Gene Therapy for Metabolic Disorders

Abstract

The present disclosure provides insulin-glucagon-like peptide 1 (GLP-1) polynucleotide for delivery to pancreatic islet cells, as well as compositions comprising said polynucleotide and methods for treating obesity-related metabolic disorders.Many blood glucose-regulating hormones have two characteristics that make their pharmacological application in disease treatment difficult: (1) short half-life, and (2) side effects of nausea and vomiting when present in circulation at high levels for a long period of time.To overcome these problems, the present disclosure provides, in some aspects, a method for producing transgenic blood glucose-regulating hormones sustainably and in response to nutrition, so that their endogenous production is mimicked.

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. § 119 of U.S. Provisional Application No. 63 / 289,850, filed December 15, 2021, and U.S. Provisional Application No. 63 / 423,411, filed November 7, 2022, the contents of each of which are incorporated by reference in their entirety herein.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (F085770000WO00-SEQ-HJD.xml; size: 103,314 bytes; creation date: December 13, 2022) are incorporated herein by reference in their entirety. [Background technology]

[0003] background Metabolic diseases such as type 2 diabetes (T2D) account for significant morbidity and mortality worldwide. Approximately 50% of the estimated 27 million people diagnosed with T2D in the United States have inadequate disease control despite the availability of over 60 approved medications, and an estimated 50 million people in the United States are projected to be living with T2D by 2035. The majority of currently available treatments attempt to treat metabolic disease by managing symptoms rather than treating the underlying cause of the disease. Summary of the Invention [Means for solving the problem]

[0004] Abstract The primary role of pancreatic beta cells is to produce and secrete endogenous insulin in response to nutrients. Insulin production and secretion from beta cells is a multi-step complex process that involves significant post-translational processing and trafficking of the insulin gene product to load functional insulin hormone peptide into nutrient-responsive secretory vesicles of the beta cell (see Figure 1). These vesicles fuse with the plasma membrane to release functional insulin in response to nutrients, particularly glucose.

[0005] Glycemic control hormones, including but not limited to GLP-1, are often produced and secreted in response to dietary nutrients, and affect the function of pancreatic beta cells in a variety of ways.The main effects of glycemic control hormones on beta cells include stimulating insulin secretion and production, and positively affecting the health of beta cells.The reported improvements in beta cell health for GLP-1 include maintaining beta cell mass by increasing cell proliferation, inhibiting beta cell neogenesis, and / or apoptosis.Applying these beneficial functions of glycemic control hormones to the treatment of diabetes and related disorders has become a successful clinical strategy and remains an active area of ​​therapeutic research.

[0006] Many blood glucose-regulating hormones have two characteristics that make their pharmacological application in disease treatment difficult: (1) short half-life, and (2) side effects of nausea and vomiting when present in circulation at high levels for long periods of time. To overcome these problems, the present disclosure provides, in some embodiments, a method for producing transgenic blood glucose-regulating hormones sustainedly and in response to nutrition, so as to mimic their endogenous production. This mode of production limits sustained high levels in circulation. To achieve this, the technology described herein utilizes pancreatic beta cells for the production and secretion of transgenic blood glucose-regulating hormones. Pancreatic beta cells are utilized because these cells already perform similar functions in terms of endogenous insulin production and secretion. Furthermore, the local production of blood glucose-regulating hormones by pancreatic beta cells allows these hormones to be produced in one of their major sites of action, thereby reducing the circulating levels of hormones required to achieve the desired effect on beta cell function.

[0007] The disclosed method provides a way to code transgenic glucose-regulating hormones that are functionally active and loaded into nutrient-responsive secretory vesicles, similar to endogenous insulin. The glucose-regulating hormone (payload) is fused into a copy of the transgenic insulin sequence, allowing post-translational processing, loading into secretory vesicles, and nutrient-responsive payload secretion by beta cells (see FIG. 2). This payload needs to be processed into a functional and independent peptide. Thus, the constructs presented herein are strategically designed both with respect to the position where the in-frame insertion of the transgenic insulin sequence occurs, as well as the adjacent sequences that facilitate the processing of the transgenic glucose-regulating hormone into a functional peptide, independent of the transgenic insulin peptide.

[0008] Some aspects provide an endoscopic gene therapy method comprising advancing a drug delivery device comprising at least one drug delivery element to at least one pancreatic delivery site in a patient having a metabolic and / or pancreatic disease, and delivering an effective amount of a therapeutic agent to the at least one pancreatic delivery site through the at least one drug delivery element, wherein the therapeutic agent comprises an adeno-associated virus (AAV) vector comprising an AAV vector genome, and the AAV vector genome comprises a polynucleotide comprising a human islet beta cell-specific promoter operably linked to (a) a human GLP-1 receptor agonist coding sequence, optionally a human GLP-1 coding sequence, or (b) a peptide tyrosine tyrosine (PYY) coding sequence.

[0009] In some embodiments, the at least one medication delivery element includes at least one needle positioned at a distal end of the medication delivery device.

[0010] In some embodiments, the tip of the drug delivery device is delivered into the patient via the mouth and advanced through the wall of the gastrointestinal tract to a position adjacent to the pancreas, and, optionally, (a) the drug delivery device is delivered through a working channel of a gastrointestinal endoscope delivered via the patient's mouth, or (b) the drug delivery device is delivered alongside a gastrointestinal endoscope delivered via the patient's mouth.

[0011] In some embodiments, the metabolic disease is selected from the group consisting of type 1 diabetes; type 2 diabetes; non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); obesity; and combinations thereof, or the pancreatic disease is selected from the group consisting of pancreatitis; pancreatic cancer; hyperinsulinemia; and combinations thereof.

[0012] In some embodiments, at least one pancreatic delivery site is selected from the group consisting of: the parenchymal lumen; the pararenal lumen; the ductal lumen; the arterial lumen of an artery that drains at least a portion of the pancreas; and combinations thereof, and preferably, at least one pancreatic delivery site is the parenchymal lumen.

[0013] In some embodiments, the step of delivering the treatment agent includes at least a first delivery of a minimal volume of the treatment agent into the pancreatic parenchyma, the minimal volume of the treatment agent including a volume sufficient to cause at least a portion of the volume of the treatment agent to exit into the pararenal space, diffuse, and re-enter the pancreas, and optionally, the method further includes at least a second delivery of the treatment agent to at least one additional delivery site proximate the tail of the pancreas.

[0014] In some embodiments, the step of delivering the treatment agent includes at least a first delivery that delivers a minimum volume of the treatment agent into the pancreatic parenchyma, the minimum volume of the treatment agent including a volume of at least 2 ml, at least 3 ml, and / or at least 5 ml.

[0015] In some embodiments, the delivery device is advanced to at least one pancreatic delivery site under image guidance, where optional image guidance includes endoscopic ultrasound guidance; computed tomography (CT) guidance; and / or magnetic resonance imaging (MRI) guidance.

[0016] In some embodiments, at least one pancreatic delivery site includes a location within 10 cm, within 7.5 cm, within 5 cm, and / or within 3 cm of a portion of the pancreas, the portion of the pancreas including the tail, neck, body, head, and / or uncinate process.

[0017] In some embodiments, the therapeutic agent and / or the at least one delivery element are configured to be visualized by an imaging device, and the method further includes visualizing the therapeutic agent and / or the at least one delivery element with the imaging device to confirm proper delivery of the therapeutic agent.

[0018] In some embodiments, the method further includes delivering a contrast agent through the at least one delivery element and visualizing the delivery of the contrast agent using an imaging device to subsequently confirm proper delivery of the treatment agent.

[0019] In some embodiments, the method further comprises pre-loading the drug delivery device with a therapeutic agent, optionally wherein the therapeutic agent is loaded into the drug delivery device through a tip of the drug delivery device.

[0020] In some embodiments, the step of delivering the therapeutic agent is carried out under a pressure of at least 3 mmHg and / or no more than 25 mmHg.

[0021] In some embodiments, the step of delivering the therapeutic agent is carried out at a flow rate of at least 1 ml per minute and / or no more than 5 ml per minute.

[0022] In some embodiments, at least one medication delivery element includes multiple openings along its length.

[0023] In some embodiments, the method further includes verifying that the at least one delivery element is in proper position prior to the step of delivering the treatment agent.

[0024] In some embodiments, the method further comprises delivering a permeability enhancer prior to and / or simultaneously with the step of delivering the therapeutic agent, optionally wherein the step of delivering the permeability enhancer is performed locally and / or intravenously, optionally wherein the permeability enhancer comprises an agent selected from the group consisting of hyaluronidase; collagenase; losartan; and combinations thereof, optionally wherein the therapeutic agent comprises a co-formulation of the therapeutic agent and the permeability enhancer.

[0025] In some embodiments, the method further comprises warming tissue adjacent to at least one pancreatic delivery site to a temperature greater than 39° C. before, during, and / or after delivery of the therapeutic agent.

[0026] In some embodiments, the method further includes delivering a seeding blocking material configured to prevent undesired seeding of the treatment agent to non-target locations, optionally wherein the seeding blocking material comprises a viscous substance and / or a polymer.

[0027] In some embodiments, the method further includes positioning a blocking element within the patient, the blocking element configured to prevent undesired seeding of the treatment agent to non-target locations.

[0028] In some embodiments, the method further includes removing at least a portion of the therapeutic agent from the location of the delivery site after delivery of the therapeutic agent has begun.

[0029] In some embodiments, the method further comprises removing all of the treatment agent.

[0030] Some aspects relate to polynucleotides that include a blood glucose-regulating hormone coding sequence and an insulin coding sequence.

[0031] In some embodiments, the blood glucose regulating hormone coding sequence and the insulin coding sequence are arranged such that a functional blood glucose regulating hormone and functional insulin are produced in vivo following administration to a subject.

[0032] In some embodiments, the blood glucose regulating hormone coding sequence is nested within the insulin coding sequence.

[0033] In some embodiments, the insulin coding sequence comprises, from 5' to 3', a signal peptide coding sequence, a B chain coding sequence, a C peptide coding sequence, and an A chain coding sequence, optionally wherein (a) the blood glucose regulating hormone coding sequence is located between the signal peptide coding sequence and the B chain coding sequence; (b) the blood glucose regulating hormone coding sequence is nested within the B chain coding sequence; (c) the blood glucose regulating hormone coding sequence is located between the B chain coding sequence and the C peptide coding sequence; (d) the blood glucose regulating hormone coding sequence is nested within the C peptide coding sequence; (e) the blood glucose regulating hormone coding sequence is located between the C peptide coding sequence and the A chain coding sequence; (f) the blood glucose regulating hormone coding sequence is nested within the A chain coding sequence; or (g) the blood glucose regulating hormone coding sequence is downstream of the A chain coding sequence.

[0034] In some embodiments, (a) the blood glucose-regulating hormone coding sequence is flanked by a first PCSK1 (PC1 / 3) and / or PCSK2 (PC2) enzyme processing sequence, optionally a native PCSK1 or PCSK2 enzyme processing sequence or an artificial PCSK1 or PCSK2 enzyme processing sequence; or (b) the blood glucose-regulating hormone coding sequence is flanked by a first PCSK1 and / or PCSK2 enzyme processing sequence and a second PCSK1 and / or PCSK2 enzyme processing sequence, optionally a native PCSK1 or PCSK2 enzyme processing sequence or an artificial PCSK1 or PCSK2 enzyme processing sequence.

[0035] In some embodiments, the polynucleotide comprises, from 5' to 3', (a) a signal peptide coding sequence, a blood glucose regulating hormone coding sequence, a B chain coding sequence, a first PCSK1 or PCSK2 enzyme processing sequence, a C peptide coding sequence, a second PCSK1 or PCSK2 enzyme processing sequence, and an A chain coding sequence; (b) a signal peptide coding sequence, a B chain coding sequence, a blood glucose regulating hormone coding sequence, a first PCSK1 or PCSK2 enzyme processing sequence, a C peptide coding sequence, a second PCSK1 or PCSK2 enzyme processing sequence, and an A chain coding sequence; (c) a signal peptide coding sequence, a B chain coding sequence, a first PCSK1 or PCSK2 enzyme processing sequence, a blood glucose regulating hormone coding sequence, a C peptide coding sequence, a second PCSK1 or PCSK2 enzyme processing sequence, and an A chain coding sequence; (d) a signal peptide coding sequence, a B chain coding sequence, a first PCSK1 or PCSK2 enzyme processing sequence, , a blood glucose regulating hormone coding sequence nested within the C-peptide coding sequence, a second PCSK1 or PCSK2 enzyme processing sequence, and an A-chain coding sequence; (e) a signal peptide coding sequence, a B-chain coding sequence, a first PCSK1 or PCSK2 enzyme processing sequence, a C-peptide coding sequence, a blood glucose regulating hormone coding sequence, a second PCSK1 or PCSK2 enzyme processing sequence, and an A-chain coding sequence; (f) a signal peptide coding sequence, a B-chain coding sequence, a first PCSK1 or PCSK2 enzyme processing sequence, a C-peptide coding sequence, a second PCSK1 or PCSK2 enzyme processing sequence, a blood glucose regulating hormone coding sequence, and an A-chain coding sequence; or (g) a signal peptide coding sequence, a B-chain coding sequence, a first PCSK1 or PCSK2 enzyme processing sequence, a C-peptide coding sequence, a second PCSK1 or PCSK2 enzyme processing sequence, an A-chain coding sequence, and a blood glucose regulating hormone coding sequence.

[0036] In some embodiments, the blood glucose regulating hormone is selected from glucagon, GLP-1, oxyntomodulin, glicentin, glicentin-related polypeptide (GRPP), major proglucagon fragment, intervening peptide 1 (IP-1), intervening peptide 2 (IP-2), GLP-2, glucose-dependent insulinotropic peptide (GIP), peptide tyrosine tyrosine (PYY), cholecystokinin (CCK), somatostatin, oxyntomodulin, ghrelin, amylin, glucagon, leptin, follistatin, insulin-like growth factor 1 (IGF1), vasoactive intestinal peptide (VIP), and growth hormone 1 (GH1), and peptides, variants, and fusions thereof, optionally selected from any one of SEQ ID NOs: 31-58, 93, and 94.

[0037] In some embodiments, the blood glucose regulating hormone is wild-type human GLP-1.

[0038] In some embodiments, the GLP-1 is a variant human GLP-1, optionally comprising a Gly8 substitution compared to wild-type human GLP-1.

[0039] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the nucleotide sequence of any one of SEQ ID NOs:59-71.

[0040] In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs:72-84.

[0041] In some embodiments, the blood glucose regulating hormone is wild-type human PYY.

[0042] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the nucleotide sequence of any one of SEQ ID NOs:85-87.

[0043] In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 88-90.

[0044] In some embodiments, the polynucleotide is operably linked to a promoter sequence, preferably a pancreatic islet cell promoter sequence, more preferably a pancreatic islet beta cell promoter sequence, optionally selected from the human insulin promoter, mouse insulin 2 promoter, mouse insulin 1 promoter, rat insulin 2 promoter, rat insulin 1 promoter, Slc2a, IAPP, NKX6.1, DLK1, MafA, Slc30a8 / Znt8, PCSK1, and ADCYAP1.

[0045] In some embodiments, the polynucleotide further comprises an enhancer sequence.

[0046] Some embodiments relate to a vector comprising the polynucleotide of any one of the preceding paragraphs.

[0047] In some embodiments, the vector is a non-viral vector, optionally a plasmid, a bacterial artificial chromosome, a yeast artificial chromosome, or a minicircle.

[0048] In some embodiments, the vector is a viral vector, optionally selected from a retroviral vector, an adenoviral vector, a herpes simplex virus (HSV) vector, and an adeno-associated virus (AAV) vector.

[0049] Another aspect relates to a recombinant adeno-associated virus (AAV) vector genome comprising the polynucleotide of any one of the preceding paragraphs.

[0050] In some embodiments, the recombinant AAV vector genome optionally further comprises inverted terminal repeat (ITR) sequences flanking the polynucleotide, and further optionally, the ITR sequences are selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 (AAVrh10), and AAV11 ITR sequences.

[0051] A further embodiment relates to a recombinant adeno-associated virus (AAV) vector comprising (a) a recombinant AAV vector genome of any one of the preceding paragraphs and (b) a capsid protein, wherein, optionally, 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, and variants thereof.

[0052] Yet another embodiment relates to a fusion protein encoded by the polynucleotide of any one of the preceding paragraphs.

[0053] Some embodiments relate to fusion proteins comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 72-84 and 88-90.

[0054] Other aspects relate to a host cell comprising a polynucleotide of any one of the preceding paragraphs, a vector of any one of the preceding paragraphs, a recombinant AAV vector genome of any one of the preceding paragraphs, a recombinant AAV vector of any one of the preceding paragraphs, or a fusion protein of any one of the preceding paragraphs.

[0055] Some embodiments relate to a method comprising administering to a pancreatic islet cell a polynucleotide of any one of the preceding paragraphs, a vector of any one of the preceding paragraphs, a recombinant AAV vector genome of any one of the preceding paragraphs, a recombinant AAV vector of any one of the preceding paragraphs, or a fusion protein of any one of the preceding paragraphs.

[0056] Other embodiments relate to a method comprising administering to a subject a polynucleotide of any one of the preceding paragraphs, a vector of any one of the preceding paragraphs, a recombinant AAV vector genome of any one of the preceding paragraphs, a recombinant AAV vector of any one of the preceding paragraphs, or a fusion protein of any one of the preceding paragraphs, optionally wherein the subject has an obesity-related metabolic disorder selected from the group consisting of prediabetes, type 2 diabetes, cardiovascular disease, polycystic ovary syndrome (PCOS), nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH).

[0057] Some embodiments relate to a method of treating an obesity-related metabolic disorder, comprising administering to a subject in need thereof an effective amount of a polynucleotide of any one of the preceding paragraphs, a vector of any one of the preceding paragraphs, a recombinant AAV vector genome of any one of the preceding paragraphs, a recombinant AAV vector of any one of the preceding paragraphs, or a fusion protein of any one of the preceding paragraphs, wherein optionally the obesity-related metabolic disorder is selected from the group consisting of pre-diabetes, type 2 diabetes, cardiovascular disease, polycystic ovary syndrome (PCOS), non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH).

[0058] In some embodiments, the administering step is by endoscopic delivery to the pancreas or an area near the pancreas, and optionally: (a) an effective amount that restores glycemic durability in the subject; (b) an effective amount that reduces fasting blood glucose by at least 50% compared to baseline; (c) an effective amount that increases fasting insulin by at least 2-fold compared to baseline; (d) an effective amount that significantly improves glucose tolerance compared to baseline; and / or (e) an effective amount that significantly improves glucose-stimulated insulin secretion compared to baseline.

[0059] In some embodiments, the subject's body weight does not change significantly compared to baseline.

[0060] In some embodiments, the effective amount is a single dose, which single dose is sufficient for long-term restoration of beta cell function and / or reduced therapeutic burden.

[0061] Some embodiments relate to an endoscopic gene therapy method comprising: advancing a drug delivery device comprising at least one drug delivery element to at least one pancreatic delivery site in a patient having a metabolic and / or pancreatic disease; and delivering an effective amount of a therapeutic agent to the at least one pancreatic delivery site through the at least one drug delivery element, wherein the therapeutic agent comprises a recombinant AAV vector genome of any one of the preceding paragraphs, or a recombinant AAV vector of any one of the preceding paragraphs.

[0062] In some embodiments, the at least one medication delivery element includes at least one needle positioned at a distal end of the medication delivery device.

[0063] In some embodiments, the tip of the drug delivery device is delivered into the patient via the mouth and advanced through the wall of the gastrointestinal tract to a position adjacent to the pancreas, and, optionally, (a) the drug delivery device is delivered through a working channel of a gastrointestinal endoscope delivered via the patient's mouth, or (b) the drug delivery device is delivered alongside a gastrointestinal endoscope delivered via the patient's mouth.

[0064] In some embodiments, the metabolic disease is selected from the group consisting of type 1 diabetes; type 2 diabetes; non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); obesity; and combinations thereof, or the pancreatic disease is selected from the group consisting of pancreatitis; pancreatic cancer; hyperinsulinemia; and combinations thereof.

[0065] In some embodiments, at least one pancreatic delivery site is selected from the group consisting of: the parenchymal lumen; the pararenal lumen; the ductal lumen; the arterial lumen of an artery that drains at least a portion of the pancreas; and combinations thereof, and preferably, at least one pancreatic delivery site is the parenchymal lumen.

[0066] In some embodiments, the step of delivering the treatment agent includes at least a first delivery of a minimal volume of the treatment agent into the pancreatic parenchyma, the minimal volume of the treatment agent including a volume sufficient to cause at least a portion of the volume of the treatment agent to exit into the pararenal space, diffuse, and re-enter the pancreas, and optionally, the method further includes at least a second delivery of the treatment agent to at least one additional delivery site proximate the tail of the pancreas.

[0067] In some embodiments, the step of delivering the treatment agent includes at least a first delivery that delivers a minimum volume of the treatment agent into the pancreatic parenchyma, the minimum volume of the treatment agent including a volume of at least 2 ml, at least 3 ml, and / or at least 5 ml.

[0068] In some embodiments, the delivery device is advanced to at least one pancreatic delivery site under image guidance, where optional image guidance includes endoscopic ultrasound guidance; computed tomography (CT) guidance; and / or magnetic resonance imaging (MRI) guidance.

[0069] In some embodiments, at least one pancreatic delivery site includes a location within 10 cm, within 7.5 cm, within 5 cm, and / or within 3 cm of a portion of the pancreas, the portion of the pancreas including the tail, neck, body, head, and / or uncinate process.

[0070] In some embodiments, the therapeutic agent and / or the at least one delivery element are configured to be visualized by an imaging device, and the method further includes visualizing the therapeutic agent and / or the at least one delivery element with the imaging device to confirm proper delivery of the therapeutic agent.

[0071] In some embodiments, the method further includes delivering a contrast agent through the at least one delivery element and visualizing the delivery of the contrast agent using an imaging device to subsequently confirm proper delivery of the treatment agent.

[0072] In some embodiments, the method further comprises pre-loading the drug delivery device with a therapeutic agent, optionally wherein the therapeutic agent is loaded into the drug delivery device through a tip of the drug delivery device.

[0073] In some embodiments, the step of delivering the therapeutic agent is carried out under a pressure of at least 3 mmHg and / or no more than 25 mmHg.

[0074] In some embodiments, the step of delivering the therapeutic agent is carried out at a flow rate of at least 1 ml per minute and / or no more than 5 ml per minute.

[0075] In some embodiments, at least one medication delivery element includes multiple openings along its length.

[0076] In some embodiments, the method further includes verifying that the at least one delivery element is in proper position prior to the step of delivering the treatment agent.

[0077] In some embodiments, the method further comprises delivering a permeability enhancer prior to and / or simultaneously with delivery of the therapeutic agent, optionally where delivery of the permeability enhancer is performed locally and / or intravenously, optionally where the permeability enhancer comprises an agent selected from the group consisting of hyaluronidase; collagenase; losartan; and combinations thereof, optionally where the therapeutic agent comprises a co-formulation of the therapeutic agent with the permeability enhancer.

[0078] In some embodiments, the method further comprises warming tissue adjacent to at least one pancreatic delivery site to a temperature greater than 39° C. before, during, and / or after delivery of the therapeutic agent.

[0079] In some embodiments, the method further includes delivering a seeding blocking material configured to prevent undesired seeding of the treatment agent to non-target locations, optionally wherein the seeding blocking material comprises a viscous substance and / or a polymer.

[0080] In some embodiments, the method further includes positioning a blocking element within the patient, the blocking element configured to prevent undesired seeding of the treatment agent to non-target locations.

[0081] In some embodiments, the method further includes removing at least a portion of the therapeutic agent from the location of the delivery site after delivery of the therapeutic agent has begun.

[0082] In some embodiments, the method further comprises removing all of the treatment agent. [Brief description of the drawings]

[0083] [Figure 1]1A-1B show a schematic diagram of endogenous insulin production (Figure 1A) and the corresponding subcellular localization (Figure 1B). [Diagram 2] Figures 2A-2B show a schematic diagram of AAV-based transgene production (Figure 2A) and the corresponding subcellular localization (Figure 2B). [Diagram 3] 3A-3B show schematic diagrams of preproinsulin (INS) (FIG. 3A) and preproglucagon (GCG) (FIG. 3B). [Figure 4] Figure 4 shows a schematic diagram of the putative site of insertion of a blood glucose-regulating hormone into the preproinsulin gene. The arrow indicates the fusion site. [Diagram 5] FIG. 5 is a graph showing total GLP-1 detected in the supernatant of MIN-6 cells (mouse insulinoma cell line) transfected with buffer only (TE buffer), an empty plasmid (pUC18 plasmid), a plasmid expressing enhanced green fluorescent protein (GFP), a plasmid expressing human preproglucagon coding sequence (GCG), or a plasmid expressing any of the INS-GLP-1 hybrids #01, #02, #03, #04, #05, #06, #07, #08, #09, #10, #11, #12, or #13, 5 hours after high glucose treatment (i.e., high glucose (25 mM + 0.100 mM 3-isobutyl-1-methylxanthine (IBMX)) treatment). [Figure 6] FIG. 6 is a graph showing the fold increase in total GLP-1 detected in the supernatant of MIN-6 cells transfected with cells alone (control), empty plasmid (control), a plasmid expressing GFP, a plasmid expressing GCG, a plasmid expressing INS-GLP-1 hybrid #1, or a plasmid expressing INS-GLP-1 hybrid #2 after treatment with either low glucose (2 mM) or high glucose (25 mM + 0.100 mM 3-isobutyl-1-methylxanthine (IBMX)). [Figure 7]FIG. 7 is a graph showing human insulin detected in the supernatant of MIN-6 cells transfected with cells alone (control), empty plasmid (control), a plasmid expressing GFP, a plasmid expressing GCG, a plasmid expressing INS-GLP-1 hybrid #1, or a plasmid expressing INS-GLP-1 hybrid #2 after treatment with either low glucose (2 mM) or high glucose (25 mM + 0.100 mM IBMX). [Figure 8A] 8A-8B are graphs showing total GLP-1 in the supernatant (FIG. 8A) and cell lysates (FIG. 8B) of EndoC-BH5 cells (human beta cells) 48 hours after transfection with various expression plasmids. [Figure 8B] Same as above. [Figure 9] 9A-9B are graphs showing a dose-dependent sustained reduction in glycemia (FIG. 9A) and increase in fasting insulinemia (FIG. 9B). Statistics: **P, 0.01, ***P<0.001 vs. vehicle, MIP-eGFP 1012 group; *P<0.05 vs. vehicle only; one-way ANOVA, post-hoc Tukey's test. [Figure 10A-B] Figures 10A-10C are graphs showing the results of the intraperitoneal glucose tolerance test (IPGTT) (Figure 10A), area under the curve (Figure 10B), and insulin secretion (Figure 10C) on day 39. Statistics: For Figures 10A-10B, one-way ANOVA; post hoc Tukey's multiple comparison test, *P<0.05, ****P<0.0001. For Figure 10C, two-way ANOVA mixed effects model [REML]; post hoc Tukey's multiple comparison test, a P<0.005 vs. vehicle, b P<0.05 vs. eGFP control. [Figure 10C] Same as above. [Figure 11A] 11A-11B are graphs showing absolute body weight (FIG. 11A) and change in body weight (FIG. 11B) over time. [Figure 11B] Same as above. [Figure 12A]Figures 12A-12C show that GLP-1RA protein (ie, exendin-4) is expressed in the pancreas by immunohistochemical staining (Figure 12A), percent islet expression (Figure 12B), and total pancreatic protein expression (Figure 12C). [Figure 12B-C] Same as above. [Figure 13] FIG. 13 shows PYY in the supernatant of MIN-6 cells transfected with various expression plasmids including control plasmid (empty and eGFP), human PYY CDS, and INS-PYY hybrid (#1-3) after treatment with either low glucose (2 mM) or high glucose (25 mM + 0.01 mM IBMX) (n=3 independent experiments). [Figure 14A] Figures 14A-14B are graphs showing the identification of top functional GLP-1RA (i.e., exendin-4) producers in the MIN-6 beta cell line. Figure 14A shows total GLP-1RA secretion upon 25 mM glucose stimulation. Figure 14B shows cAMP signaling in the CHO-K1 hGLP-1R Gs cell line. [Figure 14B] Same as above. [Figure 15] Figures 15A-15B are graphs showing improved insulin secretion from ex vivo primary BKS db / db islets following treatment with AAV delivering GLP-1RA (i.e., exendin-4) (compared to AAV-eGFP control). Figure 15A shows total GLP-1 content and Figure 15B shows glucose-stimulated insulin secretion. [Figure 16] Figure 16 shows insulin levels in the human beta cell line EndoC-BH5 after AAV-mediated delivery of GLP-1RA (i.e., exendin-4). Treatment with exendin-9 (Ex9) peptide, a potent inhibitor of GLP-1R, demonstrates that the increase in INS secretion caused by AAV-GLP-1RA is due to the action of AAV-GLP-1RA on the GLP-1R. [Figure 17]Figures 17A-17B are graphs showing the change in fasting blood glucose (Figure 17A) and quantification of exendin-4 in serum and pancreas (Figure 17B) of BKS db / db mice 4 weeks after administration of AAV-MIP-Ex-4 (AAV-based exendin-4 treatment) or vehicle. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0084] Detailed Description Metabolic diseases result from disruptions to normal metabolism, the process that converts inputs (food and drink) into outputs (energy). In general, ingested proteins, carbohydrates, and fats are broken down by chemicals in the body and converted into energy that is used immediately or stored for later use. Metabolic disorders such as prediabetes, type 2 diabetes (T2D), cardiovascular disease, polycystic ovarian syndrome (PCOS), and nonalcoholic fatty liver disease (NAFLD) are conditions that increase the risk of heart disease, stroke, and death. These diseases are becoming more prevalent, with up to one-third of Americans estimated to have at least one. Despite advances in treatment over the past 50 years, metabolic diseases in general, and T2D in particular, remain a major contributor to morbidity and mortality today.

[0085] T2D is a disorder in which blood glucose is elevated, caused by a number of factors that lead to two parallel progressive disease processes in the body: insulin resistance and insulin deficiency. Insulin resistance is the inability of the body to properly respond to insulin signals to remove glucose from the bloodstream, while insulin deficiency is the gradual inability of the pancreas to produce enough insulin to meet the body's needs. Current guidelines focus on managing the blood glucose-related symptoms of T2D, often measured by blood levels of glycosylated hemoglobin, or HbA1c, rather than attempting to correct the underlying pathological deviations in the body that cause insulin resistance and insulin deficiency. Thus, patients make significant dietary and lifestyle changes that they must adhere to for the rest of their lives, as well as sustained drug therapy. For some, this approach to care is overwhelming, leaving many patients at risk, potentially resulting in chronically elevated blood glucose that increases the likelihood of microvascular and macrovascular complications of T2D, and even death.

[0086] There are currently no approved treatments for T2D that are disease-modifying, ie, provide continuous, long-lasting protection of the insulin-producing capacity of the pancreas even after treatment has stopped.

[0087] A new approach is described herein. Instead of treating the patient's symptoms, the compositions and methods described herein are used to treat the underlying cause of the disease using gene therapy. A gene therapy approach is described herein for treating metabolic disorders (e.g., T2D) by restoring insulin production with the goal of achieving long-term remission. Briefly, gene therapy compositions and methods are provided for key metabolic hormones required for proper insulin production in pancreatic beta cells. As an example, a glucagon-like peptide-1 (GLP-1) coding sequence can be nested within an insulin (preproinsulin, proinsulin, or insulin) coding sequence, and the resulting polynucleotide can be delivered to the pancreas (e.g., in the vicinity of beta cells) by any means known in the art. In one embodiment, the polynucleotide is packaged as an adeno-associated virus (AAV) vector and delivered locally using an endoscope. Without wishing to be bound by theory, it is believed that enhanced GLP-1 receptor activation in the pancreas can lead to reduced blood glucose. Other configurations are possible and are described in more detail below.

[0088] Insulin - Blood sugar regulating hormone polynucleotide Polynucleotides comprising blood glucose-regulating hormone coding sequences and insulin (preproinsulin, proinsulin, or insulin) coding sequences are presented herein. As used herein, "coding sequence" refers to a nucleotide sequence that directly defines the amino acid sequence of a protein product (i.e., the protein encoded by the nucleotide coding sequence). The boundaries of a coding sequence are generally determined by an open reading frame (hereinafter "ORF"). An open reading frame (ORF) is a contiguous 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). An ORF generally codes for a protein (e.g., a blood glucose-regulating hormone or insulin). A polynucleotide comprises nucleic acids (nucleotides). The nucleic acid may be or may comprise, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA including LNA having a β-D-ribo configuration, α-LNA (a diastereomer of LNA) having an α-L-ribo configuration, 2′-amino-LNA having a 2′-amino functionalization, and 2′-amino-α-LNA having a 2′-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) and / or chimeras, and / or combinations thereof.

[0089] As used herein, "glycemic regulating hormone" refers to a hormone involved in the modulation of circulating blood glucose levels. Two hormones involved in the regulation of blood glucose are typically known: insulin, which lowers blood glucose levels, and glucagon, which increases blood glucose levels. Thus, both insulin and glucagon are glycemic regulating hormones, and additional hormones that affect the secretion or function of insulin and / or glucagon may also fall into the category of glycemic regulating hormones. In some embodiments, glycemic regulating hormones act directly; i.e., they directly participate in the regulation of blood glucose by modulating insulin and / or glucagon, while in other embodiments, the effect of glycemic regulating hormones is indirect; i.e., the activity of the hormone indirectly affects blood glucose by modulating insulin and / or glucagon. Examples of blood glucose regulating hormones include preproglucagon derived peptides (e.g., glucagon, GLP-1, oxyntomodulin, glicentin, glicentin-related polypeptide (GRPP), major proglucagon fragment, intervening peptide 1 (IP-1), intervening peptide 2 (IP-2), and GLP-2), incretins (e.g., GLP-1 and glucose-dependent insulinotropic peptide (GIP)), enteroendocrine cell derived peptides (e.g., GLP-1, peptide tyrosine (PYY), cholecystokinin (CCK), GIP, somatostatin, oxyntomodulin, and ghrelin), and hormones produced in the pancreas (e.g., insulin, amylin, somatostatin, glucagon, and GLP-1). In some embodiments, the blood glucose regulating hormone comprises an agent that mimics the action of GLP-1. In some embodiments, the agent comprises a GLP-1 receptor agonist (e.g., an agent that binds to and activates the GLP-1 receptor and reduces blood glucose levels). In some embodiments, the GLP-1 receptor agonist is a polypeptide agonist of the GLP-1 receptor (e.g., exendin-4 and variants thereof).Exendin-4 (present in the saliva of the Gila monster, Heloderma suspectum) is a long-acting GLP-1 analogue that is an agonist of the GLP-1 receptor. In some embodiments, the GLP-1 receptor agonist is exendin-4. Other endocrine hormones with blood glucose regulating activity contemplated herein include, without limitation, leptin, follistatin, insulin-like growth factor 1 (IGF1), vasoactive intestinal peptide (VIP), and growth hormone 1 (GH1). See Table 1 for exemplary proteins and coding sequences. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0090] The term "glycemic regulating hormone" should be understood to encompass any of the aforementioned example functional peptides and polypeptides, as well as functional variants thereof, meaning peptides and / or variants capable of affecting blood glucose control by either a direct or indirect function.

[0091] Thus, amino acid modifications (e.g., substitutions) can be made to the blood glucose-regulating hormones presented 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 substitution of alanine with glycine at amino acid position 8 (GLP-1-Gly8) of the GLP-1(1-37) sequence, which confers resistance to cleavage by dipeptidyl peptidase-IV (DPP4 or DPPIV) to an inactive form. Other modifications, and thus other variants, are also contemplated herein.

[0092] In some embodiments, the polynucleotide comprises multiple coding sequences, each for a different protein. In some embodiments, the polynucleotide comprises one or more coding sequences for 1-10 blood glucose regulating hormones. For example, the polynucleotide may have one or more coding sequences for 1-3, 1-4, or 1-5 different blood glucose regulating hormones. In some embodiments, the polynucleotide comprises one or more coding sequences for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more blood 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.

[0093] GLP-1 is a polypeptide derived from the proglucagon protein. Under physiological conditions, GLP-1 is produced and secreted by enteroendocrine L-cells and certain neurons in the nucleus of the solitary tract in the brainstem upon feeding. GLP-1 is rapidly metabolized and inactivated by dipeptidyl peptidase IV (an enzyme) even before the hormone leaves the intestine. GLP-1 stimulates insulin secretion (acting as an incretin hormone) and inhibits glucagon secretion. GLP-1 also inhibits gastrointestinal motility and secretion. Thus, the protein acts as an enterogastrone and as part of the "ileal brake" mechanism. GLP-1 also plays a role as a physiological regulator of appetite and food intake. Decreased secretion of GLP-1 may lead to the development of obesity. In some embodiments, the polynucleotide encodes a full-length GLP-1 sequence (e.g., SEQ ID NO: 33) or a truncated GLP-1 sequence (e.g., any one of SEQ ID NOs: 53-58), or other functional variants or fragments thereof.

[0094] The polynucleotides described herein also include insulin (preproinsulin, proinsulin, insulin) coding sequences. Preproinsulin is 110 amino acids long and is a biologically inactive precursor of insulin. Insulin mRNA is translated as a single-chain precursor, preproinsulin, whose signal peptide is removed during insertion into the endoplasmic reticulum to generate proinsulin. Insulin is produced and secreted by beta cells of the pancreas. Proinsulin and preproinsulin contain three domains: an amino-terminal B chain, a carboxy-terminal A chain, and an intermediate connecting peptide known as the C peptide. In the endoplasmic reticulum, proinsulin is exposed to several specific endopeptidases that cleave off the C peptide, thereby generating the mature form of insulin, consisting of the A and B chains. Insulin and free C peptide are packaged in the Golgi into secretory granules that accumulate in the cytoplasm. In some embodiments, the insulin protein comprises the amino acid sequence of SEQ ID NO: 46, or a variant thereof. Unless otherwise specified, the term "insulin" should be understood to encompass the various forms of insulin, including preproinsulin, proinsulin, and insulin.

[0095] In some embodiments, the blood glucose-regulating hormone coding sequence and the insulin coding sequence are arranged such that functional blood glucose-regulating hormone and functional insulin are produced in vivo after administration to a subject. As used herein, "functional" refers to a protein having biological activity (e.g., enzymatic activity). That is, the functional protein produced has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity (e.g., enzymatic activity) compared to the corresponding wild-type protein. Biological activity can be measured by any method known in the art, for example, by in vitro activity assay or by in vivo measurement of enzymatic by-products (e.g., C-peptide) or other related components (e.g., glucose levels). In some embodiments, functional insulin has about the same activity as wild-type insulin (e.g., promoting glucose uptake, glycogen production, lipogenesis, and / or protein synthesis in skeletal muscle and / or adipose tissue through tyrosine kinase receptor pathways). In some embodiments, functional insulin has greater activity than wild-type insulin. In some embodiments, functional blood glucose regulating hormones have about the same activity as the corresponding wild-type blood glucose regulating hormones (e.g., maintaining circulating glucose concentrations within a physiological range). In some embodiments, functional blood glucose regulating hormones have greater activity than the corresponding wild-type blood glucose regulating hormones.

[0096] In some embodiments, the blood glucose regulating coding sequence is nested within the insulin coding sequence; i.e., the blood glucose regulating coding sequence is located between the 5' and 3' ends of the insulin coding sequence. As used herein, "nested" refers to the positional relationship between two coding sequences: one is located within the other, and the sequences are in-frame and positioned to result in the production of insulin and blood glucose regulating hormones (e.g., GLP-1) in vivo. That is, the nesting does not result in one or more frameshift mutations.

[0097] The blood glucose regulating hormone coding sequence may be nested within the insulin protein coding sequence or may be adjacent to either of the insulin protein coding sequences. The insulin protein coding sequence includes, from 5' to 3', a B chain coding sequence, a C peptide coding sequence, and an A chain coding sequence. Thus, in some embodiments, the blood glucose regulating hormone coding sequence is nested within the C peptide coding sequence. In some embodiments, the blood glucose regulating hormone coding sequence is nested between the B chain coding sequence and the C peptide coding sequence. In some embodiments, the blood glucose regulating hormone coding sequence is nested between the C peptide coding sequence and the A chain coding sequence.

[0098] In some embodiments, the blood glucose-regulating hormone coding sequence is flanked by one or more sequences, such as an intervening peptide sequence or a cleavage / enzymatic processing site. As used herein, "flanked" refers to the addition of a polynucleotide sequence adjacent to the 5'-end and / or 3'-end of the coding sequence (e.g., within 5, 10, 15, 20, 25, or 30 nucleotides of the 5'-end and / or 3'-end of the coding sequence). In some embodiments, the blood glucose-regulating hormone coding sequence is flanked at the 5'-end of the blood glucose-regulating hormone coding sequence. In some embodiments, the blood glucose-regulating hormone coding sequence is flanked at the 3'-end of the blood glucose-regulating hormone coding sequence. In some embodiments, the blood glucose-regulating hormone coding sequence is flanked at the 3'-end and 5'-end of the blood glucose-regulating hormone coding sequence.

[0099] In some embodiments, the flanking sequence comprises a processing or cleavage signal sequence, so that the resulting blood glucose-regulating hormone is cleaved from the resulting insulin. In some embodiments, the flanking sequence comprises a PCSK1 (also known as prohormone convertase 1 / 3 (PC1 / 3)) processing sequence and / or a PCSK2 (also known as prohormone convertase 2 (PC2)) enzyme processing sequence. PCSK1 and PCSK2 are enzymes that work together to process proinsulin. PCSK1 cleaves the PCSK1 sequence (generally either a lysine-arginine (KR) amino acid sequence or an arginine-arginine (RR) amino acid sequence), while PCSK2 cleaves the PCSK2 sequence (also either a KR amino acid sequence or an RR amino acid sequence). There is certainly some redundancy that is not fully understood with respect to additional adjacent amino acid sequences surrounding a given "KR" or "RR" enzymatic processing site, or additional considerations such as protein cofactors or secondary or tertiary protein structure that confer additional specificity to either PCSK1 or PCSK2 for a given "KR" or "RR" enzymatic processing site. Thus, without wishing to be bound by theory, it is believed that the inclusion of one or two of the PCSK1 and / or PCSK2 enzymatic processing sequences adjacent to the blood glucose regulating hormone sequence results in excision of the blood glucose regulating hormone from proinsulin.

[0100] Other cleavage sites, such as furin cleavage sites (eg, RXXR, RXKR, and RXRR), are also contemplated herein.

[0101] In some embodiments, the blood glucose-regulating hormone coding sequence is flanked by a first PCSK1 and / or PCSK2 enzyme processing sequence (either at the 5' or 3' end). In other embodiments, the blood glucose-regulating hormone coding sequence is flanked by a first PCSK1 and / or PCSK2 enzyme processing sequence and a second PCSK1 and / or PCSK2 enzyme processing sequence (there are flanking sequences at the 5' and 3' ends of the blood glucose-regulating hormone coding sequence). In some embodiments, the PCSK1 and / or PCSK2 enzyme processing sequence is a native (wild-type) sequence. In other embodiments, the PCSK1 and / or PCSK2 enzyme processing sequence is an artificial (engineered) sequence. As used herein, an "artificial sequence" is a nucleotide sequence (or amino acid sequence) that does not occur in nature (e.g., a polynucleotide that does not have 100% identity to a naturally occurring protein or fragment thereof). In a configuration or arrangement comprising two adjacent PCSK1 and / or PCSK2 enzyme processing sequences, both PCSK1 and / or PCSK2 enzyme processing sequences may be artificial, both may be native, or one may be artificial and the other native.

[0102] In some embodiments, one or both of the flanking sequences may further comprise a preproglucagon intervening peptide (IP) sequence (e.g., IP-1 or IP-2). In native (wild-type) proglucagon, GLP-1 is flanked by two IP sequences: IP-1 (at the 5' end) and IP-2 (at the 3' end). In some embodiments, the blood glucose-regulating hormone coding sequence is flanked by a first IP sequence (at either the 5' or 3' end). In other embodiments, the blood glucose-regulating hormone coding sequence is flanked by a first IP sequence and a second IP sequence (there are flanking sequences at the 5' and 3' ends of the blood glucose-regulating hormone coding sequence). In some embodiments, the IP sequence is a native (wild-type) sequence (e.g., IP-1 or IP-2). In other embodiments, the IP sequence is an artificial (engineered) sequence. In configurations or arrangements that include two flanking IP sequences, both IP sequences may be artificial, both may be native, or one may be artificial and the other may be native.

[0103] In some embodiments, the blood glucose-regulating hormone coding sequence is flanked by an IP sequence and a processing sequence (e.g., PCSK1 and / or PCSK2 enzyme processing sequence). For example, the blood glucose-regulating hormone coding sequence is flanked on both ends by a PCSK1 and / or PCSK2 enzyme processing sequence, and each PCSK1 and / or PCSK2 enzyme processing sequence is flanked by an IP sequence. That is, the polynucleotide comprises, from 5' to 3', a first IP sequence (e.g., encoding IP-1), a first PCSK1 and / or PCSK2 enzyme processing sequence, a blood glucose-regulating hormone coding sequence, a second PCSK1 and / or PCSK2 enzyme processing sequence, and a second IP sequence (e.g., encoding IP-2).

[0104] Different configurations or arrangements of adjacent sequences or sequences within a polynucleotide are possible. In some embodiments, the polynucleotide comprises, from 5' to 3', a signal peptide coding sequence, a B chain coding sequence, a first PCSK1 and / or PCSK2 enzymatic processing site, a C peptide coding sequence, a blood glucose regulating hormone coding sequence, a second PCSK1 and / or PCSK2 enzymatic processing site, and an A chain coding sequence. In another embodiment, the polynucleotide comprises, from 5' to 3', a signal peptide coding sequence, a B chain coding sequence, a first PCSK1 and / or PCSK2 enzymatic processing site, a C peptide coding sequence, a second PCSK1 and / or PCSK2 enzymatic processing site, a first preproglucagon IP sequence, a third PCSK1 and / or PCSK2 enzymatic processing site, a blood glucose regulating hormone coding sequence, a fourth PCSK1 and / or PCSK2 enzymatic processing site, a second preproglucagon IP, a fifth PCSK1 and / or PCSK2 enzymatic processing site, and an A chain coding sequence.

[0105] "Identity" refers to the relatedness between two sequences or between three or more sequences (e.g., amino acid or nucleotide sequences) as determined by comparing the sequences with each other. Identity also refers to the degree of sequence relatedness between sequences as determined by the number of matches between a series of amino acids (polypeptides) or a series of nucleotides (polynucleotides). Identity is a measure of the percentage of identical matches of the smaller of two or more sequences, with gap alignment (if any), as positioned by a particular mathematical model or computer program (e.g., "algorithm"). The identity of related polypeptides and polynucleotides can be readily calculated by known methods. "Percent (%) identity" as 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 the residues of a second polypeptide or polynucleotide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity.

[0106] Methods and computer programs for alignment are well known in the art. Identity is dependent on the calculation of percent identity, but it is understood that the value may vary due to gaps and penalties introduced in the calculation. In general, a particular polynucleotide or polypeptide variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity with a particular wild type, native or reference sequence, as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include, but are not limited to, those in the BLAST suite (Altschul, SF, et al. Nucleic Acids Res. 1997;25:3389-3402); and those based on the Smith-Waterman algorithm (Smith, TF & Waterman, MSJ Mol. Biol. 1981;147:195-197). A popular global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, SB & Wunsch, CDJ Mol. Biol. 1920;48:443-453). The Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has also been developed, which purportedly produces global alignments of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm.

[0107] In some embodiments, the polynucleotides presented herein further comprise at least one promoter sequence. As used herein, a "promoter sequence" is a nucleotide sequence located at the 5' end of a polynucleotide to which a polymerase specifically binds and initiates transcription of the remainder of the polynucleotide. In some embodiments, the promoter is a pancreatic islet cell promoter sequence, such as a pancreatic islet beta cell promoter sequence. In some embodiments, the promoter sequence is an insulin promoter sequence, such as a human insulin promoter, a mouse insulin 1 promoter, a mouse insulin 2 promoter, a rat insulin 2 promoter, or a rat insulin 1 promoter. Additional exemplary promoters include, but are not limited to, Slc2a, IAPP, NKX6.1, DLK1, MafA, Slc30a8 / Znt8, PCSK1, and ADCYAP1.

[0108] In some embodiments, the polynucleotide presented herein further comprises an enhancer sequence. An "enhancer sequence" is a nucleotide sequence that can stimulate promoter activity by enhancing the level of tissue specificity of the promoter, and is located between the promoter and the coding sequence of the polynucleotide. Exemplary enhancer sequences include, but are not limited to, CMV enhancer, synthetic enhancer, liver-specific enhancer, vascular-specific enhancer, brain-specific enhancer, nervous system cell-specific enhancer, lung-specific enhancer, muscle-specific enhancer, kidney-specific enhancer, pancreas-specific enhancer, and islet cell-specific enhancer. In some embodiments, the enhancer sequence is an islet cell-specific enhancer.

[0109] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the nucleotide sequence of SEQ ID NO:59. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the nucleotide sequence of SEQ ID NO:60. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the nucleotide sequence of SEQ ID NO:61. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the nucleotide sequence of SEQ ID NO:62. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the nucleotide sequence of SEQ ID NO:63. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the nucleotide sequence of SEQ ID NO: 64. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the nucleotide sequence of SEQ ID NO: 65. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the nucleotide sequence of SEQ ID NO: 66. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the nucleotide sequence of SEQ ID NO: 67.In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the nucleotide sequence of SEQ ID NO:68. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the nucleotide sequence of SEQ ID NO:69. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the nucleotide sequence of SEQ ID NO:70. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the nucleotide sequence of SEQ ID NO:71. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the nucleotide sequence of SEQ ID NO:85. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the nucleotide sequence of SEQ ID NO: 86. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the nucleotide sequence of SEQ ID NO: 87.

[0110] In some embodiments, the polynucleotide encodes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the polynucleotide encodes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the polynucleotide encodes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 74. In some embodiments, the polynucleotide encodes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the polynucleotide encodes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 76. In some embodiments, the polynucleotide encodes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 77. In some embodiments, the polynucleotide encodes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 78. In some embodiments, the polynucleotide encodes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, the polynucleotide encodes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 80.In some embodiments, the polynucleotide encodes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO:81. In some embodiments, the polynucleotide encodes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO:82. In some embodiments, the polynucleotide encodes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO:83. In some embodiments, the polynucleotide encodes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO:84. In some embodiments, the polynucleotide encodes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO:88. In some embodiments, the polynucleotides encode an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the polynucleotides encode an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO:90. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0111] Delivery Systems and Routes In some embodiments, the polynucleotides of the present disclosure are further formatted for delivery. In some embodiments, the present disclosure provides a vector comprising any one of the polynucleotides described herein.

[0112] Non-viral vectors In some embodiments, the vector is a non-viral vector, such as a plasmid, a bacterial artificial chromosome, a yeast artificial chromosome, or a minicircle.

[0113] In some embodiments, the polynucleotide is delivered as a plasmid vector. In some embodiments, the polynucleotide is part of a nucleic acid cassette, which contains the elements necessary for the expression of the encoded polypeptide in the cassette. Thus, in some aspects, a plasmid for the expression of an encoded polypeptide is provided, which includes an expression cassette, also referred to as a transcription unit, that includes the coding sequence of the polypeptide. When the plasmid is placed in an environment suitable for expression, the polypeptide and others encoded in the construct are expressed from the transcription unit. The transcription unit includes a transcription control sequence transcriptionally linked to a cellular immune response element coding sequence. The transcription control sequence may include a promoter / enhancer sequence, such as a cytomegalovirus (CMV) promoter / enhancer sequence, such as those described above. However, one of skill in the art will understand that a variety of other promoter sequences suitable for expression in mammalian cells, including human patient cells, are known and can also be used in the constructs disclosed herein. The level of expression of the polypeptide depends on the presence and activation of the associated promoter and associated enhancer element.

[0114] In some embodiments, the polynucleotides can be cloned into an expression plasmid that contains regulatory elements for transcription, translation, RNA stability, and replication (including, for example, transcriptional control sequences). Such expression plasmids are well known in the art, and one of skill in the art would be able to design appropriate expression constructs for in vivo production of the recombinant polypeptides described herein.

[0115] In some embodiments, the polynucleotides can be formatted as bacterial artificial chromosomes (BACs). BACs are engineered DNA molecules used to clone DNA sequences into bacterial cells (e.g., E. coli). On average, DNA sequences ranging from 30,000 base pairs to about 300,000 base pairs can be inserted into BACs. The BACs with DNA inserted can be incorporated into bacterial cells. As bacterial cells grow and divide, the number of bacterial cell copies of BAC DNA per bacterial cell becomes very low (e.g., one copy per cell) and is stably maintained under certain conditions.

[0116] In some embodiments, polynucleotide is formatted as yeast artificial chromosome (YAC). YAC is a genetically modified circular chromosome that contains elements from yeast chromosome (e.g., yeast) and foreign DNA (e.g., polynucleotide described herein). YAC vector contains specific structural components for replicating in yeast, including but not limited to centromere, telomere, autonomously replicating sequence (ARS), yeast selection marker (e.g., TRP1, URA3 and SUP4), and cloning site for inserting large segments of exogenous DNA of more than 50kb.

[0117] In some embodiments, the polynucleotides are formatted as minicircles. Minicircle (mcDNA)-based gene transfer can also be adapted for in vivo delivery of encoded polypeptides to tissues. Because plasmid DNA can cause undesirable inflammatory responses, some embodiments use minicircles that do not contain the same elements as plasmid DNA and are less immunogenic. In minicircles, immunogenic bacterial control regions, such as origins of replication and antibiotic resistance genes, are eliminated from the gene delivery vector during the process of plasmid production. Thus, a "parent" plasmid is recombined into a "minicircle" that generally contains the polypeptide to be delivered (in this case, the polynucleotide coding sequence) and the appropriate control regions for its expression. Thus, the polynucleotide embodiments described herein can be processed into the form of minicircle DNA. Minicircle DNA relates to a small (2-4 kb) circular plasmid derivative that is devoid of all prokaryotic parts of the vector.

[0118] Viral Vectors In some embodiments, the vector is a viral vector, such as a retroviral vector, an adenoviral vector, a herpes simplex virus (HSV) vector, and an adeno-associated virus (AAV) vector.

[0119] In some embodiments, the vector is a retroviral vector. The polynucleotide is inserted into the viral genome in place of a certain viral sequence to generate a replication-defective virus. To produce virions, a packaging cell line is constructed that contains gag, pol and env genes but does not have LTR (long terminal repeat) and psi components (Mann et al., Cell, 33:153-159(1983)). When a recombinant plasmid containing a polynucleotide, LTR and psi is introduced into this cell line, the psi sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles and then secreted into culture medium. The medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene delivery system.

[0120] In some embodiments, the retroviral vector for use in the present disclosure is a lentiviral vector, where lentivirus refers to a genus of retrovirus that can infect dividing and non-dividing cells and generally produces high viral titers.Some examples of lentivirus include HIV (human immunodeficiency virus: HIV type 1 and HIV type 2); equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).In some embodiments, the retroviral vector is based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), and combinations thereof.Other retroviral vectors that can also be used in the present disclosure include, for example, vectors based on human foamy virus (HFV) or other viruses of the spumavirus genus.

[0121] In some embodiments, retroviral vector contains all of the cis-acting sequences required for packaging and integration of viral genome, such as (a) long terminal repeats (LTRs) or parts thereof at each end of the vector; (b) primer binding sites for minus and plus strand DNA synthesis; and (c) packaging signals required for genomic RNA assembly into virions.In some embodiments, retrovirus is a recombinant replication-competent retrovirus that includes a nucleotide sequence encoding retroviral GAG protein; a nucleotide sequence encoding retroviral POL protein; a nucleotide sequence encoding retroviral envelope; an oncoretroviral polynucleotide sequence that includes long terminal repeat (LTR) sequences at the 5' and 3' ends of the oncoretroviral polynucleotide sequence; a cassette that includes an internal ribosome entry site (IRES) operably linked to the polynucleotide described herein, the cassette being located 5' to the U3 region of 3'LTR and 3' to the sequence encoding retroviral envelope; and cis-acting sequences for reverse transcription, packaging and integration in target cells (e.g., pancreatic beta cells).

[0122] In some embodiments, the vector is a herpes simplex virus (HSV) vector. HSV-based vectors are either replication-defective viruses that have been eliminated from cytotoxicity by deletion of viral gene products, or amplicon vectors that are plasmids packaged into HSV particles using helper viruses (Lachmann, Int J Exp Pathol, 2004; 85(4): 177-190; Warnock et al. (2011) Methods Mol. Biol. 737:1-25). Herpes simplex virus (HSV) 1 and 2 are members of the Herpesviridae family and infect humans. The HSV genome contains two distinct regions, called unique long (UL) and unique short (US) regions. Each of these regions is flanked by a pair of terminal inverted repeats, which can be replaced with the polynucleotides described herein.

[0123] In some embodiments, the vector is an adenovirus vector (AdV). AdV is a non-enveloped, double-stranded DNA virus that does not integrate into the host genome and does not replicate during cell division. "Adenovirus expression vector" refers to a vector that contains sufficient adenovirus sequences to (a) support packaging of the construct and (b) express the polynucleotides described herein. Adenoviruses are typically used as gene delivery vectors due to their moderate genome size, ease of manipulation, high titer, wide target cell range, and high infectivity. Both ends of the viral genome contain 100-200 bp ITRs (inverted terminal repeats), which are cis elements necessary for viral DNA replication and packaging. The E1 region (E1A and E1B) of the genome encodes proteins involved in regulating transcription of the viral genome and a few cellular genes. The E2 region (E2A and E2B) encodes proteins involved in viral DNA replication. In some embodiments, a polynucleotide sequence can be inserted into the DA promoter region.

[0124] In some embodiments, the vector comprises an adeno-associated virus (AAV) vector. AAV (or recombinant AAV, "rAAV") is a small, non-enveloped, single-stranded DNA virus that can infect both dividing and non-dividing cells. In contrast to AdV-mediated gene transfer, which generally has limited persistence, transgene expression can persist for several years after intramuscular recombinant AAV (rAAV) vector delivery.

[0125] In general, recombinant AAV viruses are produced by co-transfecting a plasmid containing a gene of interest (e.g., a polynucleotide) flanked by two AAV terminal repeats and an expression plasmid containing a wild-type AAV coding sequence without terminal repeats. AAV expression vectors with polynucleotides bound to AAV ITRs can be constructed by directly inserting selected sequences into the AAV genome from which the main AAV open reading frame ("ORF") has been excised. In some embodiments, the ITR sequences are selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 (AAVrhlO) and AAV11 ITR sequences. In some embodiments, the ITRs are designed for single-stranded or self-complementary AAV genomes. For self-complementary AAV genomes, the TRS (terminal release site) located within the 3'ITR is deleted.

[0126] In some embodiments, the AAV vector comprises the above-mentioned recombinant AAV vector genome and a nucleotide sequence encoding a capsid protein. Capsid protein is related to the determination of the tissue-specific targeting ability of 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 protein and variants thereof.

[0127] For eukaryotic cells, expression control sequences generally include promoters, enhancers, such as those derived from immunoglobulin genes, SV40, cytomegalovirus, etc. (see above), and polyadenylation sequences that may include splice donor and acceptor sites. The polyadenylation sequence is generally inserted after the transgene sequence and before the 3'ITR sequence. In some embodiments, the polyadenylation sequence includes SV40 polyA or bovine GH polyA sequences. 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. The selection of these and other common vectors and regulatory elements is routine, and many such sequences are available. Those skilled in the art can select among these expression control sequences without departing from the scope of the present disclosure. Those skilled in the art can select appropriate promoter / enhancer sequences using the guidance provided by this application, and such selection is routine and is not intended to be limiting of the molecule or construct.

[0128] Exemplary publications regarding AAV vectors and virions include, but are not limited to, U.S. Patent Application Publication Nos. 2020 / 0024616, 2015 / 0176027, 2015 / 0023924, 2014 / 0348794, 2014 / 0242031, and 2012 / 0164106, all of which are incorporated by reference in their entireties herein.

[0129] Delivery route The polynucleotides and vectors described herein can be delivered by a variety of routes, including, but not limited to, intraparenchymal, intraCSF, intramuscular, and systemic (e.g., intravenous or intraarterial) delivery.

[0130] In some embodiments, polynucleotides of the present disclosure can be delivered to the intestine or pancreas of a subject by a minimally invasive endoscopic procedure (e.g., using a catheter). A variety of endoscopic procedures and devices are known and contemplated herein.

[0131] In some embodiments, a delivery device including a delivery element is used to deliver a polynucleotide of the present disclosure to a delivery site, such as a pancreatic delivery site.

[0132] In some embodiments, the drug delivery device includes a device for implanting, placing, seeding, inserting, spraying, locally applying, and / or otherwise delivering a polynucleotide or pharmaceutical composition comprising a polynucleotide of the present disclosure to a "drug delivery site" of a patient. The drug delivery device includes one or more needles positioned at a tip of the drug delivery device. In some embodiments, the tip of the drug delivery device is delivered into the patient via the mouth and advanced through the wall of the gastrointestinal tract to a position adjacent to the pancreas. The drug delivery device can be delivered, for example, through a working channel of a gastrointestinal endoscope delivered from the patient's mouth. The drug delivery device can be delivered, for example, along a gastrointestinal endoscope delivered from the patient's mouth. In some embodiments, the pancreatic drug delivery site includes one or more sites selected from the group consisting of the parenchymal lumen; the pararenal lumen; the ductal lumen; the arterial lumen of an artery that drains at least a portion of the pancreas; and combinations thereof. Exemplary drug delivery devices and systems are described, for example, in WO2022 / 174091 (e.g., the REJUVA® System) and WO2016 / 011269, the contents of each of which are incorporated by reference in their entirety herein.

[0133] In some embodiments, the step of delivering the polynucleotide of the present disclosure comprises at least a first delivery of a pharmaceutical composition comprising the polynucleotide to the pancreatic parenchyma in a minimal volume, the minimal volume of the composition comprising a volume sufficient to cause 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 comprising the polynucleotide to one or more additional delivery sites proximate the tail of the pancreas.

[0134] In some embodiments, the step of delivering the pharmaceutical composition includes at least a first delivery that delivers the pharmaceutical composition into the pancreatic parenchyma in a minimal volume, the minimal volume of the pharmaceutical composition including a volume of at least 2 ml, at least 3 ml, and / or at least 5 ml.

[0135] In some embodiments, the delivery device is advanced to the selected pancreatic delivery site or sites under image guidance, which may include endoscopic ultrasound guidance; CT guidance; and / or MRI guidance.

[0136] In some embodiments, the therapeutic benefit is realized for a period of at least six months.

[0137] In some embodiments, the one or more selected pancreatic delivery sites include locations within 10 cm, within 7.5 cm, within 5 cm, and / or within 3 cm of a portion of the pancreas, the portion of the pancreas including the tail, neck, body, head, and / or uncinate process.

[0138] In some embodiments, the pharmaceutical composition and / or the at least one drug delivery element are configured to be visualized by an imaging device, and the method further includes visualizing the pharmaceutical composition and / or the at least one drug delivery element with the imaging device to confirm proper delivery of the pharmaceutical composition.

[0139] In some embodiments, the method further includes delivering a contrast agent through the at least one delivery element and visualizing the delivery of the contrast agent using an imaging device to subsequently confirm proper delivery of the pharmaceutical composition.

[0140] In some embodiments, the method further comprises pre-loading the drug delivery device with a pharmaceutical composition. The pharmaceutical composition may be loaded into the drug delivery device through a tip of the drug delivery device.

[0141] In some embodiments, the step of delivering the pharmaceutical composition is performed under a pressure of at least 3 mmHg. In some embodiments, the step of delivering the pharmaceutical composition is performed under a pressure of 25 mmHg or less.

[0142] In some embodiments, the step of delivering the pharmaceutical composition is performed at a flow rate of at least 1 ml per minute. In some embodiments, the step of delivering the pharmaceutical composition is performed at a flow rate of 5 ml per minute or less.

[0143] In some embodiments, at least one medication delivery element includes multiple openings along its length.

[0144] In some embodiments, the method further comprises the step of verifying that the at least one drug delivery element is in proper position prior to the step of delivering the pharmaceutical composition.

[0145] In some embodiments, the method further comprises delivering a permeability enhancer prior to and / or simultaneously with the delivery of the pharmaceutical composition. The delivery of the permeability enhancer can be performed locally and / or intravenously. The permeability enhancer can comprise an agent selected from the group consisting of hyaluronidase; collagenase; losartan; and combinations thereof. The pharmaceutical composition can comprise a co-formulation of the pharmaceutical composition and the permeability enhancer.

[0146] In some embodiments, the method further comprises warming tissue adjacent to the selected pancreatic delivery site or sites to a temperature greater than 39° C. before, during, and / or after delivery of the pharmaceutical composition.

[0147] In some embodiments, the method further comprises delivering a seeding blocking material configured to prevent undesired seeding of the pharmaceutical composition to non-target locations. The seeding blocking material may comprise a viscous material and / or a polymer.

[0148] In some embodiments, the method further comprises positioning a blocking element within the patient, the blocking element configured to prevent undesired seeding of the pharmaceutical composition to non-target locations.

[0149] In some embodiments, the method further comprises removing at least a portion of the pharmaceutical composition from the location of the drug delivery site after initiation of delivery of the pharmaceutical composition.

[0150] Additional aspects and embodiments of the delivery devices and methods are described in International Publication No. WO2022 / 174091 (International Application No. PCT / US2022 / 016200), the entire contents of which are incorporated by reference herein.

[0151] host cell The present disclosure provides, in some embodiments, a host cell comprising a polynucleotide, vector, vector genome, recombinant AAV vector, or fusion protein described herein. In some embodiments, the host cell comprises a pancreatic islet cell, such as a beta cell.

[0152] When the gene delivery system is constructed based on a viral vector construction, delivery can be performed using conventional infection methods known in the art. Physical methods for enhancing the delivery of both viral and non-viral polynucleotides or polypeptides include electroporation, gene guns, sonoporation, magnetofection, hydrodynamic delivery, and the like, all of which are known to those skilled in the art. As hosts for the expression of the polypeptides disclosed herein, eukaryotic and prokaryotic host cells, including mammalian cells, are well known in the art, including many immortalized cell lines available from the American Type Culture Collection (ATCC), such as Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells, and some other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. Other cell lines that can be used are bacterial and fungal cells, including yeast and filamentous fungal cells.

[0153] therapeutic use Gene therapy as described herein can be used to treat metabolic disorders, such as obesity-related metabolic disorders.Exemplary obesity-related metabolic disorders include, but are not limited to, prediabetes, type 2 diabetes (T2D), cardiovascular disease, polycystic ovarian syndrome (PCOS), nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH).

[0154] In some embodiments, the polynucleotides or polypeptides (e.g., GLP-1-encoding) described herein (fusion proteins) are used to treat T2D. In some embodiments, the treatment of T2D can include reducing HbA1c, reducing fasting blood glucose, improving the duration of blood glucose levels within normal range, and / or improving hyperglycemia. The treatment of T2D can also include reducing or eliminating the need for exogenous insulin, reducing or eliminating the need for exogenous GLP-1 receptor agonists without causing increased rates of nausea, diarrhea, vomiting, constipation, or abdominal pain.

[0155] In some embodiments, reducing blood glucose also results in additional beneficial outcomes, including reducing the rate or progression of diabetes-associated retinopathy, nephropathy, neuropathy, myocardial infarction, microvascular disease, and prevention or incidence of end-stage renal disease.

[0156] In some embodiments, additional potential benefits include a reduced 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.

[0157] Other forms of diabetes in which, in principle, production of blood glucose-regulating hormones in pancreatic beta cells could be helpful include "double diabetes" (when T1D patients also develop T2D); gestational diabetes; and prediabetes.

[0158] Other consequences of T2D that may be ameliorated include, for example, hypertension, hypertriglyceridemia, hypercholesterolemic heart disease, diabetic heart disease, heart failure, diabetic heart failure, and / or diastolic dysfunction.

[0159] Other diseases thought to occur with and be associated with T2D and obesity (but are not treated with insulin unless T2D is indicated) include, for example, polycystic ovary syndrome (PCOS), hyperandrogenism, fertility problems, menstrual disorders, hirsutism, dementia, Alzheimer's disease, cognitive decline, cancer, e.g., liver cancer, ovarian cancer, breast cancer, uterine cancer, cholangiocarcinoma, adenocarcinoma, glandular tissue tumors, stomach cancer, colon cancer, and / or prostate cancer, psoriasis, hypogonadism, insufficient total testosterone levels, and / or insufficient free testosterone levels.

[0160] In some embodiments, the polynucleotides or polypeptides (eg, encoding PYY) described herein (fusion proteins) are used to treat NAFLD.

[0161] In some embodiments, the polynucleotides or polypeptides (eg, encoding PYY) described herein (fusion proteins) are used to treat NASH.

[0162] In some embodiments, the polynucleotide or polypeptide (e.g., encoding PYY) described herein (fusion protein) is used to treat obesity.Other diseases that occur with obesity and are therefore predicted to be improved by 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.

[0163] As used herein, "treating" refers to administering one or more therapeutic agents (e.g., polynucleotides, vectors, or fusion proteins) to a subject with the expectation that a benefit may result from the administration. The subject may be any mammal, including non-human primates and human subjects. Generally, the subject is a human subject. In some embodiments, the amount of polynucleotide, vector, or fusion protein administered is an effective amount. An "effective amount" or a "therapeutically effective amount" of a composition (e.g., a pharmaceutical composition comprising a polynucleotide, vector, and / or fusion protein presented herein) is based, at least in part, on the target tissue, the target cell type, the means of administration, the physical characteristics of the pharmaceutical composition, other pharmaceutical compositions, and other determinants, such as the age, weight, height, sex, and overall health of the subject. Generally, an effective amount of a pharmaceutical composition is one that results in an improvement in glucose (blood sugar) levels in the subject.

[0164] In some embodiments, the effective amount is one that restores glycemic continuity in a subject. As used herein, "glycemic continuity" refers to a period during which a subject's blood glucose level is within a physiological range (e.g., "ideal glycemic control" or "optimal glycemic control"). According to the American Diabetes Association, the recommended Hb1Ac cut point for diagnosing diabetes is 6.5% and the individual is at high risk (Gillett et al., Diabetes Care. 2009;32:1327-34). In some embodiments, the physiological range of blood glucose is a glycosylated 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 amount is one that results in the maintenance of a HbA1c value of less than 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.

[0165] In some embodiments, the effective amount is one that reduces fasting blood glucose compared to baseline. In some embodiments, the effective amount is one that 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 amount is one that reduces fasting blood glucose by at least 50% compared to baseline. In some embodiments, the effective amount is one that reduces fasting blood glucose by at least 55% compared to baseline. In some embodiments, the effective amount is one that reduces fasting blood glucose by at least 60% compared to baseline. According to the WHO, normal fasting blood glucose is between 70 mg / dL and 100 mg / dL, with 100 mg / dL to 125 mg / dL representing a prediabetic state, and fasting blood glucose above 126 mg / dL indicating that the subject has diabetes (WHO, "Mean fasting blood glucose," who.int / data / gho / indicator-metadata-registry / imr-details / 2380). In some embodiments, an effective amount reduces fasting blood glucose in a subject to less than 130 mg / dL, less than 126 mg / dL, less than 120 mg / dL, less than 115 mg / dL, less than 110 mg / dL, less than 105 mg / dL, or less than 100 mg / dL. As used herein, "fasting blood glucose" refers to blood glucose values ​​(concentration of glucose in venous plasma) determined when a subject has been fasting (without any food other than water) for at least 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24 hours or longer (see, e.g., WHO, "Mean fasting blood glucose"). "Baseline" refers to the subject's level (e.g., blood glucose level) before treatment begins.

[0166] In some embodiments, the effective amount is one that increases fasting insulin compared to baseline. In some embodiments, the effective amount is one that increases fasting insulin at least 1-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 4-fold, 5-fold, or more compared to baseline. In some embodiments, the effective amount is one that increases fasting insulin 2-fold compared to baseline. In some embodiments, the effective amount is one that increases fasting insulin 2.8-fold, or at least 2.8-fold compared to baseline. In some embodiments, the effective amount is one that increases fasting insulin 3-fold compared to baseline. As used herein, "fasting insulin" refers to insulin levels determined when a subject has fasted (has not consumed any food other than water) for at least 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24 hours or more. "Baseline" refers to the subject's levels (e.g., insulin levels) before treatment begins.

[0167] In some embodiments, an effective amount is one that significantly improves glucose tolerance compared to baseline. Glucose tolerance refers to a subject's ability to control plasma glucose and / or plasma insulin levels when glucose intake fluctuates. Glucose tolerance can be measured using any method in the art, including an oral glucose tolerance test (OGTT), e.g., a glucose challenge test in which a subject drinks a glass of concentrated glucose solution (e.g., 50 g glucose dissolved in 250-300 mL water) and measures blood glucose levels in the subject's blood at least one hour later. In some embodiments, glucose tolerance is measured by comparing fasting blood glucose levels to blood glucose levels 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, the subject's blood glucose level is less than 200 mg / dL, less than 190 mg / dL, less than 180 mg / dL, less than 170 mg / dL, less than 160 mg / dL, less than 150 mg / dL, less than 140 mg / dL, less than 130 mg / dL, or less. In some embodiments, the subject's blood glucose level after treatment is less than 140 mg / dL. In some embodiments, the subject's blood glucose level is reduced 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 than baseline.

[0168] In some embodiments, effective amount is that which significantly improves glucose stimulated insulin secretion compared to baseline.Glucose stimulated insulin secretion (GSIS) can be measured using any method known in the art, such as hyperinsulinemic euglycemic clamp, hyperglycemic clamp, or extrapolation from surrogate measures of insulin sensitivity (e.g., intravenous glucose tolerance test data, fasting blood samples, and quantitative insulin sensitivity test index).In some embodiments, GSIS increases from baseline after treatment. In some embodiments, GSIS is increased by at least 1 fold, 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 2.6 fold, 2.7 fold, 2.8 fold, 2.9 fold, 3 fold, 3.1 fold, 3.2 fold, 3.3 fold, 3.4 fold, 3.5 fold, 4 fold, 5 fold, or more compared to baseline.

[0169] In some embodiments, subject's body weight does not change significantly compared to baseline.In some embodiments, subject's body weight changes less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% compared to baseline.In some embodiments, subject's body weight remains the same compared to baseline.

[0170] In some embodiments, an effective amount of the pharmaceutical composition is one that brings a subject with T2D into remission (i.e., the subject maintains physiological levels of blood glucose). In some embodiments, the effective amount is a single dose, two doses, three doses, four doses, five doses, six doses, or more. In some embodiments, the effective amount is sufficient for long-term restoration of beta cell function and / or reduction in treatment burden (e.g., the health care workload experienced by the subject and its impact on the subject's well-being). In some embodiments, "long-term restoration" means 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or longer, including complete and permanent remission.

[0171] In some embodiments, in addition to the pharmaceutical composition provided herein, additional treatment is administered.Exemplary additional treatment includes treatment for T2D, such as amylinomimetics, alpha-glucosidase inhibitors, biguanides, dopamine agonists, dipeptidyl peptidase-4 (DPP-4) inhibitors, GLP-1 receptor agonists, meglitinides, statins, sodium-glucose transporter (SGLT) 2 inhibitors, sulfonylureas, thiazolidinediones, insulin, and combinations thereof.In some embodiments, no additional treatment is administered to subject.

[0172] In some embodiments, the polynucleotide, vector, or fusion protein may be administered as part of a pharmaceutical composition. The term "pharmaceutical composition" refers to an active agent combined with a carrier, inert or active, that renders the composition particularly suitable for therapeutic use in vivo or ex vivo. A "pharmaceutical acceptable carrier" is one that does not cause undesirable physiological effects after or upon administration to a subject. A carrier in a pharmaceutical composition must also be "acceptable" in the sense that it may be compatible with the active ingredient and capable of stabilizing the active ingredient. One or more solubilizing agents may be utilized as pharmaceutical carriers for delivering the active agent. Examples of pharmaceutical acceptable carriers include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to obtain a composition that can be used in a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences.

[0173] Pharmaceutical compositions may further comprise one or more pharma- ceutically acceptable excipients.In addition to conventional excipients such as any and all solvents, dispersion media, diluents or other liquid vehicles, dispersion or suspension aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, etc., pharma-ceutically acceptable excipients may include, without limitation, lipid-like substances, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, RNA-transfected cells (e.g., for transplantation into subjects), hyaluronidase, nanoparticle mimics, and combinations thereof.In some embodiments, pharmaceutical compositions comprise at least one additional active substance, such as, for example, a therapeutically active substance.

[0174] The composition may be sterile, pyrogen-free, or sterile and pyrogen-free. For the purposes of this disclosure, the phrase "active ingredient" generally refers to the polynucleotide, vector, or fusion protein described herein. The formulations of the compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preliminary methods include the steps of bringing the active ingredient (e.g., polynucleotide) into association with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desired, dividing, shaping, and / or packaging the product into the desired single or multiple unit doses. The relative amounts of the active ingredient, pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition according to the present disclosure will vary depending on the identity, size, and / or condition of the subject being treated, and will also depend on the route by which the composition is administered. By way of example, the composition may contain between 0.1% and 100%, for example between 0.5% and 50%, between 1% and 30%, between 5% and 80%, at least 80% (w / w) active ingredient.

[0175] The polynucleotides, vectors, or fusion proteins described herein can be administered by any route that results in a therapeutically effective outcome. These include, but are not limited to, intradermal, intramuscular, intranasal, and / or subcutaneous administration. In some embodiments, the polynucleotides, vectors, or fusion proteins described herein are delivered locally, rather than systemically. In some embodiments, the polynucleotides, vectors, or fusion proteins are delivered to pancreatic islet cells (e.g., beta cells). In some embodiments, the polynucleotides, vectors, or fusion proteins are delivered to pancreatic islet cells by endoscopic procedures.

[0176] The disclosure also contemplates combination therapy, for example, using the REVITA® System, which is a minimally invasive, outpatient, endoscopic, one-procedure treatment. The REVITA® System includes a specially designed control console and a novel disposable balloon catheter. The catheter is used by the physician to apply heat to the duodenum while the console is used to monitor the procedure. In some embodiments, the REVITA® System can be used as an adjunctive combination therapy.

[0177] Additional Embodiments Additional embodiments are described in the following numbered paragraphs: 1. A polynucleotide comprising a blood glucose-regulating hormone coding sequence and an insulin coding sequence. 2. The polynucleotide of item 1, wherein the blood glucose-regulating hormone coding sequence and the insulin coding sequence are arranged such that a functional blood glucose-regulating hormone and functional insulin are produced in vivo after administration to a subject. 3. The polynucleotide of item 1, wherein the blood glucose-regulating hormone coding sequence is nested within the insulin coding sequence. 4. The polynucleotide of item 1, wherein the insulin coding sequence comprises, from 5' to 3', a signal peptide coding sequence, a B chain coding sequence, a C peptide coding sequence, and an A chain coding sequence. 5. The polynucleotide of item 4, wherein the blood glucose-regulating hormone coding sequence is located between the signal peptide coding sequence and the B chain coding sequence. 6. The polynucleotide of item 4, wherein the blood glucose-regulating hormone coding sequence is nested within the B chain coding sequence. 7. The polynucleotide of item 4, wherein the blood glucose-regulating hormone coding sequence is located between the B chain coding sequence and the C peptide coding sequence. 8. The polynucleotide of item 4, wherein the blood glucose-regulating hormone coding sequence is nested within the C-peptide coding sequence. 9. The polynucleotide of item 4, wherein the blood glucose-regulating hormone coding sequence is located between the C-peptide coding sequence and the A-chain coding sequence. 10. The polynucleotide of item 4, wherein the blood glucose-regulating hormone coding sequence is nested within the A chain coding sequence. 11. The polynucleotide of item 4, wherein the blood glucose-regulating hormone coding sequence is located downstream of the A chain coding sequence. 12. The polynucleotide of any one of items 1 to 11, wherein the blood glucose-regulating hormone coding sequence is flanked by a first PCSK1 (PC1 / 3) and / or PCSK2 (PC2) enzyme processing sequence, optionally a native PCSK1 or PCSK2 enzyme processing sequence or an artificial PCSK1 or PCSK2 enzyme processing sequence. 13. The polynucleotide of any one of items 1 to 11, wherein the blood glucose-regulating hormone coding sequence is flanked by a first PCSK1 and / or PCSK2 enzyme processing sequence and a second PCSK1 and / or PCSK2 enzyme processing sequence, optionally a native PCSK1 or PCSK2 enzyme processing sequence or an artificial PCSK1 or PCSK2 enzyme processing sequence. 14. The polynucleotide of any one of items 1 to 3, comprising, from 5' to 3', a signal peptide coding sequence, a blood glucose regulating hormone coding sequence, a B chain coding sequence, a first PCSK1 or PCSK2 enzyme processing sequence, a C peptide coding sequence, a second PCSK1 or PCSK2 enzyme processing sequence, and an A chain coding sequence. 15. The polynucleotide of any one of items 1 to 3, comprising, from 5' to 3', a signal peptide coding sequence, a B chain coding sequence, a blood glucose regulating hormone coding sequence, a first PCSK1 or PCSK2 enzyme processing sequence, a C peptide coding sequence, a second PCSK1 or PCSK2 enzyme processing sequence, and an A chain coding sequence. 16. The polynucleotide of any one of items 1 to 3, comprising, from 5' to 3', a signal peptide coding sequence, a B chain coding sequence, a first PCSK1 or PCSK2 enzyme processing sequence, a blood glucose regulating hormone coding sequence, a C peptide coding sequence, a second PCSK1 or PCSK2 enzyme processing sequence, and an A chain coding sequence. 17. The polynucleotide of any one of items 1 to 3, comprising, from 5' to 3', a signal peptide coding sequence, a B chain coding sequence, a first PCSK1 or PCSK2 enzymatic processing sequence, a blood glucose regulating hormone coding sequence nested within the C peptide coding sequence, a second PCSK1 or PCSK2 enzymatic processing sequence, and an A chain coding sequence. 18. The polynucleotide of any one of items 1 to 3, comprising, from 5' to 3', a signal peptide coding sequence, a B chain coding sequence, a first PCSK1 or PCSK2 enzyme processing sequence, a C peptide coding sequence, a blood glucose regulating hormone coding sequence, a second PCSK1 or PCSK2 enzyme processing sequence, and an A chain coding sequence. 19. The polynucleotide of any one of items 1 to 3, comprising, from 5' to 3', a signal peptide coding sequence, a B chain coding sequence, a first PCSK1 or PCSK2 enzyme processing sequence, a C peptide coding sequence, a second PCSK1 or PCSK2 enzyme processing sequence, a blood glucose regulating hormone coding sequence, and an A chain coding sequence. 20. The polynucleotide of any one of items 1 to 3, comprising, from 5' to 3', a signal peptide coding sequence, a B chain coding sequence, a first PCSK1 or PCSK2 enzyme processing sequence, a C peptide coding sequence, a second PCSK1 or PCSK2 enzyme processing sequence, an A chain coding sequence, and a blood glucose-regulating hormone coding sequence. 21. The polynucleotide of any one of the preceding items, wherein the blood glucose regulating hormone is selected from glucagon, GLP-1, oxyntomodulin, glicentin, glicentin-related polypeptide (GRPP), major proglucagon fragment, intervening peptide 1 (IP-1), intervening peptide 2 (IP-2), GLP-2, glucose-dependent insulinotropic peptide (GIP), peptide tyrosine (PYY), cholecystokinin (CCK), somatostatin, oxyntomodulin, ghrelin, amylin, glucagon, leptin, follistatin, insulin-like growth factor 1 (IGF1), vasoactive intestinal peptide (VIP), and growth hormone 1 (GH1), and peptides, variants and fusions thereof, optionally selected from any one of SEQ ID NOs: 30-58. 22. The polynucleotide of item 21, wherein the blood glucose-regulating hormone is wild-type human GLP-1. 23. The polynucleotide of item 21, wherein the GLP-1 is a variant human GLP-1 comprising a Gly8 substitution compared to wild-type human GLP-1. 24. The polynucleotide of any one of the preceding items, comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the nucleotide sequence of any one of SEQ ID NOs: 59-71. 25. The polynucleotide of any one of the preceding items, comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 72-84. 26. The polynucleotide of any one of the preceding items, wherein the polynucleotide is operably linked to a promoter sequence, preferably a pancreatic islet cell promoter sequence, more preferably a pancreatic islet beta cell promoter sequence, optionally selected from the human insulin promoter, the mouse insulin 2 promoter, the mouse insulin 1 promoter, the rat insulin 2 promoter, the rat insulin 1 promoter, Slc2a, IAPP, NKX6.1, DLK1, MafA, Slc30a8 / Znt8, PCSK1, and ADCYAP1. 27. The polynucleotide of any one of the preceding items, further comprising an enhancer sequence. 28. A vector comprising the polynucleotide of any one of the preceding items. 29. The vector of item 28, which is a non-viral vector, optionally a plasmid, a bacterial artificial chromosome, a yeast artificial chromosome, or a minicircle. 30. The vector of item 28, which is optionally a viral vector selected from a retroviral vector, an adenoviral vector, a herpes simplex virus (HSV) vector, and an adeno-associated virus (AAV) vector. 31. A recombinant adeno-associated virus (AAV) vector genome comprising any one of the polynucleotides of items 1 to 27. 32. The recombinant AAV vector genome of item 31, further comprising inverted terminal repeat (ITR) sequences flanking the polynucleotide as needed. 33. The recombinant AAV vector genome of item 32, wherein the ITR sequences are selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 (AAVrh10), and AAV11 ITR sequences. 34. A recombinant adeno-associated virus (AAV) vector comprising (a) the recombinant AAV vector genome of any one of items 31 to 33 and (b) a capsid protein. 35. The recombinant AAV vector of item 34, wherein 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, and variants thereof. 36. A fusion protein encoded by any one of the polynucleotides of items 1 to 27. 37. A fusion protein comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 72 to 84. 38. A host cell comprising the polynucleotide of any one of items 1 to 27, the vector of any one of items 28 to 30, the AAV vector genome of any one of items 31 to 33, the recombinant AAV vector of item 34 or 35, or the fusion protein of item 36 or 37. 39. A method comprising delivering to a pancreatic islet cell the polynucleotide of any one of items 1 to 27, the vector of any one of items 28 to 30, the AAV vector genome of any one of items 31 to 33, the recombinant AAV vector of item 34 or 35, or the fusion protein of item 36 or 37. 40. A method comprising administering to a subject the polynucleotide of any one of items 1 to 27, the vector of any one of items 28 to 30, the AAV vector genome of any one of items 31 to 33, the recombinant AAV vector of item 34 or 35, or the fusion protein of item 36 or 37. 41. The method of item 40, wherein the subject has an obesity-related metabolic disorder selected from the group consisting of prediabetes, type 2 diabetes, cardiovascular disease, polycystic ovary syndrome (PCOS), nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH). 42. A method for treating an obesity-related metabolic disorder, comprising administering to a subject in need thereof an effective amount of the polynucleotide of any one of items 1 to 27, the vector of any one of items 28 to 30, the AAV vector genome of any one of items 31 to 33, the recombinant AAV vector of item 34 or 35, or the fusion protein of item 36 or 37. 43. The method of item 40, wherein the obesity-related metabolic disorder is selected from the group consisting of prediabetes, type 2 diabetes, cardiovascular disease, polycystic ovary syndrome (PCOS), nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH). 44. The method of any one of items 39 to 43, wherein the administering step is by endoscopic delivery to the pancreas or an area near the pancreas. 45. The method of item 42, wherein the effective amount restores glycemic continuity in the subject. 46. ​​The method of item 42, wherein the effective amount reduces fasting blood glucose by at least 50% compared to baseline. 47. The method of item 42, wherein the effective amount increases fasting insulin by at least 2-fold compared to baseline. 48. The method of item 42, wherein the effective amount significantly improves glucose tolerance compared to baseline. 49. The method of item 42, wherein the effective amount significantly improves glucose-stimulated insulin secretion compared to baseline. 50. A method according to item 48 or 49, in which the subject's weight does not change significantly compared to baseline. 51. The method of item 42, wherein the effective amount is a single dose, and the single dose is sufficient for long-term restoration of beta cell function and / or reduction in therapeutic burden. 52. A method for treating an obesity-related metabolic disorder, comprising administering to a subject in need thereof an effective amount of an adeno-associated virus (AAV) vector genome, wherein the AAV vector genome comprises a polynucleotide comprising a human pancreatic islet beta cell specific promoter operably linked to a human GLP-1 receptor agonist coding sequence, and optionally a human GLP-1 coding sequence; (a) the effective amount is one that restores glycemic continuity in a subject; (b) the effective amount reduces fasting blood glucose by at least 50% compared to baseline; (c) the effective amount increases fasting insulin by at least two-fold compared to baseline; (d) the effective amount significantly improves glucose tolerance compared to baseline; (e) the effective amount significantly improves glucose-stimulated insulin secretion compared to baseline; (f) the subject's body weight does not change significantly compared to baseline; and / or (g) the effective amount is a single dose, and the single dose is sufficient for long-term restoration of beta cell function and / or reduction in therapeutic burden; method. 53. The method of item 52, comprising administering to a subject in need thereof an effective amount of an AAV vector comprising an AAV vector genome and capsid proteins. 54. The method of item 53, wherein 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, and variants thereof. 55. An endoscopic gene therapy method comprising: advancing a drug delivery device including at least one drug delivery element to at least one pancreatic drug delivery site in a patient having a metabolic and / or pancreatic disease; delivering an effective amount of a therapeutic agent to at least one pancreatic delivery site through at least one delivery element; Including, A method, wherein the treatment agent comprises an adeno-associated virus (AAV) vector comprising an AAV vector genome, the AAV vector genome comprising a polynucleotide comprising a human pancreatic islet beta cell specific promoter operably linked to (a) a human GLP-1 receptor agonist coding sequence, optionally a human GLP-1 coding sequence, or (b) a peptide tyrosine tyrosine (PYY) coding sequence. 56. The method of item 55, wherein the at least one medication delivery element includes at least one needle positioned at a distal end of the medication delivery device. 57. The method of items 55 or 56, wherein the tip of the drug delivery device is delivered into the patient through the mouth and advanced through the wall of the gastrointestinal tract to a position adjacent to the pancreas, and, optionally, (a) the drug delivery device is delivered through a working channel of a gastrointestinal endoscope delivered through the patient's mouth, or (b) the drug delivery device is delivered along a gastrointestinal endoscope delivered through the patient's mouth. 58. The method of any one of items 55 to 57, wherein the metabolic disease is selected from the group consisting of type 1 diabetes; type 2 diabetes; non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); obesity; and combinations thereof, or the pancreatic disease is selected from the group consisting of pancreatitis; pancreatic cancer; hyperinsulinemia; and combinations thereof. 59. The method of any one of items 55 to 58, wherein at least one pancreatic drug delivery site is selected from the group consisting of: the parenchymal lumen; the pararenal lumen; the ductal lumen; the arterial lumen of an artery that drains at least a portion of the pancreas; and combinations thereof, and it is preferred that at least one pancreatic drug delivery site is the parenchymal lumen. 60. The method of any one of items 55 to 59, wherein the step of delivering the treating agent includes at least a first delivery of a minimal volume of the treating agent into the pancreatic parenchyma, the minimal volume of the treating agent including a volume sufficient to cause at least a portion of the volume of the treating agent to exit into the anterior pararenal space, diffuse and re-enter the pancreas, and optionally, the method further includes at least a second delivery of the treating agent to at least one additional delivery site proximate the tail of the pancreas. 61. The method of any one of items 55 to 60, wherein the step of delivering the treatment agent includes at least a first delivery that delivers a minimal volume of the treatment agent into the pancreatic parenchyma, the minimal volume of the treatment agent including a volume of at least 2 ml, at least 3 ml, and / or at least 5 ml. 62. The method of any one of items 55 to 61, wherein the delivery device is advanced to at least one pancreatic delivery site under image guidance, and optionally, the image guidance includes endoscopic ultrasound guidance; computed tomography (CT) guidance; and / or magnetic resonance imaging (MRI) guidance. 63. Any one of the methods of items 55 to 62, wherein at least one pancreatic delivery site includes a location within 10 cm, 7.5 cm, 5 cm, and / or 3 cm of a portion of the pancreas, the portion of the pancreas including the tail, neck, body, head, and / or uncinate process. 64. The method of any one of items 55 to 63, wherein the therapeutic agent and / or the at least one delivery element are configured to be visualized by an imaging device, and the method further comprises a step of visualizing the therapeutic agent and / or the at least one delivery element using the imaging device to confirm proper delivery of the therapeutic agent. 65. The method of any one of items 55 to 64, further comprising the steps of delivering a contrast agent through at least one delivery element and visualizing the delivery of the contrast agent using an imaging device to subsequently confirm proper delivery of the treatment agent. 66. The method of any one of items 55 to 65, further comprising the step of preloading the drug delivery device with the treatment agent, whereby, if necessary, the treatment agent is loaded into the drug delivery device from a tip of the drug delivery device. 67. The method of any one of items 55 to 66, wherein the step of delivering the treatment agent is carried out under a pressure of at least 3 mmHg and / or no more than 25 mmHg. 68. The method of any one of items 55 to 67, wherein the step of delivering the treatment agent is carried out at a flow rate of at least 1 ml per minute and / or no more than 5 ml per minute. 69. The method of any one of items 55 to 68, wherein at least one medication delivery element includes a plurality of openings along its length. 70. The method of any one of items 55 to 69, further comprising the step of verifying that at least one delivery element is in proper position prior to the step of delivering the therapeutic agent. 71. The method of any one of items 55 to 70, further comprising delivering a permeability enhancer prior to and / or simultaneously with delivery of the therapeutic agent, optionally wherein delivery of the permeability enhancer is performed locally and / or intravenously, optionally wherein the permeability enhancer comprises an agent selected from the group consisting of hyaluronidase; collagenase; losartan; and combinations thereof, and optionally wherein the therapeutic agent comprises a co-formulation of the therapeutic agent and the permeability enhancer. 72. The method of any one of items 55 to 71, further comprising the step of warming tissue adjacent to at least one pancreatic delivery site to a temperature greater than 39° C. before, during, and / or after delivery of the therapeutic agent. 73. The method of any one of items 55 to 72, further comprising the step of delivering a seeding blocking material configured to prevent undesired seeding of the treatment agent to non-target locations, optionally wherein the seeding blocking material comprises a viscous substance and / or a polymer. 74. The method of any one of items 55 to 73, further comprising the step of positioning a blocking element within the patient, the blocking element configured to prevent undesired seeding of the treatment agent to non-target locations. 75. The method of any one of items 55 to 74, further comprising the step of removing at least a portion of the therapeutic agent from the location of the drug delivery site after delivery of the therapeutic agent has commenced. 76. The method of any one of items 55 to 75, further comprising the step of removing all of the treatment agent. EXAMPLES

[0178] Example 1 In vitro experiments with mouse insulinoma (MIN-6) cells DNA expression plasmids were generated to allow for transient transfection of different transgenes into a mouse pancreatic beta cell model to test the ability of each transgene to code for functional GLP-1 peptide production and secretion. The transgenes tested are presented in Tables 2 and 3 and are shown diagrammatically in Figure 4. Briefly, INS-GLP-1 hybrids (#01-#13) were cloned into an expression plasmid under the control of the human cytomegalovirus (CMV) promoter. The coding sequence of the human preproglucagon (GCG) gene, which produces GLP-1 as well as additional peptides including peptides containing GLP-2 and glucagon sequences, served as a positive control. Other controls included: no transfection (cells only), an empty plasmid, and a plasmid expressing the green fluorescent protein (GFP) transgene.

[0179] The above expression plasmids were transfected into the mouse MIN-6 cell line, a commonly used model of beta cells. 48 hours after transfection, the cells were exposed to 25 mM glucose + 0.100 mM 3-isobutyl-1-methylxanthine (IBMX), and then the levels of GLP-1 in the supernatant were measured. The results are shown in Figure 5 and demonstrate that transfection with multiple INS-GLP-1 hybrid expression plasmids resulted in the secretion of GLP-1.

[0180] In another experiment, after 48 hours of incubation with the above expression vectors, cells were exposed to either low glucose (2 mM) or high glucose (25 mM glucose + 0.100 mM IBMX) for 5 hours to investigate nutrient-responsive transgene secretion into the supernatant of transfected cells. The results are shown in Figures 6 and 7, and show that the two hybrid transgene constructs produced GLP-1 (Figure 6) and human insulin (Figure 7), which were selectively secreted in response to high glucose conditions in beta cells. [Table 3-1] [Table 3-2]

[0181] Example 2 In vitro experiments with human EndoC-BH5 cell model An expression vector containing the transgene described in Example 1 was prepared and used to transfect human pancreatic beta cells (EndoC-BH5 cells). 48 hours after transfection, the levels of GLP-1 in the supernatant and cell lysate were measured. As shown in Figure 8A, transfection of both hybrid transgenes resulted in GLP-1 production in the supernatant. As shown in Figure 8B, similar results were observed in the cell lysate, indicating that the hybrid transgenes were able to successfully produce and secrete GLP-1. The levels of total GLP-1 detected in the supernatant of EndoC-BH5 cells (Figure 8A), indicating glucose-responsive secretion of the transgene, were significantly higher than the levels of total GLP-1 detected in the cells (Figure 8B), suggesting robust processing of the transgenic glucose-regulating hormone and subsequent nutrient-responsive transgene secretion.

[0182] Example 3 In vivo experiments in a mouse model of type 2 diabetes AAV-based gene therapy candidates were tested in the db / db T2D mouse model to determine their impact 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 relevant AAV composition (vehicle control, MIP-eGFP (10 12 VG / Animal), MIP-Ex4 (2.5×10 12 VG / animal), or MIP-Ex4(10 12 Mice were treated with 100 mg / animal of 100 mg / kg of 10 ...

[0183] Dose-dependent and long-lasting glycemic control was observed over 10 weeks after injection in db / db mice. Mice treated with high dose MIP-Ex4 showed a 59% reduction in fasting blood glucose [Δ304 mg / dL] (p<0.0001) (Figure 9A) and a 2.8-fold increase in fasting insulin (p=0.004) (Figure 9B). Intraperitoneal glucose tolerance test (IPGTT) (Figure 10C) showed significant improvement in both glucose tolerance (p<0.0001) (Figures 10A and 10B) and glucose-stimulated insulin secretion (p<0.05), with no effect on body weight (Figures 11A-11B). Immunohistochemical analysis showed that GLP-1RA protein was expressed in the pancreas (Figure 12C) and restricted to islet cells (Figures 12A and 12B).

[0184] Example 4 In vitro experiment with mouse insulinoma (MIN-6) cells (peptide tyrosine tyrosine) To allow transient transfection of different transgenes into a mouse pancreatic beta cell model, DNA expression plasmids were generated to test the ability of each transgene to encode functional peptide tyrosine tyrosine (PYY) secretion. The transgenes tested are presented in Tables 2 and 3. Briefly, INS-PYY hybrids (#01-#03) were cloned into an expression plasmid under the control of the human cytomegalovirus (CMV) promoter. The following groups were included: no transfection (cells only), empty plasmid, and a plasmid expressing the enhanced green fluorescent protein (eGFP) transgene.

[0185] The above expression plasmids were transfected into mouse MIN-6 cell line, a commonly used model of beta cells. After transfection, cells were exposed to 25 mM glucose + 0.100 mM 3-isobutyl-1-methylxanthine (IBMX) (high glucose) or 2 mM glucose (low glucose), and then the levels of PYY in the supernatant were measured. The results are shown in Figure 13 and demonstrate that transfection with multiple INS-PYY hybrid expression plasmids resulted in the secretion of PYY.

[0186] Example 5 In vitro experiments with mouse insulinoma (MIN-6) cells A DNA construct library containing a plasmid encoding GLP-1RA was transfected into mouse MIN-6 cell line.After transfection, cells were exposed to 25mM glucose, and then the level of GLP-1RA secretion was measured in the cell supernatant.The results are shown in Figure 14A, demonstrating that transfection with multiple GLP-1RA constructs resulted in the secretion of GLP-1RA compared to control (Tris-EDTA buffer).

[0187] The supernatants from the conditions that showed high levels of GLP-1RA secretion after stimulation with glucose were then exposed to the CHO-K1 hGLP-1R Gs cell line to evaluate the functional activity of each GLP-1RA. Cyclic AMP signaling was measured relative to the GFP control. The results are shown in Figure 14B. Eight of the nine constructs tested had significantly higher levels of cAMP signaling compared to the control.

[0188] Example 6 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. Four days after transduction, insulin secretion was measured. Significantly more GLP-1 (FIG. 15A) and glucose-stimulated insulin secretion (FIG. 15B) was observed in islet cells transduced with the AAV-GLP-1RA construct compared to islet cells transduced with AAV-eGFP (control).

[0189] Using the human beta cell line EndoC-BH5, AAV-mediated delivery of GLP-1RA was found to enhance GLP-1RA-dependent insulin secretion (Figure 16). Notably, in the presence of glucose, there was a significant difference in the amount of insulin secreted by AAV-GLP-1RA transduced cells 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. Statistically significant difference between groups.

[0190] Example 7 In vivo localization test BKS db / db mice were injected with an AAV expressing a beta cell-restricted exendin-4 transgene, AAV-MIP-Ex4 (7.5 × 10 12Mice were administered AAV-MIP-Ex4 (VG / animal), or vehicle control (n=3 / group). Note that "Ex4" refers to "exendin-4," a glucagon-like peptide 1 (GLP-1) receptor agonist, and "MIP" refers to mouse insulin promoter. After 4 weeks, fasting blood glucose was measured (after 4-6 h fasting). The results are shown in Figure 17A. A significant reduction was observed in mice administered AAV-MIP-Ex4 compared to vehicle. In addition, the levels of exendin-4 in serum and pancreas of each mouse were measured. The results are shown in Figure 17B. AAV-based exendin-4 production was not detectable in serum in any group of animals; 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.

[0191] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter cited, and in some cases may include the entire document.

[0192] The indefinite articles "a" and "an," as used in the specification and claims, should be understood to mean "at least one," unless clearly indicated to the contrary. It should also be understood that in any method claimed in the present invention that includes more than one step or act, unless clearly indicated to the contrary, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are described.

[0193] In the claims, as well as in the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0194] The terms "about" and "substantially" preceding a numerical value mean ±10% of the stated numerical value.

[0195] Where a range of values ​​is presented, each value therebetween, including the upper and lower limits, is specifically contemplated and described herein.

Claims

1. 1. A composition comprising a therapeutic agent for use in an endoscopic gene therapy method, the method comprising: advancing a drug delivery device including at least one drug delivery element to at least one pancreatic drug delivery site in a patient with a metabolic and / or pancreatic disease; delivering an effective amount of the therapeutic agent to the at least one pancreatic delivery site through the at least one delivery element; Including, 1. A composition, wherein the therapeutic agent comprises an adeno-associated virus (AAV) vector comprising an AAV vector genome, the AAV vector genome comprising a polynucleotide comprising a human pancreatic islet beta cell-specific promoter operably linked to a human GLP-1 receptor agonist coding sequence, and optionally a human GLP-1 coding sequence.

2. 10. The composition of claim 1, wherein the at least one medication element comprises at least one needle positioned at a distal end of the medication delivery device.

3. The composition of claim 1, wherein the tip of the drug delivery device is delivered into the patient through the mouth and advanced through the wall of the gastrointestinal tract to a position adjacent to the pancreas, and optionally (a) the drug delivery device is delivered through a working channel of a gastrointestinal endoscope delivered through the patient's mouth, or (b) the drug delivery device is delivered along a gastrointestinal endoscope delivered through the patient's mouth.

4. 2. The composition of claim 1, wherein the metabolic disease is selected from the group consisting of type 1 diabetes; type 2 diabetes; non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); obesity; and combinations thereof, or the pancreatic disease is selected from the group consisting of pancreatitis; pancreatic cancer; hyperinsulinemia; and combinations thereof.

5. The composition of claim 1, wherein the at least one pancreatic drug delivery site is selected from the group consisting of the parenchymal lumen; the pararenal lumen; the ductal lumen; the arterial lumen of an artery that drains into at least a portion of the pancreas; and combinations thereof, and it is preferred that the at least one pancreatic drug delivery site is the parenchymal lumen.

6. The composition of claim 1, wherein the step of delivering the therapeutic agent includes at least a first delivery of a minimum volume of the therapeutic agent into the pancreatic parenchyma, the minimum volume of the therapeutic agent including a volume sufficient to cause at least a portion of the volume of the therapeutic agent to exit into the anterior pararenal space, diffuse, and re-enter the pancreas, and optionally, the method further includes at least a second delivery of the therapeutic agent to at least one additional delivery site proximate the tail of the pancreas.

7. The composition of claim 1, wherein the step of delivering the treatment agent includes at least a first delivery that delivers a minimum volume of the treatment agent into the pancreatic parenchyma, and the minimum volume of the treatment agent includes a volume of at least 2 ml, at least 3 ml, and / or at least 5 ml.

8. The composition of claim 1, wherein the delivery device is advanced to the at least one pancreatic delivery site under image guidance, and optionally the image guidance includes endoscopic ultrasound guidance; computed tomography (CT) guidance; and / or magnetic resonance imaging (MRI) guidance.

9. 2. The composition of claim 1, wherein the at least one pancreatic drug delivery site comprises a location within 10 cm, 7.5 cm, 5 cm, and / or 3 cm of a portion of the pancreas, the portion of the pancreas comprising the tail, neck, body, head, and / or uncinate process.

10. 10. The composition of claim 1, wherein the therapeutic agent and / or the at least one medication delivery element is configured to be visualized by an imaging device, and the method further comprises the step of visualizing the therapeutic agent and / or the at least one medication delivery element with the imaging device to confirm proper delivery of the therapeutic agent.

11. The composition of claim 1, wherein the method further comprises the steps of delivering a contrast agent through the at least one drug delivery element and visualizing the delivery of the contrast agent using an imaging device to subsequently confirm proper delivery of the therapeutic agent.

12. The composition described in claim 1, wherein the method further includes a step of pre-loading the treatment agent into the medication device, and if necessary, the treatment agent is loaded into the medication device from the tip of the medication device.

13. The composition described in claim 1, wherein the step of delivering the therapeutic agent is carried out under a pressure of at least 3 mmHg and / or not more than 25 mmHg.

14. The composition of claim 1, wherein the step of delivering the therapeutic agent is carried out at a flow rate of at least 1 ml per minute and / or not more than 5 ml per minute.

15. The composition of claim 1 , wherein the at least one medication delivery element comprises a plurality of openings along its length.

16. The composition described in claim 1, wherein the method further includes a step of verifying that the at least one medication element is in the appropriate position prior to the step of delivering the therapeutic agent.

17. The composition of claim 1, wherein the method further comprises the step of delivering a permeability enhancer prior to and / or simultaneously with the step of delivering the therapeutic agent, wherein, optionally, the step of delivering the permeability enhancer is performed topically and / or intravenously, wherein, optionally, the permeability enhancer comprises an agent selected from the group consisting of hyaluronidase; collagenase; losartan; and combinations thereof, and wherein, optionally, the therapeutic agent comprises a co-formulation of the therapeutic agent and the permeability enhancer.

18. The composition of claim 1, wherein the method further comprises the step of warming tissue adjacent to the at least one pancreatic drug delivery site to a temperature above 39°C before, during, and / or after delivery of the therapeutic agent.

19. The composition of claim 1, wherein the method further comprises the step of delivering a seeding blocking material configured to prevent undesired seeding of the treatment agent to non-target locations, and optionally the seeding blocking material comprises a viscous substance and / or a polymer.

20. The composition described in claim 1, wherein the method further comprises the step of positioning a blocking element within the patient, the blocking element configured to prevent undesired seeding of the treatment agent to non-target locations.

21. The composition described in claim 1, wherein the method further comprises the step of removing at least a portion of the therapeutic agent from the location of the drug delivery site after delivery of the therapeutic agent has begun.

22. The composition of claim 1, wherein the method further comprises the step of removing all of the treatment agent.

23. A composition comprising a therapeutic agent for use in an endoscopic gene therapy method, said method comprising: advancing a medication delivery device including a medication delivery element to at least one pancreatic medication delivery site in a patient who has type 2 diabetes and / or is obese, the medication delivery element including a needle positioned at a distal end of the medication delivery device; delivering an effective amount of the therapeutic agent through the needle to the at least one pancreatic drug delivery site; Including, The composition, wherein the therapeutic agent comprises an adeno-associated virus (AAV) vector comprising (i) an AAV vector genome, and (ii) an AAV9 capsid protein, and the AAV vector genome comprises a polynucleotide comprising a human pancreatic islet beta cell-specific promoter operably linked to a human GLP-1 coding sequence.

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