WO2022006551A2 - Modified insulin and glucokinase nucleic acids for treating diabetes - Google Patents

Modified insulin and glucokinase nucleic acids for treating diabetes Download PDF

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Publication number
WO2022006551A2
WO2022006551A2 PCT/US2021/040366 US2021040366W WO2022006551A2 WO 2022006551 A2 WO2022006551 A2 WO 2022006551A2 US 2021040366 W US2021040366 W US 2021040366W WO 2022006551 A2 WO2022006551 A2 WO 2022006551A2
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Prior art keywords
seq
polynucleotide
nucleic acid
sequence
vector
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English (en)
French (fr)
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WO2022006551A3 (en
Inventor
Nachi GUPTA
Weiran SHEN
Miquel Garcia Martinez
Veronica Jimenez Cenzano
Maria Fatima Bosch Tubert
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Universitat Autonoma de Barcelona UAB
Kriya Therapeutics Inc
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Universitat Autonoma de Barcelona UAB
Kriya Therapeutics Inc
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Priority to CN202180052115.3A priority Critical patent/CN116234904A/zh
Priority to KR1020237003717A priority patent/KR20230087436A/ko
Priority to IL299545A priority patent/IL299545A/en
Priority to AU2021300450A priority patent/AU2021300450A1/en
Priority to MX2023000099A priority patent/MX2023000099A/es
Priority to US18/003,980 priority patent/US20240279300A1/en
Priority to EP21833939.8A priority patent/EP4176060A4/en
Priority to JP2023524498A priority patent/JP7837963B2/ja
Application filed by Universitat Autonoma de Barcelona UAB, Kriya Therapeutics Inc filed Critical Universitat Autonoma de Barcelona UAB
Priority to CA3174156A priority patent/CA3174156A1/en
Publication of WO2022006551A2 publication Critical patent/WO2022006551A2/en
Publication of WO2022006551A3 publication Critical patent/WO2022006551A3/en
Anticipated expiration legal-status Critical
Priority to JP2025256145A priority patent/JP2026034616A/ja
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5412—IL-6
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01002—Glucokinase (2.7.1.2)

Definitions

  • Certain aspects of the disclosure are directed to a polynucleotide comprising a nucleic acid encoding a human insulin (Ins) protein, wherein the nucleic acid comprises an open reading frame (ORF) comprising: a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 43-57, 110-122, or 150-159.
  • the polynucleotide comprises at least two nucleic acid sequences encoding a human Ins protein.
  • the encoded human Ins protein comprises a signal sequence and a proinsulin polypeptide.
  • the encoded human Ins protein comprises the amino acid sequence of any of amino acids 25-110 of SEQ ID NO: 41, amino acids 25- 110 of SEQ ID NO: 144, or amino acids 25-110 of SEQ ID NO: 145.
  • the encoded human Ins protein is a preproinsulin.
  • the encoded human Ins protein comprises the amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO: 145.
  • the polynucleotide further comprises a cleavage site (e.g., a furin cleavage site).
  • a cleavage site e.g., a furin cleavage site
  • the one or more amino acid modifications at P52, K53, R55, and/or L86 comprise P52D, K53R, R55K, L86R, or any combination thereof (or the one or more modifications in the proinsulin B-chain or C-chain comprise a proline (P) to aspartic acid (D) at position B28 of the proinsulin B-chain, a lysine (K) to arginine (R) at position B29 of the proinsulin B-chain, arginine (R) to lysine (K) at position Cl of the proinsulin C-chain, leucine (L) to arginine (R) at position C32 of the proinsulin C-chain, or any combination thereof).
  • Certain aspects of the disclosure are directed to a polynucleotide comprising a nucleic acid encoding a human glucokinase (Gck) protein, wherein the nucleic acid comprises an ORF comprising: a nucleotide sequence at least 85%, 86%, 87%, 88%,
  • the polynucleotide or nucleic acid sequence encoding a Gck protein further comprises a 5’ UTR and/or a 3’ UTR.
  • the nucleic acid further comprises a 5’ UTR comprising a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 42, 5-329 of SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148.
  • the nucleic acid further comprises a 3’ UTR comprising a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 60, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, or SEQ ID NO: 149.
  • FIG. 1A shows a listing of nucleic acid sequence constructs including the human insulin (hlns) unmodified nucleic acid sequence (SEQ ID NO: 1, SEQ ID NO: 127, and SEQ ID NO: 160) and modified hlns nucleic acid sequences (SEQ ID NOs: 2-16, 84-88, 123-126, and 128-141).
  • the sequences include 3’ UTR, ORF, and 5’ UTR nucleic acid sequences.
  • Certain sequences also include an IRES sequence.
  • Exemplary pAAV-Ins plasmids transfected into HEK cells are shown in the right-hand column.
  • FIGs. IB and 1C are graphs showing insulin secretion from HEK cells transfected with either 0.5ug/well (FIG. IB) or 0. lug/well (FIG. 1C) pAAV-insulin plasmids.
  • the Insulin expression level of each plasmid was compared with the control plasmid (AAVl-CMV-hInsB10D_2).
  • FIG. 2A shows a listing of nucleic acid sequence constructs including the human glucokinase (hGcK) wild-type nucleic acid sequence (SEQ ID NO: 19 and SEQ ID NO: 163) and modified hGcK nucleic acid sequences (SEQ ID NOs: 20-39 and 89-96).
  • the sequences include 3’ UTR, ORF, and 5’ UTR nucleic acid sequences.
  • Exemplary pAAV- Gck plasmids transfected into HEK cells are shown in the right-hand column.
  • FIG. 2B is a graph showing glucokinase expression in HEK cells transfected with
  • FIG. 3A is assay 1
  • FIG. 3B is assay 2
  • FIG. 3C is assay 3.
  • FIGs. 4A-4C are graphs showing human insulin mRNA expression levels in
  • FIGs. 5A-5C are graphs showing secreted human insulin (mU/L) levels measured after three independent infection studies of 2v6.11 cells with vectors AAV1-CMV- hlnsBlOD (SEQ ID NO: 110), AAV1-CMV-Ins5 (SEQ ID NO: 87) and AAV1-CMV- Ins7 (SEQ ID NO: 88) at three different MOIs (1000 vg/cell (IK), 2000 vg/cell (2K) and 4000 vg/cell (4K)).
  • FIG. 5A is assay 1
  • FIG 5B is assay 2
  • FIG. 5C is assay 3.
  • FIGs. 6A-6C are graphs showing the functionality of secreted human insulin measured after three independent infection studies of 2v6.11 cells with vectors A AAV1- CMV-hlnsBlOD (SEQ ID NO: 110), AAV1-CMV-Ins5 (SEQ ID NO: 87) and AAV1- CMV-Ins7 (SEQ ID NO: 88) at three different MOIs (1000 vg/cell (IK), 2000 vg/cell (2K) and 4000 vg/cell (4K)). Activity is expressed as ng/ml using recombinant human insulin (Life Technologies) as the standard reference.
  • FIG. 6A is assay 1
  • FIG 6B is assay 2
  • FIG. 6C is assay 3.
  • FIGs. 7A-7C are graphs showing intracellular AAVl-hGlucokinase vector genome (vg) quantities in cell extracts of 2v6.11 cells infected with vectors AAV1-CMV- hGckWT (Wild type) (SEQ ID NO: 19), AAV1-CMV-Gck8 (SEQ ID NO: 93) and AAV1-CMV-Gckl2 (SEQ ID NO: 95) at three different MOIs (1000 vg/cell (IK), 2000 vg/cell (2K) and 4000 vg/cell (4K)) in three independent assays.
  • FIG. 7A is assay 1
  • FIG 7B is assay 2
  • FIG. 7C is assay 3.
  • FIGs. 8A-8C are graphs showing human glucokinase mRNA expression levels in
  • FIGs. 10A-10C are graphs showing glucokinase enzymatic activity (mU/mg) measured in cell extracts after three independent infection studies of 2v6.11 cells with vectors AAVl-CMV-hGckWT (Wild type) (SEQ ID NO: 19), AAV1-CMV-Gck8 (SEQ ID NO: 93) and AAV1-CMV-Gckl2 (SEQ ID NO: 95) at three different MOIs (1000 vg/cell (IK), 2000 vg/cell (2K) and 4000 vg/cell (4K)) in three independent assays.
  • FIG. 10A is assay 1
  • FIG 10B is assay 2K
  • FIG. IOC is assay 3.
  • FIG. 12 is a graph showing mean glucose levels under fed or fasted conditions in
  • FIGs. 14A-14D are graphs showing expression levels of insulin in HEK293 cells transfected with pAAV comprising modified insulin nucleic acid sequences.
  • FIG. 15 is a graph showing secreted insulin levels for HEK293 cells transfected with pAAV comprising modified insulin nucleic acid sequences.
  • FIG. 24B levels in control or STZ-treated mice after administration of B10H AAVl- Gck (low dose), B10H AAVl-Gck (high dose), Ins-BIOD AAVl-Gck (low dose), Ins- BIOD AAVl-Gck (mid dose), Ins-B10H+IL6 AAVl-Gck (low dose), Ins-B10H+IL6 AAVl-Gck (high dose), or vehicle controls.
  • polypeptide encompasses both peptides and proteins, unless indicated otherwise.
  • DNA or RNA region (the transcribed region) which “encodes” a particular protein, e.g., such as an insulin or a glucokinase.
  • a coding sequence is transcribed (DNA) and translated (RNA) into a polypeptide, in vitro or in vivo , when placed under the control of an appropriate regulatory region, such as a promoter. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a coding sequence can include, but is not limited to, cDNA from prokaryotes or eukaryotes, genomic DNA from prokaryotes or eukaryotes, and synthetic DNA sequences.
  • a transcription termination sequence can be located 3' to the coding sequence.
  • the transgene allows for the increased expression or over-expression of the gene, e.g., an insulin and/or a glucokinase.
  • the transgene can comprise sequences that are native to the cell, comprise sequences that do not naturally occur in the cell, or it can comprise combinations of both.
  • derived from refers to a component that is isolated from or made using a specified molecule or organism, or information (e.g., amino acid or nucleic acid sequence) from the specified molecule or organism.
  • a nucleic acid sequence e.g., a modified human insulin gene
  • a second nucleic acid sequence e.g., a wild-type human insulin gene
  • mutants, analogs or derivatives can be derived from a wild-type sequence.
  • an "AAV vector” includes a derivative of a known AAV vector.
  • an "AAV vector” includes a modified or an artificial AAV vector.
  • the terms "AAV genome” and "AAV vector” can be used interchangeably.
  • AAV Cap means AAV Cap proteins, VP1, VP2 and VP3 and analogs thereof.
  • the terms "effective amount,” “therapeutically effective amount,” and a “sufficient amount” of, e.g., a gene therapy composition comprising a polynucleotide disclosed herein, refer to a quantity sufficient to, when administered to the subject, including a human, effect beneficial or desired results, including clinical results, and, as such, an "effective amount” or synonym thereto depends on the context in which it is being applied.
  • the amount of a given therapeutic agent or composition will correspond to such an amount will vary depending upon various factors, such as the given agent, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, and/or weight) or host being treated, and the like.
  • the disclosure provides modified nucleic acids encoding wild- type or mutant insulin and/or wild-type glucokinase or a functional fragment thereof.
  • the disclosure also provides nucleic acid constructs that include as part of their sequence the modified nucleic acid(s) encoding wild-type or mutant insulin and/or wild-type glucokinase or a functional fragment thereof.
  • the disclosure includes expression cassettes, plasmids and/or other vectors that include the modified nucleic acid sequence(s) along with other elements, such as regulatory elements.
  • the disclosure also provides gene therapy methods in which the modified nucleic acid sequence(s) encoding wild-type or mutant insulin and/or wild-type glucokinase or a functional fragment thereof is/are administered to a subject, e.g., as a component of one or more vectors and/or packaged as a component of one or more viral gene delivery vehicles. Treatment can, for example, be effected to treat or reduce the symptoms of diabetes in a subject in need thereof.
  • Treatment can, for example, be effected to treat or reduce the symptoms of diabetes in a subject in need thereof.
  • the encoded human Ins protein comprises a signal sequence and a proinsulin polypeptide. In some aspects, the encoded human Ins protein comprises the amino acid sequence of any of amino acids 25-110 of SEQ ID NO: 41, amino acids 25-110 of SEQ ID NO: 144, or amino acids 25-110 of SEQ ID NO: 145. In some aspects, the modified nucleic acid sequence encodes a human preproinsulin (e.g., SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO: 145). In some aspects, the modified nucleic acid sequence encodes a human Gck (e.g., SEQ ID NO: 82).
  • the modified nucleic acids are codon optimized.
  • the codon optimization includes modifying codons in the open reading frame of the nucleic acid encoding insulin or glucokinase.
  • the modified nucleic acids comprise reduced CpG content relative to the corresponding wild-type sequence and/or unmodified sequence.
  • the modified nucleic acid has reduced innate immunogenicity relative to the corresponding wild-type sequence and/or unmodified sequence. In some aspects, the modified nucleic acid has increased expression relative to the corresponding wild-type sequence and/or unmodified sequence. In some aspects, the modified nucleic acid has decreased expression relative to the corresponding wild-type sequence and/or unmodified sequence. In some aspects, the modified sequences are developed through in silico methods followed by manual sequence examination. Nucleic acids of the disclosure can be produced using molecular biology techniques, e.g., modified cDNAs encoding insulin or glucokinase can be obtained by PCR amplification or cDNA cloning techniques.
  • the codon modifications can reduce the immunogenicity of the insulin and/or glucokinase encoding polynucleotides relative to a corresponding wild-type polynucleotide and/or unmodified polynucleotide. In some aspects, the codon modifications improve the expression of the insulin or glucokinase encoding polynucleotide relative to a corresponding wild-type and/or unmodified polynucleotide.
  • the modified nucleic acids of the disclosure can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • the modified nucleic acids can be isolated.
  • a nucleic acid is “isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art, see e.g. F. Ausubel, etal ., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York.
  • a modified nucleic acid of the disclosure can be, for example, DNA or RNA and may or may not contain intron sequences.
  • the nucleic acid can be a cDNA molecule.
  • a polynucleotide or nucleic acid sequence disclosed herein is modified relative to a wild-type (SEQ ID NO: 147) and/or unmodified human insulin (Ins) or a human Ins mutant or analogue (e.g., SEQ ID NO: 110 or SEQ ID NO: 111).
  • the polynucleotide or nucleic acid is modified relative to a sequence including a 5’ UTR, an ORF, and/or a 3’ UTR , e.g., corresponding to SEQ ID NO: 1 or SEQ ID NO: 127.
  • the modified nucleic acid encodes wild-type human insulin (SEQ ID NO: 41), variants or mutants thereof (e.g., SEQ ID NO: 144 or SEQ ID NO: 145) or a functional fragment thereof.
  • Insulin includes two polypeptide chains, the A- and B- chains, linked together by disulfide bonds. It is first synthesized as a single polypeptide called preproinsulin. “Preproinsulin” is the primary translational product of the insulin gene. It is a peptide that is 110 amino acids in length. Preproinsulin includes a proinsulin molecule with a signal peptide attached to its N-terminus. Part of the N-terminus including the signal peptide of the preproinsulin is cleaved off, leaving the remaining amino acids as “proinsulin”.
  • Amino acids 1-30 of the resulting cleaved sequence is the “B chain”, and here “B10” corresponds to position 34 of preproinsulin.
  • a “B10” proinsulin mutation corresponds to a H34 mutation in preproinsulin.
  • “B10H” refers the wild-type histidine amino acid at the B10 position (also referenced as H34 in the wild-type preproinsulin sequence).
  • the preproinsulin and proinsulin also include a C-peptide between the A- and B- chains. In the mature insulin protein, the C-peptide is proteolytically cleaved and the A- and B- chains are linked by disulfide bonds.
  • the modified nucleic acid sequence comprises a cleavage site, e.g., a furin endoprotease cleavage site.
  • the signal sequence of wild-type preproinsulin is replaced with a non-insulin secretion peptide, e.g., an IL-6 signal sequence (e.g, MNSFSTSAFGPVAFSLGLLLVLPAAFPAP (SEQ ID NO: 166)) or a fibronectin signal sequence (e.g,
  • the modified nucleic acid encodes a human insulin comprising an amino acid modification selected from H34D, H34I, or H34V corresponding to wild-type preproinsulin amino acid positions (or a histidine (H) to aspartic acid (D), isoleucine (I) or valine (V) at position B10 of the proinsulin B-chain).
  • the modified nucleic acid encodes a human insulin comprising an amino acid modification H34D corresponding to wild-type preproinsulin amino acid positions (or a histidine (H) to aspartic acid (D) at position B10 of the proinsulin B-chain).
  • the modified nucleic acid encodes a human insulin comprising amino acid modifications K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to the proinsulin lysine (K) to arginine (R) at position B29, arginine (R) to lysine (K) at position Cl, and leucine (L) to arginine (R) at position C32).
  • the modified nucleic acid encodes a human insulin comprising amino acid modifications K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to the proinsulin lysine (K) to arginine (R) at position B29, arginine (R) to lysine (K) at position Cl, and leucine (L) to arginine (R) at position C32), wherein the human insulin optionally comprises a cleavage site, e.g., a furin cleavage site, and a signal peptide (e.g., a wild-type preproinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence).
  • a cleavage site e.g., a furin cleavage site
  • a signal peptide e.g., a wild-type preproinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence
  • the modified nucleic acid encodes a human insulin comprising amino acid modifications H34D, K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to the proinsulin a histidine (H) to aspartic acid (D) at position B10, lysine (K) to arginine (R) at position B29, arginine (R) to lysine (K) at position Cl, and leucine (L) to arginine (R) at position C32).
  • the modified nucleic acid encodes a human insulin comprising amino acid modifications H34D, K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to the proinsulin a histidine (H) to aspartic acid (D) at position B10, lysine (K) to arginine (R) at position B29, arginine (R) to lysine (K) at position Cl, and leucine (L) to arginine (R) at position C32), wherein the human insulin optionally comprises a cleavage site, e.g., a furin cleavage site, and a signal peptide (e.g., a wild-type preproinsulin signal sequence, an IL- 6 signal sequence, or a fibronectin signal sequence).
  • a cleavage site e.g., a furin cleavage site
  • a signal peptide e.g., a wild-type
  • the modified nucleic acid encodes a human insulin comprising amino acid modifications H34I, K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to the proinsulin a histidine (H) to isoleucine (I) at position B10, lysine (K) to arginine (R) at position B29, arginine (R) to lysine (K) at position Cl, and leucine (L) to arginine (R) at position C32).
  • the modified nucleic acid encodes a human insulin comprising amino acid modifications H34V, K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to the proinsulin a histidine (H) to valine (V) at position B10, lysine (K) to arginine (R) at position B29, arginine (R) to lysine (K) at position Cl, and leucine (L) to arginine (R) at position C32).
  • the modified nucleic acid encodes a human insulin comprising amino acid modifications H34V, K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to the proinsulin a histidine (H) to valine (V) at position B10, lysine (K) to arginine (R) at position B29, arginine (R) to lysine (K) at position Cl, and leucine (L) to arginine (R) at position C32), wherein the human insulin optionally comprises a cleavage site, e.g., a furin cleavage site, and a signal peptide (e.g., a wild-type preproinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence).
  • a cleavage site e.g., a furin cleavage site
  • a signal peptide e.g., a wild-type preproinsul
  • the encoded human Ins protein (e.g., a preproinsulin or variant thereof) comprises an amino acid modification selected from (i) H34D, H34I, or H34V (or a histidine (H) to aspartic acid (D), isoleucine (I) or valine (V) at position BIO of the proinsulin B-chain), and/or (ii) one or more amino acid modifications at P52, K53, R55, and/or L86 relative to the wild-type preproinsulin sequence (or positions B28 and/or B29 of the proinsulin B-chain or positions Cl and/or C32 of the proinsulin C-chain).
  • an amino acid modification selected from (i) H34D, H34I, or H34V (or a histidine (H) to aspartic acid (D), isoleucine (I) or valine (V) at position BIO of the proinsulin B-chain), and/or (ii) one or more amino acid modifications at P52, K53, R55, and
  • a polynucleotide of the disclosure comprises an open reading frame (ORF) comprising a nucleic acid sequence having a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to nucleic acids 73-330 of any of SEQ ID NOs: 43-57, 110-116, 150-151, 154-155, and 157-159, nucleic acids 88-345 of any of SEQ ID NOs: 117-122, 152, and 156, or nucleic acids 79-336 of SEQ ID NO: 153.
  • the ORF further comprises a nucleic acid sequence encoding a signal peptide.
  • a polynucleotide of the disclosure comprises an open reading frame (ORF) comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 122.
  • ORF open reading frame
  • a polynucleotide of the disclosure comprises an open reading frame comprising a nucleic acid having the sequence of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 150, SEQ ID NO: 151,
  • a polynucleotide of the disclosure comprises two or more ORFs selected from the group consisting of a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO:
  • one of the two or more ORFs have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 122.
  • the two or more ORFS are selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID
  • ORFs are operably linked by an IRES.
  • the IRES comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 142 or SEQ ID NO: 143.
  • the IRES comprises a nucleic acid sequence of SEQ ID NO: 142 or SEQ ID NO: 143.
  • SEQ ID NO: 16 SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87,
  • a polynucleotide of the disclosure comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 138.
  • a polynucleotide of the disclosure comprises an ORF comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO:
  • the polynucleotide of the disclosure comprises an open reading frame comprising a nucleic acid having the sequence of SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68,
  • SEQ ID NO: 69 SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73,
  • SEQ ID NO: 74 SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78,
  • polynucleotide comprises an ORF sequence present or referenced in Table 2 and/or FIG. 2 A.
  • a polynucleotide of the disclosure further comprises a modified
  • a polynucleotide of the disclosure further comprises a 5’ UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42, nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148.
  • the polynucleotide further comprises a Kozak consensus sequence (Kozak consensus or Kozak sequence).
  • the 5’ UTR comprises a nucleic acid having the sequence of SEQ ID NO: 42, nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148.
  • the polynucleotide comprises a 5’ UTR sequence present or referenced in Table 2 and/or FIG. 2A.
  • a polynucleotide of the disclosure further comprises a modified
  • nucleic acid sequence encodes a human glucokinase protein (e.g., SEQ ID NO: 82) or functional fragment thereof.
  • a nucleic acid construct having a eukaryotic promoter operably linked to a DNA of interest can be used in the disclosure.
  • the constructs containing the DNA sequence (or the corresponding RNA sequence), which can be used in accordance with the disclosure, can be any eukaryotic expression construct containing the DNA or the RNA sequence of interest.
  • a plasmid or viral construct e.g., an AAV vector
  • the construct is capable of replication in both eukaryotic and prokaryotic hosts.
  • the exogenous DNA used in the disclosure is obtained from suitable cells, and the constructs prepared using techniques known in the art.
  • techniques for obtaining expression of exogenous DNA or RNA sequences in a genetically altered host cell are known in the art (see e.g., Kormal et ah, Proc. Natl. Acad. Sci. USA, 84:2150-2154 (1987); Sambrook et al. Molecular Cloning: a Laboratory Manual, 2nd Ed., 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; each of which are hereby incorporated by reference with respect to methods and compositions for eukaryotic expression of a DNA of interest).
  • the DNA construct contains a promoter to facilitate expression of the DNA of interest (e.g., a modified nucleic acid encoding an insulin, a glucokinase, a combination thereof, or a fragment thereof) within a secretory cell.
  • the promoter is a strong, eukaryotic promoter such as a promoter from cytomegalovirus (CMV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RS V), or adenovirus.
  • promoters include, but are not limited to the promoter from the immediate early gene of human CMV (Boshart et al., Cell 41:521-530 (1985) and the promoter from the long terminal repeat (LTR) of RSV (Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777-6781 (1982)).
  • the promoter used can be a tissue-specific promoter.
  • the constructs of the disclosure can also include other components such as a marker (e.g., an antibiotic resistance gene (such as an ampicillin resistance gene) or b- galactosidase) to aid in selection of cells containing and/or expressing the construct, an origin of replication for stable replication of the construct in a bacterial cell (preferably, a high copy number origin of replication), a nuclear localization signal, or other elements which facilitate production of the DNA construct, the protein encoded thereby, or both.
  • a marker e.g., an antibiotic resistance gene (such as an ampicillin resistance gene) or b- galactosidase
  • an origin of replication for stable replication of the construct in a bacterial cell preferably, a high copy number origin of replication
  • a nuclear localization signal e.g., a nuclear localization signal, or other elements which facilitate production of the DNA construct, the protein encoded thereby, or both.
  • the DNA of interest e.g., a modified nucleic acid encoding an insulin, a glucokinase, a combination thereof, or a fragment thereof
  • a construct so that the therapeutic molecule (e.g., a protein) is expressed as a fusion protein (e.g., a fusion protein having b-galactosidase or a portion thereof at the N-terminus and the therapeutic protein at the C-terminal portion).
  • a fusion protein e.g., a fusion protein having b-galactosidase or a portion thereof at the N-terminus and the therapeutic protein at the C-terminal portion.
  • Production of a fusion protein can facilitate identification of transformed cells expressing the protein (e.g., by enzyme-linked immunosorbent assay (ELISA) using an antibody which binds to the fusion protein).
  • ELISA enzyme-linked immunosorbent assay
  • the vectors for delivery of the DNA of interest can be either viral or non-viral, or can be composed of naked DNA admixed with an adjuvant such as viral particles (e.g., AAV particle) or cationic lipids or liposomes.
  • an adjuvant such as viral particles (e.g., AAV particle) or cationic lipids or liposomes.
  • An "adjuvant” is a substance that does not by itself produce the desired effect, but acts to enhance or otherwise improve the action of the active compound. The precise vector and vector formulation used will depend upon several factors such as the cell and/or organ targeted for gene transfer.
  • Suitable promoters include cytomegalovirus (CMV) intermediate early promoter, viral long terminal repeat promoters (LTRs), such as those from murine moloney leukaemia virus (MMLV) rous sarcoma virus, or HTLV-1, the simian virus 40 (SV 40) early promoter, RSV promoter, and the herpes simplex virus thymidine kinase promoter.
  • CMV cytomegalovirus
  • LTRs viral long terminal repeat promoters
  • MMLV murine moloney leukaemia virus
  • HTLV-1 HTLV-1
  • SV 40 simian virus 40
  • RSV promoter herpes simplex virus thymidine kinase promoter
  • the promoter is a cell-specific and/or a tissue-specific promoter.
  • the promoter is used together with an intronic sequence.
  • the promoter is tissue specific.
  • the promoter is a CMV promoter.
  • the expression cassette comprises a promoter operably linked to a modified nucleic acid sequence comprising an ORF having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49,
  • SEQ ID NO: 50 SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54,
  • SEQ ID NO: 55 SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO:
  • SEQ ID NO: 116 SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, or SEQ ID NO: 159, wherein the modified nucleic acid sequence encodes a human insulin protein (e.g., SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO: 145) or a functional fragment thereof.
  • a human insulin protein e.g., SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO: 1405 or a functional fragment thereof.
  • the polynucleotide of the disclosure comprises an open reading frame comprising a nucleic acid having the sequence of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50,
  • SEQ ID NO: 56 SEQ ID NO: 57, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO:
  • the polynucleotide of the disclosure comprises an open reading frame comprising a nucleic acid having the sequence of SEQ ID NO: 122.
  • the polynucleotide comprises an ORF sequence present or referenced in Table 1, Table 13, and/or FIG. 1A
  • the expression cassette comprises a modified nucleic acid further comprising a 5’ UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42, nucleic acids 5-329 of SEQ ID NO: 42, or SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148.
  • the 5’ UTR comprises a nucleic acid having the sequence of SEQ ID NO: 42, nucleic acids 5- 329 of SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148.
  • the polynucleotide comprises a 5’ UTR sequence present or referenced in Table 1 and/or FIG. 1A.
  • the expression cassette comprises a modified nucleic acid further comprising a 3’ UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 60, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, or SEQ ID NO: 149.
  • the 3’ UTR comprises a nucleic acid having the sequence of SEQ ID NO: 60, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, or SEQ ID NO: 149.
  • the 3’ UTR comprises a restriction site selected from the group consisting of BamHl, /xoRI, Ndel, EcoEN, Spe I, Xbal, Nhel, Vspl, Nsil, Seal , Kpnl , Sspl, and Pad , and any combination thereof.
  • the polynucleotide comprises a 3’ UTR sequence present or referenced in Table 1 and/or FIG. 1A
  • the expression cassette comprises a modified nucleic acid having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 138.
  • the expression cassette comprises a modified nucleic acid having the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87 SEQ ID NO: 88, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO
  • the expression cassette comprises a modified nucleic acid having the sequence of SEQ ID NO: 138. In some aspects, the expression cassette comprises a modified nucleic acid having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to nucleic acids 5-957 of a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:
  • the expression cassette comprises a promoter operably linked to a modified nucleic acid sequence comprising an ORF having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67,
  • SEQ ID NO: 73 SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77,
  • SEQ ID NO: 70 SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74,
  • polynucleotide comprises an ORF sequence present or referenced in Table 2 and/or FIG. 2A.
  • the expression cassette comprises a modified nucleic acid further comprising a 5’ UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42, nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148.
  • the 5’ UTR comprises a nucleic acid having the sequence of SEQ ID NO: 42, nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148.
  • the polynucleotide comprises a 5’ UTR sequence present or referenced in Table 2 and/or FIG. 2A.
  • the expression cassette comprises a modified nucleic acid further comprising a 3’ UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 60, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, or SEQ ID NO: 149.
  • the 3’ UTR comprises a nucleic acid having the sequence of SEQ ID NO: 60, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, or SEQ ID NO: 149.
  • the 3’ UTR comprises a restriction site selected from the group consisting of BamHl, EcoBl, Ndel, EcoKV , Spel, Xbal, Nhe I, Vspl, Nsil, Seal, Kpnl, Sspl, and Pad, or any combination thereof.
  • the polynucleotide comprises a 3’ UTR sequence present or referenced in Table 2 and/or FIG. 2A.
  • the expression cassette comprises a modified nucleic acid having the sequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34,
  • SEQ ID NO: 89 SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93,
  • SEQ ID NO: 94 SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 163, or SEQ ID NO:
  • the expression cassette comprises a modified nucleic acid comprising nucleic acids 5-2025 of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27,
  • SEQ ID NO: 28 SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32,
  • SEQ ID NO: 34 SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or
  • the expression cassette comprises modified nucleic acid comprising a 5’ UTR, an ORF, and a 3’ UTR present or referenced in Table 2 and/or FIG. 2 A.
  • the expression cassette comprises a first modified nucleic acid comprising a first ORF having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to
  • SEQ ID NO: 43 SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47,
  • SEQ ID NO: 110 SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, or SEQ ID NO: 159 and a second modified nucleic acid sequence comprising a second ORF having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 9
  • the expression cassette comprises a promoter operably linked to a modified nucleic acid, wherein each the first and second modified nucleic acids are linked to a first and a second promoter, respectively.
  • the first modified nucleic acid sequence comprising a first ORF is selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO:
  • SEQ ID NO: 110 SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, and SEQ ID NO: 159 and the second modified nucleic acid sequence comprising a second ORF is selected from the group consisting of SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO:
  • the first modified nucleic acid sequence encodes a human insulin comprising a wild-type preproinsulin secretion signal peptide. In some aspects, the first modified nucleic acid sequence does not encode a wild-type preproinsulin secretion signal peptide. In some aspects, the wild-type preproinsulin is replaced by a non-insulin secretion signal. In some aspects, the first modified nucleic acid sequence encodes a human preproinsulin comprising an interleukin 6 (IL-6) secretion signal peptide. In some aspects, the first modified nucleic acid sequence encodes a human preproinsulin comprising a fibronectin secretion signal peptide.
  • IL-6 interleukin 6
  • SEQ ID NO: 98 SEQ ID NO 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, or SEQ ID NO: 149.
  • the expression cassette comprises a first modified nucleic acid having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO:
  • SEQ ID NO: 20 SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO:
  • SEQ ID NO: 12 SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 160, or SEQ ID NO: 161 and the second nucleic acid sequence is selected from the group consisting of SEQ ID NO: 20, S
  • the first modified nucleic acid sequence comprises a 5’ UTR, an ORF, and a 3’ UTR present or referenced in Table 1 and/or FIG. 1A and the second modified nucleic acid sequence comprises a 5’ UTR, an ORF, and a 3’ UTR present or referenced in Table 2 and/or FIG. 2A.
  • an expression construct e.g., a vector.
  • the expression construct comprises an expression cassette.
  • the expression construct further comprises a genome that is able to stabilize and remain episomal in a cell.
  • a cell or host cell can encompass a cell used to make the construct or a cell to which the construct is administered.
  • a construct is capable of integrating into a cell's genome, e.g. through homologous recombination or otherwise.
  • the expression construct is one wherein a nucleotide sequence encoding an insulin and/or a glucokinase as disclosed herein, is operably linked to a promoter as provided herein wherein the promoter is capable of directing expression of the nucleotide sequence(s) (i.e. coding sequence(s)) in a cell.
  • an expression cassette as used herein comprises or consists of a nucleotide sequence encoding an insulin and/or a nucleotide sequence encoding a glucokinase, in each case the nucleotide sequence is operably linked to a promoter wherein the promoter is capable of directing expression of said nucleotide sequences.
  • a viral expression construct is an expression construct that is intended to be used in gene therapy. It can be designed to comprise part of a viral genome as disclosed herein.
  • the expression construct further comprises one or more of: an
  • ITR sequence e.g., AAV2 ITRs
  • polyA sequence e.g., a SV40 polyadenylation signal, a bGH polyadenylation signal
  • enhancer sequence e.g., a SV40 enhancer sequence
  • expression constructs disclosed herein are prepared using recombinant techniques in which modified nucleic acid sequences encoding an insulin and/or a glucokinase are expressed in a suitable cell, e.g. cultured cells or cells of a multicellular organism, such as described in Ausubel et al., “Current Protocols in Molecular Biology”, Greene Publishing and Wiley-Interscience, New York (1987) and in Sambrook and Russell (2001, supra); both of which are incorporated herein by reference in their entirety. Also see, Kunkel (1985) Proc. Natl. Acad. Sci. 82:488 (describing site directed mutagenesis) and Roberts et al. (1987) Nature 328:731-734 or Wells, J. A., et al. (1985) Gene 34: 315 (describing cassette mutagenesis).
  • the present disclosure also provides vectors comprising any of the modified nucleic acids, polynucleotides, or expression cassettes described herein.
  • the delivery vector is a viral vector, a non-viral vectors, a plasmid, a lipid, or a lysosome.
  • the delivery vector is a viral vector.
  • the viral vector is an adeno-associated virus (AAV) expression vector.
  • AAV adeno-associated virus
  • a modified nucleic acid or nucleotide sequence encoding an insulin and/or a glucokinase are used in an expression construct or expression vector.
  • expression vector generally refers to a nucleotide sequence that is capable of effecting expression of a gene in a host compatible with such sequences.
  • These expression vectors can include at least suitable promoter sequences and optionally, transcription termination signals. An additional factor necessary or helpful in effecting expression can also be used as disclosed herein.
  • a modified nucleic acid or DNA or codon-optimized nucleotide sequence encoding an insulin and/or a glucokinase can be incorporated into an expression vector capable of introduction into and expression in an in vitro cell culture.
  • the expression vector is suitable for replication in a prokaryotic host, such as bacteria, e.g., E. coli, or can be introduced into a cultured mammalian, plant, insect, (e.g., Sf9), yeast, fungi or other eukaryotic cell lines.
  • a prokaryotic host such as bacteria, e.g., E. coli
  • the expression construct is suitable for expression in vivo.
  • the delivery vector comprises an expression cassette comprises a promoter operably linked to a modified nucleic acid sequence comprising an ORF having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID
  • nucleic acid sequence encodes a human insulin protein (e.g., SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO: 145) or a functional fragment thereof.
  • the delivery vector comprises an expression cassette comprises a promoter operably linked to a modified nucleic acid sequence comprising an ORF having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 122.
  • the modified nucleic acid comprises an ORF having the sequence of SEQ ID NOs: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO:
  • the modified nucleic acid comprises an ORF having the sequence of SEQ ID NO: 122.
  • the polynucleotide comprises an ORF sequence present or referenced in Table 1, Table 13, and/or FIG. 1A.
  • delivery vector comprises an expression cassette comprising a modified nucleic acid sequence encodes a human insulin comprising a wild-type preproinsulin secretion signal peptide.
  • delivery vector comprises an expression cassette comprising a modified nucleic acid sequence does not encode a wild- type preproinsulin secretion signal peptide.
  • the wild-type preproinsulin is replaced by a non-insulin secretion signal.
  • delivery vector comprises an expression cassette comprising a modified nucleic acid sequence encodes a human preproinsulin comprising an interleukin 6 (IL-6) secretion signal peptide.
  • delivery vector comprises an expression cassette comprising a modified nucleic acid sequence encodes a human preproinsulin comprising a fibronectin secretion signal peptide.
  • IL-6 interleukin 6
  • the delivery vector comprises an expression cassette comprising a modified nucleic acid further comprising a 5’ UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, 100% sequence identity to SEQ ID NO: 42, nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148.
  • the delivery vector comprises an expression cassette comprising a modified nucleic acid further comprising a 3’ UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 60, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, or SEQ ID NO: 149.
  • the 3’ UTR comprises a restriction site selected from the group consisting of BamHl, /xoRI, Ndel, EcoKV, Spe I, Xbal, Nhe I, Vspl, Nsil, Seal, Kpnl, Sspl, and Pad, and any combination thereof.
  • the 3’ UTR comprises a nucleic acid having the sequence of SEQ ID NO: 60, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, or SEQ ID NO: 149.
  • the polynucleotide comprises a 3’ UTR sequence present or referenced in Table 1 and/or FIG. 1A.
  • the delivery vector comprises an expression cassette comprising a modified nucleic acid having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 123, SEQ ID NO: 1, SEQ ID NO
  • the delivery vector comprises an expression cassette comprising a modified nucleic acid having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 138.
  • the delivery vector comprises an expression cassette comprising a modified nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to nucleic acids 5-957 of a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.
  • the delivery vector comprises an expression cassette comprising a modified nucleic acid having the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88,
  • the delivery vector comprises an expression cassette comprising a modified nucleic acid having the sequence of SEQ ID NO: 138.
  • the delivery vector comprises an expression cassette comprising a modified nucleic acid comprising nucleic acids 5-957 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:
  • the delivery vector comprises an expression cassette comprising a modified nucleic acid comprising a 5’ UTR, an ORF, and a 3’ UTR present or referenced in Table 1 and/or FIG. 1A.
  • the delivery vector comprises an expression cassette comprising a promoter operably linked to a modified nucleic acid comprising an ORF sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65,
  • SEQ ID NO: 66 SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70,
  • SEQ ID NO: 76 SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, or
  • the delivery vector comprises an expression cassette comprising a promoter operably linked to a modified nucleic acid having the sequence of SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65,
  • SEQ ID NO: 66 SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70,
  • SEQ ID NO: 76 SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, or
  • the polynucleotide comprises an ORF sequence present or referenced in Table 2 and/or FIG. 2A.
  • the delivery vector comprises an expression cassette comprising a modified nucleic acid further comprising a 5’ UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42, nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148.
  • the delivery vector comprises an expression cassette comprising a modified nucleic acid further comprising a 3’ UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 60, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, or SEQ ID NO: 149.
  • SEQ ID NO: 30 SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35,
  • SEQ ID NO: 35 SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39,
  • SEQ ID NO: 94 SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 163, or SEQ ID NO:
  • each the first and second modified nucleic acids are linked to a first and a second promoter, respectively.
  • the first modified nucleic acid sequence comprising a first ORF is selected from the group consisting of SEQ ID NOs: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO:
  • the first modified nucleic acid sequence encodes a human insulin comprising a wild-type preproinsulin secretion signal peptide. In some aspects, the first modified nucleic acid sequence does not encode a wild-type preproinsulin secretion signal peptide. In some aspects, the wild-type preproinsulin is replaced by a non-insulin secretion signal. In some aspects, the first modified nucleic acid sequence encodes a human preproinsulin comprising an interleukin 6 (IL-6) secretion signal peptide. In some aspects, the first modified nucleic acid sequence encodes a human preproinsulin comprising a fibronectin secretion signal peptide.
  • IL-6 interleukin 6
  • the first and second modified nucleic acid sequences further comprise a 5’ UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, 100% sequence identity to SEQ ID NO: 42, nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148.
  • the 5’ UTR comprises a nucleic acid having the sequence of SEQ ID NO: 42, nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148.
  • the first and second modified nucleic acid sequences further comprise a 3’ UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 60, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, or SEQ ID NO: 149.
  • the delivery vector comprises an expression cassette comprising a first modified nucleic acid having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, least 99%, or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 123, SEQ ID NO: 124,
  • the first modified nucleic acid has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to nucleic acids 5-957 of a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:
  • SEQ ID NO: 4 SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO:
  • the first modified nucleic acid sequence comprising nucleic acids 5-957 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16 and the second modified nucleic acid sequence comprising nucleic acids 5-2025 of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO:
  • the first modified nucleic acid sequence comprises a 5’ UTR, an ORF, and a 3’ UTR present or referenced in Table 1 and/or FIG. 1A and the second modified nucleic acid sequence comprises a 5’ UTR, an ORF, and a 3’ UTR present or referenced in Table 2 and/or FIG. 2A.
  • the delivery vectors can comprise sequences encoding a protein
  • modified nucleic acid is placed into a functional relationship with another nucleic acid sequence.
  • regulatory sequence includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the protein. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology, Methods in Enzymology 185, Academic Press, San Diego, CA (1990)).
  • the expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the protein, and are typically appropriate to the host cell used to express the protein.
  • the transcriptional and translational regulatory sequences may include promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
  • expression vectors can contain a selection gene or marker to allow the selection of transformed host cells containing the expression vector. Selection genes are known in the art and will vary with the host cell used.
  • the gene therapy vector includes an Adenoviral and Adeno- associated virus (AAV) vector. These vectors infect a wide number of dividing and non dividing cell types including synovial cells and liver cells. The episomal nature of the adenoviral and AAV vectors after cell entry makes these vectors suited for therapeutic applications. (Russell, 2000, J. Gen. Virol. 81: 2573-2604; Goncalves, 2005, Virol J. 2(1):43) as indicated above. AAV vectors can result in very stable long term expression of transgene expression (up to 9 years in dog (Niemeyer et al, Blood. 2009 Jan. 22;
  • the gene therapy vector includes a retroviral vector.
  • the retroviral vector is a lentiviral based expression construct. Lentiviral vectors have the ability to infect and to stably integrate into the genome of dividing and non dividing cells (Amado and Chen, 1999 Science 285: 674-6). Methods for the construction and use of lentiviral based expression constructs are described in U.S. Pat. Nos. 6,165,782, 6,207,455, 6,218,181, 6,277,633 and 6,323,031 and in Federico (1999, Curr Opin Biotechnol 10: 448-53) and Vigna et al. (2000, J Gene Med 2000; 2: 308-16).
  • the gene therapy vector comprises a modified nucleotide sequence encoding an insulin and/or a glucokinase, whereby each of said modified nucleotide sequence is operably linked to the appropriate regulatory sequences.
  • Such regulatory sequence can at least comprise a promoter sequence.
  • Suitable promoters for expression of a nucleotide sequence encoding an insulin and/or a glucokinase from gene therapy vectors can include e.g.
  • the gene therapy vector includes a further nucleotide sequence coding for a further polypeptide.
  • a further polypeptide can be a (selectable) marker polypeptide that allows for the identification, selection and/or screening for cells containing the expression construct.
  • suitable marker proteins for this purpose are e.g.
  • the fluorescent protein GFP and the selectable marker genes HSV thymidine kinase (for selection on HAT medium), bacterial hygromycin B phosphotransferase (for selection on hygromycin B), Tn5 aminoglycoside phosphotransferase (for selection on G418), and dihydrofolate reductase (DHFR) (for selection on methotrexate), CD20, the low affinity nerve growth factor gene.
  • HSV thymidine kinase for selection on HAT medium
  • bacterial hygromycin B phosphotransferase for selection on hygromycin B
  • Tn5 aminoglycoside phosphotransferase for selection on G418)
  • DHFR dihydrofolate reductase
  • a modified nucleic acid, polynucleotide, or expression construct of the disclosure can be administered using a non-viral vector.
  • Non-viral vector " as used herein is meant to include naked DNA, chemical formulations containing naked DNA (e.g., a formulation of DNA and cationic compounds (e.g., dextran sulfate)), and naked DNA mixed with an adjuvant such as a viral particle (i.e., the DNA of interest is not contained within the viral particle, but the transforming formulation is composed of both naked DNA and viral particles (e.g., AAV particles) (see e.g., Curiel et ah, Am. J. Respir. Cell Mol. Biol. 6:247-52 (1992)).
  • the "non-viral vector” can include vectors composed of DNA plus viral particles where the viral particles do not contain the DNA of interest within the viral genome.
  • a modified nucleic acid, polynucleotide, or expression construct of the disclosure can be complexed with polycationic substances such as poly-L-lysine or DEAC-dextran, targeting ligands, and/or DNA binding proteins (e.g., histones).
  • DNA- or RNA-liposome complex formulations comprise a mixture of lipids which bind to genetic material (DNA or RNA) and facilitate delivery of the nucleic acid into the cell.
  • Liposomes which can be used in accordance with the disclosure include DOPE (dioleyl phosphatidyl ethanol amine), CUDMEDA (N-(5-cholestrum-3-P-ol 3-urethanyl)-N',N'- dimethylethylene diamine).
  • DOPE dioleyl phosphatidyl ethanol amine
  • CUDMEDA N-(5-cholestrum-3-P-ol 3-urethanyl)-N',N'- dimethylethylene diamine.
  • a modified nucleic acid, polynucleotide, or expression construct of the disclosure can also be administered as a chemical formulation of DNA or RNA coupled to a carrier molecule (e.g., an antibody or a receptor ligand) which facilitates delivery to host cells for the purpose of altering the biological properties of the host cells.
  • a carrier molecule e.g., an antibody or a receptor ligand
  • the term "chemical formulations" refers to modifications of nucleic acids to allow coupling of the nucleic acid compounds to a carrier molecule such as a protein or lipid, or derivative thereof.
  • Exemplary protein carrier molecules include antibodies specific to the target cells, i.e., molecules capable of interacting with receptors associated with a cell targeted for delivery.
  • Adeno Associated Virus Vector AAV vector
  • the modified nucleic acids, polynucleotides, or expression constructs of disclosed herein can be administered as a component of a packaged viral vector.
  • packaged viral vectors include a viral vector packaged in a capsid.
  • Part of an AAV genome can contain the inverted terminal repeats (ITR) derived from an adeno-associated virus serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVRH10, AAV11, AAV 12, and others.
  • ITR inverted terminal repeats
  • a vector genome requires the use of flanking 5' and a 3' ITR sequences to allow for efficient packaging of the vector genome into the rAAV capsid.
  • the rAAV genome present in a rAAV vector comprises at least the nucleotide sequences of the inverted terminal repeat regions (ITR) of one of the AAV serotypes (e.g., of serotype AAV2 as disclosed earlier herein), or nucleotide sequences substantially identical thereto, and a modified nucleic acid sequence encoding an insulin and/or a glucokinase under control of a suitable regulatory element (e.g., a promoter), wherein the regulatory element and modified nucleic acid sequence(s) are inserted between the two ITRs.
  • ITR inverted terminal repeat regions
  • the complete genome of several AAV serotypes and corresponding ITR has been sequenced (Chiorini et al. 1999, J. of Virology Vol. 73, No. 2, p 1309-1319). They can be either cloned or made by chemical synthesis as known in the art, using for example an oligonucleotide synthesizer as supplied e.g. by Applied Biosystems Inc. (Fosters, Calif., USA) or by standard molecular biology techniques.
  • the ITRs can be cloned from the AAV viral genome or excised from a vector comprising the AAV ITRs.
  • the ITR nucleotide sequences can be either ligated at either end to the nucleotide sequence encoding one or more therapeutic proteins using standard molecular biology techniques, or the wild type AAV sequence between the ITRs can be replaced with the desired nucleotide sequence.
  • the viral capsid component of the packaged viral vectors can be a parvovirus capsid, e.g., AAV Cap and/or chimeric capsids.
  • suitable parvovirus viral capsid components are capsid components from the family Parvoviridae, such as an autonomous parvovirus or a Dependovirus.
  • the viral capsid may be an AAV capsid (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRH8 AAV 9, AAV10, AAVRH10, AAV11 or AAV12 capsid; one skilled in the art would know there are likely other variants not yet identified that perform the same or similar function), or may include components from two or more AAV capsids.
  • a full complement of AAV Cap proteins includes VP1, VP2, and VP3.
  • the ORF comprising nucleotide sequences encoding AAV VP capsid proteins can comprise less than a full complement AAV Cap proteins or the full complement of AAV Cap proteins can be provided.
  • One or more of the AAV Cap proteins can be a chimeric protein, including amino acid sequences AAV Caps from two or more viruses, preferably two or more AAVs.
  • the chimeric virus capsid can include an AAV1 Cap protein or subunit and at least one AAV2 Cap or subunit.
  • the rAAV genome as present in a rAAV vector does not comprise any nucleotide sequences encoding viral proteins, such as the rep (replication) or cap (capsid) genes of AAV.
  • This rAAV genome may further comprise a marker or reporter gene, such as a gene for example encoding an antibiotic resistance gene, a fluorescent protein (e.g. gfp) or a gene encoding a chemically, enzymatically or otherwise detectable and/or selectable product (e.g. lacZ, aph, etc.) known in the art.
  • suitable 3' untranslated sequence can also be operably linked to the modified nucleic acid sequences encoding an insulin and/or a glucokinase.
  • Suitable 3' untranslated regions can be those naturally associated with the nucleotide sequence or can be derived from different genes, such as for example the bovine growth hormone 3' untranslated region (e.g., bGH polyadenylation signal, SV40 polyadenylation signal,
  • additional nucleotide sequences can be operably linked to the modified nucleic acid sequence(s) encoding an insulin and/or a glucokinase, such as nucleotide sequences encoding signal sequences, nuclear localization signals, expression enhancers, and the like.
  • rAAV parvovirus and AAV
  • packaging vectors expressing the parvovirus Rep and/or Cap sequences transiently and stably transacted packaging cells.
  • Such techniques are known to those skilled in the art. See, e g., SAMBROOK et ah, MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, N.Y., 1989); AUSUBEL el ah, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley Sons, Inc., New York).
  • Lentiviruses are complex retroviruses that in addition to the common retroviral genes gag, pol and env, contain other genes with regulatory or structural function. The higher complexity enables the lentivirus to modulate the life cycle thereof, as in the course of latent infection.
  • a typical lentivirus is the human immunodeficiency virus (HIV), the etiologic agent of AIDS.
  • HIV human immunodeficiency virus
  • MDM monocyte-derived macrophages
  • HeLa-Cd4 human immunodeficiency virus
  • T lymphoid cells arrested in the cell cycle by treatment with aphidicolin or g irradiation.
  • Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (the Psi site). If the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the cis defect prevents encapsidation of genomic RNA. However, the resulting mutant remains capable of directing the synthesis of all virion proteins.
  • the recombinant lentivirus is capable of infecting a non-dividing cell by transfecting a suitable host cell with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat.
  • vectors lacking a functional tat gene are desirable.
  • a first vector can provide a nucleic acid encoding a viral gag and a viral pol and another vector can provide a nucleic acid encoding a viral env to produce a packaging cell.
  • Introducing a vector providing a heterologous gene, identified as a transfer vector, into that packaging cell yields a producer cell which releases infectious viral particles carrying the foreign gene of interest.
  • gag, pol and env genes of the vectors of interest also are known in the art.
  • the relevant genes are cloned into the selected vector and then used to transform the target cell of interest.
  • the second vector can provide a nucleic acid encoding a viral envelope (env) gene.
  • env gene can be derived from any virus, including retroviruses.
  • the env preferably is an amphotropic envelope protein which allows transduction of cells of human and other species.
  • Retroviral vectors can be made target-specific by inserting, for example, a gly colipid or a protein. Targeting often is accomplished by using an antigen-binding portion of an antibody or a recombinant antibody -type molecule, such as a single chain antibody, to target the retroviral vector.
  • retroviral-derived env genes include, but are not limited to: Moloney murine leukemia virus (MoMuLV or MMLV), Harvey murine sarcoma virus (HaMuSV or HSV), murine mammary tumor virus (MuMTV or MMTV), gibbon ape leukemia virus (GaLV or GALV), human immunodeficiency virus (HIV) and Rous sarcoma virus (RSV).
  • Other env genes such as Vesicular stomatitis virus (VSV) protein G (VSV G), that of hepatitis viruses and of influenza also can be used.
  • VSV Vesicular stomatitis virus
  • VSV G Vesicular stomatitis virus
  • the vector providing the viral env nucleic acid sequence is associated operably with regulatory sequences, e.g., a promoter or enhancer.
  • the regulatory sequence can be any eukaryotic promoter or enhancer, including for example, the Moloney murine leukemia virus promoter-enhancer element, the human cytomegalovirus enhancer or the vaccinia P7.5 promoter. In some cases, such as the Moloney murine leukemia virus promoter-enhancer element, the promoter-enhancer elements are located within or adjacent to the LTR sequences.
  • the lentiviral genome as present in said lentiviral vector further comprises a promoter sequence operably linked to the nucleotide sequence encoding an insulin and/or a glucokinase.
  • the promoter sequences are promoters which confer expression in muscle cells and/or muscle tissues. Examples of such promoters include a CMV and a RSV promoters as disclosed herein.
  • suitable 3' untranslated sequence can also be operably linked to the modified nucleic acid sequences encoding an insulin and/or a glucokinase.
  • Suitable 3' untranslated regions can be those naturally associated with the nucleotide sequence or can be derived from different genes, such as for example the bovine growth hormone 3' untranslated region (e.g., bGH polyadenylation signal, SV40 polyadenylation signal,
  • additional nucleotide sequences can be operably linked to the modified nucleic acid sequence(s) encoding an insulin and/or a glucokinase, such as nucleotide sequences encoding signal sequences, nuclear localization signals, expression enhancers, and the like.
  • the present disclosure also provides host cells comprising the modified nucleic acid sequences, polynucleotides, expression cassettes, vectors, or expression constructs disclosed herein.
  • the host cell is a mammalian cell.
  • a DNA sequence that is operably linked are contiguous, and, in the case of a signal sequence, both contiguous and in reading frame.
  • enhancers need not be contiguous with a coding sequence whose transcription they control. Linking is accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof, or by gene synthesis.
  • the selection of an appropriate promoter sequence generally depends upon the host cell selected for the expression of a DNA segment.
  • suitable promoter sequences include prokaryotic, and eukaryotic promoters well known in the art (see, e.g. Sambrook and Russell, 2001, supra).
  • a transcriptional regulatory sequence typically includes a heterologous enhancer or promoter that is recognized by the host.
  • the selection of an appropriate promoter depends upon the host, but promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters are known and available (see, e.g. Sambrook and Russell, 2001, supra).
  • An expression vector includes the replication system and transcriptional and translational regulatory sequences together with the insertion site for the polypeptide encoding segment can be employed. In most cases, the replication system is only functional in the cell that is used to make the vector (bacterial cell as E. Coli). Most plasmids and vectors do not replicate in the cells infected with the vector. Examples of workable combinations of cell lines and expression vectors are described in Sambrook and Russell (2001, supra) and in Metzger et al. (1988) Nature 334: 31-36. For example, suitable expression vectors can be expressed in, yeast, e.g. S.
  • a cell may thus be a prokaryotic or eukaryotic host cell.
  • a cell may be a cell that is suitable for culture in liquid or on solid media.
  • helper functions can include helper virus elements needed for establishing active infection of the packaging cell, which is required to initiate packaging of the viral vector.
  • helper virus elements needed for establishing active infection of the packaging cell, which is required to initiate packaging of the viral vector.
  • examples include functions derived from adenovirus, baculovirus and/or herpes virus sufficient to result in packaging of the viral vector.
  • adenovirus helper functions will typically include adenovirus components Ela, Elb, E2a, E4, and VA RNA.
  • the packaging functions can be supplied by infection of the packaging cell with the required virus.
  • the packaging functions can be supplied together or separately to the packaging cell using a genetic construct such as a plasmid or an amplicon.
  • the packaging functions can exist extrachromosomally within the packaging cell, or can be integrated into the cell's chromosomal DNA.
  • helper virus functions may be employed.
  • packaging cells are insect cells
  • baculovirus can serve as a helper virus.
  • Herpes virus can also be used as a helper virus in AAV packaging methods.
  • any suitable permissive or packaging cell known in the art can be employed in the production of the packaged viral vector.
  • Mammalian cells or insect cells are preferred.
  • Examples of cells useful for the production of packaging cells in the practice of the invention include, for example, human cell lines or primate cells, such as VERO, WI38, MRC5, A549, 293 cells, B-50 or any other HeLa cells, HepG2, Saos-2, HuH7, and HT1080 cell lines.
  • the packaging cells can include one or more viral vector functions along with helper functions and packaging functions sufficient to result in replication and packaging of the viral vector. These various functions can be supplied together or separately to the packaging cell using a genetic construct such as a plasmid or an amplicon, and they can exist extrachromosomally within the cell line or integrated into the cell's chromosomes.
  • the present disclosure also provides pharmaceutical compositions comprising the modified nucleic acid sequences, polynucleotides, expression cassettes, vectors, or expression constructs disclosed herein.
  • a composition comprising an expression construct or a delivery vector (e.g., a viral vector packaged in an AAV capsid) comprising a modified nucleic acid sequence encoding an insulin and/or glucokinase as disclosed herein.
  • a composition is a gene therapy composition.
  • the composition is a pharmaceutical composition said pharmaceutical composition comprising a pharmaceutically acceptable carrier, adjuvant, diluents, solubilizer, filler, preservative and/or excipient.
  • Such pharmaceutically acceptable carrier, filler, preservative, solubilizer, diluent and/or excipient may for instance be found in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins, 2000.
  • the modified nucleic acid, expression construct, delivery vector and/or composition is used for preventing, reducing or ameliorating the symptoms of, delaying, reverting, curing and/or treating a diabetes, when said the modified nucleic acid, expression construct, delivery vector and/or composition is able to exhibit an anti diabetes effect.
  • An anti-diabetes effect can be reached when glucose disposal in blood is increased and/or when glucose tolerance is improved. This can be assessed using techniques known to the skilled person.
  • “increase” means at least a detectable increase (respectively a detectable improvement) using an assay known to the skilled person or using assays as carried out in the experimental part.
  • An anti-diabetes effect can also be observed when the progression of a typical symptom (i.e. insulitis, beta cell loss) has been slowed down as assessed by a physician.
  • a typical symptom i.e. insulitis, beta cell loss
  • a decrease of a typical symptom associated with diabetes can mean a slowdown in progression of symptom development or a complete disappearance of symptoms.
  • Symptoms, and also a decrease in symptoms can be assessed using a variety of methods, to a large extent the same methods as used in diagnosis of diabetes, including clinical examination and routine laboratory tests. Such methods include both macroscopic and microscopic methods, as well as molecular methods, biochemical, immunohistochemical and others.
  • a medicament as defined herein is preferably able to alleviate one symptom or one characteristic of a patient or of a cell, tissue or organ of said diabetes patient if after at least one week, one month, six month, one year or more of treatment using the modified nucleic acid, viral expression construct, viral vector, or composition disclosed herein, said symptom or characteristic is decreased or no longer detectable.
  • a modified nucleic acid, expression construct, delivery vector, or composition as disclosed herein for use in preventing, reducing or ameliorating the symptoms of, delaying, reverting, curing and/or treating a diabetes can be suitable for administration to a cell, tissue and/or an organ in vivo of individuals affected by or at risk of developing a diabetes, and may be administered in vivo, ex vivo or in vitro.
  • Said combination and/or composition can be directly or indirectly administrated to a cell, tissue and/or an organ in vivo of an individual affected by or at risk of developing a diabetes, and may be administered directly or indirectly in vivo, ex vivo or in vitro.
  • the administration mode is intramuscular.
  • the modified nucleic acid, expression construct, delivery vector, or composition as disclosed herein can be directly or indirectly administered using suitable means known in the art.
  • the modified nucleic acid, expression construct, delivery vector, or composition as disclosed herein can be delivered as is to an individual, a cell, tissue or organ of said individual. Depending on the disease or condition, a cell, tissue or organ of said individual may be as earlier defined herein.
  • the modified nucleic acid, expression construct, delivery vector, or composition as disclosed herein is dissolved in a solution that is compatible with the delivery method.
  • the solution may be a physiological salt solution.
  • administration is intramuscular administration.
  • intramuscular administration is carried out using a multineedle.
  • a therapeutically effective dose of the modified nucleic acid, expression construct, the vector, or the composition as described herein is administered in a single and unique dose hence avoiding repeated periodical administration.
  • the single dose is administered to muscle tissue.
  • the single dose is administered to skeletal muscle tissue.
  • the single dose comprise multiple injections (e.g., two, three, four, or five) to one or more muscles (e.g., multiple muscle groups).
  • a compound can be present in a composition of the invention.
  • the compound can help in delivery of the modified nucleic acid or composition comprising the same.
  • the compound is a compound capable of forming complexes, nanoparticles, micelles, liposomes that deliver each constituent as defined herein, complexed or trapped in a vesicle or liposome through a cell membrane, or combinations thereof. Many of these compounds are known in the art.
  • the further compound is polyethylenimine (PEI), or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives, synthetic amphiphiles (SAINT-18), LipofectinTM, DOTAP, or combinations thereof.
  • the present disclosure also provides a method for preventing, reducing or ameliorating the symptoms of, delaying, reverting, curing and/or treating diabetes comprising administering to a subject in need thereof any of a modified nucleic acid, a polynucleotide, an expression cassette, a delivery vectors, or expression construct disclosed herein.
  • the diabetes can be T1DM.
  • the diabetes can be T2DM.
  • the method is a gene therapy.
  • the methods of the disclosure comprise administration (e.g., intramuscular administration) of any of a modified nucleic acid, a polynucleotide, an expression cassette, a delivery vectors, or expression construct disclosed herein to a cell, tissue, or subject in need thereof.
  • the methods comprise adiminstration of (i) a modified (or wild-type or unmodified) nucleic acid, a polynucleotide, an expression cassette, a delivery vectors, or expression construct encoding a human insulin (Ins) protein (e.g., a preproinsulin or variant thereof) and/or (ii) a modified (or wild-type or unmodified) nucleic acid, a polynucleotide, an expression cassette, a delivery vectors, or expression construct comprising a nucleic acid encoding a human glucokinase (Gck) protein.
  • a modified (or wild-type or unmodified) nucleic acid e.g., a preproinsulin or variant thereof
  • Gck human glucokinase
  • Certain aspects of the disclosure are directed methods of use comprising administering a polynucleotide encoding a human insulin (Ins) protein (e.g., a preproinsulin or variant thereof) comprising (i) a nucleotide sequence encoding a signal peptide, optionally wherein the signal peptide is not a wild-type preproinsulin signal sequence, and (ii) a nucleotide sequence encoding a proinsulin polypeptide comprising an amino acid modification at a position selected from amino acid B10, B28, and/or B29 of the human insulin B-chain, Cl and/or C32 of the human insulin C-chain, or any combination thereof relative to the corresponding amino acid position in wild-type proinsulin, and optionally the polynucleotide further comprises a cleavage site.
  • the signal peptide is a wild-type preproinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence.
  • Certain aspects of the disclosure are directed to methods of use comprising administering a polynucleotide comprising a nucleic acid encoding a human insulin (Ins) protein, wherein the nucleic acid comprises an open reading frame (ORF) comprising: (i) a nucleotide sequence encoding a signal peptide and (ii) a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to nucleic acids 73-330 of any of SEQ ID NOs: 43-57, 110-116, 150-151, 154-155, and 157-159, nucleic acids 88-345 of any of SEQ ID NOs: 117-122, 152, and 156, or nucleic acids 79-336 of SEQ ID NO: 153.
  • ORF open reading frame
  • Certain aspects of the disclosure are directed to a method of use comprising administering a polynucleotide comprising a nucleic acid encoding a human insulin (Ins) protein, wherein the nucleic acid comprises an open reading frame (ORF) comprising: a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 43-57, 110-122, or 150-159.
  • the polynucleotide comprises at least two nucleic acid sequences encoding a human Ins protein.
  • the polynucleotide comprises at least two ORF nucleotide sequences at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 43-57, 110-122, or 150-159, wherein the two ORF nucleotide sequences can be the same or different.
  • the polynucleotide further comprises an IRES sequences.
  • the at least two ORF nucleotide sequences are separated by an IRES sequences.
  • the encoded human Ins protein comprises a signal sequence and a proinsulin polypeptide.
  • the encoded human Ins protein comprises the amino acid sequence of any of amino acids 25-110 of SEQ ID NO: 41, amino acids 25-110 of SEQ ID NO: 144, or amino acids 25-110 of SEQ ID NO: 145.
  • the encoded human Ins protein is a preproinsulin.
  • the encoded human Ins protein comprises the amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO: 145.
  • the polynucleotide or nucleic acid sequence further comprises a 5’ UTR and/or a 3’ UTR.
  • the polynucleotide or nucleic acid comprises: a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 1-16, 84-88, 123-141, or 160-161.
  • the proinsulin polypeptide comprises the amino acid sequence of any of amino acids 25-110 of SEQ ID NO: 41, amino acids 25-110 of SEQ ID NO: 144, or amino acids 25-110 of SEQ ID NO: 145.
  • the polynucleotide further comprises a cleavage site (e.g., a furin cleavage site).
  • the nucleic acid further comprises a 3’ UTR comprising a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 60, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, or SEQ ID NO: 149.
  • the polynucleotide or nucleic acid comprises: a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 20-39 and 89-96, and 163-164.
  • the nucleic acid is operably linked to a promoter (e.g., a eukaryotic promoter).
  • a promoter e.g., a eukaryotic promoter
  • Certain aspects of the disclosure are directed to an expression cassette comprising a polynucleotide of the disclosure and a heterologous expression control sequence operably linked to the nucleic acid sequence.
  • the nucleic acid is operably linked to a polyadenylation (poly A) element.
  • Certain aspects of the disclosure are directed to methods of use comprising administering a vector (e.g., viral vector, a non-viral vector, a plasmid, a lipid, or a lysosome) comprising a polynucleotide or an expression cassette of the disclosure.
  • a vector e.g., viral vector, a non-viral vector, a plasmid, a lipid, or a lysosome
  • the vector is an adeno-associated virus (AAV) vector or a Lenti virus vector.
  • rAAV recombinant AAV
  • WO 2012/007458 discloses the generation of two viral vectors, one expressing the insulin gene and one expressing the glucokinase gene as a treatment of diabetes. Furthermore, WO 2016/110518 discloses single-vector gene constructs comprising insulin and glucokinase genes. In certain aspects, the present disclosure provides improved nucleic acid sequences, expression constructs, and/or delivery vectors for diabetes treatment or prevention having increased expression of insulin and/or glucokinase, decreased adverse immune reaction, and/or allowing for administration of a lower dose of viral vector.
  • the methods of the disclosure alleviates or reduces one or more symptom(s) of diabetes in an individual, in a cell, tissue or organ of said individual or alleviates or reduces one or more characteristic(s) or symptom(s) of a cell, tissue or organ of said individual, the method comprising administering to said individual one or more of the modified nucleic acids, polynucleotides, expression cassettes, vectors, or expression constructs disclosed herein.
  • Insulin plays a central role in the regulation of lipid metabolism in liver, adipose, and gut (Verges B. Insulin sensitivity and lipids. Diabetes Metab. 2001 Apr;27(2 Pt 2):223-7. PMID: 11452214.).
  • patients are unable to utilize glucose, requiring an alternative fuel source.
  • insulin inhibits hormone-sensitive lipase normally promoting storage of triglycerides in the adipocytes and reducing release of free fatty acids from adipose tissue in the circulation.
  • lipoprotein catabolism Taskinen MR. Lipoprotein lipase in diabetes.
  • the methods of the disclosure reduce the level of a triglyceride-rich lipoprotein (e.g., chylomicrons or VLDLs) in a subject (e.g., a subject suffering from diabetes), in a cell, tissue or organ of said subject, the method comprising administering to the subject one or more of the modified nucleic acids, polynucleotides, expression cassettes, vectors, or expression constructs disclosed herein.
  • a triglyceride-rich lipoprotein e.g., chylomicrons or VLDLs
  • ketones In the liver, ketone bodies (b-hydroxybutyrate (b-HB) and acetoacetate (AcAc)) are produced by the b-oxidation of fatty acids.
  • b-HB b-hydroxybutyrate
  • AcAc acetoacetate
  • ketones serve as a source of alternative energy in glucose limiting conditions and can provide up to 80% of the brain's energy requirements.
  • a chronic elevation of circulating ketones can produce unwanted effects in the brain, kidney, liver, and microvasculature (Kanikarla-Marie P, Jain SK. Hyperketonemia and ketosis increase the risk of complications in type 1 diabetes. Free Radic Biol Med. 95:268-277, 2016.
  • the methods of the disclosure reduce the level of a ketones in a subject (e.g., a subject suffering from diabetes), in a cell, tissue or organ of said subject, the method comprising administering to the subject one or more of the modified nucleic acids, polynucleotides, expression cassettes, vectors, or expression constructs disclosed herein
  • the methods of the disclosure provide (i) reduction and/or regulation of glycated blood hemoglobin (HbAlc) levels in the subject; (ii) reduction in circulating ketones in the subject, (iii) reduction in triglycerides in the subject, or (iv) any combination thereof.
  • HbAlc glycated blood hemoglobin
  • the method or use is performed in vitro , for instance using a cell culture. In some aspects, the method or use is performed in vivo. In some aspects, a modified nucleic acid, a polynucleotide, an expression cassette, a delivery vectors, or expression construct disclosed herein is combined with an additional compound known to be used for treating diabetes in an individual. In some aspects, the method further comprises administering recombinant insulin, e.g., via regular injections. [0243] In some aspects, the method disclosed herein is not repeated. In some aspects, the method disclosed herein is repeated each year or each 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.
  • the method comprises administering a therapeutically effective dose of the modified nucleic acid, expression construct, the vector, or the composition as described herein, wherein the administration is a single, e.g., avoiding repeated periodical administration.
  • the single dose is administered to muscle tissue.
  • the single dose is administered to skeletal muscle tissue.
  • the single dose comprise multiple injections (e.g., two, three, four, or five) to one or more muscles (e.g., multiple muscle groups).
  • the 5’UTRs having the sequence of SEQ ID NO: 42 was further modified to remove the CTAG at positions 1-4. Accordingly, in certain constructs the 5’UTR included nucleic acids 5-329 of SEQ ID NO: 42. In some constructs, an IRES sequence was added between two insulin ORF sequences, the IRES sequences are shown in BOLD and italicized (SEQ ID NO: 143).
  • the nucleic acids include codon optimized and CpG reduced sequences relative to wild-type and/or unmodified human insulin nucleic acid sequences (e.g., SEQ ID NO: 1).
  • the modified nucleic acids were chemically synthesized, prepared in expression cassettes including a CMV promoter, and cloned into an expression plasmid. The modified sequences were confirmed by Sanger sequencing.
  • AAVl-CMV-hInsB10D_2 (SEQ ID NO: 160), AAVl-CMV-unmodified hlns (SEQ ID NO: 1), AAVl-CMV-modified hlns22 (SEQ ID NO: 123), AAVl-CMV-modified hlns6 (SEQ ID NO: 6), AAVl-CMV-modified hlns8 (SEQ ID NO: 8), AAVl-CMV-modified hlns23 (SEQ ID NO: 124), AAV1-CMV- modified hlns24 (SEQ ID NO: 125), AAVl-CMV-modified hlns25 (SEQ ID NO: 126), AAVl-CMV-modified hlns_2 (SEQ ID NO: 160), AAVl-CMV-unmodified hlns (SEQ ID NO: 1), AAVl-CMV-modified hlns22 (SEQ ID NO
  • the following modified human glucokinase (Gck) nucleic acid sequences (shown in Table 2) corresponding to SEQ ID Nos: 20-39 89-96, and 163-164 were designed in silico.
  • 5’UTR sequences (SEQ ID NO: 42 or SEQ ID NO: 83) are in BOLD, ORF sequences are underlined (SEQ ID NOs: 61-80 and 162), and 3’ UTR are italicized (SEQ ID NO: 60, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, or SEQ ID NO: 109).
  • the 5’UTRs having the sequence of SEQ ID NO: 42 was further modified to remove the CTAG at positions 1-4. Accordingly, in certain constructs the 5’UTR included nucleic acids 5-329 of SEQ ID NO: 42.
  • the nucleic acids include codon optimized and CpG reduced sequences relative to wild-type and/or unmodified human Gck nucleic acid sequences (e.g., SEQ ID NO: 19).
  • the modified nucleic acids were chemically synthesized, prepared in expression cassettes including a CMV promoter, and cloned into an expression plasmid. The modified sequences were confirmed by Sanger sequencing.
  • pAAV-GcK plasmids AAVl-CMV-hGckWT (SEQ ID NO: 19), AAV1-CMV- hGckWT_2 (SEQ ID NO: 163), AAVl-CMV-modified hGck9 (SEQ ID NO: 68), AAV1- CMV-modified hGcklO (SEQ ID NO: 69), AAVl-CMV-modified hGckl 1 (SEQ ID NO: 70), AAV 1 -CMV -modified hGckl2 (SEQ ID NO: 71), AAVl-CMV-modified hGckl3 (SEQ ID NO: 72), AAVl-CMV-modified hGckl4 (SEQ ID NO: 73), AAV1-CMV- modified hGckl5 (SEQ ID NO: 74),
  • AAVl-CMV-hlnsulin vectors bearing insulin variants (SEQ ID NO: 1, SEQ ID NO: 1
  • SEQ ID NO: 87 and SEQ ID NO: 88 were studied for infectivity and potency (insulin mRNA expression, insulin secretion into cell media and biological activity of the secreted insulin).
  • 2v6.11 cells were infected with AAVl-CMV-hlnsWT (SEQ ID NO: 110), AAV1-CMV-Ins5 (SEQ ID NO: 87) and AAV1-CMV-Ins7 (SEQ ID NO: 88) vectors at different MOIs.
  • AAVl-CMV-hlnsWT SEQ ID NO: 110
  • AAV1-CMV-Ins5 SEQ ID NO: 87
  • AAV1-CMV-Ins7 SEQ ID NO: 88
  • a primer-probe mix targeting the SV40 polyA signal was used (Forward primer: AGC AAT AGC ATC ACA AAT TTC ACA A; Reverse primer: CAG ACA TGA TAA GAT ACA TTG ATG AGT T; Probe: /56-FAM/ AGC ATT TTT TT/ZEN/CAC TGC ATT CTA GTT GTG GTT TGT C /3IABkFQ/).
  • a primer-probe mix for housekeeping gene hRplpO was used to normalize (forward primer: CAG ACA GAC ACT GGC AAC AT; Reverse primer: GCA GCA TCT ACA ACC CTG AA;
  • Probe /5HEX/AA CTC TGC A/ZEN/TT CTC GCT TCC TGG A/3IABkFQ).
  • 2v6.11 cells were infected with AAVl-hlnsulin vectors bearing the hlnsulin variants Ins5 and Ins7 and the WT hlnsulin to assess vector infectivity and potency in three independent studies.
  • the levels of secreted insulin mediated by vector AAV1-CMV-Ins7 were significantly lower than those provided by AAVl-CMV-hlnsBlOD (SEQ ID NO: 1) and AAV1-CMV-Ins5 (SEQ ID NO: 87) vectors (FIGs. 5A-5C and Table 6).
  • Statistical analysis was performed with data from infection at 2K and 4K since data from IK was below the lowest standard and could not be quantified.
  • Table 6 Statistical significance (adjusted P value) of secreted hlnsulin for the different AAVl-hlnsulin vectors.
  • Example 4 In vitro comparative analysis of hGlucokinase vectors
  • AAVl-CMV-hGlucokinase vectors bearing the wild-type (SEQ ID NO: 19) and the Gck8 (SEQ ID NO: 93) and Gckl2 (SEQ ID NO: 95) hGlucokinase variants were studied for infectivity and potency (mRNA expression, protein content and biological activity).
  • 2v6.11 cells were infected with AAVl-CMV-hGckWT (SEQ ID NO: 19), AAV 1 -CMV-Gck8 (SEQ ID NO: 93) and AAV1-CMV-Gckl2 (SEQ ID NO: 95) at different MOIs.
  • the infectivity assay the intracellular content of vector genomes was quantified.
  • human glucokinase mRNA expression, glucokinase intracellular content and the glucokinase activity were measured.
  • RNeasy Mini Kit Qiagen
  • RNase-free DNase I Qiagen
  • qPCR was performed in triplicate using Taqman Probes Master (Roche) and 2 m ⁇ of sample (diluted 1/10).
  • a primer-probe mix targeting the SV40 polyA signal was used (Forward primer: AGC AAT AGC ATC AC A AAT TTC AC A A; Reverse primer: CAG ACA TGA TAA GAT ACA TTG ATG AGT T; Probe: /56-FAM/ AGC ATT TTT TT/ZEN/CAC TGC ATT CTA GTT GTG GTT TGT C GIABkFQ/).
  • a primer-probe mix for housekeeping gene hRplpO was used to normalize (forward primer: CAG ACA GAC ACT GGC AAC AT; Reverse primer: GCA GCA TCT ACA ACC CTG AA;
  • Probe /5HEX/AA CTC TGC A/ZEN/TT CTC GCT TCC TGG A/3IABkFQ).
  • hGlucokinase was measured in duplicate (Standard and samples) using the Human glucokinase ELISA Kit (Abeam). Samples were diluted 1/20 using lx diluent N provided by the ELISA kit. Glucokinase content was normalized by total protein content, which was quantified in duplicate in cell extracts by the BCA method using a 1/10 dilution of samples in milliQ water.
  • AAVl-hGlucokinase vectors AAVl-CMV-hGckWT (SEQ ID NO: 19),
  • AAV 1 -CMV-Gck8 (SEQ ID NO: 93) and AAV1-CMV-Gckl2 (SEQ ID NO: 95) showed no consistent significant differences in their ability to infect 2v6.11 cells at the 3 MOIs tested (1000 vg/cell (IK), 2000 vg/cell (2K) and 4000 vg/cell (4K)) as indicated by the intracellular vector genome content (vg/ng DNA) in three different assays (FIGs. 7A- 7C and Table 9)
  • Table 9 Statistical significance (adjusted P value) of the infectivity assays for the different AAVl-hGlucokinase vectors.
  • AAV1-CMV-Gckl2 (SEQ ID NO: 95), containing Glucokinase variant Gckl2, showed a tendency to mediate a lower mRNA expression when compared to AAVl-CMV-hGckWT (SEQ ID NO: 19) and specially AAV1-CMV-Gck8 (SEQ ID NO: 93), although not consistently among assays and MOIs (FIGs. 8A-8C and Table 10)
  • Table 10 Statistical significance (adjusted P value) of hGlucokinase mRNA expression for the different AAVl-hGlucokinase vectors.
  • Table 11 Statistical significance (adjusted P value) of intracellular glucokinase content for the different AAVl-hGlucokinase vectors.
  • preproinsulin variants were designed (shown in Table 13), corresponding to SEQ ID NOs: 150-159.
  • the ORFs encode preproinsulin variants comprising amino acid mutations in the B chain and/or C chain, substitutions in the signal sequence, addition of furin endoprotease cleavage sites, or combinations thereof.
  • each insulin variant was first cloned in AAV plasmids (pAAV) under the control of the miniCMV promoter (pAAV-miniCMV-InsX, where X indicates the specific ORF, i.e ., SEQ ID NO: 150-159).
  • pAAV AAV plasmids
  • pAAV-miniCMV-InsX pAAV-miniCMV-InsX
  • X indicates the specific ORF, i.e ., SEQ ID NO: 150-159.
  • the plasmid name and corresponding ORF sequence are listed in Table 14.
  • AAV expression cassettes were obtained by cloning, between the ITRs of AAV2, the human preproinsulin variants under the control of the miniCMV promoter (pAAV- miniCMV-InsX, where X indicates the specific insulin variant).
  • AAVl-InsX Single-stranded AAV vectors of serotype 1 encoding preproinsulin variants under the control of the miniCMV promoter and human glucokinase under the control of the RSV promoter (AAVl-InsX, where X indicates the specific proinsulin variant) were produced by triple transfection of HEK293 cells according to standard methods (Ayuso,
  • AAV were purified with an optimized method based on a polyethylene glycol precipitation step and two consecutive cesium chloride (CsCl) gradients. This second- generation CsCl-based protocol reduced empty AAV capsids and DNA and protein impurities dramatically (Ayuso, E. et al., 2010. Curr Gene Ther. 10(6):423-36).
  • Purified AAV vectors were dialyzed against PBS, filtered and stored at -80°C. Titers of viral genomes were determined by quantitative PCR following the protocol described for the AAV2 reference standard material using linearized plasmid DNA as standard curve (Lock M, et al., Hum. Gene Ther. 2010; 21:1273-1285). The vectors were constructed according to molecular biology techniques well known in the art.
  • AAV1 vectors encoding the preproinsulin variants [0299] To evaluate biological activity, AAV1 vectors encoding the preproinsulin variants
  • AAVl-Insl, AAV1-Ins3, AAV1-Ins4, AAV1-Ins5 or AAV1-Ins6 were generated and their efficacy to enhance glucose disposal in vivo was assessed in healthy mice.
  • CD1 mice were treated with 3xl0 u viral genomes (vg) of AAV-Ins or AAVl-Null vectors. Mice were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg).
  • TLR9 stimulation was reduced in cells transduced with modified AAV1-GCK constructs including (1) a CMV promoter and modified hGck8 coding sequence (AAVl-hGck8) and (2) a CMV promoter and modified hGckl2 coding sequence (AAVl-hGckl2) compared to wildtype AAV1-GCK control construct including a CMV promoter and hGckWT coding sequence (AAVl-hGckWT) (FIG. 18B).
  • modified AAV1-GCK constructs including (1) a CMV promoter and modified hGck8 coding sequence (AAVl-hGck8) and (2) a CMV promoter and modified hGckl2 coding sequence (AAVl-hGckl2) compared to wildtype AAV1-GCK control construct including a CMV promoter and hGckWT coding sequence (AAVl-hGckWT) (FIG. 18B).
  • TLR9 stimulation was similar among cells transduced with control AAVl-CMV-hInsBIOD and modified AAVl-Ins constructs including (1) a CMV promoter and modified hlns5 coding sequence (AAVl-hIns5) and (2) a CMV promoter and modified hlns7 coding sequence (AAVl-hIns7) (FIG. 18C).
  • Slopes of TLR9 stimulation are provided in Table 15. The results show reduced stimulation of TLR9 with constructs including modified GcK coding sequences and suggest reduced immune activation with the modified constructs.
  • AAV1 vector constructs expressing rat glucokinase and human insulin was evaluated in a streptozotocin-induced model of diabetic C57BL/6J mice following administration of six intramuscular injections.
  • Two cohorts of mice (8-9 weeks of age at initiation of dosing) were obtained.
  • One cohort was administered 5 daily i.p. doses of streptozotocin (50 mg/kg; STZ) to induce diabetes.
  • the second cohort was administered 5 daily i.p. doses of Na-Citrate buffer to serve as a non-diabetic controls.
  • AAV1-926+AAV1-927 (a 1:1 mixture of rat GcK and human Insulin, respectively, which had been previously described in a published study from the Bosch laboratory (Mas et al 2006) and U.S. Patent No. 9,309,534 which is hereby incorporated by reference), was administered as a control to for comparison.
  • Vectors were administered at either high, middle (mid), or low doses.
  • Body weight and non-fasted blood glucose were determined weekly. For consistency, every measurement was made at the same time of day (between 1-3 PM). A fasted blood glucose was determined at week 4 and an oral glucose tolerance test (OGTT) performed at week 8.
  • OGTT oral glucose tolerance test
  • blood samples were collected from all animals to measure blood glucose, mouse and human circulating insulin, Fib Ale and other metabolic parameters. Tissue samples of muscles, liver, and pancreas were collected, weighed, and preserved for evaluation of mRNA, protein levels and protein activity. Baseline values for all mice treated with either STZ or Na Citrate buffer are shown in Table 17.
  • test articles were effective in significantly lowering blood glucose to the level of non-diabetic controls by day 33 (FIG. 19). Once normoglycemia was achieved, the effect was maintained throughout the course of the study. Treatment appeared to protect against weight loss often associated with untreated type 1 diabetes and restored a number of metabolic parameters to levels associated with normoglycemia. Optimal profiles for kinetics and glucose lowering effects were demonstrated with the B10H construct containing the native insulin signal peptide (High Dose) and the B10H construct containing the IL-6 signal peptide (Low Dose).
  • mice injected with hInsBIOD included coding sequence of SEQ ID NO: 110
  • AAV1-GCK AAV926
  • hInsB10H+IL6 included coding sequence of SEQ ID NO:
  • the animals administered hInsBIOH included coding sequence of SEQ ID NO: 111) + AAV1-GCK (AAV926) (high dose) and hInsB10H+IL6 (included coding sequence of SEQ ID NO: 120)+ AAV1-GCK (AAV926) (low dose) both reduced and maintained blood glucose at or near the level of non-Diabetic control mice for the duration of the study. Circulating human insulin values for these mice were 1.33 ⁇ 0.14 and 0.6 ⁇ 0.1 ng/mL respectively (FIGs. 20A and 20B). These data show that the coadministration of hlNS and hGCK to skeletal muscle of STZ-treated mice can effectively control blood glucose when circulating insulin levels are within the range of normal fasting (i.e., basal) levels.
  • the blood glucose of non-STZ controls increased 189 ⁇ 17 mg/dL with a peak at 15 min and return to near-control levels in 90 mins.
  • STZ treated mice increased 264 ⁇ 37 mg/dL with a peak occurring nearer to 30 min and fail to return to control levels even at 120 mins.
  • a goal for Type 1 diabetes is to normalize glycemic control with no change in body weight while preventing diabetic ketoacidosis and medically consequential hypoglycemia. Since Type 1 diabetics have very little or no circulating insulin, they must take insulin every day to stay alive. Further, it has been reported that the hyperglycemia produced by streptozotocin (STZ)-induced diabetes, leads to progressive insulin resistance of the peripheral tissues (Ordonez P, Moreno M, Alonso A, Fernandez R, Diaz F and Gonzalez C. Insulin sensitivity in streptozotocin-induced diabetic rats treated with different doses of 17beta-oestradiol or progesterone. Exp Physiol 92:241-9, 2007. doi: 10.1113/expphysiol.2006.035006. Epub 2006 Oct 26.) The results shown here support that being able to provide an alternative supply of insulin to muscle in addition to improving insulin sensitivity of peripheral tissues provides multiple means to restore glycemic control.
  • HbAlc Glycated blood hemoglobin
  • HbAlc The normal range of HbAlc in non-diabetic mice is 4%-5.6% with diabetes defined as an HbAlc >6.5%. Data from this study are shown in FIG. 23.
  • Administration of STZ produced a significant increase in HbAlc (9.48 ⁇ 0.64%; p ⁇ 0.001) compared to non-diabetic controls.
  • Treatment with vector constructs hInsBIOH + AAV1-GCK (included coding sequence of SEQ ID NO: 111) (AAV926) high dose (4.89 ⁇ 0.15) and hInsB10H+IL6 (included coding sequence of SEQ ID NO: 120) + AAV1-GCK (AAV926) low dose (5.03 ⁇ 0.27%) significantly reduced blood glucose and HbAlc to the level of non diabetic controls.
  • HbAlc is an integrated signal reflecting average glycemia over a period of time; in the case of a mouse, the red blood cell ti/2 is ⁇ 14 days. Clinically, this test is the major tool for assessing glycemic control and has strong predictive value for diabetes and comorbidities.
  • a goal of diabetes therapy is to maintain HbAlc in the normal range ( ⁇ 6.5%), and most marketed products lower HBA1C between 0.5 and 1.25%.
  • chemical induction of type 1 diabetes in mice with STZ increased HbAlc from 4.3 to 9.48 %.
  • IM Injections with AAV1 vectors containing hlNS and rGCK essentially normalized HbAlc to those of non-diabetic controls within 8 wks. Further, these vectors produced a >4% reduction in HbAlc compared to STZ controls. All the groups lowered HbAlc, however only hInsBIOH (included coding sequence of SEQ ID NO: 111) + AAVl-Gck (high dose) and hIns-B10H-IL6 (included coding sequence of SEQ ID NO: 120) + AAVl-Gck (low dose) survived to the 8 week blood draw meaning that the HbAlc levels are the average over the 8 weeks.
  • HbAlc along with the temporal measurements of weekly blood glucose provided a quantitative measure of both an improved post-prandial glucose exposure over a period of time and a reduced degree of glycemic variability suggesting that both factors can be normalized with this treatment.
  • the results support that hlns and GcK AAV constructs co-administered IM to a Type 1 Diabetic patient could represent a single dose reversal of this chronic and debilitating disease.
  • qPCR analysis was used to assess mRNA expression in STZ-induced diabetic mice after administration of AAV-Ins and AAV-Gck constructs.
  • hINS mRNA levels in the liver was assessed to determine whether the AAV-1 vectors escaped the muscle, entered the circulation and transduced the liver, subsequently becoming transcribed at a detectible level. Samples were not measured in non-Diabetic Control or STZ-Control mice since no vectors were injected in those groups. Animals who experienced unexpected deaths or showed distress were not analyzed. The results are shown in Table 18 and FIG. 25. Table 18. Liver Expression of hINS
  • results of this assay show a D Ct of >5 cycles suggesting very low or no hINS mRNA present in the liver of mice administered the three constructs tested (AAV1- mWTIns + AAVl-rGck (AAV926) high dose; AAVl-mWTIns (Ins 17) + AAVl-rGck (AAV926) low dose; and AAVl-mWTIns (Ins 17) + AAVl-rGck (AAV926) high dose) and therefore the IM injected AAVs remain mostly in the target muscles.

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US18/003,980 US20240279300A1 (en) 2020-07-03 2021-07-02 Modified insulin and glucokinase nucleic acids for treating diabetes
CN202180052115.3A CN116234904A (zh) 2020-07-03 2021-07-02 用于治疗糖尿病的经修饰的胰岛素和葡萄糖激酶核酸
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WO2025096954A1 (en) * 2023-11-01 2025-05-08 Kriya Therapeutics, Inc. Aav mediated insulin and glucokinase gene therapy for treating diabetes

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