Compositions and methods for reprogramming skin tissue to have insulin producing and delivering functions - Patent Application 20070122999

Abstract

Disclosed herein are compositions and in vitro and in vivo methods for reprogramming postnatal (adult and juvenile) tissues into insulin-producing cells. These compositions and methods are useful for a variety of purposes, including the development of diabetes treatments.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 045,440, filed June 29, 2020, the disclosure of which is expressly incorporated herein.

[0002] Incorporation by Reference of Electronically Submitted Material The computer-readable nucleotide / amino acid sequence listing, submitted concurrently herewith and identified as follows, is incorporated by reference in its entirety: A 23 kilobyte ACII (text) file created on June 9, 2021, and named "337064_ST25.txt." [Background technology]

[0003] Diabetes mellitus currently afflicts at least 200 million people worldwide. Type 1 diabetes accounts for approximately 10% of this number and results from the autoimmune destruction of insulin-secreting beta cells in the pancreatic islets of Langerhans. Survival depends on multiple daily insulin injections. Type 2 diabetes accounts for the remaining 90% of affected individuals and is increasing in prevalence. Type 2 diabetes is often, but not always, associated with obesity and was formerly called late-onset or adult-onset diabetes, but is now increasingly occurring in younger individuals. Type 2 diabetes is caused by a combination of insulin resistance and insufficient insulin secretion.

[0004] Diabetes, particularly type 2 diabetes, has emerged as a global epidemic in the 21st century. Numerous long-term complications result from poorly controlled diabetes, including those affecting the kidneys, legs, feet, eyes, heart, nerves, and circulation. Prevention of these conditions requires comprehensive treatment, involving lifestyle modifications and drug therapy. Several effective antidiabetic drugs are available and are generally safe and well-tolerated. However, all currently available drug therapies become less effective as the disease progresses, and most patients eventually require insulin.

[0005] The occurrence of diabetes is associated with a substantial decrease in pancreatic islet mass.At the time of diagnosis, more than 90% of pancreatic islet mass is lost in type 1 diabetes (T1D) patients, and approximately 50% is lost in type 2 diabetes (T2D) patients.Many attempts have been made to find a promising stimulus for pancreatic islet neogenesis, which is considered to be the optimal treatment for both T1D and T2D.As disclosed herein, compositions and methods are provided for converting patient's own skin tissue into insulin-producing and insulin-producing cells.It is believed that such compositions and methods provide an alternative or supplementary method for treating diabetes to existing treatments. Summary of the Invention [Means for solving the problem]

[0006] According to the present disclosure, compositions and in vivo methods are provided for reprogramming somatic cells of postnatal (adult and juvenile) tissues, including non-pancreatic somatic cells such as skin cells, to be insulin-producing and release insulin into the patient's bloodstream. In one embodiment, postnatal skin tissues are reprogrammed in vivo to become insulin-producing and optionally exhibit the properties of pancreatic β cells, including the production of insulin and C-peptide (i.e., pancreatic β-like cells). More particularly, somatic cells are transfected with a nucleic acid sequence encoding a cocktail of β-cell-associated peptides or a unique cocktail of said β-cell-associated peptides, so that the transfected somatic cells (e.g., skin tissues) are insulin-producing and / or can be induced to produce insulin and / or insulin C-peptide in cells that do not otherwise produce insulin and / or C-peptide.

[0007] According to one embodiment, postnatal skin tissue is reprogrammed to be insulinogenic by transfecting cells of the postnatal mammalian skin tissue with nucleic acid sequences that initiate or enhance expression of Pancreatic And Duodenal Homeobox 1 (PDX-1), transcription factor MafA, Glucagon-like peptide 1 receptor (GLP-1R), and optionally, Fibroblast Growth Factor 21 (FGF21) in the transfected cells. In one embodiment, postnatal skin tissue is transfected with a first nucleic acid sequence encoding PDX-1, a second nucleic acid sequence encoding the transcription factor MafA, a third nucleic acid sequence encoding GLP-1R, and optionally a fourth nucleic acid sequence comprising a nucleic acid sequence encoding FGF21, wherein each of the first, second, third, and optional fourth nucleic acid sequences is operably linked to a regulatory sequence that allows expression (transcription and translation) of the proteins PDX-1, MafA, GLP-1R, and optionally FGF21 in the transfected cell. According to one embodiment, postnatal skin tissue is transfected with a composition comprising the first, second, third, and fourth nucleic acid sequences, wherein optionally each of the first, second, third, and fourth nucleic acid sequences is provided on a separate plasmid. In one embodiment, postnatal skin tissue is transfected with a composition comprising a first, second, third, and fourth nucleic acid sequence, wherein two or more of said first, second, third, and fourth nucleic acids are located on a single plasmid, and in one embodiment, all four of said first, second, third, and fourth nucleic acid sequences are located on a single plasmid.

[0008] According to one aspect, a reprogramming cocktail is provided comprising: a first nucleic acid sequence comprising a sequence encoding a peptide having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO:2; a second nucleic acid sequence comprising a sequence encoding a peptide having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO:4; a third nucleic acid sequence comprising a sequence encoding a peptide having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO:6; and optionally a fourth nucleic acid sequence comprising a sequence encoding a peptide having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO:8, wherein each of the first, second, third, and fourth nucleic acid sequences is operably linked to regulatory sequences that allow expression (transcription and translation) of the respective proteins PDX-1, MafA, GLP-1R, and FGF21 when the nucleic acid sequences are transfected into a mammalian cell. In one embodiment, the first, second, third, and fourth nucleic acid sequences are operably linked to a heterologous promoter that is operable in a mammalian cell but that is different from the native promoter operably linked to the human genes encoding the PDX-1, MafA, GLP-1R, and optionally FGF21 proteins.

[0009] In one embodiment, a composition for reprogramming somatic tissue (e.g., skin tissue) to be insulin-producing and release insulin from the inside of the cells of the tissue to the outside of the cells is provided. In one embodiment, the reprogramming composition comprises a first nucleic acid sequence comprising a sequence having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO: 1, a second nucleic acid sequence comprising a sequence having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO: 3, a third nucleic acid sequence comprising a sequence having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO: 5, and a fourth nucleic acid sequence comprising a sequence having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO: 7. In one embodiment, a non-viral vector is provided comprising each of the first, second, third, and fourth nucleic acid sequences, each of which is operably linked to a control sequence that enables expression of the encoded protein in a mammalian cell. In one embodiment, the non-viral vector comprises a single eukaryotic promoter operably linked to a multiple coding sequence comprising two or more of said first, second, third, and fourth nucleic acid sequences, wherein said multiple coding sequence further comprises an internal ribosome entry site present before each of said first, second, third, and fourth nucleic acid sequences; in one embodiment, the eukaryotic promoter is a heterologous promoter.

[0010] According to the present disclosure, target postnatal skin tissue can be transfected with any of the reprogramming cocktails disclosed herein using any transformation technique known to those of skill in the art. According to one embodiment, the nucleic acids of the reprogramming cocktail are introduced into the cytosol of skin cells in vivo by nanotransfection (TNT), more particularly using the TNT device described in Example 1 and shown in Figures 2A-2D.

[0011] According to one embodiment, a kit is provided for in vivo transfection of somatic tissue to induce cells of the somatic tissue to become insulin-producing and release insulin into a patient's circulation. In one embodiment, the transfected cells exhibit characteristics of pancreatic beta cells, including insulin production and release. In one embodiment, the kit includes a disposable nanotransfection device and a reprogramming cocktail. In one embodiment, the nanotransfection device includes a hollow microneedle array having one or more compartments for receiving a reprogramming cocktail solution or a cartridge containing a reprogramming cocktail. In one embodiment, the hollow microneedle array includes an electrode (i.e., an optionally gold- or silver-plated, cathode) positioned for contact with a solution loaded into a compartment of the device, and a needle counter-electrode (i.e., an anode) positioned for intradermal insertion into a patient's skin. In one embodiment, the reprogramming cocktail solution comprises a first nucleic acid sequence encoding pancreatic duodenal homeobox 1 (PDX-1), a second nucleic acid sequence encoding the transcription factor MafA, a third nucleic acid sequence encoding glucagon-like peptide 1 receptor (GLP-1R), and optionally a fourth nucleic acid sequence comprising a nucleic acid sequence encoding fibroblast growth factor 21 (FGF21). According to one embodiment, the nanotransfection device is pre-loaded with the reprogramming cocktail solution.

[0012] According to one embodiment, a method for treating type 1 or type 2 diabetes is provided, wherein a reprogramming cocktail solution comprising a first nucleic acid sequence encoding pancreatic duodenal homeobox 1 (PDX-1), a second nucleic acid sequence encoding the transcription factor MafA, a third nucleic acid sequence encoding glucagon-like peptide 1 receptor (GLP-1R), and optionally a fourth nucleic acid sequence comprising a nucleic acid sequence encoding fibroblast growth factor 21 (FGF21) is introduced into the cytosol of somatic cells in vivo, optionally by transfection with transgenic mice. In one embodiment, a method for treating diabetes and / or controlling blood glucose levels in a patient in need thereof comprises transfecting the reprogramming cocktail of the present disclosure into cells of the patient's skin tissue in vivo once monthly, every 8-12 weeks, every 10-15 weeks, or every 15-18 weeks.

[0013] In one embodiment, a method for normalizing blood glucose levels in a subject with diabetes is provided, the method comprising reprogramming target skin cells in vivo to produce insulin, the method comprising contacting the target skin cells with a reprogramming composition under conditions that enhance cellular uptake of the reprogramming composition components. In one embodiment, the transfection composition comprises a first nucleic acid sequence encoding a peptide having at least 95% sequence identity to SEQ ID NO:2, a second nucleic acid sequence encoding a peptide having at least 95% sequence identity to SEQ ID NO:4, a third nucleic acid sequence encoding a peptide having at least 95% sequence identity to SEQ ID NO:6, and optionally a fourth nucleic acid sequence encoding a peptide having at least 95% sequence identity to SEQ ID NO:8, wherein the first, second, third, and additional nucleic acid sequences are operably linked to regulatory sequences that enable expression of the encoded proteins upon introduction into human skin cells. In one embodiment, cellular uptake of the nucleic acid sequences is induced through the use of nanotransfection (TNT). [Brief explanation of the drawings]

[0014] [Figure 1] This is a schematic diagram of a hollow microneedle-mediated TNT process based on a tip with a hollow microneedle array performed on exfoliated skin. A plasmid DNA solution (5) is held in a reservoir (1) and is in fluid communication with multiple microneedles (2) of the hollow microneedle array. The plasmid DNA solution (5) is delivered to the skin tissue, including the epidermis layer (3) and dermis layer (4), under square electric pulses applied at the microsecond level. [Figure 2A] Figures 2A-2D provide schematic diagrams of TNT tips with various nanochannels and microneedle arrays. Figure 2A shows a TNT tip lacking any needle structure at all. Figure 2B shows a Type I hollow microneedle array with a flat tip. Figure 2C shows a Type II hollow microneedle array with a sharp tip and a central perforation. Figure 2D shows a Type III hollow microneedle array with a sharp tip and an off-center perforation. Cross-sectional views are also shown for each type of TNT tip. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Figure 3A] Figures 3A and 3B are graphs of two separate experiments showing the effectiveness of a transfection cocktail ("PMGF" cocktail) containing nucleic acid sequences encoding pancreatic duodenal homeobox 1 (PDX-1), transcription factor MafA, glucagon-like peptide 1 receptor (GLP-1R), and fibroblast growth factor 21 (FGF21) in lowering blood glucose levels in streptozotocin (STZ)-induced diabetic mice. Skin cell uptake of the PMGF cocktail was induced by the use of lentiviral particles. Administration of streptozotocin (STZ) and lentiviral particles is indicated by arrows. Blood glucose levels were significantly reduced in streptozotocin (STZ)-induced diabetic mice receiving the lentiviral particle-mediated PMGF cocktail (LentiPMGF) compared to controls. [Figure 3B] Same as above. [Figure 4A] Figures 4A-4C are graphs from three separate experiments showing the effectiveness of a transfection cocktail ("PMGF" cocktail) containing nucleic acid sequences encoding pancreatic duodenal homeobox 1 (PDX-1), transcription factor MafA, glucagon-like peptide 1 receptor (GLP-1R), and fibroblast growth factor 21 (FGF21) in lowering blood glucose levels in streptozotocin (STZ)-induced diabetic mice. Mice were divided into two different groups: 1) control and 2) mice receiving TNT-mediated reprogramming factors (TNTPMGF). In the reprogramming cocktail, 37.5 μg of each component (P / M / G / F) was used. An equal amount of control plasmid was delivered to the control group. The data demonstrate that TNT-mediated delivery of the PMGF reprogramming factor cocktail results in tissue reprogramming, resulting in the formation of insulin-producing cells in the postnatal skin, which leads to lower blood glucose levels in a mouse model of streptozotocin-induced diabetes. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 5A]Figures 5A-5C are graphs showing the results of an intraperitoneal glucose tolerance test (IPGTT). The IPGTT is used to test the clearance of an intraperitoneally injected glucose load from the body. This test detects disorders in glucose metabolism and insulin secretion. For this experiment, mice were fasted and fasting blood glucose levels were determined before a glucose solution (D-glucose, 2 g / kg body weight) was administered via intraperitoneal (IP) injection. Blood glucose levels were then measured from the tail vein at different time points (0, 15, 30, 60, 90, and 120 min) over the next 120 min. Intraperitoneal injection of glucose at 2 g / kg body weight caused an increase in blood glucose levels, which returned to basal levels within 120 min in the TNTPMGF group but not in the control group. Note that this experiment was performed on STZ-induced diabetic animals (Figures 4A-C) followed for 7 weeks after TNT intervention. The control group was an STZ-induced diabetic group treated with a mock (control) plasmid by TNT, and no glucose reduction was observed in that group (Figures 4A-C). Therefore, in the control group in Figure 5A, no blood glucose-lowering effect was observed, and the mice exhibited elevated blood glucose in the hyperglycemic range. Therefore, the baseline glucose levels for these mice were in the range of approximately 550 mg / dL compared to the TNT-PMGF group (approximately 300 mg / dL). The baseline blood glucose levels were much lower (approximately 200-250 mg / dL) in the high-responders shown in Figures 5B and 5C. [Figure 5B] Same as above. [Figure 5C] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0015] definition In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

[0016] As used herein, the term "about" means ten percent greater than or less than a stated value or range of values, but is not intended to limit any value or range of values ​​to this broader definition. Each value or range of values ​​preceded by the term "about" is also intended to encompass aspects of the stated absolute value or range of values.

[0017] As used herein, the term "purified" and similar terms refer to the isolation of a molecule or compound in a form that is substantially free from contaminants normally associated with the molecule or compound in its native or natural environment. As used herein, the term "purified" does not require absolute purity; rather, it is intended as a relative definition. The term "purified polypeptide" is used herein to describe a polypeptide that has been separated from other compounds, including, but not limited to, nucleic acid molecules, lipids, and carbohydrates.

[0018] The term "isolated" requires that the referenced material be removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide present in a living animal is not isolated, but the same polynucleotide separated from some or all of the coexisting materials in its natural system is isolated.

[0019] Tissue nanotransfection (TNT) is an electroporation-based technique capable of delivering nucleic acid sequences and proteins into the cytosol of cells at the nanoscale. More specifically, TNT uses a very strong and focused electric field through an array of nanochannels to gently form nanopores in adjacent tissue cell members, electrophoretically transporting cargo (e.g., nucleic acids or proteins) into the cells.

[0020] As used herein, a "regulatory element" or "control sequence" is a non-translated region of a functional gene, including enhancers, promoters, 5' and 3' untranslated regions, that interacts with host cell proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. A "eukaryotic control sequence" is a non-translated region of a functional gene, including enhancers, promoters, 5' and 3' untranslated regions, that interacts with eukaryotic host cell proteins to carry out transcription and translation in eukaryotic cells, including mammalian cells.

[0021] As used herein, a "promoter" is one or more sequences of DNA that function when in a fixed location relative to the transcription start site of a gene. A "promoter" contains core elements required for basic interaction of RNA polymerase and transcription factors, and can contain upstream elements and response elements.

[0022] As used herein, an "enhancer" is a DNA sequence that functions regardless of distance from the transcription start site and can be located either 5' or 3' of a transcription unit. Furthermore, enhancers can be located within introns as well as within the coding sequence itself. They are usually between 10 bp and 300 bp in length and function in cis. Enhancers function to increase transcription from nearby promoters. Like promoters, enhancers often contain response elements that mediate the control of transcription. Enhancers often determine the control of expression.

[0023] An "endogenous" enhancer / promoter is one that is naturally linked to a given gene in the genome. An "exogenous" or "heterologous" enhancer / promoter is one that has been placed in proximity to a gene using genetic engineering (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer / promoter. As used herein, an exogenous sequence, with respect to a cell, is a sequence that has been introduced into the cell from a source external to the cell.

[0024] As used herein, the term "non-coded (non-standard) amino acid" includes any amino acid that is not the L-isomer of any of the following 20 amino acids: Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr.

[0025] As used herein, the term "identity" refers to the similarity between two or more sequences. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the result by 100 to obtain a percentage. Thus, two copies of the exact same sequence will have 100% identity, while two sequences that have amino acid deletions, additions, or substitutions compared to each other will have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity, such as those that use algorithms such as BLAST (Basic Local Alignment Search Tool, Altschul et al. (1993) J. Mol. Biol. 215:403-410).

[0026] As used herein, the term "stringent hybridization conditions" means that hybridization will generally occur if there is at least 95%, preferably at least 97%, sequence identity between the probe and target sequence. An example of stringent hybridization conditions is overnight incubation in a solution containing 50% formamide, 5xSSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5xDenhardt's solution, 10% dextran sulfate, and 20 μg / ml denatured, sheared carrier DNA, such as salmon sperm DNA, followed by washing the hybridization support in 0.1xSSC at approximately 65°C. Other hybridization and washing conditions are well known and are exemplified in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, NY (1989), particularly Chapter 11.

[0027] As used herein, the term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate-buffered saline, water, emulsions such as oil / water or water / oil emulsions, and various types of wetting agents. The term also encompasses any agent approved by a federal regulatory agency or listed in the United States Pharmacopoeia for use in animals, including humans.

[0028] As used herein, the term "phosphate buffered saline" or "PBS" refers to an aqueous solution containing sodium chloride and sodium phosphate. While different formulations of PBS are known to those skilled in the art, for purposes of this invention, the phrase "standard PBS" refers to a solution having final concentrations of 137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, and a pH of 7.2 to 7.4.

[0029] As used herein, the term "treating" includes alleviating the symptoms associated with a particular injury or condition and / or preventing or eliminating said symptoms. As used herein, an "effective" amount or a "therapeutically effective amount" of a drug refers to a nontoxic but sufficient amount of a drug to provide the desired effect. The amount that is "effective" will vary from subject to subject, or even within a subject over time, depending on the age and general condition of the individual, the mode of administration, and the like. Thus, it is not always possible to specify an exact "effective amount." However, an appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation.

[0030] As used herein, an amino acid "substitution" refers to the replacement of one amino acid residue with a different amino acid residue. As used herein, the term "conservative amino acid substitution" is defined herein as an exchange within one of the following five groups: I. Small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro, Gly; II. Polar negatively charged residues and their amides: Asp, Asn, Glu, Gln; III. Polar positively charged residues: His, Arg, Lys; Ornithine (Orn) IV. Large aliphatic nonpolar residues: Met, Leu, Ile, Val, Cys, norleucine (Nle), homocysteine ​​(hCys) V. Large aromatic residues: Phe, Tyr, Trp, acetylphenylalanine, naphthylalanine (Nal) As used herein, the term "patient" without further specification is intended to include any warm-blooded vertebrate domesticated animal (e.g., including, but not limited to, livestock, horses, cats, dogs, and other pets) and human beings receiving therapeutic care, whether or not under medical supervision.

[0031] The term "carrier" means a compound, composition, object, or structure that, when combined with a compound or composition, aids or facilitates the preparation, storage, administration, delivery, efficacy, selectivity, or any other characteristic of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0032] The term "inhibit" refers to a decrease in an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. It can also include, for example, a 10% decrease in the activity, response, condition, or disease compared to native or control levels. Thus, the decrease can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between compared to native or control levels.

[0033] The term "polypeptide" refers to amino acids linked together by peptide bonds or modified peptide bonds, such as peptide isosteres, and may contain modified amino acids other than the 20 gene-encoded amino acids. Polypeptides may be modified by either natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. The same type of modification can be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may have many types of modifications. Modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of a flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, disulfide bond formation, demethylation, formation of cysteine ​​or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, PEGylation, protein processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer RNA-mediated addition of amino acids to proteins such as arginylation (Proteins—Structure and Molecular Properties, 2nd ed., TECreighton, W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, edited by B.C. Johnson, Academic Press, New York, pp. 1-12 (1983).

[0034] The term "amino acid sequence" refers to a string of two or more amino acids linked together by peptide bonds, the order of which is indicated by a list of abbreviations, letters, symbols, or words representing the amino acid residues. Amino acid abbreviations used herein are the conventional single-letter codes for amino acids, represented as follows: A, alanine; B, asparagine or aspartic acid; C, cysteine; D, aspartic acid; E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.

[0035] As used herein, the phrase "nucleic acid" refers to a naturally occurring or synthetic oligonucleotide or polynucleotide capable of hybridizing with a complementary nucleic acid through Watson-Crick base pairing, whether DNA, RNA, or a DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense. Nucleic acids can also include nucleotide analogs (e.g., BrdU) and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids can include, but are not limited to, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof.

[0036] As used herein, a "nucleotide" is a molecule containing a base moiety, a sugar moiety, and a phosphate moiety. Nucleotides can be linked together through their phosphate and sugar moieties to form an internucleoside linkage. The term "oligonucleotide" is sometimes used to refer to a molecule containing two or more nucleotides linked together. The base moiety of a nucleotide can be adenine-9-yl (A), cytosine-1-yl (C), guanine-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T). The sugar moiety of a nucleotide is ribose or deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. Non-limiting examples of nucleotides are 3'-AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate). A nucleotide analog is a nucleotide containing certain modifications to the base moiety, sugar moiety, and / or phosphate moiety. Modifications to nucleotides are well known in the art and include, for example, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, and 2-aminoadenine, as well as modifications in the sugar or phosphate moieties.

[0037] Nucleotide substitutes are molecules that have similar functional properties to nucleotides, such as peptide nucleic acids (PNAs), but do not contain a phosphate moiety. Nucleotide substitutes recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but are linked together through moieties other than the phosphate moiety. Nucleotide substitutes can conform to a double-helix structure when interacting with the appropriate target nucleic acid.

[0038] The terms "vector" or "construct" refer to a DNA molecule used as a vehicle to carry foreign genetic material into another cell, where it can be replicated and / or expressed. The term "expression vector" includes any vector (e.g., a plasmid, cosmid, or phage chromosome) that contains a genetic construct in a form suitable for expression by the cell (e.g., linked to transcriptional regulatory elements). Because the plasmid is a commonly used form of vector, "plasmid" and "vector" are used interchangeably. Furthermore, the invention is intended to include other vectors that serve equivalent functions.

[0039] The term "delivery vehicle" defines any moiety that facilitates the uptake of nucleic acids by cells and includes both viral and non-viral delivery systems such as nucleic acid-containing cationic polymers, liposomes, exosomes, and nanoparticles.

[0040] The term "operably linked" refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcription and translation termination sites, and other signal sequences are examples of nucleic acid sequences that can be operably linked with other sequences. For example, the operably linked relationship of DNA with a transcriptional regulatory element refers to the physical and functional relationship between DNA and a promoter such that the transcription of such DNA is initiated from the promoter by RNA polymerase that specifically recognizes, binds to, and transcribes the DNA.

[0041] As used herein, the abbreviation "PMGF" refers to a combination of one or more plasmids containing nucleic acid sequences encoding the proteins PDX1, MafA, GLP1R, and FGF21.

[0042] Aspects As disclosed herein, compositions and methods are provided for transfecting tissues and cells to convert non-insulin-producing postnatal tissues into tissues that produce and deliver functional insulin peptides to the patient's circulatory system.The present disclosure is based on the discovery that cells modified to express a combination of proteins including PDX1, MafA, GLP1R, and FGF21 express insulin and C-peptide, characteristic beta cell markers.Therefore, it has been found that increasing the cellular concentrations of the proteins PDX1, MafA, GLP1R, and FGF21 is effective in non-invasively reprogramming skin to insulin production.Furthermore, overexpression of PDX1, MafA, GLP1R, and FGF21 in mammalian skin cells reprograms skin tissue into insulin-producing tissue in vivo, and the level of insulin production in such reprogrammed tissue can be sufficient to normalize blood glucose levels in streptozotocin-induced diabetic mice.

[0043] The amino acid sequences (Table 1) and nucleic acid sequences (Table 2) encoding the transcription factors PDX1, MafA, GLP1R, and FGF21 are known in the art. Although human sequences are disclosed herein, other mammalian forms of these proteins, including human forms, are known in the art and can be used in the disclosed methods.

[0044] Amino acid sequences having at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence shown in Table 1 are encompassed by the present invention.

[0045] Nucleotide sequences that hybridize under stringent hybridization conditions to the nucleic acid sequences set forth in Table 2 are included in the present invention.

[0046] Table 1-1

[0047] Table 1-2

[0048] Table 2-1

[0049] Table 2-2

[0050] Table 2-3

[0051] Table 2-4

[0052] Table 2-5

[0053] Table 2-6

[0054] Table 2-7

[0055] Table 2-8

[0056] Polynucleotide can be delivered to skin tissue by gene gun, microparticle or nanoparticle suitable for such delivery, liposome or other membrane-bound vesicle suitable for such delivery, naked DNA or virus-based vector injection, or by electroporation transfection using three-dimensional nanochannel electroporation, tissue nanotransfection (TNT) device or deep local tissue nanoelectroinjection device.In some embodiments, viral vector can be used.However, in other embodiments, polynucleotide is not delivered by viral.

[0057] The compositions and methods of the present invention for reprogramming postnatal somatic tissues, including non-pancreatic somatic tissues such as skin tissue, into insulin-producing cells can be applied both in vitro and in vivo.

[0058] Electroporation is a technique in which an electric field is applied to cells to increase the permeability of the cell membrane and allow the introduction of cargo (e.g., reprogramming factors) into the cells (see FIG. 1). Electroporation is a common technique for introducing foreign DNA into cells. Figures 2A-2D provide examples of microchannel and microneedle arrays that can be used to transfect somatic cells in vivo. Additional details regarding such devices are described in U.S. Patent Application Nos. 62 / 903,298 and 62 / 877,060, the disclosures of which are expressly incorporated by reference.

[0059] Tissue nanotransfection allows for direct cytosolic delivery of cargo (e.g., reprogramming factors) into cells by applying a very strong and focused electric field through arrayed nanochannels, which gently form nanopores in the lining tissue cell members and electrophoretically transport the cargo into the cells.

[0060] In one embodiment, the disclosed compositions are administered at a dose equivalent to parenteral administration of about 0.1 ng to about 100 g per kg body weight, about 10 ng to about 50 g per kg body weight, about 100 ng to about 1 g per kg body weight, about 1 μg to about 50 mg per kg body weight, about 1 mg to about 500 mg per kg body weight, and about 1 mg to about 50 mg per kg body weight. Alternatively, the amount of the disclosed compositions administered to achieve a therapeutically effective dose is about 0.1 ng, 1 ng, 10 ng, 100 ng, 1 μg, 10 μg, 100 μg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 500 mg, or more per kg of body weight.

[0061] To express a polypeptide or functional nucleic acid, the nucleotide coding sequence can be inserted into an appropriate expression vector. Thus, also disclosed is a non-viral vector comprising a polynucleotide comprising three or more nucleic acid sequences encoding a protein selected from the group consisting of PDX1, MafA, GLP1R, and FGF21, wherein the three or more nucleic acid sequences are operably linked to an expression control sequence. In some embodiments, the nucleic acid sequences are operably linked to a single expression control sequence, and each coding sequence is preceded by a eukaryotic internal ribosome entry site. In other embodiments, the nucleic acid sequence is operably linked to three or more separate expression control sequences. In some embodiments, the non-viral vector comprises a plasmid.

[0062] Methods for constructing expression vectors containing gene sequences and appropriate transcriptional and translational regulatory elements are well known in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Press, Plainview, NY, 1989), and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York, NY, 1989).

[0063] In some embodiments, the nucleic acid sequences encoding PDX-1, MafA, GLP1R, and optionally FGF21 are each separately linked to eukaryotic expression control sequences, and optionally, each of the nucleic acid sequences encoding PDX-1, MafA, GLP1R, and FGF21 is linked to a heterologous eukaryotic promoter.

[0064] In one embodiment, an internal ribosome entry site (IRES) element is used to create a multigene or polycistronic construct. The IRES element can bypass the ribosome scanning model of 5' methylated cap-dependent translation and initiate translation at an internal site. The IRES element can be linked to a heterologous open reading frame. Multiple open reading frames can be transcribed together, each separated by an IRES, resulting in a polycistronic message. The IRES allows each open reading frame to access ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message.

[0065] Disclosed is a non-viral vector containing one or more polynucleotides disclosed herein, optionally operably linked with expression control sequences.Examples of such non-viral vectors include oligonucleotides alone or in combination with suitable protein, polysaccharide, or lipid preparations.Non-viral methods offer certain advantages over viral methods, and simple large-scale production and low host immunogenicity are just two of them.Previously, the low level of gene transfection and expression has made non-viral methods a disadvantage; however, recent advances in vector technology have produced molecules and technologies with transfection efficiency similar to that of viruses.Examples of suitable non-viral vectors are known to those skilled in the art.

[0066] In one embodiment, the nucleic acids encoding PDX-1, MafA, GLP1R, and FGF21 are delivered to the cytosol of the cell in the absence of a delivery vehicle. In one embodiment, electroporation is used to stimulate uptake of the nucleic acids encoding PDX-1, MafA, GLP1R, and FGF21.

[0067] The compositions disclosed herein can be used therapeutically in combination with a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means that the substance is not biologically or otherwise undesirable, i.e., the substance can be administered to a subject together with the nucleic acid or vector without causing any undesirable biological effects or interacting in a harmful manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier will naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as is well known to those skilled in the art.

[0068] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.), edited by A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic.

[0069] Examples of pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably from about 5 to about 8, more preferably from about 7 to about 7.5. Further carriers include sustained-release preparations such as semipermeable matrices of solid hydrophobic polymers containing the nucleic acid, which matrices are in the form of shaped articles, e.g., films, liposomes, or microparticles. For example, it will be apparent to those skilled in the art that certain carriers will be more preferable depending on the route of administration and concentration of the composition to be administered.

[0070] Pharmaceutical carriers are known to those skilled in the art.These are most typically the standard carriers for administering drugs to humans, and include solutions such as sterile water, saline and buffer solutions at physiological pH.Compositions can be administered intramuscularly or subcutaneously.Other compounds are administered according to the standard procedures used by those skilled in the art.

[0071] In addition to the molecule of choice, pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surfactants, and the like. Pharmaceutical compositions may also include one or more active ingredients such as antibacterial agents, anti-inflammatory agents, anesthetics, and the like.

[0072] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may be necessary or desirable.

[0073] Some of the compositions may be administered as pharmaceutically acceptable acid or base addition salts formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl, and aryl amines, and substituted ethanol amines.

[0074] The compositions, including pharmaceutical compositions, disclosed herein can be administered in several ways, depending on whether local or systemic treatment is desired and the area to be treated. For example, the disclosed compositions can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. The compositions can be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, externally, ophthalmically, vaginally, rectally, intranasally, topically, etc., including topical intranasal administration or administration by inhalation.

[0075] According to one embodiment, the patient's somatic cells are reprogrammed to be insulinogenic by enhancing the intracellular concentrations of proteins PDX1, MafA, GLP1R, and FGF21 in target tissues.The intracellular concentrations of PDX1, MafA, GLP1R, and FGF21 can be enhanced using any standard molecular biology technique known to those skilled in the art.In one embodiment, the intracellular concentrations of PDX1, MafA, GLP1R, and FGF21 can be enhanced by introducing control elements (for example, heterologous promoters or enhancer elements) into each native PMGF gene, or by introducing other factors, such as gene silencers or epigenetic manipulators that target DNA methylation and chromatin remodeling.In one embodiment, the native genes encoding each PMGF protein are modified to enhance their expression using standard gene editing techniques, including, for example, the use of CRISPR technology. Alternatively, the intracellular concentrations of PDX1, MafA, GLP1R, and FGF21 polypeptides can also be achieved by introducing exogenous components (e.g., proteins and nucleic acids) into the cytosol of skin cells, where the exogenous components directly or indirectly enhance the intracellular concentrations of PDX1, MafA, GLP1R, and FGF21. In one embodiment, the introduced exogenous components include nucleic acid sequences (e.g., DNA, mRNA, miRNA, and RNAi) that enhance the expression of genes encoding PDX1, MafA, GLP1R, and FGF21 polypeptides. In one embodiment, the exogenous components introduced into cells are DNAs encoding each of PDX1, MafA, GLP1R, and FGF21 polypeptides.

[0076] According to the present invention, nucleic acids and / or proteins are introduced into the cytosol of postnatal somatic cells, such as skin cells, to induce reprogramming of target cells. Any standard technique for introducing macromolecules into cells can be used in accordance with the present invention. Known delivery methods can be broadly classified into two types. In the first type, membrane-disruption-based methods, including mechanical, thermal, or electrical means, can be used to disrupt the continuity of the cell membrane and enhance permeability for direct entry of the desired macromolecules. In the second type, carrier-based methods, using various viruses, exosomes, vesicles, and nanoparticle capsules, enable the uptake of carriers through endocytosis and cell fusion processes for delivery of the carrier payload.

[0077] Among the methods of permeabilization-based disruptive delivery, electroporation has already been established as a universal tool.By carefully adjusting the electric field distribution, high-efficiency delivery can be achieved with minimal cytotoxicity.In one embodiment, the nucleic acid sequences encoding PDX1, MafA, GLP1R and FGF21 polypeptides are delivered into the cytosol of somatic cells through the use of tissue nanotransfection (TNT).

[0078] Tissue nanotransfection (TNT) is an electrogenic gene transfer technique that delivers plasmids, RNA, and oligonucleotides into living tissues, resulting in direct conversion of tissue function under in vivo immune surveillance without the need for any experimental procedures. Unlike viral gene transfer, which is commonly used for in vivo tissue reprogramming, TNT eliminates the need for viral delivery vehicles, thus minimizing the risk of genomic integration or cell transformation.

[0079] Current methods of in vivo reprogramming can involve transfecting cells in vivo or in vitro followed by implantation. One aspect of the present invention involves reprogramming cells in vitro followed by transplantation, but cell implantation often suffers from low survival rates and poor tissue integration. Furthermore, transfecting cells in vitro involves additional regulatory and experimental challenges.

[0080] According to one embodiment, somatic cells are transfected in vivo with a reprogramming cocktail as disclosed herein. Common methods for bulk in vivo transfection include delivery via viral delivery vehicles, non-viral delivery vehicles, or electroporation. While viral vectors can be used in accordance with the present disclosure to deliver reprogramming cocktails to non-pancreatic somatic cells, viral vectors suffer from the drawback of potentially eliciting an undesirable immune response. In addition, many viral vectors induce long-term gene expression, which is useful for some gene therapy applications, but excludes applications where persistent gene expression is not necessary or desired and transient transfection is a viable option. Viral vectors also involve insertional mutagenesis and genomic integration, which can result in undesirable side effects. However, according to one embodiment, certain non-viral carriers, such as liposomes or exosomes, can be used to deliver reprogramming cocktails to somatic cells in vivo.

[0081] TNT provides a method for localized gene delivery that causes direct changes in tissue function under immune surveillance in vivo without the need for any experimental procedures. By using TNT with a plasmid, it is possible to regulate gene overexpression temporally and spatially. Spatial regulation with TNT allows for the transfection of a target area, such as a portion of skin tissue, without transfecting other tissues.

[0082] As disclosed in more detail in the Examples, hollow needle array structures have been designed that enable efficient dermal delivery of loaded drugs, including nucleic acid sequences. Three different types of silicon hollow needle arrays, with perforation diameters ranging from nm to μm (as shown schematically in Figures 2B-2D), can be prepared for TNT applications. In less than one second, the silicon hollow needle arrays disclosed herein enable delivery of active agents to specific depths in mouse, rat, and human tissues.

[0083] According to one embodiment, a composition is provided for reprogramming cells and tissues, more particularly for reprogramming skin tissues in vivo.In one embodiment, the composition comprises: a first nucleic acid sequence encoding pancreatic duodenal homeobox 1 (PDX-1); a second nucleic acid sequence encoding transcription factor MafA; a third nucleic acid sequence encoding glucagon-like peptide 1 receptor (GLP-1R); and optionally a fourth nucleic acid sequence comprising the nucleic acid sequence encoding fibroblast growth factor 21 (FGF21), wherein each of the first, second, third and any fourth nucleic acid sequences is operably linked with a control sequence that allows the expression of the encoded protein in eukaryotic cells, including mammalian cells.In one embodiment, the composition comprises each of the first, second, third and fourth nucleic acid sequences. In one embodiment, the composition consists of a first, second, third, and fourth nucleic acid sequence and a pharmaceutically acceptable carrier, optionally wherein each of said first, second, third, and fourth nucleic acid sequences is operably linked to a heterologous promoter.

[0084] According to one embodiment, a reprogramming cocktail solution is provided, the solution comprising: a first nucleic acid sequence encoding a peptide having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO: 2; a second nucleic acid sequence encoding a peptide having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO: 4; a third nucleic acid sequence encoding a peptide having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO: 6; and an optional fourth nucleic acid sequence encoding a peptide having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO: 8. In one embodiment, the reprogramming cocktail solution comprises purified or isolated nucleic acid sequences encoding the proteins of SEQ ID NOs: 2, 4, 6, and 8.

[0085] According to one embodiment, a reprogramming cocktail solution is provided, the solution comprising: a first nucleic acid sequence encoding a peptide having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO:2; a second nucleic acid sequence encoding a peptide having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO:4; a third nucleic acid sequence encoding a peptide having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO:6; and a fourth nucleic acid sequence encoding a peptide having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO:8.

[0086] According to one embodiment, a reprogramming cocktail solution is provided, the solution comprising: a first nucleic acid sequence encoding a peptide of SEQ ID NO: 2; a second nucleic acid sequence encoding a peptide of SEQ ID NO: 4; a third nucleic acid sequence encoding a peptide of SEQ ID NO: 6; and a fourth nucleic acid sequence encoding a peptide of SEQ ID NO: 8, optionally wherein each of the first, second, third, and fourth nucleic acid sequences is operably linked to a heterologous promoter.

[0087] According to one embodiment, the reprogramming cocktail solution comprises multiple non-viral expression vectors comprising first, second, third, and fourth nucleic acid sequences. In one embodiment, the reprogramming cocktail solution comprises four separate plasmids, each comprising one of the first, second, third, and fourth nucleic acid sequences operably linked to a promoter and other regulatory sequences, enabling expression of the encoded protein in a eukaryotic cell. In one embodiment, two or more of the first, second, third, and fourth nucleic acids are located on an expression vector, the expression vector comprising a single promoter operably linked to multiple coding sequences, the multiple coding sequences comprising two or more of the first, second, third, and fourth nucleic acid sequences, and an internal ribosome entry site preceding each of two or more of the first, second, third, and fourth nucleic acid sequences.

[0088] In one embodiment, the reprogramming cocktail solution comprises only one distinct type of plasmid / expression vector, wherein the plasmid / expression vector comprises all four of the first, second, third, and fourth nucleic acid sequences linked together to form a multiple coding sequence, wherein the multiple coding sequence comprises all four of the first, second, third, and fourth nucleic acid sequences, each driven by an internal ribosome entry site, and all operably linked to the single promoter operable in mammalian cells. In one embodiment, the plasmid / expression vector is a non-viral expression vector. According to one embodiment, the reprogramming cocktail solution further comprises a reagent that enhances the efficiency of electroporation to deliver nucleic acids into eukaryotic cells or mammalian tissues.

[0089] One embodiment of the present disclosure is directed to a polynucleotide comprising three or more nucleic acid sequences encoding transcription factors / proteins selected from the group consisting of PDX-1, MafA, GLP1R, and optionally, FGF21. The PDX-1, MafA, GLP1R, and FGF21 proteins can be mammalian proteins, such as human proteins. In one embodiment, the encoded PDX-1, MafA, GLP1R, and FGF21 proteins comprise the amino acid sequences of SEQ ID NOs:2, 4, 6, and 8, respectively, or peptides that differ from the amino acid sequences of SEQ ID NOs:2, 4, 6, and 8 by 1 to 10, 1 to 5, or 1 to 3 amino acid substitutions, insertions, or deletions, or peptides that differ from the amino acid sequences of SEQ ID NOs:2, 4, 6, and 8 by 1 to 10, 1 to 5, or 1 to 3 amino acid substitutions, optionally conservative amino acid substitutions.

[0090] In one embodiment, a reprogramming cocktail solution is provided that includes a non-viral vector, the vector comprising a polynucleotide including three or more nucleic acid sequences encoding proteins selected from the group consisting of PDX-1, MafA, GLP1R, and FGF21, and the three or more nucleic acid sequences are optionally operably linked to expression control sequences. Each of the nucleic acid sequences can be individually operably linked to a single promoter and other control sequences required for expression in eukaryotic cells, or alternatively, multiple nucleic acid sequences can be expressed under the control of a single promoter.

[0091] In one embodiment, a reprogramming cocktail solution is provided that comprises a peptide, more particularly, in one embodiment, a composition comprising: a peptide having at least 95% sequence identity to SEQ ID NO:2; a peptide having at least 95% sequence identity to SEQ ID NO:4; a peptide having at least 95% sequence identity to SEQ ID NO:6; A peptide having at least 95% sequence identity to SEQ ID NO:8; and Optionally, a reagent that enhances the efficiency of protein delivery into the interior of a eukaryotic cell.

[0092] A further embodiment is directed to a method for reprogramming somatic cells into insulin-producing cells, optionally cells having the insulin-producing properties of pancreatic beta cells (i.e., pancreatic beta-like cells), by (a) delivering into the somatic cells a polynucleotide encoding the proteins PDX-1, MafA, GLP1R, and optionally FGF21, or the proteins PDX-1, MafA, GLP1R, and optionally FGF21. In one embodiment, the somatic cells are skin cells, and more particularly, the transfected cells are skin cells of skin tissue transfected in vivo with a reprogramming cocktail solution, optionally in the absence of a viral delivery vehicle. In one embodiment, the polynucleotides encoding the PDX-1, MafA, GLP1R, and optionally FGF21 proteins, or the PDX-1, MafA, GLP1R, and optionally FGF21 proteins, are delivered into the cells using any standard technique known to those skilled in the art. In one embodiment, intracellular delivery is via viral vectors or other delivery vehicles that can interact with cell membranes and deliver contents into cells.In one embodiment, intracellular delivery is via three-dimensional nanochannel electroporation, tissue nanotransfection device delivery, or deep local tissue nanoelectroinjection device delivery.In one embodiment, reprogramming cocktail is delivered to the cytosol of postnatal skin tissue cells in vivo through tissue nanotransfection (TNT) using silicon hollow needle array.

[0093] In one embodiment, a method for reprogramming non-pancreatic somatic tissue, and optionally reprogramming cells of postnatal skin tissue in vivo, to produce insulin and C-peptide comprises delivering any of the reprogramming cocktail solutions disclosed herein into cells of the non-pancreatic somatic tissue, optionally with TNT. In one embodiment, the reprogramming cocktail solution comprises or consists of naked DNA, wherein the naked DNA comprises: a first nucleic acid sequence encoding a peptide having at least 95% sequence identity with SEQ ID NO:2; a second nucleic acid sequence encoding a peptide having at least 95% sequence identity with SEQ ID NO:4; a third nucleic acid sequence encoding a peptide having at least 95% sequence identity with SEQ ID NO:6; and optionally a fourth nucleic acid sequence encoding a peptide having at least 95% sequence identity with SEQ ID NO:8. In one embodiment, the reprogramming cocktail solution comprises or consists of naked DNA, wherein the naked DNA comprises each of the first, second, third, and fourth nucleic acids. Some of the first, second, third, and fourth nucleic acid sequences disclosed herein can be located on separate plasmids or expression vectors, or can be clustered together in individual plasmids or expression vectors. In one embodiment, each of the first, second, third, and fourth nucleic acid sequences is located on a single plasmid or expression vector as separate genes under the control of individual promoters, or as a single multigene construct under the control of a single promoter.

[0094] In one embodiment, the reprogramming cocktail solution comprises one or more separate expression vectors, each of which comprises two or more of the first, second, third, and fourth nucleic acids as part of an expression vector, wherein the expression vector comprises a single eukaryotic promoter operably linked to a multiple coding sequence comprising two or more of the first, second, third, and fourth nucleic acid sequences, wherein the multiple coding sequence further comprises an internal ribosome entry site preceding each of two or more of the first, second, third, and fourth nucleic acid sequences. In one embodiment, each of the first, second, third, and fourth nucleic acids are located on a single expression vector as part of a multiple coding sequence, wherein the multiple coding sequence further comprises an internal ribosome entry site preceding each of two or more of the first, second, third, and fourth nucleic acid sequences, and a single promoter driving transcription of the multiple coding sequence.

[0095] In one embodiment, a method for normalizing blood glucose levels in a subject with diabetes is provided, comprising reprogramming target postnatal skin tissue in vivo to produce insulin. In one embodiment, the method comprises delivering any of the reprogramming cocktail solutions of the present disclosure into the cytosol of cells in target skin tissue. Any known technique for transfecting cells can be used, including, for example, TNT. In one embodiment, the reprogramming cocktail solution comprises a first nucleic acid sequence encoding a peptide having at least 95% sequence identity with SEQ ID NO:2; a second nucleic acid sequence encoding a peptide having at least 95% sequence identity with SEQ ID NO:4; and a third nucleic acid sequence encoding a peptide having at least 95% sequence identity with SEQ ID NO:6; and optionally, a fourth nucleic acid sequence encoding a peptide having at least 95% sequence identity with SEQ ID NO:8. In one embodiment, the reprogramming cocktail solution comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 1; a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 3; a third nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 5; and, optionally, a fourth nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 7. In one embodiment, the reprogramming cocktail solution comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 1; a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 3; a third nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 5; and a fourth nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 7.

[0096] In one embodiment, a method is provided for treating a diabetic or prediabetic patient by direct tissue reprogramming of somatic tissue (i.e., skin or fat or another non-pancreatic somatic tissue, or pancreatic somatic tissue) to convert somatic cells into insulin-producing cells, optionally wherein the reprogrammed cells have the properties of pancreatic β cells (i.e., pancreatic β-like cells). The reprogrammed cells produced by the methods as disclosed herein secrete at least 15%, or at least 25%, or at least 30% of the insulin secreted by endogenous β cells, or alternatively, in some embodiments, the reprogrammed cells exhibit at least two properties of endogenous pancreatic β cells, such as secreting insulin and being positive for applicable biomarkers, including detection of insulin C-peptide.

[0097] According to one embodiment, a method is provided for treating type 1 or type 2 diabetes and / or reducing blood glucose levels to normal levels (i.e., between 70 mg / dL and 100 mg / dL), in which a patient's somatic tissue is induced to produce elevated intracellular concentrations of the polypeptide pancreatic duodenal homeobox 1 (PDX-1), transcription factor MafA, glucagon-like peptide 1 receptor (GLP-1R), and fibroblast growth factor 21 (FGF21). In one embodiment, increased levels of these polypeptides are achieved by introducing into the cytosol of skin cells in vivo a first nucleic acid sequence encoding the polypeptide pancreatic duodenal homeobox 1 (PDX-1), a second nucleic acid sequence encoding the transcription factor MafA, a third nucleic acid sequence encoding the glucagon-like peptide 1 receptor (GLP-1R), and a fourth nucleic acid sequence encoding fibroblast growth factor 21 (FGF21). In one embodiment, the nucleic acid sequence is introduced into the cytosol of cells of the target tissue by nanotransfection (TNT).

[0098] According to one embodiment, a kit is provided for in vivo transfection of postnatal skin tissue to induce the skin tissue to become insulin-producing and, optionally, exhibit pancreatic beta cell characteristics. In one embodiment, the kit includes a disposable nanotransfection device and a reprogramming cocktail. In one embodiment, the nanotransfection device includes a silicon wafer including a series of microchannels. In one embodiment, the nanotransfection device includes a plurality of shafts, each of which has an electrically conductive outer surface and is electrically coupled to one another, each of which extends from a proximal end to a distal end, each of which defines a primary channel interior to a corresponding shaft extending from the proximal end to the distal end, the primary channel being open at the proximal end and closed at the distal end, each of which further defines one or more microchannels, each of which extends from the primary channel to a wall of the corresponding shaft, and each of the one or more microchannels having a diameter of less than 10 micrometers. The kit may further include a plurality of electrodes, each of which is electrically coupled to one another of the plurality of electrodes, such that when a voltage is applied between the plurality of shafts and the plurality of electrodes, an electric field is generated perpendicular to the axis of each of the plurality of shafts.

[0099] According to one embodiment, a kit for in vivo transfection of postnatal skin tissue to induce the skin tissue to become insulin-producing is provided, the kit comprising a disposable nanotransfection device and a reprogramming cocktail, the nanotransfection device comprising a hollow microneedle array having one or more compartments for receiving the reprogramming cocktail solution. In one embodiment, the nanotransfection device is selected from the group consisting of a type I hollow microneedle array with a flat tip, a type II hollow microneedle array with a sharp tip, and a type III hollow microneedle array with a sharp tip and off-center perforations, as shown in Figures 2B-2D. In one embodiment, the cylindrical needles of the type I, type II, and type III microneedle arrays have a length of approximately 210 μm, an outer diameter of approximately 50 μm, and a hollow channel located at the center of the needle with a diameter of approximately 6 μm. The spacing between two adjacent needles is approximately 150 μm. In one embodiment, the diameter of the backhole holes is approximately 20 μm, and their spacing is the same as that of the hollow microneedles. Type II and Type III microneedles are expected to produce similar delivery results, but with additional functionality. Unlike flat tips, the sharpness of Type II needle arrays yields better performance in reducing the insertion force required for insertion into tissue. The Type III silicon hollow needle array shown in FIG. 2D has sharp tips and off-center perforations. The hollow perforations are designed to be approximately 15 μm off-center from the needle center to reduce the incidence of tissue clogging during insertion.

[0100] In one embodiment, the hollow microneedle array comprises an electrode (i.e., an optionally gold- or silver-plated, cathode) positioned for contact with the solution loaded into a compartment of the device, and a needle counter-electrode (i.e., an anode) positioned for intradermal insertion into the patient's skin. In one embodiment, the reprogramming cocktail solution comprises a first nucleic acid sequence encoding pancreatic duodenal homeobox 1 (PDX-1), a second nucleic acid sequence encoding the transcription factor MafA, a third nucleic acid sequence encoding the glucagon-like peptide 1 receptor (GLP-1R), and optionally a fourth nucleic acid sequence comprising a nucleic acid sequence encoding fibroblast growth factor 21 (FGF21). According to one embodiment, the nanotransfection device is pre-loaded with the reprogramming cocktail solution.

[0101] According to aspect 1, there is provided a method of reprogramming postnatal cells of a somatic tissue to produce insulin and C-peptide, said method comprising delivering into cells of said somatic tissue, optionally in the absence of a viral delivery vehicle, DNA comprising: a first nucleic acid sequence encoding a peptide having at least 95% sequence identity to SEQ ID NO:2; a second nucleic acid sequence encoding a peptide having at least 95% sequence identity to SEQ ID NO:4; a third nucleic acid sequence encoding a peptide having at least 95% sequence identity to SEQ ID NO:6; and optionally A fourth nucleic acid sequence encoding a peptide having at least 95% sequence identity to SEQ ID NO:8.

[0102] According to embodiment 2, there is provided a method according to embodiment 1, wherein the first, second, third, and fourth nucleic acid sequences are each simultaneously delivered in vivo to the cytosol of cells of the somatic tissue.

[0103] According to aspect 3, there is provided a method according to aspect 1 or 2, wherein one or more expression vectors are transfected into cells of the somatic tissue, the expression vectors comprising the first, second, third, and fourth nucleic acid sequences.

[0104] According to aspect 4, two or more of the first, second, third, and fourth nucleic acid sequences are part of an expression vector, wherein the expression vector comprises a single eukaryotic promoter operably linked to a multiple coding sequence comprising two or more of the first, second, third, and fourth nucleic acid sequences, wherein the multiple coding sequence further comprises an internal ribosome entry site present before each of two or more of the first, second, third, and fourth nucleic acid sequences, and optionally, wherein the first nucleic acid sequence comprises a sequence encoding a peptide having at least 95%, 99%, or 100% sequence identity to SEQ ID NO:2; and wherein the second nucleic acid sequence comprises a sequence encoding a peptide having at least 95%, 99%, or 100% sequence identity to SEQ ID NO:4. The method of any one of aspects 1-3 is provided, wherein the first nucleic acid sequence comprises a sequence encoding a peptide having 95%, 99%, or 100% sequence identity to SEQ ID NO:6; the third nucleic acid sequence comprises a sequence encoding a peptide having at least 95%, 99%, or 100% sequence identity to SEQ ID NO:6; and the fourth nucleic acid sequence comprises a sequence encoding a peptide having at least 95%, 99%, or 100% sequence identity to SEQ ID NO:8; optionally, the first nucleic acid sequence comprises the sequence of SEQ ID NO:1, the second nucleic acid sequence comprises the sequence of SEQ ID NO:3, the third nucleic acid sequence comprises the sequence of SEQ ID NO:5, and the fourth nucleic acid sequence comprises the sequence of SEQ ID NO:17.

[0105] According to aspect 5, there is provided a method according to any one of aspects 1 to 4, wherein each of the first, second, third, and fourth nucleic acid sequences are located on a single expression vector, optionally wherein the expression vector comprises a single eukaryotic promoter operably linked to multiple coding sequences comprising each of the first, second, third, and fourth nucleic acid sequences, and wherein an internal ribosome entry site is present before each of the first, second, third, and fourth nucleic acid sequences.

[0106] According to a sixth aspect, there is provided a method according to any one of the first to fifth aspects, wherein the somatic cells are skin cells. According to embodiment 7, there is provided a method according to any one of embodiments 1 to 6, wherein the intracellular delivery is via tissue nanotransfection.

[0107] According to an embodiment 8, there is provided a method according to any one of embodiments 1 to 7, wherein the cells are skin cells of skin tissue that are transfected in vivo. According to aspect 9, there is provided a method of lowering blood glucose levels to normal levels in a subject with diabetes, the method comprising a step of reprogramming target skin tissue in vivo to produce insulin, wherein the reprogramming step comprises contacting cells of the target skin tissue with a reprogramming composition under conditions that enhance cellular uptake of reprogramming composition components, the reprogramming composition comprising: a first nucleic acid sequence encoding a peptide having at least 95%, 99%, or 100% sequence identity to SEQ ID NO:2; a second nucleic acid sequence encoding a peptide having at least 95%, 99%, or 100% sequence identity to SEQ ID NO:4; and a third nucleic acid sequence encoding a peptide having at least 95%, 99%, or 100% sequence identity to SEQ ID NO:6; and optionally a fourth nucleic acid sequence encoding a peptide having at least 95%, 99%, or 100% sequence identity to SEQ ID NO:8.

[0108] According to aspect 10, there is provided a composition for use in reprogramming postnatal cells of somatic tissue to produce insulin and C-peptide. In one aspect, the composition comprises: a first nucleic acid sequence encoding pancreatic duodenal homeobox 1 (PDX-1), optionally wherein the first nucleic acid sequence encodes a peptide having at least 95%, 99%, or 100% sequence identity to SEQ ID NO:2; a second nucleic acid sequence encoding the transcription factor MafA, optionally wherein said second nucleic acid sequence encodes a peptide having at least 95%, 99%, or 100% sequence identity to SEQ ID NO:4; a third nucleic acid sequence encoding a glucagon-like peptide 1 receptor (GLP-1R), optionally wherein said third nucleic acid sequence encodes a peptide having at least 95%, 99%, or 100% sequence identity to SEQ ID NO: 6; and optionally, a fourth nucleic acid sequence encoding fibroblast growth factor 21 (FGF21), optionally wherein said fourth nucleic acid sequence encodes a peptide having at least 95%, 99%, or 100% sequence identity to SEQ ID NO:8. wherein each of said first, second, third, and optional fourth nucleic acid sequences is operably linked to a eukaryotic regulatory sequence.

[0109] According to aspect 11, the first nucleic acid sequence encodes a peptide having at least 95% sequence identity to SEQ ID NO:2; the second nucleic acid sequence encodes a peptide having at least 95% sequence identity to SEQ ID NO:4; the third nucleic acid sequence encodes a peptide having at least 95% sequence identity to SEQ ID NO:6; and the optional fourth nucleic acid sequence encodes a peptide having at least 95% sequence identity to SEQ ID NO:8; A composition according to embodiment 10 is provided.

[0110] According to a twelfth aspect, there is provided a composition according to any one of the tenth and eleventh aspects, comprising said first, second, third and fourth nucleic acid sequences. According to Aspect 13, there is provided a composition according to any one of Aspects 10 to 12, wherein two or more of the first, second, third, and fourth nucleic acid sequences are part of an expression vector, wherein the expression vector comprises a single eukaryotic promoter operably linked to a multiple coding sequence comprising the two or more first, second, third, and fourth nucleic acid sequences, and wherein the multiple coding sequence further comprises an internal ribosome entry site present before each of the two or more first, second, third, and fourth nucleic acid sequences.

[0111] According to embodiment 14, there is provided the composition of any one of embodiments 10 to 13, wherein the multiple coding sequence comprises all four of the first, second, third, and optional fourth nucleic acid sequences, each preceded by an internal ribosome entry site and operably linked to the single eukaryotic promoter.

[0112] According to embodiment 15, there is provided a composition according to any one of embodiments 10 to 14, wherein the first, second, third and fourth nucleic acid sequences are part of a non-viral vector. According to aspect 16, there is provided a kit for in vivo transfection of postnatal skin tissue to induce said skin tissue to be insulin-producing, said kit comprising: a disposable nanotransfection device; and A reprogramming cocktail solution comprising a first nucleic acid sequence encoding pancreatic duodenal homeobox 1 (PDX-1), a second nucleic acid sequence encoding a transcription factor MafA, a third nucleic acid sequence encoding a glucagon-like peptide 1 receptor (GLP-1R); and optionally, a fourth nucleic acid sequence comprising a nucleic acid sequence encoding fibroblast growth factor 21 (FGF21). A kit is provided comprising:

[0113] According to embodiment 17, there is provided a kit according to embodiment 16, wherein the nanotransfection device comprises a hollow microneedle array having one or more compartments for receiving said reprogramming cocktail solution.

[0114] Aspects According to embodiment 1, there is provided a method of reprogramming cells of a somatic tissue to produce insulin and C-peptide, comprising the steps of: a first nucleic acid sequence encoding a peptide having at least 95% sequence identity to SEQ ID NO:2; a second nucleic acid sequence encoding a peptide having at least 95% sequence identity to SEQ ID NO:4; a third nucleic acid sequence encoding a peptide having at least 95% sequence identity to SEQ ID NO:6; and optionally A fourth nucleic acid sequence encoding a peptide having at least 95% sequence identity to SEQ ID NO:8. a DNA comprising the steps of:

[0115] According to embodiment 2, there is provided a method according to embodiment 1, wherein the first, second, third, and fourth nucleic acid sequences are each simultaneously delivered in vivo to the cytosol of cells of the somatic tissue.

[0116] According to aspect 3, there is provided a method according to aspect 1 or 2, wherein one or more expression vectors are transfected into cells of the somatic tissue, the expression vectors comprising the first, second, third, and fourth nucleic acid sequences.

[0117] According to Aspect 4, there is provided a method according to any one of Aspects 1 to 3, wherein two or more of the first, second, third, and fourth nucleic acid sequences are part of an expression vector, wherein the expression vector comprises a single eukaryotic promoter operably linked to a multiple coding sequence comprising the two or more first, second, third, and fourth nucleic acid sequences, and wherein the multiple coding sequence further comprises an internal ribosome entry site present before each of the two or more first, second, third, and fourth nucleic acid sequences.

[0118] According to aspect 5, there is provided a method according to any one of aspects 1 to 4, wherein each of the first, second, third, and fourth nucleic acid sequences is located on a single expression vector. According to a sixth aspect, there is provided a method according to any one of the first to fifth aspects, wherein the somatic cells are skin cells.

[0119] According to embodiment 7, there is provided a method according to any one of embodiments 1 to 6, wherein the intracellular delivery is via tissue nanotransfection. According to embodiment 8, there is provided a method according to embodiment 7, wherein the cells are skin cells of skin tissue that are transfected in vivo.

[0120] According to aspect 9, there is provided a method of normalizing blood glucose levels in a subject having diabetes, said method comprising reprogramming target skin tissue in vivo to produce insulin, said method comprising: contacting cells of said target skin tissue with a reprogramming composition under conditions that enhance cellular uptake of reprogramming composition components, said reprogramming composition comprising: a first nucleic acid sequence encoding a peptide having at least 85%, 95%, or 99% sequence identity to SEQ ID NO:2; a second nucleic acid sequence encoding a peptide having at least 85%, 95%, or 99% sequence identity to SEQ ID NO:4; and a third nucleic acid sequence encoding a peptide having at least 85%, 95%, or 99% sequence identity to SEQ ID NO:6; and optionally, A fourth nucleic acid sequence encoding a peptide having at least 85%, 95%, or 99% sequence identity to SEQ ID NO:8. A method is provided, comprising:

[0121] According to embodiment 10, the reprogramming composition comprises: a first nucleic acid sequence encoding a peptide comprising SEQ ID NO:2; a second nucleic acid sequence encoding a peptide comprising SEQ ID NO:4; a third nucleic acid sequence encoding a peptide comprising SEQ ID NO:6; and A fourth nucleic acid sequence encoding a peptide comprising SEQ ID NO:8. The method of embodiment 9 is provided, comprising:

[0122] According to aspect 11, a first nucleic acid sequence encoding pancreatic duodenal homeobox 1 (PDX-1); a second nucleic acid sequence encoding the transcription factor MafA; a third nucleic acid sequence encoding a glucagon-like peptide 1 receptor (GLP-1R); and optionally, A fourth nucleic acid sequence encoding fibroblast growth factor 21 (FGF21) wherein each of the first, second, third, and optional fourth nucleic acid sequences is operably linked to a eukaryotic regulatory sequence.

[0123] According to aspect 12, the first nucleic acid sequence encodes a peptide having at least 85%, 95%, or 99% sequence identity to SEQ ID NO:2; the second nucleic acid sequence encodes a peptide having at least 85%, 95%, or 99% sequence identity to SEQ ID NO:4; and the third nucleic acid sequence encodes a peptide having at least 85%, 95%, or 99% sequence identity to SEQ ID NO:6; and optionally, the fourth nucleic acid sequence encodes a peptide having at least 85%, 95%, or 99% sequence identity to SEQ ID NO:8; A composition according to embodiment 11 is provided.

[0124] According to embodiment 13, the reprogramming composition comprises a first nucleic acid sequence encoding a peptide comprising SEQ ID NO:2; a second nucleic acid sequence encoding a peptide comprising SEQ ID NO:4; a third nucleic acid sequence encoding a peptide comprising SEQ ID NO:6; and A fourth nucleic acid sequence encoding a peptide comprising SEQ ID NO:8. 13. The method of claim 11 or 12, comprising:

[0125] According to aspect 14, the first nucleic acid sequence encodes a peptide having at least 95% sequence identity to SEQ ID NO:2; the second nucleic acid sequence encodes a peptide having at least 95% sequence identity to SEQ ID NO:4; a third nucleic acid sequence encoding a peptide having at least 95% sequence identity to SEQ ID NO:6; and optionally, the fourth nucleic acid sequence encodes a peptide having at least 95% sequence identity to SEQ ID NO:8; A composition according to embodiment 11 is provided.

[0126] According to aspect 15, there is provided a composition according to aspect 14, comprising the first, second, third, and fourth nucleic acid sequences. According to Aspect 16, there is provided the composition of any one of Aspects 11 to 15, wherein two or more of the first, second, third, and fourth nucleic acid sequences are part of an expression vector, wherein the expression vector comprises a single eukaryotic promoter operably linked to a multiple coding sequence comprising the two or more first, second, third, and fourth nucleic acid sequences, and wherein the multiple coding sequence further comprises an internal ribosome entry site present before each of the two or more first, second, third, and fourth nucleic acid sequences.

[0127] According to embodiment 17, there is provided the composition of embodiment 16, wherein the multiple coding sequence comprises all four of the first, second, third, and optionally fourth nucleic acid sequences, each preceded by an internal ribosome entry site and operably linked to the single eukaryotic promoter.

[0128] According to an eighteenth aspect, there is provided a composition according to any one of aspects 11 to 17, wherein the first, second, third and fourth nucleic acid sequences are part of a non-viral vector. According to aspect 19, there is provided a kit for in vivo transfection of postnatal skin tissue to induce said skin tissue to be insulin-producing, said kit comprising: a disposable nanotransfection device; and a reprogramming cocktail comprising a first nucleic acid sequence encoding pancreatic duodenal homeobox 1 (PDX-1), a second nucleic acid sequence encoding the transcription factor MafA, a third nucleic acid sequence encoding the glucagon-like peptide 1 receptor (GLP-1R), and a fourth nucleic acid sequence comprising a nucleic acid sequence encoding fibroblast growth factor 21 (FGF21). A kit is provided comprising:

[0129] According to an embodiment 20, there is provided a kit according to embodiment 19, wherein the nanotransfection device comprises a hollow microneedle array having one or more compartments for receiving said reprogramming cocktail solution. [Example]

[0130] Example 1 Reprogramming skin tissue to become insulin-producing Detailed steps: In vivo tissue reprogramming (lentivirus-mediated and TNT-mediated) Diabetes was induced in 8-week-old male mice (C57Bl / 6, Jackson Laboratories, Catalog No. 000664) by intraperitoneal injection of 50 mg / kg streptozotocin (STZ, Catalog No. S0130, Millipore Sigma) for 5 consecutive days. STZ, a drug that selectively destroys beta cells in pancreatic islets, induces elevated blood glucose levels (up to 400-500 mg / dL) in mice, leading to the development of diabetes. Blood glucose monitoring was performed on mice after a 6-hour fast, and blood glucose was measured every 7 days using a Contour blood glucose meter (Cat. No. 9545C) and test strips (Cat. No. 7099).

[0131] For lentivirus-mediated PMGF reprogramming, mice in the PMGF group were injected intradermally into the posterior dorsal skin every other day (days 1, 3, and 5) for 3 days with lentiviruses overexpressing PDX-1, MafA, GLP-1R, and FGF21 (10 for each reprogramming factor). 7 Control mice were injected with a control vector containing lentivirus without any reprogramming factors (10 7(100 μl per mouse at a titer of 100 particles / mL). Mouse lentivirus was purchased from Applied Biological Materials Inc., Richmond, BC, Canada, catalog number LV002.

[0132] For TNT-mediated PMGF reprogramming, the area to be treated was first denuded 24–48 h before TNT. The skin was then stripped to remove the dead / keratinized cell layer in the epidermis and expose nucleated cells. The TNT device was placed directly on the stripped skin surface. PMGF plasmid cocktail was loaded into the reservoir at a concentration of 0.05–0.1 μg / μl. A gold-plated electrode (i.e., the cathode) was immersed in the plasmid solution, while a 24G needle counterelectrode (i.e., the anode) was inserted intradermally, juxtaposed to the TNT platform surface. Pulse electrical stimulation (i.e., 10 pulses with an amplitude of 250 V and a duration of 10 seconds per pulse) was then applied to the electrode to form nanopores in the exposed cell membrane and transport the plasmid cargo through the nanochannels into the cells. PMGF (PM:G:F) plasmids were mixed in a 1:1:1 molar ratio. In the reprogramming cocktail, 37.5 μg of each component PM / G / F was used.

[0133] Equal amounts of control plasmid were delivered to control groups of mice. Unless otherwise specified, control samples included TNT treatment with blank phosphate-buffered saline (PBS) / mock plasmid solution. Mock (empty vector) PDX-1-MafA, GLP-1R, and FGF-21 plasmids were prepared using a plasmid DNA purification kit (ZymoPURE II Plasmid Midiprep Kit, catalog number D4201), and DNA concentrations were obtained using a Nanodrop 2000c spectrophotometer (Thermoscientific). PDX-1-MafA, GLP-1R, and FGF-21 plasmids were constructed with GFP (PDX-1-MafA), td-Tomato (GLP-1R), or CFP (FGF-21) from Applied Biological Materials Inc., Richmond, BC, Canada, catalog number C315. For blood glucose monitoring, mice were fasted for 6 hours and blood glucose was measured every 7 days using a Contour blood glucose meter (cat. no.-9545C) and test strips (cat. no.-7099).

[0134] Intraperitoneal glucose tolerance test (IPGTT) IPGTT was used to test the clearance of intraperitoneally injected glucose load from the body. This test was performed 7 weeks after TNT intervention. This test detects impairments in glucose metabolism and insulin secretion. For this experiment, mice were fasted for 6 hours before a glucose solution (D-glucose, Gibco, Cat. No. 15023-021, 2g / kg body weight) was administered intraperitoneally (IP) to determine fasting blood glucose levels. Blood glucose levels were then measured from the tail vein at different time points (0, 15, 30, 60, 90, and 120 minutes) over the next 120 minutes.

[0135] Immunohistochemistry and microscopy For histological studies, excised skin and pancreas from euthanized mice were embedded in paraffin and processed for immunohistochemistry with antibodies specific for insulin-producing cells, insulin (Abcam, ab7842, 1:100 dilution), and C-peptide (Abcam, ab14181, 1:100 dilution). Subsequent incubation with appropriate fluorescently tagged secondary antibodies (Alexa488-tagged anti-guinea pig, 1:200; Alexa568-tagged anti-rabbit, 1:200) and counterstaining with DAPI allowed visualization. Images were captured using a laser scanning confocal microscope (Olympus FV 1000 filter / spectrum).

[0136] Confocal images demonstrated the formation of insulin and C-peptide in the reprogrammed skin. For histological examination, excised skin and pancreas from euthanized mice were embedded in paraffin and processed for immunohistochemistry with antibodies specific for insulin-producing cells, insulin (Abcam, ab7842, 1:100 dilution) and C-peptide (Abcam, ab14181, 1:100 dilution). The reprogrammed skin exhibited insulin-producing cells that were pancreatic islet-like clusters in morphology and produced abundant insulin and C-peptide, characteristic markers of pancreatic islet beta cells. C-peptide expression in the skin provided evidence of de novo insulin formation in the reprogrammed skin. Interestingly, no such structures were found in control skin. Thus, the data demonstrated that the PMGF reprogramming factor cocktail resulted in tissue reprogramming, resulting in the formation of insulin-producing cells in postnatal skin, which regulated blood glucose levels in a mouse streptozotocin-induced diabetic model. Confocal microscopy images of mouse skin 24 hours after TNT treatment revealed expression of the PDX-1-MafA pancreatic transcription factor. In one aspect, the present invention may be as follows. [Embodiment 1] A method for reprogramming cells of a somatic tissue to produce insulin and C-peptide, comprising: a first nucleic acid sequence encoding a peptide having at least 95% sequence identity to SEQ ID NO:2; a second nucleic acid sequence encoding a peptide having at least 95% sequence identity to SEQ ID NO:4; a third nucleic acid sequence encoding a peptide having at least 95% sequence identity to SEQ ID NO:6; and optionally The above method, comprising the step of delivering DNA comprising a fourth nucleic acid sequence encoding a peptide having at least 95% sequence identity to SEQ ID NO:8 into a cell of a somatic tissue. [Embodiment 2] The method of embodiment 1, wherein the first, second, third, and fourth nucleic acid sequences are each simultaneously delivered in vivo to the cytosol of cells of somatic tissue. [Embodiment 3] A method according to embodiment 1 or 2, wherein one or more expression vectors are transfected into cells of the somatic tissue, and the expression vectors comprise first, second, third, and fourth nucleic acid sequences. [Aspect 4] The method of aspect 2, wherein two or more of the first, second, third, and fourth nucleic acid sequences are part of an expression vector, the expression vector comprising a single eukaryotic promoter operably linked to a multiple coding sequence comprising the two or more first, second, third, and fourth nucleic acid sequences, and the multiple coding sequence further comprising an internal ribosome entry site present before each of the two or more first, second, third, and fourth nucleic acid sequences. [Aspect 5] The method described in aspect 4, wherein each of the first, second, third, and fourth nucleic acid sequences is located in a single expression vector. [Aspect 6] The method of aspect 5, wherein the somatic cells are skin cells. [Aspect 7] The method of aspect 1, wherein the intracellular delivery is via tissue nanotransfection. [Aspect 8] The method described in aspect 7, wherein the cells are skin cells of skin tissue that are transfected in vivo. [Aspect 9] A method of normalizing blood glucose levels in a subject with diabetes, the method comprising reprogramming target skin tissue to produce insulin in vivo, the method comprising: contacting cells of a target skin tissue with a reprogramming composition under conditions that enhance cellular uptake of reprogramming composition components, wherein the reprogramming composition: a first nucleic acid sequence encoding a peptide having at least 95% sequence identity to SEQ ID NO:2; a second nucleic acid sequence encoding a peptide having at least 95% sequence identity to SEQ ID NO:4; and a third nucleic acid sequence encoding a peptide having at least 95% sequence identity to SEQ ID NO:6; and optionally, The above method, further comprising a fourth nucleic acid sequence encoding a peptide having at least 95% sequence identity to SEQ ID NO:8. [Aspect 10] a first nucleic acid sequence encoding pancreatic duodenal homeobox 1 (PDX-1); a second nucleic acid sequence encoding the transcription factor MafA; a third nucleic acid sequence encoding a glucagon-like peptide 1 receptor (GLP-1R); and optionally, A fourth nucleic acid sequence comprising a nucleic acid sequence encoding fibroblast growth factor 21 (FGF21). wherein each of the first, second, third, and optional fourth nucleic acid sequences is operably linked to a eukaryotic control sequence. [Embodiment 11] The first nucleic acid sequence encodes a peptide having at least 95% sequence identity to SEQ ID NO:2; the second nucleic acid sequence encodes a peptide having at least 95% sequence identity to SEQ ID NO:4; the third nucleic acid sequence encodes a peptide having at least 95% sequence identity to SEQ ID NO:6; and the optional fourth nucleic acid sequence encodes a peptide having at least 95% sequence identity to SEQ ID NO:8; 11. The composition of embodiment 10. [Aspect 12] The composition of aspect 10 or 11, comprising first, second, third, and fourth nucleic acid sequences. [Aspect 13] The composition described in Aspect 12, wherein two or more of the first, second, third, and fourth nucleic acid sequences are part of an expression vector, the expression vector comprising a single eukaryotic promoter operably linked to a multiple coding sequence comprising the two or more first, second, third, and fourth nucleic acid sequences, and the multiple coding sequence further comprising an internal ribosome entry site present before each of the two or more first, second, third, and fourth nucleic acid sequences. [Embodiment 14] The composition of embodiment 13, wherein the multiple coding sequence comprises all four of the first, second, third, and optionally fourth nucleic acid sequences, each preceded by an internal ribosome entry site and operably linked to a single eukaryotic promoter. [Aspect 15] The composition of aspect 13 or 14, wherein the first, second, third, and fourth nucleic acid sequences are part of a non-viral vector. [Embodiment 16] A kit for in vivo transfection of postnatal skin tissue to induce the skin tissue to be insulin-producing, the kit comprising: a disposable nanotransfection device; and a reprogramming cocktail comprising a first nucleic acid sequence encoding pancreatic duodenal homeobox 1 (PDX-1), a second nucleic acid sequence encoding the transcription factor MafA, a third nucleic acid sequence encoding the glucagon-like peptide 1 receptor (GLP-1R), and a fourth nucleic acid sequence comprising a nucleic acid sequence encoding fibroblast growth factor 21 (FGF21). The above kit. [Embodiment 17] The kit described in embodiment 16, wherein the nanotransfection device comprises a hollow microneedle array having one or more compartments for receiving a reprogramming cocktail solution.

Claims

1. A composition for reprogramming postnatal skin tissue to produce insulin and C-peptide, comprising: a nucleic acid having a first nucleic acid sequence encoding a pancreatic duodenal homeobox 1 (PDX-1) peptide having the amino acid sequence of SEQ ID NO:2; a nucleic acid having a second nucleic acid sequence encoding a transcription factor MafA peptide having the amino acid sequence of SEQ ID NO: 4; a nucleic acid having a third nucleic acid sequence encoding a glucagon-like peptide 1 receptor (GLP-1R) peptide having the amino acid sequence of SEQ ID NO:6; and a nucleic acid having a fourth nucleic acid sequence encoding a fibroblast growth factor 21 (FGF21) peptide having the amino acid sequence of SEQ ID NO:8; and a DNA comprising: the DNA is delivered into cells of postnatal skin tissue; each of the nucleic acids having the first, second, third, and fourth nucleic acid sequences is operably linked to a control sequence; The above composition.

2. The composition of claim 1, wherein the nucleic acids having the first, second, third, and fourth nucleic acid sequences are each simultaneously delivered to the cytosol of cells of postnatal skin tissue in vivo.

3. 3. The composition of claim 1 or 2, wherein one or more expression vectors are transfected into cells of postnatal skin tissue, and the expression vectors comprise nucleic acids having first, second, third, and fourth nucleic acid sequences.

4. 3. The composition of claim 2, wherein two or more of the nucleic acids having the first, second, third, and fourth nucleic acid sequences are part of an expression vector, the expression vector comprising a single eukaryotic promoter operably linked to multiple coding sequences comprising nucleic acids having the two or more first, second, third, and fourth nucleic acid sequences, the multiple coding sequences further comprising an internal ribosome entry site present before each of the nucleic acids having the two or more first, second, third, and fourth nucleic acid sequences.

5. 5. The composition of claim 4, wherein each of the nucleic acids having the first, second, third, and fourth nucleic acid sequences is located on a single expression vector.

6. The composition of claim 1 , wherein the intracellular delivery is via tissue nanotransfection.

7. The composition of claim 6, wherein the cells are skin cells of skin tissue that are transfected in vivo.

8. A nucleic acid having a first nucleic acid sequence encoding pancreatic duodenal homeobox 1 (PDX-1) having the amino acid sequence of SEQ ID NO: 2; a nucleic acid having a second nucleic acid sequence encoding the transcription factor MafA having the amino acid sequence of SEQ ID NO: 4; a nucleic acid having a third nucleic acid sequence encoding a glucagon-like peptide 1 receptor (GLP-1R) having the amino acid sequence of SEQ ID NO: 6; and a nucleic acid having a fourth nucleic acid sequence comprising a nucleic acid sequence encoding fibroblast growth factor 21 (FGF21) having the amino acid sequence of SEQ ID NO:8; wherein each of the nucleic acids having the first, second, third, and fourth nucleic acid sequences is operably linked to a eukaryotic control sequence for expression in a eukaryotic cell.

9. 9. The composition of claim 8, wherein two or more of the nucleic acids having the first, second, third, and fourth nucleic acid sequences are part of an expression vector, the expression vector comprising a single eukaryotic promoter operably linked to multiple coding sequences comprising nucleic acids having the two or more first, second, third, and fourth nucleic acid sequences, the multiple coding sequences further comprising an internal ribosome entry site present before each of the nucleic acids having the two or more first, second, third, and fourth nucleic acid sequences.

10. 10. The composition of claim 9, wherein the multiple coding sequence comprises all four of the nucleic acids having first, second, third, and fourth nucleic acid sequences, each preceded by an internal ribosome entry site and operably linked to a single eukaryotic promoter.

11. 11. The composition of claim 9 or 10, wherein the nucleic acid having the first, second, third, and fourth nucleic acid sequences is part of a non-viral vector.

12. 1. A kit for in vivo transfection of postnatal skin tissue to induce the skin tissue to be insulin-producing, the kit comprising: a disposable nanotransfection device; and 1. A reprogramming cocktail comprising: a nucleic acid having a first nucleic acid sequence encoding pancreatic duodenal homeobox 1 (PDX-1) having the amino acid sequence of SEQ ID NO:2; a nucleic acid having a second nucleic acid sequence encoding a transcription factor MafA having the amino acid sequence of SEQ ID NO:4; a nucleic acid having a third nucleic acid sequence encoding a glucagon-like peptide 1 receptor (GLP-1R) having the amino acid sequence of SEQ ID NO:6; and a nucleic acid having a fourth nucleic acid sequence comprising a nucleic acid sequence encoding fibroblast growth factor 21 (FGF21) having the amino acid sequence of SEQ ID NO:

8. Including, each of the nucleic acids having the first, second, third, and fourth nucleic acid sequences is operably linked to a control sequence; The above kit.

13. 13. The kit of claim 12, wherein the nanotransfection device comprises a hollow microneedle array having one or more compartments for receiving the reprogramming cocktail solution.

Images