Gene therapy for metabolic disorders

Abstract

This disclosure provides methods and compositions for treating metabolic disorders, including type 2 diabetes, obesity, metabolic dysfunction-related fatty liver disease, and other obesity-related metabolic disorders, by combining local delivery of gene therapy to pancreatic tissue. In some embodiments, the gene therapy composition is delivered in an amount effective to maintain approximately 5% weight loss over a year. In some embodiments, the gene therapy composition comprises an adeno-associated virus (AAV) vector genome containing a pancreatic islet beta cell-specific promoter operably linked to a GLP-1 receptor agonist coding region.

Description

[Technical Field]

[0001] Related applications This application claims the benefits under Section 119(e) of U.S. Patent Act, as stipulated in U.S. Provisional Application No. 63 / 508,251 filed on 14 June 2023, U.S. Provisional Application No. 63 / 594626 filed on 31 October 2023, and U.S. Provisional Application No. 63 / 603,577 filed on 28 November 2023, each of which is incorporated herein by reference in whole.

[0002] Reference to electronic sequence listings The contents of the electronic sequence listing (F085770001WO00-SEQ-JXV.xml, size: 63,088 bytes, and creation date: June 12, 2024) are incorporated in their entirety hereby by reference. [Background technology]

[0003] Obesity, often associated with or causing a variety of metabolic disorders, is a globally widespread problem, reaching an estimated 650 million adults and over 340 million children and adolescents worldwide. This condition is associated with numerous serious health conditions, including but not limited to heart disease, diabetes, and certain types of cancer. Current treatments primarily involve lifestyle modifications such as dietary changes and increased physical activity, pharmacological interventions, or, in severe cases, bariatric surgery. While these treatments can be effective, they often come with significant side effects and, in some cases, their effectiveness diminishes over time due to factors such as drug tolerance or patients reverting to previously unhealthy behaviors. Most currently available therapies aim to treat metabolic disorders by managing symptoms rather than addressing the underlying causes. There is a keen need for innovative, effective, and safer treatment strategies for obesity and other metabolic disorders that can overcome current challenges and provide sustainable and accessible care for those suffering from these debilitating conditions.

[0004] Type 2 diabetes is a form of diabetes characterized by high blood glucose levels due to the body's inability to effectively use or produce sufficient insulin. The pancreas is an endocrine gland that produces insulin and other hormones that regulate blood glucose levels. Insulin helps transport glucose from the bloodstream to cells, where it can be used for energy. In type 2 diabetes, the body develops resistance to the effects of insulin, meaning it can no longer effectively use insulin to lower blood glucose levels. This can lead to high blood glucose levels, which, if left untreated, can cause a variety of health problems. Type 2 diabetes, along with other obesity-related metabolic disorders, accounts for significant morbidity and mortality worldwide. Of the estimated 27 million people diagnosed with type 2 diabetes in the United States, approximately 50% are poorly controlled despite the availability of more than 60 approved medications, and it is projected that by 2035, an estimated 50 million people in the United States will be living with type 2 diabetes. [Overview of the Initiative]

[0005] Some aspects of this disclosure relate to methods for treating metabolic disorders by directly delivering gene therapy to pancreatic tissue. Other aspects of this disclosure relate to methods for reducing body weight in subjects, for example, subjects with metabolic diseases. The methods may include, for example, delivering a single dose or two or fewer doses of a gene therapy composition to the pancreatic endocrine tissue of a subject with a metabolic disease. In some embodiments, the gene therapy composition is delivered in an amount effective to maintain a body weight loss of about 5% over one year. In some embodiments, the gene therapy composition comprises an adeno-associated virus (AAV) vector genome containing a pancreatic islet beta cell-specific promoter operably linked to a GLP-1 receptor agonist coding region.

[0006] In some embodiments, the subject's body weight is maintained at a reduction of at least 20% on day 40 after delivery of a single dose of the gene therapy composition.

[0007] In some embodiments, the single dose is approximately 5 × 10 12 ~Approx. 1.5×10 14 AAV vector genome (VG), for example, approximately 1 × 10⁻⁶ 13 ~Approx. 5×10 13 It contains AAV VG. In some embodiments, the single dose is about 1 × 10 13 Includes AAV VG.

[0008] In some embodiments, the total volume of a single dose is approximately 1 ml to 3 ml.

[0009] In some embodiments, the gene therapy composition is delivered via infusion (e.g., single infusion).

[0010] In some embodiments, a single dose is delivered using an endoscopic ultrasound-guided fine-needle infusion (EUS-FNI) procedure.

[0011] In some embodiments, the islet beta cell-specific promoter includes the human insulin promoter or a region thereof (e.g., the core region).

[0012] In some embodiments, at least 15% of the endocrine tissue is transduced with an AAV vector.

[0013] In some embodiments, the effective dose restores glucose persistence in the subject, significantly reduces fasting blood glucose compared to baseline, significantly increases fasting insulin compared to baseline, significantly improves glucose tolerance compared to baseline, and / or significantly improves glucose-stimulated insulin secretion compared to baseline.

[0014] In some embodiments, a single dose is sufficient for long-term restoration of islet beta cell function and / or reduction of the treatment burden.

[0015] In some embodiments, the serum lipase level in the subject is within three times the upper limit of the normal serum lipase level on days 1 to 7 after delivery of the gene therapy composition.

[0016] In other embodiments, the human GLP-1 receptor agonist is present in the pancreas at levels at least 50% higher than those detected in the target brain and / or serum after delivery.

[0017] In some embodiments, fewer than one vector copy per diploid genome of the AAV vector genome can be detected in the target liver, heart, spleen, or kidney after delivery of the gene therapy composition, for example, 3 to 4 weeks after delivery.

[0018] In some embodiments, fewer than one to about five vector copies per diploid genome of the AAV vector genome are detectable in the target liver, heart, spleen, or kidney after delivery of the gene therapy composition, for example, 3 to 4 weeks after delivery.

[0019] In some embodiments, the effective dose significantly reduces the target liver weight and / or liver triglycerides compared to baseline.

[0020] In some embodiments, the effective dose significantly reduces the subject's body fat mass and / or significantly increases the subject's lean body mass compared to baseline.

[0021] In some embodiments, the effective dose significantly reduces the target plasma leptin level compared to baseline.

[0022] In some embodiments, the effective dose significantly reduces the subject's total cholesterol compared to baseline.

[0023] In some embodiments, the effective dose significantly reduces the target low-density lipoprotein (LDL) level compared to baseline.

[0024] In some embodiments, metabolic disorders are selected from obesity, diabetes mellitus, lipid metabolism disorders, congenital metabolic disorders, lysosomal storage diseases, glycogen storage diseases, mitochondrial diseases, purine-pyrimidine diseases, urea cycle disorders, fructose metabolism disorders, amino acid metabolism disorders, mineral metabolism disorders, porphyria, lactose intolerance and Wilson's disease, polycystic ovary syndrome, metabolic dysfunction-related fatty liver disease (non-alcoholic fatty liver disease), and non-alcoholic steatohepatitis.

[0025] In some embodiments, the metabolic disorder is type 2 diabetes. In some embodiments, the metabolic disorder is obesity.

[0026] In some embodiments, subjects have a body mass index (BMI) of 25.0 to less than 30. In other embodiments, subjects have a BMI of 30.0 or greater, and optionally have a BMI of 30 to less than 35 (Class 1), 35 to less than 40 (Class 2), or 40 or greater (Class 3).

[0027] In some embodiments, subjects receive and then discontinue another weight-loss therapy (e.g., semaglutide or dietary therapy) within 3, 6, 9, 12, or 18 months of delivery of the gene therapy composition, and optionally, subjects lose weight (decrease) while receiving the other weight-loss therapy.

[0028] In some embodiments, the AAV vector genome includes a 5' inverted terminal repeat (ITR) sequence, a 5' untranslated region (UTR), an open reading frame encoding a GLP-1 receptor agonist, an insulin gene promoter and enhancer element operably linked to a nucleic acid containing a 3' UTR, a polyadenylation signal, and a 3' ITR.

[0029] In some embodiments, the insulin gene promoter is the human insulin gene promoter or its core region.

[0030] In some embodiments, the enhancer element is a cytomegalovirus enhancer element, e.g., CMV upstream genome region (CMVugr), the 5'UTR contains 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 contains a modified woodchuck hepatitis virus post-transcriptional regulator (WPRE) element, optionally mut6.WPRE, and the polyadenylation signal is a bovine growth hormone polyadenylation signal.

[0031] In some embodiments, the AAV vector genome is a single-stranded AAV vector genome, for example, a self-complementary AAV vector genome. [Brief explanation of the drawing]

[0032] [Figure 1] This graph shows dose-dependent sustained and decreased blood glucose (A) and elevated fasting insulinemia (B) in db / db mice after AAV injection (day 0). Statistical analysis: **P, 0.01, ***P<0.001 for the MIP-eGFP 10e12 group compared to the vehicle; *P<0.05 compared to the vehicle only; one-way ANOVA, post-hoc Tukey test. [Figure 2A] Figures 2A, 2B, and 2C show the results of the intraperitoneal glucose tolerance test (IPGTT) 39 days after AAV injection in db / db mice. Statistical analysis: Figures 2A-2B use one-way ANOVA, post-hoc Tukey multiple comparison tests, *P<0.05, ****P<0.0001. [Figure 2B]Figures 2A, 2B, and 2C show the results of the intraperitoneal glucose tolerance test (IPGTT) 39 days after AAV injection in db / db mice. Statistical analysis: Figures 2A-2B use one-way ANOVA, post-hoc Tukey multiple comparison tests, *P<0.05, ****P<0.0001. [Figure 2C] Figure 2A shows the results of the intraperitoneal glucose tolerance test (IPGTT) 39 days after AAV injection in db / db mice, along with graphs showing the area under the curve (Figure 2B) and insulin secretion (Figure 2C). Statistics: Figure 2C uses a two-way mixed-effects model [REML] for analysis of variance, with a=P<0.005 for vehicle and b=P<0.05 for eGFP. [Figure 3A] These graphs show the absolute body weight (Figure 3A) and changes in body weight (Figure 3B) over time after AAV injection in db / db mice. [Figure 3B] These graphs show the absolute body weight (Figure 3A) and changes in body weight (Figure 3B) over time after AAV injection in db / db mice. [Figure 4A] The GLP-1RA protein (i.e., exendin-4) is expressed in the pancreas after AAV injection in db / db mice via immunohistochemical staining (Figure 4A), as well as the islet expression percentage (Figure 4B) and total pancreatic protein expression (Figure 4C) as determined by LCMS. [Figure 4B] Figure 4A shows the expression of GLP-1RA protein (i.e., exendin-4) in the pancreas after AAV injection in db / db mice via immunohistochemical staining, as well as the islet expression percentage (Figure 4B) and total pancreatic protein expression (Figure 4C) as determined by LCMS. Figures 4B and 4C show the data mean ± SEM. One-way ANOVA, post-hoc Tukey test**P<0.01,****P<0.0001. [Figure 4C]Figure 4A shows the expression of GLP-1RA protein (i.e., exendin-4) in the pancreas after AAV injection in db / db mice via immunohistochemical staining, as well as the islet expression percentage (Figure 4B) and total pancreatic protein expression (Figure 4C) as determined by LCMS. Figures 4B and 4C show the data mean ± SEM. One-way ANOVA, post-hoc Tukey test**P<0.01,****P<0.0001. [Figure 5] This graph shows the improvement in ex vivo insulin secretion from primary BKS db / db pancreatic islets after treatment with AAV delivering a GLP-1RA (i.e., exendin-4) (compared to an AAV-eGFP control). A shows the total GLP-1 content determined by ELISA, and B shows glucose-stimulated insulin secretion. Both graphs show the mean ± standard deviation. Unpaired t-test, *P<0.05. [Figure 6] This shows insulin levels after AAV-mediated delivery of GLP-1RA (i.e., exendin-4) in the human beta cell line EndoC-BH5. Treatment with exendin-9 (Ex9) peptide, a potent inhibitor of GLP-1RA, demonstrates that the increased INS secretion attributable to AAV-GLP-1RA is due to its action on GLP-1RA. Mean ± standard deviation. Two-way ANOVA, post-hoc Tukey test. P<0.0001. [Figure 7] This graph shows the changes in fasting blood glucose (A) and the quantification of exendin-4 in serum and pancreas by LCMS (B) in BKS db / db mice four weeks after administration of AAV-MIP-Ex-4 (AAV-based exendin-4 therapy) or vehicle. [Figure 8A] Graphs show the percentage of green fluorescent protein (GFP) expression in exocrine (Figures 8A and 8C) and endocrine (Figures 8B and 8D) tissues of either the whole pancreas (Figures 8A and 8B) or the targeted splenic lobe (Figures 8C and 8D) of the pancreas of Yucatan pigs after endoscopic ultrasound-guided fine-needle infusion (EUS-FNI) of scAAV9-CMV-eGFP at various indicated doses. Intervals were calculated using individual standard deviations. N = 2–4 pigs per dose tested. [Figure 8B] Graphs show the percentage of green fluorescent protein (GFP) expression in exocrine (Figures 8A and 8C) and endocrine (Figures 8B and 8D) tissues of either the whole pancreas (Figures 8A and 8B) or the targeted splenic lobe (Figures 8C and 8D) of the pancreas of Yucatan pigs after endoscopic ultrasound-guided fine-needle infusion (EUS-FNI) of scAAV9-CMV-eGFP at various indicated doses. Intervals were calculated using individual standard deviations. N = 2–4 pigs per dose tested. [Figure 8C] Graphs show the percentage of green fluorescent protein (GFP) expression in exocrine (Figures 8A and 8C) and endocrine (Figures 8B and 8D) tissues of either the whole pancreas (Figures 8A and 8B) or the targeted splenic lobe (Figures 8C and 8D) of the pancreas of Yucatan pigs after endoscopic ultrasound-guided fine-needle infusion (EUS-FNI) of scAAV9-CMV-eGFP at various indicated doses. Intervals were calculated using individual standard deviations. N = 2–4 pigs per dose tested. [Figure 8D] Graphs show the percentage of green fluorescent protein (GFP) expression in exocrine (Figures 8A and 8C) and endocrine (Figures 8B and 8D) tissues of either the whole pancreas (Figures 8A and 8B) or the targeted splenic lobe (Figures 8C and 8D) of the pancreas of Yucatan pigs after endoscopic ultrasound-guided fine-needle infusion (EUS-FNI) of scAAV9-CMV-eGFP at various indicated doses. Intervals were calculated using individual standard deviations. N = 2–4 pigs per dose tested. [Figure 9] This graph shows vector copies per diploid genome of AAV DNA in targeted splenic lobes of porcine pancreas after EUS-FNI of scAAV9 at various indicated doses. Each data point represents one pig, and the bars represent the mean + / - standard deviation. [Figure 10A] This graph shows data indicating that the number of EUS-FNI-mediated injections into the pancreas of Yucatan pigs alters the in vivo distribution of AAV vectors at a 5 × 10¹³ vector genome (VG) dose. [Figure 10B]The graph shows data indicating that the number of EUS-FNI infusions in the pancreas of Yucatan pigs alters the in vivo distribution of AAV vectors at higher doses of 1 × 10¹⁴ VG. [Figure 11A] The graphs show serum lipase levels after single or multiple infusions of various indicated doses of AAV vector into Yucatan pig pancreases. The number of EUS-FNI infusions into the pig pancreas correlates with an increase in serum lipase levels. [Figure 11B] The graphs show serum lipase levels after single or multiple infusions of various indicated doses of AAV vector into Yucatan pig pancreases. It demonstrates that infusion volumes of 1–5 mL of EUS-FNI into pig pancreases at a flow rate of 1 mL / min do not affect serum lipase levels. [Figure 11C] The graphs show serum lipase levels after single or multiple infusions of various indicated doses of AAV vector into Yucatan pig pancreases. The results indicate that AAV dose does not correlate with the elevation of serum lipase using either single or triple infusions of EUS-FNI into the pig pancreas. [Figure 11D] The graphs show serum lipase levels after single or multiple infusions of various indicated doses of AAV vector into Yucatan pig pancreases. The results indicate that AAV dose does not correlate with the elevation of serum lipase using either single or triple infusions of EUS-FNI into the pig pancreas. [Figure 12] Graphs show baseline and NFL levels of neurofilament light chains (NFLs) after a single infusion of two different doses of AAV9-CMV-eGFP or AAV9-INSp-eGFP into Yucatan pig pancreases. [Figure 13]Graphs show the change in body weight of db / db mice over 4 weeks after administration of a single dose vehicle control, semaglutide (10 nmol / kg), and one of two different embodiments of AAV-INS-GLP1RA at doses of 10¹² or 5¹² VG. Mean ± SEM is shown. ****p<0.0001, n=4-16 per group. AAV=adeno-associated virus, Gen=generation, GLP1RA=glucagon-like peptide-1 receptor agonist, INS=insulin promoter, Sema=semaglutide. [Figure 14] The graphs show fasting blood glucose levels and fasting insulin levels in db / db mice 8 weeks after administration of a single dose vehicle control, semaglutide (10 nmol / kg), and one of two different forms of AAV-INS-GLP1RA at doses of 10¹² or 5¹² VG, respectively, during a 4-5 hour fast. Mean ± SEM is shown. ****p<0.0001, n=4-16 per group. AAV=adeno-associated virus, Gen=generation, GLP1RA=glucagon-like peptide-1 receptor agonist, INS=insulin promoter, Sema=semaglutide. [Figure 15] Graphs show disease progression and persistence in db / db mice over 64 days after administration of a single dose vehicle control, semaglutide (10 nmol / kg), and one of two different embodiments of AAV-INS-GLP1RA at doses of 10¹² or 5¹² VG. AAV = adeno-associated virus, FBG = fasting blood glucose, Gen = generation, GLP1RA = glucagon-like peptide 1 receptor agonist, INS = insulin promoter. [Figure 16] Graphs (A) and (B) show the percentage change in body weight and food intake in mice treated with GLP-1-based pancreatic gene therapy or semaglutide (sema). Statistics: Two-way ANOVA, post-hoc Tukey test: $$p<0.01, ****p<0.0001, mean ± SEM. [Figure 17] The graphs show liver weight (left) and liver triglycerides (right) of mice treated with GLP-1-based pancreatic gene therapy or vehicle. [Figure 18A]The graphs show the body composition analysis of mice treated with GLP-1-based pancreatic gene therapy (s.AAV004), vehicle, semaglutide, or semaglutide followed by GLP-1-based pancreatic gene therapy after 8 weeks. Body weight (Figure 18A), fat mass (Figure 18B), peripheral plasma leptin (Figure 18C), and lean body mass (Figure 18D) are shown. [Figure 18B] The graphs show the body composition analysis of mice treated with GLP-1-based pancreatic gene therapy (s.AAV004), vehicle, semaglutide, or semaglutide followed by GLP-1-based pancreatic gene therapy after 8 weeks. Body weight (Figure 18A), fat mass (Figure 18B), peripheral plasma leptin (Figure 18C), and lean body mass (Figure 18D) are shown. [Figure 18C] The graphs show the body composition analysis of mice treated with GLP-1-based pancreatic gene therapy (s.AAV004), vehicle, semaglutide, or semaglutide followed by GLP-1-based pancreatic gene therapy after 8 weeks. Body weight (Figure 18A), fat mass (Figure 18B), peripheral plasma leptin (Figure 18C), and lean body mass (Figure 18D) are shown. [Figure 18D] The graphs show the body composition analysis of mice treated with GLP-1-based pancreatic gene therapy (s.AAV004), vehicle, semaglutide, or semaglutide followed by GLP-1-based pancreatic gene therapy after 8 weeks. Body weight (Figure 18A), fat mass (Figure 18B), peripheral plasma leptin (Figure 18C), and lean body mass (Figure 18D) are shown. [Figure 19] The quantitative analysis of terminal pancreatic (A) and serum (B) exendin-4 after 8 weeks is shown. [Figure 20] The exendin-4 expression endocrine region (pancreatic islet region) in mouse pancreas, as measured by immunohistochemistry, is shown. Note that exendin-4 levels were not analyzed for groups 1, 3, or 4 (as they were vehicle groups and therefore assumed to have 0% expression). [Figure 21A]Figure 21D shows total cholesterol (Figure 21A), LDL (Figure 21B), HDL (Figure 21C), and triglycerides (Figure 21D) in DIO mice in a direct comparison study between GLP-1RA PGTx and semaglutide. [Figure 21B] Figure 21D shows total cholesterol (Figure 21A), LDL (Figure 21B), HDL (Figure 21C), and triglycerides (Figure 21D) in DIO mice in a direct comparison study between GLP-1RA PGTx and semaglutide. [Figure 21C] Figure 21D shows total cholesterol (Figure 21A), LDL (Figure 21B), HDL (Figure 21C), and triglycerides (Figure 21D) in DIO mice in a direct comparison study between GLP-1RA PGTx and semaglutide. [Figure 21D] Figure 21D shows total cholesterol (Figure 21A), LDL (Figure 21B), HDL (Figure 21C), and triglycerides (Figure 21D) in DIO mice in a direct comparison study between GLP-1RA PGTx and semaglutide. [Figure 22] Results of a direct comparison study between GLP-1RA PGTx and semaglutide in db / db mice. Fasting blood glucose (A), fasting plasma insulin (B), and change in body weight from baseline (C) are shown on day 29. [Figure 23] The graphs show blood glucose (left graph) and plasma insulin (right graph) in DIO mice treated with GLP-1-based pancreatic gene therapy (s.AAV004), vehicle, semaglutide, or semaglutide followed by GLP-1-based pancreatic gene therapy after 8 weeks. [Figure 24] This shows the HOMA-IR (Hostasis Model Assessment of Insulin Resistance) levels measured in DIO mice treated with GLP-1-based pancreatic gene therapy (ss.AAV004), vehicle, semaglutide, or semaglutide followed by GLP-1-based pancreatic gene therapy. [Modes for carrying out the invention]

[0033] Metabolic disorders arise from disruptions in normal metabolism, the process of converting input (food and drink) into output (energy). Typically, chemicals in the body break down ingested proteins, carbohydrates, and fats, converting them into energy for immediate use or storing them for later use. Metabolic disorders include conditions that increase the risk of heart disease, stroke, and death. The disease is becoming more prevalent, with an estimated one-third of adults in the United States having at least one. Despite advances in treatment over the past 50 years, metabolic disorders in general, particularly obesity and type 2 diabetes, remain leading causes of morbidity and mortality today.

[0034] Glucose-regulating hormones, including but not limited to glucagon-like peptide-1 (GLP-1), are often produced and secreted in response to nutrients in food and affect the function of islet beta cells in numerous ways. Among their primary effects on islet beta cells are stimulation of insulin secretion and production, as well as positive effects on islet beta cell health. Reported improvements in beta cell health with GLP-1 include increased cell proliferation, islet beta cell regeneration, and / or maintenance of islet beta cell aggregates through inhibition of apoptosis. Applying these beneficial functions of glucose-regulating hormones to the treatment of metabolic disorders, including obesity and diabetes, is a successful clinical strategy and remains an active area of ​​therapeutic research.

[0035] However, the effective delivery of such glucose-regulating hormones to the pancreas is associated with many challenges. For example, the effectiveness of systemic delivery of such drugs via viral vectors (e.g., adeno-associated virus (AAV)) is limited by immune response, transduction efficiency, size, pre-existing immunity, and route of administration. In addition, many glucose-regulating hormones have two characteristics that make their pharmacological application to disease treatment difficult: (1) a short half-life, and (2) nausea and vomiting as side effects when present in chronically high levels in circulation. To overcome these challenges, the disclosure provides, in some embodiments, a method for producing transgenic glucose-regulating hormones in a localized and sustained manner. Such production methods limit the maintenance of high levels of the hormone in circulation. To achieve this, in some embodiments, the techniques described herein utilize pancreatic islet beta cells for the localized production and secretion of transgenic glucose-regulating hormones such as GLP-1 and GLP-1 analogs, because these cells already perform similar functions for endogenous insulin production and secretion. Furthermore, although not constrained by theory, local delivery to the pancreas and local production of glucose-regulating hormones (or more) by islet beta cells achieve the desired effect (or more) on islet beta cell function while minimizing the circulating levels of hormones.

[0036] For many metabolic disorders, including obesity and type 2 diabetes, there are no FDA-approved therapies that provide disease modification (i.e., continued and sustained preservation of pancreatic insulin production capacity even after discontinuation of therapy).

[0037] Instead of treating the patient's symptoms, the methods and compositions provided herein (e.g., gene therapy compositions) are used in some embodiments to treat the underlying cause(s) of the disease. Gene therapy approaches to restore insulin production in individuals (subjects) with metabolic disorders (e.g., obesity and / or type 2 diabetes) are described herein, with the aim of achieving long-term remission. Accordingly, gene therapy compositions and methods related to the delivery of key metabolic hormones necessary for adequate insulin production in pancreatic islet beta cells are provided herein in some embodiments. As a non-limiting example, the GLP-1 coding sequence may be packaged in an adeno-associated virus (AAV) vector and delivered locally, for example, by endoscopic ultrasound-guided fine-needle infusion. While not wishing to be bound by theory, it is thought that locally enhancing GLP-1 receptor activation in the pancreatic endocrine tissue, e.g., in the splenic lobe, would result in a reduction in blood glucose levels. Other configurations are also possible and are described in more detail below.

[0038] Remarkably, the data provided herein demonstrate that localized islet beta cell production of GLP-1 analogs in the pancreas results in significant, measurable, and sustained improvement in symptoms associated with metabolic diseases such as type 2 diabetes and obesity (e.g., decreased fasting blood glucose levels, increased fasting insulin, decreased weight gain, increased lean body mass, decreased plasma leptin levels, decreased cholesterol levels, and / or decreased liver weight and triglyceride content) without adverse effects on the brain or serum of the subject. In other words, these preclinical analyses in animal models demonstrate that the gene therapies provided herein achieve localized production of gene products in the pancreas at levels sufficient to improve symptoms of metabolic diseases such as type 2 diabetes and obesity without exposing the brain or serum to harmful levels of gene therapy or gene products.

[0039] metabolic disease In some aspects, this disclosure relates to methods for reducing body weight in subjects, for example, subjects having metabolic disorders (e.g., diagnosed with metabolic disorders). Metabolic disorders include a diverse group of disorders that affect the body's ability to process carbohydrates, fats, and proteins obtained through diet. Non-specific examples of metabolic diseases include obesity, diabetes (type 1 diabetes, type 2 diabetes, gestational diabetes), lipid metabolism disorders (familial hypercholesterolemia, hypertriglyceridemia), congenital metabolic disorders (phenylketonuria (PKU), galactosemia, Tay-Sachs disease, maple syrup urine disease (MSUD), Gaucher disease, Fabry disease, glucose-6-phosphate dehydrogenase (G6PD) deficiency), lysosomal storage diseases (Hunter syndrome, Harler syndrome, Niemann-Pick disease), glycogen storage diseases (von Gilke disease (type I), Pompe disease (type II), Koli's disease (type III), Andersen's disease (type IV), McArdle disease (type V)), mitochondrial diseases (Kerns-Sayre syndrome, MELAS, MERRF), and purine-pyrimidine diseases (Lessi These include Nyhan syndrome (orotic aciduria), urea cycle disorders (ornithine transcarbamylase deficiency, argininosuccinic aciduria, citrullinemia), fructose metabolism disorders (hereditary fructose intolerance, essential fructosuria), amino acid metabolism disorders (homocystinuria, tyrosinemia), mineral metabolism disorders (primary hyperparathyroidism, hypophosphatasia), porphyria (acute intermittent porphyria (AIP), patent cutaneous porphyria (PCT)), lactose intolerance and Wilson's disease, polycystic ovary syndrome (PCOS), metabolic dysfunction-related fatty liver disease (MASLD, formerly known as non-alcoholic fatty liver disease [NAFLD]), and non-alcoholic steatohepatitis (NASH).

[0040] In some embodiments, the subject is obese. Obesity is a medical condition characterized in part by an excess of body fat. Obesity is often measured using the Body Mass Index (BMI). A standard BMI is calculated by dividing a person's weight in kilograms by the square of their height in meters. A higher BMI may suggest a higher amount of body fat. Generally, a BMI of 30 or higher is considered obese. A subject's BMI less than 18.5 is within the underweight range. A subject's BMI between 18.5 and less than 25 is within the healthy weight range. A subject's BMI between 25.0 and less than 30 is within the overweight range. A subject's BMI of 30.0 or higher is within the obese range. Obesity is often subdivided into the following categories: Class 1 is a BMI between 30 and less than 35, Class 2 is a BMI between 35 and less than 40, and Class 3 is a BMI of 40 or higher. Class 3 obesity may be classified as “severe” obesity.

[0041] This condition is not only a cosmetic concern, but also a serious health problem as it increases the risk of diseases and health problems such as heart disease, diabetes, hypertension, and certain types of cancer. Obesity is usually caused by a combination of genetic factors combined with environmental factors, diet, and levels of physical activity. Obesity 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 occur with obesity and are therefore expected to improve with weight loss include, for example, gastroesophageal reflux disease (GERD), sleep apnea, arthritis, hypertension, coronary artery disease (e.g., as secondary prevention), stroke, transient ischemic attack (TIA), diastolic dysfunction, myocardial infarction, and heart failure.

[0042] In some embodiments, the subjects have metabolic disorder-associated fatty liver disease (MASLD) (formerly known as non-alcoholic fatty liver disease [NAFLD]). MASLD is characterized by the presence of hepatic fat accumulation in the absence of secondary causes of fatty liver (e.g., excessive alcohol consumption, other liver diseases, and / or long-term use of lipolytic drugs). MASLD is the most common cause of chronic liver disease and is a 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 first course of treatment (e.g., changes in diet and / or exercise). As MASLD progresses to cirrhosis, medications for treating diabetes and other metabolic conditions may be administered.

[0043] In some embodiments, the subjects have obesity and MASLD. In some embodiments, the subjects have diabetes and MASLD. In some embodiments, the subjects have obesity, diabetes, and MASLD.

[0044] In some embodiments, the subject has diabetes, which is a group of metabolic disorders characterized by chronic hyperglycemia (high blood glucose levels) resulting from a defect in insulin secretion, the action of insulin, or both. Various types of diabetes include prediabetes, type 1 diabetes, type 2 diabetes, and gestational diabetes. Other types may result from specific genetic conditions, surgery, medications, infections, and other diseases. In some embodiments, the subject has type 2 diabetes, typically resulting from the body's ineffective use of insulin, often compounded with relative insulin deficiency. Type 2 diabetes may be characterized by elevated blood glucose levels that can be caused by two parallel, progressive disease processes in the body: insulin resistance and insulin insufficiency. Insulin resistance involves a lack of the body's ability to adequately respond to insulin signals to remove glucose from the bloodstream, while insulin insufficiency involves a gradual failure of the pancreas to produce enough insulin to meet the body's needs. The guidelines focus on managing the glycemic symptoms of type 2 diabetes, often measured by glycated hemoglobin, or HbA1c, rather than attempting to correct the underlying pathology in the body that causes insulin resistance and insulin deficiency. Therefore, patients undergo radical dietary and lifestyle changes that require lifelong patient adherence and sustained medication. For some, this approach to care is unmanageable, putting many patients at risk and potentially increasing the likelihood of microvascular and macrovascular complications in type 2 diabetes, or even leading to chronically elevated blood glucose levels that can be fatal.

[0045] Glucose-regulating hormones Glucose-regulating hormones include hormones involved in regulating circulating blood glucose levels. Two hormones known to be regularly involved in blood glucose regulation are insulin, which lowers blood glucose levels, and glucagon, which raises blood glucose levels. Therefore, both insulin and glucagon are glucose-regulating hormones. Additional hormones that affect the secretion or function of insulin and / or glucagon may also be classified as glucose-regulating hormones. In some embodiments, glucose-regulating hormones act directly, i.e., they are directly involved in regulating blood glucose through insulin and / or glucagon regulation. In other embodiments, the effect of glucose-regulating hormones is indirect, i.e., the activity of the hormones indirectly affects blood glucose through insulin and / or glucagon regulation. Examples of glucose-regulating hormones include preproglucagon-derived peptides (e.g., glucagon, GLP-1, oxytomodulin, glycentin, glycentin-related polypeptide (GRPP), major proglucagon fragments, intermediary peptide 1 (IP-1), intermediary peptide 2 (IP-2), and GLP-2), incretins (e.g., GLP-1 and glucose-dependent insulinotropic polypeptide (GIP)), enteroendocrine cell-derived peptides (e.g., GLP-1, peptide tyrosine (PYY), cholecystokinin (CCK), GIP, somatostatin, oxytomodulin, and ghrelin), and hormones produced in the pancreas (e.g., insulin, amylin, somatostatin, glucagon, and GLP-1).

[0046] Naturally occurring GLP-1 is a polypeptide derived from the proglucagon protein. Under physiological conditions, it is produced and secreted by enteroendocrine L cells and certain neurons in the nucleus tractus solitarius of the brainstem upon food intake. GLP-1 is rapidly metabolized and inactivated by dipeptidyl peptidase IV (an enzyme) even before the hormone leaves the intestines. 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 functions as part of the enterogastron and "ileal brake" mechanisms. GLP-1 also plays a role as a physiological regulator of appetite and food intake. Reduced GLP-1 secretion may lead to the development of obesity. In some embodiments, the polynucleotide encodes the full-length GLP-1 sequence (e.g., SEQ ID NO: 33). In other embodiments, the polynucleotide encodes a cleaved GLP-1 sequence (e.g., any one of SEQ ID NOs: 53-58). In yet another embodiment, the polynucleotide encodes a functional variant or fragment of GLP-1.

[0047] In some embodiments, the glucose-regulating hormone is GLP-1. In some embodiments, the glucose-regulating hormone includes a drug that mimics the action of GLP-1. In some embodiments, the drug includes a GLP-1 receptor agonist (e.g., a drug that binds to and activates the GLP-1 receptor, thereby reducing blood glucose levels). In some embodiments, the GLP-1 receptor agonist is a polypeptide agonist against the GLP-1 receptor (e.g., exendin-4 and its variants). Exendin-4 (found in the saliva of the Gila monster Heloderma suspectum) is a long-acting GLP-1 analog that is an agonist of the GLP-1 receptor. In some embodiments, the GLP-1 receptor agonist is exendin-4. Other endocrine hormones having glucose-regulating activity as intended herein include, but are not limited to, leptin, follistatin, insulin-like growth factor 1 (IGF1), vasoactive intestinal peptide (VIP), and growth hormone 1 (GH1). For example proteins and coding sequences, please refer to Table 1.

[0048] The polynucleotides described herein include, in some embodiments, insulin (preproinsulin, proinsulin, insulin) coding sequences. Preproinsulin, 110 amino acids long, is a biologically inactive precursor to insulin. Insulin mRNA is translated as preproinsulin, a single-stranded precursor, and proinsulin is produced by the removal of its signal peptide during insertion into the endoplasmic reticulum. Insulin is produced and secreted by pancreatic islet beta cells in the pancreas. Proinsulin and preproinsulin contain three domains, namely, an amino-terminal B chain, a carboxy-terminal A chain, and an intermediate linkage peptide known as the C peptide. Within the endoplasmic reticulum, proinsulin is exposed to certain endopeptidases that cleave the C peptide, producing a mature form of insulin consisting of the A and B chains. Insulin and the free C peptide are packaged in the Golgi within secretory granules that accumulate in the cytoplasm. In some embodiments, the insulin protein includes the amino acid sequence of SEQ ID NO: 46, or a variant thereof. Unless otherwise specified, the term “insulin” encompasses various forms of insulin, including preproinsulin, proinsulin, and insulin.

[0049] Polynucleotides (i.e., nucleic acids) containing a coding region for a glucose-regulating hormone (e.g., GLP-1), also referred to as a coding sequence, are provided herein. The coding sequence includes a nucleotide sequence that directly defines the amino acid sequence of the product (e.g., the protein encoded by the coding sequence). The boundaries of the coding sequence are generally determined by an open reading frame. An open reading frame includes a continuous stretch of DNA or RNA that begins with a start codon (e.g., methionine (ATG or AUG)) and ends with a stop codon (e.g., TAA, TAG or TGA, or UAA, UAG or UGA). The open reading typically encodes a protein (e.g., a glucose-regulating hormone or insulin). Polynucleotides may be, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA containing LNA having a β-D-ribo configuration, α-LNA having an α-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having 2'-amino functionalization, and 2'-amino-α-LNA having 2'-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) and / or chimeras and / or combinations thereof, or may include them.

[0050] The term "glucose-regulating hormone" includes any of the functional peptides and polypeptides described in the above examples, as well as their functional variants, meaning that the peptides, polypeptides, and / or variants can influence blood glucose control either through direct or indirect functions.

[0051] Therefore, amino acid modifications (e.g., substitutions) may be performed on the glucose-regulating hormones provided herein. In some embodiments, the modified amino acid sequence confers beneficial properties for protein production and / or function. For example, the GLP-1 peptide sequence may include a glycine substitution of alanine at 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 therefore other variants, are contemplated herein.

[0052] In some embodiments, the polynucleotide comprises multiple coding sequences, each encoding a different protein. In some embodiments, the polynucleotide comprises one or more coding sequences of 1 to 10 glucose-regulating hormones. For example, the polynucleotide may comprise one or more coding sequences of 1 to 3, 1 to 4, or 1 to 5 different glucose-regulating hormones. In some embodiments, the polynucleotide comprises one or more coding sequences and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more glucose-regulating hormones. In some embodiments, the polynucleotide comprises a GLP-1 coding sequence. In some embodiments, the polynucleotide comprises a GLP-1 coding sequence, an IP-1 coding sequence, and / or an IP-2 coding sequence.

[0053] In some embodiments, the glucose-regulating hormone coding sequence encodes a functional glucose-regulating hormone that is produced in vivo after administration to a subject. As used herein, “functional” refers to a protein that possesses biological activity (e.g., enzymatic activity). That is, the produced functional protein has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more activity (e.g., enzymatic activity) compared to the 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 measurement of an enzyme byproduct (e.g., C-peptide) or other related component (e.g., glucose levels). In some embodiments, the functional glucose-regulating hormone has substantially the same activity as a naturally occurring glucose-regulating hormone (e.g., promotion of glucose uptake, glycolysis, adipogenesis, and / or protein synthesis in skeletal muscle and / or adipose tissue via the tyrosine kinase receptor pathway, and / or maintenance of circulating glucose concentration within a physiological range). In some embodiments, functional glucose-regulating hormones have greater activity than naturally occurring glucose-regulating hormones.

[0054] "Identity" refers to the relationship between two or more sequences (e.g., amino acid sequences or nucleotide sequences) determined by comparing the sequences to one another. Identity also refers to the degree of sequence relevance between sequences, determined by the number of matches between amino acid chains (polypeptides) or nucleotide chains (polynucleotides). Identity is a measure of the percentage of identical matches between the smaller of two or more sequences that have gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., "algorithms"). The identity of related polypeptides and polynucleotides can be readily calculated by known methods. The "percent identity (%)" applied to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid or nucleic acid residues) in a candidate (first) polypeptide or polynucleotide sequence that are identical to residues in the second polypeptide or polynucleotide sequence, after the sequences have been aligned and gaps introduced as necessary to achieve the maximum possible percentage identity.

[0055] Methods and computer programs for alignment are well known in the art. Identity depends on the calculation of identity percentages, but it is understood that values ​​may differ due to gaps and penalties introduced into the calculation. Generally, variants of a particular polynucleotide or polypeptide have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a particular wild-type, natural, or reference sequence, but less than 100%, as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Tools for such alignment include, but are not limited to, tools from the BLAST suite (Altschul, SF, et al. Nucleic Acids Res. 1997;25:3389-3402) and tools based on the Smith-Waterman algorithm (Smith, TF & Waterman, MSJ Mol. Biol. 1981;147:195-197). A common global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, S.B. & Wunsch, CDJ Mol. Biol. 1920; 48: 443-453). The Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has also been developed, which is said to generate global alignments of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm.

[0056] In some embodiments, the polynucleotides provided herein further comprise at least one promoter. The promoter comprises a nucleotide sequence located at the 5' end of the polynucleotide, which a polymerase specifically binds to the remainder of the polynucleotide and initiates its transcription. In some embodiments, the promoter is an islet cell promoter, such as an islet beta cell promoter. In some embodiments, the promoter is an insulin promoter, such as a human insulin promoter, a mouse insulin-1 promoter, a mouse insulin-2 promoter, a rat insulin-1 promoter, or a rat insulin-2 promoter. Additional exemplary promoters include, but are not limited to, Slc2a, IAPP, NKX6.1, DLK1, MafA, Slc30a8 / Znt8, PCSK1, and ADCYAP1.

[0057] In some embodiments, the polynucleotides provided herein further comprise an enhancer. The enhancer comprises a nucleotide sequence capable of stimulating promoter activity by increasing the tissue specificity level of the promoter, and is positioned between the promoter and the coding sequence of the polynucleotide. Exemplary enhancers include, but are not limited to, a CMV enhancer, a synthetic enhancer, a liver-specific enhancer, a vascular-specific enhancer, a brain-specific enhancer, a neuronal-specific enhancer, a lung-specific enhancer, a muscle-specific enhancer, a kidney-specific enhancer, a pancreas-specific enhancer, and a pancreatic islet cell-specific enhancer. In some embodiments, the enhancer is a pancreatic islet cell-specific enhancer. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11]

[0058] Delivery system and route of administration In some embodiments, the polynucleotides of this disclosure are formulated, for example, for delivery in vivo. In some embodiments, this disclosure provides a vector comprising any one of the polynucleotides described herein.

[0059] In some embodiments, the 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.

[0060] In some embodiments, the vector comprises an adeno-associated virus (AAV) vector. AAV (or "rAAV" for recombinant AAV) is a small, non-enveloped, single-stranded DNA virus capable of infecting both dividing and non-dividing cells. In contrast to the generally limited persistence of AdV-mediated gene transfer, transgene expression can persist for many years after intramuscular recombinant AAV (rAAV) vector delivery.

[0061] An "adenovirus expression vector" or "AAV vector" comprises a vector that (a) supports construct / genome packaging and (b) contains an adenovirus or AAV sequence sufficient to express the polynucleotides described herein.

[0062] Typically, recombinant AAV viruses are constructed by co-transfecting an expression plasmid containing a target gene (e.g., a polynucleotide) adjacent to two AAV terminal repeats with an expression plasmid containing a wild-type AAV coding sequence that does not contain terminal repeats. AAV expression vectors containing polynucleotides linked by AAV ITRs can be constructed by directly inserting a selected sequence(s) into an AAV genome that already contains the excised major AAV open reading frame ("ORF"). In some embodiments, the ITR sequence is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 (AAVrh10), and AAV11 ITR sequences. In some embodiments, the ITR is designed for a single-stranded AAV genome or a self-complementary AAV genome. For self-complementary AAV genomes, the TRS (terminal dissociation site) located at the 3' ITR is deleted.

[0063] In some embodiments, the AAV vector comprises the recombinant AAV vector genome described above (e.g., an AAV genome comprising a human islet beta cell-specific promoter operably linked to a human GLP-1 receptor agonist coding sequence) and a nucleotide sequence encoding a capsid protein. The capsid protein is relevant to determining the tissue-specific targeting ability of the AAV and is known in the art. In some embodiments, the capsid protein is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, AAV-2i8, AAV-DJ, AAV-LK03, AAV-KP1, AAV-KP2, and AAV-KP3 capsid proteins, as well as their variants.

[0064] In some embodiments, the AAV vector genome is single-stranded. In some embodiments, the AAV vector genome is self-complementary.

[0065] For eukaryotic cells, the expression regulatory sequence typically includes a promoter, an immunoglobulin gene, an enhancer (see above) such as one derived from SV40 or cytomegalovirus, and a polyadenylated sequence that may contain splice donor and acceptor sites. The polyadenylated sequence is generally inserted after the transgene sequence and before the 3'ITR sequence. In some embodiments, the polyadenylated sequence includes an SV40 polyA or bovine GH polyA sequence. In some embodiments, the AAV vector includes a 5'UTR between the promoter and the coding sequence. In some embodiments, the 5'UTR sequence includes an intron. In some embodiments, the intron is artificial, derived from the insulin 5'UTR, or derived from the hemoglobin subunit beta (HBB) locus.

[0066] In some embodiments, the vector is a retroviral vector. Therefore, in some embodiments, the gene therapy composition includes a retroviral vector genome comprising a pancreatic islet beta cell-specific promoter operably linked to a GLP-1 receptor agonist coding region. Non-limiting examples of retroviral vectors include mouse leukemia virus vectors and lentiviral vectors (e.g., derived from human immunodeficiency virus).

[0067] In some embodiments, the vector is a herpes simplex virus (HSV) vector. Thus, in some embodiments, the gene therapy composition comprises an HSV vector genome containing a pancreatic islet beta cell-specific promoter operably linked to a GLP-1 receptor agonist coding region.

[0068] In some embodiments, the vector is an adenovirus vector (AdV). Thus, in some embodiments, the gene therapy composition comprises an AdV vector genome containing a pancreatic islet beta cell-specific promoter operably linked to a GLP-1 receptor agonist coding region.

[0069] In some embodiments, the vector is a non-viral vector such as a plasmid, bacterial artificial chromosome, yeast artificial chromosome, or minicircle.

[0070] The gene therapy compositions of this disclosure are, in some embodiments, delivered directly to the pancreas, which has both exocrine and endocrine functions, producing digestive enzymes and hormones, respectively. The human pancreas has three main anatomical parts: the head, body, and tail. The head of the pancreas is the widest part and is located to the right of the midline. It is located within the duodenum, which is the first part of the small intestine. The common bile duct extends through the head of the pancreas, and the body of the pancreas is located behind the stomach and extends toward 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 toward the spleen. The two main ducts of the human pancreas are the pancreatic duct and the accessory duct. The pancreatic duct extends the length of the pancreas and is the duct that transports digestive enzymes and bicarbonates from the pancreas to the duodenum, while the accessory duct is a smaller duct that can be discharged into the duodenum. The two types of cells of the pancreas are called the islets of Langerhans and the acini. The islets of Langerhans are clusters of endocrine cells that produce hormones such as insulin, glucagon, and somatostatin, which regulate blood glucose levels, while the acini are clusters of exocrine cells that produce digestive enzymes such as amylase, lipase, and protease, which help break down carbohydrates, fats, and proteins in the small intestine. Together, the pancreas has a unique and complex structure that allows it to perform both exocrine and endocrine functions essential for digestion and metabolism.

[0071] In contrast, the pig pancreas has a unique structure with four lobes: the head, body, tail, and splenic lobe. In humans, the pancreas has a more homogeneous structure without distinct lobes. The region in humans that corresponds to the splenic lobe of the pig pancreas is the tail of the pancreas. Therefore, the tail of the human pancreas is equivalent to the splenic lobe of the pig pancreas.

[0072] In a preferred embodiment, the gene therapy composition is delivered directly to the pancreas (e.g., the endocrine tissue of the pancreas) via an endoscopic delivery method. For example, the gene therapy composition may be delivered locally to the tail of the pancreas or the body of the pancreas near the tail terminal via an endoscopic delivery method. However, in other embodiments, the gene therapy composition is delivered via intraparenchymal delivery, intra-CSF delivery, intramuscular delivery, or systemic delivery (e.g., intravenous or intra-arterial delivery).

[0073] In some embodiments, the gene therapy composition is delivered to the target intestine or pancreas via a minimally invasive endoscopic procedure (e.g., using a catheter). Various endoscopic procedures and devices are known and are discussed herein.

[0074] In some embodiments, the gene therapy composition of the present disclosure is delivered using an endoscopic ultrasound-guided fine-needle injection (EUS-FNI) procedure. Endoscopic ultrasound-guided fine-needle injection (EUS-FNI) is a medical procedure used to inject a drug (e.g., the gene therapy composition of the present disclosure) into the body while being guided by endoscopic ultrasound. This is a minimally invasive procedure in which a small needle passes through an endoscope and is guided by ultrasound to a target area in the body (e.g., a pancreatic deposit site). The drug, such as the gene therapy composition, is then injected directly into the targeted site using this needle. In some embodiments, the needle is a 20-30 g needle with a diameter of 0.4-0.6 mm. In some embodiments, the needle is a 25 g needle with a diameter of 0.515 mm.

[0075] In some embodiments, a deposition device containing deposition elements is used to deliver (e.g., inject) the gene therapy composition of the present disclosure to a deposition site, such as a pancreatic deposition site (e.g., endocrine tissue in the tail of the pancreas).

[0076] In some embodiments, the deposition device includes a device for implanting, positioning, seeding, inserting, spraying, topically applying, and / or otherwise depositing the gene therapy composition of the present disclosure into a “deposition site” of a target. The deposition device may include one or more needles positioned in the distal portion of the deposition device. In some embodiments, the distal end of the deposition device is delivered to the target via an orifice and advances through the wall of the digestive tract to a position close to the pancreas. The deposition device may be delivered, for example, via the working channel of a gastrointestinal endoscope delivered through the orifice of the target. The deposition device may be delivered, for example, together with a gastrointestinal endoscope delivered through the orifice of the target. In some embodiments, the pancreatic deposition site includes one or more sites selected from the group consisting of intraparenchymal space, pre-pararenal space, intraluminal space, intraarterial space of an artery supplying at least a portion of the pancreas, and combinations thereof. Exemplary deposition devices and systems are described, for example, in WO2022 / 174091 (e.g., REJUVA® system) and WO2016 / 011269, the entire contents of which are incorporated herein by reference.

[0077] In some embodiments, the delivery of the gene therapy composition comprises at least a first delivery in which a minimum volume of the gene therapy composition is delivered to the pancreatic parenchyma, the minimum volume of the composition being sufficient to allow at least a portion of the volume of the composition to exit into the anterior pararenal space, diffuse, and re-enter the pancreas. The method may further comprise at least a second delivery of the composition to one or more additional deposition sites adjacent to the tail of the pancreas.

[0078] In some embodiments, the delivery of the gene therapy composition comprises at least a first delivery in which a minimum volume of the gene therapy composition is delivered to the pancreatic parenchyma, the minimum volume of the gene therapy composition comprising a volume of at least 2 ml, at least 3 ml, and / or at least 5 ml.

[0079] In some embodiments, the deposition device is advanced to one or more selected pancreatic deposition sites under image guidance. Image guidance may include endoscopic ultrasound guidance, CT guidance, and / or MRI guidance.

[0080] In some embodiments, therapeutic benefits are achieved over a period of at least six months. In some embodiments, therapeutic benefits are achieved over a period of at least one week, at least two weeks, at least three weeks, at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, or at least one year.

[0081] In some embodiments, one or more pancreatic deposition sites include locations within 10 cm, 7.5 cm, 5 cm, and / or 3 cm of a portion of the pancreas, and the portion of the pancreas includes the tail, neck, body, head, and / or uncinate process.

[0082] In some embodiments, the gene therapy composition and / or at least one deposition element is configured to be visualized by an imaging device, and the method further includes visualizing the gene therapy composition and / or at least one deposition element with an imaging device to confirm proper delivery of the gene therapy composition.

[0083] In some embodiments, the method further includes delivering an imaging agent through at least one deposition element, visualizing the delivery of the imaging agent using an imaging device, and then confirming the proper delivery of the gene therapy composition.

[0084] In some embodiments, the method further includes pre-loading a gene therapy composition into a deposition device. The gene therapy composition can be loaded into the deposition device from the distal end of the deposition device.

[0085] In some embodiments, the gene therapy composition is delivered at a pressure of at least 3 mmHg (e.g., at least 4 mmHg, at least 5 mmHg, at least 10 mmHg, at least 15 mmHg, or at least 20 mmHg) (e.g., via injection). In some embodiments, the gene therapy composition is delivered 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 (e.g., via injection). In some embodiments, the gene therapy composition is delivered at a pressure of 25 mmHg or less.

[0086] In some embodiments, the gene therapy composition is delivered at a flow rate of about 0.5 ml / min to about 10 ml / min (e.g., 0.5 ml / min, 1.0 ml / min, 1.5 ml / min, 2.0 ml / min, 2.5 ml / min, 3.0 ml / min, 3.5 ml / min, 4.0 ml / min, 4.5 ml / min, 5.0 ml / min, 5.5 ml / min, 6.0 ml / min, 6.5 ml / min, 7.0 ml / min, 7.5 ml / min, 8.0 ml / min, 8.5 ml / min, 9.0 ml / min, 9.5 ml / min, or 10.0 ml / min) (e.g., via injection). In some embodiments, the gene therapy composition is delivered at a flow rate of about 1.0 ml / min to about 5.0 ml / min (e.g., via injection). In some embodiments, the gene therapy composition is delivered at a flow rate of at least 1 ml / min (e.g., via injection). In some embodiments, the gene therapy composition is delivered at a flow rate of 5 ml / min or less.

[0087] In some embodiments, at least one sedimentary element includes a plurality of openings along its length.

[0088] In some embodiments, the method further includes ensuring that at least one deposition element is in the appropriate position before delivering the gene therapy composition.

[0089] In some embodiments, the method further includes the delivery of a permeability enhancer before and / or concurrently with the delivery of the gene therapy composition. The delivery of the permeability enhancer can be done locally and / or intravenously. The permeability enhancer may include agents selected from the group consisting of hyaluronidase, collagenase, losartan, and combinations thereof. The gene therapy composition may include a co-formulation of the gene therapy composition and the permeability enhancer.

[0090] In some embodiments, the method further includes heating tissue adjacent to one or more selected pancreatic deposition sites to a temperature above 39°C before, during, and / or after delivery of the gene therapy composition.

[0091] In some embodiments, the method further includes delivering a seeding blocker material configured to prevent the undesirable seeding of the gene therapy composition to non-target sites. The seeding blocker material may include viscous substances and / or polymers.

[0092] In some embodiments, the method further includes positioning a blocking element in a subject, wherein the blocking element is configured to prevent undesirable seeding of the gene therapy composition at non-target sites.

[0093] In some embodiments, the method further includes removing at least a portion of the gene therapy composition from the deposition site after delivery of the gene therapy composition has commenced.

[0094] Additional aspects and embodiments of the delivery devices and methods are described in International Publication No. WO2022 / 174091 (International Application No. PCT / US2022 / 016200), which is incorporated herein by reference in whole.

[0095] therapeutic use The gene therapy compositions described herein (e.g., comprising an AAV vector genome, a polynucleotide encoding a glucose regulatory hormone(s), and a glucose regulatory hormone) 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 (e.g., prediabetes, type 1 diabetes, type 2 diabetes), lipid metabolism disorders, congenital metabolic disorders, lysosomal storage diseases, glycogen storage diseases, mitochondrial diseases, purine-pyrimidine diseases, urea cycle disorders, fructose metabolism disorders, amino acid metabolism disorders, mineral metabolism disorders, porphyria, lactose intolerance and Wilson's disease, polycystic ovary syndrome, metabolic dysfunction-related fatty liver disease (non-alcoholic fatty liver disease), and non-alcoholic steatohepatitis.

[0096] Treatment of metabolic disorders involves administering or delivering one or more gene therapy compositions in an amount effective to alleviate one or more symptoms of the metabolic disorder, which in some embodiments is a single dose within a specific time window. The amount effective (used interchangeably with effective amount) to achieve a particular outcome (also referred to herein as the therapeutic effective amount) may depend at least in part on the target tissue, target cell type, means of administration, e.g., the physical characteristics of the gene therapy composition, and / or other determinants such as the subject's age, weight, height, sex, and general health. In a preferred embodiment, the effective amount of gene therapy composition results in an improvement in glucose (blood sugar) levels in the subject.

[0097] Treatment of type 2 diabetes may, in some embodiments, include reducing HbA1c, reducing fasting blood glucose, improving the time spent at normal blood glucose levels, and / or improving hyperglycemia. This treatment may also include reducing or eliminating the need for exogenous insulin, reducing or eliminating the need for exogenous GLP-1 receptor agonists, without increasing the rate of nausea, diarrhea, vomiting, constipation, or abdominal pain. Other consequences of type 2 diabetes that may be improved include, for example, hypertension, hypertriglyceridemia, hypercholesterolemia-induced heart disease, diabetic heart disease, heart failure, diabetic heart failure, and / or diastolic dysfunction. Other conditions that occur with and are thought to be associated with type 2 diabetes and obesity (but are not treated with insulin unless type 2 diabetes is indicated) include, for example, polycystic ovary syndrome (PCOS), androgen hypertension, infertility, menstrual irregularities, hirsutism, dementia, Alzheimer's disease, cognitive impairment, cancer, such as liver cancer, ovarian cancer, breast cancer, uterine cancer, cholangiocarcinoma, adenocarcinoma, glandular tumor(s), gastric cancer, colorectal cancer, and / or prostate cancer, psoriasis, hypogonadism, insufficient total testosterone levels, and / or insufficient free testosterone levels. Other forms of diabetes in which glucose-regulating hormone production in pancreatic islet beta cells may, in principle, be useful include "double diabetes" (when a T1D patient also develops type 2 diabetes), gestational diabetes, and prediabetes.

[0098] In some embodiments, a single dose or two or fewer doses of the gene therapy composition are sufficient to achieve a specific effect in a subject within a specific time window. This time window may be, for example, within 3 months after administering the gene therapy composition to the subject (e.g., delivering the gene therapy composition to the subject's pancreas), within 6 months after administering the gene therapy composition to the subject (e.g., delivering the gene therapy composition to the subject's pancreas), within 9 months after administering the gene therapy composition to the subject (e.g., delivering the gene therapy composition to the subject's pancreas), within 12 months / 1 year after administering the gene therapy composition to the subject (e.g., delivering the gene therapy composition to the subject's pancreas), or within 18 months after administering the gene therapy composition to the subject (e.g., delivering the gene therapy composition to the subject's pancreas). In any one of the embodiments described herein, the delivery of a single dose of the gene therapy composition may include the delivery of only a single dose of the gene therapy composition within a specific period. Alternatively, the delivery of the gene therapy composition may include, for example, the delivery of two or fewer doses (i.e., only two doses) within a period of one year or less. Therefore, in some embodiments, a method comprising delivering a single dose or two or fewer doses of a gene therapy composition (for example, to the pancreatic endocrine tissue of a subject with a metabolic disorder) in an amount effective to maintain a weight loss of approximately 5% (compared to baseline) over a year is achieved within one year of delivery of the single dose or two or fewer doses of the gene therapy composition, and no other doses of the gene therapy composition are administered within that one-year time window.

[0099] In some embodiments, the total volume of a single dose is 1 ml or less. In other embodiments, the total volume of a single dose is approximately 1 ml to approximately 5 ml. In some embodiments, the total volume of a single dose is approximately 1 ml, approximately 2 ml, approximately 3 ml, approximately 4 ml, or approximately 5 ml.

[0100] The methods and compositions of this disclosure are, in some embodiments, used to maintain the weight loss of a subject, for example, a subject who has undergone weight loss therapy (e.g., weight loss medication such as semaglutide or treatment with a specific diet) but has discontinued the use of such therapy. Maintaining weight loss, in some embodiments, refers to the process of successfully losing weight over a long period of time, for example, 6 months, 12 months, or longer (e.g., several years or a lifetime), without regaining the lost weight. Maintaining weight can be as difficult as the weight loss process itself, due to various physiological, psychological, and environmental factors. The methods and compositions of this disclosure are, in some embodiments, used to maintain (e.g., ±1-5%, ±1-4%, ±1-3%, or ±1-2%) or further reduce (e.g., 1-5% of total body weight) the weight of a subject who has already lost weight.

[0101] In some embodiments, the gene therapy composition (e.g., a single dose of the gene therapy composition) is delivered to the subject's pancreatic endocrine tissue (e.g., directly to the pancreatic tail) in an amount effective in reducing the subject's body weight (e.g., baseline) 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, for example, 3, 6, 9, 12, or 18 months. The percentage of weight loss and the length of time to weight loss depend at least in part on the subject's baseline (initial) body weight.

[0102] In some embodiments, the gene therapy composition (e.g., as a single dose or a gene therapy composition in two or fewer doses) is delivered to the target pancreatic endocrine tissue (e.g., near the tail of the pancreas) in an amount effective in reducing the subject's body weight (relative to baseline) by at least 1%, at least 2%, at least 3%, or at least 4% within 3, 6, 9, 12, or 18 months, for example, after receiving a single or first dose of the gene therapy composition. In some embodiments, the gene therapy composition (e.g., as a single dose or a gene therapy composition in two or fewer doses) is delivered to the target pancreatic endocrine tissue (e.g., near the tail of the pancreas) in an amount effective in reducing the subject's body weight (relative to baseline) by at least 5% within 3, 6, 9, 12, or 18 months, for example, after receiving a single or first dose of the gene therapy composition. In some embodiments, a gene therapy composition (e.g., as a single dose or a gene therapy composition in two or fewer doses) is delivered to the target pancreatic endocrine tissue (e.g., near the tail of the pancreas) in an amount effective in reducing the target's body weight (relative to baseline) by at least 10% within, for example, 3, 6, 9, 12, or 18 months after receiving a single or first dose of the gene therapy composition. In some embodiments, the weight loss is maintained for at least 3 months (e.g., within 1-2% of the weight loss). In some embodiments, the weight loss is maintained for at least 6 months (e.g., within 1-2% of the weight loss). In some embodiments, the weight loss is maintained for at least 9 months (e.g., within 1-2% of the weight loss). In some embodiments, the weight loss is maintained for at least 12 months (e.g., within 1-2% of the weight loss). In some embodiments, the weight loss is maintained for at least 18 months (e.g., within 1-2% of the weight loss).

[0103] In other embodiments, the subject's body weight is reduced 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% compared to a control (e.g., without gene therapy or semaglutide) or baseline. In some embodiments, the subject's body weight decreases by approximately 5–30%, 5–25%, 5–20%, 5–15%, 5–10%, 10–30%, 10–25%, 10–20%, 10–15%, 15–30%, 15–25%, 15–20%, 20–30%, 20–25%, or 25–30% compared to a control (e.g., without gene therapy or semaglutide) or baseline.

[0104] In some embodiments, the subject's weight does not change significantly compared to baseline. In some embodiments, the subject's weight changes by less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to baseline. In some embodiments, the subject's weight remains the same compared to baseline.

[0105] In some embodiments, the weight gain by the subject is reduced 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% compared to a control (e.g., without gene therapy or semaglutide) or baseline. In some embodiments, the weight gain by a subject is approximately 5–30%, 5–25%, 5–20%, 5–15%, 5–10%, 10–30%, 10–25%, 10–20%, 10–15%, 15–30%, 15–25%, 15–20%, 20–30%, 20–25%, or 25–30% lower than a control (e.g., without gene therapy or semaglutide) or baseline. In some embodiments, the weight gain by a subject is approximately 5%, 10%, 20%, or 25% lower than a control (e.g., without gene therapy or semaglutide) or baseline.

[0106] In some embodiments, the effective dose increases the lean body mass of the subject. In some embodiments, the lean body mass of the subject is measured as a percentage of the subject's body weight. In some embodiments, the gene therapy composition (e.g., as a single-dose gene therapy composition) is delivered to the subject's pancreatic endocrine tissue (e.g., directly to the pancreatic tail) in an amount effective in increasing the subject's lean body mass. The subject's lean body mass may be determined by any method known in the art, for example, using dual-energy X-ray absorptiometry (DEXA). In some embodiments, the effective dose increases the subject's lean body mass by 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 compared to baseline. In some embodiments, the effective amount results in lean body mass within the normal physiological range (60-90%), i.e., 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.

[0107] In some embodiments, the effective dose reduces the body fat mass of the subject. In some embodiments, the body fat mass of the subject is measured as a percentage of the subject's body weight. In some embodiments, the gene therapy composition (e.g., as a single-dose gene therapy composition) is delivered to the subject's pancreatic endocrine tissue (e.g., directly to the pancreatic tail) in an amount effective in reducing the subject's body fat mass. Body fat mass may be determined by any method known in the art, for example, using a skin sebum meter, body circumference measurement, hydrostatic pressure measurement, bioelectrical impedance, air-displaced plethysmography, and / or 3D body scan. In some embodiments, the effective dose reduces the target body fat mass by 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, compared to baseline. In some embodiments, the effective dose results in lean body mass within normal physiological values ​​(12–30%), i.e., at least 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, or more percent of body fat mass.

[0108] In some embodiments, an effective dose restores blood glucose persistence in the subject. In some embodiments, the gene therapy composition (e.g., as a single-dose gene therapy composition) is delivered to the subject's pancreatic endocrine tissue (e.g., directly to the pancreatic tail) in an amount effective in restoring blood glucose persistence in the subject. The term blood glucose persistence includes the period during which the subject's blood glucose levels are within a physiological range (e.g., "ideal blood glucose control" or "optimal blood glucose control"). According to the American Diabetes Association, the recommended Hb1c cut-off point for diagnosing diabetes is 6.5%, at which point individuals are at high risk (Gillett et al., Diabetes Care. 2009;32:1327-34). In some embodiments, the blood glucose physiological range is a glycated hemoglobin (HbA1c) 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, the effective dose results in maintaining an HbA1c level below 7%. In some embodiments, 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, optimal glycemic control is maintained without substitution and / or addition of other hypoglycemic agents.

[0109] In some embodiments, the effective dose reduces fasting blood glucose compared to baseline. In some embodiments, the gene therapy composition (e.g., as a single-dose gene therapy composition) is delivered to the target pancreatic endocrine tissue (e.g., directly to the pancreatic tail) in an amount effective to significantly reduce fasting blood glucose. In some embodiments, the gene therapy composition (e.g., as a single-dose gene therapy composition) is delivered to the target pancreatic endocrine tissue (e.g., directly to the pancreatic tail) in an amount effective to reduce fasting blood glucose by, for example, at least 10% compared to baseline. In some embodiments, the effective dose 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 compared to baseline. In some embodiments, the effective dose reduces fasting blood glucose by at least 50% compared to baseline. In some embodiments, the effective dose reduces fasting blood glucose by at least 55% compared to baseline. In some embodiments, the effective dose reduces fasting blood glucose by at least 60% compared to baseline. According to the WHO, the normal range for fasting blood glucose is 70 mg / dL to 100 mg / dL, with 100 mg / dL to 125 mg / dL indicating a prediabetic state, and fasting blood glucose above 126 mg / dL indicating diabetes in the subject (WHO, "Mean fasting blood glucose," who.int / data / gho / indicator-metadata-registry / imr-details / 2380). In some embodiments, the effective dose reduces fasting blood glucose in the 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” means the blood glucose level (concentration of glucose in venous plasma) determined when a subject fasts for at least 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24 hours or longer (without any food except water) (see, e.g., WHO, “Mean fasting blood glucose”). A reduction in blood glucose also results in additional beneficial outcomes, in some embodiments, including a reduction in the rate or progression of retinopathy, nephropathy, neuropathy, myocardial infarction, diabetes-related microvascular disease, and prevention or reduction of the development of end-stage renal disease. In some embodiments, additional possible benefits include a reduction in the rate of cognitive decline and / or a reduction in major adverse cardiovascular (CV) events (MACE), e.g., a reduction in a composite of CV death, non-fatal myocardial infarction (MI), and / or non-fatal stroke.

[0110] In some embodiments, the effective dose increases fasting insulin compared to baseline. In some embodiments, the gene therapy composition (e.g., as a single-dose gene therapy composition) is delivered to the target pancreatic endocrine tissue (e.g., directly to the pancreatic tail) in an amount effective to significantly increase fasting insulin. In some embodiments, the gene therapy composition (e.g., as a single-dose gene therapy composition) is delivered to the target pancreatic endocrine tissue (e.g., directly to the pancreatic tail) in an amount effective to increase fasting insulin by, for example, at least twice compared to baseline. In some embodiments, the effective dose increases fasting insulin by at least 1x, 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 4x, 5x, or more compared to baseline. In some embodiments, the effective dose doubles fasting insulin compared to baseline. In some embodiments, the effective dose increases fasting insulin by 2.8x, or at least 2.8x, compared to baseline. In some embodiments, the effective dose triples fasting insulin compared to baseline. As used herein, “fasting insulin” refers to the insulin level determined when a subject fasts for at least 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24 hours, or longer (without any food except water). “Baseline” refers to the level of a measurable component or characteristic of a subject (e.g., insulin, blood glucose, body weight, etc.) before initiating treatment with a topical gene therapy such as those provided herein.

[0111] In some embodiments, the effective dose significantly improves glucose tolerance compared to baseline. In some embodiments, the gene therapy composition (e.g., as a single-dose gene therapy composition) is delivered to the subject's pancreatic endocrine tissue (e.g., directly to the pancreatic tail) in an amount effective in significantly improving glucose tolerance compared to baseline. Glucose tolerance refers to the subject's ability to control plasma glucose levels and / or plasma insulin levels when glucose intake changes. Glucose tolerance may be measured using any method in the art, including an oral glucose tolerance test (OGTT), such as a glucose challenge test, in which the subject drinks a glass of concentrated glucose solution (e.g., 50 g of glucose dissolved in 250-300 ml of water) and the subject's blood glucose level is measured in the blood at least one hour later. In some embodiments, glucose tolerance is measured by comparing the fasting blood glucose level to the blood glucose level 1-3 hours after ingestion of 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 to 199 mg / dL indicates prediabetes, and 200 mg / dL or higher indicates diabetes (diabetes.org / diabetes / a1c / diagnosis). In some embodiments, subjects have blood glucose levels 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 lower. In some embodiments, the subject's blood glucose level is less than 140 mg / dL after treatment. In some embodiments, the target blood glucose level decreases by 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 compared to baseline.

[0112] In some embodiments, the effective dose significantly improves glucose-stimulated insulin secretion compared to baseline. In some embodiments, the gene therapy composition (e.g., as a single-dose gene therapy composition) is delivered to the target pancreatic endocrine tissue (e.g., directly to the pancreatic tail) in an amount effective in significantly improving glucose-stimulated insulin secretion compared to baseline. Glucose-stimulated insulin secretion (GSIS) can be measured using any method known in the art, e.g., the hyperinsulinemia-hyperglycemia clamp method, the hyperglycemia clamp method, or by estimation from surrogate measures of insulin sensitivity (e.g., intravenous glucose tolerance test data, fasting blood samples, and quantitative insulin sensitivity check indices). In some embodiments, GSIS increases from baseline after treatment. In some embodiments, GSIS increases by at least 1x, 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 4x, 5x, or more compared to the baseline.

[0113] In some embodiments, the effective dose significantly improves liver triglyceride levels compared to baseline. In some embodiments, the gene therapy composition (e.g., as a single-dose gene therapy composition) is delivered to the target pancreatic endocrine tissue (e.g., directly to the pancreatic tail) in an amount effective to significantly improve liver triglyceride levels compared to baseline. Liver triglycerides can be measured using any method known in the art, for example, by magnetic resonance imaging proton density lipid fraction (MRI-PDFF). In some embodiments, liver triglycerides are reduced by, for example, at least 10% compared to baseline. In some embodiments, the effective dose 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 compared to baseline. In some embodiments, the effective dose reduces liver triglycerides by at least 30% compared to baseline. In some embodiments, the effective dose reduces liver triglycerides by at least 35% compared to baseline. In some embodiments, the effective dose reduces liver triglycerides by at least 40% compared to baseline. In some embodiments, the effective dose reduces liver triglycerides by at least 45% compared to baseline. In some embodiments, the effective dose reduces liver triglycerides by at least 50% compared to baseline. In some embodiments, the effective dose reduces liver triglycerides by at least 55% compared to baseline. In some embodiments, the effective dose reduces liver triglycerides by at least 60% compared to baseline. In some embodiments, the effective dose reduces liver triglycerides by at least 65% compared to baseline. In some embodiments, the effective dose reduces liver triglycerides by at least 70% compared to baseline.

[0114] In some embodiments, the effective dose reduces the target leptin level (e.g., plasma leptin level) compared to baseline. In some embodiments, the gene therapy composition (e.g., as a single-dose gene therapy composition) is delivered to the target pancreatic endocrine tissue (e.g., directly to the pancreatic tail) in an amount effective in reducing the target leptin level (e.g., plasma leptin level). The target leptin level can be determined by any method known in the art, for example, by the use of an immunoassay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). In some embodiments, the effective dose reduces the target leptin level 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 dose results in leptin serum levels 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.

[0115] In some embodiments, the total cholesterol level of the subject is reduced compared to baseline (e.g., by 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more compared to baseline). In some embodiments, the gene therapy composition (e.g., as a single-dose gene therapy composition) is delivered to the subject's pancreatic endocrine tissue (e.g., directly to the pancreatic tail) in an amount effective in reducing the subject's total cholesterol level. The subject's total cholesterol level can be determined by any method known in the art, for example, using a blood test. In some embodiments, the effective amount reduces the subject's total cholesterol level to a physiological level (e.g., less than 239 mg / dL or less than 200 mg / dL). For several applications, the effective doses are 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, and 220 mg / dL. This results in total cholesterol levels of 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, less than 200 mg / dL, or less than 199 mg / dL.

[0116] In some embodiments, the effective amount increases the level of high-density lipoprotein (HDL) in the subject (e.g., 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more compared to the baseline). For example, in some embodiments, the effective amount increases the subject's HDL level to more 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 more than 60 mg / dL.

[0117] "Baseline" refers to the level (e.g., blood glucose level) of the subject before starting treatment, for example, before receiving a dose of the gene therapy composition.

[0118] In some embodiments, the effective amount of the gene therapy composition is about 5×10 12 ~ about 1.5×10 14 vector genomes (VG), for example, AAV VG. For example, the effective amount is about 5×10 12 , 5.5×10 12 , 6×10 12 , 6.5×10 12 , 7×10 <故 12 , 7.5×10 12 , 8×10 12 , 8.5×10 12 , 9×10 12 , 9.5×10 12 , 1×10 13 , 1.5×10 13 , 2×10 13 , 2.5×10 13 , 3×10 13 , 3.5×10 13 , 4×10 13 , 4.5×10 13 , 5×10 13 , 5.5×10 It should be noted that there is an error in the original text at "故0000012", which is likely a misrepresentation. I have translated it as is while pointing out this potential error.13 , 6×10 13 , 6.5×10 13 , 7×10 13 , 7.5×10 13 , 8×10 13 , 8.5×10 13 , 9×10 13 , 9.5×10 13 , 1 x 10 14 , or 1.5 × 10 14 It is possible. In some embodiments, the effective amount is about 5 × 10 12 ~Approx. 1×10 13 It is VG. In some embodiments, the effective amount is about 5 × 10 12 ~Approx. 5×10 13 It is VG. In some embodiments, the effective amount is about 5 × 10 12 ~Approx. 1×10 14 It is VG. In some embodiments, the effective amount is about 1 × 10⁻⁶. 13 ~Approx. 5×10 13 It is VG. In some embodiments, the effective amount is about 1 × 10⁻⁶. 13 ~Approx. 1×10 14 It is VG.

[0119] In some embodiments, the effective dose of the gene therapy composition is a single dose, for example, about 5 × 10⁻⁶ 12 ~Approx. 1.5×10 14 This is a single dose of a vector genome (VG), such as AAV VG. For example, the effective dose is approximately 5 × 10⁻⁶. 12 , 5.5×10 12 , 6×10 12 , 6.5×10 12 , 7×10 12 , 7.5×10 12 , 8×10 12 , 8.5×10 12 , 9×10 12 , 9.5×10 12 , 1 x 10 13 , 1.5×10 13 , 2×10 13 , 2.5×10 13 , 3 x 10 13 , 3.5×10 13 , 4×10 13 , 4.5×1013 , 5×10 13 , 5.5×10 13 , 6×10 13 , 6.5×10 13 , 7×10 13 , 7.5×10 13 , 8×10 13 , 8.5×10 13 , 9×10 13 , 9.5×10 13 , 1×10 14 , or 1.5×10 14 may be a single dose. In some embodiments, the effective amount is from about 5×10 12 to about 1×10 13 VG per single dose. In some embodiments, the effective amount is from about 5×10 12 to about 5×10 13 VG per single dose. In some embodiments, the effective amount is from about 5×10 12 to about 1×10 14 VG per single dose. In some embodiments, the effective amount is from about 1×10 13 to about 5×10 13 VG per single dose. In some embodiments, the effective amount is from about 1×10 13 to about 1×10 14 VG per single dose.

[0120] In some embodiments, in a subject requiring treatment for a metabolic disorder, a method for treating the metabolic disorder includes delivering an effective amount of a gene therapy composition to the endocrine tissue of the pancreatic lobe of the subject's pancreas using an endoscopic ultrasound-guided fine needle injection (EUS-FNI) technique, the gene therapy composition includes an adeno-associated virus (AAV) vector encoding a human GLP-1 receptor agonist, and the effective amount is from about 5×10 12 to about 1.5×10 14 vector genomes (VG) per single dose. For example, the effective amount is about 5×10 12 , 5.5×10 12 , 6×10 12 , 6.5×10 12 , 7×10 12 , 7.5×10 12 , 8×10 12 , 8.5×1012 , 9×10 12 , 9.5×10 12 , 1 x 10 13 , 1.5×10 13 , 2×10 13 , 2.5×10 13 , 3 x 10 13 , 3.5×10 13 , 4×10 13 , 4.5×10 13 , 5×10 13 , 5.5×10 13 , 6×10 13 , 6.5×10 13 , 7×10 13 , 7.5×10 13 , 8×10 13 , 8.5×10 13 , 9×10 13 , 9.5×10 13 , 1 x 10 14 , or 1.5 × 10 14 This could be a single dose.

[0121] In some embodiments, in subjects requiring treatment for obesity, a method for treating obesity comprises delivering an effective amount of a gene therapy composition to the endocrine tissue of the pancreatic lobe of the subject's pancreas using an endoscopic ultrasound-guided fine-needle infusion (EUS-FNI) technique, wherein the gene therapy composition comprises an adeno-associated virus (AAV) vector encoding a human GLP-1 receptor agonist, and the effective amount is approximately 5 × 10⁻⁶ 12 ~Approx. 1.5×10 14 This is a single dose of vector genome (VG). For example, the effective dose is approximately 5 × 10⁻⁶. 12 , 5.5×10 12 , 6×10 12 , 6.5×10 12 , 7×10 12 , 7.5×10 12 , 8×10 12 , 8.5×10 12 , 9×10 12 , 9.5×10 12 , 1 x 10 13 , 1.5×10 13 , 2×10 13 , 2.5×10 13 , 3 x 1013 , 3.5×10 13 , 4×10 13 , 4.5×10 13 , 5×10 13 , 5.5×10 13 , 6×10 13 , 6.5×10 13 , 7×10 13 , 7.5×10 13 , 8×10 13 , 8.5×10 13 , 9×10 13 , 9.5×10 13 , 1 x 10 14 , or 1.5 × 10 14 This could be a single dose.

[0122] In some embodiments, in subjects requiring treatment for obesity, a method for treating obesity comprises delivering an effective amount of a gene therapy composition to the endocrine tissue of the pancreatic lobe of the subject's pancreas using an endoscopic ultrasound-guided fine-needle infusion (EUS-FNI) technique, wherein the gene therapy composition comprises an adeno-associated virus (AAV) vector encoding a human GLP-1 receptor agonist, and the effective amount is approximately 5 × 10⁻⁶ 12 ~Approx. 1.5×10 14 This is a single dose of vector genome (VG). For example, the effective dose is approximately 5 × 10⁻⁶. 12 , 5.5×10 12 , 6×10 12 , 6.5×10 12 , 7×10 12 , 7.5×10 12 , 8×10 12 , 8.5×10 12 , 9×10 12 , 9.5×10 12 , 1 x 10 13 , 1.5×10 13 , 2×10 13 , 2.5×10 13 , 3 x 10 13 , 3.5×10 13 , 4×10 13 , 4.5×10 13 , 5×10 13 , 5.5×10 13 , 6×10 13 , 6.5×1013 , 7×10 13 , 7.5×10 13 , 8×10 13 , 8.5×10 13 , 9×10 13 , 9.5×10 13 , 1 x 10 14 , or 1.5 × 10 14 This could be a single dose.

[0123] In some embodiments, the serum lipase level in the subject is within three times the upper limit of the normal serum lipase level on days 1 to 7 after delivery of the gene therapy composition. The normal range for adults under 60 years of age is typically about 10 to 140 U / L. The normal range for adults 60 years of age and older is typically 24 to 151 U / L. In some embodiments, the serum lipase level in the subject is less than 5 U / L on days 1 to 7 after delivery of the gene therapy composition. Serum lipase is an enzyme produced by the pancreas that is involved in the digestion of fats. In pancreatitis, the pancreas becomes inflamed and damaged, and serum lipase and other enzymes leak into the bloodstream. Elevated serum lipase is seen in most cases of acute pancreatitis and can help confirm the diagnosis. In fact, serum lipase levels are often more sensitive and specific to pancreatitis than other diagnostic tests such as serum amylase levels. The in vivo data provided herein demonstrate that the methods described herein have minimal effect on serum lipase levels.

[0124] In some embodiments, human GLP-1 receptor agonists are present in the pancreas at levels 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 levels detected in the serum of the subject. In some embodiments, human GLP-1 receptor agonists are present in the pancreas at levels 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 levels detected in the brain of the subject. In some embodiments, human GLP-1 receptor agonists are undetectable in the serum of the subject. In some embodiments, human GLP-1 receptor agonists are undetectable in the brain of the subject.

[0125] In some embodiments, an effective dose of the gene therapy composition results in a subject with type 2 diabetes in remission (i.e., the subject maintains physiological blood glucose levels). In some embodiments, the effective dose is a single dose, two doses, three doses, four doses, five doses, six doses, or more. In some embodiments, the effective dose is sufficient for long-term recovery of islet beta cell function and / or reduction of the treatment burden (e.g., the workload of health management experienced by the subject and its impact on the subject's well-being). In some embodiments, “long-term recovery” means one, two, three, four, five, six, seven, eight, nine, ten years, or longer, and includes complete and permanent remission.

[0126] 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 amylin mimetic drugs, alpha-glucosidase inhibitors, biguanides, dopamine agonists, dipeptidyl peptidase-4 (DPP-4) inhibitors, GLP-1 receptor agonists, meglitinide, statins, sodium-glucose transporter (SGLT)2 inhibitors, sulfonylureas, thiazolidinediones, insulin, and combinations thereof. In some embodiments, no additional treatments are administered to the subject.

[0127] In some embodiments, the gene therapy composition comprises inactive or active excipients and / or carriers to make the composition particularly suitable for in vivo or ex vivo therapeutic use. The pharmaceutically acceptable excipients and / or carriers do not cause undesirable physiological effects after or when administered to the subject.

[0128] The subjects are typically humans, but they can be any mammal, including non-human primates.

[0129] In preferred embodiments, the gene therapy compositions described herein are administered topically, but other routes may be used. These include, but are not limited to, intradermal, intramuscular, intranasal, and / or subcutaneous administration. In some embodiments, the gene therapy composition is delivered topically (e.g., to the splenic lobe / tail of the pancreas) rather than systemically. In some embodiments, the gene therapy composition is delivered to islet cells (e.g., islet beta cells). In some embodiments, the gene therapy composition is delivered to islet beta cells via endoscopic treatment such as EUS-FNI.

[0130] This disclosure also envisions a combination therapy using the REVITA® system, which is, for example, minimally invasive, outpatient, endoscopic, and a one-time procedure. The REVITA® system includes a specially designed control console and a new disposable balloon catheter. The console is used to monitor the procedure, and the physician uses the catheter to apply heat to the duodenum. REVITA® may be used as an adjunctive combination therapy in some embodiments.

[0131] Additional Embodiments This disclosure also relates to additional embodiments described in the following numbered paragraphs.

[0132] 1. A method for treating type 2 diabetes in a person requiring treatment for type 2 diabetes, The procedure comprises delivering an effective dose of a gene therapy composition to the endocrine tissue of the tail and / or body of the pancreas of the subject using an endoscopic ultrasound-guided fine-needle infusion (EUS-FNI) technique, wherein the gene therapy composition comprises an adeno-associated virus (AAV) vector genome encoding a human GLP-1 receptor agonist, and optionally, the effective dose is approximately 5 × 10⁻⁶ 12 ~Approx. 1.5×10 14 The method, wherein the AAV vector genome (VG) is a single dose.

[0133] 2. The effective amount is approximately 1 × 10 13 ~Approx. 5×10 13 The method according to paragraph 1, which is a single dose of VG.

[0134] 3. The method according to paragraph 1 or 2, wherein the total volume of the single dose is 1 ml or less.

[0135] 4. The method according to any one of paragraphs 1 to 3, wherein the total volume of the single dose is approximately 1 ml to approximately 5 ml.

[0136] 5. The method according to any one of the preceding paragraphs, wherein the single dose is delivered via a single injection.

[0137] 6. The method according to any one of the preceding paragraphs, wherein the single dose is delivered via multiple infusions.

[0138] 7. The method according to any one of the preceding paragraphs, wherein the AAV vector genome is operably linked to a human GLP-1 receptor agonist coding sequence and comprises a human islet beta cell-specific promoter, optionally a human insulin promoter.

[0139] 8. The method according to any one of the preceding paragraphs, wherein the EUS-FNI procedure comprises advancing the deposition device, which has a fine needle at the distal portion of the deposition device, to a pancreatic deposition site within the pancreas, and delivering the gene therapy composition to the pancreatic deposition site through the fine needle.

[0140] 9. The method according to any one of the preceding paragraphs, wherein the distal end of the deposition device enters the mouth of the target and is advanced through the wall of the gastrointestinal tract to a position close to the pancreas, and optionally (a) the deposition device is delivered through the working channel of a gastrointestinal endoscope delivered through the mouth of the target, and (b) the deposition device is delivered together with a gastrointestinal endoscope delivered through the mouth of the target.

[0141] 10. The method according to any one of the preceding paragraphs, wherein the pancreatic deposition site is the parenchyma of the tail and / or head of the pancreas.

[0142] 11. The method according to any one of the preceding paragraphs, wherein the gene therapy composition is delivered to the pancreatic deposition site through the fine needle at a pressure of at least 3 mmHg and / or 25 mmHg or less.

[0143] 12. The method according to any one of the preceding paragraphs, wherein the gene therapy composition is delivered to the pancreatic deposition site through the fine needle at a flow rate of at least 1 ml / min and / or 5 ml / min or less.

[0144] 13. The method according to any one of the preceding paragraphs, further comprising delivering a permeability enhancer before and / or simultaneously with the delivery of the gene therapy composition, optionally, the delivery of the permeability enhancer is performed locally and / or intravenously, optionally, the permeability enhancer comprises an agent selected from the group consisting of hyaluronidase, collagenase, losartan, and combinations thereof, and optionally, the therapeutic agent comprises a co-formulation of the gene therapy composition and the permeability enhancer.

[0145] 14. The method according to any one of paragraphs 7 to 13, further comprising heating tissue adjacent to the pancreatic deposition site to a temperature above 39°C before, during, and / or after the delivery of the gene therapy composition.

[0146] 15. The method according to any one of the preceding paragraphs, further comprising delivering a seeding blockage material configured to prevent undesirable seeding of the gene therapy composition at non-target sites, wherein the seeding blockage material optionally comprises a viscous substance and / or a polymer.

[0147] 16. The method according to any one of the preceding paragraphs, further comprising positioning a blocking element in the subject, wherein the blocking element is configured to prevent undesirable seeding of the gene therapy composition at non-target sites.

[0148] 17. The method according to any one of the preceding paragraphs, wherein the serum lipase level in the subject is less than 5 U / L on days 1 to 7 after delivery of the gene therapy composition.

[0149] 18. The method according to any one of the preceding paragraphs, wherein less than one vector copy per diploid genome of the AAV vector genome is detectable in the liver, heart, spleen, or kidney of the subject 3 to 4 weeks after delivery of the gene therapy composition.

[0150] 19. The method according to any one of the preceding paragraphs, wherein more than one, more than two, or more than three vector copies per diploid genome of the AAV vector genome are detectable in the liver, heart, spleen, or kidney of the subject 3 to 4 weeks after delivery of the gene therapy composition.

[0151] 20. The method according to any one of the preceding paragraphs, wherein approximately 1 to 5 vector copies per diploid genome of the AAV vector genome are detectable in the liver, heart, spleen, or kidney of the subject 3 to 4 weeks after delivery of the gene therapy composition.

[0152] 21. The method according to any one of the preceding paragraphs, wherein the human GLP-1 receptor agonist is not detectable in the brain and / or serum of the subject.

[0153] 22. The method according to any one of the preceding paragraphs, wherein the effective amount restores blood glucose persistence in the subject.

[0154] 23. The method according to any one of the preceding paragraphs, wherein the effective dose reduces fasting blood glucose by at least 50% compared to baseline.

[0155] 24. The method according to any one of the preceding paragraphs, wherein the effective dose increases fasting insulin by at least twofold compared to baseline.

[0156] 25. The method according to any one of the preceding paragraphs, wherein the effective dose significantly improves glucose tolerance compared to baseline.

[0157] 26. The method according to any one of the preceding paragraphs, wherein the effective dose significantly improves glucose-stimulated insulin secretion compared to baseline.

[0158] 27. The method according to any one of the preceding paragraphs, wherein the subject's weight does not change significantly compared to baseline.

[0159] 28. The method according to any one of the preceding paragraphs, wherein the single dose is sufficient for long-term restoration of pancreatic islet beta cell function.

[0160] 29. The method according to any one of the preceding paragraphs, wherein the single dose is sufficient to reduce the therapeutic burden.

[0161] 30. The method according to any one of the preceding paragraphs, wherein at least 15% of the endocrine tissue is transduced with the AAV vector.

[0162] 31. A method for treating obesity in a person who requires treatment for obesity, The procedure comprises delivering an effective dose of a gene therapy composition to the endocrine tissue of the tail and / or body of the pancreas of the subject using an endoscopic ultrasound-guided fine-needle infusion (EUS-FNI) technique, wherein the gene therapy composition comprises an adeno-associated virus (AAV) vector encoding a human GLP-1 receptor agonist, and optionally, the effective dose is approximately 5 × 10⁻⁶ 12 ~Approx. 1.5×10 14 The method, wherein the vector genome (VG) is a single dose.

[0163] 32. The effective amount is approximately 1 × 10 13 ~Approx. 5×10 13 The method according to paragraph 31, which is a single dose of VG.

[0164] 33. The method according to paragraph 31 or 32, wherein the total volume of the single dose is 1 ml or less.

[0165] 34. The method according to any one of paragraphs 31 to 33, wherein the total volume of the single dose is approximately 1 ml to approximately 5 ml.

[0166] 35. The method according to any one of the preceding paragraphs, wherein the single dose is delivered via a single injection.

[0167] 36. The method according to any one of the preceding paragraphs, wherein the single dose is delivered via multiple infusions.

[0168] 37. The method according to any one of the preceding paragraphs, wherein the AAV vector genome comprises a human islet beta cell-specific promoter operably linked to a human GLP-1 receptor agonist coding sequence.

[0169] 38. The method according to any one of the preceding paragraphs, wherein the EUS-FNI procedure comprises advancing a deposition device, which has a fine needle at the distal portion of the deposition device, to a pancreatic deposition site within the pancreas, and delivering the gene therapy composition to the pancreatic deposition site through the fine needle.

[0170] 39. The method according to any one of the preceding paragraphs, wherein the distal end of the deposition device enters the mouth of the target and is advanced through the wall of the gastrointestinal tract to a position close to the pancreas, and optionally (a) the deposition device is delivered through the working channel of a gastrointestinal endoscope delivered through the mouth of the target, and (b) the deposition device is delivered together with a gastrointestinal endoscope delivered through the mouth of the target.

[0171] 40. The method according to any one of the preceding paragraphs, wherein the pancreatic deposition site is the parenchyma of the tail and / or head of the pancreas.

[0172] 41. The method according to any one of the preceding paragraphs, wherein the gene therapy composition is delivered to the pancreatic deposition site through the fine needle at a pressure of at least 3 mmHg and / or 25 mmHg or less.

[0173] 42. The method according to any one of the preceding paragraphs, wherein the gene therapy composition is delivered to the pancreatic deposition site through the fine needle at a flow rate of at least 1 ml / min and / or 5 ml / min or less.

[0174] 43. The method according to any one of the preceding paragraphs, further comprising delivering a permeability enhancer before and / or simultaneously with the delivery of the gene therapy composition, optionally, the delivery of the permeability enhancer is performed locally and / or intravenously, optionally, the permeability enhancer comprises an agent selected from the group consisting of hyaluronidase, collagenase, losartan, and combinations thereof, and optionally, the therapeutic agent comprises a co-formulation of the gene therapy composition and the permeability enhancer.

[0175] 44. The method according to any one of paragraphs 38 to 43, further comprising heating tissue adjacent to the pancreatic deposition site to a temperature above 39°C before, during, and / or after delivery of the gene therapy composition.

[0176] 45. The method according to any one of the preceding paragraphs, further comprising delivering a seeding blockage material configured to prevent undesirable seeding of the gene therapy composition at non-target sites, wherein the seeding blockage material optionally comprises a viscous substance and / or a polymer.

[0177] 46. ​​The method according to any one of the preceding paragraphs, further comprising positioning a blocking element in the subject, wherein the blocking element is configured to prevent undesirable seeding of the gene therapy composition at non-target sites.

[0178] 47. The method according to any one of the preceding paragraphs, wherein the serum lipase level in the subject is less than 5 U / L on days 1 to 7 after delivery of the gene therapy composition.

[0179] 48. The method according to any one of the preceding paragraphs, wherein less than one vector copy per diploid genome of the AAV vector genome is detectable in the liver, heart, spleen, or kidney of the subject 3 to 4 weeks after delivery of the gene therapy composition.

[0180] 49. More than 1, more than 2, or more than 3 vector copies per diploid genome of the AAV vector genome are detectable in the liver, heart, spleen, or kidney of the subject 3 to 4 weeks after delivery of the gene therapy composition, according to any one of the preceding paragraphs.

[0181] 50. 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 3 to 4 weeks after delivery of the gene therapy composition, according to any one of the preceding paragraphs.

[0182] 51. Is the human GLP-1 receptor agonist not detectable in the brain and / or serum of the subject, or is the human GLP-1 receptor agonist present in the pancreas at a level at least 50% higher than the level detected in the brain and / or the serum of the subject, according to any one of the preceding paragraphs.

[0183] 52. A method according to any one of the preceding paragraphs, (a) the weight of the subject decreases by at least 5%, at least 10%, at least 15%, at least 20%, or at least 25% compared to the baseline, or the weight of the subject decreases by about 5 - 25% compared to the baseline, or (b) the weight gain by the subject decreases by at least 5%, at least 10%, at least 15%, at least 20%, or at least 25% compared to the control, or the weight gain by the subject decreases by about 5 - 25%, according to any one of the preceding paragraphs.

[0184] 53. The method according to any one of the preceding paragraphs, wherein the effective amount restores glycemic persistence in the subject.

[0185] 54. The method according to any one of the preceding paragraphs, wherein the effective amount reduces fasting blood glucose by at least 50% compared to the baseline.

[0186] 55. The method according to any one of the preceding paragraphs, wherein the effective dose increases fasting insulin by at least twofold compared to baseline.

[0187] 56. The method according to any one of the preceding paragraphs, wherein the effective dose significantly improves glucose tolerance compared to baseline.

[0188] 57. The method according to any one of the preceding paragraphs, wherein the effective dose significantly improves glucose-stimulated insulin secretion compared to baseline.

[0189] 58. The method according to any one of the preceding paragraphs, wherein the single dose is sufficient for long-term restoration of pancreatic islet beta cell function.

[0190] 59. The method according to any one of the preceding paragraphs, wherein the single dose is sufficient to reduce the therapeutic burden.

[0191] 60. The method according to any one of the preceding paragraphs, wherein at least 15% of the endocrine tissue is transduced with the AAV vector.

[0192] 61. The method according to any one of the preceding paragraphs, wherein the subject has a body mass index (BMI) between 25.0 and less than 30.

[0193] 62. The method according to any one of the preceding paragraphs, wherein the subject has a BMI of 30.0 or higher, and optionally has a BMI of 30 to less than 35 (Class 1), 35 to less than 40 (Class 2), or 40 or higher (Class 3).

[0194] 63. An adeno-associated virus (AAV) vector genome as described in any one of the preceding paragraphs, An adeno-associated virus (AAV) vector genome as described in any one of the preceding paragraphs, comprising a 5' inverted terminal repeat (ITR) sequence, a 5' untranslated region (UTR), an open reading frame encoding a GLP-1 receptor agonist, an insulin gene promoter and enhancer element operably linked to a nucleic acid including a 3' UTR, a polyadenylation signal, and a 3' ITR.

[0195] 64. The AAV vector genome according to Embodiment 63, wherein the insulin gene promoter is a human insulin gene promoter.

[0196] 65. The AAV vector genome described in paragraph 63 or 64, wherein the insulin gene promoter is the rat insulin gene promoter.

[0197] 66. An AAV vector genome according to any one of paragraphs 63 to 65, wherein the enhancer element is a cytomegalovirus enhancer element.

[0198] 67. An AAV vector genome as described in any one of paragraphs 63-66, wherein the 5'UTR contains a modified human hemoglobin subunit beta-intron.

[0199] 68. An AAV vector genome as described in any one of paragraphs 63 to 67, wherein the GLP-1 receptor agonist is human GLP-1.

[0200] 69. An AAV vector genome as described in any one of paragraphs 63-68, wherein the GLP-1 receptor agonist is fused to a signal peptide.

[0201] 70. An AAV vector genome as described in 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).

[0202] 71. The AAV vector genome according to any one of paragraphs 63 to 70, wherein the polyadenylation signal is a bovine growth hormone polyadenylation signal.

[0203] 72. The AAV vector genome according to any one of paragraphs 63 to 71, wherein the AAV vector genome is a single-stranded AAV vector genome.

[0204] 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 the pancreatic endocrine tissue of the subject in an amount effective to maintain a weight loss of about 5% over one year, the gene therapy composition comprising an adeno-associated virus (AAV) vector genome comprising a pancreatic beta cell-specific promoter operably linked to a GLP-1 receptor agonist coding region.

[0205] 74. A method for reducing low-density lipoprotein (LDL) and / or total cholesterol in a subject, the method comprising delivering the gene therapy composition in a dose of 2 or less to the pancreatic endocrine tissue of the subject in an amount effective to maintain a weight loss of about 5% over one year, the gene therapy composition comprising an adeno-associated virus (AAV) vector genome comprising a pancreatic beta cell-specific promoter operably linked to a GLP-1 receptor agonist coding region.

[0206] 75. The method according to paragraph 73 or 74, wherein the single dose comprises about 5×10 12 ~ about 1.5×10 14 of AAV vector genome (VG).

[0207] 76. The method according to any one of paragraphs 73 to 75, wherein the single dose comprises about 1×10 13 ~ about 5×10 13 of AAV vector genome (VG).

[0208] 77. The method according to any one of paragraphs 73 to 76, wherein the total volume of the single dose is approximately 1 ml to approximately 3 ml.

[0209] 78. The method according to any one of paragraphs 73 to 77, wherein the gene therapy composition is delivered by injection.

[0210] 79. The method according to any one of paragraphs 73-78, wherein the single dose is delivered using an endoscopic ultrasound-guided fine-needle injection (EUS-FNI) procedure.

[0211] 80. The method according to any one of paragraphs 73 to 79, wherein the islet beta cell-specific promoter comprises a human insulin promoter or a region thereof.

[0212] 81. The method according to any one of paragraphs 73-80, wherein at least 15% of the endocrine tissue is transduced with the AAV vector.

[0213] 82. The AAV vector genome is The method according to any one of paragraphs 73-81, comprising a 5' inverted terminal repeat (ITR) sequence, a 5' untranslated region (UTR), an open reading frame encoding a GLP-1 receptor agonist, an insulin gene promoter and enhancer element operably linked to a nucleic acid including a 3' UTR, a polyadenylation signal, and a 3' ITR.

[0214] 83. The method according to paragraph 81 or 82, wherein the insulin gene promoter is the human insulin gene promoter or its core region.

[0215] 84. The method described in the preceding clause 82 or 83, The enhancer element is the cytomegalovirus enhancer element, or optionally the CMV upstream genome 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), optionally mut6.WPRE. The method according to paragraph 82 or 83, wherein the polyadenylation signal is a bovine growth hormone polyadenylation signal.

[0216] 85. The method according to any one of paragraphs 73 to 84, wherein the AAV vector genome is a single-stranded AAV vector genome and optionally a self-complementary AAV vector genome. [Examples]

[0217] Examples 1-2: In vivo experiments in a mouse model of type 1-2 diabetes AAV-based gene therapy candidates were tested in a db / db type 2 diabetic mouse model to determine their effects on disease progression and severity. Four-week-old db / db male mice were divided into different treatment groups (n=8 / group). On day 1, mice were treated with their respective AAV compositions (vehicle control, MIP-eGFP (10e12 VG / animal), MIP-Ex4 (2.5e12 VG / animal), or MIP-Ex4 (10e12 VG / animal)). "Ex4" refers to the exendin-4 glucagon-like peptide 1 (GLP-1) receptor agonist, and "MIP" refers to the 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 levels were measured. Insulin levels were also measured on days 1, 22, 36, 50, and 64 after the fasting period. On day 39, an intraperitoneal glucose tolerance test (IPGTT) was performed and followed for 120 minutes (0, 15, 30, 60, and 120 minutes). On day 70, tissue was collected for analysis.

[0218] Dose-dependent and sustained glycemic control was observed in db / db mice 10 weeks after injection. High-dose MIP-Ex4 treated mice showed a 59% reduction in fasting blood glucose [Δ304 mg / dL] (p<0.0001) (Figure 1A) and a 2.8-fold increase in fasting insulin (p=0.004) (Figure 1B). Significant improvements in both glucose tolerance (p<0.0001) (Figures 2A and 2B), as well as a significant improvement in glucose-stimulated insulin secretion by intraperitoneal glucose tolerance test (IPGTT) (p<0.05) (Figure 2C), were observed, with no effect on body weight (Figures 3A-3B). Immunohistochemical analysis showed that the GLP-1RA protein was expressed in the pancreas (Figure 4C) and limited to islet cells (Figures 4A and 4B).

[0219] Example 2 - Ex vivo experiments in mouse and human cells BKS db / db islet cells were isolated, cultured ex vivo, and then transduced with an AAV-based GLP-1RA construct containing exendin-4 to investigate its effect on insulin secretion. Insulin secretion was measured 4 days after transduction. Islet cells transduced with the AAV-GLP-1RA construct showed significantly higher levels of GLP-1 (Figure 5A) and glucose-stimulated insulin secretion (Figure 5B) compared to islet cells transduced with AAV-eGFP (control).

[0220] When using the human islet beta strain EndoC-BH5, AAV-mediated delivery of GLP-1RA was found to enhance insulin secretion in a GLP-1RA-dependent manner (Figure 6). In particular, in the presence of glucose, there was a significant difference in 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 the GLP-1RA antagonist exendin-9 (Ex9) peptide, there was no statistically significant difference between the groups.

[0221] Example 3 - In vivo location study BKS db / db mice were administered either AAV-MIP-Ex4 (7.5e12 VG / animal), an AAV expressing the islet beta cell restriction exendin-4 transgene, or a vehicle control (n=3 / group). "Ex4" refers to the exendin-4 glucagon-like peptide 1 (GLP-1) receptor agonist, and "MIP" refers to the mouse insulin promoter. After 4 weeks, fasting blood glucose was measured (4-6 hours after fasting), and the results are shown in Figure 7A. A significant decrease was observed in mice administered AAV-MIP-Ex4 compared to the vehicle control. In addition, serum and pancreatic exendin-4 levels were measured in each mouse, and the results are shown in Figure 7B. AAV-based exendin-4 production was not detected in the serum of any of the animal groups. However, high levels of exendin-4 were detected in the pancreas, as measured by lipid chromatography-mass spectrometry (LCMS), indicating targeted local delivery of AAV-MIP-Ex4.

[0222] Example 4 - In vivo experiment in a Yucatan pig model Three to four weeks after EUS-FNI injection of scAAV9-CMV-GFP into the pancreas of Yucatan pigs, the animals were euthanized and 8 mm biopsies were collected. 16 to 20 biopsies were analyzed from each pig, distributed across the entire target splenic lobe, as well as the duodenum and connecting lobes. GFP protein expression was detected via immunohistochemistry (IHC) analysis of the biopsies, and endocrine regions were determined via endogenous insulin protein IHC on adjacent tissue sections. Exocrine signals were determined by the percentage of GFP signal across the entire biopsy, excluding all endocrine regions defined by insulin IHC, while endocrine signals were determined by the percentage of GFP signal across 5 to 10 pancreatic islets per biopsy using the MatLab® image analysis tool. Each point in the plot represents an individual pig and is the average of either exocrine or endocrine signals across all biopsies taken from either the splenic lobe or the entire pancreas. For each AAV9 vector genome dose, the group size was 2–4 animals. Following EUS-FNI of scAAV9-CMV-GFP into the pancreas of Yucatan pigs, a significant dose-dependent increase in AAV-mediated GFP expression was observed in endocrine and exocrine tissues. See Figures 8A–8D and Figure 9.

[0223] Example 5 - In vivo vector in vivo distribution analysis Vector biodistribution analysis is performed using 5 × 10⁻⁶ vectors. 13 VG (Figure 10A) or 1.5 × 10 14Using a fixed dose of VG (Figure 10B), tissue was collected 3–4 weeks after EUS-FNI injection of AAV into the porcine pancreas by either a single or triple infusion. Vector copies per diploid genome were determined by digital PCR specific to the DNA transgene sequence within the AAV genome in (a) 7 pigs with one (n=2) or three (n=5) infusions for lower doses, or in (b) 4 pigs with one (n=2) or three (n=2) infusions for higher doses. The splenic lobe (SL) of the pancreas received an average of 14–16 biopsies per pig, the pancreatic duodenal lobe (DL) received an average of 2 biopsies per pig, the pancreatic ligamentous lobe received an average of 2–4 biopsies per pig, and other tissues received an average of 1–4 biopsies per pig.

[0224] Example 6 - In vivo toxicity study Lipase Blood was collected from the pancreas of Yucatan pigs at 0, 4, 24, 72, and 168 hours after single or triple AAV or vehicle-injected EUS-FNI, and serum was analyzed for lipase. No elevation of lipase was observed across all single injection procedures, however, a subset of animals injected at three sites showed a transient elevation of serum lipase below the threshold for acute pancreatitis (Figure 11A). In all cases, lipase levels returned to baseline within 3–7 days post-procedure.

[0225] Using a single EUS-FNI procedure, with volumes of 1 mL, 2 mL, or 5 mL at a flow rate of 0.9–1.0 mL / min, 1 × 10⁻¹⁶ 13 ~5×10 13 The VG AAV dose was delivered to the pancreas of pigs. Serum lipase levels did not rise above baseline in any of the pigs tested (Figure 11B).

[0226] Using the EUS-FNI technique, 5 × 10¹⁶ units of various AAV serotypes were injected into the pig pancreas using either a single infusion (Figure 11C) or three infusions (Figure 11D). 12 ~1.5×10 14VG AAV doses were delivered. The AAV dose did not correlate with an increase in serum lipase using either a single or triple infusion.

[0227] Neurofilament Light Chain (NFL) Using a single EUS-FNI procedure, 1 × 10 13 VG or 5x10 13 VG-mediated AAV9-CMV-eGFP (ubiquitous CMV promoter) or AAV9-INSp-eGFP (beta-cell restriction INSp promoter) were delivered to porcine pancreas. NFL levels, indicating dorsal root ganglion (DRG) toxicity, did not increase after administration of the beta-cell restriction promoter (Figure 12).

[0228] Example 7 - In vivo study of body weight, fasting blood glucose, and fasting insulin Two AAV-based GLP1RA vectors (first and second generation) containing a β-cell restriction promoter (AAV-GLP1RA) were selected for analysis in db / db mice, which are susceptible to obesity due to chronic bulimia. Over 4 weeks, 10 12 (1st generation) or 5 12 Following a single intraperitoneal injection of (second-generation) VG dose, weight gain was significantly reduced by 23% in AAV-GLP1RA-treated mice compared to vehicle-treated controls (p<0.0001) and by 19.6% compared to semaglutide (10 nmol / kg)-treated controls (Figure 13). At week 8, after 4-6 hours of fasting, gene therapy improved fasting glucose and insulin levels (Figures 14A-14B). Despite the decrease in fasting insulin with second-generation vectors, the improved blood glucose improvement suggests higher insulin sensitivity compared to first-generation vectors. GLP-1 gene therapy also shifts disease progression (Figure 15).

[0229] In further studies, 8-week-old db / db mice were administered either a single intraperitoneal injection of GLP-1RA PGTx (5e12 VG) or an AAV vehicle, or daily subcutaneous injections of semaglutide (10 nmol / kg) for 62 days. Samples were collected on days 8, 15, 22, 29, 36, 43, 50, and 57 to determine fasting blood glucose, insulin, and body weight. 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 Figures 22A-22C, demonstrating that GLP-1RA PGTx treatment improved fasting blood glucose (Figure 22A), fasting plasma insulin (Figure 22B), and reduced total body weight (Figure 22C) compared to both the semaglutide vehicle control and daily administration.

[0230] The first-generation AAV vector genome is self-complementary and drives the expression of exendin-4 (Ex4) under a mouse insulin gene promoter containing a cytomegalovirus enhancer element (CMVe). The exendin-4 transgene is generated as an exendin-4 fusion protein (mNGF-Ex4) containing a mouse nerve growth factor (mNGF) propeptide fragment and a signal peptide for protein translocation and secretion. The mNGF fragment is then cleaved, and the Ex4 molecule is available to activate GLP-1R. The first-generation AAV vector genome also contains a bovine growth hormone polyadenylation signal (bGHpA) for RNA stability.

[0231] The second-generation AAV vector genome is single-stranded and drives Ex4 expression under a rat insulin-1 gene promoter (rINS1p) with a CMV upstream genomic region (CMVugr). The exendin-4 transgene is generated as an mNGF-Ex4 fusion protein containing a signal peptide for protein translocation and secretion. The mNGF fragment is then cleaved, and the Ex4 molecule is available to activate GLP-1R. The second-generation AAV vector genome also contains a modified human hemoglobin subunit beta-intron in the 5' untranslated region (UTR), a modified woodchuck hepatitis virus post-transcriptional regulator (WPRE) element in the 3' UTR to enhance transgene expression, and a bovine growth hormone polyadenylation signal (bGHpA) for RNA stability.

[0232] Example 8 - A single dose of GLP-1-based pancreatic gene therapy induces sustained weight loss in obese mouse models. The efficacy and duration of a single-dose GLP-1-based pancreatic gene therapy (PGTx, i.e., the second-generation AAV vector genome described in Example 7) were tested in a mouse diet-induced obesity model. Briefly, C57BL / 6 mice were fed a 60% high-fat diet for 25 weeks and then randomly divided into four groups based on body weight: (1) Control group 1 received only a single intraperitoneal dose of PGTx vehicle (n=8) ("AAV vehicle"). (2) Test group 2 received a single intraperitoneal dose of PGTx (1x10). 13 (3) Group 3 received a daily subcutaneous dose of semaglutide (10 nmol / kg daily, n=5) for 28 days. On day 29, semaglutide was discontinued and only the PGTx vehicle was administered ("Sema discontinuation + vehicle"). (4) Group 4 received a daily subcutaneous dose of semaglutide (10 nmol / kg daily, n=5) for 28 days. On day 29, semaglutide was discontinued and only the PGTx vehicle was administered. 12VG (n=5) was administered ("Sema withdrawal + pssAAV.004 [5e12 VG]"). Mean body weight and food intake were measured daily for 57 days. Mean fasting peripheral liver weight, liver triglycerides, plasma leptin (as an indicator of obesity), fat mass (by EchoMRI), fasting glucose, islet and serum GLP-1 (exendin-4) protein levels, and islet histopathology scores of 0 to 5 were also evaluated.

[0233] Weight changes are shown in Figure 16A, and final weight is shown in Figure 18A. On day 28, weight decreased by 27% in test group 2 (single dose of PGTx), and by 20-21% in test groups 3 and 4 (daily semaglutide) (p<0.05). Surprisingly, weight loss was maintained for at least 57 days in test group 2 using only a single dose of PGTx (p<0.0001). The results were even more surprising in test groups 3 and 4. Without further PGTx treatment, semaglutide withdrawal in group 3 resulted in weight gain to almost baseline (-2% from baseline). In comparison, semaglutide withdrawal in Group 4, followed by a single dose of PGTx on day 29 (half the PGTx dose used in Group 2), resulted in a stabilization of weight loss at -22% below baseline (p<0.01) on day 57, as well as a significant increase in lean body mass compared to the control group, as measured by percentage of body weight (Figure 18D). Similar to fat mass (Figure 18B), mean food intake in all treatment groups (Figure 16B) correlated with weight loss or weight gain.

[0234] Liver weight and liver triglyceride levels are shown in Figure 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 as consistent as the vehicle control.

[0235] At week 8, leptin levels decreased by 67% in PGTx-Ex4 compared to the control (p<0.0001) (Figure 18C), which corresponded to a 35% reduction in body fat (r=0.93, P<0.0001). Low-dose and high-dose gene therapy resulted in PGTx-Ex4 expression in 9% and 15% of pancreatic islets, respectively (Figure 20), indicating that PGTx-Ex4 expression is restricted to the pancreatic islets. Similarly, compared to the control, dose-responsive increases of 44% and 68% were observed in serum PGTx-Ex4 levels (Figure 19B) and pancreatic PGTx-Ex4 levels (Figure 19A) (p<0.001 and p<0.0001, respectively). Serum PGTx correlated with islet expression percentage (r=0.77, p<0.0001) and weight loss percentage (r=0.75, p<0.0001). Fasting glucose decreased by 21% compared to the control group (Figure 24) (Figure 23), and HOMA-IR (Hostasis Model Assessment of Insulin Resistance) improved by 72% in the PGTx-Ex4 group (p<0.001, p<0.01). No significant evidence of inflammation was observed, and histopathology scores were less than 0.5 in all groups (data not shown).

[0236] Plasma cholesterol levels, as well as low-density lipoprotein (LDL) (Figure 21B) and high-density lipoprotein (HDL) (Figure 21C) levels, were significantly reduced after treatment with GLP-1RA PGTx compared to their respective controls (Figure 21A). Triglyceride levels did not show any statistical significance between groups (Figure 21D).

[0237] Therefore, a single dose of GLP-1-based pancreatic gene therapy can sustain weight loss without causing pancreatic inflammation, stabilize weight loss after semaglutide withdrawal, and result in improvements in liver weight and liver triglycerides. These data support GLP-1-based pancreatic gene therapy as an effective and sustained therapy for metabolic diseases (e.g., obesity and MASLD).

[0238] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter they cite, and in some cases, the entire document may be included.

[0239] The indefinite articles “a” and “an” used herein and in the claims should be understood to mean “at least one” unless explicitly indicated otherwise. Furthermore, unless explicitly indicated otherwise, it should be understood that in any method claimed herein involving multiple steps or acts, the order of the steps or acts of the method is not necessarily limited to the order in which they are described.

[0240] In the claims and in the above specification, all transitional phrases such as “comprising,” “including,” “possessing,” “having,” “containing,” “involving,” “holding,” and “composed of” should be understood as open-ended, meaning they include, but are not limited to. Only the transitional phrases “essentially from” and “essentially from” are considered restrictive or semi-restrictive transitional phrases, respectively, as described in Section 2111.03 of the U.S. Patent and Trademark Office's Examination Manual.

[0241] The terms "approximately" and "effectively" preceding a number mean ±10% of the listed number.

[0242] Where a range of values ​​is provided, each value between the upper and lower limits of the range is specifically intended and described herein.

Claims

1. A method for reducing weight in a subject, wherein the method is The method comprises delivering a single dose of a gene therapy composition to the pancreatic endocrine tissue of a subject with a metabolic disorder in an amount effective to maintain a weight loss of approximately 5% over a year, wherein the gene therapy composition comprises an adeno-associated virus (AAV) vector genome containing an islet beta cell-specific promoter operably linked to a GLP-1 receptor agonist coding region.

2. A method for reducing weight in a subject, wherein the method is The method comprises delivering a gene therapy composition in two or fewer doses to the pancreatic endocrine tissue of a subject with a metabolic disorder in an amount effective to maintain a weight loss of approximately 5% over a year, wherein the gene therapy composition comprises an adeno-associated virus (AAV) vector genome containing a pancreatic islet beta cell-specific promoter operably linked to a GLP-1 receptor agonist coding region.

3. The aforementioned single dose is approximately 5 × 10 １２ ~Approx. 1.5×10 １４ The method according to claim 1 or 2, comprising the AAV vector genome (VG).

4. The aforementioned single dose is approximately 1 × 10 １３ ~Approx. 5×10 １３ The method according to any one of the prior claims, comprising an AAV vector genome (VG).

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

6. The method according to any one of the prior claims, wherein the gene therapy composition is delivered by injection.

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

8. The method according to any one of the prior claims, wherein the islet beta cell-specific promoter comprises a human insulin promoter or a region thereof.

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

10. The effective amount is To restore blood glucose persistence in the aforementioned subjects, Compared to baseline, fasting blood glucose was significantly reduced. Compared to baseline, fasting insulin levels were significantly increased. Significantly improved glucose tolerance compared to baseline, and / or The method according to any one of the prior claims, which significantly improves glucose-stimulated insulin secretion compared to baseline.

11. The method according to any one of the prior claims, wherein the single dose is sufficient for long-term recovery of islet beta cell function and / or reduction of the therapeutic burden.

12. The method according to any one of the prior claims, wherein the serum lipase level in the subject is within three times the upper limit of the normal serum lipase level on days 1 to 7 after delivery of the gene therapy composition.

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

14. The method according to any one of the prior claims, wherein less than one vector copy per diploid genome of the AAV vector genome is detectable in the target liver, heart, spleen, or kidney three to four weeks after delivery of the gene therapy composition.

15. The method according to any one of the prior claims, wherein approximately 1 to 5 vector copies per diploid genome of the AAV vector genome are detectable in the target liver, heart, spleen, or kidney 3 to 4 weeks after delivery of the gene therapy composition.

16. The method according to any one of the prior claims, wherein the effective amount significantly reduces the liver weight and / or liver triglycerides of the subject compared to baseline.

17. The effective amount is (a) Significantly reduces the body fat mass of the subject compared to baseline, and / or (b) The method according to any one of the prior claims, which significantly increases the lean body mass of the subject compared to baseline.

18. The method according to any one of the prior claims, wherein the effective dose significantly reduces the plasma leptin level of the subject compared to baseline.

19. The method according to any one of the prior claims, wherein the effective amount significantly reduces the total cholesterol of the subject compared to baseline.

20. The method according to claim 19, wherein the effective amount significantly reduces the low-density lipoprotein (LDL) level of the subject compared to baseline.

21. The method according to any one of the prior claims, wherein the metabolic disorder is selected from obesity, diabetes mellitus, lipid metabolism disorders, congenital metabolic disorders, lysosomal storage diseases, glycogen storage diseases, mitochondrial diseases, purine-pyrimidine diseases, urea cycle disorders, fructose metabolism disorders, amino acid metabolism disorders, mineral metabolism disorders, porphyria, lactose intolerance and Wilson's disease, polycystic ovary syndrome, metabolic dysfunction-related fatty liver disease, and non-alcoholic steatohepatitis.

22. The method according to claim 21, wherein the metabolic disease is type 2 diabetes mellitus.

23. The method according to claim 21, wherein the metabolic disorder is obesity.

24. The method according to claim 21, wherein the metabolic disease is metabolic dysfunction-related fatty liver disease.

25. The method according to any one of the prior claims, wherein the subject has a body mass index (BMI) of 25.0 to less than 30.

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

27. The method according to any one of the prior claims, wherein the subject receives and then discontinues another weight-loss therapy within 3, 6, 9, 12, or 18 months from the delivery of the gene therapy composition, and optionally, the subject loses weight while receiving the other weight-loss therapy.

28. The AAV vector genome, The method according to any one of the prior claims, comprising a 5' inverted terminal repeat (ITR) sequence, a 5' untranslated region (UTR), an open reading frame encoding a GLP-1 receptor agonist, an insulin gene promoter and enhancer element operably linked to a nucleic acid including a 3' UTR, a polyadenylation signal, and a 3' ITR.

29. The method according to claim 28, wherein the insulin gene promoter is a human insulin gene promoter or its core region.

30. In the formula, R11 is The enhancer element is a cytomegalovirus enhancer element, or optionally a CMV upstream genome 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 includes a modified woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), optionally mut6.WPRE. The method according to claim 28 or 29, wherein the polyadenylation signal is a bovine growth hormone polyadenylation signal.

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

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