Gene therapies for metabolic disorders

Abstract

The disclosure provides methods and compositions for treating metabolic disorders, including Type 2 Diabetes, obesity, Metabolic Dysfunction-associated Steatotic Liver Disease, and other obesity-related metabolic disorders by combining localized delivery of a gene therapy to pancreatic tissue.

Description

[0001] 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 number 63 / 508,251, filed June 14, 2023, U.S. provisional application number 63 / 594626, filed October 31, 2023, and U.S. provisional application number 63 / 603,577, filed November 28, 2023, each of which is incorporated by reference herein in its entirety.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (F085770001WO00-SEQ-JXV.xml; Size: 63,088 bytes; and Date of Creation: June 12, 2024) is herein incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] Obesity, which often accompanies or leads to various metabolic disorders, is globally pervasive issue, which has reached epidemic proportions, affecting an estimated 650 million adults and over 340 million children and adolescents worldwide. The condition is associated with a plethora of serious health conditions, including but not limited to heart disease, diabetes, and certain types of cancers. Current treatments primarily involve lifestyle modifications such as dietary adjustments and increased physical activity, pharmacological interventions, or in severe cases, bariatric surgery. While these treatments can be effective, they are often accompanied by substantial side effects, and in some cases, their effectiveness diminishes over time due to factors such as drug resistance or patients reverting to previous unhealthy behaviors. Most of the currently available therapies aim to treat metabolic diseases by managing the symptoms, rather than treating the underlying causes of the disease. There exists a significant need for innovative, effective, and safer therapeutic strategies for obesity and other metabolic disorders that would overcome current challenges, providing a sustainable and accessible treatment to those affected by these debilitating conditions.

[0008] Type 2 Diabetes, a form of Diabetes Mellitus, is a metabolic disorder characterized by high blood sugar levels due to the body's inability to effectively use insulin or produce enough insulin. The pancreas is an endocrine gland that produces insulin, as well as other hormones that regulate blood sugar levels. Insulin helps to transport glucose from the bloodstream into cells, where it can be used for energy. With Type 2 Diabetes, the body becomes resistant to the effects of insulin, meaning that it cannot use insulin effectively to lower blood sugar levels. This can lead to high levels of glucose in the blood, which can cause a range of health problems if left untreated. Type 2 Diabetes, along with other obesity-related metabolic diseases, accounts for significant morbidity and mortality globally. Approximately 50% of the estimated 27 million people diagnosed with Type 2 Diabetes in the United States have inadequately controlled disease despite the availability of over 60 approved drugs, and an estimated 50 million people are expected to be living with Type 2 Diabetes in the United States by 2035.

[0009] SUMMARY

[0010] Some aspects of the disclosure relate to methods for treating metabolic disorders through localized delivery of a gene therapy directly to pancreatic tissue. Other aspects of the disclosure relate to methods for reducing body weight in a subject, for example, a subject having a metabolic disease. The methods may comprise, for example, delivering a single dose, or no more than two doses, of a gene therapy composition to pancreatic endocrine tissue of a subject having a metabolic disease. In some embodiments, a gene therapy composition is delivered in an amount effective to maintain a reduction in body weight of about 5% over the course of a year. In some embodiments, a gene therapy composition comprises an adeno-associated virus (AAV) vector genome comprising a pancreatic islet beta cell-specific promoter operably linked to a GLP-1 receptor agonist coding region.

[0011] In some embodiments, the body weight of a subject is maintained at a reduced weight of at least 20% at day 40 post delivery of the single dose of the gene therapy composition.

[0012] In some embodiments, a single dose comprises about 5xl012to about 1.5xl014AAV vector genomes (VG), for example, about IxlO13to about 5xl013AAV VG. In some embodiments, a single dose comprises about IxlO13AAV VG.

[0013] In some embodiments, the total volume of a single dose is about 1 ml to about 3 ml.

[0014] In some embodiments, a gene therapy composition is delivered via infusion (e.g., a single infusion).

[0015] In some embodiments, a single dose is delivered using an endoscopic ultrasound-guided fine needle injection (EUS-FNI) procedure.

[0016] In some embodiments, a pancreatic islet beta cell-specific promoter comprises a human insulin promoter or a region (e.g., core region) thereof.

[0017] In some embodiments, at least 15% of the endocrine tissue is transduced with the AAV vector.

[0018] In some embodiments, an effective amount restores glycemic durability in the subject; significantly reduces fasting blood glucose relative to baseline, significantly increases fasting insulin relative to baseline, significantly improves glucose tolerance relative to baseline, and / or significantly improves glucose- stimulated insulin secretion relative to baseline.

[0019] In some embodiments, a single dose is sufficient for long-term restoration of pancreatic islet beta cell function and / or reduction in therapeutic burden.

[0020] In some embodiments, the 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.

[0021] In other embodiments, a human GLP-1 receptor agonist is present in the pancreas at a level that is at least 50% higher than the level detected in the brain and / or sera of a subject following delivery.

[0022] In some embodiments, fewer than 1 vector copy per diploid genome of an AAV vector genome is detectable in the liver, heart, spleen, or kidney of a subject post-delivery, e.g., at 3-4 weeks post-delivery, of a gene therapy composition.

[0023] In some embodiments, fewer about 1 to about 5 vector copies per diploid genome of the AAV vector genome are detectable in the liver, heart, spleen, or kidney of a subject postdelivery, e.g., at 3-4 weeks post-delivery, of the gene therapy composition.

[0024] In some embodiments, the effective amount significantly reduces the subject’s liver weight and / or liver triglycerides relative to baseline.

[0025] In some embodiments, the effective amount significantly reduces the subject’s body fat mass relative to baseline, and / or significantly increases the subject’s lean body mass relative to baseline.

[0026] In some embodiments, the effective amount significantly reduces the subject’s plasma leptin level relative to baseline.

[0027] In some embodiments the effective amount significantly reduces the subject’s total cholesterol relative to baseline.

[0028] In some embodiments, the effective amount significantly decreases the subject’s low density lipoprotein (LDL) level relative to baseline.

[0029] In some embodiments, a metabolic disease 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 (Non- Alcoholic Fatty Liver Disease), and Non-Alcoholic Steatohepatitis. In some embodiments, a metabolic disease is Type 2 Diabetes. In some embodiments, a metabolic disease is obesity.

[0030] In some embodiments, a subject has a body mass index (BMI) of 25.0 to <30. In other embodiments, a subject has a BMI of 30.0 or higher, optionally a BMI of 30 to <35 (Class 1), 35 to <40 (Class 2), or 40 or higher (Class 3).

[0031] In some embodiments, the subject has received another weight loss therapy (e.g., treatment with Semaglutide or diet) and then discontinued the other weight loss therapy within 3, 6, 9, 12 or 18 months of the delivering of the gene therapy composition, optionally wherein the subject had loss (reduced) weight while receiving the other weight loss therapy.

[0032] In some embodiments, the AAV vector genome comprises: a 5’ inverted terminal repeat (ITR) sequence, an insulin gene promoter and an enhancer element operably linked to a nucleic acid comprising a 5’ untranslated region (UTR), an open reading frame encoding a GLP-1 receptor agonist, a 3’UTR, a polyadenylation signal, and a 3TTR.

[0033] In some embodiments, the insulin gene promoter is a human insulin gene promoter or core region thereof.

[0034] In some embodiments, the enhancer element is a cytomegalovirus enhancer element, for example, a CMV upstream genomic region (CMVugr); the 5’ UTR comprises a modified human hemoglobin subunit beta intron; the GLP-1 receptor agonist is human GLP-1; the GLP-1 receptor agonist is fused to a signal peptide; the 3’ UTR comprises a modified Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) element, optionally mut6.WPRE; and the polyadenylation signal is a bovine growth hormone polyadenylation signal.

[0035] In some embodiments, the AAV vector genome is a single-stranded AAV vector genome, for example, a self-complementary AAV vector genome.

[0036] BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIGs. 1A-1B are graphs showing dose-dependent sustained and reduced glycemia (FIG. 1A) and elevated fasting insulinemia (FIG. IB) in db / db mice after AAV injection (Day 0). Statistics: **P,0.01, ***P<0.001 vs. Vehicle, MIP-eGFP 10el2 groups; *P<0.05 vs vehicle only; One-Way ANOVA, post-hoc Tukey Test

[0038] FIGs. 2A-2C are graphs showing intraperitoneal glucose tolerance test (IPGTT) test results (FIG. 2A), the area under the curve (FIG. 2B), and insulin secretion (FIG. 2C) on Day 39 after AAV injection in db / db mice. Statistics: in FIGs. 2A-2B, One-Way ANOVA; Post-hoc Tukey’s multiple comparisons test, *P<0.05, ****P<0.0001. In FIG. 2C, Two-Way ANOVA mixed-effects model [REML]; Post-hoc Tukey’s multiple comparison test, a = P<0.005 vs.

[0039] Vehicle, b = P<0.05 vs. eGFP control.

[0040] FIGs. 3A-3B are graphs showing absolute body weight (FIG. 3A) and the change in body weight (FIG. 3B) over time after AAV injection in db / db mice.

[0041] FIGs. 4A-4C show that GLP-1RA protein (i.e., Exendin-4) is expressed in the pancreas via immunohistochemical staining (FIG. 4A), percent islet expression (FIG. 4B), and whole pancreas protein expression as determined by LCMS (FIG. 4C) after AAV injection in db / db mice. Figure 4B and 4C show the data means ± SEM. One-Way ANOVA, post-hoc Tukey Test **P<0.01, ****P<0.0001

[0042] FIGs. 5A-5B are graphs showing improved insulin secretion from primary BKS db / db islets ex vivo after treatment with an AAV delivering a GLP-1RA (i.e., Exendin-4) (compared to an AAV-eGFP control). FIG. 5A shows the total GLP-1 content as determined by ELISA while FIG. 5B shows the glucose- stimulated insulin secretion. Both graphs show the means ± Std Dev. Unpaired t-test, *P<0.05.

[0043] FIG. 6 shows insulin levels following AAV-mediated delivery of GLP-1RA (i.e., Exendin-4) in the human beta-cell line EndoC-BH5. Exendin-9 (Ex9) peptide treatment, a potent inhibitor of the GLP-1R, demonstrates that increased INS secretion due to AAV-GLP-1RA is due to its action on the GLP-1R. Means ± Std Dev. Two-Way ANOVA, post-hoc Tukey Test ****P<0.0001.

[0044] FIGs. 7A-7B are graphs showing the change in fasting blood glucose (FIG. 7A) and quantification of Exendin-4 via LCMS in the serum and pancreas (FIG. 7B) of BKS db / db mice four weeks after administration of AAV-MIP-Ex-4 (an AAV-based Exendin-4 treatment) or vehicle.

[0045] FIGs. 8A-8D show graphs of percent green fluorescent protein (GFP) expression in exocrine (FIG. 8A and FIG. 8C) and endocrine (FIG. 8B and FIG. 8D) tissue of either the entire pancreas (FIG. 8A and FIG. 8B) or the targeted splenic lobe of the pancreas (FIG. 8C and FIG. 8D) of Yucatan pigs after endoscopic ultrasound-guided fine needle injection (EUS-FNI) of scAAV9-CMV-eGFP at various indicated doses. Individual standard deviations were used to calculate the intervals. N = 2 to 4 pigs per dose tested.

[0046] FIG. 9 shows a graph of vector copies per diploid genome of AAV DNA in the targeted splenic lobe of the pig pancreas after EUS-FNI of scAAV9 at various indicated doses. Each data point represents 1 pig, with the bars depicting the mean + / - the std. dev.

[0047] FIG. 10A shows a graph of data demonstrating that the number of infusions via EUS- FNI in the pancreas of Yucatan pigs changes AAV vector biodistribution at the 5xl013vector genome (VG) dose. FIG. 10B shows a graph of data demonstrating that the number of infusions via EUS-FNI in the pancreas of Yucatan pigs also changes AAV vector biodistribution at the higher IxlO14VG dose.

[0048] FIGs. 11A-11D show graphs of lipase serum levels following a single infusion or multiple infusions of various indicated doses of AAV vector into the Yucatan pig pancreas. FIG. 11A shows that the number of EUS-FNI infusions into the pig pancreas correlates with elevated serum lipase values. FIG. 11B shows that infusion volumes between l-5mL for EUS-FNI into the pig pancreas do not impact serum lipase levels at a ImL / minute flow rate. FIG. 11C and FIG. 11D shows that AAV dose does not correlate with elevations in serum lipase using either a single infusion or three infusions for EUS-FNI into the pig pancreas.

[0049] FIG. 12 shows a graph of baseline levels of neurofilament light chain (NFL) and levels of NFL following a single infusion of two different doses of AAV9-CMV-eGFP or AAV9-INSp- eGFP into the Yucatan pig pancreas.

[0050] FIG. 13 shows a graph of body weight change in db / db mice at 4 weeks following administration of a single dose of vehicle control, Semaglutide (10 nmol / kg), and one of two different versions of AAV-INS-GLP1RA at a dose of 1012or 512VG. Mean ± SEM shown; ****p<0.0001; n=4-16 per group. AAV=adeno-associated virus, Gen=generation, GLPlRA=glucagon-like peptide 1 receptor agonist, INS=insulin promoter, Sema=semaglutide

[0051] FIGs. 14A-14B respectively show a graph of fasting blood glucose levels and a graph of fasting insulin levels in db / db mice at 8 weeks, 4-5 hours fasted, following administration of a single dose of vehicle control, Semaglutide (10 nmol / kg), and one of two different versions of AAV-INS-GLP1RA at a dose of 1012or 512VG. Mean ± SEM shown; ****p<0.0001; n=4-16 per group. AAV=adeno-associated virus, Gen=generation, GLPlRA=gluc agon-like peptide 1 receptor agonist, INS=insulin promoter, Sema=semaglutide

[0052] FIG. 15 shows a graph of disease progression and durability in db / db mice over the course of 64 days, following administration of a single dose of vehicle control, Semaglutide (10 nmol / kg), and one of two different versions of AAV-INS-GLP1RA at a dose of 1012or 512VG. AAV=adeno-associated virus, FBG=fasting blood glucose, Gen=generation, GLPlRA=glucagon-like peptide 1 receptor agonist, INS=insulin promoter

[0053] FIGs. 16A-16B show a graph of percent body weight change (FIG. 16A) and a graph of food intake (FIG. 16B) in mice treated with a GLP-1 -based pancreatic gene therapy or semaglutide (sema). Statistics: Two-Way ANOVA post-hoc Tukey Test: $$ p<0.01;

[0054] **** p<0.0001; Means ± SEM. FIG. 17 shows graphs of liver weight (left) and liver triglycerides (right) of mice treated with a GLP-1 -based pancreatic gene therapy or vehicle.

[0055] FIGs. 18A-18D show graphs of body composition analysis of mice treated with a GLP- 1-based pancreatic gene therapy (ss.AAV004), vehicle, semaglutide, or semaglutide and then the GLP-l-based pancreatic gene therapy after eight weeks. Body weight (FIG. 18A), fat mass (FIG. 18B), terminal plasma leptin (FIG. 18C), and lean mass (FIG. 18D) are shown.

[0056] FIGs. 19A-19B show the quantitation of terminal pancreas (FIG. 19A) and serum (FIG. 19B) Exendin-4 after eight weeks.

[0057] FIG. 20 shows the Exendin-4-expressing endocrine area (islet area) in mouse pancreas, as measured by immunohistochemistry. Note that Extendin-4 levels were not analyzed for groups 1, 3, or 4 (0% expression was assumed because they are vehicle groups).

[0058] FIGs. 21A-21D show the total cholesterol (FIG. 21A), LDL (FIG. 21B), HDL (FIG. 21C), and triglycerides (FIG. 21D) in DIO mice in a head-to-head study between GLP-1RA PGTx and semaglutide.

[0059] FIGs. 22A-22C results of a head-to-head study between GLP-1RA PGTx and semaglutide in db / db mice. Fasting blood glucose (FIG. 22A), fasting plasma insulin (FIG. 22B) and body weight change from baseline (FIG. 22C) at day 29 are shown.

[0060] FIG. 23 shows the blood glucose (left graph) and plasma insulin (right graph) in DIO mice treated with a GLP-l-based pancreatic gene therapy (ss.AAV004), vehicle, semaglutide, or semaglutide and then the GLP-l-based pancreatic gene therapy after eight weeks.

[0061] FIG. 24 shows HOMA-IR (Homeostatic Model Assessment for Insulin Resistance) levels measured in DIO mice treated with a GLP-l-based pancreatic gene therapy (ss.AAV004), vehicle, semaglutide, or semaglutide and then the GLP-l-based pancreatic gene therapy.

[0062] DETAILED DESCRIPTION

[0063] Metabolic diseases result from a disruption of normal metabolism, the process of converting inputs (food and drink) into an output (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. Metabolic disorders include conditions that increase the risk of heart disease, stroke, and death. The diseases are becoming more prevalent, and it is estimated that up to one-third of American adults have at least one. Despite advances in treatment over the last 50 years, metabolic diseases in general, and obesity and Type 2 Diabetes in particular, continue to be a principal driver of morbidity and mortality today. Glucoregulatory hormones, including but not limited to glucagon-like peptide- 1 (GLP-1), are often produced and secreted in response to dietary nutrients and impact pancreatic islet beta cell function in a multitude of ways. Primary among their effects on pancreatic islet beta cells include stimulation of insulin secretion and production, as well as positively impacting pancreatic islet beta cell health. Beta cell health improvements reported for GLP-1 include the maintenance of pancreatic islet beta cell mass through increased cell proliferation, pancreatic islet beta cell neogenesis, and / or the inhibition of apoptosis. Applying these beneficial functions of glucoregulatory hormones for the treatment of metabolic disorders, including obesity and diabetes, has been a successful clinical strategy and remains an active area of therapeutic research.

[0064] Effective delivery of such glucoregulatory hormones to the pancreas has been associated with many challenges, however. For example, the effectiveness of systemic delivery of such agents via viral vectors (e.g., such as adeno-associated virus (AAV)) has been limited by the immune response, transduction efficiency, size, pre-existing immunity, and route of administration. Additionally, many glucoregulatory hormones possess two properties that make their pharmacologic application to disease treatment challenging: (1) a short half-life, and (2) side effects of nausea and vomiting when present in circulation at chronically at high levels. To overcome these challenges, the present disclosure provides, in some aspects, methods for producing transgenic glucoregulatory hormone(s) in a localized and sustained way. This manner of production limits sustained high levels of the hormone(s) in circulation. To achieve this, the technology described herein leverages, in some aspects, pancreatic islet beta cells for the localized production and secretion of transgenic glucoregulatory hormones, such as GLP-1 and GLP-1 analogs, as these cells already perform a similar function for endogenous insulin production and secretion. Furthermore, without being bound by theory, local delivery to the pancreas and local production of the glucoregulatory hormone(s) by the pancreatic islet beta cells achieve the desired effect(s) on pancreatic islet beta cell function while minimizing circulating levels of the hormones.

[0065] With many metabolic disorders, including obesity and Type 2 Diabetes, there are no FDA-approved therapies that offer disease modification; that is, ongoing and durable preservation of pancreatic insulin production capacity, even after therapy is discontinued.

[0066] Instead of treating a patient’s symptoms, the methods and compositions (e.g., gene therapy compositions) provided herein, in some aspects, are used to treat the underlying cause(s) of the disease. Described herein are gene therapy approaches to restoring insulin production in individuals (subjects) with a metabolic disorder (e.g., obesity and / or Type 2 Diabetes), with the goal of achieving long-term remission. Thus, provided herein, in some aspects, are gene therapy compositions and methods relating to the delivery of key metabolic hormones necessary for proper insulin production in pancreatic islet beta cells. As a non-limiting example, a GLP-1 coding sequence may be packaged in an adeno-associated virus (AAV) vector and delivered locally using, for example, endoscopic ultrasound-guided fine needle injection. Without wishing to be bound by theory, it is thought that augmenting GLP-1 receptor activation locally in endocrine tissue of the pancreas, for example, in the splenic lobe the pancreas, leads to a reduction in the blood glucose level. Other configurations are also possible and described in greater detail below.

[0067] Surprisingly, the data provided herein demonstrate that localized pancreatic islet beta cell production of a GLP-1 analog in the pancreas results in a significant, measurable and durable improvement in symptoms associated with metabolic diseases such as Type 2 Diabetes and obesity (e.g., reduction in fasting blood glucose, increase in fasting insulin, reduction in weight gain, increase in lean mass, decrease in plasma leptin level, decrease in cholesterol levels, and / or reduction in liver weight and triglyceride content), without adversely impacting the brain or sera of the subjects. In other words, these preclinical analyses in animal models show that the gene therapy provided by the disclosure achieves localized production of the gene product in the pancreas at levels sufficient to improve symptoms of metabolic diseases such as Type 2 Diabetes and obesity without exposing the brain or sera to harmful levels of the gene therapy or the gene product.

[0068] Metabolic Diseases

[0069] The disclosure, in some aspects, relates to methods for reducing body weight in a subject, for example, a subject having (e.g., diagnosed with) a metabolic disease. Metabolic diseases include a diverse group of disorders affecting the body's ability to process the carbohydrates, fats, and proteins obtained through diet. Non-limiting examples of metabolic diseases include Obesity, Diabetes Mellitus (Type 1 Diabetes, Type 2 Diabetes, Gestational Diabetes), Disorders of Lipid Metabolism (Familial Hypercholesterolemia, Hypertriglyceridemia), Inborn Errors of Metabolism (Phenylketonuria (PKU), Galactosemia, Tay-Sachs disease, Maple Syrup Urine Disease (MSUD), Gaucher's disease, Fabry disease, Glucose-6-phosphate dehydrogenase (G6PD) deficiency), Lysosomal Storage Disorders (Hunter syndrome, Hurler syndrome, Niemann-Pick disease), Glycogen Storage Diseases (Von Gierke disease (Type I), Pompe disease (Type II), Cori disease (Type III), Andersen disease (Type IV), McArdle disease (Type V)), Mitochondrial Disorders (Kearns-Sayre syndrome, MELAS, MERRF), Purine and Pyrimidine Disorders (Lesch-Nyhan syndrome, Orotic aciduria), Urea Cycle Disorders (Ornithine transcarbamylase deficiency, Argininosuccinic aciduria, Citrullinemia), Disorders of Fructose Metabolism (Hereditary Fructose Intolerance, Essential fructosuria), Disorders of Amino Acid Metabolism (Homocystinuria, Tyrosinemia), Disorders of Mineral Metabolism (Primary hyperparathyroidism, Hypophosphatasia), Porphyrias (Acute Intermittent Porphyria (AIP), Porphyria Cutanea Tarda (PCT)), Lactose Intolerance and Wilson's Disease, Polycystic Ovary Syndrome (PCOS), Metabolic Dysfunction-associated Steatotic Liver Disease (MASLD, formerly known as Non-Alcoholic Latty Liver Disease (NAELD)), and Non-Alcoholic Steatohepatitis (NASH).

[0070] In some embodiments, a subject is obese. Obesity is a medical condition characterized, in part, 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 25.0 to <30, it falls within the overweight range. If a subject’s BMI is 30.0 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.

[0071] 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. Other diseases that co-occur with obesity, and thus would be expected to improve with weight loss include, for example, gastroesophageal reflux disease (GERD), sleep apnea, arthritis, hypertension coronary artery disease (e.g., as a secondary prevention), stroke, transient ischemic attack (TIA), diastolic dysfunction, myocardial infarction, and heart failure.

[0072] In some embodiments, a subject has Metabolic Dysfunction-associated Steatotic Liver Disease (MASLD) (formerly known as non-alcoholic fatty liver disease [NAELD]). 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). As MASLD progresses to cirrhosis, medications for the treatment of diabetes and other metabolic conditions may be administered.

[0073] In some embodiments, a subject has obesity and MASLD. In some embodiments, a subject has Diabetes Mellitus and MASLD. In some embodiments, a subject has obesity, Diabetes Mellitus, and MASLD.

[0074] In some embodiments, a subject has 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. In some embodiments, a subject has Type 2 Diabetes, which typically results from the body’s ineffective use of insulin, often combined with a relative insulin deficiency. Type 2 Diabetes can be characterized by rising blood glucose that can be caused by a multitude of factors, which lead to two parallel, progressive disease processes within the body: insulin resistance and 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. Guidelines focus on managing the blood glucose symptoms of Type 2 Diabetes, often measured by blood concentrations of glycosylated hemoglobin, or HbAlc, rather than attempting to correct 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, and even death.

[0075] Glucoregulatory Hormones

[0076] Glucoregulatory hormones include hormones involved in the modulation of circulating blood glucose levels. Two hormones known canonically to be involved in blood glucose regulation are insulin, which lowers blood glucose levels, and glucagon, which elevates blood glucose levels. Thus, both insulin and glucagon are glucoregulatory hormones. Additional hormones that impact the secretion or function of insulin and / or glucagon may also be categorized as glucoregulatory hormones. In some embodiments, a glucoregulatory hormone acts directly; that is, it is directly involved in regulating blood glucose via insulin and / or glucagon modulation. In other embodiments, the glucoregulatory hormone’s effects are indirect; that is, the activity of the hormone indirectly affects blood glucose via insulin and / or glucagon modulation. Glucoregulatory hormones include, for example, preproglucagon-derived peptides (e.g., glucagon, GLP-1, oxyntomodulin, glicentin, glicentin-related polypeptide (GRPP), major proglucagon fragment, intervening peptide 1 (IP-1), intervening peptide 2 (IP-2), and GLP-2), incretins (e.g., GLP-1 and glucose-dependent insulinotropic peptide (GIP)), gut enteroendocrine cell derived peptides (e.g., GLP-1, peptide tyrosine (PYY), cholecystokinin (CCK), GIP, somatostatin, oxyntomodulin, and ghrelin), and hormones produced in the pancreas (e.g., insulin, amylin, somatostatin, glucagon, and GLP-1).

[0077] Naturally occurring GLP-1 is a polypeptide derived from a proglucagon protein. Under physiological conditions, it is produced and secreted by intestinal enteroendocrine L-cells and certain neurons within the nucleus of the solitary tract in the brainstem upon food consumption. GLP-1 is rapidly metabolized and inactivated by dipeptidyl peptidase IV (an enzyme) even before the hormone has left the gut. GLP-1 stimulates insulin secretion (acting as an incretin hormone) and inhibits glucagon secretion. It also inhibits gastrointestinal motility and secretion. In this way, the protein acts as an enterogastrone and part of the "ileal brake" mechanism. GLP-1 also plays a role as a physiological regulator of appetite and food intake. Decreased secretion of GLP-1 can lead to the development of obesity. In some embodiments, a polynucleotide encodes a full-length GLP-1 sequence (e.g., SEQ ID NO: 33). In other embodiments, a polynucleotide encodes a truncated GLP-1 sequence (e.g., any one of SEQ ID NOs: 53-58). In yet other embodiments, a polynucleotide encodes a functional variant or fragment of a GLP-1.

[0078] In some embodiments, a glucoregulatory hormone is GLP-1. In some embodiments, a glucoregulatory hormone comprises an agent that mimics the action of GLP- 1. In some embodiments, the agent comprises a GLP-1 receptor agonist (e.g., an agent that binds and activates GLP-1 receptors, reducing blood glucose levels). In some embodiments, a GLP-1 receptor agonist is a polypeptide agonist for the GLP-1 receptor (e.g., Exendin-4 and variants thereof). Exendin-4 (present in the saliva of the Gila monster, Heloderma suspectum) is a long acting GLP-1 analogue that is an agonist for the GLP-1 receptor. In some embodiments, a GLP-1 receptor agonist is Exendin-4. Other endocrine hormones with glucoregulatory activity contemplated herein include, without limitation, leptin, follistatin, insulin-like growth factor 1 (IGF1), vasoactive intestinal peptide (VIP), and growth hormone 1 (GH1). See Table 1 for exemplary protein and coding sequences. The polynucleotides described herein, in some embodiments, comprise an insulin (preproinsulin, proinsulin, insulin) coding sequence. Preproinsulin, 110 amino acids in length, is a biologically inactive precursor to insulin. Insulin mRNA is translated as preproinsulin, a single chain precursor, and removal of its signal peptide during insertion into the endoplasmic reticulum generates proinsulin. Insulin is produced in and secreted by pancreatic islet beta cells in the pancreas. Proinsulin and preproinsulin comprise three domains: an amino-terminal B- chain, a carboxy-terminal A-chain, and a connecting peptide in the middle known as the C- peptide. Within the endoplasmic reticulum, proinsulin is exposed to several specific endopeptidases which excise the C-peptide, generating the mature form of insulin which consists of the A and B-chain. Insulin and free C-peptide are packaged in the Golgi into secretory granules which accumulate in the cytoplasm. In some embodiments, the insulin protein comprises the amino acid sequence of SEQ ID NO: 46, or a variant thereof. Unless otherwise stated, the term “insulin” encompasses the various forms of insulin, including preproinsulin, proinsulin, insulin.

[0079] Provided herein, are polynucleotides (i.e., nucleic acids) comprising a glucoregulatory hormone (e.g., GLP-1) coding region, also referred to as a coding sequence. Coding sequences include a nucleotide sequence that directly defines the amino acid sequence of a product (e.g., a protein encoded by coding sequence). The limits of the coding sequence are generally determined by an open reading frame. An open reading frame includes a continuous stretch of DNA or RNA beginning with a start codon (e.g., methionine (ATG or AUG)) and ending with a stop codon (e.g., TAA, TAG or TGA, or UAA, UAG or UGA). An open reading typically encodes a protein (e.g., a glucoregulatory hormone or insulin). Polynucleotides may be or may include, for example, deoxyribonucleic acids (DNAs), ribonucleic acids (RNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a P-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino- a-LNA having a 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) and / or chimeras and / or combinations thereof.

[0080] The term “glucoregulatory hormone” includes functional peptides and polypeptides of any of the foregoing examples as well as functional variants thereof, meaning the peptides, polypeptides, and / or variants are capable of impacting blood glucose control through either direct or indirect functions.

[0081] Thus, amino acid modifications (e.g., substitutions) can be made to the glucoregulatory hormones provided herein. In some embodiments, a modified amino acid sequence imparts a beneficial property for protein production and / or function. For example, a GLP-1 peptide sequence can include a glycine substitution for alanine at the amino acid position 8 of the GLP- 1(1-37) sequence (GLP-1-Gly8), which confers resistance to cleavage into an inactive form by dipeptidyl peptidase-IV (DPP4 or DPPIV). Other modifications and thus other variants are contemplated herein.

[0082] In some embodiments, a polynucleotide comprises multiple coding sequences, each encoding a different protein. In some embodiments, a polynucleotide comprises one or more coding sequence(s) for 1-10 glucoregulatory hormones. For example, a polynucleotide may comprise one or more coding sequence(s) for 1-3, 1-4, or 1-5 different glucoregulatory hormones. In some embodiments, a polynucleotide comprises one or more coding sequence(s) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more glucoregulatory hormones. In some embodiments, a polynucleotide comprises a GLP-1 coding sequence. In some embodiments, a polynucleotide comprises a GLP-1 coding sequence, an IP-1 coding sequence and / or an IP-2 coding sequence.

[0083] In some embodiments, a glucoregulatory hormone coding sequence encodes a functional glucoregulatory hormone produced in vivo following administration to a subject. As used herein, “functional” refers to a protein that possesses biological activity (e.g., enzymatic activity). That is, the functional protein produced has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity (e.g., enzymatic activity) compared to a corresponding naturally occurring protein. 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, the functional glucoregulatory hormone has approximately the same activity as a naturally occurring glucoregulatory 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, the functional glucoregulatory hormone has more activity than a naturally occurring glucoregulatory hormone.

[0084] “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.

[0085] 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 wild-type, native, 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); and those based on the Smith-Waterman algorithm (Smith, T.F. & Waterman, M.S. J. Mol. Biol. 1981 ; 147: 195- 197). A general global alignment technique based on dynamic programming is the Needleman- Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. J. Mol. Biol. 1920;48:443-453). 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.

[0086] In some embodiments, a polynucleotide provided herein further comprises at least one promoter. A promoter includes a nucleotide sequence located at the 5’ terminal of a polynucleotide to which a polymerase specifically binds and initiates transcription of the remainder of a polynucleotide. In some embodiments, a promoter is a pancreatic islet cell promoter, such as a pancreatic islet beta cell promoter. In some embodiments, a promoter is an insulin promoter, such as a human insulin promoter, mouse insulin- 1 promoter, mouse insulin-2 promoter, rat insulin- 1 promoter, or rat insulin-2 promoter. Additional exemplary promoters include, but are not limited to, Slc2a, IAPP, NKX6.1, DLK1, MafA, Slc30a8 / Znt8, PCSK1, and ADCYAP1.

[0087] In some embodiments, a polynucleotide provided herein further comprises an enhancer. An enhancer includes a nucleotide sequence that can stimulate promoter activity by enhancing the level of tissue specificity of a promoter and is positioned between a promoter and a coding sequences of a polynucleotide. Exemplary enhancers include, but are not limited to, CMV enhancers, synthetic enhancers, liver- specific enhancers, vascular-specific enhancers, brain- specific enhancers, neural cell-specific enhancers, lung-specific enhancers, muscle-specific enhancers, kidney- specific enhancers, pancreas-specific enhancers, and islet cell-specific enhancers. In some embodiments, an enhancer is an islet cell-specific enhancer.

[0088] Table 1. Examples of Glucoregulatory Hormone Coding Sequences and Proteins Sequences

[0089] Delivery Systems and Routes of Administration

[0090] In some embodiments, the polynucleotides of the disclosure are formulated for delivery in vivo, for example. The disclosure, in some embodiments, provides vectors comprising any one of the polynucleotides described herein.

[0091] In some embodiments, a vector is a viral vector, such as an adeno-associated virus (AAV) vector, a retroviral vector, an adenovirus vector, or a Herpes simplex virus (HSV) vector. In some embodiments, a vector comprises an adeno-associated virus (AAV) vector. AAVs (or “rAAV” for recombinant AAV) are non-enveloped small, single- stranded DNA viruses capable of infecting both dividing and non-dividing cells. In contrast to the generally limited durability of AdV-mediated gene transfer, transgene expression can persist for years following intramuscular recombinant AAV (rAAV) vector delivery.

[0092] An “adenovirus expression vector” or “AAV vector” includes vectors comprising adenovirus or AAV sequences sufficient to (a) support packaging of the construct / genome and (b) to express a polynucleotide described herein.

[0093] Typically, a recombinant AAV virus is made by co-transfecting a plasmid containing the gene of interest (e.g., a polynucleotide) flanked by the two AAV terminal repeats and an expression plasmid containing the wild type AAV coding sequences without the terminal repeats. The AAV expression vector which harbors a polynucleotide bounded by AAV ITRs, 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 therefrom. In some embodiments, an ITR sequences are selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 (AAVrhlO), and AAV11 ITR sequences. In some embodiments, an ITRs are designed for a single-stranded AAV genome or a self-complimentary AAV genome. With respect to the self-complimentary AAV genome, the TRS (terminal resolution site) located in the 3' ITR is deleted.

[0094] In some embodiments, an AAV vector comprises a recombinant AAV vector genome described above (e.g., AAV genome comprising a human pancreatic islet beta cell-specific promoter operably linked to a human GLP- 1 receptor agonist coding sequence) and a nucleotide sequence encoding a capsid protein. Capsid proteins are related to the determination of the tissue-specific targeting capabilities of an AAV and are known in the art. In some embodiments, a capsid protein is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, RhlO, Rh74, AAV-2i8, AAV-DJ, AAV-LK03, AAV-KP1, AAV-KP2, and AAV-KP3 capsid proteins and variants thereof.

[0095] In some embodiments, an AAV vector genome is single-stranded. In some embodiments, the AAV vector genome is self-complementary.

[0096] For eukaryotic cells, expression control sequences typically include a promoter, an enhancer, such as one derived from an immunoglobulin gene, SV40, cytomegalovirus, etc. (see above), and a polyadenylation sequence which may include splice donor and acceptor sites. The polyadenylation sequence generally is inserted following the transgene sequences and before the 3' ITR sequence. In some embodiments, a polyadenylation sequence comprises a SV40 polyA or bovine GH polyA sequence. In some embodiments, an AAV vector comprises a 5’ UTR between the promoter and the coding sequence. In some embodiments, a 5’ UTR sequence comprises an intron. In some embodiments, an intron is artificial, derived from an insulin 5’ UTR, or derived from the hemoglobulin subunit beta (HBB) locus.

[0097] In some embodiments, a vector is a retroviral vector. Thus, in some embodiments, the gene therapy composition comprises a retroviral vector genome comprising a pancreatic islet beta cell-specific promoter operably linked to a GLP-1 receptor agonist coding region. Nonlimiting examples of retroviral vectors include murine leukemia virus vectors and lentiviral vectors (e.g., derived from human immunodeficiency virus).

[0098] In some embodiments, a vector is a herpes simplex virus (HSV) vector. Thus, in some embodiments, the gene therapy composition comprises an HSV vector genome comprising a pancreatic islet beta cell-specific promoter operably linked to a GLP-1 receptor agonist coding region.

[0099] In some embodiments, a vector is an adenoviral vector (AdV). Thus, in some embodiments, the gene therapy composition comprises an AdV vector genome comprising a pancreatic islet beta cell-specific promoter operably linked to a GLP-1 receptor agonist coding region.

[0100] In some embodiments, a vector is a nonviral vector, such as a plasmid, bacterial artificial chromosome, yeast artificial chromosome, or minicircle.

[0101] The gene therapy compositions of the disclosure, in some aspects, are delivered directly to the pancreas, having both exocrine and endocrine functions that produce digestive enzymes and hormones, respectively. The human pancreas has three main anatomical parts: the head, body, and tail. The head of the pancreas, which is the widest part, lies to the right of the midline. It is nestled within the duodenum, which is the first part of the small intestine. The common bile duct runs through the head of the pancreas, and the body of the pancreas is located behind the stomach and extends towards the left side of the body. Finally, the tail of the pancreas is the narrowest part of the gland and is located to the left of the midline, extending towards the spleen. The two main ducts of the human pancreas are the pancreatic duct and the accessory duct. The pancreatic duct is a tube that runs the length of the pancreas and transports digestive enzymes and bicarbonate from the pancreas to the duodenum, while the accessory duct is a smaller duct that can drain into the duodenum. The two types of cells of the pancreas are referred to as islets of Langerhans and acini. Islets of Langerhans are clusters of endocrine cells that produce hormones such as insulin, glucagon, and somatostatin, which regulate blood sugar levels, while acini are clusters of exocrine cells that produce digestive enzymes, such as amylase, lipase, and proteases, which help break down carbohydrates, fats, and proteins in the small intestine. Overall, the pancreas has a unique and complex structure that enables it to perform both exocrine and endocrine functions critical for digestion and metabolism.

[0102] The porcine pancreas, by contrast, has a unique structure, with four lobes: the pancreatic head, body, tail, and splenic lobe. In humans, the pancreas has a more uniform structure without distinct lobes. The area that corresponds to the splenic lobe of the porcine pancreas in humans is the tail of the pancreas. Thus, the pancreatic tail of the human pancreas is the equivalent of the splenic lobe of the porcine pancreas.

[0103] In preferred aspects, a gene therapy composition is delivered directly to the pancreas (e.g., to endocrine tissue of the pancreas) via an endoscopic delivery method. For example, a gene therapy composition may be local delivered to the pancreatic tail of the pancreas or to the body of the pancreas near the tail end via an endoscopic delivery method. In other aspects, however, a gene therapy composition is delivered via intraparenchymal delivery, intra-CSF delivery, intramuscular delivery, or systemic delivery (e.g., intravenous or intra-arterial).

[0104] In some embodiments, a gene therapy composition is delivered to the intestine or pancreas of a subject via a minimally invasive endoscopic procedure (e.g., using a catheter). Various endoscopic procedures and devices are known and contemplated herein.

[0105] In some embodiments, an endoscopic ultrasound-guided fine needle injection (EUS-FNI) procedure is used to deliver a gene therapy composition of the present disclosure. Endoscopic ultrasound-guided fine needle injection (EUS-FNI) is a medical procedure that is used to inject medications (e.g., a gene therapy composition of the disclosure) into the body under the guidance of endoscopic ultrasound. It is a minimally invasive procedure in which a small needle is passed through the endoscope and guided by ultrasound to the target area in the body (e.g., pancreatic deposit site). The needle is then used to inject medication, such as a gene therapy composition, directly into the targeted area. In some embodiments, a needle is a 20-30g needle with a 0.4- 0.6mm diameter. In some embodiments, a needle is a 25g needle with a 0.515mm diameter.

[0106] In some embodiments, a depositing device comprising a depositing element is used to deliver (e.g., infuse) a gene therapy composition of the present disclosure to a deposit site, such as a pancreatic deposit site (e.g., endocrine tissue of the pancreatic tail).

[0107] A depositing device, in some embodiments, comprises a device for implanting, placing, seeding, inserting, spraying, topically applying, and / or otherwise depositing a gene therapy composition of the present disclosure at a “deposit site” of a subject. A depositing device may comprise one or more needle(s) positioned on a distal portion of the depositing device. In some embodiments, a distal end of the depositing device is delivered into a subject through the mouth, and advanced through a wall of the gastrointestinal tract to a location proximate the pancreas. The depositing device, for example, can be delivered through a working channel of a gastrointestinal endoscope that has been delivered through the mouth of a subject. The depositing device can be delivered alongside a gastrointestinal endoscope that has been delivered through the mouth of a subject, for example. In some embodiments, a pancreatic deposit site comprise one or more sites selected from the group consisting of: intra-parenchymal space; anterior pararenal space; intraductal space; intra-arterial space of an artery that feeds at least a portion of the pancreas; and combinations thereof. Exemplary depositing devices and systems are described, for example, in WO 2022 / 174091 (e.g., the REJUVA® System) and WO 2016 / 011269, the entire contents of each of which are incorporated herein by reference.

[0108] In some embodiments, delivering of the gene therapy composition comprises at least a first delivery in which a minimum volume of a gene therapy composition comprising the gene therapy composition is delivered into the pancreatic parenchyma, and the minimum volume of the composition comprises a volume sufficient to cause at least a portion of the volume of the composition to exit into the anterior pararenal space, spread, and re-enter the pancreas. The method can further comprise at least a second delivery of the composition comprising the gene therapy composition to one or more additional deposit sites proximate the tail of the pancreas.

[0109] In some embodiments, delivering of the gene therapy composition comprises at least a first delivery in which a minimum volume of gene therapy composition is delivered into the pancreatic parenchyma, and the minimum volume of the gene therapy composition comprises a volume of at least 2ml, at least 3ml, and / or at least 5ml.

[0110] In some embodiments, a depositing device is advanced to the selected one or more pancreatic deposit sites under image guidance. The image guidance can comprise: endoscopic ultrasound guidance; CT guidance; and / or MRI guidance.

[0111] In some embodiments, a therapeutic benefit is achieved for a time period of at least 6 months. In some embodiments, a therapeutic benefit is achieved for a time period of at least 1 week, at least 2 weeks, at least 3 weeks, 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 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least one year.

[0112] In some embodiments, one or more pancreatic deposit sites comprise locations within 10cm, 7.5cm, 5cm, and / or 3cm of a portion of the pancreas, and the portion of the pancreas comprises the tail, the neck, the body, the head, and / or the uncinate process.

[0113] In some embodiments, a gene therapy composition and / or the at least one depositing element is configured to be visualized by an imaging device, and the method further comprises visualizing the gene therapy composition and / or the at least one depositing element with the imaging device to confirm proper delivery of the gene therapy composition.

[0114] In some embodiments, a method further comprises delivering an imaging agent through the at least one depositing element and visualizing the delivery of the imaging agent with an imaging device to confirm subsequently proper delivery of the gene therapy composition.

[0115] In some embodiments, a method further comprises pre-loading the depositing device with the gene therapy composition. The gene therapy composition can be loaded into the depositing device from the distal end of the depositing device.

[0116] In some embodiments, delivering of the gene therapy composition is performed (e.g., via infusion) at a pressure of at least 3 mmHg (e.g., at least 4mmHg, at least 5mmHg, at least lOmmHg, at least 15mmHg or at least 20mmHg). In some embodiments, the delivering of the gene therapy composition is performed (e.g., via infusion) at a pressure of about 3 mmHg to about 10 mmHg, about 3 mmHg to about 15 mmHg, or about 3 mmHg to about 20 mmHg). In some embodiments, the delivering of the gene therapy composition is performed at a pressure of no more than 25mmHg.

[0117] In some embodiments, delivering of the gene therapy composition is performed (e.g., via infusion) at a flow rate of about 0.5ml / min to about lOml / min (e.g., 0.5ml / min, l.Oml / min, 1.5ml / min, 2.0ml / min, 2.5ml / min, 3.0ml / min, 3.5ml / min, 4.0ml / min, 4.5ml / min, 5.0ml / min, 5.5ml / min, 6.0ml / min, 6.5ml / min, 7.0ml / min, 7.5ml / min, 8.0ml / min, 8.5ml / min, 9.0ml / min, 9.5ml / min, or lO.Oml / min). In some embodiments, the delivering of the gene therapy composition is performed (e.g., via infusion) at a flow rate of about l.Oml / min to about 5.0ml / min. In some embodiments, delivering of the gene therapy composition is performed (e.g., via infusion) at a flow rate of at least Iml / min. In some embodiments, delivering of the gene therapy composition is performed at a flow rate of no more than 5ml / min.

[0118] In some embodiments, at least one depositing element comprises multiple fenestrations along its length.

[0119] In some embodiments, a method further comprises confirming the at least one depositing element is in a proper location prior to delivering the gene therapy composition.

[0120] In some embodiments, a method further comprises the delivery of a permeabilityenhancing agent prior to the delivery of the gene therapy composition and / or simultaneously with the delivery of the gene therapy composition. The delivery of the permeability-enhancing agent can be performed locally and / or intravenously. The permeability-enhancing agent can comprise an agent selected from the group consisting of: hyaluronidase; collagenase; losartan; and combinations thereof. The gene therapy composition can comprise a coformulation of the gene therapy composition and the permeability-enhancing agent.

[0121] In some embodiments, a method further comprises heating tissue proximate the selected one or more pancreatic deposit sites to a temperature above 39°C prior to, during, and / or after the delivery of the gene therapy composition.

[0122] In some embodiments, a method further comprises delivering a dissemination-blocking material that is configured to prevent undesired dissemination of the gene therapy composition to non-target locations. The dissemination-blocking material can comprise a viscous substance and / or a polymer.

[0123] In some embodiments, a method further comprises positioning a blocking element in a subject, and the blocking element is configured to prevent undesired dissemination of the gene therapy composition to non-target locations.

[0124] In some embodiments, a method further comprises removing at least a portion of the gene therapy composition from a deposit site location after the delivery of the gene therapy composition begins.

[0125] Additional aspects and embodiments of the delivery device and methods are described in International Publication Number WO 2022 / 174091 (International Application Number PCT / US2022 / 016200), incorporated herein by reference in its entirety.

[0126] Therapeutic Uses

[0127] The gene therapy compositions described herein (including, for example, AAV vector genomes, polynucleotides encoding glucoregulatory hormone(s), and glucoregulatory hormones) may be used to treat one or more metabolic disorders, such as obesity-related metabolic disorders. Exemplary metabolic disorders include, but are not limited to, Obesity, Diabetes Mellitus (e.g., prediabetes, Type 1 Diabetes, Type 2 Diabetes), 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 (Non-Alcoholic Fatty Liver Disease), and Non- Alcoholic Steatohepatitis.

[0128] Treatment of a metabolic disorder includes the administration or delivery of one or more gene therapy compositions in an amount effective to alleviate one or more symptoms of the metabolic disorder, in some embodiments, with a single dose within a specified window of time. 1

[0129] An amount effective (used interchangeably with effective amount) to achieve a specified result (also referred to herein as a therapeutically effective amount) may depend, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of a gene therapy composition, for example, and / or other determinants, such as age, body weight, height, sex and general health of a subject. In preferred embodiments, an effective amount of a gene therapy composition results in an improvement with respect to glucose (blood sugar) level in a subject.

[0130] 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. Other diseases that co-occur with Type 2 Diabetes and obesity and are thought to be related (but not treated with insulin unless Type 2 Diabetes indicated) include, for example, polycystic ovarian syndrome (PCOS), hyperandrogenism, fertility issues, menstrual dysfunction, hirsutism, dementia, Alzheimer's disease, cognitive decline, cancer such as liver cancer, ovarian cancer, breast cancer, endometrial cancer, cholangiocarcinoma, adenocarcinoma, glandular tissue tumor(s), stomach cancer, large bowel cancer, and / or prostate cancer, psoriasis, hypogonadism, insufficient total testosterone levels, and / or insufficient free testosterone level. Other forms of diabetes where glucoregulatory hormone production in the pancreatic islet beta cells may in principle be helpful include "double diabetes" (when T1D patients also get Type 2 Diabetes); gestational diabetes; and pre-diabetes.

[0131] 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 (e.g., delivering a gene therapy composition to the pancreas of a subject), within 6 months of administering a gene therapy composition to a subject (e.g., delivering a gene therapy composition to the pancreas of a subject), within 9 months of administering a gene therapy composition to a subject (e.g., delivering a gene therapy composition to the pancreas of a subject), within 12 months / 1 year of administering a gene therapy composition to a subject (e.g., delivering a gene therapy composition to the pancreas of a subject), or within 18 months of administering a gene therapy composition to a subject (e.g., delivering a gene therapy composition to the pancreas of 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 of time. 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 disease) 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 gene therapy composition, and no other doses of that gene therapy composition are administered during that 1 year window of time.

[0132] 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.

[0133] 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 of time, 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.

[0134] In some embodiments, a gene therapy composition (e.g., as single dose of a gene therapy composition) is delivered to pancreatic endocrine tissue (e.g., directly to the pancreatic tail) 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.

[0135] 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., near the pancreatic tail) 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 (e.g., near the pancreatic tail) 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., near the pancreatic tail) 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.

[0136] 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.

[0137] 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. 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.

[0138] In some embodiments, an effective amount increases the subject’s lean body mass. In some embodiments, the subject’s lean body mass is measured as a percentage of the subject’s 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 (e.g., directly to the pancreatic tail) of a subject in an amount effective to increase the subject’s lean mass. A subject’s lean (body) mass may be determined through any method known in the art, for example, with the use of dual-energy x-ray absorptiometry (DEXA). In some embodiments, an effective amount results in an increase in the subject’s lean (body) mass of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more relative to baseline. In some embodiments, an effective amount results in a lean body mass within normal physiological values (60-90%), that is, at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, or more percent lean body mass.

[0139] In some embodiments, an effective amount decreases the subject’s body fat mass. In some embodiments, the subject’s body fat mass is measured as a percentage of the subject’s 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 (e.g., directly to the pancreatic tail) of a subject in an amount effective to decrease the subject’s body fat mass. A body fat mass may be determined through any method known in the art, for example, with the use of skinfold calipers, body circumference measures, hydrostatic weighing, bioelectrical impedance, airdisplacement plethysmography, and / or 3D body scans. In some embodiments, an effective amount results in a decrease in the subject’s body fat mass of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more relative to baseline. In some embodiments, an effective amount results in a body fat mass within normal physiological values (12-30%), that is, at least 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, or more percent body fat mass.

[0140] 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 (e.g., directly to the pancreatic tail) 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.

[0141] 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 (e.g., directly to the pancreatic tail) 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 (e.g., directly to the pancreatic tail) 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.

[0142] 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 (e.g., directly to the pancreatic tail) 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 (e.g., directly to the pancreatic tail) 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,

[0143] 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,

[0144] 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.

[0145] 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 (e.g., directly to the pancreatic tail) 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.

[0146] 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 (e.g., directly to the pancreatic tail) 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.

[0147] 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 (e.g., directly to the pancreatic tail) 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.

[0148] In some embodiments, an effective amount decreases the subject’s leptin levels (e.g., plasma leptin 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 (e.g., directly to the pancreatic tail) of a subject in an amount effective to decrease the subject’s leptin levels (e.g., plasma leptin levels). A subject’s leptin levels may be determined through any method known in the art, for example, with the use of immunoassays, such as a radioimmunoassay (RIA) or an enzyme-linked immunosorbent assay (ELISA). In some embodiments, an effective amount results in a decrease in the subject’s leptin levels to a physiological level (e.g., 2-11 ng / mL in serum; Sultan et al., J Family Community Med., 2006 Sep-Dec; 13(3): 97-102). In some embodiments, an effective amount results in a leptin serum level of 2 ng / mL, 2.5 ng / mL, 3 ng / mL, 3.5 ng / mL, 4 ng / mL, 4.5 ng / mL, 5 ng / mL, 5.5 ng / mL, 6 ng / mL, 6.5 ng / mL, 7 ng / mL, 7.5 ng / mL, 8 ng / mL, 8.5 ng / mL, 9 ng / mL, 9.5 ng / mL, 10 ng / mL, 10.5 ng / mL, 11 ng / mL or more in the subject. In some embodiments, an effective amount decreases the subject’s total cholesterol levels relative to baseline (e.g., 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, 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 (e.g., directly to the pancreatic tail) of a subject in an amount effective to decrease the subject’s total cholesterol levels. A subject’s total cholesterol levels may be determined through any method known in the art, for example, with the use of a blood test. In some embodiments, an effective amount results in a decrease in the subject’s total cholesterol levels to a physiological level (e.g., less than 239 mg / dL or less than 200 mg / dL). In some embodiments, an effective amount results in a total cholesterol level of less than 240 mg / dL, 239 mg / dL, 238 mg / dL, 237 mg / dL, 236 mg / dL, 235 mg / dL, 234 mg / dL, 233 mg / dL, 232 mg / dL, 231 mg / dL, 230 mg / dL, 229 mg / dL, 228 mg / dL, 227 mg / dL, 226 mg / dL, 225 mg / dL, 224 mg / dL, 223 mg / dL, 222 mg / dL, 221 mg / dL, 220 mg / dL, 219 mg / dL, 218 mg / dL, 217 mg / dL, 216 mg / dL, 215 mg / dL, 214 mg / dL, 213 mg / dL, 212 mg / dL, 211 mg / dL, 210 mg / dL, 209 mg / dL, 208 mg / dL, 207 mg / dL, 206 mg / dL, 205 mg / dL, 204 mg / dL, 203 mg / dL, 202 mg / dL, 201 mg / dL, 200 mg / dL, or less than 199 mg / dL in the subject.

[0149] In some embodiments, an amount effective increases the subject’s high-density lipoprotein (HDL) level relative to baseline (e.g., 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, relative to baseline). For example, in some embodiments, an amount effective increases the subject’s HDL level to greater than 35 mg / dL, 36 mg / dL, 37 mg / dL, 38 mg / dL, 39 mg / dL, 40 mg / dL, 41 mg / dL, 42 mg / dL, 43 mg / dL, 44 mg / dL, 45 mg / dL, 46 mg / dL, 47 mg / dL, 48 mg / dL, 49 mg / dL, 50 mg / dL, 51 mg / dL, 52 mg / dL, 53 mg / dL, 54 mg / dL, 55 mg / dL, 56 mg / dL, 57 mg / dL, 58 mg / dL, 59 mg / dL, or greater than 60 mg / dL.

[0150] “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.

[0151] An effective amount of a gene therapy composition, in some embodiments, is about 5xl012to about 1.5xl014vector genomes (VG), for example AAV VG. For example, the effective amount may be about 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 5xl012to about IxlO13VG. 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. An effective amount of a gene therapy composition, in some embodiments, is a single dose, for example, of about 5xl012to about 1.5xl014vector genomes (VG), e.g., AAV VG. For example, the effective amount may be a single dose of about 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 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.

[0152] 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 5xl012to about 1.5xl014vector genomes (VG). For example, the effective amount may be a single dose of about 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.

[0153] 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 5xl012to about 1.5xl014vector genomes (VG). For example, the effective amount may be a single dose of about 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.

[0154] 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 1.5xl014vector genomes (VG). For example, the effective amount may be a single dose of about 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] Subjects are typically human subjects; however, a subject could be any mammal, including a non-human primate.

[0161] 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.

[0162] 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 single-use 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.

[0163] Additional Embodiments

[0164] The disclosure also relates to the additional embodiments set forth in the following numbered paragraphs: 1. A method for treating Type 2 Diabetes in a subject in need thereof, comprising: delivering an effective amount of a gene therapy composition to endocrine tissue of the tail and / or body of the pancreas of the subject using an endoscopic ultrasound-guided fine needle injection (EUS-FNI) procedure, wherein the gene therapy composition comprises an adeno- associated virus (AAV) vector genome encoding a human GLP-1 receptor agonist, optionally wherein the effective amount is a single dose of about 5xl012to about 1.5xl014vector genomes (VG).

[0165] 2. The method of paragraph 1, wherein the effective amount is a single dose of about IxlO13to about 5xl013VG.

[0166] 3. The method of paragraph 1 or 2, wherein the total volume of the single dose is no more than 1 ml.

[0167] 4. The method of any one of paragraphs 1-3, wherein the total volume of the single dose is about 1 ml to about 5 ml.

[0168] 5. The method of any one of the preceding paragraphs, wherein the single dose is delivered via a single infusion.

[0169] 6. The method of any one of the preceding paragraphs, wherein the single dose is delivered via multiple infusions.

[0170] 7. The method of any one of the preceding paragraphs, wherein the AAV vector genome comprises a human pancreatic islet beta cell-specific promoter, optionally a human insulin promoter, operably linked to a human GLP-1 receptor agonist coding sequence.

[0171] 8. The method of any one of the preceding paragraphs, wherein the EUS-FNI procedure comprises advancing a depositing device comprising a fine needle at a distal portion of the depositing device to a pancreatic deposit site in the pancreas, and delivering the gene therapy composition through the fine needle into the pancreatic deposit site.

[0172] 9. The method of any one of the preceding paragraphs, wherein the distal end of the depositing device is advanced into the mouth of the subject and through a wall of the gastrointestinal tract to a location proximate the pancreas, optionally wherein (a) the depositing device is delivered through a working channel of a gastrointestinal endoscope that has been delivered through the mouth of the subject of (b) the depositing device is delivered alongside a gastrointestinal endoscope that has been delivered through the mouth of the subject.

[0173] 10. The method of any one of the preceding paragraphs, wherein the pancreatic deposit site is the parenchyma of the tail and / or head of the pancreas. 11. The method of any one of the preceding paragraphs, wherein the delivering the gene therapy composition through the fine needle into the pancreatic deposit site is performed at a pressure of at least 3 mmHg and / or no more than 25mmHg.

[0174] 12. The method of any one of the preceding paragraphs, wherein the delivering the gene therapy composition through the fine needle into the pancreatic deposit site is performed at a flow rate of at least Iml / min and / or no more than 5ml / min.

[0175] 13. The method of any one of the preceding paragraphs, further comprising delivering a permeability-enhancing agent prior to the delivering of the gene therapy composition and / or simultaneously with the delivering of the gene therapy composition, optionally wherein the delivering of the permeability-enhancing agent is performed locally and / or intravenously, optionally wherein the permeability-enhancing agent comprises an agent selected from the group consisting of: hyaluronidase; collagenase; losartan; and combinations thereof, and optionally wherein the treatment agent comprises a coformulation of the gene therapy composition and the permeability-enhancing agent.

[0176] 14. The method of any one of paragraphs 7-13, further comprising heating tissue proximate the pancreatic deposit site to a temperature above 39°C prior to, during, and / or after the delivery of the gene therapy composition.

[0177] 15. The method of any one of the preceding paragraphs, further comprising delivering a dissemination-blocking material that is configured to prevent undesired dissemination of the gene therapy composition to non-target locations, optionally wherein the dissemination-blocking material comprises a viscous substance and / or a polymer.

[0178] 16. The method of any one of the preceding paragraphs, further comprising positioning a blocking element in the subject, wherein the blocking element is configured to prevent undesired dissemination of the gene therapy composition to non-target locations.

[0179] 17. The method of any one of the preceding paragraphs, wherein the serum lipase level in the subject is less than 5 U / L at day 1 through day 7 post delivery of the gene therapy composition.

[0180] 18. The method of any one of the preceding paragraphs, wherein fewer than 1 vector copy per diploid genome of the AAV vector genome is detectable in the liver, heart, spleen, or kidney of the subject at 3-4 weeks post-delivery of the gene therapy composition.

[0181] 19. The method of any one of the preceding paragraphs, wherein more than 1, more than 2 or more than 3 vector copies per diploid genome per diploid genome of the AAV vector genome are detectable in the liver, heart, spleen, or kidney of the subject at 3-4 weeks post-delivery of the gene therapy composition. 20. The method of any one of the preceding paragraphs, wherein about 1 to about 5 vector copies per diploid genome of the AAV vector genome are detectable in the liver, heart, spleen, or kidney of the subject at 3-4 weeks post-delivery of the gene therapy composition.

[0182] 21. The method of any one of the preceding paragraphs, wherein the human GLP-1 receptor agonist is not detectable in the brain and / or sera of the subject.

[0183] 22. The method of any one of the preceding paragraphs, wherein the effective amount restores glycemic durability in the subject.

[0184] 23. The method of any one of the preceding paragraphs, wherein the effective amount reduces fasting blood glucose by at least 50% relative to baseline.

[0185] 24. The method of any one of the preceding paragraphs, wherein the effective amount increases fasting insulin by at least 2-fold relative to baseline.

[0186] 25. The method of any one of the preceding paragraphs, wherein the effective amount significantly improves glucose tolerance relative to baseline.

[0187] 26. The method of any one of the preceding paragraphs, wherein the effective amount significantly improves glucose- stimulated insulin secretion relative to baseline.

[0188] 27. The method of any one of the preceding paragraphs, wherein the body weight of the subject does not change significantly relative to baseline.

[0189] 28. The method of any one of the preceding paragraphs, wherein the single dose is sufficient for long-term restoration of pancreatic islet beta function.

[0190] 29. The method of any one of the preceding paragraphs, wherein the single dose is sufficient for reduction in therapeutic burden.

[0191] 30. The method of any one of the preceding paragraphs, wherein at least 15% of the endocrine tissue is transduced with the AAV vector.

[0192] 31. A method for treating obesity in a subject in need thereof, comprising: delivering an effective amount of a gene therapy composition to endocrine tissue of the tail and / or body of the pancreas of the 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, optionally wherein the effective amount is a single dose of about 5xl012to about 1.5xl014vector genomes (VG).

[0193] 32. The method of paragraph 31, wherein the effective amount is a single dose of about IxlO13to about 5xl013VG.

[0194] 33. The method of paragraph 31 or 32, wherein the total volume of the single dose is no more than 1 ml. 34. The method of any one of paragraphs 31-33, wherein the total volume of the single dose is about 1 ml to about 5 ml.

[0195] 35. The method of any one of the preceding paragraphs, wherein the single dose is delivered via a single infusion.

[0196] 36. The method of any one of the preceding paragraphs, wherein the single dose is delivered via multiple infusions.

[0197] 37. The method of any one of the preceding paragraphs, wherein the AAV vector genome comprises a human pancreatic islet beta cell-specific promoter operably linked to a human GLP- 1 receptor agonist coding sequence.

[0198] 38. The method of any one of the preceding paragraphs, wherein the EUS-FNI procedure comprises advancing a depositing device comprising a fine needle at a distal portion of the depositing device to a pancreatic deposit site in the pancreas, and delivering the gene therapy composition through the fine needle into the pancreatic deposit site.

[0199] 39. The method of any one of the preceding paragraphs, wherein the distal end of the depositing device is advanced into the mouth of the subject and through a wall of the gastrointestinal tract to a location proximate the pancreas, optionally wherein (a) the depositing device is delivered through a working channel of a gastrointestinal endoscope that has been delivered through the mouth of the subject of (b) the depositing device is delivered alongside a gastrointestinal endoscope that has been delivered through the mouth of the subject.

[0200] 40. The method of any one of the preceding paragraphs, wherein the pancreatic deposit site is the parenchyma of the tail and / or head of the pancreas.

[0201] 41. The method of any one of the preceding paragraphs, wherein the delivering the gene therapy composition through the fine needle into the pancreatic deposit site is performed at a pressure of at least 3 mmHg and / or no more than 25mmHg.

[0202] 42. The method of any one of the preceding paragraphs, wherein the delivering the gene therapy composition through the fine needle into the pancreatic deposit site is performed at a flow rate of at least Iml / min and / or no more than 5ml / min.

[0203] 43. The method of any one of the preceding paragraphs, further comprising delivering a permeability-enhancing agent prior to the delivering of the gene therapy composition and / or simultaneously with the delivering of the gene therapy composition, optionally wherein the delivering of the permeability-enhancing agent is performed locally and / or intravenously, optionally wherein the permeability-enhancing agent comprises an agent selected from the group consisting of: hyaluronidase; collagenase; losartan; and combinations thereof, and optionally wherein the treatment agent comprises a coformulation of the gene therapy composition and the permeability-enhancing agent.

[0204] 44. The method of any one of paragraphs 38-43, further comprising heating tissue proximate the pancreatic deposit site to a temperature above 39°C prior to, during, and / or after the delivery of the gene therapy composition.

[0205] 45. The method of any one of the preceding paragraphs, further comprising delivering a dissemination-blocking material that is configured to prevent undesired dissemination of the gene therapy composition to non-target locations, optionally wherein the dissemination-blocking material comprises a viscous substance and / or a polymer.

[0206] 46. The method of any one of the preceding paragraphs, further comprising positioning a blocking element in the subject, wherein the blocking element is configured to prevent undesired dissemination of the gene therapy composition to non-target locations.

[0207] 47. The method of any one of the preceding paragraphs, wherein the serum lipase level in the subject is less than 5 U / L at day 1 through day 7 post delivery of the gene therapy composition.

[0208] 48. The method of any one of the preceding paragraphs, wherein fewer than 1 vector copy per diploid genome of the AAV vector genome is detectable in the liver, heart, spleen, or kidney of the subject at 3-4 weeks post-delivery of the gene therapy composition.

[0209] 49. The method of any one of the preceding paragraphs, wherein more than 1, more than 2 or more than 3 vector copies per diploid genome per diploid genome of the AAV vector genome are detectable in the liver, heart, spleen, or kidney of the subject at 3-4 weeks post-delivery of the gene therapy composition.

[0210] 50. The method of any one of the preceding paragraphs, wherein about 1 to about 5 vector copies per diploid genome of the AAV vector genome are detectable in the liver, heart, spleen, or kidney of the subject at 3-4 weeks post-delivery of the gene therapy composition.

[0211] 51. The method of any one of the preceding paragraphs, wherein the human GLP-1 receptor agonist is not detectable in the brain and / or sera of the subject; or wherein the human GLP-1 receptor agonist is present in the pancreas at a level that is at least 50% higher than the level detected in the brain and / or sera of the subject.

[0212] 52. The method of any one of the preceding paragraphs, wherein

[0213] (a) the body weight of the subject decreases by at least 5%, at least 10%, at least 15%, at least 20%, or at least 25% relative to baseline; or wherein the body weight of the subject decreases by about 5-25% relative to baseline; or (b) the weight gained by the subject decreases by at least 5%, at least 10%, at least 15%, at least 20%, or at least 25% relative to control; or wherein the weight gained by the subject is decrease by about 5-25%.

[0214] 53. The method of any one of the preceding paragraphs, wherein the effective amount restores glycemic durability in the subject.

[0215] 54. The method of any one of the preceding paragraphs, wherein the effective amount reduces fasting blood glucose by at least 50% relative to baseline.

[0216] 55. The method of any one of the preceding paragraphs, wherein the effective amount increases fasting insulin by at least 2-fold relative to baseline.

[0217] 56. The method of any one of the preceding paragraphs, wherein the effective amount significantly improves glucose tolerance relative to baseline.

[0218] 57. The method of any one of the preceding paragraphs, wherein the effective amount significantly improves glucose- stimulated insulin secretion relative to baseline.

[0219] 58. The method of any one of the preceding paragraphs, wherein the single dose is sufficient for long-term restoration of pancreatic islet beta function.

[0220] 59. The method of any one of the preceding paragraphs, wherein the single dose is sufficient for reduction in therapeutic burden.

[0221] 60. The method of any one of the preceding paragraphs, wherein at least 15% of the endocrine tissue is transduced with the AAV vector.

[0222] 61. The method of any one of the preceding paragraphs, wherein the subject has a body mass index (BMI) of 25.0 to <30.

[0223] 62. The method of any one of the preceding paragraphs, wherein the subject has a BMI of 30.0 or higher, optionally a BMI of 30 to <35 (Class 1), 35 to <40 (Class 2), or 40 or higher (Class 3).

[0224] 63. An adeno-associated virus (AAV) vector genome of any one of the preceding paragraphs comprising: a 5’ inverted terminal repeat (ITR) sequence, an insulin gene promoter and an enhancer element operably linked to a nucleic acid comprising a 5’ untranslated region (UTR), an open reading frame encoding a GLP-1 receptor agonist, a 3 ’UTR, poly adenylation signal, and a 3 TR.

[0225] 64. The AAV vector genome of paragraph 63, wherein the insulin gene promoter is a human insulin gene promoter.

[0226] 65. The AAV vector genome of paragraph 63 or 64, wherein the insulin gene promoter is a rat insulin gene promoter. 66. The AAV vector genome of any one of paragraphs 63-65, wherein the enhancer element is a cytomegalovirus enhancer element

[0227] 67. The AAV vector genome of any one of paragraphs 63-66, wherein the 5’ UTR comprises a modified human hemoglobin subunit beta intron.

[0228] 68. The AAV vector genome of any one of paragraphs 63-67, wherein the GLP-1 receptor agonist is human GLP-1

[0229] 69. The AAV vector genome of any one of paragraphs 63-68, wherein the GLP-1 receptor agonist is fused to a signal peptide

[0230] 70. The AAV vector genome of any one of paragraphs 63-69, wherein the 3’ UTR comprises a modified Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) element (e.g., mut6.WPRE).

[0231] 71. The AAV vector genome of any one of paragraphs 63-70, wherein the polyadenylation signal is a bovine growth hormone polyadenylation signal

[0232] 72. The AAV vector genome of any one of paragraphs 63-71, wherein the AAV vector genome is a single- stranded AAV vector genome.

[0233] 73. A method for reducing low density lipoprotein (LDL) and / or total cholesterol in a subject, the method comprising: delivering a single dose of a gene therapy composition to pancreatic endocrine tissue of a subject, in an amount effective to maintain a reduction in body weight of about 5% over the course of a year, wherein the gene therapy composition comprises an adeno-associated virus (AAV) vector genome comprising a pancreatic islet beta cell-specific promoter operably linked to a GLP-1 receptor agonist coding region.

[0234] 74. A method for reducing low density lipoprotein (LDL) and / or total cholesterol in a subject, the method comprising: delivering no more than two doses of a gene therapy composition to pancreatic endocrine tissue of a subject, in an amount effective to maintain a reduction in body weight of about 5% over the course of a year, wherein the gene therapy composition comprises an adeno-associated virus (AAV) vector genome comprising a pancreatic islet beta cell- specific promoter operably linked to a GLP-1 receptor agonist coding region.

[0235] 75. The method of paragraph 73 or 74, wherein the single dose comprises about 5xl012to about 1.5xl014AAV vector genomes (VG).

[0236] 76. The method of any one of paragraphs 73-75, wherein the single dose comprises about IxlO13to about 5xl013AAV vector genomes (VG). 77. The method of any one of paragraphs 73-76, wherein the total volume of the single dose is about 1 ml to about 3 ml.

[0237] 78. The method of any one of paragraphs 73-77, wherein the gene therapy composition is delivered via infusion.

[0238] 79. The method of any one of paragraphs 73-78, wherein the single dose is delivered using an endoscopic ultrasound-guided fine needle injection (EUS-FNI) procedure.

[0239] 80. The method of any one of paragraphs 73-79, wherein the pancreatic islet beta cellspecific promoter comprises a human insulin promoter or a region thereof.

[0240] 81. The method of any one of paragraphs 73-80, wherein at least 15% of the endocrine tissue is transduced with the AAV vector.

[0241] 82. The method of any one of paragraphs 73-81, wherein the AAV vector genome comprises: a 5’ inverted terminal repeat (ITR) sequence, an insulin gene promoter and an enhancer element operably linked to a nucleic acid comprising a 5’ untranslated region (UTR), an open reading frame encoding a GLP-1 receptor agonist, a 3 ’UTR, a poly adenylation signal, and a 3 TR.

[0242] 83. The method of paragraph 81 or 82, wherein the insulin gene promoter is a human insulin gene promoter or core region thereof.

[0243] 84. The method of paragraph 82 or 83, wherein: the enhancer element is a cytomegalovirus enhancer element, optionally a CMV upstream genomic region (CMVugr); the 5’ UTR comprises a modified human hemoglobin subunit beta intron; the GLP-1 receptor agonist is human GLP-1; the GLP-1 receptor agonist is fused to a signal peptide; the 3’ UTR comprises a modified Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) element, optionally mut6.WPRE; and the polyadenylation signal is a bovine growth hormone polyadenylation signal.

[0244] 85. The method of any one of paragraphs 73-84, wherein the AAV vector genome is a singlestranded AAV vector genome, optionally a self-complementary AAV vector genome.

[0245] EXAMPLES

[0246] Example 1 - In vivo Experiments in Type 2 Diabetes Mouse Model

[0247] An AAV-based gene therapy candidate was tested in a db / db Type 2 Diabetes mouse model to determine its impact on disease progression and severity. Lour- week-old db / db male mice were separated into different treatment groups (n=8 / group). On day 1, mice were treated with their respect AAV compositions (vehicle control, MIP-eGFP (10el2 VG / animal), MIP-Ex4 (2.5el2 VG / animal), or MIP-Ex4 (10el2 VG / animal)). Note that “Ex4” refers to “Exendin-4,” a glucagon-like peptide 1 (GLP-1) receptor agonist and “MIP” refers to a mouse insulin promoter. On days -1, 8, 15, 22, 29, 36, 43, 50, 57, and 64, mice underwent a 4 hour fasting period and blood glucose was measured. On days -1, 22, 36, 50, and 64, insulin levels were also measured following the fasting period. On day 39, intraperitoneal glucose tolerance tests (IPGTT) were administered and tracked over 120 minutes (0 minutes, 15 minutes, 30 minutes, 60 minutes, and 120 minutes). On day 70, tissue was collected for analysis.

[0248] Dose-dependent and durable glycemic control in db / db mice for 10-weeks post-injection was observed. High-dose MIP-Ex4-treated mice exhibited a 59% reduction [A 304mg / dL] in fasting blood glucose (p < 0.0001) (FIG. 1A) and a 2.8-fold increase in fasting insulin (p = 0.004) (FIG. IB). A significant improvement in both glucose tolerance (p < 0.0001) (FIGs. 2A and 2B) and glucose-stimulated insulin secretion (p < 0.05) via intraperitoneal glucose tolerance test (IPGTT) (FIG. 2C) was observed, with no effect on body weight (FIGs. 3A-3B). Immunohistochemical analysis showed that the GLP-1RA protein was expressed in the pancreas (FIG. 4C) and was restricted to the islet cells (FIGs. 4A and 4B).

[0249] Example 2 - Ex vivo Experiments in Mouse and Human Cells

[0250] BKS db / db islet cells were isolated and cultured ex vivo and then transduced with an AAV-based GLP-1RA construct comprising Exendin-4 to examine its impact on insulin secretion. Four days after transduction, insulin secretion was measured. In islet cells transduced with the AAV-GLP-1RA construct, there was significantly more GLP-1 (FIG. 5A) and glucose- stimulated insulin secretion (FIG. 5B) compared to the islet cells transduced with AAV-eGFP (control).

[0251] When a human pancreatic islet beta line, EndoC-BH5, was used, AAV-mediated delivery of GLP-1RA was found to enhance insulin secretion in a GLP- IRA-dependent manner (FIG. 6). In particular, in the presence of glucose, there was a significant difference between the amount of insulin secreted by cells transduced with AAV-GLP-1RA compared to the control (AAV-eGFP). When cells were exposed to both glucose and exendin-9 (Ex9) peptide, a GLP- IRA antagonist, there was no statistically significant difference between groups.

[0252] Example 3 - In vivo Locational Studies

[0253] BKS db / db mice were administered an AAV expressing a pancreatic islet beta cell- restricted exendin-4 transgene, AAV-MIP-Ex4 (7.5el2 VG / animal), or a vehicle control (n = 3 / group) Note that “Ex4” refers to “exendin-4,” a glucagon-like peptide 1 (GLP-1) receptor agonist and “MIP” refers to a mouse insulin promoter. Four weeks later, fasting blood glucose was measured (after 4-6 hours of fasting), and the results are shown in FIG. 7A. Significant reductions were observed in mice administered AAV-MIP-Ex4, as compared to the vehicle. In addition, levels of Exendin-4 were measured in the serum and pancreas of each mouse, and the results are shown in FIG. 7B. AAV-based Exendin-4 production was not detectable in the sera of either group of animals; however, high levels of Exendin-4 were detected in the pancreas, as measured via lipid chromatograph mass spectrometry (LCMS), indicated a targeted local delivery of AAV-MIP-Ex4.

[0254] Example 4 - In vivo Experiments in Yucatan Pig Model

[0255] Three to four weeks after EUS-FNI injection of scAAV9-CMV-GFP into the pancreas of Yucatan pigs, animals were sacrificed and 8mm biopsies collected. Sixteen to twenty biopsies were analyzed from each pig, distributed throughout the targeted splenic lobe as well as in the duodenal and connecting lobes. GFP protein expression was detected via immunohistochemical (IHC) analysis of biopsies, and endocrine area was determined via endogenous insulin protein IHC on an adjacent tissue section. Exocrine signal was determined by percent GFP signal in the entire biopsy excluding all endocrine regions defined by insulin IHC, while endocrine signal was determined by percent GFP signal across 5-10 islets per biopsy using MatLab image analysis tools. Each dot in the plot represents an individual pig, averaging either the exocrine or endocrine signal across all biopsies either taken from the splenic lobe or the entire pancreas. For each vector genome dose of AAV9, group sizes were between 2-4 animals. A significant dose dependent increase in AAV-mediated GFP expression was observed in the endocrine and exocrine tissue after EUS-FNI of scAAV9-CMV-GFP into the pancreas of the Yucatan pigs. See FIGs. 8A-8D and FIG. 9.

[0256] Example 5 - In Vivo Vector Biodistribution Analysis

[0257] A vector biodistribution analysis was conducted on tissues collected 3-4 weeks after EUS-FNI injection of AAV into in the pig pancreas, either via single infusion or three infusions using a fixed dose of 5xl013VG (FIG. 10A) or 1.5xl014VG (FIG. 10B). Vector copies per diploid genome was determined by digital PCR specific for the DNA transgene sequence in the AAV genome (a) in 7 pigs either via 1 (n = 2) or 3 infusions (n = 5) for the lower dose, or (b) in 4 pigs either via 1 (n = 2) or 3 infusions (n = 2) for the higher dose. Signal in the pancreas splenic lobe (SL) is an average of 14-16 biopsies per pig, in pancreas duodenal lobe (DL) is an average of 2 biopsies per pig, in the pancreas connecting lobe is an average of 2-4 biopsies per pig, and other tissues are between 1 to 4 biopsies per pig.

[0258] Example 6 - In Vivo Toxicity Studies

[0259] Lipase

[0260] Blood was collected at 0, 4, 24, 72, and 168 hours after single or triple EUS-FNI of AAV or vehicle into the pancreas of Yucatan pigs, and serum analyzed for lipase. No elevation in lipase was observed across all single infusion procedures, while a subset of animals injected at 3 locations demonstrated transient elevations of serum lipase, below the threshold for acute pancreatitis (FIG. 11A). In all cases, lipase levels returned to baseline by 3-7 days after the procedure.

[0261] Single EUS-FNI procedures were used to deliver between IxlO13to 5xl013VG AAV doses to the pig pancreas using a volume of 1ml, 2ml, or 5ml at a 0.9 to 1.0 mL / minute flow rate. Serum lipase levels were not elevated above baseline in any pig tested (FIG. 11B).

[0262] EUS-FNI procedures were used to deliver between 5xl012to 1.5xl014VG AAV doses to the pig pancreas using either 1 infusion (FIG. 11C) or 3 infusions (FIG. 11D) of various AAV serotypes. AAV dose did not correlate with elevations in serum lipase using either a single infusion or 3 infusions.

[0263] Neurofilament Light Chain (NFL)

[0264] Single EUS-FNI procedures were used to deliver IxlO13VG or 5xl013VG of AAV9- CMV-eGFP (ubiquitous CMV promoter) or AAV9-INSp-eGFP (beta-cell restricted INSp promoter) to the pig pancreas. NFL levels, indicative of dorsal root ganglion (DRG) toxicity, were not elevated following administration of the beta-cell restricted promoter (FIG. 12).

[0265] Example 7 - In vivo Body Weight, Fasting Blood Glucose and Fasting Insulin Studies

[0266] Two AAV-based GLP1RA vectors (1stGen and 2ndGen) with a P-cell-restricted promoter (AAV-GLP1RA) were chosen for analysis in db / db mice, which are susceptible to obesity due to chronic hyperphagia. At 4 weeks, following a single intraperitoneal injection of a 1012(1stGen) or 512(2ndGen) VG dose, body weight gain was significantly reduced by 23% in AAV-GLP1RA treated mice compared to vehicle treated controls (p<0.0001) (vehicle control) and by 19.6% compared to Semaglutide (10 nmol / kg) treated controls (FIG. 13). At 8 weeks, following a 4-6 hour fast, the gene therapy improved fasting glucose and insulin (FIGs. 14A- 14B). Despite lower fasting insulin using the 2ndgeneration vector, the enhanced improvement in blood glucose suggests greater insulin sensitivity, relative to the 1stgeneration vector. The GLP- 1 gene therapy also shifts progression of disease (FIG. 15).

[0267] In a further study, 8 week-old db / db mice were administered a single intraperitoneal injection of GLP-1RA PGTx(5el2 VG) or AAV vehicle or received daily subcutaneous injections of semaglutide (10 nmol / kg) for 62 days. Samples were taken on days 8, 15, 22, 29, 36, 43, 50, and 57 and fasting blood glucose, insulin, and weight were determined. At the end of the protocol (day 62), organ histology, pancreatic GLP-1RA protein, and serum GLP-1RA protein were measured. The results are shown in FIGs. 22A-22C and demonstrate that administration of the GLP-1RA PGTx treatment improves fasting blood glucose (FIG. 22A), fasting plasma insulin (FIG. 22B), and lowers total body weight (FIG. 22C) relative to both the vehicle control and daily administration of semaglutide.

[0268] The 1st Gen AAV vector genome is self-complementary and drives the expression of Exendin-4 (Ex4) under a mouse insulin gene promoter with a Cytomegalovirus enhancer element (CMVe). The Exendin-4 transgene is generated as a mouse nerve growth factor (mNGF) propeptide fragment and Exendin-4 fusion protein (mNGF-Ex4) that contains a signal peptide for protein translocation and secretion. The mNGF fragment is then cleaved off, and the Ex4 molecule is available to activate GLP-1R. The 1st Gen AAV vector genome also contains a bovine growth hormone poly adenylation signal (bGHpA) for RNA stability.

[0269] The 2nd Gen AAV vector genome is single- stranded and drives the expression of Ex4 under a rat Insulin- 1 gene promoter (rINSIp) with a CMV upstream genomic region (CMVugr). The Exendin-4 transgene is generated as an mNGF-Ex4 fusion protein that contains a signal peptide for protein translocation and secretion. The mNGF fragment is then cleaved off, and the Ex4 molecule is available to activate GLP-1R. The 2nd Gen AAV vector genome also contains a modified human hemoglobin subunit beta intron in the 5’ untranslated region (UTR) and a modified Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) element in the 3’ UTR to boost transgene expression, as well as a bovine growth hormone poly adenylation signal (bGHpA) for RNA stability.

[0270] Example 8 - Single Dose GLP-1 Based Pancreatic Gene Therapy Induces Durable Weight Loss in a Murine Model of Obesity

[0271] The efficacy and durability of a single dose GLP-1 -based pancreatic gene therapy (PGTx, i.e., 2nd Gen AAV vector genome described in Example 7) was tested in a murine diet-induced obesity model. Briefly, C57BL / 6 mice were fed a 60% high-fat diet for 25 weeks and then randomized by body weight into four groups: (1) control Group 1 received a single intraperitoneal dose of PGTx vehicle only (n=8) (“AAV vehicle”); (2) test Group 2 received a single intraperitoneal dose of PGTx (IxlO13VG, n=10, “pss.AAV044 [lel3 VG]”); (3) test Group 3 received a daily subcutaneous dose of semaglutide for 28 days (10 nmol / kg daily, n=5); on Day 29 semaglutide was withdrawn and PGTx vehicle only was administered (“Serna w / draw + Vehicle); and (4) test Group 4 received a daily subcutaneous dose of semaglutide for 28 days (10 nmol / kg daily, n=5); on Day 29 semaglutide was withdrawn and a single intraperitoneal dose of PGTx (5xl012VG, n=5) was administered (“Serna w / draw + pssAAV.004 [5el2 VG]”). Mean body weight and food intake were measured daily for 57 days. Fasting mean terminal liver weight, liver triglycerides, plasma leptin (as an indicator of adiposity), fat mass (by EchoMRI), fasting glucose, islet and serum GLP-1 (Exendin-4) protein levels, and 0-5-point islet histopathology scores were also assessed.

[0272] Body weight changes are shown in FIG. 16A, and final body weights are shown in FIG. 18A. At Day 28, body weight was reduced by 27% in test Group 2 (single dose PGTx) and by 20-21% in test Groups 3 and 4 (daily semaglutide) (p<0.05). Surprisingly, body weight loss was maintained for at least 57 days in test Group 2, with only a single dose of PGTx (p<0.0001). The results were even more surprising with test Groups 3 and 4. Semaglutide withdrawal in Group 3, without subsequent PGTx treatment, resulted in body weight gain to nearly baseline (-2% from baseline). By comparison, semaglutide withdrawal in Group 4, followed by a single dose of PGTx on Day 29 (¥2 of the PGTx dose used in Group 2), resulted in stabilization of body weight loss at -22% below baseline at Day 57 (p<0.01), as well as a significant increase in lean mass relative to the control groups, as measured by percent body weight (FIG. 18D). Mean food intake paralleled body weight loss or gain in all treatment groups (FIG. 16B), as did fat mass (FIG. 18B).

[0273] Liver weight and liver triglyceride levels are shown in FIG. 17. At the end of the study (Day 57), treatment with a single dose of PGTx reduced mean liver weight by 42% (p<0.01) and liver triglycerides by 67% (p<0.0001) compared to the vehicle control. The semaglutide withdrawal group was similarly consistent with the vehicle control.

[0274] At week 8, leptin levels were reduced by 67% with PGTx-Ex4 compared to control (p<0.0001) (FIG. 18C), corresponding to a 35% drop in body fat (r=0.93, P<0.0001). Nine percent and 15% of islets expressed PGTx-Ex4 with low and high-dose gene therapy, respectively (FIG. 20), demonstrating that expression of PGTx-Ex4 was islet-restricted. Similarly, dose-responsive increases of 44% and 68% in serum PGTx-Ex4 levels vs. controls were observed (p<0.001 and p<0.0001, respectively) (FIG. 19B) and pancreas PGTx-Ex4 levels (FIG. 19A). Serum PGTx was correlated with percent islet expression (r=0.77, p<0.0001) and percent body weight loss (r=-0.75, p<0.0001). Fasting glucose was reduced by 21% (FIG. 23) and HOMA-IR (Homeostatic Model Assessment for Insulin Resistance) improved by 72% with PGTx-Ex4 compared to control (FIG. 24), respectively (p<0.001, p<0.01). No significant evidence of inflammation was observed, with histopathology scores of <0.5 in all groups (data not shown).

[0275] Plasma cholesterol was significantly reduced following treatment with the GLP-1RA PGTx relative to the respective controls (FIG. 21A), as were low-density lipoprotein (LDL) (FIG. 21B) and high-density lipoprotein (HDL) (FIG. 21C) levels. Triglyceride levels did not show any statistical significance between groups (FIG. 21D).

[0276] Thus, a single dose of GLP-1 -based pancreatic gene therapy can durably reduce body weight and stabilize a body weight reduction following semaglutide withdrawal without causing pancreatic inflammation, leading to improvements in liver weight and hepatic triglycerides. These data support GLP-1 -based pancreatic gene therapy as an effective and durable therapy for metabolic diseases (e.g., obesity and MASLD).

[0277] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0278] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0279] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0280] The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value.

[0281] Where a range of values is provided, each value between and including the upper and lower ends of the range are specifically contemplated and described herein.

Claims

CLAIMSWhat is claimed is:

1. A method for reducing body weight in a subject, the method comprising: delivering a single dose of a gene therapy composition to pancreatic endocrine tissue of a subject having a metabolic disease, in an amount effective to maintain a reduction in body weight of about 5% over the course of a year, wherein the gene therapy composition comprises an adeno-associated virus (AAV) vector genome comprising a pancreatic islet beta cell-specific promoter operably linked to a GLP-1 receptor agonist coding region.

2. A method for reducing body weight in a subject, the method comprising: delivering no more than two doses of a gene therapy composition to pancreatic endocrine tissue of a subject having a metabolic disease, in an amount effective to maintain a reduction in body weight of about 5% over the course of a year, wherein the gene therapy composition comprises an adeno-associated virus (AAV) vector genome comprising a pancreatic islet beta cell-specific promoter operably linked to a GLP-1 receptor agonist coding region.

3. The method of claim 1 or 2, wherein the single dose comprises about 5xl012to about 1.5xl014AAV vector genomes (VG).

4. The method of any one of the preceding claims, wherein the single dose comprises about IxlO13to about 5xl013AAV vector genomes (VG).

5. The method of any one of the preceding claims, wherein the total volume of the single dose is about 1 ml to about 3 ml.

6. The method of any one of the preceding claims, wherein the gene therapy composition is delivered via infusion.

7. The method of any one of the preceding claims, wherein the single dose is delivered using an endoscopic ultrasound-guided fine needle injection (EUS-FNI) procedure.

8. The method of any one of the preceding claims, wherein the pancreatic islet beta cellspecific promoter comprises a human insulin promoter or a region thereof.

9. The method of any one of the preceding claims, wherein at least 15% of the endocrine tissue is transduced with the AAV vector.

10. The method of any one of the preceding claims, wherein the effective amount: restores glycemic durability in the subject; significantly reduces fasting blood glucose relative to baseline; significantly increases fasting insulin relative to baseline; significantly improves glucose tolerance relative to baseline; and / or significantly improves glucose- stimulated insulin secretion relative to baseline.

11. The method of any one of the preceding claims, wherein the single dose is sufficient for long-term restoration of pancreatic islet beta cell function and / or reduction in therapeutic burden.

12. The method of any one of the preceding claims, wherein the serum lipase level in the 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.

13. The method of any one of the preceding claims, wherein the human GLP-1 receptor agonist is present in the pancreas at a level that is at least 50% higher than the level detected in the brain and / or sera of the subject.

14. The method of any one of the preceding claims, wherein fewer than 1 vector copy per diploid genome of the AAV vector genome is detectable in the liver, heart, spleen, or kidney of the subject at 3-4 weeks post-delivery of the gene therapy composition.

15. The method of any one of the preceding claims, wherein about 1 to about 5 vector copies per diploid genome of the AAV vector genome are detectable in the liver, heart, spleen, or kidney of the subject at 3-4 weeks post-delivery of the gene therapy composition.

16. The method of any one of the preceding claims, wherein the effective amount significantly reduces the subject’s liver weight and / or liver triglycerides relative to baseline.

17. The method of any one of the preceding claims, wherein the effective amount(a) significantly reduces the subject’s body fat mass relative to baseline, and / or(b) significantly increases the subject’s lean body mass relative to baseline.

18. The method of any one of the preceding claims, wherein the effective amount significantly reduces the subject’s plasma leptin level relative to baseline.

19. The method of any one of the preceding claims, wherein the effective amount significantly reduces the subject’s total cholesterol relative to baseline.

20. The method of claim 19, wherein the effective amount significantly decreases the subject’s low density lipoprotein (LDL) level relative to baseline.

21. The method of any one of the preceding claims, wherein the metabolic disease 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.

22. The method of claim 21, wherein the metabolic disease is Type 2 Diabetes.

23. The method of claim 21, wherein the metabolic disease is obesity.

24. The method of claim 21, wherein the metabolic disease is Metabolic Dysfunction- associated Steatotic Liver Disease.

25. The method of any one of the preceding claims, wherein the subject has a body mass index (BMI) of 25.0 to <30.

26. The method of any one of the preceding claims, wherein the subject has a BMI of 30.0 or higher, optionally a BMI of 30 to <35 (Class 1), 35 to <40 (Class 2), or 40 or higher (Class 3).

27. The method of any one of the preceding claims, wherein the subject has received and then discontinued another weight loss therapy within 3, 6, 9, 12 or 18 months of the delivering of the gene therapy composition, optionally wherein the subject had reduced weight while receiving the other weight loss therapy.

28. The method of any one of the preceding claims, wherein the AAV vector genome comprises: a 5’ inverted terminal repeat (ITR) sequence, an insulin gene promoter and an enhancer element operably linked to a nucleic acid comprising a 5’ untranslated region (UTR), an open reading frame encoding a GLP-1 receptor agonist, a 3 ’UTR, a poly adenylation signal, and a 3 TR.

29. The method of claim 28, wherein the insulin gene promoter is a human insulin gene promoter or core region thereof.

30. The method of claim 28 or 29, wherein: the enhancer element is a cytomegalovirus enhancer element, optionally a CMV upstream genomic region (CMVugr); the 5’ UTR comprises a modified human hemoglobin subunit beta intron; the GLP-1 receptor agonist is human GLP-1; the GLP-1 receptor agonist is fused to a signal peptide; the 3’ UTR comprises a modified Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) element, optionally mut6.WPRE; and the polyadenylation signal is a bovine growth hormone polyadenylation signal.

31. The method of any one of the preceding claims, wherein the AAV vector genome is a single- stranded AAV vector genome, optionally a self-complementary AAV vector genome.

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