Localized in vivo electrogenic gene therapy for type 1 diabetes

Abstract

A method for controlling type 1 diabetes (T1D) comprising gene electrotransfer (GET) for the delivery of nucleic acids encoding at least one of insulin and glucokinase is provided herein. Avoiding the need for immunomodulation required for standard gene therapy approaches, the application of an electrical pulse enables plasmid uptake for insulin and glucokinase production in target cells. GET plasmid delivery provides long-term T1D treatment by controlling glucose levels.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit and priority of U.S. Provisional Application No. 63 / 506,631, filed on June 7, 2023, and U.S. Provisional Application No. 63 / 559,928, filed on March 1, 2024, the entire contents of each of which are incorporated herein by reference.

Background Art

[0002] Type 1 diabetes (T1D) affects nearly 1.6 million Americans, with 64,000 new diagnoses each year and a lifetime cost to the healthcare system of $800 billion. Patients with T1D require insulin injections to survive, but this treatment does not control blood sugar in all cases. Chronic hyperglycemia can cause diabetes - related microvascular, macrovascular, and neurological complications. Therefore, precise control of glucose homeostasis is a major challenge in diabetes management. Islet transplantation and stem cell transplantation have been investigated clinically, but access to human islets and the required immunosuppressive therapies pose limitations to this type of treatment.

[0003] Genetic manipulation is an attractive approach for managing hyperglycemia through the introduction of insulin or glucokinase transcripts into cells. Traditional approaches for insulin and / or glucokinase gene therapy include adeno - associated virus (AAV) vector delivery and transfection. However, a major drawback of AAV - vector - mediated delivery is the need for immunosuppressive drugs to avoid inducing unwanted immune responses.

[0004] Therefore, there is a need to develop therapies for blood sugar control that avoid the expensive, cumbersome, ineffective, or harmful aspects of existing T1D treatments.

Summary of the Invention

Problems to be Solved by the Invention

[0005] This disclosure provides a system and method for overcoming the aforementioned drawbacks associated with T1D gene therapy by co-delivering insulin and glucokinase gene transcripts to cells using a non-viral, non-integrating localized in vivo electrogene therapy (LiveGT) approach. [Means for solving the problem]

[0006] In one embodiment, a method is provided for treating type 1 diabetes in a subject, the method comprising (a) administering to the subject at a site of administration at least one of a nucleic acid encoding insulin and a nucleic acid encoding glucokinase in a therapeutically effective amount, and (b) applying an electrical pulse to the site of administration. The method may also comprise administering both the nucleic acid encoding insulin and the nucleic acid encoding glucokinase.

[0007] The administration site may be within the skeletal muscle.

[0008] The electrical pulse may be applied using a single-pole electrode.

[0009] The electrical pulse may be a single-phase pulse. The electrical pulse may be approximately 1V to approximately 1.5kV. The electrical pulse may be approximately 90V. The electrical pulse may be applied for a period of approximately 50μs to approximately 200ms. The electrical pulse may be applied for a period of approximately 150ms. The electrical pulse is applied 1 to 100 times.

[0010] The nucleic acids encoding insulin and the nucleic acids encoding glucokinase may each be provided on a plasmid. In some embodiments, the nucleic acids encoding insulin and the nucleic acids encoding glucokinase are provided on separate plasmids. In some embodiments, the nucleic acids encoding insulin and the nucleic acids encoding glucokinase are provided together on the same plasmid.

[0011] Step (a) may be performed by intramuscular injection.

[0012] The method described above may further include (c) repeating steps (a) and (b) at least every six months. Step (c) may be performed between every six months and every twelve months.

[0013] These embodiments are not limiting. Other embodiments and features of the systems and methods described herein are provided below. [Brief explanation of the drawing]

[0014] [Figure 1] Figure 1 shows plasmids encoding insulin and glucokinase.

[0015] [Figure 2A] Figures 2A and 2B show glucose consumption in B16F10 melanoma cells transfected with insulin and glucokinase over time (Figure 2A) and on day 2 (Figure 2B). [Figure 2B] Figures 2A and 2B show glucose consumption in B16F10 melanoma cells transfected with insulin and glucokinase over time (Figure 2A) and on day 2 (Figure 2B).

[0016] [Figure 3A] Figures 3A and 3B show glucose consumption in insulin and glucokinase-transfected C2C12 myoblasts over time (Figure 3A) and on day 2 (Figure 3B). [Figure 3B] Figures 3A and 3B show glucose consumption in insulin and glucokinase-transfected C2C12 myoblasts over time (Figure 3A) and on day 2 (Figure 3B).

[0017] [Figure 4A]Delivery of plasmid DNA encoding insulin (Figure 4A), glucokinase (Figure 4B), insulin and glucokinase on separate plasmids (Figure 4C), insulin and glucokinase on the same plasmid (Figure 4D), and a DNA-free control (Figure 4E). [Figure 4B] Delivery of plasmid DNA encoding insulin (Figure 4A), glucokinase (Figure 4B), insulin and glucokinase on separate plasmids (Figure 4C), insulin and glucokinase on the same plasmid (Figure 4D), and a DNA-free control (Figure 4E). [Figure 4C] Delivery of plasmid DNA encoding insulin (Figure 4A), glucokinase (Figure 4B), insulin and glucokinase on separate plasmids (Figure 4C), insulin and glucokinase on the same plasmid (Figure 4D), and a DNA-free control (Figure 4E). [Figure 4D] Delivery of plasmid DNA encoding insulin (Figure 4A), glucokinase (Figure 4B), insulin and glucokinase on separate plasmids (Figure 4C), insulin and glucokinase on the same plasmid (Figure 4D), and a DNA-free control (Figure 4E). [Figure 4E] Delivery of plasmid DNA encoding insulin (Figure 4A), glucokinase (Figure 4B), insulin and glucokinase on separate plasmids (Figure 4C), insulin and glucokinase on the same plasmid (Figure 4D), and a DNA-free control (Figure 4E). [Figure 4F] Figure 4F shows the transfection efficiency calculated from five fields of view.

[0018] [Figure 5] Figure 5 is a plot of single-phase pulse parameters according to some aspects of the present disclosure.

[0019] [Figure 6A]Figures 6A and 6B illustrate proposed mechanisms of gene electrotransfer (GET) of pDNA encoding insulin and glucokinase. Figure 6A is a schematic diagram of the GET transfection process according to several aspects of this disclosure. [Figure 6B] Figure 6B is a schematic diagram of insulin and glucokinase synthesis and the resulting downstream processes according to some aspects of the present disclosure.

[0020] [Figure 7A] GET significantly enhances plasmid DNA delivery. Immunofluorescence images show the expression of human insulin (Figure 7A), human glucokinase (Figure 7B), insulin and glucokinase on the same plasmid (Figure 7C), and insulin and glucokinase on separate plasmids (Figure 7D). [Figure 7B] GET significantly enhances plasmid DNA delivery. Immunofluorescence images show the expression of human insulin (Figure 7A), human glucokinase (Figure 7B), insulin and glucokinase on the same plasmid (Figure 7C), and insulin and glucokinase on separate plasmids (Figure 7D). [Figure 7C] GET significantly enhances plasmid DNA delivery. Immunofluorescence images show the expression of human insulin (Figure 7A), human glucokinase (Figure 7B), insulin and glucokinase on the same plasmid (Figure 7C), and insulin and glucokinase on separate plasmids (Figure 7D). [Figure 7D] GET significantly enhances plasmid DNA delivery. Immunofluorescence images show the expression of human insulin (Figure 7A), human glucokinase (Figure 7B), insulin and glucokinase on the same plasmid (Figure 7C), and insulin and glucokinase on separate plasmids (Figure 7D). [Figure 7E] Figure 7E shows the transfection efficiency calculated from three fields of view. [Figure 7F] Figure 7F shows the colocalization of insulin and glucokinase under two codelivery conditions.

[0021] [Figure 8A] Monophase GET delivery of insulin and glucokinase significantly increases cellular glucose consumption. Glucose consumption over 6 days in GET-treated cells compared to glucose metabolism in controls, over time (Figure 8A) and on day 3 (Figure 8B). [Figure 8B] Monophase GET delivery of insulin and glucokinase significantly increases cellular glucose consumption. Glucose consumption over 6 days in GET-treated cells compared to glucose metabolism in controls, over time (Figure 8A) and on day 3 (Figure 8B).

[0022] [Figure 9A] GET significantly enhances plasmid DNA delivery. Fluorescence images show protein expression in the same plasmid group under both high-glucose conditions (Figure 9A) and low-glucose conditions (Figure 9B), as well as in different plasmid groups in high-glucose medium (Figure 9C) and low-glucose medium (Figure 9D), compared to high-glucose controls (Figure 9E) and low-glucose controls (Figure 9F). [Figure 9B] GET significantly enhances plasmid DNA delivery. Fluorescence images show protein expression in the same plasmid group under both high-glucose conditions (Figure 9A) and low-glucose conditions (Figure 9B), as well as in different plasmid groups in high-glucose medium (Figure 9C) and low-glucose medium (Figure 9D), compared to high-glucose controls (Figure 9E) and low-glucose controls (Figure 9F). [Figure 9C] GET significantly enhances plasmid DNA delivery. Fluorescence images show protein expression in the same plasmid group under both high-glucose conditions (Figure 9A) and low-glucose conditions (Figure 9B), as well as in different plasmid groups in high-glucose medium (Figure 9C) and low-glucose medium (Figure 9D), compared to high-glucose controls (Figure 9E) and low-glucose controls (Figure 9F). [Figure 9D]GET significantly enhances plasmid DNA delivery. Fluorescence images show protein expression in the same plasmid group under both high-glucose conditions (Figure 9A) and low-glucose conditions (Figure 9B), as well as in different plasmid groups in high-glucose medium (Figure 9C) and low-glucose medium (Figure 9D), compared to high-glucose controls (Figure 9E) and low-glucose controls (Figure 9F). [Figure 9E] GET significantly enhances plasmid DNA delivery. Fluorescence images show protein expression in the same plasmid group under both high-glucose conditions (Figure 9A) and low-glucose conditions (Figure 9B), as well as in different plasmid groups in high-glucose medium (Figure 9C) and low-glucose medium (Figure 9D), compared to high-glucose controls (Figure 9E) and low-glucose controls (Figure 9F). [Figure 9F] GET significantly enhances plasmid DNA delivery. Fluorescence images show protein expression in the same plasmid group under both high-glucose conditions (Figure 9A) and low-glucose conditions (Figure 9B), as well as in different plasmid groups in high-glucose medium (Figure 9C) and low-glucose medium (Figure 9D), compared to high-glucose controls (Figure 9E) and low-glucose controls (Figure 9F). [Figure 9G] Protein expression was measured using an immunofluorescence microscope (Figure 9G). [Figure 9H] Insulin expression was significantly enhanced in the culture medium as measured by ELISA (Figures 9H-9I). [Figure 9I] Insulin expression was significantly enhanced in the culture medium as measured by ELISA (Figures 9H-9I).

[0023] [Figure 10A] Glucokinase acts as a glucose sensor that prevents hypoglycemia. Figure 10A shows glucose consumption under high and low glucose conditions for cells exposed to GET using the same plasmid and different plasmid schemes. [Figure 10B]The changes in glucose consumption under high-glucose and low-glucose conditions in cells exposed to GET using the same plasmid scheme (Figure 10B) and a different plasmid scheme (Figure 10C) are shown. [Figure 10C] The changes in glucose consumption under high-glucose and low-glucose conditions in cells exposed to GET using the same plasmid scheme (Figure 10B) and a different plasmid scheme (Figure 10C) are shown.

[0024] [Figure 11] Figure 11 shows plots of single-phase pulse parameters according to several aspects of this disclosure.

[0025] [Figure 12A] Single-phase electrotransfer enhances gene delivery. Luciferase expression was observed in C2C12 cells treated with applied electric fields of 1300 V / cm (Figure 12A) and 600 V / cm (Figure 12B), lipofectamine (Figure 12C), and control (Figure 12D). [Figure 12B] Single-phase electrotransfer enhances gene delivery. Luciferase expression was observed in C2C12 cells treated with applied electric fields of 1300 V / cm (Figure 12A) and 600 V / cm (Figure 12B), lipofectamine (Figure 12C), and control (Figure 12D). [Figure 12C] Single-phase electrotransfer enhances gene delivery. Luciferase expression was observed in C2C12 cells treated with applied electric fields of 1300 V / cm (Figure 12A) and 600 V / cm (Figure 12B), lipofectamine (Figure 12C), and control (Figure 12D). [Figure 12D] Single-phase electrotransfer enhances gene delivery. Luciferase expression was observed in C2C12 cells treated with applied electric fields of 1300 V / cm (Figure 12A) and 600 V / cm (Figure 12B), lipofectamine (Figure 12C), and control (Figure 12D). [Figure 12E] Figure 12E shows luciferase expression over a 5-day period. [Figure 12F] The transfection efficiency is shown in Figure 12F.

[0026] [Figure 13A] Single-phase electrotransfer enhances the delivery of plasmid DNA encoding insulin (Figure 13A), glucokinase (Figure 13B), insulin and glucokinase on separate plasmids (Figure 13C), insulin and glucokinase on the same plasmid (Figure 13D), and a DNA-free control (Figure 13E). [Figure 13B] Single-phase electrotransfer enhances the delivery of plasmid DNA encoding insulin (Figure 13A), glucokinase (Figure 13B), insulin and glucokinase on separate plasmids (Figure 13C), insulin and glucokinase on the same plasmid (Figure 13D), and a DNA-free control (Figure 13E). [Figure 13C] Single-phase electrotransfer enhances the delivery of plasmid DNA encoding insulin (Figure 13A), glucokinase (Figure 13B), insulin and glucokinase on separate plasmids (Figure 13C), insulin and glucokinase on the same plasmid (Figure 13D), and a DNA-free control (Figure 13E). [Figure 13D] Single-phase electrotransfer enhances the delivery of plasmid DNA encoding insulin (Figure 13A), glucokinase (Figure 13B), insulin and glucokinase on separate plasmids (Figure 13C), insulin and glucokinase on the same plasmid (Figure 13D), and a DNA-free control (Figure 13E). [Figure 13E] Single-phase electrotransfer enhances the delivery of plasmid DNA encoding insulin (Figure 13A), glucokinase (Figure 13B), insulin and glucokinase on separate plasmids (Figure 13C), insulin and glucokinase on the same plasmid (Figure 13D), and a DNA-free control (Figure 13E). [Figure 13F] Transfection efficiency was calculated from five fields of view (Figure 13F). [Figure 13G] Figure 13G shows the decrease in culture medium glucose over 3 days in C2C12 cells, and Figure 13H shows the decrease in culture medium glucose on day 3. [Figure 13H] Figure 13G shows the decrease in culture medium glucose over 3 days in C2C12 cells, and Figure 13H shows the decrease in culture medium glucose on day 3.

[0027] [Figure 14] Gene expression (luciferase) in skeletal muscle over a 6-month period of electrotransfer.

[0028] [Figure 15A] Co-delivery of human insulin and glucokinase via LiveGT is safe. Figure 15A shows the Kaplan-Meier survival plot of animals subjected to LiveGT. [Figure 15B] Figure 15B is a plot of the body weight of animals subjected to LiveGT over time.

[0029] [Figure 16A] Co-delivery of human insulin and glucokinase via LiveGT significantly reduced serum glucose over 21 days. Figure 16A shows serum exogenous human insulin levels. [Figure 16B] Figure 16B shows serum glucose levels. [Modes for carrying out the invention]

[0030] This disclosure provides an innovative nonviral, non-embedded LiveGT approach for delivering insulin and glucokinase-encoding DNA as a treatment for T1D.

[0031] In one embodiment, a method for treating type 1 diabetes in a subject is provided herein, comprising (a) administering to the subject at an administration site at least one of a nucleic acid encoding a therapeutically effective amount of insulin and a nucleic acid encoding a therapeutically effective amount of glucokinase, and (b) applying an electrical pulse to the administration site.

[0032] Type 1 diabetes is a chronic autoimmune disease in which the pancreas is unable to produce normal levels of insulin. As used herein, the terms “treating” and “to treat” mean alleviating symptoms, eliminating the cause of symptoms that result temporarily or permanently, and / or preventing or delaying the onset of symptoms resulting from a specified disease or disorder, or reversing their progression or severity. The terms “treating” and “to treat” further include reducing one or more symptoms associated with type 1 diabetes, such as excessive hunger or thirst, frequent urination, unexplained weight loss, fatigue, blurred vision, delayed healing of cuts and erosions, and vaginal yeast infection.

[0033] Insulin is a peptide hormone produced by beta cells of the pancreas that regulates the metabolism of carbohydrates, fats, and proteins by promoting the absorption of glucose from the blood into the cells of the liver, fat, and skeletal muscle. Glucose production and secretion by the liver are strongly inhibited by high concentrations of insulin in the blood. A decrease or absence of insulin activity leads to diabetes mellitus. The insulin nucleic acid administered in the manner described herein may encode human insulin or any other form of insulin having the desired effect. The nucleic acid may encode sustained-release insulin.

[0034] Glucokinase is an enzyme that promotes the phosphorylation of glucose to glucose-6-phosphate. It acts as a glucose sensor, playing a crucial role in regulating carbohydrate metabolism by triggering shifts in metabolism or cellular function in response to increases or decreases in glucose levels.

[0035] Figures 6A and 6B illustrate nucleic acid (pDNA) uptake and resulting glucose regulation in cells exposed to the GET method described herein. The electrical pulse induces permeabilization of the cell membrane, allowing nucleic acids to enter the cell (Figure 6A). Once inside the cell, the nucleic acids are transported into the nucleus.

[0036] Referring to Figure 6B, nucleic acids are transcribed and then translated into insulin and glucokinase proteins. Insulin is secreted by cells and binds to extracellular insulin receptors on nearby cells, initiating phosphoinositide 3-kinase (PI3K) signaling, which activates glucose transporter type IV (GLUT-4) and moves it to the cell membrane. GLUT-4 is an insulin-regulated glucose transporter that facilitates the diffusion of circulating glucose into cells along the concentration gradient. Once inside the cell, glucose binds to glucokinase to produce glucose-6-phosphate, which is involved in further metabolic pathways.

[0037] As used herein, the term “administering” an agent, such as a therapeutic entity like an insulin-encoding nucleic acid or a glucokinase-encoding nucleic acid, to a subject or cell is intended to mean dispensing, delivering, or applying the substance to a target intended by any appropriate route for delivery, including parenteral / oral routes, intramuscular injection, subcutaneous / intradermal injection, intravenous injection, retroorbital injection, subarachnoid administration, oral administration, transdermal delivery, topical administration, and administration via nasal or airway routes. Nucleic acids may be administered to any tissue or cell type expressing GLUT4, such as skeletal muscle, adipose connective tissue (adipocytes), and liver tissue (hepatocytes). In exemplary embodiments, nucleic acids are administered by intramuscular injection into skeletal muscle.

[0038] The terms “nucleic acid,” “nucleic acid sequence,” “polynucleotide,” and “polynucleotide sequence” refer to polymers of nucleotides, oligonucleotides, polynucleotides (these terms may be used interchangeably), or any fragment thereof. Polynucleotides may refer to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base. There is no intentional distinction in length between the terms “nucleic acid,” “oligonucleotide,” and “polynucleotide,” and these terms are used interchangeably. These terms refer only to the primary structure of molecules. Therefore, these terms include double-stranded DNA and single-stranded DNA, as well as double-stranded RNA and single-stranded RNA. For use in this composition and method, oligonucleotides may also include nucleotide analogs modified with bases, sugars, or phosphate backbones, as well as non-purine or non-pyrimidine nucleotide analogs. These terms also refer to genomes, DNA or RNA of natural or synthetic origin (which may be single-stranded or double-stranded, and may represent sense or antisense strands).

[0039] As used herein, “therapeutic polynucleotide” refers to a DNA sequence encoding a polypeptide or RNA that, when expressed, induces a positive therapeutic effect. A therapeutic polynucleotide may comprise several operablely linked fragments, e.g., a promoter, a 5' leader sequence, a coding sequence, and a 3' untranslated sequence (e.g., a sequence encoding a polyadenylation site). “Expression” of a polynucleotide refers to the process by which a gene is transcribed into RNA and / or translated into a protein.

[0040] The nucleic acids described herein may be provided in constructs. The terms “construct,” “nucleic acid construct,” and “expression construct” are used herein to refer to recombinant polynucleotides, i.e., polynucleotides artificially formed by combining at least two polynucleotide components from different sources (natural or synthetic). For example, the constructs described herein include a coding region of a target transgene ("therapeutic polynucleotide") operably ligated to a promoter that is (1) related to another gene found in the same genome, (2) derived from the genome of a different species, or (3) synthetic. Constructs can be produced using conventional recombinant DNA methods. “Transgene” refers to a gene introduced into a host cell. A transgene may include sequences native to the host cell, sequences not naturally present in the host cell, or a combination thereof. A transgene may include sequences encoding one or more proteins, which may be operably ligated to regulatory sequences suitable for the expression of the coding sequence in the host cell.

[0041] A “promoter” or “transcriptional regulatory sequence” refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, such as a therapeutic polynucleotide sequence, and is typically located upstream of the direction of transcription of the coding sequence. Promoters are structurally identified by the presence of a DNA-dependent RNA polymerase binding site, a transcription start site, and any other DNA sequences (including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites), as well as any other nucleotide sequences (including, e.g., attenuators or enhancers, and silencers) known to those skilled in the art to act directly or indirectly to control the amount of transcription from the promoter. A “constitutive” promoter is a promoter that is active under most physiological and developmental conditions. An “inducible” promoter is a promoter that is physiologically or developmentally controlled, for example, by the application of a chemical inducer.

[0042] A construct may be part of a vector. A “vector” is a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. The four main types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Certain vectors can autonomously replicate in the host cell to which they are introduced. Other vectors can be incorporated into the host cell’s genome upon introduction into the host cell, thereby replicating with the host genome (e.g., lentiviral vectors). Furthermore, certain vectors can direct the expression of exogenous genes to which they are operably ligated. Suitable vectors are known in the art and contain the elements necessary for the gene encoded within the vector to be expressed as a protein in the host cell. The terms “plasmid,” and also “minicircle DNA” and “nanoplasmid” refer to a circular double-stranded DNA loop to which additional DNA segments (in particular additional DNA segments encoding the mutant α-gal protein) may be ligated. The term “viral vector” is used to describe a viral particle used to deliver genetic material (e.g., the construct of the present invention) to a cell, to which additional DNA segments may be ligated into the viral genome. Viral vectors include replication-deficient retroviruses (including lentiviruses), adenoviruses, and adeno-associated viruses (AAVs) that perform equivalent functions. In exemplary embodiments, the construct is provided on a plasmid. In some embodiments, the nucleic acid encoding insulin and the nucleic acid encoding glucokinase are provided on plasmids, respectively. Insulin and glucokinase may be encoded on separate plasmids. Both insulin and glucokinase may be encoded on the same plasmid.

[0043] Nucleic acids encoding insulin and glucokinase may be formulated into one or two pharmaceutical compositions. As used herein, the term “pharmaceutical composition” refers to a chemical or biological composition suitable for administration to a mammal. Such compositions typically include an active agent and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” includes saline, solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders suitable for pharmaceutical administration. Auxiliary active compounds may also be incorporated into the composition. Examples of compositions suitable for such therapeutic use include preparations for intramuscular administration, such as sterile suspensions and emulsions. In some cases, a pharmaceutical composition suitable for therapeutic use may be mixed with one or more pharmaceutically acceptable excipients, diluents, or carriers, such as sterile water, saline, or glucose.

[0044] As used herein, the terms “protein,” “polypeptide,” or “peptide” may be used interchangeably to refer to polymers of amino acids. Typically, a “polypeptide” or “protein” is defined as a longer polymer of amino acids, typically with a length of 50, 60, 70, 80, 90, or 100 amino acids. A “peptide” is defined as a shorter polymer of amino acids, typically with a length of 50, 40, 30, 20, or less amino acids. Proteins typically include polymers of naturally occurring or unnaturally occurring amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine).

[0045] "Subject" or "Subject in need" refers to a subject in need of treatment for a disease or disorder. The disease or disorder may be type 1 diabetes or a disorder associated with type 1 diabetes. The term "subject" may be used interchangeably with the terms "individual" and "patient" and includes human and non-human mammalian subjects. In a preferred embodiment, the subject is human.

[0046] In preferred embodiments, nucleic acids encoding insulin and nucleic acids encoding glucokinase are administered to an administration site in skeletal muscle, and then an electrical pulse is applied to or near the administration site so that an electric field generated by electrodes covers the entire volume of the administration site. Intramuscular injection refers to the injection of nucleic acids into muscle. However, the electrical pulse may be applied externally (e.g., to the skin) at or near the administration site.

[0047] As used herein, the terms “therapeutic effective dose” and “effective dose” refer to the amount or dosage of insulin and the nucleic acid encoding glucokinase that provides the desired effect. In some embodiments, the effective dose is the amount or dosage of the drug administered to a subject, either once or multiple times, that provides the desired effect to the subject under diagnosis or treatment.

[0048] The process of applying an electrical pulse to the administration site refers to the application of an electric field that induces a transmembrane voltage across the cell membrane of the cells at the application site. The transmembrane voltage must be strong enough to temporarily permeate the cell membrane, allowing the injected nucleic acids encoding insulin and glucokinase to enter the cells.

[0049] The electrical pulse may be applied using one or more electrodes. The electrodes may be unipolar electrodes. The electrodes may be platinum electrodes. In an exemplary embodiment, the electrodes are 10 mm 2 This is a platinum monopolar electrode. Other electrodes, such as needle electrodes, bipolar electrodes, calipers, or multi-electrode arrays, may also be used.

[0050] In one embodiment, a single-phase electrical pulse of approximately 1V to approximately 1.5kV is applied. The single-phase electrical pulse may be approximately 90V. The single-phase electrical pulse may be applied for a period of approximately 50μs to approximately 200ms. For example, the single-phase electrical pulse may be applied for a period of approximately 150ms. The single-phase electrical pulse may be applied 1 to 100 times.

[0051] The method described above may be performed more than once. For example, the method may be performed in a regimen of approximately once every six months or once every twelve months, or at any interval in between. The administration site may differ each time the method is performed. For example, subsequent administrations may be performed at a site not covered by the electric field applied during the first administration.

[0052] As used herein and in the claims, the singular forms "a," "an," and "the" include the plural form unless the context clearly indicates otherwise.

[0053] Where used herein, “about,” “approximately,” “substantially,” and “significantly” are understood by those skilled in the art and vary to some extent depending on the context in which they are used. In cases where the use of a term is unclear to those skilled in the art, “about” and “approximately” mean up to ±10% of a particular term, and “substantially” and “significantly” mean more than ±10% of a particular term.

[0054] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as “open” transitional clauses that allow for the inclusion of additional components beyond those described in the claims. The terms “consist” and “consist of” should be interpreted as “closed” transitional clauses that do not allow for the inclusion of additional components other than those described in the claims. The term “consisting essentially of” is partially closed and should be interpreted as allowing the inclusion of only additional components that do not fundamentally alter the nature of the claimed subject matter.

[0055] The phrase "such as" should be interpreted as "for example, including." Furthermore, the use of any and all illustrative language, including but not limited to "such as," is merely intended to better illustrate the invention and does not limit its scope unless otherwise specifically asserted.

[0056] Furthermore, where conventions similar to “at least one of A, B, and C, etc.” are used, such configurations are generally intended to be understood by those skilled in the art (for example, “a system having at least one of A, B, and C” includes, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together). It will further be understood by those skilled in the art that substantially any separate words and / or phrases presenting two or more alternative terms, whether in the specification or in the drawings, should be understood to intend the possibility of including one of these terms, either one or both of these terms. For example, the phrase “A or B” is understood to include the possibilities of “A” or “B” or “A and B”.

[0057] All terms such as "up to," "at least," "greater than," and "less than" refer to a range that includes the number stated and can then be divided into ranges and subranges. A range includes individual elements. Therefore, for example, a group with 1 to 3 elements refers to a group with 1, 2, or 3 elements. Similarly, a group with 6 elements refers to a group with 1, 2, 3, 4, or 6 elements, and so on.

[0058] The modal verb "may" refers to a preferred use or selection of one or more options or choices from among several described embodiments or features contained herein. If no options or choices are disclosed with respect to a particular embodiment or feature contained herein, the modal verb "may" refers to an affirmative action regarding a method of manufacturing or using one aspect of a described embodiment or feature contained herein, or a definitive decision to use a particular technique relating to a described embodiment or feature contained herein. In this latter context, the modal verb "may" has the same meaning and implications as the modal verb "can".

[0059] The following examples illustrate, and are not intended to limit, the use of the methods described herein.

[0060] Examples

[0061] Example 1. Exogenous insulin and glucokinase expression enhance glucose depletion.

[0062] Lipofectamine transfection was performed on plasmid DNA encoding insulin and glucokinase, and their effects on glucose reduction were observed.

[0063] method

[0064] Cells were seeded at a density of 2000 cells / well and allowed to stand for 24 hours with mitomycin C (which causes DNA crosslinking and prevents DNA replication). Cells were lipofectamine-transfected with Nanoplasmid® DNA encoding a) insulin, b) glucokinase, c) insulin and glucokinase on different plasmids, or d) insulin and glucokinase on the same plasmid. Plasmids are shown in Figure 1. Glucose was measured using GlucCell® (mg / dL). The medium was changed after measurement and measured daily for 3 days. Immunofluorescence was performed to observe transfection efficiency (one-way ANOVA and Tukey's multiple comparison test).

[0065] result

[0066] As shown in Figures 2A and 2B, co-expression of insulin and glucokinase resulted in a significantly higher decrease in medium glucose over three days in B16F10 melanoma cells (Figure 2A), with peak expression on day 2 (Figure 2B). Similar results were observed in C2C12 myoblasts (Figures 3A and 3B). Figures 4A to 4F show the efficiency of insulin and glucokinase lipofectamine transfection.

[0067] Consideration

[0068] Exogenous co-expression of insulin and glucokinase led to a significant enhancement of glucose reduction compared to controls.

[0069] Glucokinase or insulin delivery alone enhanced glucose reduction, but to a lesser degree. The most significant reduction across all test groups occurred on day 2, with both insulin and glucokinase delivery alone and co-expression showing significantly different culture glucose levels compared to untreated cells. Cells expressing both insulin and glucokinase delivered on the same plasmid had the lowest transfection rate, likely due to increased plasmid size and minimized endocytosis. These observations are consistent with our hypothesis that glucose transporter 4-expressing cells may be reprogrammed to regulate glucose in an insulin-dependent manner, potentially leading to a non-immunomodulatory T1D treatment.

[0070] Example 2. Gene electrotransfer increases glucose consumption in mammalian cells in an insulin-dependent manner.

[0071] method

[0072] Experimental groups. In the in vitro experiment, four experimental groups of C2C12 myoblasts treated with different Nanoplasmid® plasmids (5 μg) were used. These groups, as shown in Figure 1, included (1) insulin alone (IN), (2) glucokinase alone (GK), (3) IN and GK in the same plasmid, and (4) IN and GK in different plasmids.

[0073] Gene electrotransfer. Prior to gene electrotransfer (GET), replication of C2C12 myoblasts was inactivated using mitomycin C (10 μg / mL). GET parameters were based on previously optimized experiments using reporter gene expression, as shown in Figure 5. Specifically, 5 μg of plasmid DNA was added to the cells, and the cells were subjected to six monophase pulses lasting 100 μs at 130 V, with 250 ms intervals between pulses.

[0074] Next, the cells were incubated in either high-glucose medium (450 mg / dL) or low-glucose medium (100 mg / dL). The medium glucose levels were measured using the GlucCell® system.

[0075] Immunofluorescence (IF) microscopy imaging and analysis. Immunofluorescence microscopy was performed on cells after 7 days. Human insulin ELISA was performed to quantify insulin expression. Transfection efficiency and co-localization percentages were calculated. The groups were compared using standard two-way ANOVA and Tukey's multiple comparison test, with p<0.05 considered statistical significance. The proposed mechanism of plasmid DNA delivery is illustrated in Figures 6A and 6B.

[0076] result

[0077] As shown in Figures 7A–7F, GET significantly enhanced plasmid DNA delivery in each treatment group, as indicated by glucokinase and insulin immunofluorescence. Transfection efficiency, calculated from three fields of view, was significantly higher than in the control group, which received plasmid DNA (pDNA) encoding insulin and glucokinase without pulse (Figure 7E). High co-localization of insulin and glucokinase was observed under both co-delivery conditions (Figure 7F).

[0078] Monophase GET delivery of insulin and glucokinase significantly increased cellular glucose consumption, as shown in Figures 8A and 8B. Figure 8A shows glucose consumption over 6 days compared to glucose metabolism in the control group. The highest level of culture glucose decrease is observed on day 3 (Figure 8B).

[0079] Figures 9A–9I further demonstrate that GET significantly enhanced plasmid DNA delivery. Fluorescence images show increased protein expression in the treatment groups where insulin and glucokinase were delivered on the same plasmid in high-glucose medium (Figure 9A), in low-glucose medium (Figure 9B), in high-glucose medium (Figure 9C), and in low-glucose medium (Figure 9F), compared to the control group (C2C12 cells not exposed to plasmid or electrotransfer). Insulin and glucokinase expression increased in all treatment groups in both high-glucose and low-glucose medium (Figure 9G). Media insulin levels for cells maintained in high-glucose and low-glucose medium are shown in Figures 9H and 9I, respectively.

[0080] As shown in Figures 10A to 10C, glucokinase transfer prevented hypoglycemic glucose levels in cells.

[0081] Consideration

[0082] Exogenous co-expression of insulin and glucokinase led to a significant enhancement of glucose reduction compared to controls. Glucokinase or insulin delivery alone also enhanced glucose reduction, but to a lesser degree. The most significant reductions across all test groups occurred on days 3 and 4, and co-expression of insulin and glucokinase on the same plasmid, and co-expression on different plasmids, differed significantly from the medium glucose levels of untreated cells. No measured differences were observed between protein expression across all groups. When stimulating a fasting state, glucokinase regulated glucose consumption, keeping medium glucose levels above hypoglycemia. These observations are consistent with our hypothesis that glucose transporter-expressing cells may be reprogrammed to regulate glucose in an insulin-dependent manner, potentially leading to a non-immunomodulatory T1D treatment.

[0083] Example 3. Insulin and glucokinase delivery via gene electrotransfer regulates glucose.

[0084] method

[0085] For single-phase electrotransfer delivery of plasmid DNA, cells were seeded at 12,000 cells / well with 10 μg of plasmid DNA (containing a luciferin reporter). The electrotransfer parameters tested were 1300 V / cm and 600 V / cm. MIRUS lipofectamine transfection was used as a positive control. Cells without added DNA and applied voltage were used as a negative control. Luciferin luminescence was measured over 5 days in cells treated with luciferase. Immunofluorescence was measured (one-way ANOVA and Tukey's multiple comparison test).

[0086] To evaluate glucose control, cells were seeded at a density of 12,000 cells / well with 10 μg of plasmid DNA. A voltage of 1300 V / cm was applied. The plasmids tested were insulin, glucokinase, insulin and glucokinase (different plasmids), and insulin and glucokinase (same plasmid) (Figure 1). Cells without added DNA and applied voltage were used as negative controls. Medium glucose levels (GlucCell) were measured daily for three consecutive days. Immunofluorescence was measured (one-way ANOVA and Tukey's multiple comparison test).

[0087] For electrotransfer delivery of plasmid DNA, cells were seeded at 12,000 cells / well with 10 μg of plasmid DNA (containing a luciferin reporter). Monophase wave (1300 V / cm) was used. Cells without added DNA and applied voltage were used as negative controls. Luciferin luminescence was measured over 5 days in cells treated with luciferase. Immunofluorescence was measured (one-way ANOVA and Tukey multiple comparison test). The applied pulse protocol is shown in Figure 11.

[0088] result

[0089] As shown in Figure 12, the conventional single-phase pulse protocol enhanced gene delivery. Applied electric fields of 1300 V / cm and 600 V / cm resulted in delivery and expression comparable to lipofectamine in C2C12 cells (Figures 12A-12D). Both GET and lipofectamine enhanced luciferase expression over 5 days (Figure 12E) and increased transfection efficiency (Figure 12F).

[0090] As shown in Figures 13A to 13D, co-expression of insulin and glucokinase regulated extracellular glucose. Co-expression of insulin and glucokinase resulted in a significantly higher decrease in medium glucose over 3 days in C2C12 cells (Figure 13G), with peak expression occurring on day 3 (Figure 13H).

[0091] Consideration

[0092] The results show that conventional monophase pulse parameters significantly enhance luciferase, insulin, and glucokinase gene delivery in C2C12 cells. Extracellular glucose levels were significantly reduced by exogenous co-delivery of insulin and glucose-encoding plasmid DNA using the conventional monophase pulse protocol.

[0093] The conventional monophase pulse parameters used proved effective in effector gene delivery experiments, with both 1300 V / cm and 600 V / cm significantly increasing expression. Exogenous co-expression of both insulin and glucokinase significantly reduced culture glucose levels compared to their respective controls, indicating that GET delivery of insulin and glucokinase is a viable therapeutic pathway. C2C12 cells expressing glucose transporter 4 regulate glucose in an insulin-dependent manner. These observations are consistent with our hypothesis that skeletal muscle cells can be reprogrammed to regulate blood glucose levels, potentially leading to a treatment for type 1 diabetes without the use of immunomodulatory agents.

[0094] Example 4. Skeletal muscle LiveGT enhances therapeutic expression levels over several months.

[0095] method

[0096] In vivo gene electrotransfer procedure. Non-diabetic spray-dorry rats were anesthetized with isoflurane inhalation. Both flanks were carefully shaved to remove as much hair as possible to allow direct electrode contact with the skin. The animals were placed on their sides, and the return plate electrode was placed under the opposite flank. Ultrasonic gel was applied between the skin and the contact plate to ensure contact. A 50 μl intradermal injection of 2 mg / ml plasmid DNA encoding firefly luciferase, or pDNA encoding both human insulin and glucokinase (Figure 1), was administered. Immediately after injection, 10 mm 2 A single-phase pulse was applied using a single-pole electrode.

[0097] Bioluminescence imaging. Bioluminescence imaging was performed 1, 2, 7, 14, 21, 28, 91, and 182 days after injection. After induction of isoflurane inhalation anesthesia, animals received a subcutaneous injection of 150 mg / kg of D-luciferin (Gold Biotechnology, St. Louis, Missouri) at the treatment site. Bioluminescence signals were captured and quantified using an in vivo imaging system (PerkinElmer, Akron, Ohio). Peak flux was recorded for each pulse condition (n=4). The groups were compared using standard two-way ANOVA and Tukey's multiple comparison test, with p<0.05 considered statistical significance.

[0098] Sustained glucose response. Postprandial blood glucose was measured using a blood glucose meter 1, 2, 7, 14, 21, 28, and 91 days after injection. Serum human insulin levels were measured by ELISA (n=4). The groups were compared using standard two-way ANOVA and Tukey's multiple comparison test, with p<0.05 considered statistical significance. Kaplan-Meier survival analysis was performed, and body weight was recorded.

[0099] result

[0100] As shown in Figure 14, gene electrotransfer significantly enhanced the delivery and expression of the luciferase-encoding gene in skeletal muscle over a period of 6 months. Monopolar monophase pulses resulted in the highest expression (more than a 1000-fold increase compared to the injection-only group).

[0101] Figures 15A and 15B demonstrate the safety of co-delivery of human insulin and glucokinase via LiveGT (localized in vivo electrogenic gene therapy). The Kaplan-Meier survival analysis in Figure 15A shows no significant difference in survival between LiveGT animals and control animals (p=0.3173). As shown in Figure 15B, individual rats continued to grow normally throughout the experimental period.

[0102] Referring to Figures 16A and 16B, co-delivery of human insulin and glucokinase via LiveGT significantly reduced serum glucose over 21 days. Serum exogenous (human) insulin levels were significantly elevated over 21 days (Figure 16A). Serum glucose levels were significantly lower with co-delivery of insulin and glucokinase via LiveGT (Figure 16B). Exogenous insulin and glucokinase gene delivery mediated by LiveGT significantly reduced blood glucose levels without causing hypoglycemia.

[0103] Consideration The proposed approach enables insulin independence, eliminates the need for daily insulin protein injections, and eliminates the need for immunosuppressants, potentially minimizing the treatment burden on patients. Results demonstrate that co-expression and glucose control can be maintained for several months. Since plasmid DNA delivery is well-established as non-integrational and non-immunogenic, repeated treatments are feasible. Furthermore, LiveGT can be used as a platform technology as an alternative to other protein replacement therapies.

Claims

1. A method for treating type 1 diabetes, (a) administering to the subject at the administration site at least one of the nucleic acids encoding a therapeutically effective amount of insulin and the nucleic acid encoding a therapeutically effective amount of glucokinase, (b) Applying an electrical pulse to the administration site. Methods that include...

2. The method according to claim 1, wherein step (a) includes administering both the nucleic acid encoding insulin and the nucleic acid encoding glucokinase.

3. The method according to claim 1 or claim 2, wherein the administration site is located within skeletal muscle.

4. The method according to any one of claims 1 to 3, wherein the electrical pulse is applied using a single electrode.

5. The method according to any one of claims 1 to 4, wherein the electrical pulse is a single-phase pulse.

6. The method according to claim 5, wherein the electrical pulse is approximately 1V to approximately 1.5kV.

7. The method according to claim 6, wherein the electrical pulse is approximately 90V.

8. The method according to any one of claims 5 to 7, wherein the electrical pulse is applied for a period of about 50 μs to about 200 ms.

9. The method according to claim 8, wherein the electrical pulse is applied for approximately 150 ms.

10. The method according to any one of claims 5 to 9, wherein the electrical pulse is applied 1 to 100 times.

11. The method according to any one of claims 1 to 10, wherein the nucleic acid encoding insulin and the nucleic acid encoding glucokinase are each provided on a plasmid.

12. The method according to claim 11, wherein the nucleic acid encoding insulin and the nucleic acid encoding glucokinase are provided on separate plasmids.

13. The method according to claim 11, wherein the nucleic acid encoding insulin and the nucleic acid encoding glucokinase are both provided on the same plasmid.

14. The method according to any one of claims 1 to 13, wherein step (a) is performed by intramuscular injection.

15. (c) The method according to any one of claims 1 to 14, further comprising repeating steps (a) and (b) at least every six months.

16. The method according to claim 15, wherein step (c) is performed at intervals of approximately 6 months to approximately 12 months.

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