Recombinant active peptide compositions and methods of use thereof

Abstract

Disclosed herein are compositions comprising recombinant polypeptides and expression vectors encoding thereof and methods of using the same for treating a subject having a disorder, for example including but not limited to metabolic and brain disorders.

Description

[0001] Docket No. 103362-094WO1

[0002] RECOMBINANT ACTIVE PEPTIDE COMPOSITIONS AND METHODS OF USE THEREOF

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U. S. Provisional Patent Application 63 / 762,762, filed February 25, 2025, which is incorporated herein by reference in entirety.

[0005] FIELD OF THE INVENTION

[0006] This invention is in the area of improvements in compositions comprising recombinant polypeptide or vectors encoding thereof that are highly expressed and secreted to target specific tissues which improve one or more phenotypes associated with diseases, disorders, or cancers, while also suppressing proinflammatory effects.

[0007] INCORPORATION BY REFERENCE

[0008] The contents of the xml file named “103362-094WO1-ST26” which was created on February 10, 2026, and is 174,949 bytes in size, are hereby incorporated by reference in their entirety.

[0009] BACKGROUND OF THE INVENTION

[0010] The past century has seen tremendous advancement in peptide drug discovery with more than 80 peptide drugs approved for a large range of diseases including diabetes, obesity, osteoporosis, cancer, multiple sclerosis, chronic pain, and HIV infection. However, many peptide drugs require frequent injection and long-term use, posing challenges of patient coherence, risk of side effects, and high cost. As such, gene therapy, particularly based on the adeno-associated virus (AAV), may have significant advantages compared to current standard of care for some peptide drugs.

[0011] There is an urgent need to develop new compositions and methods for enhanced disease therapeutics.

[0012] SUMMARY OF THE INVENTION

[0013] It has been surprisingly discovered herein that a recombinant polypeptide platform, or an expression vector comprising a transgene encoding thereof, provides one or more therapeutic benefits in a subject having a disease, a disorder, or a cancer. For example, it was surprisingly discovered herein that a recombinant Exendin-4 polypeptide encoded by anDocket No. 103362-094WO1

[0014] adipotropic AAV vector as described herein led to improved glucose tolerance and anti-diabetic effect and durable weight loss in obese subjects compared to control (see, e.g., FIGs. 4, 7A, 8A, 9, 13A-13B of Example 1), while in addition synergistically limiting a proinflammatory effect (see, e.g., FIGs. 5A-5B, 6A-6B of Example 1) in obesogenic condition which negatively impacts the efficacy of gene therapies. The improved recombinant polypeptides and expression vectors encoding thereof as described herein also provide additional therapeutic effects, for example including but not limited to limiting food intake (see, e.g., FIG. 7B, 13C of Example 1) while reducing adiposity and / or increasing lean mass relative to body weight (see, e.g., FIG.

[0015] 8B of Example 1).

[0016] The deficiency in the state of the art was the therapeutic efficacy of peptide-based drugs which are limited by frequent administration, poor patient coherence, side effect risks, and high costs. In contrast, the recombinant polypeptides, or expression vectors as described herein are provide highly-targeted gene transfer in specific tissues, leading to enhanced expression and secretion of the recombinant polypeptides as described herein. In some cases, the recombinant polypeptides as disclosed herein are expressed at levels greatly exceeding that of endogenous peptide, for example 10-fold or greater (see, e.g., FIG. 20-21 of Example 3), even while being administered at low doses (e.g., 2E10 vg / mouse or 5x1011vg / kg) (see, e.g., FIG. 32B of Example 5). In some cases, the recombinant polypeptides are engineered forms from the natural gene sequence, or a fragment thereof, to achieve desirable functions (see, e.g., mature forms of BDNF and BMP7 of Example 3 and Example 4, respectively).

[0017] New compositions and methods of delivering thereof are urgently needed since peptide therapeutics are limited by several efficacy-impacting challenges. The novel recombinant polypeptides as described herein address the gap in the state of human therapy art that needs to be met to enable high expression and secretion of a therapeutic peptide, for example including but not limited to Exendin-4, GLP-l(7-36), BDNF, and / or BMP7.

[0018] The result is remarkable in that the recombinant polypeptides, or expression vectors encoding thereof, as described herein provides one or more of:

[0019] (i) successful expression and secretion of recombinant polypeptide in vitro and in vivo (see, e.g., FIGs. 2A-2B of Example 1; FIGs. 17, 20, 21, 25 of Example 3; FIGs. 27A-27C of Example 4);

[0020] (ii) substantially increased expression (for example, 10-fold or greater) of recombinant polypeptide relative to endogenous peptide expression (see, e.g., FIG. 20-21 of Example 3);Docket No. 103362-094WO1

[0021] (iii) robustly improved glucose tolerance which leads to an antidiabetic effect in obese subjects (see, e.g., FIGs. 4, 9 of Example 1; FIG. 27 of Example 3; FIGs. 32G-32H and 37F-37G of Example 5);

[0022] (iv) significant downregulation of proinflammatory genes in multiple tissues, including but not limited to the liver, hypothalamus, and adipose tissue (see, e.g., FIGs. 5A-5B, 6A-6B of Example 1);

[0023] (v) durably and significantly reduced weight gain in obese subjects that were genetics-induced (see, e.g., FIG. 7Aand 13A-13B of Example 1; FIGs. 18Aand 25 of Example 3; FIGs. 32C-32F of Example 5) as well as in diet-induced obesity (see, e.g., FIGs. 37A-37B of Example 5), while advantageously retaining muscle mass (see, e.g., FIGs. 33H and 45G of Example 5);

[0024] (vi) suppressed food intake over several weeks (see, e.g., FIG. 7B and 13C of Example 1; FIG. 37C of Example 5);

[0025] (vii) robustly reduced adiposity while increasing lean mass relative to body weight (see, e.g., FIG. 8B of Example 1; FIGs. 43A-43B of Example 5);

[0026] (viii) increased locomotion and exploratory behavior (see, e.g., FIG. 11 A of Example 1; FIGs. 37H and 44A of Example 5); and / or

[0027] (ix) retained efficacy even when administered at low doses, for example at doses of 2E10 vg / mouse or 5x1011vg / kg (see, e.g., FIG. 32B of Example 5).

[0028] Furthermore, an AAV vector gene therapy (e.g., Rec2) encoding recombinant peptide Exendin-4 is an efficacious and safe for treatment of genetic- and / or dietary-induced forms of obesity, for example Prader Willi Syndrome (see, e.g., Example 5 herein). Indeed, it was surprisingly discovered herein that intraperitoneal administration of Rec2-Ex4 gene therapy at a low dose, for example at doses of 2E10 vg / mouse (or 5x1011vg / kg), normalizes metabolic dysfunction and improves exploratory behavior in the PWS model of Magel2-nvX\ mice. This dose is much lower than the standard systemic dosing of AAV. It was also unexpectedly discovered an unusually long expression of transgene by gene delivery of the adipotropic AAV vectors disclosed herein (e.g., Rec2-leptin gene therapy in Example 9), where restoration of normal leptin levels was achieved and body weight was normalized through 14 months following only a one-time subcutaneous administration at a low dose in congenital leptin deficiency model ob / ob mice (e.g., 2E10 vg / mouse) (see, e.g., FIGs. 60-63 of Example 9). Remarkably, AAV genome DNA and transgene mRNA were detected in the targeted adipose tissue while absent in liver and other peripheral organs / tissues. These data suggest the adiposetargeting AAV compositions and methods of use thereof can achieve long-lasting and stableDocket No. 103362-094WO1

[0029] transgene expression which is also highly specific. The novel recombinant polypeptide compositions and methods of delivering thereof as disclosed herein may even be effectively dosed as infrequently as once every year, for example, once every two years, once every three years, once every 4 years, once every 5 years, once every 6 years, once every 7 years, once every 8 years, once every 9 years, or once every 10 years. These unexpected improvements offer a promising approach with several advantages over current standard of care therapies which require continuous repeated administration of protein / peptide drugs.

[0030] These findings indicate that the novel platform design of the recombinant polypeptides as disclosed herein, or expression vectors encoding thereof, provide several therapeutic enhancements which result in improved disease phenotypes (including but not limited to weight gain, glucose tolerance, food intake, fat / lean mass percentages, locomotion and exploratory behavior) in a subject having a disorder (e.g., a metabolic disorder), disease, or a cancer, while synergistically limiting inflammatory effects to enhance the therapy.

[0031] This improvement provides a significant advance in the state of the art of human therapy. Specific aspects of the invention as described herein include one or more of

[0032] (i) a recombinant polypeptide comprising: (a) a signal peptide sequence, for example a leptin signal peptide sequence; (b) a linker peptide comprising between 1 to 30 amino acid residues, for example a linker peptide comprising a valine residue; and (c) a therapeutic peptide, wherein the therapeutic peptide comprises a peptide hormone, a GLP1 receptor agonist peptide, a glucagon-like peptide, a biomimetic peptide, an immunomodulatory peptide, a neurotoxic peptide, a neurotrophic factor peptide, a bone morphogenetic peptide, a guanylate cyclase-C (G-CC) agonist peptide, a calcitonin receptor inhibitor, a GnRH receptor inhibitor, a 20S proteasome inhibitor, a N0D2 inhibitor, a VIP1 receptor inhibitor, an OT inhibitor, a TRH receptor inhibitor, an MC receptor inhibitor, a PTH1 receptor inhibitor, a guanylate cyclase C inhibitor, an NPR- A inhibitor, an ATI receptor inhibitor, a beta2-receptor inhibitor, a gp41 inhibitor, a GHRH receptor inhibitor, an N-type calcium channel inhibitor, a thrombopoietin receptor inhibitor, a human erythropoietin receptor inhibitor, a pulmonary surfactant inhibitor, a CaSR inhibitor, an MC 1 receptor inhibitor, a somatostatin receptor inhibitor, a melanocortin-4 receptor inhibitor, or an immune checkpoint inhibitor peptide, or an analog thereof;

[0033] (ii) a recombinant polypeptide comprising: (a) a signal peptide sequence, for example a leptin signal peptide sequence; (b) a linker peptide comprising between 1 to 30 aminoDocket No. 103362-094WO1

[0034] acid residues, for example a linker peptide comprising a valine residue; and (c) a GLP1 receptor agonist peptide;

[0035] (iii) a recombinant polypeptide comprising: (a) a signal peptide sequence, for example a leptin signal peptide sequence; (b) a linker peptide comprising between 1 to 30 amino acid residues, for example a linker peptide comprising a valine residue; and (c) a GLP1 receptor agonist peptide; wherein the GLP1 receptor agonist peptide comprises an exendin-4 peptide, optionally wherein the exenin-4 peptide further comprises one or more additional polypeptides conjugated to the amino and / or carboxy termini (e.g., HA tag);

[0036] (iv) a recombinant polypeptide comprising: (a) a signal peptide sequence, for example a leptin signal peptide sequence; (b) a linker peptide comprising between 1 to 30 amino acid residues, for example a linker peptide comprising a valine residue; and (c) a GLP1 receptor agonist peptide; wherein the GLP1 receptor agonist peptide comprises a GLP- 1 peptide;

[0037] (v) a recombinant polypeptide comprising: (a) a signal peptide sequence, for example a leptin signal peptide sequence; (b) a linker peptide comprising between 1 to 30 amino acid residues, for example a linker peptide comprising a valine residue; and (c) a brain- derived neurotrophic factor (BDNF) peptide;

[0038] (vi) a recombinant polypeptide comprising: (a) a signal peptide sequence, for example a leptin signal peptide sequence; (b) a linker peptide comprising between 1 to 30 amino acid residues, for example a linker peptide comprising a valine residue; and (c) a peptide derived from bone morphogenetic protein 7 (BMP7);

[0039] (vii) an expression vector comprising a nucleic acid encoding for a recombinant polypeptide as described herein;

[0040] (viii) an adeno-associated viral vector (AAV) comprising a nucleic acid encoding for a recombinant polypeptide as described herein;

[0041] (ix) a pharmaceutical composition comprising at least one recombinant polypeptide as described herein, at least one expression vector as described herein, or at least one AAV as described herein, and a pharmaceutically acceptable carrier;

[0042] (x) a method for gene transfer of a nonnative, recombinant polypeptide to adipose tissue in a subject in need thereof, comprising administering a therapeutically effective amount of an AAV as described herein;

[0043] (xi) a method for reducing blood glucose in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide asDocket No. 103362-094WO1

[0044] described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein;

[0045] (xii) a method for increasing blood glucose tolerance in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein;

[0046] (xiii) a method for reducing expression of proinflammatory genes in one or more tissues in a subject receiving an AAV therapy, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein;

[0047] (xiv) a method for treating a subject having a metabolic disorder, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein;

[0048] (xv) a method for treating a subject having diabetes, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein;

[0049] (xvi) a method for treating a subject having obesity, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein;

[0050] (xvii) a method for reducing one or more metabolic disorders in a subject having a body mass index (BMI) of equal to or greater than 27.5, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein.

[0051] (xviii) a method for reducing one or more metabolic disorders in a subject having a body mass index (BMI) of equal to or greater than 30, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein;Docket No. 103362-094WO1

[0052] (xix) method for reducing one or more metabolic disorders in a subject having a body mass index (BMI) of equal to or greater than 40, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein;

[0053] (xx) a method for reducing weight of a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein;

[0054] (xxi) a method for treating a neurodevel opmental disease in a subject in need thereof, including but not limited to Prader-Willi syndrome, in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein;

[0055] (xxii) a method for treating Prader-Willi syndrome in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein;

[0056] (xxiii) a method for treating a neurodegenerative disease in a subject in need thereof, including but not limited to Alzheimer’s disease and Parkinson’s disease, in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein;

[0057] (xxiv) a method for treating major depression in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein;

[0058] (xxv) a method for treating osteoporosis in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein;

[0059] (xxvi) a method for enhancing dental regeneration in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide asDocket No. 103362-094WO1

[0060] described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein;

[0061] (xxvii) a method for treating an inflammatory disease in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein;

[0062] (xxviii)a method for improving cellular plasticity in a neurological disorder in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein; and (xxviv)a method for treating cancer, for example obesity-associated colon cancer, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein.

[0063] A summary of embodiments of the invention is described in further detail below.

[0064] BRIEF DESCRIPTION OF THE DRAWINGS

[0065] 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 upon request and payment of the necessary fee.

[0066] FIG. 1 shows Exendin-4 transgene sequences and encoded products thereof.

[0067] FIG. 2A shows a western blot stained for Exendin-4 of cell lysates and media of HEK293 cells transfected with AAV expression plasmid-CBA-Exendin-4 run by SDS-PAGE. Exendin-4 was detected in cell lysates and in medium harvested at 72 hours post-transfection. Signal peptide + Exendin-4 (3-39 aa): 11.16 kDa; Exendin-4 (3-39 aa): 8.46 kDa.

[0068] FIG. 2B shows a western blot stained for Exendin-4 of cell lysates of HEK293 cells transfected with AAV-V7-CBA-Exendin-4 run by SDS-PAGE. Exendin-4 was detected in cell lysates. Signal peptide + Exendin-4 (3-39 aa): 11.16 kDa; Exendin-4 (3-39 aa): 8.46 kDa.

[0069] FIG. 3 A shows a graph of body weight of mice receiving AAV-V7-Exendin-4 (salmon) versus control (black). Mice were administered a single intraperitoneal (IP) injection of V7-Exendin-4 or green fluorescent protein (GFP) as a control, each at a concentration of 8E10 viral genome particle (vg) per mouse. Body weight as measured in grams (g) is represented on the y-axis, while time after AAV injection is represented on the x-axis in weeks.Docket No. 103362-094WO1

[0070] FIG. 3B shows a graph of body composition — percentage of fat versus lean muscle of mice receiving AAV- V7-Exendin-4 (salmon) versus control (black). Mice were administered a single IP injection of V7-Exendin-4 or GFP as a control, each at a concentration of 8E10 vg per mouse. Percent of body weight (%) is represented on the y-axis, while fat or lean mass is represented on the x-axis.

[0071] FIG. 4 shows the antidiabetic effect of AAV-V7-Exendin-4 in mice receiving AAV-V7-Exendin-4 (salmon) versus control (black). Mice were administered a single IP injection of V7-Exendin-4 or GFP as a control, each at a concentration of 8E10 vg per mouse. Blood glucose concentration (mg / dL) is represented on the y-axis, while time in minutes (min) after injection is represented on the x-axis. **, P < 0.01.

[0072] FIG. 5A shows that mice administered AAV-V7-Exendin-4 (salmon) exhibited significantly downregulated expression of liver proinflammatory genes compared to control (black). Relative levels of mRNA are represented on the y-axis for different proinflammatory genes represented on the x-axis. *, P < 0.05; **, P < 0.01.

[0073] FIG. 5B shows that mice administered AAV-V7-Exendin-4 (salmon) exhibited significantly downregulated expression of hypothalamus proinflammatory genes compared to control (black). Relative levels of mRNA are represented on the y-axis for different proinflammatory genes represented on the x-axis.

[0074] FIG. 6A shows that mice administered AAV-V7-Exendin-4 (salmon) exhibited significantly downregulated expression of brown adipose tissue (BAT) proinflammatory genes compared to control (black). Relative levels of mRNA are represented on the y-axis for different proinflammatory genes represented on the x-axis.

[0075] FIG. 6B shows that mice administered AAV-V7-Exendin-4 (salmon) exhibited significantly downregulated expression of epididymal white adipose tissue (eWAT) proinflammatory genes compared to control (black). Relative levels of mRNA are represented on the y-axis for different proinflammatory genes represented on the x-axis.

[0076] FIG. 7A shows mice administered AAV-Rec2-Exendin-4 had durably and significantly reduced weight gain when fed on a high fat diet compared to control mice. Mice were fed on a high fat diet and randomized to receive a single IP injection of Rec-2-Exendin-4 (4E10 vg per mouse; red) or AAV buffer as control (blue). Body weight as measured in grams (g) is represented on the y-axis, while time after AAV injection is represented on the x-axis in weeks. n=5 per group. Rec2-Exendin-4: 4xIO10vg / mouse, IP. ****, P < 0.0001.Docket No. 103362-094WO1

[0077] FIG. 7B shows mice administered Rec2 -Exendin-4 ate substantially less food when fed on a high fat diet compared to control mice. Mice were fed on a high fat diet and randomized to receive a single IP injection of AAV-Rec-2-Exendin-4 (4E10 vg per mouse; red) or AAV buffer as control (blue). Average food intake as measured in grams per day (g / day) is represented on the y-axis, while time after AAV injection in weeks is represented on the x-axis.

[0078] FIG. 8A shows mice administered AAV-Rec2-Exendin-4 had significantly reduced weight gain within 11 weeks when fed on a high fat diet compared to control mice. Mice were fed on a high fat diet and randomized to receive a single IP injection of Rec-2 -Exendin-4 (4E10 vg per mouse; red) or AAV buffer as control (blue). Body weight as measured in grams (g) is represented on the y-axis, while different treatment groups are represented on the x-axis. **, P < 0.01; ***, P< 0.001.

[0079] FIG. 8B shows mice administered AAV-Rec2-Exendin-4 had significantly reduced body fat percent of body weight, and concomitantly, significantly increased lean mass percent of body weight when fed on a high fat diet compared to control mice. Mice were fed on a high fat diet and randomized to receive a single IP injection of Rec-2-Exendin-4 (4E10 vg per mouse; red) or AAV buffer as control (blue). Fat and lean mass as percentages of body weight were assessed at 5 weeks post AAV injection by echoMRI. Percent of body weight (%) is represented on the y-axis, while fat or lean mass is represented on the x-axis. ***, P < 0.001.

[0080] FIG. 9 shows mice administered AAV-Rec2-Exendin-4 had significantly improved glucose tolerance compared to control mice. Mice were fed on a high fat diet and randomized to receive a single IP injection of Rec-2-Exendin-4 (4E10 vg per mouse; red) or AAV buffer as control (blue). Blood glucose concentration (mg / dL) is represented on the y-axis, while time in minutes (min) after glucose injection is represented on the x-axis. **, P < 0.01; ****, p < 0.0001.

[0081] FIG. 10A shows that mice administered AAV-Rec2-Exendin-4 had similar oxygen consumption compared to control mice. Mice were fed on a high fat diet and randomized to receive a single IP injection of Rec-2-Exendin-4 (4E10 vg per mouse; red) or AAV buffer as control (blue). Indirect calorimetry was assessed 12-weeks post AAV injection. VO2 as measured in milliliters per kilogram per hour (ml / kg / h) is represented on the y-axis, while time in hours (h) is represented on the x-axis.

[0082] FIG. 10B shows that mice administered AAV-Rec2 -Exendin-4 had similar heat energy expenditure compared to control mice. Mice were fed on a high fat diet and randomized to receive a single IP injection of Rec-2-Exendin-4 (4E10 vg per mouse; red) or AAV buffer as control (blue). Indirect calorimetry was assessed 12-weeks post AAV injection. Heat asDocket No. 103362-094WO1

[0083] measured in kilocalories per hour (kcal / hr) is represented on the y-axis, while time in hours (h) is represented on the x-axis.

[0084] FIG. 10C shows that mice administered AAV-Rec2 -Exendin-4 had a similar respiratory exchange ratio (RER) compared to control mice. Mice were fed on a high fat diet and randomized to receive a single IP injection of Rec-2 -Exendin-4 (4E10 vg per mouse; red) or AAV buffer as control (blue). Indirect calorimetry was assessed 12-weeks post AAV injection. RER is represented on the y-axis, while time in hours (h) is represented on the x-axis.

[0085] FIG. 10D shows that mice administered AAV-Rec2 -Exendin-4 had similar locomotor activity compared to control mice. Mice were fed on a high fat diet and randomized to receive a single IP injection of Rec-2-Exendin-4 (4E10 vg per mouse; red) or AAV buffer as control (blue). Indirect calorimetry was assessed 12-weeks post AAV injection. Ambulation as measured in counts per hour (counts / h) is represented on the y-axis, while time in hours (h) is represented on the x-axis.

[0086] FIG. 11A-11C show Rec2-Exendin-4 treatment improves locomotion and exploratory behavior in open field test.

[0087] FIG. 11 A shows Rec2 -Exendin-4 treatment significantly increased locomotion and exploratory activity as measured by total distance traveled. Mice were fed on a high fat diet and randomized to receive a single IP injection of Rec-2-Exendin-4 (4E10 vg per mouse; red) or AAV buffer as control (blue). At 13 weeks post AAV injection, mice were subjected to open field test to assess exploratory behavior, locomotion, and anxiety-like behavior. Total distance traveled as measured in millimeters (mm) is represented on the y-axis for different treatment conditions as represented on the x-axis. **, P < 0.01.

[0088] FIG. 11B shows no difference in anxiety-like behavior was observed as measured by ratio of center / total distance. Mice were fed on a high fat diet and randomized to receive a single IP injection of Rec-2 -Exendin-4 (4E10 vg per mouse; red) or AAV buffer as control (blue). At 13 weeks post AAV injection, mice were subjected to open field test to assess exploratory behavior, locomotion, and anxiety-like behavior. At 13 weeks post AAV injection, mice were subjected to open field test to assess exploratory behavior, locomotion, and anxietylike behavior. The ratio of center distance divided by total distance traveled is represented by a percentage on the y-axis for different treatment conditions as represented on the x-axis.

[0089] FIG. 11C shows no difference in anxiety-like behavior was observed as measured by peripheral / total distance. Mice were fed on a high fat diet and randomized to receive a single IP injection of Rec-2-Exendin-4 (4E10 vg per mouse; red) or AAV buffer as control (blue). TheDocket No. 103362-094WO1

[0090] ratio of peripheral distance divided by total distance traveled is represented by a percentage on the y-axis for different treatment conditions as represented on the x-axis.

[0091] FIG. 12 shows Rec2 -Exendin-4 treatment has no effect on recognition memory in novel recognition test. Mice were fed on a high fat diet and randomized to receive a single IP injection of Rec-2-Exendin-4 (4E10 vg per mouse; red) or AAV buffer as control (blue). At 14 weeks post AAV injection, novel object recognition test was performed to assess recognition memory. Discrimination Index is represented on the x-axis for different treatment conditions which are represented on the x-axis.

[0092] FIG. 13A-13C show Rec2 -Exendin-4 treatment reduces food intake and weight gain in Afoge / 2-null mouse model of Prader-Willi Syndrome (PWS).

[0093] FIG. 13 A shows Rec2 -Exendin-4 treatment reduced body weight starting at one week post AAV injection in Mage! 2 -nu\\ PWS model mice compared to control. M age! 2 -nu\\ mice were randomized to receive a single IP injection of Rec2 -Exendin-4 (4E10 vg per mouse; salmon) or Rec2-GFP (4E10 vg per mouse; black) as control. All mice were fed on high fat diet. Body weight as measured in grams (g) is represented on the y-axis, while time after AAV injection in weeks is represented on the x-axis in weeks. n=5 per group.

[0094] FIG. 13B shows Rec2 -Exendin-4 treatment significantly reduced body weight at two weeks post AAV injection in M age! 2 -nu\\ PWS model mice compared to control. Magel2-rax\\ mice were randomized to receive a single IP injection of Rec2 -Exendin-4 (4E10 vg per mouse; salmon) or Rec2-GFP (4E10 vg per mouse; black) as control. All mice were fed on high fat diet. Body weight change as measured in percent of baseline weight is represented on the y-axis, while time after AAV injection in weeks is represented on the x-axis. n=5 per group. *, P < 0.05.

[0095] FIG. 13C shows Rec2 -Exendin-4 treatment reduces food intake in Magel2-nu\\ PWS model mice compared to control. Mage!2-nu\\ mice were randomized to receive a single IP injection of Rec2 -Exendin-4 (4E10 vg per mouse; salmon) or Rec2-GFP (4E10 vg per mouse; black) as control. Average food intake as measured in grams per day (g / day) is represented on the y-axis, while time after AAV injection in weeks is represented on the x-axis. All mice were fed on high fat diet. n=5 per group.

[0096] FIGs. 14A-14B show GLP-1 transgene in an obesity model.

[0097] FIG. 14A shows GLP-1 transgene sequences and encoded products thereof.

[0098] FIG. 14B shows Rec2-GLP-1 in diet induced obesity model. n=5 per group.

[0099] FIG. 15 shows mature BDNF (mBDNF) transgene sequences and encoded products thereof.Docket No. 103362-094WO1

[0100] FIG. 16 shows TAT-mature BDNF (TAT-mBDNF) transgene sequences and encoded products thereof.

[0101] FIG. 17 shows a western blot stained for HA tag and beta-actin of cell lysates and media of HEK293 cells transfected with AAV expression plasmid containing mBDNF transgenes run by SDS-PAGE. Beta-actin staining was included as a positive control of protein expression and consistent sample loading. Recombinant mBDNF protein was detected in cell lysates and in medium harvested at 48 hours and 72 hours post-transfection. N-terminal TAT affected pattern of mBDNF. Signal peptide was cleaved without TAT at N-terminus of mBDNF.

[0102] FIG. 18 A shows a graph of body weight of ob / + mice receiving two doses of IP inj ection of AAV-Rec2-mBDNF at 2E10 vg per mouse (salmon) and at 4E10 vg per mouse (teal) versus control AAV buffer (black). Mice were maintained on normal chow diet throughout the 6-weeks experiment. Body weight change as measured in percent of baseline weight is represented on the y-axis, while different treatment groups are represented on the x-axis. *, P < 0.05.

[0103] FIG. 18B shows glucose tolerance of ob / + mice receiving two doses of IP injection of AAV-Rec2 -mBDNF at 2E10 vg per mouse (salmon) and at 4E10 vg per mouse (teal) versus control AAV buffer (black). Mice were maintained on normal chow diet throughout the 6-weeks experiment. Blood glucose concentration (mg / dL) is represented on the y-axis, while time in minutes (min) after glucose injection is represented on the x-axis.

[0104] FIG. 19A shows exploratory behavior of ob / + mice receiving two doses of IP injection of AAV-Rec2-mBDNF at 2E10 vg per mouse (salmon) and at 4E10 vg per mouse (teal) versus control AAV buffer (black). Mice were maintained on normal chow diet throughout the 5-weeks experiment. Total distance traveled as measured in millimeters (mm) is represented on the x-axis for different treatment conditions which are represented on the x-axis.

[0105] FIG. 19B shows an open field test of ob / + mice receiving two doses of IP injection of AAV-Rec2 -mBDNF at 2E10 vg per mouse (salmon) and at 4E10 vg per mouse (teal) versus control AAV buffer (black). Mice were maintained on normal chow diet throughout the 5-weeks experiment. Percent from center over total distance traveled is represented on the y-axis for different treatment conditions which are represented on the x-axis.

[0106] FIG. 19C shows novel object recognition of ob / + mice receiving two doses of IP injection of AAV-Rec2-mBDNF at 2E10 vg per mouse (salmon) and at 4E10 vg per mouse (teal) versus control AAV buffer (black). Mice were maintained on normal chow diet throughout the 5-weeks experiment. Novel Object Discrimination Index is represented on the x-axis for different treatment conditions which are represented on the x-axis.Docket No. 103362-094WO1

[0107] FIG. 20 shows a dose-dependent increase in serum levels of BDNF in mice receiving AAV-Rec2-mBDNF compared to control. ob / + mice received two doses of IP injection of AAV-Rec2-mBDNF at 2E10 vg per mouse (salmon) and at 4E10 vg per mouse (teal) versus control AAV buffer (black). Mice were maintained on normal chow diet throughout the experiment. Serum BDNF as measured in nanograms per milliliter (ng / ml) as measured by ELISA is represented on the y-axis for different treatment conditions which are represented on the x-axis. *, P< 0.05; **, P< 0.01, ****, P < 0.0001.

[0108] FIG. 21 shows mBDNF protein levels in epidi dymal white adipose tissue (eWAT) and liver by Western blotting. Western blot stained for HA tag and GAPDH of white visceral adipose tissue (eWAT) and of liver in ob / + mice receiving two doses of IP injection of AAV-Rec2 -mBDNF at 2E10 vg per mouse or 4E10 vg per mouse versus control AAV buffer. Antibody detects HA tag in the mBDNF transgene. GAPDH staining was included as a sample loading control. Recombinant mBDNF protein was detected only in tissue samples from mice administered AAV-Rec2 -mBDNF.

[0109] FIG. 22 shows Rec2-mBDNF treatment altered gene expression in the hypothalamus in a dose-dependent manner. Hypothalamus gene expression profiles were assayed by qRT-PCR in ob / + mice receiving two doses of IP injection of AAV-Rec2-mBDNF at 2E10 vg per mouse (salmon) and at 4E10 vg per mouse (teal) versus control AAV buffer (black). Relative mRNA levels are represented on the y-axis for different genes on the x-axis. *, P < 0.05; **, P < 0.01.

[0110] FIG. 23 shows Rec2-mBDNF treatment altered gene expression in the liver in a dosedependent manner. Liver gene expression profiles were assayed by qRT-PCR in ob / + mice receiving two doses of IP injection of AAV-Rec2-mBDNF at 2E10 vg per mouse (salmon) and at 4E10 vg per mouse (teal) versus control AAV buffer (black). Relative mRNA levels are represented on the y-axis for different genes on the x-axis. *, P < 0.05; **, P < 0.01.

[0111] FIG. 24 shows Rec2-mBDNF in db / db mice. n=4 Buffer, n=5 Rec2-mBDNF 2E10. FIG. 25 shows Rec2-mBDNF in db / db mice post crossover dosing of Rec2-mBDNF 5E10 vg / mouse. n=5 Rec2-mBDNF 2E10, n=4 Rec2-mBDNF 5E10, * P<0.05.

[0112] FIG. 26 shows Rec2-mBDNF in db / db mice post crossover dosing of Rec2-mBDNF 5E10 vg / mouse. n=5 Rec2-mBDNF 2E10, n=4 Rec2-mBDNF 5E10.

[0113] FIG. 27 shows Rec2-mBDNF in db / db mice post crossover dosing of Rec2-mBDNF 5E10 vg / mouse. * P<0.05, ** P<0.01

[0114] FIG. 28 shows mature BDNF-TAT (mBDNF-TAT) transgene sequences and encoded products thereof.Docket No. 103362-094WO1

[0115] FIG. 29 Western blotting detection of mBDNF-TAT after transfection to HEK293 cells. Western blot stained for HA, GFP, and beta-actin of cell lysates and media of HEK293 cells transfected with a vector encoding mBDNF-TAT harvested 72 hr post transfection and run by SDS-PAGE. TAT at N-terminus of BDNF and BDNF without TAT were included, GFP as negative control. Lane 1: TAT-mBDNF (TAT at N-terminus); Lane 2: mBDNF (w / o TAT); Lane 3: GFP; Lane 4: mBNDF-TAT (TAT at C-terminus). Beta-actin staining was included as a sample loading control. Recombinant mBDNF transgene product was detected in cell lysates and in medium.

[0116] FIG. 30 shows mature BMP7 (mBMP7) transgene sequences and encoded products thereof.

[0117] FIG. 31Ashows a western blot stained for HA tag (1:1000) of lysates ofHEK293 cells transfected with a vector encoding mBMP7 or GFP control and run by SDS-PAGE. Antibody detects HA tag in the mBMP7 transgene. Recombinant mBMP7 transgene product, an estimated 18 kDa, was detected in lysate. Cell lysates were collected 72 hr after transfection.

[0118] FIG. 3 IB shows a western blot stained for beta-tubulin of lysates of HEK293 cells transfected with a vector encoding mBMP7 or GFP control and run by SDS-PAGE. Beta tubulin, an estimated 50 kDa, was detected in lysate at equal amounts in different wells, suggesting the samples were equally loaded in the gel. Cell lysates were collected 72 hr after transfection.

[0119] FIG. 31C shows a western blot stained for HA tag (1:500) of cell culture media collected at 48 hours and 72 hours after HEK293 cell transfection with a vector encoding mBMP7 or GFP control and run by SDS-PAGE. Recombinant mBMP7 transgene product, an estimated 18 kDa, was detected in cell culture media indicating recombinant mBMP7 was secreted from transfected cells.

[0120] FIGs. 32A-32K show Intraperitoneal administration of Rec2-Exendin4 normalizes metabolic function in Magel2-nu\\ mice. Data are means ± SEM. Sample size: WT GFP n=6, WT Ex4 n=5, M age! 2 -nu\\ GFP n=5, M age! 2 -nu\\ Ex4 n=7. Individual values are shown in graph. Two-way ANOVAs with u ey' poslhoc test. * <0.05, ** <0.01, *** P<0.001, **** P<0.0001, ns not significant. Scatter plots not connected by same letter are significantly different.

[0121] FIG. 32A shows Amino acid sequence of Exendin-4 transgene (SEQ ID NO: 4).

[0122] FIG. 32B shows Experimental timeline of metabolic and behavioral parameters.

[0123] FIG. 32C shows Body weight.

[0124] FIG. 32D shows Area under the curve of body weight.Docket No. 103362-094WO1

[0125] FIG. 32E shows Weight gain.

[0126] FIG. 32F shows Total weight gain.

[0127] FIG. 32G shows Glucose tolerance test at 4 weeks post AAV injection.

[0128] FIG. 32H shows Area under the curve of the glucose tolerance test.

[0129] FIG. 321 shows Blood glucose after overnight fasting.

[0130] FIG. 32J shows Relative fat mass as measured by EchoMRI at 8.5 weeks post AAV injection.

[0131] FIG. 32K shows Relative lean mass as measured by EchoMRI at 8.5 weeks post injection.

[0132] FIGs. 33A-33H show Rec2-Exendin4 gene therapy reverses genotype-driven fat mass increase in Mage!2-nu\\ mice. Data are means ± SEM. Sample size: WT GFP n=6, WT Ex4 n=5, M age! 2 -nu\\ GFP n=5, M age! 2 -nu\\ Ex4 n=7. Individual values are shown in graph. Two-way A NOVAs with Tukey’s s / Z / oc test. * P<0.05, ** <0.01, *** <0.001, **** O. OOOl, ns not significant. Scatter plots not connected by same letter are significantly different.

[0133] FIG. 33 A shows Body weight at time of euthanasia.

[0134] FIG. 33B shows Brown adipose tissue (BAT) weight.

[0135] FIG. 33C shows Inguinal white adipose tissue (iWAT) weight.

[0136] FIG. 33D shows Epididymal white adipose tissue (eWAT) weight.

[0137] FIG. 33E shows Retroperitoneal white adipose tissue (rWAT) weight.

[0138] FIG. 33F shows Liver weight.

[0139] FIG. 33G shows Pancreas weight.

[0140] FIG. 33H shows Gastrocnemius muscle weight.

[0141] FIGs. 34A-34J show Rec2-Exendin4 gene therapy improves circulating markers of systemic metabolism in Magel2-nu\\ mice. Data are means ± SEM. Sample size: WT GFP n=6, WT Ex4 n=5, M age! 2 -nu\\ GFP n=5, M age! 2 -nu\\ Ex4 n=7. Individual values are shown in graph. Two-way ANOVAs with Tukey’s / ioVAoc test. * P<0.05, **P<0.01, *** P<0.001, **** O. OOOl, ns not significant. Scatter plots not connected by same letter are significantly different.

[0142] FIG. 34A shows Serum exendin4. * P<0.05 WT Ex4 versus Mage / 2 -nu\\ Ex4.

[0143] FIG. 34B shows Serum glucose.

[0144] FIG. 34C shows Serum insulin.

[0145] FIG. 34D shows Homeostatic model assessment for insulin resistance index (HOMA-IR).

[0146] FIG. 34E shows Serum leptin.Docket No. 103362-094WO1

[0147] FIG. 34F shows Serum adiponectin.

[0148] FIG. 34G shows Adiponectin to leptin ratio (with adiponectin level expressed in pg / mL and leptin level expressed in ng / mL).

[0149] FIG. 34H shows Serum triglyceride.

[0150] FIG. 341 shows Serum alanine transaminase (ALT).

[0151] FIG. 34J shows Serum aspartate transferase (AST).

[0152] FIGs. 35A-35B show Rec2-Exendin4 gene therapy regulates adipose gene expression in Magel2-nu\\ mice. Data are means ± SEM. Sample size: n=5 per group. Individual values are shown in graph. Two-way ANOVAs with Tukey’s post hoc test. * <0.05, ** <0.01 *** O. OOl, ns not significant, p values trending toward significance were shown where applicable. Scatter plots not connected by same letter are significantly different.

[0153] FIG. 35 A shows Epidi dymal white adipose tissue (eWAT) gene expression.

[0154] FIG. 35B shows Brown adipose tissue (BAT) gene expression.

[0155] FIGs. 36A-36B show Rec2-Exendin4 gene therapy alters hypothalamic and hepatic gene expression in Magel2-nu\\ mice. Data are means ± SEM. Sample size: n=5 per group. Individual values are shown in graph. Two-way ANOVAs with Tukey’s post hoc test. * <0.05, ** O. Ol **** P<0.0001, ns not significant, p values trending toward significance were shown where applicable. Scatter plots not connected by same letter are significantly different.

[0156] FIG. 36A shows Hypothalamic gene expression.

[0157] FIG. 36B shows Liver gene expression.

[0158] FIGs. 37A-37J show Rec2-Exendin4 gene therapy improves metabolic and behavioral functions in diet-induced obesity model (5 months study). Data are means ± SEM. Sample size: n=5 per group. Individual values are shown in graph. Time course data (BWs, GTT) were analyzed using Two-way RM ANOVA with Sidak’s multiple comparisons test. Unpaired t test for other data analyses. * <0.05, ** <0.01, *** <0.001, **** O. OOOl. Scale bar: 100 pm.

[0159] FIG. 37A shows Body weight.

[0160] FIG. 37B shows Total weight gain of 5 months.

[0161] FIG. 37C shows Average food intake measured week 1 to week 5 post AAV injection. FIG. 37D shows Absolute fat mass and absolute lean mass at 5 weeks post AAV injection.

[0162] FIG. 37E shows Relative fat mass and relative lean mass at 5 weeks post AAV injection. FIG. 37F shows Glucose tolerance test at 6 weeks post AAV injection.Docket No. 103362-094WO1

[0163] FIG. 37G shows Area under the curve of the glucose tolerance test.

[0164] FIG. 37H shows Open field test at 13 weeks post AAV injection.

[0165] FIG. 371 shows Novel object recognition test at 13 weeks post AAV injection.

[0166] FIG. 37J shows H& E staining of livers.

[0167] FIGs. 38A-38H show Rec2-Exendin4 and Rec2-Exendin4-HA reverses diet-induced obesity and associated metabolic dysfunction.

[0168] FIG. 38A shows Body weight. Mixed ANOVA with Sidak’s multiple comparisons test. * P<0.05 Ex4 and Ex4-HA versus GFP

[0169] FIG. 38B shows Final body weight.

[0170] FIG. 38C shows Weight gain. Two-way RM ANOVA with Sidak’s multiple comparisons test. *** O. OOl Ex4 and Ex4-HA versus GFP Data are means ± SEM. Sample size: GFP n=4, Ex4 n=5, Ex4-HAn=5. Individual values are shown in graph. One-way ANOVA with ukey's post hoc test. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

[0171] FIG. 38D shows Total weight gain.

[0172] FIG. 38E shows Average food intake measured week 1 to week 4 post AAV injection. FIG. 38F shows Glucose tolerance test at 6 weeks post AAV injection.

[0173] FIG. 38G shows Area under the curve of the glucose tolerance test.

[0174] FIG. 38H shows Indirect calorimetry at 7-8 weeks post injection.

[0175] FIGs. 39A-39L show Rec2-Exendin4 and Rec2-Exendin4-HA alleviates obesity-associated fatty liver, insulin resistance, and hyperleptinemia. Data are means ± SEM. Sample size: GFP n=4, Ex4 n=5, Ex4-HAn=5. Individual values are shown in graph. One-way ANOVA with ukey’s post hoc test. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

[0176] FIG. 39A shows Tissue mass. BAT: brown adipose tissue, eWAT: epididymal white adipose tissue, iWAT: inguinal white adipose tissue, rWAT, retroperitoneal white adipose tissue.

[0177] FIG. 39B shows Serum glucose.

[0178] FIG. 39C shows Serum insulin.

[0179] FIG. 39D shows Homeostatic model assessment for insulin resistance index (HOMA-IR).

[0180] FIG. 39E shows Serum leptin.

[0181] FIG. 39F shows Serum adiponectin.

[0182] FIG. 39G shows Adiponectin to leptin ratio (with adiponectin level expressed in pg / mL and leptin level expressed in ng / mL).

[0183] FIG. 39H shows Serum triglyceride.Docket No. 103362-094WO1

[0184] FIG. 391 shows Hepatic triglyceride.

[0185] FIG. 39J shows Serum exendin4. * <0.05 Ex4 versus Ex4-HA.

[0186] FIG. 39K shows Western blotting of liver and eWAT samples from Ex4-HA group. FIG. 39L shows Quantification of Western blotting in (K).

[0187] FIGs. 40A-40B show Exendin4 transgene in vitro validation.

[0188] FIG. 40A shows Western blotting of HEK293 cell lysate and culture media 72 h after transfection.

[0189] FIG. 40B shows Ex4 concentration in cell culture media collected 0, 24, 48, and 72 h after transfection measured by ELISA. Data are means of duplication of transfection.

[0190] FIGs. 41A-41C show pilot study in Magel2-null mice receiving an intraperitoneal injection of Rec2-Ex4 at the dose of 4x1010vg per mouse. Data are means ± SEM. Sample size: n=5 per group. Two-way RM ANO VA with Sidak’s multiple comparisons test. * P<0.05.

[0191] FIG. 41 A shows Body weight.

[0192] FIG. 4 IB shows Weight change from baseline.

[0193] FIG. 41C shows Average food intake.

[0194] FIGs. 42A-42D show energy expenditure measured by indirect calorimetry in Magel2-null mice at 7-8 weeks post AAV injection at the dose of 2x1010vg per mouse. Data are means ± SEM. Sample size: WT GFP n=4, WT Ex4 n=4, Magel2-null GFP n=5, Magel2-null Ex4 n=5. Individual values are shown in graph. Two-way ANOVAs with Tukey’s post hoc test. *** PO. OOl, **** PO. OOOl, ns not significant. Scatter plots not connected by same letter are significantly different.

[0195] FIG. 42A shows heat, respiratory exchange ratio (RER), and ambulation during the second 24 h in the metabolic chamber. Two-way RM ANOVA, groups: ns.

[0196] FIG. 42B shows Average hourly heat, average hourly RER, and average hourly ambulation over 24 h.

[0197] FIG. 42C shows Absolute fat mass measured by EchoMRI at 8.5 weeks post AAV injection.

[0198] FIG. 42D shows Absolute lean mass measured by EchoMRI at 8.5 weeks post AAV injection.

[0199] FIGs. 43A-43B show food intake in Magel2-null mice between 7- and 10-weeks post AAV injection. Mice were singly housed starting at 7-weeks post AAV injection. Food intake was recorded on weekly basis till the euthanasia 10-weeks post AAV injection excluding time spent in the metabolic chamber. Data are means ± SEM. Sample size: WT GFP n=6, WT Ex4 n=5, Magel2-null GFP n=5, Magel2-null Ex4 n=7. Individual values are shown in graph. Two-Docket No. 103362-094WO1

[0200] way ANOVAs with Tukey’s post hoc test. * P<0.05, ns not significant. Scatter plots not connected by same letter are significantly different.

[0201] FIG. 43 A shows Average food intake.

[0202] FIG. 43B shows Relative food intake calibrated to body weight.

[0203] FIGs. 44A-44D show behavioral tests in Magel2-null mice at 5-weeks post AAV injection. Data are means ± SEM. Sample size: WT GFP n=6, WT Ex4 n=5, Magel2-null GFP n=5, Magel2-null Ex4 n=7. Individual values are shown in graph. Two-way ANOVAs with Tukey’s post hoc test. * P<0.05, **** P<0.0001, ns not significant, p values trending toward significance were shown where applicable. Scatter plots not connected by same letter are significantly different.

[0204] FIG. 44A shows Total distance traveled in the open field arena.

[0205] FIG. 44B shows Percentage distance traveled in the center of the open field arena. FIG. 44C shows Percentage distance traveled in the periphery of the open field arena. FIG. 44D shows Novel object discrimination index.

[0206] FIGs. 45A-45G show relative tissue mass calibrated to body weight. Data are means ± SEM. Sample size: WT GFP n=6, WT Ex4 n=5, Magel2-null GFP n=5, Magel2-null Ex4 n=7. Individual values are shown in graph. Two-way ANOVAs with Tukey’s post hoc test. * P<0.05, ** P<0.01, **** PO. OOOl, ns not significant. Scatter plots not connected by same letter are significantly different.

[0207] FIG. 45A shows Relative brown adipose tissue (BAT) weight.

[0208] FIG. 45B shows Relative inguinal white adipose tissue (iWAT) weight.

[0209] FIG. 45C shows Relative epididymal white adipose tissue (eWAT) weight.

[0210] FIG. 45D shows Relative retroperitoneal white adipose tissue (rWAT) weight.

[0211] FIG. 45E shows Relative liver weight.

[0212] FIG. 45F shows Relative pancreas weight.

[0213] FIG. 45G shows Relative gastrocnemius muscle weight.

[0214] FIG. 46 shows energy expenditure measured by indirect calorimetry in diet-induced obesity mice at 19-weeks post AAV injection at the dose of 4x1010vg per mouse. Heat, respiratory exchange ratio (RER), and ambulation during the second 24 h in the metabolic chamber. Two-way RM ANOVA, groups: ns.

[0215] FIGs. 47A-47B show HA tagged Exendin4 transgene sequence and in vitro validation. FIG. 47A shows Amino acid sequence of Ex4-HA transgene (SEQ ID NO: 64).

[0216] FIG. 47B shows Western blotting of HEK293 cell lysate and culture media 72 h after transfection.Docket No. 103362-094WO1

[0217] FIGs. 48A-48L show additional data of diet-induced obesity model treated with Rec2-Exendin4 or Rec2-Exendin4-HA. Data are means ± SEM. Sample size: GFP n=4, Ex4 n=5, Ex4- HAn=5. Individual values are shown in graph. One-way ANOVA with Tukey’s post hoc test. * P<0.05, ** P<0.01, **** PO. OOOl.

[0218] FIG. 48A shows Relative brown adipose tissue (BAT) weight.

[0219] FIG. 48B shows Relative inguinal white adipose tissue (iWAT) weight.

[0220] FIG. 48C shows Relative epididymal white adipose tissue (eWAT) weight.

[0221] FIG. 48D shows Relative retroperitoneal white adipose tissue (rWAT) weight.

[0222] FIG. 48E shows Relative liver weight.

[0223] FIG. 48F shows Absolute and relative pancreas weight.

[0224] FIG. 48G shows Absolute and relative heart weight.

[0225] FIG. 48H shows Absolute and relative gastrocnemius muscle weight.

[0226] FIG. 481 shows Serum alanine transaminase (ALT).

[0227] FIG. 48J shows Serum aspartate transferase (AST).

[0228] FIG. 48K shows Relative WPRE expression in the liver.

[0229] FIG. 48L shows Relative WPRE expression in the eWAT.

[0230] FIG. 49 shows Rec2-Exendin 4 in obesity-associated colon cancer model. Mice were fed on high fat diet for 3 weeks and randomized to receive intraperitoneal injection of either Rec2 -Exendin 4 (4E10 vg / mouse) or AAV buffer. Five days after AAV injection, MC38 colon cancer cells were subcutaneously implanted on the flank (4E5 cells / mouse). Mice were maintained on high fat diet throughout the 3-weeks experiment. n=8 per group.

[0231] FIG. 50 shows Rec2 -Exendin 4 in obesity-associated colon cancer model. n=8 per group. ** P<0.01, **** PO. OOOl, ns not significant. ND not detected.

[0232] FIG. 51 shows Rec2 -Exendin 4 in congenital generalized lipodystrophy model of aP2-SREBPlc transgenic mice via subcutaneous injection (1E10 vg / mouse). ns, not significant.

[0233] FIG. 52 shows Rec2-Exendin 4 in congenital generalized lipodystrophy model of SHREBPlc transgenic mice via subcutaneous injection (1E10 vg / mouse).

[0234] FIG. 53 shows Rec2-Exendin 4 in congenital generalized lipodystrophy model of SHREBPlc transgenic mice via subcutaneous injection (1E10 vg / mouse). ns, not significant.

[0235] FIG. 54 shows Rec2 -Exendin 4 in congenital generalized lipodystrophy model of aP2-SREBPlc transgenic mice via subcutaneous injection (1E10 vg / mouse). ns, not significant.

[0236] FIG. 55 shows Rec2 -Exendin 4 in congenital generalized lipodystrophy model of aP2-SREBPlc transgenic mice via subcutaneous injection (1E10 vg / mouse). * P<0.05.

[0237] FIG. 56 shows GLP-lR / GIPR / GcgR Triagonist transgene sequence.Docket No. 103362-094WO1

[0238] FIG. 57 shows Western blotting detection of HA-tagged GLP-lR / GIPR / GcgR triagonist protein after transfection to HEK 293 cells. Antibody detects HA tag in the Exendin-4 transgene and GLP-l / GIPR / GcgR triagonist 72 hrs post transfection to HEK 293 cells.

[0239] FIG. 58 shows Secretory enhanced green fluorescent protein transgene sequence. FIG. 59 shows Western blotting detection of GFP protein after transfection to HEK 293 cells. GFP detected in cell lysates but not in culture medium 72 hrs post transfection to HEK 293 cells.

[0240] FIG. 60 shows Rec2-Leptin was subcutaneously injected to area where the inguinal fat depot locates (1E10 vg each side, 2E10 vg per mouse). Rec2-Leptin treatment prevented the development of obesity in ob / ob mice throughout the 14-months study.

[0241] FIG. 61 shows Rec2-Leptin was subcutaneously injected to area where the inguinal fat depot locates (1E10 vg each side, 2E10 vg per mouse). Rec2-Leptin treatment restored circulating leptin levels in ob / ob mice throughout the 14-months study.

[0242] FIG. 62 shows viral DNA detected only in inguinal white adipose tissue.

[0243] FIG. 63 shows transgene mRNA detected only in inguinal white adipose tissue.

[0244] DETAILED DESCRIPTION

[0245] Before the present compositions and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0246] Definitions

[0247] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

[0248] As used in the specification and claims, the singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.Docket No. 103362-094WO1

[0249] As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient" is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

[0250] Administration" to a subject includes any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra- articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques), and the like. " Concurrent administration", "administration in combination", "simultaneous administration" or "administered simultaneously" as used herein, means that the compounds are administered at the same point in time or essentially immediately following one another. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time. " Systemic administration" refers to the introducing or delivering to a subject an agent via a route which introduces or delivers the agent to extensive areas of the subject's body (e.g. greater than 50% of the body), for example through entrance into the circulatory or lymph systems. By contrast, "local administration" refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration but are undetectable or detectable at negligible amounts in distal parts of the subject's body. Administration includes self-administration and the administration by another.

[0251] As used here, the terms “therapeutic agent,” “beneficial agent,” and “active agent” are used interchangeably herein to refer to a chemical compound or composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, i.e., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, i.e., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides,Docket No. 103362-094WO1

[0252] prodrugs, active metabolites, isomers, fragments, analogs, and the like. When the terms “beneficial agent” or “active agent” are used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0253] As used herein, the terms “treating” or “treatment” of a subject includes the administration of a drug to a subject with the purpose of preventing, curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing or affecting a disease or disorder, or a symptom of a disease or disorder. The terms “treating” and “treatment” can also refer to reduction in severity and / or frequency of symptoms, elimination of symptoms and / or underlying cause, prevention of the occurrence of symptoms and / or their underlying cause, and improvement or remediation of damage.

[0254] As used herein, the term “preventing” a disorder or unwanted physiological event in a subject refers specifically to the prevention of the occurrence of symptoms and / or their underlying cause, wherein the subject may or may not exhibit heightened susceptibility to the disorder or event.

[0255] By the term “effective amount” of a therapeutic agent is meant a nontoxic but sufficient amount of a beneficial agent to provide the desired effect. The amount of beneficial agent that is “effective” will vary from subject to subject, depending on the age and general condition of the subject, the particular beneficial agent or agents, and the like. Thus, it is not always possible to specify an exact “effective amount”. However, an appropriate “effective’ amount in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of a beneficial can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts.

[0256] An “effective amount” of a drug necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0257] As used herein, a “therapeutically effective amount” of a therapeutic agent refers to an amount that is effective to achieve a desired therapeutic result, and a “prophylactically effective amount” of a therapeutic agent refers to an amount that is effective to prevent an unwanted physiological condition. Therapeutically effective and prophylactically effective amounts of aDocket No. 103362-094WO1

[0258] given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term “therapeutically effective amount" can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the drug and / or drug formulation to be administered (e.g., the potency of the therapeutic agent (drug), the concentration of drug in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art.

[0259] As used herein, the term “pharmaceutically acceptable” component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When the term “pharmaceutically acceptable" is used to refer to an excipient, it is generally implied that the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U. S. Food and Drug Administration.

[0260] " Pharmaceutically acceptable carrier" (sometimes referred to as a "carrier") means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.

[0261] As used herein, “pharmaceutically acceptable salt” is a derivative of the disclosed compound in which the parent compound is modified by making inorganic and organic, nontoxic, acid or base addition salts thereof. The salts of the present compounds can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the like), or by reacting free base forms of these compounds with aDocket No. 103362-094WO1

[0262] stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. Generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are typical, where practicable. Salts of the present compounds further include solvates of the compounds and of the compound salts.

[0263] Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, conventional non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, HOOC-(CH2)n- COOH where n is 0-4, and the like, or using a different acid that produces the same counterion. Lists of additional suitable salts may be found, e.g., in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985).

[0264] Also, as used herein, the term “pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.

[0265] As used herein, the term “subject” or “host” can refer to living organisms such as mammals, including, but not limited to humans, livestock, dogs, cats, and other mammals. Administration of the therapeutic agents can be carried out at dosages and for periods of time effective for treatment of a subject. In some embodiments, the subject is a human. Chemical Definitions

[0266] As used herein, the term “composition” is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a mixture containing 2 parts by weight of component XDocket No. 103362-094WO1

[0267] and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the mixture.

[0268] A weight percent (wt.%) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

[0269] Compositions

[0270] In one aspect, provided herein is a recombinant polypeptide comprising: (a) a signal peptide sequence; (b) a linker peptide comprising between 1 to 30 amino acid residues; and (c) a therapeutic peptide.

[0271] In some embodiments, the signal peptide sequence comprises a leptin signal peptide sequence. In some embodiments, the leptin signal peptide sequence comprises SEQ ID NO: 9, or an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the leptin signal peptide sequence comprises SEQ ID NO: 9.

[0272] In some embodiments, the leptin signal peptide sequence is encoded by a nucleic acid sequence comprising SEQ ID NO: 2, or a nucleic acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the leptin signal peptide sequence is encoded by a nucleic acid sequence comprising SEQ ID NO: 2.

[0273] In some embodiments, the linker peptide is a valine residue. In some embodiments, the linker peptide comprises a glycine / serine linker. In some embodiments, the gly cine / serine linker comprises (GS)x, (GGS)x, (GGGS)x (SEQ ID NO: 46), (GGGGS)x (SEQ ID NO: 47), or (GGGGGS)x (SEQ ID NO: 48), wherein x is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0274] In some embodiments, the linker peptide comprises an amino acid sequence selected from any one of 40-41, 49-50, 55, or 60, or an amino acid sequence at least about 70% identical thereto.

[0275] In some embodiments, the recombinant polypeptide further comprises one or more additional peptides. In some embodiments, the one or more additional peptides comprise a TAT cell-penetrating peptide. In some embodiments, the TAT cell-penetrating peptide comprises SEQ ID NO: 22, or an amino acid at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least aboutDocket No. 103362-094WO1

[0276] 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the TAT cell-penetrating peptide comprises SEQ ID NO: 22.

[0277] In some embodiments, the TAT cell-penetrating peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 20, or a nucleic acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the TAT cell-penetrating peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 20.

[0278] In some embodiments, the signal peptide is derived from GLP-1, PDX-1, GIP, GLP-2, insulin, growth hormone, leptin, prolactin, tissue plasminogen activator (tPA), calcitonin, luteinising hormone, parathyroid hormone, somatostatin, thyroid stimulating hormone, vasoactive intestinal polypeptide, trefoil factors, cell and tissue repair factors, transforming growth factor P, keratinocyte growth factor, a structural group 1 cytokine adopting an antiparallel 4a helical bundle structure, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL- 9, IL-10, IL-11, IL-12, IL-13, GM-CSF, M-CSF, SCF, IFN-y, EPO, G-CSF, LIF, OSM, CNTF, GH, PRL, IFNa / p, a structural group 2 cytokine, a TNF-family cytokine, TNFa, TNFp, CD40, CD27, FAS ligands, an IL-l-family cytokine, a fibroblast growth factor, a platelet derived growth factor, transforming growth factor p, a nerve growth factor, a structural group 3 cytokine comprising a short chain a / P molecule, an epidermal growth factor-family cytokine, a C-C or C-X-C chemokine, an insulin-related cytokine, a structural group 4 cytokine, a heregulins, a neuregulins, EGF, immunoglobulin-like domain, kringle domain, one or more fragments thereof, analogs thereof, or combinations thereof.

[0279] In some embodiments, the therapeutic peptide comprises a peptide hormone, a GLP1 receptor agonist peptide, a glucagon-like peptide, a biomimetic peptide, an immunomodulatory peptide, a neurotoxic peptide, a neurotrophic factor peptide, a bone morphogenetic peptide, a guanylate cyclase-C (G-CC) agonist peptide, a calcitonin receptor inhibitor, a GnRH receptor inhibitor, a 20S proteasome inhibitor, a N0D2 inhibitor, a VIP1 receptor inhibitor, an OT inhibitor, a TRH receptor inhibitor, an MC receptor inhibitor, a PTH1 receptor inhibitor, a guanylate cyclase C inhibitor, an NPR-A inhibitor, an ATI receptor inhibitor, a beta2 -receptor inhibitor, a gp41 inhibitor, a GHRH receptor inhibitor, an N-type calcium channel inhibitor, a thrombopoietin receptor inhibitor, a human erythropoietin receptor inhibitor, a pulmonary surfactant inhibitor, a CaSR inhibitor, an MCI receptor inhibitor, a somatostatin receptor inhibitor, a melanocortin-4 receptor inhibitor, or an immune checkpoint inhibitor peptide, or an analog thereof.Docket No. 103362-094WO1

[0280] In some embodiments, the therapeutic peptide comprises a peptide hormone. In some embodiments, the peptide hormone is insulin. In some embodiments, the peptide hormone is oxytocin. In some embodiments, the peptide hormone is leptin. In some embodiments, the peptide hormone is vasopressin. In some embodiments, the peptide hormone is teriparatide. In some embodiments, the peptide hormone comprises an amino acid sequence derived from liraglutide.

[0281] In some embodiments, the therapeutic peptide is derived from P-Galactosidase 1 (GLB1), Niemann-Pick Cl (NPC1), Apolipoprotein E (APOE), GD3 synthase, huntingtin (Htt), interleukin (IL)- 10 (IL- 10), Myelin Oligodendrocyte Glycoprotein (MOG), mitogen-activated protein kinase 8 interacting protein 3 (MAPKA8IP3), survival motor neuron (SMN) 1 (SMN1), SMN2, Cas9, P-Glucocerebrosidase (GBA), Sphingomyelin phosphodiesterase 1 (SMPD1), beta-hexosaminidase A (HEXA), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin (NT) 3 (NT-3), NT-4 / 5, NT-6, Glial Cell Derived Neurotrophic Factor (GDNF), Ciliary Neurotrophic Factor (CNTF), Leukemia inhibitory factor (LIF), Insulin-like growth factor (IGF) 1 (IGF-1), P-fibroblast growth factor (FGF), neurturin, persephin, artemin, transforming growth factor (TGF) alpha (TGFa), TGFP, IGF-2, platelet derived growth factor (PDGF), epidermal growth factor (EGF), cardiotropin, vascular endothelial growth factor (VEGF), Sonic hedgehog (SHH), bone morphogenic proteins (BMP), FGF20, Vasoactive Intestinal Peptide (VIP), pleiotrophin (PTN), Aromatic L-amino Acid Decarboxylase (AADC), TH, 5-hydroxytryptamine (5HT), hepatocyte growth factor (HGF). In some embodiments, the therapeutic peptide is derived from a BDNF. In some embodiments, the therapeutic peptide is derived from a BMP.

[0282] In some embodiments, the therapeutic peptide comprises a glucagon-like peptide. In some embodiments, the glucagon-like peptide is dulaglutide. In some embodiments, the glucagon-like peptide is semaglutide. In some embodiments, the glucagon-like peptide is teduglutide. In some embodiments, the glucagon-like peptide comprises an amino acid sequence derived from liraglutide.

[0283] In some embodiments, the therapeutic peptide comprises a neurotoxic peptide. In some embodiments, the neurotoxic peptide is ziconotide.

[0284] In some embodiments, the therapeutic peptide comprises a biomimetic peptide. In some embodiments, the biomimetic peptide comprises enfuvirtide.

[0285] In some embodiments, the therapeutic peptide comprises an immunomodulatory peptide. In some embodiments, the immunomodulatory peptide is glatiramer.Docket No. 103362-094WO1

[0286] In some embodiments, the therapeutic peptide comprises a G-CC agonist peptide. In some embodiments, the G-CC agonist peptide is linaclotide.

[0287] In some embodiments, the therapeutic peptide comprises a GLP1 receptor agonist peptide. In some embodiments, the therapeutic peptide comprises a neurotrophic factor peptide. In some embodiments, the therapeutic peptide comprises a bone morphogenetic peptide.

[0288] Exendin-4

[0289] In some embodiments, the therapeutic peptide comprises Exendin-4 (GenBank ID: U77613.1; UniProt ID P26349). Exendin 4 has 53% homology with the biological active form of glucagon-like peptide, GLP-l(7-37), but acts as a full agonist of the GLP1 receptor, and therefore has been developed as a drug for diabetes (marketed as Byetta). Several other GLP-1 receptor agonists have become breakthrough drugs for obesity treatment such as semaglutide (marketed as OZEMPIC® and WEGOVY®).

[0290] In one aspect, provided herein is a recombinant polypeptide comprising: (a) a signal peptide sequence; (b) a linker peptide comprising between 1 to 30 amino acid residues; and (c) a GLP1 receptor agonist peptide. In some embodiments, the GLP1 receptor agonist peptide comprises an exendin-4 peptide or a GLP-1 peptide. In some embodiments, the linker peptide comprises a valine residue.

[0291] In another aspect, provided herein is a recombinant polypeptide comprising: (a) a signal peptide sequence; (b) a linker peptide comprising between 1 to 30 amino acid residues; and (c) a GLP1 receptor agonist peptide; wherein the GLP1 receptor agonist peptide comprises an exendin-4 peptide. In some embodiments, the linker peptide comprises a valine residue.

[0292] In some embodiments, the exendin-4 peptide comprises SEQ ID NO: 7, or an amino acid at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the exendin-4 peptide comprises SEQ ID NO: 7.

[0293] In some embodiments, the exendin-4 peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 3, or a nucleic acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the exendin-4 peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 3.Docket No. 103362-094WO1

[0294] In some embodiments, the recombinant polypeptide comprises SEQ ID NO: 4, or an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the recombinant polypeptide comprises SEQ ID NO: 4.

[0295] In some embodiments, the recombinant polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1, or a nucleic acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the recombinant polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1.

[0296] In some embodiments, the recombinant polypeptide further comprises an additional peptide. In some embodiments, the additional peptide comprises a hemagglutinin (HA) tag. In some embodiments, the HA tag is conjugated to the C-terminus of the exendin-4 peptide. In some embodiments, the HA tag comprises the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence at least about 70% identical thereto. In some embodiments, the HA tag is encoded by the nucleic acid sequence of SEQ ID NO: 15, or a nucleic acid sequence at least about 70% identical thereto.

[0297] In some embodiments, the recombinant polypeptide comprises the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the recombinant polypeptide comprises SEQ ID NO: 64.

[0298] GLP1

[0299] In some embodiments, the therapeutic peptide comprises GLP-1. GLP-1 is cleaved from preproglucagon encoded by the GCG gene which is expressed in pancreatic a-cells, in enteroendocrine L-cells throughout the gut, and in a population of neurons in the brainstem. Posttranslational processing of proglucagon results in smaller peptide hormones / molecules including glicentin, glicentin-related pancreatic polypeptide, glucagon, oxyntomodulin, the major proglucagon fragment, GLP-1, and GLP-2. The expression of specific prohormone convertase enzymes determines which smaller peptide hormones are formed. These smaller peptides derived from proglucagon exert important physiological and pharmacological effects.Docket No. 103362-094WO1

[0300] Of note, these peptides can have opposing effects, e.g. glucagon increases whereas GLP-1 decreases blood glucose. As such, the expression and cleavage of proglucagon and secretion of the various proglucagon derived peptides must be precisely controlled in a cell-specific process.

[0301] For therapeutic application, it is difficult to achieve the goal of producing active GLP-1 peptide by overexpressing GCG gene. Moreover, several forms of GLP-1 are processed from proglucagon with varying ability to enhance glucose-induced insulin secretion including GLP-1 (l-36amide), GLP-1 (7-36amide) and GLP-1 (7-37). In humans, -80% of circulating GLP-1 is GLP-1 (7-36amide) and -20% is GLP-1 (7-37). The two truncated forms of GLP-1 have equal insulinotropic efficacy.

[0302] In one aspect, provided herein is a recombinant polypeptide comprising: (a) a signal peptide sequence; (b) a linker peptide comprising between 1 to 30 amino acid residues; and (c) a GLP1 receptor agonist peptide; wherein the GLP1 receptor agonist peptide comprises a GLP-1 peptide. In some embodiments, the GLP-1 peptide is selected from GLP-1 (l-36amide), GLP-1 (7-36amide), or GLP-1 (7-37). In some embodiments, the GLP-1 peptide is GLP-1 (7-36amide).

[0303] In some embodiments, the GLP-1 peptide comprises SEQ ID NO: 12, or an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the GLP-1 peptide comprises SEQ ID NO: 12.

[0304] In some embodiments, the GLP-1 peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 10, or a nucleic acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the GLP-1 peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 10.

[0305] In some embodiments, the GLP-1 peptide has one or more mutations to increase stability. In some embodiments, the recombinant polypeptide comprises SEQ ID NO: 11, or an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the recombinant polypeptide comprises SEQ ID NO: 11.Docket No. 103362-094WO1

[0306] In some embodiments, the recombinant polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 8, or a nucleic acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the recombinant polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 8.

[0307] In some embodiments, the therapeutic peptide comprises a GLP-1R peptide, a GIPR peptide, a GcgR peptide, or a combination thereof. In some embodiments, the therapeutic peptide is a triagonist polypeptide comprising a GLP-1R peptide, a GIPR peptide, and a GcgR peptide. In some embodiments, the triagonist polypeptide comprises the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the triagonist polypeptide comprises the amino acid sequence of SEQ ID NO: 68.

[0308] In some embodiments, the triagonist polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 66, or a nucleic acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the triagonist polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 66.

[0309] In some embodiments, the recombinant polypeptide comprises the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the recombinant polypeptide comprises SEQ ID NO: 67.

[0310] In some embodiments, the recombinant polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 65, or a nucleic acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the recombinant polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 65.Docket No. 103362-094WO1

[0311] BDNF

[0312] In some embodiments, the therapeutic peptide comprises brain-derived neurotrophic factor (BDNF) (GenBank ID: M37762.1; UniProt ID: P23560). BDNF, which has a diverse range of functions in brain development and plasticity, is one of the most extensively studied molecules. Human BDNF gene contains eleven noncoding exons, each with distinct promoter regions, resulting in alternatively spliced mRNA isoforms. BDNF is initially synthesized as precursor form — pro-BDNF, that is processed by proteases intracellularly to produce c-terminal mature form of BDNF. The BDNF isoforms bind to different types of receptors leading to a large variety of signaling cascades and functional consequences. Mature BDNF (mBDNF) binds to the high affinity neurotrophin receptor tropomyosin receptor kinase B (also known as tyrosine receptor kinase B, TrkB), regulating cell function, growth, survival, and synaptic plasticity. Pro-BDNF is not an inactive precursor but rather a signaling protein mediating diverse responses by binding the p75 neurotrophin receptor (p75NTR).

[0313] Pro-BDNF and mBDNF have distinct or sometimes opposing effects on regulation of neurophysiological processes. Hence, BDNF actions are regulated by the form of BDNF (mature or pro) secreted by cells, by extracellular proteolytic process, and by the presence of different types of receptors. This complexity allows proper control of signaling pathways critical for maintaining a dynamic balance between stimulating and inhibitory effects of BDNF that are exerted upon processes of brain development, synaptic plasticity, and brain regeneration after injury.

[0314] In one aspect, provided herein is a recombinant polypeptide comprising: (a) a signal peptide sequence; (b) a linker peptide comprising between 1 to 30 amino acid residues; and (c) a brain-derived neurotrophic factor (BDNF) peptide. In some embodiments, the recombinant polypeptide further comprises a TAT cell-penetrating peptide. In some embodiments, the TAT cell-penetrating peptide is operatively conjugated to the amino terminus of the BDNF peptide. In some embodiments, the TAT cell-penetrating peptide is operatively conjugated to the carboxy terminus of the BDNF peptide.

[0315] In some embodiments, the TAT cell-penetrating peptide comprises SEQ ID NO: 22, or an amino acid at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the TAT cell-penetrating peptide comprises SEQ ID NO: 22. In some embodiments, the TAT cell-penetrating peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 20, or a nucleic acid sequence atDocket No. 103362-094WO1

[0316] least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the TAT cell-penetrating peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 20.

[0317] In some embodiments, the BNDF peptide comprises pro-BDNF. In some embodiments, the BNDF peptide comprises mature BDNF (mBDNF). In some embodiments, the mBDNF peptide comprises SEQ ID NO: 17, or an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the mBDNF peptide comprises SEQ ID NO: 17.

[0318] In some embodiments, the mBDNF peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 14, or a nucleic acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the mBDNF peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 14.

[0319] In some embodiments, the recombinant polypeptide comprises SEQ ID NO: 16, or an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the recombinant polypeptide comprises SEQ ID NO: 16.

[0320] In some embodiments, the recombinant polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 13, or a nucleic acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the recombinant polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 13.

[0321] In some embodiments, the recombinant polypeptide comprises SEQ ID NO: 21 or SEQ ID NO: 24, or an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the recombinant polypeptide comprises SEQ ID NO: 21. In some embodiments, the recombinant polypeptide comprises SEQ ID NO: 24.Docket No. 103362-094WO1

[0322] In some embodiments, the recombinant polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 19 or SEQ ID NO: 23, or a nucleic acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the recombinant polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 19. In some embodiments, the recombinant polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 23.

[0323] BMP7

[0324] In some embodiments, the therapeutic peptide comprises bone morphogenetic protein 7 (BMP7) (GenBank ID: X51801.1; UniProt ID: P18075). BMP7, also known as osteogenic protein 1, is a member of the transforming growth factor-beta superfamily, that plays important roles in diverse biological processes including embryogenesis, tissue development, and maintenance of homeostasis. BMP7 has gained attention for its potential application in osteoporosis, dental regeneration, inflammatory diseases including cardiovascular diseases, diabetes, obesity, and cellular plasticity to neurological disorders.

[0325] The BMP7 protein consists of 431 amino acids: an N-terminal 29-amino acid signal peptide, a 263 -amino acid pro-peptide, and a 139-amino acid C-terminal mature peptide. During posttranslational processing, pro-BMP7 is hydrolyzed by furin-like proteinase in the cell converting into mature BMP7 and secreted into the extracellular matrix. Clinically, osteogenic implants containing mature BMP7 have been widely used to treat long bone nonunions, spinal fusions, and acute fractures. Recent findings have revealed the role of BMP7 in regulation of appetite, energy expenditure, adipogenesis, and insulin sensitivity. For applications as metabolic modulator, sustained expression of BMP7 within adipose tissue is desirable.

[0326] In one aspect, provided herein is a recombinant polypeptide comprising: (a) a signal peptide sequence; (b) a linker peptide comprising between 1 to 30 amino acid residues; and (c) a peptide derived from bone morphogenetic protein 7 (BMP7). In some embodiments, the peptide derived from BMP7 comprises pro-BMP7. In some embodiments, the peptide derived from BMP7 comprises mature BMP7 (mBMP7).

[0327] In some embodiments, the mBMP7 peptide comprises SEQ ID NO: 28, or an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least aboutDocket No. 103362-094WO1

[0328] 98%, or at least about 99% identical thereto. In some embodiments, the mBMP7 peptide comprises SEQ ID NO: 28.

[0329] In some embodiments, the mBMP7 peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 26, or a nucleic acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the mBMP7 peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 26.

[0330] In some embodiments, the recombinant polypeptide comprises SEQ ID NO: 27, or an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the recombinant polypeptide comprises SEQ ID NO: 27.

[0331] In some embodiments, the recombinant polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 25, or a nucleic acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the recombinant polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 25.

[0332] Signal Sequence

[0333] Signal sequence peptides typically include amino acid residues at the amino terminus of a polypeptide to guide the newly translated polypeptide to endoplasmic reticulum and / or through the secretory pathway of a cell. Signal sequences are described, for example, in Kapp, Katja, et al. Post-targeting functions of signal peptides. Protein transport into the endoplasmic reticulum 1 (2009): 1-16., incorporated herein by reference. In some embodiments, the signal sequence peptide is cleaved by a signal peptidase in the host or subject. In some embodiments, the signal sequence is a membrane-spanning molecule.

[0334] In some embodiments, the signal sequence peptide is derived from GLP-1, PDX-1, GIP, GLP-2, insulin, growth hormone, leptin, prolactin, tissue plasminogen activator (tPA), calcitonin, luteinising hormone, parathyroid hormone, somatostatin, thyroid stimulating hormone, vasoactive intestinal polypeptide, trefoil factors, cell and tissue repair factors, transforming growth factor P, keratinocyte growth factor, a structural group 1 cytokine adopting an antiparallel 4a helical bundle structure, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL- 9,Docket No. 103362-094WO1

[0335] IL-10, IL-11, IL-12, IL-13, GM-CSF, M-CSF, SCF, IFN-y, EPO, G-CSF, LIF, OSM, CNTF, GH, PRL, IFNa / , a structural group 2 cytokine, a TNF-family cytokine, TNFa, TNFp, CD40, CD27, FAS ligands, an IL-l-family cytokine, a fibroblast growth factor, a platelet derived growth factor, transforming growth factor p, a nerve growth factor, a structural group 3 cytokine comprising a short chain a / 0 molecule, an epidermal growth factor-family cytokine, a C-C or C-X-C chemokine, an insulin-related cytokine, a structural group 4 cytokine, a heregulins, a neuregulins, EGF, immunoglobulin-like domain, kringle domain, one or more fragments thereof, analogs thereof, or combinations thereof.

[0336] In some embodiments, the signal sequence peptide is derived from the signal sequence of interleukin-2 (IL2), CD5, Immunoglobulin Kappa light chain, trypsinogen, serum albumin, leptin, tPA, vesicular stomatitis virus G-protein (VSV-G), or prolactin.

[0337] In some embodiments, the signal sequence peptide is a leptin signal sequence peptide. The leptin signal sequence peptide facilitates the secretion of leptin, or a protein to which the leptin signal sequence peptide is conjugated to, as well as the binding thereof to the leptin receptor (LepRb).

[0338] In some embodiments, the signal peptide sequence comprises a leptin signal peptide sequence. In some embodiments, the leptin signal peptide sequence comprises SEQ ID NO: 9, or an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the leptin signal peptide sequence comprises SEQ ID NO: 9.

[0339] In some embodiments, the leptin signal peptide sequence is encoded by a nucleic acid sequence comprising SEQ ID NO: 2, or a nucleic acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the leptin signal peptide sequence is encoded by a nucleic acid sequence comprising SEQ ID NO: 2.

[0340] In some embodiments, the leptin signal sequence peptide comprises an amino acid sequence selected from MCWRPLCRFLWLWSYLSYVQA (SEQ ID NO: 5), MHWGTLCGFLWLWPYLFYVQA (SEQ ID NO: 29), MHWGTLCGFLWLWPYLFYVSPS (SEQ ID NO: 30), MHWGTLCGFLWLWPYLFYVSP (SEQ ID NO: 31), MHWGTLCGFLWLWPYLFYVSPA (SEQ ID NO: 32), or MHWGTLCGFLWLWPYLFYVSNS (SEQ ID NO: 33), or an amino acid sequence at least about 70% identical thereto, for example at least about 75%, at least about 80%, at least aboutDocket No. 103362-094WO1

[0341] 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the leptin signal sequence peptide comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the leptin signal sequence peptide comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the leptin signal sequence peptide comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the leptin signal sequence peptide comprises the amino acid sequence of SEQ ID NO: 31. In some embodiments, the leptin signal sequence peptide comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the leptin signal sequence peptide comprises the amino acid sequence of SEQ ID NO: 33.

[0342] In some embodiments, the leptin signal peptide is conjugated to a linker peptide. In some embodiments, the linker peptide comprises a valine residue. In some embodiments, the leptin signal peptide conjugated to a linker peptide comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the leptin signal peptide conjugated to a linker peptide comprises the amino acid sequence of SEQ ID NO: 6.

[0343] In some embodiments, the signal sequence peptide is a tPA signal sequence peptide. In some embodiments, the tPA signal sequence peptide comprises an amino acid sequence selected from MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 34), MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 35), MDAMKRGLCCVLLLCGAVFVSNS (SEQ ID NO: 36), or an amino acid sequence at least about 70% identical thereto, for example at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the tPA signal sequence peptide comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the tPA signal sequence peptide comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the tPA signal sequence peptide comprises the amino acid sequence of SEQ ID NO: 36.

[0344] In some embodiments, the signal sequence peptide is a chimera of a leptin signal sequence and a tPA signal sequence. In some embodiments, the leptin / tPA chimeric signal sequence peptide comprises an amino acid sequence selected from MDAMKRGLCCVLLLCGAVFVSPA (SEQ ID NO: 37), MHWGTLCCVLLLCGAVFVSPS (SEQ ID NO: 38), or MHWGTLCCVLLLCGAVFVSP (SEQ ID NO: 39), or an amino acidDocket No. 103362-094WO1

[0345] sequence at least about 70% identical thereto, for example at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the leptin / tPA chimeric signal sequence peptide comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the leptin / tPA chimeric signal sequence peptide comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the leptin / tPA chimeric signal sequence peptide comprises the amino acid sequence of SEQ ID NO: 39.

[0346] In some embodiments, the signal peptide sequence comprises an insulin signal peptide sequence. In some embodiments, the leptin signal peptide sequence comprises MALWMRLLPLLALLALWGPDPAAA (SEQ ID NO: 73), or an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the leptin signal peptide sequence comprises SEQ ID NO: 73.

[0347] In some embodiments, the insulin signal peptide sequence is encoded by the nucleic acid sequence of SEQ ID NO: 70, or a nucleic acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the insulin signal peptide sequence is encoded by the nucleic acid sequence of SEQ ID NO: 70

[0348] Linker

[0349] In some embodiments, the signal peptide sequence is covalently linked to the therapeutic peptide via a linker. Any linker is optional; i.e., any linker may simply be a bond. In some embodiments, the linker serves a spacer function. In some embodiments, the linker comprises from 1 to 30 or less amino acids linked by peptide bonds. In some embodiments, the linker comprises between 1 to 30, between 2 to 25, between 3 and 22, between 5 and 20, between 1 and 10, between 10 and 20, or between 10 and 15 amino acids linked by peptide bonds.

[0350] In some embodiments, the amino acids of the linker are selected from the 20 naturally occurring amino acids. In some embodiments, non-natural amino acids are incorporated into the linker either by chemical synthesis, post-translational chemical modification, or by in vivo incorporation by recombinant expression in a host cell. In some embodiments, these amino acids are glycosylated.Docket No. 103362-094WO1

[0351] In some embodiments, the 1 to 30 or less amino acids are selected from glycine (G), alanine (A), proline (P), asparagine (N), glutamine (Q), lysine (K), aspartate(D), and glutamate (E). In some embodiments, the linker comprises a majority of sterically unhindered amino acids selected from glycine, alanine, serine, or a combination thereof.

[0352] In some embodiments, the linker comprises one or more stretches of polyglycines, e.g., GGG, GGGG (SEQ ID NO: 40), GGGGG (SEQ ID NO: 41), of polyalanines, or of polydipeptides selected from (GA)x, (GS)x, (GE)x, (GK)x, (GD)x, or (GR)x motifs, wherein x is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the linker is (GGGKGGGG)x (SEQ ID NO: 42); (GGGNGSGG)x (SEQ ID NO: 43); (GGGCGGGG)x (SEQ ID NO: 44); or (GPNGG)x (SEQ ID NO: 45), wherein x is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0353] In some embodiments, the linker comprises a combinations of Gly and Ala or combinations of Gly and Ser. In some embodiments, the linker is selected from GGG, (GS)x, (GGS)x, (GGGS)x (SEQ ID NO: 46), or (GGGGS)x (SEQ ID NO: 47), where x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, for example, (GGGS)i (SEQ ID NO: 46), (GGGGS)i (SEQ ID NO: 47), (GGGS)4 = GGGSGGGSGGGSGGGS (SEQ ID NO: 49), or (GGGGS)3= GGGGSGGGGSGGGGS (SEQ ID NO: 50).

[0354] In some embodiments, the linker comprises an uncharged linker. As used herein, a linker having “uncharged” amino acids is defined as amino acids that do not have an overall charge. Uncharged amino acids include alanine, glycine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, cysteine, methionine, serine, threonine, asparagine, and glutamine. Uncharged amino acids can be either polar or nonpolar. Polar, uncharged amino acids include serine, threonine, glutamine, and asparagine.

[0355] In some embodiments, charged linkers may be used. Charged amino acids include aspartate, glutamate, lysine, serine, and histidine. Such charged linkers may contain a substantial number of acidic residues (e.g., D, E, and the like), or may contain a significant number of basic residues (e.g., K, R, and the like), such that the linker has a pl lower than 7 or greater than 7, respectively. As understood by the person of skill in the art, and all other things being equal, the greater the relative amount of acidic or basic residues in a given linker, the lower or higher, respectively, the pl of the linker will be. Such linkers may impart advantages to the engineered polypeptides disclosed herein, such as improving solubility and / or stability characteristics of such polypeptides at a particular pH, such as a physiological pH (e.g., between pH 7.2 and pH 7.6, inclusive), or a pH of a pharmaceutical composition comprising such polypeptides.Docket No. 103362-094WO1

[0356] For example, an “acidic linker” is a linker that has a pl of less than 7; between 6 and 7, inclusive; between 5 and 6, inclusive; between 4 and 5, inclusive; between 3 and 4, inclusive; between 2 and 3, inclusive; or between 1 and 2, inclusive. Similarly, a “basic linker” is a linker that has a pl of greater than 7; between 7 and 8, inclusive; between 8 and 9, inclusive; between 9 and 10, inclusive; between 10 and 11, inclusive; between 11 and 12 inclusive, or between 12 and 13, inclusive.

[0357] In some embodiments, the acidic linker is selected from (GE)x, (GGE)x, (GGGE)x (SEQ ID NO: 51); (GGGGE)x (SEQ ID NO: 52), (GD)x, (GGD)x, (GGGD)x (SEQ ID NO: 53); (GGGGD)x (SEQ ID NO: 54) wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, the acidic linker is (GGE)e = GGEGGEGGEGGEGGEGGE (SEQ ID NO: 55).

[0358] In some embodiments, the basic linker is selected from (GK)x, (GGK)x, (GGGK)x (SEQ ID NO: 56); (GGGGK)x (SEQ ID NO: 57), (GR)x, (GGR)x, (GGGR)x (SEQ ID NO: 58), or (GGGGR)x (SEQ ID NO: 59) where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, the linker is (GGR)5 = GGRGGRGGRGGRGGR (SEQ ID NO: 60).

[0359] In some embodiments, the linker peptide comprises an amino acid sequence selected from any one of 40-41, 49-50, 55, or 60, or an amino acid sequence at least about 70% identical thereto.

[0360] In some additional embodiments, linkers may be prepared which possess certain structural motifs or characteristics, such as an a helix. In some embodiments, a linker may contain an sequence that is selected from the group consisting of (EAAAK)x (SEQ ID NO: 61).

[0361] In some embodiments, the linker comprises a non-peptidic linker. In some embodiments, the non-peptide linker is a PEG linker. PEG linkers are known to those of skill in the art.

[0362] In some embodiments, the linker is a cleavable linker. Cleavable peptide linkers are known in the art (see, e.g., Alas, M. et al. Peptide-Drug Conjugates with Different Linkers for Cancer Therapy. J Med Chem. 2021 Jan 14;64(l):216-232).

[0363] In some embodiments, the recombinant polypeptides as described herein comprise more than one linker, and each such linker may possess one or more of the characteristics described above.

[0364] Molecules can be produced that resemble peptides, but which are not connected via a natural peptide linkage. For example, linkages for amino acids or amino acid analogs can include CH2NH—, — CH2S—, — CH2— CH2 —, — CH=CH— (cis and trans), — COCH2 —, — CH(OH)CH2 —, and — CHHzSO— (These and others can be found in Spatola, A. F. inDocket No. 103362-094WO1

[0365] Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci (1980) pp.

[0366] 463-468; Hudson, D. et al., Int J Pept Prot Res 14: 177-185 (1979) (— CH2NH—, CH2CH2— ); Spatola et al. Life Sci 38:1243-1249 (1986) ( — CHH2 — S); Hann J. Chem. Soc Perkin Trans.

[0367] 1307-314 (1982) (— CH— CH—, cis and trans); Almquist et al. J. Med. Chem. 23:1392-1398 (1980) (— COCH2— ); Jennings-White et al. Tetrahedron Lett 23:2533 (1982) (— COCH2— ). Szelke et al. European Appln, EP 45665 CA (1982): 97:39405 (1982) (— CH(OH)CH2— ), Holladay et al. Tetrahedron. Lett 24:4401-4404 (1983) ( — C(OH)CH2 — ); and Hruby Life Sci 31:189-199 (1982) ( — CH2 — S — ), each of which is incorporated herein by reference. A particularly preferred non-peptide linkage is — CH2NH —. It is understood that peptide analogs can have more than one atom between the bond atoms, such as b-alanine, g-aminobutyric acid, and the like.

[0368] Additional Peptides

[0369] In some embodiments, the recombinant polypeptide further comprises a cell penetrating peptide. The term "cell penetrating peptide" as used herein refers to short peptides that facilitate the transport of molecular cargo across plasma membranes found in a cell. In some embodiments, the cell penetrating peptides are conjugated to the recombinant polypeptide via any number of means, including covalent bonds and / or non-covalent bonds. In a number of instances, however, such cell penetrating peptides will often include a relatively high concentration of positively-charged amino acids, such as lysine and arginine. In some embodiments, the cell penetrating peptide has a sequence containing an alternating pattern of charged (polar) and non-charged amino acids.

[0370] In some embodiments, the recombinant polypeptide further comprises the transactivating transcriptional activator (TAT) cell penetrating peptide. In some embodiments, the TAT cell penetrating peptide comprises the sequence of SEQ ID NO: 20, or an amino acid sequence at least about 70% identical thereto, for example at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto.

[0371] Another leader sequence includes that of the sequence derived from penetratin (AP) comprising the sequence of RQIKIWFQNRRMKWKK (SEQ ID NO: 62), or an amino acid sequence at least about 70% identical thereto, for example at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at leastDocket No. 103362-094WO1

[0372] about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the recombinant polypeptide further comprises an amino acid sequence encoding the N-terminal poly-lysine domain of the al subunit of Na / K-ATPase (AIN), which is represented by the sequence of KKGKKGKK (SEQ ID NO: 63), or an amino acid sequence at least about 70% identical thereto, for example at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. Those of skill will appreciate though that other leader sequences, including other cell penetrating peptides, can also be used in conjunction with the presently-disclosed recombinant polypeptides.

[0373] In certain embodiments of the present invention, a TAT cell penetrating peptide is linked either directly or via a linker to the peptides of the present invention.

[0374] In some embodiments, the recombinant polypeptide further comprises a hemagglutinin (HA) tag. In some embodiments, the HA tag comprises the amino acid sequence of YPYDVPDYA (SEQ ID NO: 18), or an amino acid sequence at least about 70% identical thereto, for example at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the HA tag is conjugated to the C-terminus of the therapeutic peptide. In some embodiments, the HA tag is encoded by the nucleic acid sequence of SEQ ID NO: 15, or a nucleic acid sequence at least about 70% identical thereto, for example at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto.

[0375] Gene Transfer of Transgenes Encoding

[0376]

[0377] In another aspect, provided herein is an expression vector comprising a nucleic acid encoding for a recombinant polypeptide as described herein. In some embodiments, the expression vector is a non-viral vector. In some embodiments, the non-viral expression vector is selected from a DNA plasmid, a miniplasmid, an exosome, a liposome, or a lipid nanoparticle.

[0378] In some embodiments, the expression vector is a viral vector. In some embodiments, the viral expression vector is selected from a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector (AAV). In some embodiments, the viral expression vector is an AAV.

[0379] In another aspect, provided herein is an adeno-associated viral vector (AAV) comprising a nucleic acid encoding for a recombinant polypeptide as described herein. In someDocket No. 103362-094WO1

[0380] embodiments, the AAV is recombinant and / or engineered. In some embodiments, the AAV has decreased tropism towards liver. In some embodiments, the AAV has enhanced tropism towards adipose tissue. In some embodiments, the AAV is a V7 AAV capsid vector. In some embodiments, the AAV has enhanced tropism towards neural tissue. In some embodiments, the AAV is a Rec2 AAV capsid vector.

[0381] As used herein, plasmid or viral vectors transport a nucleic acid encoding the recombinant polypeptide as described herein into the cell without degradation and include a promoter yielding expression of the nucleic acid in the cells into which it is delivered. Viral vectors include, for example, but are not limited to Adenovirus, Adeno-associated virus, Herpes virus, Vaccinia virus, Polio virus, AIDS virus, neuronal trophic virus, Sindbis and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors.

[0382] Retroviruses include Murine Maloney Leukemia virus, MMLV, and retroviruses that express the desirable properties of MMLV as a vector. Retroviral vectors are able to carry a larger genetic payload, i.e., a transgene or marker gene, than other viral vectors, and for this reason are a commonly used vector. However, they are not as useful in non-proliferating cells.

[0383] Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation, and can transfect non-dividing cells. Pox viral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature.

[0384] Viral vectors can have higher transaction (ability to introduce genes) abilities than chemical or physical methods to introduce genes into cells. Typically, viral vectors contain, nonstructural early genes, structural late genes, an RNA polymerase III transcript, inverted terminal repeats necessary for replication and encapsidation, and promoters to control the transcription and replication of the viral genome. When engineered as vectors, viruses typically have one or more of the early genes removed and a gene or gene / promotor cassette is inserted into the viral genome in place of the removed viral DNA. Constructs of this type can carry up to about 8 kb of foreign genetic material. The necessary functions of the removed early genes are typically supplied by cell lines which have been engineered to express the gene products of the early genes in trans.Docket No. 103362-094WO1

[0385] Adeno-associated virus (AAV)

[0386] Another type of viral vector is based on an adeno-associated virus (AAV). This defective parvovirus is a preferred vector because it can infect many cell types and is nonpathogenic to humans. AAV type vectors can transport about 4 to 5 kb and wild type AAV is known to stably insert into chromosome 19. Vectors which contain this site specific integration property are preferred. In some embodiments, this type of vector is the P4.1 C vector produced by Avigen, San Francisco, CA, which can contain the herpes simplex virus thymidine kinase gene, HSV-tk, and / or a marker gene, such as the gene encoding the green fluorescent protein, GFP.

[0387] In another type of AAV virus, the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell-specific expression operably linked to a heterologous gene. Heterologous in this context refers to any nucleotide sequence or gene which is not native to the AAV or B 19 parvovirus.

[0388] Typically the AAV and B19 coding regions have been deleted, resulting in a safe, noncytotoxic vector. The AAV ITRs, or modifications thereof, confer infectivity and sitespecific integration, but not cytotoxicity, and the promoter directs cell-specific expression. United States Patent No. 6,261,834 is herein incorporated by reference for material related to the AAV vector.

[0389] In one aspect, an engineered hybrid serotype Rec2 vector comprising a transgene encoding a recombinant polypeptide as described herein leads to high transduction of adipose tissue superior to naturally occurring serotypes (e.g., AAV1 and AAV8) and other engineered serotypes (e.g., Reel, Rec3, Rec4). The Rec2 AAV vector is described in United States Patent Publication No. 20230330267 Al, incorporated herein by reference herein for all purposes. The administration route substantially influences the tropism and efficacy of Rec2 vector. In some embodiments, intravenous administration of Rec2 vector comprising a transgene encoding a recombinant polypeptide as described herein primarily transduces liver. In some embodiments, oral administration of Rec2 vector comprising a transgene encoding a recombinant polypeptide as described herein leads to preferential transduction of brown fat with absence of transduction in the gastrointestinal track. In some embodiments, administration of a Rec2 vector comprising a transgene encoding a recombinant polypeptide as described herein leads to preferential transduction of visceral adipose tissue in a subject in need thereof.

[0390] In another aspect, provided herein an engineered variant Rec2 capsid vector comprising a transgene encoding a recombinant polypeptide as described herein leads to high transduction of adipose tissue. In some embodiments, the engineered variant Rec2 capsid vector has one orDocket No. 103362-094WO1

[0391] more amino acid substitutions of the capsid that occur at residues that promote hepatic intracellular AAV degradation, ablate liver tropism, and / or disrupt AAV binding to hepatocytes. In some embodiments, the engineered variant Rec2 capsid vector is Rec2 variant V7. Methods of making and using the engineered Rec2 variant V7 capsid vector are disclosed in, for example, Huang W, Bates R, Appana B, Mohammed T, Cao L. Development of an adiposetropic AAV capsid ablating liver tropism. iScience. 2024 Sep 17;27(10):110930, incorporated herein by reference.

[0392] To prevent transgene expression in an off-target tissue, such as the liver, an AAV expression plasmid containing two expression cassettes: one comprising the transgene encoding a recombinant polypeptide as described herein (for example, a nucleic acid encoding Exendin-4), and the other cassette uses a liver-specific promoter (for example, an albumin promoter) to drive a specific RNA silencing element (such as, for example, microRNA) targeting a regulatory element (such as, for example, WPRE) sequence which only exists in the transgene expression cassette.

[0393] The inserted genes in viral and retroviral usually contain promoters, and / or enhancers to help control the expression of the desired gene product (for example, a chicken beta-actin (CBA) promoter). A promoter is generally a sequence or sequences of DNAthat function when in a relatively fixed location in regard to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.

[0394] Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. a beta-actin promoter, such as, for example, the chicken beta-actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers etal., Nature, 273: 113 (1978)). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment (Greenway, PJ. et al., Gene 18: 355360 (1982)). Of course, promoters from the host cell or related species also are useful herein. Thus, in one aspect, disclosed herein are engineered adeno-associated virus (AAV) vector comprising two expression cassettes; wherein the first cassette comprises a regulatory element and a transgene operatively linked to a promoter (such as, for example, the chicken beta-actin promoter); and wherein the second cassette comprisesDocket No. 103362-094WO1

[0395] a liver-specific albumin promoter and microRNAthat targets the regulatory element in the first expression cassette.

[0396] Enhancer generally refers to a sequence of DNAthat functions at no fixed distance from the transcription start site and can be either 5' (Laimins, L. etal., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3' (Lusky, M. L., et al., Mol. Cell Bio. 3: 1108 (1983)) to the transcription unit. Furthermore, enhancers can be within an intron (Banerji, J. L. et al., Cell 33: 729 (1983)) as well as within the coding sequence itself (Osborne, T. F., et al., Mol. Cell Bio. 4: 1293 (1984)). They are usually between 10 and 300 bp in length, and they function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100 270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0397] The promotor and / or enhancer may be specifically activated either by light or specific chemical events which trigger their function. Systems can be regulated by reagents such as tetracycline and dexamethasone. There are also ways to enhance viral vector gene expression by exposure to irradiation, such as gamma irradiation, or alkylating chemotherapy drugs.

[0398] In certain embodiments the promoter and / or enhancer region can act as a constitutive promoter and / or enhancer to maximize expression of the region of the transcription unit to be transcribed. In certain constructs the promoter and / or enhancer region be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time. A preferred promoter of this type is the CMV promoter (650 bases). In some embodiments, the promoter is selected from an SV40 promoter, a cytomegalovirus promoter, a CMV early enhancer / chicken beta actin (CAG) promoter, or a retroviral vector LTR.

[0399] It has been shown that all specific regulatory elements can be cloned and used to construct expression vectors that are selectively expressed in specific cell types. For example, the glial fibrillary acetic protein (GFAP) promoter has been used to selectively express genes in cells of glial origin. In some embodiments, an adipose tissue-specific promoter is used to drive expression in adipose tissue and is selected from an aP2 promoter, an adiponectin promoter, or an hAGPAT2 promoter. In some embodiments, a neural tissue-specific promoterDocket No. 103362-094WO1

[0400] is used to drive expression in adipose tissue and is selected from a synapsin promoter, an neuron-specific enolase (NSE) promoter, a CBh promoter, an MeCP2 promoter, or a Thyl promoter. In some embodiments, a heart tissue-specific promoter is used to drive expression in heart tissue (e.g., cardiomyocytes) and is selected from an alpha-Myosin Heavy chain (alpha-MHC) promoter, a myosin light chain 2 (MLC2v) promoter, or a cardiact troponin T (cTnT) promoter. In another aspect, a liver-specific promoter such as an albumin promoter, a thyroxine binding globin promoter, a phosphoenolpyruvatecarboxykinase (PEPCK) promoter, a tyrosine aminotransgerase (TAT) promoter, or a human alpha 1 -antitrypsin (hAAT) promoter can be used to drive expression in the liver. In some embodiments, the tissue-specific promoter is used to drive expression of a transgene encoding a recombinant polypeptide as described herein.

[0401] In some embodiments, the second cassette of an engineered AAV comprises a tissue specific promoter operatively linked to an RNA silencing element, expression of an RNA silencing element (such as, for example microRNA) which targets the regulatory element in the first cassette can be used to limit off-target transduction of the transgene (for example Exendin-4 and / or GLPl(7-37)) encoding a recombinant polypeptide as described herein. For example, provided herein is an engineered adeno-associated virus (AAV) vectors comprising two expression cassettes; wherein the first cassette comprises a regulatory element and a transgene (such as, for example, Exendin-4 and / or GLPl(7-37)) operatively linked to a promoter (such as, for example CBA); and wherein the second cassette comprises a tissue specific promoter, for example, a liver specific promoter (such as, for example, albumin or hAAT) and a RNA silencing element (such as, for example, microRNA) that targets the regulatory element in the first expression cassette. It is understood and herein contemplated that the tissue-specific promoter such as the liver specific promoter in the second cassette is operatively linked to the RNA silencing element such that transcription of the RNA silencing element only occurs when in the appropriate tissue for the tissue specific promoter. Thus, as noted above, the disclosed engineered AAV can restrict off-target transduction of a transgene in the liver.

[0402] Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells) may also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated regions also include transcription termination sites. It is preferred that the transcription unit also contains a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported likeDocket No. 103362-094WO1

[0403] mRNA. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that homologous polyadenylation signals be used in the transgene constructs. In certain transcription units, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the transcribed units contain other standard sequences alone or in combination with the above sequences improve expression from, or stability of, the construct.

[0404] The viral vectors can include nucleic acid sequence encoding a marker product. This marker product is used to determine if the gene has been delivered to the cell and once delivered is being expressed. Preferred marker genes are the E. Coli lacZ gene, which encodes B galactosidase, and green fluorescent protein.

[0405] In some embodiments the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure.

[0406] The second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, (Southern P. and Berg, P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan, R. C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B. et al., Mol. Cell. Biol. 5: 410413 (1985)). The three examples employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and puramycin.

[0407] In an alternative aspect, provided herein is a pharmaceutical composition comprising at least one recombinant polypeptide as described herein, at least one expression vector as described herein, or at least one AAV as described herein, and a pharmaceutically acceptable carrier.

[0408] The recombinant polypeptide compositions, or expression vectors encoding thereof, as described herein can be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious mannerDocket No. 103362-094WO1

[0409] with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

[0410] The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant. As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector. Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation. The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

[0411] Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U. S. Patent No. 3,610,795, which is incorporated by reference herein.

[0412] In some embodiments, an AAV vector comprising a transgene encoding a recombinant polypeptide as described herein is administered to a neural tissue wherein the AAV vector is administered systemically (such as, for example, intravenously, including but not limited to, i.v. injection or i.v. drip; and / or retro-orbitally); or via cerebrospinal fluid injection.

[0413] In some embodiments, the AAV vector comprising a transgene encoding a recombinant polypeptide as described herein are administered once every year, once every two years, once every three years, once every 4 years, once every 5 years, once every 6 years, once every 7 years, once every 8 years, once every 9 years, or once every 10 years.

[0414] In some embodiments, the AAV is formulated in a dose of between about IxlO10vg / kg to about IxlO12vg / kg, for example between about IxlO11vg / kg to about IxlO12vg / kg, betweenDocket No. 103362-094WO1

[0415] about IxlO10vg / kg to about IxlO11vg / kg, between about 5xlO10vg / kg to about 5x1011vg / kg, between about 2.5xlO10vg / kg to about 9xlOnvg / kg, between about 2.5xlO10vg / kg to about 7.5xlO10vg / kg, between about IxlO11vg / kg to about 9xlOnvg / kg, between about 2xlOnvg / kg to about 8x1011vg / kg, between about 2x1010vg / kg to about 8x1010vg / kg, or between about 6xlO10vg / kg to about 9xlOnvg / kg. In some embodiments, the AAV is formulated in a dose of about 5x1011vg / kg.

[0416] The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K. D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)}. In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991).

[0417] The compositions can be used therapeutically in combination with a pharmaceutically acceptable carrier.Docket No. 103362-094WO1

[0418] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

[0419] Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0420] Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.

[0421] The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.

[0422] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also beDocket No. 103362-094WO1

[0423] present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0424] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0425] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.

[0426] Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.

[0427] Methods of Treatment

[0428] In one aspect, the presently-disclosed subject matter includes methods for treating disorders, diseases, or cancers, including but not limited to obesity or a brain disorder, with a recombinant polypeptide, or an expression vector encoding thereof, including but not limited to an AAV, as described herein, or a pharmaceutical composition as disclosed herein.

[0429] The disclosed recombinant polypeptides as described herein, or vectors encoding thereof (e.g., AAV vectors), can be used to rescue deficiencies of gene / protein expression that lead to a disease.

[0430] In one aspect, the recombinant polypeptides as described herein, or vectors encoding thereof (e.g., AAV vectors), can be used to treat obesity, diabetes, lipodystrophy (such as, for example, congenital generalized lipodystrophy (also known as Beradinelli-Seip syndrome), familial partial lipodystrophy, acquired partial lipodystrophy (also known as Barraquer-Simons syndrome), acquired generalized lipodystrophy, centrifugal abdominal lipodystrophy, lipoatrophia annularis, localized lipodystrophy, and HIV-associated lipodystrophy), or metabolic syndromes.

[0431] Obesity and body fat distribution are important risk factors for type II diabetes, lipodystrophy, and other metabolic syndromes. Visceral adipose tissue (VAT) plays distinctiveDocket No. 103362-094WO1

[0432] roles in metabolic homeostasis and disturbance. The type of obesity characterized by increased VAT is strongly associated with adverse metabolic outcomes whereas accumulation of subcutaneous fat is thought to have neutral or even beneficial effects on metabolism. This relates to intrinsic functional differences of adipocytes in different depots, including insulin sensitivity, glucose uptake, rate of lipolysis, and adipokine and cytokine secretion. Thus, a gene delivery tool to deliver a recombinant polypeptide which targets visceral fat has great potential for applications in gene therapy.

[0433] In another aspect, provided herein is a method for gene transfer of a nonnative, recombinant polypeptide to adipose tissue in a subject in need thereof, comprising administering a therapeutically effective amount of an expression vector as described herein. In some embodiments, the expression vector is a non-viral vector. In some embodiments, the non-viral expression vector is selected from a DNA plasmid, a miniplasmid, an exosome, a liposome, or a lipid nanoparticle. In some embodiments, the expression vector is an AAV. In some embodiments, the AAV is administered by intraperitoneal or intravenous injection.

[0434] In another aspect, provided herein is a method for gene transfer of a nonnative, recombinant polypeptide to brain tissue in a subject in need thereof, comprising administering a therapeutically effective amount of an expression vector as described herein. In some embodiments, the expression vector is a non-viral vector. In some embodiments, the non-viral expression vector is selected from a DNA plasmid, a miniplasmid, an exosome, a liposome, or a lipid nanoparticle. In some embodiments, the expression vector is comprised in a lipid nanoparticle. In some embodiments, the expression vector is an AAV. In some embodiments, the AAV is administered by intracranial, intraparenchymal, or intravenous injection.

[0435] As used herein a “nanoparticle” or “lipid nanoparticle” is any composition comprising one or more materials that can be formulated into nanoparticles that are capable of encapsulating or otherwise retaining (e.g., by adsorption or otherwise) the first and the one or more additional nucleic acid sequences. The nanoparticle carrier may be formulated out of any suitable material, including, but not limited to a polymer-lipid, a lipid (to form a lipid nanoparticle (LNP)), or gold. In certain embodiments, the nanoparticle carrier is a lipid.

[0436] In some embodiments, the expression vector is a viral vector. In some embodiments, the viral expression vector is selected from a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector (AAV). In some embodiments, the viral expression vector is an AAV. In some embodiments, the AAV is administered by intraperitoneal or intravenous injection. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from Exendin-4. In some embodiments, the recombinant polypeptide comprises aDocket No. 103362-094WO1

[0437] therapeutic peptide that is derived from a GLP-1 peptide or a GLP-1 receptor agonist peptide. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BDNF. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BMP7.

[0438] In another aspect, provided herein is a method for reducing blood glucose in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from Exendin-4. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from a GLP-1 peptide or a GLP-1 receptor agonist peptide. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BDNF. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BMP7.

[0439] In another aspect, provided herein is a method for increasing blood glucose tolerance in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein. In some embodiments, the subject is obese. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from Exendin-4. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from a GLP-1 peptide or a GLP-1 receptor agonist peptide. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BDNF. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BMP7.

[0440] In another aspect, provided herein is a method for reducing expression of proinflammatory genes in one or more tissues in a subject receiving an AAV therapy, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein. In some embodiments, the one or more tissues are selected from liver, hypothalamus, brown adipose tissue, white adipose tissue, or a combination thereof.

[0441] In another aspect, provided herein is a method for treating a subject having a metabolic disorder, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as describedDocket No. 103362-094WO1

[0442] herein, or a pharmaceutical composition as described herein. In some embodiments, the metabolic disorder is selected from diabetes, obesity, chronic inflammation, or sleep apnea. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from Exendin-4. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from a GLP-1 peptide or a GLP-1 receptor agonist peptide. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BDNF. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BMP7.

[0443] In another aspect, provided herein is a method for treating a subject having diabetes, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from Exendin-4. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from a GLP-1 peptide or a GLP-1 receptor agonist peptide. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BDNF. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BMP7.

[0444] In another aspect, provided herein is a method for treating a subject having obesity, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from Exendin-4. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from a GLP-1 peptide or a GLP-1 receptor agonist peptide. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BDNF. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BMP7.

[0445] In another aspect, provided herein is a method for reducing one or more metabolic disorders in a subject having a body mass index (BMI) of equal to or greater than about 27.5, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein.Docket No. 103362-094WO1

[0446] In another aspect, provided herein is a method for reducing one or more metabolic disorders in a subject having a body mass index (BMI) of equal to or greater than about 30, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein.

[0447] In another aspect, provided herein is a method for reducing one or more metabolic disorders in a subject having a body mass index (BMI) of equal to or greater than about 40, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein.

[0448] In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from Exendin-4. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from a GLP-1 peptide or a GLP-1 receptor agonist peptide. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BDNF. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BMP7.

[0449] In some embodiments, the one or more metabolic disorders are selected from obesity, diabetes, lipodystrophy, congenital generalized lipodystrophy (Beradinelli-Seip syndrome), familial partial lipodystrophy, acquired partial lipodystrophy (Barraquer-Simons syndrome), acquired generalized lipodystrophy, centrifugal abdominal lipodystrophy, lipoatrophia annularis, localized lipodystrophy, or HIV-associated lipodystrophy.

[0450] In another aspect, provided herein is a method for reducing weight of a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from Exendin-4. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from a GLP-1 peptide or a GLP-1 receptor agonist peptide. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BDNF. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BMP7.

[0451] In some embodiments, the subject, prior to being administered the recombinant polypeptide or expression vector encoding thereof, had a BMI of equal to or greater than about 27.5, equal to or greater than about 30, equal to or greater than about 32.5, equal to or greater than about 37.5, equal to or greater than about 40, or more than about 40.Docket No. 103362-094WO1

[0452] In some embodiments, the subject experiences a weight loss of equal to or greater than about 5% of the baseline weight of the subject prior to being administered the recombinant polypeptide or expression vector encoding thereof. In some embodiments, the subject experiences a weight loss of equal to or greater than about 10% of the baseline weight of the subject prior to being administered the recombinant polypeptide or expression vector encoding thereof. In some embodiments, the subject experiences a weight loss of equal to or greater than about 20% of the baseline weight of the subject prior to being administered the recombinant polypeptide or expression vector encoding thereof. In some embodiments, the subject experiences a weight loss of equal to or greater than about 30% of the baseline weight of the subject prior to being administered the recombinant polypeptide or expression vector encoding thereof. In some embodiments, the subject has a reduced body fat percentage following being administered the recombinant polypeptide or expression vector encoding thereof. In some embodiments, the subject has an increased relative lean mass calibrated to body weight following being administered the recombinant polypeptide or expression vector encoding thereof.

[0453] In one aspect, provided herein is a method for treating Prader-Willi syndrome in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from Exendin-4. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from a GLP-1 peptide or a GLP-1 receptor agonist peptide. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BDNF. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BMP7.

[0454] In one aspect, provided herein is a method for treating Prader-Willi syndrome in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide comprising (a) a leptin signal peptide sequence; (b) a linker peptide comprising a valine residue; and (c) an exendin-4 peptide, or an expression vector or AAV encoding thereof. In some embodiments, the exendin-4 peptide comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid at least about 95% identical thereto. In some embodiments, the exendin-4 peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 3, or a nucleic acid sequence at least about 95% identical thereto. In some embodiments, the recombinant polypeptide comprises the amino acid sequence of SEQ ID NO:Docket No. 103362-094WO1

[0455] 4, or an amino acid sequence at least about 95% identical thereto. In some embodiments, the recombinant polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1, or a nucleic acid sequence at least about 95% identical thereto. In some embodiments, the recombinant polypeptide further comprises one or more additional peptides. In some embodiments, the one or more additional peptides comprise a TAT cell-penetrating peptide and / or an HA tag peptide. In some embodiments, the recombinant polypeptide comprises the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence at least about 95% identical thereto.

[0456] In one aspect, provided herein is a method for treating a subject having lipodystrophy, comprising administering a therapeutically effective amount of a recombinant polypeptide as disclosed herein, an expression vector as disclosed herein, an AAV as disclosed herein, or a pharmaceutical composition as disclosed herein.

[0457] Accordingly, in one aspect, disclosed herein are methods of treating obesity, diabetes, cancer, lipodystrophy, and / or metabolic syndromes in a subject comprising administering to the subject a recombinant polypeptide or an engineered adeno-associated virus (AAV) vector encoding thereof. In some embodiments, the AAV vector comprises two expression cassettes; wherein the first cassette comprises a regulatory element (such as, for example WPRE) and a therapeutic transgene operatively linked to a promoter (such as a CB A promoter); and wherein the second cassette comprises a tissue specific promoter (such as the liver specific albumin promoter) and a RNA silencing element (such as, for example, microRNA) that targets the regulatory element in the first expression cassette; wherein the RNA silencing element is operatively linked to the liver specific promoter.

[0458] In some embodiments, the AAV is formulated in a dose of between about IxlO10vg / kg to about IxlO12vg / kg, for example between about IxlO11vg / kg to about IxlO12vg / kg, between about IxlO10vg / kg to about IxlO11vg / kg, between about 5xlO10vg / kg to about 5x1011vg / kg, between about 2.5xlO10vg / kg to about 9xlOnvg / kg, between about 2.5xlO10vg / kg to about 7.5xlO10vg / kg, between about IxlO11vg / kg to about 9xlOnvg / kg, between about 2xlOnvg / kg to about 8x1011vg / kg, between about 2x1010vg / kg to about 8x1010vg / kg, or between about 6xlO10vg / kg to about 9xlOnvg / kg. In some embodiments, the AAV is formulated in a dose of about 5x1011vg / kg.

[0459] Also disclosed herein are methods of targeting a nucleic acid encoding a recombinant polypeptide as described herein to visceral adipose tissue in a subject comprising administering to the subject an engineered adeno-associated virus (AAV) vector comprising two expression cassettes; wherein the first cassette comprises a regulatory element and a therapeuticDocket No. 103362-094WO1

[0460] polypeptide-encoding transgene operatively linked to a promoter; and wherein the second cassette comprises a liver specific promoter (such as, for example, albumin) and a RNA silencing element (such as, for example, microRNA) that targets the regulatory element in the first expression cassette; wherein the RNA silencing element is operatively linked to the liver specific promoter; and wherein the AAV vector is administered intraperitoneally.

[0461] Also disclosed herein are methods of treating, inhibiting, decreasing, reducing, ameliorating, and / or preventing a neurodevelopmental disorder (such as, for example, Prader-Willi syndrome) in a subject comprising administering to the subject the recombinant polypeptide, or an expression vector encoding thereof (e.g., an engineered AAV vector). In some embodiments, the recombinant polypeptide comprises a therapeutic peptide derived from P-Galactosidase 1 (GLB1), Niemann-Pick Cl (NPC1), Apolipoprotein E (APOE), GD3 synthase, huntingtin (Htt), interleukin (IL)- 10 (IL- 10), Myelin Oligodendrocyte Glycoprotein (MOG), mitogen-activated protein kinase 8 interacting protein 3 (MAPKA8IP3), survival motor neuron (SMN) 1 (SMN1), SMN2, Cas9, P-Glucocerebrosidase (GBA), Sphingomyelin phosphodiesterase 1 (SMPD1), beta-hexosaminidase A (HEXA), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin (NT) 3 (NT-3), NT-4 / 5, NT-6, Glial Cell Derived Neurotrophic Factor (GDNF), Ciliary Neurotrophic Factor (CNTF), Leukemia inhibitory factor (LIF), Insulin-like growth factor (IGF) 1 (IGF-1), P-fibroblast growth factor (FGF), neurturin, persephin, artemin, transforming growth factor (TGF) alpha (TGFa), TGFP, IGF-2, platelet derived growth factor (PDGF), epidermal growth factor (EGF), cardiotropin, vascular endothelial growth factor (VEGF), Sonic hedgehog (SHH), bone morphogenic proteins (BMP), FGF20, Vasoactive Intestinal Peptide (VIP), pleiotrophin (PTN), Aromatic L-amino Acid Decarboxylase (AADC), TH, 5-hydroxytryptamine (5HT), hepatocyte growth factor (HGF). In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from Exendin-4. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from a GLP-1 peptide or a GLP-1 receptor agonist peptide. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BDNF. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BMP7.

[0462] In some embodiments, the neurodevelopmental disorder is selected from Fragile-X Syndrome, X-Linked Intellectual Disability-Hypotonia-Facial Dysmorphism- Aggressive Behaviour Syndrome, Kleefstra Syndrome, Hunters Syndrome (MPS II), ADNP (activity dependent neuroprotector homeobox) syndrome, Rett syndrome, Autism Spectrum Disorders, Prader-Willi Syndrome, Angelman Syndrome, Brunner syndrome, Cri du Chat syndrome,Docket No. 103362-094WO1

[0463] Cornelia de Lange syndrome, Smith-Lemli-Opitz syndrome, Smith-Magenis syndrome, Tuberous Sclerosis Complex, CHARGE syndrome, sotos syndrome, attention-deficit / hyperactivity disorder (ADHD), Cerebral Palsy, Down Syndrome, or PTEN associated disorder.

[0464] Also disclosed herein are methods of treating, inhibiting, decreasing, reducing, ameliorating, and / or preventing a neurological disease or neurodegenerative disease (such as, for example, Alzheimer’s disease, Parkinson’s disease, Multiple Systems Atrophy (MSA), Lysosomal Storage Disease (LSD), and / or muscular dystrophy) in a subject comprising administering to the subject the recombinant polypeptide, or an expression vector encoding thereof (e.g., an engineered AAV vector). In some embodiments, the recombinant polypeptide comprises a therapeutic peptide derived from P-Galactosidase 1 (GLB1), Niemann-Pick Cl (NPC1), Apolipoprotein E (APOE), GD3 synthase, huntingtin (Htt), interleukin (IL)- 10 (IL-10), Myelin Oligodendrocyte Glycoprotein (MOG), mitogen-activated protein kinase 8 interacting protein 3 (MAPKA8IP3), survival motor neuron (SMN) 1 (SMN1), SMN2, Cas9, P-Glucocerebrosidase (GBA), Sphingomyelin phosphodiesterase 1 (SMPD1), betahexosaminidase A (HEXA), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin (NT) 3 (NT-3), NT-4 / 5, NT-6, Glial Cell Derived Neurotrophic Factor (GDNF), Ciliary Neurotrophic Factor (CNTF), Leukemia inhibitory factor (LIF), Insulin-like growth factor (IGF) 1 (IGF-1), P-fibroblast growth factor (FGF), neurturin, persephin, artemin, transforming growth factor (TGF) alpha (TGFa), TGFP, IGF-2, platelet derived growth factor (PDGF), epidermal growth factor (EGF), cardiotropin, vascular endothelial growth factor (VEGF), Sonic hedgehog (SHH), bone morphogenic proteins (BMP), FGF20, Vasoactive Intestinal Peptide (VIP), pleiotrophin (PTN), Aromatic L-amino Acid Decarboxylase (AADC), TH, 5-hydroxytryptamine (5HT), hepatocyte growth factor (HGF). In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from Exendin-4. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from a GLP-1 peptide or a GLP-1 receptor agonist peptide. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BDNF. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BMP7.

[0465] In some embodiments, the neurodegenerative disease is selected from Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia 2 (SCA2), Parkinson’s disease, chronic traumatic encephalopathy (CTE), multiple sclerosis, depression, epilepsy, spinocerebellar ataxia type 1, Machado- Joseph Disease, Down's syndrome, DementiaDocket No. 103362-094WO1

[0466] puglistica, Pick's disease, progressive supranuclear palsy (PSP), Guam parkinsonism dementia complex, Fronto-temporal dementia (FTD), Cortico-Basal Degeneration (CBD), Pallido-Pontal-Nigral Degeneration, Progressive Nuclear Palsy (PNP), Parkinsonism of Chromosome 17 (FTDP-17), Dementia with Lewy bodies, Huntington's disease, Multiple System Atrophy, fatty liver disease (liver steatosis), al-anti-trypsin deficiency, muscle diseases, sporadic inclusion body myositis, limb girdle muscular dystrophy type 2B, prion disease, Creutzfeldt-Jakob disease, or Miyoshi myopathy.

[0467] In another aspect, provided herein is a method for treating Alzheimer’s disease in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from Exendin-4. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from a GLP-1 peptide or a GLP-1 receptor agonist peptide. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BDNF. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BMP7.

[0468] In another aspect, provided herein is a method for improving cellular plasticity in a neurological disorder in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from Exendin-4. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from a GLP-1 peptide or a GLP-1 receptor agonist peptide. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BDNF. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BMP7.

[0469] In another aspect, provided herein is a method for treating major depression in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from Exendin-4. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from a GLP-1 peptide or a GLP-1 receptor agonist peptide. In some embodiments, theDocket No. 103362-094WO1

[0470] recombinant polypeptide comprises a therapeutic peptide that is derived from BDNF. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BMP7.

[0471] In another aspect, provided herein is a method for treating osteoporosis in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from Exendin-4. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from a GLP-1 peptide or a GLP-1 receptor agonist peptide. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BDNF. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BMP7.

[0472] In another aspect, provided herein is a method for enhancing dental regeneration in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from Exendin-4. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from a GLP-1 peptide or a GLP-1 receptor agonist peptide. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BDNF. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BMP7.

[0473] In another aspect, provided herein is a method for treating an inflammatory disease in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from Exendin-4. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from a GLP-1 peptide or a GLP-1 receptor agonist peptide. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BDNF. In some embodiments, the recombinant polypeptide comprises a therapeutic peptide that is derived from BMP7. In some embodiments, the inflammatory disease is selected from cardiovascular disease, diabetes, or obesity.Docket No. 103362-094WO1

[0474] In another aspect, provided herein is a method for treating cancer, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein. In some embodiments, the cancer is selected from lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin’s Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, lung cancers such as small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, colon cancer, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon cancer, and / or rectal cancer) in a subject by administering an AAV vector encoding a recombinant polypeptide (for example an exendin-4, IL-15, or immune checkpoint inhibitor peptide) as described herein. In some embodiments, the cancer is selected from adenocarcinoma of the esophagus, breast cancer, colon cancer, rectum cancer, uterine cancer, gallbladder cancer, upper stomach cancer, kidney cancer, liver cancer, ovary cancer, pancreatic cancer, thyroid cancer, meningioma, or multiple myeloma. In some embodiments, the cancer comprises obesity-associated cancer. In some embodiments, the obesity-associated cancer comprises obesity-associated colon cancer.

[0475] In one aspect, provided herein is a method for treating obesity-associated colon cancer in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide as described herein, an expression vector as described herein, an AAV as described herein, or a pharmaceutical composition as described herein. In some embodiments, the recombinant polypeptide comprises exendin-4.

[0476] There are a number of compositions and methods which can be used to deliver nucleic acids to cells, either in vitro or in vivo. These methods and compositions can largely be broken down into viral- and non-viral based delivery systems. For example, the recombinant polypeptides as described herein can be encoded and or delivered via a number of vectors and / or direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes.Docket No. 103362-094WO1

[0477] EMBODIMENTS

[0478] 1. A recombinant polypeptide comprising:

[0479] (a) a signal peptide sequence;

[0480] (b) a linker peptide comprising between 1 to 30 amino acid residues; and

[0481] (c) a therapeutic peptide, wherein the therapeutic peptide comprises a peptide hormone, a GLP1 receptor agonist peptide, a glucagon-like peptide, a biomimetic peptide, an immunomodulatory peptide, a neurotoxic peptide, a neurotrophic factor peptide, a bone morphogenetic peptide, a guanylate cyclase- C (G-CC) agonist peptide, a calcitonin receptor inhibitor, a GnRH receptor inhibitor, a 20S proteasome inhibitor, a NOD2 inhibitor, a VIP1 receptor inhibitor, an OT inhibitor, a TRH receptor inhibitor, an MC receptor inhibitor, a PTH1 receptor inhibitor, a guanylate cyclase C inhibitor, an NPR-A inhibitor, an ATI receptor inhibitor, a beta2 -receptor inhibitor, a gp41 inhibitor, a GHRH receptor inhibitor, an N-type calcium channel inhibitor, a thrombopoietin receptor inhibitor, a human erythropoietin receptor inhibitor, a pulmonary surfactant inhibitor, a CaSR inhibitor, an MCI receptor inhibitor, a somatostatin receptor inhibitor, a melanocortin-4 receptor inhibitor, or an immune checkpoint inhibitor peptide, or an analog thereof.

[0482] 2. The recombinant polypeptide of embodiment 1, wherein the signal peptide is derived from GLP-1, PDX-1, GIP, GLP-2, insulin, growth hormone, leptin, prolactin, tissue plasminogen activator (tPA), calcitonin, luteinising hormone, parathyroid hormone, somatostatin, thyroid stimulating hormone, vasoactive intestinal polypeptide, trefoil factors, cell and tissue repair factors, transforming growth factor P, keratinocyte growth factor, a structural group 1 cytokine adopting an antiparallel 4a helical bundle structure, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL- 9, IL-10, IL-11, IL-12, IL-13, GM-CSF, M-CSF, SCF, IFN-y, EPO, G-CSF, LIF, OSM, CNTF, GH, PRL, IFNa / p, a structural group 2 cytokine, a TNF-family cytokine, TNFa, TNFp, CD40, CD27, FAS ligands, an IL-1- family cytokine, a fibroblast growth factor, a platelet derived growth factor, transforming growth factor p, a nerve growth factor, a structural group 3 cytokine comprising a short chain a / P molecule, an epidermal growth factor-family cytokine, a C-C or C-X-C chemokine, an insulin-related cytokine, a structural group 4 cytokine, a heregulins, a neuregulins, EGF, immunoglobulin-like domain, kringle domain, one or more fragments thereof, analogs thereof, or combinations thereof.Docket No. 103362-094WO1

[0483] 3. The recombinant polypeptide of embodiment 1 or 2, wherein the signal peptide sequence comprises a leptin signal peptide sequence.

[0484] . The recombinant polypeptide of embodiment 3, wherein the leptin signal peptide sequence comprises SEQ ID NO: 9, or an amino acid sequence at least 95% identical thereto.

[0485] 5. The recombinant polypeptide of embodiment 3 or 4, wherein the leptin signal peptide sequence is encoded by a nucleic acid sequence comprising SEQ ID NO: 2, or a nucleic acid sequence at least 95% identical thereto.

[0486] 6. The recombinant polypeptide of any one of embodiments 1-5, wherein the linker peptide is a valine residue.

[0487] 7. The recombinant polypeptide of any one of embodiments 1-5, wherein the linker peptide comprises a glycine / serine linker.

[0488] 8. The recombinant polypeptide of embodiment 7, wherein the glycine / serine linker comprises (GS)x, (GGS)x, (GGGS)x (SEQ ID NO: 46), (GGGGS)x (SEQ ID NO: 47), or (GGGGGS)x (SEQ ID NO: 48), wherein x is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0489] 9. The recombinant polypeptide of any one of embodiments 1-8, further comprising one or more additional peptides.

[0490] 10. The recombinant polypeptide of embodiment 9, wherein the one or more additional peptides comprise a TAT cell-penetrating peptide.

[0491] 11. The recombinant polypeptide of embodiment 10, wherein the TAT cell-penetrating peptide comprises SEQ ID NO: 22, or an amino acid at least 95% identical thereto. 12. The recombinant polypeptide of embodiment 10 or 11, wherein the TAT cellpenetrating peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 20, or a nucleic acid sequence at least 95% identical thereto.

[0492] 13. The recombinant polypeptide of any one of embodiments 1-8, wherein the therapeutic peptide comprises a GLP1 receptor agonist peptide.

[0493] 14. The recombinant polypeptide of any one of embodiments 1-8, wherein the therapeutic peptide comprises a neurotrophic factor peptide.

[0494] 15. The recombinant polypeptide of any one of embodiments 1-8, wherein the therapeutic peptide comprises a bone morphogenetic peptide.

[0495] 16. A recombinant polypeptide comprising:

[0496] (a) a signal peptide sequence;

[0497] (b) a linker peptide comprising between 1 to 30 amino acid residues; andDocket No. 103362-094WO1

[0498] (c) a GLP1 receptor agonist peptide.

[0499] The recombinant polypeptide of embodiment 16, wherein the GLP1 receptor agonist peptide comprises an exendin-4 peptide or a GLP-1 peptide.

[0500] A recombinant polypeptide comprising:

[0501] (a) a signal peptide sequence;

[0502] (b) a linker peptide comprising between 1 to 30 amino acid residues; and

[0503] (c) a GLP1 receptor agonist peptide; wherein the GLP1 receptor agonist peptide comprises an exendin-4 peptide.

[0504] The recombinant polypeptide of embodiment 16 or 17, wherein the exendin-4 peptide comprises SEQ ID NO: 7, or an amino acid at least 95% identical thereto.

[0505] The recombinant polypeptide of any one of embodiments 17-19, wherein the exendin-4 peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 3, or a nucleic acid sequence at least 95% identical thereto.

[0506] The recombinant polypeptide of any one of embodiments 17-20, comprising SEQ ID NO: 4, or an amino acid sequence at least 95% identical thereto.

[0507] The recombinant polypeptide of any one of embodiments 17-21, encoded by a nucleic acid sequence comprising SEQ ID NO: 1, or a nucleic acid sequence at least 95% identical thereto.

[0508] A recombinant polypeptide comprising:

[0509] (a) a signal peptide sequence;

[0510] (b) a linker peptide comprising between 1 to 30 amino acid residues; and

[0511] (c) a GLP1 receptor agonist peptide; wherein the GLP1 receptor agonist peptide comprises a GLP-1 peptide.

[0512] The recombinant polypeptide of embodiment 23, wherein the GLP-1 peptide is selected from GLP-1 (l-36amide), GLP-1 (7-36amide), or GLP-1 (7-37).

[0513] The recombinant polypeptide of embodiment 23 or 24, wherein the GLP-1 peptide is GLP-1 (7-36amide).

[0514] The recombinant polypeptide of any one of embodiments 23-25, wherein the GLP-1 peptide comprises SEQ ID NO: 12, or an amino acid sequence at least 95% identical thereto.

[0515] The recombinant polypeptide of any one of embodiments 23-26, wherein the GLP-1 peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 10, or a nucleic acid sequence at least 95% identical thereto.Docket No. 103362-094WO1

[0516] 28. The recombinant polypeptide of any one of embodiments 23-27, wherein the GLP-1 peptide has one or more mutations to increase stability.

[0517] 29. The recombinant polypeptide of any one of embodiments 23-28, comprising SEQ ID NO: 11, or an amino acid sequence at least 95% identical thereto.

[0518] 30. The recombinant polypeptide of any one of embodiments 23-29, encoded by a nucleic acid sequence comprising SEQ ID NO: 8, or a nucleic acid sequence at least 95% identical thereto.

[0519] 31. A recombinant polypeptide comprising:

[0520] (a) a signal peptide sequence;

[0521] (b) a linker peptide comprising between 1 to 30 amino acid residues; and

[0522] (c) a brain-derived neurotrophic factor (BDNF) peptide.

[0523] 32. The recombinant polypeptide of embodiment 31, further comprising a TAT cellpenetrating peptide.

[0524] 33. The recombinant polypeptide of embodiment 32, wherein the TAT cell-penetrating peptide is operatively conjugated to the amino terminus of the BDNF peptide.

[0525] 34. The recombinant polypeptide of embodiment 32, wherein the TAT cell-penetrating peptide is operatively conjugated to the carboxy terminus of the BDNF peptide.

[0526] 35. The recombinant polypeptide of any one of embodiments 32-34, wherein the TAT cellpenetrating peptide comprises SEQ ID NO: 22, or an amino acid at least 95% identical thereto.

[0527] 36. The recombinant polypeptide of any one of embodiments 32-35, wherein the TAT cellpenetrating peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 20, or a nucleic acid sequence at least 95% identical thereto.

[0528] 37. The recombinant polypeptide of any one of embodiments 31-36, wherein the BNDF peptide comprises pro-BDNF.

[0529] 38. The recombinant polypeptide of any one of embodiments 31-36, wherein the BNDF peptide comprises mature BDNF (mBDNF).

[0530] 39. The recombinant polypeptide of embodiment 38, wherein the mBDNF peptide comprises SEQ ID NO: 17, or an amino acid sequence at least 95% identical thereto.

[0531] 40. The recombinant polypeptide of embodiment 38 or 39, wherein the mBDNF peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 14, or a nucleic acid sequence at least 95% identical thereto.

[0532] 41. The recombinant polypeptide of any one of embodiments 38-40, comprising SEQ ID NO: 16, or an amino acid sequence at least 95% identical thereto.Docket No. 103362-094WO1

[0533] The recombinant polypeptide of any one of embodiments 38-41, encoded by a nucleic acid sequence comprising SEQ ID NO: 13, or a nucleic acid sequence at least 95% identical thereto.

[0534] The recombinant polypeptide of any one of embodiments 38-40, comprising SEQ ID NO: 21 or SEQ ID NO: 24, or an amino acid sequence at least 95% identical thereto. The recombinant polypeptide of any one of embodiments 38-40 or 43, encoded by a nucleic acid sequence comprising SEQ ID NO: 19 or SEQ ID NO: 23, or a nucleic acid sequence at least 95% identical thereto.

[0535] A recombinant polypeptide comprising:

[0536] (a) a signal peptide sequence;

[0537] (b) a linker peptide comprising between 1 to 30 amino acid residues; and

[0538] (c) a peptide derived from bone morphogenetic protein 7 (BMP7).

[0539] The recombinant polypeptide of embodiment 45, wherein the peptide derived from BMP7 comprises pro-BMP7.

[0540] The recombinant polypeptide of embodiment 46, wherein the peptide derived from BMP7 comprises mature BMP7 (mBMP7).

[0541] The recombinant polypeptide of embodiment 47, wherein the mBMP7 peptide comprises SEQ ID NO: 28, or an amino acid sequence at least 95% identical thereto. The recombinant polypeptide of embodiment 47 or 48, wherein the mBMP7 peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 26, or a nucleic acid sequence at least 95% identical thereto.

[0542] The recombinant polypeptide of any one of embodiments 47-49, comprising SEQ ID NO: 27, or an amino acid sequence at least 95% identical thereto.

[0543] The recombinant polypeptide of any one of embodiments 47-50, encoded by a nucleic acid sequence comprising SEQ ID NO: 25, or a nucleic acid sequence at least 95% identical thereto.

[0544] The recombinant polypeptide of any one of embodiments 16-51, wherein the signal peptide sequence comprises a leptin signal peptide sequence.

[0545] The recombinant polypeptide of embodiment 52, wherein the leptin signal peptide sequence comprises SEQ ID NO: 9, or an amino acid sequence at least 95% identical thereto.

[0546] The recombinant polypeptide of embodiment 51 or 52, wherein the leptin signal peptide sequence is encoded by a nucleic acid sequence comprising SEQ ID NO: 2, or a nucleic acid sequence at least 95% identical thereto.Docket No. 103362-094WO1

[0547] 55. The recombinant polypeptide of any one of embodiments 16-54, wherein the linker peptide is a valine residue.

[0548] 56. The recombinant polypeptide of any one of embodiments 16-54, wherein the linker peptide comprises a glycine / serine linker.

[0549] 57. The recombinant polypeptide of embodiment 56, wherein the glycine / serine linker comprises (GS)x, (GGS)x, (GGGS)x (SEQ ID NO: 46), (GGGGS)x (SEQ ID NO: 47), or (GGGGGS)x (SEQ ID NO: 48), wherein x is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0550] 58. The recombinant polypeptide of any one of embodiments 16-57, further comprising one or more additional peptides.

[0551] 59. An expression vector comprising a nucleic acid encoding for a recombinant polypeptide of any one of embodiments 1-58.

[0552] 60. The expression vector of embodiment 59, wherein expression vector is a non-viral vector.

[0553] 61. The expression vector of embodiment 60, wherein the non-viral expression vector is selected from a DNA plasmid, a miniplasmid, an exosome, a liposome, or a lipid nanoparticle.

[0554] 62. The expression vector of embodiment 59, wherein expression vector is a viral vector.

[0555] 63. The expression vector of embodiment 62, wherein the viral expression vector is selected from a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector (AAV).

[0556] 64. The expression vector of embodiment 62, wherein the viral expression vector is an AAV

[0557] 65. An adeno-associated viral vector (AAV) comprising a nucleic acid encoding for a recombinant polypeptide of any one of embodiments 1-58.

[0558] 66. The AAV of embodiment 65, wherein the AAV is recombinant and / or engineered. 67. The AAV of embodiment 65 or 66, wherein the AAV has decreased tropism towards liver.

[0559] 68. The AAV of any one of embodiments 65-67, wherein the AAV has enhanced tropism towards adipose tissue.

[0560] 69. The AAV of any one of embodiments 65-68, wherein the AAV is a V7 AAV capsid vector.

[0561] 70. The AAV of any one of embodiments 65-67, wherein the AAV has enhanced tropism towards neural tissue.Docket No. 103362-094WO1

[0562] 71. The AAV of any one of embodiments 65-68 or 70, wherein the AAV is a Rec2 AAV capsid vector.

[0563] 72. A pharmaceutical composition comprising at least one recombinant polypeptide of any one of embodiments 1-58, at least one expression vector of any one of embodiments 59-64, or at least one AAV of any one of embodiments 65-71, and a pharmaceutically acceptable carrier.

[0564] 73. A method for gene transfer of a nonnative, recombinant polypeptide to adipose tissue in a subject in need thereof, comprising administering a therapeutically effective amount of an AAV of any one of embodiments 65-71.

[0565] 74. The method of embodiment 73, wherein the AAV is administered by intraperitoneal, subcutaneous, or intravenous injection.

[0566] 75. A method for gene transfer of a nonnative, recombinant polypeptide to brain tissue in a subject in need thereof, comprising administering a therapeutically effective amount of an AAV of any one of embodiments 65-71.

[0567] 76. The method of embodiment 75, wherein the AAV is administered by intracranial, intraparenchymal, or intravenous injection.

[0568] 77. A method for reducing blood glucose in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of embodiments 1-58, an expression vector of any one of embodiments 59-64, or an AAV of any one of embodiments 65-71.

[0569] 78. A method for increasing blood glucose tolerance in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of embodiments 1-58, an expression vector of any one of embodiments 59-64, or an AAV of any one of embodiments 65-71.

[0570] 79. The method of embodiment 77 or 78, wherein the subject is obese.

[0571] 80. A method for reducing expression of proinflammatory genes in one or more tissues in a subject receiving an AAV therapy, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of embodiments 1-58, an expression vector of any one of embodiments 59-64, or an AAV of any one of embodiments 65-71.

[0572] 81. The method of embodiment 80, wherein the one or more tissues are selected from liver, hypothalamus, brown adipose tissue, white adipose tissue, or a combination thereof.

[0573] 82. A method for treating a subject having a metabolic disorder, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one ofDocket No. 103362-094WO1

[0574] embodiments 1-58, an expression vector of any one of embodiments 59-64, or an AAV of any one of embodiments 65-71.

[0575] 83. The method of embodiment 82, wherein the metabolic disorder is selected from diabetes, obesity, chronic inflammation, or sleep apnea.

[0576] 84. A method for treating a subject having diabetes, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of embodiments 1-58, an expression vector of any one of embodiments 59-64, or an AAV of any one of embodiments 65-71.

[0577] 85. A method for treating a subject having obesity, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of embodiments 1-58, an expression vector of any one of embodiments 59-64, or an AAV of any one of embodiments 65-71.

[0578] 86. A method for reducing one or more metabolic disorders in a subject having a body mass index (BMI) of equal to or greater than 27.5, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of embodiments 1-58, an expression vector of any one of embodiments 59-64, or an AAV of any one of embodiments 65-71.

[0579] 87. A method for reducing one or more metabolic disorders in a subject having a body mass index (BMI) of equal to or greater than 30, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of embodiments 1-58, an expression vector of any one of embodiments 59-64, or an AAV of any one of embodiments 65-71.

[0580] 88. A method for reducing one or more metabolic disorders in a subject having a body mass index (BMI) of equal to or greater than 40, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of embodiments 1-58, an expression vector of any one of embodiments 59-64, or an AAV of any one of embodiments 65-71.

[0581] 89. The method of any one of embodiments 85-87, wherein the one or more metabolic disorders are selected from obesity, diabetes, lipodystrophy, congenital generalized lipodystrophy (Beradinelli-Seip syndrome), familial partial lipodystrophy, acquired partial lipodystrophy (Barraquer-Simons syndrome), acquired generalized lipodystrophy, centrifugal abdominal lipodystrophy, lipoatrophia annularis, localized lipodystrophy, or HIV-associated lipodystrophy.Docket No. 103362-094WO1

[0582] 90. A method for reducing weight of a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of embodiments 1-58, an expression vector of any one of embodiments 59-64, or an AAV of any one of embodiments 65-71.

[0583] 91. The method of embodiment 90, wherein the subject, prior to being administered the recombinant polypeptide or expression vector encoding thereof, had a BMI of equal to or greater than 27.5, equal to or greater than 30, equal to or greater than 32.5, equal to or greater than 37.5, equal to or greater than 40, or more than 40.

[0584] 92. The method of embodiment 90 or 91, wherein the subject experiences a weight loss of equal to or greater than 5% of the baseline weight of the subject prior to being administered the recombinant polypeptide or expression vector encoding thereof. 93. The method of any one of embodiments 90-92, wherein the subject experiences a weight loss of equal to or greater than 10% of the baseline weight of the subject prior to being administered the recombinant polypeptide or expression vector encoding thereof.

[0585] 94. The method of any one of embodiments 90-93, wherein the subject experiences a weight loss of equal to or greater than 20% of the baseline weight of the subject prior to being administered the recombinant polypeptide or expression vector encoding thereof.

[0586] 95. The method of any one of embodiments 90-94, wherein the subject experiences a weight loss of equal to or greater than 30% of the baseline weight of the subject prior to being administered the recombinant polypeptide or expression vector encoding thereof.

[0587] 96. The method of any one of embodiments 90-95, wherein the subject has a reduced body fat percentage following being administered the recombinant polypeptide or expression vector encoding thereof.

[0588] 97. The method of any one of embodiments 90-96, wherein the subject has an increased relative lean mass calibrated to body weight following being administered the recombinant polypeptide or expression vector encoding thereof.

[0589] 98. A method for treating a neurodevelopmental disease in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of embodiments 1-58, an expression vector of any one of embodiments 59-64, or an AAV of any one of embodiments 65-71.Docket No. 103362-094WO1

[0590] The method of embodiment 98, wherein the neurodevelopmental disease comprises Prader-Willi syndrome.

[0591] A method for treating a neurodegenerative disease in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of embodiments 1-58, an expression vector of any one of embodiments 59-64, or an AAV of any one of embodiments 65-71.

[0592] The method of embodiment 100, wherein the neurodegenerative disease comprises Alzheimer’s disease.

[0593] The method of embodiment 100, wherein the neurodegenerative disease comprises Parkinson’s disease.

[0594] A method for treating major depression in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of embodiments 1-58, an expression vector of any one of embodiments 59-64, or an AAV of any one of embodiments 65-71.

[0595] A method for treating osteoporosis in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of embodiments 1-58, an expression vector of any one of embodiments 59-64, or an AAV of any one of embodiments 65-71.

[0596] A method for enhancing dental regeneration in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of embodiments 1-58, an expression vector of any one of embodiments 59-64, or an AAV of any one of embodiments 65-71.

[0597] A method for treating an inflammatory disease in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of embodiments 1-58, an expression vector of any one of embodiments 59-64, or an AAV of any one of embodiments 65-71.

[0598] The method of embodiment 103, wherein the inflammatory disease is selected from cardiovascular disease, diabetes, or obesity.

[0599] A method for improving cellular plasticity in a neurological disorder in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of embodiments 1-58, an expression vector of any one of embodiments 59-64, or an AAV of any one of embodiments 65-71.

[0600] A method for treating cancer, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of embodiments 1-58, an expressionDocket No. 103362-094WO1

[0601] vector of any one of embodiments 59-64, or an AAV of any one of embodiments 65- 71.

[0602] 110. The method of any one of embodiments 73-109, wherein the subject is a human.

[0603] EXAMPLES

[0604] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0605] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. While the invention has been described with reference to particular embodiments and implementations, it will be understood that various changes and additional variations may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention or the inventive concept thereof. In addition, many modifications may be made to adapt a particular situation or device to the teachings of the invention without departing from the essential scope thereof. Such equivalents are intended to be encompassed by the following claims. It is intended that the invention not be limited to the particular implementations disclosed herein, but that the invention will include all implementations falling within the scope of the appended claims.

[0606] Example 1. Exendin-4

[0607] Adipose tissue, one of the largest organs of the body, has not been at the forefront of AAV-based gene therapy largely due to the poor transduction efficiency of natural AAV serotypes. A novel recombinant AAV capsid was recently developed that exhibits exclusive adipo-tropism and de-targeting liver upon systemic administration (via intraperitoneal or intravenous injection). This AAV capsid vector (referred as V7) platform provides a powerful vehicle to genetically manipulating adipose tissue for producing therapeutic molecules including peptide drugs.

[0608] For adipose gene transfer of a nonnative peptide, a proof-of-concept study was conducted using adipo-tropic V7 capsid vector to deliver Exendin-4, a peptide isolated from the venom of Gila monster, and tested its therapeutic efficacy in a diet-induced obesity model.Docket No. 103362-094WO1

[0609] Atransgene was designed in which the Exendin-4 sequence (SEQ ID NO: 3) (encoding a 39-amino acid (SEQ ID NO: 7)) was linked to the 21 -amino acid signal peptide sequence of leptin gene (SEQ ID NO: 5) with an extra Valine added (SEQ ID NO: 6) (FIG. 1). This transgene Exendin-4 (SEQ ID NO: 1) was then cloned to an AAV expression plasmid driven by the CBA promoter. An in vitro transfection experiment showed that Exendin-4 protein was detected in the supernatant of HEK293 cells by ELISA and Western blotting, suggesting the Exendin-4 transgene product being secreted from the transfected cells (FIG. 2A-2B).

[0610] Next, the Exendin 4 expression cassette was packaged to adipo-tropic V7 capsid and tested its therapeutic efficacy in a diet-induced obesity model.

[0611] Mice were fed on high fat diet (60% calorie from fat) and randomized to receive a single intraperitoneal (IP) injection of V7-Exendin-4 or green fluorescent protein (GFP) as a control, 8E10 viral genome particle (vg) per mouse. Although body weight or body composition were not significantly altered (FIG. 3A-3B), V7-Exendin-4 treatment robustly improved glucose tolerance in obese mice. This indicates that the Exendin-4 transgene product was secreted from the adipocytes and exerted a robust antidiabetic effect in vivo (FIG. 4).

[0612] Obesity is associated with chronic low-grade inflammation contributing to many obesity-associated complications. The adipo-tropic V7 capsid comprising the Exendin 4 expression cassette was tested for its effect on inflammatory genes in different tissues. V7-Exendin-4 treatment significantly downregulated the expression of proinflammatory genes in the liver (FIG. 5A), in the hypothalamus (FIG. 5B), in the brown adipose tissue (FIG. 6A) and white adipose tissue (FIG. 6B).

[0613] The biodistribution of Exendin 4 transgene, its circulating level, and impact on hepatic steatosis was then analyzed.

[0614] To test the efficacy of Exendin-4 gene delivery in tissues beyond adipose tissue, the same Exendin-4 expression cassette as previously used (SEQ ID NO: 1) was packaged to the Rec2 capsid that potently transduces both adipose tissue and liver by IP injection. Mice fed on high fat diet were randomized to receive a single IP injection of Rec2 -Exendin-4 (4E10 vg per mouse) or AAV buffer as control. Rec2 -Exendin-4 treatment durably and significantly reduced weight gain (FIG. 7A) and suppressed food intake over several weeks (FIG. 7B), starting as early as the first week post AAV injection. For example, the average body weight of Rec2-Exendin-4 mice was 30% lower than that of control mice by 11 weeks post AAV inj ection (FIG.

[0615] 7 A). Rec2 -Exendin-4 treatment reduced weight gain by 66% at 11 weeks post AAV injection (FIG. 8A).Docket No. 103362-094WO1

[0616] At 5 weeks post AAV injection, body composition was assessed by echoMRI. Rec2-Exendin-4 treatment robustly reduced the adiposity and increased the relative lean mass calibrated to body weight (FIG. 8B). In addition, at 6 weeks post AAV inj ection, mice receiving Rec2 -Exendin-4 displayed significantly improved glucose tolerance (FIG. 9).

[0617] At 12 weeks post AAV injection, mice were subjected to indirect calorimetry. Rec2-Exendin-4 treatment did not alter the oxygen consumption (FIG. 10 A), energy expenditure as heat (kcal / hr) (FIG. 10B), the respiratory exchange ratio (RER) (FIG. 10C), or locomotor activity (FIG. 10D), indicating that the weight loss induced by Rec2 -Exendin-4 was due to reduced food intake.

[0618] At 13 weeks post AAV injection, mice were subjected to open field test to assess exploratory behavior, locomotion, and anxiety-like behavior. Rec2-Exendin-4 treatment significantly increased locomotion and exploratory activity as measured by total distance travelled (FIG. 11 A), while no difference in anxiety-like behavior was observed as measured by ratio of center / total distance (FIG. 1 IB) or peripheral / total distance (FIG. 11C). At 14 weeks post AAV injection, novel object recognition test was performed to assess recognition memory. No significant difference was found (FIG. 12).

[0619] Up to date, in vivo data as described herein have demonstrated that Rec2-Exendin-4 treatment significantly reverses diet-induced obesity and impaired glucose metabolism associated with obesity as well as certain behaviors.

[0620] Additional in vivo assessments such as behavioral assessments will be conducted. To test the therapeutic potential of Exendin-4 gene therapy in genetic form of obesity, a proof-of-concept study was conducted in the Magel2-nu\\ mouse model of Prader-Willi Syndrome (PWS). PWS is a rare genetic disease, occurring in approximately 1 in 15,000 individuals, and is caused by the loss of function of paternally inherited copy of chromosome 15ql 1 -ql 3. PWS patients display developmental delays, cognitive impairment, behavioral and neuropsychiatric abnormalities, endocrine dysfunction, hypothalamic hypogonadism, and constant sense of hunger (hyperphagia) that often leads to obesity and many obesity-related complications.

[0621] MAGEL2 (MAGE Family Member L2) is one of the five protein-coding genes in the PWS-critical domain on chromosome 15qll-ql3 and its loss contributes to disturbed energy homeostasis in PWS. Magel2-nu\\ mice recapitulate some aspects of PWS pathophysiology including disturbance in the hypothalamic leptin-proopiomelanocortin (POMC) pathway and feeding circuitry, increased adiposity, impaired glucose metabolism, and alterations in neuroendocrine responses.Docket No. 103362-094WO1

[0622] Magel2-mA\ mice were randomized to receive a single IP injection of Rec2 -Exendin-4 (4E10 vg per mouse) or Rec2-GFP (4E10 vg per mouse) as control. All mice were fed on high fat diet. Rec2 -Exendin-4 treatment decreased food intake (FIG. 13C) and weight gain as early as the first week post AAV injection (FIG. 13 A). Two out of five Magel2-null mice receiving Rec2-Exendin-4 lost >40% body weight in two weeks and therefore were sacrificed due to extreme low body weight (12-15 g).

[0623] The in vivo data demonstrate the PWS mouse model Magel2-nu\\ mice are highly sensitive to Rec2 -Exendin-4 treatment and a dose-deescalating study is warranted. The study will be expanded in Magel2-nu\\ mice to generate preclinical data for developing Rec2-Exendin-4 gene therapy as a novel gene therapy for PWS.

[0624] Example 2. Active GLP-1 Peptide

[0625] For therapeutic application, it is difficult to achieve the goal of producing active GLP-1 peptide by overexpressing GCG gene. Moreover, several forms of GLP-1 are processed from proglucagon with varying ability to enhance glucose-induced insulin secretion including GLP-1 (l-36amide), GLP-1 (7-36amide) and GLP-1 (7-37). In humans, -80% of circulating GLP-1 is GLP-1 (7-36amide) and -20% is GLP-1 (7-37). The two truncated forms of GLP-1 have equal insulinotropic efficacy.

[0626] A transgene was designed (SEQ ID NO: 8) in which the human GLP-1 sequence (SEQ ID NO: 10) encoding GLP-1 (7-36amide, (SEQ ID NO: 12)) was linked to the 21-amino acid signal peptide sequence of leptin gene (SEQ ID NO: 5) with an extra Valine added (SEQ ID NO: 6) (FIG 14A).

[0627] Native GLP-1 has a very short half-life around 1-2 min due to rapid degradation by dipeptidyl peptidase 4 (DPP4) at the N-terminal dipeptide. As such, a residue substitution from Ala to Gly (see, e.g., bolded residue of FIG. 14A) was introduced to the GLP-1 amino acid sequence (SEQ ID NO: 12) to increase stability of GLP-1.

[0628] The GLP-1 expressing plasmid was transfected to the HEK293 cells. Western blotting did not detect GLP-1 peptide in the cell lysate likely due to the failure of the GLP-1 antibody used. Optimizing the western blotting method will be attempted.

[0629] Results

[0630] To assess whether AAV-mediated gene transfer of active GLP-1 can alter systemic metabolism, the GLP-1 transgene (SEQ ID NO: 12) was packaged to the Rec2 capsid vectorDocket No. 103362-094WO1

[0631] and intraperitoneally injected to diet induced obesity mice at the dose of 4E10 vg per mouse. Mice were maintained on high fat diet throughout the 6-weeks experiment.

[0632] Rec2-GLP-1 vector resulted in no effect on body weight or glucose tolerance at 5 weeks post AAV injection (FIG. 14B). The preliminary data suggest Rec2-GLP-1 at the dose of 4E10 vg per mouse was ineffective. Modification of GLP-1 transgene and / or dose adjustment are warranted.

[0633] Example 3. Mature BDNF Peptide

[0634] Therapeutic use of BDNF protein has been largely unsuccessful due to unfavorable pharmacokinetics and the need for fine delivery localization and repeated dosing in wide range of neurological and psychiatric disorders.

[0635] Here, a transgene (SEQ ID NO: 13) was designed in which the human mBDNF sequence (SEQ ID NO: 14) encoding a 118-amino acid (SEQ ID NO: 17) was linked to the 21-amino acid signal peptide sequence of leptin gene (SEQ ID NO: 5) with an extra Valine (SEQ ID NO: 6). An HA tag sequence (SEQ ID NO: 15) — encoding a 9-amino acid derived from the human influenza hemagglutinin protein (SEQ ID NO: 18) was added to the C-terminus of mBDNF to distinguish transgene-derived mBDNF from endogenous mBDNF (FIG. 15).

[0636] In addition, a TAT cell-penetrating peptide sequence (SEQ ID NO: 20) (encoding an 11 -amino acid TAT tag (SEQ ID NO: 22)) was added to the N-terminus of mBDNF to enhance trafficking to the brain (FIG. 16). Both mBDNF transgenes were cloned into an AAV expression plasmid in which expression was driven by the CB A promoter.

[0637] An in vitro transfection experiment showed that mBDNF protein was detected in the supernatant of HEK293 cells by Western blotting, suggesting the mBDNF transgene product being secreted from the transfected cells (FIG. 17). Of note, the presence of TAT peptide at the N-terminus of mBDNF affected the expression and secretion of mBDNF. The signal peptide was cleaved from the mBDNF transgene product in absence of TAT peptide at the N-terminus. Therefore, the mBDNF transgene without TAT at the N-terminus was advanced for a proof-of-concept in vivo study.

[0638] To assess whether AAV-mediated gene transfer of mBDNF can alter systemic metabolism and behaviors, the mBDNF transgene (SEQ ID NO: 13) (FIG. 15) was packaged to the Rec2 capsid vector and intraperitoneally injected to ob / + mice at two doses (2E10 and 4E10 vg per mouse). Mice were maintained on normal chow diet throughout the 6-weeks experiment. Rec2-mBDNF vector at higher dose of 4E10 vg / mouse led to significant weightDocket No. 103362-094WO1

[0639] loss in contrast to the weight gain in mice receiving buffer (FIG. 18 A). No difference was found in the glucose tolerance test at 4 weeks post AAV injection (FIG. 18B).

[0640] At 5 weeks post AAV injection, an open field test was conducted to assess exploratory behavior and anxiety -like behavior followed by a cognitive behavioral test novel object recognition test. No significant difference was observed in any metric (FIG. 19A-19C). It is noteworthy that the number of mice per group was likely too small to assess the behavioral effect. Future studies using models of neurological diseases with sufficient power will be conducted to evaluate whether Rec2-mBDNF can alleviate aberrant behaviors.

[0641] At the termination of the experiment, serum BDNF level was measured by an ELISA kit that detects both mouse BDNF and human BDNF. The circulating BDNF level in mice receiving Rec2-mBDNF at lower dose (2E10 vg / mouse) was approximately 5 times higher than the endogenous mouse BDNF level in the mice receiving buffer. Rec2-mBDNF at higher dose (4E10 vg / mouse) resulted in an even higher circulating BDNF level, approximately 10 times of the level in control mice (FIG. 20).

[0642] The presence of mBDNF transgene product in the subject was further confirmed by Western blotting in the visceral adipose tissue and liver (FIG. 21). Gene expression was profiled by quantitative RT-PCR. Rec2-mBDNF treatment altered gene expression in the hypothalamus (FIG. 22) and in the liver (FIG. 23) in a dose-dependent manner.

[0643] To evaluate whether Rec2-mBDNF can alleviate hyperglycemia in a genetic model of type 2 diabetes and obesity, Rec2-mBDNF was intraperitoneally injected into leptin receptor deficient db / db mice at the dose of 2E10 vg per mouse, db / db mice receiving AAV buffer served as control. Rec2-mBDNF (2E10 vg / mouse) did not alter body weight or glucose tolerance at 4 weeks post AAV injection. Nonfasted blood glucose levels were measured 3 weeks post AAV injection, and no difference was found (FIG. 24). Circulating BDNF level was 0.078±0.014 ng / ml, indicating the dose of 2E10 vg was ineffective in db / db mice.

[0644] To assess whether increasing dose can improve outcome, Rec2 -mBDNF was injected to control db / db mice (previously receiving AAV buffer) at the dose of 5E10 vg / mouse. Higher dose of Rec2-mBDNF led to significant reduction in weight gain (FIG. 25) and improved insulin tolerance at 4 weeks post crossover dosing as compared to lower dose of Rec2-mBDNF (FIG. 26). Moreover, nonfasted blood glucose levels were reduced by 46.5±1.5% at 11 days post injection of higher dose Rec2-mBDNF (FIG. 27). The improved outcomes were associated with higher circulating BDNF levels (FIG. 27).

[0645] In summary, these pilot studies have shown that mBDNF transgene product was released to circulation and exerted biological effects in a dose-dependent manner, includingDocket No. 103362-094WO1

[0646] metabolic phenotypes such as body weight gain, insulin sensitivity, and blood glucose, and triggered molecular changes in the brain and in the periphery.

[0647] In addition, another mBDNF transgene (SEQ ID NO: 23) was designed by placing the TAT cell-penetrating peptide sequence (SEQ ID NO: 20) to the C-terminus of the mBDNF (SEQ ID NO: 17) (FIG. 28). An in vitro transfection experiment showed that mBDNF transgene product (SEQ ID NO: 24) was detected in the cell lysates and supernatants by Western blotting (FIG. 29). The mBDNF-TAT transgene (SEQ ID NO: 23) will be packaged to Rec2 capsid vector to be tested in the ob / + mice at two doses (2E10, 4E10 vg / mouse) mirroring the abovedescribed study on Rec2-mBDNF (without TAT).

[0648] Example 4. Mature BMP7 Peptide

[0649] Recent findings have revealed the role of BMP7 in regulation of appetite, energy expenditure, adipogenesis, and insulin sensitivity. For applications as metabolic modulator, sustained expression of BMP7 within adipose tissue is desirable.

[0650] As such, a mature BMP7 transgene (SEQ ID NO: 25) was designed consisting of signal peptide sequence from leptin gene (SEQ ID NO: 5) with an additional Valine added (SEQ ID NO: 6), human mature BMP7 (SEQ ID NO: 26) (139-amino acid (SEQ ID NO: 28)), and an HA tag (SEQ ID NO: 15) (9-amino acid (SEQ ID NO: 18)) at the C-terminus of mature BMP7 to distinguish transgene-derived mature BMP7 from endogenous mature BMP7 (FIG. 30).

[0651] An in vitro transfection experiment confirmed the expression and secretion of mature BMP7 transgene product in the cell lysates and cell culture supernatants (FIG. 31A-31C).

[0652] An adipose-tropic V7 capsid vector will be generated to deliver the mature BMP7 transgene specifically to the adipose tissue in proof-of-concept study in models of obesity and diabetes.

[0653] In summary, the disclosed transgene design may be applied to other nonnative peptide / protein molecules for research and therapeutic purposes. Moreover, the transgene design disclosed here can be coupled with other viral vehicles (e.g. other AAV capsids, adenoviral vector, etc.) or nonviral gene transfer methods.Docket No. 103362-094WO1

[0654] Example 5. Low dose systemic AAV-Exendin4 gene therapy for Prader-Willi syndrome and dietary obesity

[0655] Prader-Willi syndrome (PWS) patients display developmental delays, endocrine dysfunction, excessive eating, central obesity, and various behavioral abnormalities. Effective and sustained treatments are limited, highlighting the need for new therapeutic strategies. Glucagon-like peptide-1 receptor agonists (GLP-1RA) have revolutionized obesity treatment while their efficacy in PWS population remains inconsistent and data in PWS animal models are lacking. Here, we assessed the efficacy of a newly developed AAV platform to deliver a GLP-1RA exendin-4 via an engineered hybrid capsid Rec2. Intraperitoneal administration of Rec2-exendin-4 at the dose of 2x1010viral genome per mouse normalized metabolic dysfunction and impaired exploratory behavior in the Magel2-null mouse model of PWS. Systemic Rec2-exendin-4 treatment reversed genotype-driven excessive adiposity, impaired glycemic control, hyperleptinemia, and adipose gene expression signatures. Moreover, intraperitoneal injection of Rec2-exendin-4 (4x1010viral genome / mouse) exerted high levels of efficacy in diet-induced obesity model — decreasing food intake; preventing excessive weight gain and obesity; improving glucose metabolism and insulin sensitivity; and reversing fatty liver. Metabolic improvements were maintained at least 5 months. These data demonstrate the therapeutic potential of a systemic AAV-mediated exendin-4 gene therapy for PWS-related metabolic abnormalities and dietary obesity.

[0656] PWS is a rare genetic disease that occurs in approximately 1 in 15,000 individuals, due to the loss of function of paternally inherited copy of chromosome 15qll-ql3. Individuals with PWS display developmental delays, cognitive impairment, behavioral and neuropsychiatric abnormalities such as obsessive-compulsive behavior and temper tantrums, endocrine dysfunction, hypothalamic hypogonadism, and constant sense of hunger leading to obesity and associated metabolic syndromes. Up to date, effective and sustained treatments are limited to address the metabolic dysregulation, highlighting the urgent need for new therapeutic strategies.

[0657] MAGEL2 (MAGE Family Member L2) is one of the five protein-coding genes in the PWS-critical domain on chromosome 15qll-ql3. Loss of function oiMAGEL2 is thought to contribute to some aspects of PWS pathophysiology. Magel2-nvX\ mice, one of the most commonly used animal models for PWS, recapitulate key aspects of PWS pathophysiology including disturbance in the hypothalamic leptin-proopiomelanocortin (POMC) pathway and feeding circuitry, elevated adiposity, impaired glucose metabolism, and alterations in neuroendocrine responses.Docket No. 103362-094WO1

[0658] Previous research has demonstrated that a hypothalamic adeno-associated virus (AAV) based brain-derived neurotrophic factor (BDNF) gene therapy normalized metabolic disturbance and improved some behavioral abnormalities in the Magel2-nu\\ mice with high level of efficacy and safety. However, the translation of a hypothalamic BDNF gene therapy remains challenging. Thus, systemic gene therapy approaches are worthy of investigation for this genetic disease potentially requiring lifelong treatment.

[0659] Incretin mimetics are agonists at one or more satiety-related receptors that have revolutionized obesity treatment such as glucagon-like peptide- 1 receptor agonist (GLP-1RA) semaglutide. However, the efficacy of incretin mimetics in individuals with PWS remains inconsistent and scarce evidence in PWS relevant animal models is available. Incretin mimetics, like many peptide drugs, require frequent injection and long-term use, positing challenges of patient compliance, risk of side effects, and high cost. As such, AAV-based gene therapy may have advantages compared to current standard of care for some peptide drugs such as GLP-1RA. Exendin-4 (Ex4) is a peptide isolated from the venom of Gila monster which has 53% homology with the biological active form of GLP-l(7-37), but acts as a full agonist of GLP-1R and therefore has been developed as a drug for diabetes. To assess the therapeutic potential of a systemic incretin mimetics gene therapy as a novel, long lasting, and safe treatment for PWS obesity, we designed a transgene of Ex4 whose peptide product can be secreted from transduced cells. This transgene was packaged to an engineered hybrid AAV capsid Rec2 which can achieve high level transgene expression by intraperitoneal (IP) injection. In this study, we investigated the therapeutic effects of this Rec2-Ex4 gene therapy via IP administration in the preclinical model of PWS — Afage / 2-null mice, and diet-induced obesity model in wildtype mice.

[0660] Results

[0661] Ex4 transgene design and in vitro validation

[0662] We designed a transgene of Ex4 in which the Ex4 sequence was linked to the signal peptide sequence of leptin gene with an extra Valine (FIG. 32A) and inserted this transgene to AAV cis plasmid under the control of CB A promoter. After transfection to HEK293 cells, Ex4 was detected in the cell lysates and the cell culture media by Western blotting and ELISA (FIGs.

[0663] 40A-40B) indicating the Ex4 peptide product being secreted from cells.Docket No. 103362-094WO1

[0664] Pilot study of Rec2-Ex4 in Magel2-null mice

[0665] To estimate the dose of Rec2-Ex4, we conducted a pilot study by IP injection of Rec2-Ex4 in M age! 2 -nu\\ mice or Rec2-GFP as a control, at the dose of 4x1010viral genome (vg) per mouse. Mice receiving Rec2-Ex4 exhibited a sharp drop in food intake, 35% in week-1 and 65% in week-2 post AAV injection as compared to mice receiving Rec2-GFP (FIG. 41C). Two out of five Rec2-Ex4-treated mice lost >40% body weight within two weeks and the experiment was terminated due to extreme lowbody weight (12-15 g) (FIGs. 41 A-41B). These data suggest Magel2-nu\\ mice are highly sensitive to Rec2-Ex4, and the dose of 4x1010vg per mouse was too high. Thus, we lowered the dose to 2x1010vg per mouse in a study examining the genotype and Ex4 treatment effects.

[0666] IP injection of Rec2-Ex4 normalizes genotype-driven metabolic abnormalities in Magel2-null mice

[0667] Male Magel2-null mice and age-matched wildtype (WT) littermates were randomized to receive an IP injection of either Rec2-Ex4 or Rec2-GFP at the dose of 2x1010vg per mouse and subject to in vivo metabolic assessments and behavioral testing as outlined in FIG. 32B. The baseline body weight of Magel2-nu\\ mice was significantly higher than WT counterpart (Two-way ANOVA, Genotype <0.01) prior to vector administration. Magel2-nu\\ mice receiving Rec2-GFP maintained higher body weight as compared to WT mice throughout the experiment (FIGs. 32C-32D). In contrast, Mage! 2 -nu\\ mice receiving Rec2-Ex4 displayed significant weight loss (FIGs. 32E-32F), alleviating the genotype-driven increase in body weight (FIGs. 32C-32D). Rec2-Ex4 treatment resulted in milder body weight effect in WT mice than that observed in the M age! 2 -nu\\ mice (FIGs. 32C-32F).

[0668] At 4-weeks post AAV injection, a glucose tolerance test (GTT) was performed to assess glycemic control. M age! 2 -nu\\ mice receiving Rec2-GFP exhibited impaired GTT over WT controls (FIGs. 32G-32H). This genotype-driven deficit was rescued by Rec2-Ex4 treatment as Rec2-Ex4 treated M age! 2 -nu\\ mice performed similarly to Rec2-GFP treated WT mice (FIGs. 32G-32H). Of note, Rec2-Ex4 treated WT mice displayed further improvement in GTT as compared to Rec2-GFP treated WT mice (FIGs. 32G-32H) in the absence of significant change in body weight (FIGs. 32C-32D). Magel2-nu\\ mice exhibited elevated fasting blood glucose, which was ameliorated by Rec2-Ex4 treatment (FIG. 321).

[0669] Energy expenditure was examined by indirect calorimetry between 7- and 8-weeks post AAV injection. Energy expenditure or heat, RER (respiratory exchange ratio, calculated as VCO2 / VO2), and ambulation over 24 h showed no significant difference among the four groupsDocket No. 103362-094WO1

[0670] (FIG. 42A). No genotype or treatment effects were found in the average heat, average RER, or average ambulation (FIG. 42B).

[0671] Body composition was determined by EchoMRI following the completion of indirect calorimetry. Magel2-nu\\ mice treated with Rec2-GFP displayed significant increase in absolute fat mass (FIG. 42C) and relative fat mass (FIG. 32J) as compared to WT mice. This genotype-driven increase in fat mass was reversed by Rec2-Ex4 treatment (FIGs. 32J, 42C). Absolute lean mass was similar among the four groups (FIG. 42D) whereas relative lean mass was increased by Rec2-Ex4 treatment regardless of genotypes (FIG. 32K).

[0672] It is reported that Magel2-nu\\ mice do not exhibit hyperphagia fed on normal diet, despite manifestation of various other metabolic abnormalities. Mice were singly housed prior to indirect calorimetry to collect food intake data till the termination of the study. Two-way ANOVA revealed a genotype effect although post-hoc multiple comparisons were not significant (FIG. 43 A). No Ex4 treatment effects on average food intake (FIG. 43 A). As food intake was recorded after the body weight changes occurred, relative food intake was calibrated to body weight and no Ex4 treatment effects were observed (FIG. 43B).

[0673] Behavioral assessments in Magel2-null mice

[0674] Our previous research has demonstrated that hypothalamic BDNF gene therapy has no adverse behavioral effects and instead normalizes genotype-driven behavior in female Magel2-null mice including open field test and novel object recognition test. These two behavioral tests were performed at 5-weeks post AAV injection to assess safety and determine whether Rec2-Ex4 alters behavior in male Magel2-nu\\ mice. In the open field test assessing exploratory activity and anxiety-like behavior, a significant genotype-driven decrease in total distance traveled was observed in Magel2-nu\\ mice (FIG. 44A). Rec2-Ex4 treatment resulted in a trending but not significant increase in exploratory activity (FIG. 44A, Treatment effect P=0.06). Nevertheless, Rec2-Ex4 treatment ameliorated the genotype-driven deficit in exploratory activity (not significant, Magel2-nu\\ Ex4 versus WT GFP). No significant genotype or treatment effects on the percentage of distance travelled in the center of the open field arena were observed indicating no changes in anxiety-like behavior.

[0675] In addition, novel object recognition test was performed as previous report has shown that female Magel2-nu\\ mice are averse to novel objects and environments, manifesting a reduced discrimination index. However, no genotype effect was observed in the male Magel2-null mice (FIG. 44D). Rec2-Ex4 treatment had no significant effect on novel object discrimination index (FIG. 44D).Docket No. 103362-094WO1

[0676] Rec2-Ex4 gene therapy reduces adipose tissue mass and improves circulating biomarkers in Magel2-null mice

[0677] Mice were euthanized at 10-weeks post AAV injection and tissues were collected. Both genotype- and treatment-effect was observed regarding the final body weight (FIG. 33A). Rec2-Ex4 ameliorated excessive weight in the Magel2-nu\\ mice. Consistent with increase in adiposity measured by EchoMRI (FIGs. 32J, 42C) due to loss of Magel2, Rec2-GFP treated Magel2-nu\\ mice exhibited increased mass of the brown adipose tissue (BAT) (FIG. 33B) and three white adipose tissue (WAT) depots — the inguinal white adipose tissue (iWAT, FIG. 33C), gonadal white adipose tissue (gWAT, FIG. 33D), and retroperitoneal adipose tissue (rWAT, FIG. 33E) — compared to WT counterparts, ranging from 72% to 163% increase. Increases in Magel2-nu\\ adipose depots were reversed following Rec2-Ex4 gene therapy (FIGs. 33B-33E). Relative BAT and WAT weights calibrated to body weight were also normalized by Rec2-Ex4 treatment (FIGs. 45A-45D). A significant Ex4 treatment effect on liver weight was observed (FIG. 33F). The liver weight in Rec2-Ex4 treated Magel2-nu\\ mice was significantly reduced as compared to Rec2-GFP treated Magel2-nu\\ mice (FIG. 33F). Relative liver weight was significantly affected by both genotype and Ex4 treatment (FIG. 45E). Pancreas weight was increased in Rec2-Ex4 treated mice regardless of genotypes (FIGs. 33G, 45F) consistent with previous report that GLP-lRAs including Ex4 increase pancreas mass through induction of protein synthesis. Muscle loss has become a concern of GLP-1RA as weight loss treatment. Rec2-Ex4 did not alter the absolute gastrocnemius muscle weight (FIG. 33H) and ameliorated the decrease in relative gastrocnemius weight in Magel2-nu\\ mice (FIG. 45G).

[0678] Serum levels of Ex4 were measured by ELISA validating the secretion of Ex4 transgene product in Rec2-Ex4 treated mice (FIG. 34A, WT Ex4: 28.5+5.4 ng / mL; Afoge / 2-null Ex4: 10.6+2.6 ng / mL). Serum biomarkers were profiled to assess changes in systemic metabolism affected by genotype and Rec2-Ex4 treatment. No genotype-induced changes were observed in circulating glucose levels (FIG. 34B). Rec2-Ex4 treatment resulted in significant reduction in serum glucose levels after 4-h fast, an effect more prominent in WT mice (FIG. 34B). No genotype or Ex4 treatment effects on fasting serum insulin were observed (FIG. 34C). Similar results were found in the HOMA-IR (homeostatic model assessment for insulin resistance) index (FIG. 34D). Loss o Magel2 causes leptin resistance in the hypothalamic POMC neurons contributing to the metabolic dysfunctions in Magel2-nu\\ mice and hyperleptinemia. Indeed, Magel2-roi\\ mice treated with Rec2-GFP exhibited 3,4-folds increase in serum leptin over WTDocket No. 103362-094WO1

[0679] counterparts (FIG. 34E). The genotype-driven hyperleptinemia was reversed by Rec2-Ex4 treatment (FIG. 34E). Adiponectin is another key adipokine that plays important roles in insulin sensitivity, glucose homeostasis, and systemic metabolic function. The adiponectin / leptin ratio (with adiponectin level expressed in pg / mL and leptin level expressed in ng / mL) is negatively correlated with insulin resistance and low-grade chronic inflammation. This index is considered a predictive marker for adipose dysfunction and cardiometabolic risk associated with obesity better than leptin or adiponectin alone. Rec2-Ex4 treatment resulted in a significant increase in the adiponectin / leptin ratio, and the effect was more robust in the WT mice (FIG. 34G). No treatment-induced changes in serum triglyceride were observed (FIG.

[0680] 34H). Liver function markers alanine transaminase (ALT) and aspartate aminotransferase (AST) were not affected by genotype (FIGs. 34L34J). Rec2-Ex4 treatment resulted in a significant decrease in the serum ALT level, and the effect was more prominent in the Magel2-null mice (FIG. 341).

[0681] Rec2-Ex4 gene therapy normalizes adipose gene expression in Magel2-null mice

[0682] Given the robust effects on adipose tissue mass induced by both genotype and Ex4 treatment, gene expression was profiled in the largest visceral WAT depot, the eWAT (FIG.

[0683] 35 A) by qRT-PCR. Magel2-nu\\ mice showed a trend of Adipoq (encoding adiponectin) downregulation in the eWAT, which was reversed by Rec2-Ex4 treatment. Similarly, a genotype-driven downregulation of Cfd (encoding complement factor D, or adipsin) was rescued by Rec2-Ex4 treatment. Magel2-nu\\ mice exhibited a significant upregulation of Lep (encoding leptin) in eWAT as compared to WT, suggesting transcription changes contributing to the hyperleptinemia in addition to increases in total fat mass. Rec2-Ex4 treatment normalized the Lep expression level to the level in WT. Consistent with the alteration in Lep expression, a genotype-induced reduction in Adrb3 (encoding adrenoceptor P3), mediating sympathetic regulation, was observed, which was rescued by Rec2-Ex4 treatment. Pparg (encoding peroxisome proliferator-activated receptor gamma), a master regulator of adipogenesis and adipose function, was downregulated in the Magel2-nu\\ mice, which was also corrected by Rec2-Ex4 treatment. A genotype-driven downregulation of Vegfa (encoding vascular endothelial growth factor A), playing a role in induction of thermogenic competent cells in the WAT, was observed in Magel2-nu\\ mice. Rec2-Ex4 treatment resulted in a trend to higher Vegfa expression but not reach significance. No genotype- or treatment-induced changes in Ap2 (encoding adipocyte protein 2) and Ppargcla (encoding peroxisome proliferator-activatedDocket No. 103362-094WO1

[0684] receptor gamma coactivator 1 -alpha), a transcriptional coactivator regulating genes involved in energy metabolism, were observed in eWAT (FIG. 35 A).

[0685] Similarly to findings in the eWAT, Ex4 treatment-induced upregulations of Adipoq, CfcL and Vegfa were observed in the BAT (FIG. 35B). Interestingly, BAT exhibited genotype-induced upregulation of Adrb3, opposing to the downregulation in eWAT (FIG. 35 A). Nevertheless, Rec2-Ex4 treatment reversed this genotype-driven change (FIG. 35B). Ucpl (encoding uncoupling protein 1), the key thermogenic protein, was significantly upregulated by Rec2-Ex4 treatment. Moreover, Cidea (encoding cell death-inducing DNA fragmentation factor A), a brown fat protein, and Prdml6 (encoding PR-domain-containing 16), a transcription coregulator determining the formation and function of brown adipocytes, were downregulated in Magel2-nu\\ mice and these genotype-driven changes were rescued by Rec2-Ex4 treatment (FIG. 35B).

[0686] Rec2-Ex4 treatment alters hypothalamic and hepatic gene expression in the Magel2-null mice We previously reported that hypothalamic BDNF gene therapy modulated hypothalamic gene expression in Magel2-nu\\ mice. However, all mice received stereotaxic injection which could be a confounding factor. Here, a set of genes playing roles in regulating feeding and energy balance were profiled. No genotype- or treatment-induced changes were found in Bdnf, Mc4r (encoding melanocortin 4 receptor), and Glplr (encoding GLP-1 receptor). The expression of BDNF receptor — TrkB-FL (encoding tropomyosin receptor kinase B full length) was upregulated by Rec2-Ex4 treatment. Obrb (encoding leptin receptor long-form) expression was trending up following Rec2-Ex4 treatment but did not reach significance. Trh (encoding thyrotropin releasing hormone) was downregulated in Magel2-nu\\ mice while not altered by Rec2-Ex4 treatment (FIG. 36A).

[0687] As Rec2-Ex4 treatment led to a decrease in liver weight (FIG. 33F), we examined the gene expression of enzymes and signaling molecules critical for glucose and lipid metabolism (FIG. 36B). Rec2-Ex4 treatment led to significant downregulation of Fasn (encoding fatty acid synthase). No genotype- or gene therapy-induced changes were observed in the expression of Srebplc (encoding sterol regulatory element-binding protein 1, isoform c) or Scd2 (encoding stearoyl-coenzyme A desaturase 2). Protein kinase Cs (PKCs) is implicated in lipid-induced insulin resistance in the liver and subsequent impaired insulin-induced suppression of hepatic gluconeogenesis. A gene therapy-induced downregulation of Prkce (encoding PKCs) was observed in concomitant with a decrease in the expression of G6pc, encoding glucose-6-Docket No. 103362-094WO1

[0688] phosphatase, the major gluconeogenic enzyme (FIG. 36B), suggesting regulation of hepatic glucose production, possibly in part, contributing to the improved glucose tolerance in Rec2-Ex4 treated mice. No genotype-driven changes in Gck (encoding glucokinase) were observed while a trend toward downregulation was associated with Rec2-Ex4 treatment.

[0689] We previously reported that Rec2 capsid vector primarily transduced liver and visceral fat via IP injection. Consistent with previous findings, WPRE within the transgene transcript was detected in the liver and eWAT but not in the BAT or hypothalamus (data not shown).

[0690] IP administration of Rec2-Ex4 gene therapy ameliorates diet-induced obesity and associated metabolic syndromes

[0691] Next, the therapeutic potential of Rec2-Ex4 was evaluated in a nongenetic obesity model. C57BL / 6 mice were randomized to receive Rec2-Ex4 (4 x IO10vg per mouse) or AAV buffer as control via IP injection. Mice were maintained on high fat diet throughout the experiment. Mice receiving Rec2-Ex4 displayed significantly lower body weight as early as 1-week post AAV injection and sustained throughout the 21-weeks experiment (FIG. 37A). The average total weight gain was reduced by 39% following Rec2-Ex4 treatment as compared to control mice (FIG. 37B). The decrease in weight gain was associated with significant reduction in food intake (FIG. 37C) but not changes in energy expenditure (FIG. 46). At 5 -weeks post AAV injection, relative fat mass in Rec2-Ex4 treated mice was 76% lower than that in control mice (FIG. 37E). At 6-weeks post AAV injection, Rec2-Ex4 treated mice exhibited robust improvements in fasting blood glucose level and glucose tolerance (FIGs. 37F-37G).

[0692] Behavioral assessments were conducted between 13 - and 14-weeks post AAV injection. Rec2-Ex4 treated mice showed a significant increase in exploratory activity in the open field test while had no effect on anxiety-like behavior (FIG. 37H). Rec2-Ex4 did not alter novel object discrimination index in the novel object recognition test (FIG. 371).

[0693] At 21-weeks post AAV injection, a pyruvate tolerance test (PTT) was performed to examine hepatic glucose production. Out of the five control mice, one died during the PTT and two more died overnight likely due to pyruvate-induced torpor in obese mice. In contrast, all Rec2-Ex4 treated mice remained healthy. The experiment was terminated because the remaining two control mice appeared in risk of death. Livers from control mice were visibly pale. H& E staining revealed vast liver steatosis in the control mice, which was completely prevented by Rec2-Ex4 treatment (FIG. 37J).Docket No. 103362-094WO1

[0694] HA-tagged Ex4 gene therapy prevents dietary obesity and related metabolic dysfunction Because the long-term DIO experiment was terminated unexpectedly, we repeated the Rec2-Ex4 gene therapy in DIO model to collect tissues properly. Moreover, we added a hemagglutinin (HA) tag to the c-terminus of Ex4 transgene as a proxy for Ex4 transgene expression allowing better detection by immunoblotting. The amino acid sequence of the Ex4-HA transgene was shown in (FIG. 47A). HA signal was detected in the cell lysate and culture media confirming secretion of the Ex4-HA peptide (FIG. 47B).

[0695] C57BL / 6 mice were fed on HFD for five weeks and then randomized to three groups: Rec2-GFP, Rec2-Ex4, and Rec2-Ex4-HA, at the dose of 4x1010vg per mouse via IP injection. Mice were maintained on HFD throughout the experiment. Rec2-GFP treated mice continued to gain weight while both Rec2-Ex4 and Rec2-Ex4-HA treated mice lost weight within the first week after injection. HFD-induced excessive weight gain was completely prevented by Rec2-Ex4 or Rec2-Ex4-HA treatment (FIGs. 38A-38C). The average total weight gain over 9 weeks was reduced by approximately 6-folds in Rec2-Ex4 mice and 13-folds in Rec2-Ex4-HA mice as compared to Rec2-GFP mice (FIG. 38D). Consistent with findings in previous DIO model, food intake was significantly reduced in Rec2-Ex4 and Rec2-Ex4-HA treated mice (FIG. 38E) while no significant changes in energy expenditure were observed (FIG. 38H). A GTT at 6-weeks post AAV injection revealed significantly improved glycemic control in Rec2-Ex4 treated mice. Interestingly, mice receiving Rec2-Ex-HA exhibited further improvement over mice receiving Rec2-Ex4 (FIGs. 38F-38G).

[0696] Tissues were collected at 9-weeks post AAV injection. The weight of BAT and all three WAT depots were robustly reduced in both Rec2-Ex4 and Rec2-Ex-HA treated mice (FIG.

[0697] 39A). Enlarged livers were observed in obese mice receiving Rec2-GFP while the liver weights in Rec2-Ex4 and Rec2-Ex-HA treated mice were decreased by approximately 40% (FIG. 39A). Pancreas, heart, and gastrocnemius muscle weights were not different among the three groups (FIGs. 48F-48H). Relative tissue weights are shown in (FIG. 48A-48L).

[0698] Serum glucose and insulin levels were significantly lower in the Rec2-Ex4 and Rec2-Ex4-HA mice as compared to GFP controls (FIGs. 39B-39C). HOMA-IR was significantly improved in Rec2-Ex4 mice and Rec2-Ex4-HA mice suggesting enhanced insulin sensitivity (FIG. 39D). Rec2-Ex4 led to a 3-folds reduction in serum leptin level as compared to GFP control (FIG. 39E). Rec2-Ex4-HA appeared to furtherly reduce serum leptin, a 10-folds reduction as compared to GFP control (FIG. 39E). Although serum triglyceride levels were not changed (FIG. 39H), liver triglyceride content was significantly reduced by 7-folds in the Rec2-Docket No. 103362-094WO1

[0699] Ex4 mice and by 13-folds in the Rec2-Ex4-HA mice (FIG. 391). No changes in serum ALT and AST levels were observed (FIGs. 48I-48J).

[0700] Serum Ex4 levels were quantified by ELISA (FIG. 39J, Ex4: 49.5+6.8 ng / mL; Ex4-HA: 25.6+3.3 ng / mL). Immunoblotting of HA tag was performed as a proxy for Ex4 transgene expression in the liver and eWAT from mice treated with Rec2-Ex4-HA (FIGs. 39K-39L). To determine whether the lower serum Ex4 in Rec2-Ex4-HA treated mice was due to lower transgene expression in targeted tissues, WPRE mRNA levels were measured by qRT-PCR. Transgene transcript levels in the liver and eWAT from Rec2-Ex4-HA mice were not lower than their counterparts from Rec2-Ex4 mice (FIGs. 48K-48L). It is plausible that the HA-tagged Ex4 had altered antibody binding and thereby underestimating the serum Ex4 level in Rec2-Ex4-HA mice determined by ELISA (FIG. 48J).

[0701] Discussion

[0702] Growth hormone therapy and strict monitoring of daily food intake continue to be the standard of care for patients diagnosed with PWS. Growth hormone therapy has several drawbacks, such as the need for patient compliance, lacking efficacy evidence in older adults, and the exclusion of patients who have common comorbidities such as severe obesity, uncontrolled diabetes, or active psychosis. Caregivers for individuals with PWS experience high levels of caregiver burden due to strict supervision of their daily food intake and management of their emotional reactivity, which may surpass that of caregivers for patients with Alzheimer’s disease and traumatic brain injury. Melanotan II and setmelanotide have been among the recent pharmacological developments targeting the hypothalamic leptin-POMC pathway. Additional categories of therapeutics have been evaluated in animal models and clinical trials at various stages. Diazoxide choline extended-release tablet has recently been approved by FDA to treat severe hyperphagia in patients with PWS. However, this therapy is ineffective in patients without severe baseline hyperphagia and may be contraindicated for subsets of PWS patients due to serious side effects including hyperglycemia, diabetic ketoacidosis, and peripheral edema. Bariatric surgeries in PWS patients have much higher levels of complications than those observed in non-PWS obese patients and require postoperative compliance from the patient or caregiver to ensure adequate weight loss. Behavioral and exercise therapies have shown benefits in managing some symptoms but long-term patient compliance is difficult to achieve. As such, new therapeutic strategies for PWS remain an urgent and unmet need.Docket No. 103362-094WO1

[0703] Previous work on an autoregulatory BDNF vector represents the first AAV-based gene therapy for PWS. In addition to the PWS model Magel2-nu\\ mice, the hypothalamic BDNF gene therapy has shown high efficacy and safety in various genetic and dietary models of obesity including MC4R deficiency, leptin receptor deficiency, DIO, and normal aging model. However, brain-targeted gene therapies may subject to more stringent consideration of the risk / benefit ratio and administration to the hypothalamus remains highly challenging.

[0704] In this study, an IP injection of Rec2-Ex4 at a low dose of 2x1010vg per mouse normalized the metabolic abnormalities induced by loss of Magel2 including excessive adiposity, impaired glycemic control, and hyperleptinemia. IP Rec2-Ex4 gene therapy achieved metabolic improvements comparable to the efficacy of hypothalamic AAV1-BDNF gene therapy in the same PWS model. It is worthy of noting that the dose of this systemic gene therapy is equivalent to the dose of hypothalamic injection, 2x1010vg / mouse. When converted to a per Kg basis, the effective dose of Rec2-Ex4 is approximately 5x1011vg / Kg, one to two orders lower as compared to clinical systemic gene therapies that are often dosed 1013-1014vg / Kg. Magel2-nu\\ mice appeared highly sensitive to Rec2-Ex4 treatment exhibiting drastic weight loss and suppression in food intake at the dose of 4x1010vg / mouse. We recently report an IP injection of an adipose-targeting Rec2-leptin vector rescues congenital leptin deficiency at a dose as low as 1x109vg / mouse. It is plausible that the effective dose of Rec2-Ex4 can be furtherly reduced in the Magel2-nu\\ model of PWS, and a dose-deescalating study is warranted.

[0705] GLP-1RA drugs require repeated dosing and weight regain occurs following drug discontinuation. A recent study of a large US cohort with overweight or obesity finds that most patients discontinued GLP- IRA within one year, 47% of patients with and 65% without type 2 diabetes. The dropping-out rate is reported to increase to 85% after two years. The high discontinuation rates are associated with high cost, side effects, loss of reward, and other factors, causing concerns about the sustainability of weight loss to achieve long-term health benefits. On this note, an AAV-based GLP- IRA gene therapy that can be administered once to achieve long-term efficacy could offer a therapeutic option to patients with genetic obesity (e.g. PWS) who might need continuous treatment. Moreover, gene therapy induces a stable circulating GLP- IRA level that might be associated with milder or different pattern of side effects as compared to repeated bogus dosing of a peptide drug, warranting further investigation.

[0706] One new finding of the present study is revealing genotype-driven gene expression signature in the adipose tissue. In the visceral WAT, loss o Magel2 induced downregulation ofDocket No. 103362-094WO1

[0707] Adrb3, Adipoq, Cfd, Vegfa and upregulation of Lep, suggesting decreased sympathetic tone to the WAT. Importantly, Rec2-Ex4 treatment reversed this genotype-driven gene expression signature (FIG. 35 A). In the BAT, a cluster of thermogenic genes were downregulated in the Afoge / 2-null mice, which was restored by Rec2-Ex4 treatment (FIG. 35B), also indicating normalizing sympathetic tone to the BAT. Glplr expression in BAT and WAT was extremely low or undetectable by qRT-PCR. While not wishing to be bound by any one theory, the effect of Ex4 on adipose gene expression is unlikely due to direct effect on adipose tissue but rather possibly mediated by modulating the sympathetic tone. It is reported that GLP-1RA stimulates BAT thermogenesis independent of nutrient intake through hypothalamic mechanisms. Rec2-Ex4 gene therapy increased BDNF receptor TrkB FL and long-form leptin receptor Obrb expression in the hypothalamus (FIG. 36A). As aberrant hypothalamic leptin-POMC pathway underlies metabolic dysregulation in the Magel2-nu\\ model and BDNF acts downstream of this circuit regulating food intake and energy balance, data of present study raises the possibility of addressing the root cause of metabolic abnormalities in PWS through a systemic Ex4 gene therapy.

[0708] Scarce data regarding hepatic phenotypes in the Magel2-nu\\ model has been reported. In this study, genotype-driven changes in the liver were unremarkable. However, Rec2-Ex4 treatment resulted in a significant decrease in liver weight and downregulation of genes involved in de novo lipogenesis and hepatic glucose production. Whether and how hepatic regulation mediates the whole-body metabolic improvement following Rec2-Ex4 treatment requires further investigation.

[0709] In this proof-of-concept study, behavioral assessments were limited to the two tests that were altered in the M age! 2 -nu\\ mice following hypothalamic BDNF gene therapy. In the open field test, genotype-driven reduction in exploratory behavior was mitigated by Rec2-Ex4 treatment similarly to the observation in the hypothalamic BDNF study. In contrast to previous data in female Magel2-nu\\ mice, neither genotype-driven deficit nor treatment-induced changes were observed in novel object recognition in the male mice of the present study. Current data indicate that a low dose systemic gene therapy of Rec2-Ex4 was sufficient to rescue the genotype-driven deficit in exploratory behavior and modulate hypothalamic gene expression in male mice, supporting further investigation on behavioral effects. Emerging evidence has revealed potential therapeutic effect of GLP-lRAon mental health and psychiatric diseases. Future research will expand preclinical assessments to female Magel2-nu\\ mice, increase the number of mice, and perform more comprehensive behavioral phenotyping to investigate potential therapeutic benefits beyond metabolic improvement.Docket No. 103362-094WO1

[0710] MAGEL2 is one of many genes within the PWS deletion region of chromosome 15ql 1 -ql3. Notably, Magel2-mA\ mice do not mirror the hyperphagia of human PWS when being maintained on normal chow diet. It is interesting to see whether Rec2-Ex4 exerts more pronounced effects in models or conditions manifesting hyperphagia. Moreover, testing in other preclinical models of PW S such as Snordll6 and Ndn will be evaluated to further confirm the therapeutic potential of Rec2-Ex4 gene therapy. In addition, Schaaf-Yang syndrome is a rare genetic disease driven by loss-of-function of MAGEL2, displaying many PWS-like phenotypes but lack of hyperphagia and obesity.

[0711] In addition to genetic obesity such as the PWS model o Magel2 deficiency, Rec2-Ex4 via IP administration was highly effective in the dietary obesity model — reversing excessive weight gain, decreasing food intake, reducing adiposity, improving glucose metabolism, enhancing insulin sensitivity, and preventing hepatic steatosis, at a low dose of 4x1010vg per mouse. Given the potent therapeutic effects and high circulating levels of Ex4, we predict the effective dose can decrease further. We are planning a dose-finding study in DIO models prior to expanding investigations to additional preclinical models of diseases related to obesity. AAV5-mediated Ex4 gene therapy targeting salivary glands improved insulin sensitivity but did not decrease food intake or body weight in DIO mice. Notably, IP delivery of Rec2-Ex4, at 6-folds lower dose, resulted in 50-folds higher circulating Ex4 level as compared to the AAV5-Ex4 targeting salivary glands. Further optimizing the AAV-Ex4 vector will improve translational potential.

[0712] Adding HA tag to the c-terminus of Ex4 did not interfere with the actions of Ex4 transgene product in vivo. On the contrary, Rec2-Ex4-HA exerted better effects over Rec2-Ex4 in GTT and serum leptin level. Whether the HA tag alters the property of Ex4 peptide remains unknown. It is reported that hydrodynamic delivery of a fusion gene in which a-antitrypsin was fused to the c-terminus of Ex4 was effective in DIO model. Further engineering the c-terminus of Ex4 might be worthy of consideration to improve therapeutic efficacy.

[0713] Development and optimization of AAV capsid vector and minimally invasive administration methods continue to be an area of focus on the path to clinical development. We have developed a Rec2 capsid mutant with three residue substitutions (named V7) that exclusively transduces adipose tissue while ablating liver tropism, thereby eliminating AAV sequestering in the liver and minimizing severe adverse events related to liver toxicity that has been a major challenge to the field of systemic gene therapies. Moreover, subcutaneous injection of V7 vector carrying human leptin gene (V7-LEP) restored leptin levels andDocket No. 103362-094WO1

[0714] normalized metabolic syndromes in the ob / ob mice at the dose of 4x1010vg per mouse, equivalent to the IP delivery in the same animal model. In an ongoing experiment, one-time subcutaneous injection of V7-LEP to extremely obese ob / ob mice leads to stable circulating human leptin level and weight loss for at least 12 months. We suppose that adipose-targeting gene delivery may have advantages as compared to other peripheral tissues such as liver: 1) as human adipose tissue has a lifespan of 10 years in contrast to 3 years for liver, a one-time dosing of adipose-targeting AAV is expected to be effective 5-10 years; 2) accessible administration as subcutaneous drug delivery (e.g. GLP-1 drugs) in humans is in fact injected to subcutaneous fat; 3) fat tissues can be easily removed if treatment cessation is needed. To leverage these new technological advancements, we will refine the Ex4 transgene design and develop an adipose-tropic AAV vector to deliver the therapeutic gene via subcutaneous administration in models of genetic and acquired metabolic diseases.

[0715] Admittedly, Rec2-Ex4 treatment, although likely long-lasting, is not permanent. It is reported that in vivo genome editing targeting liver led to elevation of Ex4 and blunted weight gain for 28 weeks in DIO mice. Of note, the circulating Ex4 levels in the Ex4 knock-in mice were sharply reduced at 2-weeks post injection of Crispr / Cas9 plasmids-loaded lipid nano particles and stabilized at ~ 0.1 ng / mL throughout the 28-weeks study, which was approximately 500-fold lower than levels achieved with Rec2-Ex4 treatment (49.5+6.8 ng / mL). It would be interesting to investigate whether Rec2 vector could improve in vivo genome editing particularly targeting adipose tissue. A recent report demonstrates that subcutaneous delivery of Ex4 via a lipid nanoparticle-based DNA delivery system improved metabolic outcomes in DIO mice, suggesting a potential alternative to viral vectors. However, circulating Ex4 concentrations were not reported, and therapeutic efficacy was assessed only up to 8 weeks. Direct comparison of AAV-based and lipid nanoparticle-based platforms is warranted to determine relative efficacy, durability, and safety.

[0716] Furthermore, this vector system is being developed as a platform for delivery of therapeutic peptides particularly those not derived from a native mammalian gene. For example, transgenes are designed to express engineered BDNF peptide variants that might improve the unfavorable pharmacokinetics of native or recombinant BDNF protein as an alternative to hypothalamic AAV-BDNF gene therapy for Magel2-nu\\ mouse model of PWS.

[0717] In summary, presented herein are preclinical data that suggest intraperitoneal administration of Rec2-Ex4 gene therapy at a low dose normalizes metabolic dysfunction and improves exploratory behavior in the PWS model of Mage! 2 -nu\\ mice. Furthermore, Rec2-Docket No. 103362-094WO1

[0718] Ex4 gene therapy is efficacious and safe for treatment of dietary obesity. These proof-of-concept data indicate Rec2-Ex4 as a potential molecular therapy for genetic and acquired forms of obesity.

[0719] Materials and Methods

[0720] Animals

[0721] Magel2-n\A\ mice harbor a maternally inherited imprinted / silenced wild type allele and a paternally inherited Magel2-lacZ knock-in allele that abolishes endogenous Magel2 gene function. Male mice containing the Magel2-lacZ allele (Jackson Labs #009062) were bred with female C57BL / 6 mice to produce both wildtype mice and Magel2-nu\\ littermates. Mice were genotyped from ear notch biopsies. Identification of mutant offspring was performed by polymerase chain reaction genotyping with Magel2 and LacZ oligonucleotide primers (common forward, 5'-ATGGCTCCATCAGGAGAAC (SEQ ID NO: 131); Magel2 reverse, 5'-GATGGAAAGACCCTTGAGGT (SEQ ID NO: 132); and LacZ reverse, RW4237, 5'-GGGATAGGTCACGTTGGTGT (SEQ ID NO: 133)). Genotype was further confirmed by qRT-PCR detecting Magel2 transcript in the hypothalamus. For diet-induced obesity experiment, C57BL / 6 mice were purchased from Jackson Laboratory. All mice had ad libitium access to water and normal chow diet (11% fat, caloric density 3.4 kcal / g, Teklad) or high-fat diet (60% kcal from fat; caloric density 5.2 kcal / g, Research Diets, Inc. #D 12492) as specified in each experiment. Mice were housed in standard laboratory cages within temperature (22-23°C) and humidity (30-70%) controlled rooms under a 12:12 light:dark cycle. All animal experiments were in accordance with the regulations of The Ohio State University’s Institutional Animal Care and Use Committee (IACUC).

[0722] Ex4 transgene design and in vitro validation

[0723] Ex4 transgene was designed in which the Exendin-4 sequence (encoding 39-amino acid) was linked to the 21 -amino acid signal peptide sequence of mouse leptin gene with an extra Valine (FIG. 32A). This transgene Ex4 was then cloned to an AAV expression plasmid driven by a cytomegalovirus enhancer and a chicken B-actin promoter (CB A promoter). In vitro transfection experiment was carried out in HEK293 cells by Lipofectamine 3000 (Invitrogen).

[0724] A A V vector construction, packaging, and administration

[0725] Transgenes: Ex4, Ex4-HA or GFP were inserted in the AAV expression cassette contains a hybrid cytomegalovirus-chicken P-actin (CBA) promoter, woodchuckDocket No. 103362-094WO1

[0726] posttranscriptional regulatory element (WPRE) and bovine growth hormone polyadenylation signal (BGH poly A) flanked by two AAV2-inverted terminal repeats (ITR). Plasmids used for AAV packaging were prepared by using EndoFree plasmid Maxi and Mega Kit (Qiagen). Human embryonic kidney 293 cells were co-transfected with three plasmids — AAV cis-plasmid containing transgene, AAV trans-plasmid encoding rep and Rec2 cap genes and adenoviral helper pF A6 — using standard CaPO4transfection protocol. AAV vector was purified from the cell lysate by ultracentrifugation through an iodixanol density gradient (OptiPrep Density Gradient Medium, D1556, Sigma). Vector titers were determined by quantitative PCR as previously described. Some AAV vectors were packaged by VectorBuilder. AAV vectors were administered by IP injection in 150 pL AAV dilution buffer.

[0727] Magel2-null mice experiment

[0728] Pilot study: Magel2-nu\\ mice (4-5 months of age) were randomized to receive an IP injection of either Rec2-GFP or Rec2-Ex4 at the dose of 4x1010vg per mouse and fed on HFD after AAV injection. Four female mice and one male mouse each group. The experiment was terminated two weeks after AAV injection due to extreme weight loss.

[0729] Study with lower dose: Male Magel2-nu\\ mice (4-19 months of age) and sex- and age-matched wildtype littermates were randomized to receive an IP injection of either Rec2-GFP or Rec2-Ex4 at the dose of 2x1010vg per mouse. Mice were maintained on normal chow throughout the experiment. Metabolic and behavioral assessments were conducted as indicated in FIG 32B.

[0730] DIO model

[0731] Long-term experiment: Male C57BL / 6 mice (19 weeks of age) were randomized to receive an IP injection of Rec2-Ex4 (4x1010vg per mouse) or AAV buffer. Mice were fed on HFD throughout the 21 -weeks experiment.

[0732] Short-term experiment: Male C57BL / 6 mice (5 weeks of age) were fed on HFD for 5 weeks and then randomized to receive an IP injection of Rec2-Ex4, Rec2-Ex4-HA, or Rec2-GFP at the dose of 4x1010vg per mouse. Mice were maintained on HFD till euthanasia 9 weeks post AAV injection.

[0733] Body weight and food intake measurement

[0734] Body weight was recorded weekly. Food intake was measured at the cage level on a weekly basis. Food intake data collected in weeks during which invasive assessments occurredDocket No. 103362-094WO1

[0735] that disrupted normal feeding patterns (e.g., removal from home cages for indirect calorimetry monitoring and fasting periods for glucose tolerance tests) were excluded. Average food intake was calculated on a per-mouse per-day basis.

[0736] Body composition assessment

[0737] Body composition analysis was performed with a 3-in-l Analyzer (EchoMRI LLC, Houston, TX) according to manufacturer instructions. Mice were subjected to a 5-Gauss magnetic field and whole-body masses of fat, lean, free water, and total water were determined during separate cycles by manufacturer software comparison to a canola oil standard.

[0738] Glucose tolerance test (GTT)

[0739] Mice were overnight fasted with water ad libitum. The baseline blood glucose level was measured via tail niche with a portable glucose meter using default manufacturer settings (Bayer Contour Next). Then, mice were IP injected with glucose solution at a dose of Img glucose per gram body weight. Blood glucose concentrations were measured at 15, 30, 60, 90, and 120 min after glucose injection. Following blood collection, styptic powder was used to stop bleeding.

[0740] Indirect calorimetry

[0741] Mice underwent indirect calorimetry using a Comprehensive Laboratory Animal Monitoring System (CLAMS, Columbus Instruments, Columbus, OH) at CLAMS core, Davis Heart and Lung Research Institute of The Ohio State University Medical Center. Mice were placed in the calorimetry chamber individually with an access to water and standard chow diet for Magel2-nvX\ study or high fat diet for DIO study. The various physiological and behavioral parameter (VO2, VCO2, respiratory exchange ratio, energy expenditure, and ambulation) were recorded for 48 hrs. The first 24-h period is considered as habituation time and therefore excluded from data analysis. Mice were returned to their home cages after indirect calorimetry was performed.

[0742] Open field test

[0743] Mice were individually placed into the center of an open square arena (60 cm x 60 cm, enclosed by walls of 48 cm). Each mouse was allowed to explore the arena for 10 min, during which time and locomotion — in the center and the periphery of the open field — was recordedDocket No. 103362-094WO1

[0744] and analyzed via TopScan (Clever Sys, Inc.) software. Between each trial, the arena was cleaned with 70% ethanol to remove odor cues.

[0745] Novel object recognition test

[0746] For the test habituation period, mice were placed in an open arena (30 x 30 cm, enclosed by walls of 50 cm) with no objects present and allowed to explore the empty arena for 5 minutes. The habituation period was typically conducted 24 hours prior to the test. For the familiarization period, mice were placed in the open arena with two identical objects (either two Falcon tubes filled with water or two stacks of large Legos). Mice were allowed to explore the identical objects for 10 minutes while being recorded with an overhead camera. Mice were returned to their home cage while the arena / objects were cleaned with 70% ethanol. During the novel object recognition test session, the two training objects were replaced with one matched item from the training session and a novel item. Mice were placed in the arena and allowed to explore both the novel and learned object for 10 minutes while being recorded with an overhead camera setup. Mice were returned to their home cages after 10 minutes and the arena / objects were cleaned with 70% ethanol. Video recording was analyzed using a “huggingface” open-source event-logging software. When scoring the familiarization and testing period recordings, we monitored the time spent interacting with each object until 20 s of cumulative exploration time was achieved between both objects, with a maximal time of 10 min to reach the criteria; those that did not meet the criteria were excluded from statistical analyses. Time spent exploring each respective object was recorded. Exploration activity was defined as “directing the nose toward the object at a distance less than or equal to 2 cm” and time spent climbing on or chewing on objects was not deemed exploration activity. The discrimination index was calculated by dividing the novel object exploration time by the total amount of object exploration during the test.

[0747] Tissue collection

[0748] Mice were euthanized following a 4 h fast. Mice were anesthetized with 2.5% isoflurane (1.0 L / min) and then decapitated to collect trunk blood. Hypothalamus was collected under a dissection microscope. Adipose tissues (BAT, iWAT, eWAT, rWAT), liver, heart, pancreas, and gastrocnemius muscle were collected and weighed. Tissues to be used for mRNA and protein analyses were flash frozen on dry ice and stored at -80°C until further analysis.Docket No. 103362-094WO1

[0749] Serum harvest and analysis

[0750] Trunk blood was collected at euthanasia, clotted on ice, and centrifuged at 10,000 rpm for 10 min at 4°C. The serum component was collected and stored at -20°C until further analysis. ELISAs were performed to assay serum leptin (R and D Systems Cat# DY498), serum adiponectin (R and D Systems Cat# DY1119), serum insulin (Alpco Diagnostics Cat# 80-INSMSU-E01), and serum exendin-4 (Phoenix Pharmaceuticals Cat#EK-070-94). Caymen Chemical colorimetric assay kit was used to assay glucose (#10009582) and triglycerides (#10010303). HOMA-IR (homeostatic model assessment for insulin resistance) index was calculated as [fasting serum glucose (mmol / L) x fasting serum insulin (pmol / L) / 22.5], Alanine transaminase (ALT) and aspartate aminotransferase (AST) levels were determined by Comparative Pathology & Digital Imaging Shared Resource at Ohio State University Comprehensive Cancer Center Department of Veterinary Biosciences.

[0751] Liver triglycerides assay

[0752] Hepatic lipid was extracted from 15-30 mg of wet liver tissues by chloroform / methanol (2: 1 v / v), followed by rinse in 50 mM NaCl and CaCl₂ (0.36M) / Methanol (1: 1 v / v). An aliquot of the extract was mixed with Triton X100 and cold acetone, then was dried up and redissolved in PBS. Hepatic triglyceride quantification was performed with a Caymen Chemical triglyceride assay kit (Caymen Chemical, #10010303).

[0753] Histology

[0754] Liver tissues were fixed in 10% formalin. Paraffin embedding, sectioning, and H& E staining were conducted by Comparative Pathology & Digital Imaging Shared Resource at Ohio State University Comprehensive Cancer Center Department of Veterinary Biosciences.

[0755] Quantitative RT-PCR

[0756] Following tissue sonication, RNA was isolated using the RNeasy Mini kit (QIAGEN #74804) with RNase-free DNase treatment. cDNA was reverse transcribed using Taqman Reverse Transcription Reagents (Applied Biosystems #N8080234). qRT-PCR was completed on the StepOnePlus Real-Time PCR System using Power SYBR Green PCR Master Mix (Applied Biosystems #A25742). Data were calibrated to endogenous controls — Hprtl for hypothalamus, Ppia for liver, Actb for adipose tissue. The relative gene expression was quantified using the 2-ΔΔCTmethod.Docket No. 103362-094WO1

[0757] Western blotting

[0758] Tissue samples were homogenized in RIPA buffer (Pierce #89901) containing 1x PhosSTOP (Roche #4906845001) and protease inhibitor cocktail III (Calbiochem #539134). Tissue lysates were separated by gradient gel (4-20%, Mini-PROTEAN TGX, Bio-Rad #4561096) and then transferred to a nitrocellulose membrane (Bio-Rad #1620115). Blots were incubated overnight at 4 °C with the following primary antibodies: GAPDH (Millipore Sigma Cat#CB1001-500UG, 1:1000), HA-tag (Cell Signaling Technology #3724, 1:1000), Exendin-4 (Bioss Antibodies Cat#bs-4121R, 1:500), P-Actin (Cell Signaling Technology # 4970, 1:1000). Blots were rinsed and incubated with HRP-conjugated secondary antibodies (BioRad, 1:3000). Chemiluminescence signal was detected and visualized by Odyssey Fc imaging (LI-COR Biotechnology, Lincoln, NE). Quantification analysis was carried out with Image Studio software version 5.2 (LI-COR Biotechnology).

[0759] Statistical Analysis

[0760] Statistical analyses were performed using GraphPad Prism 10 software (GraphPad, San Diego, CA, USA). Unpaired t test with Welch’s correction was utilized for comparisons between two groups. One-way ANOVA with Tukey’s post hoc test were used for comparisons between three groups. Two-way ANOVAs with Tukey’s post hoc test were used for comparisons between four groups (genotype and Ex4 treatment as factors). Time course data (BWs, GTT, indirect calorimetry) were analyzed using Two-way RM ANOVA and area under the curve calculations were performed where applicable. Normality was tested using the Shapiro-Wilk method. Significance was defined as P < 0.05. Data are reported as means ± SEM. Sample sizes (n) can be found in each figure’s respective legend.

[0761] Example 6. Obesity associated colon cancer model

[0762] Obesity is a major risk factor for 13 types of cancer including colon cancer. To assess whether gene delivery of Exendin-4 can alter colon cancer progression in obese condition, Rec2 -Exendin-4 was intraperitoneally injected to diet induced obese mice at the dose of 4E10 vg per mouse. Obese mice receiving AAV buffer served as control. Five days after AAV injection, syngenic MC38 colon cancer cells were implanted subcutaneously at the flank of all mice (4E5 cells per mouse). Mice were maintained on high fat diet throughout the experiment and euthanized 3 weeks post AAV injection and 16 days post MC38 cancer cell implantation.Docket No. 103362-094WO1

[0763] Rec2 -Exendin-4 treatment resulted in significant weight loss as early as one week post AAV injection in contrast to the continuous weight gain in control mice (FIG. 49). Consistent with weight loss, liver mass and visceral fat mass were significantly reduced in Rec2-Exendin-4 treated mice. The robust metabolic effects were associated with high level of circulating Exendin-4. However, MC38 tumor mass was not different between the groups (FIG. 50). Molecular and pathological analyses of the tumors are underway.

[0764] The pilot study demonstrated Rec2 -Exendin-4 potently alleviated obesity but exerted no effect on subcutaneous implanted MC38 tumor growth within a short treatment window. Future studies will employ better colon cancer models such as orthotopic implantation models and genetic models to assess the therapeutic potential of Exendin-4 gene therapy for prevention and treatment of obesity-associated colon cancer.

[0765] Example 7. Congenital generalized lipodystrophy model

[0766] Lipodystrophy syndromes comprise a heterogeneous group of inherited or acquired disorders characterized by abnormal or degenerative adipose tissue. Lipodystrophy is associated with significant metabolic complications including diabetes, severe insulin resistance, dyslipidemia, hypertension, fatty liver, and accelerated atherosclerosis. GLP-1 drugs have been proposed to manage lipodystrophy-induced metabolic syndrome.

[0767] aP2-SREBPlc transgenic mice overexpress human nuclear sterol regulatory elementbinding protein-lc (nSREBP-lc / ADDl) specifically in adipose tissue. This transgenic mouse strain exhibits many of the features of congenital generalized lipodystrophy (CGL), including disorderly differentiation of adipose tissue, marked insulin resistance, and fatty liver. To assess whether gene delivery of Exendin 4 can improve metabolic outcomes in lipodystrophy model, aP2-SREBPlc transgenic mice, both sexes, were randomized to receive either Rec2-GFP or Rec2-Exendin 4 via subcutaneous injection where the interscapular brown adipose tissue resides. To avoid substantial weight loss in the lipodystrophy model, a lower dose of 1E10 vg / mouse was used in contrast to 4E10 vg / mouse in models of obesity.

[0768] Rec2-Exendin 4 exerted no significant effect on weight gain or food intake in both male and female mice (FIG. 51). At 4 weeks post AAV injection, an insulin tolerance test was conducted to assess insulin sensitivity. Female transgenic mice treated with Rec2 -Exendin 4 showed a trend towards improved insulin sensitivity (FIG. 52), which was not observed in male mice (FIG. 53). Sex-dependent response was also observed in glucose tolerance test at 5 weeks post AAV injection. Rec2 -Exendin 4 led to significantly improved glycemic control in male transgenic mice but not in the females (FIGs. 54-55). Circulating exendin-4 levels wereDocket No. 103362-094WO1

[0769] 1.08±0.24 ng / ml in male mice and 1.07±0.12 ng / ml in female mice, at least 10-folds lower than the exendin-4 levels in obesity models by intraperitoneal injection at 4E10 vg / mouse.

[0770] These data suggest congenital generalized lipodystrophy model aP2-SREBPlc transgenic mice were sensitive to a low dose subcutaneous Rec2 -Exendin 4 gene therapy in a sex-dependent manner. A dose finding study with larger number of mice is warranted.

[0771] Example 8. Unimolecular GLP-lR / GIPR / GcgR Triagonist

[0772] A unimolecular peptide agonist acting on GLP-1 receptor (GLP-1R), glucosedependent insulin-tropic peptide receptor (GIPR), and glucagon receptor (GcgR) has demonstrated potential for greater weight loss in broader population, superior correction of metabolic syndromes, with less risk of insufficient glycemic control (Tschop et al. Cell Metab 2016, 24: 51-62). A transgene was designed in which the GLP-1 R / GIPR / GcgR triagonist sequence encoding a 41 -amino acid was linked to the 21 -amino acid signal peptide sequence of leptin gene (SEQ ID NO: 5) with an extra Valine (SEQ ID NO: 6). An HA tag sequence (SEQ ID NO: 15) — encoding a 9-amino acid derived from the human influenza hemagglutinin protein (SEQ ID NO: 18) was added to the C-terminus of triagonist to detect transgene product (FIG 56).

[0773] Triagonist transgene was cloned into an AAV expression plasmid in which expression was driven by the CBA promoter. An in vitro transfection experiment showed that the HA-tagged triagonist protein was detected in the cell culture medium of HEK 293 cells by Western blotting, indicating the triagonist transgene product being secreted from the transfected cells (FIG. 57). Of note, the transgene product levels in the cell lysate and the supernatant were comparable between HA-tagged Exendin 4 and HA-tagged triagonist in in vitro transfection experiment.

[0774] Example 9. Secretory Enhanced green fluorescent protein

[0775] To test additional signal peptide, a reporter transgene (SEQ ID NO: 69) was designed consisting of a nucleic acid sequence (SEQ ID NO: 70) encoding a signal peptide sequence from human insulin gene (SEQ ID NO: 73) and a nucleic acid sequence (SEQ ID NO: 71) encoding an enhanced green fluorescent protein (SEQ ID NO: 74), termed secretory GFP (SEQ ID NO: 72) (FIG. 58). An in vitro transfection experiment confirmed transgene protein product in cell lysates by Western blotting. However, no GFP protein was detected in the supernatants 72 hours post transfection to HEK 293 cells (FIG. 59) indicating failure in secretion.Docket No. 103362-094WO1

[0776] Transfection experiments using additional cell lines will be conducted to validate the efficacy of insulin signal peptide.

[0777] To assess the long-term durability of adipose-targeting AAV gene therapy, Rec2 vector harboring mouse leptin gene was injected subcutaneously to ob / ob mice, a congenital leptin deficiency model, at the dose of 2E10 vg per mouse. Rec2 -Leptin gene therapy restored circulating leptin level and normalized body weight throughout the experiment of 14 months (FIGs. 60-61). AAV genome DNA and transgene mRNA were detected in the targeted subcutaneous adipose tissue while absent in liver and other peripheral organs / tissues (FIGs.

[0778] 62-63). Remarkably, circulating leptin levels remained stable from 4-weeks to 14-months post one-time subcutaneous administration (FIG. 61) without a substantial drop that is often observed in liver-targeting AAV-mediated gene delivery.

[0779] These data suggest adipose-targeting AAV can achieve long-lasting and stable transgene expression. Given the 10-year lifespan of human adipocytes, a single dose of adipose-targeting AAV gene therapy is expected to be effective for 5-10 years, offering a promising alternative with potential advantages over current standard of care requiring continuous repeated administration of protein / peptide drugs.

Claims

1. Docket No. 103362-094WO1CLAIMSWhat is claimed is:

1. A recombinant polypeptide comprising:(a) a leptin signal peptide sequence;(b) a linker peptide comprising a valine residue; and(c) a therapeutic peptide, wherein the therapeutic peptide is selected from a peptide hormone, a GLP1 receptor agonist peptide, a glucagon-like peptide, a biomimetic peptide, an immunomodulatory peptide, a neurotoxic peptide, a neurotrophic factor peptide, a bone morphogenetic peptide, a guanylate cyclase- C (G-CC) agonist peptide, a calcitonin receptor inhibitor, a GnRH receptor inhibitor, a 20S proteasome inhibitor, a N0D2 inhibitor, a VIP1 receptor inhibitor, an OT inhibitor, a TRH receptor inhibitor, an MC receptor inhibitor, a PTH1 receptor inhibitor, a guanylate cyclase C inhibitor, an NPR-A inhibitor, an ATI receptor inhibitor, a beta2 -receptor inhibitor, a gp41 inhibitor, a GHRH receptor inhibitor, an N-type calcium channel inhibitor, a thrombopoietin receptor inhibitor, a human erythropoietin receptor inhibitor, a pulmonary surfactant inhibitor, a CaSR inhibitor, an MCI receptor inhibitor, a somatostatin receptor inhibitor, a melanocortin-4 receptor inhibitor, an immune checkpoint inhibitor peptide, or an analog thereof.

2. The recombinant polypeptide of claim 1, wherein the leptin signal peptide sequence comprises SEQ ID NO: 5, or an amino acid sequence at least about 95% identical thereto.

3. The recombinant polypeptide of claim 1 or 2, wherein the leptin signal peptide sequence is encoded by a nucleic acid sequence comprising SEQ ID NO: 2, or a nucleic acid sequence at least about 95% identical thereto.

4. The recombinant polypeptide of any one of claims 1-3, further comprising one or more additional peptides.

5. The recombinant polypeptide of claim 4, wherein the one or more additional peptides comprise a TAT cell-penetrating peptide and / or an HA tag peptide.

6. The recombinant polypeptide of any one of claims 1-5, wherein the therapeutic peptide comprises an exendin-4 peptide, a GLP1 receptor agonist peptide, a neurotrophic factor peptide, a bone morphogenetic peptide, a GLP-1R peptide, a GIPR peptide, a GcgR peptide, or a combination thereof.

7. A recombinant polypeptide comprising:Docket No. 103362-094WO1(a) a leptin signal peptide sequence;(b) a linker peptide comprising a valine residue; and(c) an exendin-4 peptide.

8. The recombinant polypeptide of claim 7, wherein the exendin-4 peptide comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid at least about 95% identical thereto.

9. The recombinant polypeptide of claim 7 or 8, wherein the exendin-4 peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 3, or a nucleic acid sequence at least about 95% identical thereto.

10. The recombinant polypeptide of any one of claims 7-9, comprising the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence at least about 95% identical thereto.

11. The recombinant polypeptide of any one of claims 7-10, encoded by a nucleic acid sequence comprising SEQ ID NO: 1, or a nucleic acid sequence at least about 95% identical thereto.

12. The recombinant polypeptide of any one of claims 7-11, further comprising one or more additional peptides.

13. The recombinant polypeptide of claim 12, wherein the one or more additional peptides comprise a TAT cell-penetrating peptide and / or an HA tag peptide.

14. The recombinant polypeptide of claim 12 or 13, comprising the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence at least about 95% identical thereto.

15. A recombinant polypeptide comprising:(a) a leptin signal peptide sequence;(b) a linker peptide comprising a valine residue; and(c) a GLP-1 peptide.

16. The recombinant polypeptide of claim 15, wherein the GLP-1 peptide is selected from GLP-1 (l-36amide), GLP-1 (7-36amide), or GLP-1 (7-37).

17. The recombinant polypeptide of claim 15 or 16, wherein the GLP-1 peptide is GLP-1 (7-36amide).

18. The recombinant polypeptide of any one of claims 15-17, wherein the GLP-1 peptide comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence at least about 95% identical thereto.Docket No. 103362-094WO119. The recombinant polypeptide of any one of claims 15-18, wherein the GLP-1 peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 10, or a nucleic acid sequence at least about 95% identical thereto.

20. The recombinant polypeptide of any one of claims 15-19, wherein the GLP-1 peptide comprises one or more mutations to increase stability.

21. The recombinant polypeptide of any one of claims 15-20, comprising the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence at least about 95% identical thereto.

22. The recombinant polypeptide of any one of claims 15-21, encoded by a nucleic acid sequence comprising SEQ ID NO: 8, or a nucleic acid sequence at least about 95% identical thereto.

23. A recombinant polypeptide comprising:(a) a leptin signal peptide sequence;(b) a linker peptide comprising a valine residue; and(c) a brain-derived neurotrophic factor (BDNF) peptide.

24. The recombinant polypeptide of claim 23, further comprising a TAT cell-penetrating peptide.

25. The recombinant polypeptide of claim 24, wherein the TAT cell-penetrating peptide comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid at least about 95% identical thereto.

26. The recombinant polypeptide of claim 24 or 25, wherein the TAT cell-penetrating peptide is operatively conjugated to the amino terminus and / or the carboxy terminus of the BDNF peptide.

27. The recombinant polypeptide of any one of claims 23-26, wherein the BNDF peptide comprises pro-BDNF or mature BDNF (mBDNF).

28. The recombinant polypeptide of claim 27, wherein the mBDNF peptide comprises the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence at least about 95% identical thereto.

29. The recombinant polypeptide of claim 27 or 28, wherein the mBDNF peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 14, or a nucleic acid sequence at least about 95% identical thereto.

30. The recombinant polypeptide of any one of claims 23-29, comprising the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence at least about 95% identical thereto.Docket No. 103362-094WO131. The recombinant polypeptide of any one of claims 23-30, encoded by a nucleic acid sequence comprising SEQ ID NO: 13, or a nucleic acid sequence at least about 95% identical thereto.

32. The recombinant polypeptide of any one of claims 24-31, comprising the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 24, or an amino acid sequence at least about 95% identical thereto.

33. The recombinant polypeptide of any one of claims 24-30 or 32, encoded by a nucleic acid sequence comprising SEQ ID NO: 19 or SEQ ID NO: 23, or a nucleic acid sequence at least about 95% identical thereto.

34. A recombinant polypeptide comprising:(a) a leptin signal peptide sequence;(b) a linker peptide comprising a valine residue; and(c) a peptide derived from bone morphogenetic protein 7 (BMP7).

35. The recombinant polypeptide of claim 34, wherein the peptide derived from BMP7 comprises pro-BMP7 or mature BMP7 (mBMP7).

36. The recombinant polypeptide of claim 35, wherein the mBMP7 peptide comprises the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence at least about 95% identical thereto.

37. The recombinant polypeptide of claim 35 or 36, wherein the mBMP7 peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 26, or a nucleic acid sequence at least about 95% identical thereto.

38. The recombinant polypeptide of any one of claims 34-37, comprising the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence at least about 95% identical thereto.

39. The recombinant polypeptide of any one of claims 34-38, encoded by a nucleic acid sequence comprising SEQ ID NO: 25, or a nucleic acid sequence at least about 95% identical thereto.

40. A recombinant polypeptide comprising:(a) a leptin signal peptide sequence;(b) a linker peptide comprising a valine residue; and(c) a triagonist peptide comprising a GLP-1R peptide, a GIPR peptide, and a GcgR peptide.Docket No. 103362-094WO141. The recombinant peptide of claim 40, wherein the triagonist peptide comprises the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence at least about 95% identical thereto.

42. The recombinant polypeptide of claim 40 or 41, wherein the triagonist peptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 66, or a nucleic acid sequence at least about 95% identical thereto.

43. The recombinant polypeptide of any one of claims 40-42, further comprising one or more additional peptides.

44. The recombinant polypeptide of claim 43, wherein the one or more additional peptides comprise a TAT cell-penetrating peptide and / or an HA tag peptide.

45. The recombinant polypeptide of any one of claims 40-44, comprising the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence at least about 95% identical thereto.

46. The recombinant polypeptide of any one of claims 40-45, encoded by a nucleic acid sequence comprising SEQ ID NO: 65, or a nucleic acid sequence at least about 95% identical thereto.

47. An expression vector comprising a nucleic acid encoding for a recombinant polypeptide of any one of claims 1-46.

48. The expression vector of claim 47, wherein the expression vector is a non-viral vector.

49. The expression vector of claim 48, wherein the non-viral expression vector is selected from a DNA plasmid, a miniplasmid, an exosome, a liposome, or a lipid nanoparticle.

50. The expression vector of claim 47, wherein the expression vector is a viral vector.

51. The expression vector of claim 50, wherein the viral expression vector is selected from a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector (AAV).

52. The expression vector of claim 50, wherein the viral expression vector is an AAV.

53. An adeno-associated viral vector (AAV) comprising a nucleic acid encoding for a recombinant polypeptide of any one of claims 1-46.

54. The AAV of claim 53, wherein the AAV is recombinant and / or engineered.

55. The AAV of claim 53 or 54, wherein the AAV has decreased tropism towards liver and / or enhanced tropism towards adipose tissue.

56. The AAV of any one of claims 53-55, wherein the AAV is a V7 AAV capsid vector.

57. The AAV of any one of claims 53-55, wherein the AAV has enhanced tropism towards neural tissue.Docket No. 103362-094WO158. The AAV of any one of claims 53-55 or 57, wherein the AAV is a Rec2 AAV capsid vector.

59. The AAV of any one of claims 53-58, wherein the AAV is formulated in a dose between about IxlO10vg / kg to about IxlO12vg / kg.

60. The AAV of any one of claims 53-58, wherein the AAV is formulated in a dose of about 5x1011vg / kg.

61. The AAV of any one of claims 53-60, wherein the AAV is formulated for an injection selected from subcutaneous injection, intraperitoneal injection, intravenous injection, intracranial injection, or intraparenchymal injection.

62. A pharmaceutical composition comprising at least one recombinant polypeptide of any one of claims 1-46, at least one expression vector of any one of claims 47-52, or at least one AAV of any one of claims 53-61, and a pharmaceutically acceptable carrier.

63. A method for gene transfer of a nonnative, recombinant polypeptide to adipose tissue in a subject in need thereof, comprising administering a therapeutically effective amount of an AAV of any one of claims 53-61.

64. The method of claim 63, wherein the AAV is administered by subcutaneous injection, intraperitoneal injection, intravenous injection, intracranial injection, or intraparenchymal injection.

65. A method for gene transfer of a nonnative, recombinant polypeptide to brain tissue in a subject in need thereof, comprising administering a therapeutically effective amount of an AAV of any one of claims 53-61.

66. The method of claim 65, wherein the AAV is administered by intracranial injection, intraparenchymal injection, or intravenous injection.

67. A method for reducing blood glucose in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of claims 1-46, an expression vector of any one of claims 47-52, or an AAV of any one of claims 53-61.

68. A method for increasing blood glucose tolerance in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of claims 1-46, an expression vector of any one of claims 47- 52, or an AAV of any one of claims 53-61.

69. The method of claim 67 or 68, wherein the subject is obese.

70. A method for reducing expression of proinflammatory genes in one or more tissues in a subject receiving an AAV therapy, comprising administering a therapeuticallyDocket No. 103362-094WO1effective amount of a recombinant polypeptide of any one of claims 1-46, an expression vector of any one of claims 47-52, or an AAV of any one of claims 53-61.

71. The method of claim 70, wherein the one or more tissues are selected from liver, hypothalamus, brown adipose tissue, white adipose tissue, or a combination thereof.

72. A method for treating a subject having a metabolic disorder, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of claims 1-46, an expression vector of any one of claims 47-52, or an AAV of any one of claims 53-61.

73. The method of claim 72, wherein the metabolic disorder is selected from diabetes, obesity, chronic inflammation, or sleep apnea.

74. A method for treating a subject having diabetes, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of claims 1- 46, an expression vector of any one of claims 47-52, or an AAV of any one of claims 53-61.

75. A method for treating a subject having obesity, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of claims 1- 46, an expression vector of any one of claims 47-52, or an AAV of any one of claims 53-61.

76. A method for reducing one or more metabolic disorders in a subject having a body mass index (BMI) of equal to or greater than 27.5, equal to or greater than 30, or equal to or greater than 40, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of claims 1-46, an expression vector of any one of claims 47-52, or an AAV of any one of claims 53-61.

77. The method of claim 76, wherein the one or more metabolic disorders are selected from obesity, diabetes, lipodystrophy, congenital generalized lipodystrophy (Beradinelli- Seip syndrome), familial partial lipodystrophy, acquired partial lipodystrophy (Barraquer-Simons syndrome), acquired generalized lipodystrophy, centrifugal abdominal lipodystrophy, lipoatrophia annularis, localized lipodystrophy, or HIV- associated lipodystrophy.

78. A method for reducing weight of a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of claims 1- 46, an expression vector of any one of claims 47-52, or an AAV of any one of claimsDocket No. 103362-094WO179. The method of claim 78, wherein the subject, prior to being administered the recombinant polypeptide or expression vector encoding thereof, had a BMI of equal to or greater than 27.5, equal to or greater than 30, equal to or greater than 32.5, equal to or greater than 37.5, equal to or greater than 40, or more than 40.

80. The method of claim 78 or 79, wherein the subject experiences a weight loss of equal to or greater than about 5%, equal to or greater than about 10%, equal to or greater than about 20%, equal to or greater than about 30%, or more than about 30%, of the baseline weight of the subject prior to being administered the recombinant polypeptide or expression vector encoding thereof.

81. The method of any one of claims 78-80, wherein the subject has a reduced body fat percentage following being administered the recombinant polypeptide or expression vector encoding thereof.

82. The method of any one of claims 78-81, wherein the subject has an increased relative lean mass calibrated to body weight following being administered the recombinant polypeptide or expression vector encoding thereof.

83. A method for treating a neurodevel opmental disease in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of claims 1-46, an expression vector of any one of claims 47- 52, or an AAV of any one of claims 53-61.

84. The method of claim 83, wherein the neurodevelopmental disease comprises Prader- Willi syndrome (PWS).

85. A method for treating a subject having Prader-Willi syndrome (PWS), comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of claims 1-46, an expression vector of any one of claims 47-52, or an AAV of any one of claims 53-61.

86. A method for treating a subject having lipodystrophy, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of claims 1- 46, an expression vector of any one of claims 47-52, or an AAV of any one of claims 53-61.

87. A method for treating a neurodegenerative disease in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of claims 1-46, an expression vector of any one of claims 47- 52, or an AAV of any one of claims 53-61.Docket No. 103362-094WO188. The method of claim 87, wherein the neurodegenerative disease comprises Alzheimer’s disease.

89. The method of claim 87, wherein the neurodegenerative disease comprises Parkinson’s disease.

90. A method for treating major depression in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of claims 1-46, an expression vector of any one of claims 47-52, or an AAV of any one of claims 53-61.

91. A method for treating osteoporosis in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of claims 1-46, an expression vector of any one of claims 47-52, or an AAV of any one of claims 53-61.

92. A method for enhancing dental regeneration in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of claims 1-46, an expression vector of any one of claims 47-52, or an AAV of any one of claims 53-61.

93. A method for treating an inflammatory disease in a subj ect in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of claims 1-46, an expression vector of any one of claims 47-52, or an AAV of any one of claims 53-61.

94. The method of claim 93, wherein the inflammatory disease is selected from cardiovascular disease, diabetes, or obesity.

95. A method for improving cellular plasticity in a neurological disorder in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of claims 1-46, an expression vector of any one of claims 47-52, or an AAV of any one of claims 53-61.

96. A method for treating cancer, comprising administering a therapeutically effective amount of a recombinant polypeptide of any one of claims 1-46, an expression vector of any one of claims 47-52, or an AAV of any one of claims 53-61.

97. The method of claim 96, wherein the cancer is selected from adenocarcinoma of the esophagus, breast cancer, colon cancer, rectum cancer, uterine cancer, gallbladder cancer, upper stomach cancer, kidney cancer, liver cancer, ovary cancer, pancreatic cancer, thyroid cancer, meningioma, or multiple myeloma.

98. The method of claim 96, wherein the cancer comprises an obesity-associated cancer.Docket No. 103362-094WO199. The method of claim 98, wherein the obesity-associated cancer comprises obesity- associated colon cancer.

100. The method of any one of claims 63-99, wherein the subject is a human.

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