Methods to treat hyperglycemia and prevent the onset of type 1 diabetes

Abstract

We provide effective immunotherapy for the treatment of autoimmune diseases such as type 1 diabetes. [Solution] A method is provided for reversing hyperglycemia and suppressing the onset of diabetes in patients at risk of developing type 1 diabetes. In particular, a vector system comprising a first expression cassette encoding BCL2-related X apoptosis regulator (BAX) and a permethylated second expression cassette encoding secreted glutamate decarboxylase 65 (sGAD55) is administered to the patient to induce an immune tolerance-inducing response.

Description

[Technical Field]

[0001] background Type 1 diabetes mellitus (T1D) is a condition in which insulin-producing β cells in the pancreatic islets are regulated by autoantigen-specific polyclonal T lymphocytes that have escaped the control of immune tolerance. It is an autoimmune disease in which the body is destroyed by an immune attack [1, 2]. The field of immunotherapy addresses the lost tolerance process with vaccine-like immunotherapies that avoid the undesirable effects inherent in broad-acting immunosuppressive therapies. A promising class of immunotherapies utilizes apoptosis, a natural cell death process, which is a natural non-inflammatory tolerance induction pathway [3-6]. Antigen-presenting cells (APCs), such as dendritic cells (DCs), engulf apoptotic cells and then immune... It becomes tolerogenic; this is because the processed apoptotic cells For the stimulation or inactivation of regulatory T cells (Tregs) by their own antigen (without co-stimulation) This enables the presentation of the information to autoreactive memory effector T cells (Teff) [3-6].

[0002] The need remains for the development of effective immunotherapies for the treatment of autoimmune diseases such as T1D. [Overview of the project]

[0003] overview Reversing hyperglycemia and initiating diabetes in patients at risk of developing type 1 diabetes. A method for suppressing this is provided. In particular, (a) encoding the BCL2-related X apoptosis regulator (BAX) A vector system comprising (b) a first expression cassette; and (b) a permethylated second expression cassette encoding the secreted form of glutamate decarboxylase 65 (e.g., sGAD55) is administered to the patient. is given, thereby inducing an immune tolerance-inducing response, resulting in an increase in the immune tolerance-inducing dendritic cell population and an increase in the number of GAD-specific regulatory T cells in the draining lymph node to occur. The methods described herein are effective for reversing hyperglycemia and suppressing the onset of type 1 diabetes .

[0004] In one aspect, a method for reversing hyperglycemia in a patient at risk of developing type 1 diabetes is provided, the method comprising administering a therapeutically effective amount of a vector system comprising (a) a first expression cassette comprising a polynucleotide encoding BAX ; and (b) a hypermethylated second expression cassette comprising a polynucleotide encoding a secreted form of glutamic acid decarboxylase 65 (GAD65). .

[0005] In another aspect, a method for suppressing the onset of diabetes in a patient at risk of developing type 1 diabetes is provided, the method comprising administering a therapeutically effective amount of a vector system comprising (a) a first expression cassette comprising a polynucleotide encoding BAX ; and (b) a hypermethylated second expression cassette comprising a polynucleotide encoding a secreted form of glutamic acid decarboxylase 65 (GAD65). .

[0006] In yet another aspect, a method for increasing the number of immune tolerance-inducing dendritic cells and GAD-specific regulatory T cells in a patient at risk of developing type 1 diabetes is provided, the method comprising administering an effective amount of a vector system comprising a first expression cassette comprising a polynucleotide encoding BCL2-related X apoptosis regulator (BAX) and a second expression cassette comprising a hypermethylated polynucleotide encoding a secreted form of glutamic acid decarboxylase 65 (e.g., sGAD55). ​​​​The process includes the giving of a second expression. In any of the above embodiments, the first expression cassette may further include a promoter operably linked to a polynucleotide encoding BAX, and the second expression The cassette contains a pro- A motor may be further included. In one embodiment, the first expression cassette encodes BAX. The expression cassette comprises a CMV promoter or SV-40 promoter operably ligated to a polynucleotide. In one embodiment, the second expression cassette comprises an SV-40 promoter operably ligated to a polynucleotide encoding the secreted form of GAD65.

[0007] In any of the embodiments described above, the secreted form of GAD65 may be encoded as msGAD55.

[0008] In any of the embodiments described above, the vector system is (a) a first expression cassette expressing BAX (b) a first vector containing; and (b) a permethylated second vector containing a second expression cassette expressing the secreted form of GAD65. In some embodiments, the first and second vectors are administered in a ratio in the range of 1:1 to 1:8, any ratio within this range, such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 or 1:8. In some embodiments, the first and second vectors are administered in a ratio of 1:2.

[0009] In any of the embodiments described above, the patient may have mild, moderate, or severe hyperglycemia. In one embodiment, the patient has severe hyperglycemia and the first vector —and the second vector is administered in a 1:2 ratio.

[0010] In any of the embodiments described above, the patient may have a quantity of insulin-producing pancreatic β-cells less than 50%, less than 60%, less than 70%, or less than 80% of the reference quantity of β-cells for a non-diabetic subject. In some embodiments, the patient has lost any quantity within this range, such as 50% to 80% of β-cells, for example, 50%, 55%, 60%, 65%, 70%, 75%, or 80% of β-cells.

[0011] In any of the embodiments described above, the patient may be a human being.

[0012] In another context, in patients at risk of developing type 1 diabetes, immune tolerance induction trees A method is provided for increasing the number of cellular cells and GAD-specific regulatory T cells, the method comprising a first expression cassette containing a polynucleotide encoding a BCL2-related X apoptosis regulator (BAX) Administer an effective dose of a vector system containing a second expression cassette that includes a permethylated polynucleotide encoding the secreted form of glutamate decarboxylase 65 (e.g., sGAD55). Includes the process. [Brief explanation of the drawing]

[0013] Brief explanation of the drawing [Figure 1]Figures 1A-1D. ADi-100-induced tol-DC subset in the aspiration region lymph nodes of NOD mice. A group of 8-week-old female NOD mice were vaccinated with either vector plasmid DNA alone (control) or ADi-100 1:4 (BAX 10 μg + msGAD 40 μg). Four days after vaccination, leukocytes were isolated from the aspiration region lymph nodes (inguinal region), and the DC population was analyzed by flow cytometry. The DC population was phenotypically defined. Figure 1A shows the total classical DC population, MHC class II+ / CD11c+. Figure 1B shows the tol-DC lymphoid tissue populations MHC class II+ / CD11c+ / CD8α+ ("CD8α+"), MHC class II+ / CD11c+ / CD11b+ / CD103+ ("CD11b+ / CD103+"), and the tissue-migrating / non-lymphoid tissue tol-DC population MHC class II+ / CD11c+ / CD207+ ("CD207+"). Figure 1C shows the plasma cell-like DC population MHC class II(IAg7)+ / CD11c- / PDCA+. Compared to the vector-controlled cohort, * p < 0.001 (two-tailed t-test). CD11c+ (cDC; CD11c+ CD8+ integrin αvβ8+) and CD11c- (plasma cell-like DC, pDC; CD11c- / PDCA+) immune tolerance-inducing DC populations prepared from vector-control or ADi-100 1:2-treated NOD mouse spleen cells were cultured for 72 hours with GAD-stimulated (3-day) CD4+ T lymphocytes and rhIL-2 from untreated NOD mice, and proliferation was evaluated by CSFE staining and flow cytometry (Figure 1D). Cell division was analyzed using FlowJo software, and proliferation was calculated as the percentage of dividing cells per total CD4+ T cells. [Figure 2]Figure 2. Two ADi-100 formulations containing different BAX and msGAD55 content suppressed the incidence of diabetes in NOD mice when treating mild hyperglycemia (≥140 mg / dL). A group of 8-week-old female NOD mice were monitored weekly for fasting blood glucose (FBG) levels, and on day 1, when FBG was ≥140 mg / dL (day 0; mild hyperglycemia), the mice were given id injections (50 μL) once a week for 8 weeks of formulations containing a total of 50 μg of different amounts of empty vectors (Vb, BAX vector; mVa, permethylated antigen vector) and vectors containing BAX or msGAD55 [8]. Untreated mice received no injections. The study was terminated once 100% of the mice in the untreated cohort were diagnosed with diabetes (i.e., 2 FBG readings were ≥300 mg / dL at least 7 days apart). The percentage of mice that remained diabetes-free in each cohort is shown. The raw FBG data per mouse used to calculate the disease incidence for the first five cohorts (but not ADi-100 1:2) was obtained from the dataset revealed in our previous publication [8], but that was only presented as raw FBG data in a long-term form (age of the mouse); note here that the data is presented in the form of “diabetes incidence” including further ADi-100 1:2 data not included in the previous publication. Compared to the untreated cohort, * p < 0.001. [Figure 3]Figure 3. Increased efficacy of ADi-100 containing a higher BAX plasmid content when administered to severely hyperglycemic NOD mice. Female NOD mice were monitored weekly for morning blood glucose (mBG) levels, and each mouse received its first ADi-100 dose (day 0) of either two ADi-100 formulations, 1:4 or 1:2 id injections, if mBG was ≥180 mg / dL at least twice or upon the first occurrence of mBG ≥200 mg / dL. On day 0, the mean ±SEM mBG of all 31 mice was 244 ± 12 mg / dL. Mice received weekly ADi-100 injections, followed by a total of five injections. Daily mBG monitoring continued, and mice were diagnosed with diabetes if mBG was ≥300 mg / dL at least twice, separated by at least 7 days. Percentages of mice that remained diabetes-free in each cohort are shown. Compared to the untreated cohort, p < 0.035 for ADi-100 1:4 and p < 0.001 for ADi-100 1:2. [Figure 4] Figure 4. Immunohistochemical analysis of insulin in pancreatic islet samples from representative untreated NOD mice that were non-diabetic (left panel) or diabetic (right panel) (upper panel), as well as hematoxylin and eosin (H&E; insulinitis) (lower panel) staining. Scale bar = 50 μm. [Modes for carrying out the invention]

[0014] Detailed explanation Reversing hyperglycemia and initiating diabetes in patients at risk of developing type 1 diabetes. A method for suppressing is provided. In particular, to induce an immune tolerance-inducing response which may include increasing the immune tolerance-inducing dendritic cell population in inflow region lymph nodes and increasing the number of GAD-specific regulatory T cells, a vector system comprising (a) a first expression cassette encoding BCL2-related X apoptosis regulator (BAX); and (b) a second permethylated expression cassette encoding secreted glutamate decarboxylase 65 (e.g., sGAD55) is administered to the patient. The method described herein is effective in reversing hyperglycemia and suppressing the onset of type 1 diabetes. be.

[0015] Before describing the compositions, methods, and kits, it is understood that the present invention is not limited to the specific methods or compositions described and is therefore naturally subject to change. Since the scope of the present invention is limited solely by the appended claims, it is also understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit them.

[0016] If a range of values ​​is provided, unless the context explicitly indicates otherwise, the values ​​between the upper and lower limits of that range, up to one-tenth of the lower limit unit, are also explicitly provided. It is understood that the following are shown: Each smaller range between any stated values ​​or any other stated values ​​or values ​​between any stated range and any other stated values ​​within the stated range are included in the present invention. The upper and lower limits of these smaller ranges may be included in or excluded from the range, either or neither of them, or Each of the ranges that both fall within a smaller range is also included in the present invention and belongs to any specifically excluded limit within the range described. If the range described includes one or both of the limits, the ranges that exclude either or both of these limits are also included in the present invention.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the invention, but several potential and preferred methods and materials are described herein. All publications referenced herein disclose and describe such methods and / or materials in the context in which such publications are referenced herein. This disclosure is incorporated herein by reference. This disclosure is understood to negate any disclosures in the incorporated publications to the extent that they are inconsistent.

[0018] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has other components and features that can be readily separated from or combined with features of any of several other embodiments without departing from the scope or spirit of the invention. Any described method may be carried out in the order of the described events or in any other logically possible order.

[0019] As used herein and in the appended claims, the singular forms "a" and "an" And "the" can refer to multiple objects unless the context clearly indicates otherwise. It must be noted that, for example, a reference to "a vector" includes multiple such vectors, and a reference to "the cell" includes one or more cells. This includes references to cells, etc.

[0020] The publications discussed herein are provided only for disclosures prior to the filing date of this application. Nothing herein should be construed as an acceptance that the present invention is not granted prior rights to such publications by prior invention. Furthermore, the publication dates provided may differ from the actual publication dates which may need to be independently verified.

[0021] definition "Tolerogenicity" refers to the ability to suppress or downregulate the adaptive immune response. It means.

[0022] The term "immune tolerance-inducing dendritic cells" refers to dendritic cells that have the ability to induce immunological tolerance. Immune tolerance-inducing dendritic cells have a low ability to activate effector T cells, however... It has a high ability to induce and activate regulatory T cells.

[0023] When used herein to describe nucleic acid molecules, “recombinant” means a polynucleotide of genomic, cDNA, viral, semi-synthetic, or synthetic origin that, by its origin or manipulation, is not related in nature to all or part of the polynucleotide to which it relates. When the term “recombinant” is used in relation to proteins or polypeptides, it means a polypeptide produced by the expression of a recombinant polynucleotide. Generally, the gene of interest is cloned and then expressed in a transformed organism as further described below. The host organism expresses the exogenous gene to produce the protein under expression conditions.

[0024] The term "transformation" refers to the insertion of exogenous polynucleotides into host cells, regardless of the method used for insertion. Examples include direct incorporation, transduction, or f-mating. Exogenous polynucleotides can also be incorporated as non-integrated vectors, such as plasmids. It may be present or alternatively integrated into the host genome.

[0025] The terms “recombinant host cell,” “host cell,” “cell,” “cell line,” “cell culture,” and other such terms, which refer to higher eukaryotic cell lines cultured as microorganisms or single-cell entities, may be used as recipients for recombinant vectors or other transferred DNA. This refers to the cells used, including the original offspring of the transfected original cells.

[0026] A "coding sequence," or a sequence that "codes" a selected polypeptide, is a nucleic acid molecule that, when controlled by an appropriate regulatory sequence (or "regulatory factor"), is transcribed (in the case of DNA) and translated into a polypeptide in vivo (in the case of mRNA). The coding sequence boundary The coding sequence can be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. Coding sequences may include, but are not limited to, viral cDNA, prokaryotic or eukaryotic mRNA, genomic DNA sequences derived from viral or prokaryotic DNA, and even synthetic DNA sequences. The transcription termination sequence is located 3' relative to the coding sequence. obtain.

[0027] Typical "regulatory factors" include, but are not limited to, transcription promoters, transcription enhancers, transcription termination signals, polyadenylation sequences (located 3' relative to the translation stop codon), sequences for optimizing translation initiation (located 5' relative to the coding sequence), and translation termination sequences.

[0028] "Operationally linked" refers to an alignment of factors configured such that the components described as such perform their normal functions. Therefore, a given promoter operably linked to a coding sequence can perform the expression of the coding sequence if the appropriate enzyme is present. The promoter does not need to be contiguous with the coding sequence, insofar as it functions to direct the expression of the coding sequence. Thus, for example, an untranslated but transcribed sequence may exist between the promoter sequence and the coding sequence, and the promoter sequence may still be considered "operationally linked" to the coding sequence.

[0029] "Encoded by" refers to a nucleic acid sequence that encodes a polypeptide sequence, where the polypeptide sequence or a portion thereof is at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, derived from the polypeptide encoded by the nucleic acid sequence, and even more preferably It contains an amino acid sequence of at least 15-20 amino acids.

[0030] An "expression cassette" or "expression construct" is a set that can direct the expression of one or more sequences or one or more genes of interest. Generally, an expression cassette directs the transcription of one or more sequences or one or more genes of interest, as described above. It includes control factors such as promoters that are operablely linked to ( ), and is often polyadeny This also includes the chemical sequence. Within certain aspects of the present invention, the expression cassette described herein may be included in a plasmid construct. In addition to the components of the expression cassette, the plasmid construct may include one or more The selectable markers above allow the plasmid construct to exist as single-stranded DNA. This may also include a signal (e.g., an M13 replication origin), at least one multicloning site, and a “mammalian” replication origin (e.g., SV40 or an adenovirus replication origin).

[0031] "Purified polynucleotide" means a polynucleotide or fragment of which the polynucleotide essentially does not contain the naturally associated protein, for example, containing less than about 50%, preferably less than about 70%, and more preferably less than about 90% of the protein. Techniques for purifying target polynucleotides are well known in the art, for example, disruption of cells containing polynucleotides using chaotropic agents, as well as ion exchange chromatography, affinity chromatography, and density-based precipitation of polynucleotides (1 Includes the isolation of proteins (or multiple proteins).

[0032] The term "transfection" is used to refer to the uptake of foreign DNA by cells. Cells are "transfected" when exogenous DNA is introduced into the cell membrane. Several transfection techniques are generally known in the art. See, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA portions into suitable host cells. The term refers to the stable development of genetic material. This refers to both transient uptake and uptake, including the uptake of peptides or antibody-bound DNA.

[0033] A "vector" is a device that can transfer nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors). (Vectors, granular carriers, and liposomes). Typically, these are referred to as "vector constructs" or "expression vectors." The terms "constructor" and "gene transfer vector" refer to any nucleic acid construct capable of directing the expression of a desired nucleic acid and transferring a nucleic acid sequence to target cells. Therefore, the terms include cloning and expression vehicles as well as viral vectors.

[0034] "Genetic transfer" or "gene delivery" refers to a method or system for reliably inserting target DNA or RNA into a host cell. Such a method involves the transient transfer of unintegrated transferred DNA. Expression, extrachromosomal replication, and expression of transposable replicons (e.g., episomes), or insertion of transposable gene material into the host cell's genomic DNA may occur. As a gene delivery expression vector, it is limited. While not definitively defined, examples include bacterial plasmid vectors, viral vectors, non-viral vectors, alphaviruses, poxviruses, and vaccinia virus-derived vectors.

[0035] The polynucleotides "derived" from the indicated sequence are approximately at least about six nucleotides, preferably identical or complementary to the region of the indicated nucleotide sequence. is at least about 8 nucleotides, more preferably at least about 10-12 nucleotides and More preferably, a polynucleotide sequence comprising a continuous sequence of at least about 15 to 20 nucleotides. The derived polynucleotide is not necessarily physically derived from the target nucleotide sequence, but is not limited to, chemical synthesis, replication, reverse transcription, etc., based on information provided by the sequence of bases in the region(s) from which the polynucleotide originates. This can be prepared in any manner, such as by transcription. Thus, it can present either the sense or antisense direction of the original polynucleotide.

[0036] The “reference level” or “reference value” of a biomarker refers to the level of a biomarker (e.g., blood glucose level or the number of pancreatic β-islets) that indicates a specific disease state, phenotype, or predisposition or deficiency to develop a specific disease state or phenotype, and a combination of such conditions. A “positive” reference level of a biomarker refers to the level that indicates a specific disease state or phenotype. A “negative” reference level of a biomarker refers to the level that indicates a deficiency in a specific disease state or phenotype. The “reference level” of a biomarker can also refer to the absolute or relative amount or concentration of the biomarker, the presence or absence of the biomarker, a range of amounts or concentrations of the biomarker, the minimum and / or maximum amounts or concentrations of the biomarker, or the biomarker itself. The average amount or concentration of the marker and / or the median amount or concentration of the biomarker It can be a degree; furthermore, the “reference level” of a biomarker combination can also be two or more This can be the absolute or relative amount or concentration ratio of biomarkers relative to each other. Appropriate positive and negative reference levels for a biomarker for a particular disease state, phenotype, or deficiency involve measuring the level of the desired biomarker in one or more suitable subjects. This can be determined by, and such reference levels can be adjusted for a particular population of subjects (e.g.) For example, reference levels can be age-matched or sex-matched, so that comparisons can be made between biomarker levels in samples from subjects of a specific age or sex and reference levels for a specific disease state, phenotype, or deficiency in a specific age or sex group. The level may also vary depending on the specific technique used to measure the level of the biomarker in the sample (e.g., Fluorescence-mediated cell analysis (FACS), immunoassays (e.g., ELISA), and mass spectrometry (e.g., LC-MS) are used for cell analysis and separation. It can be adapted for methods such as GC-MS, tandem mass spectrometry, NMR, biochemical or enzymatic assays, PCR, and microarray analysis.

[0037] The terms “quantity,” “amount,” and “level” are used interchangeably herein and may refer to the absolute quantification of an analyte in a molecule, cell (e.g., pancreatic islets), or sample, or the relative quantification of an analyte in a molecule or sample, i.e., against another value, such as a reference value taught herein or a range of values ​​for a biomarker. These values ​​or ranges may be obtained from a single patient or a group of patients.

[0038] When used herein, “diagnosis” generally includes a determination of whether a subject is likely to suffer from a given disease, disorder, or dysfunction. Those skilled in the art often know one or more such conditions. Diagnosis is made based on diagnostic indicators, i.e., biomarkers whose presence, absence, or quantity indicates the presence or absence of disease, disorder, or dysfunction.

[0039] As commonly used herein, “prognosis” means the expected course and outcome of a clinical condition or disease. A patient’s prognosis is usually made by evaluating the factors or symptoms of the disease that indicate a favorable or unfavorable course or outcome of the disease. It should be understood that the term “prognosis” does not necessarily mean the ability to predict the course or outcome of a condition with 100% accuracy. Rather, those skilled in the art will understand that the term “prognosis” means a high probability that a particular course or outcome will occur; that is, that the course or outcome is more likely to occur in a patient exhibiting a given condition compared to those individuals not exhibiting the condition.

[0040] The terms "treatment," "treating," and "treat" are used herein to generally refer to obtaining desired pharmacological and / or physiological effects. It is used to completely or partially eliminate a disease or its symptoms (one or more). It may be preventive in terms of preventing the disease and / or therapeutic in terms of partial or complete stabilization or cure of the disease and / or adverse effects resulting from the disease. The term “treatment” encompasses any treatment of a disease in mammals, in particular humans: (a) disease (b) To prevent the occurrence of disease and / or symptoms (one or more) in subjects who are susceptible to the symptoms but have not yet been diagnosed as having them; (b) disease and (c) suppressing one or more symptoms, i.e., stopping their occurrence; or (c) reducing one or more disease symptoms, i.e., causing a regression or reversal of the disease and / or symptoms. Those requiring treatment include those already affected (e.g., those with hyperglycemia or prediabetes) and those for which prevention is desirable (e.g., those with high susceptibility to diabetes, those with a genetic predisposition to developing diabetes). Examples include those that have a cause. The terms "treatment," "the act of treating," and "therapy" are examples of sugars. This may include suppressing the onset of urinary tract diseases.

[0041] The term "suppressing the onset of diabetes" is used herein to refer to a type of treatment that generally prevents or delays the onset of diabetes. Delaying the onset of diabetes includes delays of one day or more, one week or more, one month or more, or longer. Preventing the onset of the disease includes preventing the onset of diabetes over a specific period of time, or preventing the onset of diabetes over an unlimited period of time. The onset of diabetes can be identified by any appropriate measurement, such as measuring blood glucose levels or measuring insulin production.

[0042] Hyperglycemia, as used herein, refers to a condition in which there is an excess of sugar in the bloodstream. Hyperglycemia is also called prediabetes or stage 2 disglycemia. Hyperglycemia can be characterized as mild, moderate, or severe based on blood glucose levels. In people without diabetes, a healthy fasting blood glucose level is approximately 70-100 milligrams (mg / dL) per deciliter of blood. Yes, it exists. Hyperglycemia is diagnosed when the fasting blood glucose level is approximately 100 mg / dL to 125 mg / dL. Fasting blood glucose levels higher than 126 mg / dL indicate the development of clinical diabetes. In the NOD mouse model, mild cases... Mild hyperglycemia refers to hyperglycemia where the fasting blood glucose level or morning blood glucose level is approximately 140 mg / dL, while severe hyperglycemia refers to hyperglycemia where the fasting blood glucose level or morning blood glucose level is approximately 180 mg / dL or higher. Individuals with severe hyperglycemia may also be referred to as "highly hyperglycemic." Moderate hyperglycemia is defined in the NOD mouse model as having a fasting or morning blood glucose level ranging from mild to severe. This refers to hyperglycemia that falls within a range such as approximately 140 mg / dL to approximately 180 mg / dL.

[0043] Therapeutic treatments are those in which the subject is suffering prior to administration, while prophylactic treatments are those in which the subject is not suffering prior to administration. In some embodiments, the subject is suspected to have a high probability of developing or developing suffering prior to treatment. In some embodiments, the subject is suspected to have a high probability of developing suffering. Methods for administering therapeutic treatments are well known in the art and include oral, topical, transdermal or intradermal, inhalation, parenteral, sublingual, buccal, rectal, transvaginal, and intranasal administration. The term “parenteral” as used herein includes subcutaneous injection (e.g., transdermal or intradermal injection), intravenous, intramuscular, intrasternal injection or infusion techniques. In some embodiments, administration includes administration by a route selected from intradermal and mucosal.

[0044] In particular, with respect to a given quantity, the term "approximately" means to include a deviation of plus or minus 5%.

[0045] The terms “recipient,” “individual,” “subject,” “host,” and “patient” are used interchangeably herein and refer to any mammalian subject, particularly humans, for which diagnosis, treatment, or therapy is desired. “Mammal” for therapeutic purposes refers to any animal classified as a mammal, e.g., humans, domestic and farm animals, as well as zoo, sports, or pet animals, e.g., dogs, horses, cats, cows, sheep, goats, pigs, etc. In some embodiments, the mammal is human.

[0046] The "therapeutic effective dose" or "therapeutic dose" is the dose that produces the desired clinical outcome (i.e., the therapeutic dose). This is a sufficient amount to achieve efficacy. The therapeutically effective dose may be administered in one or more doses.

[0047] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably in this specification to refer to polymers of amino acid residues. These terms refer to polymers of one or more amino acids. The definition also applies to amino acid polymers, which are artificial chemical mimics of corresponding naturally occurring amino acids, as well as naturally occurring and non-naturally occurring amino acid polymers. Both full-length proteins and their fragments are included in this definition. The term also includes post-expression modifications of polypeptides, such as phosphorylation, glycosylation, acetylation, hydroxylation, and oxidation.

[0048] The terms “polynucleotide,” “oligonucleotide,” “nucleic acid,” and “nucleic acid molecule” are used herein to include polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers to the primary structure of a molecule. It refers only to the structure. Therefore, the term refers to triple-stranded, double-stranded, and single-stranded DNA, as well as triple-stranded DNA. This includes single-stranded, double-stranded, and single-stranded RNA. This also includes modified and unmodified forms, such as those by methylation and / or capping of polynucleotides. More specifically, the terms “polynucleotide,” “oligonucleotide,” “nucleic acid,” and “nucleic acid molecule” refer to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and N- or C-glycosides of purine or pyrimidine bases. This includes other types of polynucleotides. There is no intended distinction in length between the terms "polynucleotide," "oligonucleotide," "nucleic acid," and "nucleic acid molecule," and these terms are used interchangeably.

[0049] When referring to proteins, polypeptides, or peptides, "isolated" means that the molecule in question is a distinct entity separated from the whole organism found in nature, or exists in the substantial absence of other biological macromolecules of the same kind. With respect to polynucleotides, the term "isolated" refers to a nucleic acid molecule that is entirely or partially lacking the sequence that would normally bind to it in nature; or a heterologous sequence that would bind to it but remains naturally present; or a molecule separated from a chromosome.

[0050] This disclosure provides the following aspects. Apparatus 1. A method for suppressing the onset of diabetes in patients at risk of developing type 1 diabetes. That is, (a) A first expression cassette containing a polynucleotide encoding a BCL2-related X apoptosis regulator (BAX); and (b) A permethylated second expression cassette containing a polynucleotide encoding the secreted form of glutamate decarboxylase 65 (GAD65). A method comprising the step of administering a therapeutically effective dose of a vector system containing the vector system. Embodiment 2. The method of Embodiment 1, further comprising a promoter operably ligated to a polynucleotide encoding BAX in a first expression cassette. Embodiment 3. The method of Embodiment 2, wherein the first expression cassette comprises a CMV promoter or SV-40 promoter operably ligated to a polynucleotide encoding BAX. Embodiment 4. The method of any embodiment 1 to 3, further comprising a second expression cassette which is operably linked to a polynucleotide encoding the secreted form of GAD65. Embodiment 5. The method of Embodiment 4, wherein the second expression cassette comprises an SV-40 promoter operably ligated to a polynucleotide encoding the secreted form of GAD65. Embodiment 6. Any method of Embodiments 1 to 5, wherein the secretory form of GAD65 is encoded as msGAD55. Appearance 7. The vector system is: (a) A first vector comprising a first expression cassette expressing BAX; and (b) A permethylated second vector containing a second expression cassette expressing the secreted form of GAD65. A method including any of embodiments 1 to 6. Embodiment 8. The method of Embodiment 7, wherein the second vector is permethylated with a CpG motif. Embodiment 9. The method of Embodiment 7 or 8, wherein the first vector and the second vector are administered in a ratio in the range of 1:1 to 1:8. Embodiment 10. The method of Embodiment 9, wherein the first vector and the second vector are administered in a 1:2 ratio. Appearance 11. A patient has mild, moderate, or severe hyperglycemia. One of the following methods (1-10). Embodiment 12. The method of Embodiment 11, wherein the patient has mild hyperglycemia. Embodiment 13. The method of Embodiment 11, wherein the patient has severe hyperglycemia. Embodiment 14. The method of Embodiment 13, wherein the first vector and the second vector are administered in a 1:2 ratio. Apparatus 15. Diabetes onset is identified by measurement of blood glucose levels or insulin production. or any of the methods described in aspects 1 to 14. Embodiment 16. The method of Embodiment 15, wherein the blood glucose level is the fasting blood glucose level or the morning blood glucose level. Embodiment 17. Any method from Embodiments 1 to 16, wherein the onset of diabetes is delayed for one day or more, one week or more, one month or more, or longer. Apparatus 18: The patient has less than 50% of the reference amount of insulin in pancreatic β-cells compared to a non-diabetic subject. A method according to any of embodiments 1 to 17, having a quantity of pancreatic β-cells produced. Embodiment 19. The method of Embodiment 18, wherein the patient has lost 50% to 80% of insulin-producing pancreatic β-cells. Embodiment 20. Any method of Embodiments 1 to 19, wherein the administration results in an increase in the number of immune tolerance-inducing dendritic cells and / or GAD-specific regulatory T cells. Apparatus 21. Total CD11c in the draining inguinal lymph node + Dendritic cells CD8α in a population + The method according to embodiment 20, wherein the proportion of immune tolerance-inducing dendritic cells increases by approximately 13 times. Apparatus 22. Total CD11c in the inflow region of the inguinal lymph nodes. + CD11b against dendritic cell populations + / CD103 + The method according to embodiment 20, wherein the proportion of immune tolerance-inducing dendritic cells increases by approximately twofold. Apparatus 23. Total CD11c in the inflow region of the inguinal lymph nodes. + CD207 in dendritic cell populations + Induction of immune tolerance The method according to embodiment 20, wherein the proportion of sex dendritic cells increases by approximately 2.5 times. Embodiment 24. Any of Embodiments 1 to 23, wherein the patient is a human being. Embodiment 25. Any method of Embodiments 1 to 24, wherein the vector system is administered intradermally or mucosally. Apparatus 26. A method for reversing hyperglycemia in patients at risk of developing type 1 diabetes: (a) A first expression cassette containing a polynucleotide encoding a BCL2-related X apoptosis regulator (BAX); and (b) A permethylated second expression cassette containing a polynucleotide encoding the secreted form of glutamate decarboxylase 65 (GAD65). A method comprising the step of administering a therapeutically effective dose of a vector system containing the vector system. Embodiment 27. The first expression cassette is operably linked to a polynucleotide encoding BAX. The method according to embodiment 26, further comprising a promoter. Embodiment 28. The first expression cassette is operably linked to a polynucleotide encoding BAX. The method according to embodiment 27, comprising a CMV promoter or SV-40 promoter. Embodiment 29. The second expression cassette is modifiable to a polynucleotide encoding the secreted form of GAD65. A method of any one of embodiments 26 to 28, further comprising a promoter linked to the No. Embodiment 30. The second expression cassette is operable to encode a polynucleotide of the secreted form of GAD65. The method according to embodiment 29, comprising an SV-40 promoter linked to a function. Embodiment 31. Any method of Embodiments 26 to 30, wherein the secreted form of GAD65 is encoded as msGAD55. Appearance 32. The vector system is: (a) A first vector comprising a first expression cassette expressing BAX; and (b) A hypermethylated second vector containing a second expression cassette that expresses the secreted form of GAD65 The method according to any one of aspects 26 to 31, comprising Aspect 33. The method according to aspect 32, wherein the second vector is hypermethylated with CpG motifs. Aspect 34. The method according to aspect 32 or 33, wherein the first vector and the second vector are administered in a ratio ranging from 1:1 to 1:8. The method according to aspect 32 or 33, wherein the first vector and the second vector are administered in a ratio of 1:2. Aspect 35. The method according to aspect 34, wherein the first vector and the second vector are administered in a ratio of 1:2. Aspect 36. The method according to any one of aspects 26 to 35, wherein the patient has mild hyperglycemia, moderate hyperglycemia or severe hyperglycemia. The method according to any one of aspects 26 to 35, wherein the patient has mild hyperglycemia. Aspect 37. The method according to aspect 36, wherein the patient has mild hyperglycemia. Aspect 38. The method according to aspect 36, wherein the patient has severe hyperglycemia. Aspect 39. The method according to aspect 38, wherein the first vector and the second vector are administered in a ratio of 1:2. Aspect 40. The method according to any one of aspects 26 to 39, wherein the patient has an amount of insulin-producing pancreatic β-cells less than 50% of the reference amount of pancreatic β-cells for non-diabetic subjects. Aspect 41. The method according to aspect 40, wherein the patient has lost 50% to 80% of insulin-producing pancreatic β-cells. Aspect 42. The method according to any one of aspects 26 to 41, wherein the administration results in an increase in the number of immune tolerance-inducing dendritic cells and / or GAD-specific regulatory T cells. Aspect 43. Total CD11c in the draining inguinal lymph nodes + CD8α to the dendritic cell population + Immune tolerance induction The method according to aspect 42, wherein the proportion of immune tolerance-inducing dendritic cells increases by about 13-fold. Aspect 44. Total CD11c in the draining inguinal lymph nodes + CD11b to the dendritic cell population + / CD103 + The method according to aspect 42, wherein the proportion of immune tolerance-inducing dendritic cells increases by about 2-fold. <00​+ CD207 in dendritic cell populations + Induction of immune tolerance The method according to embodiment 42, wherein the proportion of sex dendritic cells increases by approximately 2.5 times. Embodiment 46. Any of Embodiments 26 to 45, wherein the patient is human. Embodiment 47. Any method of Embodiments 26 to 46, wherein the vector system is administered intradermally or mucousally. Apparatus 48. A method for increasing the number of immune tolerance-inducing dendritic cells and GAD-specific regulatory T cells in patients at risk of developing type 1 diabetes: (a) A first expression cassette expressing BCL2-related X apoptosis regulator (BAX); and (b) A permethylated second expression cassette expressing the secreted form of glutamate decarboxylase 65 A method comprising the step of administering an effective amount of a vector system containing the vector system. Embodiment 49. The first expression cassette is operably linked to a polynucleotide encoding BAX. A method according to embodiment 48, further comprising a promoter. Embodiment 50. The first expression cassette is operably linked to a polynucleotide encoding BAX. A method according to embodiment 49, comprising a CMV promoter or an SV-40 promoter. Embodiment 51. The second expression cassette is modifiable to a polynucleotide encoding the secreted form of GAD65. A method according to any one of embodiments 48 to 50, further comprising a promoter linked to the Noh. Embodiment 52. The second expression cassette is modifiable to a polynucleotide encoding the secreted form of GAD65. A method according to embodiment 51, comprising an SV-40 promoter linked to a function. Embodiment 53. Any method of Embodiments 48 to 52, wherein the secretory form of GAD65 is encoded as msGAD55. Appearance 54. The vector system is: (a) A first vector comprising a first expression cassette expressing BAX; and (b) A permethylated second vector containing a second expression cassette expressing the secreted form of GAD65. A method including any of embodiments 48 to 53. Embodiment 55. The method of Embodiment 54, wherein the second vector is permethylated with a CpG motif. Embodiment 56. The first vector and the second vector are administered in a ratio in the range of 1:1 to 1:8. Method 54 or 55. Embodiment 57. The method of Embodiment 56, wherein the first vector and the second vector are administered in a ratio of 1:2. Embodiment 58. Total CD11c in the inflow region inguinal lymph nodes. + CD8α against dendritic cell populations + Induction of immune tolerance A method according to any of embodiments 48 to 57, wherein the proportion of sex dendritic cells increases by approximately 13 times. Appearance 59. Total CD11c in the infiltrating inguinal lymph nodes. + CD11b against dendritic cell populations + / CD103 + A method in which the proportion of immune tolerance-inducing dendritic cells increases by approximately twofold, as described in any of embodiments 48 to 58. Apparatus 60. Total CD11c in the inflow region of the inguinal lymph node + CD207 in dendritic cell populations + Induction of immune tolerance A method according to any of embodiments 48 to 59, wherein the proportion of sex dendritic cells increases by approximately 2.5 times. Embodiment 61. Any of the methods described in Embodiments 48 to 60, wherein the patient is human. Embodiment 62. Any method of Embodiments 48 to 61, wherein the vector system is administered intradermally or mucousally.

[0051] This invention is described with respect to specific embodiments found or presented by the inventors to include preferred modes for carrying out the invention. In light of this disclosure, it will be understood by those skilled in the art that many modifications and changes can be made in the specific embodiments described without departing from the intended scope of the invention. All such modifications are intended to fall within the scope of the appended claims.

[0052] It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the present invention. [Examples]

[0053] Examples The following examples are described to provide a complete disclosure and description to those skilled in the art of how the present invention is made and used, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to indicate that the following experiments are all or only those that have been performed. While efforts have been made to ensure accuracy with respect to the figures used (e.g., quantity, temperature, etc.), some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is degrees Celsius, and pressure is atmospheric pressure or close to it.

[0054] Example 1 Reversal of hyperglycemia and type 1 diabetes in NOD mice using apoptotic DNA immunotherapy Suppression ADi-100, a unique and potent immunotherapy consisting of two DNA plasmids, has been developed. One plasmid expresses BAX, an intracellular apoptosis-inducing signaling molecule, while the other expresses island autoantigens. A certain secreted glutamate decarboxylase 65 (sGAD55) was expressed [3, 7, 8]. The efficacy of ADi-100 in a non-obese diabetic (NOD) mouse model of T1D has been previously shown to be significantly increased when the sGAD55 plasmid is hypermethylated [8], which may reduce inflammation caused by a non-methylated CpG motif, a ligand for Toll-like receptor 9 expressed on some APCs. ADi-100 treatment also reduces total CD11c + Along with DC, NOD mice are being introduced. It increases sGAD-specific Treg levels in regional lymph nodes [7-9]; however, it is unknown whether these DCs have an immune tolerance-inducing phenotype. We have shown that ADi-100 treatment induces immune tolerance We found that increasing tolerable DCs (tol-DCs) and increasing apoptosis-inducing BAX content enhances the effectiveness of reversing hyperglycemia when administered to NOD mice during late hyperglycemia, a pre-diabetic stage relevant to the corresponding clinical diagnostic stage in human T1D.

[0055] ADi-100: Plasmid DNA construct Two DNA plasmids, including the previously described ADi-100 formulation [8], have a modified CMV promoter. The pSG5-GAD55 plasmid contains a cDNA construct encoding secreted human GAD65 (sGAD55) under transcriptional control of the SV-40 promoter in the pND2-BAX vector (Stratagene, San Diego, CA, USA) and a pSG5 vector (Stratagene, San Diego, CA, USA). The pSG5-GAD plasmid was permethylated at the CpG motif (msGAD55) in E. coli strain ER1821 by the activity of SssI methylase (New England BioLabs, Ipswich, MA, USA). This method has been shown to result in 85%–100% methylation of the CpG motif in the plasmid (Jimenez-Useche et al., Biophys J. 107(7) 1629-1636). (See reference). Plasmid DNA was dissolved in sterile saline immediately before intradermal (id) injection. bax sequence insertion All plasmids, including ADi-100, induced significant and substantial levels of apoptosis in human HeLa cells (using 1 ug / mL DNA per cluster; data not shown), confirming the activity of the BAX-induced apoptosis-tolerant delivery system.

[0056] animal Eight-week-old female NOD mice were used in a study at Loma Linda University (Loma Linda, CA, USA). Animals were purchased from Taconic Farms (NOD / MrkTac; Germantown, NY, USA)[8] and from The Jackson Laboratory (NOD / ShiLtJ; Sacramento, CA, USA) for testing at Stanford University (Palo Alto, CA, USA). All animals were housed in enclosures under pathogen-free conditions at their respective locations, and the experiments were approved by the respective Institutional Animal Care and Use Committees.

[0057] Dendritic cell isolation and characterization Eight-week-old female NOD mice (Taconic Farms) were given two id injections of either 50 μg plasmid DNA alone (vector) or ADi-100 1:4 (BAX 10 μg + msGAD 40 μg) in the ventral flank region, seven days apart. Four days after the second injection, leukocytes were isolated from the inguinal lymph nodes in the influx region, and single-cell suspensions were prepared by flow cytometry at that time for analysis of various DC phenotypic populations. These newly isolated cells (10 6 The rats were incubated on ice for 30 minutes with one or more of the following conjugate antibodies (1 μg; see below) and evaluated using FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA) as previously described [7]; rat anti-mouse CD317 / PDCA-1, clone 129C1, PE conjugate (BioLegend, San Diego, CA, USA); hamster anti-mouse CD11c, clone N418, FITC conjugate (BioLegend, San Diego, CA, USA); rat anti-mouse MHC class II, clone M5 / 114.15.2, APC conjugate (R&D Systems, Minneapolis, MN, USA); Rat anti-mouse CD8a, clones 53-6.7, PE conjugate (BioLegend, San Diego, CA, USA); Rat anti-mouse integrin αM / CD11b, clone M1 / 70 Alexa Fluor 647 conjugate (R&D Systems, Minneapolis, MN, USA); rat anti-mouse CD103, clone M290, PE conjugate (BD Biosciences, Franklin Lakes, NJ, USA); rat anti-mouse CD207, clone 4C7, PE conjugate (BioLegend, San Diego, CA, USA).

[0058] GAD-specific T lymphocyte proliferation To evaluate the ADi-100-induced immune tolerance-inducing properties of DCs, pooled spleen cells from eight ADi-100-vaccinated NOD mice (as described above) were used, and CD11c-positive and mPDCA-1-positive kits (Miltenyi, Auburn, CA, USA) were used, respectively. + (cDC;CD11c + CD8 + Inte Grin αvβ8 + ) and CD11c - (Plasma cell-like DC, pDC; CD11c- / PDCA+) tol-DC population Released. GAD-stimulated CD4+ lymphocytes were derived from 10 8-week-old female NOD mice. 6 Each lymph node cell was cultured in 1 mL of culture medium supplemented with 10% thermoinactivated fetal bovine serum (FBS; HyClone, Logan, UT, USA), 2 mM L-glutamine, 1 mM sodium pyruvate, and 0.11 mM sodium bicarbonate (high glucose). The cells were prepared by culturing them for 3 days in Dulbecco's Modified Eagle Medium (DMEM; Sigma, St. Louis, MO, USA) with GAD (20 μg / mL), and then, as previously described [7], CD4+ T cells were enriched as non-contact cells using negative selection with anti-CD8, -CD11b, -CD16, -CD56, -CD19, and -CD36 mAbs (Miltenyi Biotec, Auburn, CA, USA). T cell purity was >95% as assessed by flow cytometry (data not shown). Before culturing with DCs, GAD-stimulated CD4+ T cells were cultured in 1.5 μM CFSE (Invitrogen, Carlsbad, CA, USA). Stained with . In a triple well of a 96-well plate, DC (5 × 10 4 ) in the presence or absence of sGAD (20 μg / mL) CD4 + T cells (5 x 10 4 Cells were cultured with ) and hrIL-2 (20 U / mL; PeproTech, Rocky Hill, NJ, USA). After 72 hours of culture, cells were examined with anti-CD4-PE mAb and green nucleic acid staining dead cell indicators. Using a certain SYTOX® (Invitrogen, Carlsbad, CA, USA), follow the manufacturer's instructions. Therefore, CFSE is obtained by flow cytometry. + CD4 + SYTOX - Cell proliferation was detected. Proliferation data was collected using FlowJo 7.6.5 software (Becton, Dickinson, & Co., Ashland, OR, USA). The percentage of divided CD4+ T cells analyzed indicates the degree of proliferation. The percentage of divided cells in the absence of sGAD antigen was <1% (not shown).

[0059] Diabetes study in NOD mice To demonstrate the strength of ADi-100 efficacy, two NOD mouse diabetes studies were conducted in two separate laboratories: the first study, using female NOD mice with mild hyperglycemia, was conducted at Loma Linda University (Loma Linda, CA, USA) [8], and the second study, using female NOD mice with severe hyperglycemia, was conducted at Stanford University (Palo Alto, CA, USA). All animals were purchased at 8 weeks of age. As previously described [8], blood glucose levels are monitored weekly using a glucometer (Bayer Contour Glucose Meter; Ascensia Diabetes Care, Parsippany, NJ, USA) At the first reading of ≥140 mg / dL (fasting blood glucose, FBG, mild hyperglycemia test) or at least two readings of ≥180 mg / dL or at the first occurrence of ≥200 mg / dL (morning) Animals were randomly assigned to a cohort to receive either a first weekly injection of ADi-100 (50 μg) or a control vector for blood glucose, mBG, and severe hyperglycemia tests. Animals received a 50 μL id injection in the flank of the abdomen, as previously described [8], and their blood glucose levels were monitored weekly. Here, diabetes was diagnosed if blood glucose levels were ≥300 mg / dL on two separate occasions at least 7 days apart. In the mild hyperglycemia test, each diabetic mouse was conditioned when FBG reached ≥600 mg / dL. Mice that were not euthanized and did not have diabetes were euthanized at 50 weeks of age. In the severe hyperglycemia test, tissue samples were obtained at the same time point for comparison, and all animals were euthanized 5 weeks after treatment. They were euthanized. Mild and severe hyperglycemia tests included blood glucose assessments as FBG and mBG respectively, so the true mean and SEM difference were calculated. This was determined to be 17.9 ± 10 mg / dL, and FBG was intuitively lower than the respective mBG readings due to fasting (two mBG readings were assessed the day before and the day after each FBG reading, this was done for four non-diabetic mice). Each of these was evaluated once a week for 7 weeks; that is, in the mean difference induction, 28 FBG (Using the total of 56 mBG readings that produced the 56Δ value).

[0060] immunohistochemistry At the end of the experiment, the animals were euthanized, the pancreases were harvested and embedded in OCT compound (Tissue Tek, Torrance, CA, USA) or paraffin, as previously described

[10] , with rat anti-insulin primary antibody ((1:100, #MAB1417, R & D systems, Minneapolis, MN, USA) and donkey anti-rat IgG secondary antibody (1:500, Invitrogen, Carlsbad, CA, USA) conjugated to Alexa488. Insulin was stained using [this method].

[0061] statistical analysis Plot Kaplan-Meier estimations of survival curves for disease-free individuals and perform log-rank tests. The differences between groups were tested using the Wilcoxon test for comparing continuous variables between groups, and Fisher's exact test for comparing categorical variables. All data were analyzed using Stata Release 15.2 (StataCorp LP, College Station, TX, USA). 0.05 The significance level was used. A two-tailed t-test (Prism, GraphPad Software, Inc, San Diego, CA, USA) was used to compare means.

[0062] Tol-DC subset analysis in lymph nodes in the lymphatic region after ADi-100 treatment The BAX component of ADi-100 is designed to induce the migration of tol-DCs to inflow region lymph nodes, which then present antigens to stimulate the number and function of GAD-specific Treg cells. It was calculated that the delivery of plasmids containing BAX and sGAD55 actually occurred in the influx region lymph nodes and Total CD11c in the spleen + In addition to the increase in the number of DCs [9], in NOD mice, the inflow area lymph It has been previously shown that functional GAD-specific Treg cells were induced in the junction [7]. Here, we have shown that tol-DC populations were induced in the inguinal region of the influx of phosphorus phosphate four days after the second injection of ADi-100 1:4 twice a week. By evaluating the "immune tolerance-inducing" phenotype of these DCs using flow cytometry analysis of lymph nodes, we further defined the different tol-DC phenotypes examined in [11, 12] (see Figure 1). Total CD11c per lymph node + / MHC Class II + While the DC population was confirmed to have increased only threefold (Figure 1A), the total CD11c + Collective CD8α + The proportion of tol-DC increased by only 13 times, while CD11c + Group CD11b + / CD103 + and CD207 + The proportion of tol-DC increased by 2x and 2.5x, respectively. (Figure 1B). Furthermore, immune tolerance-inducing plasma cell-like DCs (pDCs;CD11c) per lymph node. - / PDCA + The number of ) increased by only 2.5 times (Figure 1C). These results suggest that ADi-100 was administered to the flank injection site of the abdomen. This indicates a significant and substantial increase in the migration of tol-DCs to the inguinal inflow lymph nodes. These phenotypically defined DC populations are immune to GAD-specific CD4+ T lymphocyte proliferation. The inducible activity was further evaluated. CD11c prepared from spleen cells of vector-control-or ADi-100-treated NOD mice. + (cDC;CD11c + CD8 + Integrin αvβ8 + ) and CD11c - (Plastic Zuma cell-like DCs, pDCs; CD11c- / PDCA + Both tol-DC populations exhibited an immune tolerance-inducing phenotype and And so, GAD stimulation CD4 + They lost their ability to support the proliferation of T lymphocytes (Figure 1D).

[0063] Efficacy of ADi-100 containing increased BAX plasmid content to reverse hyperglycemia in mildly hyperglycemic NOD mice. Since BAX-induced apoptosis enhances immune tolerance, the inventors of the ADi-100 formulation have developed a BAX-induced apoptosis. Increased smid content is effective in reversing hyperglycemia in female NOD mice with mild hyperglycemia. We evaluated whether it could increase the effect of lower doses (10 μg BAX plasmid and 40 μg msGAD55). Plasmid; i.e., a 1:4 ratio of BAX:msGAD55) or higher amounts (17 μg BAX + 33 μg msGAD55, 1:2 ratio, 50 μg total) of BAX content in two ADi-100 formulations, where FBG >140 mg / dL It was administered when the condition was present. The untreated and empty vector-treated cohorts reached 100% diabetes incidence (except for mVa + BAX, which reached 90%), while the ADi-100 1:2 and 1:4-treated cohorts had significantly lower diabetes incidence, only 20% at 15 and 23 weeks, respectively (see Figure 2). None of the ADi-100-treated mice developed diabetes during the first 8 weeks of ADi-100 administration. No disease developed. In patients with mild hyperglycemia, administered in a relatively early stage, there was no difference in efficacy between ADi-100 1:4 and 1:2 formulations, with a mean ±SEM FBG of 173 ± 4 mg / dL on day 0 of treatment. Therefore, we evaluated the possible differences in efficacy by treating mice in the later stages of the disease process with significantly higher blood glucose levels.

[0064] Increased BAX plasmid content in ADi-100 1:2 formulation indicates late-stage autoimmune diabetes (i.e., It showed greater efficacy in cases of severe hyperglycemia. In the treatment of NOD mice to reverse hyperglycemia and suppress the onset of diabetes The difficulty lies in treating only mice that are likely to develop diabetes and determining the timing of treatment when the degree of β-cell loss still allows for a reversal of hyperglycemia at the onset of the disease. The goal is to ensure that the treatment is administered within the preceding "pre-symptomatic" hyperglycemia stage. The inventors induced a hyperglycemia threshold of 4 × SD above the mean normal mBG level in aged, non-diabetic female mice (mean ± SD, 113 ± 17 mg / dL; n = 685, 5 naturally non-diabetic mice). The daily mBG reading of the inventors (note that 20% of the inventors' colonies remained diabetes-free), was 180 mg / dL. Non-diabetic mice are very unlikely to have mBG readings above this level (p = 0.00003). In fact, Mathews et al.

[13] recently recommended that mBG values ​​should be used instead of FBG to avoid any unfavorable effects of fasting on the course of disease progression. To determine the efficacy of ADi-100 when administered relatively late in the disease process during high hyperglycemia, each NOD mouse was given the initial ADi-100 dose with mBG ≥ 180 mg / dL and then the weekly dose for all five injections thereafter. The study involved the following: The mean ± SEM mBG on day 0 for all 31 mice was 244 ± 12 mg / dL, which was significantly higher than the mean ± SEM FBG of 173 ± 4 mg / dL in the mild hyperglycemia study (p < (0.001). The inherent difference between FBG and mBG at 18 ± 10 mg / dL is large compared to the mean values ​​on day 0. Note that the difference will not be explained.

[0065] The untreated cohort gradually developed diabetes, reaching 100% incidence by 5 weeks (i.e., day 35, end of the study), while the ADi-100 1:4 treated cohort remained at 17 days until the end of the 5-week study. This resulted in a 50% reduction in disease incidence (see Figure 3; vs. untreated, p = 0.035). Importantly, the ADi-100 1:2 treatment cohort showed an 80% reduction in disease incidence from day 31 to the end of the study. This was highly significant compared to the untreated group (p = 0.001), and the statistical significance was considerably higher than that of the ADi-100 1:4 treatment group (p = 0.035). The probability between the ADi-100 1:2 and 1:4 groups was, The p-value was 0.17 due to an insufficient number of mice that developed diabetes. The acceptance criterion of "severely hyperglycemic" (≥180 mg / dL) resulted in a substantially shorter time to 100% diabetes incidence in the severe hyperglycemia trial compared to the untreated control group; 5 vs. 23 weeks, respectively (see Figures 2 and 3). Therefore, it is clear that this resulted in a substantially later initiation of treatment in the disease process for mild hyperglycemia trials.

[0066] Further differences exist between the two ADi-100 formulations: (1) The efficacy of ADi-100 1:4 group was better than that of 5 non- While the responsive mice appeared to show a higher bias in day 0 mBG values, the subject is that mouse #5 developed diabetes despite having an exceptionally high mBG level of 286 mg / dL on day 0. Therefore, it did not appear to be a case using the protected ADi-100 1:2 formulation (see Table 1 below); (2) ADi-100 1:2 compared to ADi-100 1:4 in terms of time from day 0 to T1D diagnosis (3) All 5 mice ADi-100 1:4 The mBG levels of non-responding diabetic mice were ≥600 mg / dL, while the mBG levels of two mice derived from ADi-100 1:2 were ≥600 mg / dL. (4) Pancreatic islet insulin expression analysis showed that ADi-100 1:2 responders (i.e., non-diabetic mice on day 35) were controlled to insulin The results were positive for insulin staining, while all three samples available from the ADi-100 1:4 responder were negative (see Table 1; examples of positive and negative insulin staining in Figure 4). Interestingly, these insulin-negative samples from the three ADi-100 1:4 responders correlated with final mBG levels in the hyperglycemic range (≥180 mg / dL), while those from the ADi-100 1:2 responder were below this threshold, indicating that ADi-100 1:2, rather than ADi-100 1:4, reversed the correlation with hyperglycemia (see Table 1). Therefore, this correlation between insulin staining and blood glucose levels was observed in the ADi-100 1:4 formulation group. This suggests that the responding mice could eventually develop diabetes if the treatment was continued for more than 35 days. This was observed in samples from untreated diabetic control mice and all ADi-100 non-responder (diabetic) mice. The samples were negative for insulin staining at the end of the test (see Table 1; some low signals from insulin staining were observed in 2 out of 10 untreated diabetic mice at the end of the test). (Not shown; not indicated). [Table 1]

[0067] Table 1 shows the mBG analysis of ADi-100-treated NOD female mice that showed very high hyperglycemia on the first day of treatment (day 0). Female NOD mice were monitored daily for mBG, and the results are shown here individually. These mice were deemed to have mBG levels of ≥180 mg / dL at least twice, or the first occurrence of mBG was ≥2 If the blood glucose level was 00 mg / dL, the first ADi-100 dose (day 0) was administered. Mice received weekly ADi-100 injections, followed by all five subsequent injections. Daily mBG monitoring was continued for at least 7 days. If the blood glucose level was ≥300 mg / dL on two separate occasions, the mouse was diagnosed with diabetes. a The values ​​are from two mBG measurements. (This shows the age at the initial point in time.) The gray shaded cells are diabetes "non-responders," and the shaded cells are Cells that did not show a reaction were non-diabetic "responders." The study was terminated on day 35, when 100% of diabetes cases occurred in the untreated group (see Figure 2 for diabetes incidence figures). b For comparisons of mean age, p = 0.008 (two-sided unpaired Wilcoxon test), and for comparisons of mean mBG incidence against the mean of each of the 6-10 non-diabetic responder mice in the ADi-100 1:4 group, p < 0.001 (Poisson regression). The untreated control group (n = 12) shows mean ± SEM mBG and age on day 0 (282 ± 29 mg / dL and 120 ± 9 days), as well as age at diagnosis of type 1 diabetes (T1D) at 136 ± 12 days. nd indicates non-diabetic. c The animals were euthanized at the end of the experiment, their pancreases were collected, and insulin was administered to them. The samples were then stained (see examples of positive and negative insulin staining in Figure 4).

[0068] In summary, these results suggest that ADi-100 is transferred to lymph nodes in the inflow area of ​​the tol-DC subset. It induced a reaction and predicted the reversal of hyperglycemia and the onset of diabetes in two independent trials. The inhibitory effect was strong and effective; since none of the plasmids were effective alone, they were antigen-specific and exhibited a mechanism reliant on the apoptosis-inducing factor BAX. Importantly, the strong efficacy of ADi-100 is evident in the reproducible results of experiments conducted at two different institutions, a concept proposed by the T1D Research Committee

[14] . For comparison with the ADi-100 1:4 formulation, enhanced efficacy was achieved by increasing the BAX content in the ADi-100 1:2 formulation while decreasing the msGAD55 content to maintain a total dose of 50 μg. Furthermore, the msGAD55 plasmid was permethylated with a CpG motif to avoid induction of inflammatory signaling. However, the BAX plasmid was not permethylated (i.e., hypomethylated) to ensure that CMV promoter activity was not impaired [8]. It may seem counterintuitive that an increase in the content of such hypomethylated plasmids would result in enhanced efficacy, but it has been shown that relatively small amounts of unmethylated CpG oligonucleotides added to immune tolerance-inducing immunotherapy can increase the expression of the anti-inflammatory cytokine IL-10, thereby promoting the development of tol-DCs and Treg cells and immune tolerance

[15] . Furthermore, ADi-100 The permethylation used in its development is similar to that of a single plasmid immunotherapy (expressing proinsulin II) containing a motif recombinantly modified from CpG to CpC to avoid induction of inflammation

[16] , which has shown promising efficacy in T1D clinical trials

[17] in addition to hyperglycemia. The NOD mouse population was reversed.

[0069] Immunotherapy involving different tolerance delivery systems (TDSs) and autoantigens has been shown to prevent diabetes when administered to young, prehyperglycemic NOD mice, which is similar to stage 1 in human T1D (i.e., autoantibody-positive titers without signs of dysglycemia;

[18] discussed). However, there are very few published studies showing that such immunotherapy (as monotherapy) can “reverse hyperglycemia” (i.e., stage 2) in NOD mice

[19] . Several nonspecific immunomodulators, such as anti-CD3 mAbs, have successfully reversed hyperglycemia in NOD mice, either alone or in combination with immunotherapy [13, 19-21], and have recently been shown to be effective in delaying the loss of insulin production in prediabetic (i.e., dysglycemia, stage 2) subjects

[22] . However, these nonspecific therapies cannot induce sustained tolerance and therefore require long-term administration with safety concerns. DNA-based immunotherapy, including proinsulin II

[16] or secreted GAD, such as our ADi-100[7,8], is a monotherapy. When used as such, it demonstrated successful reversal of hyperglycemia in NOD mice. The divalent IgG Fc-MHC / GAD65 fusion protein DEF-GAD also showed similar efficacy

[23] . The remarkable efficacy of these monotherapies for reversing hyperglycemia, combined with the unique characteristics of each TDS, This may be due to the prolonged presence of the antigen in vivo.

[0070] Female NOD mice have a population of <100% depending on the colony and laboratory; i.e., typically 70% to 9 Diabetic hyperglycemia developed spontaneously with a 0% incidence rate

[24] . Such unpredictability makes it difficult to “prevent disease” using young non-diabetic mice by increasing the number per cohort. This can be statistically explained in the study. However, if mice are selected based on their likelihood of developing diabetes, fewer mice may be used in the "hyperglycemia reversal" study. Empirically, 180 mg / dL mBG is the upper limit of the true normal mBG range from female mice that did not develop the disease. The derived hyperglycemia threshold predictably led to the development of diabetes. In fact, this threshold model was confirmed with 100% diabetes development in a group of 12 untreated mice. Using this threshold, the accurate prediction of diabetes development in female NOD mice was <170 mg / mL

[16] . Or, consistent with others that derived a normal mBG range of <175 mg / dL

[13] , we used two consecutive values ​​≥300 mg / dL or ≥400 mg / dL, respectively (almost all diabetic mice in our tests terminated with mBG ≥500 mg / dL). Although these glycemic stages in NOD mice cannot be precisely converted to those in human T1D, ADi-100 was used before overt clinical diabetes (stage 3). Targeting treatment during clinically detectable abnormal blood glucose (i.e., stage 2 disease including hyperglycemia

[25] ) It is clear that this is possible.

[0071] Several preventive or interventional clinical trials using immunotherapy generally focus on insulin production ( That is, storage of stimulated C peptide and blood glucose criteria (HbA1c and insulin use) The results were disappointing in terms of improvement.

[26] Most of these immunotherapies are administered orally or mucosally. Delivered via the intranasal route, consisted only of autoantigens that could be considered weak TDSs, or other weak or irrelevant TDSs such as Alum (e.g., GAD-Alum; Diamyd Therapeutics;

[27] ) or incomplete Freund's adjuvant (IFA) [28, 29]. Alum It does not appear to induce a focused Treg response, so it may not be the most effective TDS. However, it can rather induce significant Th2 responses and even pathogenic Th1 and Th17 responses (as discussed in [30, 31]). Of note, GAD-Alum (Diamyd Therapeutics) In several Phase I and II trials using subjects who were positive for anti-GAD65 antibodies (stage 2) or newly initiated (stage 3), it was evaluated that there was a tendency toward preservation of residual insulin secretion, particularly in subjects with autoimmune diabetes mellitus (LADA) initiated in late adulthood

[27] , but III This trend disappeared in phase 1 trials (failed) [32, 33]. This clinical experience was that GAD-Alum was not tested in animal models before clinical evaluation, and the positive results of the GAD65 efficacy evaluation in NOD mouse efficacy studies were in a “preventive” setting using young (4-6 week old) NOD mice, but hyperkalemia The failure to show reversal of the stage 2 state of diabetes

[30] was a major factor in the preclinical development of immunotherapy. The key issue is highlighted. Interestingly, in the expected NOD trial, clinical GAD-Alum analysis The product did not prevent diabetes in NOD mouse models

[30] . Therefore, different immune responses were found. Tolerance-inducing agents, such as rapamycin, aryl hydrocarbon receptor ligands, and retinoic acid. It is important to develop more control-specific and potent TDSs, such as soluble or granular tolerance vehicles (e.g., nanoparticles, microspheres, and liposomes) containing vitamin D3 and cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-β [31, 34]. Other TDSs are available in vivo tol-DCs or Tregs produced ex vivo. They are either cellular in nature that are introduced [35, 36] or genetically modified gastroenteric bacterial strains that express autoantigens and immune tolerance-inducing cytokines

[37] . Note that apoptosis tolerance vehicles fall into this cellular category of TDSs.

[0072] Apoptosis-based immunotherapy uses “natural” tolerance systems rather than synthetic tolerance systems to avoid the risk of inducing a pathogenic autoimmune response due to the non-inflammatory immune tolerance-inducing nature of apoptotic cells (unlike synthetic particles which tend to trigger inflammatory processes

[38] ). (This is true.) In fact, in the development of apoptosis-based immunotherapy using different approaches... There is currently great interest in this. One immunotherapy involves the use of a surface marker called glycohol. It selectively binds to erythrocytes (i.e., red blood cells, RBCs) via nucleotide A. It is a soluble therapeutic agent composed of recombinant autoantigens conjugated to linker molecules, which showed potent efficacy in preventing diabetes in NOD mice upon systemic delivery [5, 39]. RBCs bound to autoantigens undergo their natural apoptosis process (for non-nuclear RBCs, ellipsoidal When they enter ptosis, immune tolerance-inducing APCs recognize them for interaction with T cells. Recognizing and processing. RBCs have an exceptionally high metabolic turnover rate of approximately 100 billion cells per day, so high levels of autoantigen-binding apoptotic vesicles are produced in each ASI administration. Note that this may potentially be delivered to immune tolerance-inducing APCs. Before reinfusion, remove the autoantigen. In order to covalently bind to RBCs in cytovivo, the transpeptidase saltase is used. Another RBC-based apoptosis therapy also showed efficacy in preventing diabetes in NOD mice [6]. In addition, ex vivo chemical induction of mouse spleen cells or human peripheral blood monocytes (PBMCs) Apoptosis [4] has shown efficacy in experimental autoimmune encephalomyelitis and T1D in mice, as well as in autoimmune conditions of multiple myelopathy in human studies

[40] . Others include phosphatidylserine to deliver autoantigens to tol-DCs derived from human T1D subjects. The technology uses liposomes containing immune tolerance-inducing apoptosis mimics.

[41]

[0073] The disclosed method is not only highly effective but also possesses other beneficial characteristics, such as the availability of a non-cellular therapeutic approach, low production costs, a favorable storage profile, and the ability to be administered frequently over long periods to achieve tolerance. The following are examples of aspects of the present invention. Item 1 A method for suppressing the onset of diabetes in patients at risk of developing type 1 diabetes, (a) A first expression cassette containing a polynucleotide encoding a BCL2-related X apoptosis regulator (BAX); and (b) A permethylated second expression cassette containing a polynucleotide encoding the secreted form of glutamate decarboxylase 65 (GAD65). A method comprising the step of administering a therapeutically effective dose of a vector system containing the vector system. Section 2 The method according to item 1, further comprising a promoter operably ligated to a polynucleotide encoding BAX in a first expression cassette. Section 3 The first expression cassette is a CMV operably ligated to a polynucleotide encoding BAX. The method described in item 2, comprising a promoter or an SV-40 promoter. Section 4 The method according to any one of claims 1 to 3, further comprising a second expression cassette which is a promoter operably ligated to a polynucleotide encoding the secreted form of GAD65. Section 5 The method according to item 4, wherein the second expression cassette comprises an SV-40 promoter operably ligated to a polynucleotide encoding the secreted form of GAD65. Section 6 The secretory form of GAD65 is coded as msGAD55, as described in any of sections 1-5. Section 7 Vector systems: (a) A first vector comprising a first expression cassette expressing BAX; and (b) A permethylated second vector containing a second expression cassette expressing the secreted form of GAD65. A method including any of the methods described in items 1 to 6. Section 8 The method described in section 7, wherein the second vector is permethylated with a CpG motif. Section 9 The method according to paragraph 7 or 8, wherein the first vector and the second vector are administered in a ratio ranging from 1:1 to 1:8. Section 10 The method according to item 9, wherein the first vector and the second vector are administered in a 1:2 ratio. Section 11 The method described in any of items 1 to 10, for patients who have mild, moderate, or severe hyperglycemia. Section 12 The method described in paragraph 11, wherein the patient has mild hyperglycemia. Section 13 The method described in paragraph 11, wherein the patient has severe hyperglycemia. Section 14 The method according to item 13, wherein the first vector and the second vector are administered in a 1:2 ratio. Section 15 The method described in any of sections 1 to 14, wherein the onset of diabetes is identified by measurement of blood glucose levels or measurement of insulin production. Item 16 The method described in paragraph 15, wherein the blood glucose level is the fasting blood glucose level or the morning blood glucose level. Section 17 The onset of diabetes was delayed by more than one day, more than one week, more than one month, or longer. The method described in any of items 1 to 16. Section 18 The patient had insulin-producing pancreatic β-cells less than 50% of the reference amount for non-diabetic subjects. A method according to any one of items 1 to 17, having a quantity of visceral β-cells. Section 19 The method described in paragraph 18, wherein the patient has lost 50% to 80% of insulin-producing pancreatic β-cells. Section 20 The administration produced an increase in the number of immune tolerance-inducing dendritic cells and / or GAD-specific regulatory T cells. The method described in any of items 1 to 19. Section 21 The method described in item 20, wherein the proportion of CD8α+ immune tolerance-inducing dendritic cells to the total CD11c+ dendritic cell population in the draining inguinal lymph node increases by approximately 13 times. Section 22 Induction of CD11b+ / CD103+ immune tolerance to the total CD11c+ dendritic cell population in the infiltrating inguinal lymph nodes. The method described in item 20, which increases the proportion of sex dendritic cells by approximately twofold. Section 23 The method described in item 20, wherein the proportion of CD207+ immune tolerance-inducing dendritic cells to the total CD11c+ dendritic cell population in the infiltrating inguinal lymph nodes increases by approximately 2.5 times. Section 24 The patient is human, as described in any of items 1-23. Section 25 The method described in any of sections 1 to 24, wherein the vector system is administered intradermally or mucousally. Section 26 A method for reversing hyperglycemia in patients at risk of developing type 1 diabetes: (a) A first expression cassette containing a polynucleotide encoding a BCL2-related X apoptosis regulator (BAX); and (b) A permethylated second expression cassette containing a polynucleotide encoding the secreted form of glutamate decarboxylase 65 (GAD65). A method comprising the step of administering a therapeutically effective dose of a vector system containing the vector system. Section 27 The method according to item 26, further comprising a promoter in which a first expression cassette is operably ligated to a polynucleotide encoding BAX. Section 28 The first expression cassette is a CMV operably ligated to a polynucleotide encoding BAX. The method described in item 27, comprising a promoter or an SV-40 promoter. Section 29 The method according to any one of sections 26 to 28, further comprising a second expression cassette which is a promoter operably ligated to a polynucleotide encoding the secreted form of GAD65. Section 30 The method according to item 29, wherein the second expression cassette comprises an SV-40 promoter operably ligated to a polynucleotide encoding the secreted form of GAD65. Section 31 The secretory form of GAD65 is coded as msGAD55, as described in any of sections 26-30. Section 32 Vector systems: (a) A first vector comprising a first expression cassette expressing BAX; and (b) A permethylated second vector containing a second expression cassette expressing the secreted form of GAD65. A method described in any of paragraphs 26 to 31, including the method described in paragraphs 26 to 31. Item 33 The method described in section 32, wherein the second vector is permethylated with a CpG motif. Section 34 The method according to claim 32 or 33, wherein the first vector and the second vector are administered in a ratio ranging from 1:1 to 1:8. Section 35 The method according to item 34, wherein the first vector and the second vector are administered in a 1:2 ratio. Section 36 The method described in any of paragraphs 26-35, for patients with mild, moderate, or severe hyperglycemia. Section 37 The method described in paragraph 36, for patients with mild hyperglycemia. Section 38 The method described in paragraph 36, for patients with severe hyperglycemia. Section 39 The method according to paragraph 38, wherein the first vector and the second vector are administered in a 1:2 ratio. Section 40 The patient had insulin-producing pancreatic β-cells less than 50% of the reference amount for non-diabetic subjects. A method according to any one of items 26 to 39, having a quantity of visceral β-cells. Section 41 The method described in paragraph 40, wherein the patient has lost 50% to 80% of insulin-producing pancreatic β-cells. Section 42 The administration produced an increase in the number of immune tolerance-inducing dendritic cells and / or GAD-specific regulatory T cells. The method described in any of sections 26-41. Section 43 The method described in item 42, wherein the proportion of CD8α+ immune tolerance-inducing dendritic cells to the total CD11c+ dendritic cell population in the infiltrating inguinal lymph nodes increases by approximately 13 times. Section 44 Induction of CD11b+ / CD103+ immune tolerance to the total CD11c+ dendritic cell population in the infiltrating inguinal lymph nodes. The method described in item 42, which increases the proportion of sex dendritic cells by approximately twofold. Section 45 The method described in item 42, wherein the proportion of CD207+ immune tolerance-inducing dendritic cells to the total CD11c+ dendritic cell population in the infiltrating inguinal lymph nodes increases by approximately 2.5 times. Section 46 The patient is human, as described in any of sections 26-45. Section 47 The method described in any of sections 26 to 46, wherein the vector system is administered intradermally or mucousally. Section 48 A method for increasing the number of immune tolerance-inducing dendritic cells and GAD-specific regulatory T cells in patients at risk of developing type 1 diabetes: (a) A first expression cassette expressing BCL2-related X apoptosis regulator (BAX); and (b) A permethylated second expression cassette expressing the secreted form of glutamate decarboxylase 65 A method comprising the step of administering an effective amount of a vector system containing the vector system. Section 49 The method according to section 48, further comprising a promoter operably ligated to a polynucleotide encoding BAX in a first expression cassette. Item 50 The first expression cassette is a CMV operably ligated to a polynucleotide encoding BAX. The method described in item 49, comprising a promoter or an SV-40 promoter. Section 51 The method according to any one of sections 48 to 50, further comprising a second expression cassette which is a promoter operably ligated to a polynucleotide encoding the secreted form of GAD65. Section 52 The method according to item 51, wherein the second expression cassette comprises an SV-40 promoter operably ligated to a polynucleotide encoding the secreted form of GAD65. Section 53 The secretory form of GAD65 is coded as msGAD55, as described in any of sections 48-52. Section 54 Vector systems: (a) A first vector comprising a first expression cassette expressing BAX; and (b) A permethylated second vector containing a second expression cassette expressing the secreted form of GAD65. A method described in any of paragraphs 48 to 53, including the method described in paragraphs 48 to 53. Section 55 The method described in section 54, wherein the second vector is permethylated with a CpG motif. Section 56 The method according to paragraph 54 or 55, wherein the first vector and the second vector are administered in a ratio ranging from 1:1 to 1:8. Section 57 The method according to item 56, wherein the first vector and the second vector are administered in a ratio of 1:2. Section 58 The method described in any of sections 48-57, wherein the proportion of CD8α+ immune tolerance-inducing dendritic cells to the total CD11c+ dendritic cell population in the infiltrating inguinal lymph nodes increases by approximately 13 times. Section 59 Induction of CD11b+ / CD103+ immune tolerance to the total CD11c+ dendritic cell population in the infiltrating inguinal lymph nodes. The method described in any of sections 48-58, which increases the proportion of sexual dendritic cells by approximately twofold. Item 60 The method described in any of sections 48-59, wherein the proportion of CD207+ immune tolerance-inducing dendritic cells to the total CD11c+ dendritic cell population in the infiltrating inguinal lymph nodes increases by approximately 2.5 times. Section 61 The patient is human, as described in any of items 48-60. Section 62 The method described in any of sections 48 to 61, wherein the vector system is administered intradermally or mucousally.

[0074] reference [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] Table 2-6

Claims

[Claim 1] A method for suppressing the onset of diabetes in patients at risk of developing type 1 diabetes, (a) A first expression cassette containing a polynucleotide encoding a BCL2-related X apoptosis regulator (BAX); and (b) A permethylated second expression cassette containing a polynucleotide encoding the secreted form of glutamate decarboxylase 65 (GAD65). A method comprising the step of administering a therapeutically effective dose of a vector system containing the vector system.

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