Modified insulin and glucokinase nucleic acids for treating diabetes

Abstract

Providing modified insulin and glucokinase nucleic acids for treating diabetes SOLUTION: Reducing hyperglycaemia and maintaining euglycaemia are the goals of all therapeutic approaches to the T1DM and T2DM. Current therapy for most diabetics is based on regular subcutaneous injections of both short-acting and long-acting insulin preparations. The present disclosure relates to modified nucleic acid sequences encoding insulin and glucokinase, expression cassettes and delivery vectors comprising the same, and methods for their delivery to treat diabetes.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 047,965, filed July 3, 2020, U.S. Provisional Application No. 63 / 054,162, filed July 20, 2020, U.S. Provisional Application No. 63 / 067,264, filed August 18, 2020, U.S. Provisional Application No. 63 / 141,918, filed January 26, 2021, and U.S. Provisional Application No. 63 / 188,788, filed May 14, 2021, each of which is incorporated by reference in its entirety.

[0002] Reference to an electronically submitted sequence listing The contents of the Sequence Listing submitted with this application, submitted electronically as an ASCII text file (4525_016PC05_Seqlisting_ST25, size: 240,360 bytes, and created on June 30, 2021), are hereby incorporated by reference in their entirety. [Background technology]

[0003] background The two main forms of diabetes mellitus are type 1 (T1DM) and type 2 (T2DM) (Diabetes care, 1997, 20-1183-1197).

[0004] T1DM is characterized by a severe deficiency in insulin production due to the specific destruction of pancreatic beta cells. The loss of beta cells in T1DM is the result of an autoimmune-mediated process in which chronic inflammation, called insulitis, leads to the destruction of beta cells (Eizirik DL et al, 2001, Diabetologia, 44:2115-2133 and Mathis D et al, 2001, Nature, 414: 792-798). T1DM is one of the most common endocrine and metabolic conditions in children. The incidence is rising particularly rapidly among young children. T1DM is diagnosed when autoimmune-mediated beta cell destruction is nearly complete and patients require insulin replacement therapy for survival. T1DM in adults can manifest similarly to T2DM, with a slow deterioration of metabolic control followed by progression to an insulin-dependent state. This form has been termed latent autoimmune diabetes mellitus in adults (LADA) (Diabetes Atlas 4th Edition). edition, 2009, International Diabetes Federation).

[0005] T2DM is the most common form of diabetes mellitus and is thought to result from interactions between genetic, environmental, and behavioral risk factors. T2DM is characterized by insulin insensitivity, reduced insulin production, and eventual pancreatic beta cell failure (Olokoba, A. et al., 2012, Oman Med. J. 27(4):269-273).

[0006] Lifelong insulin treatment is often the therapy of choice for both T1DM and T2DM. Although lifelong exogenous insulin treatment has been largely successful in managing diabetes, diabetic complications can still occur due to the difficulty of maintaining strict glycemic control. Prolonged hyperglycemic states can lead to serious microvascular or macrovascular complications, most commonly manifesting as retinopathy, neuropathy, nephropathy, cerebrovascular accident, or myocardial infarction. These serious complications can be prevented by improving glycemic control. Furthermore, unstable diabetes, particularly the labile form, can be extremely difficult to manage even with lifelong exogenous insulin.

[0007] Furthermore, in many developing countries, access to self-care tools and insulin can be limited, which can lead to significant handicaps and premature death in children with diabetes (Diabetes Atlas 4th edition, 2009, International Diabetes Federation, Beran D. et al 2006, Lancet, 368: 1689-1695, and Gale EA, et al, 2006, Lancet, 368: 1626-1628). Lack of access to insulin is the most common cause of death in children with diabetes worldwide. Therefore, the availability of a one-time gene therapy approach could have a tremendous impact in settings where access to insulin is limited (Greenwood HL et al, 2006, PLoS Med 3.e381). Reducing hyperglycemia and maintaining normoglycemia are the goals of all therapeutic approaches for T1DM and T2DM. Current therapy for most diabetic patients is based on regular subcutaneous injections of both short- and long-acting insulin preparations. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Diabetes care, 1997, 20-1183-1197 [Non-patent document 2] Eizirik DL et al, 2001, Diabetologia, 44:2115-2133 [Non-patent document 3] Mathis D et al, 2001, Nature, 414: 792-798 [Non-patent document 4] Diabetes Atlas 4th edition, 2009, International Diabetes Federation [Non-patent document 5] Olokoba, A. et al, 2012, Oman Med. J. 27(4):269-273 [Non-patent document 6] Beran D. et al 2006, Lancet, 368: 1689-1695 [Non-Patent Document 7] Gale EA, et al, 2006, Lancet, 368: 1626-1628 [Non-patent document 8] Greenwood HL et al, 2006, PLoS Med 3.e381

[0009] Field of Disclosure The present disclosure relates to the medical field, including gene therapy compositions comprising modified nucleic acids encoding insulin and / or glucokinase for use in the treatment of diabetes. Summary of the Invention

[0010] A brief overview Certain aspects of the present disclosure are directed to polynucleotides encoding human insulin (Ins) proteins (e.g., preproinsulin or variants thereof), the polynucleotides comprising: (i) a nucleotide sequence encoding a signal peptide, where optionally, 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 acids B10, B28, and / or B29 of the human insulin B chain, C1 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. In some aspects, the signal peptide is a wild-type preproinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence. In some aspects, the cleavage site is a furin cleavage site.

[0011] Certain aspects of the present disclosure are directed to a polynucleotide comprising a nucleic acid encoding a human insulin (Ins) protein, the nucleic acid comprising 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.

[0012] In some embodiments, the encoded human Ins protein comprises (i) a signal peptide (e.g., a wild-type preproinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence) and (ii) 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 embodiments, the encoded human insulin protein further comprises a cleavage site (e.g., a furin cleavage site).

[0013] Certain embodiments of the present 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. In some embodiments, the polynucleotide comprises at least two nucleic acid sequences encoding a human Ins protein. In some embodiments, 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, and the two ORF nucleotide sequences may be the same or different. In some embodiments, the polynucleotide further comprises an IRES sequence. In some embodiments, the at least two ORF nucleotide sequences are separated by an IRES sequence.

[0014] In some embodiments, the encoded human Ins protein comprises a signal sequence and a proinsulin polypeptide. In some embodiments, 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 embodiments, the encoded human Ins protein is preproinsulin. In some embodiments, the encoded human Ins protein comprises the amino acid sequence of SEQ ID NO:41, SEQ ID NO:144, or SEQ ID NO:145.

[0015] In some embodiments, the polynucleotide or nucleic acid sequence further comprises a 5' UTR and / or a 3' UTR. In some embodiments, 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.

[0016] Certain aspects of the present disclosure are directed to polynucleotides comprising a nucleic acid encoding a human insulin (Ins) protein (e.g., preproinsulin or a variant thereof), the nucleic acid comprising: (i) a nucleotide sequence encoding a signal peptide (e.g., a wild-type preproinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence); and (ii) a nucleotide sequence encoding a proinsulin polypeptide comprising an amino acid modification at a position selected from amino acids B10, B28, and / or B29 of the human insulin B chain, C1 and / or C32 of the human insulin C chain, or any combination thereof, relative to the corresponding amino acid in wild-type proinsulin (or an amino acid modification at a position selected from amino acids H34, P52, K53, R55, L86, or any combination thereof, relative to the corresponding amino acid in wild-type preproinsulin).

[0017] In some embodiments, the signal peptide is not the wild-type preproinsulin signal sequence (e.g., the wild-type preproinsulin sequence is replaced with an IL-6 signal sequence or a fibronectin signal sequence). In some embodiments, 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.

[0018] In some aspects, the polynucleotide further comprises a cleavage site (e.g., a furin cleavage site).

[0019] In some embodiments, the encoded human Ins protein (e.g., preproinsulin or a 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 B10 of the proinsulin B chain); and / or (ii) one or more amino acid modifications at P52, K53, R55, and / or L86 (or at positions B28 and / or B29 of the proinsulin B chain or C1 and / or C32 of the proinsulin C chain) relative to the wild-type preproinsulin sequence. In some embodiments, 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, an arginine (R) to lysine (K) at position C1 of the proinsulin C chain, a leucine (L) to arginine (R) at position C32 of the proinsulin C chain, or any combination thereof).

[0020] Certain embodiments of the present 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%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to nucleic acids 1-1398 of any of SEQ ID NOs: 61-80 or 162, or any of SEQ ID NOs: 61-80 and 162. In some embodiments, the encoded human Gck protein comprises the amino acid sequence of SEQ ID NO: 82.

[0021] In some embodiments, the polynucleotide or nucleic acid sequence encoding the Gck protein further comprises a 5' UTR and / or a 3' UTR. In some embodiments, 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. In some embodiments, 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. In some embodiments, the polynucleotide or nucleic acid comprises a nucleotide sequence that is 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, 89-96, and 163-164.

[0022] In some aspects, the nucleic acid is operably linked to a promoter (e.g., a eukaryotic promoter). Certain aspects of the present disclosure are directed to expression cassettes comprising a polynucleotide of the present disclosure and a heterologous expression control sequence operably linked to the nucleic acid sequence. In some aspects, the nucleic acid is operably linked to a polyadenylation (polyA) element.

[0023] Certain aspects of the present disclosure are directed to vectors (e.g., viral vectors, non-viral vectors, plasmids, lipids, or lysosomes) comprising the polynucleotides or expression cassettes of the present disclosure. In some aspects, the vector is an adeno-associated virus (AAV) vector or a lentiviral vector. Certain aspects of the present disclosure are directed to recombinant AAV (rAAV) particles comprising an AAV capsid and a vector genome comprising the polynucleotides or expression cassettes of the present disclosure. In some aspects, the AAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRH8, AAVrh9, AAV9, AAVrh10, AAV10, AAVRH10, AAV11, and AAV12. Certain aspects of the present disclosure are directed to host cells (e.g., mammalian cells) comprising the polynucleotides, expression cassettes, vectors, or rAAV particles of the present disclosure.

[0024] Certain embodiments of the present disclosure are directed to a method for producing human Ins protein and / or human Gck protein in a subject, comprising administering to the subject a polynucleotide, expression cassette, vector, or rAAV particle of the present disclosure, thereby producing human Ins protein and / or human Gck in the subject. Certain embodiments of the present disclosure are directed to a method for treating or ameliorating symptoms associated with diabetes in a subject in need thereof, comprising delivering a therapeutically effective amount of a polynucleotide, expression cassette, vector, or rAAV particle of the present disclosure to the subject, thereby treating diabetes in the subject. In some embodiments, the diabetes is type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM).

[0025] In some embodiments, the methods disclosed herein include administering to a subject a plurality of polynucleotides, including a first polynucleotide encoding human insulin and a second polynucleotide encoding human Gck; a plurality of expression cassettes, including a first expression cassette comprising a polynucleotide encoding human insulin and a second expression cassette comprising a polynucleotide encoding human Gck; a plurality of vectors, including a first vector comprising an expression cassette comprising a polynucleotide encoding human insulin and a second vector comprising an expression cassette comprising a polynucleotide encoding human Gck; or a plurality of rAAV particles, including a first rAAV particle comprising an expression cassette comprising a polynucleotide encoding human insulin and a second rAAV particle comprising an expression cassette comprising a polynucleotide encoding human Gck. In some embodiments, the plurality of polynucleotides, expression cassettes, vectors, or rAAV particles are administered simultaneously or sequentially. In some embodiments, the delivery and / or administration of the polynucleotides, expression cassettes, vectors, or rAAV particles of the present disclosure is intramuscular. In some aspects, the methods of the present disclosure result in (i) a reduction and / or modulation of blood glycated hemoglobin (HbA1c) levels in a subject, (ii) a reduction in circulating ketones in a subject, (iii) a reduction in triglycerides in a subject, or (iv) any combination thereof. [Brief explanation of the drawings]

[0026] [Figure 1A-1] Figure 1A shows a list of nucleic acid constructs containing the unmodified human insulin (hIns) nucleic acid sequence (SEQ ID NO:1, SEQ ID NO:127, and SEQ ID NO:160) and modified hIns nucleic acid sequences (SEQ ID NOs:2-16, 84-88, 123-126, and 128-141). The sequences include the 3' UTR, ORF, and 5' UTR nucleic acid sequences. Certain sequences also include an IRES sequence. The right column shows exemplary pAAV-Ins plasmids transfected into HEK cells. [Figure 1A-2]Figure 1A shows a list of nucleic acid constructs containing the unmodified human insulin (hIns) nucleic acid sequence (SEQ ID NO:1, SEQ ID NO:127, and SEQ ID NO:160) and modified hIns nucleic acid sequences (SEQ ID NOs:2-16, 84-88, 123-126, and 128-141). The sequences include the 3' UTR, ORF, and 5' UTR nucleic acid sequences. Certain sequences also include an IRES sequence. The right column shows exemplary pAAV-Ins plasmids transfected into HEK cells. [Figure 1A-3] Figure 1A shows a list of nucleic acid constructs containing the unmodified human insulin (hIns) nucleic acid sequence (SEQ ID NO:1, SEQ ID NO:127, and SEQ ID NO:160) and modified hIns nucleic acid sequences (SEQ ID NOs:2-16, 84-88, 123-126, and 128-141). The sequences include the 3' UTR, ORF, and 5' UTR nucleic acid sequences. Certain sequences also include an IRES sequence. The right column shows exemplary pAAV-Ins plasmids transfected into HEK cells.

[0027] [Figure 1B] Figures 1B and 1C are graphs showing insulin secretion from HEK cells transfected with either 0.5 μg / well (Figure 1B) or 0.1 μg / well (Figure 1C) of the pAAV-insulin plasmid. The insulin expression levels of each plasmid were compared with the control plasmid (AAV1-CMV-hInsB10D_2). [Figure 1C] Figures 1B and 1C are graphs showing insulin secretion from HEK cells transfected with either 0.5 μg / well (Figure 1B) or 0.1 μg / well (Figure 1C) of the pAAV-insulin plasmid. The insulin expression levels of each plasmid were compared with the control plasmid (AAV1-CMV-hInsB10D_2).

[0028] [Figure 2A-1]Figure 2A shows a list of nucleic acid constructs containing the wild-type nucleic acid sequence of human glucokinase (hGcK) (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 the 3' UTR, ORF, and 5' UTR nucleic acid sequences. The right column shows an exemplary pAAV-Gck plasmid transfected into HEK cells. [Figure 2A-2] Figure 2A shows a list of nucleic acid constructs containing the wild-type nucleic acid sequence of human glucokinase (hGcK) (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 the 3' UTR, ORF, and 5' UTR nucleic acid sequences. The right column shows an exemplary pAAV-Gck plasmid transfected into HEK cells.

[0029] [Figure 2B] Figure 2B is a graph showing glucokinase expression in HEK cells transfected with 2.5 μg / well of pAAV-Gck plasmid. Gck expression levels for each plasmid were compared with the control plasmid (AAV1-CMV-hGcKWT_2).

[0030] [Figure 3A-3C] Figures 3A-3C are graphs showing the amount of intracellular AAV1-hInsulin vector genome (vg) in cell extracts of 2v6.11 cells infected with vectors AAV1-CMV-hInsB10D-9 (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 (1K), 2000 vg / cell (2K), and 4000 vg / cell (4K)) in three independent studies. Figure 3A is Assay 1, Figure 3B is Assay 2, and Figure 3C is Assay 3.

[0031] [Figures 4A-4C]Figures 4A-4C are graphs showing human insulin mRNA expression levels in 2v6.11 cells infected with vectors AAV1-CMV-hInsB10D (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 (1K), 2000 vg / cell (2K), and 4000 vg / cell (4K)) in three independent studies. Figure 4A is Assay 1, Figure 4B is Assay 2, and Figure 4C is Assay 3.

[0032] [Figures 5A-5C] Figures 5A-5C are graphs showing the levels of secreted human insulin (mU / L) measured after three independent infection studies of 2v6.11 cells with vectors AAV1-CMV-hInsB10D (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 (1K), 2000 vg / cell (2K), and 4000 vg / cell (4K). Figure 5A is Assay 1, Figure 5B is Assay 2, and Figure 5C is Assay 3.

[0033] [Figures 6A-6C] Figures 6A-6C are graphs showing the functionality of secreted human insulin measured after three independent infection studies of 2v6.11 cells with vectors AAV1-CMV-hInsB10D (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 (1K), 2000 vg / cell (2K), and 4000 vg / cell (4K)). Activity is expressed as ng / ml using recombinant human insulin (Life Technologies) as a standard control. Figure 6A is Assay 1, Figure 6B is Assay 2, and Figure 6C is Assay 3.

[0034] [Figure 7A-7C]Figures 7A-7C are graphs showing the amount of intracellular AAV1-human glucokinase (hGlucokinase) vector genome (vg) in cell extracts of 2v6.11 cells infected with the vectors AAV1-CMV-hGckWT (wild-type) (SEQ ID NO: 19), AAV1-CMV-Gck8 (SEQ ID NO: 93), and AAV1-CMV-Gck12 (SEQ ID NO: 95) at three different MOIs (1000 vg / cell (1K), 2000 vg / cell (2K), and 4000 vg / cell (4K)) in three independent assays. Figure 7A is Assay 1, Figure 7B is Assay 2, and Figure 7C is Assay 3.

[0035] [Figures 8A-8C] Figures 8A-8C are graphs showing human glucokinase mRNA expression levels in 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-Gck12 (SEQ ID NO: 95) at three different MOIs (1000 vg / cell (1K), 2000 vg / cell (2K), and 4000 vg / cell (4K)) in three independent assays. Figure 8A is Assay 1, Figure 8B is Assay 2, and Figure 8C is Assay 3.

[0036] [Figures 9A-9C] Figures 9A-9C are graphs showing intracellular glucokinase levels (ng / mg) measured after three independent infection studies of 2v6.11 cells with vectors AAV1-CMV-hGckWT (wild-type) (SEQ ID NO: 19), AAV1-CMV-Gck8 (SEQ ID NO: 93), and AAV1-CMV-Gck12 (SEQ ID NO: 95) at three different MOIs (1000 vg / cell (1K), 2000 vg / cell (2K), and 4000 vg / cell (4K)) in three independent assays. Figure 9A is Assay 1, Figure 9B is Assay 2, and Figure 9C is Assay 3.

[0037] [Figures 10A-10C]Figures 10A-10C are graphs showing glucokinase enzyme activity (mU / mg) measured in cell extracts after three independent infection studies of 2v6.11 cells with vectors AAV1-CMV-hGckWT (wild-type) (SEQ ID NO: 19), AAV1-CMV-Gck8 (SEQ ID NO: 93), and AAV1-CMV-Gck12 (SEQ ID NO: 95) at three different MOIs (1000 vg / cell (1K), 2000 vg / cell (2K), and 4000 vg / cell (4K)) in three independent assays. Figure 10A is Assay 1, Figure 10B is Assay 2, and Figure 10C is Assay 3.

[0038] [Figure 11A] 11A-11C are graphs showing glucose levels for individual C57Blk6 mice injected with AAV1 containing the human insulin gene (SEQ ID NO: 1) and AAV1 containing the rat glucokinase gene under fed or fasted conditions at 3 weeks (FIG. 11A), 4 weeks (FIG. 11B), and 5 weeks (FIG. 11C) after injection. The 4-week time point in FIG. 11B was collected under fasted conditions. [Figure 11B] 11A-11C are graphs showing glucose levels for individual C57Blk6 mice injected with AAV1 containing the human insulin gene (SEQ ID NO: 1) and AAV1 containing the rat glucokinase gene under fed or fasted conditions at 3 weeks (FIG. 11A), 4 weeks (FIG. 11B), and 5 weeks (FIG. 11C) after injection. The 4-week time point in FIG. 11B was collected under fasted conditions. [Figure 11C] 11A-11C are graphs showing glucose levels for individual C57Blk6 mice injected with AAV1 containing the human insulin gene (SEQ ID NO: 1) and AAV1 containing the rat glucokinase gene under fed or fasted conditions at 3 weeks (FIG. 11A), 4 weeks (FIG. 11B), and 5 weeks (FIG. 11C) after injection. The 4-week time point in FIG. 11B was collected under fasted conditions.

[0039] [Figure 12]12 is a graph showing the average glucose levels in C57Blk6 mice injected with AAV1 containing the human insulin gene (SEQ ID NO: 1) and AAV1 containing the rat glucokinase gene under fed or fasted conditions, tested over a 5-week period after injection. These time points were collected under fed conditions except for the 4-week time point, which was collected under fasted conditions.

[0040] [Figures 13A-13B] 13A-13B are graphs showing intracellular insulin content and insulin secretion levels for HEK293 cells transfected with AAV plasmids (pAAV) containing modified insulin nucleic acid sequences.

[0041] [Figures 14A-14B] 14A-14D are graphs showing insulin expression levels in HEK293 cells transfected with pAAV containing a modified insulin nucleic acid sequence. [Figure 14C-14D] 14A-14D are graphs showing insulin expression levels in HEK293 cells transfected with pAAV containing a modified insulin nucleic acid sequence.

[0042] [Figure 15] FIG. 15 is a graph showing insulin secretion levels for HEK293 cells transfected with pAAV containing modified insulin nucleic acid sequences.

[0043] [Figures 16A-16B] 16A-16F are graphs showing blood glucose levels after an oral glucose tolerance test in CD1 mice treated with AAV vectors containing modified insulin nucleic acid sequences. [Figures 16C-16D] 16A-16F are graphs showing blood glucose levels after an oral glucose tolerance test in CD1 mice treated with AAV vectors containing modified insulin nucleic acid sequences. [Figures 16E-16F]16A-16F are graphs showing blood glucose levels after an oral glucose tolerance test in CD1 mice treated with AAV vectors containing modified insulin nucleic acid sequences.

[0044] [Figure 17] 17 is a graph showing fasting glucose levels in healthy mice treated with AAV vectors containing modified insulin nucleic acid sequences. Measurements were taken 3 weeks after AAV administration.

[0045] [Figures 18A-18C] Figures 18A-18C are graphs showing TLR9 stimulation in HEK cells modified to overexpress human TLR9 and then transduced with AAV-ratGck or AAV-hInsB10_2 (Figure 18A); AAV1-hGckWT, AAV1-hGck8, or AAV1-hGck12 (Figure 18B); and AAV1-CMV-hInsB10D, AAV1-hIns5, or AAV1-hIns7 (Figure 18C).

[0046] [Figure 19] 19 is a graph showing mean blood glucose levels over time in an STZ-induced mouse model of type 1 diabetes. Filled circles represent non-STZ + PBS vehicle controls, open circles represent STZ + PBS controls, open triangles represent AAV1_926 + AAV1_927 (high dose), dashed filled triangles represent B10H AAV1-Gck (high dose), filled diamonds represent Ins-B10H + IL6 AAV1-Gck (low dose), solid filled triangles represent B10H AAV1-Gck (medium dose), and open diamonds represent B10H + IL6 AAV1-Gck (high dose).

[0047] [Figure 20A]Figures 20A and 20B are graphs showing circulating human insulin levels (ng / mL) in fasted control or STZ-treated mice 4 weeks after intramuscular (im) administration of AAV1_926+AAV1_927 (high dose), B10D AAV1-Gck2 (low dose), B10H-IL6 AAV1-Gck (low dose), B10H-IL6 AAV1-Gck (high dose), or PBS control. Circulating insulin levels were not determined in animals that died or were euthanized due to poor health / hypoglycemia. The dashed line in Figure 20B represents circulating insulin levels in non-diabetic C57BL / 6 mice under fasting conditions. [Figure 20B] Figures 20A and 20B are graphs showing circulating human insulin levels (ng / mL) in fasted control or STZ-treated mice 4 weeks after intramuscular (im) administration of AAV1_926+AAV1_927 (high dose), B10D AAV1-Gck2 (low dose), B10H-IL6 AAV1-Gck (low dose), B10H-IL6 AAV1-Gck (high dose), or PBS control. Circulating insulin levels were not determined in animals that died or were euthanized due to poor health / hypoglycemia. The dashed line in Figure 20B represents circulating insulin levels in non-diabetic C57BL / 6 mice under fasting conditions.

[0048] [Figure 21] Figure 21 is a graph showing the results of an oral glucose tolerance test in control or STZ-treated mice performed 8 weeks after administration of B10H AAV1-Gck (high dose), Ins-B10H-IL6 AAV1-Gck (low dose), or vehicle control.

[0049] [Figure 22] Figure 22 is a graph showing the area under the curve (AUC) of blood glucose levels calculated from 0 to 120 minutes after a glucose load in control or STZ-treated mice performed 8 weeks after administration of B10H AAV1-Gck (high dose), B10H+IL6 AAV1-Gck (low dose), or vehicle control.

[0050] [Figure 23A] Figures 23A and 23B are graphs showing HbA1c levels in control and STZ-treated mice 8 weeks after administration of B10H AAV1-Gck (high dose), Ins-B10D AAV-Gck (low dose), Ins-B10D AAV-Gck (medium dose), Ins-B10H+IL6 AAV1-Gck (low dose), Ins-B10H+IL6 AAV1-Gck (high dose), or vehicle control (Figure 23A), or B10H-AAV1-Gck (high dose), Ins-B10H+IL6 AAV1-Gck (low dose), or vehicle control (Figure 23B). HbA1c levels were not determined in animals that died or were euthanized due to ill health / hypoglycemia. [Figure 23B] Figures 23A and 23B are graphs showing HbA1c levels in control and STZ-treated mice 8 weeks after administration of B10H AAV1-Gck (high dose), Ins-B10D AAV-Gck (low dose), Ins-B10D AAV-Gck (medium dose), Ins-B10H+IL6 AAV1-Gck (low dose), Ins-B10H+IL6 AAV1-Gck (high dose), or vehicle control (Figure 23A), or B10H-AAV1-Gck (high dose), Ins-B10H+IL6 AAV1-Gck (low dose), or vehicle control (Figure 23B). HbA1c levels were not determined in animals that died or were euthanized due to ill health / hypoglycemia.

[0051] [Figure 24A] Figures 24A-24B are graphs showing serum triglyceride (Figure 24A) or ketone (Figure 24B) levels in control or STZ-treated mice after administration of B10H AAV1-Gck (low dose), B10H AAV1-Gck (high dose), Ins-B10D AAV1-Gck (low dose), Ins-B10D AAV1-Gck (medium dose), Ins-B10H+IL6 AAV1-Gck (low dose), Ins-B10H+IL6 AAV1-Gck (high dose), or vehicle control. [Figure 24B]Figures 24A-24B are graphs showing serum triglyceride (Figure 24A) or ketone (Figure 24B) levels in control or STZ-treated mice after administration of B10H AAV1-Gck (low dose), B10H AAV1-Gck (high dose), Ins-B10D AAV1-Gck (low dose), Ins-B10D AAV1-Gck (medium dose), Ins-B10H+IL6 AAV1-Gck (low dose), Ins-B10H+IL6 AAV1-Gck (high dose), or vehicle control.

[0052] [Figure 25] Figure 25 is a graph showing hINS expression in the liver of STZ-treated mice after administration of AAV1mTWhIns + AAV1rGck (KT1 + AAV926; high dose), AAV1mWTINS + AAV1rGck (INS-17 + AAV926; low dose), or AAV1mWThINS + AAV1rGck (INS-17 + AAV926; high dose). DETAILED DESCRIPTION OF THE INVENTION

[0053] Detailed Description of Disclosure Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present application, including definitions, will control. Unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular.

[0054] Throughout this disclosure, the term "a" or "an" attached to an entity refers to one or more of that entity. For example, "a polynucleotide" is understood to refer to one or more polynucleotides. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0055] Furthermore, when used herein, "and / or" shall be construed as a specific disclosure of each of the two specified features or components, whether or not the other is included. Thus, the term "and / or" used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0056] The term "about" is used herein to mean approximately, in the region of, roughly, or within a range thereof. When used in conjunction with a numerical range, the term "about" modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" is used herein to modify a numerical value above or below (higher or lower) by a variance of 10 percent above or below the stated value, unless otherwise specified.

[0057] The term "at least" preceding a number or series of numbers is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that may be logically included as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the specified property. When "at least" precedes a series of numbers or a range, it is understood that "at least" can modify each number in the series or range. Also, "at least" is not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, and 5.18%, regardless of the number of significant digits).

[0058] Nucleotide sequences are presented herein in a single strand only, from left to right in a 5' to 3' orientation, unless specifically stated otherwise. Nucleotides and amino acids are designated herein by the IUPAC-IUB Biochemical Nomenclature Amino acids are represented either in the manner recommended by the Commission, or (in the case of amino acids) by the one-letter or three-letter code, both in accordance with 37 CFR §1.822 and established usage.

[0059] "Polynucleotide" or "nucleic acid," as used herein, refers to a sequence of nucleotides joined by phosphodiester bonds. Polynucleotides are presented herein in a 5' to 3' orientation. Polynucleotides of the present disclosure can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules. Nucleotide bases are referred to herein by the single-letter code: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I), and uracil (U).

[0060] As used herein, the term "polypeptide" encompasses both peptides and proteins, unless otherwise specified.

[0061] The terms "coding sequence" or "sequence encoding" are used herein to refer to a DNA or RNA region (transcribed region) that "encodes" a particular protein, e.g., insulin or glucokinase. A coding sequence can be transcribed (DNA) and translated (RNA) into a polypeptide, either in vitro or in vivo, when placed under the control of appropriate regulatory regions, 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 prokaryotic or eukaryotic sources, genomic DNA from prokaryotic or eukaryotic sources, and synthetic DNA sequences. A transcription termination sequence can be located 3' to the coding sequence.

[0062] A gene can include several operably linked fragments, such as a promoter, a 5' leader sequence, introns, a coding sequence, and a 3' untranslated sequence, such as a polyadenylation site or signal sequence. As used herein, "expression of a gene" refers to the process by which a gene is transcribed into RNA and / or translated into an active protein.

[0063] As used herein, an open reading frame (ORF) is a portion of a reading frame that has the ability to be translated. An ORF is a continuous stretch of codons that begins with a start codon and ends with a stop codon. In some embodiments, an ORF sequence can be shown or referenced with or without a start codon sequence and / or a stop codon sequence.

[0064] The Kozak consensus sequence, Kozak consensus, or Kozak sequence occurs in eukaryotic mRNA and is known as the sequence having the consensus (gcc)gccRccAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another "G." In some embodiments, a polynucleotide comprises a nucleic acid sequence having at least 95%, at least 99%, or more sequence identity to the Kozak consensus sequence. In some embodiments, a polynucleotide comprises a Kozak consensus sequence.

[0065] The term "sequence identity" is used herein to mean the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In certain embodiments, sequence identity is calculated based on the full length of two given SEQ ID NOs or portions thereof. A portion thereof may mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NOs, or any other specified percentage. The term "identity" can also refer to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences.

[0066] In certain embodiments, methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs.

[0067] When referring to a nucleic acid or a fragment thereof, "substantial homology" or "substantial similarity" means that when optimally aligned with another nucleic acid (or its complementary strand) using appropriate nucleotide insertions or deletions, the sequences share at least about 95-99% nucleotide sequence identity.

[0068] As used herein, and unless otherwise specified, the term "complementary" when used to describe a first nucleic acid sequence in the context of a second nucleic acid sequence refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleic acid sequence to hybridize to an oligonucleotide or polynucleotide comprising the second nucleic acid sequence to form a duplex structure under certain conditions, as understood by those of skill in the art. Such conditions may be, for example, stringent conditions, which may include the following: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C, for 12-16 hours, followed by washing (see, e.g., "Molecular Cloning: A Laboratory Manual," Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). (See, for example, ). Other conditions may be used, such as physiologically relevant conditions that may occur inside an organism. Those skilled in the art will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences according to the ultimate use of the hybridized nucleotides.

[0069] The term "promoter" is used herein to mean a nucleic acid sequence or fragment that functions to control the transcription of one or more genes (or coding sequences), is located upstream of the transcription start site of the gene relative to the direction of transcription, and is structurally distinguished by the presence of any other DNA sequences, including, but not limited to, a DNA-dependent RNA polymerase binding site, a transcription start site, and transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to those skilled in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active under most physiological and developmental conditions. An "inducible" promoter is a promoter that is regulated in response to physiological or developmental conditions. A "tissue-specific" promoter is preferentially active in specific types of differentiated cells / tissues.

[0070] As used herein, the term "enhancer" refers to a cis-acting element that stimulates or inhibits the transcription of adjacent genes. Enhancers that inhibit transcription are also called "silencers." Enhancers can function (e.g., associated with coding sequences) in either direction, over distances of up to several kilobase pairs (kb) from the coding sequence and downstream of the transcribed region.

[0071] The terms "operably linked," "operably inserted," "operably positioned," "under control," or "under transcriptional control" mean that the promoter, in relation to the nucleic acid, is in the correct position and orientation to control RNA polymerase initiation and gene expression. The term "operably linked" means that the DNA sequence and regulatory sequence are connected in such a manner that gene expression is possible when the appropriate molecule (e.g., a transcriptional activator protein) binds to the regulatory sequence. The term "operably inserted" means that a DNA of interest introduced into a cell is positioned adjacent to a DNA sequence that directs the transcription and translation of the introduced DNA (i.e., facilitates the production of a polypeptide encoded by the DNA of interest, for example).

[0072] The term "transgene" is used herein to refer to a gene or nucleic acid molecule that is introduced into a cell. One example of a transgene is a nucleic acid encoding a therapeutic polypeptide (e.g., a gene encoding insulin and / or a gene encoding glucokinase). In some embodiments, a gene may be present in a cell, but in some cases is not normally expressed in the cell or is expressed at an insufficient level in the cell. In this context, "insufficient" means that the gene, e.g., insulin and / or glucokinase, is normally expressed in the cell, but a condition and / or disease disclosed herein (e.g., diabetes) may still occur. In certain aspects, a transgene allows for increased expression or overexpression of a gene, e.g., insulin and / or glucokinase. A transgene may include a sequence native to the cell, a sequence that does not naturally occur in the cell, or a combination of both. In certain embodiments, the transgene can include modified sequences encoding insulin, glucokinase, both insulin and glucokinase, and / or additional proteins, which may be operably linked to appropriate regulatory sequences for expression in the cell of the sequences encoding insulin, glucokinase, or both insulin and glucokinase. In some embodiments, the transgene does not integrate into the genome of the host cell.

[0073] The terms "modified gene," "modified nucleic acid," and the like are used interchangeably herein and refer to the introduction of one or more modifications or changes to the native sequence of a gene or nucleic acid sequence. Such modifications may or may not result in mutations to the encoded protein sequence. In some embodiments, the modified nucleic acid encodes a wild-type or mutant protein sequence or a fragment thereof.

[0074] The term "derived from," as used herein, refers to a component that is isolated from or made using a particular molecule or organism, or information (e.g., amino acid or nucleic acid sequence) from a particular molecule or organism. For example, a nucleic acid sequence (e.g., a modified human insulin gene) derived from a second nucleic acid sequence (e.g., a wild-type human insulin gene) can contain a nucleotide sequence, or a portion thereof, that is identical or substantially similar to the nucleotide sequence of the second nucleic acid sequence. In some aspects, mutants, analogs, or derivatives can be derived from the wild-type sequence.

[0075] In the case of polynucleotides, the derived species can be obtained, for example, by naturally occurring mutagenesis, artificially directed mutagenesis, or artificially random mutagenesis. The mutagenesis used to derive the polynucleotides can be deliberately directed or deliberately random, or a mixture of each.

[0076] As used herein, the term "delivery vector" or "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., that can replicate and transfer genes or nucleic acid sequences between cells when associated with appropriate control elements. Thus, the term includes cloning and expression vehicles and viral vectors. In some aspects, a useful vector is envisioned as a vector in which the nucleic acid segment to be transcribed is placed under the transcriptional control of a promoter. In some aspects, the delivery vector is selected from the group consisting of a viral vector, a plasmid, a lipid, and a lysosome.

[0077] In some aspects, biological vectors include viruses, particularly attenuated and / or replication-defective viruses. In some embodiments, chemical vectors include lipid complexes and naked DNA constructs.

[0078] As used herein, terms such as "naked DNA" or "naked nucleic acid" refer to a nucleic acid molecule that is not contained in a viral particle, bacterial cell, or other encapsulation means that facilitates delivery of the nucleic acid to the cytoplasm of a target cell. Naked nucleic acid may be associated with a means to facilitate delivery of the nucleic acid to the site of the target cell (e.g., to facilitate movement of the nucleic acid through the digestive tract into the target cell, to protect the nucleic acid from stomach acid, and / or to aid in penetration of intestinal mucus), and / or to facilitate delivery of the nucleic acid to the surface of a target epithelial cell.

[0079] "Viral genome" or "vector genome" or "viral vector" refers to a sequence comprising one or more polynucleotide regions that encode or contain a molecule or molecules of interest, such as proteins, peptides, and polynucleotides. Viral vectors are used to deliver genetic material into cells. Viral vectors can be modified for specific uses. In some embodiments, the delivery vector comprises a viral vector selected from the group consisting of an adeno-associated viral (AAV) vector, an adenoviral vector, a lentiviral vector, or a retroviral vector.

[0080] The term "adeno-associated virus vector" or "AAV vector" as used herein refers to any vector that contains or is derived from components of an adeno-associated vector and is suitable for infecting mammalian cells, preferably human cells. The term AAV vector generally refers to an AAV-type virus particle or virion that contains a payload. AAV vectors can be derived from various serotypes, including combinations of serotypes (i.e., "pseudotyped" AAV), or can be derived from various genomes (e.g., single-stranded or self-complementary). Furthermore, AAV vectors can be replication-deficient and / or targeting. As used herein, the term "adeno-associated virus" (AAV) refers to any of AAV types 1, 2, 3 (including types 3A and 3B), 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, AAVrh8, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 11 The term "AAV vector" includes, but is not limited to, the AAV serotypes and clades known in the art, as well as any other AAV. See, for example, FIELDS et al. VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). In some embodiments, "AAV vector" includes derivatives of known AAV vectors. In some embodiments, "AAV vector" includes modified or artificial AAV vectors. The terms "AAV genome" and "AAV vector" can be used interchangeably.

[0081] As used herein, an "AAV particle" is an AAV virus comprising an AAV vector having at least one payload region (e.g., a polynucleotide encoding insulin and / or Gck) and at least one inverted terminal repeat (ITR) region. In some embodiments, the term "AAV vector of the present disclosure" or "AAV vector disclosed herein" refers to an AAV vector comprising a polynucleotide or nucleic acid disclosed herein, for example, encoding insulin, GcK, or a combination thereof, packaged in an AAV particle.

[0082] "Transduction" of a cell by a virus means that a nucleic acid is transferred from the viral particle to the cell. In some embodiments, transduction refers to the delivery of one or more nucleic acids encoding insulin and / or glucokinase to a recipient host cell by a viral vector. For example, transduction of a target cell by an rAAV vector of the present disclosure results in the transfer of the rAAV genome (e.g., comprising a polynucleotide of the present disclosure) contained in the vector into the transduced cell.

[0083] "Transfection" of a cell means the introduction of genetic material into a cell for the purpose of genetically modifying the cell. Transfection can be accomplished by various means known in the art, such as transduction or electroporation.

[0084] "Vector," as used herein, means a recombinant plasmid or virus containing a polynucleotide that is delivered into a host cell either in vitro or in vivo.

[0085] The terms "host cell" or "target cell" are used herein to refer to cells to which polynucleotides are delivered, either in vitro or in vivo. AAV vectors can transduce both dividing and non-dividing cells.

[0086] "Recombinant" means distinguishable from that normally found in nature.

[0087] A "serotype" for a vector or viral capsid is defined by a distinguishable immunological profile based on capsid protein sequence and capsid structure.

[0088] "AAV Cap" refers to the AAV Cap proteins, VP1, VP2, and VP3, and analogs thereof.

[0089] "AAV Rep" refers to the AAV Rep protein and analogs thereof.

[0090] "Flanked" with respect to a sequence flanked by other elements indicates that one or more flanking elements are present upstream and / or downstream, i.e., 5' and / or 3', relative to the sequence. The term "flanked" is not intended to indicate that multiple sequences are necessarily contiguous. For example, there may be intervening sequences between the nucleic acid encoding the transgene and the flanking elements. A sequence (e.g., a transgene) "flanked" by two other elements (e.g., ITRs) indicates that one element is located 5' and the other is located 3' of the sequence, although there may be intervening sequences.

[0091] As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount," e.g., of a gene therapy composition comprising a polynucleotide disclosed herein, refer to an amount sufficient to effect beneficial or desired results, including clinical results, when administered to a subject, including a human; thus, "effective amount" or its synonyms will depend on the context in which it is applied.

[0092] The amount of a given therapeutic agent or composition corresponds to an amount that will vary depending on a variety of factors, such as the given agent, pharmaceutical formulation, route of administration, type of disease or disorder, the personality (e.g., age, sex, and / or weight) of the subject or host being treated, etc.

[0093] As used herein, the term "gene therapy" refers to the insertion of a nucleic acid sequence (e.g., a nucleic acid comprising a promoter operably linked to a polynucleotide encoding a therapeutic molecule as defined herein) into the cells and / or tissues of an individual to treat a disease or condition. Gene therapy also includes the insertion of transgenes that are inhibitory in nature, i.e., transgenes that inhibit, decrease, or reduce the expression, activity, or function of an endogenous gene or protein, such as an unwanted or abnormal (e.g., pathogenic) gene or protein. Such transgenes may be exogenous. An exogenous molecule or sequence is understood to be a molecule or sequence that does not normally occur in the cells, tissues, and / or individual to be treated. Both acquired and congenital diseases may be amenable to gene therapy.

[0094] In some aspects, the present disclosure provides modified nucleic acids encoding wild-type or mutant insulin and / or wild-type glucokinase or functional fragments thereof. The present disclosure also provides nucleic acid constructs that include, as part of their sequence, modified nucleic acids encoding wild-type or mutant insulin and / or wild-type glucokinase or functional fragments thereof. For example, the present disclosure includes expression cassettes, plasmids, and / or other vectors that include modified nucleic acid sequences in conjunction with other elements, such as regulatory elements. In some aspects, the present disclosure provides packaged gene delivery vehicles, such as viral capsids, that include modified nucleic acid sequences encoding wild-type or mutant insulin and / or wild-type glucokinase or functional fragments thereof. The present disclosure also includes methods for expressing wild-type or mutant insulin and / or wild-type glucokinase or functional fragments thereof by delivering modified nucleic acid sequences into cells in conjunction with the necessary elements to promote expression in the cells. The present disclosure also provides a method of gene therapy in which a modified nucleic acid sequence encoding wild-type or mutant insulin and / or wild-type glucokinase or a functional fragment thereof is administered to a subject, for example, as a component of one or more vectors, and / or packaged as a component of one or more viral gene delivery vehicles. Treatment can be performed, for example, to treat or reduce diabetic symptoms in a subject in need thereof. Each of these aspects of the present disclosure is discussed in further detail herein.

[0095] modified nucleic acid In some aspects, the present disclosure provides polynucleotides comprising modified (e.g., codon-optimized and / or reduced CpG content) nucleic acids encoding insulin, glucokinase, or a combination thereof. In some aspects, the modified nucleic acid encodes human insulin (e.g., preproinsulin or proinsulin, or a mutant, analog, or variant thereof). In some aspects, the modified nucleic acid encodes human glucokinase (e.g., Gck, or a mutant, analog, or variant thereof). In some aspects, modifications to the coding sequence preserve the wild-type or mutant amino acid sequence of insulin and / or glucokinase. In some aspects, the encoded human Ins protein comprises a signal sequence and a proinsulin polypeptide. In some aspects, the encoded human Ins protein comprises any of the amino acid sequences 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 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 human Gck (eg, SEQ ID NO: 82).

[0096] In some embodiments, the modified nucleic acid is codon-optimized. In some embodiments, the codon optimization comprises modifying codons in the open reading frame of the nucleic acid encoding insulin or glucokinase. In some embodiments, the modified nucleic acid comprises a reduced CpG content relative to the corresponding wild-type sequence and / or unmodified sequence.

[0097] In some embodiments, the modified nucleic acids have reduced natural immunogenicity relative to the corresponding wild-type and / or unmodified sequences. In some embodiments, the modified nucleic acids have increased expression relative to the corresponding wild-type and / or unmodified sequences. In some embodiments, the modified nucleic acids have decreased expression relative to the corresponding wild-type and / or unmodified sequences. In some embodiments, the modified sequences are developed by in silico methods followed by manual sequence inspection. The nucleic acids of the present disclosure can be produced using molecular biology techniques; for example, modified cDNAs encoding insulin or glucokinase can be obtained by PCR amplification or cDNA cloning techniques.

[0098] In some aspects, nucleic acid sequence is modified to reduce CpG content, for example, to minimize inflammatory response through TLR9 dimerization and related pathways.In some aspects, certain CpG motifs inhibit or neutralize their inflammatory effects.In some embodiments, one or more of these motifs can be conserved.In some aspects, such CpG motifs can be introduced into nucleic acid sequence to inhibit the downstream effect of TLR9 dimerization.

[0099] In some aspects, codon modifications may reduce the immunogenicity of a polynucleotide encoding insulin and / or glucokinase relative to the corresponding wild-type and / or unmodified polynucleotide. In some aspects, codon modifications improve expression of a polynucleotide encoding insulin or glucokinase relative to the corresponding wild-type and / or unmodified polynucleotide. In some aspects, codon modifications may reduce the immunogenicity of a polynucleotide encoding glucokinase relative to the corresponding wild-type and / or unmodified Gck polynucleotide.

[0100] The modified nucleic acids of the present disclosure may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. The modified nucleic acids may be isolated. A nucleic acid is "isolated" or "substantially purified" when purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art (see, e.g., F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York). In some embodiments, the modified nucleic acids of the present disclosure may be, for example, DNA or RNA, and may or may not contain intronic sequences. In some embodiments, the nucleic acid may be a cDNA molecule.

[0101] Modified insulin nucleic acid In some aspects, the polynucleotide or nucleic acid sequences disclosed herein are modified relative to wild-type (SEQ ID NO: 147) and / or unmodified human insulin (Ins) or a human Ins mutant or analog (e.g., SEQ ID NO: 110 or SEQ ID NO: 111). In some aspects, the polynucleotide or nucleic acid is modified relative to a sequence including the 5' UTR, ORF, and / or 3' UTR, e.g., corresponding to SEQ ID NO: 1 or SEQ ID NO: 127. In some aspects, the modified nucleic acid encodes wild-type human insulin (SEQ ID NO: 41), a variant or mutant thereof (e.g., SEQ ID NO: 144 or SEQ ID NO: 145), or a functional fragment thereof.

[0102] Insulin contains two polypeptide chains, the A chain and the B chain, linked together by a disulfide bond. It is initially synthesized as a single polypeptide called preproinsulin. "Preproinsulin" is the primary translation product of the insulin gene. It is a 110-amino acid-long peptide. Preproinsulin contains a proinsulin molecule with a signal peptide attached to its N-terminus. A portion of the N-terminus of preproinsulin, including the signal peptide, is cleaved, leaving the remaining amino acids as "proinsulin." Amino acids 1-30 of the resulting cleaved sequence are the "B chain," where "B10" corresponds to position 34 of preproinsulin. Thus, for example, a "B10" proinsulin mutation corresponds to the H34 mutation in preproinsulin. In certain embodiments, as referred to herein, "B10H" refers to the wild-type histidine amino acid at position B10 (also referred to as H34 in the wild-type preproinsulin sequence). Preproinsulin and proinsulin also contain a C-peptide between the A chain and the B chain. In the mature insulin protein, the C-peptide is proteolytically cleaved and the A and B chains are linked by a disulfide bond.

[0103] In some aspects, the modified coding sequences disclosed herein encode preproinsulin mutants comprising one or more mutations at positions H34, P52, K53, R55, and / or L86 relative to the corresponding positions in wild-type preproinsulin (SEQ ID NO: 41). In some aspects, the modified coding sequences encode preproinsulin mutants comprising one or more of the mutations H34D, H34I, H34V, P52D, K53R, R55K, and / or L86R relative to the corresponding positions in SEQ ID NO: 41. In some aspects, the modified coding sequences encode preproinsulin mutants comprising the mutations H34D, H34I, H34V, P52D, K53R, R55K, and / or L86R relative to the corresponding positions in SEQ ID NO: 41. In some embodiments, the modified coding sequence encodes a preproinsulin mutant comprising the mutations P52D, K53R, R55K, and / or L86R relative to the corresponding positions in SEQ ID NO: 41. In some embodiments, the modified coding sequence encodes an amino acid sequence that is at least 90%, 95%, 99% or 100% similar to an amino acid sequence selected from SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO: 145. In some embodiments, the modified coding sequence encodes an amino acid sequence that is at least 90%, 95%, 99% or 100% similar to an amino acid sequence selected from SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO: 145, wherein the amino acid sequence comprises one or more of the mutations H34D, H34I, H34V, P52D, K53R, R55K, and / or L86R relative to the corresponding positions in SEQ ID NO: 41. In some embodiments, the modified coding sequence encodes an amino acid sequence that does not comprise the H34 mutation relative to the corresponding positions in SEQ ID NO: 41.

[0104] In some aspects, the modified nucleic acid sequence comprises a cleavage site, for example, a furin endoprotease cleavage site.

[0105] In some embodiments, the modified nucleic acid sequence comprises a nucleic acid encoding a signal peptide (e.g., a wild-type preproinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence). In some embodiments, the preproinsulin comprises a wild-type insulin signal sequence (e.g., MALWMRLLPLLALLALWGPDPAAA (SEQ ID NO: 165) or amino acids 1-24 of SEQ ID NO: 41). In some embodiments, the signal sequence of wild-type preproinsulin has been replaced with a non-insulin-secreting peptide, e.g., an IL-6 signal sequence (e.g., MNSFSTSAFGPVAFSLGLLLVLPAAFPAP (SEQ ID NO: 166)) or a fibronectin signal sequence (e.g., MLRGPGPGLLLLAVQCLGTAVPSTGA (SEQ ID NO: 167)).

[0106] In some embodiments, the modified nucleic acid encodes human insulin comprising an amino acid modification selected from H34D, H34I, or H34V corresponding to the wild-type preproinsulin amino acid position (or a histidine (H) to aspartic acid (D), isoleucine (I), or valine (V) at position B10 of the proinsulin B chain). In some embodiments, the modified nucleic acid encodes human insulin comprising an amino acid modification H34D corresponding to the wild-type preproinsulin amino acid position (or a histidine (H) to aspartic acid (D) at position B10 of the proinsulin B chain). In some embodiments, the modified nucleic acid encodes human insulin containing an amino acid modification selected from H34D, H34I, or H34V corresponding to the 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), which optionally includes a cleavage site, e.g., a furin cleavage site, and a signal peptide (e.g., the wild-type preproinsulin signal sequence, the IL-6 signal sequence, or the fibronectin signal sequence).

[0107] In some embodiments, the modified nucleic acid encodes human insulin containing the amino acid modifications K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to lysine (K) to arginine (R) at position B29, arginine (R) to lysine (K) at position C1, and leucine (L) to arginine (R) at position C32 of proinsulin). In some embodiments, the modified nucleic acid encodes human insulin containing the amino acid modifications K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to lysine (K) to arginine (R) at position B29, arginine (R) to lysine (K) at position C1, and leucine (L) to arginine (R) at position C32 of proinsulin), optionally including a cleavage site, e.g., a furin cleavage site, and a signal peptide (e.g., the wild-type preproinsulin signal sequence, the IL-6 signal sequence, or the fibronectin signal sequence).

[0108] In some embodiments, the modified nucleic acid encodes human insulin containing the amino acid modifications H34D, K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to histidine (H) to aspartic acid (D) at position B10, lysine (K) to arginine (R) at position B29, arginine (R) to lysine (K) at position C1, and leucine (L) to arginine (R) at position C32 of proinsulin). In some embodiments, the modified nucleic acid encodes human insulin including the amino acid modifications H34D, K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to histidine (H) to aspartic acid (D) at position B10, lysine (K) to arginine (R) at position B29, arginine (R) to lysine (K) at position C1, and leucine (L) to arginine (R) at position C32 of proinsulin), which optionally include a cleavage site, e.g., a furin cleavage site, and a signal peptide (e.g., the wild-type preproinsulin signal sequence, the IL-6 signal sequence, or the fibronectin signal sequence).

[0109] In some embodiments, the modified nucleic acid encodes human insulin containing the amino acid modifications H34I, K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to histidine (H) to isoleucine (I) at position B10, lysine (K) to arginine (R) at position B29, arginine (R) to lysine (K) at position C1, and leucine (L) to arginine (R) at position C32 of proinsulin). In some embodiments, the modified nucleic acid encodes human insulin including the amino acid modifications H34I, K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to histidine (H) to isoleucine (I) at position B10, lysine (K) to arginine (R) at position B29, arginine (R) to lysine (K) at position C1, and leucine (L) to arginine (R) at position C32 of proinsulin), which optionally include a cleavage site, e.g., a furin cleavage site, and a signal peptide (e.g., the wild-type preproinsulin signal sequence, the IL-6 signal sequence, or the fibronectin signal sequence).

[0110] In some embodiments, the modified nucleic acid encodes human insulin containing the amino acid modifications H34V, K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to histidine (H) to valine (V) at position B10, lysine (K) to arginine (R) at position B29, arginine (R) to lysine (K) at position C1, and leucine (L) to arginine (R) at position C32 of proinsulin). In some embodiments, the modified nucleic acid encodes human insulin including the amino acid modifications H34V, K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to histidine (H) to valine (V) at position B10, lysine (K) to arginine (R) at position B29, arginine (R) to lysine (K) at position C1, and leucine (L) to arginine (R) at position C32 of proinsulin), which optionally include a cleavage site, e.g., a furin cleavage site, and a signal peptide (e.g., the wild-type preproinsulin signal sequence, the IL-6 signal sequence, or the fibronectin signal sequence).

[0111] In some embodiments, the modified nucleic acid encodes human insulin containing the amino acid modifications P49D, K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to proline (P) to aspartic acid (D) at position B28, lysine (K) to arginine (R) at position B29, arginine (R) to lysine (K) at position C1, and leucine (L) to arginine (R) at position C32 of proinsulin). In some embodiments, the modified nucleic acid encodes human insulin containing the amino acid modifications P49D, K53R, R55K, and L86R (corresponding to wild-type preproinsulin amino acid positions), which optionally include 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).

[0112] In some embodiments, the modified nucleic acid encodes a human insulin (Ins) protein (e.g., preproinsulin or a variant thereof), wherein the nucleic acid comprises (i) a nucleotide sequence encoding a signal peptide (e.g., a wild-type preproinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence) and (ii) a nucleotide sequence encoding a proinsulin polypeptide comprising an amino acid modification at a position selected from amino acids B10, B28, and / or B29 of the human insulin B chain, C1 and / or C32 of the human insulin C chain, or any combination thereof, relative to the corresponding amino acid in wild-type proinsulin (or an amino acid modification at a position selected from amino acids H34, P52, K53, R55, L86, or any combination thereof, relative to the corresponding amino acid in wild-type preproinsulin). In some embodiments, the signal peptide is not the wild-type preproinsulin signal sequence, e.g., the wild-type preproinsulin sequence is replaced with the IL-6 signal sequence or the fibronectin signal sequence. In some embodiments, the polynucleotide further comprises a cleavage site (e.g., a furin cleavage site). In some embodiments, the encoded human Ins protein (e.g., preproinsulin or a 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 B10 of the proinsulin B chain); and / or (ii) one or more amino acid modifications at P52, K53, R55, and / or L86 (or at positions B28 and / or B29 of the proinsulin B chain or C1 and / or C32 of the proinsulin C chain) relative to the wild-type preproinsulin sequence.In some embodiments, 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, an arginine (R) to lysine (K) at position C1 of the proinsulin C chain, a leucine (L) to arginine (R) at position C32 of the proinsulin C chain, or any combination thereof).

[0113] In some embodiments, the modified nucleic acid encodes a variant or mutant human insulin protein or a functional fragment thereof. In some embodiments, the human insulin protein comprises an amino 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% identity to 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 embodiments, the human insulin protein comprises an amino 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% identity to SEQ ID NO:41, SEQ ID NO:144, or SEQ ID NO:145. In some embodiments, the human insulin protein comprises an insertion, deletion, substitution, or a combination thereof relative to wild-type human insulin. In some embodiments, the human insulin protein comprises at least one substitution. In some embodiments, at least one substitution is a conservative substitution. In some embodiments, at least one substitution is a non-conservative substitution.

[0114] In some embodiments, a polynucleotide of the present disclosure comprises an open reading frame (ORF) comprising a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequence 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. In some embodiments, the ORF further comprises a nucleic acid sequence encoding a signal peptide.

[0115] In some aspects, the polynucleotides of the disclosure include 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, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, The modified nucleic acid sequence 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 a sequence selected from 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. In some aspects, a polynucleotide of the present 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.In some aspects, 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, 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. In some aspects, a polynucleotide of the disclosure comprises an open reading frame comprising a nucleic acid having the sequence of SEQ ID NO:122. In some embodiments, the polynucleotide comprises an ORF sequence present in or referenced in Table 1, Table 13, and / or Figure 1A.

[0116] In some aspects, the polynucleotides of the disclosure include 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, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:15 ... The nucleic acid sequence comprises two or more ORFs selected from the group consisting of nucleic acid sequences 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: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and any combination thereof. In some embodiments, one of the two or more ORFs 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 SEQ ID NO:122.In some embodiments, 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 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:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, and any combination thereof. In some embodiments, one of the two or more ORFs has the nucleic acid sequence of SEQ ID NO:122.

[0117] In some embodiments, two or more ORFs are operably linked. In some embodiments, the ORFs are operably linked by an IRES. In some embodiments, 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. In some embodiments, the IRES comprises the nucleic acid sequence of SEQ ID NO: 142 or SEQ ID NO: 143.

[0118] In some embodiments, the two or more ORFs linked by the IRES comprise a nucleic acid sequence having at least 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 comprising SEQ ID NO:110 and SEQ ID NO:53, SEQ ID NO:47 and SEQ ID NO:54, SEQ ID NO:49 and SEQ ID NO:56, SEQ ID NO:111 and SEQ ID NO:114, SEQ ID NO:112 and SEQ ID NO:115, SEQ ID NO:113 and SEQ ID NO:116, SEQ ID NO:120 and SEQ ID NO:114, SEQ ID NO:121 and SEQ ID NO:115, or SEQ ID NO:122 and SEQ ID NO:116. In some embodiments, the two or more ORFs linked through an IRES comprise a nucleic acid sequence comprising SEQ ID NO:110 and SEQ ID NO:53, SEQ ID NO:47 and SEQ ID NO:54, SEQ ID NO:49 and SEQ ID NO:56, SEQ ID NO:111 and SEQ ID NO:114, SEQ ID NO:112 and SEQ ID NO:115, SEQ ID NO:113 and SEQ ID NO:116, SEQ ID NO:120 and SEQ ID NO:114, SEQ ID NO:121 and SEQ ID NO:115, or SEQ ID NO:122 and SEQ ID NO:116.

[0119] In some embodiments, the polynucleotides of the present disclosure further comprise a modified 5' UTR nucleic acid sequence. In some embodiments, the polynucleotides of the present disclosure 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%, 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. In some embodiments, the polynucleotide further comprises a Kozak consensus sequence (Kozak consensus or Kozak sequence). In some embodiments, 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. In some embodiments, the polynucleotide comprises a 5'UTR nucleic acid sequence present in or referenced in Table 1 and / or Figure 1A.

[0120] In some embodiments, the polynucleotides of the present disclosure further comprise a modified 3'UTR nucleic acid sequence. In some embodiments, the polynucleotides of the present disclosure 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, or SEQ ID NO:149. In some embodiments, 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. In some embodiments, the 3'UTR comprises a restriction site selected from the group consisting of BamHI, EcoRI, NdeI, EcoRV, SpeI, XbaI, NheI, VspI, NsiI, ScaI, KpnI, SspI, and PacI, and any combination thereof. In some embodiments, the polynucleotide comprises a 3'UTR nucleic acid sequence present in or referenced in Table 1 and / or Figure 1A.

[0121] In some aspects, the polynucleotides of the disclosure are selected from the group consisting 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:136, SEQ ID NO:137, SEQ ID NO: and 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:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:160, or SEQ ID NO:161, wherein the 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. In some embodiments, a polynucleotide of the present 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.In some embodiments, 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 a nucleic acid 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. In some aspects, a polynucleotide of the present disclosure comprises a nucleic acid having a 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: 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. In some aspects, a polynucleotide of the present disclosure comprises a nucleic acid having a sequence of SEQ ID NO: 138. In some embodiments, a polynucleotide of the disclosure comprises nucleic acid 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. In some embodiments, a polynucleotide comprises a modified nucleic acid comprising a 5' UTR, ORF, and 3' UTR presented in Table 1 and / or Figure 1A.

[0122] In some aspects, the polynucleotides of the present disclosure encode human insulin comprising a wild-type preproinsulin secretory signal peptide. In some aspects, the polynucleotides of the present disclosure do not encode a wild-type preproinsulin secretory signal peptide. In some aspects, the wild-type preproinsulin is replaced by a non-insulin secretory signal. In some aspects, the polynucleotides of the present disclosure encode human preproinsulin comprising an interleukin-6 (IL-6) secretory signal peptide. In some aspects, the polynucleotides of the present disclosure encode human preproinsulin comprising a fibronectin secretory signal peptide.

[0123] Modified glucokinase nucleic acid In some aspects, the polynucleotides or nucleic acid sequences disclosed herein are modified relative to wild-type and / or unmodified human glucokinase (Gck), including, for example, the 5' UTR, ORF, and / or 3' UTR, nucleic acid sequences, e.g., those corresponding to SEQ ID NO: 19. In some aspects, the modified nucleic acid encodes wild-type human glucokinase (SEQ ID NO: 82) or a functional fragment thereof.

[0124] In some aspects, 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:78, SEQ ID NO:79, SEQ ID NO:80, or SEQ ID NO:162, wherein the nucleic acid sequence encodes a human glucokinase protein (SEQ ID NO:82) or a functional fragment thereof. In some aspects, a polynucleotide of the disclosure comprises an open reading frame comprising a nucleic acid having a 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, SEQ ID NO:79, SEQ ID NO:80, or SEQ ID NO: 162. In some aspects, a polynucleotide comprises an ORF sequence present in or referenced in Table 2 and / or Figure 2A.

[0125] In some embodiments, the polynucleotides of the present disclosure further comprise a modified 5' UTR nucleic acid sequence. In some embodiments, the polynucleotides of the present disclosure 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%, 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. In some embodiments, the polynucleotide further comprises a Kozak consensus sequence (Kozak consensus or Kozak sequence). In some embodiments, 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. In some embodiments, the polynucleotide comprises a 5'UTR sequence present in or referenced in Table 2 and / or Figure 2A.

[0126] In some embodiments, the polynucleotides of the present disclosure further comprise a modified 3' UTR nucleic acid sequence. In some embodiments, the polynucleotides of the present disclosure further comprise a 3' UTR comprising a nucleic acid sequence that is 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 identical 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. In some embodiments, 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. In some embodiments, the 3'UTR comprises a restriction site selected from the group consisting of BamHI, EcoRI, NdeI, EcoRV, SpeI, XbaI, NheI, VspI, NsiI, ScaI, KpnI, SspI, and PacI, and any combination thereof. In some embodiments, the polynucleotide comprises a 3'UTR sequence present in or referenced in Table 2 and / or Figure 2A.

[0127] In some aspects, 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 a sequence selected from the group consisting 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, SEQ ID NO:39, 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:164, wherein the nucleic acid sequence encodes a human glucokinase protein (e.g., SEQ ID NO:82) or a functional fragment thereof. In some aspects, polynucleotides of the disclosure include nucleic acids 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 nucleic acid 5-2025 of a sequence selected from 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:39.In some embodiments, polynucleotides of the disclosure comprise nucleic acids 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:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, 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: 164. In some embodiments, polynucleotides of the disclosure comprise nucleic acids 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:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39. In some embodiments, the polynucleotide is a modified nucleic acid present in or referenced in Table 2 and / or Figure 2A.

[0128] Expression constructs In some aspects, the present disclosure also provides expression cassettes comprising a nucleic acid sequence, e.g., a modified nucleic acid sequence disclosed herein encoding insulin, glucokinase, a combination thereof, or a functional fragment thereof, and a heterologous control sequence operably linked to the nucleic acid sequence. In some aspects, the heterologous control sequence is a promoter.

[0129] A nucleic acid construct having a eukaryotic promoter operably linked to a DNA of interest can be used in the present disclosure. The construct containing a DNA sequence (or corresponding RNA sequence) that can be used according to the present disclosure can be any eukaryotic expression construct containing a DNA or RNA sequence of interest. For example, a plasmid or viral construct (e.g., an AAV vector) can be cleaved to produce a linear DNA with ligatable ends. These ends can be combined with exogenous DNA with complementary, similarly ligatable ends to produce an intact replicon and a biologically functional recombinant DNA molecule with the desired phenotypic characteristics. In some embodiments, the construct can be replicated in both eukaryotic and prokaryotic hosts.

[0130] In some embodiments, the exogenous DNA used in the present disclosure is obtained from a suitable cell, and the construct is prepared using techniques known in the art. Similarly, techniques for achieving expression of exogenous DNA or RNA sequences in genetically modified host cells are known in the art (e.g., Kormal et al., 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, NY, USA). See Spring Harbor, NY; each of these is a method for determining the identity of a target DNA in eukaryotes. (which is incorporated herein by reference with respect to methods and compositions for expression in

[0131] In some embodiments, the DNA construct contains a promoter to facilitate expression of the DNA of interest (e.g., a modified nucleic acid encoding insulin, glucokinase, a combination thereof, or a fragment thereof) in secretory cells. In some embodiments, the promoter is a strong eukaryotic promoter, such as a promoter from cytomegalovirus (CMV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), or adenovirus. Exemplary promoters include the promoter from the immediate early gene of human CMV (Boshart et al., Cell 41:521-530 (1985)) and the long terminal repeat (LT) of RSV. Examples of promoters that can be used include, but are not limited to, promoters derived from the recombinant human ovarian fungus (R. cerevisiae) (Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777-6781 (1982)). Alternatively, the promoter used may be a tissue-specific promoter.

[0132] The constructs of the present disclosure may also include other components, such as a marker to aid in the selection of cells containing and / or expressing the construct (e.g., an antibiotic resistance gene (such as an ampicillin resistance gene) or β-galactosidase), an origin of replication (preferably a high copy number origin of replication) for stable replication of the construct in bacterial cells, a nuclear localization signal, or other elements that facilitate production of the DNA construct, the protein encoded thereby, or both.

[0133] For eukaryotic expression, the construct can contain, at a minimum, a eukaryotic promoter operably linked to the DNA of interest (e.g., a modified nucleic acid encoding insulin, glucokinase, a combination thereof, or a fragment thereof), which in turn is operably linked to a polyadenylation sequence. The polyadenylation signal sequence may be selected from any of a variety of polyadenylation signal sequences known in the art. In some embodiments, the polyadenylation signal sequence is the SV40 early polyadenylation signal sequence. The construct may also contain one or more introns, which may increase the expression level of the DNA of interest, particularly when the DNA of interest is cDNA (e.g., does not contain introns of the naturally occurring sequence). Any of a variety of introns known in the art may be used (e.g., a human β-globin intron inserted into the construct 5' to the DNA of interest).

[0134] The DNA of interest (e.g., a modified nucleic acid encoding insulin, glucokinase, a combination thereof, or a fragment thereof) may be inserted into the construct such that the therapeutic molecule (e.g., a protein) is expressed as a fusion protein (e.g., a fusion protein having β-galactosidase or a portion thereof at the N-terminus and a therapeutic protein at the C-terminus). Production of the fusion protein can facilitate identification of transformed cells expressing the protein (e.g., by enzyme-linked immunosorbent assay (ELISA) using an antibody that binds to the fusion protein).

[0135] Vectors for delivery of the DNA of interest (e.g., modified nucleic acids encoding insulin, glucokinase, combinations thereof, or fragments thereof) can be viral or non-viral, or can consist of naked DNA mixed with adjuvants such as viral particles (e.g., AAV particles) or cationic lipids or liposomes. An "adjuvant" is a substance that does not itself produce the desired effect but acts to enhance or otherwise improve the action of an active compound. The exact vector and vector formulation used will depend on several factors, such as the cells and / or organs targeted for gene transfer.

[0136] Examples of suitable promoters include the cytomegalovirus (CMV) intermediate early promoter, viral long terminal repeat (LTR) promoters, such as those derived from murine Moloney leukemia virus (MMLV), Rous sarcoma virus, or HTLV-1, the simian virus 40 (SV40) early promoter, the RSV promoter, and the herpes simplex virus thymidine kinase promoter. In some embodiments, the promoter is a cell-specific promoter and / or a tissue-specific promoter. In some embodiments, the promoter is used with an intron sequence. In some embodiments, the promoter is tissue-specific. In some embodiments, the promoter is a CMV promoter. In some embodiments, the CMV promoter is a miniCMV promoter.

[0137] In some aspects, the expression cassette 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: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: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. In some embodiments, 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 SEQ ID NO:122.In some aspects, 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, 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. In some aspects, a polynucleotide of the disclosure comprises an open reading frame comprising a nucleic acid having the sequence of SEQ ID NO:122. In some embodiments, the polynucleotide comprises an ORF sequence present in or referenced in Table 1, Table 13 and / or Figure 1A.

[0138] In some aspects, the expression cassette comprises a polynucleotide encoding human insulin comprising a wild-type preproinsulin secretory signal peptide. In some aspects, the polynucleotide of the present disclosure does not encode a wild-type preproinsulin secretory signal peptide. In some aspects, the wild-type preproinsulin is replaced by a non-insulin secretory signal. In some aspects, the expression cassette comprises a polynucleotide encoding human preproinsulin comprising an interleukin-6 (IL-6) secretory signal peptide. In some aspects, the expression cassette comprises a polynucleotide encoding human preproinsulin comprising a fibronectin secretory signal peptide.

[0139] In some embodiments, 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. In some embodiments, the polynucleotide comprises a 5' UTR sequence present in or referenced in Table 1 and / or FIG. 1A.

[0140] In some embodiments, 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. In some embodiments, 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. In some embodiments, the 3'UTR comprises a restriction site selected from the group consisting of BamHI, EcoRI, NdeI, EcoRV, SpeI, XbaI, NheI, VspI, NsiI, ScaI, KpnI, SspI, and PacI, and any combination thereof. In some embodiments, the polynucleotide comprises a 3'UTR sequence present in or referenced in Table 1 and / or Figure 1A.

[0141] In some embodiments, the expression cassette is selected from the group consisting 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 and modified nucleic acids 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: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, 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. In some embodiments, 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.In some embodiments, 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 acid 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. In some embodiments, an 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: 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. In some embodiments, an expression cassette comprises a modified nucleic acid having the sequence of SEQ ID NO: 138. In some embodiments, 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 acid 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.In some embodiments, the expression cassette comprises a modified nucleic acid comprising a 5'UTR, an ORF, and a 3'UTR present in or referenced in Table 1 and / or Figure 1A.

[0142] In some embodiments, 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: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, SEQ ID NO:79, SEQ ID NO:80, or SEQ ID NO:162. In some embodiments, the expression cassette comprises a promoter operably linked to a modified nucleic acid having a 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, SEQ ID NO:79, SEQ ID NO:80, or SEQ ID NO: 162. In some embodiments, the polynucleotide comprises an ORF sequence present in or referenced in Table 2 and / or Figure 2A.

[0143] In some embodiments, 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. In some embodiments, the polynucleotide comprises a 5' UTR sequence present in or referenced in Table 2 and / or Figure 2A.

[0144] In some embodiments, 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. In some embodiments, 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. In some embodiments, the 3'UTR comprises a restriction site selected from the group consisting of BamHI, EcoRI, NdeI, EcoRV, SpeI, XbaI, NheI, VspI, NsiI, ScaI, KpnI, SspI, and PacI, or any combination thereof. In some embodiments, the polynucleotide comprises a 3'UTR sequence present in or referenced in Table 2 and / or Figure 2A.

[0145] 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 a sequence selected from 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, SEQ ID NO:39, 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:164, wherein the nucleic acid sequence encodes a human glucokinase protein (e.g., SEQ ID NO:82) or a functional fragment thereof. In some embodiments, 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 a nucleic acid 5-2025 of a sequence selected from 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:39.In some embodiments, 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:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, 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: 164. In some embodiments, the expression cassette comprises a modified nucleic acid comprising a nucleic acid 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:39. In some embodiments, the expression cassette comprises a modified nucleic acid comprising a 5'UTR, ORF, and 3'UTR present in or referenced in Table 2 and / or Figure 2A.

[0146] In some embodiments, the expression cassette has a sequence identical 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: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 by 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%, or %, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to 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, SEQ ID NO:79, SEQ ID NO:80, or SEQ ID NO:162.

[0147] In some embodiments, the expression cassette comprises a promoter operably linked to the modified nucleic acid, and each of the first and second modified nucleic acids is linked to the first and second promoter, respectively. In some embodiments, the first modified nucleic acid sequence comprising the 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: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:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, 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 and the second modified nucleic acid sequence comprising the 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: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, SEQ ID NO:79, SEQ ID NO:80, and SEQ ID NO:162.

[0148] In some embodiments, the first modified nucleic acid sequence encodes human insulin containing a wild-type preproinsulin secretory signal peptide. In some embodiments, the first modified nucleic acid sequence does not encode a wild-type preproinsulin secretory signal peptide. In some embodiments, the wild-type preproinsulin is replaced by a non-insulin secretory signal. In some embodiments, the first modified nucleic acid sequence encodes human preproinsulin containing an interleukin-6 (IL-6) secretory signal peptide. In some embodiments, the first modified nucleic acid sequence encodes human preproinsulin containing a fibronectin secretory signal peptide.

[0149] In some embodiments, 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%, 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. In some embodiments, 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.

[0150] In some embodiments, 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. In some embodiments, the 3' UTR comprises a restriction site selected from the group consisting of BamHI, EcoRI, NdeI, EcoRV, SpeI, XbaI, NheI, VspI, NsiI, ScaI, KpnI, SspI, and PacI, or any combination thereof. In some embodiments, 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, 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.

[0151] In some embodiments, the expression cassette comprises a sequence selected from the group consisting 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: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 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 a first modified nucleic acid having at least 99%, or 100%, sequence identity with 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, SEQ ID NO:39, 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, and a second 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 SEQ ID NO: 163, or SEQ ID NO: 164, wherein each of the first and second modified nucleic acids is linked to a first and second promoter, respectively.In some embodiments, the first modified nucleic acid sequence 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 acid 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, and the second 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 acid 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, or SEQ ID NO:16. A nucleic acid having a sequence selected from 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:39 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 acid 5-2025 of a sequence selected from 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:39.In some embodiments, the first modified nucleic acid sequence is selected from the group consisting 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:136, SEQ ID NO:137, SEQ ID NO:138, and the second nucleic acid sequence is selected from the group consisting of 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, 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, SEQ ID NO:39, 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:164. In some embodiments, the first modified nucleic acid comprises 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 comprises 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:39. In some embodiments, the first modified nucleic acid sequence comprises a 5'UTR, ORF, and 3'UTR present in or referenced in Table 1 and / or Figure 1A, and the second modified nucleic acid sequence comprises a 5'UTR, ORF, and 3'UTR present in or referenced in Table 2 and / or Figure 2A.

[0152] Certain aspects of the present disclosure are directed to expression constructs, e.g., vectors. In some aspects, the expression construct comprises an expression cassette. In some aspects, the expression construct further comprises a genome that can be stabilized and maintained episomal in the cell. In the context of the present disclosure, in some aspects, a cell or host cell can include a cell used to generate the construct or a cell to which the construct is administered. In some aspects, the construct is capable of integrating into the genome of a cell, for example, by homologous recombination or otherwise. In some aspects, the expression construct comprises a nucleotide sequence encoding insulin and / or glucokinase as disclosed herein operably linked to a promoter as provided herein, wherein the promoter is capable of directing expression of the nucleotide sequence (i.e., coding sequence) in the cell. In some aspects, an expression cassette, as used herein, comprises or consists of a nucleotide sequence encoding insulin and / or a nucleotide sequence encoding glucokinase, in each case operably linked to a promoter, wherein the promoter is capable of directing expression of the nucleotide sequence. In some aspects, the viral expression construct is an expression construct intended for use in gene therapy, which can be designed to include portions of the viral genomes disclosed herein.

[0153] In some embodiments, the expression construct further comprises one or more of an ITR sequence (e.g., an AAV2 ITR), a polyA sequence (e.g., an SV40 polyadenylation signal, a bGH polyadenylation signal), and an enhancer sequence (e.g., an SV40 enhancer sequence).

[0154] In some aspects, the expression constructs disclosed herein are prepared using recombinant techniques, in which modified nucleic acid sequences encoding insulin and / or glucokinase are transfected into suitable cells, e.g., cultured cells or cells of a multicellular organism, e.g., by recombinant techniques, such as those described in Ausubel et al., "Current Protocols in Molecular Biology," Greene Publishing and Wiley-Interscience, New York (1987) and Sambrook and Russell (2001, supra), both of which are incorporated herein by reference in their entireties. Kunkel (1985) Proc. Natl. Acad. Sci. 82:488 (partial text missing or illegible) (1987) Nature 328:731-734 or Wells, JA, et al. (1985) Gene 34: 315 (Cassette insertion) See also the NIH Guidelines for Genetic Modification (which describes spontaneous mutagenesis).

[0155] Delivery Vector The present disclosure also provides a vector comprising any of the modified nucleic acids, polynucleotides or expression cassettes described herein.In some aspects, the delivery vector is a viral vector, a non-viral vector, a plasmid, a lipid or a lysosome.In some aspects, the delivery vector is a viral vector.In some aspects, the viral vector is an adeno-associated virus (AAV) expression vector.

[0156] In some embodiments, modified nucleic acids or nucleotide sequences encoding insulin and / or glucokinase are used in expression constructs or expression vectors. The term "expression vector" generally refers to a nucleotide sequence capable of effecting gene expression in a host compatible with the nucleotide sequence. These expression vectors contain at least a suitable promoter sequence and, optionally, a transcription termination signal. Additional factors necessary or useful in effecting expression may also be used as disclosed herein. Modified nucleic acids or DNA or codon-optimized nucleotide sequences encoding insulin and / or glucokinase can be incorporated into expression vectors that can be introduced into in vitro cell cultures and expressed. In some embodiments, the expression vectors are suitable for replication in prokaryotic hosts such as bacteria, e.g., E. coli, or can be introduced into cultured mammalian, plant, insect (e.g., Sf9), yeast, fungi, or other eukaryotic cell systems. In some embodiments, the expression constructs are suitable for in vivo expression.

[0157] In some embodiments, the delivery vector 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: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:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, The present invention also includes an expression cassette comprising 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: 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. In some embodiments, the delivery vector comprises an expression cassette comprising 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.In some embodiments, the modified nucleic acid comprises an ORF 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, 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. In some embodiments, the modified nucleic acid comprises an ORF having the sequence of SEQ ID NO:122. In some embodiments, the polynucleotide comprises an ORF sequence present in or referenced in Table 1, Table 13, and / or FIG. 1A.

[0158] In some embodiments, the delivery vector comprises an expression cassette comprising a modified nucleic acid sequence encoding human insulin comprising a wild-type preproinsulin secretory signal peptide. In some embodiments, the delivery vector comprises an expression cassette comprising a modified nucleic acid sequence that does not encode the wild-type preproinsulin secretory signal peptide. In some embodiments, the wild-type preproinsulin has been replaced by a non-insulin secretory signal. In some embodiments, the delivery vector comprises an expression cassette comprising a modified nucleic acid sequence encoding human preproinsulin comprising an interleukin-6 (IL-6) secretory signal peptide. In some embodiments, the delivery vector comprises an expression cassette comprising a modified nucleic acid sequence encoding human preproinsulin comprising a fibronectin secretory signal peptide.

[0159] In some embodiments, 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. In some embodiments, 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. In some embodiments, the polynucleotide comprises a 5' UTR sequence present in or referenced in Table 1 and / or FIG. 1A.

[0160] In some embodiments, the delivery vector comprises an expression cassette comprising a modified nucleic acid further comprising a nucleic acid sequence having a 3' UTR of 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. In some embodiments, the 3' UTR comprises a restriction site selected from the group consisting of BamHI, EcoRI, NdeI, EcoRV, SpeI, XbaI, NheI, VspI, NsiI, ScaI, KpnI, SspI, and PacI, and any combination thereof. In some embodiments, 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. In some embodiments, the polynucleotide comprises a 3'UTR sequence present in or referenced in Table 1 and / or Figure 1A.

[0161] In some embodiments, the delivery vector is selected from the group consisting 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: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, 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. In some embodiments, 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.In some embodiments, 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 acid 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. In some embodiments, a delivery vector comprises an expression cassette comprising a modified nucleic acid sequence 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: 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. In some embodiments, a delivery vector comprises an expression cassette comprising a modified nucleic acid sequence having the sequence of SEQ ID NO: 138. In some embodiments, the delivery vector comprises an expression cassette comprising a modified nucleic acid comprising nucleic acid 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. In some embodiments, the delivery vector comprises an expression cassette comprising a modified nucleic acid comprising a 5' UTR, ORF, and 3' UTR present in or referenced in Table 1 and / or Figure 1A.

[0162] In some embodiments, 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: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, SEQ ID NO:79, SEQ ID NO:80, or SEQ ID NO:162. In some embodiments, 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: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, SEQ ID NO:79, SEQ ID NO:80, or SEQ ID NO: 162. In some embodiments, the polynucleotide comprises an ORF sequence present in or referenced in Table 2 and / or Figure 2A.

[0163] In some embodiments, 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. In some embodiments, the polynucleotide comprises a 5' UTR sequence present in or referenced in Table 2 and / or Figure 2A.

[0164] In some embodiments, 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. In some embodiments, the 3' UTR comprises a restriction site selected from the group consisting of BamHI, EcoRI, NdeI, EcoRV, SpeI, XbaI, NheI, VspI, NsiI, ScaI, KpnI, SspI, and PacI, and any combination thereof. In some embodiments, 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. In some embodiments, the polynucleotide comprises a 3'UTR sequence present in or referenced in Table 2 and / or Figure 2A.

[0165] In some aspects, 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: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, SEQ ID NO:39, 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:164, wherein the nucleic acid sequence encodes a human glucokinase protein (e.g., SEQ ID NO:82) or a functional fragment thereof. In some embodiments, 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 nucleic acid 5-2025 of a sequence selected from 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:39.In some embodiments, a delivery vector comprises an expression cassette comprising 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:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, 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: 164. In some embodiments, a delivery vector comprises an expression cassette comprising a modified nucleic acid comprising a nucleic acid 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:39. In some embodiments, the delivery vector comprises the 5'UTR sequence, ORF and 3'UTR present in or referenced in Table 2 and / or Figure 2A.

[0166] In some embodiments, the delivery vector is 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%, or at least slightly identical 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: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 an expression cassette comprising a first modified nucleic acid comprising a first ORF having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to 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, SEQ ID NO:79, SEQ ID NO:80, or SEQ ID NO:162.In some embodiments, 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: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, SEQ ID NO:79, SEQ ID NO:80, or SEQ ID NO:162, wherein each of the first and second modified nucleic acids is linked to the first and second promoters, respectively. In some embodiments, the first modified nucleic acid sequence comprising the 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: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, The second modified nucleic acid sequence comprising the second ORF is selected from the group consisting of 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 the 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: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, SEQ ID NO:79, and SEQ ID NO:80.

[0167] In some embodiments, the first modified nucleic acid sequence encodes human insulin containing a wild-type preproinsulin secretory signal peptide. In some embodiments, the first modified nucleic acid sequence does not encode a wild-type preproinsulin secretory signal peptide. In some embodiments, the wild-type preproinsulin is replaced by a non-insulin secretory signal. In some embodiments, the first modified nucleic acid sequence encodes human preproinsulin containing an interleukin-6 (IL-6) secretory signal peptide. In some embodiments, the first modified nucleic acid sequence encodes human preproinsulin containing a fibronectin secretory signal peptide.

[0168] In some embodiments, 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. In some embodiments, 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.

[0169] In some embodiments, 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. In some embodiments, the 3' UTR comprises a restriction site selected from the group consisting of BamHI, EcoRI, NdeI, EcoRV, SpeI, XbaI, NheI, VspI, NsiI, ScaI, KpnI, SspI, and PacI, and any combination thereof. In some embodiments, 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, 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.

[0170] In some embodiments, the delivery vector is selected from the group consisting 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: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 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 (leas t) a first modified nucleic acid having 99% or 100% sequence identity and a first modified nucleic acid having 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, SEQ ID NO:39, 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 an expression cassette comprising a second 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 SEQ ID NO: 164, wherein each of the first and second modified nucleic acids is linked to a first and second promoter, respectively.In some embodiments, 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 acid 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, and the second modified nucleic acid sequence 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 acid 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, or SEQ ID NO:16. A nucleic acid having a sequence selected from 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:39 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 acid 5-2025 of a sequence selected from 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:39.In some embodiments, the first modified nucleic acid sequence is selected from the group consisting 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:136, SEQ ID NO:137, SEQ ID NO:138, 8, 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, 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, SEQ ID NO:39, 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:164. In some embodiments, the first modified nucleic acid sequence comprises 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 comprises 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:39. In some embodiments, the first modified nucleic acid sequence comprises a 5'UTR, ORF, and 3'UTR present in or referenced in Table 1 and / or Figure 1A, and the second modified nucleic acid sequence comprises a 5'UTR, ORF, and 3'UTR present in or referenced in Table 2 and / or Figure 2A.

[0171] In some embodiments, a delivery vector may include a sequence encoding a protein (e.g., insulin and / or Gck) operably linked to control or regulatory sequences, selectable markers, optional fusion partners, and / or additional elements. In certain embodiments, the modified nucleic acid is placed in a functional relationship with another nucleic acid sequence. The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of a protein. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology, Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). In some embodiments, an expression vector includes transcriptional and translational regulatory nucleic acids operably linked to the protein-encoding nucleic acid, typically appropriate for the host cell used to express the protein. Generally, 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. As is known in the art, expression vectors can also contain a selection gene or marker that allows for the selection of transformed host cells containing the expression vector. Selection genes are known in the art and vary depending on the host cell used. For example, typically, a selectable marker gene confers resistance to drugs such as G418, hygromycin, or methotrexate on host cells into which the vector has been introduced. In some embodiments, selectable marker genes include a dihydrofolate reductase (DHFR) gene (for use with methotrexate selection / amplification in dhfr-host cells) and a neo gene (for G418 selection).

[0172] In some aspects, the delivery vector is a viral vector comprising a viral expression construct or a gene therapy vector, hi certain aspects, the viral vector or gene therapy vector is a vector suitable for gene therapy.

[0173] In some embodiments, gene therapy vectors include adenovirus vectors and adeno-associated virus (AAV) vectors.These vectors infect many dividing and non-dividing cell types, including synovial cells and liver cells.The episomal nature of adenovirus vectors and AAV vectors after entering cells makes these vectors suitable for therapeutic use, as mentioned above (Russell, 2000, J. Gen. Virol. 81: 2573-2604; Goncalves, 2005, Virol J. 2(1):43). AAV vectors can provide very stable, long-term transgene expression (up to 9 years in dogs (Niemeyer et al, Blood. 2009 Jan. 22; 113(4):797-806) and up to 2 years in humans (Nathwani et al, N Engl J Med. 2011 Dec. 22; 365(25):2357-65; Simonelli et al, Mol Ther. 2010 March; 18(3):643-50. Epub 2009 Dec. 1.)). In some embodiments, adenoviral vectors The vector is modified to reduce the host response, as reviewed by Russell (2000, supra). Methods for gene therapy using AAV vectors are described by Wang et al. al., 2005, J Gene Med. March 9 (Epub ahead of print), Mandel et al., 2004, Curr Opin Mol Ther. 6(5):482-90, and Martin et al., 2004, Eye 18(11):1049-55, Nathwani et al, N Engl J Med. 2011 Dec. 22; 365(25):2357-65, Apparaily et al, Hum Gene Ther. 2005 April; 16(4):426-34.

[0174] In some embodiments, the gene therapy vector comprises a retroviral vector. In some embodiments, the retroviral vector is a lentiviral-based expression construct. Lentiviral vectors have the ability to infect dividing and non-dividing cells and stably integrate into their genomes (Amado and Chen, 1999 Science 285: 674-6). Methods for the construction and use of lentiviral-based expression constructs are described in U.S. Patent Nos. 6,165,782, 6,207,455, 6,218,181, 6,277,633, and 6,323,031, as well as Federico (1999, Curr Opin Biotechnol 10: 448-53) and Vigna et al. (2000, J Gene Med 2000; 2: 308-16).

[0175] In some aspects, the gene therapy vector is a herpes virus vector, a polyoma virus vector, or a vaccinia virus vector.

[0176] In some embodiments, the gene therapy vector comprises a modified nucleotide sequence encoding insulin and / or glucokinase, each of which is operably linked to an appropriate regulatory sequence. Such regulatory sequence may include at least a promoter sequence. Suitable promoters for expressing the nucleotide sequence encoding insulin and / or glucokinase from the gene therapy vector may include, for example, the cytomegalovirus (CMV) intermediate early promoter, viral long terminal repeat (LTR) promoters, such as those derived from murine Moloney leukemia virus (MMLV), Rous sarcoma virus, or HTLV-1, the simian virus 40 (SV40) early promoter, and the herpes simplex virus thymidine kinase promoter.

[0177] In some embodiments, the gene therapy vector comprises an additional nucleotide sequence encoding an additional polypeptide. The additional polypeptide can be a (selectable) marker polypeptide that allows for identification, selection, and / or screening of cells containing the expression construct. In some embodiments, suitable marker proteins for this purpose are, for example, the fluorescent protein GFP, as well as the selectable marker genes HSV thymidine kinase (for selection in HAT medium), bacterial hygromycin B phosphotransferase (for selection in hygromycin B), Tn5 aminoglycoside phosphotransferase (for selection in G418), and dihydrofolate reductase (DHFR) (for selection in methotrexate), CD20, the low-affinity nerve growth factor gene. Sources for obtaining these marker genes and methods for their use are described in Sambrook and Russell (2001) "Molecular Cloning: A Laboratory Manual (3 rd (edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York It is being done.

[0178] Non-viral vectors In some embodiments, the modified nucleic acids, polynucleotides, or expression constructs of the present disclosure can be administered using non-viral vectors. "Non-viral vector," as used herein, is meant to include naked DNA, chemical formulations containing naked DNA (e.g., formulations 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 a viral particle, but the transforming formulation is composed of both naked DNA and a viral particle (e.g., an AAV particle) (see, e.g., Curiel et al., Am. J. Respir. Cell Mol. Biol. 6:247-52 (1992)). Therefore, the term "non-viral vector" can include a vector constructed by adding a viral particle to DNA, where the viral particle does not contain the DNA of interest within the viral genome.

[0179] In some embodiments, the modified nucleic acids, polynucleotides, or expression constructs of the present 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 contain a mixture of lipids that bind to genetic material (DNA or RNA) to facilitate delivery of the nucleic acid into cells. Liposomes that can be used in accordance with the present disclosure include DOPE (dioleylphosphatidylethanolamine) and CUDMEDA (N-(5-cholestrum-3-β-ol 3-urethanyl)-N',N'-dimethylethylenediamine).

[0180] In some embodiments, the modified nucleic acids, polynucleotides, or expression constructs of the present disclosure can also be administered as chemical formulations of DNA or RNA coupled to carrier molecules (e.g., antibodies or receptor ligands) that facilitate delivery to host cells in order to modify the biological properties of the host cells. The term "chemical formulation" refers to a modification of a nucleic acid that allows the nucleic acid compound to be coupled to a carrier molecule such as a protein or lipid, or a derivative thereof. Exemplary protein carrier molecules include target cell-specific antibodies, i.e., molecules that can interact with receptors associated with the cells targeted for delivery.

[0181] Adeno-associated virus vector (AAV vector) In some aspects, the modified nucleic acids, polynucleotides, or expression constructs disclosed herein can be administered as a component of a packaged viral vector. Generally, a packaged viral vector comprises a viral vector packaged within a capsid.

[0182] In some embodiments, the viral vector is an AAV vector. In some embodiments, the AAV vector as used herein can include recombinant AAV vector (rAAV). As used herein, "rAAV vector" refers to a recombinant vector that includes a portion of the AAV genome encapsidated in a protein shell of a capsid protein derived from the AAV serotype disclosed herein. The portion of the AAV genome can contain an inverted terminal repeat (ITR) derived from an adeno-associated virus serotype such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVRH10, AAV11, AAV12, etc. In some embodiments, the ITR is derived from AAV2.

[0183] Typically, vector genomes require the use of flanking 5' and 3' ITR sequences for efficient packaging of the vector genome into an rAAV capsid. In some embodiments, the rAAV genome present in the rAAV vector comprises at least the nucleotide sequence of the inverted terminal repeat region (ITR) of one of the AAV serotypes (e.g., serotype AAV2 previously disclosed herein), or a nucleotide sequence substantially identical thereto, and a modified nucleic acid sequence encoding insulin and / or glucokinase under the control of a suitable regulatory element (e.g., a promoter), wherein the regulatory element and modified nucleic acid sequence are inserted between the two ITRs.

[0184] The complete genomes and corresponding ITRs of several AAV serotypes have been sequenced (Chiorini et al. 1999, J. of Virology Vol. 73, No. 2, p 1309-1319). They can be cloned or produced by chemical synthesis, as known in the art, using an oligonucleotide synthesizer such as those supplied by Applied Biosystems Inc. (Fosters, Calif., USA), or by standard molecular biology techniques. ITRs can be cloned from the AAV viral genome or excised from a vector containing AAV ITRs. Using standard molecular biology techniques, the ITR nucleotide sequences can be ligated at either end to a nucleotide sequence encoding one or more therapeutic proteins, or the wild-type AAV sequence between the ITRs can be replaced with a desired nucleotide sequence.

[0185] The viral capsid component of the packaged viral vector can be a parvovirus capsid, such as an AAV Cap and / or a chimeric capsid. Examples of suitable parvovirus viral capsid components include capsid components from Parvoviridae, such as autonomous parvoviruses or dependoviruses. For example, the viral capsid can be an AAV capsid (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRH8, AAV9, AAV10, AAVRH10, AAV11, or AAV12 capsid; those skilled in the art will recognize that there are likely other unidentified variants that perform the same or similar functions), or it can contain components from two or more AAV capsids. The full complement of AAV Cap proteins includes VP1, VP2, and VP3. An ORF comprising a nucleotide sequence encoding an AAV VP capsid protein can contain less than the full complement of AAV Cap proteins, or can provide the full complement of AAV Cap proteins.

[0186] One or more of the AAV Cap proteins may be chimeric proteins containing AAV Cap amino acid sequences derived from two or more viruses, preferably two or more AAVs. For example, a chimeric viral capsid may contain an AAV1 Cap protein or subunit and at least one AAV2 Cap or subunit. In some embodiments, the rAAV genome present in the rAAV vector does not contain any nucleotide sequences encoding viral proteins, such as the AAV rep (replication) or cap (capsid) genes. The rAAV genome may further contain a marker or reporter gene, such as a gene encoding an antibiotic resistance gene, a gene encoding 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.), as known in the art.

[0187] In some embodiments, the rAAV genome present in the rAAV vector further comprises a promoter sequence operably linked to the nucleotide sequence encoding insulin and / or glucokinase.In some embodiments, the promoter sequence is a promoter that confers expression in muscle cell and / or muscle tissue.Examples of such promoters include the CMV promoter and RSV promoter as disclosed herein.

[0188] In some embodiments, a suitable 3' untranslated sequence may be operably linked to the modified nucleic acid sequence encoding insulin and / or glucokinase. Suitable 3' untranslated regions may be those naturally associated with the nucleotide sequence or may be derived from various genes, such as the bovine growth hormone 3' untranslated region (e.g., bGH polyadenylation signal, SV40 polyadenylation signal, SV40 polyadenylation signal, and enhancer sequence).

[0189] In some embodiments, additional nucleotide sequences, such as nucleotide sequences encoding signal sequences, nuclear localization signals, expression enhancers, etc., may be operably linked to the modified nucleic acid sequence encoding insulin and / or glucokinase.

[0190] Except as otherwise noted, recombinant parvovirus and AAV (rAAV) constructs, packaging vectors expressing parvovirus Rep and / or Cap sequences, and transient and stably engineered packaging cells can be constructed using methods known to those skilled in the art. Such techniques are known to those skilled in the art. See, for example, SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, NY, 1989); AUSUBEL el al., CURRENT PROTOCOLS IN MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, NY, 1989); BIOLOGY (Green Publishing Associates, Inc. and John Wiley Sons, Inc., New York).

[0191] Lentiviral expression constructs Lentiviruses are complex retroviruses that contain the common retroviral genes gag, pol, and env as well as other genes with regulatory or structural functions. This high complexity allows lentiviruses to modulate their life cycle during latent infection.

[0192] A typical lentivirus is the human immunodeficiency virus (HIV), the causative agent of AIDS. In vivo, HIV can infect terminally differentiated cells that divide infrequently, such as lymphocytes and macrophages. In vitro, HIV can infect primary cultures of monocyte-derived macrophages (MDMs) and HeLa-Cd4 or T lymphoid cells whose cell cycle has been arrested by treatment with aphidicolin or gamma irradiation.

[0193] Infection of cells depends on the active nuclear import of HIV preintegration complexes through the target cell's nuclear pores. This occurs through the interaction of multiple, partially overlapping, molecular determinants within the complex with the target cell's nuclear import machinery. Identified determinants include a functional nuclear localization signal (NLS) in the gag matrix (MA) protein, the karyophilic virion-associated protein, vpr, and a C-terminal phosphotyrosine residue in the gag MA protein.

[0194] Lentiviral genomes and proviral DNA contain three genes found in retroviruses: gag, pol, and env, flanked by two long terminal repeat (LTR) sequences. The gag gene encodes internal structural (matrix, capsid, and nucleocapsid) proteins; the pol gene encodes RNA-dependent DNA polymerase (reverse transcriptase), protease, and integrase; and the env gene encodes viral envelope glycoproteins. The 5' and 3' LTRs serve to promote transcription and polyadenylation of virion RNA. The LTRs contain all other cis-acting sequences necessary for viral replication. Lentiviruses possess additional genes, including vif, vpr, tat, rev, vpu, nef, and vpx (in HIV-1, HIV-2, and / or SIV).

[0195] The 5' LTR is flanked by sequences required for reverse transcription of the genome (tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (Psi site). When sequences required for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, a cis defect prevents encapsidation of genomic RNA. However, the resulting mutant is still capable of directing the synthesis of all virion proteins.

[0196] In some embodiments, recombinant lentiviruses can infect non-dividing cells by transfecting suitable host cells with two or more vectors carrying packaging functions, i.e., gag, pol, and env, and rev and tat. In some instances, vectors lacking a functional tat gene are desirable. Thus, for example, to produce packaging cells, one vector can provide nucleic acid encoding viral gag and viral pol, and another vector can provide nucleic acid encoding viral env. Introducing a vector, identified as a transfer vector, that provides a heterologous gene into the packaging cells results in producer cells that release infectious viral particles carrying the foreign gene of interest.

[0197] The gag, pol, and env genes of a vector of interest are also known in the art. Thus, the relevant genes are cloned into a selected vector, which is then used to transform a target cell of interest.

[0198] According to the above configuration of vector and foreign gene, the second vector can provide a nucleic acid encoding a viral envelope (env) gene. The env gene can be derived from any virus, including retroviruses. Preferably, the env is an amphotropic envelope protein that allows transduction of cells of human and other species.

[0199] It may be desirable to target recombinant viruses by linking the envelope protein with an antibody or a specific ligand for targeting a receptor on a specific cell type. Inserting a sequence of interest (including a regulatory region) into a viral vector, for example, along with another gene encoding a ligand for a receptor on a specific target cell, makes the vector target-specific. Retroviral vectors can be made target-specific by inserting, for example, glycolipids or proteins. Targeting is often achieved by targeting retroviral vectors using the antigen-binding portion of an antibody or recombinant antibody-type molecule, such as a single-chain antibody. Those skilled in the art will know, or can easily ascertain, specific methods for achieving delivery of retroviral vectors to specific targets without undue experimentation.

[0200] Examples of env genes from retroviruses include, but are not limited to, Moloney murine leukemia virus (MoMuLV or MMLV), Harvey murine sarcoma virus (HaMuSV or HSV), mouse 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 those from vesicular stomatitis virus (VSV) G protein (VSV G), hepatitis virus, and influenza virus, can also be used.

[0201] The vector that provides the viral env nucleic acid sequence is operably associated with a regulatory sequence, for example, a promoter or enhancer.The regulatory sequence can be any eukaryotic promoter or enhancer, including, for example, Moloney murine leukemia virus promoter enhancer element, human cytomegalovirus enhancer, or vaccinia P7.5 promoter.In some cases, a promoter enhancer element, such as Moloney murine leukemia virus promoter enhancer element, is located in or adjacent to the LTR sequence.

[0202] In some embodiments, the lentiviral genome present in the lentiviral vector further comprises a promoter sequence that is operably linked to the nucleotide sequence encoding insulin and / or glucokinase.In some embodiments, the promoter sequence is a promoter that confers expression in muscle cell and / or muscle tissue.Examples of such promoters include CMV promoter and RSV promoter as disclosed herein.

[0203] In some embodiments, a suitable 3' untranslated sequence may be operably linked to the modified nucleic acid sequence encoding insulin and / or glucokinase. Suitable 3' untranslated regions may be those naturally associated with the nucleotide sequence or may be derived from various genes, such as the bovine growth hormone 3' untranslated region (e.g., bGH polyadenylation signal, SV40 polyadenylation signal, SV40 polyadenylation signal, and enhancer sequence).

[0204] In some embodiments, additional nucleotide sequences, such as nucleotide sequences encoding signal sequences, nuclear localization signals, expression enhancers, etc., may be operably linked to the modified nucleic acid sequence encoding insulin and / or glucokinase.

[0205] Unless otherwise stated, lentiviral constructs, vectors, and transient and stably engineered packaging cells can be constructed using methods known to those skilled in the art. Such techniques are known to those skilled in the art. See, for example, SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, NY, 1989), AUSUBEL el al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley Sons, Inc., New York). It refers to light.

[0206] host cell In some aspects, the present disclosure also provides host cells comprising the modified nucleic acid sequences, polynucleotides, expression cassettes, vectors, or expression constructs disclosed herein. In some aspects, the host cells are mammalian cells.

[0207] Constructs prepared for introduction into a particular host may include a replication system recognized by the host, a DNA segment intended to encode the desired polypeptide, and transcriptional and translational initiation and termination regulatory sequences operably linked to the polypeptide-encoding segment. The term "operably linked" has been defined herein. For example, a promoter or enhancer is operably linked to a coding sequence if it stimulates the transcription of the sequence. Signal sequence DNA is operably linked to polypeptide-encoding DNA if it is expressed as a preprotein involved in the secretion of the polypeptide. Generally, operably linked DNA sequences are contiguous, and in the case of a signal sequence, contiguous and in reading frame. However, enhancers need not be contiguous with the coding sequence whose transcription they control. Linking is accomplished by ligation at convenient restriction sites or adapters or linkers inserted in lieu thereof, or by gene synthesis.

[0208] The selection of an appropriate promoter sequence generally depends on the host cell selected for the expression of the DNA segment. Examples of suitable promoter sequences include prokaryotic and eukaryotic promoters well known in the art (see, e.g., Sambrook and Russell, 2001, supra). The transcriptional regulatory sequence typically includes a heterologous enhancer or promoter recognized by the host. The selection of an appropriate promoter depends on the host, but promoters such as trp, lac, and phage promoters, tRNA promoters, and glycolytic enzyme promoters are known and available (see, e.g., Sambrook and Russell, 2001, supra). The expression vector contains a replication system, and transcriptional and translational regulatory sequences can be used in conjunction with the insertion site of the polypeptide-encoding segment. In most cases, the replication system is only functional in the cells (bacterial cells such as E. coli) used to produce the vector. Most plasmids and vectors do not replicate in cells infected with the vector. Examples of possible combinations of cell lines and expression vectors are described in Sambrook and Russell (2001, supra) and Metzger et al. (1988) Nature 334: 31-36. For example, suitable expression vectors can be expressed in yeast, e.g., S. cerevisiae, insect cells, e.g., Sf9 cells, mammalian cells, e.g., CHO cells, and bacterial cells, e.g., E. coli. Thus, the cell can be a prokaryotic or eukaryotic host cell. The cell can be a cell suitable for culturing in liquid or solid medium.

[0209] The method of introducing exogenous nucleic acid into host cells is well known in the art and varies depending on the host cell used.Techniques include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, calcium chloride treatment, polyethyleneimine-mediated transfection, polybrene-mediated transfection, protoplast fusion, electroporation, virus or phage infection, encapsulation of polynucleotide in liposome, and direct microinjection of DNA into nucleus.For mammalian cells, transfection can be either transient or stable.

[0210] The host cell can be a yeast cell, such as S. cerevisiae, an insect cell, such as Sf9 cell, a mammalian cell, such as CHO cell, and a bacterial cell, such as E. coli. Thus, the cell can be a prokaryotic or eukaryotic host cell. The cell can be a cell suitable for culturing in liquid or solid medium. Alternatively, the host cell is a cell that is part of a multicellular organism, such as a transgenic plant or animal. In some embodiments, the host cell is a mammalian cell.

[0211] In some aspects, methods can be used to introduce viral vectors containing modified nucleic acids disclosed herein into cellular hosts for replication and packaging, including, but not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes in combination with nuclear localization signals. In embodiments in which viral vector function is provided by transfection with a viral vector, standard methods for effecting viral infection can be used.

[0212] In some embodiments, the packaging function may include genes for replication and packaging of the viral vector. Thus, for example, the packaging function may include functions necessary for viral gene expression, viral vector replication, viral vector recovery from an integrated state, viral gene expression, and packaging of the viral vector into viral particles, as needed. The packaging function may be provided together or separately in the packaging cell using a gene construct such as a plasmid or amplicon. The packaging function may be present extrachromosomally in the packaging cell or integrated into the chromosomal DNA of the cell. Examples include genes encoding AAV Rep and Cap proteins.

[0213] In some embodiments, helper functions may include helper virus elements necessary to establish active infection of packaging cells, which is required to initiate packaging of the viral vector. Examples include functions derived from adenovirus, baculovirus, and / or herpesvirus that are sufficient to result in packaging of the viral vector. For example, adenovirus helper functions typically include the adenovirus components E1a, E1b, E2a, E4, and VA RNA. Packaging functions can be provided by infecting packaging cells with the necessary viruses. Packaging functions can be provided together or separately in packaging cells using genetic constructs such as plasmids or amplicons. Packaging functions can be present extrachromosomally in the packaging cells or integrated into the chromosomal DNA of the cells.

[0214] Any suitable helper virus function can be used.For example, when packaging cell is insect cell, baculovirus can function as helper virus.Herpesvirus can also be used as helper virus in AAV packaging method.

[0215] Any method for introducing nucleotide sequences with helper functions into a cellular host for replication and packaging may be used, including, but not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes in combination with nuclear localization signals. In embodiments in which helper functions are provided by transfection with a viral vector or infection with a helper virus, standard methods for effecting viral infection may be used.

[0216] Any suitable permissive or packaging cell known in the art can be used to produce packaged viral vectors. Mammalian or insect cells are preferred. Examples of cells useful for producing packaging cells in the practice of the present invention include 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.

[0217] In some embodiments, the cell line used as packaging cell is an insect cell line.Any insect cell that allows AAV replication and can be maintained in culture can be used in accordance with the present invention.Examples include Spodoptera frugiperda, Drosophila spp. cell line, such as Sf9 or Sf21 cell line, or mosquito cell line, such as Aedes albopictus cell line.A preferred cell line is Spodoptera frugiperda Sf9 cell line. The following references are incorporated herein for their teachings regarding the use of insect cells for expression of heterologous polypeptides, methods for introducing nucleic acids into such cells, and methods for maintaining such cells in culture: Methods in Molecular Biology, ed. Richard, Humana Press, NJ (1995); O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al., J. Vir. 63:3822-8 (1989); Kajigaya et al., Proc. Nat'l. Acad. Sci. USA 88: 4646-50 (1991); Ruffing et al., J. Vir. 66:6922-30 (1992); Kimbauer et al., Vir. 219:37-44 (1996); Zhao et al., Vir. 272:382-93 (2000), and Samulsk i et al., U.S. Patent No. 6,204,059.

[0218] During production, packaging cells may contain one or more viral vector functions along with helper and packaging functions sufficient to effect replication and packaging of the viral vector. These various functions may be provided to the packaging cell together or separately using genetic constructs such as plasmids or amplicons, and may be present extrachromosomally within the cell line or integrated into the cell's chromosome.

[0219] The cells may be supplied with any one or more of the functions already integrated, for example, a cell line in which one or more vector functions have been integrated extrachromosomally or into the cell's chromosomal DNA, a cell line in which one or more packaging functions have been integrated extrachromosomally or into the cell's chromosomal DNA, or a cell line in which helper functions have been integrated extrachromosomally or into the cell's chromosomal DNA.

[0220] Pharmaceutical Composition In some aspects, the present disclosure also provides pharmaceutical compositions comprising the modified nucleic acid sequences, polynucleotides, expression cassettes, vectors, or expression constructs disclosed herein. In some aspects, compositions are provided comprising an expression construct or delivery vector (e.g., a viral vector packaged in an AAV capsid) comprising the modified nucleic acid sequence encoding insulin and / or glucokinase as disclosed herein. In some aspects, the composition is a gene therapy composition. In some aspects, the composition is a pharmaceutical composition, and the pharmaceutical composition comprises a pharmaceutically acceptable carrier, adjuvant, diluent, solubilizer, filler, preservative, and / or excipient.

[0221] Such pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, diluents and / or excipients are described, for example, in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins, 2000. This can be done.

[0222] In some embodiments, the composition is for use as a medicine. In some embodiments, the medicine is used to prevent, reduce or ameliorate, delay, cure, reverse, and / or treat the symptoms of diabetes. In some embodiments, the diabetes can be type 1 diabetes, type 2 diabetes, or monogenic diabetes. In some embodiments, the subject to be treated is a mammal, for example, a cat, a rodent (mouse, rat, gerbil, guinea pig, mouse or rat), a dog, or a human.

[0223] In some embodiments, the modified nucleic acids, expression constructs, delivery vectors and / or compositions are used to prevent, reduce or ameliorate, delay, reverse, cure, and / or treat the symptoms of diabetes when the modified nucleic acids, expression constructs, delivery vectors and / or compositions are capable of exhibiting an anti-diabetic effect. An anti-diabetic effect is observed when glucose disposal in the blood is increased and / or glucose tolerance is improved. ) which can be assessed using techniques known to those skilled in the art. In this context, an "increase" (respectively, an "improvement") means at least an increase that is detectable using an assay known to those skilled in the art or using an assay performed in the experimental part (respectively, a detectable improvement).

[0224] An antidiabetic effect can also be observed when a physician assesses a slowing of the progression of typical symptoms (i.e., insulitis, beta cell loss). A reduction in typical symptoms associated with diabetes can mean a slowing of the progression of symptom development or a complete disappearance of symptoms. Symptoms, and also their reduction, can be assessed using a variety of methods, most often the same methods used to diagnose diabetes, including clinical examinations and routine laboratory tests. Such methods include both macroscopic and microscopic methods, as well as molecular, biochemical, immunohistochemical, and other methods.

[0225] A medicament (modified nucleic acid, expression construct, delivery vector, composition, etc.) as defined herein is preferably capable of alleviating a symptom or characteristic of a patient, or of a cell, tissue or organ of said diabetic patient, if said symptom or characteristic is reduced or no longer detectable at least one week, one month, six months, one year or more after treatment with a modified nucleic acid, viral expression construct, viral vector, or composition disclosed herein.

[0226] The modified nucleic acids, expression constructs, delivery vectors, or compositions disclosed herein that are used to prevent, reduce, or ameliorate, delay, reverse, cure, and / or treat the symptoms of diabetes may be suitable for administration to cells, tissues, and / or organs in vivo of an individual suffering from diabetes or at risk of developing diabetes, and can be administered in vivo, ex vivo, or in vitro. The combinations and / or compositions can be administered directly or indirectly to cells, tissues, and / or organs in vivo of an individual suffering from diabetes or at risk of developing diabetes, and can be administered directly or indirectly in vivo, ex vivo, or in vitro. In some embodiments, the mode of administration is intramuscular.

[0227] In some embodiments, the modified nucleic acids, expression constructs, delivery vectors, or compositions disclosed herein can be administered directly or indirectly using suitable means known in the art. In some embodiments, the modified nucleic acids, expression constructs, delivery vectors, or compositions disclosed herein can be delivered directly to an individual, or to the individual's cells, tissues, or organs. Depending on the disease or condition, the individual's cells, tissues, or organs may be as previously defined herein. In some embodiments, the modified nucleic acids, expression constructs, delivery vectors, or compositions disclosed herein are dissolved in a solution compatible with the delivery method. For intravenous, subcutaneous, intramuscular, intrathecal, intraarticular, and / or intraventricular administration, the solution may be saline. In some embodiments, the administration is intramuscular. In some embodiments, the intramuscular administration is performed using a multi-needle. In some embodiments, a therapeutically effective dose of the modified nucleic acids, expression constructs, vectors, or compositions described herein is administered in a single and unique dose, thereby avoiding regular repeated administration. In some embodiments, the single dose is administered to muscle tissue. In some embodiments, a single dose is administered to skeletal muscle tissue. In some embodiments, a single dose comprises multiple injections (e.g., 2, 3, 4, or 5) into one or more muscles (e.g., multiple muscle groups).

[0228] In some embodiments, a compound may be present in the composition of the present invention. The compound may be useful for the delivery of modified nucleic acid or a composition comprising the same. In some embodiments, the compound is a compound capable of forming a complex, nanoparticle, micelle, liposome that delivers each component defined herein complexed or entrapped in vesicles or liposomes through the cell membrane, or a combination thereof. Many of these compounds are known in the art. In some embodiments, the additional compound is polyethyleneimine (PEI) or similar cationic polymers, including polypropyleneimine or polyethyleneimine copolymers (PEC) and derivatives, synthetic amphiphiles (SAINT-18), Lipofectin™, DOTAP, or a combination thereof.

[0229] How to use The present disclosure also provides methods for preventing, reducing or ameliorating, delaying, reversing, curing, and / or treating the symptoms of diabetes, comprising administering any of the modified nucleic acids, polynucleotides, expression cassettes, delivery vectors, or expression constructs disclosed herein to a subject in need thereof. In some embodiments, the diabetes may be T1DM. In some embodiments, the diabetes may be T2DM. In some embodiments, the method is gene therapy. In certain embodiments, the methods of the present disclosure comprise administering (e.g., intramuscularly) any of the modified nucleic acids, polynucleotides, expression cassettes, delivery vectors, or expression constructs disclosed herein to a cell, tissue, or subject in need thereof. In certain embodiments, the method comprises administering (i) a modified (or wild-type or unmodified) nucleic acid, polynucleotide, expression cassette, delivery vector, or expression construct encoding a human insulin (Ins) protein (e.g., preproinsulin or a variant thereof), and / or (ii) a modified (or wild-type or unmodified) nucleic acid, polynucleotide, expression cassette, delivery vector, or expression construct comprising a nucleic acid encoding a human glucokinase (Gck) protein. In some embodiments, the hIns nucleic acid sequence is a modified hIns sequence disclosed herein, and the hGck nucleic acid sequence is a modified hGck sequence disclosed herein. In some embodiments, the hIns nucleic acid sequence is a wild-type or unmodified hIns sequence disclosed herein, and the hGck sequence is a modified hGck sequence disclosed herein. In certain embodiments, the administration of (i) and (ii) is concurrent or sequential.

[0230] Certain aspects of the present disclosure are directed to methods of use comprising administering a polynucleotide encoding a human insulin (Ins) protein (e.g., preproinsulin or a variant thereof), the polynucleotide comprising: (i) a nucleotide sequence encoding a signal peptide, where optionally, 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 acids B10, B28, and / or B29 of the human insulin B chain, C1 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 comprising a cleavage site. In some aspects, the signal peptide is a wild-type preproinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence. In some aspects, the cleavage site is a furin cleavage site.

[0231] Certain aspects of the present disclosure are directed to methods of use comprising administering a polynucleotide comprising a nucleic acid encoding a human insulin (Ins) protein, the nucleic acid comprising 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. In some embodiments, the encoded human Ins protein comprises (i) a signal peptide (e.g., a wild-type preproinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence) and (ii) 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 embodiments, the encoded human insulin protein further comprises a cleavage site (e.g., a furin cleavage site).

[0232] Certain embodiments of the present 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 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. In some embodiments, the polynucleotide comprises at least two nucleic acid sequences encoding human Ins proteins. In some embodiments, 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, and the two ORF nucleotide sequences may be the same or different. In some embodiments, the polynucleotide further comprises an IRES sequence. In some embodiments, the at least two ORF nucleotide sequences are separated by an IRES sequence. In some embodiments, the encoded human Ins protein comprises a signal sequence and a proinsulin polypeptide. In some embodiments, the encoded human Ins protein comprises any of the amino acid sequences 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 embodiments, the encoded human Ins protein is preproinsulin. In some embodiments, the encoded human Ins protein comprises the amino acid sequence of SEQ ID NO:41, SEQ ID NO:144, or SEQ ID NO:145. In some embodiments, the polynucleotide or nucleic acid sequence further comprises a 5' UTR and / or a 3' UTR. In some embodiments, 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.

[0233] Certain aspects of the present disclosure are directed to methods of use comprising administering a polynucleotide comprising a nucleic acid encoding a human insulin (Ins) protein (e.g., preproinsulin or a variant thereof), the nucleic acid comprising (i) a nucleotide sequence encoding a signal peptide (e.g., a wild-type preproinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence) and (ii) a proinsulin polypeptide comprising an amino acid modification at a position selected from amino acids B10, B28, and / or B29 of the human insulin B chain, C1 and / or C32 of the human insulin C chain, or any combination thereof, relative to the corresponding amino acid in wild-type proinsulin (or an amino acid modification at a position selected from amino acids H34, P52, K53, R55, L86, or any combination thereof, relative to the corresponding amino acid in wild-type preproinsulin). In some aspects, the signal peptide is not the wild-type preproinsulin signal sequence (e.g., the wild-type preproinsulin sequence is replaced with the IL-6 signal sequence or the fibronectin signal sequence). In some embodiments, 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. In some embodiments, the polynucleotide further comprises a cleavage site (e.g., a furin cleavage site).

[0234] Certain embodiments of the present disclosure are directed to methods of use comprising administering 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%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 61-80 and 162. In some embodiments, the encoded human Gck protein comprises the amino acid sequence of SEQ ID NO: 82. In some embodiments, the polynucleotide or nucleic acid sequence encoding the Gck protein further comprises a 5' UTR and / or a 3' UTR. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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, 89-96, and 163-164. In some aspects, the nucleic acid is operably linked to a promoter (e.g., a eukaryotic promoter). Certain aspects of the present disclosure are directed to expression cassettes comprising a polynucleotide of the present disclosure and a heterologous expression control sequence operably linked to the nucleic acid sequence. In some aspects, the nucleic acid is operably linked to a polyadenylation (polyA) element.

[0235] Certain aspects of the present disclosure are directed to methods of use that include administering a vector (e.g., a viral vector, a non-viral vector, a plasmid, a lipid, or a lysosome) comprising a polynucleotide or expression cassette of the present disclosure. In some aspects, the vector is an adeno-associated virus (AAV) vector or a lentiviral vector. Certain aspects of the present disclosure are directed to methods of administering a recombinant AAV (rAAV) particle comprising an AAV capsid and a vector genome comprising a polynucleotide or expression cassette of the present disclosure. In some aspects, the AAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRH8, AAVrh9, AAV9, AAVrh10, AAV10, AAVRH10, AAV11, and AAV12.

[0236] Certain advantages of the gene therapy methods disclosed herein include the possibility of administering the modified nucleic acids, polynucleotides, expression cassettes, delivery vectors, or expression constructs disclosed herein to result in expression of a therapeutic gene throughout the diabetic subject's life. WO 2012 / 007458 discloses the generation of two viral vectors, one expressing an insulin gene and one expressing a glucokinase gene, as a treatment for diabetes. Furthermore, WO 2016 / 110518 discloses a single-vector gene construct containing an insulin gene and a glucokinase gene. In certain aspects, the present disclosure provides improved nucleic acid sequences, expression constructs, and / or delivery vectors for the treatment or prevention of diabetes that increase insulin and / or glucokinase expression, reduce adverse immune responses, and / or allow for the administration of lower doses of viral vectors.

[0237] In some aspects, the methods of the present disclosure alleviate or reduce one or more symptoms of diabetes in an individual, a cell, tissue, or organ of said individual, or alleviate or reduce one or more characteristics or symptoms 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.

[0238] Treatment recommendations for adults with diabetes generally target HbA1c<7.0% without significant hypoglycemia. In some embodiments, the "normal range" of HbA1c is <7.0%, e.g., <6.5%, <6.0%, <5.7%, e.g., between about 5.0% and about 6.5%. Most commercially available products reduce HbA1c to between 0.5% and 1.50%. In some embodiments, the methods of the present disclosure normalize HbA1c levels in treated diabetic subjects to those in non-diabetic subjects, e.g., within 8 weeks. In some embodiments, the methods of the present disclosure allow for the reduction and / or regulation of blood glycated hemoglobin (HbA1c) levels in subjects. In some embodiments, the HbA1c level in the subject after treatment is <7.0% (e.g., <6.5%, <6.0%, <5.7%, e.g., between 5.0% and 6.5%), e.g., within 8 weeks after treatment.

[0239] Insulin plays a central role in regulating lipid metabolism in the liver, adipose tissue, and intestine. (Verges B. Insulin sensitivity and lipids. Diabetes Metab. 2001 Apr;27(2 Pt 2):223-7. PMID: 11452214.) In uncontrolled type 1 diabetes, patients Glucose is unavailable and an alternative fuel source is required. In adipose tissue, insulin inhibits hormone-sensitive lipase, which normally promotes triglyceride storage in adipocytes and reduces the release of free fatty acids from adipose tissue into the circulation. Low circulating insulin levels significantly reduce lipoprotein catabolism (Taskinen MR. Lipoprotein lipase in diabetes. Diabetes Metab Rev. 3:551-570. 1987 doi: 10.1002 / dmr.5610030208. 1987). Lipolysis occurs, resulting in the release of circulating triglycerides. Elevated levels of triglyceride-rich lipoproteins (chylomicrons, VLDL) lead to hypertriglyceridemia. In some aspects, the disclosed methods reduce the level of triglyceride-rich lipoproteins (e.g., chylomicrons or VLDL) in a subject (e.g., a subject suffering from diabetes), cells, tissues, or organs of the subject, the methods comprising administering to the subject one or more of the modified nucleic acids, polynucleotides, expression cassettes, vectors, or expression constructs disclosed herein.

[0240] In the liver, ketone bodies (β-hydroxybutyrate (β-HB) and acetoacetate (AcAc)) are produced by the β-oxidation of fatty acids. During fasting or dietary carbohydrate restriction, ketones can serve as an alternative energy source in glucose-restricted conditions and provide up to 80% of the brain's energy requirements. Although beneficial in the short term, chronically high circulating ketones can produce undesirable 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. doi:10.1016 / j.freeradbiomed.2016.03.020), which can be fatal. In some aspects, the methods of the present disclosure reduce ketone levels in a subject (e.g., a subject with diabetes), a cell, tissue, or organ of the 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.

[0241] In some aspects, the methods of the present disclosure result in (i) a reduction and / or modulation of blood glycated hemoglobin (HbA1c) levels in a subject, (ii) a reduction in circulating ketones in a subject, (iii) a reduction in triglycerides in a subject, or (iv) any combination thereof.

[0242] In some embodiments, the method or use is carried out in vitro, for example, using cell culture. In some embodiments, the method or use is carried out in vivo. In some embodiments, the modified nucleic acid, polynucleotide, expression cassette, delivery vector, or expression construct disclosed herein is combined with an additional compound known to be used to treat diabetes in an individual. In some embodiments, the method further comprises administering recombinant insulin, for example, via regular injection.

[0243] In some embodiments, the methods disclosed herein are not repeated. In some embodiments, the methods disclosed herein are repeated every year, or every two, three, four, five, six, seven, eight, nine, or ten years.

[0244] In some embodiments, the method comprises administering a therapeutically effective dose of a modified nucleic acid, expression construct, vector, or composition described herein, wherein the administration is single, e.g., avoiding periodic repeated administration. In some embodiments, the single dose is administered to muscle tissue. In some embodiments, the single dose is administered to skeletal muscle tissue. In some embodiments, the single dose comprises multiple injections (e.g., 2, 3, 4, or 5) into one or more muscles (e.g., multiple muscle groups).

[0245] It is understood that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections describe one or more exemplary embodiments of the invention as contemplated by the inventors, but are not exhaustive, and thus are not intended to limit the scope of the invention and the appended claims in any manner.

[0246] The breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0247] Having described the invention, it is further illustrated in the following examples, which are included herein for illustrative purposes only and are not intended to limit the invention. [Example]

[0248] Example 1 Modified insulin nucleic acid The following modified human insulin nucleic acid sequences (shown in Table 1) were designed in silico, corresponding to SEQ ID NOS: 1-16, 84-88, 123-141, and 160-161. The 5' UTR sequence (SEQ ID NOS: 42, 83, 146, or 148) is in bold, the ORF sequence is underlined (SEQ ID NOS: 43-57, 110-122), and the 3' UTR sequence is in italics (SEQ ID NOS: 60, 97, 98, 99, 100, 101, or 149). The 5' UTR, having the sequence of SEQ ID NOS: 42, was further modified to remove CTAG at positions 1-4. Thus, in one particular construct, the 5' UTR contained nucleic acids 5-329 of SEQ ID NOS: 42. In some constructs, an IRES sequence was added between the two insulin ORF sequences, and the IRES sequence is shown in bold and italics (SEQ ID NO: 143). [Table 1-1] Table 1-2 Table 1-3 Table 1-4 Table 1-5 Table 1-6 Table 1-7 Table 1-8 Table 1-9 Table 1-10 Table 1-11 Table 1-12 Table 1-13 Table 1-14 Table 1-15 Table 1-16 Table 1-17 Table 1-18 [Table 1-19] [Table 1-20] [Table 1-21] [Table 1-22] [Table 1-23] [Table 1-24] [Table 1-25] [Table 1-26]

[0249] In some embodiments, the nucleic acid comprises a codon-optimized and CpG-reduced sequence relative to the wild-type and / or unmodified human insulin nucleic acid sequence (e.g., SEQ ID NO: 1). The modified nucleic acid was chemically synthesized, prepared in an expression cassette containing a CMV promoter, and cloned into an expression plasmid. The modified sequence was confirmed by Sanger sequencing.

[0250] Insulin secretion from HEK cells transfected with pAAV-insulin plasmids was tested. The pAAV-insulin plasmids AAV1-CMV-hInsB10D_2 (SEQ ID NO: 160), AAV1-CMV-unmodified hIns (SEQ ID NO: 1), AAV1-CMV-modified hIns22 (SEQ ID NO: 123), AAV1-CMV-modified hIns6 (SEQ ID NO: 6), AAV1-CMV-modified hIns8 (SEQ ID NO: 8), AAV1-CMV-modified hIns23 (SEQ ID NO: 124), AAV1-CMV-modified h ...B10D_2 (SEQ ID NO: 160), AAV1-CMV-unmodified hIns (SEQ ID NO: 1), AAV1-CMV-modified hInsB10D_2 (SEQ ID NO: 160), AAV1-CMV-unmodified hInsB10D_2 (SEQ ID NO: 160), AAV1-CMV-unmodified hInsB10D_2 (SEQ ID NO: 160 AAV1-CMV-modified hIns24 (SEQ ID NO: 125), AAV1-CMV-modified hIns25 (SEQ ID NO: 126), AAV1-CMV-modified hIns_2 (SEQ ID NO: 127), AAV1-CMV-modified hIns27 (SEQ ID NO: 128), AAV1-CMV-modified hIns28 (SEQ ID NO: 129), AAV1-CMV-modified hIns29 (SEQ ID NO: 130), AAV1-CMV-modified AAV1-CMV-modified hIns30 (SEQ ID NO: 131), AAV1-CMV-modified hIns31 (SEQ ID NO: 132), AAV1-CMV-modified hIns32 (SEQ ID NO: 133), AAV1-CMV-modified hIns33 (SEQ ID NO: 134), AAV1-CMV-modified hIns34 (SEQ ID NO: 135), AAV1-CMV-modified hIns35 (SEQ ID NO: 136), AAV1-CMV-modified hIns36 (SEQ ID NO: 137), HEK293 cells were transfected with 0.5 μg (FIG. 1B) or 0.1 μg (FIG. 1C) of AAV1-CMV-modified hIns37 (column #137), AAV1-CMV-modified hIns38 (column #139), AAV1-CMV-modified hIns39 (column #140), or AAV1-CMV-modified Ins40 (column #141) in 24-well plates. Extracellular insulin levels were determined by ELISA assay. The pAAV-insulin plasmid exhibits insulin expression after transfection.

[0251] Example 2 Modified Gck nucleic acid The following modified human glucokinase (Gck) nucleic acid sequences (shown in Table 2) were designed in silico, corresponding to SEQ ID NOS: 20-39, 89-96, and 163-164. The 5' UTR sequence (SEQ ID NOS: 42 or 83) is in bold, the ORF sequence is underlined (SEQ ID NOS: 61-80 and 162), and the 3' UTR is in italics (SEQ ID NOS: 60, 102, 103, 104, 105, 106, 107, 108, or 109). The 5' UTR, having the sequence of SEQ ID NOS: 42, was further modified to remove CTAG at positions 1-4. Thus, in one particular construct, the 5' UTR contained nucleic acids 5-329 of SEQ ID NOS: 42. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] Table 2-13 Table 2-14 Table 2-15 Table 2-16 Table 2-17 Table 2-18 Table 2-19 Table 2-20 Table 2-21 Table 2-22 Table 2-23 Table 2-24 Table 2-25 Table 2-26 Table 2-27 Table 2-28 Table 2-29 [Table 2-30] [Table 2-31] [Table 2-32] [Table 2-33] [Table 2-34] [Table 2-35]

[0252] In some embodiments, the nucleic acid comprises a codon-optimized and CpG-reduced sequence relative to the wild-type and / or unmodified human Gck nucleic acid sequence (e.g., SEQ ID NO: 19). The modified nucleic acid is chemically synthesized, prepared in an expression cassette containing a CMV promoter, and cloned into an expression plasmid. The modified sequence is confirmed by Sanger sequencing.

[0253] GcK expression was examined in HEK cells transfected with the pAAV-Gck plasmid. HEK293 cells were transfected with pAAV-GcK plasmids AAV1-CMV-hGckWT (SEQ ID NO: 19), AAV1-CMV-hGckWT_2 (SEQ ID NO: 163), AAV1-CMV-modified hGck9 (SEQ ID NO: 68), AAV1-CMV-modified hGck10 (SEQ ID NO: 69), AAV1-CMV-modified hGck11 (SEQ ID NO: 70), AAV1-CMV-modified hGck12 (SEQ ID NO: 71), AAV1-CMV-modified hGck13 (SEQ ID NO: 72), AAV1-CMV-modified hGck14 (SEQ ID NO: 73), AAV1-CMV-modified hGck15 (SEQ ID NO: 74), and AAV1-CMV-modified hGck16 (SEQ ID NO: 75) at 2.5 μg per well in a 6-well plate. Cell pellets were collected 48 hours after transfection, and intracellular insulin levels were determined by ELISA. The pAAV-GcK plasmid exhibited GcK expression after transfection.

[0254] Example 3 Comparative in vitro analysis of human insulin vectors The infectivity and efficacy (insulin mRNA expression, insulin secretion into the cell culture medium, and biological activity of the secreted insulin) of AAV1-CMV-human insulin vectors carrying insulin variants (SEQ ID NO: 1, SEQ ID NO: 87, and SEQ ID NO: 88) were investigated. 2v6.11 cells were infected with AAV1-CMV-hInsWT (SEQ ID NO: 110), AAV1-CMV-Ins5 (SEQ ID NO: 87), and AAV1-CMV-Ins7 (SEQ ID NO: 88) vectors at different MOIs. Infectivity assays quantified the intracellular content of vector genomes. Potency assays measured human insulin mRNA expression and insulin secreted into the cell culture medium. The secreted insulin was also evaluated for functionality. method

[0255] 3.1 Infection of 2v6.11 cells with the AAV1-CMV-human insulin vector The day before infection, 2v6.11 cells were seeded into 24-well plates at a density of 2E+05 cells / well. Cells were grown at 37°C and 8.5% CO in growth medium (DMEM + 10% FBS) supplemented with antibiotics (penicillin = 10,000 U / ml, streptomycin = 10,000 μg / ml) and 1 μg / ml ponasterone A.

[0256] Prior to infection, cells were assessed for adequate cell confluence (70-80%) under a bright-field microscope. To assess cell count, cells from four wells were trypsinized and quantified using a Scepter 2.0 Handheld Automated Cell Counter (Merck-Millipore). Cells were infected with the AAV1-CMV-human insulin vectors AAV1-CMV-hInsB10D (wild-type) (SEQ ID NO: 110), AAV1-CMV-Ins5 (SEQ ID NO: 87), and AAV1-CMV-Ins7 (SEQ ID NO: 88) (Table 3) at MOIs of 1000, 2000, and 4000 vg / cell. Cells were infected in quadruplicate for both infectivity and potency assays. Uninfected 2v6.11 cells (NI) were used as a negative control. Infections were repeated three times on different days using cells at different cell passages. [Table 3]

[0257] 3.2 Infectivity test Twenty-four hours after infection, cells were observed for viability under a bright-field microscope. The cell culture medium was then aspirated, and the cells were washed twice with 500 μl of 1× PBS and collected in 200 μl of 1× PBS using a cell scraper. Intracellular vector genomes were extracted using DNeasy Blood & Tissue Kits (Qiagen) and amplified by Taqman qPCR using an oligo set targeting the ITR2 sequence. Vector genomes were quantified by interpolation from a standard curve generated using serial dilutions of standard DNA.

[0258] 3.3 Sample Collection for Potency Testing Forty-eight hours after infection and after assessing cell viability, the cell culture medium was aspirated and replaced with 400 μl of DMEM + 1% BSA warmed to 37°C, and the plate was returned to the incubator. After 5 hours of incubation, for protein readings, 350 μl of cell culture medium was collected into a 1.5 ml microtube and centrifuged at 600 × g for 10 minutes at 4°C, and 300 μl of the supernatant was transferred to a new tube. For mRNA expression, the remaining medium was aspirated from the wells, and the cells were gently washed with 500 μl of 1× PBS and collected in 350 μl of RLT + β-mercaptoethanol (10 μl / ml) (RNeasy Mini Kit, Qiagen). Samples were stored at -80°C until processing.

[0259] 3.4 Human insulin mRNA expression RNA was extracted using the RNeasy Mini Kit (Qiagen) and RNase-free DNase I (Qiagen) according to the manufacturer's protocol, except that on-column DNAse I digestion was extended from the standard 15 minutes to 30 minutes to ensure adequate degradation of the infectious AAV vector genome. One microgram of each RNA sample was reverse transcribed using the Transcriptor FirstStrand cDNA Synthesis Kit (Roche). qPCR was performed in triplicate using Taqman Probes Master (Roche) and 2 μl of sample (diluted 1 / 10). To quantify expression, a primer-probe mix targeting the SV40 poly(A) signal (a sequence common to all human insulin plasmids) 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 the housekeeping gene hRplp0 was used for normalization (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).

[0260] 3.5 Quantitative analysis of secreted human insulin Secreted insulin was measured in duplicate in medium samples diluted 1 / 10 with milliQ water using an Insulin ELISA Kit (Crystal Chem).

[0261] 3.6 Functional analysis of secreted human insulin The bioactivity of insulin produced by infected cells and secreted into the cell culture medium was measured using iLite Insulin Assay Ready Cells (Svar Life Science). Briefly, 40 μl of standard or infected cell culture medium and 40 μl of iLite Insulin Assay Ready Cells, previously thawed and resuspended in RPMI supplemented with 9% FBS (heat-inactivated) and antibiotics (penicillin = 10,000 U / ml, streptomycin = 10,000 μg / ml), were added to a 96-well white plate. After 5 hours of incubation at 37°C and 5% CO2, the plate was equilibrated to room temperature, and 80 μl of ONE-Glo Luciferase Assay Reagent was added to the wells. Cells were lysed for 10 minutes, and luminescence was measured using a plate reader. Recombinant human insulin (Life Technologies) was used for the standard curve. 3.7 Statistical analysis

[0262] Each MOI was analyzed independently for the three studies using Anova and Tukey's multiple comparison test.

[0263] result 2v6.11 cells were infected with AAV1-human insulin vectors carrying human insulin variants Ins5 and Ins7 and WT human insulin, and vector infectivity and efficacy were assessed in three independent studies.

[0264] 3.8 Infectivity assay Three independent infection studies performed in 2v6.11 cells with the AAV1-human insulin vectors AAV1-CMV-hInsB10D (SEQ ID NO: 1), AAV1-CMV-Ins5 (SEQ ID NO: 87), and AAV1-CMV-Ins7 (SEQ ID NO: 88) showed no significant differences in the ability of the different vectors to infect cells at the three MOIs tested: 1000 vg / cell (1K), 2000 vg / cell (2K), and 4000 vg / cell (4K), as indicated by intracellular vector genome content (vg / ng DNA) (Figures 3A-3C and Table 4). [Table 4-1] [Table 4-2]

[0265] Infectivity data corresponding to each MOI and each assay were analyzed by Anova and Tukey's multiple comparison test.

[0266] 3.9 Potency Assays 3.9.1 Human insulin mRNA expression Quantification of human insulin expression levels revealed no significant difference between vectors AAV1-CMV-hInsB10D (SEQ ID NO: 1) and AAV1-CMV-Ins5 (SEQ ID NO: 87) in the three studies performed (Figs. 4A-4C and Table 5). In contrast, mRNA expression levels mediated by infection with vector AAV1-CMV-Ins7 (SEQ ID NO: 88) were significantly lower than those mediated by AAV1-CMV-hInsB10D (SEQ ID NO: 110) and AAV1-CMV-Ins5 (SEQ ID NO: 87) (Figs. 4A-4C and Table 5). [Table 5]

[0267] Human insulin mRNA expression data corresponding to each MOI and each assay were analyzed by Anova and Tukey's multiple comparison test. Bold and italic text indicates statistical significance.

[0268] 3.9.2 Quantitative analysis of secreted human insulin When insulin secreted into the cell culture medium by 2v6.11 cells infected with different AAV1-human insulin vectors was quantified using an Insulin ELISA kit (Crystal Chem), no significant difference was observed between the vectors AAV1-CMV-hInsB10D (SEQ ID NO: 110) and AAV1-CMV-Ins5 (SEQ ID NO: 87) (Figures 5A-5C and Table 6). As observed for mRNA expression readouts (Figures 4A-4C and Table 5), the level of insulin secretion mediated by the vector AAV1-CMV-Ins7 (SEQ ID NO: 88) was significantly lower than that mediated by the vectors AAV1-CMV-hInsB10D (SEQ ID NO: 1) and AAV1-CMV-Ins5 (SEQ ID NO: 87) (Figures 5A-5C and Table 6). Because data from the 1K infection were below the minimum standard and could not be quantified, statistical analysis was performed using data from the 2K and 4K infections. [Table 6]

[0269] Data for secreted human insulin corresponding to each MOI and each assay were analyzed by Anova and Tukey's multiple comparison test. Bold and italic text indicates statistical significance.

[0270] 3.9.3 Functional analysis of secreted human insulin The activity of insulin produced by infected cells and secreted into the cell culture medium was measured using iLite Insulin Assay Ready Cells (Svar Life Science). According to the results obtained for mRNA expression and human insulin protein readouts (Figures 4A-4C and 5A-5C and Tables 5 and 6), the insulin activity observed for vectors AAV1-CMV-hIns_B10D (SEQ ID NO: 1) and AAV1-CMV-Ins5 (SEQ ID NO: 87) was not significantly different, while AAV1-CMV-Ins7 (SEQ ID NO: 88) still showed significantly lower insulin activity (Figures 6A-6C and Table 7). [Table 7]

[0271] The human insulin activity data corresponding to each MOI and each assay were analyzed by Anova and Tukey's multiple comparison test. Bold and italic text indicates statistical significance.

[0272] Example 4 Comparative in vitro analysis of human glucokinase vectors The infectivity and efficacy (mRNA expression, protein content, and biological activity) of AAV1-CMV-human glucokinase vectors carrying wild-type (SEQ ID NO: 19) and human glucokinase variants Gck8 (SEQ ID NO: 93) and Gck12 (SEQ ID NO: 95) were investigated. To this end, 2v6.11 cells were infected with AAV1-CMV-hGckWT (SEQ ID NO: 19), AAV1-CMV-Gck8 (SEQ ID NO: 93), and AAV1-CMV-Gck12 (SEQ ID NO: 95) at different MOIs. Infectivity assays quantified the intracellular content of vector genomes. Efficacy assays measured human glucokinase mRNA expression, glucokinase intracellular content, and glucokinase activity.

[0273] method 4.1 Infection of 2v6.11 cells with the AAV1-CMV-human glucokinase vector The day before infection, 2v6.11 cells were seeded into 24-well plates at a density of 2E+05 cells / well. Cells were grown at 37°C and 8.5% CO in growth medium (DMEM + 10% FBS) supplemented with antibiotics (penicillin = 10,000 U / ml, streptomycin = 10,000 μg / ml) and 1 μg / ml ponasterone A.

[0274] Prior to infection, cells were assessed for adequate cell confluence (70-80%) under a bright-field microscope. To assess cell count, cells from four wells were trypsinized and quantified using a Scepter 2.0 Handheld Automated Cell Counter (Merck-Millipore). Cells were infected with the AAV1-CMV-human glucokinase vectors AAV1-CMV-hGckWT, AAV1-CMV-Gck8, and AAV1-CMV-Gck12 (Table 8) at MOIs of 1000, 2000, and 4000 vg / cell. Cells were infected in quadruplicate for each specific analysis: infectivity, human glucokinase mRNA expression, intracellular human glucokinase, and glucokinase activity. Uninfected 2v6.11 cells (NI) were used as a negative control. Infections were repeated three times on different days using cells at different cell passages. [Table 8]

[0275] 4.2 Infectivity assay Twenty-four hours after infection, cells were observed for viability under a bright-field microscope. The cell culture medium was then aspirated, and the cells were washed twice with 500 μl of 1× PBS and collected in 200 μl of 1× PBS using a cell scraper. Intracellular vector genomes were extracted using DNeasy Blood & Tissue Kits (Qiagen) and amplified by Taqman qPCR using an oligo set targeting the ITR2 sequence. Vector genomes were quantified by interpolation from a standard curve generated using serial dilutions of standard DNA.

[0276] 4.3 Human glucokinase mRNA expression Forty-eight hours after infection and after assessing cell viability, cells were gently washed with 500 μl of 1x PBS and collected in 350 μl of RLT + β-mercaptoethanol (10 μl / ml) (RNeasy Mini Kit, Qiagen). RNA was extracted using the RNeasy Mini Kit (Qiagen) and RNase-free DNase I (Qiagen) according to the manufacturer's protocol, except that the on-column DNAse I digestion was extended from the standard 15 minutes to 30 minutes to ensure adequate degradation of the infectious AAV vector genome. 1 μg of each RNA sample was reverse transcribed using the Transcriptor FirstStrand cDNA Synthesis Kit (Roche). qPCR was performed in triplicate using Taqman Probes Master (Roche) and 2 μl of sample (diluted 1 / 10). To quantify expression, a primer-probe mix targeting the SV40 poly(A) signal (a sequence common to all human glucokinase variants) 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 the housekeeping gene hRplp0 was used for normalization (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).

[0277] 4.4 Quantitative analysis of intracellular glucokinase content Forty-eight hours after infection, cells were gently washed with 500 μl of 1× PBS per well. Then, cells were scraped with 200 μl of ice-cold 1× PBS and collected in a microfuge tube. To obtain cell extracts, cells were frozen (liquid nitrogen) and thawed (37°C water bath) three times, centrifuged at 5000 × g for 10 minutes at 4°C, and the supernatant was saved and stored at -80°C. Human glucokinase was measured in duplicate (standard and sample) using a Human Glucokinase ELISA Kit (Abcam). Samples were diluted 1 / 20 using 1× diluent N provided by the ELISA kit. Glucokinase content was normalized by the total protein content, quantified in duplicate in cell extracts by the BCA method, using a 1 / 10 dilution of the sample in milliQ water.

[0278] 4.5 Glucokinase activity assay 48 hours after infection, cells were gently washed with 500 μl of 1×PBS / well. Then, 250 μl of trypsin was gently added to the wells, mixed, and excess trypsin was removed by aspiration. After a 2-minute incubation at room temperature, cells were pipetted up and down. The cells were collected in 750 μl of DMEM + 10% FBS by pipetting and transferred to a 1.5 ml microtube. The cells were pelleted at 600 × g for 10 minutes at 4°C, the supernatant was aspirated, and the cell pellet was stored at -80°C until processing.

[0279] Glucokinase activity was measured using a Glucokinase Activity Assay Kit (AssayGenie). The manufacturer's protocol was followed, except that the cell pellet was sonicated in 250 μl of Gck assay buffer containing 2.5 mM DTT. 10 μl of a 10-fold dilution of the sample was used in the assay.

[0280] 4.6 Statistical analysis Each MOI was analyzed independently for the three studies using Anova and Tukey's multiple comparison test.

[0281] result 2v6.11 cells were infected with AAV1-human glucokinase vectors carrying the human glucokinase variants Gck8 and Gck12 and WT hGck, and vector infectivity and efficacy were assessed in three independent studies.

[0282] 4.7 Infectivity assay The three AAV1-human glucokinase vectors, AAV1-CMV-hGckWT (SEQ ID NO: 19), AAV1-CMV-Gck8 (SEQ ID NO: 93), and AAV1-CMV-Gck12 (SEQ ID NO: 95), showed no consistent significant differences in their ability to infect 2v6.11 cells at the three MOIs tested (1000 vg / cell (1K), 2000 vg / cell (2K), and 4000 vg / cell (4K)), as indicated by intracellular vector genome content (vg / ng DNA) in three different assays (Figures 7A-7C and Table 9). [Table 9]

[0283] Infectivity data corresponding to each MOI and each assay were analyzed by Anova and Tukey's multiple comparison test. Bold and italic text indicates statistical significance.

[0284] 4.8 Potency Assays 4.8.1 Human glucokinase mRNA expression Quantification of human glucokinase expression levels revealed no consistent significant differences between vectors AAV1-CMV-hGckWT (SEQ ID NO: 19) and AAV1-CMV-Gck8 (SEQ ID NO: 93), carrying hGckWT and the glucokinase variant Gck8, respectively (Figures 8A-8C and Table 10). AAV1-CMV-Gck12 (SEQ ID NO: 95), containing the glucokinase variant Gck12, tended to mediate lower mRNA expression, although not consistently across assays and MOIs, compared with AAV1-CMV-hGckWT (SEQ ID NO: 19) and especially AAV1-CMV-Gck8 (SEQ ID NO: 93) (Figures 8A-8C and Table 10). [Table 10]

[0285] Human glucokinase mRNA expression data corresponding to each MOI and each assay were analyzed by Anova and Tukey's multiple comparison test. Bold and italic text indicates statistical significance.

[0286] 4.8.2 Quantitative analysis of intracellular glucokinase content When the intracellular glucokinase content of 2v6.11 cells infected with different AAV1-human glucokinase vectors was quantified using a Glucokinase ELISA kit (Abcam), no significant differences were observed between vectors across multiple MOIs and assays (Figures 9A-9C and Table 11). [Table 11]

[0287] The data for intracellular glucokinase content corresponding to each MOI and each assay were analyzed by Anova and Tukey's multiple comparison test. Bold and italic text indicates statistical significance.

[0288] 4.8.3 Glucokinase Activity Assay Glucokinase activity in cell extracts of infected cells was measured using a Glucokinase Activity Assay Kit (AssayGenie). Consistent with the results observed for mRNA expression and protein readouts (Figures 8A-8C and 9A-9C and Tables 10 and 11), the glucokinase activity observed for vectors AAV1-CMV-hGckWT (SEQ ID NO: 19), AAV1-CMV-Gck8 (SEQ ID NO: 93), and AAV1-CMV-Gck12 (SEQ ID NO: 95) did not significantly differ between vectors across multiple MOIs and assays (Figures 10A-10C and Table 12). [Table 12]

[0289] Glucokinase activity data corresponding to each MOI and each assay were analyzed by Anova and Tukey's multiple comparison test. Bold and italic text indicates statistical significance.

[0290] Example 5 Reversal of type 1 diabetic mice through expression of insulin and glucokinase in skeletal muscle To evaluate the efficacy of a single administration of AAV vector insulin and glucokinase constructs in removing glucose from the blood, C57Blk6 mice were injected into the skeletal muscles (quadriceps, gastrocnemius, and cranial tibia) of both hind limbs with AAV1 containing the human insulin gene (SEQ ID NO: 1) and AAV1 containing the rat glucokinase gene.

[0291] Mice were first treated with 40 mg of streptozotocin (STZ) for five consecutive days to deplete beta cells in the pancreas, thereby eliminating the production of natural mouse insulin and allowing blood glucose levels to reach approximately 600 mg / dL. The animals were then administered an equal mixture of AAV1-insulin (SEQ ID NO: 1) and AAV1-rat glucokinase in three distinct muscles in both hind limbs. In separate groups, the same total dose of AAV1-insulin (SEQ ID NO: 1) and AAV1-rat glucokinase was administered in a lower total volume to two hind limb muscles (quadriceps and gastrocnemius). These animals were compared with STZ-vehicle-treated and non-diabetic vehicle-treated animals (n = 10–11 / group). STZ-treated diabetic mice receiving AAV1-insulin (SEQ ID NO: 1) and AAV1-rat glucokinase restored and maintained normoglycemia and HbA1C levels under fed and fasted conditions, as observed in previous studies.

[0292] Administration of gene therapy to two muscles was equally effective as administration to three muscles. Streptozotocin-treated diabetic mice receiving AAV1-insulin (SEQ ID NO: 1) and AAV1-rat glucokinase restored normoglycemia under fed (Figures 11A-11C and 12) and fasted conditions (data from week 4 are shown) and maintained normoglycemia for up to 5 weeks after injection. Furthermore, mice treated with higher concentrations of vector in only two large muscle groups (quadriceps and gastrocnemius) appeared to perform equally or better than mice treated with three muscle groups (Figures 11A-11C and 12). These results indicate that muscle anatomy and blood flow may be considerations in allometric translation. Therapeutic effects were observed as early as 1 week after dosing, with significant reductions in hyperglycemia observed by 2 weeks within the range of untreated control animals. The combined action of AAV-mediated basal insulin production and glucokinase activity may generate a "glucose sensor" in skeletal muscle that allows for proper regulation of glucose in diabetic animals and may be responsible for the complete reversal of diabetes in treated animals.

[0293] Example 6 Development of further proinsulin variants Ten additional modified nucleic acid human insulin ORF sequences encoding preproinsulin variants were designed (shown in Table 13), corresponding to SEQ ID NOs: 150-159. These ORFs encode preproinsulin variants containing amino acid mutations in the B and / or C chains, substitutions in the signal sequence, addition of a furin endoprotease cleavage site, or combinations thereof. [Table 13-1] [Table 13-2]

[0294] Example 7 In vitro evaluation of preproinsulin variants To evaluate the level of insulin production and secretion mediated by SEQ ID NOs: 150-159 in vitro, each insulin variant was first cloned into an AAV plasmid (pAAV) under the control of the miniCMV promoter (pAAV-miniCMV-InsX, where X represents a particular ORF, i.e., SEQ ID NOs: 150-159). The names of the plasmids and the corresponding ORF sequences are listed in Table 14. [Table 14]

[0295] AAV expression cassettes were obtained by cloning human preproinsulin variants under the control of the miniCMV promoter between the ITRs of AAV2 (pAAV-miniCMV-InsX, where X indicates the specific insulin variant).

[0296] Serotype 1 single-stranded AAV vectors (AAV1-InsX, where X indicates the specific proinsulin variant) encoding a preproinsulin variant under the control of a miniCMV promoter and human glucokinase under the control of an RSV promoter were produced by triple transfection of HEK293 cells according to standard methods (Ayuso, E. et al., 2010. Curr Gene Ther. 10(6):423-36). Ten roller bottles (850 cm) were transfected with 1000 ribosomal RNA (RIRNA) and 1000 ribosomal RNA (RIRNA) for 10 min. 2 Cells were grown to 80% confluence in DMEM 10% FBS in a flat-bottom plate (Corning™, Sigma-Aldrich Co., Saint Louis, MO, US) and cotransfected with a plasmid carrying an expression cassette flanked by AAV2 ITRs, a helper plasmid carrying the AAV2 rep gene and the cap gene of serotype 1 AAV, and a plasmid carrying adenovirus helper functions by the calcium phosphate method. A non-coding plasmid was used to produce a null vector (pAAV-null). AAV was purified using an optimized method based on polyethylene glycol precipitation steps and two successive cesium chloride (CsCl) gradients. This second-generation CsCl-based protocol dramatically reduced empty AAV capsids and DNA and protein impurities (Ayuso, E. et al., 2010. Curr Gene Ther. 10(6):423-36). The purified AAV vector was dialyzed against PBS, filtered, and stored at -80°C. The viral genome titer was determined by quantitative PCR according to the protocol described for the AAV2 reference standard (Lock M, et al., Hum. Gene Ther. 2010; 21:1273-1285), using linearized plasmid DNA as a standard curve. The vector was constructed according to molecular biology techniques well known in the art.

[0297] First, HEK293 cells were transfected with equimolar amounts of pAAV-miniCMV-Ins1-8 plasmids. HEK293 cells were cultured in 24-well plates and transfected with 0.8 µg of DNA per well using Lipofectamine 2000 according to the manufacturer's instructions (Thermo Fisher Scientific). Untransfected HEK293 cells and HEK293 cells transfected with an AAV plasmid containing no transgene (pAAV-null) served as controls. The pAAV-miniCMV-Ins3 plasmid mediated both the highest intracellular insulin content and insulin secretion into the culture medium (Figures 13A-13B). These results were not attributable to the enhanced insulin expression levels in HEK293 cells transfected with the pAAV-miniCMV-Ins3 plasmid compared to the remaining variants (Figures 14A-14D).

[0298] Next, HEK293 cells were transfected with pAAV-miniCMV-Ins3, pAAV-miniCMV-Ins9, or pAAV-miniCMV-Ins10. HK293 cells transfected with pAAV-null plasmid were used as a control. Consistent with previous observations, cells transfected with pAAV-miniCMV-Ins3 performed better than those transfected with pAAV-miniCMV-Ins9 or pAAV-miniCMV-Ins9 (Figure 15).

[0299] Example 8 In vivo evaluation of the biological activity of preproinsulin variants To assess biological activity, AAV1 vectors encoding preproinsulin variants, AAV1-Ins1, AAV1-Ins3, AAV1-Ins4, AAV1-Ins5, or AAV1-Ins6 (see Table 13), were generated and their efficacy in improving glucose disposal in vivo was evaluated in healthy mice. For this purpose, CD1 mice were cultured at 3×10 11Mice were treated with either the AAV-Ins or AAV1-null vectors of the viral genome (vg). Mice were anesthetized with an intraperitoneal injection of ketamine (100 mg / kg) and xylazine (10 mg / kg). Hind limbs were shaved, and vectors were administered intramuscularly in six injection sites distributed across the quadriceps, gastrocnemius, and cranial tibia of each hind limb, with a total volume of 180 μl.

[0300] Three weeks after AAV administration, a glucose tolerance test was performed. To perform the glucose tolerance test, awake mice were fasted overnight (16 hours) and then administered an intraperitoneal injection of glucose (2 g / kg body weight). Blood glucose was measured in tail vein blood samples at the indicated time points.

[0301] No differences in blood glucose levels after an intraperitoneal glucose tolerance test were observed between cohorts of control mice treated with AAV1 vectors encoding WT preproinsulin, preproinsulin variants 1, or 4 (Figures 16A-16C and 17). Meanwhile, mice receiving the AAV1-Ins3 vector exhibited improved glucose tolerance compared to healthy mice (Figures 16D and 17). Treatment with AAV1-Ins6 partially improved glucose disposal (Figures 16E and 17). 6×10 11 vg of AAV-Ins6 vector-treated mice, and 3 × 10 11 The results for control mice treated with vg AAV1-Ins3 vector are shown in Figure 16F and Figure 17.

[0302] Example 9 In vitro evaluation of AAV-Ins or AAV-Gck stimulation of TLR9 Differences in TLR9 stimulation were detected in HEK-Dual™ hTLR9 cells transduced with AAV1-ratGck or AAV1-hInsB10D_2 (Fig. 18A). TLR9 stimulation was reduced in cells transduced with modified AAV1-GCK constructs (1) containing the CMV promoter and a modified hGck8 coding sequence (AAV1-hGck8) and (2) containing the CMV promoter and a modified hGck12 coding sequence (AAV1-hGck12) compared with the wild-type AAV1-GCK control construct (AAV1-hGckWT) containing the CMV promoter and the hGckWT coding sequence (Fig. 18B). TLR9 stimulation was similar between cells transduced with the control AAV1-CMV-hInsB10D and modified AAV1-Ins constructs (1) containing the CMV promoter and a modified hIns5 coding sequence (AAV1-hIns5) and (2) containing the CMV promoter and a modified hIns7 coding sequence (AAV1-hIns7) (Figure 18C). The slopes of TLR9 stimulation are shown in Table 15. The results show reduced stimulation of TLR9 by the construct containing the modified GcK coding sequence, suggesting reduced immune activation by the modified constructs. [Table 15-1] [Table 15-2]

[0303] Example 10 In vivo evaluation of AAV-Ins and AAV-Gck vectors in diabetic mice The efficacy of AAV1 vector constructs expressing rat glucokinase and human insulin was evaluated in a streptozotocin-induced model of diabetic C57BL / 6J mice after administration of six intramuscular injections. Two cohorts of mice (8–9 weeks old at the start of dosing) were obtained. One cohort was administered five daily intraperitoneal (ip) doses of streptozotocin (50 mg / kg; STZ) to induce diabetes. The second cohort was administered five daily intraperitoneal doses of sodium citrate buffer to serve as nondiabetic controls.

[0304] After induction, animals were fed food ad libitum and provided with municipal tap water, which had been reverse osmosis treated and chlorinated to maintain 1-6 ppm chlorine, via an automated water system. Animals were allowed to acclimate to the animal facility for 4 days before determining baseline body weight, nonfasting blood glucose, and circulating mouse insulin levels. Animals were then blocked and assigned to treatment groups to ensure there were no significant differences within or between groups for any of these parameters.

[0305] Before treatment, animals were anesthetized with isoflurane in oxygen, and 30 microliter (μL) doses were administered by direct injection with an insulin syringe into the quadriceps, gastrocnemius, and tibialis anterior muscles of each hind limb. Construct AAV1-926+AAV1-927 (a 1:1 mixture of rat GcK and human insulin, respectively, as previously reported in a study by the Bosch laboratory (Mas et al., 2006) and in U.S. Patent No. 9,309,534, hereby incorporated by reference) was administered as a control for comparison. The vector was administered at either a high, medium (medium), or low dose. The high dose was 3-fold higher than the low dose and 1.5-fold higher than the medium dose. Dosing was consistent across all experiments. For each injection (6 total per mouse), the total volume of vector suspension (30 μL) per injection was injected bilaterally into a selected set of muscles (tibialis anterior, quadriceps, and gastrocnemius). Treatment groups are summarized in Table 16. [Table 16]

[0306] Body weight and nonfasting blood glucose were determined weekly. For consistency, all measurements were performed at the same time of day (1–3 p.m.). Fasting blood glucose was determined at week 4, and an oral glucose tolerance test (OGTT) was performed at week 8. At the end of the study, blood samples were collected from all animals to measure blood glucose, mouse and human circulating insulin, HbA1c, and other metabolic parameters. Muscle, liver, and pancreatic tissue samples were collected, weighed, and stored for evaluation of mRNA, protein levels, and protein activity. Baseline values ​​for all mice treated with either STZ or sodium citrate buffer are shown in Table 17. [Table 17]

[0307] The test article was effective in significantly lowering blood glucose to the level of nondiabetic controls by day 33 (Figure 19). Once normoglycemia was achieved, this effect was maintained throughout the course of the study. Treatment appeared to prevent the weight loss often associated with untreated type 1 diabetes and restored several metabolic parameters to levels associated with normoglycemia. The optimal profile of kinetics and glucose-lowering effect was 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).

[0308] These data further demonstrated that AAV1 vectors can be used to express insulin and glucokinase in skeletal muscle. Treatment with these vectors in a mouse model of type 1 diabetes rapidly restored normoglycemia under fed and fasted conditions and maintained it for 8 weeks.

[0309] 10.1 Circulating insulin levels in diabetic mice after administration of AAV-Ins and AAV-Gck vectors Administration of STZ abolished all detectable circulating mouse insulin. To evaluate the efficacy of AAV1 vector constructs containing either the native or IL-6 signal sequence-containing coding sequences for hInsB10D or hInsB10H, circulating human insulin was measured in STZ mice after a 6-hour fast 4 weeks after intramuscular (i.m.) injection of the AAV1 vector constructs. All samples were analyzed by a validated plate-based ELISA method.

[0310] Fasting circulating insulin levels in C57Bl / 6 mice reported in the literature typically range from 0.75 to 1.0 ng / mL. By week 4, mice injected with AAV1-926 + AAV1-927 (high dose) and hINSB10D (containing the coding sequence of SEQ ID NO: 110) + AAV1-GCK (AAV926) (medium dose) were euthanized due to persistent hypoglycemia. At the censored endpoint, the mean circulating human insulin levels in these groups were 10.1 ng / mL and 4.5 ng / mL, respectively. At week 4, circulating human insulin levels in mice injected with hInsB10D (containing the coding sequence of SEQ ID NO: 110) + AAV1-GCK (AAV926) (low dose) and hInsB10H + IL6 (containing the coding sequence of SEQ ID NO: 120) + AAV1-GCK (AAV926) (high dose) reached levels of 5.27 ± 0.34 and 3.39 ± 0.25 ng / ml, respectively (Figure 20A). Eventually, these two groups were also euthanized due to hypoglycemia.

[0311] As shown in Figure 19, animals administered hInsB10H (comprising the coding sequence of SEQ ID NO: 111) + AAV1-GCK (AAV926) (high dose) and hInsB10H + IL6 (comprising the coding sequence of SEQ ID NO: 120) + AAV1-GCK (AAV926) (low dose) both reduced blood glucose and maintained it at or near the level of nondiabetic control mice over the study period. Circulating human insulin levels in these mice were 1.33 ± 0.14 and 0.6 ± 0.1 ng / mL, respectively (Figures 20A and 20B). These data indicate that coadministration of hINS and hGCK into the skeletal muscle of STZ-treated mice can effectively control blood glucose when circulating insulin levels are within the normal fasting (i.e., basal) range.

[0312] 10.2 Oral glucose tolerance in diabetic mice after administration of AAV-Ins and AAV-Gck vectors After the final feeding glucose measurement on day 54 and before the 12-hour dark period, animals were placed in clean cages. Food was removed, but access to water was provided throughout. After a 4-6 hour fast, animals were weighed and administered a glucose solution (0.2 mg / mL glucose at 10 mL / kg) by oral gavage at a dose of 2 g / kg glucose. Blood glucose was then determined using a handheld blood glucose meter using a second drop of blood (5-10 μL) obtained from the tail via tail snip. Measurements were taken at the following time points relative to glucose dosing: T = 0 (just before glucose dosing), 15 min, 30 min, 60 min, 90 min, and 120 min. After the final blood glucose measurement, food was returned to the cage.

[0313] Fasting blood glucose (T = 0) in nondiabetic control mice was significantly lower than that in STZ-treated animals (Figure 21). Treatment with the AAV1 vector construct significantly reduced fasting glucose compared to the levels in STZ-control mice and nondiabetic controls. After oral gavage of glucose (2 g / kg), blood glucose in non-STZ controls increased by 189 ± 17 mg / dL, peaked at 15 minutes, and returned to near-control levels by 90 minutes. In contrast, blood glucose in STZ-treated mice increased by 264 ± 37 mg / dL, peaking near 30 minutes and not returning to control levels even at 120 minutes. This data indicated that STZ-treated mice were unable to regulate glucose disposal normally after the postprandial excursion. Similar to nondiabetic controls, intramuscular injection of the vector constructs hInsB10H (containing the coding sequence of SEQ ID NO: 111) + AAV1-GCK (AAV926) (high dose) and hInsB10H + IL6 (containing the coding sequence of SEQ ID NO: 120) + AAV1-GCK (AAV926) (low dose) resulted in peak glucose excursions of 154 ± 16 and 218 ± 22 mg / dL, respectively, 15 minutes after challenge. Both treatments returned blood glucose to T = 0 levels within 60 minutes. Results from ANCOVA analysis utilizing AUC support these interpretations (Figure 22). Overall, these data suggest that intramuscular injection of these constructs can not only control fasting glucose levels but also smooth the large postprandial glucose excursions typically observed in diabetic patients.

[0314] The goal for type 1 diabetes is to normalize glycemic control without changing body weight and prevent diabetic ketoacidosis and the medical consequences of hypoglycemia. Patients with type 1 diabetes have little or no circulating insulin and must take insulin daily to stay alive. Furthermore, it has been reported that hyperglycemia resulting from streptozotocin (STZ)-induced diabetes progressively renders peripheral tissues insulin-resistant (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 presented here support the idea that providing an alternative supply of insulin to muscle, in addition to improving insulin sensitivity in peripheral tissues, offers multiple avenues for restoring glycemic control.

[0315] 10.3 HbA1c levels in diabetic mice after administration of AAV-Ins and AAV-Gck vectors Blood samples were obtained during the final non-fasting blood collection 8 weeks after injection. Blood glycated hemoglobin (HbA1c) was determined from the second drop of blood (5-10 μL) from the tail snip using a handheld HbA1c meter. This method is based on the National Institute of Allergy and Immunology (NIA) as the gold standard. Validated by comparative testing against a Glycohemoglobin Standardization Program (NGSP) certified method. HbA1c was not determined in animals that died or were euthanized due to poor health / hypoglycemia (AAV1-Ins + AAV1 GCK High Dose).

[0316] The normal range for HbA1c in nondiabetic mice is 4% to 5.6%, with diabetes defined as HbA1c >6.5%. Data from this study are shown in Figure 23. HbA1c values ​​in non-STZ control mice were 4.3 ± 0.05%. Administration of STZ resulted in a significant increase in HbA1c (9.48 ± 0.64%; p < 0.001) compared to nondiabetic controls. Treatment with the vector constructs hInsB10H + AAV1-GCK (containing the coding sequence of SEQ ID NO: 111) (AAV926) at a high dose (4.89 ± 0.15%) and hInsB10H + IL6 (containing the coding sequence of SEQ ID NO: 120) + AAV1-GCK (AAV926) at a low dose (5.03 ± 0.27%) significantly reduced blood glucose and HbA1c to the levels of nondiabetic controls.

[0317] HbA1c is a comprehensive signal that reflects the average blood glucose level over a period of time. 1 / 2 The response time is approximately 14 days. Clinically, this test is a primary tool for assessing glycemic control and has strong predictive value for diabetes and comorbidities. The goal of diabetes therapy is to maintain HbA1c in the normal range (<6.5%), and most commercially available products lower HbA1c to 0.5–1.25%. Here, chemical induction of type 1 diabetes with STZ in mice increased HbA1c from 4.3% to 9.48%. Intramuscular injection of AAV1 vectors containing hINS and rGCK virtually normalized HbA1c to that of nondiabetic controls within 8 weeks. Furthermore, these vectors resulted in a >4% reduction in HbA1c compared to STZ controls. All groups experienced a reduction in HbA1c, but only hInsB10H (containing the coding sequence of SEQ ID NO: 111) + AAV1-Gck (high dose) and hIns-B10H-IL6 (containing the coding sequence of SEQ ID NO: 120) + AAV1-Gck (low dose) survived to the week 8 blood draw, meaning that HbA1c levels were averaged over the 8 week period.

[0318] HbA1c, combined with weekly blood glucose measurements, provides a quantitative measure of both improved postprandial glucose exposure over time and reduced magnitude of glycemic excursions, suggesting that both factors can be normalized with this treatment. These results support the potential of intramuscularly co-administered hIns and GcK AAV constructs in type 1 diabetic patients to reverse this chronic and debilitating disease with a single dose.

[0319] 10.4 Serum triglyceride and ketone body levels in diabetic mice after administration of AAV-Ins and AAV-Gck vectors A mouse model of STZ-induced diabetes exhibited elevated blood triglyceride and ketone levels compared with nondiabetic controls, resulting in a shift to primarily lipid-based energy sources. Intramuscular injection of a high dose of the vector construct hInsB10H (containing the coding sequence of SEQ ID NO: 111) + AAV1-GCK (AAV926) and a low dose of hInsB10H + IL6 (containing the coding sequence of SEQ ID NO: 120) + AAV1-GCK (AAV926) reduced circulating triglyceride (Figure 24A) and ketone (Figure 24B) levels to or below those of nondiabetic controls. These data demonstrated that multiple metabolic endpoints were normalized after administration of the AAV-hInsB10H (containing either the native sequence or the IL-6 signal sequence) and AAV-GCK vectors.

[0320] 10.5 Hepatic hINS mRNA levels in diabetic mice after administration of AAV-Ins and AAV-Gck vectors qPCR analysis was used to evaluate mRNA expression in STZ-induced diabetic mice after administration of AAV-Ins and AAV-Gck constructs. hINS mRNA levels in the liver were evaluated to determine whether the AAV-1 vector escaped muscle, entered the circulation, transduced the liver, and was subsequently transcribed at detectable levels. Because non-diabetic control or STZ control mice were not injected with the vector, samples from these groups were not measured. Animals that died unexpectedly or showed distress were not analyzed. The results are shown in Table 18 and Figure 25. [Table 18]

[0321] The results of this assay showed a ΔCt of over 5 cycles, suggesting that the abundance of hINS mRNA in the livers of mice administered the three constructs tested (AAV1-mWTIns + AAV1-rGck(AAV926) high dose; AAV1-mWTIns(Ins17) + AAV1-rGck(AAV926) low dose; and AAV1-mWTIns(Ins17) + AAV1-rGck(AAV926) high dose) was very low or absent, thus suggesting that intramuscularly injected AAV remained largely in the target muscle. These results indicate that intramuscular delivery of the AAV1-Ins construct resulted in the observed normalization of blood glucose, HbA1c, ketones, and triglycerides due to transduction of the AAV-Ins and AAV-Gck vectors and protein expression in the muscle. In certain embodiments, for example, the following are provided: (Item 1) Item 2: A polynucleotide encoding a human insulin (Ins) protein, 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 acids B10, B28, and / or B29 of the human insulin B chain, C1 and / or C32 of the human insulin C chain, or any combination thereof, relative to the corresponding amino acid position in wild-type proinsulin, wherein optionally the polynucleotide further comprises a furin cleavage site. A polynucleotide comprising a nucleic acid encoding a human insulin (Ins) protein, wherein the nucleic acid comprises (a) nucleic acids 73 to 330 of any of SEQ ID NOs: 43 to 57, 110 to 116, 150 to 151, 154 to 155, or 157 to 159, nucleic acids 88 to 345 of any of SEQ ID NOs: 117 to 122, 152, or 156, or nucleic acids 79 to 336 of SEQ ID NO: 153, or (b) an open reading frame (ORF) comprising a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 43 to 57, SEQ ID NOs: 110 to 122, or SEQ ID NOs: 150 to 159. (Item 3) 3. The polynucleotide according to Item 2, wherein the encoded human Ins protein comprises amino acids 25 to 110 of SEQ ID NO: 41, amino acids 25 to 110 of SEQ ID NO: 144, amino acids 25 to 110 of SEQ ID NO: 145, any one of the amino acid sequences of SEQ ID NO: 41, SEQ ID NO: 144, and SEQ ID NO: 145. (Item 4) 4. The polynucleotide according to item 2 or 3, wherein the human Ins protein comprises a signal peptide. (Item 5) 5. The polynucleotide of any one of items 1 or 4, wherein the signal peptide is a wild-type preproinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence. (Item 6) 6. The polynucleotide according to item 5, wherein the signal peptide comprises amino acids 25 to 110 of SEQ ID NO: 41, amino acids 25 to 110 of SEQ ID NO: 144, or amino acids 25 to 110 of SEQ ID NO: 145. (Item 7) 7. The polynucleotide according to any one of items 1 to 6, wherein the human Ins protein further comprises a cleavage site. (Item 8) 8. The polynucleotide of any of items 1 to 7, wherein the nucleic acid further comprises a 5'UTR comprising a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to nucleic acids 5 to 329 of SEQ ID NO: 42. (Item 9) 9. The polynucleotide of any of items 1 to 8, wherein 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, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. (Item 10) 10. The polynucleotide of any of items 1 to 9, wherein 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, or SEQ ID NO:101, or SEQ ID NO:149. (Item 11) 11. The polynucleotide of any of items 1 to 10, wherein the 3'UTR further comprises a restriction site selected from the group consisting of BamHI, EcoRI, NdeI, EcoRV, SpeI, XbaI, NheI, VspI, NsiI, ScaI, KpnI, SspI, and PacI. (Item 12) 12. The polynucleotide of any one of items 1 to 11, comprising at least two open reading frames (ORFs) encoding human Ins proteins. (Item 13) 13. The polynucleotide of item 12, wherein the at least two ORFs are operably linked by an IRES sequence. (Item 14) 14. The polynucleotide of item 13, wherein the IRES sequence comprises a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 142 or SEQ ID NO: 143. (Item 15) The nucleic acid (a) a nucleotide sequence that is at least 85%, 90%, 95%, 99%, or 100% identical to a sequence selected from any of SEQ ID NOs: 1 to 16, or any combination thereof 11. The polynucleotide according to any one of items 1 to 10, comprising: (Item 16) The nucleic acid (a) a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to a sequence selected from any of SEQ ID NOs: 84 to 88, or any combination thereof 12. The polynucleotide of any one of items 1 to 11, comprising: (Item 17) The nucleic acid (a) a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to a sequence selected from any of SEQ ID NOs: 124 to 126, 130 to 132, and 139 to 141, or any combination thereof; 15. The polynucleotide according to any one of items 12 to 14, comprising: (Item 18) 18. The polynucleotide of any of items 1 to 17, wherein the nucleic acid is operably linked to a promoter. (Item 19) 19. An expression cassette comprising the polynucleotide of any one of items 1 to 18 and a heterologous expression control sequence operably linked to the sequence of the nucleic acid. (Item 20) 20. The expression cassette of item 19, wherein the heterologous expression control sequence is a promoter. (Item 21) 21. The polynucleotide or expression cassette of item 20, wherein the promoter is a eukaryotic promoter. (Item 22) 22. The polynucleotide or expression cassette of item 21, wherein the promoter is a CMV promoter. (Item 23) 10. The polynucleotide or expression cassette of any preceding item, wherein the nucleic acid is operably linked to a polyadenylation (polyA) element. (Item 24) A vector comprising a polynucleotide or expression cassette according to any of the preceding items. (Item 25) 25. The vector of item 24, wherein the vector is a viral vector, a non-viral vector, a plasmid, a lipid, or a lysosome. (Item 26) 25. The vector of item 24, wherein the vector is an adeno-associated virus (AAV) vector or a lentivirus vector. (Item 27) 24. A recombinant AAV (rAAV) particle comprising an AAV capsid and a vector genome comprising the polynucleotide or expression cassette of any of items 1 to 23. (Item 28) 28. The vector or rAAV particle of item 26 or 27, wherein the AAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRH8, AAVrh9, AAV9, AAVrh10, AAV10, AAVRH10, AAV11, and AAV12. (Item 29) The polynucleotide, expression cassette, vector, or rAAV particle described in any of the preceding items, wherein the signal peptide is an IL-6 signal peptide or a fibronectin signal peptide. (Item 30) A host cell comprising the polynucleotide, expression cassette, vector, or rAAV particle of any of the preceding items. (Item 31) 31. The host cell according to item 30, which is a mammalian cell. (Item 32) 30. A method for producing human Ins protein in a cell, the method comprising contacting the cell with and / or transforming the cell with the polynucleotide, expression cassette, vector, or rAAV particle described in any one of items 1 to 29, thereby producing the human Ins protein in the cell. (Item 33) 30. A method for producing human Ins protein in a subject, comprising administering to the subject a polynucleotide, expression cassette, vector, or rAAV particle described in any of items 1 to 29, thereby producing the human Ins protein in the subject. (Item 34) 30. A method of treating or ameliorating symptoms associated with diabetes in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of a polynucleotide, expression cassette, vector, or rAAV particle described in any one of items 1 to 29, thereby treating diabetes in the subject. (Item 35) 35. The method of item 34, wherein the diabetes is type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM). (Item 36) A polynucleotide comprising a nucleic acid encoding a human glucokinase (Gck) protein, said nucleic acid comprising: (a) a nucleotide sequence that is at least 85%, 90%, 95%, 99%, or 100% identical to (a) any of nucleic acids 1-1398 of SEQ ID NOs: 61-80 or 162, or (b) a sequence selected from any of SEQ ID NOs: 61-80 and 162, or any combination thereof. A polynucleotide comprising an ORF comprising: (Item 37) 37. The polynucleotide of item 36, wherein the encoded human Gck protein comprises the amino acid sequence of SEQ ID NO: 82. (Item 38) 38. The polynucleotide of claim 36 or 37, wherein the nucleic acid further comprises a 5'UTR comprising a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to nucleic acids 5-329 of SEQ ID NO: 42. (Item 39) 40. The polynucleotide of any one of items 36 to 38, wherein 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, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. 40. The polynucleotide of any of items 36 to 39, wherein 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: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. (Item 41) 41. The polynucleotide of any of items 36 to 40, wherein the 3'UTR further comprises a restriction site selected from the group consisting of BamHI, EcoRI, NdeI, EcoRV, SpeI, XbaI, NheI, VspI, NsiI, ScaI, KpnI, SspI, and PacI. (Item 42) The nucleic acid (a) a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to a sequence selected from any one of SEQ ID NOs: 20 to 39, or any combination thereof 41. The polynucleotide according to any of items 36 to 40, comprising: (Item 43) The nucleic acid (a) a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to a sequence selected from any of SEQ ID NOs: 89-96 and 163-164, or any combination thereof 41. The nucleotide according to any one of items 36 to 40, comprising: (Item 44) 44. The polynucleotide according to any of items 36 to 43, wherein the nucleic acid is operably linked to a promoter. (Item 45) 45. An expression cassette comprising the polynucleotide of any one of items 36 to 44 and a heterologous expression control sequence operably linked to the sequence of the nucleic acid. (Item 46) 46. ​​The expression cassette of item 45, wherein the heterologous expression control sequence is a promoter. (Item 47) 47. The polynucleotide or expression cassette according to item 46, wherein the promoter is a eukaryotic promoter. (Item 48) 47. The polynucleotide or expression cassette of item 46, wherein the promoter is a CMV promoter. (Item 49) 49. The polynucleotide or expression cassette according to any of items 36 to 48, wherein the nucleic acid is operably linked to a polyadenylation (polyA) element. (Item 50) 50. A vector comprising the polynucleotide or expression cassette according to any of items 36 to 49. (Item 51) 51. The vector of item 50, which is a viral vector, a non-viral vector, a plasmid, a lipid, or a lysosome. (Item 52) 52. The vector of item 51, which is an adeno-associated virus (AAV) vector or a lentivirus vector. (Item 53) 50. A recombinant AAV (rAAV) particle comprising an AAV capsid and a vector genome comprising the polynucleotide or expression cassette of any of items 36 to 49. (Item 54) 54. The vector or rAAV particle of item 52 or 53, wherein the AAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRH8, AAVrh9, AAV9, AAVrh10, and AAV10, AAV11, AAV12. (Item 55) 55. A host cell comprising the polynucleotide, expression cassette, vector, or rAAV particle of any of items 36 to 54. (Item 56) 56. The host cell of item 55, which is a mammalian cell. (Item 57) 55. A method for producing human Gck protein in a cell, comprising contacting the cell with a polynucleotide, expression cassette, vector, or rAAV particle described in any one of items 36 to 54 and / or transforming the cell with a polynucleotide, expression cassette, vector, or rAAV particle described in any one of items 36 to 54, thereby producing the human Gck protein in the cell. (Item 58) 55. A method for producing human Gck protein in a subject, comprising administering to the subject a polynucleotide, expression cassette, vector, or rAAV particle described in any of items 36 to 54, thereby producing the human Gck protein in the subject. (Item 59) 55. A method of treating or ameliorating symptoms associated with diabetes in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of a polynucleotide, expression cassette, vector, or rAAV particle described in any of items 36 to 54, thereby treating diabetes in the subject. (Item 60) 60. The method of item 59, wherein the diabetes is type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM). (Item 61) 54. A method of treating or ameliorating a symptom associated with diabetes in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of (i) a polynucleotide, expression cassette, vector, or rAAV particle described in any one of items 1 to 29, and (ii) a polynucleotide, expression cassette, vector, or rAAV particle described in any one of items 36 to 54, thereby treating diabetes in the subject. (Item 62) A method for producing human Ins protein and human Gck protein in a subject in need thereof and / or treating or ameliorating symptoms associated with diabetes in a subject in need thereof, the method comprising administering to the subject a plurality of polynucleotides, a plurality of expression cassettes, a plurality of vectors, or a plurality of rAAV particles comprising (i) a polynucleotide, expression cassette, vector, or rAAV particle described in any one of items 1 to 29, and (ii) a polynucleotide, expression cassette, vector, or rAAV particle described in any one of items 36 to 54, thereby producing human Ins protein and human Gck protein in the subject and / or treating diabetes. (Item 63) 63. The method of item 61 or 62, wherein the diabetes is type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM). (Item 64) 64. The method of any of items 61 to 63, wherein (i) the polynucleotide, expression cassette, vector, or rAAV particle of any of items 1 to 29, and (ii) the polynucleotide, expression cassette, vector, or rAAV particle of any of items 36 to 54, are administered together or sequentially. (Item 65) 65. The method of any of items 32 to 35, 57 to 60, and 61 to 64, wherein the delivery and / or administration is intramuscular. (Item 66) 66. The method of any of items 32 to 35, 57 to 60, and 61 to 65, wherein (i) blood glycated hemoglobin (HbA1c) levels are reduced and / or regulated in the subject, (ii) circulating ketones are reduced in the subject, (iii) triglycerides are reduced in the subject, or (iv) any combination thereof.

Claims

1. A polynucleotide comprising a nucleic acid encoding a human glucokinase (Gck) protein, wherein the nucleic acid encoding the human Gck protein comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 61-80 or 162.

2. The polynucleotide of claim 1, wherein the nucleic acid encoding the human Gck protein comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

68.

3. The polynucleotide described in claim 1, wherein the human Gck protein comprises the sequence of SEQ ID NO:

82.

4. The polynucleotide of claim 1, further comprising a 5'UTR comprising a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to nucleic acids 5-329 of SEQ ID NO:

42.

5. The polynucleotide of claim 1, further comprising a 5'UTR comprising an sequence at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NO:42, SEQ ID NO:83, SEQ ID NO:146, or SEQ ID NO:

148.

6. The polynucleotide of claim 1, further comprising a 3'UTR comprising a sequence at least 90%, at least 95%, at least 99%, or 100% identical to any one 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.

7. A polynucleotide described in claim 1, comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 20-39, 89-96, and 163-164.

8. A polynucleotide described in claim 1, comprising a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

27.

9. An expression cassette comprising the polynucleotide described in claim 1 and a promoter operably linked to the nucleic acid sequence.

10. An expression cassette as described in claim 9, wherein the promoter is a constitutive promoter.

11. An expression cassette as described in claim 10, wherein the promoter is a CMV promoter.

12. An expression cassette as described in claim 9, wherein the nucleic acid is operably linked to a polyadenylation (polyA) element.

13. A vector comprising the polynucleotide described in claim 1.

14. A vector comprising the expression cassette described in claim 9.

15. The vector described in claim 13 or 14, wherein the vector is a viral vector, a non-viral vector, a plasmid, a lipid, or a lysosome.

16. The vector described in claim 15, which is an adeno-associated virus (AAV) vector.

17. A recombinant AAV (rAAV) vector comprising an AAV capsid and a vector genome containing the polynucleotide described in claim 1.

18. A recombinant AAV (rAAV) vector comprising an AAV capsid and a vector genome containing the expression cassette described in claim 9.

19. An rAAV vector described in claim 17 or 18, wherein the serotype of the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh9, AAV9, AAVrhlO, AAV10, AAV11, and AAV12.

20. A host cell comprising a polynucleotide, expression cassette, vector, or rAAV vector described in any of claims 1, 9, 13, 14, 17, or 18.

21. The host cell described in claim 20, wherein the host cell is a mammalian cell.

22. A composition for use in a method for producing the human Gck protein in a cell, the composition comprising a polynucleotide, expression cassette, vector, or rAAV vector described in any one of claims 1, 9, 13, 14, 17, or 18, the method comprising contacting the cell with the polynucleotide, expression cassette, vector, or rAAV vector and / or transforming the cell with the polynucleotide, expression cassette, vector, or rAAV vector, thereby producing the human Gck protein in the cell.

23. A composition for producing the human Gck protein, comprising the host cell described in claim 20.

24. A composition for treating or ameliorating symptoms associated with diabetes in a subject in need thereof, the composition comprising a polynucleotide, expression cassette, vector, or rAAV vector described in any of claims 1, 9, 13, 14, 17, or 18.

25. The composition of claim 24, wherein the composition is formulated for intramuscular administration or delivery.

26. The composition of claim 24, characterized in that (i) blood glycated hemoglobin (HbA1c) levels are reduced and / or regulated in a subject after administration, (ii) circulating ketones are reduced in a subject after administration, (iii) triglycerides are reduced in a subject after administration, or (iv) any combination thereof.

27. The composition of claim 24 for use in combination therapy, wherein the combination therapy further comprises separate administration of recombinant insulin to the subject.

28. The composition described in claim 27, characterized in that the composition comprising the polynucleotide, the expression cassette, the vector, or the rAAV vector is administered in a single dose, and the recombinant insulin is administered via conventional injection.

29. A recombinant AAV1 (rAAV1) vector comprising an AAV1 serotype capsid and a vector genome comprising a nucleic acid encoding a human Gck protein, wherein the nucleic acid encoding the human Gck protein comprises a sequence at least 99%, or 100%, identical to SEQ ID NO: 68, and the nucleic acid encoding the human Gck protein is operably linked to a CMV promoter.

30. A composition for use in combination therapy for treating or ameliorating symptoms associated with diabetes in a subject, the composition comprising the rAAV1 vector of claim 29, and the combination therapy further comprising administering a separate composition comprising recombinant insulin.

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