providing an srRNA with a 5' cap disclosed herein or a 5' cap analog may be achieved by in vitro transcription, in which a 5' cap isco-transcriptionally expressed into an RNA strand, or may be attached to an RNApost-transcriptionally using capping enzymes. Accordingly, in some embodiments, 5' capping of srRNA can be performed co-transcriptionally with chemical cap analogs (i.e., capping during in vitro transcription). For example, CleanCap® technology provides high efficiency capping (90%+) in a co-transcriptional reaction using commercially available reagents with an AG initiator to provide a natural Cap 1 structure with a 2'-O-methyl group and N7 methyl on separate guanine components. As another example, the Anti-Reverse Cap Analog (ARCA) cap contains a 5 '-5 '-triphosphate guanine-guanine linkage where one guanine contains an N7 methyl group as well as a 2'-O-methyl group. Alternatively, in some embodiments, the srRNA of the disclosure may also be enzymatically capped post-transcriptionally. These may generate a more authentic 5' cap structure that more closely mimics, either structurally or functionally, the endogenous 5' cap which have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5' endonucleases and / or reduced 5' decapping. Numerous synthetic 5' cap analogs have been developed and are known in the art to enhance mRNA stability and translatability (see, e.g., Grudzien-Nogalska E. et al., Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, P. H. Ed), 2013). Accordingly, in some embodiments of the disclosure, the srRNA is a capped srRNA comprising a 5 ’-cap. In some embodiments, the 5 ’-cap is a cap-0, cap-1, cap-2, or cap analogue. In some embodiments, the capped srRNA is a co-transcriptionally capped srRNA. In some embodiments, the capped srRNA is an enzymatically capped srRNA.

[0114] Thus, in some embodiments, the srRNA of the disclosure is co-transcriptionally capped srRNA. Exemplary co-transcriptionally capped srRNAs include, without limitation,Attorney Docket No.: 058462-523001WOanti-reverse cap analogs (ARCAs) and CleanCap® srRNA.

[0115] In other embodiments, the srRNA of the disclosure is enzymatically capped srRNA. Exemplary capping enzymes include, without limitation, Vaccinia virus capping enzyme (VCE) and Faustovirus capping enzyme (FCE).

[0116] In some embodiments, the modified alphavirus genome or srRNA includes a poly(A) tail, and wherein: (a) the poly(A) tail does not comprise a 3’ non-A residue; and / or (b) the lengthened sequence encoding the poly(A) tail is longer than 34 residues. In some embodiments, the sequence encoding the modified alphavirus genome or srRNA further includes an additional restriction site incorporated at the end of the sequence encoding the poly(A) tail of the alphavirus genome or srRNA. In some embodiments, the lengthened poly(A) tail has a length ranging from about 30 to about 120 adenylate residues.

[0117] In some embodiments of the disclosure, the modified alphavirus genome or srRNA disclosed herein include a synthetic adaptor molecule configured for facilitating insertion of a heterologous sequence into the modified alphavirus genome or replicon RNA, and wherein the synthetic adaptor molecule having the Formula I:[ 5 ’flanking domain ]- [ restriction site ]n -[ 3 ’flanking domain ] F ormul a I

[0118] wherein a) n is an integer from 1 to 6; b) the restriction site is cleavable by a restriction endonuclease; and c) the 5’ flanking domain and 3’ flanking domain each include a nucleic acid sequence predicted to have minimal secondary structure.

[0119] In some embodiments, the 5’ flanking domain does not include a sequence which encodes an RNA sequence capable of forming a stem-loop structure. In some embodiments, the sequences of the 5’ flanking domain has a folding AG value of the minimum free energy (MFE) structure higher than a predefined threshold value. In some embodiments, the modified alphavirus genome or srRNA disclosed herein include an adaptor sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98, or at least 99% sequence identity to SEQ ID NO: 52. In some embodiments, the modified alphavirus genome or srRNA disclosed herein include an adaptor sequence having the sequence of SEQ ID NO: 52. In some embodiments, the modified alphavirus genome or srRNA disclosed herein include an adaptor sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98, or at least 99% sequence identity to SEQ ID NO: 53. In some embodiments, the modified alphavirus genome or srRNA disclosed herein include an adaptor sequence having the sequence of SEQ ID NO: 53.

[0120] In some embodiments, the modified alphavirus genome or srRNA of the disclosureAttorney Docket No.: 058462-523001WOincludes one or more modified nucleotides, e.g., modified nucleoside triphosphates (NTPs). Mammalian base-modifications may be used to enable linear mRNA and self-replicating RNA (srRNA) to evade detection by the immune system during delivery in a non-viral delivery system such as an LNP. On a molecular level these base-modifications alter the stability, base-pair accessibility, change hydrogen bonding and hydrophobicity, and may alter primary and secondary RNA structure such that RNA-protein interactions are either stabilized or inhibited. These RNA-RNA and RNA-protein interactions are essential for srRNA function and previously it has been shown that only certain base-modifications can be incorporated into srRNA and the ability to replicate and express encoded proteins remains viable. In some embodiments, the one or more modified nucleotides suppress interferon (IFN) response and maintain translation of the modified alphavirus genome or srRNA. In some embodiments, the one or more modified nucleotides confer innate immune evasion and robust long-term protein expression from the modified alphavirus genome or srRNA, which in turn enable prolonged expression at low dose. Additional information in this regard can be found in McGee JE et al., Nat Biotechnol. 2025 May;43(5):720-726; Epub 2024 Jul 8, which is incorporated by reference herein in its entirety.

[0121] In some embodiments, the coding sequence of the polypeptide construct of interest (PCI) can be redesigned and / or optimized for a desired property, such as increased stability, potency, and expression (e.g., translation efficiency), which in turns can maximize the impact of producing, delivering, and administering biotherapeutic. For example, in some embodiments, the coding sequence of the PCI is optimized for expression at a level higher than the expression level of a reference coding sequence. With respect to sequenceoptimization of nucleotide sequences, degeneracy of the genetic code provides the possibility to substitute at least one base of the protein encoding sequence of a gene with a different base without causing the amino acid sequence of the polypeptide produced from the gene to be changed. Hence, the nucleic acid constructs of the present disclosure may also have any base sequence that has been changed from any polynucleotide sequence disclosed herein by substitution in accordance with degeneracy of the genetic code. References describing codon usage are readily publicly available. In some embodiments, polynucleotide sequence variants can be produced for a variety of reasons, e.g., to optimize expression for a particular host (e.g., changing codon usage in the alphavirus mRNA to those preferred by other organisms such as human, non-human primates, hamster, mice, or monkey). Accordingly, in some embodiments, the coding sequence of the PCI is optimized for expression in a target host cellAttorney Docket No.: 058462-523001WOthrough the use of codons optimized for expression. The techniques for the construction of synthetic nucleic acid sequences encoding PCI using preferred codons optimal for host cell expression may be determined by computational methods analyzing the commonality of codon usage for encoding native proteins of the host cell genome and their relative abundance by techniques well known in the art. The codon usage database (www.kazusa.or.jp / codon) may be used for generation of codon optimized sequences in mammalian cell environments. Furthermore, a variety of software tools are available to convert sequences from one organism to the optimal codon usage for a different host organism such as the JCat Codon Optimization Tool (www.jcat.de), Integrated DNA Technologies (IDT) Codon Optimization Tool (www.idtdna.com / CodonOpt) or the Optimizer online codon optimization tool (genomes.urv.es / OPTIMIZER). Such synthetic sequences may be constructed by techniques known in the art for the construction of synthetic nucleic acid molecules and may be obtained from a variety of commercial vendors. Accordingly, in some embodiments, the coding sequence of the PCI is optimized for expression at a level higher than the expression level of a reference coding sequence, such as, for example, a coding sequence that has not been codon-optimized. In some embodiments, the codon-optimized sequence of the PCI results in an increased expression level by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% compared to a reference coding sequence that has not been codon-optimized. In some embodiments, the codon-optimized sequence of the PCI results in an increased expression level by at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold compared to a reference coding sequence that has not been codon-optimized.

[0122] In some embodiments, the coding sequence of the PCI is optimized for enhanced RNA stability and / or expression. The stability of RNA generally relates to the “half-life” of RNA. “Half-life” relates to the period of time which is needed to eliminate half of the activity, amount, or number of molecules. In the context of the present disclosure, the halflife of an RNA is indicative for the stability of said RNA. The half-life of RNA may influence the “duration of expression” of the RNA. Additional information regarding principles, strategies, and methods for use in enhancing RNA stability can be found in, for example, Leppek K. et al., Nat. Commun. 13, 1536 (2022).

[0123] In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected fromAttorney Docket No.: 058462-523001WOthe group consisting of SEQ ID NOS: 1-25. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 6. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10.

[0124] In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at leastAttorney Docket No.: 058462-523001WO99%, or 100% sequence identity to SEQ ID NO: 12. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 14. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 15. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 16. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 17. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20.

[0125] In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity SEQ ID NO: 21. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 22. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24. In someAttorney Docket No.: 058462-523001WOembodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 25.

[0126] In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 26-51. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 27. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30.

[0127] In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at leastAttorney Docket No.: 058462-523001WO97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 36. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 40.

[0128] In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 41. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 43. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 44. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at leastAttorney Docket No.: 058462-523001WO97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 45. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 47. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 48. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 49. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 50. In some embodiments, the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 51.

[0129] Nucleic acid sequences having a high degree of sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to a sequence selected from the group consisting of SEQ ID NOS: 26-51 can be identified and / or isolated by using the sequences identified herein (e.g., SEQ ID NOS: 26-51) or any others as they are known in the art, by viral genome sequence analysis, hybridization, and / or PCR with degenerate primers or gene-specific primers from sequences identified in the respective SEQ ID NOS.

[0130] The molecular techniques and methods by which these new nucleic acid constructs were assembled and characterized are described more fully in the Examples of the present application. In some embodiments, the nucleic acid molecules disclosed herein can be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning, etc. or chemical synthesis. Nucleic acid molecules as disclosed herein include natural nucleic acid molecules and homologs thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which one or more nucleotideAttorney Docket No.: 058462-523001WOresidues have been inserted, deleted, and / or substituted, in such a manner that such modifications provide the desired property in effecting a biological activity as described herein. Accordingly, in some embodiments, the nucleic acid molecules of the disclosure are recombinant nucleic acid molecules.

[0131] One skilled in the art will appreciate that nucleic acid molecules, including variants of naturally-occurring nucleic acid sequences, can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., In: Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1989)). The sequence of a nucleic acid molecule can be modified with respect to a naturally-occurring sequence from which it is derived using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as but not limited to site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, PCR amplification and / or mutagenesis of selected regions of a nucleic acid sequence, recombinational cloning, and chemical synthesis, including chemical synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules, and combinations thereof. Nucleic acid molecule homologs can be selected from a mixture of modified nucleic acid molecules by screening for the function of the protein or the srRNA encoded by the nucleic acid molecule and / or by hybridization with a wild-type gene or fragment thereof, or by PCR using primers having homology to a target or wild-type nucleic acid molecule or sequence.B. Recombinant cells

[0132] The nucleic acid constructs of the present disclosure can be introduced into a host cell or an animal to produce a recombinant cell or transgenic animal containing the nucleic acid molecule. Accordingly, prokaryotic or eukaryotic cells that contain a nucleic acid construct encoding a modified alphavirus genome or srRNA as described herein are also features of the disclosure. In a related aspect, some embodiments disclosed herein relate to introducing into a host cell, such as an animal cell, a nucleic acid construct as provided herein, and then selecting or screening for a transformed cell and / or transgenic animals. Introduction of the nucleic acid constructs of the disclosure into cells and / or animals can be achieved by methods known to those skilled in the art such as, for example, viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection,Attorney Docket No.: 058462-523001WOcalcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, direct micro-injection, nanoparticle-mediated nucleic acid delivery, and the like.

[0133] Accordingly, some embodiments of the disclosure relate to recombinant cells, for example, recombinant animal cells that include a nucleic acid construct described herein. The nucleic acid construct can be stably integrated in the host genome, or can be episomally replicating, or present in the recombinant host cell as a mini -circle expression vector for a stable or transient expression. Accordingly, in some embodiments of the disclosure, the nucleic acid construct is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid construct is stably integrated into the genome of the recombinant cell. Stable integration can be completed using classical random genomic recombination techniques or with more precise genome editing techniques such as using guide RNA directed CRISPR / Cas9 or TALEN genome editing. In some embodiments, the nucleic acid construct present in the recombinant host cell as a mini -circle expression vector for a stable or transient expression. In some embodiments, the recombinant cells and / or transgenic animals include an srRNA molecule as described herein, e.g., RNA molecule that contains all of the genetic information required for directing its own amplification or self-replication within a permissive cell and / or animal.

[0134] In some embodiments, the recombinant cell is a prokaryotic cell, such as the bacterium E. coli, or a eukaryotic cell, such as an insect cell (e.g., a mosquito cell or a Sf21 cell), or mammalian cells e.g., COS cells, NIH 3T3 cells, or HeLa cells). In some embodiments, the cell is in vivo. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in intro. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a vertebrate animal cell or an invertebrate animal cell. In some embodiments, the recombinant cell is a mammalian cell. Non-limiting examples of recombinant cells suitable for the methods and compositions of the disclosure include monkey kidney CV1 cells transformed by SV40 (e.g., COS-7 cells), human embryonic kidney cells (e.g., HEK 293 or HEK 293 cells) or derivative cells thereof (e.g., BHK-21 or BHK-570 cells), baby hamster kidney cells (BHK), mouse sertoli cells (e.g., TM4 cells), monkey kidney cells (e.g., CV1 cells), human cervical carcinoma cells (e.g., HeLa cells), canine kidney cells (MDCK cells), buffalo rat liver cells (e.g., BRL 3 A cells), human lung cell (e.g., W138 cells), human liver cell (e.g., Hep G2 cells), human fat cell or adipocyte, mouse fat cell or adipocyte mouseAttorney Docket No.: 058462-523001WOmammary tumor (e.g., MMT 060562 cells), TRI cells, FS4 cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cell (e.g., Vero cells), human A549 cells, human cervix cells, human CHME5 cells, human PER.C6 cells, NSO murine myeloma cells, human epidermoid larynx cells, human fibroblast cells, human HUH-7 cells, human MRC-5 cells, human muscle cells, human endothelial cells, human astrocyte cells, human macrophage cells, human RAW 264.7 cells, mouse 3T3 cells, mouse L929 cells, mouse connective tissue cells, mouse muscle cells, and rabbit kidney cells. In some embodiments, the recombinant cell is a cell derived from a cell described above (i.e., a derivative cell of an original cell described herein) such as, for example, a cell that is either expanded from a clone of the original cell, an engineered version of the original cell, or a reclassification of the original cell after it has undergone extensive passaging, or has been passaged through another host.

[0135] In some embodiments, the recombinant cell is an insect cell, e.g., cell of an insect cell line. In some embodiments, the recombinant insect cell is a Sf21 cell. Additional suitable insect cell lines include, but are not limited to, cell lines established from insect orders Diptera, Lepidoptera and Hemiptera, and can be derived from different tissue sources. In some embodiments, the recombinant cell is a cell of a lepidopteran insect cell line. In the past few decades, the availability of lepidopteran insect cell lines has increased at about 50 lines per decade. More information regarding available lepidopteran insect cell lines can be found in, e.g., Lynn D.E., Available lepidopteran insect cell lines. Methods Mol Biol.2007;388: 117-38, which is herein incorporated by reference. In some embodiments, the recombinant cell is a mosquito cell, e.g., a cell of mosquito species within Anopheles (An.), Culex (Cx.) and Aedes (Stegomyia) (Ae.) genera. Exemplary mosquito cell lines suitable for the compositions and methods described herein include cell lines from the following mosquito species: Aedes aegypti, Aedes albopictus, Aedes pseudoscutellaris, Aedes triseriatus, Aedes vexans, Anopheles gambiae, Anopheles stephensi, Anopheles albimanus, Culex quinquefasciatus, Culex theileri, Culex tritaeniorhynchus, Culex bitaeniorhynchus, and Toxorhynchites amboinensis. Suitable mosquito cell lines include, but are not limited to, CCL-125, Aag-2, RML-12, C6 / 26, C6 / 36, C7-10, AP-61, A t. GRIP-1, A t. GRIP-2, UM-AVE1, Mos.55, SualB, 4a-3B, Mos.43, MSQ43, and LSB-AA695BB. In some embodiments, the mosquito cell is a cell of a C6 / 26 cell line.

[0136] In another aspect, provided herein are cell cultures including at least one recombinant cell as disclosed herein, and a culture medium. Generally, the culture medium can be any suitable culture medium for culturing the cells described herein. Techniques forAttorney Docket No.: 058462-523001WOtransforming a wide variety of the above-mentioned host cells and species are known in the art and described in the technical and scientific literature. Accordingly, cell cultures including at least one recombinant cell as disclosed herein are also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.C. Transgenic animals

[0137] Also provided, in another aspect, are transgenic animals including a nucleic acid construct as described herein and / or a recombinant cell as disclosed herein. In some embodiments, the transgenic animal is a vertebrate animal or an invertebrate animal. In some embodiments, the transgenic animal is an insect. In some embodiments, the insect is a mosquito. In some embodiments, the transgenic animal is a mammalian. In some embodiments, the transgenic mammalian is a non-human mammalian. In some embodiments, the transgenic animal produces the polypeptide constructs, and / or GLP-1R agonists as described herein.

[0138] The transgenic non-human host animals of the disclosure are prepared using standard methods known in the art for introducing exogenous nucleic acid into the genome of a non-human animal. In some embodiments, the non-human animals of the disclosure are non-human primates. Other animal species suitable for the compositions and methods of the disclosure include animals that are (i) suitable for transgenesis and / or (ii) capable of rearranging immunoglobulin gene segments to produce an antibody response. Examples of such species include but are not limited to mice, rats, hamsters, rabbits, chickens, goats, pigs, sheep and cows. Approaches and methods for preparing transgenic non-human animals are known in the art. Exemplary methods include pronuclear microinjection, DNA microinjection, lentiviral vector mediated DNA transfer into early embryos and sperm-mediated transgenesis, adenovirus mediated introduction of DNA into animal sperm (e.g., in pig), retroviral vectors (e.g., avian species), somatic cell nuclear transfer e.g., in goats). The state of the art in the preparation of transgenic domestic farm animals is reviewed in Niemann, H. et al. (2005) Rev. Sci. Tech. 24:285-298.

[0139] In some embodiments, the animal is a vertebrate animal or an invertebrate animal. In some embodiments, the animal is an insect. In some embodiments, the insect is a mosquito. In some embodiments, the animal is a mammalian subject. In some embodiments, the mammalian animal is a non-human animal. In some embodiments, the mammalian animalAttorney Docket No.: 058462-523001WOis a non-human primate. In some embodiments, the transgenic animals of the disclosure can be made using classical random genomic recombination techniques or with more precise techniques such as guide RNA-directed CRISPR / Cas genome editing, or DNA-guided endonuclease genome editing with NgAgo (Natronobacterium gregoryi Argonaute), or TALENs genome editing (transcription activator-like effector nucleases). In some embodiments, the transgenic animals of the disclosure may be made using transgenic microinjection technology and do not require the use of homologous recombination technology and thus are considered to be easier to prepare and select than approaches using homologous recombination.D. Pharmaceutical compositions

[0140] The nucleic acid constructs and / or recombinant cells of the disclosure can be incorporated into compositions, including pharmaceutical compositions. Such compositions generally include one or more of the nucleic acid constructs and / or recombinant cells described and provided herein, and a pharmaceutically acceptable excipient, e.g., carrier. In some embodiments, the compositions of the disclosure are formulated for the prevention, treatment, or management of a health condition such as a metabolic disorder or disease associated with GLP-1R signaling (e.g., diabetes, obesity, hypertension, hyperlipidemia, a cardiovascular diseases, and musculoskeletal diseases). In some embodiments, the metabolic disorder is diabetes, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or obesity. In some embodiments, the diabetes is Type I diabetes, Type II diabetes, or gestational diabetes. In some embodiments, the diabetes is diabetes Type II. For example, the compositions of the disclosure can be formulated as a prophylactic composition, a therapeutic composition, or a pharmaceutical composition comprising a pharmaceutically acceptable excipient, or a mixture thereof that are useful for treating and / or preventing a metabolic disorder associated with GLP-1R signaling, such as obesity and obesity-related disorders.

[0141] Accordingly, in one aspect, provided herein are pharmaceutical compositions including a pharmaceutically acceptable excipient and: (a) a nucleic acid construct of the disclosure, and / or b) a recombinant cell of the disclosure.

[0142] Non-limiting exemplary embodiments of the pharmaceutical compositions of the disclosure can include one or more of the following features. In some embodiments, provided herein are compositions including a nucleic acid construct as disclosed herein and aAttorney Docket No.: 058462-523001WOpharmaceutically acceptable excipient. In some embodiments, provided herein are compositions including a recombinant cell as disclosed herein and a pharmaceutically acceptable excipient.

[0143] In some embodiments, the nucleic acid constructs of the disclosure (e.g., vectors or srRNA molecules) can be used in a naked form or formulated with a delivery vehicle.Exemplary delivery vehicles suitable for the compositions and methods of the disclosure include, but are not limited to exosomes, liposomes (e.g., neutral or anionic liposomes), microspheres, immune stimulating complexes (ISCOMS), lipid-based nanoparticles (LNP), solid lipid nanoparticles (SLN), polyplexes, polymer nanoparticles, viral replicon particles (VRP), or conjugated with bioactive ligands, which can facilitate delivery. These compounds are readily available to one skilled in the art; for example, see Liposomes: A Practical Approach, RCP New Ed, IRL Press (1990) and Mendes B.B. et al. Nanodelivery of nucleic acids, Nat. Rev. Methods Primers 2, 24, (2022). Accordingly, in some embodiments of the disclosure, the compositions of the disclosure that formulated in a liposome. Liposomes are enclosed nanoparticles with a bilayer membrane which show excellent performance in delivery of nucleic acid drugs (e.g., DNAs and RNAs such as srRNA) as well as different drug types. More information in this regard can be found in, for example, Hsairat H. et al., OpenNano, Vol. 11, May 2023, and Gao Y. et al. Pharmaceutics, 15(1): 178, Jan 2023.Adjuvants other than liposomes and the like are also used and are known in the art. A skilled artisan will appreciate that adjuvants may protect the antigen (e.g., nucleic acid constructs, vectors, srRNA molecules) from rapid dispersal by sequestering it in a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system.

[0144] The composition of the disclosure can be formulated in a format to be compatible with its intended route of administration, such as an exosome, liposome, lipid-based nanoparticle (LNP), polymer nanoparticle, polyplex, viral replicon particle (VRP), microsphere, immune stimulating complex (ISCOM), conjugate of bioactive ligand, or a combination of any thereof. For example, in some embodiments, the compositions of the disclosure that formulated in a liposome. In some embodiments, the compositions of the disclosure that formulated in a lipid-based nanoparticle (LNP). In some embodiments, the compositions of the disclosure that formulated in a polymer nanoparticle.

[0145] In some embodiments, the srRNA-based pharmaceutical compositions disclosed herein are administered therapeutically, e.g., for use in treating a subject who has, who isAttomey Docket No.: 058462-523001WOsuspected of having, or who may be at high risk for developing one or more relevant health conditions, disorders, or diseases associated with GLP-1R signaling. In some embodiments, the pharmaceutical compositions of the present disclosure can be administered prophylactically to prevent one or more relevant health conditions.

[0146] As described in greater detail below, in some embodiments of the disclosure, the compositions as disclosed herein can be formulated with a delivery vehicle into a delivery system, wherein the delivery system can include one or more of the following: a physiologic buffer, a liposome, a viral replicon particle (VRP), a lipid-based nanoparticle (LNP), a polymer nanoparticle, a physiologic buffer, a microsphere, an immune stimulating complex (ISCOM), a conjugate of bioactive ligand, and a combination of any thereof.

[0147] In some embodiments, the nucleic acid constructs of the disclosure can be delivered to a cell or a subject by a lipid-based nanoparticle (LNP). LNP are generally less immunogenic than viral particles. While many humans have preexisting immunity to viral particles there is no pre-existing immunity to LNP. In addition, adaptive immune response against LNP is unlikely to occur which enables repeat dosing of LNP.

[0148] The lipids suitable for the compositions and methods described herein can be cationic lipids, ionizable cationic lipids, anionic lipids, or neutral lipids.

[0149] In some embodiments, the LNP of the disclosure can include one or more ionizable lipids. As used herein, the term "ionizable lipid" refers to a lipid that is cationic or becomes ionizable (protonated) as the pH is lowered below the pKa of the ionizable group of the lipid, but is more neutral at higher pH values. At pH values below the pKa, the lipid is then able to associate with negatively charged nucleic acids (e.g., oligonucleotides). As used herein, the term "ionizable lipid" includes lipids that assume a positive charge on pH decrease from physiological pH, and any of a number of lipid species that carry a net positive charge at a selective pH, such as physiological pH. Permanently cationic lipids such as DOTMA have proven too toxic for clinical use. The ionizable lipid can be present in lipid formulations according to other embodiments, preferably in a ratio of about 30 to about 70 Mol%, in some embodiments, about 30 Mol%, in other embodiments, about 40 Mol%, in other embodiments, about 45 Mol% in other embodiments, about 47.5 Mol% in other embodiments, about 50 Mol%, in still other embodiments, and about 60 Mol% in yet others (“Mol%” means the percentage of the total moles that is of a particular component). The term “about” in this paragraph signifies a plus or minus range of 5 Mol%. DODMA, or 1 ,2-dioleyloxy-3-dimethylaminopropane, is an ionizable lipid, as is DLin-MC3-DMA or 0-(Z,Z,Z,Z-Attorney Docket No.: 058462-523001WOheptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N,N- dimethylamino) (“MC3”).

[0150] In some embodiments, the LNP of the disclosure can include one or more ionizable cationic lipids. Several suitable ionizable cationic lipids have been developed for use in manufacturing of LNP. These include Cl 2-200, MC3, LN16, and MD1 among others. Any of these cationic lipids can be used to formulate LNP for delivery of the nucleic acid constructs of the disclosure. For example, in one type of LNP of the disclosure, a GalNAc moiety can be attached to the outside of the LNP and acts as a ligand for uptake into the liver via the asialoglycoprotein receptor.

[0151] In some embodiments, LNPs can be manufactured from cationic, anionic, or neutral lipids. Neutral lipids, such as the fusogenic phospholipid DOPE or the membrane component cholesterol, can be included in LNPs as 'helper lipids' to enhance transfection activity and nanoparticle stability. This is because in some instances, limitations of cationic lipids include low efficacy owing to poor stability and rapid clearance, as well as the generation of inflammatory or anti-inflammatory responses. LNPs can also have hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.

[0152] In some embodiments, the lipids or combination of lipids that are known in the art can be suitably used to produce LNPs include DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Non-limiting examples of suitable cationic lipids include 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Nonlimiting examples of suitable neutral lipids include DPSC, DPPC, POPC, DOPE, and SM. Non-limiting examples of suitable PEG-modified lipids include PEG-DMG, PEG-CerC14, and PEG-CerC20.

[0153] In some embodiments, the lipids can be combined in any number of molar ratios to produce LNPs of the disclosure. In addition, the polynucleotide(s) can be combined with lipid(s) in a wide range of molar ratios to produce LNPs of the disclosure. For example, in some embodiments, the lipid of the LNP delivery system is present in mass ratio of lipid to RNA from about 100:1 to about 4:1. In some embodiments, the delivery system includes lipid-based nanoparticles having an average diameter of less than 1000 nm, less than 500 nm, less than 250 nm, less than 200 nm, less than 150 nm, less than 100 nm, less than 75 nm, less than 50 nm, or less than 25 nm. In some embodiments, the LNP delivery system includes lipid-based nanoparticles having an average diameter of about 1-1000 nm, about 1-1000 nm, about 1-500 nm, about 1-250 nm, about 25-200 nm, about 25-100 nm, about 35-75 nm, orAttorney Docket No.: 058462-523001WOabout 25-60 nm.

[0154] In some embodiments, the pharmaceutical compositions are formulated for one or more of the following administration routes: intranasal administration, transdermal administration, intrathecal administration, intraperitoneal administration, intramuscular administration, intratracheal administration, intranodal administration, intratumoral administration, intraarticular administration, intravenous administration, subcutaneous administration, intravaginal administration, intraocular administration, rectal administration, and oral administration. In some particular embodiments of the disclosure, the composition is formulated intramuscular administration.

[0155] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™. (BASF, Parsippany, N. J.), or phosphate buffered saline (PBS). In these cases, the composition should be sterile and should be fluid to the extent that easy syringeability and / or injectability exists. It can be stable under the conditions of manufacture and storage, and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. In the compositions of the disclosure, the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, e.g., sodium dodecyl sulfate. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be generally to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and / or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0156] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.Attorney Docket No.: 058462-523001WO

[0157] In some embodiments, the pharmaceutical compositions of the disclosure, e.g., vaccine compositions, are formulated for inhalation, such as an aerosol, spray, mist, liquid, or powder. Administration by inhalation may be in the form of either dry powders or aerosol formulations, which are inhaled by a subject (e.g., a patient) either through use of an inhalation device, e.g., a microspray, a pressurized metered dose inhaler, or nebulizer.

[0158] In some embodiments, the composition is formulated for one or more of the following administration routes: intranasal administration, intrathecal administration, transdermal administration, intramuscular administration, intranodal administration, intravenous administration, intraperitoneal administration, oral administration, intravaginal, and intra-cranial administration. In some embodiments, the administered composition results in in slowing of gastric emptying, reducing the release of glucagon, decreasing blood glucose, enhancing insulin sensitivity, and / or stimulating insulin production in the subject.METHODS OF THE DISCLOSURE

[0159] Administration of any one of the therapeutic compositions described herein, e.g., nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions, can be used in the treatment of relevant health conditions associated with GLP-1R signaling, such as diabetes, obesity, hypertension, hyperlipidemia, a cardiovascular diseases, and musculoskeletal diseases. In some embodiments, the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions as described herein can be useful for modulating, e.g., eliciting or suppressing a pharmacodynamic effect in a subject. Non-limiting examples of pharmacodynamic effect include immunogenicity effects, biomarker responses, therapeutic effects, prophylactic effects, desired effects, undesired effects, adverse effects, and effects in a disease or disease model. Accordingly, one aspect of the disclosure relates to methods for modulating a pharmacodynamic effect in a subject in need thereof, the methods include administering to the subject a composition including one or more of the following: (a) a nucleic acid construct as described herein (for example a replicon, e.g., self-replicating RNA construct), (b) a recombinant cell as described herein; and (c) a pharmaceutical composition as described herein. In some embodiments, the pharmacodynamic effect includes one or more of the following: immunogenicity effect, a biomarker response, a therapeutic effect, a prophylactic effect, a desired effect, an undesired effect, an adverse effect, and effect in a disease model.

[0160] In some embodiments, the nucleic acid constructs, recombinant cells, and / orAttorney Docket No.: 058462-523001WOpharmaceutical compositions as described herein can be incorporated into therapeutic agents for use in methods of treating a subject who has, who is suspected of having, or who may be at high risk for developing one or more relevant health conditions, disorders, or diseases associated with GLP-1R signaling (e.g., diabetes, obesity, hypertension, hyperlipidemia, a cardiovascular diseases, and musculoskeletal diseases). Accordingly, in another aspect, provided herein are methods for preventing or treating a health condition in a subject, the methods include prophylactically or therapeutically administering to the subject a composition including one or more of the following: (a) a nucleic acid construct as described herein (for example a replicon, e.g., self-replicating RNA construct; (b) a recombinant cell as described herein; and (c) a pharmaceutical composition as described herein. In some embodiments, the administered composition results in slowing of gastric emptying, reducing the release of glucagon, decreasing blood glucose, enhancing insulin sensitivity, and / or stimulating insulin production in the subject. In some embodiments, the subject has been previously treated with one or more therapies and has developed at least a partial resistance to said one or more therapies.

[0161] Non-limiting examples of health conditions and diseases associated with GLP-1R signaling that can be suitably treated or prevented in the methods of the disclosure include Type I diabetes (T1D), Type I diabetes (T2D), gestational diabetes, pre-diabetes, idiopathic T1D (Type lb), LADA (latent autoimmune diabetes in adults), EOD (early-onset T2DM), YOAD (youth-onset atypical diabetes), MODY (maturity onset diabetes of the young), malnutrition-related diabetes, hyperglycemia, insulin resistance, hepatic insulin resistance, impaired glucose tolerance, diabetic neuropathy, diabetic nephropathy, kidney disease, diabetic retinopathy, adipocyte dysfunction, visceral adipose deposition, sleep apnea, obesity, eating disorders, weight gain from use of other agents, excessive sugar craving, dyslipidemia, hyperinsulinemia, non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH).

[0162] Additional health conditions associated with GLP-1R signaling that can be suitably treated or prevented in the methods of the disclosure include fibrosis, cirrhosis, hepatocellular carcinoma, cardiovascular disease, atherosclerosis, coronary artery disease, peripheral vascular disease, hypertension, endothelial dysfunction, impaired vascular compliance, congestive heart failure, myocardial infarction, stroke, hemorrhagic stroke, ischemic stroke, traumatic brain injury, pulmonary hypertension, restenosis after angioplasty, intermittent claudication, post-prandial lipemia, metabolic acidosis, ketosis, arthritis, osteoporosis,Attorney Docket No.: 058462-523001WOParkinson's Disease, left ventricular hypertrophy, peripheral arterial disease, macular degeneration, cataract, glomerulosclerosis, chronic renal failure, metabolic syndrome, syndrome X, premenstrual syndrome, angina pectoris, thrombosis, atherosclerosis, transient ischemic attacks, vascular restenosis, impaired glucose metabolism, conditions of impaired fasting plasma glucose, hyperuricemia, gout, erectile dysfunction, skin and connective tissue disorders, psoriasis, foot ulcerations, ulcerative colitis, hyper apo B lipoproteinemia, Alzheimer's Disease, schizophrenia, impaired cognition, inflammatory bowel disease, short bowel syndrome Crohn's disease, colitis, and irritable bowel syndrome.

[0163] In some embodiments, the health condition is health condition, disorder, or disease associated with GLP-1R signaling, such as diabetes, obesity, hypertension, hyperlipidemia, a metabolic disorder, a cardiovascular disease, or a musculoskeletal disease. In some embodiments, the metabolic disorder is diabetes, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or obesity. In some embodiments, the diabetes is Type I diabetes, Type II diabetes, or gestational diabetes. In some embodiments, the diabetes is diabetes Type II.

[0164] In some embodiments, the disclosed composition is formulated to be compatible with its intended route of administration. For example, the nucleic acid constructs (e.g., srRNA constructs), recombinant cells, and / or pharmaceutical compositions of the disclosure may be given orally or by inhalation, but it is more likely that they will be administered through a parenteral route. Examples of parenteral routes of administration include, for example, intramuscular, intratumoral, intraocular, intravenous, intranodal, intradermal, subcutaneous, transdermal (topical), transmucosal, intravaginal, and rectal administration. In some embodiments, the composition is administered intramuscularly. In some embodiments, the composition is administered intratum orally. Solutions or suspensions used for parenteral application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, phosphates, tris, sucrose and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as mono- and / or di-basic sodium phosphate, hydrochloric acid or sodium hydroxide (e.g., to a pH of about 7.2-7.8, e.g., 7.5). The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple doseAttorney Docket No.: 058462-523001WOvials made of glass or plastic.

[0165] Dosage, toxicity and therapeutic efficacy of such subject nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions of the disclosure can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are generally suitable. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0166] For example, the data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (e.g., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0167] The therapeutic compositions described herein, e.g., nucleic acid constructs, e.g., srRNA constructs, recombinant cells, and / or pharmaceutical compositions, can be administered one or more times. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the pharmaceutical compositions of the disclosure can include a single treatment or can include a series of treatments. In some embodiments, the compositions are administered one or more times, followed by a rest period of 2 to 56 days, e.g., 28 days, followed by additional administration every 2-12 months. Repeated administration can be performed every 1-10 years. With regard to nucleic acid constructs (e.g., srRNA constructs),Attorney Docket No.: 058462-523001WOthe therapeutically effective amount of a nucleic acid construct of the disclosure (e.g., an effective dosage) depends on the nucleic acid construct selected. For instance, single dose amounts in the range of approximately 0.001 to 1 mg / kg of patient body weight can be administered. In some embodiments, about 0.05, 0.1, 0.55 mg / kg may be administered. In some embodiments, single dose amounts in the range of approximately 0.001 pg to 500 pg / kg of patient body weight can be administered. In some embodiments, single dose amounts in the range of approximately 0.3 mg to 3 mg / kg of patient body weight can be administered.

[0168] As discussed supra, a therapeutically effective amount includes an amount of a therapeutic composition that is sufficient to promote a particular effect when administered to a subject, such as one who has, is suspected of having, or is at risk for a relevant health condition, e.g., diabetes or obesity. In some embodiments, an effective amount includes an amount sufficient to prevent or delay the development of a symptom of the disease or health condition, alter the course of a symptom of the disease or health condition (for example but not limited to, slow the progression of a symptom of the disease or health condition), or reverse a symptom of the disease or health condition. It is understood that for any given case, an appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation.

[0169] The efficacy of a treatment including a disclosed therapeutic composition for the treatment of disease or health condition can be determined by the skilled clinician. However, a treatment is considered effective treatment if at least any one or all of the signs or symptoms of disease or health condition are improved or ameliorated. Efficacy can also be measured by failure of an individual to worsen as assessed by hospitalization or need for medical interventions (e.g., progression of the disease or health condition is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease or health condition in a subject or an animal (some non-limiting examples include a human, or a mammal) and includes: (1) inhibiting the disease, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.

[0170] As described above, pharmaceutically acceptable carriers suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In these cases, the composition must be sterile and must be fluid to the extent that easy syringeability exists.Attorney Docket No.: 058462-523001WOThe composition must further be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, efc.), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.

[0171] Sterile injectable solutions can be prepared by incorporating the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

[0172] When the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions are suitably protected, as described above, they may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the individual's diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

[0173] As discussed supra, in some embodiments of the disclosure, the compositions as disclosed herein, e.g., nucleic acid constructs of the disclosure, can be formulated with a delivery vehicle into a delivery system, wherein the delivery system can include one or more of the following: a physiologic buffer, a liposome, a viral replicon particle (VRP), a lipid-based nanoparticle (LNP), a polymer nanoparticle, a physiologic buffer, a microsphere, an immune stimulating complex (ISCOM), a conjugate of bioactive ligand, and a combination of any thereof.

[0174] In some embodiments, the nucleic acid constructs of the disclosure can be delivered to a cell or a subject by a lipid-based nanoparticle (LNP). LNP are generally less immunogenic than viral particles. While many humans have preexisting immunity to viral particles there is no pre-existing immunity to LNP. In addition, adaptive immune response against LNP is unlikely to occur which enables repeat dosing of LNP.Attorney Docket No.: 058462-523001WO

[0175] In some embodiments, the therapeutic compositions described herein, e.g., nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions are incorporated into therapeutic compositions for use in methods of preventing or treating a subject who has, who is suspected of having, or who may be at high risk for developing a health condition, disorder, or disease associated with GLP-1R signaling, such as diabetes, obesity, hypertension, hyperlipidemia, a metabolic disorder, a cardiovascular disease, or a musculoskeletal disease. In some embodiments, the metabolic disorder is diabetes, nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or obesity. In some embodiments, the diabetes is Type I diabetes, Type II diabetes, or gestational diabetes. In some embodiments, the diabetes is diabetes Type II. In some embodiments, the subject is a patient under the care of a physician.Additional therapies

[0176] In some embodiments, a pharmaceutical composition according to the present disclosure can be administered to the subject individually as the sole active agent, e.g., in a single therapy (monotherapy) or as a first therapy in combination with at least one additional therapies (e.g., second therapy). In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, targeted therapy, and surgery. In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, or surgery.

[0177] In some embodiments, the one or more additional therapeutic agents are selected from antidiabetic agents, anti-obesity agents (including lipid-lowering agents and pro-satiety agents), anti-atherosclerotic agents, anti-inflammatory agents, antioxidants, antifibrotic agents, anti-hypertensive agents, and combinations thereof. Antidiabetic agents include without limitation: AMP-activated protein kinase (AMPK) agonists, including biguanides (e. g., buformin and metformin); peroxisome proliferator-activated receptor gamma (PPARy) agonists, including thiazolidinediones (e.g., balaglitazone, ciglitazone, darglitazone, englitazone, lobeglitazone, netoglitazone, pioglitazone, rivoglitazone, rosiglitazone and troglitazone), MSDC- 0602K and saroglitazar (dual PPAR-a / g agonist); non-polypeptide GLP-1R agonists, including taspoglutide, CNT0736, CNT03649, HM11260C (LAPS-Exendin), NN9926 (OG9S7GT), TT401 and ZYOG1; dipeptidyl peptidase 4 (DPP -4) inhibitors, including alogliptin, anagliptin, dutogliptin, evogliptin, gemigliptin, gosogliptin,Attorney Docket No.: 058462-523001WOlinagliptin, omarigliptin, saxagliptin, septagliptin, sitagliptin, teneligliptin, trelagliptin and vildagliptin; sodium-glucose transport protein 2 (SGLT2) inhibitors, including canagliflozin (also inhibits SGLT1), dapagliflozin, empagliflozin, ertugliflozin, ipragliflozin, luseogliflozin, remogliflozin etabonate, sotagliflozin (also inhibits SGLT1) and tofogliflozin; blockers of ATP-dependent K+ (KATP) channels on pancreatic beta cells, including meglitinides (e.g., mitiglinide, nateglinide and repagiinide) and sulfonylureas (including first generation (e.g., acetohexamide, carbutamide, chlorpropamide, giycyclamide [tolhexamide], metahexamide, tolazamide and tolbutamide) and second generation (e.g., glibenclamide, glyburide, glibomuride, gliclazide, glimepiride, glipizide, gliquidone, glisoxepide and glyclopyramide); insulin and analogs thereof, including fast-acting insulin (e.g., insulin aspari insulin glulisine and insulin lispro), intermediate-acting insulin (e.g., NPH insulin), and long-acting insulin e.g., insulin degludec, insulin detemir and insulin glargine); and / or, analogs, derivatives and salts thereof.

[0178] In some embodiments, the antidiabetic agent is or includes a biguanide e.g., metformin), a thiazolidinedione (e.g., pioglitazone or rosiglitazone) or a SGLT2 inhibitor (e.g., empagliflozin or tofogliflozin), or any combination thereof. Anti-obesity agents include, but are not limited to: appetite suppressants (anorectics), including amphetamine, dexamphetamine, amfepramone, clobenzorex, mazindol, phentermine (with or without topiramate) and lorcaserin; pro-satiety agents, including ciliary neurotrophic factor (e.g., axokine) and longer-acting analogs of amylin, calcitonin, cholecystokinin (CCK), GLP-1, leptin, oxyntomodulin, pancreatic polypeptide (PP), peptide YY (PYY) and neuropeptide Y (NPY); lipase inhibitors, including caulerpenyne, cetilistat, ebelactone A and B, esterastin, lipstatin, orlistat, percyquinin, panclicin A-E, valilactone and vibralactone; antihyperlipidemic agents; and analogs, derivatives and salts thereof. Antihyperlipidemic agents include without limitation: HMG-CoA reductase inhibitors, including statins (e.g., atorvastatin, cerivastatin, fluvastatin, mevastatin, monacolins (e.g., monacolin K (lovastatin), pitavastatin, pravastatin, rosuvastatin and simvastatin} and flavanones (e.g., naringenin); squalene synthase inhibitors, including lapaquistat, zaragozic acid and RPR-107393; acetyl-CoA carboxylase (ACC) inhibitors, including anthocyanins, avenaciolides, chloroacetylated biotin, cyclodim, diclofop, haloxyfop, soraphens (e.g., soraphen Aia), 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA), CP-640186, GS-0976, NDI-010976; 7-(4-propyl oxy-phenylethynyl)-3, 3 -dimethyl-3, 4dihydro-2H- benzo[b][l,4]dioxepine; N-ethyl-N’-(3-{ [4-(3, 3-dimethyl -l-oxo-2-oxa-7-azaspiro[4.5]dec-7- yl)piperidin-l-yl]-carbonyl}-l-benzothien-Attorney Docket No.: 058462-523001WO2-yl)urea; 5-(3-acetamidobut-l-ynyl)-2-(4- propyl oxyphenoxy )thiazole; and l-(3-{[4-(3,3-dimethyl-l-oxo-2-oxa-7-azaspiro[4.5]dec-7- yl)piperidin-l-yl]-carbonyl}-5-(pyridin-2-yl)-2-thienyl)-3 -ethylurea; PPAR-a agonists, including fibrates (e.g., bezafibrate, ciprofibrate, clinofibrate, clofibric acid, clofibrate, aluminum clofibrate [alfibrate], clofibride, etofibrate, fenofibric acid, fenofibrate, gemfibrozil, ronifibrate and simfibrate), isoflavones (e.g., daidzein and genistein), and perfluoroalkanoic acids (e.g., perfluorooctanoic acid and perfluorononanoic acid); PPAR-6 agonists, including elafibranor (dual PPAR-a / y agonist), GFT505 (dual PPAR- a / y agonist), GW0742, GW501516 (dual PPAR-p / 8 agonist), sodelglitazar (GW677954), MBX- 8025, and isoflavones (e.g., daidzein and genistein);PPAR-y agonists, including thiazolidinediones (supra), saroglitazar (dual PPAR-a / y agonist), 4- oxo-2-thioxothiazolines (e.g, rhodanine), berberine, honokiol, perfluorononanoic acid, cyclopentenone prostaglandins (e.g, cyclopentenone 15-deoxy-A-prostaglandin h [15d-PGJ2]), and isoflavones (e.g., daidzein and genistein); liver X receptor (LXR) agonists, including endogenous ligands (e.g., oxysterols such as 22(i)-hydroxycholesterol, 24(A)-hydroxy cholesterol, 27-hydroxycholesterol and cholestenoic acid) and synthetic agonists (e.g., acetyl- podocarpic dimer, hypocholamide, A(X-di methyl -3 b- hydroxy-cholenamide [DMHCA], GW3965 and T0901317); retinoid X receptor (RXR) agonists, including endogenous ligands (e.g., 9-cis-retinoic acid) and synthetic agonists (e.g., bexarotene, AGN 191659, AGN 191701, AGN 192849, BMS649, LG100268, LG100754 and LGD346); inhibitors of acyl-CoA cholesterol acyltransferase (ACAT, aka sterol G-acyl transferase [SOAT], including ACAT1 [SOAT1] and ACAT2 [SOAT2]), including avasimibe, pactimibe, pellitorine, terpendole C and flavanones (e.g., naringenin); inhibitors of stearoyl-CoA desaturase- 1 (SCD-1, aka stearoyl-CoA delta-9 desaturase) activity or expression, including aramchol, CAY-10566, CVT-11127, SAR- 224, SAR-707, XEN-103; 3-(2-hydroxyethoxy)-4-methoxy-N-[5-(3- trifluoromethylbenzyl)thiazol-2-yl]benzamide and 4-ethylamino-3-(2-hydroxyethoxy)-N-[5-(3- trifluoromethylbenzyl)thiazol-2-yl]benzamide; 1'-{6-[5-(pyridin-3-ylmethyl)-l,3,4-oxadiazol-2- yl]pyridazin-3-yl}-5-(trifluoromethyl)-3,4-dihydrospiro[chromene-2,4'-piperidine]; 5-fluoro-l'- {6-[5-(pyridin-3-ylmethyl)-l,3,4-oxadiazol-2-yl]pyridazin-3-yl}-3,4-dihydrospiro[chromene-2,4'- piperidine]; 6-[5-(cyclopropylmethyl)-4,5-dihydro-l'H,3H-spiro[l,5-benzoxazepine-2,4'- pi peri din] -l'-yl ]-N-(2-hydroxy-2-pyri din-3 -yl ethyl )pyridazine-3 -carboxamide; 6-[4-(2-methylbenzoyl)piperidin- 1 -yl]pyridazine-3 -carboxylic acid (2-hydroxy-2-pyri din-3 -Attorney Docket No.: 058462-523001WOylethyl)amide; 4-(2-chlorophenoxy)-N-[3 -(methyl carbarnoyl)phenyl]piperi dine- 1 -carboxamide; the cis-9,trans-ll isomer and the trans-10,cis-12 isomer of conjugated linoleic acid, substituted heteroaromatic compounds disclosed in WO 2009 / 129625 Al, anti-sense polynucleotides and peptide-nucleic acids (PNAs) that target mRNA for SCD-1, and SCD-1 -targeting siRNAs; cholesterylester transfer protein (CETP) inhibitors, including anacetrapib, dalcetrapib, evacetrapib, torcetrapib and AMG 899 (TA-8995); inhibitors of microsomal triglyceride transfer protein (MTTP) activity or expression, including implitapide, lomitapide, dirlotapide, mitratapide, CP-346086, JTT-130, SLx-4090, anti-sense polynucleotides and PNAs that target mRNA for MTTP, MTTP -targeting microRNAs (e.g., miRNA-30c), and MTTP -targeting siRNAs; GLP-1 receptor agonists; fibroblast growth factor 21 (FGF21) and analogs and derivatives thereof, including BMS-986036 (pegylated FGF21); inhibitors of pro-protein eonvertase subtilisin / kexin type 9 (PCSK9) activity or expression, including berberine (reduces PC8K9 level), annexin A2 (inhibits PCSK9 activity), anti-PCSK9 antibodies (e.g., alirocumab, bococizumab, evolocumab, LGT-209, LY3015014 and RG7652), peptides that mimic the epidermal growth factor- A (EGF-A) domain of the LDL receptor which binds to PCSK9, PCSK9-binding adnectins (e.g., BMS-962476), anti-sense polynucleotides and PNAs that target mRNA for PCSK9, and PCSK9-targeting siRNAs (e.g, inclisiran [ALN-PCS] and ALN-PCS02); apolipoprotein mimetic peptides, including apoA-I mimetics (e.g., 2F, 3F, 3F-1, 3F-2, 3F-14, 4F, 4F-P-4F, 4F-IHS-4F, 4F2, 5F, 6F, 7F, 18F, 5A, 5A-C1, 5A-CH1, 5A-CH2, 5A-H1, 18 A, 37pA [18A-P-18A], ELK, ELK-1A, ELK- IF, ELK-1K1A1E, ELK-1L1K, ELK- 1W, ELK-2A, ELK- 2A2K2E, ELK-2E2K, ELK-2F, ELK-3 E3EK, ELK-3E3K3 A, ELK-3E3LK, ELK-PA, ELK- P2A, ELKA, ELKA-CH2, ATI-5261, CS-6253, ETC-642, FAMP, FREL and KRES and apoE mimetics (e.g., Ac-hE18A-NH2, AEM-28, Ac-[R]hEl 8 A-NH2, AEM-28-14, EpK, hEp, mR18L, COG-112, COG-133 and COG- 1410); omega-3 fatty acids, including docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), eicosapentaenoic acid (EP A), a-linolenic acid (ALA), fish oils (which contain, e.g., DHA and EP A), and esters (e.g., glyceryl and ethyl esters) thereof; and analogs, derivatives and salts thereof. In certain embodiments, the anti -obesity agent is or includes a lipase inhibitor (e.g., orlistat) or / and an antihyperlipidemic agent (e.g., a statin such as atorvastatin, or / and a fibrate such as fenofibrate).

[0179] Antihypertensive agents include without limitation: antagonists of the renin-angiotensin-aldosterone system (RAAS), including renin inhibitors (e.g., aliskiren), angiotensin-converting enzyme (ACE) inhibitors (e.g., benazepril, captopril, enalapril,Attorney Docket No.: 058462-523001WOfosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril and trandolapril), angiotensin II receptor type 1 (ATII1) antagonists (e.g., azilsartan, candesartan, eprosartan, fimasartan, irbesartan, losartan, olmesartan medoxomil, olmesartan, telmisartan and valsartan), and aldosterone receptor antagonists (e.g., eplerenone and spironolactone); diuretics, including loop diuretics (e.g., bumetanide, ethacrynic acid, furosemide and torsemide), thiazide diuretics (e.g., bendroflumethiazide, chlorothiazide, hydrochlorothiazide, epitizide, methyclothi azide and polythiazide), thiazide-like diuretics (e.g., chlorthalidone, indapamide and metolazone), cicletanine (an early distal tubular diuretic), potassium-sparing diuretics e.g., amiloride, eplerenone, spironolactone and triamterene), and theobromine; calcium channel blockers, including dihydropyridines (e.g., amlodipine, levamlodipine, cilni dipine, clevidipine, felodipine, isradipine, lercanidipine, nicardipine, nifedipine, nimodipine, nisoldipine and nitrendipine) and non-dihydropyri dines (e.g., diltiazem and verapamil); a2-adrenoreceptor agonists, including clonidine, guanabenz, guanfacine, methyldopa and moxonidine; a 1 -adrenoreceptor antagonists (alpha blockers), including doxazosin, indoramin, nicergoline, phenoxybenzamine, phentolamine, prazosin, terazosin and tolazoline; b -adrenoreceptor (bf or / and b2) antagonists (beta blockers), including atenolol, betaxolol, bisoprolol, carteolol, carvedilol, labetalol, metoprolol, nadolol, nebivolol, oxprenolol, penbutolol, pindolol, propranolol and timolol; mixed alpha / beta blockers, including bucindolol, carvedilol and labetalol; endothelin receptor antagonists, including selective ETA receptor antagonists e.g., ambrisentan, atrasentan, edonentan, sitaxentan, zibotentan andBQ-123) and dual ETA / ETB antagonists (e.g., bosentan, macitentan and tezosentan); other vasodilators, including hydralazine, minoxidil, theobromine, sodium nitroprusside, organic nitrates (e.g., isosorbide mononitrate, isosorbide dinitrate and nitroglycerin, which are converted to nitric oxide in the body), endothelial nitric oxide synthase (eNOS) stimulators e.g., cicletanine), activators of soluble guanylate cyclase e.g., cinaciguat and riociguat), phosphodiesterase type 5 (PDE5) inhibitors e.g., avanafil, benzamidenafil, dasantafil, dynafil, lodenafil, mirodenafil, sildenafil, tadalafil, udenafil, vardenafil, dipyridamole, papaverine, propentofylline, zaprinast and T-1032), prostaglandin Ei (alprostadil) and analogs thereof (e.g., limaprost amd misoprostol), prostacyclin and analogs thereof (e.g., ataprost, beraprost [e.g., esuberaprost], 5,6,7-trinor-4,8-inter-w-phenylene-9-fluoro-PG12, carbacyclin, isocarbacyclin, clinprost, ciprostene, eptaloprost, cicaprost, iloprost, pimilprost, SM- 10906 (des-m ethyl pimilprost), naxaprostene, taprostene, treprostinil, CS-570, OP -2507 and TY-11223), non prostanoid prostacyclin receptor agonists (e.g., 1-phthalazinol, ralinepag,Attorney Docket No.: 058462-523001WOselexipag, ACT- 333679 [MRE-269, active metabolite of selexipag], and TRA-418), phospholipase C (PLC) inhibitors, and protein kinase C (PKC) inhibitors (e.g., BIM-1, BIM-2, BIM-3, BIM-8, chelerythrine, cicletanine, gossypol, miyabenol C, myricitrin, ruboxistaurin and verbascoside; minerals, including magnesium and magnesium sulfate; and analogs, derivatives and salts thereof. In certain embodiments, the antihypertensive agent is or includes a thiazide or thiazide like diuretic (e.g., hydrochlorothiazide or chlorthalidone), a calcium channel blocker (e.g., amlodipine or nifedipine), an ACE inhibitor (e.g., benazepril, captopril or perindopril) or an angiotensin II receptor antagonist (e.g, olmesartan medoxomil, olmesartan, telmisartan or valsartan), or any combination thereof.

[0180] In some embodiments, a pharmaceutical composition according to the present disclosure is used in combination with one or more additional therapeutic agents to treat NAFLD, such as NASH. In some embodiments, the one or more additional therapeutic agents are selected from antidiabetic agents, anti-obesity agents, anti-inflammatory agents, antifibrotic agents, antioxidants, anti hypertensive agents, and combinations thereof.Therapeutic agents that can be used to treat NAFLD (e.g, NASH) include without limitation: PPAR agonists, including PPAR-6 agonists (e.g., MBX-8025, elafibranor [dual PPAR-a / 8 agonist] and GW501516 [dual PPAR-p / 6 agonist]) and PPAR-y agonists (e.g., thiazolidinediones such as pioglitazone, and saroglitazar [dual PPAR-a / y agonist]) - PPAR-6 and PPAR-y agonism increases insulin sensitivity, PPAR-a agonism reduces liver steatosis and PPAR-6 agonism inhibits activation of macrophages and Kupffer cells; famesoid X receptor (FXR) agonists, such as obeticholic acid and nonsteroidal FXR agonists like GS-9674 reduce liver gluconeogenesis, lipogenesis, steatosis and fibrosis; fibroblast growth factor 19 (FGF19) and analogs and derivatives thereof, such as NGM- 282 - FGF19 analogs reduce liver gluconeogenesis and steatosis; fibroblast growth factor 21 (FGF21) and analogs and derivatives thereof, such as BMS- 986036 (pegylated FGF21) - FGF21 analogs reduce liver steatosis, cell injury and fibrosis; HMG-CoA reductase inhibitors, including statins (e.g., rosuvastatin) - statins reduce steatohepatitis and fibrosis; ACC inhibitors, such as NDL 010976 (liver-targeted) and GS-0976 - ACC inhibitors reduce de novo lipogenesis and liver steatosis; SCD-1 inhibitors, such as aramchol - SCD-1 inhibitors reduce liver steatosis and increase insulin sensitivity; SGLT2 inhibitors, such as canagliflozin, ipragliflozin and luseogliflozin - SGLT2 inhibitors reduce body weight, liver ALT level and fibrosis; antagonists of CCR2 or / and CCR5, such as cenicriviroc - antagonists of CCR2 (binds toAttorney Docket No.: 058462-523001WOCCL2 [MCP1]) and CCR5 (binds to CCL5 [RANTES]) inhibit activation and migration of inflammatory cells (e.g., macrophages) to the liver and reduce liver fibrosis; apoptosis inhibitors, including apoptosis signal-regulating kinase 1 (ASK1) inhibitors (e.g., selonsertib) and caspase inhibitors (e.g., emricasan [pan-caspase inhibitor]) - apoptosis inhibitors reduce liver steatosis and fibrosis; lysyl oxidase-like 2 (L0XL2) inhibitors, such as simtuzumab -L0XL2 is a key matrix enzyme in collagen formation and is highly expressed in the liver; galectin-3 inhibitors, such as GR-MD-02 and TD139 - galectin-3 is critical for development of liver fibrosis; antioxidants, including vitamin E (e.g., a-tocopherol) and scavengers of reactive oxygen species (ROS) and free radicals (e.g., cysteamine, glutathione, melatonin and pentoxifylline [also anti-inflammatory via inhibition of TNF-a and phosphodiesterases]) -vitamin E reduces liver steatosis, hepatocyte ballooning and lobular inflammation; and, analogs, derivatives and salts thereof. In some embodiments, a peptide product described herein is used in conjunction with a PPAR agonist (e.g., a PPAR-6 agonist such as elafibranor or / and a PPAR-y agonist such as pioglitazone), a HMG-CoA reductase inhibitor (c.g, a statin such as rosuvastatin), an FXR agonist (e.g., obeticholic acid) or an antioxidant (e.g., vitamin E), or any combination thereof, to treat NAFLD (c.g, NASH). In certain embodiments, the one or more additional therapeutic agents for treatment of NAFLD (e.g., NASH) are or include vitamin E or / and pioglitazone. Other combinations may also be used as would be understood by those of ordinary skill in the art.

[0071] Pharmacokinetic (“PK”) parameters can be estimated using Phoenix® WinNonlin® version 8.1 or higher (Certara USA, Inc., Princeton, New Jersey).

[0181] In some embodiments, the first therapy and the second therapy are administered concomitantly. In some embodiments, the first therapy is administered at the same time as the second therapy. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first therapy is administered before the second therapy. In some embodiments, the first therapy is administered after the second therapy. In some embodiments, the first therapy is administered before and / or after the second therapy. In some embodiments, the first therapy and the second therapy are administered in rotation. In some embodiments, the first therapy and the second therapy are administered together in a single formulation.KITS

[0182] Also provided herein are various kits for the practice of a method described hereinAttorney Docket No.: 058462-523001WOas well as written instructions for making and using the same. In particular, some embodiments of the disclosure provide kits for eliciting a pharmacodynamic effect in a subject. Some embodiments of the disclosure provide kits for activating GLP-1 receptor in a subject. Some other embodiments relate to kits for the prevention of a relevant health condition in a subject in need thereof. Some other embodiments relate to kits for methods of treating a relevant health condition in a subject in need thereof. For example, in some embodiments, the present disclosure provides kits that include one or more of the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions as provided and described herein, as well as written instructions for making and using the same. In some embodiments, the kits are for the prevention and / or treatment of one or more health conditions, disorders, or diseases associated with GLP-1R signaling as described above. In some particular embodiments, the metabolic disorder is diabetes, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or obesity. In some embodiments, the diabetes is Type I diabetes, Type II diabetes, or gestational diabetes. In some embodiments, the diabetes is diabetes Type II.

[0183] In some embodiments, the kits of the disclosure further include one or more means useful for the administration of any one of the provided nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions to a subject. For example, in some embodiments, the kits of the disclosure further include one or more syringes (including pre-filled syringes) and / or catheters (including pre-filled syringes) used to administer any one of the provided nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions to a subject. In some embodiments, a kit can have one or more additional therapeutic agents that can be administered simultaneously or sequentially with the other kit components for a desired purpose, e.g., for activating GLP-1 receptor signaling, preventing, and / or treating a health condition in a subject in need thereof.

[0184] Any of the above-described kits can further include one or more additional reagents, where such additional reagents can be selected from: dilution buffers, reconstitution solutions, wash buffers, control reagents, control expression vectors, negative controls, positive controls, reagents suitable for in intro production and / or administration of the provided nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions of the disclosure.

[0185] In some embodiments, the components of a kit can be in separate containers. In some other embodiments, the components of a kit can be combined in a single container.Attorney Docket No.: 058462-523001WOAccordingly, in some embodiments of the disclosure, the kit includes one or more of the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions as provided and described herein in one container (e.g., in a sterile glass or plastic vial) and a further therapeutic agent in another container (e.g., in a sterile glass or plastic vial).

[0186] In another embodiment, the kit includes a combination of the compositions described herein, including one or more nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions of the disclosure in combination with one or more further therapeutic agents formulated together, optionally, in a pharmaceutical formulation, in a single, common container.

[0187] In some embodiments wherein the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit can include a device (e.g., an injection device or catheter) for performing such administration. For example, the kit can include one or more hypodermic needles or other injection devices as discussed above containing one or more nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions of the disclosure.

[0188] In some embodiments, a kit can further include instructions for using the components of the kit to practice one or more of the methods disclosed herein. For example, the kit can include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding a combination of the disclosure may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer / distributor information and intellectual property information.

[0189] The instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastic, etc. The instructions can be present in the kit as a package insert, in the labeling of the container of the kit or components thereof (e.g., associated with the packaging or subpackaging), etc. The instructions can be present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In some instances, the actual instructions are not present in the kit, but means for obtaining theAttorney Docket No.: 058462-523001WOinstructions from a remote source (e.g., via the internet), can be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate or recording medium.

[0190] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0191] No admission is made that any reference cited herein constitutes prior art. The discussion of the references states what their authors assert, and the Applicant reserves the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of information sources, including scientific journal articles, patent documents, and textbooks, are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0192] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure, and are to be included within the spirit and purview of this application.

[0193] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.EXAMPLES

[0194] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005).Attorney Docket No.: 058462-523001WONonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, C A: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. etal. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference.

[0195] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.EXAMPLE 1Construction of modified alphavirus vectors

[0196] This Example describes experiments performed to construct a base alphavirus vector (e.g., without a heterologous gene) that are subsequently used for construction of alphavirus vectors encoding polypeptide constructs of interest, e.g., polypeptide constructs comprising one or more of GLP-1R agonists

[0197] The base VEEV vector was synthesized de novo in four ~4 kb parts (Twist Bioscience, Thermo Fisher GeneArt) from a TC-83 strain reference sequence (Genbank L01443) with a silent A2087G mutation, and a unique Spel restriction enzyme cut site in place of the coding sequence of the VEEV structural genes (where the 5’ A is the next nucleotide after a P2A sequence following nucleotide 93 of the structural polyprotein gene, and the 3’ T matches the location of the structural polyprotein’s stop codon TGA). A 5’ adaptor sequence (5’- CTGGAGACGTGGAGGAGAACCCTGGACCT-3’; SEQ ID NO: 52) was inserted upstream of the Spel site, and a 3’ adaptor sequence (5’-GACCGCTACGCCCCAATGACCCGACCAGC-3’; SEQ ID NO: 53) was inserted downstream of the Spel site for subsequent Gibson Assembly® procedures. A bacteriophage T7 RNA polymerase promoter (5’-TAATACGACTCACTATAG-3’; SEQ ID NO: 54) was included upstream of the VEEV genome sequence, and downstream contained a poly(A)Attomey Docket No.: 058462-523001WOsequence followed by a SapI site, which cuts upstream of the recognition site. Immediately downstream of the SapI site is a T7 terminator sequence (5’-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3’; SEQ ID NO: 55) followed by a unique Not! restriction enzyme cut site. The parts were combined in a five-piece Gibson Assembly® reaction (e.g., a linearized pYL backbone and the four synthesized fragments) to result in the VEEV base vector.

[0198] The GLP-1R agonists, GCGR agonist, and GIPR agonist genes were codon optimized for human expression in silico (IDT) and synthesized de novo. The synthetic products were amplified using primers which added either 5’ and 3’ adaptor sequences to the ends of the genes, or primers which added sequences of homology to neighboring gene inserts. The A e I -linearized base vector and the PCR products were combined by Gibson Assembly® procedure to result in the final vectors. A summary of twenty -two (22) exemplary nucleic acid constructs (either monogenic or multigenic) expressing GLP-1R agonists are shown in, e.g., FIGS. 1-2, 9-10, and Table 1 (see also, Table 2). In each construct, the numbers of GLP-1R agonists and their ordinality in the expression cassette are shown. In FIGS. 1-2, 9-10, and Tables 1-2, while some constructs contain coding sequences for multiple polypeptides operably linked to one another within a single open reading frame (e.g., in a polycistronic ORF), others include coding sequences for multiple polypeptides configured into two ORFs that are connected to one another by a sequence for an internal ribosomal entry site (IRES) (see, e.g., Rep-877, Rep-897, Rep-898, and Rep-899). P2A: autoproteolytic peptide sequence from porcine teschovirus-1 2 A. In these figures, the heterologous signal peptides, the GLP-1R agonists, the autoproteolytic peptide P2A sequences, and internal ribosomal entry sites (IRES) are shown in N-terminus to C-terminus direction (i.e., in 5’ to 3’ direction) of the corresponding coding sequences.EXAMPLE 2Evaluation of srRNA constructs expressing GLP-1R agonists with heterologous signal peptides

[0199] This Example describes in vitro experiments that were performed to evaluate expression levels of srRNA constructs described in Example 1 above, and to illustrate that that GLP-1R agonists with heterologous signal peptides can be encoded into srRNA vectors capable of RNA replication in transfected BHK-21 cells (see, e.g., FIG. 2).

[0200] In vitro transcription'. RNA was prepared by in vitro transcription from a SapI-Attorney Docket No.: 058462-523001WOlinearized plasmid template with bacteriophage T7 RNA polymerase (Hi Scribe™ T7 High Yield RNA Synthesis Kit, NEB) followed by addition of a 5’ cap 1 (Vaccinia Capping System, mRNA Cap 2'-O-Methyltransferase, NEB). RNA was purified using LiCl precipitation, followed by a 70% ethanol wash and resuspended in 1 mM sodium citrate pH 6.4. RNA concentration was determined by absorbance at 260 nm (Nanodrop, Thermo Fisher Scientific).

[0201] srRNA replication and cargo expression'. The srRNA-based bioactivity was measured by detection of the replication intermediate dsRNA in BHK-21 cells. RNA was transformed into cells by electroporation (4D-Nucleofector™, Lonza). At 15-22 hours following transformation, the cells were fixed and permeabilized (eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen) and stained using a PE-conjugated anti-dsRNA mouse monoclonal antibody (J2, Scicons) to quantify the frequency of dsRNA+ cells by fluorescence flow cytometry. The media from the cells was collected and used to quantify peptide or protein expression by cell-based assay or ELISA.

[0202] FIG. 2 summarizes the results of experiments performed to illustrate that the srRNA vectors are capable of RNA replication. The frequency of dsRNA+ BHK-21 cells is shown plotted for each construct, showing that the GLP-1RA srRNA constructs exhibit RNA replication.

[0203] FIG. 3 summarizes the results of experiments performed to illustrate that the signal peptide identity in srRNA vector affects secretion of the expressed GLP-1R agonist. The Y-axis represents the concentration of GLP-1 in cell culture media after BHK-21 cells were transfected with the corresponding srRNA. In these experiments, it was observed that not all signal peptides result in detectable secreted GLP-1R agonist (GLP-1), as determined by quantification using the HEK GLP-1 R-Luciferase cell-based assay (BPS Bioscience).

[0204] FIG. 4 summarizes the results of additional experiments performed to illustrate that the signal peptide identity in srRNA vector affects secretion of another exemplary GLP-1R agonist, glucagon (GCG). The Y-axis represents the concentration of GCG in cell culture media after BHK-21 cells were transfected with the corresponding srRNA. In these experiments, it was observed that not all signal peptides result in detectable secreted GCG, as determined by ELISA.

[0205] FIGS. 5A-5B summarize the results of experiments performed to illustrate that the signal peptide identity in srRNA vector affects expression of another exemplary GLP-1RAttorney Docket No.: 058462-523001WOagonist, Exenatide, in transfected BEK-21 cells. The Y-axes represent the concentration of Exenatide in cell culture media after BHK-21 cells were transfected with the corresponding srRNA. In FIG. 5A, the functional concentration of Exenatide was determined by quantification using the HEK GLP-IR-Luciferase cell-based assay (BPS Bioscience). In FIG.5B, the concentration of Exenatide was determined by ELISA assay using Exendin-4 as a reference.

[0206] FIGS. 6A-6B summarize the results of experiments performed to illustrate that the signal peptide identity in srRNA vector affects expression of another exemplary GLP-1R agonist, GLP-l-Fc in transfected BHK-21 cells. The Y-axes represent the concentration of GLP-l-Fc in cell culture media after BHK-21 cells were transfected with the corresponding srRNA. In FIG. 6A, the functional concentration of GLP-l-Fc was determined by using the HEK GLP-IR-Luciferase cell-based assay (BPS Bioscience). In FIG. 6B, the concentration of GLP-l-Fc was determined by ELISA.

[0207] These data demonstrate that srRNA which encode GLP-1RA and GCGR agonist (GCGRA) are functional (e.g., capable of RNA replication) and can be used to produce detectable peptide or protein. The functionality of GLP-lRAs were confirmed using a cellbased assays. Importantly, the use of signal peptides can affect protein production from srRNA, where a functional signal peptide is required to secrete the expressed protein from cells.EXAMPLE S GLP-1RA in vitro expression vs secretion in different srRNA vectors

[0208] This Example describes in vitro experiments that were performed to evaluate GLP-1RA in vitro expression versus secretion from transfected cells with different srRNA vectors.

[0209] In vitro transcription'. RNA was prepared by in vitro transcription from a Sapl-linearized plasmid template with bacteriophage T7 RNA polymerase (Hi Scribe™ T7 High Yield RNA Synthesis Kit, NEB) followed by addition of a 5’ cap 1 (Vaccinia Capping System, mRNA Cap 2'-O-Methyltransferase, NEB). RNA was purified using LiCl precipitation, followed by a 70% ethanol wash and resuspended in 1 mM sodium citrate pH 6.4. RNA concentration was determined by absorbance at 260 nm (Nanodrop, Thermo Fisher Scientific).

[0210] GLP-1RA expression'. RNA was transformed into BHK-21 cells by electroporation 4D-Nucleofector™, Lonza). At 15-22 hours following transformation, the cells wereAttorney Docket No.: 058462-523001WOcollected and lysed, and the cell culture media was collected. These samples were used to quantify peptide or protein expression by ELISA.

[0211] FIGS. 7A-7B summarize the results of experiments illustrating in vitro expression and secretion of GLP-l-Fc and Exenatide from transfected cells with different srRNA-based vectors. The Y-axes represent the concentration of GLP-lRAs in cell lysates or cell culture media (“Secreted”) after BHK-21 cells were transfected with the corresponding srRNA.

[0212] These data demonstrate that different srRNA vectors result in differential amounts of GLP-1RA polypeptides produced and the relative intracellular versus secreted GLP-1RA.EXAMPLE 4GLP-1RA in vivo expression from monogenic EEEV vector by multiple routes of administration

[0213] This Example describes experiments that were performed to evaluate GLP-1RA in vivo expression from monogenic EEEV vector by multiple routes of administration.

[0214] LNP formulation . srRNA was formulated in lipid nanoparticles using a microfluidics mixer and analyzed for particle size, poly dispersity using dynamic light scattering and encapsulation efficiency. Lipids were suspended in ethanol. RNA was suspended in 100 mM NaOAc pH 4.0 at a concentration of 82 ug / ml, and was mixed at a flow rate of 3 : 1 (aqueous:organic).

[0215] Mice and injections. BALB / c mice were purchased from Charles River Labs, Envigo, or Jackson Laboratories. On the day of dosing, 5 pg of LNP was injected subcutaneously or intramuscularly either into one or split into both quadricep muscles. One group with each route of LNP administration were also administered 5 mg / kg dexamethasone intraperitoneally. In a control group, 600 pg / kg recombinant GLP-l-Fc was injected subcutaneously. Serum was collected 3 days after injection and the concentration of GLP-l-Fc was quantified by ELISA. Animals were monitored for body weight and other general observations throughout the course of the study.

[0216] FIG. 8 schematically summarizes the results of experiments performed to evaluate GLP-l-Fc’s in vivo expression from a monogenic EEEV srRNA-based vector by multiple routes of administration.

[0217] These data demonstrate that srRNA-encoded GLP-1RA can be administered via an intramuscular or subcutaneous injection and result in detectable protein in serum.EXAMPLE 5Attorney Docket No.: 058462-523001WOGLP-1RA in vitro expression from different multigenic srRNA-based vectors with different heterologous signal peptides

[0218] This Example describes experiments that were performed to evaluate GLP-1RA in vitro expression and secretion from different multigenic srRNA-based vectors with different heterologous signal peptides from transfected cells.

[0219] As discussed above, Table 2 provides a list of exemplary nucleic acid constructs expressing a GLP-1R agonist (GLP-l-Fc), either alone or in combination with a GIPR agonist and / or a GCGR agonist in accordance with some embodiments of the disclosure.

[0220] FIGS. 9A-9C summarizes the results of experiments performed to evaluate GLP-1-Fc’s expression and secretion from different monogenic and multigenic srRNA-based vectors with heterologous signal peptides from msIgGK or insulin (INS). The Y-axes represent the concentration of the secreted GLP-l-Fc in cell culture media after BHK-21 cells were transfected with the corresponding srRNA, as determined by ELISA.

[0221] These data demonstrate that GLP-1RA expression using either msIgGK or INS heterologous signal peptides results in detectable secreted GLP-1RA from srRNA encoding GLP-1RA as a monogenic construct or multigenic in combination with GIPR or GCGR agonists.EXAMPLE 6Expression and secretion of GIPR and GCGR agonists in vitro from multigenic srRNA vectors

[0222] This Example describes experiments that were performed to evaluate in vitro expression and secretion of GCG and GIP receptor agonists, co-encoded with GLP-1RA, from different multigenic srRNA-based vector with different heterologous signal peptides.

[0223] In vitro transcription'. RNA was prepared by in vitro transcription from a Sapl-linearized plasmid template with bacteriophage T7 RNA polymerase (Hi Scribe™ T7 High Yield RNA Synthesis Kit, NEB) followed by addition of a 5’ cap 1 (Vaccinia Capping System, mRNA Cap 2'-O-Methyltransferase, NEB). RNA was purified using LiCl precipitation, followed by a 70% ethanol wash and resuspended in 1 mM sodium citrate pH 6.4. RNA concentration was determined by absorbance at 260 nm (Nanodrop, Thermo Fisher Scientific).

[0224] GLP-1RA expression'. RNA was transformed into BHK-21 cellsby electroporation 4D-Nucleofector™, Lonza). At 15-22 hours following transformation, the cells wereAttorney Docket No.: 058462-523001WOcollected and lysed, and the cell culture media was collected. These samples were used to quantify peptide or protein expression by ELISA.

[0225] FIGS. 9B-9C and 10B-10C summarize the results of experiments performed to evaluate expression and secretion of GCGR and GIPR agonists from different multigenic srRNA-based vectors along with GLP-1RA with different heterologous signal peptides.

[0226] In FIGS. 9B-9C, the Y-axis represent the concentration of GLP-Fc expressed with a msIgGK signal peptide (FIG. 9B) or an Insulin signal peptide (FIG. 9C) in cell culture media quantified by ELISA after BHK-21 cells were transfected with the corresponding srRNA.

[0227] In FIGS. 10B-10C, the Y-axis represent the concentration of secreted glucagon (FIG. 10B) or secreted GIP (FIG. 10C) quantified by ELISA in cell culture media after BHK-21 cells were transfected with the corresponding srRNA. In these experiments, it was observed that Rep-897 and Rep-899 conferred best GCG and GIP expression.

[0228] These data demonstrate that GLP-1, GCG, and GIPR agonists can be co-encoded and expressed from a single srRNA construct.EXAMPLE 7Expression and secretion of GLP-lRAs. GIPR, and GCGR agonists in vitro from multigenic srRNA vectors

[0229] This Example describes experiments that are performed to evaluate in vitro expression and secretion of multiple GLP-lRAs, with or without GCG and GIP receptor agonists, from different multigenic srRNA-based vectors with heterologous signal peptides.

[0230] Vector construction'. Gene cassettes are inserted into base vectors like the VEEV base vector described in Example 1. Gene cassettes contain coding sequences for multiple polypeptides operably linked to one another within a single open reading frame (e.g., in a polycistronic ORF), others include coding sequences for multiple polypeptides configured into two ORFs that are connected to one another by a sequence for an internal ribosomal entry site (IRES). In this manner, one or more GLP-lRAs, with or without GCG and GIP receptor agonists are encoded in the same srRNA vector.

[0231] In vitro transcription'. RNA is prepared by in vitro transcription from a Sap -linearized plasmid template with bacteriophage T7 RNA polymerase (Hi Scribe™ T7 High Yield RNA Synthesis Kit, NEB) followed by addition of a 5’ cap 1 (Vaccinia Capping System, mRNA Cap 2'-O-Methyltransferase, NEB). RNA is purified using LiClAttorney Docket No.: 058462-523001WOprecipitation, followed by a 70% ethanol wash and resuspended in 1 mM sodium citrate pH 6.4. RNA concentration is determined by absorbance at 260 nm (Nanodrop, Thermo Fisher Scientific).

[0232] GLP-1RA expression'. RNA is transformed into BHK-21 cells by electroporation 4D-Nucleofector™, Lonza). At 15-22 hours following transformation, the cell culture media is collected and peptide or protein expression is quantified by ELISA.These experiments demonstrate that multiple GLP-lRAs can be encoded and be expressed from a single vector, with or without GCG and GIP receptor agonists.

Claims

Attorney Docket No.: 058462-523001WOCLAIMS WHAT IS CLAIMED IS:

1. A nucleic acid construct comprising a nucleic acid sequence encoding a modified alphavirus genome or self-replicating RNA (srRNA), wherein at least a portion of the nucleic acid sequence encoding the viral structural proteins of the modified alphavirus genome or srRNA has been replaced by a coding sequence for a polypeptide construct comprising one or more glucagon-like peptide-1 receptor (GLP-1R) agonists.

2. The nucleic acid construct of claim 1, wherein the nucleic acid sequence further comprises coding sequences for one or more glucagon receptor (GCGR) agonists, glucosedependent insulinotropic peptide (GIP), GIP receptor (GIPR) agonists, and / or G-protein-coupled receptor (GPCR) agonists.

3. The nucleic acid construct of any one of claims 1-2, wherein the nucleic acid sequence further comprises coding sequences for one or more heterologous signal peptides that directs secretion of the one or more GLP-1R agonists, alone or in combination with GCGR agonist, GIP, and / or GPCR agonist.

4. The nucleic acid construct of claim 3, wherein the one or more heterologous signal peptides is selected from signal peptides of human AACT, human FGF21, human FZD3, mouse IGKV3-10, human IGKV3-20, human INS, human INSR, human IPSP, human PORIM, human SAMP, and a combination of any thereof.

5. The nucleic acid construct of any one of claims 1-4, wherein the one or more agonists of GLP-1R, GCGR, GPCR, GIPR, and GIP are operably linked to one another within a single open reading frame (i.e., in a polycistronic ORF).

6. The nucleic acid construct of any one of claims 1-5, wherein the one or more agonists of GLP-1R, GCGR, GPCR, GIPR, and GIP are operably linked to one another by one or more linkers.

7. The nucleic acid construct of any one of claims 1-6, wherein at least one of the one or more agonists of GLP-1R, GCGR, GPCR, GIPR, and GIP comprises a structural modification to increase half-life.Attorney Docket No.: 058462-523001WO8. The nucleic acid construct of claim 7, wherein the structural modification comprises one or more mutations, N-extension, and / or C-terminal extensions.

9. The nucleic acid construct of any one of claims 7-8, wherein the structural modification comprises fusion to a Fc region of an immunoglobulin.

10. The nucleic acid construct of any one of claims 1-9, wherein the modified alphavirus genome or srRNA comprises no nucleic acid sequence encoding viral structural proteins.

11. The nucleic acid construct of any one of claims 1-10, wherein the nucleic acid sequence encoding the polypeptide construct is operably linked to a promoter sequence.

12. The nucleic acid construct of claim 11, wherein the promoter sequence is a 26S subgenomic (.g) promoter.

13. The nucleic acid construct of any one of claims 1-12, wherein the srRNA is a capped srRNA comprising a 5 ’-cap.

14. The nucleic acid construct of claim 13, wherein the 5’-cap is a cap-0, cap-1, cap-2, or cap analogue.

15. The nucleic acid construct of claim 13, wherein the capped srRNA is a co-transcriptionally capped srRNA.

16. The nucleic acid construct of claim 13, wherein the capped srRNA is an enzymatically capped srRNA.

17. The nucleic acid construct any one of claims 1-16, wherein the modified alphavirus genome or srRNA comprises a poly(A) tail, and wherein:(a) the poly(A) tail does not comprise a 3’ non-A residue; and / or(b) the lengthened sequence encoding the poly(A) tail is longer than 34 residues.

18. The nucleic acid construct of claim 17, further comprising an additional restriction site engineered into the sequence encoding the poly(A) tail of the alphavirus genome or srRNA.

19. The nucleic acid construct of any one of claims 17-18, further comprising an additional restriction site incorporated at the end of the sequence encoding the poly(A) tail of theAttorney Docket No.: 058462-523001WOalphavirus genome or srRNA.

20. The nucleic acid construct of any one of claims 17-19, wherein the lengthened poly(A) tail has a length ranging from about 30 to about 120 adenylate residues.

21. The nucleic acid construct any one of claims 1-20, wherein the modified alphavirus genome or srRNA is of an alphavirus belonging to the VEEV / EEEV group, or the SFV group, or the SINV group.

22. The nucleic acid of claim 21, wherein the alphavirus is Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), Chikungunya virus (CHIKV), Madariaga virus (MADV), Western equine encephalitis virus (WEEV), or Sindbis virus (SINV).

23. The nucleic acid construct of any one of claims 1-22, wherein the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-25.

24. The nucleic acid construct of any one of claims 1-23, wherein the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 26-51.

25. A recombinant cell comprising a nucleic acid construct according to any one of any one of claims 1-24.

26. The recombinant cell of claim 25, wherein the recombinant cell is a eukaryotic cell.

27. The recombinant cell of claim 25, wherein the recombinant cell is an animal cell.

28. The recombinant cell of claim 27, wherein the animal cell is a vertebrate animal cell or an invertebrate animal cell.

29. The recombinant cell of claim 28, wherein the recombinant cell is an insect cell.Attorney Docket No.: 058462-523001WO30. The recombinant cell of claim 29, wherein the recombinant insect cell is a mosquito cell.

31. The recombinant cell of claim 28, wherein the recombinant cell is a mammalian cell.

32. The recombinant cell of claim 28, wherein the recombinant cell is selected from the group consisting of a monkey kidney CV1 cell transformed by SV40, a human embryonic kidney cell (HEK), a baby hamster kidney cell (BHK) or a derivative cell thereof, a mouse sertoli cell, a monkey kidney cell, a human cervical carcinoma cell, a canine kidney cell, a buffalo rat liver cell, a human lung cell, a human liver cell, a human fat cell or adipocyte, a mouse fat cell or adipocyte a mouse mammary tumor, a TRI cell, a FS4 cell, a Chinese hamster ovary cell (CHO), an African green monkey kidney cell, a human A549 cell, a human cervix cell, a human CHME5 cell, a human PER.C6 cell, aNSO murine myeloma cell, a human epidermoid larynx cell, a human fibroblast cell, a human HUH-7 cell, a human MRC-5 cell, a human muscle cell, a human endothelial cell, a human astrocyte cell, a human macrophage cell, a human RAW 264.7 cell, a mouse 3T3 cell, a mouse L929 cell, a mouse connective tissue cell, a mouse muscle cell, and a rabbit kidney cell.

33. A cell culture comprising at least one recombinant cell according to any one of claims 25-32 and a culture medium.

34. A transgenic animal comprising:(a) a nucleic acid construct according to any one of claims 1-24; and / or(b) a recombinant cell according to any one of claims 25-32.

35. The transgenic animal of claim 34, wherein the transgenic animal is a vertebrate animal or an invertebrate animal.

36. The transgenic animal of claim 34, wherein the transgenic animal is an insect.

37. The transgenic animal of claim 34, wherein the transgenic animal is a mammalian.

38. The transgenic animal of claim 37, wherein the mammalian is a non-human mammalian.

39. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and:(a) a nucleic acid construct according to any one of any one of claims 1-24; and / orAttorney Docket No.: 058462-523001WO(b) a recombinant cell according to any one of claims 25-32.

40. The pharmaceutical composition of claim 39, wherein the pharmaceutical composition comprises a nucleic acid construct of any one of claims 1-24, and a pharmaceutically acceptable excipient.

41. The pharmaceutical composition of any one of claims 39-40, the composition is formulated with a delivery vehicle into a delivery system, wherein the delivery system comprises a liposome, a viral replicon particle (VRP), a lipid-based nanoparticle (LNP), a polymer nanoparticle, a physiologic buffer, a microsphere, an immune stimulating complex (ISCOM), a conjugate of bioactive ligand, or a combination of any thereof.

42. The pharmaceutical composition of claim 41, wherein the LNP delivery system comprises a cationic lipid, an ionizable cationic lipid, an anionic lipid, or a neutral lipid.

43. The pharmaceutical composition of claim 42, wherein the lipid of the LNP delivery system is present in mass ratio of lipid to RNA from about 100:1 to about 4:1.

44. The pharmaceutical composition of claim 43, wherein the LNP delivery system comprises lipid-based nanoparticles having an average diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm.

45. The pharmaceutical composition of claim 43, wherein the LNP delivery system comprises lipid-based nanoparticles having an average diameter of about 1-1000 nm, about 1-1000 nm, about 1-500 nm, about 1-250 nm, about 25-200 nm, about 25-100 nm, about 35-75 nm, or about 25-60 nm.

46. The pharmaceutical composition of any one of claims 39-45, wherein the pharmaceutical composition is formulated for one or more of intranasal administration, intrathecal administration, transdermal administration, intraperitoneal administration, intramuscular administration, intratracheal administration, intranodal administration, intratumoral administration, intraarticular administration, intravenous administration, subcutaneous administration, intravaginal administration, intraocular administration, rectal administration, and oral administration.Attorney Docket No.: 058462-523001WO47. The pharmaceutical composition of claim 46, wherein the pharmaceutical composition is formulated intramuscular administration.

48. A method for activating GLP-1 receptor signaling in a subject, the method comprises administering to the subject a composition comprising:a) a nucleic acid construct of any one of claims 1-24;b) a recombinant cell of any one of claims 25-32; and / ord) a pharmaceutical composition of any one of claims 39-47.

49. A method for eliciting a pharmacodynamic effect in a subject in need thereof, the method comprises administering to the subject a composition comprising:a) a nucleic acid construct of any one of claims 1-24;b) a recombinant cell of any one of claims 25-32; and / ord) a pharmaceutical composition of any one of claims 39-47.

50. The method of claim 49, wherein the pharmacodynamic effect comprises one or more of the following: immunogenicity effect, a biomarker response, a therapeutic effect, a prophylactic effect, a desired effect, an undesired effect, an adverse effect, and effect in a disease model.

51. The method of claim 50, wherein the pharmacodynamic effect comprises eliciting an immune response in the subject.

52. A method for preventing and / or treating a health condition in a subject in need thereof, the method comprises prophylactically or therapeutically administering to the subject a composition comprising:a) a nucleic acid construct of any one of claims 1-24;b) a recombinant cell of any one of claims 25-32; and / ord) a pharmaceutical composition of any one of claims 39-47.

53. The method of any one of claim 48-52, wherein the administered composition results in slowing of gastric emptying, reducing the release of glucagon, decreasing blood glucose, enhancing insulin sensitivity, and / or stimulating insulin production in the subject.

54. The method of any one of claim 48-53, wherein the subject is having or suspected of having a metabolic disorder associated with GLP-1R signaling.Attorney Docket No.: 058462-523001WO55. The method of claim 54, wherein the metabolic disorder associated with GLP-1R signaling is diabetes, obesity, hypertension, hyperlipidemia, a cardiovascular disease, or a musculoskeletal disease.

56. The method of any one of claim 48-55, wherein the composition is administered to the subject individually as a single therapy (monotherapy) or as a first therapy in combination with at least one additional therapies.

57. A kit comprising:a) a nucleic acid construct of any one of claims 1-24;b) a recombinant cell of any one of claims 25-32; and / ord) a pharmaceutical composition of any one of claims 39-47,and instructions for performing the method of any one of claims 48-56.

58. The kit of claim 57, wherein the health condition is a metabolic disorder associated with GLP-1R signaling.

59. The kit of claim 58, wherein the metabolic disorder associated with GLP-1R signaling is diabetes, obesity, hypertension, hyperlipidemia, a cardiovascular disease, or a musculoskeletal disease.

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