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WO2024254549A1 - Thérapie électrogénique in vivo localisée pour le diabète de type 1 - Google Patents

Thérapie électrogénique in vivo localisée pour le diabète de type 1 Download PDF

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Publication number
WO2024254549A1
WO2024254549A1 PCT/US2024/033138 US2024033138W WO2024254549A1 WO 2024254549 A1 WO2024254549 A1 WO 2024254549A1 US 2024033138 W US2024033138 W US 2024033138W WO 2024254549 A1 WO2024254549 A1 WO 2024254549A1
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insulin
glucokinase
nucleic acid
glucose
acid encoding
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Anna Bulysheva
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University of South Florida
University of South Florida St Petersburg
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University of South Florida
University of South Florida St Petersburg
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/327—Applying electric currents by contact electrodes alternating or intermittent currents for enhancing the absorption properties of tissue, e.g. by electroporation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0016—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the nucleic acid is delivered as a 'naked' nucleic acid, i.e. not combined with an entity such as a cationic lipid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01002—Glucokinase (2.7.1.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous

Definitions

  • Type 1 Diabetes affects nearly 1.6 million Americans, with 64,000 newly diagnosed each year, at a $800B lifetime cost to the healthcare system. Patients with T1D require insulin injections to survive, but this treatment does not regulate glycemia in all cases. Chronic hyperglycemia can cause diabetes-associated microvascular, macrovascular, and neurologic complications. Thus, precise regulation of glucose homeostasis is a major challenge in diabetes management. Islet and stem cell transplantations have been explored in the clinic, but access to human islets and the necessary immunosuppressive therapy present limitations to this treatment type.
  • AAV vector-mediated delivery is the need for immunosuppressive drugs to avoid triggering an unwanted immune response.
  • the present disclosure provides systems and methods that overcome the aforementioned drawbacks associated with T1D gene therapy by co-delivering genetic transcripts for insulin and glucokinase into cells using the non-viral, non-integrating, localized in vivo electro gene therapy (liveGT) approach.
  • liveGT localized in vivo electro gene therapy
  • a method for treating type 1 diabetes in a subject comprising: (a) administering to the subject at least one of a therapeutically effective amount of a nucleic acid encoding insulin and a therapeutically effective amount of a nucleic acid encoding glucokinase at an administration site; and (b) applying an electrical pulse to the administration site.
  • the method may comprise administering both the nucleic acid encoding insulin and the nucleic acid encoding glucokinase.
  • the administration site may be in skeletal muscle.
  • the electrical pulse may be applied using a monopolar electrode.
  • the electrical pulse may be a monophasic pulse.
  • the electrical pulse may be between about 1 V and about 1.5 kV.
  • the electrical pulse may be about 90V.
  • the electrical pulse may be applied for between about 50 ps and about 200 ms.
  • the electrical pulse may be applied for about 150 ms.
  • the electrical pulse is applied between 1 and 100 times.
  • the nucleic acid encoding insulin and the nucleic acid encoding glucokinase may each be provided on a plasmid. In embodiments, the nucleic acid encoding insulin and the nucleic acid encoding glucokinase are provided on separate plasmids. In embodiments, the nucleic acid encoding insulin and the nucleic acid encoding glucokinase are both provided on the same plasmid.
  • Step (a) may be performed by intramuscular injection.
  • the method may further comprise (c) repeating steps (a) and (b) at least about every six months.
  • Step (c) may be performed at between about every six months and about every twelve months.
  • FIG. 1 illustrates plasmids encoding insulin and glucokinase.
  • FIGS. 2A-2B show glucose consumption in B16F10 melanoma cells transfected with insulin and glucokinase over time (FIG. 2A), and on day 2 (FIG. 2B).
  • FIGS. 3A-3B show glucose consumption in C2C12 myoblasts transfected with insulin and glucokinase over time (FIG. 3 A), and on day 2 (FIG. 3B).
  • FIGS. 4A-4F delivery of plasmid DNA encoding insulin (FIG. 4A), glucokinase (FIG. 4B), insulin and glucokinase on separate plasmids (FIG. 4C), insulin and glucokinase on the same plasmid (FIG. 4D), and no DNA control (FIG. 4E).
  • FIG. 4F shows the transfection efficiency calculated from five fields of view.
  • FIG. 5 is a plot of a monophasic pulse parameter, according to aspects of the present disclosure.
  • FIGS. 6A-6B illustrate the proposed mechanism of gene electrotransfer (GET) of pDNA encoding insulin and glucokinase.
  • FIG. 6A is a schematic of the GET transfection process, according to aspects of the present disclosure.
  • FIG. 6B is a schematic of insulin and glucokinase synthesis and resulting downstream processes, according to aspects of the present disclosure.
  • FIGS 7A-7F GET significantly enhances plasmid DNA delivery.
  • Immunofluorescence images show expression of human insulin (FIG. 7A), human glucokinase (FIG. 7B), insulin and glucokinase on the same plasmid (FIG. 7C), and insulin and glucokinase on separate plasmids (FIG. 7D).
  • FIG. 7E shows transfection efficiency calculated from three fields of view.
  • FIG. 7F shows co-localization of insulin and glucokinase for the two co-delivery conditions.
  • FIGS. 8A-8B Monophasic GET delivery of insulin and glucokinase significantly increases cellular glucose consumption. Glucose consumption over six days in cells treated with GET compared to glucose metabolism in controls over time (FIG. 8A), and on day 3 (FIG. 8B).
  • FIGS 9A-9I GET significantly enhances plasmid DNA delivery.
  • the fluorescence images show protein expression in the same plasmid group in both high (FIG. 9A) and low (FIG. 9B) glucose conditions and different plasmid groups for in high glucose media (FIG. 9C) and low glucose media (FIG. 9D), when compared to high (FIG. 9E) and low (FIG. 9F) glucose controls.
  • Protein expression was measured using immunofluorescence microscopy (FIG. 9G). Insulin expression was significantly enhanced in media as measured via ELISA (FIGS. 9H-9I).
  • FIGS. 10A-10C Glucokinase acts as a glucose sensor preventing hypoglycemia.
  • FIG. 10A-10C Glucokinase acts as a glucose sensor preventing hypoglycemia.
  • FIG. 10A shows glucose consumption is shown in high and low glucose conditions for cells exposed to GET with the same plasmid and different plasmid schemes.
  • FIG. 10B shows glucose consumption in high and low glucose conditions for cells exposed to GET with the same plasmid scheme
  • FIG. 10C different plasmid scheme
  • FIG. 11 is a plot of monophasic pulse parameters, according to aspects of the present disclosure.
  • FIGS. 12A-12F Monophasic electrotransfer enhances gene delivery. Luciferase expression shown in applied electric field of 1300 V/cm (FIG. 12A) and 600 V/cm (FIG. 12B), lipofectamine (FIG. 12C), and control (FIG. 12D) treatment groups in C2C12 cells. FIG. 12E shows luciferase expression over five days. Transfection efficiency is shown in FIG. 12F.
  • FIGS. 13A-13H Monophasic electrotransfer enhances delivery of plasmid DNA encoding insulin (FIG. 13 A), glucokinase (FIG. 13B), insulin and glucokinase on separate plasmids (FIG. 13C), insulin and glucokinase on the same plasmid (FIG. 13D), and no DNA control (FIG. 13E). Transfection efficiency was calculated from five fields of view (FIG. 13F). Media glucose depletion in C2C12 cells over three days is shown in FIG. 13G, and on day 3 in FIG. 13H.
  • FIG. 14 Gene expression (luciferase) after electrotransfer in skeletal muscle over 6 months.
  • FIGS. 15A-15B Human insulin and glucokinase co-delivery via UveGT is safe.
  • FIG. 15A is a Kaplan Meier Survival plot of animals subjected to UveGT.
  • FIG. 15B is a plot of weight over time of animals subject to UveGT.
  • FIGS. 16A-16B LiveGT mediated human insulin and glucokinase co-delivery significantly reduced serum glucose over 21 days.
  • FIG. 16A shows exogenous human insulin levels in serum.
  • FIG. 16B shows serum glucose levels.
  • This disclosure provides an innovative, non-viral, non-integrating, liveGT approach to deliver insulin- and glucokinase-encoding DNA as a treatment for T ID.
  • a method for treating type 1 diabetes in a subject comprising: (a) administering to the subject at least one of: a therapeutically effective amount of a nucleic acid encoding insulin and a therapeutically effective amount of a nucleic acid encoding glucokinase at an administration site; and (b) applying an electrical pulse to the administration site.
  • Type 1 diabetes is a chronic autoimmune disease in which the pancreas is unable to produce normal levels of insulin.
  • the terms “treating” and “to treat” mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and/or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disease or disorder.
  • the terms “treating” and “to treat” further include the reduction in one or more symptom associated with type 1 diabetes, for example excessive hunger or thirst, frequent urination, unexplained weight loss, fatigue, blurred vision, slow healing of cuts and sores, vaginal yeast infections.
  • Insulin is a peptide hormone produced by beta cells of the pancreas that regulates the metabolism of carbohydrates, fats, and protein by promoting the absorption of glucose from the blood into cells of the liver, fat, and skeletal muscles. Glucose production and secretion by the liver are strongly inhibited by high concentrations of insulin in the blood. Decreased or absent insulin activity results in diabetes.
  • the insulin nucleic acid administered in the methods described herein may encode human insulin or any other form of insulin having the desired effect.
  • the nucleic acid may encode long-lasting insulin.
  • Glucokinase is an enzyme that facilitates phosphorylation of glucose to glucose-6- phosphate. It plays an important role in the regulation of carbohydrate metabolism by acting as a glucose sensor, triggering shifts in metabolism or cell function in response to rising or falling levels of glucose.
  • FIGS. 6A-6B illustrate the nucleic acid (pDNA) uptake and resulting glucose regulation in a cell exposed to the GET methods described herein.
  • the electrical pulse induces cell membrane permeabilization, allowing the nucleic acid to enter the cell (FIG. 6A). Once in the cell, the nucleic acid is transported into the nucleus.
  • the nucleic acid is transcribed, then translated into the insulin and glucokinase proteins.
  • the insulin is secreted by the cell and binds to extracellular insulin receptors on neighboring cells, initiating phosphoinositide 3-kinase (PI3K) signaling to activate and translocate glucose transporter type 4 (GLUT -4) to the cell membrane.
  • GLUT-4 is an insulin-regulated glucose transporter that facilitates the diffusion of circulating glucose down its concentration gradient into cells. Once inside the cell, glucose binds to glucokinase to produce glucose-6-phosphate for participation in further metabolic pathways.
  • administering an agent, such as a therapeutic entity like a nucleic acid encoding insulin or a nucleic acid encoding glucokinase, to a subject or cell, is intended to refer to dispensing, delivering or applying the substance to the intended target by any suitable route for delivery, including delivery by either the parenteral/oral route, intramuscular injection, subcutaneous/intradermal injection, intravenous injection, retro- orbital injection, intrathecal administration, buccal administration, transdermal delivery, topical administration, and administration by the intranasal or respiratory tract route.
  • the nucleic acids may be administered to any tissue or cell type that expresses GLUT4, such as skeletal muscle, adipose connective tissue (fat cells), and liver tissue (hepatocytes).
  • the nucleic acids are administered by intramuscular injection into the skeletal muscle.
  • nucleic acid refers to a polymer of nucleotides, an oligonucleotide, a polynucleotide (which terms may be used interchangeably), or any fragment thereof.
  • a polynucleotide may refer to a polydeoxyribonucleotide (containing 2-deoxy-D-ribose), a polyribonucleotide (containing D-ribose), and to any other type of polynucleotide that is an N glycoside of a purine or pyrimidine base.
  • nucleic acid refers only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA.
  • an oligonucleotide also can comprise nucleotide analogs in which the base, sugar, or phosphate backbone is modified as well as nonpurine or non-pyrimidine nucleotide analogs.
  • DNA or RNA of genomic, natural, or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand).
  • a "therapeutic polynucleotide” as used herein refers to DNA sequence encoding a polypeptide or an RNA that induces a positive therapeutic effect when expressed.
  • a therapeutic polynucleotide may comprise several operably linked fragments, such as a promoter, a 5' leader sequence, a coding sequence and a 3' non-translated sequence, such as sequence encoding a polyadenylation site.
  • "Expression” of a polynucleotide refers to the process wherein a gene is transcribed into an RNA and/or translated into a protein.
  • nucleic acids described herein may be provided in a construct.
  • construct “nucleic acid construct” and “expression construct” are used herein to refer to a recombinant polynucleotide, i.e., a polynucleotide that was formed artificially by combining at least two polynucleotide components from different sources (natural or synthetic).
  • the constructs described herein comprise the coding region of a transgene of interest (a “therapeutic polynucleotide”) operably linked to a promoter that (1) is associated with another gene found within the same genome, (2) is from the genome of a different species, or (3) is synthetic. Constructs can be generated using conventional recombinant DNA methods.
  • transgene refers to a gene that has been introduced into a host cell.
  • the transgene may comprise sequences that are native to the cell, sequences that do not occur naturally in the cell, or combinations thereof.
  • a transgene may contain sequences coding for one or more proteins that may be operably linked to appropriate regulatory sequences for expression of the coding sequences in the cell.
  • a “promoter” or “transcription 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 with respect to the direction of transcription of the coding sequence.
  • a promoter is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter, including e.g. attenuators or enhancers, but also silencers.
  • 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 regulated, e.g. by the application of a chemical inducer.
  • the construct may be part of a vector.
  • a “vector” is a nucleic acid molecule capable of transporting another nucleic acid to which it is linked.
  • the four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced.
  • Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g., lentiviral vectors).
  • certain vectors are capable of directing the expression of exogenous genes to which they are operatively linked.
  • Suitable vectors are known in the art and contain the necessary elements in order for the gene encoded within the vector to be expressed as a protein in the host cell.
  • plasmid and also “minicircle DNA” as well as “nanoplasmid” refers to a circular double stranded DNA loop into which additional DNA segments may be ligated, specifically exogenous DNA segments encoding the mutant a-gal protein.
  • viral vector is used to describe a virus particle that is used to deliver genetic material (e.g., the constructs of the present invention) into cells, wherein additional DNA segments may be ligated into the viral genome.
  • Viral vectors include replication defective retroviruses (including lentiviruses), adenoviruses, and adeno-associated viruses (AAV)), which serve equivalent functions.
  • the construct is provided on a plasmid.
  • the nucleic acid encoding insulin and the nucleic acid encoding glucokinase are each provided on a plasmid. Insulin and glucokinase may be encoded on separate plasmids. Both insulin and glucokinase be encoded on the same plasmid.
  • compositions refers to a chemical or biological composition suitable for administration to a mammal. Such compositions typically include the active agent and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. Examples of compositions appropriate for such therapeutic applications include preparations for intramuscular administration, such as sterile suspensions and emulsions. In some cases, pharmaceutical compositions appropriate for therapeutic applications may be in admixture with one or more pharmaceutically acceptable excipients, diluents, or carriers such as sterile water, physiological saline, glucose or the like.
  • protein or “polypeptide” or “peptide” may be used interchangeable to refer to a polymer of amino acids.
  • a “polypeptide” or “protein” is defined as a longer polymer of amino acids, of a length typically of greater than 50, 60, 70, 80, 90, or 100 amino acids.
  • a “peptide” is defined as a short polymer of amino acids, of a length typically of 50, 40, 30, 20 or less amino acids.
  • a protein typically comprises a polymer of naturally or non-naturally 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).
  • 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).
  • a “subject” or “subject in need thereof’ 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 preferred embodiments, the subject is a human.
  • the nucleic acid encoding insulin and the nucleic acid encoding glucokinase are administered to an administration site in skeletal muscle, after which the electrical pulse is applied to or near the administration site such that the electrical field produced by the electrode covers the entire volume of the administration site.
  • An intramuscular injection refers to an injection of the nucleic acids into the muscle.
  • the electrical pulse may be applied externally (e.g. to the skin) at or near the administration site.
  • the terms “therapeutically effective amount” and “effective amount” refer to the amount or dose of the nucleic acids encoding insulin and glucokinase that provides the desired effect.
  • the effective amount is the amount or dose of the agent, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment.
  • the step of applying an electrical pulse to the administration site refers to the application of an electric field resulting in induction of a transmembrane voltage across the plasma membrane of cells at the site of application.
  • the transmembrane voltage must be sufficiently strong to render the cell membrane transiently permeable, allowing the entry of the injected nucleic acids encoding insulin and glucokinase into the cells.
  • the electrical pulse may be applied using one or more electrodes.
  • the electrode may be a monopolar electrode.
  • the electrode may be a platinum electrode.
  • the electrode is a 10 mm 2 platinum monopolar electrode.
  • Other electrodes may be used, e.g. needle electrodes, bipolar electrodes, calipers, or multi -el ectrode arrays.
  • a monophasic electrical pulse of between about 1 V and about 1.5 kV is applied.
  • the monophasic electrical pulse may be about 90V.
  • the monophasic electrical pulse may be applied for between about 50 ps and about 200 ms.
  • the monophasic electrical pulse may be applied for about 150 ms.
  • the monophasic electrical pulse may be applied between 1 and 100 times.
  • the method may be performed more than one time.
  • the method may be performed on a regimen of once every about six months or about every twelve months, or at any interval in between.
  • the administration site may be different.
  • the second and subsequent administrations may be done at a site that is not covered by the electrical field applied at the first administration.
  • the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.”
  • the terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims.
  • the terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims.
  • the term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.
  • the modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use an aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”
  • Example 1 Exogenous Insulin and Glucokinase Expression Enhance Glucose Depletion.
  • Cells were plated at a density of 2000 cells/well and settled for 24 hours with mitomycin C (causes DNA crosslinking, not allowing DNA replication). Cells were lipofectamine transfected with Nanoplasmid® DNA encoding for a) insulin, b) glucokinase, c) insulin and glucokinase on different plasmids, or d) insulin and glucokinase on the same plasmid. The plasmids are shown at FIG. 1. Glucose was measured using GlucCellTM (mg/dL). Media was replaced after measurement and taken every day for three days. Immunofluorescence was performed to observe transfection efficiency (One-way ANOVA and Tukey multiple comparison test).
  • FIGS. 2A-2B co-expression of insulin and glucokinase led to significantly higher media glucose depletion over 3 days in Bl 6F 10 melanoma cells (FIG. 2A), with peak expression on day 2 (FIG. 2B). Similar results were observed in C2C12 myoblast cells (FIGS. 3A-3B).
  • FIGS. 4A-4F show the efficiency of insulin and glucokinase lipofectamine transfection.
  • glucose depletion compared to controls. Glucokinase or insulin delivery alone, also enhanced glucose depletion, however to a lower degree. The most significant decrease between all test groups appeared on day two as both insulin and glucokinase delivery alone and coexpression was significantly different than media glucose levels of untreated cells. Cells expressing both insulin and glucokinase delivered on the same plasmid had the lowest transfection rate, likely due to the increased size of the plasmid and minimized endocytosis. These observations are consistent with our hypothesis that glucose transporter 4 expressing cells can be reprogramed to modulate glucose in an insulin dependent manner, leading to an immunomodulatory drug-free, potential treatment for T1D.
  • Example 2 Gene Electrotransfer Increases Mammalian Cell Glucose Consumption in an Insulin-Dependent Manner.
  • FIGS. 7A-7F GET significantly enhanced plasmid DNA delivery in each treatment group as shown by glucokinase and insulin immunofluorescence.
  • the transfection efficiency calculated from three fields of view was significantly higher over a control group in which plasmid DNA (pDNA) encoding insulin and glucokinase were provided with no pulses (FIG. 7E).
  • pDNA plasmid DNA
  • FIG. 7F High co-localization of insulin and glucokinase was observed for both co-delivery conditions.
  • FIGS. 8A-8B Monophasic GET delivery of insulin and glucokinase significantly increased cellular glucose consumption as seen in FIGS. 8A-8B.
  • FIG. 8 A shows the glucose consumption over six days compared to glucose metabolism in controls. The highest levels of media glucose depletion are seen on day three (FIG. 8B).
  • FIGS. 9A-9I further show that GET significantly enhanced plasmid DNA delivery.
  • the fluorescence images show increased protein expression in the treatment group in which the insulin and glucokinase were provided on the same plasmid in high glucose media (FIG. 9A) and low glucose media (FIG. 9B), and in the treatment group in which the insulin and glucokinase were provided on different plasmids in high glucose media (FIG. 9C) and low glucose media (FIG. 9D), when compared to a control group (C2C12 cells not exposed to plasmid or electrotransfer) in high glucose media (FIG. 9E) and low glucose media (FIG. 9F). Insulin and glucokinase expression were increased in all treatment groups in both high and low glucose media (FIG. 9G). Media insulin levels for cells maintained in high glucose and low glucose are shown in FIGS. 9H and 91, respectively.
  • Example 3 Gene Electrotransfer Mediated Insulin and Glucokinase Delivery Modulates Glucose
  • plasmid DNA For monophasic electrotransfer delivery of plasmid DNA, cells were plated at 12,000 cells/well with 10 pg plasmid DNA (with luciferin reporter). The electrotransfer parameters tested were 1300 V/cm and 600 V/cm. MIRUS Lipofectamine transfection was used as a positive control. Cells with DNA and no applied voltage were used as a negative control. Luciferin luminescence was measured over five days when treated with luciferase. Immunofluorescence was measured (One-way ANOVA and Tukey multiple comparison test). [0086] For assessing glucose regulation, cells were plated at a density of 12,000 cells/well with 10 pg plasmid DNA. 1300 V/cm was applied.
  • the plasmid groups tested were: insulin, glucokinase, insulin and glucokinase (different plasmids), insulin and glucokinase (same plasmid) (FIG. 1). Cells with DNA and no applied voltage were used as a negative control. Media glucose levels (GlucCell) were measured each day for three consecutive days. Immunofluorescence was measured (One-way ANOVA and Tukey multiple comparison test). [0087] For electrotransfer delivery of plasmid DNA, cells were plated at 12,000 cells/well with 10 pg plasmid DNA (with luciferin reporter). A monophasic wave (1300 V/cm) was used. Cells with DNA and no applied voltage were used as a negative control. Luciferin luminescence was measured over five days when treated with luciferase. Immunofluorescence was measured (One-way ANOVA and Tukey multiple comparison test). The applied pulse protocols are shown at FIG. 11.
  • FIG. 12 traditional monophasic pulse protocols enhanced gene delivery. Applied electric fields of 1300 V/cm and 600 V/cm yielded comparable delivery and expression as lipofectamine in C2C12 cells. (FIGS. 12A-12D). Both GET and lipofectamine enhanced luciferase expression over five days (FIG. 12E) and transfection efficiency (FIG. [0090] As shown in FIGS. 13A-13D, insulin and glucokinase co-expression modulated extracellular glucose. Co-expression of insulin and glucokinase led to significantly higher media glucose depletion over three days in C2C12 cells (FIG. 13G), with peak expression on day 3 (FIG. 13H).
  • Non-diabetic Sprague Dawley rats were anesthetized with isoflurane inhalation. Both flanks were carefully shaved to remove as much hair as possible to allow for direct electrode contact with the skin. The animals were placed on their side, and the return plate electrode was placed under the opposite flank with ultrasound gel applied between the skin and grounding plate to ensure contact.
  • a 50 pl intradermal injection of 2 mg/ml plasmid DNA encoding firefly luciferase, or pDNA encoding both human insulin and glucokinase (FIG. 1) was administered. Immediately after injection, a 10 mm 2 monopolar electrode was used to apply monophasic pulses.
  • FIGS. 15A-15B show that human insulin and glucokinase co-delivery via (localized in vivo electro gene therapy) HveGT is safe.
  • FIGS. 16A-16B LiveGT mediated human insulin and glucokinase codelivery significantly reduced serum glucose over 21 days. Exogenous (human) insulin levels in serum were significantly elevated over 21 days (FIG. 16A). Serum glucose levels were significantly lower with insulin and glucokinase co-deliver via HveGT (FIG. 16B). Exogenous insulin and glucokinase gene delivery mediated with HveGT significantly reduced blood glucose levels without hypoglycemia.
  • the presented approach allows for insulin independence, and potentially substantially minimizes the treatment burden for patients eliminating the need for daily insulin protein injection and eliminating the need for immunosuppressive drugs.
  • the results indicate that coexpression and glucose control can be maintained for several months. Since it is well established that plasmid DNA delivery is non-integrating and non-immunogenic, repeat treatments are feasible. Additionally, HveGT can be used as a platform technology as an alternative to other protein replacement therapies.

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Abstract

La présente divulgation concerne des méthodes de régulation du diabète de type 1 (T1D) comprenant l'électrotransfert génique (GET) pour l'administration d'acide nucléique codant pour l'insuline et/ou la glucokinase. En évitant le besoin d'immunomodulation comme requis pour des approches de thérapie génique standard, l'application d'impulsions électriques permet une absorption de plasmide pour la production d'insuline et de glucokinase dans des cellules cibles. L'administration de plasmide GET fournit un traitement du T1D à long terme par régulation des taux de glucose.
PCT/US2024/033138 2023-06-07 2024-06-07 Thérapie électrogénique in vivo localisée pour le diabète de type 1 Pending WO2024254549A1 (fr)

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Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
"Advances in Genetics", vol. 89, 1 January 2015, ACADEMIC PRESS, US, ISSN: 0065-2660, article YOUNG JENNIFER L. ET AL: "Electroporation-Mediated Gene Delivery", pages: 49 - 88, XP093191583, DOI: 10.1016/bs.adgen.2014.10.003 *
BATISTA NAPOTNIK TINA ET AL: "Cell death due to electroporation - A review", BIOELECTROCHEMISTRY, ELESEVIER, AMSTERDAM, NL, vol. 141, 6 June 2021 (2021-06-06), XP086713914, ISSN: 1567-5394, [retrieved on 20210606], DOI: 10.1016/J.BIOELECHEM.2021.107871 *
BOSCH I TUBERT FÀTIMA: "GENE THERAPY APPROACHES FOR DIABETES MELLITUS CENTRED ON GENETIC MANIPULATION OF THE PANCREAS AND EXTRAPANCREATIC TISSUES", V SYMPOSIUM, 1 January 2004 (2004-01-01), XP093191527, Retrieved from the Internet <URL:https://statics.ccma.cat/recursos/marato/simposiums/marato1998/eng/p_27.html#> *
BULYSHEVA ANNA ET AL: "Monopolar gene electrotransfer enhances plasmid DNA delivery to skin", BIOELECTROCHEMISTRY, ELESEVIER, AMSTERDAM, NL, vol. 140, 17 April 2021 (2021-04-17), XP086602727, ISSN: 1567-5394, [retrieved on 20210417], DOI: 10.1016/J.BIOELECHEM.2021.107814 *
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