Viral vector mediated transduction into adipocytes for weight loss

Abstract

Provided herein are compositions with viral vectors that mediate overexpression of Gastric Inhibitory Polypeptide Receptor (GIPR), a Glucagon Receptor (GCGR), or a Glucagon-Like Peptide 1 Receptor (GLP-1R), or ligands thereof in adipocytes. Also provided here are methods of treating obesity and other metabolic disorders using these compositions.

Description

TITLEVIRAL VECTOR MEDIATED TRANSDUCTION INTO ADIPOCYTES FOR WEIGHT LOSSCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 664,052 filed 25 June 2024, and titled “VIRAL VECTOR MEDIATED GIP-R TRANSDUCTION INTO ADIPOCYTES FOR WEIGHT LOSS,” which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] This instant application contains a Sequence Listing which has been submitted in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 24, 2025, is named UTSD.P4367WO_SEQ_LISTING.xml and is 179,550 bytes in size.BACKGROUND1. Field

[0003] The present disclosure relates to nucleic acid compositions for administration into the adipocytes of a subject, and their use in studying and treating obesity.2. Discussion of Related Art

[0004] The prevalence of obesity has substantially increased. The World Health Organization recognizes obesity as a worldwide epidemic and a major disease of the 21st century; with >4 million people dying in 2017. Obesity is characterized as excessive fat accumulation which results from a dysregulation in energy homeostasis ( / .e. caloric intake exceeding energy expenditure) and presents a health risk. This leads to an increase in lipid storage, primarily in white adipose tissue (WAT). Managing obesity through lifestyle intervention, such as increased physical activity and / or caloric restriction, is extremely challenging to sustain longterm. As such, there is an urgent requirement for effective strategies to curb the rising prevalence of obesity. While effective in weight reduction, bariatric surgery is an invasive and irreversible treatment, often inaccessible to a segment of patients who require it the most. Therefore, pharmacological interventions play an increasingly prominent role in therapeutic regimens. Identifying novel mechanisms that increase energy expenditure and reduce caloric intake to elicit sustainable weight loss can be a powerful approach to manage excess adiposity.

[0005] There is thus a renewed interest in controlling obesity by exploiting pathways in white adipose tissue (WAT) to increase energy expenditure, suppress appetite, promote weight loss, and prevent weight gain.SUMMARY

[0006] In some aspects, the current disclosure encompasses a nucleic acid comprising a nucleic acid sequence encoding any one or more of: a) a Gastric Inhibitory Polypeptide Receptor (GIPR), a Glucagon Receptor (GCGR), or a Glucagon-Like Peptide 1 Receptor (GLP-1 R), or a functional variant thereof, or any combination thereof; or b) a ligand of GIPR, GCGR, or GLP-1 R, or a functional variant thereof, or any combination thereof; wherein the nucleic acid sequence is operably linked to at least one adipocyte-specific promoter. In some aspects, the adipocyte-specific promoter is a constitutive promoter or an inducible promoter. In some aspects, the adipocyte-specific promoter is an adiponectin promoter. In some aspects, the adipocyte-specific promoter is an engineered adiponectin promoter. In some aspects, the adiponectin promoter comprises the nucleic acid sequence as set forth in SEQ ID NO: 7 or SEQ ID NO: 8, or a sequence at least, at most or about 60%, 61 %, 62%. 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical hereto.

[0007] In some aspects, the disclosed ligand comprises a gastric inhibitory polypeptide (GIP), a glucagon peptide (GCG), a glucagon like peptide 1 (GLP1), exendin-4 (EX4), a tirzepeptide- like ligand, or a retatrutide(RET)-like ligand, or a functional variant thereof. In some aspects, the functional variant can bind more than one of GIPR, GCGR, and / or GLP-1 R receptors. In some aspects, the nucleic acid sequence encodes one or more repeats of the ligand. In some aspects, the nucleic acid sequence encodes one or more distinct ligands. In some aspects, the nucleic acid sequence further encodes one of more leader sequences. In some aspects, the leader sequence is a GIP, GCG, GLP1 leader sequence, or any variant thereof.

[0008] In some aspects, the disclosed nucleic acid sequence further encodes one or more cleavage sites. In some aspects, the one or more cleavage sites comprise a furin cleavage site, or a variant thereof.

[0009] In some aspects, the nucleic acid further comprises a 5' UTR sequence. In some aspects, the 5' UTR comprises a nucleic acid sequence as set forth in SEQ ID NO: 17 or a sequence at least, at most, or about, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical thereto.

[0010] In some aspects, the nucleic acid sequence comprises a sequence as set forth in any one of SEQ ID NOS: 1 , 2, 4, 5, 11 , or 13, or a sequence at least, at most or about, 60%, 61 %, 62%. 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical hereto. In some aspects, the nucleic acid sequence encodes an amino acid sequence as set forth in any one of SEQ ID NOS: 3, 6, 12, or 14, or a sequence at least, at most, or about, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical hereto. In some aspects, the nucleic acid sequence may comprise a sequence as set forth in SEQ ID NOS: 9, 10, 15, or 16, or a sequence at least, at most, or about, 60%, 61%, 62%. 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical hereto. In some aspects, the nucleic acid sequence comprises a sequence as set forth in any one of SEQ ID NOS: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100, or a sequence at least, at most, or about 80% (or any derivable range therein) identical thereto. In some aspects, the nucleic acid sequence encodes an amino acid sequence as set forth in any one of SEQ ID NOS: 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99, or 101 , or a sequence at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical thereto.

[0011] In some aspects, the one of more leader sequences are encoded by a sequence as set forth in any one of SEQ ID NOS: 18 or 20, or a sequence at least, at most, or about 60%, 61 %, 62%. 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical thereto. In some aspects, the one of more leader sequences comprise an amino acid sequence as set forth in any one of SEQ ID NOS: 19 or 21 , or an amino acid sequence at least, at most, or about, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical thereto.

[0012] In some aspects, the one of more furin cleavage sites are encoded by a sequence as set forth in any one of SEQ ID NOS: 22, 24, 26, 28, 30, 32, 34, or 36, or a sequence at least, at most, or about, 60%, 61 %, 62%. 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivablerange therein) identical thereto. In some aspects, the one of more furin cleavage sites comprise an amino acid sequence as set forth in any one of SEQ ID NOS: 23, 25, 27, 29, 31 , 33, or 35, or an amino acid sequence at least, at most, or about, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical thereto.

[0013] In some aspects, the nucleic acid further encodes a carrier protein as set forth in any one of SEQ ID NOS: 103 or 105, or an amino acid sequence at least about 60%, 61 %, 62%. 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. In some aspects, the nucleic acid further comprises a nucleic acid sequence as set forth in any one of SEQ ID NOS: 103 or 105, or an amino acid sequence at least about 60%, 61 %, 62%. 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0014] In some aspects, the nucleic acid is functional in expressing the one or more of GIPR, GCGR, or GLP-1 R or the one or more ligands thereof in the adipocytes.

[0015] In some aspects, the current disclosure also encompasses a vector comprising a nucleic acid as disclosed herein. In some aspects, the vector is a viral vector.

[0016] Disclosed herein is a viral vector comprising a nucleic acid comprising a nucleic acid sequence encoding any one or more of: a) a Gastric Inhibitory Polypeptide Receptor (GIPR), a Glucagon Receptor (GCGR), or a Glucagon-Like Peptide 1 Receptor (GLP-1 R), or a functional variant thereof, or any combination thereof; or b) a ligand of GIPR, GCGR, or GLP- 1 R, or a functional variant thereof, or any combination thereof; wherein the nucleic acid sequence is operably linked to at least one adipocyte-specific promoter. In some aspects, the viral vector is an AAV vector. In some aspects, the viral vector is a AAV vector of serotype 9.

[0017] In some aspects, disclosed herein are delivery particles comprising any of the disclosed nucleic acids or disclosed viral vectors. In some aspects, the delivery particle is a AAV viral particle. In some aspects, the AAV particle comprises a capsid protein comprising a AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhIO, AAV11 , AAV12, AAV LK03, AAV2R471A, AAV2 / 2-7m8, AAV DJ, an AAV DJ8 capsid, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, goat AAV, AAV1 / AAV2 chimeric, bovine AAV, mouse AAV, AAV Rec2, rAAV2 / HBoV1, AAV2HBKO, AAVPHP.B, or AAVPHP.eB serotype capsid, or a variant thereof. In some aspects, the delivery particle is a lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles,microparticle, peptides, proteins, cells transfected with viral vectors, or nanoparticle mimics, or any combinations thereof.

[0018] Disclosed herein is a host cell comprising any of the disclosed nucleic acids or any one the disclosed viral vectors. In some aspects, the host cell is an HEK293, HEK293T, A549 Hela, CHO, Sf9, SP2 or NSO CELL or any derivative cell line thereof.

[0019] In some aspects, the current disclosure also encompasses a composition comprising any of the nucleic acids, viral vectors, delivery particles or host cells as disclosed herein, and at least one excipient.

[0020] In some aspects, the current disclosure also encompasses a method for preventing and / or treating a metabolic disease in a subject in need thereof, comprising administering to the subject a disclosed composition.

[0021] Also disclosed herein is a method for preventing and / or treating a metabolic disease, wherein the administering to the subject in need thereof comprises injecting the composition into the adipose tissue of the subject, wherein the adipose tissue may be (a) in the subject's body during injection or (b) outside of the subject's body and subsequently transplanted into the subject after injection. In some aspects, the metabolic disease is obesity.

[0022] Also disclosed herein is a method of preventing weight gain and / or inducing weight loss in a subject in need thereof, comprising administering to the subject a disclosed composition. In some aspects of the method, the administering to the subject in need thereof comprises injecting a disclosed composition into an adipose tissue of the subject, wherein the adipose tissue may be (a) in the subject's body during injection or (b) outside of the subject's body and subsequently transplanted into the subject after injection.

[0023] Disclosed herein is a method for suppressing the appetite of a subject in need thereof, comprising administering to the subject any of the disclosed compositions. In some aspects, the subject is a mammal. In some aspects, the subject is administered at least one additional therapy. In some aspects, the administration of the composition results in a adipocyte-specific overexpression of one or more of GIPR, GCGR, or GLP-1 R. In some aspects, the administration of the composition results in a adipocyte-specific overexpression of one or more of gastric inhibitory polypeptide (GIP), a glucagon peptide (GOG), a glucagon like peptide 1 (GLP1), exendin-4 (EX4), a tirzepeptide-like ligand, or a retatrutide(RET)-like ligand, or a functional variant thereof. In some aspects, the adipocyte-specific overexpression is turned off or ceases after 4 weeks, after 6 weeks, after 8 weeks, after 10 weeks, after 12 weeks, after 14 weeks, after 16 weeks, after 20 weeks, after 22 weeks, or after 24 weeks, post administration. In some aspects, the adipocyte-specific overexpression is turned on or induced after about 1 day, after 1 week, after 2 weeks, after 4 weeks, after 6 weeks, after 8 weeks,after 10 weeks, after 12 weeks, after 14 weeks, after 16 weeks, after 20 weeks, after 22 weeks, or after 24 weeks post administration.

[0024] Also disclosed herein is a method of remodeling adipose tissue for esthetical purposes, and / or to support plastics surgery, comprising injecting into a specific adipose tissue depot in the subject in need thereof, a disclosed composition, wherein the adipose tissue may be (a) in the subject's body during injection or (b) outside of the subject's body and subsequently transplanted into the subject after injection.

[0025] Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.

[0026] Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office by request and payment of the necessary fee.

[0028] Aspects of the present disclosure are illustrated by way of example in which:

[0029] FIG. 1A shows GIPR expression data for Cohort A. Violin-plot showing the expression of the GIPR in different cell-types (Clusters 1-16) from single-nuclei RNA-Seq of human abdominal WAT. n=12. Cluster numbers were defined by supervised analysis of established markers and include: mature white adipocytes (Clusters 0 and 9), adipogenic progenitor cells (APCs) (1 , 6), endothelial cells (2, 10), macrophages (3), neutrophils (14), lymphatic endothelial cells (13), T / NK / B cells (11), smooth-muscle cells (4), mast cells (16), pericytes (12) and mesothelial cells (7). Highlighted in dashed circles are cells that predominantly express high levels of GIPR, namely: i) Cluster 0 (high adiponectin-expressing mature adipocytes; red circle), ii) Cluster 7 (mesothelial cells; green circle), and iii) Cluster 12 (pericytes; purple circle).

[0030] FIG. 1B shows GIPR expression data for Cohort B. Violin-plot showing GIPR expression in various cell-types from single-nuclei RNA-Seq of human WAT. The dashed circles highlight adipocytes, mesothelial cells, and pericytes, respectively.

[0031] FIG. 1C shows that GIPR is expressed in specific sub-populations of human white adipocytes. Left: Dot-plot of normalized RNA expression in sub-clusters of 25,817 human white adipocytes from single-nuclei RNA-Sequencing data. Both subcutaneous and visceral adipocytes are included in the analysis. The dot-plot shows the average expression andpercent expression of leptin (LEP) and the GIPR in distinct sub-populations of human adipocytes (hAd1-7). The red dashed boxes highlight the expression of GIPR in the hAd7 subpopulation of adipocytes. Right: Feature-plots of normalized LEP and GIPR expression in human white adipocytes. The plots demonstrate the clusters of leptin-positive and Gl PR- positive expressing white fat cells. The dashed red circle around the central cluster of adipocytes highlights the distinct human adipocyte 7 (hAd7) sub-population, whereas the right cluster shows the hAd6 sub-population.

[0032] FIG. 1D shows GIPR gene expression levels (relative to p-actin) in subcutaneous white adipose tissue (sWAT), gonadal white adipose tissue (gWAT), mesenteric white adipose tissue (mWAT), brown adipose tissue (BAT), and liver from male C57 / BL6 wild-type (WT) mice. n=3-11. Data are shown as mean ± SEM. * p<0.05; ** p<0.01.

[0033] FIG. 1E provides breeding strategy for the induction of the GIPR specifically in white adipose tissue. Fat cell-specific adiponectin promoter-driven rtTA mice were bred with TRE- GIPR mice. Following Dox chow-diet (600 mg / kg Dox) feeding to the resulting GIPR-Adip mice, the GIPR was exclusively induced in adipose tissue depots.

[0034] FIG. 1F shows GIPR gene expression in sWAT, gWAT, mWAT, BAT, and liver from GIPR-Adip mice following 1 week of Dox-chow feeding. n=6-12. Data are shown as mean ± SEM. * p<0.05; ** p<0.01 ; *** p<0.001.

[0035] FIG. 1G provides: [Left] GIPR gene expression in gWAT from WT and GIPR-Adip mice during a time course of Dox-HFD feeding (0-24 h). [Right] A magnification of the 1-3 hour timepoints to highlight the initial acute difference in GIPR expression between groups. n=6-12. Data are shown as mean ± SEM. ***p<0.001.

[0036] FIG. 1H shows cyclic AMP (cAMP) levels in sWAT from WT and GIPR-Adip mice after 2 weeks of Dox-HFD feeding. n=7-10. Data are shown as mean ± SEM. *** p<0.001.

[0037] FIG. 11 shows circulating levels of total GIP in WT and GIPR-Adip mice following Dox- HFD feeding, before and after pharmacological treatment with vehicle control, or long-acting GIPR agonist-085 (GIPRA-085) (300 nmol / kg; single subcutaneous injection -12-14 hours prior to blood collection). n=6. Data are shown as mean ± SEM. *** p<0.001.

[0038] FIGs. 2A-2H demonstrate that GIPR in adipose tissue prevents diet-induced obesity and triggers weight loss. FIG. 2A displays Body weight (g) and % body weight change in WT and GIPR-Adip mice during high-dose 600 mg / kg Dox-HFD feeding. n=9. FIG. 2B shows low- dose 50 mg / kg Dox-HFD feeding. n=7-9. FIG. 2C shows % body weight change in diet-induced obese (DIO) WT and GIPR-Adip mice during Dox-HFD feeding. n=6-8. FIG. 2D shows [Left] fat-mass and [Right] lean body mass of WT and GIPR-Adip mice following 2 weeks of Dox-HFD feeding. n=6. FIG. 2E shows tissue mass (mg tissue / body weight [g]) of sWAT, gWAT, mWAT, BAT, liver, heart, and soleus muscle in WT and GIPR-Adip mice after 2 weeks of Dox- HFD feeding. The dashed box highlights the adipose tissue depots. n=6. FIG. 2F shows the circulating levels of leptin in WT and GIPR-Adip mice. n=7. FIG. 2G shows representative H&E images of sWAT, gWAT and BAT from WT and GIPR-Adip mice following Dox-HFD. Scale bar: 50 pm. FIG. 2H shows body weight change (%) in WT and GIPR-Adip mice injected with vehicle control (WT vehicle: blue line; GIPR-Adip vehicle: orange line) or with the GLP-1 R agonist semaglutide (30 nmol / kg s.c injection) (WT Semaglutide: green line; GIPR-Adip Semaglutide: red line). First segment: all mice were maintained on a HFD (no Dox, thus no GIPR induction), with daily injections of vehicle or semaglutide. Second segment: all mice were switched to a doxycycline-containing HFD (Dox-HFD) to allow GIPR activation in the adipocyte, while maintaining daily injections of vehicle or semaglutide. Third segment: all vehicle and semaglutide injections were stopped, while all mice were maintained on Dox-HFD (GIPR induction in the adipocyte). n=5-9. Data are shown as mean ± SEM. * p<0.05; ** p<0.01 ; *** p<0.001.

[0039] FIGs. 3A-3K demonstrate that adipose tissue GIPR suppresses food intake. FIG. 3A shows [Left] daily food intake and [Right] body weight of pair-fed lean WT and GIPR-Adip mice during Dox-HFD (600 mg / kg Dox). [Left] WT (blue) and GIPR-Adip (red) ad libitum (ad lib) fed mice. n=7. [right] body weight (g) of ad lib-fed WT mice (blue), ad lib-fed GIPR-Adip mice (red), and pair-fed WT mice (green). Note the pair-fed WT group (green) were fed equal amounts of food as ad lib-fed GIPR-Adip mice (red), as such, one red line (left food intake graph) represents both groups. FIG. 3B shows daily food intake over a period of 4 days during metabolic cage analyses of WT and GIPR-Adip mice following 4 days of prior Dox-HFD feeding. n=6. FIG. 3C shows food intake during ad lib Dox-HFD fasting and re-feeding. WT and GIPR-Adip mice were gavaged Dox (600 mg / kg), then immediately fasted for 12-14 h. Following the fast, ad lib re-feeding was measured after 1-2 h, then after a total of 24 h. n=6. FIG. 3D shows daily food intake in WT and GIPR-Adip mice during Dox-HFD feeding. n=6-8. FIG. 3E shows daily food intake in HFD-fed WT mice following treatment with vehicle control (blue) or long-acting GIPR agonist (GIPRA) (300 nmol / kg) (pink). The dashed box highlights the transient, yet significant suppression in food intake with GIPR agonist treatment. n=7. FIG. 3F shows [Left] daily food intake and [Right] % body weight change in WT and GIPR-Adip mice fed either Dox-HFD alone (green and orange, respectively) or rosiglitazone (Rosi)- containing Dox-HFD (blue and red, respectively). n=9. FIG. 3G displays schematic diagram of donor gWAT transplantation into WT recipient mice. Donor WT and GIPR-Adip mice were maintained on chow diet. Several pieces (~50 mg / piece; total of -200 mg) of gWAT were harvested from donor WT control mice (blue) or donor GIPR-Adip mice (red) and thentransplanted and grafted onto the endogenous gWAT depot of recipient WT mice. Dox-HFD was fed to the recipient WT mice harboring either WT or transgenic gWAT transplants. A representative photograph shows the donor gWAT (dashed outline) transplanted and grafted onto an endogenous WT gWAT depot. n=4-6. FIG. 3H shows H&E staining of the: i) endogenous recipient WT gWAT surrounding the WT graft, ii) transplanted donor WT gWAT, iii) endogenous recipient WT gWAT surrounding the grafted GIPR-Adip gWAT and, iv) transplanted GIPR-Adip gWAT. Scale: 50 pm. FIG. 31 shows GIPR gene expression levels in gWAT of endogenous (endo) recipient WT mice, transplanted (trans) gWAT from donor WT or GIPR-Adip mice in addition to endogenous sWAT from recipient WT mice harboring the grafted WT or GIPR-Adip gWAT. n=4-6. FIG. 3J shows [Left] daily food intake and [Right] body weight in recipient WT mice harboring either donor WT or GIPR-Adip gWAT transplants. n=4-6. FIG. 3K depicts [Left] an illustration demonstrating the construction of the adeno- associated virus (AAV) TRE-GIPR vector and the control AAV TRE-mmR2F vector (a membrane-targeted mRuby2-based ‘spaghetti monster’ fluorescent protein). The strategy shows that HFD-fed adiponectin-rtTA mice were directly injected with either AAV TRE-GIPR, or control AAV TRE-mmR2F into sWAT. The use of adiponectin-rtTA mice ensured restriction of GIPR transgene expression from the TRE promoter specifically to mature adipocytes. Following injection and recovery, mice were fed Dox-HFD (600 mg / kg Dox). [Right] Daily food intake and % body weight change of adiponectin-rtTA mice during Dox-HFD feeding, following local injection of control AAV TRE-mmR2F (Ctl-AAV; blue) or AAV TRE-GIPR (GIPR-AAV; pink) in sWAT. n=6. Data are shown as mean ± SEM. * p<0.05; ** p<0.01 ; *** p<0.001.

[0040] FIG. 3L shows [left] GIPR mRNA expression levels in sWAT, gWA, and liver from Dox- HFD-fed adiponectin-rtTA mice following Ctl-AAV injection (black) or GIPR-AAV injection (red). In all mice, the left sWAT depot (sWAT-L) had no AAV injection (i.e., served as an internal negative control) while the right sWAT depot (sWAT-R) received the AAV injection. [Center] GIPR gene expression only in sWAT-L and sWAT-R in mice that received Ctl-AAV. [right] GIPR mRNA expression levels in sWAT-L and sWAT-R in mice that were injected with GIPR-AAV. n=5-6. Data are shown as mean ± SEM. * p<0.05; ** p<0.01.

[0041] FIGs. 3M-3O demonstrate that leptin is not responsible for the transient suppression in food intake in GIPR-Adip mice. FIG. 3M shows a timecourse (hours) of the circulating levels of leptin in WT and GIPR-Adip mice during Dox-HFD feeding. The dashed box indicates the time-point at which leptin levels significantly drop in transgenic mice. n=6-7. FIG. 3N illustrates the breeding strategy for the co-induction of both leptin and the GIPR specifically in adipose tissue. Fat cell-specific GIPR-Adip mice were bred with inducible TRE-leptin mice. Following Dox-HFD feeding, the resulting Lep-GIPR-Adip mice co-overexpressed both leptin and the GIPR exclusively in fat. FIG. 30 shows [Left] daily food intake and [Right] % body weightchange in WT mice (blue), GIPR-Adip mice (orange) (GIPR only induction), Lep-GIPR-Adip mice (red) (both leptin and GIPR co-induction), and Lep mice (green) (leptin only induction) during Dox-HFD feeding. n=9. Data are shown as mean ± SEM. * p<0.05; ** p<0.01.

[0042] FIGs. 4A-4M demonstrate that the GIPR in adipose tissue increases whole-body energy expenditure, promotes lipid uptake and oxidation in fat and enhances energy demand. Metabolic cage analyses were performed in WT and GIPR-Adip mice over a 5 day period. All mice were exposed to 4 days of Dox-HFD feeding before to the initiation of metabolic cage assessment ( / .e. before body weights diverged between WT and GIPR-Adip groups). n=5-6. FIG. 4A shows the locomotor activity in WT and GIPR-Adip mice over a 5 day period. [Left] Total X- and Y-beam breaks and [Right] Total Z-beam breaks. n=5-6. FIG. 4B shows the average oxygen consumption (VO2). FIG. 4C the carbon dioxide (CO2) production and total heat production, and FIG. 4D the respiratory exchange ratio (RER). FIG. 4E shows3H-triolein lipid oxidation (% / g tissue) in WT and GIPR-Adip tissues (sWAT, gWAT, mWAT, BAT, liver, heart, and soleus muscle) (2 pCi / mouse in 100 pl of 5% Intralipid; single retro-orbital injection) from mice fed Dox-HFD for 2 weeks. The dashed box highlights the WAT depots. n=6. FIG. 4F-4H Seahorse analyses of adipose tissues from WT and GIPR-Adip mice fed Dox-HFD for 2 weeks. Approximately 10 mg of sWAT and mWAT was utilized, whereas 4 mg of BAT was used. FIG. 4F shows oxygen consumption rates (OCRs) (pmol / min / mg tissue) in sWAT. The bar graph shows the basal state, the FCCP-stimulated maximal oxidative respiration, and the spare capacity; the latter calculated by the maximal OCR minus the basal OCR, which reflects the ability of surplus energy that can be generated in case of a sudden increase in energy demand. n=4-5. FIG. 4G those in gWAT and mWAT. OCRs are shown in the basal state (DM EM media containing 25 mM glucose, 1 mM sodium pyruvate, and 1 mM L-glutamine), then in response to sequential additions of 4 pM oligomycin, 8 pM carbonyl cyanide 4- (trifluoromethoxy) phenylhydrazone (FCCP), and 10 pM antimycin A + 3 pM rotenone. FIG. 4H shows the energy demand in [Left] WT and GIPR-Adip sWAT and gWAT as well as [Right] mWAT and BAT (right). The glycolytic potential (x-axis) is measured by the extracellular acidification rate (ECAR), whereas the OCR is on the y-axis. The open squares represent the basal unstimulated state, whereas the shift to the filled squares demonstrates the FCCP- stimulated maximal state. The four quadrants in the grid are: i) the quiescent state in the bottom left, ii) the aerobic state in the top left, iii) the glycolytic state in the bottom right, and iv) the energetic state in the top right. The dashed circles highlight the increased energetic state in transgenic fat. n=4-5. FIG. 4I shows the circulating levels of triglycerides (TGs) in WT and GIPR-Adip mice following 2 weeks or 8 weeks of Dox-HFD feeding. n=6-7. FIG. 4J shows a TG clearance test in WT and GIPR-Adip mice following 3 days Dox-HFD. Mice were fasted 14-16 hours and then gavaged 20% Intralipid (15 pl / g body weight). Prior to fasting, mice weresubcutaneously injected with vehicle control or long-acting GIPR agonist (300 nmol / kg). n=5. FIG. 4K shows the whole body3H-triolein lipid clearance rate (rate / sec) in WT and GIPR-Adip mice (2 pCi / mouse in 100 pl of 5% Intralipid; single tail-vein injection) from mice fed Dox-HFD for 2 weeks. n=6. FIG. 4L shows the3H-triolein lipid uptake (% / g tissue) in WT and GIPR-Adip tissues (sWAT, gWAT, mWAT, BAT, liver, heart, and soleus muscle) (2 pCi / mouse in 100 pl of 5% Intralipid; single tail-vein injection) from mice that were fed Dox-HFD for 2 weeks. The dashed box highlights the WAT depots. n=6. FIG. 4M shows non-esterified fatty acid (NEFA) levels and glycerol levels in WT and GIPR-Adip mice during a p3-adrenergic receptor (P3-AR) agonist test following 2 weeks of Dox-HFD feeding. n=7. Data are shown as mean ± SEM. * p<0.05; ** p<0.01 ; *** p<0.001.

[0043] FIGs. 5A-5C demonstrate that adipose tissue GIPR activates a futile calcium cycling pathway to increase energy expenditure and promote weight loss. FIG. 5A shows heat maps of the top 100 differentially-regulated genes in sWAT from WT mice (blue) and GIPR-Adip mice (red) following [Left] 3 days or [Right] 2 weeks of Dox-HFD feeding. n=9. FIG. 5B shows the number and % of genes that overlap and are differentially regulated in sWAT (as identified by RNA-Seq) following 3 days versus 2 weeks of Dox-HFD. FIG. 5C shows the top upregulated pathways in GIPR-Adip sWAT (relative to WT sWAT) following [Top] 3 days or [Bottom] 2 weeks of Dox-HFD. The red dashed boxes highlight the common upregulated calcium signaling pathway, p-values (-log ) are shown on the bottom of each graph.

[0044] FIGs. 5D-5G demonstrate that the GIPR does not utilize browning / beiging or the futile creatine cycle pathways to increase energy expenditure in adipose tissue. FIG. 5D shows gene expression levels of browning / beiging or BAT markers (Ucp1, Prdm16, Pgc1a1, Metrnl, Dio2, Cidea, / 33-AR, Tbx1, Lhx8, and Otopl) in WT and GIPR-Adip sWAT following 2 weeks of Dox-HFD feeding. n=8-9. FIG. 5E shows gene expression levels of futile creatine cycle markers (Ckmtl, Ckmt2, Gamt, Gatm, Slc6a8 / [CrT], and Alp!) in WT and GIPR-Adip sWAT following 2 weeks of Dox-HFD feeding. n=9. FIG. 5F shows creatine kinase activity in WT and GIPR sWAT after 2 weeks of Dox-HFD feeding. n=6-7. FIG. 5G shows gene expression levels of Sercal, Serca2a, and Serca2b in sWAT of WT and GIPR-Adip mice after 2 weeks of Dox- HFD feeding. n=6-9. Data are shown as mean ± SEM. * p<0.05; *** p<0.001.

[0045] FIGs. 5H-5K detail protein expression. FIG. 5H shows SERCA1 protein expression levels in WT and GIPR sWAT. FIG. 5I shows sarcolipin protein expression levels in WT and GIPR sWAT. n=4-5. FIG. 5J shows [Left] SERCA2 and [Right] SERCA2a protein expression levels in gWAT of WT and GIPR-Adip mice. n=4-5. Data are shown as mean ± SEM. ** p<0.01 ; *** p<0.001. FIG. 5K displays representative immunofluorescence staining images of SERCA2 protein in gWAT of WT and GIPR-Adip mice following Dox-HFD feeding. Scale: 20X.

[0046] FIGs. 5L-5O demonstrate the requirement of SERCA2 for the effects of adipocyte GIPR overexpression on energy expenditure. FIG. 5L shows [Left] an illustration showing the adipocyte-targeted GIPR overexpression and Serca2 gRNA CRISPR / Cas9 AAV constructs. The sfGV-CAAX and non-targeting gRNA CRISPR / Cas9 AAV constructs are not shown. The use of an engineered human adiponectin-promoter ensured restriction of concomitant protein overexpression and gene knockout to mature adipocytes. [Center] A schematic illustration of the strategy utilized to record sWAT tissue temperature, i) sfGV-CAAX + non-targeting gRNA AAVs, ii) GIPR + non-targeting gRNA AAVs, iii) GIPR + Serca2 gRNA AAVs, or iv) sfGV- CAAX + Serca2 gRNA AAVs were co-injected directly into the sWAT fat-pad of obese WT HFD-fed mice. Following AAV injection and recovery, mice were maintained on HFD. Temperature probes were attached to the sWAT fat-pad, then tissue temperature was recorded. [Right] sWAT tissue temperature following injection of sfGV-CAAX + non-targeting gRNA AAVs (blue), GIPR + non-targeting gRNA AAVs (red), GIPR + Serca2 gRNA AAVs (orange), or sfGV-CAAX + Serca2 gRNA AAVs (green). n=4. FIG. 5M shows [Left] energy expenditure (kcal / hr) rhythms and [Right] average levels of obese WT HFD-fed mice that were injected with Control AAV+Control-CRISPR (blue), GIPR AAV+Control-CRISPR AAV (red), GIPR AAV+Serca2-CRISPR AAVs (orange), or Control AAV+Serca2-CRISPR AAV (green). The p value shown in the left graph is the ANOVA effect. All values were non-adjusted for body weight. The experiment was performed under thermoneutral (~30°C) conditions. n=3. FIG. 5N shows representative infrared thermal images of WT and GIPR-Adip mice at baseline (t=0 min) or following 30 min or 60 min of a single oral gavage of glucose (2.5 g / kg body weight). FIG. 50 shows core body temperature of WT and GIPR-Adip mice at thermoneutrality (~30°C) during ad lib feeding, 12 hours of fasting, and 6 hours of re-feeding Dox-HFD. n=4-5. Data are shown as mean ± SEM. * p<0.05; ** p<0.01.

[0047] FIGs. 5P-5R outline Seahorse analyses. FIG. 5P shows OCRs (pmol / min / pg protein) in mitochondria (5 pg) isolated from WT and GIPR-Adip gWAT, in response to sequential additions of DMEM media (basal measurements with media containing FCCP, pyruvate, malate, and rotenone (complex I inhibitor)), succinate (complex II substrate), antimycin A (complex III inhibitor), and ascorbate and TMPD (cytochrome c substrate). n=4-5. Data are shown as mean ± SEM. * p<0.05; ** p<0.01 ; *** p<0.001. FIG. 5Q shows [Left] oxygenconsumption rates (OCRs) (pmol / min / pg / pl protein) in differentiated white adipocytes from WT and GIPR-Adip mice. OCR values were normalized to adipocyte protein concentrations (pg / pl). OCRs are shown in the basal state (DMEM media containing glucose, sodium pyruvate and L-glutamine), then in response to sequential additions of oligomycin, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) and antimycin A / rotenone. n=4-5. [Right] Cellular energy demand in differentiated white adipocytes from WT and GIPR-Adipmice. Prior to measurement, WT and GIPR-Adip adipocytes were treated for 1 hour with vehicle (WT vehicle in green; GIPR vehicle in purple) or the SERCA inhibitor thapsigargin (Thap) (300 nM) (WT Thap in blue; GIPR Thap in red). The x-axis highlights the extracellular acidification rate (ECAR), whereas the y-axis highlights the OCRs. The open squares represent the basal unstimulated state, whereas the shift to the filled squares demonstrates the FCCP-stimulated maximal state. The four quadrants in the grid are: i) the quiescent state in the bottom left), ii) the aerobic state in the top left, iii) the glycolytic state in the bottom right and, iv) the energetic state in the top right. The dashed circle highlights the reduction in energy demand in GIPR overexpressing adipocytes treated with thapsigargin. n=4-5. FIG. 5R shows [Left] OCRs (pmol / min / mg tissue) (left) and [Right] energy demand (right) in gWAT perfused (for 30 min) with vehicle control, or the SERCA inhibitor thapsigargin (1 pM), in of WT and GIPR-Adip mice following 2- weeks of Dox-HFD feeding.

[0048] FIG. 5S shows a diagram of a mechanism of GIPR action in the white adipocyte.

[0049] FIGs. 6A-6H demonstrate that the GIPR in adipose triggers a metabolic memory effect to maintain weight loss through a sarcolipin axis. FIG. 6A shows [Left] weekly body weight (g) and [Right] % body weight change of WT mice (black) and GIPR-Adip mice (red) during Dox- HFD feeding at thermoneutrality (~30°C). Note, all mice were lean at the beginning of the time course. The vertical dashed lines indicate whether Dox was present (green) or absent (pink) at each time phase. The green text highlights induction of GIPR in adipose tissue with Dox- HFD feeding. The pink text indicates that the GIPR was turned off in adipose tissue by switching to a HFD-only diet (no Dox). First segment: Mice began Dox-HFD feeding for 12 weeks, inducing the GIPR in adipose tissue (green text). Second segment: Dox was then removed by switching mice to HFD-only feeding for the next 12 weeks, turning off GIPR overexpression in adipose tissue (pink text). Third segment: Dox-HFD was then used to switch GIPR overexpression in adipose tissue back on (green text) following the 12 weeks of imprinting the “metabolic memory” effect. The blue arrow highlights the 12 week timeframe that produced the Gl PR-driven metabolic memory effect on body weight. n=9. FIG. 6B shows % body weight change in DIO WT mice (black) or GIPR-Adip mice (orange). Note, all mice were obese (-35-40 g) at the beginning of the time course. First segment: Obese mice were fed Dox-HFD for 8 weeks, inducing the GIPR in adipose tissue (green text). Second segment: Mice were switched back to HFD-only (no Dox) for the next 8 weeks, turning off GIPR overexpression in adipose tissue (pink text). The blue arrow indicates the 8 week duration that produced the Gl PR-driven metabolic memory effect. n=6-10. FIG. 6C shows [Left] food intake in weeks and [Right] in days of obese WT mice (black) or GIPR-Adip mice (orange) following 8 weeks of Dox-HFD feeding (first segment, green text), followed by 8 weeks of HFD-only (no Dox) feeding (second segment, pink text). n=6-10. FIG. 6D shows GIPR mRNA levels in sWATof obese WT and GIPR-Adip mice following 8 weeks of Dox-HFD (GIPR on), then 8 weeks of HFD (GIPR off). n=4-9. FIG. 6E shows sarcolipin protein expression levels in WT and GIPR- Adip sWAT following 8 weeks of metabolic memory. n=4-5. FIG. 6F shows % body weight change in obese WT or GIPR-Adip mice at thermoneutrality (~30°C). First segment: For ~3 weeks, WT and GIPR-Adip mice were fed either HFD alone (no Dox) (green and orange, respectively) or Dox-HFD (red and blue, respectively). The WT group fed Dox-HFD (blue) were placed on a 50% food-restricted diet (50 % rest.) in an attempt to achieve a comparable rate of weight loss to ad lib-fed GIPR-Adip mice (red). The light blue arrow highlights that “no metabolic memory” was achieved following 3 weeks of Dox-HFD exposure (initial GIPR induction). n=8. FIG. 6G shows GIPR mRNA levels in sWAT from obese WT HFD-fed mice (green), GIPR-Adip HFD-fed mice (orange), WT Dox-HFD-fed mice (50% rest.) (blue) and Dox-HFD-fed GIPR-Adip mice (ad lib) (red) following ~3 weeks of corresponding diet. All mice were then placed on HFD alone (no Dox) for ~5 weeks. n=6-8. FIG. 6H shows sarcolipin protein expression levels in WT and GIPR-Adip sWAT following 3 weeks of “no metabolic memory” effect. n=3. Data are shown as mean ± SEM. *p<0.05; **p<0.01 ; ***p<0.001.

[0050] FIG. 6I demonstrated that there is no metabolic memory effect on body weight or food intake following 5 weeks of GIPR induction in adipose tissue. [Left] Body weight change (%) and [Right] food intake in obese WT or GIPR-Adip mice. First segment: For ~5 weeks, WT and GIPR-Adip mice were fed either HFD alone (no Dox; green and orange, respectively) or Dox-HFD (red and blue, respectively). Second segment: Following these 5 weeks, all mice were switched to a HFD alone diet (no Dox; GIPR transgene switched off). The light blue arrow highlights that “no metabolic memory” was achieved after 5 weeks of Dox-HFD exposure (GIPR induction). n=7-9. Data are shown as mean ± SEM.***p<0.001.

[0051] FIG. 7 provides results of an oral triglyceride tolerance test after adipocyte-specific glucagon receptor (‘AdipoGIcR’ model) overexpression for 3 months in mice on Dox-HFD. n=6 Data are shown as mean ± SEM.*p<0.05.

[0052] FIG. 8 shows the % body weight change in diet-induced obese wild-type mice retro- orbitally injected with control AAVs, GIP AAVs, GLP-1 AAVs, GOG AAVs, or a combination of these. n=6. Data are shown as mean ± SEM.DETAILED DESCRIPTION

[0053] The following detailed description references the accompanying drawings that illustrate various aspects of the present disclosure. The drawings and description are intended to describe aspects of the present disclosure in sufficient detail to enable those skilled in the art to practice the present disclosure. Other components can be utilized and changes can bemade without departing from the scope of the present disclosure. The following description is, therefore, not to be taken in a limiting sense.

[0054] The present disclosure is based, in part, on the surprising discovery that targeted overexpression of the Gastric Inhibitory Polypeptide Receptor (GIPR), Glucagon Receptor (GCGR), and / or Glucagon-Like Peptide 1 Receptor (GLP-1 R) in adipocytes exerts beneficial effects on body weight and energy balance providing a means of selectively targeted interventions within the adipocyte to treat metabolic disorders like obesity.

[0055] GIPR, GCGR, and GLP-1 R are class B G protein-coupled receptors that mediate the effects of their respective peptide hormones — incretins (GIP, GLP-1) and glucagon — and together orchestrate key metabolic pathways. Located in the pancreas, liver, adipose tissue, central nervous system, and other organs, these receptors regulate postprandial insulin secretion, glycemic control, lipid metabolism, energy expenditure, appetite, and body weight. GIPR binds the incretin hormone GIP, secreted by intestinal K cells in response to nutrient ingestion, and potentiates glucose-dependent insulin secretion from pancreatic p-cells. Beyond islets, GIPR is expressed in the hypothalamus and other CNS regions, where its activity lowers food intake and promotes weight loss in preclinical obesity models. GCGR is expressed mainly in liver, but also in kidney, adipose, and CNS and mediates glucagon's role in fasting metabolism by promoting hepatic gluconeogenesis and lipid oxidation. GCGR signaling suppresses triglyceride synthesis, increases fatty acid oxidation during fasting, and supports hepatic response to nutrient scarcity. GLP-1 R is found on pancreatic p-cells, central and peripheral neurons, and other tissues, and is activated by the incretin GLP-1 (30 amino acids) released postprandially from intestinal L-cells. Its activation enhances glucose-dependent insulin secretion, inhibits glucagon release, delays gastric emptying, suppresses appetite, increases satiety, and supports p-cell survival and proliferation. While the role of these receptor related pathways on obesity, diabetes, and associated metabolic disorders has been explored, targeted expression in the adipocytes for therapeutic use have not been explored thus far.I. Terminology

[0056] The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. For example, the use of a singular term, such as, “a” is not intended as limiting of the number of items. Also, the use of relational terms such as, but not limited to, “top,” “bottom,” “left,” “right,” “upper,” “lower,” “down,” “up,” and “side,” are used in the description for clarity in specific reference to the figures and are not intended to limit the scope of the present disclosure or the appended claims.

[0057] Any term of degree such as, but not limited to, “substantially” as used in the description and the appended claims, should be understood to include an exact, or a similar, but not exact configuration. For example, “a substantially planar surface” means having an exact planar surface or a similar, but not exact planar surface. Similarly, the terms “about” or “approximately,” as used in the description and the appended claims, should be understood to include the recited values or a value that is three times greater or one third of the recited values. For example, about 3 mm includes all values from 1 mm to 9 mm, and approximately 50 degrees includes all values from 16.6 degrees to 150 degrees. For example, they can refer to less than or equal to ± 5%, such as less than or equal to ± 2%, such as less than or equal to ± 1 %, such as less than or equal to ± 0.5%, such as less than or equal to ± 0.2%, such as less than or equal to ± 0.1%, such as less than or equal to ± 0.05%.

[0058] The terms “comprising,” “including,” and “having” are used interchangeably in this disclosure. The terms “comprising,” “including,” and “having” mean to include, but not necessarily be limited to the things so described.

[0059] The terms “or” and “and / or,” as used herein, are to be interpreted as inclusive or meaning any one or any combination. Therefore, “A, B, or C” or “A, B, and / or C” mean any of the following: “A,” “B,” or “C;” “A and B;” “A and C;” “B and C;” or “A, B, and C.” An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.

[0060] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991), all of which are incorporated by reference herein. As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0061] The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. When introducing elements of the present disclosure or the preferred aspects(s) thereof, the articles “a,” “an,” “the,” and “said” are intended to mean that there are one or more of the elements. The terms “comprising,” “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. Wherever the terms “comprising” or “including” are used, it should be understood the disclosure also expressly contemplates and encompasses additional aspects“consisting of” the disclosed elements, in which additional elements other than the listed elements are not included.

[0062] The term “about” or “approximately,” as used herein, can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” can mean an acceptable error range for the particular value, such as 10% of the value modified by the term “about.” As used herein, the term “about,” can mean relative to the recited value, e.g., amount, dose, temperature, time, percentage, etc., ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1 %.

[0063] Further, as the present disclosure is susceptible to aspects of many different forms, it is intended that the present disclosure be considered as an example of the principles of the present disclosure and not intended to limit the present disclosure to the specific aspects shown and described. Any one of the features of the present disclosure may be used separately or in combination with any other feature. References to the terms “aspect,” “aspects,” and / or the like in the description mean that the feature and / or features being referred to are included in, at least, one aspect of the description. Separate references to the terms “aspect,” “aspects,” and / or the like in the description do not necessarily refer to the same aspect and are also not mutually exclusive unless so stated and / or except as will be readily apparent to those skilled in the art from the description. For example, a feature, structure, process, step, action, or the like described in one aspect may also be included in other aspects but is not necessarily included. Thus, the present disclosure may include a variety of combinations and / or integrations of the aspects described herein. Additionally, all aspects of the present disclosure, as described herein, are not essential for its practice. Likewise, other systems, methods, features, and advantages of the present disclosure will be, or become, apparent to one with skill in the art upon examination of the figures and the description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be encompassed by the claims.

[0064] The term “nucleic acid” or “polynucleotide” or refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues See, e.g., Batzer et al., Nucleic Acid Res. 19:5081 (1991), the disclosure of which is incorporated in its entirety herein.

[0065] The term “polynucleotide construct” as used herein is a synthetically designed or engineered sequence of nucleotides — DNA or RNA — that is assembled to perform a specific biological function within a host cell or organism. It typically includes one or more functional elements such as promoters (e.g., adipocyte-specific promoter), enhancers, coding sequences (e.g., any one or more of the receptors provided herein, or ligands thereof), untranslated regions (UTRs), introns, and polyadenylation signals, and may also contain regulatory elements that control expression. These constructs are commonly used in gene therapy, molecular biology research, and biotechnology to express proteins, silence genes, or modify genetic material within cells.

[0066] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[0067] Within the context of the application a protein is represented by an amino acid sequence and correspondingly a nucleic acid molecule or a polynucleotide represented by a nucleic acid sequence. Identity and similarity between sequences: throughout this application, each time one refers to a specific amino acid sequence SEQ ID NO (take SEQ ID NO: Y as example), one may replace it by: a polypeptide represented by an amino acid sequence comprising a sequence that has at least 60% sequence identity or similarity with amino acid sequence SEQ ID NO: Y. Another preferred level of sequence identity or similarity is 65%. Another preferred level of sequence identity or similarity is 70%. Another preferred level ofsequence identity or similarity is 75%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 85%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 98%. Another preferred level of sequence identity or similarity is 99%.

[0068] Each amino acid sequence described herein by virtue of its identity or similarity percentage with a given amino acid sequence respectively has in a further preferred aspect an identity or a similarity of at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% with the given nucleotide or amino acid sequence, respectively. The terms “homology,” “sequence identity” and the like are used interchangeably herein. Sequence identity is described herein as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In a preferred aspect, sequence identity is calculated based on the full length of two given SEQ ID NO’s or on a part thereof. Part thereof preferably means at least 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NO's. In the art, “identity” also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. The degree of sequence identity between two sequences can be determined, for example, by comparing the two sequences using computer programs commonly employed for this purpose, such as global or local alignment algorithms. Nonlimiting examples include BLASTp, BLASTn, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, GAP, BESTFIT, or another suitable method or algorithm. A Needleman and Wunsch global alignment algorithm can be used to align two sequences over their entire length or part thereof (part thereof may mean at least 50%, 60%, 70%, 80%, 90% of the length of the sequence), maximizing the number of matches and minimizes the number of gaps. Default settings can be used and preferred program is Needle for pairwise alignment (in an aspect, EMBOSS Needle 6.6.0.0, gap open penalty 10, gap extent penalty: 0.5, end gap penalty: false, end gap open penalty: 10 , end gap extent penalty: 0.5 is used) and MAFFT for multiple sequence alignment ( in an aspect, MAFFT v7Default value is: BLOSUM62 [bl62], Gap Open: 1.53, Gap extension: 0.123, Order: aligned, Tree rebuilding number: 2, Guide tree output: ON [true], Max iterate: 2, Perform FFTS: none is used).

[0069] “Similarity” between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. Similar algorithms used for determination of sequence identity may be used for determination of sequence similarity. Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called conservative amino acid substitutions. As used herein, “conservative” amino acid substitutions refer to the interchangeability of residues having similar side chains.

[0070] For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gin or His; Asp to Glu; Cys to Ser or Ala; Gin to Asn; Glu to Asp; Gly to Pro; His to Asn or Gin; lie to Leu or Vai; Leu to lie or Vai; Lys to Arg; Gin or Glu; Met to Leu or lie; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and Vai to lie or Leu.

[0071] An “individual” or “subject,” as used interchangeably herein, is a mammal. In certain aspects, the individual or subject is a human. In an aspect, the subject has been diagnosed with or is suspected of having a metabolic disorder, for example obesity.

[0072] “AAV” is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. The abbreviation “rAAV” refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or “rAAV vector”). The term “AAV” includes AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. “Primate AAV” refers to AAV isolated from a primate, “non-primate AAV” refers to AAV isolated from a non-primatemammal, “bovine AAV” refers to AAV isolated from a bovine mammal (e.g., a cow), etc. In an aspect, the AAV is AAV9 or a derivative thereof.

[0073] An “rAAV vector” as used herein refers to an AAV vector comprising a polynucleotide sequence not of AAV origin (i.e. a polynucleotide heterologous to AAV), typically a sequence of interest for introducing into a target cell. In general, the heterologous polynucleotide is flanked by at least one, and generally by two AAV inverted terminal repeat sequences (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids.

[0074] An “AAV virus” or “AAV viral particle” or “rAAV vector particle” refers to a viral particle composed of at least one AAV capsid protein (typically by all of the capsid proteins of a wildtype AAV) and an encapsidated polynucleotide rAAV vector. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an “rAAV vector particle” or simply an “rAAV vector”. Thus, production of rAAV particle necessarily includes production of rAAV vector, as such a vector is contained within an rAAV particle.

[0075] “Packaging” refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle.

[0076] AAV “rep” and “cap” genes refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV “packaging genes.”

[0077] A “helper virus” for AAV refers to a virus that allows AAV (e.g. wild-type AAV) to be replicated and packaged by a mammalian cell. A variety of such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein- Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC.

[0078] “Helper virus function(s)” refers to function(s) encoded in a helper virus genome which allow AAV replication and packaging (in conjunction with other requirements for replication and packaging described herein). As described herein, “helper virus function” may be provided in a number of ways, including by providing helper virus or providing, for example, polynucleotide sequences encoding the requisite function(s) to a producer cell in trans.

[0079] An “infectious” virus or viral particle is one that comprises a polynucleotide component which it is capable of delivering into a cell for which the viral species is tropic. The term does not necessarily imply any replication capacity of the virus. As used herein, an “infectious” virus or viral particle is one that can access a target cell, can infect a target cell, and can express a heterologous nucleic acid in a target cell. Thus, “infectivity” refers to the ability of a viral particle to access a target cell, infect a target cell, and express a heterologous nucleic acid in a target cell. Infectivity can refer to in vitro infectivity or in vivo infectivity. Assays for counting infectious viral particles are described elsewhere in this disclosure and in the art. Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Total viral particles can be expressed as the number of viral genome (vg) copies. The ability of a viral particle to express a heterologous nucleic acid in a cell can be referred to as “transduction.” The ability of a viral particle to express a heterologous nucleic acid in a cell can be assayed using a number of techniques, including assessment of a marker gene, such as a green fluorescent protein (GFP) assay (e.g. where the virus comprises a nucleotide sequence encoding GFP), where GFP is produced in a cell infected with the viral particle and is detected and / or measured; or the measurement of a produced protein, for example by an enzyme-linked immunosorbent assay (ELISA). Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Methods of determining the ratio of infectious viral particle to total viral particle are known in the art.

[0080] A “replication-competent” virus (e.g., a replication-competent AAV) refers to a phenotypically wild-type virus that is infectious and is also capable of being replicated in an infected cell (i.e. , in the presence of a helper virus or helper virus functions). In the case of AAV, replication competence generally requires the presence of functional AAV packaging genes. In general, rAAV vectors as described herein are replication-incompetent in mammalian cells (especially in human cells) by virtue of the lack of one or more AAV packaging genes. Typically, such rAAV vectors lack any AAV packaging gene sequences in order to minimize the possibility that replication competent AAV are generated by recombination between AAV packaging genes and an incoming rAAV vector. In general, rAAV vector preparations as described herein are those which contain few if any replication competent AAV (rcAAV, also referred to as RCA).

[0081] As used herein, “regulatory elements” refer to any sequence elements that regulate, positively or negatively, the expression of an operably linked sequence. “Regulatory elements” include, without being limiting, a promoter, an enhancer, a leader, a transcription start site (TSS), a linker, 5' and 3' untranslated regions (UTRs), an intron, a polyadenylation signal, and a termination region or sequence, etc., that are suitable, necessary, or preferred for regulating or allowing expression of the gene or transcribable DNA sequence in a cell. Such additionalregulatory element(s) can be optional and used to enhance or optimize expression of the gene or transcribable DNA sequence. A regulatory sequence can, for example, be inducible, noninducible, constitutive, cell-cycle regulated, metabolically regulated, and the like. A regulatory sequence may be a promoter. As used herein, the term “promoter” refers to a DNA sequence that contains an RNA polymerase binding site, a transcription start site, and / or a TATA box and assists or promotes the transcription and expression of an associated transcribable polynucleotide sequence and / or gene (or transgene). A promoter can be synthetically produced, varied, or derived from a known or naturally occurring promoter sequence or other promoter sequence. A promoter can also include a chimeric promoter comprising a combination of two or more heterologous sequences. A promoter of the present application can thus include variants of promoter sequences that are similar in composition, but not identical to, other promoter sequence(s) known or provided herein. In some exemplary respects, the promoter sequence is adapted to enable expression of a polynucleotide in adipocytes. In an aspect, the promoter is an adiponectin promoter or a derivative thereof.

[0082] As used herein, the term “operably linked” refers to a functional linkage between a promoter or other regulatory element and an associated transcribable DNA sequence or coding sequence of a gene (or transgene), such that the promoter, etc., operates to initiate, assist, affect, cause, and / or promote the transcription and expression of the associated transcribable DNA sequence or coding sequence, at least in certain tissue(s), developmental stage(s) and / or condition(s).

[0083] The term “heterologous” when used in reference to a nucleic acid molecule (such as a coding sequence) or a polypeptide (such as an enzyme) refers to a nucleic acid molecule or a protein that is not natively found in the host organism or cell. “Heterologous” also includes a native coding region, or portion thereof, that is removed from the source organism and subsequently reintroduced into the source organism in a form that is different from the corresponding native gene, e.g., not in its natural location in the organism's genome. The heterologous nucleic acid molecule is deliberately introduced into the host cell. The term “heterologous” is also used synonymously herein with the term “exogenous”.

[0084] As used herein, the term “treating” refers to the application or administration of a composition including one or more active agents to a subject, who is in need of the treatment, for example, having a target disease or disorder, a symptom of the disease / disorder, or a predisposition toward the disease / disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward the disease or disorder. Alleviating a target disease / disorder includes delaying the development or progression of the disease or reducing disease severity. Alleviating the disease does not necessarily require curative results. As used therein,“delaying” the development of a target disease or disorder means to defer, hinder, slow, retard, stabilize, and / or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or individuals being treated. A method that “delays” or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and / or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

[0085] “Development” or “progression” of a disease means initial manifestations and / or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of a target disease or disorder includes initial onset and / or recurrence.

[0086] The term “administration” and variants thereof (e.g., “administering” a composition) in reference to a composition of the disclosure means introducing the composition or a prodrug (for example a caged or inducible polynucleotide) of the composition into the system of the subject in need of treatment. When a composition of the disclosure or prodrug thereof is provided in combination with one or more other active agents (e.g., another anti-obesity drug), “administration” and its variants are each understood to include concurrent and sequential introduction of the composition or prodrug thereof and other agents. The present disclosure includes within its scope prodrugs of the compositions of this disclosure. In general, such prodrugs will be functional derivatives of the compositions of this disclosure which are readily convertible in vivo into the required composition. Thus, in the methods of treatment of the present disclosure, the term “administering” shall encompass the treatment of the various conditions described with the composition specifically disclosed or with a composition which may not be specifically disclosed, but which converts to the specified composition in vivo after administration to the patient.

[0087] The term “therapeutically effective amount” as used herein means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.II. Nucleic acids

[0088] In some aspects, the current disclosure encompasses a nucleic acid comprising a nucleic acid sequence encoding any one or more of a Gastric Inhibitory Polypeptide Receptor (GIPR), a Glucagon Receptor (GCGR), and a Glucagon-Like Peptide 1 Receptor (GLP-1 R), or a functional variant thereof, or any combination thereof. In an aspect, the nucleic acid sequence is operably linked to an adiponectin promoter, such that one or more of the receptors are specifically expressed in the adipocytes.

[0089] In some aspects, the current disclosure encompasses a nucleic acid comprising a nucleic acid sequence encoding the GIPR protein. The nucleic acid sequence encoding the GIPR polypeptide may comprise a wild-type GIPR nucleic acid sequence (including one or more of the wild-type regulatory sequences, introns, UTRs, promoters, terminators, or any combination thereof), a GIPR cDNA sequence, or a recombinant GIPR gene sequence. The recombinant GIPR gene sequence may comprise one or more deletions, one or more insertions, one or more nucleotide substitutions, or any combination thereof, with respect to the wild-type GIPR nucleic acid sequence, or the GIPR cDNA sequence as provided in Table 1. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in any one of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 5, or a sequence at least about 60% identical thereto. In an aspect, the nucleic acid sequence may comprise a nucleic acid sequence at least about 60%, 61%, 62%. 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in any one of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 5.

[0090] In an aspect, the disclosed nucleic acid sequence may encode a GIPR polypeptide sequence or an allelic variant, splice variant, derivative, substitution, deletion, and / or insertion variant, or interspecies homolog thereof. In an aspect, the nucleic acid sequence encodes an amino acid sequence as set forth in any one of SEQ ID NO: 3 or SEQ ID NO: 6, or a functional derivative thereof, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in any one of SEQ ID NO: 3 or SEQ ID NO: 6. In an aspect, the nucleic acid sequence encodes an amino acid as set forth in SEQ ID NO: 3 or SEQ ID NO: 6.

[0091] In some aspects, the current disclosure encompasses a nucleic acid comprising a nucleic acid sequence encoding the GCGR protein. The nucleic acid sequence encoding theGCGR polypeptide may comprise a wild-type GCGR nucleic acid sequence (including one or more of the wild-type regulatory sequences, introns, UTRs, promoters, terminators, or any combination thereof), a GCGR cDNA sequence, or a recombinant GCGR gene sequence. The recombinant GCGR gene sequence may comprise one or more deletions, one or more insertions, one or more nucleotide substitutions, or any combination thereof, with respect to the GCGR cDNA sequence as provided in Table 1. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 11 , or a sequence at least about 60% identical thereto. In an aspect, the nucleic acid sequence may comprise a nucleic acid sequence at least about 60%, 61 %, 62%. 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 11.

[0092] In an aspect, the disclosed nucleic acid sequence may encode a GCGR polypeptide sequence or an allelic variant, splice variant, derivative, substitution, deletion, and / or insertion variant, or interspecies homolog thereof. In an aspect, the nucleic acid sequence encodes an amino acid sequence as set forth in SEQ ID NO: 12, or a functional derivative thereof, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 12.

[0093] In some aspects, the current disclosure encompasses a nucleic acid comprising a nucleic acid sequence encoding the GLP-1 R protein. The nucleic acid sequence encoding the GLP-1 R polypeptide may comprise a wild-type GLP-1 R nucleic acid sequence (including one or more of the wild-type regulatory sequences, introns, UTRs, promoters, terminators, or any combination thereof), a GLP-1 R cDNA sequence, or a recombinant GLP-1 R gene sequence. The recombinant GLP-1 R gene sequence may comprise one or more deletions, one or more insertions, one or more nucleotide substitutions, or any combination thereof, with respect to the GLP-1 R cDNA sequence as provided in Table 1. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 13, or a sequence at least about 60% identical thereto. In an aspect, the nucleic acid sequence may comprise a nucleic acid sequence at least about 60%, 61%, 62%. 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 13.

[0094] In some aspects, the disclosed nucleic acid sequence may encode a GLP-1 R polypeptide sequence or an allelic variant, splice variant, derivative, substitution, deletion,and / or insertion variant, or interspecies homolog thereof. In an aspect, the nucleic acid sequence encodes an amino acid sequence as set forth in SEQ ID NO: 14, or a functional derivative thereof, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 14.

[0095] In some aspects, the nucleic acid may further comprise one or more regulatory sequences operably linked to the GIPR, the GCGR, or the GLP-1 R encoding sequence. Regulatory elements are well known in the art and are also disclosed herein. In some aspects, the regulatory sequence is a promoter. In some aspects, the promoter is an adipocyte-specific promoter, which drive the expression of the introduced gene (transgene) only in adipocytes.

[0096] In an aspect, the nucleic acid sequence encoding the GIPR, the GCGR, and / or the GLP-1 R polypeptide is operably linked to an adiponectin promoter. In an aspect, the adiponectin promoter is operable to bring about cell specific expression of GIPR, GCGR, and / or GLP-1 R in adipocytes. In an aspect, the adiponectin promoter comprises a nucleic acid sequence as set forth in SEQ ID NO: 7 or SEQ ID NO: 8, or a functional derivative thereof. In an aspect, the adiponectin promoter is a full-length promoter comprising all the regulatory elements in the promoter (SEQ ID NO: 7), or a sequence with at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. In an aspect, the adiponectin promoter is a minimal promoter, needed for expression of the operably linked GIPR, GCGR, and / or GLP-1 R nucleic acid sequence in adipocytes. In an aspect, the adiponectin promoter comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in any one of SEQ ID NO: 8.

[0097] In an aspect, the nucleic acid sequence encoding the GIPR, GCGR and / or GLP-1 R polypeptide may further comprise a 5' UTR sequence. In an aspect, the 5' UTR comprises a sequence as set forth in SEQ ID NO: 17 or a sequence with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identity thereto.

[0098] In an aspect, the nucleic acid sequence may comprise a minimal adiponectin promoter as disclosed herein, operably linked to the GIPR encoding sequence and a 3' UTR sequence as disclosed herein. In an aspect, the nucleic acid comprises the minimal promoter as set forth in SEQ ID NO: 8, or a sequence at least about 80% identical thereto, operably linked to the GIPR nucleic acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 4, or SEQID NO: 5, or a sequence at least about 60% identical thereto, and further comprising a 5' UTR sequence set forth in SEQ ID NO: 17, or a sequence at least about 90% identical thereto. In an aspect, the nucleic acid comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to the nucleic acid sequence as set forth in any one of SEQ ID NO: 9 or SEQ ID NO: 10.

[0099] In an aspect, the nucleic acid sequence may comprise a minimal adiponectin promoter as disclosed herein, operably linked to the GCGR encoding sequence and a 3’ UTR sequence as disclosed herein. In an aspect, the nucleic acid comprises the minimal promoter as set forth in SEQ ID NO: 8, or a sequence at least about 80% identical thereto, operably linked to the GCGR nucleic acid sequence set forth in SEQ ID NO: 12, or a sequence at least about 60% identical thereto, and further comprising a 5' UTR sequence set forth in SEQ ID NO: 17, or a sequence at least about 90% identical thereto. In an aspect, the nucleic acid comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in any one of SEQ ID NO: 15.

[0100] In an aspect, the nucleic acid sequence may comprise a minimal adiponectin promoter as disclosed herein, operably linked to the GLP1-R encoding sequence and a 3' UTR sequence as disclosed herein. In an aspect, the nucleic acid comprises the minimal promoter as set forth in SEQ ID NO: 8, or a sequence at least about 80% identical thereto, operably linked to the GLP-1 R nucleic acid sequence set forth in SEQ ID NO: 13, or a sequence at least about 60% identical thereto, and further comprising a 5' UTR sequence set forth in SEQ ID NO: 17, or a sequence at least about 90% identical thereto. In an aspect, the nucleic acid comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in any one of SEQ ID NO: 16.

[0101] In some aspects, the current disclosure also encompasses a nucleic acid comprising a nucleic acid sequence encoding one or more ligand of the GIPR, GCGR, or GLP-1 R, or a functional variant thereof, or any combination thereof. In an aspects, the nucleic acid sequence is operably linked to an adiponectin promoter, such that one or more ligands of the GIPR, GCGR, or GLP-1 R, are specifically expressed in the adipocytes.

[0102] In some aspects, the current disclosure encompasses a nucleic acid comprising a nucleic acid sequence encoding the Gastric Inhibitory Polypeptide (GIP) peptide. The nucleic acid sequence encoding the GIP peptide may comprise a wild-type GIP nucleic acid sequence, or a recombinant GIP sequence. The recombinant GIP peptide coding sequencemay comprise one or more deletions, one or more insertions, one or more nucleotide substitutions, or any combination thereof, with respect to the GIP peptide coding sequence as provided in Table 1. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 38, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 38. In some aspects, the nucleic acid sequence encodes at least two GIP ‘G’ peptides. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 40, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 40.

[0103] In some aspects, the disclosed nucleic acid sequence may encode a GIP peptide or derivative, substitution, deletion, and / or insertion variant, or interspecies homolog thereof. In an aspect, the nucleic acid sequence encodes an amino acid sequence as set forth in SEQ ID NO: 39, or a functional derivative thereof, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 39. In some aspects, the nucleic acid sequence encodes at least two GIP ‘G’ peptides. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 41.

[0104] In some aspects, the current disclosure encompasses a nucleic acid comprising a nucleic acid sequence encoding the Glucagon (GCG) peptide. The nucleic acid sequence encoding the GCG peptide may comprise a wild-type GCG nucleic acid sequence, or a recombinant GCG sequence. The recombinant GCG peptide coding sequence may comprise one or more deletions, one or more insertions, one or more nucleotide substitutions, or any combination thereof, with respect to the GCG peptide coding sequence as provided in Table 1. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 42, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 42. In some aspects, the nucleic acid sequence encodes at least two GCG peptides. In an aspect, the nucleic acid comprises anucleic acid sequence as set forth in SEQ ID NO: 44, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 44.

[0105] In some aspects, the disclosed nucleic acid sequence may encode a GCG peptide or derivative, substitution, deletion, and / or insertion variant, or interspecies homolog thereof. In an aspect, the nucleic acid sequence encodes an amino acid sequence as set forth in SEQ ID NO: 43, or a functional derivative thereof, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 43. In some aspects, the nucleic acid sequence encodes at least two GCG peptides. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 45.

[0106] In some aspects, the current disclosure encompasses a nucleic acid comprising a nucleic acid sequence encoding the Glucagon-Like Peptide 1 (GLP-1) peptide. The nucleic acid sequence encoding the GLP-1 peptide may comprise a wild-type GLP-1 nucleic acid sequence or a recombinant GLP-1 gene sequence. The recombinant GLP-1 sequence may comprise one or more deletions, one or more insertions, one or more nucleotide substitutions, or any combination thereof, with respect to the GLP-1 sequence as provided in Table 1 . In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 46, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 46. In some aspects, the nucleic acid sequence encodes at least two GLP-1 ‘G’ peptides. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 48, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 48.

[0107] In some aspects, the disclosed nucleic acid sequence may encode a GLP-1 peptide or derivative, substitution, deletion, and / or insertion variant, or interspecies homolog thereof. In an aspect, the nucleic acid sequence encodes an amino acid sequence as set forth in SEQID NO: 47, or a functional derivative thereof, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 47. In some aspects, the nucleic acid sequence encodes at least two GLP-1 ‘G’ peptides. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 49.

[0108] In some aspects, the current disclosure encompasses a nucleic acid comprising a nucleic acid sequence encoding the Exendin-4 (EX4) peptide. The nucleic acid sequence encoding the EX4 peptide may comprise a wild-type EX4 coding sequence, or a recombinant EX4 coding sequence. The recombinant EX4 sequence may comprise one or more deletions, one or more insertions, one or more nucleotide substitutions, or any combination thereof, with respect to the EX4 sequence as provided in Table 1. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 50, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 50. In some aspects, the nucleic acid sequence encodes at least two EX4 peptides. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 52, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 52.

[0109] In some aspects, the disclosed nucleic acid sequence may encode an EX4 peptide or derivative, substitution, deletion, and / or insertion variant, or interspecies homolog thereof. In an aspect, the nucleic acid sequence encodes an amino acid sequence as set forth in SEQ ID NO: 51 , or a functional derivative thereof, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 51. In some aspects, the nucleic acid sequence encodes at least two EX4 peptides. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 53.

[0110] In some aspects, the current disclosure also encompasses a nucleic acid encoding one or more multi-receptor agonist, for example tirzepatide(TIR)-like or retatrutide(RET)-like peptides, or a functional variant thereof, or any combination thereof. Tirzepatide is a synthetic peptide drug that functions as a dual agonist of the GLP-1 R and the GIPR receptors. It mimics both GLP-1 and GIP, two incretin hormones involved in regulating blood glucose, insulin secretion, appetite, and body weight. Retatrutide is a synthetic peptide designed for the treatment of obesity and related metabolic disorders. It is a multi-receptor agonist, specifically engineered to simultaneously activate three key receptors involved in metabolic regulation GLP-1 R, GIPR, and GCGR. By targeting these three receptors, retatrutide harnesses the complementary metabolic effects of each pathway, resulting in enhanced weight loss and improved glycemic control. Tirzepatide and retatrutide contain non-natural amino acids that cannot be encoded genetically. To allow genetic expression in adipocytes, the TIR and RET sequences were modified to replace these non-natural and modified amino acids with natural ones. Multiple different replacements were generated in the current disclosure and these are referred to as tirzepatide(TIR)-like and retatrutide(RET)-like (RET) peptides. For clarity, each variant is further referred to with letters in single quotation marks indicating the one letter amino acid code for the replacements. TIR ‘GAK’, for instance, indicates that the three non- natural amino acids in tirzepatide are replaced with glycine (G), alanine (A), and lysine (K), while RET ‘GLKK’ indicates that the four non-natural amino acids in retatrutide were replaced with glycine (G), leucine (L), lysine (K), and lysine (K).

[0111] In some aspects, the current disclosure encompasses a nucleic acid comprising a nucleic acid sequence encoding the TIR ‘GAK’ peptide. The recombinant TIR ‘GAK’ sequence may comprise one or more deletions, one or more insertions, one or more nucleotide substitutions, or any combination thereof, with respect to the TIR ‘GAK’ sequence as provided in Table 1. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 54, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 54. In some aspects, the nucleic acid sequence encodes at least two TIR ‘GAK’ peptides. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 56, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 56.

[0112] In some aspects, the disclosed nucleic acid sequence may encode a Tl R ‘GAK’ peptide or a derivative, substitution, deletion, and / or insertion variant thereof. In an aspect, the nucleic acid sequence encodes an amino acid sequence as set forth in SEQ ID NO: 55, or a functional derivative thereof, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 55. In some aspects, the nucleic acid sequence encodes at least two TIR ‘GAK’ peptides. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 57.

[0113] In some aspects, the current disclosure encompasses a nucleic acid comprising a nucleic acid sequence encoding the TIR ‘GAQ’ peptide. The recombinant TIR ‘GAQ’ sequence may comprise one or more deletions, one or more insertions, one or more nucleotide substitutions, or any combination thereof, with respect to the Tl R ‘GAQ’ sequence as provided in Table 1. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 58, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 58. In some aspects, the nucleic acid sequence encodes at least two TIR ‘GAQ’ peptides. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 60, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 60.

[0114] In some aspects, the disclosed nucleic acid sequence may encode a TIR ‘GAQ’ peptide or a derivative, substitution, deletion, and / or insertion variant thereof. In an aspect, the nucleic acid sequence encodes an amino acid sequence as set forth in SEQ ID NO: 59, or a functional derivative thereof, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 59. In some aspects, the nucleic acid sequence encodes at least two TIR ‘GAQ’ peptides. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 61.

[0115] In some aspects, the current disclosure encompasses a nucleic acid comprising a nucleic acid sequence encoding the TIR ‘GYK’ peptide. The recombinant TIR ‘GYK’ sequence may comprise one or more deletions, one or more insertions, one or more nucleotide substitutions, or any combination thereof, with respect to the TIR ‘GYK’ sequence as provided in Table 1. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 62, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 62. In some aspects, the nucleic acid sequence encodes at least two TIR ‘GYK’ peptides. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 64, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 64.

[0116] In some aspects, the disclosed nucleic acid sequence may encode a Tl R ‘GYK’ peptide or a derivative, substitution, deletion, and / or insertion variant thereof. In an aspect, the nucleic acid sequence encodes an amino acid sequence as set forth in SEQ ID NO: 63, or a functional derivative thereof, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 63. In some aspects, the nucleic acid sequence encodes at least two TIR ‘GYK’ peptides. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 65.

[0117] In some aspects, the current disclosure encompasses a nucleic acid comprising a nucleic acid sequence encoding the TIR ‘GYQ’ peptide. The recombinant TIR ‘GYQ’ sequence may comprise one or more deletions, one or more insertions, one or more nucleotide substitutions, or any combination thereof, with respect to the TIR ‘GYQ’ sequence as provided in Table 1. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 66, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the nucleic acid sequence as set forth in SEQ ID NO: 66. In some aspects, the nucleic acid sequence encodes at least two TIR ‘GYQ’ peptides. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 68, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 68.

[0118] In some aspects, the disclosed nucleic acid sequence may encode a TIR ‘GYQ’ peptide or a derivative, substitution, deletion, and / or insertion variant thereof. In an aspect, the nucleic acid sequence encodes an amino acid sequence as set forth in SEQ ID NO: 67, or a functional derivative thereof, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 67. In some aspects, the nucleic acid sequence encodes at least two TIR ‘GYQ’ peptides. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 69.

[0119] In some aspects, the current disclosure encompasses a nucleic acid comprising a nucleic acid sequence encoding the RET ‘GAIA’ peptide. The recombinant RET ‘GAIA’ sequence may comprise one or more deletions, one or more insertions, one or more nucleotide substitutions, or any combination thereof, with respect to the RET ‘GAIA’ sequence as provided in Table 1. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 70, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 70. In some aspects, the nucleic acid sequence encodes at least two RET ‘GAIA’ peptides. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 72, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 72.

[0120] In some aspects, the disclosed nucleic acid sequence may encode a RET ‘GAIA’ peptide or a derivative, substitution, deletion, and / or insertion variant thereof. In an aspect, the nucleic acid sequence encodes an amino acid sequence as set forth in SEQ ID NO: 71 , or afunctional derivative thereof, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 71. In some aspects, the nucleic acid sequence encodes at least two RET ‘GAIA’ peptides. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 73.

[0121] In some aspects, the current disclosure encompasses a nucleic acid comprising a nucleic acid sequence encoding the RET ‘GAIK’ peptide. The recombinant RET ‘GAIK’ sequence may comprise one or more deletions, one or more insertions, one or more nucleotide substitutions, or any combination thereof, with respect to the RET ‘GAIK’ sequence as provided in Table 1. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 74, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 74. In some aspects, the nucleic acid sequence encodes at least two RET ‘GAIK’ peptides. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 76, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 76.

[0122] In some aspects, the disclosed nucleic acid sequence may encode a RET ‘GAIK’ peptide or a derivative, substitution, deletion, and / or insertion variant thereof. In an aspect, the nucleic acid sequence encodes an amino acid sequence as set forth in SEQ ID NO: 75, or a functional derivative thereof, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 75. In some aspects, the nucleic acid sequence encodes at least two RET ‘GAIK’ peptides. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 77.

[0123] In some aspects, the current disclosure encompasses a nucleic acid comprising a nucleic acid sequence encoding the RET ‘GAKA’ peptide. The recombinant RET ‘GAKA’sequence may comprise one or more deletions, one or more insertions, one or more nucleotide substitutions, or any combination thereof, with respect to the RET ‘GAKA’ sequence as provided in Table 1. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 78, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 78. In some aspects, the nucleic acid sequence encodes at least two RET ‘GAKA’ peptides. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 80, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 80.

[0124] In some aspects, the disclosed nucleic acid sequence may encode a RET ‘GAKA’ peptide or a derivative, substitution, deletion, and / or insertion variant thereof. In an aspect, the nucleic acid sequence encodes an amino acid sequence as set forth in SEQ ID NO: 79, or a functional derivative thereof, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 79. In some aspects, the nucleic acid sequence encodes at least two RET ‘GAKA’ peptides. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 81.

[0125] In some aspects, the current disclosure encompasses a nucleic acid comprising a nucleic acid sequence encoding the RET ‘GAKK’ peptide. The recombinant RET ‘GAKK’ sequence may comprise one or more deletions, one or more insertions, one or more nucleotide substitutions, or any combination thereof, with respect to the RET ‘GAKK’ sequence as provided in Table 1. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 82, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 82. In some aspects, the nucleic acid sequence encodes at least two RET ‘GAKK’ peptides. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 84, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleicacid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 84.

[0126] In some aspects, the disclosed nucleic acid sequence may encode a RET ‘GAKK’ peptide or a derivative, substitution, deletion, and / or insertion variant thereof. In an aspect, the nucleic acid sequence encodes an amino acid sequence as set forth in SEQ ID NO: 83, or a functional derivative thereof, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 83. In some aspects, the nucleic acid sequence encodes at least two RET ‘GAKK’ peptides. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 85.

[0127] In some aspects, the current disclosure encompasses a nucleic acid comprising a nucleic acid sequence encoding the RET ‘GLIA’ peptide. The recombinant RET ‘GLIA’ sequence may comprise one or more deletions, one or more insertions, one or more nucleotide substitutions, or any combination thereof, with respect to the RET ‘GLIA’ sequence as provided in Table 1. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 86, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 86. In some aspects, the nucleic acid sequence encodes at least two RET ‘GLIA’ peptides. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 88, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 88.

[0128] In some aspects, the disclosed nucleic acid sequence may encode a RET ‘GLIA’ peptide or a derivative, substitution, deletion, and / or insertion variant thereof. In an aspect, the nucleic acid sequence encodes an amino acid sequence as set forth in SEQ ID NO: 87, or a functional derivative thereof, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 87. In someaspects, the nucleic acid sequence encodes at least two RET ‘GLIA’ peptides. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 89.

[0129] In some aspects, the current disclosure encompasses a nucleic acid comprising a nucleic acid sequence encoding the RET ‘GLIK’ peptide. The recombinant RET ‘GLIK’ sequence may comprise one or more deletions, one or more insertions, one or more nucleotide substitutions, or any combination thereof, with respect to the RET ‘GLIK’ sequence as provided in Table 1. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 90, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 90. In some aspects, the nucleic acid sequence encodes at least two RET ‘GLIK’ peptides. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 92, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 92.

[0130] In some aspects, the disclosed nucleic acid sequence may encode a RET ‘GLIK’ peptide or a derivative, substitution, deletion, and / or insertion variant thereof. In an aspect, the nucleic acid sequence encodes an amino acid sequence as set forth in SEQ ID NO: 91 , or a functional derivative thereof, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 91. In some aspects, the nucleic acid sequence encodes at least two RET ‘GLIK’ peptides. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 93.

[0131] In some aspects, the current disclosure encompasses a nucleic acid comprising a nucleic acid sequence encoding the RET ‘GLKA’ peptide. The recombinant RET ‘GLKA’ sequence may comprise one or more deletions, one or more insertions, one or more nucleotide substitutions, or any combination thereof, with respect to the RET ‘GLKA’ sequence as provided in Table 1. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 94, or a sequence at least about 80% identical thereto. In an aspect,the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 94. In some aspects, the nucleic acid sequence encodes at least two RET ‘GLKA’ peptides. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 96, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 96.

[0132] In some aspects, the disclosed nucleic acid sequence may encode a RET ‘GLKA’ peptide or a derivative, substitution, deletion, and / or insertion variant thereof. In an aspect, the nucleic acid sequence encodes an amino acid sequence as set forth in SEQ ID NO: 95, or a functional derivative thereof, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 95. In some aspects, the nucleic acid sequence encodes at least two RET ‘GLKA’ peptides. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 97.

[0133] In some aspects, the current disclosure encompasses a nucleic acid comprising a nucleic acid sequence encoding the RET ‘GLKK’ peptide. The recombinant RET ‘GLKK’ sequence may comprise one or more deletions, one or more insertions, one or more nucleotide substitutions, or any combination thereof, with respect to the RET ‘GLKK’ sequence as provided in Table 1. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 98, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 98. In some aspects, the nucleic acid sequence encodes at least two RET ‘GLKK’ peptides. In an aspect, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NO: 100, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid may comprise a nucleic acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in SEQ ID NO: 100.

[0134] In some aspects, the disclosed nucleic acid sequence may encode a RET ‘GLKK’ peptide or a derivative, substitution, deletion, and / or insertion variant thereof. In an aspect, the nucleic acid sequence encodes an amino acid sequence as set forth in SEQ ID NO: 99, or a functional derivative thereof, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 99. In some aspects, the nucleic acid sequence encodes at least two RET ‘GLKK’ peptides. In an aspect, the nucleic acid sequence encodes an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in SEQ ID NO: 101.

[0135] In some aspects, the disclosed nucleic acid may comprise a nucleic acid sequence encoding at least one, at least two, at least three or more of the same and / or different peptides as disclosed herein above and / or provided in Table 1.

[0136] In some aspects, any of the nucleic acid sequences encoding one or more peptide ligands as disclosed herein above, may be operably linked to an adiponectin promoter. Thus, in some aspects, the nucleic acid comprises a nucleic acid sequence as set forth in one or more of SEQ ID NOS: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100, or a sequence at least about 80% identical thereto, operably linked to a nucleic acid sequence as set forth in SEQ ID NOS: 7 or 8, or a sequence at least about 80% identical thereto.

[0137] In some aspects, any of the disclosed nucleic acid may further comprise a nucleic acid sequence encoding a carrier protein. In some aspects, the carrier protein is human albumin (ALB) or a variant thereof, or human immunoglobulin heavy chain. In some aspects, the one of more ligands disclosed herein may be expressed as a fusion protein with the carrier protein. In some aspects, the nucleic acid may comprise a nucleic acid sequence as set forth in any one of SEQ ID NOS: 102, or 104, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid comprises a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in any one of SEQ ID NO: 102 or 104. In some aspects, the nucleic acid may encode a fusion protein, and comprise a nucleic acid sequence as set forth in any one or more of SEQ ID NOS: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100, or a sequence at least about 80% identical thereto; and a nucleic acid sequence as set forth in SEQ ID NO: 102 or 104, or a sequence at least about 80% identical thereto.

[0138] In some aspects, the nucleic acid may encode an amino acid sequence as set forth in SEQ ID NOS: 103 or 105, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid encodes an amino acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in any one of SEQ ID NO: 103 or 105. In some aspects, the nucleic acid encodes a fusion polypeptide comprising the amino acid sequence as set forth in any one of SEQ ID NO: 103 or 105 and further comprising one of more amino acid sequences as set forth in any one of SEQ ID NOS: 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99, or 101 , or a sequence at least about 90% identical thereto.

[0139] In some aspects, any of the disclosed nucleic acid may further comprise a nucleic acid sequence encoding a leader sequence. In some aspects, the leader sequence may be any suitable leader sequence known in the art. In some aspects, the leader sequence may be a GIP leader sequence. In some aspects, the leader sequence may be a GCG / GLP-1 leader sequence. In some aspects, the nucleic acid may comprise a nucleic acid sequence as set forth in SEQ ID NOS: 18 or 20, or a sequence at least about 90% identical thereto. In an aspect, the nucleic acid comprises a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in any one of SEQ ID NO: 18 or 20. In some aspects, the nucleic acid may encode an amino acid sequence as set forth in SEQ ID NOS: 19 or 21 , or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid encodes an amino acid sequence at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in any one of SEQ ID NO: 19 or 20.

[0140] In some aspects, any of the disclosed nucleic acid may further comprise a nucleic acid sequence encoding one or more protease cleavage sites. In some aspects, the protease cleavage site may be any suitable protease cleavage site known in the art. In some aspects, the protease cleavage site may be a furin cleavage site. In some aspects, the nucleic acid may comprise a nucleic acid sequence as set forth in SEQ ID NOS: 22, 24, 26, 28, 30, 32, 34, or 36, or a sequence at least about 90% identical thereto. In an aspect, the nucleic acid comprises a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence as set forth in any one of SEQ ID NO: 22, 24, 26, 28, 30, 32, 34, or 36. In some aspects, the nucleic acid may encode an amino acid sequence as set forth in SEQ ID NOS: 23, 25, 27, 29, 31 , 33, 35, or 37, or a sequence at least about 80% identical thereto. In an aspect, the nucleic acid encodes an amino acid sequence at least about 80%,81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in any one of SEQ ID NO: 23, 25, 27, 29, 31 , 33, 35, or 37.

[0141] In some aspects, the nucleic acid sequences disclosed herein may be codon optimized. Codon optimization is a process used to modify a nucleic acid sequence to enhance its expression in a host cell while maintaining the native amino acid sequence. This process involves replacing codons of the native sequence with those most frequently used in the genes of the host organism. The optimization can be applied to specific regions, including upstream (5') or downstream (3') regions of an open reading frame (ORF), ensuring proper folding and efficient translation. Codon optimization methods are well-established in the field and aim to achieve multiple goals, such as matching codon frequencies between target and host organisms, biasing nucleotide content to alter stability or reduce secondary structures, and minimizing tandem repeat codons or base runs that may impair gene construction or expression. Other objectives include modifying transcriptional and translational control regions, inserting or removing protein signaling sequences, and adjusting translational rates to ensure proper protein folding.

[0142] Codon optimization can also serve functional purposes, such as eliminating cryptic start codons, splice sites, or other destabilizing elements that could interfere with transcription or translation. Additionally, optimization may reduce the frequency of CpG dinucleotides, which are known to provoke heightened immune responses, particularly in therapeutic or vaccine applications. Another benefit of codon optimization is its ability to distinguish a transgene from endogenous sequences, facilitating molecular tracking of transgene DNA and / or expressed mRNA. Codon usage may be assessed using the Codon Adaptation Index (CAI), which measures the deviation of a coding sequence from a reference gene set, and codon usage tables, which are available through databases such as the Codon Usage Database. Tools and services for codon optimization, including ATUM (Menlo Park, CA, USA), GeneArt (Life Technologies), DNA2.0, OptimumGene (GenScript), and algorithms such as DNA Works v3.2.3, provide various approaches for optimizing expression in specific species.Table 1 : List of SequencesIII. Vectors

[0143] In some aspects, the disclosed nucleic acid may be incorporated into a vector such as plasmids, or viruses, viral vectors, cosmids, and artificial chromosomes, for further applications. In an aspect, the current disclosure encompasses a polynucleotide construct, for example, a viral vector or a non-viral vector, comprising the disclosed nucleic acid. Any suitable viral vector can be used in this capacity. A viral vector can comprise any number of viral polynucleotides, alone or in combination with one or more viral proteins, which facilitate delivery, replication, and / or expression of the nucleic acid in a desired host cell. The viral vector can be a nucleic acid comprising all or part of a viral genome, a viral protein / nucleic acid conjugate, a virus-like particle (VLP), or an intact virus particle comprising viral nucleic acids. The disclosed viral vector comprises a polynucleotide construct comprising the nucleic acid encoding any one or more of a) a Gastric Inhibitory Polypeptide Receptor (GIPR), aGlucagon Receptor (GCGR), and a Glucagon-Like Peptide 1 Receptor (GLP-1 R), or a functional variant thereof, or any combination thereof; or b) a ligand of the GIPR, GCGR, or GLP-1 R, or a functional variant thereof, or any combination thereof, operably linked to at least one adipocyte-specific promoter. A viral vector can comprise a wild-type viral particle or a modified viral particle comprising the disclosed polynucleotide construct. The viral vector can be a vector which requires the presence of another vector or wild-type virus for replication and / or expression (e.g., a viral vector can be a helper-dependent virus), such as an AAV vector amplicon. Typically, such viral vectors consist of a wild-type viral particle, or a viral particle modified in its protein or nucleic acid content to increase transgene capacity or aid in transfection, expression, or both of the nucleic acid (examples of such vectors include the herpes virus / AAV amplicons). Typically, a viral vector is similar to or derived from a virus that normally infects humans. Suitable viral vector particles in this respect, include, for example, adenoviral vector particles (including any virus of or derived from a virus of the adenoviridae), adeno-associated viral vector particles (AAV vector particles) or other parvoviruses and parvoviral vector particles, papillomaviral vector particles, flaviviral vectors, alphaviral vectors, herpes viral vectors, pox virus vectors, retroviral vectors, including lentiviral vectors.

[0144] Therefore, in some aspects, disclosed herein are polynucleotide constructs such as an adeno-associated virus (AAV) construct comprising the disclosed nucleic acid.

[0145] In some aspects, polynucleotide constructs provided herein can be of different sizes. In some aspects, a construct is a plasmid and can include a total length of up to about 1 kb, up to about 2 kb, up to about 3 kb, up to about 4 kb, up to about 5 kb, up to about 6 kb, up to about 7 kb, up to about 8 kb, up to about 9 kb, up to about 10 kb, up to about 11 kb, up to about 12 kb, up to about 13 kb, up to about 14 kb, or up to about 15 kb. In some aspects, a construct is a plasmid and can have a total length in a range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 1 kb to about 9 kb, about 1 kb to about 10 kb, about 1 kb to about 11 kb, about 1 kb to about 12 kb, about 1 kb to about 13 kb, about 1 kb to about 14 kb, or about 1 kb to about 15 kb.

[0146] In some aspects, a construct is a viral construct and can have a total number of nucleotides of up to 10 kb. In some aspects, a viral construct can have a total number of nucleotides in the range of about 4.5 kb to 5 kb, or about 4.7 kb. In some aspects, a viral construct can have a total number of nucleotides in the range of about 1 kb to about 2 kb, 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 1 kb to about 9 kb, about 1 kb to about 10 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 2 kb toabout 9 kb, about 2 kb to about 10 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 3 kb to about 9 kb, about 3 kb to about 10 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 4 kb to about 9 kb, about 4 kb to about 10 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 5 kb to about 9 kb, about 5 kb to about 10 kb, about 6 kb to about 7 kb, about 6 kb to about 8 kb, about 6 kb to about 9 kb, about 6 kb to about 10 kb, about 7 kb to about 8 kb, about 7 kb to about 9 kb, about 7 kb to about 10 kb, about 8 kb to about 9 kb, about 8 kb to about 10 kb, or about 9 kb to about 10 kb.

[0147] In some aspects, a construct is a lentivirus construct and can have a total number of nucleotides of up to 8 kb. In some examples, a lentivirus construct can have a total number of nucleotides of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 6 kb to about 7 kb, about 6 kb to about 8 kb, or about 7 kb to about 8 kb.

[0148] In some aspects, a construct is an adeno associated viral (AAV) construct and can have a total number of nucleotides of up to 8 kb. In some aspects, an AAV construct can have a total number of nucleotides in the range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 6 kb to about 7 kb, about 6 kb to about 8 kb, or about 7 kb to about 8 kb.

[0149] Any of the polynucleotide constructs described herein can further include a control sequence, e.g., a control sequence selected from the group of a transcription initiation sequence, a transcription termination sequence, a promoter sequence, an enhancer sequence, an RNA splicing sequence, a polyadenylation (poly(A)) sequence, a Kozak consensus sequence, and / or additional untranslated regions which may house pre- or post- transcriptional regulatory and / or control elements. In some aspects, a promoter can be anative promoter, a constitutive promoter, an inducible promoter, and / or a tissue-specific promoter. Non-limiting examples of control sequences are described herein.AAV particles

[0150] In some aspects, AAV particles can be described as having a serotype, which is a description of the construct strain and the capsid strain. For example, in some aspects an AAV particle may be described as AAV2, wherein the particle has an AAV2 capsid and a construct that comprises characteristic AAV2 Inverted Terminal Repeats (ITRs). In some aspects, an AAV particle may be described as a pseudotype, wherein the capsid and construct are derived from different AAV strains, for example, AAV2 / 9 would refer to an AAV particle that comprises a construct utilizing the AAV2 ITRs and an AAV9 capsid. Additional examples of pseudotyped AAV vectors include, but are not limited to, AAV2 / 1 , AAV2 / 2, AAV2 / 3, AAV2 / 4, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, and AAV2 / 9.

[0151] In some aspects, AAV particles suitable for use according to the present disclosure may comprise or be derived from any natural or recombinant AAV serotype. In some aspects, an AAV according to the present disclosure can be selected from natural serotypes such as AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , and AAV12; or pseudotypes, chimeras, and variants thereof.

[0152] As used herein, the term “chimera” when referring to an AAV vector, or a “chimeric AAV vector,” refers to an AAV vector which comprises a capsid containing VP1 , VP2 and VP3 proteins from at least two different AAV serotypes; or alternatively, which comprises VP1 , VP2 and VP3 proteins, at least one of which comprises at least a portion from another AAV serotype. Examples of chimeric AAV vectors include, but are not limited to, AAV-DJ, AAV- DJ / 8, AAV2G9, AAV2i8, AAV2i8G9, AAV8G9, and AAV9i1.

[0153] In some aspects, an AAV serotype and / or pseudotype according to the present invention is selected from the group comprising or consisting of AAV1 , AAV2, AAV3, AAV 4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV Rec2, AAV106.1 / hu.37, AAV114.3 / hu.4O, AAV127.2 / hu.41 , AAV127.5 / hu.42, AAV128.1 / hu.43, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV16.12 / hu.11 , AAV16.3, AAV16.8 / hu.1O, AAV161.1O / hu.6O, AAV161.6 / hu.61 , AAV1-7 / rh.48, AAV1-8 / rh.49, AAV2i8, AAV2i8G9, AAV2-15 / rh.62, AAV223.1 , AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV2-3 / rh.61 , AAV24.1 , AAV2-4 / rh.5O, AAV2-5 / rh.51 , AAV2.5T, AAV27.3, AAV29.3 / bb.1 , AAV29.5 / bb.2, AAV2G9, AAV3B, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-11 / rh.53, AAV3-3, AAV33.12 / hu.17, AAV33.4 / hu.15, AAV33.8 / hu.16, AAV3-9 / rh.52, AAV3a, AAV3b, AAV4-19 / rh.55, AAV42.12, AAV42-10, AAV42-11 , AAV42-12, AAV42-13, AAV42-15, AAV42- 1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8,AAV42-aa, AAV43-1 , AAV43-12, AAV43-20, AAV43-21 , AAV43-23, AAV43-25, AAV43-5, AAV4-4, AAV44.1 , AAV44.2, AAV44.5, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV4-8 / rh.64, AAV4-9 / rh.54, AAV52.1 / hu.2O, AAV52 / hu.19, AAV5-22 / rh.58, AAV5-3 / rh.57, AAV54.1 / hu.21 , AAV54.2 / hu.22, AAV54.4R / hu.27, AAV54.5 / hu.23, AAV54.7 / hu.24, AAV58.2 / hu.25, AAV6.1 , AAV6.1.2, AAV6.2, AAV7m8, AAV7.2, AAV7.3 / hu.7, AAV-8b, AAV8G9, AAV-8h, AAV9i1 , AAV9.11 , AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61 , AAV9.68, AAV9.84, AAV9.9, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVcy.5R1 , AAVcy.5R2, AAVcy.5R3, AAVcy.5R4, AAVcy.6, AAVhu.1 , AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.8, AAVhu.9, AAVhu.10, AAVhu.11 , AAVhu.12, AAVhu.13, AAVhu.14 / 9, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.19, AAVhu.20, AAVhu.21 , AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31 , AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41 , AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44R1 , AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48R1 , AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51 , AAVhu.52, AAVhu.53, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61 , AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVpi.1 , AAVpi.2, AAVpi.3, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh8R R533A mutant, AAVrh8R A586R mutant, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh. 13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31 , AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.43, AAVrh.44, AAVrh.45, AAVrh.46, AAVrh.47, AAVrh.48, AAVrh.48.1 , AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.50, AAVrh.51 , AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.55, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.59, AAVrh.60, AAVrh.61 , AAVrh.62, AAVrh.64, AAVrh.64R1 , AAVrh.64R2, AAVrh.65, AAVrh.67, AAVrh.68, AAVrh.69, AAVrh.70, AAVrh.72, AAVrh.73, AAVrh.74, AAV-PHP.B, AAV-PHP.A, AAV-G2B-26, AAV-G2B-13, AAV-TH1.1-32, AAV-TH1.1-35, AAV-PHP.B2, AAV-PHP.B3, AAV-PHP.N / PHP.B-DGT, AAV-PHP.B-EST, AAV-PHP.B-GGT, AAV-PHP.B-ATP, AAV-PHP.B-ATT-T, AAV-PHP.B-DGT-T, AAV-PHP.B- GGT-T, AAV-PHP.B-SGS, AAV-PHP.B-AQP, AAV-PHP.B-QQP, AAV-PHP.B-SNP(3), AAV- PHP.B-SNP, AAV-PHP.B-QGT, AAV-PHP.B-NQT, AAV-PHP.B-EGS, AAV-PHP.B-EGT, AAV-PHP.B-DST, AAV-PHP.BDST, AAV-PHP.B-STP, AAV-PHP.B-PQP, AAV-PHP.B-SGN, AAV-PHP.B-SQP, AAV-PHP.B-QIP, AAV-PHP.B-TMP, AAV-PHP.B-TTP, AAV- PHP.S / G2A12, AAV-G2A15 / G2A3, AAV-G2B4, AAV-G2B5, AAV-PHP.S, AAAV, AAV A3.3, AAV A3.4, AAV A3.5, AAV A3.7, AAV CBr-7.3, AAV CBr-7.1 , AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-E1 , AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1 , AAV CHt-2, AAV CHt-3, AAV CHt-6.1 , AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-P1 , AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-N4, AAV CKd-1 , AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-B1 , AAV CKd-B2, AAV CKd-B3, AAV CKdB4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-H1 , AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N9, AAV CLg-F1 , AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-M9, AAV CLv-R6, AAV CLv-1 , AAV CLv1-1 , AAV CLvl-10, AAV CLv1-2, AAV CLv-12, AAV CLv1-3, AAV CLv-13, AAV CLv1-4, AAV CLv1-7, AAV CLv1-8, AAV CLv1-9, AAV CLv-2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-D1 , AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-E1 , AAV CLv-K1 , AAV CLv-K3, AAV CLv-K6, AAV CLv- L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-M1 , AAV CLv-M11 , AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLvM7, AAV CLv-M8, AAV CLv-R1 , AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-8.10, AAV CSp-1 , AAV CSp-10, AAV CSp-11 , AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp- 8.8, AAV CSp-8.9, AAV CSp-9, AAV-LK08, AAV-LK15, AAV Shuffle 100-1 , AAV Shuffle 100-2, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV SM 100-10, AAV SM 100-3, AAV SM 10-1 , AAV SM 10-2, AAV SM 10-8, AAV.VR- 355, AAV-b, AAVC1 , AAVC2, AAVC5, AAVCh.5, AAVCh.5R1 , AAV-DJ, AAV-DJ / 8, AAVF1 / HSC1 , AAVF11 / HSC11 , AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14, AVF15 / HSC15, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, AAVF9 / HSC9, AAV-h, AAVH-1 / hu.1 , AAVH2, AAVH-5 / hu.3, AAVH6, AAVhE1.1 , AAVhEr1.14, AAVhEr1.16, AAVhEr1.18, AAVhER1.23, AAVhEr1.35, AAVhEr1.36, AAVhEr1.5, AAVhEr1.7, AAVhEr1.8, AAVhEr2.16, AAVhEr2.29, AAVhEr2.30, AAVhEr2.31 , AAVhEr2.36, AAVhEr2.4, AAVhEr3.1 , AAVLG-1O / rh.4O, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVLG-9 / hu.39, AAV-LK01 , AAV-LK02, AAV-LK03, AAV-LK03, AAV-LK04, AAV-LK05, AAV- LK06, AAV-LK07, AAV-LK09, AAV-LK10, AAV-LK11 , AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK16, AAV-LK17, AAVLK18, AAV-LK19, AAVN721-8 / rh.43, AAV-PAEC, AAV-PAEC12, AAV-PAEC11 , AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAECS, Anc80, Anc80L65, Anc81 , Anc82, Anc83, Anc84, Anc94, Anc110, Anc113, Anc126, Anc127, BAAV, BNP61 AAV, BNP62 AAV, BNP63 AAV, bovine AAV, caprine AAV, Japanese AAV10 serotype, UPENN AAV10, VOY101 , and VOY201.

[0154] In some aspects, an AAV is an AAV variant that has been genetically modified, e.g., by substitution, deletion or addition of one or several amino acid residues in one or more capsid proteins. Examples of such variants include, but are not limited to, AAV2 with one ormore of Y444F, Y500F, Y730F, and / or S662V mutations; AAV3 with one or more of Y705F, Y731 F, and / or T492V mutations; and AAV6 with one or more of S663V and / or T492V mutations.

[0155] In some aspects, an AAV capsid is modified to comprise at least one surface-bound saccharide or a variant thereof. As used herein, the term “surface-bound,” when referring to the at least one saccharide, means that said at least one saccharide is bound to and exposed at the outer surface of the AAV vector. Suitable examples of saccharides include, but are not limited to, monosaccharides, oligosaccharides, polysaccharides, and variants thereof.AAV constructs

[0156] In some aspects, a polynucleotide construct comprises one or more components derived from or modified from a naturally occurring AAV genomic construct. In some aspects, a sequence derived from an AAV construct is an AAV1 construct, an AAV2 construct, an AAV3 construct, an AAV4 construct, an AAV5 construct, an AAV6 construct, an AAV7 construct, an AAV8 construct, an AAV-DJ / 8 construct, an AAV9 construct, an AAV2.7m8 construct, an AAV8BP2 construct, an AAV293 construct, an AAV-PHP.B construct, or AAV-PHP.eB construct (see e.g., Chan et al., 2017). Additional exemplary AAV constructs that can be used herein are known in the art.

[0157] In some aspects, AAV derived sequences (e.g., which are comprised in a polynucleotide construct) typically include the cis-acting 5' and 3' ITR sequences. Typical AAV2-derived ITR sequences are about 145 nucleotides in length. In some aspects, at least or exactly 80% of a typical ITR sequence (e.g., at least or exactly 85%, at least or exactly 90%, at least or exactly 95%, or at least or exactly 100%, etc.) is incorporated into a construct provided herein. The ability to modify these ITR sequences is within the skill of the art. In some aspects, any of the coding sequences and / or constructs described herein are flanked by 5' and 3' AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including presently identified AAV types.

[0158] In some aspects, polynucleotide constructs described in accordance with this disclosure and in a pattern known to the art are typically comprised of, a coding sequence or a portion thereof, at least one control sequence, and optionally 5' and 3' AAV inverted terminal repeats (ITRs). In some aspects, provided constructs can be packaged into a capsid to create an AAV particle. An AAV particle may be delivered to a selected target cell. In some aspects, provided constructs comprise an additional optional coding sequence that is a nucleic acid sequence (e.g., inhibitory nucleic acid sequence), heterologous to the construct sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., a miRNA, a miRNA inhibitor), or other gene product of interest. In some aspects, a nucleic acid coding sequenceis operatively linked to control components in a manner that permits coding sequence transcription, translation, and / or expression in a cell of a target tissue.

[0159] In some aspects, an unmodified AAV endogenous genome includes two open reading frames, “cap” and “rep,” which are flanked by ITRs. In some aspects, recombinant AAV constructs similarly comprise one or more open reading frames flanked by ITR sequences. In some aspects, an AAV construct also comprises conventional control elements that are operably linked to the coding sequence in a manner that permits its transcription, translation, and / or expression in a cell transfected with the polynucleotide construct or infected with a virus particle produced by the disclosure. In some aspects, an AAV construct optionally comprises a promoter, an enhancer, an untranslated region (e.g., a 5' UTR, a 3' UTR), a Kozak sequence, an internal ribosomal entry site (IRES), splicing sites (e.g., an acceptor site, a donor site), a polyadenylation site, or any combination thereof.

[0160] In some aspects, a construct is an AAV construct. In some aspects, an AAV construct can include at least 500 bp, at least 1 kb, at least 1 .5 kb, at least 2 kb, at least 2.5 kb, at least 3 kb, at least 3.5 kb, at least 4 kb, at least 4.5 kb, or at least 4.7 kb. In some aspects, an AAV construct can include at most 7.5 kb, at most 7 kb, at most 6.5 kb, at most 6 kb, at most 5.5 kb, at most 5 kb, at most 4.5 kb, at most 4 kb, at most 3.5 kb, at most 3 kb, or at most 2.5 kb. In some aspects, an AAV construct can include about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, or about 4 kb to about 5 kb.

[0161] Any of the constructs described herein can further include regulatory and / or control sequences, e.g., a control sequence selected from the group of a transcription initiation sequence, a transcription termination sequence, a promoter sequence, an enhancer sequence, an RNA splicing sequence, a polyadenylation (poly(A)) sequence, a Kozak consensus sequence, and / or any combination thereof. In some aspects, a promoter can be a native promoter, a constitutive promoter, an inducible promoter, and / or a tissue-specific promoter. Non-limiting examples of control sequences are described herein and others are known in the art.AAV capsids

[0162] In some aspects, the present disclosure provides one or more polynucleotide constructs packaged into an AAV capsid. In some aspects, an AAV capsid is from or is derived from an AAV capsid of an AAV2, 3, 4, 5, 6, 7, 8, 9, 10, rh8, rh 10, rh39, rh43, or ancestral (Anc) serotype, or one or more hybrids thereof. In some aspects, an AAV capsid is from an Anc serotype. In some aspects, an AAV capsid is an Anc AAV capsid. An Anc capsid is createdfrom a construct sequence that is constructed using evolutionary probabilities and evolutionary modeling to determine a probable ancestral sequence. Thus, an Anc capsid / construct sequence is not known to have existed in nature. As provided herein, in some aspects, any combination of AAV capsids and AAV constructs (e.g., comprising AAV ITRs) may be used in recombinant AAV particles of the present disclosure.Exemplary AAV construct componentsInverted terminal repeat (ITR) sequences

[0163] AAV derived sequences of a construct typically comprises the cis-acting 5' and 3' ITRs. Generally, ITRs are able to form a hairpin. The ability to form a hairpin can contribute to an ITRs ability to self-prime, allowing primase-independent synthesis of a second DNA strand. ITRs can also aid in efficient encapsidation of an AAV construct in an AAV particle.

[0164] An AAV particle of the present disclosure can comprise an AAV construct comprising a coding sequence and associated elements flanked by 5' and 3' AAV ITR sequences. In some aspects, an ITR is or comprises about 130 nucleic acids. In some aspects, an ITR is or comprises about 145 nucleic acids. In some aspects, all or substantially all of a sequence encoding an ITR is used. In some aspects, an AAV ITR sequence may be obtained from any known AAV, including presently identified mammalian AAV types. In some aspects an ITR is an AAV2 ITR. In some aspects, an ITR is an AAV9 ITR.

[0165] A non-limiting example of a polynucleotide construct of the present disclosure is a “cis- acting” construct comprising a coding sequence, in which said sequence and any associated regulatory elements are flanked by 5' or “left” and 3' or “right” AAV ITR sequences. 5' and left designations refer to a position of an ITR sequence relative to an entire construct, read left to right, in a sense direction. For example, in some aspects, a 5' or left ITR is an ITR that is closest to a promoter (e.g., as opposed to a polyadenylation sequence) for a given construct, when a construct is depicted in a sense orientation, linearly. Concurrently, 3' and right designations refer to a position of an ITR sequence relative to an entire construct, read left to right, in a sense direction. For example, in some aspects, a 3' or right ITR is an ITR that is closest to a polyadenylation sequence and / or stop codon (e.g., as opposed to a promoter sequence) for a given construct, when a construct is depicted in a sense orientation, linearly. In general, ITRs as provided herein are depicted in 5' to 3' order in accordance with a sense strand. Accordingly, one of skill in the art will appreciate that a 5' or “left” orientation ITR can also be depicted as a 3' or “right” ITR when converting from sense to anti sense direction. Further, it is well within the ability of one of skill in the art to transform a given sense ITR sequence (e.g., a 57left AAV ITR) into an antisense sequence (e.g., 37right ITR sequence).One of ordinary skill in the art would understand how to modify a given ITR sequence for use as either a 57left or 37right ITR, or an antisense version thereof.Promoters

[0166] In some aspects, a construct (e.g., an AAV construct) comprises a promoter. The term “promoter” refers to a DNA sequence recognized by enzymes / proteins that can promote and / or initiate transcription of an operably linked gene. For example, a promoter typically refers to, e.g., a nucleotide sequence to which an RNA polymerase and / or any associated factor binds and from which it can initiate transcription. Thus, in some aspects, a construct (e.g., an AAV construct) comprises a promoter operably linked to one of the non-limiting example promoters described herein.

[0167] In some aspects, a promoter is an inducible promoter, a constitutive promoter, a mammalian cell promoter, a viral promoter, a chimeric promoter, an engineered promoter, a tissue-specific promoter, or any other type of promoter known in the art. In some aspects, a promoter is an RNA polymerase II promoter, such as a mammalian RNA polymerase II promoter. In some aspects, a promoter is an RNA polymerase III promoter, including, but not limited to, a H1 promoter, a human U6 promoter, a mouse U6 promoter, or a swine U6 promoter. A promoter will generally be one that is able to promote transcription in a mammalian cell.

[0168] A variety of promoters are known in the art, which in some aspects, can be used herein. Non-limiting examples of promoters that can be used herein in some aspects include: human EF1a, human cytomegalovirus (CMV) (US Patent No. 5,168,062, which is incorporated herein by reference for the purposes described herein), human ubiquitin C (UBC), mouse phosphoglycerate kinase 1 , polyoma adenovirus, simian virus 40 (SV40), p-globin, p-actin, a- fetoprotein, y-globin, p-interferon, y-glutamyl transferase, mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), rat insulin, glyceraldehyde-3-phosphate dehydrogenase, metallothionein II (MT II), amylase, cathepsin, Ml muscarinic receptor, retroviral LTR (e.g., human T-cell leukemia virus HTLV), AAV ITR, interleukin-2, collagenase, platelet-derived growth factor, adenovirus 5 E2, stromelysin, murine MX gene, glucose regulated proteins (GRP78 and GRP94), a-2-macroglobulin, vimentin, MHC class I gene H-2K b, HSP70, proliferin, tumor necrosis factor, thyroid stimulating hormone a gene, immunoglobulin light chain, T-cell receptor, HLA DQa and DQ, interleukin-2 receptor, MHC class II, MHC class II HLA-DRa, muscle creatine kinase, prealbumin (transthyretin), elastase I, albumin gene, c-fos, c-HA-ras, neural cell adhesion molecule (NCAM), H2B (TH2B) histone, rat growth hormone, human serum amyloid (SAA), troponin I (TN I), Duchenne muscular dystrophy, human immunodeficiency virus, and Gibbon Ape Leukemia Virus (GAL V) promoters. Additionalexamples of promoters are known in the art. See, e.g., Lodish, Molecular Cell Biology, Freeman and Company, New York 2007, each of which is incorporated herein by reference for the purposes described herein. In some aspects, a promoter is the CMV immediate early promoter. In some aspects, the promoter is a CAG promoter and / or a CAG / CBA promoter.

[0169] The term “constitutive” promoter refers to a nucleotide sequence that, when operably linked with a nucleic acid encoding a gene, causes RNA to be transcribed from the nucleic acid in a cell under most or all physiological conditions. Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter (see, e.g., Boshart et al., Cell 41 :521-530, 1985, which is incorporated herein by reference for the purposes described herein), the SV40 promoter, the dihydrofolate reductase promoter, the p-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1a promoter.

[0170] Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech, and Ariad. Additional examples of inducible promoters are known in the art. Examples of inducible promoters regulated by exogenously supplied compounds include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (see e.g., WO 98 / 10088, which is incorporated herein by reference for the purposes described herein), the ecdysone-inducible promoter (see e.g., No et al., Proc. Natl. Acad Sci. U.S.A 93:3346-3351 , 1996, which is incorporated herein by reference for the purposes described herein), the tetracycline- repressible system (see e.g., Gossen et al., Proc. Natl. Acad Sci. U.S.A 89:5547-5551 , 1992, which is incorporated herein by reference for the purposes described herein), the tetracyclineinducible system (see e.g., Gossen et al., Science 268: 1766-1769, 1995, see also Harvey et al., Curr. Opin. Chem. Biol. 2:512-518, 1998, each of which is incorporated herein by reference for the purposes described herein), the RU486-inducible system (see e.g., Wang et al., Nat. Biotech. 15:239- 243, 1997, and Wang et al., Gene Ther. 4:432-441 , 1997, each of which is incorporated herein by reference for the purposes described herein), and the rapamycin- inducible system (see e.g., Magari et al., J Clin. Invest. 100:2865-2872, 1997, which is incorporated herein by reference for the purposes described herein).

[0171] The term “tissue-specific” promoter refers to a promoter that is active only in certain specific cell types and / or tissues (e.g., transcription of a specific gene occurs only within cells expressing transcription regulatory and / or control proteins that bind to the tissue-specificpromoter). In some aspects, regulatory and / or control sequences impart tissue-specific gene expression capabilities. In some cases, tissue-specific regulatory and / or control sequences bind tissue-specific transcription factors that induce transcription in a tissue-specific manner. In some aspects, a tissue-specific promoter is a adipocyte-specific promoter.Enhancers

[0172] In some aspects, a construct can include an enhancer sequence. The term “enhancer” as used herein refers to a nucleotide sequence that can increase the level of transcription of a nucleic acid encoding a protein and / or RNA molecule of interest, and / or increase or modify the translational efficiency of a transcript following transcription. In some aspects, enhancer sequences (generally 50-1500 bp in length) generally increase the level of transcription by providing additional binding sites for transcription-associated proteins (e.g., transcription factors), and / or stabilize or modify post-transcriptional regulatory machinery. In some aspects, an enhancer sequence is found within an intronic sequence. In some aspects, an enhancer sequence is found in a 3' and / or 5' UTR. In some aspects, an enhancer region is found downstream of a coding sequence comprising a transgene and proximal to a poly adenylation sequence. Unlike promoter sequences, enhancer sequences can act at much larger distance away from the transcription start site (e.g., as compared to a promoter). Non-limiting examples of enhancers include a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), RSV enhancer, a CMV enhancer, and / or a SV40 enhancer.Flanking untranslated regions (5' UTR and 3' UTR)

[0173] In some aspects, any of the polynucleotide constructs described herein can include an untranslated region (UTR), such as a 5' UTR or a 3' UTR. UTRs of a gene are transcribed but not translated. A 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon. A 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal. The regulatory and / or control features of a UTR can be incorporated into any of the constructs, particles, polynucleotide constructs, compositions, kits, or methods as described herein to enhance or otherwise modulate the expression of a gene.

[0174] Natural 5' UTRs include a sequence that plays a role in translation initiation. In some aspects, a 5' UTR can comprise sequences, like Kozak sequences, which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus sequence CCRCCAUGG, where R is a purine (A or G) three bases upstream of the start codon (AUG), and the start codon is followed by another “G”. In some aspects, 5' UTRs also form secondary structures that are involved in elongation factor binding. In some aspects, a 5' UTR is included in any of the constructs described herein.Non-limiting examples of 5' UTRs, including those from the following genes: albumin, serum amyloid A, Apolipoprotein A / B / E, transferrin, a-fetoprotein, erythropoietin, and Factor VIII, can be used to enhance expression of a nucleic acid molecule, such as an mRNA. In some aspects, the 5' UTR comprises a nucleic acid sequence as set forth in SEQ ID NO: 17, or a sequence at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0175] 3' UTRs are known to have stretches of adenosines and uridines (in the RNA form) or thymidines (in the DNA form) embedded in them. These AU-rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU-rich elements (AREs) can be separated into three classes (see e.g., Chen et al., Mol. Cell. Biol. 15:5777-5788, 1995; Chen et al., Mol. Cell Biol. 15:2010-2018, 1995, each of which is incorporated herein by reference for the purposes described herein): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. For example, c-Myc and MyoD mRNAs contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U / A) (U / A) nonamers. GM-CSF and TNF-a mRNAs are examples that contain class II AREs. Class III AREs are less well defined. These U-rich regions do not contain an AUUUA motif, two well-studied examples of this class are c-Jun and myogenin mRNAs.

[0176] Most proteins binding to AREs are known to destabilize the mRNA, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules may lead to HuR binding and thus, stabilization of the mRNA in vivo.

[0177] In some aspects, the introduction, removal, or modification of 3' UTR AREs can be used to modulate the stability of an mRNA encoding a gene of interest. In other aspects, AREs can be removed or mutated to increase the intracellular stability and thus increase translation and production of a protein of interest.

[0178] In some aspects, non-ARE sequences may be incorporated into the 5' or 3' UTRs. In some aspects, introns or portions of intron sequences may be incorporated into the flanking regions of the polynucleotide constructs in any of the constructs, particles, polynucleotide constructs, compositions, kits, and methods provided herein. Incorporation of intronic sequences may increase protein production as well as mRNA levels.Cleavage sites

[0179] In some aspects, a polynucleotide construct disclosed herein may further comprise one or more protease cleavage sites. Incorporation of protease cleavage sites may beessential for activating many precursor proteins, such as hormones, growth factors, viral and membrane-bound receptors, by converting them into their functional forms. In some aspects, the protease cleavage site can be any site known in the art. In some aspects, the protease cleavage site is a furin cleavage site. The presence of a furin cleavage site can significantly influence protein function, localization, and infectivity in the case of viruses. For example, furin cleavage sites are incorporated into Adeno-Associated Virus (AAV) vectors for several reasons related to enhancing their functionality and efficiency in gene therapy applications. In some aspects, the one or more furin cleavage sites are encoded by a sequence as set forth in any one of SEQ ID NOS: 22, 24, 26, 28, 30, 32, 34, 36, or a sequence at least about 60% identical thereto. In some aspects, the one of more furin cleavage sites comprise an amino acid sequence as set forth in any one of SEQ ID NOS: 23, 25, 27, 29, 31 , 33, 35, or an amino acid sequence at least about 90% identical thereto.Internal ribosome entry sites (IRES)

[0180] In some aspects, a construct described herein can include an internal ribosome entry site (IRES). An IRES forms a complex secondary structure that allows translation initiation to occur from any position with an mRNA immediately downstream from where the IRES is located. There are several IRES sequences known to those in skilled in the art, including those from, e.g., foot-and-mouth disease virus (FMDV), encephalomyocarditis virus (EMCV), human rhinovirus (HRV), cricket paralysis virus, human immunodeficiency virus (HIV), hepatitis A virus (HAV), hepatitis C virus (HCV), and poliovirus (PV).

[0181] In some aspects, an IRES can be utilized in an AAV construct. In some aspects, a construct can include a polynucleotide construct internal ribosome entry site (IRES). In some aspects, an IRES can be part of a composition comprising more than one construct. In some aspects, an IRES is used to produce more than one polypeptide from a single gene transcript.Ribosomal skipping (2A) peptides

[0182] In some aspects, a construct described herein can include a ribosomal skipping (2A) peptide. A 2A peptide (approximately 18 amino acids in length) can suppress peptide bond formation without termination (i.e. , ribosomal skipping) during translation of a protein. There are several 2A peptides known to those in skilled in the art, including those from, e.g., equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), porcine teschovirus (P2A), and thosea asigna virus (T2A). The ribosomal skipping activities of 2A peptides have previously been demonstrated in artificial systems including plasmids and gene therapy constructs (e.g., AAV and retroviruses).

[0183] In some aspects, a 2A peptide can be utilized in an AAV construct. In some aspects, a construct can include a peptide 2A. In some aspects, a 2A peptide can be part of acomposition comprising more than one construct. In some aspects, a 2A peptide is used to produce more than one polypeptide from a single gene transcript.Splice sites

[0184] In some aspects, any of the constructs provided herein can include splice donor and / or splice acceptor sequences, which are functional during RNA processing occurring during transcription. In some aspects, splice sites are involved in trans-splicing.Polyadenylation sequences

[0185] In some aspects, a construct provided herein can include a polyadenylation (poly(A)) signal sequence. Most nascent eukaryotic mRNAs possess a poly(A) tail at their 3' end, which is added during a complex process that includes cleavage of the primary transcript and a coupled polyadenylation reaction driven by the poly(A) signal sequence. A poly(A) tail confers mRNA stability and transferability. In some aspects, a poly(A) signal sequence is positioned 3' to a coding sequence.

[0186] As used herein, “polyadenylation” refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. A 3' poly(A) tail is a long sequence of adenine nucleotides (e.g., 50, 60, 70, 100, 200, 500, 1000, 2000, 3000, 4000, or 5000) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase. In some aspects, a poly(A) tail is added onto transcripts that contain a specific sequence, e.g., a poly(A) signal. A poly(A) tail and associated proteins aid in protecting mRNA from degradation by exonucleases. Polyadenylation also plays a role in transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation typically occurs in the nucleus immediately after transcription of DNA into RNA, but also can occur later in the cytoplasm. After transcription has been terminated, an mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase. A cleavage site is usually characterized by the presence of the base sequence AALIAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3' end at the cleavage site.

[0187] As used herein, a “poly(A) signal sequence” or “polyadenylation signal sequence” is a sequence that triggers the endonuclease cleavage of an mRNA and the addition of a series of adenosines to the 3' end of the cleaved mRNA.

[0188] There are several poly(A) signal sequences that can be used in some aspects, including those derived from bovine growth hormone (bGH), mouse p-globin, mouse a-globin, human collagen, polyoma virus, the Herpes simplex virus thymidine kinase gene (HSV TK),IgG heavy-chain gene polyadenylation signal, human growth hormone (hGH), and / or the group consisting of SV40 poly(A) site, such as the SV40 late and early poly(A) site.

[0189] In some aspects, the poly(A) signal sequence can be AATAAA. The AATAAA sequence may be substituted with other hexanucleotide sequences with homology to AATAAA and that are capable of signaling polyadenylation, including ATT AAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATATA, AAGAAA, AATAAT, AAAAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATAAC, AATAGA, AATTAA, or AATAAG. In some aspects, a poly(A) signal sequence can be a synthetic polyadenylation site.Additional sequences

[0190] In some aspects, constructs of the present disclosure may comprise a 2A element or sequence. In some aspects, constructs of the present disclosure may include one or more cloning sites. In some such aspects, cloning sites may not be fully removed prior to manufacturing for administration to a subject. In some aspects, cloning sites may have functional roles including as linker sequences, or as portions of a Kozak site. As will be appreciated by those skilled in the art, cloning sites may vary significantly in primary sequence while retaining their desired function.

[0191] In some aspects, a 2A peptide is a E2A, F2A, P2A, and / or T2 peptide. In some aspects, a 2A peptide may comprise an optional 5' linker sequence, such as but not limited to GSG (e.g., Glycine, Serine, Glycine).Destabilization domains

[0192] In some aspects, any of the constructs provided herein can optionally include a sequence encoding a destabilizing domain (“a destabilizing sequence”) for temporal and / or spatial control of protein expression. Non-limiting examples of destabilizing sequences include sequences encoding a FK506 sequence, a dihydrofolate reductase (DHFR) sequence, or other exemplary destabilizing sequences.

[0193] In the absence of a stabilizing ligand, a protein sequence operatively linked to a destabilizing sequence is degraded by ubiquitination. In contrast, in the presence of a stabilizing ligand, protein degradation is inhibited, thereby allowing the protein sequence operatively linked to the destabilizing sequence to be actively expressed. As a positive control for stabilization of protein expression, protein expression can be detected by conventional means, including enzymatic, radiographic, colorimetric, fluorescence, or other spectrographic assays, fluorescent activating cell sorting (FACS) assays, and / or immunological assays (e.g., enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry).

[0194] Additional examples of destabilizing sequences and ligands are known in the art. In some aspects, the destabilizing sequence is a FK506- and rapamycin-binding protein (FKBP12) sequence and the stabilizing ligand is Shield-1 (Shldl). In some aspects, the destabilizing sequence is a DHFR sequence and the stabilizing ligand is trimethoprim (TMP).Reporter sequences or elements

[0195] In some aspects, constructs provided herein can optionally include a sequence encoding a reporter polypeptide and / or protein (“a reporter sequence”). Non-limiting examples of reporter sequences include DNA sequences encoding: a p-lactamase, a p-galactosidase (LacZ), an alkaline phosphatase, a thymidine kinase, a blue fluorescent protein (BFP), a cyan fluorescent protein (CFP), a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a red fluorescent protein (RFP), a near-infrared fluorescent protein (niRFP), a chloramphenicol acetyltransferase (CAT), and / or a luciferase. Additional examples of reporter sequences are known in the art. When associated with control elements which drive their expression, the reporter sequence can provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence, or other spectrographic assays, fluorescent activating cell sorting (FACS) assays and / or immunological assays (e.g., enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry). In some aspects, a reporter sequence is a FLAG tag (e.g., a 3xFLAG tag), and the presence of a construct carrying the FLAG tag in a cell is detected by protein binding or detection assays (e.g., Western blots, immunohistochemistry, radioimmunoassay (RIA), mass spectrometry).

[0196] In some aspects, a reporter sequence is the LacZ gene, and the presence of a construct carrying the Lacz gene in a cell is detected by assays for p-galactosidase activity. In some aspects, a reporter sequence is a fluorescent protein (e.g., green fluorescent protein (GFP)) or luciferase. In aspects where a reporter sequence is a fluorescent protein or luciferase, the presence of a construct carrying the fluorescent protein or luciferase in a cell may be measured by fluorescent imaging techniques (e.g., fluorescent microscopy or FACS) or light production in a luminometer (e.g., a spectrophotometer or an MS imaging instrument). In some aspects, a reporter sequence can be used to verify tissue-specific targeting capabilities and / or tissue-specific promoter regulatory and / or control activity of any of the constructs described herein.

[0197] In some aspects, the current disclosure also encompasses a polynucleotide construct comprising a nucleic acid comprising a nucleic acid sequence encoding any one or more of a) a Gastric Inhibitory Polypeptide Receptor (GIPR), a Glucagon Receptor (GCGR), and a Glucagon-Like Peptide 1 Receptor (GLP-1 R), or a functional variant thereof, or any combination thereof; or b) a ligand of GIPR, GCGR, or GLP-1 R, or a functional variant thereof,or any combination thereof and one or more regulatory sequences as described above. In an aspect, the nucleic acid may comprise an engineered adiponectin promoter. In an aspect, the polynucleotide construct described herein is suitable for viral or non-viral delivery systems. Description of a viral vector suitable for use in delivery of the disclosed polynucleotide sequence is provided herein above. However, in some aspects, the current disclosure also encompasses non-viral vectors or delivery systems comprising the polynucleotide sequence encoding a GIPR, GCGR, GLP-1 R polypeptide and / or ligand of GIPR, GCGR, GLP-1 R as disclosed herein. Non-limiting examples of non-viral vectors and delivery systems include transposons, plasmids, polynucleotides formulated with delivery systems like polymers, polyplexes, lipids, lipidoids, lipoplexes, liposomes, polymer nanoparticles, nanoparticles, lipid nanoparticles (LNPs), core-shell nanoparticles, solid lipid nanoparticles, metal nanoparticles, self-assembled nucleic acid nanoparticles, hyaluronidase, nanoparticle mimics, ribonucleoproteins, positively charged peptides, small molecule RNA-conjugates, aptamer- RNA chimeras, RNA-fusion protein complexes and any combination thereof. In an aspect, the non-viral delivery system may be a nanoparticle. The nanoparticle may have a monolayer enclosing the nanoparticle core, wherein the polynucleotide is disposed within the nanoparticle core. In an aspect, the nanoparticle core includes a solid lipid (i.e. , lipid that remains solid at room temperature and body temperature) or a liquid lipid (i.e., oil, which remains liquid at room temperature and body temperature, for example, vegetable oil or a lipid extracted from human adipose tissue). In particular, aspects of the present disclosure include nanoparticles and compositions for the controlled and / or sustained release (e.g., release at a predetermined rate to maintain a certain concentration for a certain period of time) of an agent. All vector or delivery systems can be used in vitro, ex vivo, or in vivo in cell cultures, tissue culture, ex vivo cell, tissue, or organ samples, or live animals.IV. Cells and method of transduction or transfection

[0198] In some aspects the current disclosure also encompasses a cell comprising the nucleic acid, polynucleotide construct, or a vector, for example a viral vector, as disclosed herein. In some aspects, the cells are from a cell line commonly used to maintain and grow viral vectors and / or maintain polynucleotide constructs. In another aspect, contemplated are the use of host cells into which a polynucleotide construct, vector, or nucleic acid has been introduced. A polynucleotide construct encoding the disclosed receptors, or ligands thereof can be transfected into cells according to a variety of methods known in the art. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Some vectors may employ control sequences that allow it to be replicated and / or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would understand the conditions under which to incubate host cells to maintain them and to permit replication ofa vector. Also understood and known are techniques and conditions that would allow large- scale production of vectors. Host cells which may be used to maintain and produce disclosed viral vectors and / or the polynucleotide constructs include HEK293, HEK293T, HeLa, Sf9, BHK-21 , A549, Vero, CHO, PER.C6. In some aspects, the cell may be infected or transduced with the viral vector or transfected with the polynucleotide construct in vivo (for therapeutic purposes), ex vivo (for example, into patient derived cells) or in vitro for testing, production and / or maintenance.

[0199] Viral vectors may be introduced into a desired cell by direct transduction or infection, in which viral particles are simply added to the culture medium containing the target cells or administered in vivo. The viral vector binds to specific receptors on the cell surface, leading to entry via endocytosis or membrane fusion, depending on the virus type. Lentivirus, adenovirus, and adeno-associated virus (AAV) all utilize this method. The efficiency of direct infection or transduction may be optimized by changing the multiplicity of infection (MOI): the ratio of viral particles to target cells, which should be carefully adjusted to balance efficiency and toxicity, the incubation time, or inclusion of various chemicals such as polybrene, protamine sulfate, or hexadimethrine bromide which increase viral binding and internalization, particularly for retroviral and lentiviral vectors.

[0200] In some aspects, the current disclosure also encompasses a method of infecting or transducing an adipocyte with the viral vector disclosed, the method comprising, the method comprising directly into an adipocyte. In some aspects, the method comprises injecting the composition into the adipose tissue of a subject, wherein the adipose tissue may be (a) in the subject's body during injection or (b) outside of the subject's body and subsequently transplanted into the subject after injection.

[0201] In addition to viral delivery employing the viral vectors mentioned above, several non- viral methods for the transfer of polynucleotide constructs into cultured mammalian cells also are contemplated by the present disclosure. These include calcium phosphate precipitation, DEAE-dextran, electroporation, direct microinjection, DNA-loaded liposomes and lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection. Some of these techniques may be successfully adapted for in vivo or ex vivo use.

[0202] Once the polynucleotide construct has been delivered into the cell the nucleic acid encoding the gene of interest may be positioned and expressed at different sites. In certain aspects, the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement), or it may be integrated in a random, non-specific location(gene augmentation). In yet further aspects, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the polynucleotide construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of construct employed.

[0203] In yet another aspect, the polynucleotide construct may simply consist of naked recombinant DNA (for e.g. the disclosed nucleic acid) or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product.

[0204] In still another aspect for transferring a naked DNA expression construct (e.g., the disclosed nucleic acid) into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them. Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force. The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.

[0205] In some aspects, the expression construct is delivered directly to adipocytes of a subject. Adipocytes, commonly known as fat cells, are specialized cells that store energy in the form of lipids and play a critical role in maintaining metabolic homeostasis. They are primarily found in adipose tissue, which exists in two major forms: white adipose tissue (WAT) and brown adipose tissue (BAT). White adipocytes are characterized by a single large lipid droplet and are involved in long-term energy storage, insulation, and endocrine signaling through the secretion of adipokines such as leptin and adiponectin. Brown adipocytes, in contrast, contain multiple small lipid droplets and abundant mitochondria, enabling them to generate heat through non-shivering thermogenesis through uncoupling protein 1 (LICP1)- dependent and -independent mechanisms. In some aspects, the disclosed nucleic acid or vector, or viral vector comprising the nucleic acid, or a pharmaceutical composition of the instant disclosure may be transduced or transfected directly in adipocytes. In some aspects, the adipocytes are white adipocytes. In some aspects, the adipocytes may be transduced or transfected ex vivo by administering the nucleic acid or vector, or viral vector comprising the nucleic acid, or a pharmaceutical composition into isolated adipocytes or adipose tissues obtained from a subject. In some aspects, the adipocytes may be transduced or transfected in vivo by administering the nucleic acid or vector, or viral vector comprising the nucleic acid,or a pharmaceutical composition into the subject. This may require parenteral delivery, localized delivery, and / or localized surgical exposure of the tissue or cells.V. Pharmaceutical compositions

[0206] Typically, the pharmaceutical composition comprises the nucleic acid, polynucleotide construct, vector or the host cell as disclosed herein and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness.

[0207] The compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans. The mode of administration may be parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In one aspect, the vector is administered by intravenous infusion or injection. In another aspect, the vector is administered by intramuscular or subcutaneous injection. In another aspect, the vector is administered periodically. In an aspect, the vector is delivered to a specific location using stereostatic delivery. In an aspect, the vector is delivered directly into the adipose tissue. In an aspect, the vector may be transfected into a host cell (for example, an adipocyte) and injected into the adipose tissue. In an aspect, the vector is designed to be therapeutically effective only in adipocytes. For example, the nucleic acid sequence encoding the any one or more of GIPR, GCGR, or GLP-1 R, or one or more of the disclosed ligands may be operably linked to an adipocyte-specific promoter, or an inducible promoter such that GIPR, GCGR, GLP-1 R, or ligand expression is restricted to adipocytes. In an aspect, the delivery system may be caged such that the GIPR, GCGR, GLP-1 R, and / or ligand is expressed only in the adipocytes. In a further aspect, the expression of the GIPR, GCGR, GLP-1 R, and / or ligand may be turned off or cease after 4 weeks, after 6 weeks, after 8 weeks, after 10 weeks,after 12 weeks, after 14 weeks, after 16 weeks, after 20 weeks, after 22 weeks, or after 24 weeks post administration.

[0208] Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., rAAV, polynucleotide, non-viral vector, nanoparticle etc.) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

[0209] Generally, dispersions are prepared by incorporating the active composition into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile, lyophilized powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and spraydrying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution 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. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0210] The vector of the present disclosure can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results. In certain aspects, the active composition may be prepared with a carrier that will protect the composition against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. The pharmaceutical compositions of the disclosure may include a “therapeutically effective amount” or a “prophylactically effective amount” of the vectors of the disclosure. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the vector may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the vector to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of thevector are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0211] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the active composition and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.VI. Method of treatment

[0212] In an aspect, the current disclosure encompasses a method for preventing and / or treating a metabolic disease in a subject in need thereof, comprising administering to the subject the pharmaceutical composition as disclosed herein. In some aspects, the method for preventing and / or treating a metabolic disease encompasses administering the nucleic acid, viral vector, and / or pharmaceutical composition into an adipose tissue of the subject. In an aspect, the adipose tissue may be in the subject's body during injection. In an aspect, the adipose tissue may be outside of the subject's body and subsequently transplanted into the subject after injection. In some aspects, the adipose tissue may be allogenic or xenogenic to the subject. In some aspects, the adipose tissue may be autologous to the subject.

[0213] In an aspect, the metabolic disease is obesity. Further provided are pharmaceutical compositions comprising a therapeutically effective amount of one or more of the pharmaceutical compositions disclosed herein for the treatment of a metabolic disorder in an individual. Such disorders include, but are not limited to, obesity, metabolic syndrome or syndrome X, type II diabetes, complications of diabetes such as retinopathy, hypertension, dyslipidemias, cardiovascular disease, gallstones, osteoarthritis, and certain forms of cancers. The obesity-related disorders herein are associated with, caused by, or result from obesity.

[0214] “Obesity” is a condition in which there is an excess of body fat. The operational definition of obesity is based on the body mass index (BMI), calculated as body weight per height in meters squared (kg / m2). “Obesity” refers to a condition whereby an otherwise healthy subject has a BMI greater than or equal to 30 kg / m2, or a condition whereby a subject with at least one co-morbidity has a BMI greater than or equal to 27 kg / m2. An “obese subject” is an otherwise healthy subject with a BMI greater than or equal to 30 kg / m2or a subject with at least one co-morbidity with a BMI greater than or equal to 27 kg / m2. A “subject at risk for obesity” is an otherwise healthy subject with a BMI of 25 kg / m2to less than 30 kg / m2or a subject with at least one co-morbidity with a BMI of 25 kg / m2to less than 27 kg / m2. The increased risks associated with obesity occur at a lower BMI in Asians. In Asian countries, including Japan, “obesity” refers to a condition whereby a subject with at least one obesity- induced or obesity-related co-morbidity that requires weight reduction or that would be improved by weight reduction, has a BMI greater than or equal to 25 kg / m2. In Asian countries, including Japan, an “obese subject” refers to a subject with at least one obesity-induced or obesity-related co-morbidity that requires weight reduction or that would be improved by weight reduction, with a BMI greater than or equal to 25 kg / m2. In Asian countries, a “subject at risk of obesity” is a subject with a BMI of greater than 23 kg / m2to less than 25 kg / m2. As used herein, the term “obesity” is meant to encompass all of the above definitions of obesity.

[0215] Obesity-induced or obesity-related co-morbidities include, but are not limited to, diabetes, non-insulin dependent type II diabetes, impaired glucose tolerance, impaired fasting glucose, insulin resistance syndrome, dyslipidemia, hypertension, hyperuric acidemia, gout, coronary artery disease, myocardial infarction, angina pectoris, sleep apnea syndrome, Pickwickian syndrome, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), fatty liver; cerebral infarction, cerebral thrombosis, transient ischemic attack, orthopedic disorders, arthritis deformans, lumbodynia, emmeniopathy, and infertility. In particular, co-morbidities include: hypertension, hyperlipidemia, dyslipidemia, glucose intolerance, cardiovascular disease, sleep apnea, diabetes mellitus, and other obesity-related conditions.

[0216] “Treatment” (of obesity and obesity-related disorders) refers to the administration of the pharmaceutical compositions of the present disclosure to reduce or maintain the body weight of an obese subject. One outcome of treatment may be reducing the body weight of an obese subject relative to that subject's body weight immediately before the administration of the compounds of the present disclosure. Another outcome of treatment may be preventing body weight regain of body weight previously lost as a result of diet, exercise, or pharmacotherapy. Another outcome of treatment may be decreasing the occurrence of and / or the severity of obesity-related diseases. The treatment may suitably result in a reduction infood or calorie intake by the subject, including a reduction in total food intake, or a reduction of intake of specific components of the diet such as carbohydrates or fats; and / or the inhibition of nutrient absorption; and / or the inhibition of the reduction of metabolic rate; and in weight reduction in patients in need thereof. The treatment may also result in an alteration of metabolic rate, such as an increase in metabolic rate, rather than or in addition to an inhibition of the reduction of metabolic rate; and / or in minimization of the metabolic resistance that normally results from weight loss.

[0217] “Prevention” (of obesity and obesity-related disorders) refers to the administration of the composition of the present disclosure to reduce or maintain the body weight of a subject at risk of obesity. In an aspect, prevention may comprise a method for suppressing the appetite of a subject in need thereof, comprising administering to the subject the composition as disclosed herein. In an aspect, prevention may comprise a method of preventing weight gain comprising administering to the subject the composition as disclosed herein.

[0218] One outcome of prevention may be reducing the body weight of a subject at risk of obesity relative to that subject's body weight immediately before the administration of the compositions of the present disclosure. Another outcome of prevention may be preventing body weight regain of body weight previously lost as a result of diet, exercise, or pharmacotherapy. Another outcome of prevention may be preventing obesity from occurring if the treatment is administered prior to the onset of obesity in a subject at risk of obesity. Another outcome of prevention may be decreasing the occurrence and / or severity of obesity- related disorders if the treatment is administered prior to the onset of obesity in a subject at risk of obesity. Moreover, if treatment is commenced in already obese subjects, such treatment may prevent the occurrence, progression or severity of obesity-related disorders, such as, but not limited to, arteriosclerosis, type II diabetes, polycystic ovarian disease, cardiovascular diseases, osteoarthritis, dermatological disorders, hypertension, insulin resistance, hypercholesterolemia, hypertriglyceridemia, and cholelithiasis.

[0219] The obesity-related disorders herein are associated with, caused by, or result from obesity. Examples of obesity-related disorders include overeating and bulimia, hypertension, diabetes, elevated plasma insulin concentrations and insulin resistance, dyslipidemias, hyperlipidemia, endometrial, breast, prostate, and colon cancer, osteoarthritis, obstructive sleep apnea, cholelithiasis, gallstones, heart disease, abnormal heart rhythms and arrythmias, myocardial infarction, congestive heart failure, coronary heart disease, sudden death, stroke, polycystic ovarian disease, craniopharyngioma, the Prader-Willi Syndrome, Frohlich's syndrome, GH-deficient subjects, normal variant short stature, Turner's syndrome, and other pathological conditions showing reduced metabolic activity or a decrease in resting energy expenditure as a percentage of total fat-free mass, e.g., children with acute lymphoblasticleukemia. Further examples of obesity-related disorders are metabolic syndrome, also known as syndrome X, insulin resistance syndrome, sexual and reproductive dysfunction, such as infertility, hypogonadism in males and hirsutism in females, gastrointestinal motility disorders, such as obesity-related gastro-esophageal reflux, respiratory disorders, such as obesityhypoventilation syndrome (Pickwickian syndrome), cardiovascular disorders, inflammation, such as systemic inflammation of the vasculature, arteriosclerosis, hypercholesterolemia, hyperuricemia, lower back pain, gallbladder disease, gout, and kidney cancer. The compositions of the present disclosure are also useful for reducing the risk of secondary outcomes of obesity, such as reducing the risk of left ventricular hypertrophy.

[0220] In an aspect, the current disclosure also encompasses a method of preventing weight gain and / or inducing weight loss comprising administering into a subject in need thereof, a composition as provided herein. In an aspect, the composition may be administered systemically using any method known in the art or disclosed herein. In an aspect, the method of preventing weight gain and / or inducing weight loss comprises injecting into an adipose tissue of the subject the composition disclosed herein. In an aspect, the adipose tissue may be in the subject's body during injection. In an aspect, the adipose tissue may be outside of the subject's body and subsequently transplanted into the subject after injection. In an aspect, the administration of the disclosed composition may suppress the appetite of the subject.

[0221] In an aspect, the current disclosure also encompasses a method of remodeling adipose tissue for esthetical purposes, and / or to support plastics surgery, comprising administering into the subject a composition disclosed herein. In an aspect, the method of remodeling adipose tissue for esthetical purposes, and / or to support plastics surgery comprises injecting into a specific adipose tissue depot in the subject in need thereof, a composition as disclosed herein. In an aspect, the adipose tissue may be in the subject's body during injection. In an aspect, the adipose tissue may be outside of the subject's body and subsequently transplanted into the subject after injection.Other Indications1. Diabetes

[0222] In some aspects, the condition, disease or disorder is diabetes. Non-limiting examples of diabetes include type 1 diabetes mellitus, type 2 diabetes mellitus (e.g., diet-treated type 2- diabetes, sulfonylurea-treated type 2-diabetes, a far-advanced stage type 2-diabetes, longterm insulin-treated type 2-diabetes), diabetes mellitus (e.g., non-insulin-dependent diabetes mellitus, insulin-dependent diabetes mellitus), gestational diabetes, obese diabetes, autoimmune diabetes, and borderline type diabetes. In some aspects, the condition, disease or disorder is type 2 diabetes mellitus (e.g., diet-treated type 2-diabetes, sulfonylurea-treatedtype 2-diabetes, a far-advanced stage type 2-diabetes, long-term insulin-treated type 2- diabetes).

[0223] In some aspects, compositions and methods for treating a subject with a condition, disease, or disorder (e.g., type 2 diabetes mellitus) described herein reduce fasting plasma glucose levels. In some aspects, compositions and methods for treating a subject with a condition, disease, or disorder (e.g., type 2 diabetes mellitus) described herein reduce nonfasting plasma glucose levels. In some aspects, compositions and methods for treating a subject with a condition, disease, or disorder (e.g., type 2 diabetes mellitus) described herein reduce HbA1c levels. In some aspects, compositions and methods for treating a subject with a condition, disease, or disorder (e.g., type 2 diabetes mellitus) described herein reduce glucagon levels. In some aspects, compositions and methods for treating a subject with a condition, disease, or disorder (e.g., type 2 diabetes mellitus) described herein increase insulin levels. In some aspects, compositions and methods for treating a subject with a condition, disease, or disorder (e.g., type 2 diabetes mellitus) described herein reduce BMI.

[0224] In some aspects, a reduction in fasting plasma glucose levels of about 5% to about 95% indicates treatment of type 2 diabetes mellitus. In some aspects, a reduction in fasting plasma glucose levels of about 15% to about 80% indicates treatment of type 2 diabetes mellitus. In some aspects, a reduction in fasting plasma glucose levels of about 25% to about 60% indicates treatment of type 2 diabetes mellitus. In some aspects, a reduction in fasting plasma glucose levels to about or below 126 mg / dL, about or below 110 mg / dL, or about or below 90 mg / dL indicates treatment of the type 2 diabetes mellitus.

[0225] In some aspects, a reduction in non-fasting plasma glucose levels of about 5% to about 95% indicates treatment of type 2 diabetes mellitus. In some aspects, a reduction in nonfasting plasma glucose levels of about 15% to about 80% indicates treatment of type 2 diabetes mellitus. In some aspects, a reduction in non-fasting plasma glucose levels of about 25% to about 60% indicates treatment of type 2 diabetes mellitus. In some aspects, a reduction in non-fasting plasma glucose levels to about or below 200 mg / dL, about or below 150 mg / dL, or about or below 130 mg / dL indicates treatment of type 2 diabetes mellitus.

[0226] In some aspects, a reduction in HbA1c levels of about 5% to about 95% indicates treatment of type 2 diabetes mellitus. In some aspects, a reduction in HbA1c levels of about 15% to about 80% indicates treatment of type 2 diabetes mellitus. In some aspects, a reduction in HbA1c levels of about 25% to about 60% indicates treatment of type 2 diabetes mellitus. In some aspects, reduction in HbA1c levels to about or below 6.5%, about or below 6.0%, or about or below 5.0% indicates treatment of type 2 diabetes mellitus.

[0227] In some aspects, a reduction in glucagon levels of about 5% to about 95% indicates treatment of type 2 diabetes mellitus. In some aspects, a reduction in glucagon levels of about 15% to about 80% indicates treatment of type 2 diabetes mellitus. In some aspects, a reduction in glucagon levels of about 25% to about 60% indicates treatment of type 2 diabetes mellitus. In some aspects, an increase in insulin levels of about 5% to about 95% indicates treatment of type 2 diabetes mellitus. In some aspects, an increase in insulin levels of about 15% to about 80% indicates treatment of type 2 diabetes mellitus. In some aspects, an increase in insulin levels of about 25% to about 60% indicates treatment of type 2 diabetes mellitus.

[0228] In some aspects, a reduction in BMI of about 5% to about 95% indicates treatment of type 2 diabetes mellitus. In some aspects, a reduction in BMI of about 15% to about 80% indicates treatment of the type 2 diabetes mellitus. In some aspects, a reduction in BMI of about 25% to about 60% indicates treatment of type 2 diabetes mellitus. In some aspects, a reduction in BMI of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% indicates treatment of type 2 diabetes mellitus. In some aspects, a reduction in BMI to about or below 40, about or below 30, or about or below 20 indicates treatment of type 2 diabetes mellitus.

[0229] In some aspects, the condition, disease or disorder is associated with diabetes (e.g., a complication of diabetes). Non-limiting examples of disorders associated with diabetes include obesity, obesity-related disorders, metabolic syndrome, neuropathy, nephropathy (e.g., diabetic nephropathy), retinopathy, diabetic cardiomyopathy, cataract, macroangiopathy, osteopenia, hyperosmolar diabetic coma, infectious disease (e.g., respiratory infection, urinary tract infection, gastrointestinal infection, dermal soft tissue infections, inferior limb infection), diabetic gangrene, xerostomia, hypacusis, cerebrovascular disorder, diabetic cachexia, delayed wound healing, diabetic dyslipidemia, peripheral blood circulation disorder, cardiovascular risk factors (e.g., coronary artery disease, peripheral artery disease, cerebrovascular disease, hypertension, and risk factors related to unmanaged cholesterol and / or lipid levels, and / or inflammation), NASH, bone fracture, and cognitive dysfunction.

[0230] Other non-limiting examples of disorders related to diabetes include pre-diabetes, hyperlipidemia (e.g., hypertriglyceridemia, hypercholesterolemia, high LDL-cholesterolemia, low HDL-cholesterolemia, postprandial hyperlipemia), metabolic syndrome (e.g., metabolic disorder where activation of GLP-1 R is beneficial, metabolic syndrome X), hypertension, impaired glucose tolerance (IGT), insulin resistance, and sarcopenia.

[0231] In some aspects, the condition, disease, or disorder is diabetes and obesity (diabesity). In some aspects, the compounds described herein are also useful in improving the therapeutic effectiveness of metformin.2. Disorders of metabolically-important tissues

[0232] In some aspects, the condition, disease or disorder is a disorder of a metabolically important tissue. Non-limiting examples of metabolically important tissues include liver, fat, pancreas, kidney, and gut.

[0233] In some aspects, the condition, disease or disorder is a fatty liver disease. Fatty liver diseases include, but are not limited to, non-alcoholic fatty acid liver disease (NAFLD), steatohepatitis, non-alcoholic steatohepatitis (NASH), fatty liver disease resulting from hepatitis, fatty liver disease resulting from obesity, fatty liver disease resulting from diabetes, fatty liver disease resulting from insulin resistance, fatty liver disease resulting from hypertriglyceridemia, Abetalipoproteinemia, glycogen storage diseases, Weber-Christian disease, Wolman’s disease, acute fatty liver of pregnancy, and lipodystrophy.

[0234] Other non-limiting examples of disorders in metabolically important tissues include joint disorders (e.g., osteoarthritis, secondary osteoarthritis), steatosis (e.g. in the liver); gall stones; gallbladder disorders; gastroesophageal reflux; sleep apnea; hepatitis; fatty liver; bone disorder characterized by altered bone metabolism, such as osteoporosis, including postmenopausal osteoporosis, poor bone strength, osteopenia, Paget's disease, osteolytic metastasis in cancer subjects, osteodystrophy in liver disease and the altered bone metabolism caused by renal failure or hemodialysis, bone fracture, bone surgery, aging, pregnancy, protection against bone fractures, and malnutrition polycystic ovary syndrome; renal disease (e.g., chronic renal failure, glomerulonephritis, glomerulosclerosis, nephrotic syndrome, hypertensive nephrosclerosis, end-stage renal disease); muscular dystrophy, angina pectoris, acute or chronic diarrhea, testicular dysfunction, respiratory dysfunction, frailty, sexual dysfunction (e.g., erectile dysfunction), and geriatric syndrome. In some aspects, compositions described herein can be used for treating surgical trauma by improving recovery after surgery and / or by preventing the catabolic reaction caused by surgical trauma.3. Cardiovascular and vascular diseases

[0235] In some aspects, the condition, disease or disorder is a cardiovascular disease. Nonlimiting examples of cardiovascular disease include congestive heart failure, atherosclerosis, arteriosclerosis, coronary heart disease, coronary artery disease, congestive heart failure, coronary heart disease, hypertension, cardiac failure, cerebrovascular disorder (e.g., cerebral infarction), vascular dysfunction, myocardial infarction, elevated blood pressure (e.g., 130 / 85mm Hg or higher), and prothrombotic state (exemplified by high fibrinogen or plasminogen activator inhibitor in the blood).

[0236] In some aspects, the condition, disease or disorder is related to a vascular disease. Non-limiting examples of vascular diseases include peripheral vascular disease, macrovascular complications (e.g., stroke), vascular dysfunction, peripheral artery disease, abdominal aortic aneurysm, carotid artery disease, cerebrovascular disorder (e.g., cerebral infarction), pulmonary embolism, chronic venous insufficiency, critical limb ischemia, retinopathy, nephropathy, and neuropathy.4. Insulin-related conditions

[0237] In some aspects, the condition, disease or disorder is impaired fasting glucose (IFG), impaired fasting glycemia (IFG), hyperglycemia, insulin resistance (impaired glucose homeostasis), hyperinsulinemia, elevated blood levels of fatty acids or glycerol, a hypoglycemic condition, insulin resistant syndrome, paresthesia caused by hyperinsulinemia, hyperlipidaemia, hypercholesteremia, impaired wound healing, leptin resistance, glucose intolerance, increased fasting glucose, dyslipidemia (e.g., hyperlipidemia, atherogenic dyslipidemia characterized by high triglycerides and low HDL cholesterol), glucagonoma, hyperuricacidemia, hypoglycemia (e.g., nighttime hypoglycemia), and concomitant comatose endpoint associated with insulin.

[0238] In some aspects, compositions described herein can reduce or slow down the progression of borderline type, impaired fasting glucose or impaired fasting glycemia into diabetes.5. Autoimmune disorders

[0239] In some aspects, the condition, disease or disorder is an autoimmune disorder. Nonlimiting examples of autoimmune disorders include multiple sclerosis, experimental autoimmune encephalomyelitis, autoimmune disorder is associated with immune rejection, graft versus host disease, uveitis, optic neuropathies, optic neuritis, transverse myelitis, inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, myasthenia gravis, and Graves’ disease.6. Stomach and intestine-related disorders

[0240] In some aspects, the condition, disease or disorder is a stomach or intestine related disorder. Non-limiting examples of these disorders include ulcers of any etiology (e.g. peptic ulcers, Zollinger-Ellison syndrome, drug-induced ulcers, ulcers related to infections or other pathogens), digestion disorders, malabsorption, short bowel syndrome, cul-de-sac syndrome, inflammatory bowel diseases (Crohn's disease and ulcerative colitis), celiac sprue,hypogammaglobulinemic sprue, chemotherapy and / or radiation therapy-induced mucositis and diarrhea, gastrointestinal inflammation, short bowel syndrome, colitis ulcerosa, gastric mucosal injury (e.g., gastric mucosal injury caused by aspirin), small intestinal mucosal injury, and cachexia (e.g., cancerous cachexia, tuberculous cachexia, cachexia associated with blood disease, cachexia associated with endocrine disease, cachexia associated with infectious disease, and cachexia caused by acquired immunodeficiency syndrome).7. Body weight

[0241] In some aspects, compositions described herein can be used to reduce body weight (e.g., excess body weight), prevent body weight gain, induce weight loss, decrease body fat, or reduce food intake in a subject (e.g., a subject in need thereof). In some aspects, the weight increase in a subject may be attributed to excessive ingestion of food or unbalanced diets or may be weight increase derived from a concomitant drug (e.g., insulin sensitizers having a PPARy agonist-like action, such as troglitazone, rosiglitazone, englitazone, ciglitazone, pioglitazone, and the like). In some aspects, the weight increase may be weight increase before reaching obesity or may be weight increase in an obese subject. In some aspects, the weight increase may also be medication-induced weight gain or weight gain subsequent to cessation of smoking.

[0242] In some aspects, the condition, disease or disorder is an eating disorder, such as hyperphagia, binge eating, bulimia, or compulsive eating.8. Inflammatory diseases

[0243] In some aspects, the condition, disease or disorder is an inflammatory disorder. Nonlimiting examples of inflammatory disorders include chronic rheumatoid arthritis, spondylitis deformans, arthritis deformans, lumbago, gout, post-operational or post-traumatic inflammation, bloating, neuralgia, laryngopharyngitis, cystitis, pneumonia, pancreatitis, enteritis, inflammatory bowel disease (including inflammatory large bowel disease), inflammation in metabolically important tissues including liver, fat, pancreas, kidney and gut, and a proinflammatory state (e.g., elevated levels of proinflammatory cytokines or markers of inflammation-like C-reactive protein in the blood).9. Hypothalamic-pituitary disorders

[0244] In some aspects, the condition, disease or disorder is related to the hypothalamic- pituitary-gonadal axis. Hypothalamic-pituitary-gonadal axis diseases include, but are not limited to, hypogonadism, polycystic ovary syndrome, hypothyroidism, hypopituitarism, sexual dysfunction, and Cushing's disease.

[0245] In some aspects, the condition, disease or disorder associated with diabetes is related to the hypothalamic-pituitary-gonadal axis.10. Pulmonary disease

[0246] In some aspects, the condition, disease or disorder is related to a pulmonary disease. Pulmonary diseases include, but are not limited to, asthma, idiopathic pulmonary fibrosis, pulmonary hypertension, obstructive sleep apnea-hypopnea syndrome, and chronic obstructive pulmonary disease (COPD) (e.g., emphysema, chronic bronchitis, and refractory (non-reversible) asthma).

[0247] In some aspects, the condition, disease or disorder associated with diabetes is a pulmonary disease.VII. Methods of administration

[0248] Methods of localized injection of compositions are well known in the art. For example, in an aspect, the injection into the adipose tissue as disclosed herein may involve a dermal incision to facilitate transdermal insertion of a cannula or equivalent for administration of the composition into the subcutaneous, submuscular, or supraperiosteal adipose tissue. The composition can be injected in small aliquots throughout the area requiring treatment, rather than as a single bolus, by manipulating the cannula into a different tract after each injection, using a tunnelling technique. In an aspect, the adipose tissue depot may be in any part of the subject’s body, for example under the skin (subcutaneous fat), packed around internal organs (visceral fat), between muscles, within bone marrow, and in breast tissue.

[0249] In some aspects, the current disclosure also encompasses injecting more than one disclosed composition simultaneously or in tandem into the adipose tissue. For example, in some aspects, more than one nucleic acids (for example, viral vectors) may be injected, wherein the nucleic acids encode at least two different receptors disclosed herein. The more than one composition may be injected at the same time. The more than one composition may be injected separated by a certain time for example after about 1 day to after about 1 week, after about 1 week to after about 2 weeks, after about 2 weeks to after about 4 weeks, after about 4 weeks to after about 6 weeks, after about 6 weeks to after about 8 weeks, after about 8 weeks to after about 10 weeks, after about 12 weeks to after about after 14 weeks, after about 14 weeks to after about 16 weeks, after about 16 weeks to after about 20 weeks, after about 20 weeks to after about 22 weeks, or after 24 weeks post administration of the initial composition.

[0250] The compositions disclosed herein may be used in a pharmaceutical composition. Such compositions comprise a therapeutically-effective amount of one or more of thepolynucleotides, viral or non-viral vectors disclosed herein and a pharmaceutically acceptable carrier.

[0251] In another aspect, this disclosure relates to the use of a disclosed pharmaceutical composition according to the disclosure combined with at least one additional active, for preparing a medicament which is suitable for the treatment or prevention of diseases or conditions. This is preferably a disease in the context of the metabolic syndrome, particularly one of the diseases or conditions listed above, most particularly diabetes or obesity or complications thereof. The compositions disclosed herein may be administered in combination with one or more active substances simultaneously, separately, or sequentially. The use of the composition disclosed herein in combination with another active substance may take place simultaneously or at staggered times, but particularly within a short space of time. If they are administered simultaneously, the two active substances are given to the patient together; if they are used at staggered times, the two active substances are given to the patient within a period of less than or equal to 12 hours, but particularly less than or equal to 6 hours.

[0252] In some aspects, any suitable mode of administration can be used depending on the disease condition and the formulation. Suitable modes of administration are known in the art and further provided herein including but not limited to intravenous, subcutaneous, intranasal route, cranial, transmucosal, intestinal, and / or parenteral delivery. In an aspect, the composition may be administered directly into the adipose tissue. In an aspect, the composition may be used to transfected a host cell (for example, an adipocyte) and the host cell may be injected into the subject in need thereof. In an aspect, the host cell may be injected into the adipose tissue. In an aspect, the vector is designed to be therapeutically effective only in the adipocytes. For example, the nucleic acid sequence encoding any one or more of GIPR, GCGR, or GLP-1 R, or one or more of the disclosed ligands may be operably linked to an adipocyte-specific promoter, or an inducible promoter such that the heterologous protein expression if restricted to the adipocytes. In an aspect, the delivery system may be caged such that the heterologous protein is expressed only in adipocytes. In an aspect, the compositions may be administered at least yearly, monthly, fortnightly, daily for a length of time. In an aspect, the administration may be stopped after one or more weeks. In a further aspect, the overexpression of heterologous protein may be turned off or cease after 4 weeks, after 6 weeks, after 8 weeks, after 10 weeks, after 12 weeks, after 14 weeks, after 16 weeks, after 20 weeks, after 22 weeks, or after 24 weeks post administration. In an aspect, the overexpression of heterologous protein may be turned on or induced after a suitable interval after administration, for example after about 1 day to after about 1 week, after about 1 week to after about 2 weeks, after about 2 weeks to after about 4 weeks, after about 4 weeks to after about 6 weeks, after about 6 weeks to after about 8 weeks, after about 8 weeks to afterabout 10 weeks, after about 12 weeks to after about after 14 weeks, after about 14 weeks to after about 16 weeks, after about 16 weeks to after about 20 weeks, after about 20 weeks to after about 22 weeks, or after 24 weeks post administration. In an aspect, the overexpression of heterologous protein may be turned on or induced for 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 20 weeks, 22 weeks, or 24 weeks or more after a suitable interval after administration and subsequently turned off.

[0253] In some aspects, a subject in need includes a human, a livestock animal, a companion animal, a lab animal, or a zoological animal. In some aspects, the human includes man, woman, children, elderly, adults, and teens. In some other aspects, the human is an adult human patient, or a pediatric human patient. In some aspects, the subject may be a rodent, e.g., a mouse, a rat, a guinea pig, etc. In some aspects, the subject may be a livestock animal. Non-limiting examples of suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas, and alpacas. In some aspects, the subject may be a companion animal. Nonlimiting examples of companion animals may include pets such as dogs, cats, rabbits, and birds. In some aspects, the subject may be a zoological animal. As used herein, a “zoological animal” refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears. In a specific aspect, the animal is a laboratory animal. Non-limiting examples of a laboratory animal may include rodents, canines, felines, and non- human primates. In certain aspects, the animal is a rodent. Non-limiting examples of rodents may include mice, rats, guinea pigs, etc. In some exemplary aspects the subject is a human.

[0254] For any formulation used in the methods of the present disclosure, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays and or screening platforms disclosed herein. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0255] In some aspects, toxicity and therapeutic efficacy of the active ingredients disclosed herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. In some aspects, data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in a human subject. In some aspects, a dosage for use herein may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics,” Ch. 1).VIII. Additional therapies

[0256] In some aspects, this disclosure contemplates using the disclosed methods and products in combination with additional therapy regimens.

[0257] In some aspects, the methods described herein can further include administering the disclosed pharmaceuticals with one or more additional therapies (e.g., one or more additional therapeutic agents and / or one or more therapeutic regimens). In some aspects, the additional therapy may be administered concurrently or in tandem with the disclosed compositions.

[0258] In some aspects, the methods described herein include administering a composition described herein in combination with one or more of a diet therapy (e.g., dietary monitoring, diet therapy for diabetes), an exercise therapy (e.g., physical activity), blood sugar monitoring, gastric electrical stimulation (e.g., TANTALUS®), and diet modifications. In some aspects, the additional therapy may comprise administering a weight loss drug, drugs to control blood pressure, blood glucose levels, cardiovascular diseases etc. Non-limiting examples of such drugs include semaglutide (WEGOVY®), liraglutide (SAXENDA®), tirzepatide (ZEPBOUND®), orlistat (ALLI®, XENICAL®), phentermine-topiramate (Qsymia®), naltrexone-bupropion (Contrave®), metformin, empagliflozin (Jardiance®), dapagliflozin (Farxiga®), sitagliptin (Januvia®), insulin (Lantus®, Humalog®, Novolog®), atorvastatin (Lipitor®), rosuvastatin (Crestor®), ezetimibe (Zetia®), PCSK9 inhibitors (Repatha®, Praluent®), ACE inhibitors (Lisinopril, Ramipril), p-blockers (Metoprolol, Carvedilol), anticoagulants (Warfarin, Apixaban), and aspirin (low dose for heart health).EXAMPLES

[0259] The following examples are included to demonstrate preferred aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0260] The Examples provided herein delineate the functional role and mechanistic action of the Gastric Inhibitory Polypeptide Receptor (GIPR), GCGR and GLP-1 R in energy homeostasis. Without being bound by theory, these data provide a rationale for using GIPR, GCGR and GLP-1 R overexpression using viral vectors as a mechanism for combatingmetabolic disorders. GIPR, GCGR and GLP-1 R were selectively and specifically induced in adipocytes. A novel mouse model of doxycycline (Dox)-inducible fat cell-specific overexpression of the GIPR was also generated. Given that GIPR / GLP-1 R co-agonism outperforms GLP-1 R agonism in energy balance, it was hypothesized that activation of the GIPR in the white adipocyte elicits favorable effects on body weight, energy expenditure and food intake in the obese state. Extending this further, overexpression of ligands of these receptors were tested for their impact on body weight and energy homeostasis.Example 1: The GIPR is Expressed by Adipocytes, Mesothelial Cells, and Pericytes

[0261] Applying unbiased single-nuclei isolation methodologies to examine different cell-types within human WAT, expression data from two independent patient cohorts (Cohorts A and B) was analyzed. It was found that the GIPR is expressed in mature white adipocytes, mesothelial cells, and pericytes (FIG. 1A (Cohort A) and FIG. 1 B (Cohort B)). Additional evidence was found that showed that the GIPR is also present in white adipocytes. Upon further analysis of single-nuclei RNA-Seq data from Cohort B, GIPR-expressing clusters were identified within specific sub-populations of white adipocytes; namely human adipocyte sub-set 6 (hAd6) and 7 (hAd7) (FIG. 1C). These unique sub-populations of adipocytes are positively associated with metabolic health. When compared with the adipokine leptin, the percent expression of the GIPR in hAd7 adipocytes is lower (FIG. 1C). However, the comparative expression of the GIPR in the hAd7 sub-population versus other sub-populations is markedly higher than that observed for leptin (FIG. 1C). The GIPR may also serve as a defining marker for the hAd6 and hAd7 sub-set of white adipocytes. It is further demonstrated that the GIPR is expressed in murine adipose tissues at levels significantly higher in fat depots, relative to the liver; the latter an organ that does not express the GIPR (FIG. 1 D). Taken together, by analyzing two independent single-nuclei RNA-Seq cohorts, it was shown that the Gl PR is expressed by white adipocytes.

[0262] To determine whether activation of the GIPR in fat cells impacts energy balance, a mouse model of GIPR induction specifically in the adipocyte was generated. The expression of the GIPR is driven by a tetracycline-responsive element promoter (TRE), i.e., a TRE-GIPR mouse (FIG. 1 E). For an operational promoter element, the ‘Tet-On’ transcription factor rtTA is required. To achieve fat cell specificity, a mouse that harbors rtTA under the control of the adiponectin promoter; highly specific for the adipocyte, was used. Upon crossing TRE-GIPR mice with adiponectin-rtTA mice (‘GIPR-Adip’ model) (FIG. 1 E), following doxycycline (Dox)- chow feeding, a highly specific, significant induction of GIPR expression in sWAT, gWAT, mWAT, and BAT was achieved, as early as 1 hour post Dox (FIG. 1 F and 1G). Cyclic AMP (cAMP) levels, a downstream component of GIPR signaling, are significantly increased in GIPR-Adip sWAT (FIG. 1 H); confirming activation of the GIPR pathway. No markeddifferences in the circulating levels of GIP were observed in GIPR-Adip mice (FIG. 11); eliminating the possibility that any phenotypic differences observed between WT and GIPR- Adip mice are due to compensatory increases in GIP levels.Example 2: Adipose Tissue GIPR Prevents Obesity and Promotes Weight Loss

[0263] To determine whether induction of the GIPR specifically in adipocytes impacts energy balance, WT and GIPR-Adip mice were subjected to diet-induced obesity. Strikingly, during Dox-HFD feeding (600 mg / kg Dox), GIPR-Adip mice gained significantly less body weight (-56% at 12 weeks Dox-HFD), than WT littermates (FIG. 2A). To demonstrate the titratable nature of our in vivo system, a low-dose of Dox-HFD (50 mg / kg) was utilized to induce the GIPR in adipose to a lesser extent. GIPR-Adip mice maintained a significant reduction in body weight gain (-40%) (FIG. 2B); confirming that the anti-obesity effects of the GIPR are preserved, even at a lower degree of GIPR induction in fat. Combined, this identifies that activation of the GIPR only in adipose tissue prevents diet-induced obesity.

[0264] It was next determined whether induction of the GIPR in fat cells drives weight loss in obese mice. WT and GIPR-Adip mice were first fed a HFD (no Dox; no GIPR transgene induction). Once obese, all mice were switched to Dox-HFD to activate the GIPR in transgenic fat. Obese GIPR-Adip mice exhibited massive, rapid, and sustained weight loss; dropping as early as 2 days following receptor induction, with a significant reduction in body weight (-35% after 2 weeks) (FIG. 2C). Importantly, weight loss was underlined by a significant decrease in whole-body fat mass and marked increase in lean mass (FIG. 2D). At the tissue level, transgenic mice exhibited significantly less white and brown fat mass (FIG. 2E). Consistent with this, GIPR-Adip mice had significantly decreased circulating levels of leptin (FIG. 2F); an adipokine that positively correlates with body fat and obesity. No marked differences in tissue morphology between WT and GIPR-Adip sWAT, gWAT, or BAT were observed (FIG. 2G), suggesting healthy adipose tissue function. Data demonstrate that in vivo, induction of the GIPR in the adipocyte provides protection from diet-induced obesity and triggers massive weight loss in an obese setting.

[0265] It is widely established that GLP-1 R agonists are effective weight loss agents. We thus examined i) the relationship between systemic GLP-1 R agonism versus GIPR activation in the white adipocyte and ii) whether induction of the GIPR in the fat cell enhances the weight loss efficacy of GLP-1 R agonism. Diet-induced obese (DIO) WT and GIPR-Adip mice were dosed daily with vehicle or a long-acting GLP-1 R agonist (LAGLP-1 RA). Initially, all mice were maintained on a HFD (i.e., no Dox, thus no GIPR transgene induction in the adipocyte). As expected, the LAGLP-1 RA significantly reduced body weight (-20%) following 2-weeks of treatment (FIG. 2H; first segment). Importantly, activation of the GIPR in the adipocyte (i.e.,switching all mice to Dox-HFD) enhanced the weight loss (-30%) induced by the LAGLP-1 RA, while weight loss plateaued in obese mice only treated with the LAGLP-1 RA (FIG. 2H; second segment). This suggests a GLP-1 R-independent mechanism of weight loss that is additive to our adipocyte-specific GIPR-mediated effects. Interestingly, upon stopping treatment with the LAGLP-1 RA, WT mice rapidly re-gained body weight, whereas in the adipocyte GIPR overexpressing mice, weight loss was preserved (FIG. 2H; third segment).

[0266] The thermal neutral zone for mice is between 27-31 °C depending on genetic background, thus when housed at normal vivarium temperatures (18-24°C), mice are in a state of thermal stress, and as such, expend additional energy to maintain body temperature. This can become relevant for some genetically modified mouse models and can lead to protection from diet-induced obesity. To confirm that protection from obesity in GIPR-Adip mice was not secondary to thermal stress, animals were housed at thermoneutrality (27°C). Data shows that under thermoneutral conditions, GIPR-Adip mice maintained significant protection from obesity (FIG. 6A, first phase); confirming that the adipose GIPR-driven prevention from weight gain is not caused by thermal stress.Example 3: The GIPR in Adipose Tissue Suppresses Food Intake

[0267] Next experiments were conducted to identify how the GIPR in adipose tissue protects mice from diet-induced obesity and triggers rapid weight loss in obese mice. In terms of promoting a negative energy balance, studies were performed to test whether the GIPR in the adipocyte: i) suppresses food intake and / or ii) increases energy expenditure. To address the former, obesity-prevention studies were performed by pair-feeding lean mice with Dox-HFD. WT mice pair-fed with GIPR-Adip mice were provided with an equal amount of food to that consumed by ad libitum fed transgenic mice. Interestingly, pair-fed WT mice gained significantly less body weight during Dox-HFD feeding, compared with WT ad libitum fed mice (FIG. 3A). A significant reduction in daily food intake was evident in GIPR-Adip mice, specifically between days 2-4 (FIG. 3A). The overlap in body weight during this timeframe between WT pair-fed mice and GIPR-Adip ad libitum fed mice suggests the reduced food intake in transgenic mice is initially responsible for the negative energy balance, while enhanced energy expenditure may take over to induce further weight loss. Notably, given that a significant upregulation in GIPR message is observed in adipose tissue as early as 1 hour post Dox (FIG. 1G), indicating that adipose GIPR is induced prior to the drop in food intake at day 2, and as such, the GIPR is a driver of this initial suppression in appetite (FIG. 3A). Metabolic cage analyses confirmed a significant reduction in food intake in transgenic mice (FIG. 3B). Similarly, following an overnight fast, food intake was significantly lower in GIPR- Adip mice during “re-feeding” (FIG. 30). Given ad libitum food intake was reduced after 2 daysof GIPR induction (FIG. 3A), a fasting-driven increase in the kinetics of appetite suppression, shifting to 1 day of GIPR activation was observed (FIG. 30).

[0268] In addition to preventing obesity, adipose GIPR triggers weight loss in obese mice (FIG. 20). Here, mice were first fed HFD (no Dox, thus no GIPR induction); once obese, mice were switched to Dox-HFD (activating the GIPR in fat). Upon initiation of Dox-HFD, obese GIPR-Adip mice exhibited a rapid significant reduction in food intake (FIG. 3D), which after 5 days, gradually normalized; albeit remaining significant (FIG. 3D). This indicates that in vivo, adipocyte GIPR induction alone is sufficient to potently suppress food intake in obese mice. In a pharmacological setting, a single subcutaneous dose of a long-acting GIPR agonist was administered to obese WT mice and a significant transient reduction in food intake was observed, which normalized thereafter (FIG. 3E). This suggests that both pharmacological and genetic approaches of GIPR agonism or induction, respectively, transiently suppress caloric intake.

[0269] Thiazolidinediones (TZDs) are anti-diabetic drugs that target the adipocyte through activation of peroxisome prol iterator-activated receptor y (PPARy). While TZDs are effective insulin-sensitizers, a primary side-effect is body weight gain and hyperphagia, and as such, much research has focused on add-on therapies to correct this undesirable consequence. Interestingly, the promoter region of the GIPR gene contains functional PPARy response elements; moreover, activation of PPARy with TZDs increases gene expression levels of the GIPR in murine and human adipocytes. It was therefore examined whether GIPR activation in the adipocyte alleviates TZD-mediated weight gain. Obese GIPR-Adip mice were fed Dox- HFD supplemented with rosiglitazone. Surprisingly, the combination of rosiglitazone and GIPR induction in fat cells further enhanced the existing suppression in food intake in obese GIPR- Adip mice with rosiglitazone significantly potentiating appetite suppression until week 5 (FIG. 3F). Similarly, the addition of rosiglitazone to obese GIPR-Adip mice increased weight loss efficacy (FIG. 3F). This finding raises the intriguing prospect of “combination therapies” of TZDs paired with GIPR-based therapies that maximize GIPR-mediated appetite suppression and weight loss.

[0270] To further demonstrate the effectiveness that adipose GIPR has on food intake, a small amount of donor GIPR-Adip fat was transplanted into WT recipient mice. Gonadal fat-pads were surgically removed from obese donor WT or GIPR-Adip mice. Small pieces of donor fatpads were transplanted and grafted onto isogenic gWAT depots of WT recipient mice. These WT recipient mice (with WT or transgenic fat-grafts) were switched to Dox-HFD to activate the GIPR only in gWAT grafts, which were now embedded into an endogenous WT environment (FIG. 3G). Gross examination of grafted donor gWAT post implantation revealed the formation of blood vessels, suggesting successful generation of a blood supply and incorporation of thegraft within the endogenous WT recipient fat depot (FIG. 3G). Consistent with this, H&E revealed viable healthy fat-grafts, when compared with endogenous fat (FIG. 3H). Gene expression confirmed significant GIPR induction only in transgenic fat grafted onto WT recipient gWAT (FIG. 31). No GIPR activation was evident in i) HFD-fed donor fat (no Dox), ii) Dox-HFD-fed grafted donor WT gWAT, iii) endogenous WT recipient gWAT or, iv) WT or transgenic sWAT (FIG. 31); confirming the specificity and efficacy of the gWAT transplantation system. Interestingly, grafting just a small amount (-200 mg in total) of donor GIPR-Adip fat onto WT recipient gWAT significantly reduced their food intake and body weight gain during Dox-HFD (FIG. 3J). This indicates that activation of the GIPR in just a small amount of transplanted adipose tissue is sufficient to elicit beneficial effects on food intake and body weight.

[0271] To further evaluate the metabolic benefits of GIPR in fat, adeno-associated viruses (AAVs) were utilized to target a sub-set of adipocytes, as opposed to induction of GIPR in all fat-depots. A novel AAV TRE-GIPR vector was engineered along with a control AAV TRE- mmR2F vector. The use of the TRE promoter restricted GIPR transgene expression from the specifically to mature rtTA-expressing adipocytes. A woodchuck post-transcriptional regulatory element 3 (WPRE3) was included to enhance transgene expression (FIG. 3K). For both vectors, AAVs of the adipocyte-targeting serotype Rec2 were produced and injected directly into the right sWAT depot of HFD-fed adiponectin-rtTA mice (FIG. 3K). The left sWAT depot was not injected and thus served as an internal negative control. GIPR expression was specific to the sWAT depot injected with GIPR-AAV, with no evidence of the GIPR expressed in gWAT, liver, or sWAT injected with control-AAV (FIG. 3L). Obese adiponectin-rtTA mice injected with AAV TRE-GIPR exhibited a significant reduction in food intake between days 2- 6 of Dox-HFD initiation when compared with control AAV TRE-mmR2F injected mice (FIG. 3K). Remarkably, AAV TRE-GIPR mice gained significantly less weight with Dox-HFD (FIG. 3K). Combined, this confirms that activation of the GIPR in a small number of adipocytes, in one specific fat-depot, is sufficient to mimic the anorectic and anti-obesity efficacy in GIPR- Adip mice.

[0272] Leptin is an adipokine that promotes satiety and increases energy expenditure in preclinical models and humans. While the complete elimination of leptin or its receptor causes obesity, a partial reduction in leptin (through leptin-neutralizing antibodies, or genetic mouse models that lower leptin levels) prevents HFD-induced weight gain. To explore leptin as a GIPR-stimulated adipokine that suppresses food intake, a Dox-HFD time course was performed. Systemic levels of leptin significantly dropped in GIPR-Adip mice at 6-24 hours of Dox-HFD, with levels remaining low (FIG. 3M); a drop that occurs prior to the reduction in food intake (which occurs at 48 hours of Dox-HFD) (FIG 3A). This suggests that a GIPR-drivenpartial reduction in leptin may initiate a reduction in food intake. It was hypothesized that preventing this drop in leptin in transgenic mice, food intake would normalize, thus pinpointing leptin as an anorectic factor that the GIPR targets. TRE-leptin mice were crossed with GIPR- Adip mice, to co-overexpress both the GIPR and leptin specifically in adipose tissue (FIG. 3N). Neither food intake or body weight were significantly altered between obese GIPR-Adip mice and Lep-GIPR-Adip mice (FIG. 30), indicating that restoration of leptin levels in GIPR-Adip mice is not sufficient to counteract negative energy balance and further, leptin is not the Gl PR- induced satiety factor. In summary, an extensive battery of methodologies were utilized to assess the impact of adipocyte-specific induction of the GIPR on feeding behavior. Specifically, adipocyte GIPR drives a transient suppression in food intake i) between 2-4 days of Dox-HFD in lean mice, ii) for ~3 weeks in obese mice, iii) following pharmacological treatment with a GIPR agonist, iv) after rosiglitazone treatment, v) upon transplantation of GIPR-Adip gWAT into WT mice and, and vi) upon injection of an AAV TRE-GIPR directly into sWAT. Collectively, these studies suggest that in addition to acting centrally to suppress appetite, GIPR-based therapeutics may also function by engaging the GIPR in the fat cell to reduce caloric intake.Example 4: GIPR in Adipose Tissue Increases Energy Expenditure and Local Lipid Oxidation

[0273] After 5 days of Dox-HFD feeding, GIPR-Adip mice normalize their caloric intake (FIG. 3A); yet thereafter, weight loss is maintained (FIG. 2C) or weight gain is prevented (FIG. 3A). Experiments were conducted to examine whether activation of the GIPR in the adipocyte sustains weight loss due to an increase in whole-body metabolic rate. Notably, no significant difference in locomotor activity was evident between genotypes (FIG. 4A). Indirect calorimetry (initiated prior to body weights diverging between WT and GIPR-Adip mice) revealed transgenic mice had significantly increased oxygen consumption (FIG. 4B), carbon dioxide production, and energy expenditure when compared with WT controls (FIG. 40). This suggests that GIPR induction in fat promotes weight loss, due to an initial suppression in appetite followed by an increase in energy expenditure. Transgenic mice further exhibited a significantly lower respiratory exchange ratio (RER) (FIG. 4D), highlighting a fat cell-specific GIPR-driven preferential shift towards the utilization of lipids as a primary fuel source. Consistent with this, ex vivo whole-tissue3H-triolein lipid tracer profiling revealed a significant increase in lipid oxidation exclusively in white fat depots of GIPR-Adip mice, with no marked differences in BAT, liver, heart, or soleus muscle (FIG. 4E). Seahorse analyses revealed a significant increase in oxygen consumption rate (OCR) in transgenic WAT explants (sWAT, gWAT, and mWAT) (FIG. 4F and 4G). Specifically, transgenic fat exhibited significant increases in basal, oligomycin-resistant, carbonyl cyanide p-trifluoromethoxyphenylhydrazone(FCCP)-stimulated maximal respiration, and spare respiratory capacity (FIG. 4F). Assessment of the energetic state revealed an increase in aerobic and glycolytic processes across all transgenic white fat depots (FIG. 4H), suggesting that activation of the GIPR enhances the energy demand of the tissue.

[0274] GIP plays a key physiological role in the postprandial disposal of dietary triglycerides (TGs) in preclinical models and humans. Experiments were conducted to test whether adipose GIPR impacts systemic lipid metabolism. Transgenic animals exhibited significantly lower circulating levels of TGs with Dox-HFD feeding (FIG. 41), suggesting efficient lipid-lowering capabilities of the GIPR in the adipocyte. Consistent with this, challenging obese GIPR-Adip mice with a lipid load revealed a significant improvement in TG clearance rate, which was more prominent following pharmacological treatment with a GIPR agonist (FIG. 4J). This effect was independently confirmed using3H-triolein lipid-tracing, showing a significantly enhanced whole-body lipid clearance rate in GIPR-Adip mice (FIG. 4K). At the tissue level,3H-triolein lipid tracer injections identified a significant increase in lipid uptake only in sWAT and mWAT of transgenic mice (FIG. 4L), thus pinpointing these fat depots primarily responsible for the lipid-lowering actions of GIPR in the adipocyte. In terms of lipolysis, GIPR-Adip mice exhibited significantly lower p3-AR-stimulated NEFA and glycerol levels (FIG. 4M). To maintain the high energy demand (FIG. 4H), the GIPR in the adipocyte may lower the release of excess lipids into circulation to maintain a critical fuel source to the fat cell. Collectively, induction of adipocyte GIPR reduces body weight through a transient suppression in appetite and augmentation of whole-body energy expenditure; an effect fueled by oxidation of both stored and dietary lipids.Example 5: Mechanism by which GIPR Action in Adipose Tissue Increases Energy Expenditure is by Driving a Sarcolipin-SERCA Pathway to Promote Futile Calcium Cycling

[0275] To identify a mechanism(s) by which activation of the GIPR in adipose tissue increases energy expenditure, targeted transcriptomics were performed.

[0276] RNA-Seq was performed on whole sWAT. Out of the top 100 differentially regulated genes between WT and GIPR-Adip sWAT, 77 genes were identified that are significantly upregulated in transgenic fat at 3 days Dox-HFD and 16 genes upregulated at 2 weeks (FIG. 5A); with 6.5% of genes (153 genes) overlapping between the two time-points (FIG. 5B). The most significantly modulated pathways in GIPR-Adip sWAT at 3 days Dox-HFD included: calcium signaling, glycolysis, JAK1 and JAK3 signaling, and protein kinase A signaling (FIG. 5C). At 2 weeks Dox-HFD, the topmost upregulated pathway in GIPR-Adip fat by 7.2-fold was the calcium signaling pathway (FIG 5C). Other pathways upregulated to a lesser extent werevitamin D receptor / retinoid X receptor activation, STAT3 signaling, phospholipase C signaling, and cAMP signaling (FIG. 50). Intriguingly, the common significantly upregulated pathway identified at both timepoints in GIPR-Adip sWAT was calcium signaling (FIG. 50). Consistent with RNA-Seq pointing to calcium signaling, gene ontology and KEGG-enrichment cluster pathway analyses of single cell RNA-Seq in human white adipocytes identified two pathways that cluster significantly with the GIPR exclusively in the hAd7 sub-population of adipocytes (the sub-population that expresses the GIPR at high levels) (FIG. 10). The two pathways identified by single cell RNA-Seq were: the i) response to calcium ion (Ca2+) pathway and ii) cAMP signaling (Table 2). Collectively, both the adipose tissue RNA-Seq and the adipocyte single cell RNA-Seq (FIG. 50 and Table 2) identified calcium signaling as a major pathway activated by the GIPR, thus providing rationale to pursue this pathway in relation to energy balance.Table 2. The two pathways and associated genes identified by single cell RNA-Seq that cluster exclusively with GIPR in the human hAd7 sub-population of white adipocytes. ** p<0.01.

[0277] The topmost significant, differentially upregulated genes in the GIPR adipose tissue RNA-Seq included S100 Ca2+-binding protein G (S100g), renin 1 (Reni), renin 2 (Ren2), synaptotagmin 4 (Syt4), steroidogenic acute regulatory protein (StAR), aldehyde dehydrogenase 1 family member L2 (Aldh1l2), and sushi, nidogen, and EGF-like domains 1 (Snedl) (Table 3).Table 3. The top differentially upregulated genes identified by RNA-Seq analysis of sWAT from WT and GIPR-Adip mice following 2 weeks of Dox-HFD. Gene abbreviations, definitions, fold increases, and significance between WT and GIPR-Adip sWAT is indicated (n=9). ***P<0.001.

[0278] S100g (calbindin-D9K) and Syt4 are both Ca2+-binding proteins. The former is a protein that buffers free cytosolic Ca2+and enhances ATP-dependent Ca2+-transport. S100g is also part of a superfamily of EF-hand Ca2+-binding mediators most involved in Ca2+-signal transduction. It was therefore examined as to how many S100 family members were regulated in GIPR-Adip sWAT, and from these, which participate in Ca2+-transport and energy metabolism. Table 4 shows the S100 family of proteins in RNA-Seq GIPR-Adip sWAT.Table 4. The S100 family of Ca2+-binding proteins from the RNA-Seq of WT and GIPR-Adip sWAT following 2 weeks of Dox-HFD. (n=9). *P<0.05; **P<0.01; ***P<0.001.

[0279] Interestingly, S100a1 signals through the intracellular Ca2+-transporter protein sarco / endoplasmic reticulum (SR) Ca2+-ATPase 2a (SERCA2a) and the Ca2+-release channel ryanodine receptor 2 (RyR2); both coordinate Ca2+-transport in skeletal muscle and the heart. RyR2 is also part of the SERCA2b futile calcium cycling pathway that promotes thermogenesis in beige fat. Consistent with this, single cell RNA-Seq of white adipocytes pinpointed RyR2 as a main component that clusters with the GIPR in the hAd7 sub-population of adipocytes during Ca2+-signaling (Table 2). Moreover, RNA-Seq of adipose tissue revealed numerous Ca2+-signaling genes significantly regulated in GIPR-Adip sWAT (Table 5); with calcium signaling being the topmost upregulated pathway (FIG. 50).Table 5. Genes involved in the calcium cycling and Ca2+-signaling pathways in the RNA-Seq WT and GIPR-Adip sWAT following 2 weeks of Dox-HFD. (n=9). * p<0.05; ** p<0.01 ; *** p<0.001.

[0280] Simultaneously, other pathways known to promote thermogenesis were examined; namely, the futile creatine cycle, classical brown adipocyte thermogenesis, and beige adipocyte thermogenesis. No differences in the transcriptional signature of known markers of brown or beige adipose tissue were evident in GIPR-Adip sWAT (FIG. 5D). Similarly, markers of futile creatine cycling (Ckmtl, Ckmt2, Gamt, Gatm, Slc6a8 / [CrT] and Al pl) were not significantly altered in transgenic fat (FIG. 5E). A significant reduction in creatine kinase activity was also observed in GIPR-Adip sWAT (FIG. 5F). Combined, a mechanism of GIPR action in white adipocytes may be to drive futile calcium cycling to increase energy expenditure.

[0281] Intracellular Ca2+is regulated by SERCA, which utilizes ATP to actively pump cytosolic Ca2+into the endoplasmic reticulum (ER) lumen. Uncoupling SERCA-mediated Ca2+-transport from the energy gained through ATP hydrolysis reduces the efficiency of Ca2+-uptake, thus releasing energy as heat. The net result is futile calcium cycling, which drives thermogenesis and whole-body energy expenditure; an effect reported in skeletal muscle, heart, brown fat, and beige fat. Here, Sercal mRNA expression levels are upregulated in transgenic fat, with no differences apparent with Serca2a and Serca2b mRNA (FIG. 5G). SERCA1 protein expression has only been reported in skeletal muscle and BAT. Here, it is demonstrated that SERCA1 is also expressed in white fat, and importantly, protein expression is significantlyreduced in GIPR-Adip sWAT (FIG. 5H). This indicates that intracellular Ca2+-signaling mechanisms are regulated post-translationally. Experiments were conducted to examine whether a GIPR-mediated reduction in SERCA1 protein impacts energy expenditure. In skeletal muscle, the ATPase activity of SERCA1 is regulated by a specific member of the regulin family of SR transmembrane proteins, sarcolipin; known to mediate non-shivering thermogenic mechanisms. Sarcolipin binds competitively to SERCA1 to reduce its Cambinding affinity, thus lowering the efficiency of Ca2+-transport into the SR lumen. This binding uncouples Ca2+-transport from the ATP hydrolysis activity of SERCA1 , which promotes futile calcium cycling of the SERCA pump and slippage of Ca2+back into the cytosol. The net result creates an increase in unproductive ATP hydrolysis, heat production, and an increased energetic demand. Interestingly, sarcolipin protein expression is significantly increased in GIPR-Adip sWAT (FIG 5I), suggesting that the GIPR may trigger sarcolipin-mediated thermogenesis and potentially the increase in energy expenditure (FIG. 4B-D).

[0282] Another means of futile calcium cycling is through SERCA2 and RyR2. SERCA2b- driven Ca2+-influx into the ER is offset by Ca2+-efflux through RyR2. This results in continuous futile consumption of ATP without a net change in Ca2+compartmentalization, causing heat production, which is energetically costly. Here, total SERCA2 and SERCA2a protein levels are significantly increased in GIPR-Adip adipose tissue (FIG. 5J). SERCA2 immunofluorescence staining further showed a marked increase in SERCA2 protein levels in GIPR adipose tissue (FIG. 5K), confirming a Gl PR-stimulated increase in SERCA2 protein in the white adipocyte. This protein data is consistent with our single cell RNA-Seq analyses that showed RyR2 clusters with the GIPR in the hAd7 sub-population of human adipocytes (Table 2). Moreover, adipose tissue RNA-Seq revealed a significant increase in RyR2 expression in GIPR-Adip fat (Table 5). Combined, we hypothesized that the GIPR in the adipocyte drives SERCA2- mediated futile calcium cycling to increase energy expenditure.

[0283] To directly test this, we utilized an AAV-based approach to overexpress the GIPR while concomitantly eliminating SERCA2 specifically in the white adipocyte. We generated two new vector backbones. First, a backbone containing an engineered version of the human adiponectin promoter to drive expression of either the GIPR or sfGV-CAAX (a membrane- targeted sfGFP-based ‘spaghetti monster’ fluorescent protein; utilized as an overexpression control) with three miR-122 binding sites located in the 3' UTR to suppress expression in hepatocytes. Second, a backbone containing the same engineered human adiponectin promoter to drive the expression of Staphylococcus aureus Cas9 (SaCas9) and a human U6 promoter to drive the expression of either a Serca2 gRNA or non-targeting gRNA (utilized as a CRISPR control). This latter new compact AAV CRISPR / Cas9 design of only 4.8-kb viral genome size allowed for efficient virus production and direct application of the vector to micewithout the need to express Cas9 from either a germline transgene or a separate viral vector (FIG. 5L, left panel). We directly injected the following AAV combinations into the sWAT depot of obese WT HFD-fed mice: i) sfGV-CAAX + non-targeting gRNA, ii) GIPR + non-targeting gRNA, iii) GIPR + Serca2 gRNAs, iv) sfGV-CAAX + Serca2 gRNAs. We observed that: 1) consistent with the phenotype in GIPR-Adip transgenic mice, AAV-mediated GIPR overexpression in white adipocytes significantly increases whole-body energy expenditure when compared with sfGV-CAAX overexpression (FIG. 5M). 2) Knockout of SERCA2 in adipocytes using our CRISPR / Cas9 approach significantly reduced the effect of adipocyte GIPR overexpression on energy expenditure (FIG. 5M). This demonstrates that the mechanism of Gl PR-stimulated increase in energy expenditure, and ultimately body weight loss, is critically-dependent on SERCA2-mediated futile calcium cycling.

[0284] Infrared thermal imaging highlighted increased body temperature of GIPR-Adip mice following glucose administration (FIG. 5N). To validate that futile calcium cycling accounts for the local thermogenic activity mediated by overexpression of the GIPR in white adipocytes, we examined adipose tissue temperature in mice treated with our novel AAVs. Adipose tissue temperature is an established measure of local thermogenesis and a surrogate of energy expenditure. By directly injecting our AAV vectors into obese HFD-fed WT mice, we knocked out SERCA2 from adipocytes while overexpressing the GIPR. We observed that AAV- mediated GIPR overexpression in white adipocytes significantly increases local tissue temperature (FIG. 5L, right panel). Importantly, inhibiting futile calcium cycling through the elimination of SERCA2, abolished the thermogenic activity of GIPR overexpression in adipose tissue (FIG. 5L, right panel). This provides further evidence that SERCA2-mediated futile calcium cycling is a central component to the GIPR-driven increase in energy expenditure.

[0285] Consistent with an ER-based futile calcium cycling mechanism, mitochondria isolated from WT and GIPR-Adip gWAT displayed no difference in oxidative consumption (FIG. 5P), suggesting no dysfunction or enhancement in oxidative metabolism of mitochondria in isolation. One explanation could be that the GIPR triggers SERCA-mediated ATP hydrolysis during futile calcium cycling in the cytosol, which increases ATP consumption and energy demand (FIG. 4H). This may enhance oxidative phosphorylation, due to increased ADP availability as mitochondrial substrate for ATP synthesis. We further observed no significant difference in body temperature under thermoneutral conditions in GIPR-Adip mice (FIG. 50). This suggests that an increase in futile calcium cycling thermogenesis in transgenic mice is tightly controlled, with no thermal stress i.e., no hyperthermia is evident, as surplus heat is effectively dissipated even at thermoneutrality.

[0286] To examine the cell-autonomous effects, we treated in vitro-differentiated white adipocytes from WT and GIPR-Adip mice with thapsigargin, a pharmacological inhibitor ofSERCA, to suppress futile calcium cycling. Consistent with our in vivo findings, we observed that overexpression of the GIPR in adipocytes significantly increases oxygen consumption (FIG. 5Q). Importantly, SERCA inhibition diminished the adipocyte GIPR-driven increase in oxygen consumption, leading to a reduction of cellular energy demand in GIPR- overexpressing adipocytes (FIG. 5Q). Similar findings were observed with acute perfusion of the whole gWAT fat-depots with thapsigargin (FIG. 5R). Combined, this indicates that SERCA- mediated futile calcium cycling is required for the GIPR-driven increase in energy expenditure in white adipocytes. Taken together, FIG. 5S shows the proposed mechanism for GIPR action in the white adipocyte, in which the GIPR activates two SERCA-mediated futile calcium cycling pathways to increase energy expenditure. This promotes weight loss during obesity and protects mice from diet-induced obesity.Example 6: GIPR in Adipose Tissue Triggers a “Metabolic Memory” Effect to Sustain Weight Loss and Maintain Protection from Obesity

[0287] A major challenge of obesity management is to provide sustained clinically relevant weight loss. Currently, the most effective approaches utilized to combat excess adiposity are focused on energy intake, and do not treat the underlying disease driving the consumption of excess calories. Therefore, when the obesity therapy is removed, dramatic hyperphagia often follows, which in combination with a state of increased metabolic efficiency, leads to weight re-gain. Here, it was found that following the removal of Dox after 12 weeks (i.e. , switching mice to a non-Dox-containing HFD, thus switching off the GIPR in fat), transgenic mice did not re-gain their body weight back to WT littermate levels (FIG 6A, second phase). Human studies demonstrate that after significant weight loss with the GLP-1 R agonist liraglutide, following discontinuation of treatment, subjects rapidly re-gain body weight. Here, the fact that GIPR- Adip mice do not re-gain body weight following normalization of the GIPR transgene, suggests a phenomenon of GIPR-driven “metabolic memory”. This effect may imprint upon the adipocyte to re-wire the metabolic program of adipose tissue, such that the net outcome is to defend weight loss and prevent weight re-gain. Interestingly, upon reverting mice back to Dox- HFD (i.e., switching the GIPR transgene back on), GIPR-Adip mice exhibit an immediate drop in body weight, as early as one week, whereas WT mice continue gaining weight; with a massive -69% difference in weight change being preserved (FIG. 6A, third phase). This confirms the potent effect that the GIPR in the white adipocyte has on weight loss during obesity.

[0288] In addition to the prevention of obesity, experiments were conducted to examine whether the GIPR triggers metabolic memory to preserve the weight lost in obese mice. After switching obese mice to Dox-HFD, it was consistently observed (FIG. 20) that GIPR-Adip mice exhibit an immediate reduction in body weight (FIG. 6B). Following a shorter 8-weekexposure to Dox-HFD, as opposed to 12 weeks (FIG. 6A), GIPR-Adip maintain the metabolic memory effect, by sustaining a lower body weight with a -39% difference in weight change (FIG. 6B). Food intake is significantly reduced for up to 3-4 weeks in obese GIPR-Adip mice, however, normalizes after this period with mice even transiently overcompensating (FIG. 60). Importantly, the metabolic memory of sustained weight loss persists in GIPR-Adip mice even after the GIPR is switched off in adipose tissue, as no difference in GIPR gene expression is evident between WT and GIPR-Adip fat (FIG. 6D). This suggests that the GIPR in the white adipocyte, after a specific timeframe, drives a potent long-term metabolic memory effect, which re-wires a weight sustaining program through a permanently imprinted increase in energy expenditure.

[0289] Given that the GIPR regulates components of the futile calcium cycling pathway in white fat (FIG. 5H-J and Table 5), it was hypothesized that this mechanism could be a contributor to metabolic memory. Interestingly, a significant increase in sarcolipin protein levels in GIPR-Adip sWAT in the metabolic memory phase was observed (FIG. 6E). Given the absence of any ectopic GIPR expression from the transgene during the memory stage (FIG. 6D) (i.e., the GIPR was switched off in fat), yet sarcolipin protein levels remained high, this suggests that the GIPR exerts long-lasting metabolic memory on weight stabilization through sarcolipin as a biomarker. To probe this further, the exposure time of obese mice was titrated down to just 3 weeks of Dox-HFD, with the added condition that WT mice were placed on a 50% food- restricted diet to match the rapid kinetics of weight loss in GIPR-Adip mice (FIG. 20). Despite 50% food-restriction, WT mice do not achieve the same degree of weight loss that transgenic mice reach (FIG. 6F). No metabolic memory was observed in GIPR-Adip mice following 3 weeks of Dox-HFD, as apparent by immediate weight re-gain following the removal of Dox (FIG. 6F), which was confirmed by normalization of the GIPR transgene expression in adipose tissue (FIG. 6G). Importantly, no significant difference in sarcolipin protein levels was evident in GIPR-Adip fat after 3 weeks of initial Dox-HFD exposure, potentially permitting weight re-gain (FIG. 6H). Sarcolipin correlates with a state of increased energy expenditure, and as such, may serve as a critical biomarker for the Gl PR-driven metabolic memory program of weight loss maintenance. Given that e a body weight metabolic memory effect at 8 and 12 weeks of Dox-HFD, but not at 3 weeks Dox-HFD, a 5 week Dox-HFD timepoint was lastly examined. Similar to 3 weeks of GIPR induction, GIPR-Adip mice did not display a permanent change in their body weight set-point, albeit transgenic mice exhibiting a slower rebound rate than the 3 weeks of GIPR induction (FIG. 6I). This pinpoints that the GIPR-driven re-set point of sustained weight loss occurs between 5 and 8 weeks of GIPR induction in adipose tissue.Example 7: Discussion

[0290] Here, a mechanism for GIPR action in white adipose tissue was identified. Genetically, it is shown that the GIPR can drive energy expenditure and weight loss based on its expression in the white adipocyte. This may have important translational implications that contribute mechanistic insights as to how GIPR-based therapies such as GIPR / GLP-1 R co-agonists elicit weight loss in individuals with obesity and type II diabetes. A novel adipocyte-specific mouse model of GIPR induction was utilized to unravel the pharmacological potential of the fat cell. This allowed the maximization of the GIPR system to unveil mechanisms that current pharmacology may not detect. Specifically, it is demonstrated that 1) induction of the GIPR in white fat activates futile calcium cycling through modulation of sarcolipin, SERCA1 , and SERCA2b; 2) Gl PR-mediated futile calcium cycling increases lipid oxidation, oxidative metabolism, and energy demand to enhance whole-body energy expenditure; 3) GIPR signaling in adipocytes creates a negative energy balance, which causes weight loss in obese mice and protection from diet-induced obesity; 4) GIPR signaling in adipocytes initiates a signal from the fat cell that targets the brain to suppress appetite; 5) a novel aspect of GIPR action was identified in its unique ability to activate a “metabolic memory” effect by recalibrating adipose tissue to sustain weight loss after cessation of GIPR induction in fat; 6) it was shown that the GIPR is endogenously expressed by human white adipocytes in specific hAd6 and hAd7 sub-populations of fat cells. Taken together, the findings from activating the GIPR specifically in the white adipocyte, offer a novel mechanism by which the engagement of the GIPR improves the therapeutic profile of GLP-1 R agonism.

[0291] Multi-receptor agonists targeting the GIPR, GLP-1 R, and GCGR provide significant clinical benefits in the management of metabolic disease. However, how the GIPR contributes to the improved therapeutic profile remains to be elucidated. A major finding of this study is that activation of the GIPR in adipose tissue protects mice from diet-induced obesity and promotes rapid body weight loss (-35%) in the obese state; both occurring with a reduction in fat mass. This suggests that the GIPR in the adipocyte has a major contribution to the antiobesity efficacy of incretin-based multi-receptor agonists. Some preclinical studies show that administration of GIPR agonists causes negligible weight-lowering efficacy in obese mice. In contrast, other GIPR agonists have been reported to promote dose-dependent weight loss. This discrepancy may be based on some GIPR agonists harboring a lower receptor potency in mice, in comparison to humans. In support of this, it has been demonstrated that GIPR agonists, optimized to bind the murine GIPR, display more impressive weight loss efficacy. Similar effects were noted with other GIPR agonists that increased GLP-1 R agonist-induced weight loss in obese rodents in addition to transgenic mice with elevated levels of GIP having protection from obesity. Human studies echo the superior weight loss achieved throughGIPR / GLP-1 R co-agonism when compared with GLP-1 R agonism. Taken together, the integration of GIPR agonism into poly-receptor therapies appears essential to achieve optimal weight-lowering efficacy, thus offering much promise for incretin-based next generation antiobesity agents. Here, the adipocyte with enhanced activation of the GIPR were stimulated. This triggers massive weight loss and further highlights the powerful weight-lowering capabilities of the GIPR in white fat. This also places adipocyte GIPR in a new light as an important contender in Gl PR-based therapeutics, which may convey clinical relevance and significant translational potential, thus solidifying GIPR agonism as being therapeutically advantageous to treat obesity.

[0292] Recently, hAd7 adipocytes were identified to be a sub-population of fat cells that negatively correlate with BMI. It is demonstrated here that the GIPR clusters within this hAd7 sub-population of adipocytes, which is consistent with Gl PR-positive fat cells having the capacity to induce weight loss in GIPR-Adip mice. Could a sub-population of human adipocytes trigger such profound effects on energy balance? Indeed, it has been indicated that hAd7 adipocytes possess an outsized importance in that they are associated with metabolic health despite being relatively small in number i.e., constituting ~1% of all human adipocytes. Moreover, hAd7 adipocyte numbers inversely correlate with insulin resistance; suggesting a beneficial role in metabolism. Notably, transplantation of as little as 200 mg of GIPR-Adip adipose tissue into WT mice still causes significant beneficial effects on body weight. This supports the notion that a small number of white adipocytes expressing the GIPR can greatly impact energy balance. It may also be plausible that hAd7 adipocytes signal to other fat cells within their microenvironment to initiate beneficial metabolic cues, particularly in an obese setting with increased dysfunctional fat mass.

[0293] How does activation of the GIPR in the adipocyte trigger profound weight loss? GIPR- Adip mice exhibit increased energy expenditure, concomitant with a reduction in RER. The latter highlighting preferential utilization of lipids as an energy source, which is also apparent by the enhanced systemic TG-lowering efficacy in GIPR-Adip mice. In line with these studies, GIPR agonism significantly contributes to increased energy expenditure in a metabolic adaptation setting. GIPR / GLP-1 R co-agonism was shown to increase TG utilization >30% during caloric restriction-induced weight loss. This effect was reversed with GIPR antagonist treatment, suggesting that the GIPR plays a role in sustaining elevated energy expenditure. Independent of this, GIPR / GLP-1 R co-agonist treatment of obese mice transiently albeit significantly, increases energy expenditure. Locally, it is demonstrated that activation of the GIPR increases WAT lipid uptake and oxidation, in addition to enhancing oxidative respiration and cellular energy demand, specifically in white fat depots. In agreement with this, the GIPR is known to promote adipose tissue health and improve lipid metabolism in several preclinicaland clinical models. Of note, to date however, it is yet to be established whether GIPR agonism treatment in humans increases energy expenditure. Future clinical mechanistic studies examining whether chronic GIPR agonist treatment alone, or in combination with GLP-1 R agonism enhances energy expenditure particularly in people with obesity and / or type II diabetes, should prove illuminating.

[0294] How GIPR agonism potentiates weight loss in multi-receptor agonism is unknown. The current study pinpoints futile calcium cycling as a mechanism by which GIPR agonism in white fat contributes to weight loss. More specifically, it is known that SERCA proteins use ATP to pump Ca2+from the cytosol to ER compartments, and uncoupling Ca2+-transport from SERCA forces Ca2+to accumulate in the cytosol. This creates futile calcium cycling, whereby the energy gained through ATP hydrolysis without Ca2+transport, increases in thermogenesis and energy expenditure. Here, through RNA-Seq of mouse white fat and single cell RNA-Seq of human white adipocytes, the GIPR was identified to modulate specific downstream components of futile calcium cycling pathways such as SERCA1 and SERCA2b. Indeed, pharmacological inhibition of SERCA proteins blunts the GIPR-driven increase in oxygen consumption and cellular energy demand in white fat, confirming that the GIPR signals through SERCAs.

[0295] Sarcolipin is a protein that competes with Ca2+to bind SERCA1 during Ca2+-transport, thus reducing its efficiency. This does not inhibit the ATP hydrolysis activity of SERCA1 , rather hinders Ca2+-uptake into the ER, to allow Ca2+to slip back into the cytosol, which results in SERCA1 consuming more ATP to transport less Ca2+. In skeletal muscle and BAT this disconnect generates futile calcium cycling of the SERCA1 pump, which leads to heat production from ATP hydrolysis, enhanced thermogenesis, and increased energy expenditure. Here, it is demonstrated that in white fat the GIPR increases sarcolipin protein levels and reduces SERCA1 levels. This suggests that the GIPR triggers sarcolipin-mediated inhibition of SERCA1 Ca2+-transport, which may drive energy expenditure. Indeed, mutations in the chromosomal locus containing sarcolipin are associated with obesity, indicating that an increase in sarcolipin is anti-obesogenic. Consistent with this adipose-specific GIPR-Adip mice, muscle-specific sarcolipin mice exhibit significant weight loss, a depletion in fat mass, resistance to obesity, and increased energy expenditure. In contrast, loss of sarcolipin predisposes mice to obesity. Collectively, this suggests that increasing sarcolipin levels, as observed in GIPR-Adip mice, increases energy expenditure. Indeed, it has been proposed that a higher sarcolipin-to-SERCA ratio is energetically costly, leading to less fat deposition. As such, a GIPR-driven increase in sarcolipin may be a critical determinant controlling the basal metabolic rate of white adipose tissue and energy balance.

[0296] The alternative pathway of futile calcium cycling is through the SERCA2b pump and the Ca2+release receptor RyR2; a pathway that increases thermogenesis in beige fat. Coldstimulation and / or P3-AR agonist treatment triggers SERCA2b-mediated Ca2+influx to become offset by RyR2-mediated Ca2+efflux; resulting in SERCA ATP-consumption without a change in ER Ca2+localization. It is demonstrated here that activation of the GIPR in white fat increases SERCA2b and RyR2 levels. Importantly, the GIPR clusters specifically with the RyR2 in the hAd7 sub-population of human white adipocytes, suggesting that the GIPR may drive SERCA2b-mediated energy demand in the white adipocyte. To directly confirm that the GIPR in adipocytes drives SERCA2-mediated futile calcium cycling to increase whole-body energy expenditure, a novel white adipocyte-specific CRISPR / Cas9 AAV was generated to eliminate SERCA2 in target tissues. Following GIPR AAV and / or Serca2-CRISPR AAV coinjection directly into adipose tissue of obese WT mice, we demonstrated that the adipocyte GIPR AAV enhances whole-body energy expenditure. Remarkably, this confirmed the observations in the adipocyte-specific GIPR transgenic mice. Importantly, with SERCA2 elimination in the adipocyte, this Gl PR-driven increase in energy expenditure was abolished. The adipose tissue temperature was further assessed as a measure of local thermogenic activity to validate the whole body energy expenditure findings using the novel AAVs. As expected, AAV-mediated white adipocyte GIPR overexpression increased local thermogenesis, while inhibiting futile calcium cycling through CRISPR / Cas9 AAV elimination of SERCA2 diminished it. Direct evidence that show that SERCA2-mediated futile calcium cycling is central to the Gl PR-mediated in local thermogenesis and whole-body energy expenditure is provided. These in vivo findings were corroborated in a cell-autonomous system, as pharmacological inhibition of SERCA activity in vitro in fully differentiated white adipocytes blunted the Gl PR-stimulated increase in oxygen consumption and cellular energy demand. In vivo and in vitro findings provided herein thus identify and confirm that the mechanism of white adipocyte Gl PR-stimulated increase in whole-body energy expenditure is directly and critically dependent on SERCA2-mediated futile calcium cycling.

[0297] Taken together, a novel mechanism of GIPR action in the white adipocyte was identified, whereby the GIPR activates two futile calcium cycling, primarily through the post- translational control of two SERCA pathways. The net result yields activation of two independent SERCA-mediated futile calcium cycle pathways, i.e., both pathways are not mutually exclusive. Of note, it is also shows that the hAd6 sub-population of adipocytes cluster within GIPR-expressing fat cells. hAd6 adipocytes selectively express genes associated with thermogenesis, thus rendering them a potential thermogenic sub-population. Combined, this is consistent with the GIPR driving SERCA-mediated thermogenesis in GIPR-Adip mice. Collectively, it is shown that during obesity, activation of the GIPR drives a state of energywasting, through futile calcium cycling in the adipocyte; this may enhance the uptake and oxidation of excess nutrients in the fat-cell to yield a net result that increases whole-body energy expenditure and promote weight loss.

[0298] Through use of multiple methodologies, it is consistently demonstrated here that activation of the GIPR in the white adipocyte transiently suppresses food-intake. Like these observations, preclinical findings show that pharmacological treatment of obese mice with GIPR agonists causes a transient reduction in food intake, which occurs during the first 24-48 hours of treatment. Similarly, transgenic mice with enhanced levels of GIP exhibit reduced caloric intake. In obese mice, GIPR / GLP-1 R co-agonist treatment suppresses appetite to a greater extent than GLP-1 R agonism alone, suggesting that GIPR agonism potentiates the anorectic activity of GLP-1 R agonism.

[0299] It is demonstrated that activation of the GIPR in white fat suppresses food intake, thereafter following normalization, an increase in energy expenditure ensues to maintain weight loss. Moreover, in addition to targeting the brain, GIPR-based therapeutics may directly engage the GIPR in the fat cell to suppress appetite. Remarkably, activation of the GIPR in the white adipocyte phenocopies the majority of aspects of GIPR / GLP-1 R co-agonism. While these co-agonists unquestionably activate receptors in other tissues too, these observations highlight the impact that the white adipocyte alone can exert on energy balance.

[0300] Maintaining weight loss following obesity is incredibly challenging. Clinical studies report that -80% of individuals with obesity that lost 10% of their body weight will re-gain that weight within one year. In fact, weight loss was shown to immediately rebound following withdrawal of the GLP-1 R agonist semaglutide. Similarly, subjects that discontinued treatment with the GLP-1 R agonist liraglutide, following significant weight loss, re-gained body weight even at a higher rate than placebo. As such, obesity is now considered a chronic disease that may require continuous treatment. Sustaining weight loss after cessation of treatment is therefore essential for the long-term management of obesity. The mechanisms underlying the biological drive to re-gain weight are poorly understood, with the key therapeutic targets that maintain weight loss yet to be identified. Here, it is shown that 8-12 weeks of GIPR induction in the white adipocyte is sufficient to trigger a “metabolic memory” effect of body weight, i.e. , weight loss is sustained after GIPR induction is switched off. Albeit between 3-5 weeks of GIPR activation, no metabolic memory is achieved, weight loss is not sustained, and immediate weight re-gain ensues. Interestingly, at any given point, there is no evidence of metabolic memory in food intake, thus the Gl PR-induced metabolic reprogramming only occurs with sustained energy balance. How is this metabolic memory of weight loss achieved? Surprisingly, sarcolipin levels remain significantly increased at the Gl PR-induced 8 week metabolic memory phase of sustained weight loss, despite removal of GIPR induction in fat.Importantly, sarcolipin levels were normalized after 3 weeks of Dox-HFD, a much shorter period of GIPR induction, i.e. when no metabolic memory was achieved, hence weight re-gain. This suggests a novel mechanism of post-translational regulation of metabolic memory, in which a GIPR-sarcolipin-axis in the white adipocyte is a critical determinant to re-wire the system to preserve weight loss and defend against weight re-gain. It is thus proposed that the GIPR in the white adipocyte is a potential novel therapeutic target for the delivery of clinically relevant, sustained weight loss in the management of excess adiposity. These findings are consistent with studies performed in C. elegans, whereby less weight re-gain was evident during feeding assays with adipocytes shown to be primary responsible for transcriptional metabolic memory. Future studies examining whether the GIPR plays a critical role in weight loss maintenance in a human obesity setting should prove illuminating. In conclusion, the remarkable weight-lowering efficacy of multi-receptor agonism has captured great attention and fueled much interest in Gl PR-based therapies. Findings herein hint that GIPR activation in the white adipocyte could be a significant contributor to the profound weight loss achieved. The underestimated power of the white adipocyte to increase whole-body energy expenditure and maximize sustainable weight loss is unveiled herein. Studies also offer mechanistic insights as to how GIPR agonism increases the therapeutic profile and clinical efficacy of GIPR / GLP-1 R co-agonism during obesity.Example 8: Methods used in Examples 1-6

[0301] Mice: All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center at Dallas. The Dox-inducible GIPR mouse (TRE-GIPR mouse) was generated here. Adiponectin-rtTA mice and TRE-leptin mice were generated as previously described. All genotyping primer sequences are provided in Table 6. All experiments were performed utilizing littermate- controlled male mice in a pure C57 / BI6 background. Mice were fed either standard chow-diet (#5058, LabDiet, St. Louis, MO), Dox-chow diet (600 mg / kg Dox; BioServ, Frenchtown, NJ), high fat-diet (HFD) (60% kcal, BioServ), Dox-HFD (50 or 600 mg / kg Dox; BioServ), or a rosiglitazone-Dox-HFD (100 mg / kg rosiglitazone maleate; 600 mg / kg Dox). All experiments were initiated at -8-12 weeks of age.Table 6: List of Genotyping Primers

[0302] Collection of Human White Adipose Tissue Biopsies: WAT biopsies were collected from intra-abdominal and subcutaneous fat-depots during elective surgery at the Brigham and Women’s Hospital, Boston, MA. All participants provided informed consent.

[0303] Transplantation of Gonadal Adipose Tissue: Whole gonadal fat-pads were harvested from 8-12 week-old donor male WT and GIPR-Adip mice., Hair was removal from the gonadal area of WT recipient mice, then 10% povidone-iodine (Medline; MDS093902) was applied to the skin. A skin incision (-5-7 mm) was made and the whole gonadal fat-pad was removed. Two strings of suture (3 cm x 1 cm) (5-0; PGA suture) remained under the depot. Fat-pads were cut 10-12 times (the grafted donor fat-pads and the WT recipient fat-pads), avoiding major blood vessels in the WT recipient fat-pad. The donor fat-pad (from WT or GIPR-Adip mice) (~50 mg / piece; total of -200 mg of fat) was grafted on top of the WT recipient depot, allowing both fat-pads to be in direct contact. Two sutures were loosely tied around the combined fat-pads to maintain intact blood circulation. The entire combined fat tissue was inserted back into the abdominal chamber of the WT recipient mouse. The opening was surgically closed with a 5-0 PGA suture. The donor fat-grafts and the WT recipient fat-pads were harvested at the specified time-points.

[0304] AAV vector construction: The open reading frames of mouse Gipr (NM_001080815.1) or a membrane-targeted mRuby2-based spaghetti monster fluorescent protein (mmR2F) with multiple FLAG-tags was cloned into a custom pAAVK TRE vector. In this vector, transgene expression is driven by a tetracycline-response element promoter (TRE), thus restricted to cells expressing a (reverse) tetracycline-controlled transactivator ((r)tTA). The open reading frames of human GIPR (NM_000164.4) or a membrane-targeted superfolder GFP-based spaghetti monster fluorescent protein (sfGV-CAAX) with multiple V5-tags was cloned into a custom pAAVK mAdipoQP vector. In this vector, transgene expression is driven by a minimal human adiponectin promoter, thus restricted to mature adipocytes. Moreover, the transgene contains three mmu-miR-122 binding sites (5'-ACAAACACCATTGTCACACTCCA-3': SEQ ID NO: 112) in its 3' UTR to suppress off-target expression in the liver. A negative control guide (g) (g1 , 5'-CTTCATACTGCACCGGGCGGG-3'; SEQ ID NO: 113) and two distinct Serca2 Atp2a2) guides (g1 , 5'-CATCGATAGGTGCACCCACAT-3' (SEQ ID NO: 114); g2, 5'- CCTCGTATTTGATGAAGTTAG-3' (SEQ ID NO: 115) were selected using CRISPick, then cloned into a custom pAAVK mAdipoQP-SaCas9+U6-sgRNA vector. In this vector, Staphylococcus aureus Cas9 (SaCas9) expression is driven by a minimized human adiponectin promoter, thus restricted to mature adipocytes, whereas sgRNA expression is driven by a human U6 promoter.

[0305] AAV Vector Production: To produce adeno-associated viruses (AAVs) of the AAV2 / Rec2 serotype43, HEK293T cells were transfected with pAAVK (6 pg), pHelper (12 pg) (Cell Biolabs, San Diego, CA) and pRepCap Rec2 (24 pg) (the latter a kind gift from Deborah Young, University of Auckland, NZ), in DMEM supplemented with 5% FBS, 2 mM L-alanyl-L- glutamine dipeptide (GlutaMAX), 100 U / ml penicillin and 100 mg / ml streptomycin, using PEI (linear, MW 25000; Polysciences, Warrington, PA) at a PEI:DNA mass ratio of 3:1. Supernatants were collected 3 days post-transfection, and supernatants as well as cells were collected 5 days post-transfection. Supernatants and cells were combined and centrifuged to pellet the cells. AAVs were purified using a 3 phase partitioning protocol. Cells were resuspended in lysis buffer (50 mM Tris-HCI, 150 mM NaCI and 2 mM MgCh, pH 8.0) and lysed by three freeze-thaw cycles in liquid nitrogen and 37 °C. Cell lysates were supplemented with 50 U / ml Benzonase (Sigma-Aldrich) and 10 U / ml RNasel (ThermoFisher Scientific), incubated for 30 min at 37 °C, then supplemented with 0.5 %w / vSDS, then incubated again. Cell lysates were combined with supernatants and 500 mM NaCI and 8 %w / wPEG-8000 were added, incubated overnight at 4 °C, and centrifuged for 30 min at 4,000 x g, 4°C. Pellets were resuspended in purification buffer II (50 mM Tris-HCI, 500 mM NaCI, 2 mM MgCh, 1 %w / w sarkosyl, and 1 %v / vTriton X-100, pH 7.5), then solid (NH4)2SO4 was added to a saturation of 20% and incubated for 5 min at 37 °C. An equal volume of tert-butanol was added, then incubated for 5 min at 37 °C and centrifuged for 10 min at 4,000 x g. The lower aqueous phase was collected and washed with DPBS supplemented with 0.01 %w / v Pluronic F-68 in Amicon Ultra-15 centrifugal filter units (100 kDa MWCO; ThermoFisher Scientific), with a surplus of injection buffer (DPBS supplemented with 200 mM NaCI and 0.001 %w / v Pluronic F-68) (injection buffer). A small aliquot of the washed and concentrated AAVs were digested with DNasel (New England Biolabs), then qPCR was performed to determine AAV titers, using the primers: 5'-GGAACCCCTAGTGATGGAGTT-3': SEQ ID NO: 116 and 5'- CGGCCTCAGTGAGCGA-3': SEQ ID NO: 117.

[0306] AAV Vector Injection into Adipose Tissue: To achieve simple transgene overexpression, TRE-GIPR and TRE-mmR2F AAVs were injected directly into the sWAT fatpads of HFD-fed adiponectin-rtTA mice, thus restricting transgene expression from the TRE promoter to mature adipocytes. The mAdipoQP-GIPR and mAdipoQP-sfGV-CAAX AAVs were injected into HFD-fed WT mice, restricting gene expression from the adiponectin promoter to the adipocyte. To achieve combined transgene overexpression and endogenous gene knockout for the latter, mAdipoQP-GIPR and mAdipoQP-sfGV-CAAX AAVs were combined with either mAdipoQP-SaCas9+U6-NT g1 or mADIPOQP-SaCas9+U6-Serca2 g1+g2 AAVs, then injected HFD-fed WT mice. For the TRE-GIPR studies, for each mouse, (1x10)12genomic copies (gc) were diluted to 100 pl in injection buffer, then injected as ten 10 pl fractions directlyinto the sWAT fat-pad. For the knockout studies, for each mouse, (2x10)12gc of GIPR or sfGV- CAAX overexpressing virus were mixed with either (2x10)12gc of NT g1 or (1+1)x1012gc of Serca2 g1+g2 CRISPR virus, diluted to 100 pl in injection buffer, and delivered as 5x10 pl fractions directly into each sWAT depot.

[0307] Single-Nuclei RNA-Sequencing of Human WAT: For Cohort A, intact human WAT- derived nuclei were isolated using a glass-on-glass homogenizer. Tissue lysis buffer (Tris- HCI, MgCI2, NaCI, NP40 and RNase inhibitors) was added to -100 mg of WAT. Samples were cut into pieces and homogenized in lysis buffer (7 ml) -20 times (10 with a loose pestle, 10 with tight pestle). Following incubation on ice for 6-min, blocking buffer (5 ml) (1 % BSA in PBS with RNase inhibitors) was added to samples, then centrifuged at 500 x g for 5-min at 4°C. Pellets were washed twice in blocking buffer and nuclei were counted. An average of 16,000 nuclei were loaded onto each lane of a 10X Chromium chip. A total of 117,717 nuclei were captured across 22 samples. 10X libraries were sequenced on an Illumina sequencer at a mean depth of 50,000 reads per nucleus. For single-nuclei gene expression, data was filtered using CellRanger and CellBender to remove low quality nuclei. DoubletFinder was applied to remove doublets. High-quality nuclei were integrated via scVI, then cell clusters were visualized using Seurat in R (Seurat-R). Individual gene expression was plotted using the FeaturePlot function Seurat-R. For Cohort B, for the analysis of sub-populations of human adipocytes, WAT was obtained from human subjects undergoing surgery, then subjected to single-nuclei RNA-Sequencing (RNA-Seq). Data was integrated and clustered using Seurat. Adipocytes were computationally removed from the dataset and re-integrated utilizing Reciprocal Principal Component Analysis, using 2000 variable genes. Uniform Manifold Approximation and Projection reduction and clustering was performed using 20 PCs. Clustering was also ran using a resolution of 0.6. Plots were generated with the log normalized to the RNA assay.

[0308] RNA-Sequencing and qPCR: Total RNA was extracted from tissues (sWAT, gWAT, mWAT, BAT and liver) using Trizol (Invitrogen, Carlsbad, CA) and a RNeasy RNA extraction kit (#74106, Qiagen, Valencia, CA). Following homogenization of tissues (TissueLyser; Qiagen), RNA was isolated according to the manufacturers' protocol. The concentration of RNA was determined using a Nanodrop Spectrophotometer (N 1-1000, Thermo Fisher Scientific, Wilmington, DE). A total of 1 jig RNA was used for reverse transcriptional reactions using an iScript cDNA synthesis kit (#170-8891 , Bio-Rad Laboratories, Inc., Hercules, CA). Diluted cDNA and SYBR Green PCR Master Mix (A25742, Life Technologies, Carlsbad, CA) were used for qPCR (QuantStudio-6 Flex Real-Time PCR System; Applied BioSystems, Foster City, CA). Primer sequences are provided in Table 7. Results were calculated using the threshold cycle method. For RNA-Seq, total sWAT RNA was processed at the UTSWRNA-Seq Core. Fold-changes and significance were calculated based on three independent replicates.Table 7: List of qPCR Primers

[0309] Tissue Temperature Recording: The tissue temperature of subcutaneous fat pads was measured -10 days following AAV injection. After mice were anesthetized by 2% isoflurane, the hypodermic needle microprobe (Cat# MT-29 / 1 HT; Physitemp) was immediately pierced through the skin to reach the center of subcutaneous fat pad. The probe was connected to a TC-2000 Thermocouple Meter (Sable Systems International) to obtain temperature recordings once temperature reached stability.

[0310] 3H-Triolein Lipid Uptake and Oxidation: For triolein clearance rates, tissue-specific lipid-uptake and p-oxidation methodologies were adapted from known protocols. Briefly,3H - triolein was injected into mice via the tail-vein (2 pCi / mouse in 100 pl of 5% Intralipid) following a 16 hours fast. Blood samples were collected at specified intervals post injection. After 20 min post injection, mice were sacrificed, blood and tissues were collected, weighed and frozen. Lipids were extracted using chloroform-to-methanol-based extraction. Radioactivity content of blood and tissues were quantified as previously detailed.

[0311] Statistical Analyses: All results are provided as means ± standard errors of the mean (SEM). Statistical analysis was performed using GraphPad Prism-9 (San Diego, CA). Differences between the two groups over time were determined by a two-way analysis of variance (ANO A). For comparison between two independent groups, a Students' t-test was applied. Significance was accepted at a P-value of <0.05. For analysis of metabolomics, data were log-transformed, then a one-way ANOVA was performed to assess differences between group data distributions (unadjusted P<0.05 for significance). Fold changes and SEM were computed between treatment groups. Heat-maps were generated using Iog2 transformation of least square means using R 3.6.0.

[0312] Metabolic Tests, Treatments, and Measurements: For triglyceride (TG) clearance tests, mice were fasted (-14-16 h), then gavaged 20% Intra-lipid (15 ul / g body weight; Fresenius Kabi Clyton, L.P., Clayton, NC). Blood was collected and assayed for TG levels (Infinity; ThermoFisher Scientific). Leptin levels were measured using a commercially available assay (Millipore Linco Research, St. Charles, MO). GIP levels were measured in blood samples treated with 10 mg / ml aprotinin (Millipore, A6279) and DPP-IV inhibitor (Millipore, DPP4-010), then measured using an ELISA (Millipore, #EZRMGIP-55k). For p3- adrenergic receptor (AR)-agonist tests, blood was collected at time-points following intraperitoneal injection of 1 mg / kg CL 316,243 (Sigma-Aldrich), then assayed for NEFAs (NEFA-HR(2); Wako Pure Chemical Industries) and glycerol (Sigma-Aldrich). For long-acting GIPR agonist treatment, subcutaneous injection (GIPRA-085; 300 nmol / kg) (Eli Lilly and Company) was performed. Creatine kinase activity was measured utilizing an assay (MAK116, Sigma-Aldrich).

[0313] Metabolic Cages and Body Composition: Mouse whole-body composition, total body fat and lean mass were conducting using nuclear magnetic resonance system (Bruckner Minispec mq10; Bruker Corporation, Billerica, MA, USA). Metabolic cage studies were performed by the UTSW Mouse Metabolic Phenotyping Core using the CLAMS system (Columbus Instrument). Prior to measurements, mice were ac...

Claims

CLAIMSWhat is claimed is:1 . A nucleic acid comprising a nucleic acid sequence encoding any one or more of: a) a Gastric Inhibitory Polypeptide Receptor (GIPR), a Glucagon Receptor (GCGR), or a Glucagon-Like Peptide 1 Receptor (GLP-1 R), or a functional variant thereof, or any combination thereof; or b) a ligand of GIPR, GCGR, or GLP-1 R, or a functional variant thereof, or any combination thereof; wherein the nucleic acid sequence is operably linked to at least one adipocytespecific promoter.

2. The nucleic acid of claim 1 , wherein the adipocyte-specific promoter is a constitutive promoter or an inducible promoter.

3. The nucleic acid of claim 2, wherein the adipocyte-specific promoter is an adiponectin promoter.

4. The nucleic acid of claim 3, wherein the adipocyte-specific promoter is an engineered adiponectin promoter.

5. The nucleic acid of any one of claims 1 -3, wherein the adiponectin promoter comprises the nucleic acid sequence as set forth in SEQ ID NO: 7, or a sequence at least 60% identical hereto.

6. The nucleic acid of claim 4, wherein the engineered adiponectin promoter comprises the nucleic acid sequence as set forth in SEQ ID NO: 8, or a sequence at least 90% identical hereto.

7. The nucleic acid of any one of claims 1 -6, wherein the ligand comprises a gastric inhibitory polypeptide (GIP), a glucagon peptide (GCG), a glucagon like peptide 1 (GLP1), exendin-4 (EX4), a tirzepatide (TIR)-like ligand, or a retatrutide (RET)-like ligand, or a functional variant thereof.

8. The nucleic acid of claim 7, wherein the functional variant can bind more than one of GIPR, GCGR, and / or GLP-1 R.

9. The nucleic acid of claim 7, wherein the nucleic acid sequence encodes one or more repeats of a ligand.

10. The nucleic acid of any one of claims 7 or 9, wherein the nucleic acid sequence encodes one or more distinct ligands.1 1 . The nucleic acid of any one of claims 1 -10, wherein the nucleic acid sequence further encodes one or more leader sequences.

12. The nucleic acid of claim 11 , wherein the leader sequence is a GIP leader sequence, a GCG / GLP-1 leader sequence, or any variant thereof.

13. The nucleic acid of any one of claims 1 -12, wherein the nucleic acid sequence further encodes one or more cleavage sites.

14. The nucleic acid of claim 13, wherein the one or more cleavage sites comprise a furin cleavage site, or a variant thereof.

15. The nucleic acid of any one of claims 1 -14, further comprising a 5' UTR sequence.

16. The nucleic acid of any one of claims 1 -6 or 11 -15, wherein the nucleic acid sequence comprises a sequence as set forth in any one of SEQ ID NOS: 1 , 2, 4, 5, 11 , or 13, or a sequence at least about 60% identical hereto.

17. The nucleic acid of any one of claims 1 -6 or 11 -16, wherein the nucleic acid sequence encodes the amino acid sequence as set forth in any one of SEQ ID NOS: 3, 6, 12, or 14, or a sequence at least about 80% identical hereto.

18. The nucleic acid of any one of claims 1 -6 or claims 11-17, wherein the nucleic acid sequence comprises a sequence as set forth in SEQ ID NOS: 9, 10, 15, or 16, or a sequence at least 60% identical hereto.

19. The nucleic acid of any one of claims 9-15, wherein the nucleic acid sequence comprises a sequence as set forth in any one of SEQ ID NOS: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100, or a sequence at least about 80% identical thereto.

20. The nucleic acid of any one of claims 1-15, or claim 19, wherein the nucleic acid sequence encodes an amino acid sequence as set forth in any one of SEQ ID NOS: 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99, or 101 , or a sequence at least about 90% identical thereto.21 . The nucleic acid of claim 1 1 , wherein the one of more leader sequences are encoded by a sequence as set forth in any one of SEQ ID NOS: 18 or 20, or a sequence at least about 60% identical thereto.

22. The nucleic acid of claim 11 , wherein the one of more leader sequences comprise an amino acid sequence as set forth in any one of SEQ ID NOS: 19 or 21 , or an amino acid sequence at least about 90% identical thereto.

23. The nucleic acid of claim 15, wherein the 5' UTR comprises a nucleic acid sequence as set forth in SEQ ID NO: 17 or a sequence at least about 90% identical thereto.

24. The nucleic acid of claim 14, wherein the one of more furin cleavage sites are encoded by a sequence as set forth in any one of SEQ ID NOS: 22, 24, 26, 28, 30, 32, 34, or 36, or a sequence at least about 60% identical thereto.

25. The nucleic acid of claim 14, wherein the one of more furin cleavage sites comprise an amino acid sequence as set forth in any one of SEQ ID NOS: 23, 25, 27, 29, 31 , 33, 35, or 37, or an amino acid sequence at least about 90% identical thereto.

26. The nucleic acid of any one of claims 1 -25, wherein the nucleic acid further encodes a carrier protein as set forth in any one of SEQ I D NOS: 103 or 105, or an amino acid sequence at least about 60% identical thereto.

27. The nucleic acid of any one of claims 1 -26, wherein the nucleic acid further comprises a nucleic acid sequence as set forth in any one of SEQ ID NOS: 103 or 105, or an amino acid sequence at least about 60% identical thereto.

28. The nucleic acid of claim 1 , wherein the nucleic acid is functional in expressing the one or more of GIPR, GCGR, or GLP-1 R or the one or more ligands thereof in adipocytes.

29. A vector comprising the nucleic acid of any one of claims 1-28.

30. The vector of claim 29, wherein the vector is a viral vector.31 . A viral vector comprising a nucleic acid sequence encoding any one or more of: a) a Gastric Inhibitory Polypeptide Receptor (GIPR), a Glucagon Receptor (GCGR), or a Glucagon-Like Peptide 1 Receptor (GLP-1 R), or a functional variant thereof, or any combination thereof; or b) a ligand of GIPR, GCGR, or GLP-1 R, or a functional variant thereof, or any combination thereof; wherein the nucleic acid sequence is operably linked to at least one adipocytespecific promoter.

32. The viral vector of claim 30 or claim 31 , wherein the viral vector is an adeno-associated virus (AAV) vector.

33. The viral vector of claim 32, wherein the viral vector is an AAV vector of serotype 9.

34. A delivery particle comprising the nucleic acid of any one of claims 1 -28, or the viral vector of any one of claims 29-33.

35. The delivery particle of claim 34, wherein the delivery particle is a AAV viral particle.

36. The delivery particle of claim 35, wherein the AAV particle comprises a capsid protein comprising an AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhIO, AAV11 , AAV12, AAV LK03, AAV2R471A, AAV2 / 2-7m8, AAV-DJ, an AAV-DJ / 8, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, goat AAV, AAV1 / AAV2 chimeric, bovine AAV, mouse AAV, AAV Rec2, rAAV2 / HBoV1 , AAV2HBKO, AAV-PHP.B, or AAV-PHP.eB serotype capsid, or a variant thereof.

37. The delivery particle of claim 34, wherein the delivery particle is a lipidoid, liposome, lipid nanoparticle, polymer, lipoplex, core-shell nanoparticle, microparticle, peptide, protein, cell transfected with viral vectors, or nanoparticle mimic, or any combinations thereof.

38. A host cell comprising the nucleic acid of any one of claims 1-28, or a viral vector of any one of claims 29-33.

39. The host cell of claim 38, wherein the host cell is a HEK293, HEK293T, A549, HeLa, CHO, Sf9, SP2, or NSO cell, or any derivative cell line thereof.

40. A composition comprising the nucleic acid of any one of claims 1-29, a viral vector of any one of claims 29-33, a delivery particle of any one of claims 34-37, or a host cell of claim 38 or claim 39, and at least one excipient.

41. A method for preventing and / or treating a metabolic disease in a subject in need thereof, comprising administering to the subject the composition of claim 40.

42. The method for preventing and / or treating a metabolic disease of claim 41 , wherein the administering to the subject in need thereof comprises injecting the composition into the adipose tissue of the subject, wherein the adipose tissue may be (a) in the subject's body during injection or (b) outside of the subject's body and subsequently transplanted into the subject after injection.

43. The method of any one of claims 41 or 42, wherein the metabolic disease is obesity.

44. A method of preventing weight gain and / or inducing weight loss in a subject in need thereof, comprising administering to the subject the composition of claim 40.

45. The method of preventing weight gain and / or inducing weight loss of claim 44, wherein the administering to the subject in need thereof comprises injecting a composition of any one of claim 40 into an adipose tissue of the subject, wherein the adipose tissue may be (a) in the subject's body during injection or (b) outside of the subject's body and subsequently transplanted into the subject after injection.

46. A method for suppressing the appetite of a subject in need thereof, comprising administering to the subject the composition of claim 40.

47. The method of any one of claims 41 -46, wherein the subject is a mammal.

48. The method of any one of claims 41 -47, wherein the subject is administered at least one additional therapy.

49. The method of any one of claims 41 -48, wherein the administration of the composition results in an adipocyte-specific overexpression of one or more of GIPR, GCGR, or GLP-1 R.

50. The method of any one of claims 41 -48, wherein the administration of the composition results in an adipocyte-specific overexpression of one or more of gastric inhibitory polypeptide (GIP), glucagon (GOG), glucagon like peptide 1 (GLP1), exendin-4 (EX4), a tirzepatide (TIR)- like ligand, or a retatrutide (RET)-like ligand, or a functional variant thereof.51 . The method of claim 49 or claim 50, wherein the adipocyte-specific overexpression is turned off or ceases after 4 weeks, after 6 weeks, after 8 weeks, after 10 weeks, after 12 weeks, after 14 weeks, after 16 weeks, after 20 weeks, after 22 weeks, or after 24 weeks, post administration.

52. The method of claim 51 , wherein the adipocyte-specific overexpression is turned on or induced after about 1 day, after 1 week, after 2 weeks, after 4 weeks, after 6 weeks, after 8 weeks, after 10 weeks, after 12 weeks, after 14 weeks, after 16 weeks, after 20 weeks, after 22 weeks, or after 24 weeks post administration.

53. A method of remodeling adipose tissue for esthetical purposes, and / or to support plastics surgery, comprising injecting into a specific adipose tissue depot in the subject in need thereof, the composition of claim 40, wherein the adipose tissue may be (a) in the subject's body during injection or (b) outside of the subject's body and subsequently transplanted into the subject after injection.

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