GLP-1 gene therapies for metabolic disorders

Abstract

An adeno-associated viral gene therapy of the disclosure may include an engineered nucleic acid comprising a promoter operably linked to an open reading frame comprising a sequence encoding an intervening peptide 2 (IP-2) peptide and a sequence encoding a glucagon-like peptide 1 (GLP-1) peptide, such as a GLP- 1(1-37) peptide.

Description

[0001] GLP-1 GENE THERAPIES FOR METABOLIC DISORDERS

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 617,896, filed January 5, 2024, U.S. Provisional Application No. 63 / 723,845, filed November 22, 2024, U.S. Provisional Application No. 63 / 729,635, filed December 9, 2024, and U.S. Provisional Application No. 63 / 730,783, filed December 11, 2024, each of which is incorporated by reference herein in its entirety.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The content of the electronic sequence listing (F085770002WO00-SEQ-HJD.xml; Size: 60,565 bytes; and Date of Creation: January 2, 2025) is herein incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] Metabolic disorders disrupt normal metabolism, which is the process of converting food to energy on a cellular level. The development of therapies to treat metabolic disorders is of paramount importance, given the increasing prevalence and impact of these conditions globally. Metabolic disorders, such as diabetes, obesity, and various lipid disorders, pose significant health risks and contribute to a range of serious complications such as heart disease, stroke, and kidney failure. Traditional treatments often focus on managing symptoms rather than addressing the underlying causes, necessitating a more effective approach. Advancements in areas such gene therapy hold promise for developing targeted treatments.

[0008] SUMMARY

[0009] Aspects of the technology relate to nutrient-responsive gene therapy methods for regulating (e.g., maintaining or lowering) blood glucose levels in a subject, which involve (i) directly targeting (e.g., injecting) pancreatic tissue and (ii) specifically activating expression of a nutrient stimulated hormone (NuSH) in insulin-producing cells using an insulin-responsive gene therapy vector encoding the NuSH (e.g., GLP-1). In some embodiments, the technology relates to compositions and methods used to produce in cells e.g., in vivo) therapeutically effective levels of an active and secreted form of glucagon-like peptide 1 (GLP-1), which binds the GLP-1 receptor. These compositions and methods may be used to treat metabolic disorders including, for example, diabetes and / or obesity. The data herein demonstrate glucose-responsive secretion of GLP-1, for example, in a tissue-specific manner. In some aspects, a signal peptide is translated as part of the overall transgene translation and serves the function of directing the product encoded by the transgene to the endoplasmic reticulum and to the regulated secretory pathway. The signal peptide is cleaved at the endoplasmic reticulum by signal peptidase, and the signal peptide is recycled. The endoplasmic reticulum directs the remaining polypeptide sequence into the secretory vesicles where PC 1 / 3 cleavage occurs to liberate active GLP-1. The secretory vesicles are released into the extracellular space in a glucose-responsive way by way of normal beta-cell secretion.

[0010] The promoters used herein regulate RNA transcription in a glucose-responsive manner, such that more RNA is transcribed in high glucose conditions and therefore more transgene product is translated in high glucose conditions. Secretory vesicles are also made in a glucoseresponsive manner, such that glucose-responsive transcription and translation are desirable. The promoters also provide beta-cell specificity, so that the episomal DNA is not transcribed in off- target cells.

[0011] In some aspects, the disclosure relates to an adeno-associated virus (AAV) vector genome including an engineered nucleic acid (e.g., transgene expression cassette), which includes a promoter operably linked to an open reading frame that includes a sequence encoding an intervening peptide 2 (IP-2) peptide and a sequence encoding a GLP-1 peptide (e.g., Transgene A / 68.1, B, 04, 05, 06, 11, and 38). In some embodiments, the open reading frame further encodes a furin cleavage site (e.g., “Transgene A,” also referred to as Transgene 68.1, used interchangeably herein). In other embodiments, the open reading frame does not encode a furin cleavage site (e.g., “Transgene B”). In some embodiments, the open reading frame does not include a sequence encoding an intervening peptide 1 (IP-1) peptide.

[0012] In other aspects, the disclosure relates to an AAV vector genome including an engineered nucleic acid including a promoter, for example, a human insulin promoter sequence, operably linked to an open reading frame including the sequence of SEQ ID NO: 4 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 4. In yet other aspects, the disclosure relates to an AAV vector genome including an engineered nucleic acid including a promoter, for example, a human insulin promoter sequence, operably linked to an open reading frame including the sequence of SEQ ID NO: 5 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 5.

[0013] In some aspects, an AAV vector genome comprises an engineered nucleic acid comprising the sequence of SEQ ID NO: 20 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 20. In other aspects, an AAV vector genome comprises an engineered nucleic acid comprising the sequence of SEQ ID NO: 21 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 21.

[0014] In further aspects, the disclosure relates to an engineered nucleic acid including a promoter, for example, a human insulin promoter sequence, operably linked to an open reading frame including a sequence encoding an IP-2 peptide and a sequence encoding a GLP-1 peptide. In some embodiments, the open reading frame does not include a sequence encoding an IP-1 peptide.

[0015] In additional aspects, the disclosure relates to a transgene expression cassette comprising: at least three copies of a human insulin promoter sequence, a human insulin exon 1 sequence, a chimeric chicken P-actin (CBA) and rabbit P-globin (RBG) intron sequence, a human insulin exon 2 sequence, a human insulin signal peptide, optionally a furin cleavage site, a GLP- 1(1-6) coding sequence, a GLP- 1(7-37) coding sequence, optionally PC 1 / 3 cleavage site, an IP-2 coding sequence, a mutated woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) coding sequence, and a bovine growth hormone (BGH) polyadenylation signal.

[0016] In further aspects, the disclosure relates to a nutrient-responsive method for regulating blood glucose levels in a subject, the method comprising delivering (e.g., injecting a single dose of) a gene therapy composition to (e.g., directly to) pancreatic tissue of the subject in an amount effective to regulate (e.g., reduce or maintain low) blood glucose levels in the subject, wherein the gene therapy composition comprises an engineered nucleic acid comprising a promoter operably linked to an open reading frame comprising a sequence encoding an intervening peptide 2 (IP-2) peptide and a sequence encoding a glucagon-like peptide 1 (GLP-1) peptide.

[0017] An additional aspect of the disclosure relates to a nutrient-responsive method for regulating blood glucose levels in a subject, the method comprising administering a gene therapy composition to the subject in an amount effective to regulate (e.g., reduce or maintain low) blood glucose levels in the subject, wherein the gene therapy composition comprises an engineered nucleic acid comprising a promoter operably linked to an open reading frame comprising a sequence encoding an intervening peptide 2 (IP-2) peptide and a sequence encoding a glucagon- like peptide 1 (GLP-1) peptide.

[0018] Another aspect relates to a nutrient-responsive method for regulating blood glucose levels in a subject, the method comprising: administering a gene therapy composition to the subject in an amount effective to regulate blood glucose levels in the subject, wherein the gene therapy composition comprises an engineered nucleic acid comprising the sequence of SEQ ID NO: 35.

[0019] A further aspect relates to a nutrient-responsive method for regulating blood glucose levels in a subject, the method comprising: administering a gene therapy composition to the subject in an amount effective to regulate blood glucose levels in the subject, wherein the gene therapy composition comprises an engineered nucleic acid comprising a promoter operably linked to an open reading frame comprising a sequence of SEQ ID NO: 35 encoding a glucagon- like peptide 1 (GLP-1) peptide and optionally a sequence of SEQ ID NO: 35 encoding an intervening peptide 2 (IP-2) peptide.

[0020] Also provided herein is an engineered nucleic acid comprising the sequence of SEQ ID NO: 20. Further provided herein is an engineered nucleic acid comprising the sequence of SEQ ID NO: 21. Further still, provided herein is an engineered nucleic acid comprising the sequence of SEQ ID NO: 35.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1A includes a graph showing that inclusion of the intervening peptide 2 (IP-2) coding sequence in a transgene expression cassette (Transgene 04, 05, 06, and 11) yields higher GLP-1 activity (comparable to positive controls) than when excluded (Transgene 01, 02, 03, and 08). FIG. IB includes a graph showing that inclusion of the intervening peptide 1 (IP-1) coding sequence in a transgene expression cassette (Transgene 03, 06, 09) or exclusion of the IP-1 coding sequence (Transgene 02, 05, and 08) had no impact on GLP-1 activity.

[0023] FIG. 2 includes a graph showing that inclusion of the GLP- 1(1-6) coding sequence in a transgene cassette (Transgene 02 and 05) is important for GLP-1 activity.

[0024] FIG. 3 shows that insertion of a sequence encoding a furin cleavage site between GLP- 1(1-6) and GLP- 1(7-37) (Transgene 25) can be detrimental, highlighting the importance of the native PC 1 / 3 cleavage site, in some instances.

[0025] FIG. 4A shows a map of a transgene expression cassette (Transgene 38) that includes a human insulin promoter operably linked to a sequences encoding GLP-l(l-6), GLP-l(7-37), and IP-2, with additional regulatory elements (e.g., promoter intron, bGH polyA, WPRE). HIP.363x3 (3 copies of human insulin promoter core sequence, SEQ ID NO: 22); hINS human insulin; cBA chicken beta-actin; rBG rabbit beta-globin; hINS.sp human insulin signal peptide; FURIN.cl furin cleavage site; RR PC 1 / 3 cleavage site; * stop codon. FIG. 4B includes graphs showing that the transgene expression cassette depicted in FIG. 4A (SEQ ID NO: 20) produces functional GLP-1 protein that matches the expression and activity of CMVp-GCG (CMP promoter operably linked to GCG coding sequence).

[0026] FIG. 5A is a graph depicting the results of an ELISA of secreted GLP- 1 stimulated by two different doses of glucose. FIG. 5B is a graph depicting the results of a cAMP activity assay using supernatants from the cells in FIG. 5A.

[0027] FIG. 6A is a graph depicting the levels of fasting blood glucose (FBG) in DIO mice following administration of a single dose of Transgene 68.1, also referred to herein as Transgene A, (7.5el2 VG) or a vehicle control. FIG. 6B shows fasting blood glucose levels in DIO mice on day 95 after administration of a single dose of Transgene A (7.5el2 VG) or a vehicle control. FIG. 6C shows the vehicle-adjusted body weight reduction in DIO mice following administration of a single dose of Transgene A (7.5el2 VG) or a vehicle control over time.

[0028] FIG. 7A shows levels of AAV-9 transduction (measured as vector copy number, VCN) in pancreatic sections from porcine subjects 34 days after administration of a single dose of Transgene A (6e 13 VG). FIG. 7B shows levels of GLP-1 transgene transcription in pancreatic sections from porcine subjects 34 days after administration of a single dose of Transgene A (6el3 VG). FIG. 7C shows levels of active GLP-1 protein in pancreatic sections from porcine subjects 34 days after administration of a single dose of Transgene A (6el3 VG). FIG. 7D shows levels of active GLP-1 protein in porcine subjects 34 days after administration of a single dose of Transgene A (6el3 VG) compared to untreated controls. FIG. 7E shows the positive correlation of pancreatic vector copy number and Transgene A RNA levels in porcine subjects 34 days after administration of a single dose of Transgene A (6el3 VG); r=0.85, p<0.0001. FIG. 7F shows the positive correlation of Transgene A RNA levels and Transgene A islet-driven GLP-1 protein in porcine subjects 34 days after administration of a single dose of Transgene A (6el3 VG); r=0.85, p<0.0001.

[0029] FIG. 8 is a graph showing mean serum lipase levels over time in porcine subjects administered a single dose of Transgene A (6e 13 VG) on day 0. In the figure, “ULN” represents “ upper limit of normal.”

[0030] FIG. 9 includes graphs showing targeted infusion and specific expression restrict GLP-1 expression to the pig pancreas — total, active, and inactive GLP-1 protein was no higher than baseline in plasma. Despite abundance of GLP-1 in Transgene A treated pig pancreas, it is not detected above baseline in circulation.

[0031] FIG. 10 includes graphs showing a dose-responsive improvement in glycemic control, including a dose-responsive decrease in bodyweight loss (BWL) and fasting blood glucose (FBG) and a dose-responsive increase in fasting plasma insulin (FPI) in db / db mice dosed with Transgene A using three different doses (8.3ell vg, 2.5el2 vg, or 7.5el2 vg).

[0032] FIGs. 11A-11B include microscopy images showing that the human insulin promoter used in Transgene A (total dose of 2.5el2 vg) restricts expression to islet cells in the pancreas of db / db mice dosed with Transgene A. Within the pancreas, expression of insulin / GFP was limited to islets (FIG. 10A), and low to no expression of GFP was observed in the liver and heart compared to ubiquitous control (FIG. 10B). FIG. 12 provides data showing that Transgene A was successfully dosed (6el3 vg total) in pigs and that double infusions (same total dose of 6el3 vg) offered mildly wider AAV distribution within the splenic lobe of the pancreas.

[0033] FIGs. 13A-13B include microscopy images (FIG. 15A) and flow cytometry data (FIG. 15B) showing that the Transgene A insulin promoter derivative is highly active in human beta cells. The promoter strength is dose-responsive and exceeds CMV promoter at the same MOI (images at 72 hours post transduction at lOx magnification).

[0034] FIGs. 14A-14B include microscopy images (FIG. 16A) and flow cytometry data (FIG. 16B) showing that the Transgene A insulin promoter derivative is inactive in human HEPG2 (liver) cells. The Transgene A promoter shows very low expression at high MOI relative to CMV promoter (images at 72 hours post transduction at lOx magnification).

[0035] FIGs. 15A-15B include microscopy images (FIG. 17A) and flow cytometry data (FIG. 17B) showing that the Transgene A insulin promoter derivative is inactive in human dorsal root ganglion (DRG) cells. The Transgene A promoter shows very low expression at high MOI relative to CMV promoter (images at 72 hours post transduction at lOx magnification).

[0036] DESCRIPTION

[0037] The disclosure provides, in some aspects, gene therapy compositions and methods for treating metabolic disorders, including diabetes (e.g., Type 2 diabetes) and obesity, for example, by delivering to cells of the pancreas an active, secreted form of a glucagon-like peptide- 1 (GLP- 1) peptide, which is a GLP-1 receptor agonist. GLP-1 is normally produced in the intestinal L cell (e.g., primarily in the ileum and large intestine), where the preproglucagon (GCG) gene is active in a glucose-responsive way. The GCG gene encodes several peptide hormones that are of cell-types specifically expressed in the pancreatic islets, the distal ileum and the large intestine, as well as certain brain neuronal cells. These hormones are important in controlling blood glucose homeostasis, intestinal cell proliferation, and satiety. Notably, the major hormone generated in the pancreas (glucagon) exerts opposite effects to the ones that are produced in the intestines (GLP-1 and GLP-2).

[0038] GLP-1 receptor agonists, first approved to treat Type 2 diabetes in 2005, have been developed to yield effective compounds / preparations that have overcome certain problems, such as rapid elimination (short half-life). Currently, GLP-1 receptor agonists are administered in various forms: twice daily (e.g., exenatide), once daily (e.g., lixisenatide, liraglutide), or weekly (e.g., weekly exenatide, dulaglutide, albiglutide, semaglutide), for example. A recent FDA approval was given for a daily oral version of semaglutide, showing comparable effectiveness to the weekly subcutaneous version. GLP-1 receptor agonists work by enhancing insulin secretion when blood sugar is high, reducing glucagon release in high or normal blood sugar situations, slowing gastric emptying to manage post-meal blood sugar spikes, and aiding in calorie intake and weight reduction. Short-acting versions such as exenatide and lixisenatide are less effective in controlling overnight and fasting blood sugar levels but consistently affect gastric emptying even with long-term use. Long-acting GLP-1 receptor agonists such as liraglutide, weekly exenatide, dulaglutide, albiglutide, and semaglutide, show stronger impact on overnight and fasting blood sugar levels and hemoglobin A1C (HbAic), either alone or with basal insulin. Their impact on gastric emptying, however, diminishes over time. Due to their comparable or superior effectiveness in reducing HbAic, along with weight loss benefits and low risk of hypoglycemia, GLP-1 receptor agonists are often the first-choice injectable treatment for Type 2 diabetes, even before insulin.

[0039] Nonetheless, systemic delivery of these primarily protein-based GLP-1 receptor agonists are often associated with intolerable side effects, including nausea, vomiting, and diarrhea, often referred to as gastrointestinal adverse events. They are typically most prominent when initiating treatment with a GLP-1 receptor agonist or after increasing the dose (e.g., during recommended up-titration regimens). Because these symptoms can occur in fasting subjects, they are likely not related to the effects of GLP-1 receptor agonist treatment on gastrointestinal functions (e.g., deceleration of gastric emptying) but instead are caused by direct interactions with GLP-1 receptors in the central nervous system most likely located in the brain stem. Nausea is typically reported in up to 25% and vomiting or diarrhea in up to 10% of subjects treated with systemically delivered GLP-1 receptor agonists. The gene therapy compositions and local delivery methods provided herein, in some aspects, are aimed at delivering therapeutically effective amounts of GLP-1, specifically to the pancreas of patients in a targeted manner, thus minimizing adverse effects associated with systemic delivery. With local delivery of the gene therapy compositions described herein and insulin-dependent (e.g., meal-dependent) production and secretion of the GLP-1 peptide, a higher, more effective dose of GLP-1 can be delivered and tolerated with minimal side effects, relative to systemic, protein-based delivery of GLP-1.

[0040] Within cells, the GCG gene is processed to ultimately liberate GLP-l(l-37), which is further cleaved by the PC1 / 3 enzyme to liberate GLP-l(7-37), which is the active form of GLP- 1. While simply expressing the active form of GLP-1 (7-37) in cells might be considered the most straightforward way to deliver a therapeutically effective amount, as the studies provided herein demonstrate, that approach instead yields low levels of GLP-1 peptide with low activity. Quite unexpectedly, the inventors found that certain elements of the GCG gene encoded in the transgene expression cassettes of the disclosure significantly boosted GLP-1 activity to levels needed to achieve a therapeutic benefit. For example, inclusion of an intervening peptide 2 (IP-2) coding sequence in a transgene expression cassette yielded higher GLP-1 activity, relative to those transgene expression cassettes that excluded that IP-2 coding sequence (see Example 1, FIG. 1A). This was surprising in part because (i) there is no known function ascribed to the IP-2 peptide, (ii) IP-2 is the most divergent region within the GCG gene, and (iii) during natural protein processing of GCG in a cell, IP-2 is cleaved off, leaving only GLP-l(l-37) for further processing. Moreover, none of the GLP- 1-based drugs on the market includes an IP-2 peptide. Even more surprising, after learning the impact of the IP-2 coding sequence on GLP- 1 activity, was the data showing that inclusion of an IP-1 coding sequence had no impact (see Example 1, FIG. IB). IP-1 is more conserved than IP-2, suggesting that it may have a functional impact. Yet, inclusion (or exclusion) of an IP-1 coding sequence from the tested transgene expression cassettes did not impact the activity of the encoded GLP-1.

[0041] The inventors were also surprised by the data showing that inclusion of a GLP- 1(1-6) coding sequence in the transgene expression cassettes of the disclosure significantly increased activity of the liberated GLP- 1(7-37) peptide. This was unexpected in part because during natural protein processing of GCG in a cell, the GLP-l(l-6) peptide is cleaved off, leaving the GLP-1(7- 37) peptide as the active form. The expectation was that excluding the GLP- 1(1-6) coding sequence from the transgene expression cassettes would not negatively impact activity of the GLP- 1(7-37) peptide. This was not the case (see Example 2, FIG. 2).

[0042] Thus, as the date herein demonstrates quite unexpectedly, the most effective transgene expression constructs for delivering a therapeutically effective amount of the active, soluble form of GLP-1 are those that include an IP-2 coding sequence and coding sequences for the full-length version of the GLP-1 peptide (i.e., GLP(l-37)).

[0043] Metabolic Disorders

[0044] Some aspects of the disclosure relate to a metabolic disorder. A metabolic disorder results from a disruption of normal metabolism, which is the chemical (metabolic) process of converting food and drink into energy. Typically, chemicals in the body break down the proteins, carbohydrates, and fats consumed, turning them into energy for current use or storing it for later use. Non-limiting examples of metabolic disorders include Obesity, Diabetes Mellitus, Disorders of Lipid Metabolism, Inborn Errors of Metabolism, Lysosomal Storage Disorders, Glycogen Storage Diseases, Mitochondrial Disorders, Purine and Pyrimidine Disorders, Urea Cycle Disorders, Disorders of Fructose Metabolism, Disorders of Amino Acid Metabolism, Disorders of Mineral Metabolism, Porphyrias, Lactose Intolerance and Wilson's Disease, Polycystic Ovary Syndrome, Metabolic Dysfunction-associated Steatotic Liver Disease (MASLD), and Non- Alcoholic Steatohepatitis (NASH). In some embodiments, a metabolic disorder is Diabetes Mellitus, which includes a group of metabolic disorders characterized by chronic hyperglycemia (high blood sugar levels) resulting from defects in insulin secretion, insulin action, or both. The various types of Diabetes Mellitus include prediabetes, Type 1 Diabetes, Type 2 Diabetes, and Gestational Diabetes. There are also other types resulting from specific genetic conditions, surgery, medications, infections, and other illnesses.

[0045] In some embodiments, a metabolic disorder is Type 2 diabetes, which is a chronic condition resulting from persistently high blood sugar levels (hyperglycemia), mainly due to insulin resistance or insulin insufficiency. Insulin resistance includes the body’s inability to respond appropriately to an insulin signal to remove glucose from the bloodstream, whereas insulin insufficiency includes the gradual failure of the pancreas to produce sufficient insulin to meet the body’s needs. Current guidelines for treating Type 2 diabetes focus on managing the blood glucose symptoms, often measured by blood concentrations of glycosylated hemoglobin (HbAlc), rather than correcting the underlying pathology in the body causing insulin resistance and insulin insufficiency. Therefore, patients make drastic dietary and lifestyle changes that require lifelong patient adherence and persistence to medicines. For some, this approach to care is unmanageable and leaves many patients at risk, potentially resulting in chronic elevations in blood glucose that increase the likelihood of microvascular and macrovascular complications of Type 2 diabetes. There are no therapies that are approved today in Type 2 diabetes that offer disease modification; that is, ongoing and durable preservation of pancreatic insulin production capacity even after therapy is discontinued.

[0046] In some embodiments, a metabolic disorder is prediabetes. In other embodiments, a metabolic disorder is Type 1 Diabetes. In yet other embodiments, a metabolic disorder is Gestational Diabetes.

[0047] In some embodiments, a metabolic disorder is obesity. Obesity refers to a medical condition characterized by an excess amount of body fat. It is often measured using the body mass index (BMI). Standard BMI is a person’s weight in kilograms divided by the square of height in meters. A high BMI can indicate high body fatness. In general, a person is considered obese if their BMI is 30 or higher. If a subject’s BMI is less than 18.5, it falls within the underweight range. If a subject’s BMI is 18.5 to <25, it falls within the healthy weight range. If a subject’s BMI is 25 to <30, it falls within the overweight range. If a subject’s BMI is 30 or higher, it falls within the obesity range. Obesity is frequently subdivided into categories: Class 1 is a BMI of 30 to < 35; Class 2 is a BMI of 35 to < 40; and Class 3 is a BMI of 40 or higher. Class 3 obesity is sometimes categorized as “severe” obesity. This condition is not just a cosmetic concern but is also a serious health issue as it increases the risk of diseases and health problems such as heart disease, diabetes, high blood pressure, and certain types of cancer. Obesity is usually caused by a combination of inherited factors, combined with the environment, diet, and level of physical activity. It is a complex disease that requires a multifaceted approach to treatment, including changes in diet and exercise habits, and sometimes medication or surgery. In some embodiments, a subject has a BMI of 25 to <30. In other embodiments, a subject has a BMI of 30 or higher. In some embodiments, a subject has a BMI of 30 to < 35. In some embodiments, a subject has a BMI of 35 to < 40. In some embodiments, a subject has a BMI of 40 or higher.

[0048] In some embodiments, a subject has Metabolic Dysfunction-associated Steatotic Liver Disease (MASLD) (formerly known as non-alcoholic fatty liver disease [NAFLD]). MASLD is characterized by the presence of hepatic fat accumulation in the absence of secondary causes of hepatic steatosis (e.g., excessive alcohol consumption, other liver diseases, and / or long-term use of steatogenic medication). MASLD is the most common cause of chronic liver disease and is the leading cause of liver-related morbidity and mortality worldwide (Chan et al., J Obes Metab Syndr. 2023 Sep 30;32(3): 197-213). There are no approved pharmacological agents for the treatment of MASLD, and lifestyle changes are the initial treatment (e.g., changes in diet and / or exercise).

[0049] Preproglucagon

[0050] Preproglucagon is a preprohormone encoded by the GCG gene in humans. Preproglucagon contains a signal peptide as well as several peptide hormones, including glicitin- related polypeptide (GRPP), glucagon(l-29), intervening peptide 1 (IP-1), glucagon-like peptide - 1 (GLP- 1(1-37)), intervening peptide 2 (IP-2), and glucagon-like peptide-2 (GLP-2). Tissuespecific posttranslational processing of GCG by prohormone convertases (PC) yield the various proglucagon products. For example, PC2, which is predominately expressed in the islet a-cell, produces glucagon. Conversely, PC 1 / 3, expressed in the intestine and brain, cleaves proglucagon to yield GLP-1, GLP-2, and oxyntomodulin. Some PC1 / 3 expression has been demonstrated in a-cells as well, suggesting that a-cells can produce GLP-1 in addition to glucagon. PC1 / 3 is also expressed in islet P-cells, where it can process the GLP-1 encoded by the transgene expression cassettes of the disclosure. Exemplary sequences for the peptide hormones encoded by the GCG gene are provided in Table 1.

[0051] Generally, the peptide hormones encoded by the transgene expression cassettes of the disclosure are functional peptides. As used herein, “functional” refers to a peptide (or protein) that possesses biological activity (e.g., enzymatic activity). That is, the functional peptide produced has at least 50%, 60%, 70%, 80%, 90%, 100% or more activity (e.g., enzymatic activity) compared to a corresponding naturally occurring peptide. Biological activity can be measured by any method known in the art; for example, by an in vitro activity assay or by in vivo measurements of enzymatic byproducts (e.g., C-peptide) or other related components (e.g., glucose levels). In some embodiments, a functional peptide hormone has approximately the same activity as a naturally occurring peptide hormone (e.g., promotion of glucose uptake, glycogenesis, lipogenesis, and / or protein synthesis of skeletal muscle and / or fat tissue through the tyrosine kinase receptor pathway and / or maintenance of circulating glucose concentrations within a physiological range). In some embodiments, a functional peptide hormone has more activity than a naturally occurring peptide hormone.

[0052] Transgene Expression Cassettes

[0053] A “transgene expression cassette” herein refers to an engineered nucleic acid comprising a promoter operably linked to an open reading frame comprising one or more sequence(s) encoding one or more preproglucagon peptides, such as GLP-1, which can be expressed by a transfected or transduced cell. A transgene expression cassette, in some embodiments, includes one or more additional regulatory element(s), for example, selected from enhancers, stabilizing polyadenylation termination signals, introns, and exons. In some embodiments, a transgene expression cassette is modified for expression by a viral vector (e.g., an AAV vector) and includes a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) (or a mutated version thereof) and / or inverted terminal repeat (ITR) sequences.

[0054] In some embodiments, a transgene expression cassette has a length of about 1 kb to about 15 kb, for example, about 1 kb to about 10 kb, about 1 kb to about 5 kb, about 2 kb to about 15 kb, about 2 kb to about 10 kb, about 2 kb to about 5 kb, about 3 kb to about 15kb, about 3 kb to about lOkb, or about 3 kb to about lOkb.

[0055] In other embodiments, for example, those in which a viral vector is used as a delivery vehicle, a transgene expression cassette has a length no longer than 5 kb. Thus, in some embodiments, an engineered nucleic acid has a length of about 1 kb to about 5 kb, about 2 kb to about 5 kb, about 3 kb to about 5 kb, about 4 kb to about 5 kb, about 1 kb to about 4 kb, about 2 kb to about 4 kb, about 3 kb to about 4 kb, about 1 kb to about 3 kb, or about 1 kb to about 2 kb.

[0056] In some embodiments, a transgene expression cassette comprises a promoter operably linked to an open reading frame comprising a sequence encoding an IP-2 peptide. An exemplary human IP-2 peptide sequence is provided in Table 1. Thus, in some embodiments, an IP-2 peptide comprises the sequence of SEQ ID NO: 16. In other embodiments, an IP-2 peptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 16. In some embodiments, an IP-2 peptide comprises a sequence having at least at least 90% to the sequence of SEQ ID NO: 16. In some embodiments, an open reading frame comprises a sequence encoding an IP-2 peptide that comprises the sequence of SEQ ID NO: 16. In other embodiments, an open reading frame comprises a sequence encoding an IP-2 peptide that comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 16. In some embodiments, an open reading frame comprises a sequence encoding an IP-2 peptide that comprises a sequence having at least at least 90% to the sequence of SEQ ID NO: 16.

[0057] In some embodiments, a transgene expression cassette comprises a promoter operably linked to an open reading frame comprising a sequence encoding a GLP-1 peptide. Exemplary human GLP-1 peptide sequences are provided in Table 1. A GLP-1 peptide encoded by an open reading frame of the disclosure, in some embodiments, includes amino acids 7-37 of the human GLP-1 peptide. A GLP-1 peptide encoded by an open reading frame of the disclosure, in some embodiments, excludes amino acids 1-6 of the human GLP-1 peptide. Thus, in some embodiments, a GLP-1 peptide comprises the sequence of SEQ ID NO: 15 (GLP-1 (7-37)). In other embodiments, a GLP-1 peptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 15. In some embodiments, a GLP-1 peptide comprises a sequence having at least at least 90% to the sequence of SEQ ID NO: 15. In some embodiments, an open reading frame comprises a sequence encoding a GLP-1 peptide that comprises the sequence of SEQ ID NO: 15. In other embodiments, an open reading frame comprises a sequence encoding a GLP-1 peptide that comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 15. In some embodiments, an open reading frame comprises a sequence encoding a GLP- 1 peptide that comprises a sequence having at least at least 90% to the sequence of SEQ ID NO: 15. In some embodiments, a GLP-1 peptide comprises the sequence of SEQ ID NO: 14 (GLP-l(l-37)). In other embodiments, a GLP-1 peptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 14. In some embodiments, a GLP-1 peptide comprises a sequence having at least at least 90% to the sequence of SEQ ID NO: 14. In some embodiments, an open reading frame comprises a sequence encoding a GLP-1 peptide that comprises the sequence of SEQ ID NO: 14. In other embodiments, an open reading frame comprises a sequence encoding a GLP-1 peptide that comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 14. In some embodiments, an open reading frame comprises a sequence encoding a GLP-1 peptide that comprises a sequence having at least at least 90% to the sequence of SEQ ID NO: 14. “Identity” refers to a relationship between two or among three or more sequences (e.g., amino acid sequences or nucleotide sequences) as determined by comparing the sequences to each other. Identity also refers to the degree of sequence relatedness between or among sequences as determined by the number of matches between or among strings of amino acids (polypeptides) or strings of nucleotides (polynucleotides). Identity is a measure of the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., “algorithms”). Identity of related polypeptides and polynucleotides can be readily calculated by known methods. “Percent (%) identity” as it applies to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid or nucleic acid residues) in the candidate (first) polypeptide or polynucleotide sequence that are identical with the residues in a second polypeptide or polynucleotide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Herein, unless stated otherwise, percent identity is calculated based on a global alignment, which is an end-to-end alignment of two or more strings of sequences (using, for example, the Needleman-Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. J. Mol. Biol. 1920;48:443-453)). Contrast that to local alignment, which is an alignment of subregions within a sequence string having the highest level of similarity (highest percent identity) (using, for example, the Smith- Waterman algorithm (Smith, T.F. & Waterman, M.S. J. Mol. Biol. 1981;147:195-197)).

[0058] Methods and computer programs for the alignment are well known in the art. It is understood that identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation. Generally, variants of a particular polynucleotide or polypeptide have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular native (wild-type) or reference sequence as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include but are not limited to those of the BLAST suite (Altschul, S.F., et al. Nucleic Acids Res. 1997;25:3389-3402). A Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) also has been developed that purportedly produces global alignment of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm.

[0059] Even before the hormone has left the gut, GLP-1 is rapidly metabolized and inactivated by dipeptidyl peptidase-4 (DPP-4), which is a serine exopeptidase that cleaves X-proline or X- alanine dipeptides from the N-terminus of polypeptides. In some embodiments, a GLP-1 peptide encoded by an open reading frame of the disclosure is a modified GLP-1 peptide that comprises one or more amino acid substitution(s), relative to a wild-type GLP-1 peptide consisting of the sequence of SEQ ID NO: 14 or 15, that renders the modified GLP-1 peptide resistant to degradation by DPP-4. The modified GLP-1 peptide, in some embodiments, is selected from the group consisting of: Arg26-GLP- 1(7-37); Arg34-GLP-l(7-37); Lys36-GLP- 1(7-37); Arg26,34Lys36-GLP- 1(7-37); Arg26,34-GLP-l(7-37); Arg26,34Lys40-GLP- 1(7-37); Arg26Lys36-GLP-l(7-37); Arg34Lys36-GLP-l(7-37) Val8Arg22-GLP-l(7-37); Met8Arg22- GLP-l(7-37);Gly8His22-GLP- 1(7-37); Vai 8His22-GLP- 1(7-37); Met8His22-GLP- 1(7-37); His37-GLP- 1(7-37); Gly8-GLP- 1(7-37); Val8-GLP- 1(7-37); Met8-GLP- 1(7-37); Gly8Asp22- GLP-l(7-37); Val8Asp22-GLP-l(7-37); Met8Asp22-GLP-l(7-37); Gly8Glu22-GLP-l(7-37); Val8Glu22-GLP- 1(7-37); Met8Glu22-GLP- 1(7-37); Gly8Lys22-GLP-l(7-37); Val8Lys22-GLP- 1(7-37); Met8Lys22-GLP-l(7-37); Gly8Arg22-GLP-l(7-37); Val8Lys22His37-GLP-l(7-37); Gly8Glu22His37-GLP-l(7-37); Val8Glu22His37-GLP-l(7-37); Met8Glu22His37-GLP-l(7-37); Gly8Lys22His37-GLP-l(7-37); Met8Lys22His37-GLP-l(7-37); Gly8Arg22His37-GLP- 1(7-37); Val8Arg22His37-GLP-l(7-37); Met8Arg22His37-GLP-l(7-37); Gly8His22His37-GLP-l(7-37); Val8His22His37-GLP- 1(7-37); Met8His22His37-GLP- 1(7-37); Gly8His37-GLP-l(7-37);

[0060] Val8His37-GLP-l(7-37); Met8His37-GLP-l(7-37); Gly8Asp22His37-GLP-l(7-37); Val8Asp22His37-GLP-l(7-37); and Met8Asp22His37-GLP-l(7-37).

[0061] In some embodiments, a GLP-1 peptide consists of or consists essentially of the sequence of SEQ ID NO: 14 or 15, optionally with no more than one amino acid substitution, or no more than two amino acid substitutions, relative to a wild-type GLP-1 peptide consisting of the sequence of SEQ ID NO: 14 or 15, that renders the modified GLP-1 peptide resistant to degradation by DPP-4. In some embodiments, a modified GLP-1 peptide is Gly8-GLP- 1(7-37) - that is, a GLP-1 peptide that includes amino acids 7-37 of human GLP-1 (SEQ ID NO: 15), where amino acid position 8 is a glycine (i.e., HGEGTETSDVSSYLEGQAAKEEIAWLVKGRG (SEQ ID NO: 18)). In some embodiments, a modified GLP-1 peptide is Val8-GLP- 1(7-37) - that is, a GLP-1 peptide that includes amino acids 7-37 of human GLP-1, where amino acid position 8 is a valine (i.e., HVEGTPTSDVSSYLEGQAAKEPIAWLVKGRG (SEQ ID NO: 19)).

[0062] In some embodiments, a sequence encoding an IP-2 peptide is downstream (3’) from a sequence encoding a GLP-1 peptide. In other embodiments, a sequence encoding an IP-2 peptide is upstream (5’) from a sequence encoding a GLP-1 peptide. A sequence encoding an IP-2 peptide, in some embodiments, is adjacent to a sequence encoding a GLP-1 peptide, separated, for example, by only 1, 2, or 3 amino acids. In some embodiments, the coding sequences are separate from each other by a single PC 1 / 3 cleavage site (see, e.g., Transgenes 04, 05, 06, 12of Table 1). The proprotein convertase 1 / 3 (PC1 / 3) is expressed in the regulated secretory pathway of neural and endocrine cells. Its major function is in the post-translational processing and activation of precursor proteins, such preproglucagon. PC 1 / 3 cleaves secretory precursors at single or paired basic amino acids within a recognized cleavage site, RX(R / K)Rj, (Hosaka M. el al. J. Biol. Chem., 266 (1991), pp. 12127-12130) or RH or RR, for example. In some embodiments, an open reading frame of an engineered nucleic acid further comprises a sequence encoding a PC1 / 3 cleavage site between amino acid positions 6 and 7 of the GLP- 1(1-37) peptide (i.e., RH).

[0063] In some embodiments, an open reading frame of an engineered nucleic acid does not comprise a sequence encoding an IP-1 peptide. While the data herein demonstrates that the presence of a coding sequence for an IP-1 peptide does not impact activity of the active GLP- 1(7- 37) peptide, in some embodiments, limiting the production of active GCG peptides is preferred, for example, to limit potential adverse effects from such active peptides and / or to limit the size of the engineered nucleic acid such that it can be delivered using an AAV vector. Thus, in some embodiments, an open reading frame of an engineered nucleic acid excludes a coding sequence for an IP-1 peptide. An exemplary human IP-1 peptide sequence is provided in Table 1. In some embodiments, an open reading frame of an engineered nucleic acid does not comprise a sequence encoding an IP-1 peptide sequence of SEQ ID NO: 13.

[0064] In some embodiments, an open reading frame of an engineered nucleic acid does not comprise (excludes) a sequence encoding one or more of: a glicentin-related pancreatic polypeptide (GRPP) (e.g., SEQ ID NO: 11), a glucagon peptide (e.g., SEQ ID NO: 12), or a GLP-2 peptide (e.g., SEQ ID NO: 17). In some embodiments, an open reading frame of an engineered nucleic acid does not comprise (excludes) a sequence encoding GRPP. In some embodiments, an open reading frame of an engineered nucleic acid does not comprise (excludes) a sequence encoding a glucagon peptide. In some embodiments, an open reading frame of an engineered nucleic acid does not comprise (excludes) a sequence encoding a GLP-2 peptide. In some embodiments, an open reading frame of an engineered nucleic acid does not comprise (excludes) a sequence encoding a GRPP, a glucagon peptide, or a GLP-2 peptide.

[0065] In some embodiments, the only preproglucagon peptides encoded by the open reading frame of an engineered nucleic acid, e.g., in a transgene expression cassette, are an IP-2 peptide and a GLP-1 peptide, and optionally a signal peptide. Thus, in some embodiments, an open reading frame of a transgene cassette of the disclosure encodes GCG peptide hormones, and those GCG peptide hormones consist of or consist essentially of IP-2 and GLP-1 (e.g., GLP(1- 37) or GLP- 1(7-37)).

[0066] The engineered nucleic acids, in some embodiments, further comprise a sequence encoding a signal peptide. A signal peptide may be encoded at the N terminus or the C terminus of a peptide. In some embodiments, a signal peptide is encoded at the N terminus. In some embodiments, a signal peptide is from a human insulin gene (e.g., the signal peptide encoded by Transgene 04 or 05). In other embodiments, a signal peptide is from a preproglucagon gene (e.g., the signal peptide encoded by Transgene 08 or 11). In some embodiments, a signal peptide is from a human insulin gene and is encoded at the N terminus of a peptide, such as GLP-1 (e.g., GLP-l(l-37) or GLP-l(7-37)). In some embodiments, a signal peptide is from a human insulin gene and is encoded at the N terminus of an IP-2 peptide.

[0067] In some embodiments, an engineered nucleic acid provided herein comprises at least one promoter. A promoter includes a nucleotide sequence located at the 5’ terminus of a nucleic acid to which a polymerase specifically binds and initiates transcription of the remainder of a nucleic acid. A promoter, in some embodiments, comprises a pancreatic tissue-specific promoter sequence. A pancreatic tissues promoter sequence is a DNA sequence that controls the expression of genes (regulates expression of an open reading frame to which it is operably linked) specifically in pancreatic tissue. Non-limiting examples of pancreatic tissue-specific promoters include insulin promoters, Pdxl (Pancreatic and Duodenal Homeobox 1) promoters, elastase promoters, amylase promoters, and somatostatin promoters.

[0068] In some embodiments, a pancreatic tissue-specific promoter sequence comprises an islet beta cell-specific promoter sequence. An islet beta cell-specific promoter sequence is a DNA sequence that controls the expression of genes (regulates expression of an open reading frame to which it is operably linked) specifically in beta cells of pancreatic islets. Non-limiting examples of islet beta cell-specific promoters include insulin promoters, Pdxl (Pancreatic and Duodenal Homeobox 1) promoters, and MafA (V-Maf Musculoaponeurotic Fibrosarcoma Oncogene Homolog A) promoters. In some embodiments, an islet beta cell-specific promoter sequence comprises an insulin gene promoter sequence, such as a human insulin gene promoter sequence. In some embodiments, a human insulin gene promoter sequence is a core sequence.

[0069] A promoter may include one or more promoter sequences. Such sequences may be the same sequences, for example, one or more (e.g., 1, 2, 3, or more) copies of a promoter sequence, or the promoter sequences may differ from one another. For example, a promoter may include a human insulin promoter sequence and a human preproglucagon promoter sequence. In some embodiments, a promoter comprises three copies of a core sequence (e.g., SEQ ID NO: 22) of a human insulin gene promoter sequence. In some embodiments, a promoter comprises a sequence comprising three sequential core sequences of a human insulin gene promoter sequence (e.g., SEQ ID NO: 22). In some embodiments, the three copies of a core sequence are identical. In some embodiments, the first two core sequences of three sequential core sequences are identical, while the third core sequence of the three sequential core sequences is different (e.g., SEQ ID NOs: 27 and 31). A transgene expression cassette, in some embodiments, comprises one or more additional regulatory elements. For example, transgene expression cassette may include an enhancer. Enhancers (also referred to as enhancer elements) include a short region of DNA that can significantly increase the likelihood of transcription of a particular gene. Enhancers can be located either upstream or downstream of the gene they regulate, and in some instances within the gene itself. Enhancers can function in any orientation - forward or reversed. In some embodiments, a transgene expression cassette comprises a cytomegalovirus (CMV) enhancer.

[0070] A transgene expression cassette, in some embodiments, comprises a sequence encoding a modified Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). A WPRE is a sequence derived from the woodchuck hepatitis virus (WHV). Its primary function is to enhance the expression of genes to which it is linked by increasing the stability and export of the mRNA from the nucleus to the cytoplasm, leading to greater protein production. In some embodiments, the WPRE is a mut6.WPRE, which is a modified version in which certain mutations have been introduced to substantially increase expression levels but without promoter activity (see, e.g., Zufferey R. et al. J Virol. 1999;73(4):2886-2892 and Zanta-Boussif M.A. et al. Gene Then 2009;16(5):605-619).

[0071] A transgene expression cassette, in some embodiments, comprises a sequence encoding a polyadenylation signal. In some embodiments, the polyadenylation signal comprises bovine growth hormone poly adenylation signal (BGH poly A).

[0072] In some embodiments, an engineered nucleic acid comprises a sequence of SEQ ID NO:

[0073] 1 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 1. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 90% identity to the sequence of SEQ ID NO: 1. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 95% identity to the sequence of SEQ ID NO: 1. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 98% identity to the sequence of SEQ ID NO: 1. In some embodiments, an engineered nucleic acid comprises the sequence of SEQ ID NO: 1.

[0074] In some embodiments, an engineered nucleic acid comprises a sequence of SEQ ID NO:

[0075] 2 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 2. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 90% identity to the sequence of SEQ ID NO: 2. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 95% identity to the sequence of SEQ ID NO: 2. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 98% identity to the sequence of SEQ ID NO: 2. In some embodiments, an engineered nucleic acid comprises the sequence of SEQ ID NO: 2. In some embodiments, an engineered nucleic acid comprises a sequence of SEQ ID NO:

[0076] 4 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 4. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 90% identity to the sequence of SEQ ID NO: 4. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 95% identity to the sequence of SEQ ID NO: 4. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 98% identity to the sequence of SEQ ID NO: 4. In some embodiments, an engineered nucleic acid comprises the sequence of SEQ ID NO: 4.

[0077] In some embodiments, an engineered nucleic acid comprises a sequence of SEQ ID NO:

[0078] 5 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 5. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 90% identity to the sequence of SEQ ID NO: 5. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 95% identity to the sequence of SEQ ID NO: 5. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 98% identity to the sequence of SEQ ID NO: 5. In some embodiments, an engineered nucleic acid comprises the sequence of SEQ ID NO: 5.

[0079] In some embodiments, an engineered nucleic acid comprises a sequence of SEQ ID NO:

[0080] 6 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 6. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 90% identity to the sequence of SEQ ID NO: 6. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 95% identity to the sequence of SEQ ID NO: 6. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 98% identity to the sequence of SEQ ID NO: 6. In some embodiments, an engineered nucleic acid comprises the sequence of SEQ ID NO: 6.

[0081] In some embodiments, an engineered nucleic acid comprises a sequence of SEQ ID NO:

[0082] 7 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 7. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 90% identity to the sequence of SEQ ID NO: 7. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 95% identity to the sequence of SEQ ID NO: 7. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 98% identity to the sequence of SEQ ID NO: 7. In some embodiments, an engineered nucleic acid comprises the sequence of SEQ ID NO: 7.

[0083] In some embodiments, an engineered nucleic acid comprises a sequence of SEQ ID NO:

[0084] 8 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 8. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 90% identity to the sequence of SEQ ID NO: 8. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 95% identity to the sequence of SEQ ID NO: 8. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 98% identity to the sequence of SEQ ID NO: 8. In some embodiments, an engineered nucleic acid comprises the sequence of SEQ ID NO: 8.

[0085] In some embodiments, an engineered nucleic acid comprises a sequence of SEQ ID NO: 9 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 9. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 90% identity to the sequence of SEQ ID NO: 9. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 95% identity to the sequence of SEQ ID NO: 9. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 98% identity to the sequence of SEQ ID NO: 9. In some embodiments, an engineered nucleic acid comprises the sequence of SEQ ID NO: 9.

[0086] In some embodiments, an engineered nucleic acid comprises a sequence of SEQ ID NO:

[0087] 20 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 20. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 90% identity to the sequence of SEQ ID NO: 20. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 95% identity to the sequence of SEQ ID NO: 20. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 98% identity to the sequence of SEQ ID NO: 20. In some embodiments, an engineered nucleic acid comprises the sequence of SEQ ID NO: 20.

[0088] In some embodiments, an engineered nucleic acid comprises a sequence of SEQ ID NO:

[0089] 21 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 21. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 90% identity to the sequence of SEQ ID NO: 21. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 95% identity to the sequence of SEQ ID NO: 21. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 98% identity to the sequence of SEQ ID NO: 21. In some embodiments, an engineered nucleic acid comprises the sequence of SEQ ID NO: 21.

[0090] In some embodiments, an engineered nucleic acid comprises a sequence of SEQ ID NO: 26 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 26. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 90% identity to the sequence of SEQ ID NO: 26. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 95% identity to the sequence of SEQ ID NO: 26. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 98% identity to the sequence of SEQ ID NO: 26. In some embodiments, an engineered nucleic acid comprises the sequence of SEQ ID NO: 26.

[0091] In some embodiments, an engineered nucleic acid comprises a sequence of SEQ ID NO: 32 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 32. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 90% identity to the sequence of SEQ ID NO: 32. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 95% identity to the sequence of SEQ ID NO: 32. In some embodiments, an engineered nucleic acid comprises a sequence having at least at least 98% identity to the sequence of SEQ ID NO: 32. In some embodiments, an engineered nucleic acid comprises the sequence of SEQ ID NO: 32.1n some embodiments, an engineered nucleic acid comprises a sequence of SEQ ID NO: 33 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 33. In some embodiments, an engineered nucleic acid comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 33. In some embodiments, an engineered nucleic acid comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 33. In some embodiments, an engineered nucleic acid comprises a sequence having at least 98% identity to the sequence of SEQ ID NO: 33. In some embodiments, an engineered nucleic acid comprises the sequence of SEQ ID NO: 33.

[0092] In some embodiments, an engineered nucleic acid comprises a sequence of SEQ ID NO:

[0093] 34 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 34. In some embodiments, an engineered nucleic acid comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 34. In some embodiments, an engineered nucleic acid comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 34. In some embodiments, an engineered nucleic acid comprises a sequence having at least 98% identity to the sequence of SEQ ID NO: 34. In some embodiments, an engineered nucleic acid comprises the sequence of SEQ ID NO: 34.

[0094] In some embodiments, an engineered nucleic acid comprises a sequence of SEQ ID NO:

[0095] 35 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 35. In some embodiments, an engineered nucleic acid comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 35. In some embodiments, an engineered nucleic acid comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 35. In some embodiments, an engineered nucleic acid comprises a sequence having at least 98% identity to the sequence of SEQ ID NO: 35. In some embodiments, an engineered nucleic acid comprises the sequence of SEQ ID NO: 35. In some embodiments, an engineered nucleic acid comprises a sequence of SEQ ID NO: 36 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 36. In some embodiments, an engineered nucleic acid comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 36. In some embodiments, an engineered nucleic acid comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 36. In some embodiments, an engineered nucleic acid comprises a sequence having at least 98% identity to the sequence of SEQ ID NO: 36. In some embodiments, an engineered nucleic acid comprises the sequence of SEQ ID NO: 36.

[0096] In some embodiments, an engineered nucleic acid comprises a sequence of SEQ ID NO: 37 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 37. In some embodiments, an engineered nucleic acid comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 37. In some embodiments, an engineered nucleic acid comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 37. In some embodiments, an engineered nucleic acid comprises a sequence having at least 98% identity to the sequence of SEQ ID NO: 37. In some embodiments, an engineered nucleic acid comprises the sequence of SEQ ID NO: 37.

[0097] Table 1. Exemplary Sequences

[0098] Adeno-Associated Virus (AAV) Vector Genomes and Vectors

[0099] Any of the engineered nucleic acids e.g., transgene expression cassettes) described herein may be included in a viral vector genome. In some embodiments, the viral vector genome is an AAV vector genome, for example, an AAV9 vector genome. AAVs (or “rAAV” for recombinant AAV) are non-enveloped small, single- stranded DNA viruses capable of infecting both dividing and non-dividing cells. An AAV vector comprises sequences sufficient to (a) support packaging of the AAV vector genome and (b) to express a transgene expression cassette described herein. Thus, in some embodiments, an engineered nucleic acid further comprises two inverted terminal repeat (ITR) sequences, for example, a 5’ ITR located upstream from the regulatory elements of the engineered nucleic acid and a 3’ ITR located downstream from the regulatory elements. Generally, the sequences positions between the ITRs include a promoter, a gene of interest (e.g., GLP-1), and a terminator. In some embodiments, the ITR sequences are selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 (AAVrhlO), and AAV11 ITR sequences. In some embodiments, the ITRs are from an AAV2 serotype, and thus are referred to as AAV2 ITRs.

[0100] In some embodiments, an AAV vector genome (e.g., an AAV9 vector genome) is a single- stranded AAV (ssAAV) vector genome. In other embodiments, an AAV vector genome is a self- complementary AAV (scAAV) vector genome. In a scAAV vector, the viral genome is engineered to form a double- stranded DNA molecule. This can be achieved by using a mutated ITR sequence that allows the single- stranded genome to fold back on itself and form a doublestranded molecule - for example, the TRS (terminal resolution site) located in the 3' ITR is deleted. Thus, in some embodiments, an ITRs are designed for a single-stranded AAV genome or a self-complimentary AAV genome.

[0101] AAV vector genomes of the disclosure are typically package in an AAV vector, for example, an AAV9 vector. An AAV vector thus includes an AAV vector genome and AAV capsid proteins. An AAV capsid is primarily composed of three proteins: VP1, VP2, and VP3. These proteins are generated from the cap gene of AAV through alternative splicing and start codon usage. VP3 is the most abundant, while VP1 and VP2 are present in smaller quantities. The capsid proteins assemble into an icosahedral structure that encloses the viral genome.

[0102] Typically, a recombinant AAV vector (virus) is made by co-transfecting a plasmid containing a transgene expression cassette flanked by two AAV ITRs and an expression plasmid containing wild type AAV coding sequences without the ITRs. The AAV vector genome, which harbors the transgene expression cassette, can be constructed by directly inserting the selected sequence(s) into an AAV genome which has had the major AAV open reading frames (“ORFs”) excised. The following is a non-limiting example of an AAV vector production and purification method that may be used to produce the AAV vectors herein:

[0103] Cell platform'. HEK-293T, Sf9, or other suitable cell system can be grown on a small scale on 150 mm tissue culture-treated culture dish, hyperflasks, or shake flasks. Cells are then transfected with adenovirus helper virus, rep / cap, and ITR-transgene plasmids (e.g., comprising an engineered nucleic acid / transgene expression cassette described herein) for 293T, or infected with baculovirus for Sf9. Producer lines with integrated expression of replication / capsid (rep / cap) and ITR-transgene can be infected with adenovirus and grown to scale. Scale-up'. For larger-scale culture volumes, virus can be produced in roller bottles, continuous perfusion, or WAVE Bioreactor systems. Purification / polishing'. Affinity or heparin chromatography are optimal for isolation of virus from culture supernatants with or without cell pellet harvesting. Benzonase / DNAse treatment of eluted virus is required for removal of extraviral DNA contamination, followed by anion-exchange chromatography to fractionate ‘empty’ vs. ‘full’ AAV particles. See, e.g., Naso M.F. et al. BioDrugs 2017; 31(4): 317-334.

[0104] Other viral vectors contemplated herein include, for example, adenoviruses, retroviruses, lentiviruses, and herpes simplex viruses. Alternatively, non- viral vectors may be used to deliver the transgene expression cassettes of the disclosure, for example, in a manner that targets pancreatic tissue. Therapeutic Methods

[0105] Provided herein, in some aspects, are methods that comprises administering to a subject (e.g., a human subject) a composition comprising a viral vector genome (e.g., an AAV vector genome) of the disclosure, a viral vector (e.g., a AAV vector) of the disclosure, or a transgene expression cassette (or any engineered nucleic acid encoding a transgene) of the disclosure (referred to herein as a gene therapy composition). Administration, in some embodiments, is via injection of pancreatic tissue. For example, the administering may be achieved using an endoscopic ultrasound-guided fine needle injection (EUS-FNI) procedure, which is a minimally invasive medical procedure that combines endoscopic and ultrasound technologies to allow for precise needle injections into targeted areas within the pancreas (see, e.g., Kaur J el al. World J Gastroenterol. 2022 Jun 7; 28(21): 2383-2395).

[0106] A subject, in some embodiments, has (e.g., has been diagnosed with) a metabolic disorder, as discussed elsewhere herein. In some embodiments, a subject has Type 2 diabetes. In some embodiments, a subject is obese. In some embodiments, a subject has Metabolic Dysfunction-associated Steatotic Liver Disease.

[0107] In some embodiments, a therapeutically effective amount of a gene therapy composition is administered to a subject. Such an amount is considered therapeutically effective if it is sufficient to achieve a desired therapeutic effect in a subject (considering factors such as age, weight, sex, genetic factors, disease severity, and other medical conditions), such as alleviating one or more symptoms of the disorder being treated, optionally within a specific window of time. For example, if a subject has Type 2 diabetes, a therapeutically effective amount may be sufficient to maintain a normal level of glucose / blood sugar in the blood of a subject. Treatment of Type 2 Diabetes can include, in some embodiments, reduction in HbAlc, reduction in fasting glucose, improved time in a normal range of blood glucose levels, and / or improvement in hyperglycemia. It may also include reduction in or elimination of the need for exogenous insulin, reduction in or elimination of need for exogenous GLP-1 receptor agonists, without causing increased rates of nausea, diarrhea, vomiting, constipation, or abdominal pain. Other consequences of Type 2 Diabetes that may improve include, for example, hypertension, hypertriglyceridemia, hypercholesterolemia heart disease, diabetic heart disease, heart failure, diabetic heart failure, and / or diastolic dysfunction. A therapeutically effective amount to treat obesity may be sufficient to promote weight loss and / or prevent weight gain in a subject, for example. An effective amount, or a therapeutically effective amount of a gene therapy composition, in some embodiments, is an amount sufficient to regulate the subject’s blood glucose levels. In some embodiments, the subject’s blood glucose levels are regulated (e.g., maintained within a physiological range) in response to nutrient intake (e.g., consumption of a meal).

[0108] A single dose, or no more than two doses, of a gene therapy composition, in some embodiments, is sufficient to achieve a specific effect in a subject within a specific window of time. That window of time may be, for example, within 3 months of administering a gene therapy composition to a subject, within 6 months of administering a gene therapy composition to a subject, within 9 months of administering composition to a subject, within 12 months / 1 year of administering a gene therapy composition to a subject, or within 18 months of administering a gene therapy composition to a subject. In any one of the embodiments described herein, delivery of a single dose of a gene therapy composition may include delivery of only a single dose of a gene therapy composition within a specific period. Alternatively, delivery of a gene therapy composition may include delivery of no more than two doses (i.e., only two doses), for example, within a period of 1 year. Thus, in some embodiments, a method that comprises delivering a single dose, or no more than two doses, of a gene therapy composition (for example, to pancreatic endocrine tissue of a subject having metabolic disorder) in an amount effective to maintain a reduction in body weight of about 5% (relative to baseline) over the course of a year is achieved within a year of delivering that single dose, or only two doses, of the composition, and no other doses of that gene therapy composition are administered during that 1 year window of time.

[0109] In some embodiments, a total volume of a single dose is no more than 1 ml. In other embodiments, a total volume of the single dose is about 1 ml to about 5 ml. In some embodiments, a total volume of the single dose is about 1 ml, about 2 ml, about 3 ml, about 4 ml, or about 5 ml.

[0110] Methods and compositions of the disclosure, in some embodiments, are used to maintain weight loss in a subject, for example, a subject who has undergone a weight loss therapy (e.g., treatment with a weight loss drug such as semaglutide or a particular diet) but has discontinued use of that weight loss therapy. Maintaining weight loss, in some embodiments, refers to the process of successfully keeping off weight that has been lost, rather than regaining it, for an extended period, for example, 6 months, 12 months, or more (e.g., multiple years or lifetime). Weight maintenance can be just as challenging, if not more so, than the weight loss process itself due to various physiological, psychological, and environmental factors. Methods and compositions of the disclosure, in some embodiments, are used to maintain weight (e.g., ±1-5%, ±1-4%, ±1-3%, or ±1-2%) or further reduce weight in a subject who has already loss weight, for example, 1-5% total body weight. In some embodiments, a gene therapy composition (e.g., as single dose of a gene therapy composition) is delivered to pancreatic endocrine tissue of a subject in an amount effective to reduce body weight of the subject (e.g., relative to baseline), for example, by at least 1%, at least 2%, at least 3%, at least 5%, at least 5%, 10%, at least 15%, or at least 20% within 3 months, 6 months, 9 months, 12 months, or 18 months. The percentage of weight loss and length of time to lose the weight depends, at least in part, on the baseline starting) weight of the subject.

[0111] In some embodiments, a gene therapy composition (e.g., as a single dose or no more than two doses of a gene therapy composition) is delivered to pancreatic endocrine tissue of a subject in an amount effective to reduce body weight of the subject (relative to baseline) by at least 1%, at least 2%, at least 3%, or at least 4%, for example, within 3, 6, 9, 12, or 18 months of receiving the single or first dose of the gene therapy composition. In some embodiments, a gene therapy composition (e.g., as a single dose or no more than two doses of a gene therapy composition) is delivered to pancreatic endocrine tissue of a subject in an amount effective to reduce body weight of the subject (relative to baseline) by at least 5%, for example, within 3, 6, 9, 12, or 18 months of receiving the single or first dose of the gene therapy composition. In some embodiments, a gene therapy composition (e.g., as a single dose or no more than two doses of a gene therapy composition) is delivered to pancreatic endocrine tissue (e.g., c) of a subject in an amount effective to reduce body weight of the subject (relative to baseline) by at least 10%, for example, within 3, 6, 9, 12, or 18 months of receiving the single or first dose of the gene therapy composition. In some embodiments, the reduced body weight is maintained (e.g., within 1-2% of the reduced body weight) for at least 3 months. In some embodiments, the reduced body weight is maintained (e.g., within 1-2% of the reduced body weight) for at least 6 months. In some embodiments, the reduced body weight is maintained (e.g., within 1-2% of the reduced body weight) for at least 9 months. In some embodiments, the reduced body weight is maintained (e.g., within 1-2% of the reduced body weight) for at least 12 months. In some embodiments, the reduced body weight is maintained (e.g., within 1-2% of the reduced body weight) for at least 18 months.

[0112] In other embodiments, the body weight of a subject decreases by at least 5%, at least 6%, at least 7%, at least 8%, at least 9 %, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, or at least 25% relative to a control (e.g., no gene therapy or semaglutide) or baseline. In some embodiments, the body weight of a subject decreases by about 5-30%, about 5-25%, about 5-20%, about 5-15%, about 5-10%, about 10- 30%, about 10-25%, about 10-20%, about 10-15%, about 15-30%, about 15-25%, about 15-20%, about 20-30%, about 20-25%, or about 25-30% relative to a control (e.g., no gene therapy or semaglutide) or baseline. In some embodiments, the body weight of a subject decreases by about 5%, about 10%, about 20%, or about 25% relative to a control (e.g., no gene therapy or semaglutide) or baseline.

[0113] In some embodiments, the body weight of a subject does not change significantly relative to baseline. In some embodiments, the body weight of a subject changes by less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% relative to baseline. In some embodiments, the body weight of a subject stays the same relative to baseline.

[0114] In some embodiments, the weight gained by a subject decreases by at least 5%, at least 6%, at least 7%, at least 8%, at least 9 %, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, or at least 25% relative to a control (e.g., no gene therapy or semaglutide) or baseline. In some embodiments, the weight gained by the subject decreases by about 5-30%, about 5-25%, about 5-20%, about 5-15%, about 5-10%, about 10- 30%, about 10-25%, about 10-20%, about 10-15%, about 15-30%, about 15-25%, about 15-20%, about 20-30%, about 20-25%, or about 25-30% relative to a control (e.g., no gene therapy or semaglutide) or baseline. In some embodiments, the weight gained by a subject decreases by about 5%, about 10%, about 20%, or about 25% relative to a control (e.g., no gene therapy or semaglutide) or baseline.

[0115] In some embodiments, an effective amount restores glycemic durability in a subject. In some embodiments, a gene therapy composition (e.g., as single dose of a gene therapy composition) is delivered to pancreatic endocrine tissue of a subject in an amount effective to restore glycemic durability in the subject. The term glycemic durability includes the period of time during which a subject’s glycemic levels are within physiological ranges (e.g., “ideal glycemic control” or “optimal glycemic control”). According to the American Diabetes Association, the recommended HblAc cut-point for diagnosing diabetes is 6.5%, and individuals are at a high risk (Gillett et al., Diabetes Care. 2009;32:1327-34). In some embodiments, a glycemic physiological range is glycosylated hemoglobin (HbAlc) value of less than 10%, less than 9%, less than 8%, less than 7.5%, less than 7%, less than 6.5%, less than 6%, or less than 5%. In some embodiments, an effective amount results in the maintenance of the HbAlc value at less than 7%. In some embodiments, an optimal glycemic control is maintained for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 8 months, at least 10 months, at least 1 year, at least 1.5 years, at least 2 years, at least 2.5 years, at least 3 years, at least 3.5 years, at least 4 years, at least 4.5 years, at least 5 years, or longer. In some embodiments, an optimal glycemic control is maintained without substitution and / or addition of other glucose-lowering agents. In some embodiments, an effective amount reduces fasting blood glucose relative to baseline. In some embodiments, a gene therapy composition (e.g., as single dose of a gene therapy composition) is delivered to pancreatic endocrine tissue of a subject in an amount effective to significantly reduce fasting blood glucose. In some embodiments, a gene therapy composition (e.g., as single dose of a gene therapy composition) is delivered to pancreatic endocrine tissue of a subject in an amount effective to reduce fasting blood glucose, for example, by at least 10% relative to baseline. In some embodiments, an effective amount reduces fasting blood glucose by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more relative to baseline. In some embodiments, an effective amount reduces fasting blood glucose by at least 50% relative to baseline. In some embodiments, the effective amount reduces fasting blood glucose by at least 55% relative to baseline. In some embodiments, an effective amount reduces fasting blood glucose by at least 60% relative to baseline. According to the WHO, normal values for fasting glucose are between 70 mg / dL and 100 mg / dL, while 100 mg / dL to 125 mg / dL represents a pre-diabetic state, and fasting blood glucose above 126 mg / dL indicates a subject is diabetic (WHO, “Mean fasting blood glucose,” who.int / data / gho / indicator-metadata-registry / imr-details / 2380). In some embodiments, an effective amount reduces fasting blood glucose in a subject to less than 130 mg / dL, less than 126 mg / dL, less than 120 mg / dL, less than 115 mg / dL, less than 110 mg / dL, less than 105 mg / dL, or less than 100 mg / dL. As used herein, “fasting blood glucose” refers to the blood glucose value (glucose concentration in venous plasma) determined when a subject has fasted (without any food except water) for at least 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, or more hours (see, e.g., WHO, “Mean fasting blood glucose”). Reductions in blood glucose, in some embodiments, also leads to additional beneficial outcomes including reductions in the rates or progression of retinopathy, nephropathy, neuropathy, myocardial infarction, microvascular disease related to diabetes, and prevention or reduced incidence of end stage kidney disease. Additional possible benefits, in some embodiments, include a reduced rate of cognitive decline and / or reduction in major adverse cardiovascular (CV) events (MACE), e.g., reduction in composite of CV death, nonfatal myocardial infarction (MI), and / or nonfatal stroke.

[0116] In some embodiments, an effective amount increases fasting insulin relative to baseline. In some embodiments, a gene therapy composition (e.g., as single dose of a gene therapy composition) is delivered to pancreatic endocrine tissue of a subject in an amount effective to significantly increase fasting insulin. In some embodiments, a gene therapy composition (e.g., as single dose of a gene therapy composition) is delivered to pancreatic endocrine tissue of a subject in an amount effective to increase fasting insulin, for example, by at least 2-fold relative to baseline. In some embodiments, an effective amount increases fasting insulin by at least 1-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.1- fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.1-fold,

[0117] 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 4-fold, 5-fold, or more relative to baseline. In some embodiments, an effective amount increases fasting insulin by 2-fold relative to baseline. In some embodiments, an effective amount increases fasting insulin by, or by at least, 2.8-fold relative to baseline. In some embodiments, an effective amount increases fasting insulin by 3- fold relative to baseline. As used herein, “fasting insulin” refers to the insulin level determined when a subject has fasted (without any food except water) for at least 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, or more hours. “Baseline” refers to the level of a measurable component or characteristic (e.g., insulin, blood glucose, weight, etc.) of a subject before beginning treatment, for example, with a localized gene therapy as provided herein.

[0118] In some embodiments, an effective amount significantly improves glucose tolerance relative to baseline. In some embodiments, a gene therapy composition (e.g., as single dose of a gene therapy composition) is delivered to pancreatic endocrine tissue of a subject in an amount effective to significantly improves glucose tolerance relative to baseline. Glucose tolerance refers to a subject’s ability to control plasma glucose and / or plasma insulin levels when glucose intake varies. It may be measured using any method in the art, including oral glucose tolerance tests (OGTTs), such as a glucose challenge test during which a subject drinks a glass of concentrated glucose solution (e.g., 50 g of glucose dissolved in 250-300 mL of water) and a subject’s blood sugar level is measured in the blood at least 1 hour later. In some embodiments, glucose tolerance is measured by comparing a fasting blood glucose level to the blood glucose level 1-3 hours after consuming the concentrated glucose solution. According to the American Diabetes Association, a blood glucose (sugar) concentration of less than 140 mg / dL is normal, 140 mg / dL - 199 mg / dL indicates prediabetes, and 200 mg / dL or more indicates diabetes (diabetes.org / diabetes / alc / diagnosis). In some embodiments, a subject has a blood sugar level of less than 200 mg / dL, less than 190 mg / dL, less than 180 mg / dL, less than 170 mg / dL, less than 160 mg / dL, less than 150 mg / dL, less than 140 mg / dL, less than 130 mg / dL, or less. In some embodiments, a subject’s blood sugar level is less than 140 mg / dL after treatment. In some embodiments, a subject’s blood sugar level is reduced 5 mg / dL, 6 mg / dL, 7 mg / dL, 8 mg / dL, 9 mg / dL, 10 mg / dL, 15 mg / dL, 20 mg / dL, 25 mg / dL, 30 mg / dL, 35 mg / dL, 40 mg / dL, 45 mg / dL, 50 mg / dL, or more relative to baseline. In some embodiments, an effective amount regulates blood glucose levels in a subject, for example, in a nutrient-responsive manner. Accordingly, in some embodiments, an effective amount causes a subject to maintain a physiological concentration of blood glucose in response to nutrient intake (e.g., during and following consumption of a meal or other food). In some embodiments, an effective amount significantly improves glucose-stimulated insulin secretion relative to baseline. In some embodiments, a gene therapy composition (e.g., as single dose of a gene therapy composition) is delivered to pancreatic endocrine tissue of a subject in an amount effective to significantly improves glucose-stimulated insulin secretion relative to baseline. Glucose-stimulated insulin secretion (GSIS) can be measured using any method known in the art, for example, hyperinsulinemic-euglycemic clamp method, the hyperglycemic clamp method, or extrapolating from surrogate measures of insulin sensitivity (e.g., intravenous glucose tolerance test data, fasting blood samples, and the quantitative insulin sensitivity check index). In some embodiments, a GSIS is increased from baseline following treatment. In some embodiments, a GSIS is increased at least 1-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6- fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 4-fold, 5- fold, or more relative to baseline.

[0119] In some embodiments, an effective amount significantly improves liver (hepatic) triglyceride levels relative to baseline. In some embodiments, a gene therapy composition (e.g., as single dose of a gene therapy composition) is delivered to pancreatic endocrine tissue of a subject in an amount effective to significantly improves liver (hepatic) triglyceride levels relative to baseline. Liver triglycerides can be measured using any method known in the art, for example, by magnetic resonance imaging proton density fat fraction (MRLPDFF). In some embodiments, liver triglycerides are reduced for example, by at least 10% relative to baseline. In some embodiments, an effective amount reduces liver triglycerides by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more relative to baseline. In some embodiments, an effective amount reduces liver triglycerides by at least 30% relative to baseline. In some embodiments, an effective amount reduces liver triglycerides by at least 35% relative to baseline. In some embodiments, an effective amount reduces liver triglycerides by at least 40% relative to baseline. In some embodiments, an effective amount reduces liver triglycerides by at least 45% relative to baseline. In some embodiments, an effective amount reduces liver triglycerides by at least 50% relative to baseline. In some embodiments, an effective amount reduces liver triglycerides by at least 55% relative to baseline. In some embodiments, an effective amount reduces liver triglycerides by at least 60% relative to baseline. In some embodiments, an effective amount reduces liver triglycerides by at least 65% relative to baseline. In some embodiments, the effective amount reduces liver triglycerides by at least 70% relative to baseline.

[0120] “Baseline” refers to a subject’s levels (e.g., blood glucose levels) before beginning treatment, for example, before receiving a dose of a gene therapy composition. An effective amount of a gene therapy composition, in some embodiments, is about 5xlO10to about 1.5xl014(e.g., about 5xl012to about 1.5xl014) vector genomes (VG), for example AAV VG. For example, the effective amount may be about 5xlO10, 5.5xlO10, 6xlO10, 6.5xlO10, 7xlO10, 7.5xlO10, 8xlO10, 8.5xlO10, 9xlO10, 9.5xlO10, IxlO11, 1.5xlOn, 2xlOn, 2.5xlOn, 3xlOn, 3.5xlOn, 4xlOn, 4.5 xlO11, 5xlOn, 5.5xlOn, 6xlOn, 6.5xlOn, 7xlOn, 7.5xlOn, 8xlOn, 8.5xlOn, 9xlOn, 9.5xlOn, IxlO12, 1.5xl012, 2xl012, 2.5xl012, 3xl012, 3.5xl012, 4xl012, 4.5xl012, 5xl012, 5.5xl012, 6xl012, 6.5xl012, 7xl012, 7.5xl012, 8xl012, 8.5xl012, 9xl012, 9.5xl012, IxlO13, 1.5xl013, 2xl013, 2.5xl013, 3xl013, 3.5xl013, 4xl013, 4.5xl013, 5xl013, 5.5xl013, 6xl013, 6.5xl013, 7xl013, 7.5xl013, 8xl013, 8.5xl013, 9xl013, 9.5xl013, IxlO14, or 1.5xl014. In some embodiments, an effective amount is about IxlO10to about IxlO13VG. In some embodiments, an effective amount is about 5xlO10to about 5xl012VG. In some embodiments, an effective amount is about 5xlOnto about 7xlOnVG. In some embodiments, an effective amount is about 5xlOnVG. In some embodiments, an effective amount is about 6xlOnVG. In some embodiments, an effective amount is about 7xlOnVG. In some embodiments, an effective amount is administered every 6 months. In some embodiments, an effective amount is administered yearly.

[0121] In some embodiments, an effective amount is about 5xlO10to about 1.5xl014(e.g., about 5xl012to about 1.5xl014) VG. In some embodiments, an effective amount is about 5xl012to about 5xl013VG. In some embodiments, an effective amount is about 5xl012to about IxlO14VG. In some embodiments, an effective amount is about IxlO13to about 5xl013VG. In some embodiments, an effective amount is about IxlO13to about IxlO14VG.

[0122] An effective amount of a gene therapy composition, in some embodiments, is a single dose, for example, of about 5xlO10to about 1.5xl014(e.g., about 5xl012to about 1.5xl014) vector genomes (VG), e.g., AAV VG. For example, the effective amount may be a single dose of about 5xlO10, 5.5xlO10, 6xlO10, 6.5xlO10, 7xlO10, 7.5xlO10, 8xlO10, 8.5xlO10, 9xlO10, 9.5xlO10, IxlO11, 1.5xlOn, 2xlOn, 2.5xlOn, 3xlOn, 3.5xlOn, 4xlOn, 4.5 xlO11, 5xlOn, 5.5xlOn, 6xlOn, 6.5xlOn, 7xlOn, 7.5xlOn, 8xlOn, 8.5xlOn, 9xlOn, 9.5xlOn, IxlO12, 1.5xl012, 2xl012, 2.5xl012, 3xl012, 3.5xl012, 4xl012, 4.5xl012, 5xl012, 5.5xl012, 6xl012, 6.5xl012, 7xl012, 7.5xl012, 8xl012, 8.5xl012, 9xl012, 9.5xl012, IxlO13, 1.5xl013, 2xl013, 2.5xl013, 3xl013, 3.5xl013, 4xl013, 4.5xl013, 5xl013, 5.5xl013, 6xl013, 6.5xl013, 7xl013, 7.5xl013, 8xl013, 8.5xl013, 9xl013, 9.5xl013, IxlO14, or 1.5xl014. In some embodiments, an effective amount is a single dose of about IxlO10to about IxlO13VG. In some embodiments, an effective amount is a single dose of about 5xlO10to about 5xl012VG. In some embodiments, an effective amount is a single dose of about 5xlOnto about 7xlOnVG. In some embodiments, an effective amount is a single dose of about 5xlOnVG. In some embodiments, an effective amount is about 6xlOnVG. In some embodiments, an effective amount is a single dose of about 7xlOnVG. In some embodiments, a single dose is administered every 6 months. In some embodiments, a single dose is administered yearly.

[0123] In some embodiments, an effective amount is a single dose of about 5xl012to about IxlO13VG. In some embodiments, an effective amount is a single dose of about 5xl012to about 5xl013VG. In some embodiments, an effective amount is a single dose of about 5xl012to about IxlO14VG. In some embodiments, an effective amount is a single dose of about IxlO13to about 5xl013VG. In some embodiments, an effective amount is a single dose of about IxlO13to about IxlO14VG.

[0124] In some embodiments, a method for treating a metabolic disorder in a subject in need thereof, comprises delivering an effective amount of a gene therapy composition to endocrine tissue of a splenic lobe of the pancreas of a subject using an endoscopic ultrasound-guided fine needle injection (EUS-FNI) procedure, wherein the gene therapy composition comprises an adeno-associated virus (AAV) vector encoding a human GLP-1 receptor agonist, and the effective amount is a single dose of about 5xlO10to about 1.5xl014(e.g., about 5xl012to about 1.5xl014) vector genomes (VG). For example, the effective amount may be a single dose of about 5xlO10, 5.5xlO10, 6xlO10, 6.5xlO10, 7xlO10, 7.5xlO10, 8xlO10, 8.5xlO10, 9xlO10, 9.5xlO10, IxlO11, 1.5xlOn, 2xlOn, 2.5xlOn, 3xlOn, 3.5xlOn, 4xlOn, 4.5 xlO11, 5xlOn, 5.5xlOn, 6xlOn, 6.5xlOn, 7xlOn, 7.5xlOn, 8xlOn, 8.5xlOn, 9xlOn, 9.5xlOn, IxlO12, 1.5xl012, 2xl012, 2.5xl012, 3xl012, 3.5xl012, 4xl012, 4.5xl012, 5xl012, 5.5xl012, 6xl012, 6.5xl012, 7xl012, 7.5xl012, 8xl012, 8.5xl012, 9xl012, 9.5xl012, IxlO13, 1.5xl013, 2xl013, 2.5xl013, 3xl013, 3.5xl013, 4xl013, 4.5xl013, 5xl013, 5.5xl013, 6xl013, 6.5xl013, 7xl013, 7.5xl013, 8xl013, 8.5xl013, 9xl013, 9.5xl013, IxlO14, or 1.5xl014. In some embodiments, an effective amount is a single dose of about IxlO10to about IxlO13VG. In some embodiments, an effective amount is a single dose of about 5xlO10to about 5xl012VG. In some embodiments, an effective amount is a single dose of about 5xlOnto about 7xlOnVG. In some embodiments, an effective amount is a single dose of about 5xlOnVG. In some embodiments, an effective amount is about 6xlOnVG.

[0125] In some embodiments, an effective amount is a single dose of about 7xlOnVG. In some embodiments, a single dose is administered every 6 months. In some embodiments, a single dose is administered yearly.

[0126] In some embodiments, a method for treating obesity in a subject in need thereof, comprises delivering an effective amount of a gene therapy composition to endocrine tissue of a splenic lobe of the pancreas of a subject using an endoscopic ultrasound-guided fine needle injection (EUS-FNI) procedure, wherein the gene therapy composition comprises an adeno- associated virus (AAV) vector encoding a human GLP-1 receptor agonist, and the effective amount is a single dose of about 5xlO10to about 1.5xl014(e.g., about 5xl012to about 1.5xl014) vector genomes (VG). For example, the effective amount may be a single dose of about 5xlO10, 5.5xlO10, 6xlO10, 6.5xlO10, 7xlO10, 7.5xlO10, 8xlO10, 8.5xlO10, 9xlO10, 9.5xlO10, IxlO11, 1.5xlOn, 2xlOn, 2.5xlOn, 3xlOn, 3.5xlOn, 4xlOn, 4.5 xlO11, 5xlOn, 5.5xlOn, 6xlOn, 6.5xlOn, 7xlOn, 7.5xlOn, 8xlOn, 8.5xlOn, 9xlOn, 9.5xlOn, IxlO12, 1.5xl012, 2xl012, 2.5xl012, 3xl012, 3.5xl012, 4xl012, 4.5xl012, 5xl012, 5.5xl012, 6xl012, 6.5xl012, 7xl012, 7.5xl012, 8xl012, 8.5xl012, 9xl012, 9.5xl012, IxlO13, 1.5xl013, 2xl013, 2.5xl013, 3xl013, 3.5xl013, 4xl013, 4.5xl013, 5xl013, 5.5xl013, 6xl013, 6.5xl013, 7xl013, 7.5xl013, 8xl013, 8.5xl013, 9xl013, 9.5xl013, IxlO14, or 1.5xl014. In some embodiments, an effective amount is a single dose of about IxlO10to about IxlO13VG. In some embodiments, an effective amount is a single dose of about 5xlO10to about 5xl012VG. In some embodiments, an effective amount is a single dose of about 5xlOnto about 7xlOnVG. In some embodiments, an effective amount is a single dose of about 5xlOnVG. In some embodiments, an effective amount is about 6xlOnVG. In some embodiments, an effective amount is a single dose of about 7xlOnVG. In some embodiments, a single dose is administered every 6 months. In some embodiments, a single dose is administered yearly.

[0127] In some embodiments, a method for treating Type 2 Diabetes in a subject in need thereof, comprises delivering an effective amount of a gene therapy composition to endocrine tissue of a splenic lobe of the pancreas of a subject using an endoscopic ultrasound-guided fine needle injection (EUS-FNI) procedure, wherein the gene therapy composition comprises an adeno- associated virus (AAV) vector encoding a human GLP-1 receptor agonist, and the effective amount is a single dose of about 5xl012to about 5xlO10, 5.5xlO10, 6xlO10, 6.5xlO10, 7xlO10, 7.5xlO10, 8xlO10, 8.5xlO10, 9xlO10, 9.5xlO10, IxlO11, 1.5xlOn, 2xlOn, 2.5xlOn, 3xlOn, 3.5xlOn, 4xlOn, 4.5 xlO11, 5xlOn, 5.5xlOn, 6xlOn, 6.5xlOn, 7xlOn, 7.5xlOn, 8xlOn, 8.5xlOn, 9xlOn, 9.5xlOn, IxlO12, 1.5xl012, 2xl012, 2.5xl012, 3xl012, 3.5xl012, 4xl012,

[0128] 4.5xl012, 5xl012, 5.5xl012, 6xl012, 6.5xl012, 7xl012, 7.5xl012, 8xl012, 8.5xl012, 9xl012,

[0129] 9.5xl012, IxlO13, 1.5xl013, 2xl013, 2.5xl013, 3xl013, 3.5xl013, 4xl013, 4.5xl013, 5xl013,

[0130] 5.5xl013, 6xl013, 6.5xl013, 7xl013, 7.5xl013, 8xl013, 8.5xl013, 9xl013, 9.5xl013, IxlO14, or

[0131] 1.5xl014. In some embodiments, an effective amount is a single dose of about IxlO10to about IxlO13VG. In some embodiments, an effective amount is a single dose of about 5xlO10to about 5xl012VG. In some embodiments, an effective amount is a single dose of about 5xlOnto about 7xlOnVG. In some embodiments, an effective amount is a single dose of about 5xlOnVG. In some embodiments, an effective amount is about 6xlOnVG. In some embodiments, an effective amount is a single dose of about 7xlOnVG. In some embodiments, a single dose is administered every 6 months. In some embodiments, a single dose is administered yearly. In some embodiments, a serum lipase level in a subject is within 3 times the upper limit of a normal serum lipase level at day 1 through day 7 post delivery of the gene therapy composition. The normal range for adults younger than 60 is typically about 10 to 140 U / L. The normal range for adults ages 60 and older is typically 24 to 151 U / L. In some embodiments, a serum lipase level in a subject is less than 5 U / L at day 1 through day 7 post delivery of the gene therapy composition. Serum lipase is an enzyme that is produced by the pancreas and is involved in the digestion of fats. With pancreatitis, the pancreas becomes inflamed and damaged, leading to the leakage of serum lipase and other enzymes into the bloodstream. Elevated levels of serum lipase are seen in most cases of acute pancreatitis and can help to confirm the diagnosis. In fact, serum lipase levels are often more sensitive and specific for pancreatitis than other diagnostic tests, such as serum amylase levels. The in vivo data provided herein demonstrated that the method of the disclosure has minimal impact on serum lipase levels.

[0132] In some embodiments, a human GLP-1 receptor agonist is present in the pancreas at a level that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% higher than the level detected in the sera of a subject. In some embodiments, a human GLP-1 receptor agonist is present in the pancreas at a level that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% higher than the level detected in the brain of a subject. In some embodiments, a human GLP-1 receptor agonist is not detectable in the sera of a subject. In some embodiments, a human GLP-1 receptor agonist is not detectable in the brain of a subject.

[0133] In some embodiments, an effective amount of a gene therapy composition results in a subject having Type 2 Diabetes in remission (that is, a subject maintains physiological levels of blood glucose). In some embodiments, an effective amount is a single dose, two doses, three doses, four doses, five doses, six doses, or more doses. In some embodiments, an effective amount is sufficient for long-term restoration of pancreatic islet beta cell function and / or reduction of therapeutic burden (e.g., workload of healthcare experienced by a subject and its impact on a subject’s well-being). In some embodiments, “long term restoration” means 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or longer, including complete and permanent remission.

[0134] In some embodiments, additional treatments are administered in addition to the gene therapy compositions provided herein. Exemplary additional treatments include treatments for Type 2 Diabetes, such as amylinomimetic drugs, alpha-glucosidase inhibitors, biguanides, dopamine agonists, dipeptidyl peptidase-4 (DPP-4) inhibitors, GLP-1 receptor agonists, meglitinides, statins, sodium-glucose transporter (SGLT) 2 inhibitors, sulfonylureas, thiazolidinediones, insulin, and combinations thereof. In some embodiments, additional treatments are not administered to a subject.

[0135] In some embodiments, a gene therapy composition comprises an excipient and / or carrier, inert or active, making the composition especially suitable for therapeutic use in vivo or ex vivo. A pharmaceutically acceptable excipient and / or carrier, after administered to or upon a subject, does not cause undesirable physiological effects.

[0136] Subjects are typically human subjects; however, a subject could be any mammal, including a non-human primate.

[0137] The gene therapy compositions described herein, in preferred embodiments, are administered locally; however, other routes may be used. These include, but are not limited, to intradermal, intramuscular, intranasal, and / or subcutaneous administration. In some embodiments, a gene therapy composition is delivered locally (e.g., into the splenic lobe / pancreatic tail of the pancreas) instead of systemically. In some embodiments, a gene therapy composition is delivered to a pancreatic islet cell (e.g., a pancreatic islet beta cell). In some embodiments, a gene therapy composition is delivered to a pancreatic islet beta cell via an endoscopic procedure, such as an EUS-FNI.

[0138] The present disclosure also contemplates combination therapies using, for example, the REVITA® System, which is a minimally invasive, outpatient, endoscopic, one-time procedural therapy. The REVITA® System includes a specially designed control console and a novel singleuse balloon catheter. The console is used to monitor the procedure, while the physician uses the catheter to apply heat to the duodenum. The REVITA® System may be used, in some embodiments, as an adjunct combination therapy.

[0139] In some embodiments, for example, those in which the GLP-1 peptide encoded by a transgene expression cassette includes the native human GLP-1 peptide sequence (e.g., SEQ ID NO: 14 or 15), a DPP-4 inhibitor is administered to a subject, in combination with the transgene expression cassette. Non-limiting example of DPP-4 inhibitors include selected from Sitagliptin, Vildagliptin, Saxagliptin, Linagliptin, Gemigliptin, Anagliptin, Teneligliptin, Alogliptin, Trelagliptin, Omarigliptin, Evogliptin, Gosogliptin, Dutogliptin, Neogliptin, Retagliptin, Denagliptin, and Cofrogliptin.

[0140] In some embodiments, a DPP-4 inhibitor is administered at the same time as administration of a gene therapy composition (e.g., comprising a transgene expression cassette encoding GLP-1 or GLP-1 and IP-2) is administered. In some embodiments, a DPP-4 inhibitor is administered after a gene therapy composition (e.g., comprising a transgene expression cassette encoding GLP-1 or GLP-1 and IP-2) is administered. For example, a DPP-4 inhibitor may be administered within 1 to 23 hours, within 1 to 6 days, with 1 to 4 weeks, or within 1 (or more) month of administering a gene therapy composition (e.g., comprising a transgene expression cassette encoding GLP-1 or GLP-1 and IP-2).

[0141] In some embodiments, a DPP-4 inhibitor is administered via an oral route of administration. Non-limiting examples of oral routes of administration include tablets (e.g., chewable tablets, effervescent tablets, sublingual and buccal tablets, and orally disintegrating tablets (ODTs)), capsules, liquid solutions, powders.

[0142] Additional methods of the disclosure are described in the following numbered paragraphs:

[0143] 1. An adeno-associated virus (AAV) vector genome comprising: an engineered nucleic acid comprising a promoter comprising multiple copies of a human insulin promoter sequence operably linked to an open reading frame comprising a sequence encoding an intervening peptide 2 (IP-2) peptide and a sequence encoding a glucagon-like peptide 1 (GLP-1) peptide.

[0144] 2. An adeno-associated virus (AAV) vector genome comprising: an engineered nucleic acid comprising a promoter operably linked to an open reading frame comprising a sequence encoding an intervening peptide 2 (IP-2) peptide and a sequence encoding a glucagon-like peptide 1 (GLP-1) peptide, wherein the open reading frame does not comprise a sequence encoding an intervening peptide 1 (IP-1) peptide.

[0145] 3. The AAV vector genome of paragraph 1 or 2, wherein the IP-2 peptide comprises the sequence of SEQ ID NO: 16 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 16.

[0146] 4. The AAV vector genome of paragraph 3, wherein the IP-2 peptide consists of or consists essentially of the sequence of SEQ ID NO: 16.

[0147] 5. The AAV vector genome of any one of the preceding paragraphs, wherein the sequence encoding an IP-2 peptide is downstream from the sequence encoding a GLP-1 peptide.

[0148] 6. The AAV vector genome of any one of the preceding paragraphs, wherein the GLP-1 peptide comprises the sequence of SEQ ID NO: 14 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 14.

[0149] 7. The AAV vector genome of any one of the preceding paragraphs, wherein the GLP-1 peptide comprises the sequence of SEQ ID NO: 15 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 15.

[0150] 8. The AAV vector genome of paragraph 7, wherein the open reading frame further comprises a sequence encoding a PC 1 / 3 cleavage site, optionally between amino acid positions corresponding to amino acid position 6 and amino acid position 7 of a GLP-1 peptide comprising the amino acid sequence of SEQ ID NO: 14. 9. The AAV vector genome of any one of the preceding paragraphs, wherein the GLP-1 peptide is a modified GLP-1 peptide that comprises one or more amino acid substitution(s), relative to a wild-type GLP-1 peptide consisting of the sequence of SEQ ID NO: 14 or 15, that renders the modified GLP-1 peptide resistant to degradation by dipeptidyl peptidase-4 (DPP-4), optionally wherein the modified GLP-1 peptide is selected from the group consisting of: Arg26- GLP-l(7-37); Arg34-GLP-l(7-37); Lys36-GLP-l(7-37); Arg26,34Lys36-GLP- 1(7-37); Arg26,34-GLP-l(7-37); Arg26,34Lys40-GLP-l(7-37); Arg26Lys36-GLP- 1(7-37); Arg34Lys36- GLP- 1 (7-37) Val8Arg22-GLP- 1(7-37); Met8Arg22-GLP-l(7-37);Gly8His22-GLP-l(7-37); Val8His22-GLP-l(7-37); Met8His22-GLP-l(7-37); His37-GLP- 1(7-37); Gly8-GLP- 1(7-37); Val8-GLP- 1(7-37); Met8-GLP- 1(7-37); Gly8Asp22-GLP-l(7-37); Val8Asp22-GLP- 1(7-37); Met8Asp22-GLP-l(7-37); Gly8Glu22-GLP-l(7-37); Val8Glu22-GLP- 1(7-37); Met8Glu22-GLP- 1(7-37); Gly8Lys22-GLP-l(7-37); Val8Lys22-GLP-l(7-37); Met8Lys22-GLP-l(7-37); Gly8Arg22-GLP-l(7-37); Vai 8Lys22His37-GLP- 1(7-37); Gly8Glu22His37-GLP-l(7-37); Val8Glu22His37-GLP-l(7-37); Met8Glu22His37-GLP- 1(7-37); Gly8Lys22His37-GLP-l(7-37); Met8Lys22His37-GLP-l(7-37); Gly8Arg22His37-GLP- 1(7-37); Val8Arg22His37-GLP- 1(7-37); Met8Arg22His37-GLP-l(7-37); Gly8His22His37-GLP-l(7-37); Vai 8His22His37-GLP- 1(7-37); Met8His22His37-GLP- 1(7-37); Gly8His37-GLP- 1(7-37); Vai 8His37-GLP- 1(7-37); Met8His37- GLP- 1(7-37); Gly8Asp22His37-GLP- 1(7-37); Val8Asp22His37-GLP- 1(7-37); and Met8Asp22His37-GLP- 1(7-37).

[0151] 10. The AAV vector genome of paragraph 9, wherein the GLP-1 peptide consists of or consists essentially of the sequence of SEQ ID NO: 14 or 15, optionally with no more than one amino acid substitution relative to a wild-type GLP-1 peptide consisting of the sequence of SEQ ID NO: 1 or 2, that renders the modified GLP-1 peptide resistant to degradation by DPP-4, optionally Gly8-GLP- 1(7-37) or Val8-GLP- 1(7-37).

[0152] 11. The AAV vector genome of any one of the preceding paragraphs, wherein the open reading frame does not comprise a sequence encoding one or more of: a glicentin-related pancreatic polypeptide (GRPP), a glucagon peptide, or a GLP-2 peptide.

[0153] 12. The AAV vector genome of paragraph 11, wherein the open reading frame does not comprise a sequence encoding the GRPP, the glucagon peptide, or the GLP-2 peptide.

[0154] 13. The AAV vector genome of paragraph 12, wherein the only preproglucagon (GCG) peptides encoded by the open reading frame are the IP-2 peptide and the GLP-1 peptide.

[0155] 14. The AAV vector genome of any one of the preceding paragraphs, wherein the open reading frame further comprises a sequence encoding a signal peptide, optionally an insulin gene or preproglucagon signal peptide. 15. The AAV vector genome of any one of the preceding paragraphs, wherein the promoter comprises a pancreatic tissue- specific promoter sequence.

[0156] 16. The AAV vector genome of paragraph 15, wherein the pancreatic tissue-specific promoter sequence comprises an islet beta cell-specific promoter sequence.

[0157] 17. The AAV vector genome of paragraph 16, wherein the islet beta cell-specific promoter sequence comprises an insulin gene promoter sequence.

[0158] 18. The AAV vector genome of paragraph 17, wherein the insulin gene promoter sequence comprises a human insulin gene promoter sequence, optionally a core sequence, further optionally at least three copies of the core sequence.

[0159] 19. The AAV vector genome of any one of the preceding paragraphs, optionally an AAV9 vector genome, wherein the engineered nucleic acid further comprises: an enhancer element, optionally a cytomegalovirus (CMV) enhancer element; a sequence encoding a modified Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), optionally mut6.WPRE; and / or a sequence encoding a polyadenylation signal, optionally a bovine growth hormone polyadenylation signal.

[0160] 20. An adeno-associated virus (AAV) vector genome, optionally an AAV9 vector genome, comprising an engineered nucleic acid comprising a promoter, optionally comprising a human insulin promoter sequence, operably linked to an open reading frame comprising the sequence of SEQ ID NO: 4 or 5, or a sequence having at least 90% identity to the sequence of SEQ ID NO: 4 or 5.

[0161] 21. An adeno-associated virus (AAV) vector genome, optionally an AAV9 vector genome, comprising an engineered nucleic acid comprising the sequence of SEQ ID NO: 20, 21, or 35 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 20, 21, or 35.

[0162] 22. The AAV vector genome of the preceding paragraphs, wherein the engineered nucleic acid further comprises a 5’ inverted terminal repeat (ITR) sequence, optionally an AAV2 ITR, upstream from the promoter and a 3’ ITR, optionally an AAV2 ITR, downstream from the open reading frame.

[0163] 23. The AAV vector genome of the preceding paragraphs, wherein the AAV vector genome is a self-complementary AAV vector genome.

[0164] 24. An AAV vector, optionally an AAV9 vector, comprising (a) the AAV vector genome of any one of the preceding paragraphs and (b) AAV capsid proteins.

[0165] 25. An engineered nucleic acid comprising: a promoter operably linked to an open reading frame comprising a sequence encoding an intervening peptide 2 (IP-2) peptide and a sequence encoding a glucagon-like peptide 1 (GLP-1) peptide, wherein the open reading frame does not comprise a sequence encoding an intervening peptide 1 (IP-1) peptide.

[0166] 26. The engineered nucleic acid of paragraph 25, wherein the IP-2 peptide comprises the sequence of SEQ ID NO: 16 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 16.

[0167] 27. The engineered nucleic acid of paragraph 26, wherein the IP-2 peptide consists of or consists essentially of the sequence of SEQ ID NO: 16.

[0168] 28. The engineered nucleic acid of any one of the preceding paragraphs, wherein the sequence encoding an IP-2 peptide is downstream from the sequence encoding a GLP-1 peptide.

[0169] 29. The engineered nucleic acid of any one of the preceding paragraphs, wherein the GLP-1 peptide comprises the sequence of SEQ ID NO: 14 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 14.

[0170] 30. The engineered nucleic acid of any one of the preceding paragraphs, wherein the GLP-1 peptide comprises the sequence of SEQ ID NO: 15 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 15.

[0171] 31. The engineered nucleic acid of paragraph 30, wherein the open reading frame further comprises a sequence encoding a PC 1 / 3 cleavage site, optionally between amino acid positions corresponding to amino acid position 6 and amino acid position 7 of a GLP-1 peptide comprising the amino acid sequence of SEQ ID NO: 14.

[0172] 32. The engineered nucleic acid of any one of the preceding paragraphs, wherein the GLP-1 peptide is a modified GLP-1 peptide that comprises one or more amino acid substitution(s), relative to a wild-type GLP-1 peptide consisting of the sequence of SEQ ID NO: 1 or 2, that renders the modified GLP-1 peptide resistant to degradation by dipeptidyl peptidase-4 (DPP-4), optionally wherein the modified GLP-1 peptide is selected from the group consisting of: Arg26- GLP-l(7-37); Arg34-GLP-l(7-37); Lys36-GLP-l(7-37); Arg26,34Lys36-GLP- 1(7-37);

[0173] Arg26,34-GLP-l(7-37); Arg26,34Lys40-GLP-l(7-37); Arg26Lys36-GLP- 1(7-37); Arg34Lys36- GLP- 1 (7-37) Val8Arg22-GLP- 1(7-37); Met8Arg22-GLP-l(7-37);Gly8His22-GLP-l(7-37);

[0174] Val8His22-GLP-l(7-37); Met8His22-GLP-l(7-37); His37-GLP- 1(7-37); Gly8-GLP- 1(7-37); Val8-GLP- 1(7-37); Met8-GLP- 1(7-37); Gly8Asp22-GLP-l(7-37); Val8Asp22-GLP- 1(7-37); Met8Asp22-GLP-l(7-37); Gly8Glu22-GLP-l(7-37); Val8Glu22-GLP- 1(7-37); Met8Glu22-GLP- 1(7-37); Gly8Lys22-GLP-l(7-37); Val8Lys22-GLP-l(7-37); Met8Lys22-GLP-l(7-37);

[0175] Gly8Arg22-GLP-l(7-37); Vai 8Lys22His37-GLP- 1(7-37); Gly8Glu22His37-GLP-l(7-37);

[0176] Val8Glu22His37-GLP-l(7-37); Met8Glu22His37-GLP- 1(7-37); Gly8Lys22His37-GLP-l(7-37); Met8Lys22His37-GLP-l(7-37); Gly8Arg22His37-GLP- 1(7-37); Val8Arg22His37-GLP- 1(7-37); Met8Arg22His37-GLP-l(7-37); Gly8His22His37-GLP-l(7-37); Vai 8His22His37-GLP- 1(7-37); Met8His22His37-GLP- 1(7-37); Gly8His37-GLP- 1(7-37); Vai 8His37-GLP- 1(7-37); Met8His37- GLP- 1(7-37); Gly8Asp22His37-GLP- 1(7-37); Val8Asp22His37-GLP- 1(7-37); and Met8Asp22His37-GLP- 1(7-37).

[0177] 33. The engineered nucleic acid of paragraph 32, wherein the GLP-1 peptide consists of or consists essentially of the sequence of SEQ ID NO: 1 or 2, optionally with no more than one amino acid substitution relative to a wild-type GLP-1 peptide consisting of the sequence of SEQ ID NO: 1 or 2, that renders the modified GLP-1 peptide resistant to degradation by DPP-4, optionally Gly8-GLP- 1(7-37) or Val8-GLP- 1(7-37).

[0178] 34. The engineered nucleic acid of any one of the preceding paragraphs, wherein the open reading frame does not comprise a sequence encoding one or more of: a glicentin-related pancreatic polypeptide (GRPP), a glucagon peptide, or a GLP-2 peptide.

[0179] 35. The engineered nucleic acid of paragraph 34, wherein the open reading frame does not comprise a sequence encoding the GRPP, the glucagon peptide, or the GLP-2 peptide.

[0180] 36. The engineered nucleic acid of paragraph 35, wherein the only preproglucagon (GCG) peptides encoded by the open reading frame are the IP-2 peptide and the GLP-1 peptide.

[0181] 37. The engineered nucleic acid of any one of the preceding paragraphs, wherein the open reading frame further comprises a sequence encoding a signal peptide, optionally an insulin gene or preproglucagon signal peptide.

[0182] 38. The engineered nucleic acid of any one of the preceding paragraphs, wherein the promoter comprises a pancreatic tissue-specific promoter sequence.

[0183] 39. The engineered nucleic acid of paragraph 38, wherein the pancreatic tissue-specific promoter sequence comprises an islet beta cell-specific promoter sequence.

[0184] 40. The engineered nucleic acid of paragraph 39, wherein the islet beta cell-specific promoter sequence comprises an insulin gene promoter sequence.

[0185] 41. The engineered nucleic acid of paragraph 40, wherein the insulin gene promoter sequence comprises a human insulin gene promoter sequence, optionally a core sequence, further optionally at least three copies of the core sequence.

[0186] 42. The engineered nucleic acid of any one of the preceding paragraphs, wherein the engineered nucleic acid further comprises: an enhancer element, optionally a cytomegalovirus (CMV) enhancer element; a sequence encoding a modified Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), optionally mut6.WPRE; and / or a sequence encoding a polyadenylation signal, optionally a bovine growth hormone polyadenylation signal. 43. A method comprising administering to a subject the AAV vector genome of any one of paragraphs 1-23, the AAV vector of paragraph 24, or the engineered nucleic acid of any one of paragraphs 25-42.

[0187] 44. The method of paragraph 43, wherein the subject has a metabolic disorder.

[0188] 45. The method of paragraph 44, wherein the metabolic disorder is selected from Obesity, Diabetes Mellitus, Disorders of Lipid Metabolism, Inborn Errors of Metabolism, Lysosomal Storage Disorders, Glycogen Storage Diseases, Mitochondrial Disorders, Purine and Pyrimidine Disorders, Urea Cycle Disorders, Disorders of Fructose Metabolism, Disorders of Amino Acid Metabolism, Disorders of Mineral Metabolism, Porphyrias, Lactose Intolerance and Wilson's Disease, Polycystic Ovary Syndrome, Metabolic Dysfunction-associated Steatotic Liver Disease, and Non-Alcoholic Steatohepatitis.

[0189] 46. The method of paragraph 45, wherein the metabolic disorder is Type 2 diabetes.

[0190] 47. The method of paragraph 45, wherein the metabolic disorder is Obesity.

[0191] 48. The method of paragraph 47, wherein the metabolic disorder is Metabolic Dysfunction- associated Steatotic Liver Disease.

[0192] 49. The method of any one of paragraphs 43-48, wherein the administering is via injection of pancreatic tissue.

[0193] 50. The method of paragraph 47, wherein the administering is using endoscopic ultrasound- guided fine needle injection (EUS-FNI) procedure.

[0194] 51. The method of any one of paragraphs 53-50, wherein the viral vector genome, the viral vector, or the engineered nucleic acid is administered in a therapeutically effective amount to treat the metabolic disorder.

[0195] 52. The method of paragraph 51, wherein the therapeutically effective amount comprises about 5xl012to about 1.5xl014AAV vector genomes (VG), optionally about IxlO13to about 5X1013AAV VG.

[0196] 53. The method of any one of paragraphs 43-52 further comprising administering a DPP-4 inhibitor to the subject.

[0197] 54. The method of paragraph 53, wherein the DPP-4 inhibitor is selected from Sitagliptin, Vildagliptin, Saxagliptin , Linagliptin, Gemigliptin, Anagliptin, Teneligliptin, Alogliptin, Trelagliptin, Omarigliptin, Evogliptin, Gosogliptin, Dutogliptin, Neogliptin, Retagliptin, Denagliptin, and Cofrogliptin.

[0198] 55. A transgene expression cassette comprising: at least three copies of a human insulin promoter sequence, a human insulin exon 1 sequence, a chimeric chicken P-actin (CBA) and rabbit P-globin (RBG) intron sequence, a human insulin exon 2 sequence, a human insulin signal peptide, optionally a furin cleavage site, a GLP- 1(1-6) coding sequence, a GLP- 1(7-37) coding sequence, optionally PC 1 / 3 cleavage site, an IP-2 coding sequence, a mutated woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) coding sequence, and a bovine growth hormone (BGH) polyadenylation signal.

[0199] 56. The transgene expression cassette of paragraph 55 further comprising a cytomegalovirus (CMV) enhancer sequence.

[0200] 57. The transgene expression cassette of paragraph 55 or 56, wherein the human insulin promoter sequence comprises the sequence of SEQ ID NO: 22, the human insulin exon 1 sequence comprises the sequence of SEQ ID NO: 23, the chimeric chicken P-actin (CBA) and rabbit P-globin (RBG) intron sequence comprises the sequence of SEQ ID NO: 24, and / or the human insulin exon 2 sequence comprises the sequence of SEQ ID NO: 25.

[0201] 58. The transgene expression cassette of any one of paragraphs 55-56 comprising the sequence of SEQ ID NO: 20 or 21.

[0202] 59. The transgene expression cassette of any one of paragraphs 55-56 comprising the sequence of SEQ ID NO: 35.

[0203] 60. A nutrient-responsive method for regulating blood glucose levels in a subject, the method comprising: injecting a single dose of a gene therapy composition to pancreatic tissue of the subject in an amount effective to regulate blood glucose levels in the subject, wherein the gene therapy composition comprises an engineered nucleic acid comprising a promoter operably linked to an open reading frame comprising a sequence encoding a glucagon- like peptide 1 (GLP-1) peptide and optionally a sequence encoding an intervening peptide 2 (IP- 2) peptide.

[0204] 61. The method of paragraph 60, wherein the single dose is administered as a single infusion or as a double infusion.

[0205] 62. A nutrient-responsive method for regulating blood glucose levels in a subject, the method comprising: administering a gene therapy composition to the subject in an amount effective to regulate blood glucose levels in the subject, wherein the gene therapy composition comprises an engineered nucleic acid comprising a promoter operably linked to an open reading frame comprising a sequence encoding a glucagon- like peptide 1 (GLP-1) peptide and optionally a sequence encoding an intervening peptide 2 (IP- 2) peptide.

[0206] 63. A nutrient-responsive method for regulating blood glucose levels in a subject, the method comprising: administering a gene therapy composition to the subject in an amount effective to regulate blood glucose levels in the subject, wherein the gene therapy composition comprises an engineered nucleic acid comprising the sequence of SEQ ID NO: 35.

[0207] 65. A nutrient-responsive method for regulating blood glucose levels in a subject, the method comprising: administering a gene therapy composition to the subject in an amount effective to regulate blood glucose levels in the subject, wherein the gene therapy composition comprises an engineered nucleic acid comprising a promoter operably linked to an open reading frame comprising a sequence of SEQ ID NO: 35 encoding a glucagon-like peptide 1 (GLP-1) peptide and optionally a sequence of SEQ ID NO: 35 encoding an intervening peptide 2 (IP-2) peptide.

[0208] Additional embodiments of the disclosure are described in the following numbered embodiments:

[0209] Embodiment 1. An adeno-associated virus (AAV) vector genome comprising: an engineered nucleic acid comprising a promoter comprising multiple copies of a human insulin promoter sequence operably linked to an open reading frame comprising a sequence encoding an intervening peptide 2 (IP-2) peptide and a sequence encoding a glucagon-like peptide 1 (GLP-1) peptide.

[0210] Embodiment 2. An adeno-associated virus (AAV) vector genome comprising: an engineered nucleic acid comprising a promoter operably linked to an open reading frame comprising a sequence encoding an intervening peptide 2 (IP-2) peptide and a sequence encoding a glucagon-like peptide 1 (GLP-1) peptide, wherein the open reading frame does not comprise a sequence encoding an intervening peptide 1 (IP-1) peptide.

[0211] Embodiment 3. The AAV vector genome of Embodiment 1 or 2, wherein the IP-2 peptide comprises the sequence of SEQ ID NO: 16 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 16.

[0212] Embodiment 4. The AAV vector genome of Embodiment 3, wherein the IP-2 peptide consists of or consists essentially of the sequence of SEQ ID NO: 16.

[0213] Embodiment 5. The AAV vector genome of any one of the preceding Embodiments, wherein the sequence encoding an IP-2 peptide is downstream from the sequence encoding a GLP-1 peptide.

[0214] Embodiment 6. The AAV vector genome of any one of the preceding Embodiments, wherein the GLP-1 peptide comprises the sequence of SEQ ID NO: 14 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 14. Embodiment 7. The AAV vector genome of any one of the preceding Embodiments, wherein the GLP-1 peptide comprises the sequence of SEQ ID NO: 15 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 15.

[0215] Embodiment 8. The AAV vector genome of Embodiment 7, wherein the open reading frame further comprises a sequence encoding a PC 1 / 3 cleavage site, optionally between amino acid positions corresponding to amino acid position 6 and amino acid position 7 of a GLP-1 peptide comprising the amino acid sequence of SEQ ID NO: 14.

[0216] Embodiment 9. The AAV vector genome of any one of the preceding Embodiments, wherein the GLP-1 peptide is a modified GLP-1 peptide that comprises one or more amino acid substitution(s), relative to a wild-type GLP-1 peptide consisting of the sequence of SEQ ID NO: 14 or 15, that renders the modified GLP-1 peptide resistant to degradation by dipeptidyl peptidase-4 (DPP-4), optionally wherein the modified GLP-1 peptide is selected from the group consisting of: Arg26-GLP-l(7-37); Arg34-GLP- 1(7-37); Lys36-GLP-l(7-37); Arg26,34Lys36- GLP-l(7-37); Arg26,34-GLP-l(7-37); Arg26,34Lys40-GLP-l(7-37); Arg26Lys36-GLP- 1(7-37); Arg34Lys36-GLP-l(7-37) Val8Arg22-GLP- 1(7-37); Met8Arg22-GLP-l(7-37);Gly8His22-GLP- 1(7-37); Vai 8His22-GLP- 1(7-37); Met8His22-GLP-l(7-37); His37-GLP-l(7-37); Gly8-GLP- 1(7-37); Val8-GLP-l(7-37); Met8-GLP- 1(7-37); Gly8Asp22-GLP-l(7-37); Val8Asp22-GLP-l(7- 37); Met8Asp22-GLP-l(7-37); Gly8Glu22-GLP-l(7-37); Val8Glu22-GLP-l(7-37); Met8Glu22- GLP- 1(7-37); Gly8Lys22-GLP-l(7-37); Vai 8Lys22-GLP- 1(7-37); Met8Lys22-GLP-l(7-37); Gly8Arg22-GLP-l(7-37); Vai 8Lys22His37-GLP- 1(7-37); Gly8Glu22His37-GLP-l(7-37); Val8Glu22His37-GLP-l(7-37); Met8Glu22His37-GLP- 1(7-37); Gly8Lys22His37-GLP-l(7-37); Met8Lys22His37-GLP-l(7-37); Gly8Arg22His37-GLP- 1(7-37); Val8Arg22His37-GLP- 1(7-37); Met8Arg22His37-GLP-l(7-37); Gly8His22His37-GLP-l(7-37); Vai 8His22His37-GLP- 1(7-37); Met8His22His37-GLP- 1(7-37); Gly8His37-GLP- 1(7-37); Vai 8His37-GLP- 1(7-37); Met8His37- GLP- 1(7-37); Gly8Asp22His37-GLP- 1(7-37); Val8Asp22His37-GLP- 1(7-37); and Met8Asp22His37-GLP- 1(7-37).

[0217] Embodiment 10. The AAV vector genome of Embodiment 9, wherein the GLP-1 peptide consists of or consists essentially of the sequence of SEQ ID NO: 14 or 15, optionally with no more than one amino acid substitution relative to a wild-type GLP-1 peptide consisting of the sequence of SEQ ID NO: 1 or 2, that renders the modified GLP-1 peptide resistant to degradation by DPP-4, optionally Gly8-GLP-l(7-37) or Val8-GLP- 1(7-37).

[0218] Embodiment 11. The AAV vector genome of any one of the preceding Embodiments, wherein the open reading frame does not comprise a sequence encoding one or more of: a glicentin-related pancreatic polypeptide (GRPP), a glucagon peptide, or a GLP-2 peptide. Embodiment 12. The AAV vector genome of Embodiment 11, wherein the open reading frame does not comprise a sequence encoding the GRPP, the glucagon peptide, or the GLP-2 peptide.

[0219] Embodiment 13. The AAV vector genome of Embodiment 12, wherein the only preproglucagon (GCG) peptides encoded by the open reading frame are the IP-2 peptide and the GLP-1 peptide.

[0220] Embodiment 14. The AAV vector genome of any one of the preceding Embodiments, wherein the open reading frame further comprises a sequence encoding a signal peptide, optionally an insulin gene or preproglucagon signal peptide.

[0221] Embodiment 15. The AAV vector genome of any one of the preceding Embodiments, wherein the promoter comprises a pancreatic tissue-specific promoter sequence.

[0222] Embodiment 16. The AAV vector genome of Embodiment 15, wherein the pancreatic tissue-specific promoter sequence comprises an islet beta cell-specific promoter sequence.

[0223] Embodiment 17. The AAV vector genome of Embodiment 16, wherein the islet beta cellspecific promoter sequence comprises an insulin gene promoter sequence.

[0224] Embodiment 18. The AAV vector genome of Embodiment 17, wherein the insulin gene promoter sequence comprises a human insulin gene promoter sequence, optionally a core sequence, further optionally at least three copies of the core sequence or at least three human insulin gene promoter core sequences, wherein at least two of the human insulin gene promoter core sequences are identical.

[0225] Embodiment 19. The AAV vector genome of any one of the preceding Embodiments, wherein the engineered nucleic acid further comprises: an enhancer element, optionally a cytomegalovirus (CMV) enhancer element; a sequence encoding a modified Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), optionally mut6.WPRE; and / or a sequence encoding a polyadenylation signal, optionally a bovine growth hormone polyadenylation signal.

[0226] Embodiment 20. An adeno-associated virus (AAV) vector genome comprising an engineered nucleic acid comprising a promoter, optionally comprising a human insulin promoter sequence, operably linked to an open reading frame comprising the sequence of SEQ ID NO: 4, 5, 27, or 31, or a sequence having at least 90% identity to the sequence of SEQ ID NO: 4, 5, 27, or 31.

[0227] Embodiment 21. An adeno-associated virus (AAV) vector genome comprising an engineered nucleic acid comprising the sequence of SEQ ID NO: 20, 21, or 32-37 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 20, 21, or 32-37. Embodiment 22. The AAV vector genome of the preceding Embodiments, wherein the engineered nucleic acid further comprises a 5' inverted terminal repeat (ITR) sequence, optionally an AAV2 ITR, upstream from the promoter and a 3' ITR, optionally an AAV2 ITR, downstream from the open reading frame.

[0228] Embodiment 23. The AAV vector genome of the preceding Embodiments, wherein the AAV vector genome is a self-complementary AAV vector genome.

[0229] Embodiment 24. An AAV vector comprising (a) the AAV vector genome of any one of the preceding Embodiments and (b) AAV capsid proteins.

[0230] Embodiment 25. An engineered nucleic acid comprising: a promoter operably linked to an open reading frame comprising a sequence encoding an intervening peptide 2 (IP-2) peptide and a sequence encoding a glucagon-like peptide 1 (GLP-1) peptide, wherein the open reading frame does not comprise a sequence encoding an intervening peptide 1 (IP-1) peptide.

[0231] Embodiment 26. The engineered nucleic acid of Embodiment 25, wherein the IP-2 peptide comprises the sequence of SEQ ID NO: 16 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 16.

[0232] Embodiment 27. The engineered nucleic acid of Embodiment 26, wherein the IP-2 peptide consists of or consists essentially of the sequence of SEQ ID NO: 16.

[0233] Embodiment 28. The engineered nucleic acid of any one of the preceding Embodiments, wherein the sequence encoding an IP-2 peptide is downstream from the sequence encoding a GLP-1 peptide.

[0234] Embodiment 29. The engineered nucleic acid of any one of the preceding Embodiments, wherein the GLP-1 peptide comprises the sequence of SEQ ID NO: 14 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 14.

[0235] Embodiment 30. The engineered nucleic acid of any one of the preceding Embodiments, wherein the GLP-1 peptide comprises the sequence of SEQ ID NO: 15 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 15.

[0236] Embodiment 31. The engineered nucleic acid of Embodiment 30, wherein the open reading frame further comprises a sequence encoding a PC 1 / 3 cleavage site, optionally between amino acid positions corresponding to amino acid position 6 and amino acid position 7 of a GLP- 1 peptide comprising the amino acid sequence of SEQ ID NO: 14.

[0237] Embodiment 32. The engineered nucleic acid of any one of the preceding Embodiments, wherein the GLP-1 peptide is a modified GLP-1 peptide that comprises one or more amino acid substitution(s), relative to a wild-type GLP-1 peptide consisting of the sequence of SEQ ID NO: 1 or 2, that renders the modified GLP-1 peptide resistant to degradation by dipeptidyl peptidase-4 (DPP-4), optionally wherein the modified GLP-1 peptide is selected from the group consisting of: Arg26-GLP-l(7-37); Arg34-GLP-l(7-37); Lys36-GLP- 1(7-37); Arg26,34Lys36-GLP- 1(7-37); Arg26,34-GLP-l(7-37); Arg26,34Lys40-GLP-l(7-37); Arg26Lys36-GLP- 1(7-37); Arg34Lys36- GLP- 1 (7-37) Val8Arg22-GLP- 1(7-37); Met8Arg22-GLP-l(7-37);Gly8His22-GLP-l(7-37);

[0238] Val8His22-GLP-l(7-37); Met8His22-GLP-l(7-37); His37-GLP- 1(7-37); Gly8-GLP- 1(7-37); Val8-GLP- 1(7-37); Met8-GLP- 1(7-37); Gly8Asp22-GLP-l(7-37); Val8Asp22-GLP- 1(7-37); Met8Asp22-GLP-l(7-37); Gly8Glu22-GLP-l(7-37); Val8Glu22-GLP- 1(7-37); Met8Glu22-GLP- 1(7-37); Gly8Lys22-GLP-l(7-37); Val8Lys22-GLP-l(7-37); Met8Lys22-GLP-l(7-37);

[0239] Gly8Arg22-GLP-l(7-37); Vai 8Lys22His37-GLP- 1(7-37); Gly8Glu22His37-GLP-l(7-37);

[0240] Val8Glu22His37-GLP-l(7-37); Met8Glu22His37-GLP- 1(7-37); Gly8Lys22His37-GLP-l(7-37); Met8Lys22His37-GLP-l(7-37); Gly8Arg22His37-GLP- 1(7-37); Val8Arg22His37-GLP- 1(7-37); Met8Arg22His37-GLP-l(7-37); Gly8His22His37-GLP-l(7-37); Vai 8His22His37-GLP- 1(7-37); Met8His22His37-GLP- 1(7-37); Gly8His37-GLP- 1(7-37); Vai 8His37-GLP- 1(7-37); Met8His37- GLP- 1(7-37); Gly8Asp22His37-GLP- 1(7-37); Val8Asp22His37-GLP- 1(7-37); and Met8Asp22His37-GLP- 1(7-37).

[0241] Embodiment 33. The engineered nucleic acid of Embodiment 32, wherein the GLP-1 peptide consists of or consists essentially of the sequence of SEQ ID NO: 1 or 2, optionally with no more than one amino acid substitution relative to a wild-type GLP-1 peptide consisting of the sequence of SEQ ID NO: 1 or 2, that renders the modified GLP-1 peptide resistant to degradation by DPP-4, optionally Gly8-GLP-l(7-37) or Val8-GLP- 1(7-37).

[0242] Embodiment 34. The engineered nucleic acid of any one of the preceding Embodiments, wherein the open reading frame does not comprise a sequence encoding one or more of: a glicentin-related pancreatic polypeptide (GRPP), a glucagon peptide, or a GLP-2 peptide.

[0243] Embodiment 35. The engineered nucleic acid of Embodiment 34, wherein the open reading frame does not comprise a sequence encoding the GRPP, the glucagon peptide, or the GLP-2 peptide.

[0244] Embodiment 36. The engineered nucleic acid of Embodiment 35, wherein the only preproglucagon (GCG) peptides encoded by the open reading frame are the IP-2 peptide and the GLP-1 peptide.

[0245] Embodiment 37. The engineered nucleic acid of any one of the preceding Embodiments, wherein the open reading frame further comprises a sequence encoding a signal peptide, optionally an insulin gene or preproglucagon signal peptide.

[0246] Embodiment 38. The engineered nucleic acid of any one of the preceding Embodiments, wherein the promoter comprises a pancreatic tissue-specific promoter sequence.

[0247] Embodiment 39. The engineered nucleic acid of Embodiment 38, wherein the pancreatic tissue-specific promoter sequence comprises an islet beta cell-specific promoter sequence. Embodiment 40. The engineered nucleic acid of Embodiment 39, wherein the islet beta cell-specific promoter sequence comprises an insulin gene promoter sequence.

[0248] Embodiment 41. The engineered nucleic acid of Embodiment 40, wherein the insulin gene promoter sequence comprises a human insulin gene promoter sequence, optionally a core sequence, further optionally at least three copies of the core sequence or at least three human insulin gene promoter core sequences, wherein at least two of the human insulin gene promoter core sequences are identical.

[0249] Embodiment 42. The engineered nucleic acid of any one of the preceding Embodiments, wherein the engineered nucleic acid further comprises: an enhancer element, optionally a cytomegalovirus (CMV) enhancer element; a sequence encoding a modified Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), optionally mut6.WPRE; and / or a sequence encoding a polyadenylation signal, optionally a bovine growth hormone polyadenylation signal.

[0250] Embodiment 43. A method comprising administering to a subject the AAV vector genome of any one of Embodiments 1-23, the AAV vector of claim 24, or the engineered nucleic acid of any one of claims 25-42.

[0251] Embodiment 44. The method of Embodiment 43, wherein the subject has a metabolic disorder.

[0252] Embodiment 45. The method of Embodiment 44, wherein the metabolic disorder is selected from Obesity, Diabetes Mellitus, Disorders of Lipid Metabolism, Inborn Errors of Metabolism, Lysosomal Storage Disorders, Glycogen Storage Diseases, Mitochondrial Disorders, Purine and Pyrimidine Disorders, Urea Cycle Disorders, Disorders of Fructose Metabolism, Disorders of Amino Acid Metabolism, Disorders of Mineral Metabolism, Porphyrias, Lactose Intolerance and Wilson's Disease, Polycystic Ovary Syndrome, Metabolic Dysfunction-associated Steatotic Liver Disease, and Non-Alcoholic Steatohepatitis.

[0253] Embodiment 46. The method of Embodiment 45, wherein the metabolic disorder is Type 2 diabetes.

[0254] Embodiment 47. The method of Embodiment 45, wherein the metabolic disorder is Obesity.

[0255] Embodiment 48. The method of Embodiment 47, wherein the metabolic disorder is Metabolic Dysfunction- associated Steatotic Liver Disease.

[0256] Embodiment 49. The method of any one of Embodiments 43-48, wherein the administering is via injection of pancreatic tissue.

[0257] Embodiment 50. The method of Embodiment 47, wherein the administering is using endoscopic ultrasound-guided fine needle injection (EUS-FNI) procedure. Embodiment 51. The method of any one of Embodiments 53-50, wherein the viral vector genome, the viral vector, or the engineered nucleic acid is administered in a therapeutically effective amount to treat the metabolic disorder.

[0258] Embodiment 52. The method of Embodiment 51, wherein the therapeutically effective amount comprises about 5xl012to about 1.5xl014AAV vector genomes (VG), optionally about IxlO13to about 5xl013AAV VG.

[0259] Embodiment 53. The method of any one of Embodiments 43-52 further comprising administering a DPP-4 inhibitor to the subject.

[0260] Embodiment 54. The method of Embodiment 53, wherein the DPP-4 inhibitor is selected from Sitagliptin, Vildagliptin, Saxagliptin , Linagliptin, Gemigliptin, Anagliptin, Teneligliptin, Alogliptin, Trelagliptin, Omarigliptin, Evogliptin, Gosogliptin, Dutogliptin, Neogliptin, Retagliptin, Denagliptin, and Cofrogliptin.

[0261] Embodiment 55. A transgene expression cassette comprising: at least three sequential human insulin promoter sequences, a human insulin exon 1 sequence, a chimeric chicken P-actin (CBA) and rabbit P-globin (RBG) intron sequence, a human insulin exon 2 sequence, a human insulin signal peptide, optionally a furin cleavage site, a GLP- 1(1-6) coding sequence, a GLP- 1(7-37) coding sequence, optionally PC 1 / 3 cleavage site, an IP-2 coding sequence, a mutated woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) coding sequence, and a bovine growth hormone (BGH) polyadenylation signal.

[0262] Embodiment 56. The transgene expression cassette of Embodiment 55 further comprising a cytomegalovirus (CMV) enhancer sequence.

[0263] Embodiment 57. The transgene expression cassette of Embodiment 55 or 56, wherein the at least three sequential human insulin promoter sequences comprises three copies of the human insulin promoter sequence comprising the sequence of SEQ ID NO: 22, the human insulin exon 1 sequence comprises the sequence of SEQ ID NO: 23, the chimeric chicken P-actin (CBA) and rabbit P-globin (RBG) intron sequence comprises the sequence of SEQ ID NO: 24, and / or the human insulin exon 2 sequence comprises the sequence of SEQ ID NO: 25.

[0264] Embodiment 58. The transgene expression cassette of any one of Embodiments 55-56 comprising the sequence of SEQ ID NO: 20.

[0265] Embodiment 59. The transgene expression cassette of any one of Embodiments 55-56 comprising the sequence of SEQ ID NO: 21.

[0266] Embodiment 60. The transgene expression cassette of Embodiment 55 or 56, wherein the at least three sequential human insulin promoter sequences comprise the sequence of SEQ ID NO: 27, the human insulin exon 1 sequence comprises the sequence of SEQ ID NO: 23, the chimeric chicken P-actin (CBA) and rabbit P-globin (RBG) intron sequence comprises the sequence of SEQ ID NO: 28, and / or the human insulin exon 2 sequence comprises the sequence of SEQ ID NO: 25.

[0267] Embodiment 61. The transgene expression cassette of Embodiment 60 comprising the sequence of SEQ ID NO: 33.

[0268] Embodiment 62. The transgene expression cassette of Embodiment 60, further comprising a chicken P-actin (CBA) exon sequence.

[0269] Embodiment 63. The transgene expression cassette of Embodiment 62, wherein the CBA exon sequence comprises SEQ ID NO: 38.

[0270] Embodiment 64. The transgene expression cassette of Embodiment 63 comprising the sequence of SEQ ID NO: 32.

[0271] Embodiment 65. A transgene expression cassette comprising: at least three sequential human insulin promoter sequences, a human insulin exon 1 sequence, a human insulin intron 1 sequence, a human insulin exon 2 sequence, a human insulin signal peptide, optionally a furin cleavage site, a GLP- 1(1-6) coding sequence, a GLP- 1(7-37) coding sequence, optionally PC 1 / 3 cleavage site, an IP-2 coding sequence, a mutated woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) coding sequence, and a bovine growth hormone (BGH) polyadenylation signal.

[0272] Embodiment 66. The transgene expression cassette of Embodiment 65, wherein the at least three sequential human insulin promoter sequences comprise the sequence of SEQ ID NO: 27, the human insulin exon 1 sequence comprises the sequence of SEQ ID NO: 23, the human insulin intron 1 sequence comprises the sequence of SEQ ID NO: 29, and / or the human insulin exon 2 sequence comprises the sequence of SEQ ID NO: 25.

[0273] Embodiment 67. The transgene expression cassette of Embodiment 66 comprising the sequence of SEQ ID NO: 34.

[0274] Embodiment 68. The transgene expression cassette of Embodiment 65, wherein the at least three sequential human insulin promoter sequences comprise the sequence of SEQ ID NO: 27, the human insulin exon 1 sequence comprises the sequence of SEQ ID NO: 23, the human insulin intron 1 sequence comprises the sequence of SEQ ID NO: 30, and / or the human insulin exon 2 sequence comprises the sequence of SEQ ID NO: 25.

[0275] Embodiment 69. The transgene expression cassette of Embodiment 68 comprising the sequence of SEQ ID NO: 35.

[0276] Embodiment 70. The transgene expression cassette of Embodiment 65, wherein the at least three sequential human insulin promoter sequences comprise the sequence of SEQ ID NO: 31, the human insulin exon 1 sequence comprises the sequence of SEQ ID NO: 23, the human insulin intron 1 sequence comprises the sequence of SEQ ID NO: 30, and / or the human insulin exon 2 sequence comprises the sequence of SEQ ID NO: 25.

[0277] Embodiment 71. The transgene expression cassette of Embodiment 70 comprising the sequence of SEQ ID NO: 36.

[0278] Embodiment 72. A transgene expression cassette comprising: at least three sequential human insulin promoter sequences, a human insulin exon 1 sequence, a human insulin exon 2 sequence, a human insulin signal peptide, optionally a furin cleavage site, a GLP- 1(1-6) coding sequence, a GLP- 1(7-37) coding sequence, optionally PC 1 / 3 cleavage site, an IP-2 coding sequence, a mutated woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) coding sequence, and a bovine growth hormone (BGH) polyadenylation signal.

[0279] Embodiment 73. The transgene expression cassette of Embodiment 72, wherein the at least three sequential human insulin promoter sequences comprise the sequence of SEQ ID NO: 27, the human insulin exon 1 sequence comprises the sequence of SEQ ID NO: 23, and / or the human insulin exon 2 sequence comprises the sequence of SEQ ID NO: 25.

[0280] Embodiment 74. The transgene expression cassette of Embodiments 72 or 73 comprising the sequence of SEQ ID NO: 37.

[0281] EXAMPLES

[0282] For the following experiments in Examples 1-3, the indicated transgene expression cassettes from Table 2 were transfected into EndoC-PH5 cells. Seventy-two (72) hours later, the cell supernatants were collected as samples. The samples were incubated with CHO reporter cells engineered to express GLP-1 receptors, and a cAMP assay (DiscoverX HitHunter® cAMP Assay for Small Molecules) was performed. Briefly, cAMP antibody reagent was added to the CHO reporter cells, followed by a cAMP detection solution, which also lyses the cells. Finally, chemiluminescent substrate was added, and following an incubation period, chemiluminescence signal was measured on a plate reader.

[0283] Table 2. Transgene Expression Cassettes

[0284] Example 1: GLP-1 Transgene Expression Cassettes - IP-1 v. IP-2

[0285] The graph in FIG. 1A shows that inclusion of the intervening peptide 2 (IP-2) coding sequence in the transgene expression cassette (Transgene 04, 05, 06, and 11) yields higher GLP-1 activity (comparable to positive controls) than when excluded (Transgene 01, 02, 03, and 08). Transgene expression cassettes lacking an IP-2 coding sequence performed poorly in the cAMP assay.

[0286] The graph in FIG. IB shows that inclusion of the intervening peptide 1 (IP-1) coding sequence in the transgene expression cassette (Transgene 03, 06, 09) or exclusion of the IP-1 coding sequence (Transgene 02, 05, and 08) had no impact on GLP-1 activity.

[0287] This data was surprising in part because (i) there is no known function ascribed to the IP- 2 peptide, (ii) IP-2 is the most divergent region within the GCG gene, and (iii) during natural protein processing of GCG in a cell, IP-2 is cleaved off, leaving only GLP- 1(1-37) for further processing. Moreover, none of the GLP- 1-based drugs on the market includes an IP-2 peptide. Even more surprising, after learning the impact of the IP-2 coding sequence on GLP- 1 activity, was the data showing that inclusion of an IP-1 coding sequence had no impact. This is in part because IP-1 is more conserved than IP-2, suggesting that it may have a functional impact. Yet, inclusion (or exclusion) of an IP-1 coding sequence from the tested transgene expression cassettes did not impact the activity of the encoded GLP-1.

[0288] Example 2: GLP-1 Transgene Expression Cassettes - GLP-l(l-37) v. GLP-l(7-37)

[0289] The graph in FIG. 2 shows that inclusion of the GLP- 1(1-6) coding sequence in the transgene cassette is important for GLP-1 activity. Transgene expression cassettes that included the GLP- 1(1-6) coding sequence (Transgene 02 and 05) outperformed (higher GLP-1 activity) the transgene expression cassettes lacking the GLP- 1(1-6) coding sequence (Transgene 01 and 04). The graph in FIG. 2 also supports the findings from Example 1, demonstrating that the transgene expression cassettes that include an IP-2 coding sequence (Transgene 05) outperforms those lacking an IP-2 coding sequence (Transgene 02).

[0290] This data was unexpected in part because during natural protein processing of GCG in a cell, the GLP- 1(1-6) peptide is cleaved off, leaving the GLP- 1(7-37) peptide as the active form. The expectation was that excluding the GLP- 1(1-6) coding sequence from the transgene expression cassettes would not negatively impact activity of the GLP- 1(7-37) peptide. This was not the case, as shown by the data in FIG. 2. Example 3: GLP-1 Transgene Expression Cassettes - PC13 Cleavage v. Furin Cleavage

[0291] The graph in FIG. 3 shows that insertion of a sequence encoding a furin cleavage site between GLP-1 (1-6) and GLP-1 (7-37) can be detrimental, highlighting the importance of the native PC 1 / 3 cleavage site, in some instances. The furin cleavage site is a 3 amino acid addition of serine, lysine, and arginine (S, K, R) between GLP-l(l-6) and GLP-l(7-37); however, insertion of the furin cleavage site still retains an intact RH PC 1 / 3 site available for the original PC 1 / 3 cleavage. This data was surprising because it was thought that furin cleavage would be equally or more efficient than native PC 1 / 3 cleavage, as more PCSK family proteins can act on this site. Additionally, it was thought that because furin is more ubiquitously expressed, cleavage would occur in other cells (e.g., a-cells) that do not necessarily express PC 1 / 3, potentially enabling use of this transgene design in a wider range of cell types. Yet, this was not the case. Clearly, Transgene 24, which does not include the furin cleavage site, outperformed Transgene 25, which does include the furin cleavage site.

[0292] Example 4: Human Insulin Promoter- GLP-1 Transgene Expression Cassette

[0293] The graphs in FIG. 4B show that Transgene 38 (SEQ ID NO: 20) (FIG. 4A) produces functional GLP-1 protein that matches the expression and activity of CMVp-GCG (CMV promoter operably linked to the GCG transgene). The graph on the left shows data from a GLP-1 ELISA, and the graph on the right shows data from a cAMP activity assay. In this example, the B-cell specific insulin gene promoter is used to drive the expression of the GLP- 1(1-37) to IP-2 portion of the human glucagon (GCG) gene.

[0294] Example 5: Secreted GLP-1 Stimulated by Glucose

[0295] To produce the ELISA and cAMP data for Transgene A (Transgene 68.1 (SEQ ID NO: 35)), including a human insulin promoter sequence and sequence encoding a furin cleavage site, GLP-l(l-6), GLP-l(7-37), a PC1 / 3 cleavage site, IP-2, and other regulatory elements) and Transgene B (a variant of Transgene 68.1, without the furin cleavage site), a series of experiments were conducted using the EndoC-PH5 cell line, which are human-derived pancreatic beta cells. Initially, the cells were prepared for transfection by being cultured in a medium containing BSA, with the wells of a 96-well plate pre-coated to ensure cell adhesion and growth. After thawing, the cell suspension was centrifuged, and the resulting pellet was re-suspended in fresh medium before being seeded into the wells. Non-transfected human beta cells were used as the control.

[0296] For the transfection process, plasmid DNA was introduced into the EndoC-PH5 cells using a transfection reagent. Each well received a specific concentration of DNA (400ng), with careful preparation of DNA-reagent complexes to ensure effective transfection. These complexes were added to the cells and incubated to allow for uptake, after which the medium was replaced with fresh growth medium. The cells were then cultured for 72 hours to facilitate expression of the transfected plasmids.

[0297] Following the 72-hour incubation period, cells were starved overnight with a medium that does not contain glucose. Supernatant and cell lysates were collected the following day separately to quantify the levels of GLP-1 secretion. The ELISA assays were performed on the supernatant to detect secreted GLP-1 protein, while cell lysates were analyzed to measure intracellular GLP-1. Both plasmids, Transgene A and Transgene B, were evaluated under varying glucose concentrations (5.5 mM and 20 mM) to assess their influence on GLP-1 expression and secretion (FIG. 5A). A dose-dependent effect was observed.

[0298] In addition, cAMP assays were conducted to measure the activation of GLP-1 receptors in response to the transfection, with different glucose conditions used to assess the downstream signaling effects on cellular activity. The data from these experiments was analyzed to determine how glucose levels impacted GLP-1 expression, the efficacy of the transfection, and the comparison of GLP-1 concentrations in supernatant versus cell lysates. As is demonstrated in FIG. 5B, the assay showed that the GLP-1 proteins secreted are active.

[0299] Example 6: Single Dose of Pancreatic Gene Therapy Encoding Human GLP-1 Sequence in DIO Mice

[0300] DIO (diet-induced obesity) mice were injected with a pancreatic gene therapy encoding human GLP-1 sequence (“Transgene A”) or a vehicle control (n=8 mice per group) and fasting blood glucose and body weight were measured over time. The results demonstrated that the single dose of Transgene A durably reduced fasting blood glucose over time (FIGs. 6A and 6B) as well as body weight (FIG. 6C).

[0301] Example 7: Feasibility and Safety of Novel Endoscopic Ultrasound- Guided Delivery of Human GLP-1 Pancreatic Gene Therapy in Pigs

[0302] GLP- 1 therapy is now the cornerstone of metabolic disease treatment. Although efficacious, discontinuation is frequent, resulting in near-complete glycemic and weight rebound. This example describes a single-dose pancreatic gene therapy (PGTx) that enables durable, nutrient-responsive, islet-driven GLP-1 production (Smart GLP-1). In mice, GLP-1 PGTx improves hyperglycemia and sustains weight loss after semaglutide withdrawal. Here, the feasibility and safety of infusing human GLP-1 PGTx (Transgene A) with an endoscopic ultrasound-guided delivery system into porcine pancreas is evaluated. An echoendoscope with custom needle was used to transgastrically infuse Transgene A (AAV9 vector encoding a human insulin promoter and GLP-1 transgene, 6el3 vector genome) into the porcine pancreas (n=5). Animals were necropsied post-procedure (day 34), and pancreatic biopsies were assessed to establish vector copy number (VCN) (FIG. 7A), transgene RNA (FIG. 7B), and protein expression (FIG. 7C) as indicators of transduction efficiency and Transgene A-driven GLP-1 production. Serum lipase served as a pancreatitis marker to establish procedural safety.

[0303] The procedure was well-tolerated with mean lipase levels remaining in the normal range post-procedure (3.5±0.5 U / L SEM) (FIG. 8). Pancreatic VCN and Transgene A RNA levels were positively correlated (r=0.7, p<0.0001) (FIG. 7E). Likewise, RNA levels correlated with Transgene A islet-driven GLP-1 protein levels (r=0.5, p<0.0001) (FIG. 7F). Active GLP-1 protein expression was five-fold higher in treated vs. untreated animals (185±32 vs. 36±9 ng / g total protein SEM, p<0.02) (FIG. 7D).

[0304] Despite an abundance of GLP-1 in Transgene A treated pig pancreas, it was not detected above baseline in circulation (FIG. 9). These data demonstrate that Transgene A can be safely delivered endoscopically in a large animal model allowing for direct pancreatic targeting. Transgene A-driven human pancreatic GLP-1 production demonstrates the feasibility of singledose local gene therapy as a durable strategy for the treatment of metabolic diseases.

[0305] Example 8: Transgene A Expression in db / db Mice

[0306] In this Example, 8-week-old db / db mice were treated via IP injection with a single dose of an AAV9 transgene and studied for 4 weeks (GFP animals) or 6 weeks (GLP-1 animals):

[0307] The results of the studies are provided in FIG. 10 and FIGs. 11A-11B. There was a dose- responsive improvement in glycemic control, including a dose-responsive decrease in bodyweight loss (BWL) and fasting blood glucose (FBG) and a dose-responsive increase in fasting plasma insulin (FPI). Transgene A expression was restricted to pancreatic islet cells. Example 9: Transgene A Expression in human cell lines

[0308] In this Example, human beta cells (EndoC-BH5) (FIGs. 13A-13B), human liver cells (HEPG2) (FIGs. 14A-14B), or human DRG cells (FIGs. 15A-15B) were seeded and transduced at two-three MOIs with an AAV- KPI capsid surrogate carrying either: a ubiquitous reporter transgene in the Transgene A backbone that is driven by the CMV / CMV enhancer / promoter, or a Transgene A reporter transgene surrogate that is identical to the Transgene A sequence except for the replacement of the coding sequence with that of GFP Expression of the encoded protein was observed in human beta cells but not in the other cell types.

[0309] Human beta cells represent the on-target (ONT) cell line while DRG and HEPG2 cells represent OFT cell lines. The reporter Transgene A surrogate serves to reflect the specificity of the hINSp regulatory sequence that is used in Transgene A, using GFP as an output, while the vector modulated by the CMV / CMV promoter serves as a ubiquitous control. In each cell line, multiple MOIs were applied to assess the robustness of hINSp promoter specificity vs CMV / CMV driven expression. Human beta cells were transduced at 0, 1E3, 1E4, or 1E5 MOI, imaged 72h post transduction and subjected to flow cytometry (FIGs. 13A-13B). The hINSp sequence performed well and surpassed that of the CMV / CMV sequence; both showing a doseresponse in GFP levels.

[0310] HEPG2 cells were transduced at 0, 3E3, 1E4, or 1E5 MOI, imaged 72h post transduction and subjected to flow cytometry (FIGs. 14A-14B). While the CMV / CMV construct showed a dose-responsive increase in GFP expression, practically no expression was observed from the hINSp.

[0311] Human DRG cells were transduced at 0, 3E4, or 1E5 MOI, imaged 72h post transduction, and subjected to plate-reader analysis to gauge GFP expression (FIGs. 15A-15B). While the CMV / CMV construct showed a dose-responsive increase in robust GFP expression, only extremely low expression was observed at the high MOI from the hINSp construct. Conclusion: Human beta cells, HEPG2 cells, and DRG cells were transduced with either a GFP vector under ubiquitous CMV / CMV control or modulated by the hINSp used in Transgene A to determine the promoter’s level of specificity in the intended human context. Results indicated that the hINSp of Transgene A is highly specific in the tested cell types and thereby serve to address the risk of potential OFT expression of the Transgene A in human.

Claims

CLAIMSWhat is claimed is:

1. An adeno-associated virus (AAV) vector genome comprising: an engineered nucleic acid comprising a promoter comprising multiple copies of a human insulin promoter sequence operably linked to an open reading frame comprising a sequence encoding an intervening peptide 2 (IP-2) peptide and a sequence encoding a glucagon-like peptide 1 (GLP-1) peptide.

2. An adeno-associated virus (AAV) vector genome comprising: an engineered nucleic acid comprising a promoter operably linked to an open reading frame comprising a sequence encoding an intervening peptide 2 (IP-2) peptide and a sequence encoding a glucagon-like peptide 1 (GLP-1) peptide, wherein the open reading frame does not comprise a sequence encoding an intervening peptide 1 (IP-1) peptide.

3. The AAV vector genome of claim 1 or 2, wherein the IP-2 peptide comprises the sequence of SEQ ID NO: 16 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 16, optionally wherein the IP-2 peptide consists of or consists essentially of the sequence of SEQ ID NO: 16.

4. The AAV vector genome of any one of the preceding claims, wherein the sequence encoding an IP-2 peptide is downstream from the sequence encoding a GLP-1 peptide.

5. The AAV vector genome of any one of the preceding claims, wherein the GLP-1 peptide comprises the sequence of SEQ ID NO: 14 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 14, and / or wherein the GLP-1 peptide comprises the sequence of SEQ ID NO: 15 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 15.

6. The AAV vector genome of claim 5, wherein the open reading frame further comprises a sequence encoding a PC 1 / 3 cleavage site, optionally between amino acid positions corresponding to amino acid position 6 and amino acid position 7 of a GLP-1 peptide comprising the amino acid sequence of SEQ ID NO: 14.

7. The AAV vector genome of any one of the preceding claims, wherein the GLP-1 peptide is a modified GLP-1 peptide that comprises one or more amino acid substitution(s), relative to awild-type GLP-1 peptide consisting of the sequence of SEQ ID NO: 14 or 15, that renders the modified GLP-1 peptide resistant to degradation by dipeptidyl peptidase-4 (DPP-4), optionally wherein the modified GLP-1 peptide is selected from the group consisting of: Arg26-GLP-1(7- 37); Arg34-GLP-l(7-37); Lys36-GLP-l(7-37); Arg26,34Lys36-GLP-l(7-37); Arg26,34-GLP- 1(7-37); Arg26,34Lys40-GLP- 1(7-37); Arg26Lys36-GLP-l(7-37); Arg34Lys36-GLP-l(7-37) Val8Arg22-GLP-l(7-37); Met8Arg22-GLP-l(7-37);Gly8His22-GLP-l(7-37); Val8His22-GLP- 1(7-37); Met8His22-GLP- 1(7-37); His37-GLP- 1(7-37); Gly8-GLP- 1(7-37); Val8-GLP- 1(7-37); Met8-GLP- 1(7-37); Gly8Asp22-GLP-l(7-37); Val8Asp22-GLP-l(7-37); Met8Asp22-GLP-l(7- 37); Gly8Glu22-GLP-l(7-37); Val8Glu22-GLP-l(7-37); Met8Glu22-GLP- 1(7-37); Gly8Lys22- GLP- 1(7-37); Vai 8Lys22-GLP- 1(7-37); Met8Lys22-GLP-l(7-37); Gly8Arg22-GLP-l(7-37); Val8Lys22His37-GLP-l(7-37); Gly8Glu22His37-GLP- 1(7-37); Val8Glu22His37-GLP- 1(7-37); Met8Glu22His37-GLP-l(7-37); Gly8Lys22His37-GLP-l(7-37); Met8Lys22His37-GLP- 1(7-37); Gly8Arg22His37-GLP-l(7-37); Val8Arg22His37-GLP- 1(7-37); Met8Arg22His37-GLP- 1(7-37); Gly8His22His37-GLP-l(7-37); Val8His22His37-GLP-l(7-37); Met8His22His37-GLP- 1(7-37); Gly8His37-GLP-l(7-37); Vai 8His37-GLP- 1(7-37); Met8His37-GLP-l(7-37); Gly8Asp22His37- GLP-l(7-37); Val8Asp22His37-GLP- 1(7-37); and Met8Asp22His37-GLP-l(7-37).

8. The AAV vector genome of claim 7, wherein the GLP-1 peptide consists of or consists essentially of the sequence of SEQ ID NO: 14 or 15, optionally with no more than one amino acid substitution relative to a wild-type GLP-1 peptide consisting of the sequence of SEQ ID NO: 1 or 2, that renders the modified GLP-1 peptide resistant to degradation by DPP-4, optionally Gly8-GLP- 1(7-37) or Val8-GLP- 1(7-37).

9. The AAV vector genome of any one of the preceding claims, wherein the open reading frame does not comprise a sequence encoding one or more of: a glicentin-related pancreatic polypeptide (GRPP), a glucagon peptide, or a GLP-2 peptide, optionally wherein the open reading frame does not comprise a sequence encoding the GRPP, the glucagon peptide, or the GLP-2 peptide, and optionally wherein the only preproglucagon (GCG) peptides encoded by the open reading frame are the IP-2 peptide and the GLP-1 peptide.

10. The AAV vector genome of any one of the preceding claims, wherein the open reading frame further comprises a sequence encoding a signal peptide, optionally an insulin gene or preproglucagon signal peptide.

11. The AAV vector genome of any one of the preceding claims, wherein the promoter comprises a pancreatic tissue- specific promoter sequence, optionally wherein the pancreatic tissue-specific promoter sequence comprises an islet beta cell-specific promoter sequence, optionally wherein the islet beta cell-specific promoter sequence comprises an insulin gene promoter sequence, and optionally wherein the insulin gene promoter sequence comprises a human insulin gene promoter sequence, optionally a core sequence, further optionally at least three copies of the core sequence.

12. The AAV vector genome of any one of the preceding claims, optionally an AAV9 vector genome, wherein the engineered nucleic acid further comprises: an enhancer element, optionally a cytomegalovirus (CMV) enhancer element; a sequence encoding a modified Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), optionally mut6.WPRE; and / or a sequence encoding a polyadenylation signal, optionally a bovine growth hormone polyadenylation signal.

13. An adeno-associated virus (AAV) vector genome, optionally an AAV9 vector genome, comprising an engineered nucleic acid comprising a promoter, optionally comprising a human insulin promoter sequence, operably linked to an open reading frame comprising the sequence of SEQ ID NO: 4 or 5, or a sequence having at least 90% identity to the sequence of SEQ ID NO: 4 or 5.

14. An adeno-associated virus (AAV) vector genome, optionally an AAV9 vector genome, comprising an engineered nucleic acid comprising the sequence of SEQ ID NO: 35 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 35.

15. The AAV vector genome of the preceding claims, wherein the engineered nucleic acid further comprises a 5’ inverted terminal repeat (ITR) sequence, optionally an AAV2 ITR, upstream from the promoter and a 3’ ITR, optionally an AAV2 ITR, downstream from the open reading frame.

16. The AAV vector genome of the preceding claims, wherein the AAV vector genome is a self-complementary AAV vector genome.

17. An AAV vector, optionally an AAV9 vector, comprising (a) the AAV vector genome of any one of the preceding claims and (b) AAV capsid proteins.

18. An engineered nucleic acid comprising: a promoter operably linked to an open reading frame comprising a sequence encoding an intervening peptide 2 (IP-2) peptide and a sequence encoding a glucagon-like peptide 1 (GLP-1) peptide, wherein the open reading frame does not comprise a sequence encoding an intervening peptide 1 (IP-1) peptide.

19. The engineered nucleic acid of claim 25, wherein the IP-2 peptide comprises the sequence of SEQ ID NO: 16 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 16, optionally wherein the IP-2 peptide consists of or consists essentially of the sequence of SEQ ID NO: 16.

20. The engineered nucleic acid of any one of the preceding claims, wherein the sequence encoding an IP-2 peptide is downstream from the sequence encoding a GLP-1 peptide.

21. The engineered nucleic acid of any one of the preceding claims, wherein the GLP-1 peptide comprises the sequence of SEQ ID NO: 14 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 14, and / or wherein the GLP-1 peptide comprises the sequence of SEQ ID NO: 15 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 15, optionally wherein the open reading frame further comprises a sequence encoding a PC 1 / 3 cleavage site, optionally between amino acid positions corresponding to amino acid position 6 and amino acid position 7 of a GLP-1 peptide comprising the amino acid sequence of SEQ ID NO: 14.

22. The engineered nucleic acid of any one of the preceding claims, wherein the open reading frame does not comprise a sequence encoding one or more of: a glicentin-related pancreatic polypeptide (GRPP), a glucagon peptide, or a GLP-2 peptide; or the only preproglucagon (GCG) peptides encoded by the open reading frame are the IP-2 peptide and the GLP-1 peptide.

23. The engineered nucleic acid of any one of the preceding claims, wherein: the open reading frame further comprises a sequence encoding a signal peptide, optionally an insulin gene or preproglucagon signal peptide; and / or the promoter comprises a pancreatic tissue-specific promoter sequence, optionally wherein the pancreatic tissue-specific promoter sequence comprises an islet beta cell-specificpromoter sequence, optionally wherein the islet beta cell-specific promoter sequence comprises an insulin gene promoter sequence, and optionally wherein the insulin gene promoter sequence comprises a human insulin gene promoter sequence, optionally a core sequence, further optionally at least three copies of the core sequence.

24. The engineered nucleic acid of any one of the preceding claims, wherein the engineered nucleic acid further comprises: an enhancer element, optionally a cytomegalovirus (CMV) enhancer element; a sequence encoding a modified Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), optionally mut6.WPRE; and / or a sequence encoding a polyadenylation signal, optionally a bovine growth hormone polyadenylation signal.

25. A method comprising administering to a subject the AAV vector genome of any one of claims 1-16, the AAV vector of claim 17, or the engineered nucleic acid of any one of claims 18- 24, optionally wherein the subject has a metabolic disorder, optionally wherein the metabolic disorder is selected from Obesity, Diabetes Mellitus, Disorders of Lipid Metabolism, Inborn Errors of Metabolism, Lysosomal Storage Disorders, Glycogen Storage Diseases, Mitochondrial Disorders, Purine and Pyrimidine Disorders, Urea Cycle Disorders, Disorders of Fructose Metabolism, Disorders of Amino Acid Metabolism, Disorders of Mineral Metabolism, Porphyrias, Lactose Intolerance and Wilson's Disease, Polycystic Ovary Syndrome, Metabolic Dysfunction-associated Steatotic Liver Disease, and Non-Alcoholic Steatohepatitis, and optionally wherein the metabolic disorder is Type 2 diabetes, Obesity, or Metabolic Dysfunction- associated Steatotic Liver Disease.

26. The method of claim 25, wherein the administering is via injection of pancreatic tissue, optionally using endoscopic ultrasound-guided fine needle injection (EUS-FNI) procedure.

27. The method of claim 25 or 26, wherein the viral vector genome, the viral vector, or the engineered nucleic acid is administered in a therapeutically effective amount to treat the metabolic disorder, optionally wherein the therapeutically effective amount comprises about 5xl012to about 1.5xl014AAV vector genomes (VG), optionally about IxlO13to about 5xl013AAV VG.

28. A nutrient-responsive method for regulating blood glucose levels in a subject, the method comprising: injecting a gene therapy composition to pancreatic tissue of the subject in an amount effective to regulate blood glucose levels in the subject, wherein the gene therapy composition comprises an engineered nucleic acid comprising a promoter operably linked to an open reading frame comprising a sequence encoding a glucagon- like peptide 1 (GLP-1) peptide and optionally a sequence encoding an intervening peptide 2 (IP- 2) peptide, optionally wherein the effective amount is administered as a single infusion or as a double infusion.

29. A nutrient-responsive method for regulating blood glucose levels in a subject, the method comprising: administering a gene therapy composition to the subject in an amount effective to regulate blood glucose levels in the subject, wherein the gene therapy composition comprises an engineered nucleic acid comprising a promoter operably linked to an open reading frame comprising a sequence encoding a glucagon- like peptide 1 (GLP-1) peptide and optionally a sequence encoding an intervening peptide 2 (IP- 2) peptide.

30. A nutrient-responsive method for regulating blood glucose levels in a subject, the method comprising: administering a gene therapy composition to the subject in an amount effective to regulate blood glucose levels in the subject, wherein the gene therapy composition comprises an engineered nucleic acid comprising the sequence of SEQ ID NO: 35.

31. A nutrient-responsive method for regulating blood glucose levels in a subject, the method comprising: administering a gene therapy composition to the subject in an amount effective to regulate blood glucose levels in the subject, wherein the gene therapy composition comprises an engineered nucleic acid comprising a promoter operably linked to an open reading frame comprising a sequence of SEQ ID NO: 35 encoding a glucagon-like peptide 1 (GLP-1) peptide and optionally a sequence of SEQ ID NO: 35 encoding an intervening peptide 2 (IP-2) peptide.

32. An engineered nucleic acid comprising the sequence of SEQ ID NO: 35.

33. An AAV vector genome comprising the engineered nucleic acid of claim 32.

34. An AAV vector comprising the AAV vector genome of claim 33 and an AAV capsid.

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