Topical in vivo electrogene therapy for type 1 diabetes

Abstract

Provided herein are methods for Type 1 diabetes (T1D) modulation, including gene electrotransfer (GET) for delivery of a nucleic acid encoding at least one of insulin and glucokinase. The application of electrical pulses avoids the need for immunomodulation required for standard gene therapy methods, enabling plasmid uptake to produce insulin and glucokinase in target cells. GET plasmid delivery provides long term T1D therapy by modulating glucose levels.

Description

Cross-references to related applications

[0001] This application claims the interests and priorities of U.S. Provisional Application No. 63 / 506,631, filed June 7, 2023, and U.S. Provisional Application No. 63 / 559,928, filed March 1, 2024, the contents of which are incorporated herein by reference in their entirety. background

[0002] Type 1 diabetes (T1D) affects nearly 1.6 million Americans, with 64,000 new diagnoses each year, costing the healthcare system $800 billion in lifetime expenses. Patients with T1D require insulin injections to survive, but this treatment doesn't regulate blood sugar in all situations. Chronic hyperglycemia can lead to diabetes-related microvascular, macrovascular, and neurological complications. Therefore, precise regulation of glucose homeostasis is a major challenge in diabetes management. Islet and stem cell transplantation have been explored clinically, but access to human islets and the necessary immunosuppressive therapies limit this type of treatment.

[0003] Genetic engineering represents an attractive approach to managing hyperglycemia by introducing insulin or glucokine transcripts into cells. Traditional approaches to insulin and / or glucokine gene therapy involve 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 triggering unwanted immune responses.

[0004] Therefore, there is a need to develop therapies for regulating blood sugar that eliminate the expensive, cumbersome, ineffective, or disadvantageous aspects of existing T1D treatments. Overview

[0005] This disclosure provides systems and methods for overcoming the aforementioned drawbacks associated with T1D gene therapy by using a non-viral, non-integrative, localized in vivo electrogenic gene therapy (liveGT) approach to co-deliver the genetic transcripts of insulin and glucokine into cells.

[0006] In one aspect, a method for treating a subject with type 1 diabetes is provided, the method comprising: (a) administering to the subject at an application site at at least one of: a therapeutically effective amount of a nucleic acid encoding insulin and a therapeutically effective amount of a nucleic acid encoding glucokine; and (b) applying an electrical pulse to the application site. The method may include administering both the nucleic acid encoding insulin and the nucleic acid encoding glucokine.

[0007] It can be applied to skeletal muscle.

[0008] An electrical pulse can be applied using a single-pole electrode.

[0009] The electrical pulse can be a single-phase pulse. The electrical pulse can be between approximately 1 V and approximately 1.5 kV. The electrical pulse can be approximately 90 V. The electrical pulse can be applied for between approximately 50 µs and approximately 200 ms. The electrical pulse can be applied for approximately 150 ms.

[0010] Between 1 and 100 electrical pulses.

[0011] The nucleic acid encoding insulin and the nucleic acid encoding glucokine can both be provided on a plasmid. In one embodiment, the nucleic acid encoding insulin and the nucleic acid encoding glucokine are provided on separate plasmids. In another embodiment, the nucleic acid encoding insulin and the nucleic acid encoding glucokine are both provided on the same plasmid.

[0012] Step (a) can be performed by intramuscular injection.

[0013] The method may also include (c) repeating steps (a) and (b) at least every six months. Step (c) may be performed between approximately every six months and approximately every twelve months.

[0014] These aspects are not limiting. Other aspects and features of the systems and methods described herein will be provided below. Brief description of the attached diagram

[0015] Figure 1 The illustration shows plasmids encoding insulin and glucokine.

[0016] Figures 2A and 2B show glucose consumption over time (Figure 2A) and on day 2 (Figure 2B) in B16F10 melanoma cells transfected with insulin and glucosamine.

[0017] Figures 3A-3B show glucose consumption over time (Figure 3A) and on day 2 (Figure 3B) in C2C12 myoblasts transfected with insulin and glucokine.

[0018] Figures 4A-4F: Delivery of plasmid DNA encoding insulin (Figure 4A), plasmid DNA encoding glucokine (Figure 4B), plasmid DNA encoding insulin and glucokine on a single plasmid (Figure 4C), plasmid DNA encoding insulin and glucokine on the same plasmid (Figure 4D), and plasmid DNA encoding a controlless plasmid (Figure 4E). Figure 4F shows the transfection efficiency calculated from five fields of view.

[0019] Figure 5 It is a diagram of single-phase pulse parameters based on aspects of this disclosure.

[0020] Figures 6A-6B The proposed mechanism of gene electrotransfer (GET) of pDNA encoding insulin and glucokine is illustrated. Figure 6AThis is a schematic diagram of the GET transfection process based on aspects of this disclosure. Figure 6B This is a schematic diagram of insulin and glucosamine synthesis and the resulting downstream processes according to aspects of this disclosure.

[0021] Figures 7A-7F. GET significantly enhances plasmid DNA delivery. Immunofluorescence images show the expression of human insulin (Figure 7A), human glucokine (Figure 7B), insulin and glucokine on the same plasmid (Figure 7C), and insulin and glucokine on separate plasmids (Figure 7D). Figure 7E shows the transfection efficiency calculated from three fields of view. Figure 7F shows the co-localization of insulin and glucokine under two co-delivery conditions.

[0022] Figures 8A-8B. Monophasic GET delivery of insulin and glucokine significantly increased cellular glucose consumption. Glucose consumption in GET-treated cells over six days compared with glucose metabolism in controls over time (Figure 8A) and on day 3 (Figure 8B).

[0023] Figures 9A-9I. GET significantly enhances plasmid DNA delivery. Fluorescence images show protein expression in the same plasmid group under both high (Figure 9A) and low (Figure 9B) glucose conditions, and in different plasmid groups under high glucose (Figure 9C) and low glucose (Figure 9D) glucose conditions, compared to high (Figure 9E) and low (Figure 9F) glucose controls. Protein expression was measured using immunofluorescence microscopy. Figure 9G Insulin expression was significantly enhanced in the culture medium, as measured by ELISA (Fig. 9H-Fig. 9I).

[0024] Figures 10A-1 0°C. Glucokinase acts as a glucose sensor to prevent hypoglycemia. Figure 10A The glucose consumption of cells exposed to GET using the same plasmid protocol and different plasmid protocols is shown under high and low glucose conditions. The changes in glucose consumption of cells exposed to GET using the same plasmid protocol (Fig. 10B) and different plasmid protocols (Fig. 10C) are shown under high and low glucose conditions.

[0025] Figure 11 It is a diagram of single-phase pulse parameters based on aspects of this disclosure.

[0026] Figures 12A-12F. Monophasic electrotransfer enhances gene delivery. Luciferase expression is shown in C2C12 cells under treatment with applied electric fields of 1300 V / cm (Figure 12A) and 600 V / cm (Figure 12B), lipofectamine (Figure 12C), and a control (Figure 12D). Figure 12E shows luciferase expression over five days. Transfection efficiency is shown in Figure 12F.

[0027] Figures 13A-13H. Monophasic electrotransfer enhanced the delivery of plasmid DNA encoding insulin (Figure 13A), plasmid DNA encoding glucokine (Figure 13B), plasmid DNA encoding insulin and glucokine on separate plasmids (Figure 13C), plasmid DNA encoding insulin and glucokine on the same plasmid (Figure 13D), and plasmid DNA encoding a DNA control-free plasmid (Figure 13E). Transfection efficiency was calculated from five fields of view (Figure 13F). Glucose depletion in the culture medium in C2C12 cells over three days is shown in Figure 13G, and depletion on day 3 is shown in Figure 13H.

[0028] Figure 14 Gene expression (luciferase) during a 6-month period after electrotransfer to skeletal muscle.

[0029] Figures 15A-15B. Via liveGT Co-delivery of human insulin and glucokine is safe. Figure 15A shows the process. liveGT Kaplan Meier survival rate plot for animals. Figure 15B shows the survival rate of animals that experienced... liveGT A graph showing how the weight of an animal changes over time.

[0030] Figures 16A-16B. LiveGT The mediated co-delivery of human insulin and glucokine significantly reduced serum glucose over 21 days. Figure 16A shows the level of exogenous human insulin in serum. Figure 16B shows the serum glucose level. Detailed Explanation

[0031] This disclosure provides an innovative, non-viral, non-integrative liveGT approach to deliver DNA encoding insulin and glucokine as a treatment for T1D.

[0032] In one aspect, this article provides a method for treating a subject with type 1 diabetes, the method comprising: (a) administering to the subject at an application site at at least one of: a therapeutically effective amount of a nucleic acid encoding insulin and a therapeutically effective amount of a nucleic acid encoding glucokine; and (b) applying an electrical pulse to the application site.

[0033] 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 the reduction of symptoms, the temporary or permanent elimination of the causal relationship of acquired symptoms, and / or the prevention or slowing of the onset of acquired symptoms of the named disease or disorder or the reversal of its progression or severity. The terms “treating” and “to treat” also include the reduction of one or more symptoms associated with type 1 diabetes, such as excessive hunger or thirst, frequent urination, unexplained weight loss, fatigue, blurred vision, slow healing of wounds and ulcers, and vaginal yeast infections.

[0034] Insulin is a peptide hormone produced by pancreatic beta cells that regulates the metabolism of carbohydrates, fats, and proteins by promoting the uptake of glucose from the blood by liver, fat, and skeletal muscle cells. Hepatic glucose production and secretion are strongly inhibited by high concentrations of insulin in the blood. Reduced or absent insulin activity leads to diabetes. The insulin nucleic acid administered in the methods described herein can encode human insulin or any other form of insulin with the desired effect. The nucleic acid can encode long-acting insulin.

[0035] Glucokinase is an enzyme that promotes the phosphorylation of glucose to glucose-6-phosphate. Glucokinase plays a crucial role in the regulation of carbohydrate metabolism by acting as a glucose sensor, triggering shifts in metabolic or cellular function in response to increases or decreases in glucose levels.

[0036] Figures 6A-6B The illustration shows nucleic acid (pDNA) uptake and the resulting glucose regulation in cells exposed to the GET method described herein. Electrical pulses induce cell membrane permeability, allowing nucleic acids to enter the cell (pDNA). Figure 6A Once inside the cell, the nucleic acid is transported to the cell nucleus.

[0037] refer to Figure 6B Nucleic acid is transcribed and then translated into insulin and glucosamine kinase proteins. Insulin is secreted by the cell and binds to extracellular insulin receptors on neighboring cells, initiating phosphoinositol 3-kinase (PI3K) signaling to activate glucose transporter type 4 (GLUT-4) and translocate it to the cell membrane. GLUT-4 is an insulin-regulated glucose transporter that facilitates the diffusion of circulating glucose down its concentration gradient into the cell. Once inside the cell, glucose binds to glucosamine kinase to produce glucose-6-phosphate, which participates in further metabolic pathways.

[0038] As used herein, the term "administer" to a subject or cell, such as a therapeutic entity, like a nucleic acid encoding insulin or glucoskinase, is intended to distribute, deliver, or apply the substance to an intended target via any suitable route of delivery, including parenteral / oral administration, intramuscular injection, subcutaneous / intradermal injection, intravenous injection, retroorbital injection, intrathecal administration, sublingual administration, percutaneous delivery, topical administration, and intranasal or respiratory administration. Nucleic acids can be administered to any tissue or cell type expressing GLUT4, such as skeletal muscle, adipose connective tissue (adipocytes), and liver tissue (hepatocytes). In an exemplary embodiment, the nucleic acid is administered via intramuscular injection into skeletal muscle.

[0039] The terms “nucleic acid,” “nucleic acid sequence,” “polynucleotide,” and “polynucleotide sequence” refer to polymers of nucleotides, oligonucleotides, polynucleotides (these terms are used interchangeably), or any fragment thereof. A polynucleotide can 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. It is not intended that the terms “nucleic acid,” “oligonucleotide,” and “polynucleotide” differ in length, and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Therefore, these terms include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. For use in this composition and method, oligonucleotides may also comprise nucleotide analogs in which the base, sugar, or phosphate backbone is modified, as well as non-purine or non-pyrimidine nucleotide analogs. These phrases also refer to genomic, natural, or synthetic DNA or RNA (which may be single-stranded or double-stranded and may represent a sense or antisense strand).

[0040] As used herein, a "therapeutic polynucleotide" refers to a DNA sequence that encodes a polypeptide or RNA that, when expressed, induces a positive therapeutic effect. A therapeutic polynucleotide may contain several operatively linked segments, such as a promoter, a 5' leader sequence, a coding sequence, and a 3' untranslated sequence (such as a sequence encoding a polyadenylation site). "Expression" of a polynucleotide refers to the process in which a gene is transcribed into RNA and / or translated into a protein.

[0041] The nucleic acids described herein can 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, a construct described herein contains the coding region of a transgene of interest (“therapeutic polynucleotide”) operatively linked to a promoter that (1) is associated with another gene found in the same genome, (2) is from a genome of a different species, or (3) is synthetic. Constructs can be produced using conventional recombinant DNA methods. A “transgene” refers to a gene that has been introduced into a host cell. A transgene can contain a cell-native sequence, a sequence not naturally present in the cell, or a combination thereof. A transgene can contain a sequence encoding one or more proteins that can be operatively linked to a suitable regulatory sequence to express the coding sequence in the cell.

[0042] A “promoter” or “transcriptional regulatory sequence” is a nucleic acid fragment that functions to control the transcription of one or more coding sequences (such as therapeutic polynucleotide sequences) and is typically located upstream of the coding sequence in the transcriptional direction. Structurally, promoters are identified by the presence of a DNA-dependent RNA polymerase binding site, a transcription start site, and any other DNA sequence, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those skilled in the art that function directly or indirectly to regulate the amount of transcription from that promoter, including, for example, attenuators or enhancers, and 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, for example, by the application of a chemical inducer.

[0043] A construct can 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 main types of vectors are plasmids, viral vectors, granules, and artificial chromosomes. Some vectors are capable of autonomous replication in the host cells to which they are introduced. Other vectors can integrate into the host cell's genome after introduction into the host cell and thus replicate along with the host genome (e.g., lentiviral vectors). Furthermore, some vectors are capable of directing the expression of a foreign gene operatively linked to them. Suitable vectors are known in the art and contain elements necessary for the expression of the gene encoded within the vector as a protein in the host cell. The terms "plasmid," "small circular DNA," and "nanoplasmid" refer to circular double-stranded DNA loops in which additional DNA segments can be linked, particularly foreign DNA segments encoding mutant α-gal proteins. The term "viral vector" is used to describe viral particles used to deliver genetic material (e.g., the constructs of the present invention) into cells, in which additional DNA segments can be linked to the viral genome. Viral vectors include replication-defective retroviruses (including lentiviruses), adenoviruses, and adeno-associated viruses (AAVs) that have equivalent functions. In an exemplary embodiment, the construct is provided on a plasmid. In this embodiment, both the nucleic acid encoding insulin and the nucleic acid encoding glucokine are provided on the plasmid. Insulin and glucokine may be encoded on separate plasmids. Alternatively, both insulin and glucokine may be encoded on the same plasmid.

[0044] Nucleic acids encoding insulin and glucoskinase can be formulated in one or two pharmaceutical compositions. As used herein, the term "pharmaceutical composition" refers to a chemical or biological composition suitable for administration to mammals. Such compositions typically include an active agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceuticalally acceptable carrier" includes saline, solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents, and absorption delayers compatible with drug administration. Complementary active compounds may also be incorporated into the composition. Examples of compositions suitable for such therapeutic applications include formulations for intramuscular administration, such as sterile suspensions and emulsions. In some cases, pharmaceutical compositions suitable for therapeutic applications may be mixed with one or more pharmaceutically acceptable excipients, diluents, or carriers such as sterile water, physiological saline, glucose, etc.

[0045] As used herein, the terms “protein” or “polypeptide” or “peptide” are used interchangeably to refer to a polymer of amino acids. Generally, a “polypeptide” or “protein” is defined as a longer polymer of amino acids, typically greater than 50, 60, 70, 80, 90, or 100 amino acids in length. A “peptide” is defined as a short polymer of amino acids, typically 50, 40, 30, 20, or fewer amino acids in length. Proteins typically comprise polymers of naturally occurring 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).

[0046] "Subject" or "subject in need" refers to a subject who requires 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 both human and non-human mammal subjects. In a preferred embodiment, the subject is a human.

[0047] In a preferred embodiment, nucleic acids encoding insulin and glucoskinase are administered to an application site in skeletal muscle, followed by the application of an electrical pulse to or near the application site, such that an electric field generated by electrodes covers the entire volume of the application site. Intramuscular injection refers to injecting nucleic acids into muscle. However, the electrical pulse can be applied externally at or near the application site (e.g., to the skin).

[0048] As used herein, the terms "therapeutic effective amount" and "effective amount" refer to the quantity or dose of nucleic acids encoding insulin and glucoskinase that provide the desired effect. In some embodiments, the effective amount is the quantity or dose of the agent that provides the desired effect in a subject receiving diagnosis or treatment after administration of a single or multiple doses to the subject.

[0049] The step of applying an electrical pulse to the application site involves applying an electric field that induces a transmembrane voltage across the cell membrane at the application site. This transmembrane voltage must be strong enough to momentarily permeate the cell membrane, allowing the injected nucleic acids encoding insulin and glucoskinase to enter the cell.

[0050] One or more electrodes can be used to apply an electrical pulse. The electrodes can be monopolar electrodes. The electrodes can be platinum electrodes. In an exemplary embodiment, the electrodes are 10 mm... 2 Platinum monopolar electrode. Other electrodes, such as needle electrodes, bipolar electrodes, calipers, or multi-electrode arrays, can be used.

[0051] In this implementation, a single-phase electrical pulse between approximately 1 V and approximately 1.5 kV is applied. The single-phase electrical pulse can be approximately 90 V. The single-phase electrical pulse can be applied for between approximately 50 µs and approximately 200 ms. For example, the single-phase electrical pulse can be applied for approximately 150 ms. The single-phase electrical pulse can be applied between 1 and 100 times.

[0052] This method can be performed more than once. For example, it can be performed every six months or every twelve months, or at any interval in between. The application site can be different each time the method is performed. For example, the second and subsequent applications can be performed at sites not covered by the electric field applied in the first application.

[0053] As used in this specification and claims, the singular forms “a”, “an”, and “the” include the plural forms unless the context clearly indicates otherwise.

[0054] As used herein, “about,” “approximately,” “substantially,” and “significantly” will be understood by those skilled in the art and will vary to some extent depending on the context in which they are used. Where there is use of a term that is unclear to those skilled in the art in the context in which the term is used, “about” and “approximately” will mean an addition or subtraction of up to 10% to the particular term, and “substantially” and “significantly” will mean more than an addition or subtraction of 10% to the particular term.

[0055] 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-ended” transitional terms, which allow for the inclusion of additional components besides those recited in the claims. The terms “consist” and “consisting of” should be interpreted as “closed-ended” transitional terms, which do not allow for the inclusion of additional components besides those recited in the claims. The term “consisting essentially of” should be interpreted as partially closed and only allows for the inclusion of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0056] The phrase “such as” should be interpreted as “for example, including”. Furthermore, the use of any and all exemplary language, including but not limited to “such as”, is intended only to better illustrate the invention and does not impose a limitation on the scope of the invention unless otherwise claimed.

[0057] Furthermore, in cases where conventions such as "at least one of A, B, and C" are used, this syntactic structure is generally intended to be understood by those skilled in the art as to convey the meaning of the convention (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having a single A, a single B, a single C, A and B together, A and C together, B and C together, and / or A, B, and C together). Those skilled in the art will further understand that, in practice, any disjunctive words and / or wording presenting two or more alternative terms, whether in the specification or in the drawings, should be understood to account for the possibility of including one, any, or both terms. For example, the phrase "A or B" should be understood to include the possibility of including "A" or "B" or "A and B".

[0058] All language expressions such as “up to,” “at least,” “greater than,” and “less than” include the stated number and refer to a range that can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group with 1-3 members refers to a group with 1, 2, or 3 members. Similarly, a group with 6 members refers to a group with 1, 2, 3, 4, 5, or 6 members, and so on.

[0059] The modal verb "may" refers to the preferred use or selection of one or more options or choices among the described implementation scheme or features included therein. Where no options or choices regarding a particular implementation scheme or its included features are disclosed, the modal verb "may" refers to an affirmative action regarding how to make or use the described implementation scheme or its included features, or a clear decision regarding the use of a specific technique related to the described implementation scheme or its included features. In this latter context, the modal verb "may" has the same meaning and connotation as the auxiliary verb "can".

[0060] The following examples illustrate the use of the methods described herein and are not intended to be limiting. Example

[0061] Example 1. Enhanced expression of exogenous insulin and glucokine enhances glucose depletion.

[0062] Lipofectamine transfection was performed on plasmid DNA encoding insulin and glucoskinase to observe their effects on glucose depletion.

[0063] method Cells were plated at a density of 2000 cells / well and incubated for 24 hours in the presence of mitomycin C (which causes DNA cross-linking and prevents DNA replication). Cells were transfected with lipofectamine using Nanoplasmid® DNA encoding the following: a) insulin, b) glucoskinase, c) insulin and glucoskinase on different plasmids, or d) insulin and glucoskinase on the same plasmid. Plasmids such as... Figure 1 As shown. Glucose (mg / dL) was measured using GlucCell™. The culture medium was changed after each measurement, and samples were collected daily for three days. Immunofluorescence was performed to observe transfection efficiency (one-way ANOVA and Tukey multiple comparison test).

[0064] result As shown in Figures 2A-2B, co-expression of insulin and glucosamine in B16F10 melanoma cells resulted in significantly higher glucose depletion of the culture medium over a 3-day period (Figure 2A), with peak expression on day 2 (Figure 2B). Similar results were observed in C2C12 myoblasts (Figures 3A-3B). Figures 4A-4F show the efficiency of insulin and glucosamine transfection with lipofectamine.

[0065] discuss Co-expression of exogenous insulin and glucokine resulted in significantly enhanced glucose depletion compared to controls. Delivery of glucokine alone or insulin alone also enhanced glucose depletion, however to a lesser extent. The most significant reduction among all test groups occurred on day 2, as delivery of both insulin and glucokine alone, as well as co-expression, differed significantly from the culture medium glucose levels in untreated cells. Cells expressing both insulin and glucokine delivered on the same plasmid had the lowest transfection rates, likely due to increased plasmid size and minimized endocytosis. These observations are consistent with our hypothesis that cells expressing glucose transporter 4 can be reprogrammed to regulate glucose in an insulin-dependent manner, leading to potential immunomodulatory therapies for T1D.

[0066] Example 2. Gene electrotransfer increases glucose consumption in mammalian cells in an insulin-dependent manner.

[0067] method Experimental groups. In vitro experiments utilized C2C12 myoblasts from four experimental groups treated with different Nanoplasmid® plasmids (5 µg). The groups included (1) insulin only (IN), (2) glucoskinase only (GK), (3) IN and GK in the same plasmid, and (4) IN and GK in different plasmids, such as… Figure 1 As shown in the figure.

[0068] Gene electrotransfer. Prior to gene electrotransfer (GET), C2C12 myoblast replication was inactivated using mitomycin C (10 μg / mL). GET parameters were based on previous experiments optimized using reporter gene expression and... Figure 5 As shown in the figure. Specifically, 5 µg of plasmid DNA was added to the cells, and the cells were subjected to six monophasic pulses at 130 V for 100 µs, with an interval of 250 ms between each pulse.

[0069] The cells were then incubated with either high-glucose medium (450 mg / dL) or low-glucose medium (100 mg / dL). GlucCell was used. TM The system measures the glucose level in the culture medium.

[0070] Immunofluorescence (IF) microscopy imaging and analysis. Cells were subjected to immunofluorescence microscopy after day 7. Human insulin ELISA was performed to quantify insulin expression. Transfection efficiency and colocalization percentage were calculated. Groups were compared using conventional two-way ANOVA and Tukey's multiple comparison test; p < 0.05 was considered significant. The proposed plasmid DNA delivery mechanism was described. Figure 6A and Figure 6B The diagram in the middle is shown.

[0071] result As shown in Figures 7A-7F, GET significantly enhanced plasmid DNA delivery in each treatment group, as indicated by immunofluorescence of glucokine and insulin. Transfection efficiency calculated from the three fields of view was significantly higher than that in the control group, where plasmid DNA (pDNA) encoding insulin and glucokine was delivered without pulses (Figure 7E). High colocalization of insulin and glucokine was observed for both co-delivery conditions (Figure 7F).

[0072] Monophasic GET delivery of insulin and glucokine significantly increased cellular glucose consumption, as shown in Figures 8A-8B. Figure 8A shows glucose consumption over six days compared to glucose metabolism in the control group. The highest level of culture medium glucose depletion was observed on day 3 (Figure 8B).

[0073] Figures 9A-9I also show that GET significantly enhances plasmid DNA delivery. Fluorescence images show increased protein expression in the treatment groups where insulin and glucokine were provided on the same plasmid in high-glucose medium (Figure 9A) and low-glucose medium (Figure 9B), and in the treatment groups where insulin and glucokine were provided on different plasmids in high-glucose medium (Figure 9C) and low-glucose medium (Figure 9D), compared to the control group (C2C12 cells not exposed to plasmids or electrotransfer) in high-glucose medium (Figure 9E) and low-glucose medium (Figure 9F). In both high-glucose and low-glucose media, insulin and glucokine expression was increased in all treatment groups. Figure 9G Insulin levels in the culture medium used to maintain cells in high and low glucose environments are shown in Figures 9H and 9I, respectively.

[0074] like Figures 10A-1 As shown in 0C, the transfer of glucosamine prevents hypoglycemic glucose levels in cells.

[0075] discuss Compared to controls, exogenous co-expression of insulin and glucokine led to a significantly enhanced glucose depletion. Delivery of glucokine alone or insulin alone also enhanced glucose depletion, however to a lesser extent. The most significant reductions among all tested groups occurred on days 3 and 4, as co-expression of both insulin and glucokine on the same and different plasmids was significantly different from the glucose levels in the culture medium of untreated cells. No differences in protein expression were measured between any groups. When stimulated in a fasting state, glucokine modulates glucose consumption, ensuring that the glucose levels in the culture medium remain above a hypoglycemic state. These observations are consistent with our hypothesis that cells expressing glucose transporters can be reprogrammed to regulate glucose in an insulin-dependent manner, leading to potential immunomodulatory therapies for T1D.

[0076] Example 3. Regulation of glucose by gene electrotransfer-mediated insulin and glucokine delivery method For single-phase electrotransfer delivery of plasmid DNA, cells were plated at 12,000 cells / well with 10 µg of plasmid DNA (containing a luciferin reporter). Electrotransfer parameters were tested at 1300 V / cm and 600 V / cm. MIRUS Lipofectamine transfection was used as a positive control. Cells containing DNA without applied voltage served as a negative control. Luciferin luminescence was measured over a five-day period when treated with luciferase. Immunofluorescence was measured (using one-dimensional ANOVA and Tukey multiple comparison test).

[0077] To assess glucose regulation, cells were plated at a density of 12,000 cells / well with 10 µg of plasmid DNA. A temperature of 1300 V / cm was applied. The plasmid groups tested were: insulin, glucokine, insulin and glucokine (different plasmids), and insulin and glucokine (same plasmid). Figure 1 Cells containing DNA and without applied voltage were used as negative controls. Glucose levels in the culture medium were measured daily for three consecutive days. Immunofluorescence was measured (one-way ANOVA and Tukey multiple comparison test).

[0078] For electrotransfer delivery of plasmid DNA, cells were plated at 12,000 cells / well with 10 µg of plasmid DNA (containing a luciferin reporter). A monophasic pulse (1300 V / cm) was used. Cells with DNA but without applied voltage served as a negative control. Luciferin luminescence was measured over a five-day period when treated with luciferase. Immunofluorescence was measured (one-way ANOVA and Tukey multiple comparison test). The applied pulse protocol was... Figure 11 As shown in the image.

[0079] result As shown in Figure 12, the conventional monophasic pulsed protocol enhanced gene delivery. Applying electric fields of 1300 V / cm and 600 V / cm produced delivery and expression comparable to lipofectamine in C2C12 cells (Figures 12A-12D). Both GET and lipofectamine enhanced luciferase expression (Figure 12E) and transfection efficiency (Figure 12F) over a five-day period.

[0080] As shown in Figures 13A-13D, co-expression of insulin and glucokine regulates extracellular glucose. Co-expression of insulin and glucokine leads to significantly higher glucose depletion in the culture medium in C2C12 cells over three days (Figure 13G), with peak expression on day 3 (Figure 13H).

[0081] discuss The results showed that conventional monophasic pulse parameters significantly enhanced the delivery of luciferase, insulin, and glucokine genes in C2C12 cells. Exogenous co-delivery of plasmid DNA encoding insulin and glucokine using the conventional monophasic pulse protocol significantly reduced extracellular glucose levels.

[0082] The conventional monophasic pulse parameters used were effective for effector gene delivery experiments, as both 1300 V / cm and 600 V / cm significantly increased expression. Exogenous co-expression of insulin and glucokine significantly reduced culture medium glucose levels compared to the corresponding controls, indicating that GET delivery of insulin and glucokine is a feasible therapeutic pathway. C2C12 cells expressing glucose transporter-4 regulated 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, leading to a potential treatment for type 1 diabetes without the use of immunomodulatory drugs.

[0083] Example 4. Skeletal muscle liveGT Enhanced therapeutic expression levels over several months method In vivo gene electrotransfer procedure. Non-diabetic Sprague Dawley rats were anesthetized with isoflurane inhalation. Both sides of the body were carefully shaved to remove as much hair as possible to allow direct electrode contact with the skin. The animal was placed in a lateral decubitus position, and the return plate electrode was placed under the contralateral side. Ultrasonic gel was applied between the skin and the contact plate to ensure contact. 50 μl of plasmid DNA encoding firefly luciferase or pDNA encoding both human insulin and glucoskinase was administered intradermally at a dose of 2 mg / ml. Figure 1 Use 10 mm immediately after injection. 2 A single-phase pulse is applied to a unipolar electrode.

[0084] Bioluminescence imaging. Bioluminescence imaging was performed on days 1, 2, 7, 14, 21, 28, 91, and 182 post-injection. Following induction of isoflurane inhalation anesthesia, animals received a subcutaneous injection of 150 mg / kg of D-luciferin (GoldBiotechnology, Inc., St. Louis, MO) at the treatment site. In vivo imaging systems (PerkinElmer, Akron OH) were used to capture and quantify bioluminescence signals. Peak flux was recorded for each pulsed condition (n=4). Group comparisons were performed using conventional two-way ANOVA and Tukey's multiple comparison test; p < 0.05 was considered significant.

[0085] Blood glucose and survival. Postprandial blood glucose was measured by glucometer on days 1, 2, 7, 14, 21, 28, and 91 post-injection. Serum human insulin levels were measured by ELISA (n=4). Groups were compared using conventional two-way ANOVA and Tukey's multiple comparison test; p < 0.05 was considered significant. Kaplan-Meier survival analysis was performed, and weight was recorded.

[0086] result like Figure 14 The study showed that gene electrotransfer significantly enhanced gene delivery and expression of luciferase-encoding genes in skeletal muscle over a 6-month period. Monopolar, monophasic pulses resulted in the highest expression (>1000-fold increase compared to the injection-only group).

[0087] Figures 15A-15B illustrate the effects of (local in vivo electrogene therapy) liveGT Co-delivery of human insulin and glucokine is safe. Figure 15A shows the Kaplan-Meier survival analysis. liveGT There was no significant difference in survival between the control and rats (p=0.3173). As illustrated in Figure 15B, individual rats continued to grow normally throughout the entire duration of the experiment.

[0088] Refer to Figures 16A-16B. LiveGT The mediated co-delivery of human insulin and glucokine significantly reduced serum glucose over 21 days. Serum exogenous (human) insulin levels significantly increased over 21 days (Figure 16A). Via [the following text is incomplete and likely refers to a separate event:] via liveGT Serum glucose levels were significantly lower when insulin and glucokine were co-delivered (Figure 16B). liveGT Mediated delivery of exogenous insulin and glucokine genes significantly reduced blood glucose levels without hypoglycemia.

[0089] discuss The presented method allows for insulin independence and potentially minimizes the treatment burden on patients, eliminating the need for daily insulin injections and immunosuppressive drugs. Results indicated that co-expression and glucose control could be maintained for several months. Since it is well established that plasmid DNA delivery is non-integrative and non-immunogenic, repeated treatments are feasible. Furthermore, liveGT It can be used as a platform technology as an alternative to other protein replacement therapies.

Claims

1. A method 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 therapeutically effective amount of a nucleic acid encoding insulin and a therapeutically effective amount of a nucleic acid encoding glucokinase; and (b) applying an electrical pulse to the site of administration.

2. The method of claim 1, wherein step (a) comprises administering both a nucleic acid encoding insulin and a nucleic acid encoding glucokinase.

3. The method of claim 1 or 2, wherein the site of administration is in skeletal muscle.

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

5. The method of any one of claims 1-4, wherein the electrical pulse is a monophasic pulse.

6. The method of claim 5, wherein the electrical pulse is between about 1 V and about 1.5 kV.

7. The method of claim 6, wherein the electrical pulse is about 90 V.

8. The method of any one of claims 5-7, wherein the electrical pulse is applied for between about 50 µs and about 200 ms.

9. The method of claim 8, wherein the electrical pulse is applied for about 150 ms.

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

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

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

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

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

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

16. The method of claim 15, wherein step (c) is performed between about every six months and about every twelve months.

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