Modified insulin and glucokinase nucleic acids for treating diabetes

Abstract

The present disclosure relates to modified nucleic acid sequences encoding insulin and glucokinase, expression cassettes and delivery vectors comprising the same, and methods of delivering them to treat diabetes. Certain embodiments are directed to a polynucleotide encoding a human insulin protein, 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 comprises a cleavage site.

Description

Technical Field

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

[0002] Reference to Electronically Submitted Sequence Listing The content of the sequence listing (4525_016PC05_Seqlisting_ST25, size: 240,360 bytes, and creation date: June 30, 2021) submitted as an ASCII text file electronically with this application is hereby incorporated by reference in its entirety.

Background Art

[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 resulting from the specific destruction of pancreatic β-cells. The loss of β-cells in T1DM is a consequence of an autoimmune-mediated process in which chronic inflammation called insular pancreatitis leads to β-cell destruction (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 states in children, and its incidence rises particularly rapidly among infants. T1DM is diagnosed when the autoimmune-mediated destruction of β-cells is nearly complete and the patient requires insulin replacement therapy for survival. In adults, T1DM can present similarly to T2DM, with a slow deterioration of metabolic regulation followed by a progression to an insulin-dependent state. This form is called latent autoimmune diabetes mellitus in adults (LADA) (Diabetes Atlas 4th edition, 2009, International Diabetes Federation).

[0005] T2DM is the most common form of diabetes mellitus and is thought to be caused by the interaction of 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 chosen therapy for both T1DM and T2DM. While lifelong treatment with exogenous insulin is generally successful in managing diabetes, maintaining strict glycemic control is difficult, and diabetic complications can still occur. Prolonged hyperglycemia can lead to serious microvascular or macrovascular complications, most commonly manifesting as retinopathy, neuropathy, nephropathy, cerebrovascular accidents, or myocardial infarction. These serious complications can be prevented by improving glycemic control. Unstable diabetes, in particular, a form that is prone to deterioration, can be extremely difficult to manage even with lifelong use of exogenous insulin.

[0007] Furthermore, in many developing countries, access to self-care tools and insulin may 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). Globally, the most common cause of death in children with diabetes is lack of access to insulin. Therefore, if a one-dose gene therapy approach were available, it could be extremely effective in situations where access to insulin is limited (Greenwood HL et al, 2006, PLoS Med 3.e381). Reducing hyperglycemia and maintaining normal blood glucose levels are goals of all therapeutic approaches for T1DM and T2DM. Current therapies for most diabetic patients are based on regular subcutaneous injections of both short-acting 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] Areas of disclosure This 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. [Overview of the Initiative]

[0010] Brief overview Certain embodiments of this disclosure relate to 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, the signal peptide optionally not being a wild-type preproinsulin signal sequence, and (ii) a nucleotide sequence encoding a proinsulin polypeptide, the polynucleotide comprising amino acid modifications at positions 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 positions in wild-type proinsulin, the polynucleotide optionally further comprising a cleavage site. In some embodiments, the signal peptide is a wild-type preproinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence. In some embodiments, the cleavage site is a furin cleavage site.

[0011] Certain aspects of the present disclosure relate to polynucleotides comprising nucleic acids encoding human insulin (Ins) protein, wherein the nucleic acids comprise (i) nucleotide sequences encoding a signal peptide and (ii) open reading frames (ORFs) comprising nucleotide sequences that are 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, SEQ ID NO: 144, or 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 aspects of this disclosure relate to a polynucleotide comprising a nucleic acid encoding human insulin (Ins) protein, wherein the nucleic acid comprises an open reading frame (ORF) containing nucleotide sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of sequence numbers 43–57, 110–122, or 150–159. In some aspects, the polynucleotide comprises at least two nucleic acid sequences encoding human Ins protein. In some embodiments, the polynucleotide comprises at least two ORF nucleotide sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of sequence numbers 43-57, 110-122, or 150-159, the two ORF nucleotide sequences may be the same or different. In some embodiments, the polynucleotide further comprises an IRES sequence. In some embodiments, at least two ORF nucleotide sequences are separated by the IRES sequence.

[0014] In some embodiments, the encoded human Ins protein includes a signal sequence and a proinsulin polypeptide. In some embodiments, the encoded human Ins protein includes one of the amino acid sequences of SEQ ID NO: 41 (amino acids 25-110), SEQ ID NO: 144 (amino acids 25-110), or SEQ ID NO: 145 (amino acids 25-110). In some embodiments, the encoded human Ins protein is preproinsulin. In some embodiments, the encoded human Ins protein includes 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 contains 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. 1-16, 84-88, 123-141, or 160-161.

[0016] Certain aspects of the present disclosure relate to 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) 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 one 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 embodiments, 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 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 positions B28 and / or B29 of the proinsulin B chain or positions 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 include P52D, K53R, R55K, L86R, or any combination thereof (or one or more modifications in the proinsulin B chain or C chain include proline (P) to aspartic acid (D) at position B28 of the proinsulin B chain, lysine (K) to arginine (R) at position B29 of the proinsulin B chain, arginine (R) to lysine (K) at position C1 of the proinsulin C chain, leucine (L) to arginine (R) at position C32 of the proinsulin C chain, or any combination thereof).

[0020] Certain aspects of this disclosure relate to polynucleotides comprising nucleic acids encoding human glucokinase (Gck) protein, wherein the nucleic acids comprise an ORF containing nucleotide sequences 1-1398 of any of SEQ ID NOs. 61-80 or 162, or any of SEQ ID NOs. 61-80 and 162 that are 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 aspects, the encoded human Gck protein comprises the amino acid sequence of SEQ ID NOs. 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 containing a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 42, SEQ ID NO: 42-329, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some embodiments, the nucleic acid further comprises a 3'UTR containing 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 contains nucleotide sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the sequence numbers 20-39 and 89-96, and 163-164.

[0022] In some embodiments, the nucleic acid is operably ligated to a promoter (e.g., a eukaryotic promoter). Certain embodiments of the present disclosure relate to an expression cassette comprising the polynucleotide of the present disclosure and heterologous expression regulatory sequences operably ligated to the nucleic acid sequence. In some embodiments, the nucleic acid is operably ligated to a polyadenylated (poly-A) element.

[0023] Certain aspects of this disclosure relate to vectors (e.g., viral vectors, non-viral vectors, plasmids, lipids, or lysosomes) containing the polynucleotides or expression cassettes of this disclosure. In some aspects, the vector is an adeno-associated virus (AAV) vector or a lentiviral vector. Certain aspects of this disclosure relate to recombinant AAV (rAAV) particles comprising an AAV capsid and a vector genome containing the polynucleotides or expression cassettes of this 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, AAVH10, AAV11, and AAV12. Certain aspects of this disclosure relate to host cells (e.g., mammalian cells) containing the polynucleotides, expression cassettes, vectors, or rAAV particles of this disclosure.

[0024] Certain embodiments of the present disclosure relate to a method for causing a subject to produce human Ins protein and / or human Gck protein, comprising administering the polynucleotides, expression cassettes, vectors, or rAAV particles of the present disclosure to the subject to thereby cause the subject to produce human Ins protein and / or human Gck. Certain embodiments of the present disclosure relate to a method for causing or ameliorating diabetes-related symptoms in a subject that requires treatment or amelioration of diabetes-related symptoms, comprising delivering a therapeutically effective amount of the polynucleotides, expression cassettes, vectors, or rAAV particles of the present disclosure to the subject to thereby treat the diabetes in the subject. In some embodiments, 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 target 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, including a polynucleotide encoding human insulin and a second expression cassette, including a polynucleotide encoding human Gck; a plurality of vectors, including a first vector, including an expression cassette, including a polynucleotide encoding human insulin and a second vector, including an expression cassette, including a polynucleotide encoding human Gck; or a plurality of rAAV particles, including a first rAAV particle, including an expression cassette, including a polynucleotide encoding human insulin and a second rAAV particle, including an expression cassette, including 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 this disclosure is intramuscular. In some embodiments, the methods of the present disclosure result in (i) a decrease and / or regulation of glycated hemoglobin (HbA1c) levels in the subject, (ii) a reduction in circulating ketones in the subject, (iii) a reduction in triglycerides in the subject, or (iv) any combination thereof. [Brief explanation of the drawing]

[0026] [Figure 1A-1] Figure 1A shows a list of nucleic acid sequence constructs containing unmodified human insulin (hIns) nucleic acid sequences (SEQ ID NOs. 1, 127, and 160) and modified hIns nucleic acid sequences (SEQ ID NOs. 2–16, 84–88, 123–126, and 128–141). The sequences include 3'UTR, ORF, and 5'UTR nucleic acid sequences. Certain sequences also include IRES sequences. The right-hand column shows exemplary pAAV-Ins plasmids transfected into HEK cells. [Figure 1A-2]Figure 1A shows a list of nucleic acid sequence constructs containing unmodified human insulin (hIns) nucleic acid sequences (SEQ ID NOs. 1, 127, and 160) and modified hIns nucleic acid sequences (SEQ ID NOs. 2–16, 84–88, 123–126, and 128–141). The sequences include 3'UTR, ORF, and 5'UTR nucleic acid sequences. Certain sequences also include IRES sequences. The right-hand column shows exemplary pAAV-Ins plasmids transfected into HEK cells. [Figure 1A-3] Figure 1A shows a list of nucleic acid sequence constructs containing unmodified human insulin (hIns) nucleic acid sequences (SEQ ID NOs. 1, 127, and 160) and modified hIns nucleic acid sequences (SEQ ID NOs. 2–16, 84–88, 123–126, and 128–141). The sequences include 3'UTR, ORF, and 5'UTR nucleic acid sequences. Certain sequences also include IRES sequences. The right-hand 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 to those of 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 to those of the control plasmid (AAV1-CMV-hInsB10D_2).

[0028] [Figure 2A-1]Figure 2A shows a list of nucleic acid sequence constructs containing wild-type human glucokinase (hGcK) nucleic acid sequences (SEQ ID NOs. 19 and 163) and modified hGcK nucleic acid sequences (SEQ ID NOs. 20-39 and 89-96). The sequences include 3'UTR, ORF, and 5'UTR nucleic acid sequences. The right-hand column shows exemplary pAAV-Gck plasmids transfected into HEK cells. [Figure 2A-2] Figure 2A shows a list of nucleic acid sequence constructs containing wild-type human glucokinase (hGcK) nucleic acid sequences (SEQ ID NOs. 19 and 163) and modified hGcK nucleic acid sequences (SEQ ID NOs. 20-39 and 89-96). The sequences include 3'UTR, ORF, and 5'UTR nucleic acid sequences. The right-hand column shows exemplary pAAV-Gck plasmids 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 the pAAV-Gck plasmid. The GcK expression levels of each plasmid were compared with those of 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) in 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] [Figure 4A-4C]Figures 4A–4C are graphs showing human insulin mRNA expression levels in 2v6.11 cells infected with the 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 (1000vg / cell (1K), 2000vg / cell (2K), and 4000vg / 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 the 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 (1000vg / cell (1K), 2000vg / cell (2K), and 4000vg / cell (4K)). Figure 5A is assay 1, Figure 5B is assay 2, and Figure 5C is assay 3.

[0033] [Figure 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 vector A AAV1-CMV-hInsB10D (SEQ ID NO: 110), AAV1-CMV-Ins5 (SEQ ID NO: 87), and AAV1-CMV-Ins7 (SEQ ID NO: 88) in three different MOIs (1000vg / cell (1K), 2000vg / cell (2K), and 4000vg / cell (4K)). Activity is expressed as ng / ml, with recombinant human insulin (Life Technologies) used as the 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) in three independent assays with three different MOIs (1000vg / cell (1K), 2000vg / cell (2K), and 4000vg / cell (4K)). 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 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) in three independent assays with three different MOIs (1000vg / cell (1K), 2000vg / cell (2K), and 4000vg / cell (4K)). 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 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) in three different MOIs (1000vg / cell (1K), 2000vg / cell (2K), and 4000vg / cell (4K)) in three independent assays. Figure 9A is assay 1, Figure 9B is assay 2, and Figure 9C is assay 3.

[0037] [Figure 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 (1000vg / cell (1K), 2000vg / cell (2K), and 4000vg / 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] Figures 11A–11C are graphs showing glucose levels at 3 weeks (Figure 11A), 4 weeks (Figure 11B), and 5 weeks (Figure 11C) after injection of AAV1 containing the human insulin gene (SEQ ID NO: 1) and AAV1 containing the rat glucokinase gene in individual C57Blk6 mice injected under feeding or fasting conditions. The data for week 4 in Figure 11B was collected under fasting conditions. [Figure 11B] Figures 11A–11C are graphs showing glucose levels at 3 weeks (Figure 11A), 4 weeks (Figure 11B), and 5 weeks (Figure 11C) after injection of AAV1 containing the human insulin gene (SEQ ID NO: 1) and AAV1 containing the rat glucokinase gene in individual C57Blk6 mice injected under feeding or fasting conditions. The data for week 4 in Figure 11B was collected under fasting conditions. [Figure 11C] Figures 11A–11C are graphs showing glucose levels at 3 weeks (Figure 11A), 4 weeks (Figure 11B), and 5 weeks (Figure 11C) after injection of AAV1 containing the human insulin gene (SEQ ID NO: 1) and AAV1 containing the rat glucokinase gene in individual C57Blk6 mice injected under feeding or fasting conditions. The data for week 4 in Figure 11B was collected under fasting conditions.

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

[0040] [Figures 13A-13B] Figures 13A-13B are graphs showing intracellular insulin content and insulin secretion levels in HEK293 cells transfected with an AAV plasmid (pAAV) containing a modified insulin nucleic acid sequence.

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

[0042] [Figure 15] Figure 15 is a graph showing the insulin secretion levels of HEK293 cells transfected with pAAV containing a modified insulin nucleic acid sequence.

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

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

[0045] [Figures 18A-18C] Figures 18A-18C are graphs showing TLR9 stimulation in HEK cells that were 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] Figure 19 is a graph showing the mean blood glucose levels over time in an STZ-induced mouse model of type 1 diabetes. Black circles represent the non-STZ+PBS vehicle control, white circles represent the STZ+PBS control, white triangles represent AAV1_926+AAV1_927 (high dose), black triangles with dashed lines represent B10H AAV1-Gck (high dose), black diamonds represent Ins-B10H+IL6 AAV1-Gck (low dose), black triangles with solid lines represent B10H AAV1-Gck (medium dose), and white 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 oral glucose tolerance tests 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 0–120 minutes after glucose loading in control or STZ-treated mice, 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 poor 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 poor 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). [Modes for carrying out the invention]

[0053] Detailed explanation of disclosure Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this disclosure pertains. In case of any conflict, this application, including its definitions, shall prevail. Unless the context requires otherwise, singular terms shall include plural forms, and plural terms shall include singular forms.

[0054] Throughout this disclosure, the terms “a” or “an” attached to an entity refer to one or more of those entities. For example, “a polynucleotide” is understood to represent one or more polynucleotides. Thus, the terms “a” (or “an”), “one or more,” and “at least one” may be used synonymously herein.

[0055] Furthermore, where used herein, “and / or” shall be interpreted as the specific disclosure of each of the two identified features or components, whether or not they include the other. Accordingly, the term “and / or” as 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” as used in phrases such as “A, B, and / or C” is intended to include 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 "approximately" is used herein to mean roughly, nearly, approximately, or within that range. When used in conjunction with a numerical range, the term "approximately" modifies the range by extending the boundary above and below the stated number. Generally, unless otherwise stated herein, the term "approximately" is used to modify a number by a difference of 10 percent above or below a given value (higher or lower).

[0057] The term “at least” preceding a number or set of numbers is understood to include the number adjacent to the term “at least,” and all subsequent numbers or integers that may logically be included as is evident from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 18 nucleotides in a 21-nucleotide nucleic acid molecule” means that 18, 19, 20, or 21 nucleotides have the specified property. When “at least” precedes a set of numbers or a range, it is understood that “at least” can modify each of the numbers in that set or range. Also, “at least” is not limited to integers (for example, “at least 5%” includes 5.0%, 5.1%, and 5.18%, without considering significant figures).

[0058] Nucleotide sequences are presented herein, unless otherwise specified, as single-stranded sequences, 5' to 3' in direction, and from left to right. Nucleotides and amino acids are represented herein in accordance with 37 CFR §1.822 and established usage, either by the method recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (in the case of amino acids) by either a single-letter or three-letter code.

[0059] As used herein, “polynucleotide” or “nucleic acid” means a sequence of nucleotides linked by phosphodiester bonds. Polynucleotides are presented herein in the 5' to 3' direction. The polynucleotides in this disclosure may be deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules. Nucleotide bases are represented herein by single-letter codes: 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 mean a DNA or RNA region (transcribed region) that “codes” a particular protein, such as insulin or glucokinase. When controlled by an appropriate regulatory region, such as a promoter, a coding sequence is transcribed (DNA), translated (RNA), and converted into a polypeptide in vitro or in vivo. The boundaries of a coding sequence are determined by a 5' (amino) start codon and a 3' (carboxy) stop codon. A coding sequence may include, but is not limited to, cDNA of prokaryotic or eukaryotic origin, genomic DNA of prokaryotic or eukaryotic origin, and synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

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

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

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

[0065] The term “sequence identity” is used herein to mean a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, determined by a comparison of sequences. In certain embodiments, sequence identity is calculated based on the full lengths of two given sequence numbers or a portion thereof. A portion thereof may mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or any other specified percentage of both sequence numbers. In some cases, the term “identity” may also mean the degree of sequence relevance between amino acid or nucleic acid sequences, which is determined by matching strings of such sequences.

[0066] In certain embodiments, the method for determining identity is designed to yield the greatest possible match between the sequences being tested. Methods for determining identity and similarity are systematized in publicly available computer programs.

[0067] Where referring to nucleic acids or fragments 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 sequence has at least approximately 95–99% nucleotide sequence identity.

[0068] Where used herein, and unless otherwise stated, the term “complementary” when used to describe a first nucleic acid sequence in relation to a second nucleic acid sequence means, as will be understood by those skilled in the art, the ability of an oligonucleotide or polynucleotide containing the first nucleic acid sequence to hybridize to an oligonucleotide or polynucleotide containing the second nucleic acid sequence under certain conditions to form a double-stranded structure. Such conditions may be, for example, stringent conditions, which may include: 400 mM NaCl, 40 mM PIPES, pH 6.4, 1 mM EDTA, 50°C, or 70°C, 12–16 hours, followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual,” Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions may be used, such as physiologically reasonable conditions that can occur inside living organisms. Those skilled in the art will be able to determine the set of conditions most appropriate for testing the complementarity of the two sequences, depending on the final use of the nucleotides to be hybridized.

[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 a gene relative to the direction of transcription, and is structurally identified by the presence of any other DNA sequence, including, but not limited to, any other sequence of nucleotides known to those skilled in the art to act directly or indirectly to regulate the amount of transcription from the DNA-dependent RNA polymerase binding site, the transcription start site, as well as transcription factor binding sites, repressor and activator protein binding sites, and 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 certain 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 an adjacent gene. An enhancer that inhibits transcription is also called a “silencer.” Enhancers can function in any orientation, from a location downstream of the coding sequence and within the transcription region, at distances of up to several kilobase pairs (kb) (e.g., they can be associated with the coding sequence).

[0071] The terms “operatably linked,” “operatably inserted,” “operatably positioned,” “controlled,” or “transcriptionally regulated” mean that the promoter is in the correct position and orientation to control RNA polymerase initiation and gene expression in relation to the nucleic acid. The term “operatably linked” means that the DNA sequence and regulatory sequence are linked in such a way that gene expression becomes possible when the appropriate molecule (e.g., a transcriptional activator protein) binds to the regulatory sequence. The term “operatably inserted” means that the target DNA introduced into the cell is positioned adjacent to a DNA sequence that directs the transcription and translation of the introduced DNA (i.e., facilitates the production of the polypeptide encoded by the target DNA).

[0072] The term “transgene” is used herein to mean a gene or nucleic acid molecule that is introduced into a cell. An example of a transgene is a nucleic acid that encodes a therapeutic polypeptide (e.g., a gene encoding insulin and / or a gene encoding glucokinase). In some embodiments, the gene may be present in the cell, but in some cases, it may not normally be expressed in the cell or may be expressed at an insufficient level. In this context, “insufficient” means that the gene, e.g., insulin and / or glucokinase, is normally expressed in the cell, but the conditions and / or diseases disclosed herein (e.g., diabetes) may still occur. In certain embodiments, the transgene enables increased or overexpression of the gene, e.g., insulin and / or glucokinase. The transgene may contain cell-specific sequences, sequences that do not occur naturally in the cell, or a combination of both. In certain embodiments, the transgene may include modified sequences encoding insulin, glucokinase, both insulin and glucokinase, and / or additional proteins, which may be operably linked to appropriate regulatory sequences for the intracellular expression of sequences encoding insulin, glucokinase, or both insulin and glucokinase. In some embodiments, the transgene is not integrated into the host cell's genome.

[0073] The terms “modified gene” and “modified nucleic acid” are used synonymously herein and refer to the introduction of one or more modifications or alterations to the native sequence or nucleic acid sequence of a gene. Such modifications may or may not result in mutations in 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 isolated from or prepared using a particular molecule or organism, or information from a particular molecule or organism (e.g., amino acids or nucleic acid sequences). For example, a nucleic acid sequence derived from a second nucleic acid sequence (e.g., the wild-type human insulin gene) (e.g., a modified human insulin gene) may include a nucleotide sequence or portion thereof that is identical or substantially similar to the nucleotide sequence of the second nucleic acid sequence. In some embodiments, mutants, analogs, or derivatives may 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 oriented mutagenesis, or artificially random mutagenesis. The mutagenesis used to derive polynucleotides may be intentionally oriented, intentionally random, or a mixture of each.

[0076] As used herein, the terms “delivery vector” or “vector” include any genetic element that, when associated with an appropriate regulatory element, is replicable and capable of transmitting genes or nucleic acid sequences between cells, such as plasmids, phages, transposons, cosmids, chromosomes, artificial chromosomes, viruses, and virions. Therefore, the term includes cloning and expression vehicles, as well as viral vectors. In some embodiments, a useful vector is assumed to be a vector in which the nucleic acid segment to be transcribed is under the transcriptional control of a promoter. In some embodiments, the delivery vector is selected from the group consisting of viral vectors, plasmids, lipids, and lysosomes.

[0077] In some embodiments, the biological vector includes a virus, particularly an attenuated virus and / or a replication-deficient virus. In some embodiments, the chemical vector includes a lipid complex and a naked DNA construct.

[0078] As used herein, terms such as “naked DNA” or “naked nucleic acid” refer to nucleic acid molecules that are not contained in viral particles, bacterial cells, or other encapsulation means that facilitate delivery of nucleic acids to the cytoplasm of target cells. Naked nucleic acids may be associated with means that facilitate delivery of nucleic acids to a site in a target cell (for example, to facilitate the movement of nucleic acids through the digestive tract into the target cell, to protect nucleic acids from stomach acid, and / or to help them permeate intestinal mucus), and / or to facilitate delivery of nucleic acids to the surface of target epithelial cells.

[0079] A “viral genome,” “vector genome,” or “viral vector” refers to a sequence containing one or more polynucleotide regions that encode or contain a molecule of interest, such as a protein, peptide, or polynucleotide, or multiple such molecules. Viral vectors are used to deliver genetic material into cells. Viral vectors can be modified for specific applications. In some embodiments, the delivery vector includes a viral vector selected from the group consisting of adeno-associated virus (AAV) vectors, adenovirus vectors, lentiviral vectors, or retroviral vectors.

[0080] The terms “adeno-associated virus vector” or “AAV vector,” as used herein, refer to any vector containing or derived from adeno-associated vector components and suitable for infecting mammalian cells, preferably human cells. The term AAV vector typically refers to an AAV-type virus particle or virion containing a payload. AAV vectors may be derived from various serotypes, including combinations of serotypes (i.e., “pseudotype” AAV), or from various genomes (e.g., single-stranded or self-complementary). Furthermore, AAV vectors may be replication-deficient and / or targeting. As used herein, the term “adeno-associated virus” (AAV) includes, but is not limited to, AAV 1, AAV 2, AAV 3 (including types 3A and 3B), AAV 4, AAV 5, AAV 6, AAV 7, AAV 8, AAV 9, AAV 10, AAV 11, AAV 12, AAV 13, AAVrh8, AAVrh10, AAVrh.74, snake AAV, bird AAV, cattle AAV, dog AAV, horse AAV, sheep AAV, goat AAV, shrimp AAV, AAV serotypes and clades disclosed by Gao et al. (J. Virol. 78:6381 (2004)) and Moris et al. (Virol. 33:375 (2004)), 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” may be used synonymously.

[0081] As used herein, “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 reverse-terminal repeat (ITR) region. In some embodiments, the terms “AAV vector of the disclosure” or “AAV vector disclosed herein” refer to an AAV vector comprising a polynucleotide or nucleic acid disclosed herein, for example, encapsulated in an AAV particle, encoding insulin, GcK, or a combination thereof.

[0082] Viral "transduction" of cells means the transfer of nucleic acids from a viral particle to a 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 the rAAV vector of this disclosure leads to the transfer of the rAAV genome contained in the vector (e.g., one containing the polynucleotides of this disclosure) into the transduced cell.

[0083] Cellular "transfection" refers to the introduction of genetic material into cells for the purpose of genetic modification. Transfection can be achieved by various means known in the art, such as transduction or electroporation.

[0084] As used herein, "vector" means a recombinant plasmid or virus containing polynucleotides 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 mean the cell in which polynucleotide delivery takes place, either in vitro or in vivo. AAV vectors can be transduced into both dividing and non-dividing cells.

[0086] "Recombinant" means that it can be distinguished from what is commonly found in nature.

[0087] The “serotype” of a vector or viral capsid is defined by an immunological profile that can be distinguished based on the capsid protein sequence and capsid structure.

[0088] "AAV Cap" refers to the AAV Cap proteins, VP1, VP2, and VP3, as well as their analogues.

[0089] "AAV Rep" refers to the AAV Rep protein and its analogues.

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

[0091] As used herein, the terms “effective dose,” “therapeutic effective dose,” and “sufficient dose,” for example, of gene therapy compositions comprising polynucleotides disclosed herein, refer to an amount sufficient to produce beneficial or desired results, including clinical outcomes, when administered to a subject including humans. Therefore, “effective dose” or its synonyms depend on the context in which it is applied.

[0092] The amount of a given therapeutic agent or composition corresponds to an amount that varies depending on various factors such as the given drug, pharmaceutical formulation, route of administration, type of disease or disorder, and the individual characteristics of the person being treated or the host (e.g., age, sex, and / or weight).

[0093] As used herein, the term “gene therapy” means the insertion of a nucleic acid sequence (e.g., a nucleic acid comprising a promoter operably ligated to a polynucleotide encoding a therapeutic molecule as defined herein) into the cells and / or tissues of an individual in order to treat a disease or condition. Gene therapy also includes the insertion of transgenes that are inherently inhibitory, i.e., transgenes that inhibit, reduce, or diminish 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 suitable for gene therapy.

[0094] In some embodiments, the Disclosure provides modified nucleic acids encoding wild-type or mutant insulin and / or wild-type glucokinase or functional fragments thereof. The Disclosure also provides nucleic acid constructs that include modified nucleic acids encoding wild-type or mutant insulin and / or wild-type glucokinase or functional fragments thereof as part of a sequence. For example, the Disclosure includes expression cassettes, plasmids, and / or other vectors that include the modified nucleic acid sequence together with other elements such as regulatory elements. In some embodiments, the 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 Disclosure also includes methods for expressing wild-type or mutant insulin and / or wild-type glucokinase or functional fragments thereof by delivering the modified nucleic acid sequence into a cell together with elements necessary to promote expression in the cell. This disclosure also provides gene therapy methods in which modified nucleic acid sequences encoding wild-type or mutant insulin and / or wild-type glucokinase or functional fragments thereof are administered to a subject, for example, as components of one or more vectors and / or packaged as components of one or more viral gene delivery vehicles. The treatment may be performed, for example, to treat or reduce symptoms of diabetes in a subject that requires treatment or reduction of symptoms of diabetes. Each of these embodiments of this disclosure will be discussed in further detail herein.

[0095] modified nucleic acid In some embodiments, the disclosure provides polynucleotides comprising modified nucleic acids (e.g., codon-optimized and / or CpG-reduced) encoding insulin, glucokinase, or a combination thereof. In some embodiments, the modified nucleic acid encodes human insulin (e.g., preproinsulin or proinsulin, their mutants, analogs, or variants). In some embodiments, the modified nucleic acid encodes human glucokinase (e.g., Gck, its mutants, analogs, or variants). In some embodiments, the modification to the coding sequence preserves the amino acid sequence of the wild-type or mutant insulin and / or glucokinase. 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 SEQ ID NO: 41 (amino acids 25-110), SEQ ID NO: 144 (amino acids 25-110), or SEQ ID NO: 145 (amino acids 25-110). In some embodiments, 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 embodiments, the modified nucleic acid sequence encodes human Gck (e.g., sequence number 82).

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

[0097] In some embodiments, the modified nucleic acid has reduced innate immunogenicity compared to the corresponding wild-type and / or unmodified sequence. In some embodiments, the modified nucleic acid has increased expression compared to the corresponding wild-type and / or unmodified sequence. In some embodiments, the modified nucleic acid has decreased expression compared to the corresponding wild-type and / or unmodified sequence. In some embodiments, the modified sequence is developed by manual sequence checking after an in silico method. The nucleic acids of this disclosure can be produced using molecular biology techniques; for example, modified cDNA encoding insulin or glucokinase can be obtained by PCR amplification or cDNA cloning techniques.

[0098] In some embodiments, nucleic acid sequences are modified to reduce CpG content, for example, to minimize inflammatory responses via TLR9 dimerization and related pathways. In some embodiments, certain CpG motifs inhibit or neutralize their inflammatory effects. In some embodiments, one or more of these motifs may be conserved. In some embodiments, such CpG motifs may be introduced into nucleic acid sequences to inhibit downstream effects of TLR9 dimerization.

[0099] In some embodiments, codon modification may reduce the immunogenicity of a polynucleotide encoding insulin and / or glucokinase compared to the corresponding wild-type and / or unmodified polynucleotide. In some embodiments, codon modification may increase the expression of a polynucleotide encoding insulin or glucokinase compared to the corresponding wild-type and / or unmodified polynucleotide. In some embodiments, codon modification may reduce the immunogenicity of a polynucleotide encoding glucokinase compared to the corresponding wild-type Gck polynucleotide and / or unmodified Gck polynucleotide.

[0100] The modified nucleic acids of this disclosure may exist in whole cells, in cell lysates, or in partially purified or substantially pure forms. The modified nucleic acids may be isolated. A nucleic acid is “isolated” or “substantially purified” when it is purified from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art (see, for example, 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 this disclosure may be, for example, DNA or RNA, and may or may not contain intron sequences. In some embodiments, the nucleic acid may be a cDNA molecule.

[0101] Modified insulin nucleic acid In some embodiments, 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 human Ins mutants or analogs (e.g., SEQ ID NO: 110 or SEQ ID NO: 111). In some embodiments, the polynucleotide or nucleic acid is modified relative to a sequence including a 5'UTR, ORF, and / or 3'UTR, for example, corresponding to SEQ ID NO: 1 or SEQ ID NO: 127. In some embodiments, the modified nucleic acid encodes wild-type human insulin (SEQ ID NO: 41), its variants or mutants (e.g., SEQ ID NO: 144 or SEQ ID NO: 145), or functional fragments thereof.

[0102] Insulin contains two polypeptide chains, the A chain and the B chain, linked together by a disulfide bond. This is first synthesized as a single polypeptide called preproinsulin. "Preproinsulin" is the primary translation product of the insulin gene. It is a peptide 110 amino acids long. Preproinsulin contains a proinsulin molecule with a signal peptide bound to its N-terminus. The portion of the N-terminus containing the signal peptide of preproinsulin is cleaved, leaving the remaining amino acids as "proinsulin". Amino acids 1-30 of the resulting cleaved sequence constitute the "B chain," where "B10" corresponds to position 34 of preproinsulin. Thus, for example, a "B10" proinsulin mutation corresponds to an H34 mutation in preproinsulin. In certain embodiments, where 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 mature insulin protein, the C-peptide is proteolytically cleaved, and the A and B chains are linked by disulfide bonds.

[0103] In some embodiments, the modified coding sequences disclosed herein encode a preproinsulin mutant comprising one or more mutations at the H34, P52, K53, R55, and / or L86 positions relative to the corresponding positions in wild-type preproinsulin (SEQ ID NO: 41). In some embodiments, the modified coding sequences encode a preproinsulin mutant comprising one or more mutations among 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 sequences encode a preproinsulin mutant comprising 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 containing mutations P52D, K53R, R55K, and / or L86R at the corresponding position 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 contains one or more mutations H34D, H34I, H34V, P52D, K53R, R55K, and / or L86R at the corresponding position in SEQ ID NO: 41. In some embodiments, the modified coding sequence encodes an amino acid sequence that does not contain an H34 mutation at the corresponding position in SEQ ID NO: 41.

[0104] In some embodiments, the modified nucleic acid sequence includes cleavage sites, such as furin endoprotease cleavage sites.

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

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

[0108] In some embodiments, the modified nucleic acid encodes human insulin, including amino acid modifications H34D, K53R, R55K, and L86R corresponding to the wild-type preproinsulin amino acid positions (or modifications corresponding to histidine (H) to aspartic acid (D) at position B10 of proinsulin, 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). In some embodiments, the modified nucleic acid encodes human insulin comprising amino acid modifications H34D, K53R, R55K, and L86R corresponding to the amino acid positions of wild-type preproinsulin (or modifications corresponding to histidine (H) to aspartic acid (D) at position B10 of proinsulin, 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), wherein the human insulin optionally comprises cleavage sites, e.g., furin cleavage sites, and signal peptides (e.g., wild-type preproinsulin signal sequence, IL-6 signal sequence, or fibronectin signal sequence).

[0109] In some embodiments, the modified nucleic acid encodes human insulin, including amino acid modifications H34I, K53R, R55K, and L86R (or modifications corresponding to histidine (H) to isoleucine (I) at position B10 of proinsulin, 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). In some embodiments, the modified nucleic acid encodes human insulin comprising amino acid modifications H34I, K53R, R55K, and L86R corresponding to the amino acid positions of wild-type preproinsulin (or modifications corresponding to histidine (H) to isoleucine (I) at position B10 of proinsulin, 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), and the human insulin optionally comprising cleavage sites, e.g., furin cleavage sites, and signal peptides (e.g., wild-type preproinsulin signal sequence, IL-6 signal sequence, or fibronectin signal sequence).

[0110] In some embodiments, the modified nucleic acid encodes human insulin, including amino acid modifications H34V, K53R, R55K, and L86R (or modifications corresponding to histidine (H) to valine (V) at position B10 of proinsulin, 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). In some embodiments, the modified nucleic acid encodes human insulin comprising amino acid modifications H34V, K53R, R55K, and L86R corresponding to the amino acid positions of wild-type preproinsulin (or modifications corresponding to histidine (H) to valine (V) at position B10 of proinsulin, 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), and the human insulin optionally comprising cleavage sites, e.g., furin cleavage sites, and signal peptides (e.g., wild-type preproinsulin signal sequence, IL-6 signal sequence, or fibronectin signal sequence).

[0111] In some embodiments, the modified nucleic acid encodes human insulin containing amino acid modifications P49D, K53R, R55K, and L86R corresponding to the wild-type preproinsulin amino acid positions (or modifications corresponding to proline (P) to aspartic acid (D) at position B28 of proinsulin, 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). In some embodiments, the modified nucleic acid encodes human insulin containing amino acid modifications P49D, K53R, R55K, and L86R (corresponding to the wild-type preproinsulin amino acid positions), and the human insulin optionally includes cleavage sites, e.g., furin cleavage sites, and signal peptides (e.g., wild-type preproinsulin signal sequence, IL-6 signal sequence, or fibronectin signal sequence).

[0112] In some embodiments, the modified nucleic acid encodes a human insulin (Ins) protein (e.g., preproinsulin or a variant thereof), and 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, which includes amino acid modifications at positions 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 acids in wild-type proinsulin (or amino acid modifications at positions selected from amino acids H34, P52, K53, R55, L86, or any combination thereof, relative to the corresponding amino acids in wild-type preproinsulin). In some embodiments, the signal peptide is not a wild-type preproinsulin signal sequence, but rather the wild-type preproinsulin sequence is replaced with, for example, an IL-6 signal sequence or a fibronectin signal sequence. In some embodiments, the polynucleotide further comprises cleavage sites (e.g., furin cleavage sites). In some embodiments, the encoded human Ins protein (e.g., preproinsulin or its variants) includes (i) H34D, H34I, or H34V (or 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 to the wild-type preproinsulin sequence, selected from P52, K53, R55, and / or L86 (or positions B28 and / or B29 of the proinsulin B chain or C1 and / or C32 of the proinsulin C chain).In some embodiments, one or more amino acid modifications at P52, K53, R55, and / or L86 include P52D, K53R, R55K, L86R, or any combination thereof (or, one or more modifications in the proinsulin B chain or C chain include proline (P) to aspartic acid (D) at position B28 of the proinsulin B chain, lysine (K) to arginine (R) at position B29 of the proinsulin B chain, arginine (R) to lysine (K) at position C1 of the proinsulin C chain, 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 with amino acids 25-110 of SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO: 145. In some embodiments, the human insulin protein contains 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 with SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO: 145. In some embodiments, the human insulin protein contains insertions, deletions, substitutions, or combinations thereof with respect to wild-type human insulin. In some embodiments, the human insulin protein contains at least one substitution. In some embodiments, at least one substitution is a conserved substitution. In some embodiments, at least one substitution is a non-conserved substitution.

[0114] In some embodiments, the polynucleotides of the Disclosure include an open reading frame (ORF) containing nucleic acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to nucleic acid sequences 73–330 of any of SEQ ID NOs. 43–57, 110–116, 150–151, 154–155, and 157–159, nucleic acid sequences 88–345 of any of SEQ ID NOs. 117–122, 152, and 156, or nucleic acid sequences 79–336 of SEQ ID NO. 153. In some embodiments, the ORF further includes a nucleic acid sequence encoding a signal peptide.

[0115] In some embodiments, the polynucleotides of the present disclosure are: SEQ ID NOs: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 150, 151, 152, 153, 154, 155, 156, The modified nucleic acid sequence comprises an open reading frame (ORF) containing 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 with a sequence selected from SEQ ID NO: 157, SEQ ID NO: 158, or SEQ ID NO: 159, wherein the modified nucleic acid sequence encodes 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 polynucleotides of the present disclosure include an oven reading frame (ORF) containing 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 with SEQ ID NO: 122.In some embodiments, the polynucleotides of the Disclosure include an open reading frame containing 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 embodiments, the polynucleotides of the Disclosure include an open reading frame containing a nucleic acid having the sequence of SEQ ID NO: 122. In some embodiments, the polynucleotide includes an ORF sequence present in or referenced in Table 1, Table 13, and / or Figure 1A.

[0116] In some embodiments, the polynucleotides of the present disclosure are: SEQ ID NOs: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 150, 151, 152, The ORF 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 with column number 153, sequence number 154, sequence number 155, sequence number 156, sequence number 157, sequence number 158, sequence number 159, and any combination thereof. In some embodiments, one of 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 with sequence number 122.In some embodiments, two or more ORFs are selected from the group consisting of SEQ ID NOs: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 150, 151, 152, 153, 154, 155, 156, 157, 158, 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 contains 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 with SEQ ID NO: 142 or SEQ ID NO: 143. In some embodiments, the IRES contains the nucleic acid sequence of SEQ ID NO: 142 or SEQ ID NO: 143.

[0118] In some embodiments, two or more ORFs linked by IRES include 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 with sequences including SEQ ID NOs. 110 and SEQ ID NOs. 53, SEQ ID NOs. 47 and SEQ ID NOs. 54, SEQ ID NOs. 49 and SEQ ID NOs. 56, SEQ ID NOs. 111 and SEQ ID NOs. 112 and SEQ ID NOs. 113 and SEQ ID NOs. 116, SEQ ID NOs. 120 and SEQ ID NOs. 121 and SEQ ID NOs. 122 and SEQ ID NOs. 116 In some embodiments, two or more ORFs linked through IRES include nucleic acid sequences such as SEQ ID NOs: 110 and 53, SEQ ID NOs: 47 and 54, SEQ ID NOs: 49 and 56, SEQ ID NOs: 111 and 114, SEQ ID NOs: 112 and 115, SEQ ID NOs: 113 and 116, SEQ ID NOs: 120 and 114, SEQ ID NOs: 121 and 115, or SEQ ID NOs: 122 and 116.

[0119] In some embodiments, the polynucleotides of the Disclosure further comprise modified 5'UTR nucleic acid sequences. In some embodiments, the polynucleotides of the 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 with SEQ ID NO: 42, SEQ ID NO: 42 nucleic acid sequences 5-329, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some embodiments, the polynucleotides further comprise 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, SEQ ID NO: 42 nucleic acid sequences 5-329, 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 Disclosure further comprise a modified 3'UTR nucleic acid sequence. In some embodiments, the polynucleotides of the 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 with 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 includes 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 includes a 3'UTR nucleic acid sequence present in or referenced in Table 1 and / or Figure 1A.

[0121] In some embodiments, the polynucleotides of the present disclosure are sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 84, 85, 86, 87, 88, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, sequence number The nucleic acid sequence comprises a 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 with a sequence selected from sequence 138, sequence number 139, sequence number 140, sequence number 141, sequence number 160, or sequence number 161, wherein the nucleic acid sequence encodes human insulin protein (e.g., sequence number 41, sequence number 144, or sequence number 145) or a functional fragment thereof. In some embodiments, the polynucleotides of the present disclosure include 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 with SEQ ID NO: 138.In some embodiments, the polynucleotides of the Disclosure include 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 with nucleic acid sequences 5-957 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 polynucleotides of the Disclosure include nucleic acids 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, the polynucleotides of the Disclosure include nucleic acids having the sequence of SEQ ID NO: 138. In some embodiments, the polynucleotides of the present disclosure include 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. In some embodiments, the polynucleotides include modified nucleic acids including the 5'UTR, ORF, and 3'UTR as presented in Table 1 and / or Figure 1A.

[0122] In some embodiments, the polynucleotides of the Disclosure encode human insulin containing a wild-type preproinsulin secretion signal peptide. In some embodiments, the polynucleotides of the Disclosure do not encode a wild-type preproinsulin secretion signal peptide. In some embodiments, wild-type preproinsulin is replaced by a non-insulin secretion signal. In some embodiments, the polynucleotides of the Disclosure encode human preproinsulin containing an interleukin-6 (IL-6) secretion signal peptide. In some embodiments, the polynucleotides of the Disclosure encode human preproinsulin containing a fibronectin secretion signal peptide.

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

[0124] In some embodiments, the polynucleotides of the Disclosure comprise 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 with 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 embodiments, the polynucleotides of the Disclosure include an oven reading frame containing nucleic acids 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 polynucleotides include ORF sequences present in or referenced in Table 2 and / or Figure 2A.

[0125] In some embodiments, the polynucleotides of the Disclosure further comprise modified 5'UTR nucleic acid sequences. In some embodiments, the polynucleotides of the 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 with 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 polynucleotides further comprise 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 Disclosure further comprise a modified 3'UTR nucleic acid sequence. In some embodiments, the polynucleotides of the 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 with 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 includes 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 includes a 3'UTR sequence present in or referenced in Table 2 and / or Figure 2A.

[0127] In some embodiments, the polynucleotides of the Disclosure include 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 with a sequence selected from the group consisting of SEQ ID NOs: 20, SEQ ID NOs: 21, SEQ ID NOs: 22, SEQ ID NOs: 23, SEQ ID NOs: 24, SEQ ID NOs: 25, SEQ ID NOs: 26, SEQ ID NOs: 27, SEQ ID NOs: 28, SEQ ID NOs: 29, SEQ ID NOs: 30, SEQ ID NOs: 31, SEQ ID NOs: 32, SEQ ID NOs: 34, SEQ ID NOs: 35, SEQ ID NOs: 36, SEQ ID NOs: 37, SEQ ID NOs: 38, SEQ ID NOs: 39, SEQ ID NOs: 89, SEQ ID NOs: 90, SEQ ID NOs: 91, SEQ ID NOs: 92, SEQ ID NOs: 93, SEQ ID NOs: 94, SEQ ID NOs: 95, SEQ ID NOs: 96, SEQ ID NOs: 163, or SEQ ID NOs: 164, wherein the nucleic acid sequence encodes a human glucokinase protein (e.g., SEQ ID NOs: 82) or a functional fragment thereof. In some embodiments, the polynucleotides of the present 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 with nucleic acids 5 to 2025 of sequences selected from SEQ ID NOs. 20, SEQ ID NOs. 21, SEQ ID NOs. 22, SEQ ID NOs. 23, SEQ ID NOs. 24, SEQ ID NOs. 25, SEQ ID NOs. 26, SEQ ID NOs. 27, SEQ ID NOs. 28, SEQ ID NOs. 29, SEQ ID NOs. 30, SEQ ID NOs. 31, SEQ ID NOs. 32, SEQ ID NOs. 34, SEQ ID NOs. 35, SEQ ID NOs. 36, SEQ ID NOs. 37, SEQ ID NOs. 38, or SEQ ID NOs. 39.In some embodiments, the polynucleotides of the Disclosure include 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, the polynucleotides of the Disclosure include nucleic acids 5 to 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 polynucleotides are modified nucleic acids present in or referenced in Table 2 and / or Figure 2A.

[0128] Expression construct In some embodiments, the disclosure also provides an expression cassette comprising a nucleic acid sequence, for example, a modified nucleic acid sequence encoding insulin, glucokinase, a combination thereof, or a functional fragment thereof, as disclosed herein, and a heterogeneous control sequence operably ligated to the nucleic acid sequence. In some embodiments, the heterogeneous control sequence is a promoter.

[0129] Nucleic acid constructs having a eukaryotic promoter operably ligated to the DNA of interest can be used in this disclosure. Constructs containing a DNA sequence (or corresponding RNA sequence) that can be used in accordance with this disclosure may be any eukaryotic expression construct containing the DNA or RNA sequence of interest. For example, a plasmid or viral construct (e.g., an AAV vector) can be cleaved to yield linear DNA with ligable ends. These ends can then be ligated to complementary exogenous DNA having similar ligable ends to yield an intact replicon and a biologically functional recombinant DNA molecule with desired phenotypic properties. In some embodiments, the constructs are replicable in both eukaryotic and prokaryotic hosts.

[0130] In some embodiments, the exogenous DNA used in this disclosure is obtained from suitable cells, and the constructs are prepared using techniques known in the art. Similarly, techniques for achieving the expression of exogenous DNA or RNA sequences in genetically modified host cells are known in the art (see, for example, 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; each of these is incorporated herein by reference with respect to methods and compositions for the expression of the DNA of interest in eukaryotes).

[0131] In some embodiments, the DNA construct contains a promoter to facilitate the expression of the target DNA (e.g., insulin, glucokinase, combinations thereof, or modified nucleic acids encoding fragments thereof) within secretory cells. In some embodiments, the promoter is a potent eukaryotic promoter, such as a promoter derived from cytomegalovirus (CMV), mouse mammary tumor virus (MMTV), Roussarcoma virus (RSV), or adenovirus. Exemplary promoters include, but are not limited to, a promoter derived from the pre-early gene of human CMV (Boshart et al., Cell 41:521-530 (1985)) and a promoter derived from the long-terminal repeat (LTR) of RSV (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 markers to help select cells that contain and / or express the construct (e.g., antibiotic resistance genes (such as ampicillin resistance genes) or β-galactosidase), origins of replication for stable replication of the construct in bacterial cells (preferably high copy number origins of replication), nuclear localization signals, or other elements that facilitate the production of the DNA construct, the protein encoded thereby, or both.

[0133] For expression in eukaryotes, the construct may contain at least a eukaryotic promoter operably ligated 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 ligated to a polyadenylation sequence. The polyadenylation signal sequence may be selected from any of the various polyadenylation signal sequences known in the art. In some embodiments, the polyadenylation signal sequence is an SV40 initial polyadenylation signal sequence. The construct may contain one or more introns that can increase the expression level of the DNA of interest, especially if the DNA of interest is cDNA (e.g., does not contain introns of naturally occurring sequences). Any of the various introns known in the art may be used (e.g., a human β-globin intron inserted into the construct at the 5' position of the DNA of interest).

[0134] The target DNA (e.g., modified nucleic acids encoding insulin, glucokinase, combinations thereof, or fragments thereof) may be inserted into the construct so that a 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 the therapeutic protein at the C-terminus). The production of the fusion protein can facilitate the 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] The vector for delivering the target DNA (e.g., insulin, glucokinase, combinations thereof, or modified nucleic acids encoding fragments thereof) may be viral or nonviral, or may consist of naked DNA mixed with an adjuvant such as viral particles (e.g., AAV particles) or cationic lipids or liposomes. An “adjuvant” is a substance that does not produce the desired effect itself, but acts to enhance or otherwise enhance the action of the active compound. The exact vector and vector formulation used depend on several factors, such as the target cells and / or organs to which the gene transfer will occur.

[0136] Examples of suitable promoters include the cytomegalovirus (CMV) initial promoter, long-terminal repeat promoters (LTRs), such as those derived from mouse Moloney's leukemia virus (MMLV), Roussarcoma virus, or HTLV-1, the monkey virus 40 (SV40) initial 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 in conjunction 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 embodiments, the expression cassette includes SEQ ID NOs: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 150, 151, 152, 153, 154, 155, 156, and The modified nucleic acid sequence comprises a promoter operably ligated to a modified nucleic acid sequence containing 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 with a sequence selected from 157, SEQ ID NO: 158, or SEQ ID NO: 159, wherein the modified nucleic acid sequence encodes 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 includes a promoter operably ligated to a modified nucleic acid sequence containing 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 with SEQ ID NO: 122.In some embodiments, the polynucleotides of the Disclosure include an oven reading frame containing 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 embodiments, the polynucleotides of the Disclosure include an oven reading frame containing a nucleic acid having the sequence of SEQ ID NO: 122. In some embodiments, the polynucleotides include ORF sequences present in or referenced in Table 1, Table 13 and / or Figure 1A.

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

[0139] In some embodiments, the expression cassette further comprises a modified nucleic acid comprising a 5'UTR containing 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 with 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 contains 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 contains a 5'UTR sequence present in or referenced in Table 1 and / or Figure 1A.

[0140] In some embodiments, the expression cassette further comprises a modified nucleic acid comprising a 3'UTR containing 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 with 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 includes 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 includes a 3'UTR sequence present in or referenced in Table 1 and / or Figure 1A.

[0141] In some embodiments, the expression cassette contains SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 84, 85, 86, and 87. The modified nucleic acid comprises 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 and 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, wherein the modified nucleic acid sequence encodes 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 includes 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 with SEQ ID NO: 138.In some embodiments, the expression cassette includes nucleic acids 5-957 of sequences 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 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. In some embodiments, the expression cassette includes 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, the expression cassette includes a modified nucleic acid having the sequence of SEQ ID NO: 138. In some embodiments, the expression cassette includes nucleic acids 5-957 of sequences 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 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.In some embodiments, the expression cassette comprises a modified nucleic acid including the 5'UTR, ORF, and 3'UTR, which are present in or referenced in Table 1 and / or Figure 1A.

[0142] In some embodiments, the expression cassette includes a promoter operably ligated to a modified nucleic acid sequence containing 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 with 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 includes a promoter operably ligated 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 includes an ORF sequence present in or referenced in Table 2 and / or Figure 2A.

[0143] In some embodiments, the expression cassette further comprises a modified nucleic acid comprising a 5'UTR containing 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 with 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 contains 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 contains a 5'UTR sequence present in or referenced in Table 2 and / or Figure 2A.

[0144] In some embodiments, the expression cassette further comprises a modified nucleic acid comprising a 3'UTR containing 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 with 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 includes 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 includes a 3'UTR sequence present in or referenced in Table 2 and / or Figure 2A.

[0145] 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 with a sequence selected from SEQ ID NOs: 20, SEQ ID NOs: 21, SEQ ID NOs: 22, SEQ ID NOs: 23, SEQ ID NOs: 24, SEQ ID NOs: 25, SEQ ID NOs: 26, SEQ ID NOs: 27, SEQ ID NOs: 28, SEQ ID NOs: 29, SEQ ID NOs: 30, SEQ ID NOs: 31, SEQ ID NOs: 32, SEQ ID NOs: 34, SEQ ID NOs: 35, SEQ ID NOs: 36, SEQ ID NOs: 37, SEQ ID NOs: 38, SEQ ID NOs: 39, SEQ ID NOs: 89, SEQ ID NOs: 90, SEQ ID NOs: 91, SEQ ID NOs: 92, SEQ ID NOs: 93, SEQ ID NOs: 94, SEQ ID NOs: 95, SEQ ID NOs: 96, SEQ ID NOs: 163, or SEQ ID NOs: 164, wherein the nucleic acid sequence encodes a human glucokinase protein (e.g., SEQ ID NOs: 82) or a functional fragment thereof. In some embodiments, the expression cassette comprises nucleic acids 5 to 2025 of sequences selected from SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, or 39, 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.In some embodiments, the expression cassette includes 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 includes a modified nucleic acid containing nucleic acids 5 to 2025 of SEQ ID NO: 20, 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 including the 5'UTR, ORF, and 3'UTR, which are present in or referenced in Table 2 and / or Figure 2A.

[0146] In some embodiments, the expression cassette contains 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%, and at least 95% of SEQ ID NOs: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 150, 151, 152, 153, 154, 155, 156, 157, 158, or 159. A first modified nucleic acid containing a first ORF having sequence identity of %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and a second modified nucleic acid sequence containing a second ORF having sequence identity 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%, or at least 99%, or 100%, with 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 includes a promoter operably ligated to a modified nucleic acid, with each of the first and second modified nucleic acids ligated to the first and second promoters, respectively. In some embodiments, the first modified nucleic acid sequence containing the first ORF is sequence 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 150, 151, The second modified nucleic acid sequence containing the second ORF is selected from sequence number 152, sequence number 153, sequence number 154, sequence number 155, sequence number 156, sequence number 157, sequence number 158, and sequence number 159, and is selected from the group consisting of sequence numbers 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, and sequence number 162.

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

[0149] In some embodiments, the first and second modified nucleic acid sequences further include a 5'UTR containing 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 with SEQ ID NO: 42, SEQ ID NO: 42, 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 include a 3' UTR containing 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 with 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 includes 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 includes 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 includes SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 84, 85, 86, 87, 88, 123, 124, 125, 126, 127, 128, 129, 130, 131, SEQ ID NOs: 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 160, or 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%, less A first modified nucleic acid having at least 99% or 100% sequence identity, and SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 89, 90, 91, 92, 93, 94, 95, 96, The sequence number 163, or sequence number 164, comprises 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, wherein each of the first and second modified nucleic acids is ligated to the first and second promoters, 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 with nucleic acids 5-957 of sequences selected from SEQ ID NOs. 1, SEQ ID NOs. 2, SEQ ID NOs. 3, SEQ ID NOs. 4, SEQ ID NOs. 5, SEQ ID NOs. 6, SEQ ID NOs. 7, SEQ ID NOs. 8, SEQ ID NOs. 9, SEQ ID NOs. 10, SEQ ID NOs. 11, SEQ ID NOs. 12, SEQ ID NOs. 13, SEQ ID NOs. 14, SEQ ID NOs. 15, or SEQ ID NOs. 15, or SEQ ID NOs. 16, and the second modified nucleic acid is distributed Nucleic acids 5-2025 of a sequence selected from sequence number 20, sequence number 21, sequence number 22, sequence number 23, sequence number 24, sequence number 25, sequence number 26, sequence number 27, sequence number 28, sequence number 29, sequence number 30, sequence number 31, sequence number 32, sequence number 34, sequence number 35, sequence number 36, sequence number 37, sequence number 38, or sequence number 39 have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.In some embodiments, the first modified nucleic acid sequence is 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, The first nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 139, 140, 141, 160, or 161, and the second nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 89, 90, 91, 92, 93, 94, 95, 96, 163, or 164. In some embodiments, the first modified nucleic acid comprises nucleic acids 5 to 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 to 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 includes 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 includes a 5'UTR, ORF, and 3'UTR present in or referenced in Table 2 and / or Figure 2A.

[0152] Certain aspects of this disclosure relate to expression constructs, such as vectors. In some aspects, the expression construct includes an expression cassette. In some aspects, the expression construct further includes a genome that can be stabilized in a cell and maintain an episomal state. In relation to this disclosure, in some aspects, the cell or host cell may include the cell used to construct the construct or the cell to which the construct is administered. In some aspects, the construct can be incorporated into the genome of a cell, for example, by homologous recombination or by other means. In some aspects, the expression construct is a nucleotide sequence encoding insulin and / or glucokinase, as disclosed herein, operably linked to a promoter, as presented herein, where the promoter can direct the expression of the nucleotide sequence (i.e., coding sequence) in a cell. In some aspects, the expression cassette, as used herein, includes or consists of a nucleotide sequence encoding insulin and / or glucokinase, in each case, the nucleotide sequence is operably linked to a promoter, where the promoter can direct the expression of the nucleotide sequence. In some embodiments, the viral expression construct is an expression construct intended for use in gene therapy. This can be designed to include a portion of the viral genome disclosed herein.

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

[0154] In some embodiments, the expression constructs disclosed herein are prepared using recombinant techniques, which express modified nucleic acid sequences encoding insulin and / or glucokinase in suitable cells, e.g., cultured cells or cells of multicellular organisms, as described, for example, in Ausubel et al., "Current Protocols in Molecular Biology", Greene Publishing and Wiley-Interscience, New York (1987) and Sambrook and Russell (2001, cited above); both of these are incorporated herein by reference in their entirety. See also Kunkel (1985) Proc. Natl. Acad. Sci. 82:488 (describes site-directed mutagenesis) and Roberts et al. (1987) Nature 328:731-734 or Wells, JA, et al. (1985) Gene 34:315 (describes cassette mutagenesis).

[0155] Delivery vector This disclosure also provides vectors comprising any of the modified nucleic acids, polynucleotides, or expression cassettes described herein. In some embodiments, the delivery vector is a viral vector, a non-viral vector, a plasmid, a lipid, or a lysosome. In some embodiments, the delivery vector is a viral vector. In some embodiments, 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 producing gene expression in a host compatible with the nucleotide sequence. These expression vectors include at least a suitable promoter sequence and may optionally include a transcription termination signal. Further factors necessary or useful for producing expression may 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 for expression. In some embodiments, expression vectors are suitable for replication in prokaryotic hosts such as bacteria, e.g., Escherichia coli, or can be introduced into cultured mammals, plants, insects (e.g., Sf9), yeast, fungi, or other eukaryotic cell lines. In some embodiments, expression constructs are suitable for in vivo expression.

[0157] In some embodiments, the delivery vector is sequence numbers 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 150, 151, 152, 153, 154, 155, 156, 157, The expression cassette comprises a promoter operably ligated to a modified nucleic acid sequence containing 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 with a sequence selected from sequence number 158 or sequence number 159, wherein the modified nucleic acid sequence encodes human insulin protein (e.g., sequence number 41, sequence number 144, or sequence number 145) or a functional fragment thereof. In some embodiments, the delivery vector includes an expression cassette comprising a promoter operably ligated to a modified nucleic acid sequence containing 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 with SEQ ID NO: 122.In some embodiments, the modified nucleic acid includes 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 includes an ORF having the sequence of SEQ ID NO: 122. In some embodiments, the polynucleotide includes an ORF sequence present in or referenced in Table 1, Table 13 and / or Figure 1A.

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

[0159] In some embodiments, the delivery vector includes an expression cassette containing a modified nucleic acid further comprising a 5'UTR containing 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 contains 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 contains a 5'UTR sequence present in or referenced in Table 1 and / or Figure 1A.

[0160] In some embodiments, the delivery vector comprises an expression cassette comprising a modified nucleic acid further 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 3'UTR 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 3'UTR includes 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 includes a 3'UTR sequence present in or referenced in Table 1 and / or Figure 1A.

[0161] In some embodiments, the delivery vector is 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: 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 with a sequence selected from 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 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 includes an expression cassette containing a modified nucleic acid having sequence identity 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% with sequence identity of SEQ ID NO: 138.In some embodiments, the delivery vector includes an expression cassette containing nucleic acids 5-957 of sequences 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 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. In some embodiments, the delivery vector includes an expression cassette containing 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, the delivery vector includes an expression cassette containing a modified nucleic acid sequence having the sequence of SEQ ID NO: 138. In some embodiments, the delivery vector includes an expression cassette containing modified nucleic acids including 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. In some embodiments, the delivery vector includes an expression cassette containing modified nucleic acids including 5'UTR, ORF, and 3'UTR present in or referenced in Table 1 and / or Figure 1A.

[0162] In some embodiments, the delivery vector includes an expression cassette comprising a promoter operably ligated to a modified nucleic acid containing 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 with 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 includes an expression cassette comprising a promoter operably ligated 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 includes an expression cassette containing a modified nucleic acid further comprising a 5'UTR containing 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 with 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 contains 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 contains a 5'UTR sequence present in or referenced in Table 2 and / or Figure 2A.

[0164] In some embodiments, the delivery vector includes an expression cassette containing a modified nucleic acid further comprising a 3'UTR containing 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 with 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 contains 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 includes 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 includes a 3'UTR sequence present in or referenced in Table 2 and / or Figure 2A.

[0165] In some embodiments, the delivery vector comprises an expression cassette containing 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 with a sequence selected from SEQ ID NOs: 20, SEQ ID NOs: 21, SEQ ID NOs: 22, SEQ ID NOs: 23, SEQ ID NOs: 24, SEQ ID NOs: 25, SEQ ID NOs: 26, SEQ ID NOs: 27, SEQ ID NOs: 28, SEQ ID NOs: 29, SEQ ID NOs: 30, SEQ ID NOs: 31, SEQ ID NOs: 32, SEQ ID NOs: 34, SEQ ID NOs: 35, SEQ ID NOs: 36, SEQ ID NOs: 37, SEQ ID NOs: 38, SEQ ID NOs: 39, SEQ ID NOs: 89, SEQ ID NOs: 90, SEQ ID NOs: 91, SEQ ID NOs: 92, SEQ ID NOs: 93, SEQ ID NOs: 94, SEQ ID NOs: 95, SEQ ID NOs: 96, SEQ ID NOs: 163, or SEQ ID NOs: 164, wherein the nucleic acid sequence encodes a human glucokinase protein (e.g., SEQ ID NOs: 82) or a functional fragment thereof. In some embodiments, the delivery vector includes an expression cassette containing nucleic acids 5 to 2025 of sequences selected from SEQ ID NOs. 20, SEQ ID NOs. 21, SEQ ID NOs. 22, SEQ ID NOs. 23, SEQ ID NOs. 24, SEQ ID NOs. 25, SEQ ID NOs. 26, SEQ ID NOs. 27, SEQ ID NOs. 28, SEQ ID NOs. 29, SEQ ID NOs. 30, SEQ ID NOs. 31, SEQ ID NOs. 32, SEQ ID NOs. 34, SEQ ID NOs. 35, SEQ ID NOs. 36, SEQ ID NOs. 37, SEQ ID NOs. 38, or SEQ ID NOs. 39, 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.In some embodiments, the delivery vector includes an expression cassette containing 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 delivery vector includes an expression cassette containing a modified nucleic acid having nucleic acids 5-2025 of SEQ ID NO: 20, SEQ ID NO: 20, 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 includes a 5'UTR sequence, ORF, and 3'UTR, which are 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%, less than 85%, less than 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, less than 95%, less than 85%, less than 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%, less than 90%, less than 90%, less than 91%, at least 92%, at least 93%, at least 94%, at least 95%, less than 90%, less than 90%, less than 90%, less than 91%, at least 93%, at least 94%, at least 95%, less than 90 The expression cassette includes a first modified nucleic acid containing a first ORF having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, and a second modified nucleic acid sequence containing a second ORF having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with 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 includes 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 containing the first ORF is sequence numbers 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 150, 151, The second modified nucleic acid sequence containing the second ORF is selected from the group consisting of SEQ ID NOs: 152, 153, 154, 155, 156, 157, 158, and 159, and is selected from the group consisting of SEQ ID NOs: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, and 80.

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

[0168] In some embodiments, the first and second modified nucleic acid sequences further include a 5'UTR containing 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 with respect 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.

[0169] In some embodiments, the first and second modified nucleic acid sequences further include a 3'UTR containing 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 with 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 includes 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 includes 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 sequence number 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 84, 85, 86, 87, 88, 123, 124, 125, 126, 127, 128, 129, 130, 131, 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 SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 89, 90, 91, 92, 93, 94, 95, 96, 163, Alternatively, the expression cassette comprises 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 with SEQ ID NO: 164, each of the first and second modified nucleic acids being linked to the first and second promoters, 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 with nucleic acids 5-957 of sequences selected from SEQ ID NOs. 1, SEQ ID NOs. 2, SEQ ID NOs. 3, SEQ ID NOs. 4, SEQ ID NOs. 5, SEQ ID NOs. 6, SEQ ID NOs. 7, SEQ ID NOs. 8, SEQ ID NOs. 9, SEQ ID NOs. 10, SEQ ID NOs. 11, SEQ ID NOs. 12, SEQ ID NOs. 13, SEQ ID NOs. 14, SEQ ID NOs. 15, or SEQ ID NOs. 15, and the second modified nucleic acid sequence is distributed Nucleic acids 5-2025 of a sequence selected from sequence number 20, sequence number 21, sequence number 22, sequence number 23, sequence number 24, sequence number 25, sequence number 26, sequence number 27, sequence number 28, sequence number 29, sequence number 30, sequence number 31, sequence number 32, sequence number 34, sequence number 35, sequence number 36, sequence number 37, sequence number 38, or sequence number 39 have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.In some embodiments, the first modified nucleic acid sequence is 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: 13 The second nucleic acid sequence is selected from the group consisting of 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 includes nucleic acids 5 to 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 includes nucleic acids 5 to 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 includes 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 includes a 5'UTR, ORF, and 3'UTR present in or referenced in Table 2 and / or Figure 2A.

[0171] In some embodiments, the delivery vector may include a sequence encoding a protein (e.g., insulin and / or Gck) operably linked to a regulatory or modulogenic sequence, a selectable marker, an optional fusion partner, and / or further elements. In certain embodiments, the modified nucleic acid is placed in a functional relationship with another nucleic acid sequence. The term “modulogenic sequence” includes promoters, enhancers, and other expression regulatory elements that control the transcription or translation of a protein (e.g., polyadenylation signals). 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, the expression vector includes transcriptional and translational regulatory nucleic acids operably linked to a protein-encoding nucleic acid, and is typically suitable for host cells used to express the protein. Generally, transcriptional and translational regulatory sequences may include promoter sequences, ribosome binding sites, transcription start and stop sequences, translation start and stop sequences, and enhancer or activator sequences. Furthermore, as is well known in the art, expression vectors may contain selection genes or markers that enable the selection of transformed host cells containing the expression vector. Selection genes are well known in the art and vary depending on the host cell used. For example, typically, selectable marker genes confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector has been introduced. In some embodiments, selectable marker genes include dihydrofolate reductase (DHFR) genes (for use with methotrexate selection / amplification in dhfr-host cells) and neo genes (for G418 selection).

[0172] In some embodiments, the delivery vector is a viral vector or gene therapy vector containing a viral expression construct. In certain embodiments, 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 numerous dividing and non-dividing cell types, including synovial cells and hepatocytes. Due to the episomal properties of adenovirus and AAV vectors after entry into cells, these vectors are suitable for therapeutic use, as described above (Russell, 2000, J. Gen. Virol. 81: 2573-2604, Goncalves, 2005, Virol J. 2(1):43). AAV vectors can result in 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, adenovirus vectors are modified to reduce the host response, as outlined by Russell (2000, cited above). Methods of gene therapy using AAV vectors are described in Wang et 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, and Apparailly et al, Hum Gene Ther. 2005 April; 16(4):426-34.

[0174] In some embodiments, gene therapy vectors include retroviral vectors. In some embodiments, retroviral vectors are lentivirus-based expression constructs. Lentivirus vectors have the ability to infect dividing and non-dividing cells and to be stably integrated into their genomes (Amado and Chen, 1999 Science 285: 674-6). Methods for constructing and using lentivirus-based expression constructs are described in U.S. Patents 6,165,782, 6,207,455, 6,218,181, 6,277,633, and 6,323,031, as well as by Federico (1999, Curr Opin Biotechnol 10: 448-53) and Vigna et al. (2000, J Gene Med 2000; 2: 308-16).

[0175] In some embodiments, the gene therapy vector is a herpesvirus vector, a polyomavirus vector, or a vacciniavirus 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 a suitable regulatory sequence. Such a 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 initial promoter in cytomegalovirus (CMV), the long-terminal repeat promoter (LTR) derived from, for example, mouse Moloney's leukemia virus (MMLV), Roussarcoma virus, or HTLV-1, the initial promoter of simian virus 40 (SV40), and the herpes simplex virus thymidine kinase promoter.

[0177] In some embodiments, the gene therapy vector includes a further nucleotide sequence encoding a further polypeptide. The further polypeptide may be a (selectable) marker polypeptide that enables the 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 selectable marker genes such as 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, and the low affinity nerve growth factor gene. Sources for obtaining these marker genes and methods for their use are described in Sambrook and Russel (2001) "Molecular Cloning: A Laboratory Manual (3 rd (edition), presented in Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York.

[0178] Nonviral vectors In some embodiments, the modified nucleic acids, polynucleotides, or expression constructs of this disclosure may be administered using a non-viral vector. “Non-viral vector,” as used herein, means naked DNA, chemical formulations containing naked DNA (e.g., formulations of DNA and a cationic compound (e.g., dextran sulfate)), and naked DNA mixed with an adjuvant such as viral particles (i.e., the DNA of interest is not contained within the viral particles, but the transformed formulation consists of both naked DNA and viral particles (e.g., AAV particles) (see, e.g., Curiel et al., Am. J. Respir. Cell Mol. Biol. 6:247-52 (1992))). Thus, “non-viral vector” may include vectors composed of DNA with viral particles, where the viral particles do not contain the DNA of interest within the viral genome.

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

[0180] In some embodiments, the modified nucleic acids, polynucleotides, or expression constructs of this disclosure may also be administered as chemical formulations of DNA or RNA coupled to a carrier molecule (e.g., an antibody or receptor ligand) that facilitates delivery to a host cell, for the purpose of altering the biological properties of the host cell. The term "chemical formulation" refers to the modification of the nucleic acid that enables the coupling of the nucleic acid compound 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 target cell of delivery.

[0181] Adeno-associated virus vector (AAV vector) In some embodiments, the modified nucleic acids, polynucleotides, or expression constructs disclosed herein may be administered as components 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 may include a recombinant AAV vector (rAAV). As used herein, “rAAV vector” refers to a recombinant vector containing a portion of the AAV genome capsid-encapsulated within a protein shell of a capsid protein derived from an AAV serotype disclosed herein. The portion of the AAV genome may contain, for example, a reverse-ended repeat (ITR) derived from an adeno-associated virus serotype such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVRH10, AAV11, or AAV12. In some embodiments, the ITR is derived from AAV2.

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

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

[0185] The viral capsid component of a packaged viral vector may be a parvovirus capsid, e.g., AAV Cap and / or chimeric capsid. Examples of suitable parvovirus viral capsid components are those derived from Parvoviridae, such as autonomous parvovirus or Dependvirus. For example, the viral capsid may be an AAV capsid (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVH8, AAV9, AAV10, AAVH10, AAV11, or AAV12 capsids; those skilled in the art will see that there are likely other unidentified variants that perform the same or similar functions), or it may contain components derived from two or more AAV capsids. The complete complement of the AAV Cap protein includes VP1, VP2, and VP3. An ORF containing the nucleotide sequence encoding the AAV VP capsid protein may contain fewer AAV Cap proteins than complete complement, or it may provide complete complement for the AAV Cap protein.

[0186] One or more AAV Cap proteins may be chimeric proteins containing an amino acid sequence AAV Cap 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 AAV rep (replication) or cap (capsid) genes. This rAAV genome may further include marker or reporter genes, such as genes encoding antibiotic resistance genes, genes encoding fluorescent proteins (e.g., GFP), or genes encoding chemically, enzymatically, or otherwise detectable and / or selectable products (e.g., lacZ, aph, etc.), which are known in the art.

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

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

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

[0190] Unless otherwise stated, recombinant parvovirus and AAV (rAAV) constructs, packaging vectors expressing parvovirus Rep and / or Cap sequences, and transiently and stably processed 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) and AUSUBEL el al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley Sons, Inc., New York).

[0191] lentiviral expression constructs Lentiviruses are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain 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 virulence factor 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 aphydicin or gamma irradiation.

[0193] Cellular infection relies on the active nuclear translocation of the HIV pre-integration complex through the nuclear pores of target cells. This occurs through the interaction of multiple partially overlapping molecular determinants within the complex with the target cell's nuclear translocation mechanism. Identified determinants include the functional nuclear localization signal (NLS) in the gag matrix (MA) protein, the nuclear affinity virion-associated protein, vpr, and the C-terminal phosphotyrosine residue in the gag MA protein.

[0194] The lentiviral genome and proviral DNA possess three genes found in retroviruses: gag, pol, and env, which are flanked by two long-terminal repeat (LTR) sequences. The gag gene encodes internal structure (matrix, capsid, and nucleocapsid) proteins; the pol gene encodes RNA-dependent DNA polymerase (reverse transcriptase), proteases, and integrases; and the env gene encodes viral envelope glycoproteins. The 5'LTR and 3'LTR help facilitate virion RNA transcription and polyadenylation. The LTRs contain all other cis-acting sequences necessary for viral replication. Lentiviruses also possess further genes, including vif, vpr, tat, rev, vpu, nef, and vpx (in HIV-1, HIV-2, and / or SIV).

[0195] The 5'LTR contains adjacent sequences necessary for reverse transcription of the genome (tRNA primer binding sites) and sequences necessary for efficient capsid encapsulation of viral RNA into particles (Psi sites). If the sequences necessary for capsid encapsulation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the cis deletion prevents capsid encapsulation 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 containing packaging functions, i.e., gag, pol, and env, as well as rev and tat. In some examples, vectors lacking the functional tat gene are desirable. For example, to produce packaging cells, a first vector may provide nucleic acids encoding viral gag and viral pol, and another vector may provide nucleic acid encoding viral env. By introducing vectors identified as transport vectors, which provide heterologous genes, into these packaging cells, producer cells that release infectious viral particles carrying the desired foreign genes are obtained.

[0197] The gag, pol, and env genes of the target vector are also known in the art. Therefore, the relevant genes are cloned into the selected vector and then used to transform the target cells.

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

[0199] It may be desirable to target recombinant viruses by linking an envelope protein to an antibody or specific ligand for targeting a receptor of a particular cell type. The vector becomes target-specific by inserting the desired sequence (including regulatory regions) into the viral vector, for example, in conjunction with another gene encoding a ligand for a receptor on a specific target cell. Retroviral vectors can be made target-specific by inserting, for example, glycolipids or proteins. Targeting is often achieved by targeting the retroviral vector using an antibody, such as a single-chain antibody, or the antigen-binding portion of a recombinant antibody-type molecule. Those skilled in the art are aware of specific methods for achieving delivery of retroviral vectors to a particular target, or can readily verify them without excessive experimentation.

[0200] Examples of retrovirus-derived env genes include, but are not limited to, Moloney's mouse leukemia virus (MoMuLV or MMLV), Harvey's mouse sarcoma virus (HaMuSV or HSV), mouse mammary tumor virus (MuMTV or MMTV), gibbon leukemia virus (GaLV or GALV), human immunodeficiency virus (HIV), and Rous sarcoma virus (RSV). Other env genes can also be used, such as those of vesicular stomatitis virus (VSV) G protein (VSV G), hepatitis viruses, and influenza viruses.

[0201] A vector providing a viral env nucleic acid sequence is operably associated with a regulatory sequence, such as a promoter or enhancer. The regulatory sequence may be any eukaryotic promoter or enhancer, including, for example, the Moloney's mouse leukemia virus promoter enhancer element, the human cytomegalovirus enhancer, or the vaccinia P7.5 promoter. In some cases, the promoter enhancer element, such as the Moloney's mouse leukemia virus promoter enhancer element, is located within or adjacent to the LTR sequence.

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

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

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

[0205] Unless otherwise stated, lentiviral constructs, vectors, and transiently and stably treated 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) and AUSUBEL el al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley Sons, Inc., New York).

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

[0207] A construct prepared for introduction into a specific host may include a replication system recognized by the host, an intended DNA segment encoding a desired polypeptide, and regulatory sequences for the initiation and termination of transcription and translation operably ligated to the polypeptide-encoding segment. The term “operably ligated” has already been defined herein. For example, a promoter or enhancer is operably ligated to a coding sequence if it stimulates the transcription of the sequence. A signal sequence DNA is operably ligated to polypeptide-encoding DNA if it is expressed as a preprotein involved in polypeptide secretion. Generally, operably ligated DNA sequences are contiguous, and in the case of signal sequences, contiguous and within the reading frame. However, enhancers do not need to be contiguous to the coding sequence that controls transcription. Ligation is achieved by ligation at a convenient restriction site or an adapter or linker inserted in its place, 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, cited above). Transcriptional regulatory sequences typically contain heterologous enhancers or promoters recognized by the host. The selection of an appropriate promoter is host-dependent, but promoters such as trp, lac, and phage promoters, tRNA promoters, and glycolysis promoters are known and available (see, e.g., Sambrook and Russell, 2001, cited above). Expression vectors include a replication system and can use transcriptional and translational regulatory sequences in conjunction with the insertion site of the polypeptide-encoding segment. In most cases, the replication system is functional only in the cells used to construct the vector (bacterial cells such as E. coli). Most plasmids and vectors do not replicate in cells infected with the vector. Examples of feasible combinations of cell lines and expression vectors are described by Sambrook and Russell (2001, cited above) 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., Escherichia coli. Thus, the cells may be prokaryotic or eukaryotic host cells. The cells may be suitable for culture in liquid or solid media.

[0209] Methods for introducing exogenous nucleic acids into host cells are well known in the art and vary depending on the host cell used. These 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, viral or phage infection, encapsulation of polynucleotides into liposomes, and direct microinjection of DNA into the nucleus. In mammalian cells, transfection may be transient or stable.

[0210] The host cell may be a yeast, e.g., S. cerevisiae; an insect cell, e.g., Sf9 cell; a mammalian cell, e.g., CHO cell; or a bacterial cell, e.g., Escherichia coli. Therefore, the cell may be a prokaryotic or eukaryotic host cell. The cell may be suitable for culture in liquid or solid medium. Alternatively, the host cell may be 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 embodiments, a method for introducing a viral vector containing a modified nucleic acid disclosed herein into a cell host for replication and packaging may be used, and this method may include, but is not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes combined with nuclear localization signals. In embodiments in which viral vector functionality is provided by transfection using the viral vector, standard methods for inducing viral infection may be used.

[0212] In some embodiments, the packaging function may include genes for viral vector replication and packaging. Therefore, for example, the packaging function may, as needed, include functions necessary for viral gene expression, viral vector replication, recovery of the viral vector from its integrated state, viral gene expression, and packaging of the viral vector into viral particles. The packaging function may be supplied together with or separately to the packaging cell using a gene construct such as a plasmid or amplicon. The packaging function may be extrachromosomal within the packaging cell or integrated into the cell's chromosomal DNA. Examples include genes encoding the AAV Rep and Cap proteins.

[0213] In some embodiments, the helper function may include helper viral elements necessary to establish active infection of the packaging cell, which is required to initiate the packaging of the viral vector. Examples include functions derived from adenovirus, baculovirus, and / or herpesvirus, sufficient to result in the packaging of the viral vector. For example, an adenovirus helper function typically includes adenovirus components E1a, E1b, E2a, E4, and VA RNA. The packaging function may be supplied by infecting the packaging cell with the required virus. The packaging function may be supplied together with the packaging cell or separately using a gene construct such as a plasmid or amplicon. The packaging function may be extrachromosomal within the packaging cell or incorporated into the cell's chromosomal DNA.

[0214] Any suitable helper virus function can be used. For example, if the packaging cells are insect cells, a baculovirus can function as a helper virus. Herpesviruses can also be used as helper viruses in AAV packaging methods.

[0215] Any method may be used to introduce the nucleotide sequence having helper function into a cell host for replication and packaging, and this method includes, but is not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes combined with nuclear localization signals. In embodiments in which helper function is provided by transfection using a viral vector or infection using a helper virus, standard methods for inducing 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 cells or insect cells are preferred. Examples of cells useful for producing packaging cells in the implementation of the present invention include, for example, human cell lines or primate cells such as VERO, WI38, MRC5, A549, 293 cells, B-50 or any other HeLa cell, HepG2, Saos-2, HuH7, and HT1080 cell lines.

[0217] In some embodiments, the cell line used as packaging cells is an insect cell line. Any insect cell that can enable AAV replication and be maintained in culture can be used according to the present invention. Examples include Spodoptera frugiperda, Drosophila spp. cell lines such as Sf9 or Sf21 cell lines, or mosquito cell lines, such as those derived from Aedes albopictus. A preferred cell line is the Spodoptera frugiperda Sf9 cell line. The following references are incorporated herein for teachings on the use of insect cells for heterologous polypeptide expression, 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 Samulski 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 enable replication and packaging of the viral vector. These various functions may be supplied together or separately to the packaging cells using gene constructs such as plasmids or amplicons, and may exist extrachromosomally within the cell line or be incorporated into the cell's chromosomes.

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

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

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

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

[0223] In some embodiments, modified nucleic acids, expression constructs, delivery vectors and / or compositions are used to prevent, reduce or improve, delay, reverse, cure, and / or treat the symptoms of diabetes, if such modified nucleic acids, expression constructs, delivery vectors and / or compositions can exhibit antidiabetic effects. Antidiabetic effects may be achieved when glucose processing in the blood increases and / or glucose tolerance improves. This can be evaluated using techniques known to those skilled in the art. In this context, “increase” (each “improvement”) means an increase (each “detectable improvement”) that is at least detectable using assays known to those skilled in the art or using assays performed in the experimental part.

[0224] The antidiabetic effect may also be observed when a physician assesses that the progression of typical symptoms (i.e., isletitis, beta-cell loss) has slowed. A reduction in typical symptoms associated with diabetes may mean a slowing of the progression of symptom development or complete disappearance of symptoms. Symptoms, and also the reduction of symptoms, can be assessed using a variety of methods, which in most cases are the same methods used to diagnose diabetes, including clinical tests and standard laboratory tests. Such methods include both macroscopic and microscopic methods, as well as molecular, biochemical, and immunohistochemical methods.

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

[0226] Modified nucleic acids, expression constructs, delivery vectors, or compositions disclosed herein, used to prevent, reduce, improve, delay, reverse, cure, and / or treat the symptoms of diabetes, may be suitable for administration in vivo to cells, tissues, and / or organs of individuals with diabetes or at risk of developing diabetes, and may be administered in vivo, ex vivo, or in vitro. The combinations and / or compositions may be administered directly or indirectly to cells, tissues, and / or organs in vivo to individuals with diabetes or at risk of developing diabetes, and may 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 as is to an organism, the cells, tissues, or organs of said organism. Depending on the disease or condition, the cells, tissues, or organs of said organism 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, subarachnoid, intra-articular, and / or intraventricular administration, the solution may be saline. In some embodiments, administration is intramuscular. In some embodiments, intramuscular administration is performed using a multi-needle. In some embodiments, the therapeutically effective dose of the modified nucleic acids, expression constructs, vectors, or compositions described herein is administered in a single, unique dose, thus avoiding periodic 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., two, three, four, or five) into one or more muscles (e.g., multiple muscle groups).

[0228] In some embodiments, the compound may be present in the composition of the present invention. The compound may be useful for the delivery of modified nucleic acids or compositions containing them. In some embodiments, the compound is capable of forming a complex, nanoparticles, micelles, liposomes that deliver each component as defined herein, compounded or captured in vesicles or liposomes via a cell membrane, or a combination thereof. Many of these compounds are known in the art. In some embodiments, further compounds are polyethyleneimine (PEI), or similar cationic polymers including polypropyleneimine or polyethyleneimine copolymer (PEC) and derivatives, synthetic amphiphilic substances (SAINT-18), Lipofectin®, DOTAP, or combinations thereof.

[0229] How to use This disclosure also provides methods for preventing, reducing, improving, 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, diabetes may be T1DM. In some embodiments, diabetes may be T2DM. In some embodiments, this method is gene therapy. In certain embodiments, the method of this disclosure includes administration (e.g., intramuscular administration) of 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 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 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) may be performed together or sequentially.

[0230] Certain embodiments of this disclosure relate 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, the signal peptide optionally not being a wild-type preproinsulin signal sequence, and (ii) a nucleotide sequence encoding a proinsulin polypeptide, the polynucleotide comprising amino acid modifications at positions 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 positions in wild-type proinsulin, the polynucleotide optionally further comprising cleavage sites. In some embodiments, the signal peptide is a wild-type preproinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence. In some embodiments, the cleavage site is a furin cleavage site.

[0231] Certain aspects of this disclosure relate to methods of use comprising administering a polynucleotide comprising a nucleic acid encoding human insulin (Ins) protein, wherein the nucleic acid comprises (i) a nucleotide sequence encoding a signal peptide and (ii) an open reading frame (ORF) comprising nucleotide sequences that are 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, SEQ ID NO: 144, or 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 aspects of this disclosure relate to methods of use comprising administering a polynucleotide comprising a nucleic acid encoding human insulin (Ins) protein, wherein the nucleic acid comprises an open reading frame (ORF) containing nucleotide sequences that are 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 aspects, the polynucleotide comprises at least two nucleic acid sequences encoding human Ins protein. In some embodiments, the polynucleotide comprises at least two ORF nucleotide sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 43-57, 110-122, or 150-159, the two ORF nucleotide sequences may be the same or different. In some embodiments, the polynucleotide further comprises an IRES sequence. In some embodiments, at least two ORF nucleotide sequences are separated by the 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 SEQ ID NOs: 41 (amino acids 25-110), SEQ ID NOs: 144 (amino acids 25-110), or SEQ ID NOs: 145 (amino acids 25-110). 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 the 5'UTR and / or 3'UTR. 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: 1-16, 84-88, 123-141, or 160-161.

[0233] Certain aspects of this disclosure relate 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), 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) 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 a 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 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 polynucleotide further comprises cleavage sites (e.g., furin cleavage sites).

[0234] Certain embodiments of this disclosure relate 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 containing nucleic acid 1-1398 of any of SEQ ID NOs. 61-80 or 162, or 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. 61-80 and 162. In some embodiments, the encoded human Gck protein comprises the amino acid sequence of SEQ ID NOs. 82. In some embodiments, the polynucleotide or nucleic acid sequence encoding the Gck protein further comprises a 5'UTR and / or 3'UTR. In some embodiments, the nucleic acid further comprises a 5'UTR containing a nucleotide sequence that is at least 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 42, SEQ ID NO: 425-329, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some embodiments, the nucleic acid further comprises a 3'UTR containing a nucleotide sequence that is 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 contains 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 and 89-96, and 163-164. In some embodiments, the nucleic acid is operably ligated to a promoter (e.g., a eukaryotic promoter). Certain embodiments of the present disclosure relate to an expression cassette comprising the polynucleotide of the present disclosure and heterologous expression regulatory sequences operably ligated to the nucleic acid sequence. In some embodiments, the nucleic acid is operably ligated to a polyadenylated (poly-A) element.

[0235] Certain embodiments of this disclosure relate to methods of use that involve administering a vector (e.g., a viral vector, a non-viral vector, a plasmid, a lipid, or a lysosome) containing the polynucleotide or expression cassette of this disclosure. In some embodiments, the vector is an adeno-associated virus (AAV) vector or a lentiviral vector. Certain embodiments of this disclosure relate to methods of administering recombinant AAV (rAAV) particles comprising an AAV capsid and a vector genome containing the polynucleotide or expression cassette of this disclosure. In some embodiments, the AAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRH8, AAVrh9, AAV9, AAVrh10, AAV10, AAVH10, 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 that result in the lifetime expression of therapeutic genes in diabetic subjects. WO2012 / 007458 discloses the production of two viral vectors, one expressing an insulin gene and the other expressing a glucokinase gene, as a treatment for diabetes. Furthermore, WO2016 / 110518 discloses a single-vector gene construct containing an insulin gene and a glucokinase gene. In certain embodiments, the disclosure provides improved nucleic acid sequences, expression constructs, and / or delivery vectors for the treatment or prevention of diabetes, which increase the expression of insulin and / or glucokinase, reduce adverse immune responses, and / or allow for the administration of lower doses of viral vectors.

[0237] In some embodiments, the methods of the Disclosure reduce or alleviate one or more symptoms of diabetes in an individual, the cells, tissues, or organs of the individual, or reduce or alleviate one or more characteristics or symptoms of the cells, tissues, or organs of the individual, the methods of which include administering one or more of the modified nucleic acids, polynucleotides, expression cassettes, vectors, or expression constructs disclosed herein to the individual.

[0238] Recommendations for treatment of 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 approximately 5.0% and approximately 6.5%. Most commercially available products reduce HbA1c to between 0.5% and 1.50%. In some embodiments, the method of the disclosure normalizes the HbA1c level in a treated diabetic subject to the HbA1c level of a non-diabetic subject, for example, within 8 weeks. In some embodiments, the method of the disclosure enables reduction and / or regulation of blood glycated hemoglobin (HbA1c) levels in a subject. In some embodiments, the HbA1c level in the treated subject is, for example, <7.0% (e.g., <6.5%, <6.0%, <5.7%, e.g., between 5.0% and 6.5%) within 8 weeks after treatment.

[0239] Insulin plays a central role in regulating lipid metabolism in the liver, fat, and intestines (Verges B. Insulin sensitivity and lipids. Diabetes Metab. 2001 Apr;27(2 Pt 2):223-7. PMID: 11452214.). In uncontrolled type 1 diabetes, patients are unable to utilize glucose and require alternative fuel sources. In adipose tissue, insulin inhibits hormone-sensitive lipases, which normally promote triglyceride storage in adipocytes and reduce the release of free fatty acids from circulating adipose tissue. 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). Lipid breakdown occurs, resulting in an increase in the levels of circulating triglyceride-rich lipoproteins (chylomicrons, VLDL), leading to hypertriglyceridemia. In some embodiments, the methods of the present disclosure reduce the levels of triglyceride-rich lipoproteins (e.g., chylomicrons or VLDL) in a subject (e.g., a subject with diabetes), the subject's cells, tissues, or organs, the methods of which include administering one or more of the modified nucleic acids, polynucleotides, expression cassettes, vectors, or expression constructs disclosed herein to the subject.

[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 function as an alternative energy source under glucose-restricted conditions, providing up to 80% of the brain's energy requirements. While useful in the short term, chronically high levels of circulating ketones can lead to undesirable effects in the brain, kidneys, liver, and microvessels (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), potentially leading to fatal ketoacidosis. In some embodiments, the methods of the present disclosure reduce the level of ketones in a subject (e.g., a subject with diabetes), the subject's cells, tissues, or organs, and the methods include administering one or more of the modified nucleic acids, polynucleotides, expression cassettes, vectors, or expression constructs disclosed herein to the subject.

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

[0242] In some embodiments, the method or use is performed in vitro, for example, using cell cultures. In some embodiments, the method or use is performed in vivo. In some embodiments, the modified nucleic acids, polynucleotides, expression cassettes, delivery vectors, or expression constructs disclosed herein are combined with further compounds known to be used to treat diabetes in individuals. In some embodiments, the method further includes administering recombinant insulin, for example, via conventional injection.

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

[0244] In some embodiments, the method involves administering a therapeutically effective dose of a modified nucleic acid, expression construct, vector, or composition described herein, the administration being a single dose, and, for example, avoiding periodic repeated administration. In some embodiments, a 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., two, three, four, or five) to one or more muscles (e.g., multiple muscle groups).

[0245] It should be understood that the detailed description section, rather than the summary and abstract section, is intended to be used to interpret the claims. The summary and abstract section describes one or more exemplary embodiments of the invention as envisioned by the inventor, but not all of them, and is therefore not intended to limit the invention and the appended claims in any way.

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

[0247] Having described the present invention, it will be further explained in detail in the following examples. The examples are included herein for illustrative purposes only and are not intended to limit the present invention. [Examples]

[0248] (Example 1) Modified insulin nucleic acid The following modified human insulin nucleic acid sequences (shown in Table 1) corresponding to Array Numbers 1 to 16, 84 to 88, 123 to 141, and 160 to 161 were designed in silico. The 5’UTR sequences (Array Number 42, Array Number 83, Array Number 146, or Array Number 148) are in bold, the ORF sequences are underlined (Array Numbers 43 to 57, 110 to 122), and the 3’UTR sequences are in italics (Array Number 60, Array Number 97, Array Number 98, Array Number 99, Array Number 100, Array Number 101, or Array Number 149). The 5’UTR having the sequence of Array Number 42 was further modified to remove the CTAG at positions 1 to 4. Thus, in a specific construct, the 5’UTR contained nucleic acids 5 to 329 of Array Number 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 (Array Number 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

[0249] In some embodiments, the nucleic acid comprises a sequence that is codon-optimized and has a reduced CpG compared 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 the pAAV-insulin plasmid was tested. The pAAV-insulin plasmids included 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), and A AV1-CMV-modified hIns24 (sequence number 125), AAV1-CMV-modified hIns25 (sequence number 126), AAV1-CMV-modified hIns_2 (sequence number 127), AAV1-CMV-modified hIns27 (sequence number 128), AAV1-CMV-modified hIns28 (sequence number 129), AAV1-CMV-modified hIns29 (sequence number 130), AAV1-CMV-modified Type hIns30 (SEQ ID NO: 131), AAV1-CMV-modified type hIns31 (SEQ ID NO: 132), AAV1-CMV-modified type hIns32 (SEQ ID NO: 133), AAV1-CMV-modified type hIns33 (SEQ ID NO: 134), AAV1-CMV-modified type hIns34 (SEQ ID NO: 135), AAV1-CMV-modified type hIns35 (SEQ ID NO: 136), AAV1-CMV-modified type hIns36 (Distributed HEK293 cells were transfected with either AAV1-CMV-modified hIns37 (sequence number 138), AAV1-CMV-modified hIns38 (sequence number 139), AAV1-CMV-modified hIns39 (sequence number 140), or AAV1-CMV-modified Ins40 (sequence number 141) at a dose of 0.5 μg (Figure 1B) or 0.1 μg (Figure 1C) in a 24-well plate. Extracellular insulin levels were determined by ELISA assay. The pAAV-insulin plasmid exhibited insulin expression after transfection.

[0251] (Example 2) Modified Gck nucleic acid The following modified human glucokinase (Gck) nucleic acid sequences (shown in Table 2) corresponding to SEQ ID NOs. 20-39, 89-96, and 163-164 were designed in silico. 5'UTR sequences (SEQ ID NOs. 42 or 83) are in bold, ORF sequences are underlined (SEQ ID NOs. 61-80 and 162), and 3'UTR sequences are italicized (SEQ ID NOs. 60, 102, 103, 104, 105, 106, 107, 108, or 109). The 5'UTR containing the sequence of SEQ ID NOs. 42 was further modified to remove CTAGs at positions 1-4. Therefore, in a 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 sequence that is codon-optimized and has a reduced CpG compared to the wild-type and / or unmodified human Gck nucleic acid sequence (e.g., SEQ ID NO: 19). 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.

[0253] GcK expression was tested in HEK cells transfected with the pAAV-Gck plasmid. HEK293 cells were transfected with 2.5 μg per well in a 6-well plate using the 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). Cell pellets were collected 48 hours after transfection, and intracellular insulin levels were determined by ELISA assay. The pAAV-GcK plasmid exhibited GcK expression after transfection.

[0254] (Example 3) In vitro comparative analysis of human insulin vectors The infectivity and efficacy (insulin mRNA expression, insulin secretion into cell culture, and bioactivity of secreted insulin) of AAV1-CMV-human insulin vectors carrying insulin variants (SEQ ID NO: 1, 87, and 88) were studied. 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 the vector genome. Efficacy assays measured human insulin mRNA expression and insulin secreted into cell culture. The functionality of the secreted insulin was also evaluated. method

[0255] 3.1 Infection of 2v6.11 cells with AAV1-CMV-human insulin vector The day before infection, 2v6.11 cells were seeded in 24-well plates at a density of 2E+05 cells / well. The cells were grown at 37°C and 8.5% CO2 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 evaluated using bright-field microscopy to ensure adequate cellular confluence (70-80%). To assess cell count, four wells of cells were trypsin-treated and quantified using a Scepter 2.0 Handheld Automated Cell Counter (Merck-Millipore). Cells were infected with 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 MOI = 1000, 2000, and 4000 vg / cell. Cells were infected in quadruple chains for both infectivity and efficacy tests. Uninfected 2v6.11 cells (NI) were used as a negative control. Infection was repeated three times on different days using cells of different passage numbers. [Table 3]

[0257] 3.2 Infectivity Test Cell viability was observed under a bright-field microscope 24 hours after infection. Next, the cell culture medium was aspirated, 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 oligoset targeting the ITR2 sequence. Vector genomes were quantified by interpolation from standard curves generated using serial dilutions of standard DNA.

[0258] 3.3 Sample collection for efficacy testing 48 hours after infection and after assessing cell viability, the cell 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 medium was collected in a 1.5 ml microcentrifuge tube, centrifuged at 600 × g for 10 minutes at 4°C, and 300 μl of supernatant was transferred to a new tube. For mRNA expression, the remaining medium was aspirated from the wells, 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, with the exception that on-column DNase I digestion was extended from the standard 15 minutes to 30 minutes to ensure proper degradation of the infected AAV vector genome. 1 μg of each RNA sample was reverse transcribed using the Transcriptor FirstStrand cDNA Synthesis Kit (Roche). Triple qPCR was performed using Taqman Probes Master (Roche) and 2 μl of sample (diluted to 1 / 10). To quantify expression, a primer-probe mix targeting the SV40 polyA 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 Using an Insulin ELISA Kit (Crystal Chem), insulin secreted from culture medium samples diluted to 1 / 10 with milliQ water was measured in a double-cycle format.

[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 material or cell culture medium of infected cells and 40 μl of iLite Insulin Assay Ready Cells, which had been 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 incubation at 37°C and 5% CO2 for 5 hours, 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] Anova and Tukey's multiple comparison tests were used to independently analyze each MOI for the three studies.

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

[0264] 3.8 Infectivity assay Three independent infection studies using 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) in 2v6.11 cells showed no significant difference 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 the intracellular vector genome content (vg / ng DNA) (Figures 3A-3C and Table 4). [Table 4-1] [Table 4-2]

[0265] Infectivity data for each MOI and assay were analyzed using Anova and Tukey's multiple comparison tests.

[0266] 3.9 Efficacy assay 3.9.1 Human insulin mRNA expression Quantification of human insulin expression levels revealed no significant difference between the vectors AAV1-CMV-hInsB10D (SEQ ID NO: 1) and AAV1-CMV-Ins5 (SEQ ID NO: 87) in the three studies conducted (Figures 4A-4C and Table 5). On the other hand, 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) (Figures 4A-4C and Table 5). [Table 5]

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

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

[0269] Data on secreted human insulin corresponding to each MOI and assay were analyzed using Anova and Tukey's multiple comparison tests. Bold and italicized 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). Results obtained for mRNA expression and human insulin protein readings (Figures 4A-4C and 5A-5C, and Tables 5 and 6) showed no significant difference in insulin activity between the vectors AAV1-CMV-hIns_B10D (SEQ ID NO: 1) and AAV1-CMV-Ins5 (SEQ ID NO: 87). However, AAV1-CMV-Ins7 (SEQ ID NO: 88) exhibited significantly lower insulin activity (Figures 6A-6C and Table 7). [Table 7]

[0271] Human insulin activity data corresponding to each MOI and assay were analyzed using Anova and Tukey's multiple comparison tests. Bold and italicized text indicates statistical significance.

[0272] (Example 4) In vitro comparative analysis of human glucokinase vectors The infectivity and efficacy (mRNA expression, protein content, and bioactivity) of AAV1-CMV-human glucokinase vectors carrying wild-type (SEQ ID NO: 19), Gck8 (SEQ ID NO: 93), and Gck12 (SEQ ID NO: 95) human glucokinase variants were studied. For this purpose, 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 the vector genome. Efficacy assays measured human glucokinase mRNA expression, intracellular glucokinase content, and glucokinase activity.

[0273] method 4.1 Infection of 2v6.11 cells with AAV1-CMV-human glucokinase vector The day before infection, 2v6.11 cells were seeded in 24-well plates at a density of 2E+05 cells / well. The cells were grown at 37°C and 8.5% CO2 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 evaluated using bright-field microscopy to ensure adequate cell confluence (70-80%). To assess cell count, four wells of cells were trypsin-treated and quantified using a Scepter 2.0 Handheld Automated Cell Counter (Merck-Millipore). Cells were infected with AAV1-CMV-human glucokinase vectors AAV1-CMV-hGckWT, AAV1-CMV-Gck8, and AAV1-CMV-Gck12 (Table 8) at MOI = 1000, 2000, and 4000 vg / cell. Cells were infected in quadruple chains 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. Infection was repeated three times on different days using cells of different passage numbers. [Table 8]

[0275] 4.2 Infectivity Assay Cell viability was observed under a bright-field microscope 24 hours after infection. Next, the cell culture medium was aspirated, 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 oligoset targeting the ITR2 sequence. Vector genomes were quantified by interpolation from standard curves generated using serial dilutions of standard DNA.

[0276] 4.3 Human glucokinase mRNA expression 48 hours after infection and after assessing cell viability, 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). 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 proper degradation of the infected AAV vector genome. 1 μg of each RNA sample was reverse transcribed using the Transcriptor FirstStrand cDNA Synthesis Kit (Roche). Triple qPCR was performed using Taqman Probes Master (Roche) and 2 μl of sample (diluted to 1 / 10). To quantify expression, a primer-probe mix targeting the SV40 polyA 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 48 hours after infection, cells were gently washed with 500 μl of 1×PBS / well. The cells were then scraped with 200 μl of ice-cold 1×PBS and collected in microcentrifuge tubes. 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 diptychs (standard and sample) using the Human Glucokinase ELISA Kit (Abcam). Samples were diluted 1 / 20 using 1× diluent N provided by the ELISA kit. Glucokinase content was normalized by total protein content quantified in diptychs in cell extracts using the BCA method with a 1 / 10 dilution of the sample in milliQ water.

[0278] 4.5 Glucokinase activity assay 48 hours after infection, the cells were gently washed with 500 μl of 1×PBS / well. Then, 250 μl of trypsin was gently added to the well, stirred, and excess trypsin was removed by aspirate. After incubation at room temperature for 2 minutes, the cells were collected in 750 μl of DMEM + 10% FBS by pipetting up and down and transferred to 1.5 ml microcentrifuge tubes. 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 the Glucokinase Activity Assay Kit (AssayGenie). The protocol provided by the manufacturer 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 for the assay.

[0280] 4.6 Statistical analysis Anova and Tukey's multiple comparison tests were used to independently analyze each MOI for the three studies.

[0281] result 2v6.11 cells were infected with AAV1-human glucokinase vectors carrying human glucokinase variants Gck8 and Gck12, as well as WT hGck, and the infectivity and efficacy of the vectors were evaluated 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), did not show any consistently 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 the intracellular vector genome content (vg / ng DNA) in three different assays (Figures 7A-7C and Table 9). [Table 9]

[0283] Infectivity data for each MOI and assay were analyzed using Anova and Tukey's multiple comparison tests. Bold and italicized text indicates statistical significance.

[0284] 4.8 Efficacy Assay 4.8.1 Human glucokinase mRNA expression Quantification of human glucokinase expression levels revealed no consistent significant difference between the vectors AAV1-CMV-hGckWT (SEQ ID NO: 19) and AAV1-CMV-Gck8 (SEQ ID NO: 93), which carry hGckWT and the glucokinase variant Gck8, respectively (Figures 8A-8C and Table 10). AAV1-CMV-Gck12 (SEQ ID NO: 95), which contains the glucokinase variant Gck12, tended to mediate lower mRNA expression compared to AAV1-CMV-hGckWT (SEQ ID NO: 19) and especially AAV1-CMV-Gck8 (SEQ ID NO: 93), although this was not consistent across multiple assays and MOIs (Figures 8A-8C and Table 10). [Table 10]

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

[0286] 4.8.2 Quantitative analysis of intracellular glucokinase content When intracellular glucokinase content in 2v6.11 cells infected with different AAV1-human glucokinase vectors was quantified using the 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] Data on intracellular glucokinase content for each MOI and assay were analyzed using Anova and Tukey's multiple comparison tests. Bold and italicized text indicates statistical significance.

[0288] 4.8.3 Glucokinase activity assay Glucokinase activity in cell extracts of infected cells was measured using the Glucokinase Activity Assay Kit (AssayGenie). Consistent with the results observed for mRNA expression and protein readings (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 differ significantly among vectors across multiple MOIs and assays (Figures 10A-10C and Table 12). [Table 12]

[0289] Data on glucokinase activity corresponding to each MOI and assay were analyzed using Anova and Tukey's multiple comparison tests. Bold and italicized text indicates statistical significance.

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

[0291] First, mice were treated with 40 mg of streptozotocin (STZ) for 5 consecutive days to deplete pancreatic beta cells, thereby eliminating the production of natural mouse insulin and raising blood glucose levels to approximately 600 mg / dL. Next, three identified muscles in both hind limbs of the animals were administered an equal mixture of AAV1-insulin (SEQ ID NO: 1) and AAV1-rat glucokinase. In separate groups, the same total doses of AAV1-insulin (SEQ ID NO: 1) and AAV1-rat glucokinase were administered in a lower total volume to two hind limb muscles (quadriceps and gastrocnemius) of the animals. These animals were compared to STZ-vehicle-treated animals and non-diabetic vehicle-treated animals (n=10-11 / group). STZ-treated diabetic mice treated with AAV1-insulin (SEQ ID NO: 1) and AAV1-rat glucokinase restored and maintained euglycemic and HbA1c levels under both feeding and fasting conditions, as observed in previous studies.

[0292] Genetherapy administered to two muscle groups was as effective as administration to three muscle groups. Streptozotocin-treated diabetic mice treated with AAV1-insulin (SEQ ID NO: 1) and AAV1-rat glucokinase restored and maintained euglycemic levels up to 5 weeks after injection under both feeding conditions (Figures 11A-11C and 12) and fasting conditions (data from week 4 is shown). Furthermore, mice treated with higher concentrations of the vector in only two large muscle groups (quadriceps and gastrocnemius) appeared to function equally or better than mice treated in three muscle groups (Figures 11A-11C and 12). These results suggest that muscle anatomy and blood flow may be factors to consider in allometric translation. Therapeutic effects were observed as early as one week after administration, and a significant reduction in hyperglycemia was observed by two weeks, within the range of untreated control animals. The combined effect of AAV-mediated basal insulin production and glucokinase activity generates a "glucose sensor" in skeletal muscle that enables proper glucose regulation in diabetic animals, potentially driving the complete reversal of diabetes in treated animals.

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

[0294] (Example 7) In vitro evaluation of preproinsulin variants To evaluate the levels of insulin production and secretion mediated by sequence numbers 150–159 in vitro, each insulin variant was first cloned in an AAV plasmid (pAAV) under the control of the miniCMV promoter (pAAV-miniCMV-InsX, where X is a specific ORF, i.e., sequence numbers 150–159). The plasmid names and corresponding ORF sequences are listed in Table 14. [Table 14]

[0295] An AAV expression cassette was obtained by cloning a human prepro insulin variant under the control of the miniCMV promoter between the ITRs of AAV2 (pAAV-miniCMV-InsX, where X represents a specific insulin variant).

[0296] A single-stranded AAV vector (AAV1-InsX, where X represents a specific proinsulin variant) encoding a preproinsulin variant under the control of the miniCMV promoter and a human glucokinase under the control of the RSV promoter (AAV1-InsX, where X represents a specific proinsulin variant) was produced by triple transfection of HEK293 cells according to a standard method (Ayuso, E. et al., 2010. Curr Gene Ther. 10(6):423-36). Ten roller bottles (850 cm³) were used. 2 Cells were cultured in 10% DMEM FBS to 80% confluence in a flat-bottomed cell (Corning®, Sigma-Aldrich Co., Saint Louis, MO, US). The cells were cotransfected by calcium phosphate with plasmids containing an expression cassette flanked by AAV2 ITR, a helper plasmid containing the AAV2 rep gene and the cap gene for serotype 1 AAV, and a plasmid containing adenovirus helper function. A null vector (pAAV-null) was produced using a non-coding plasmid. AAV was purified by an optimized method based on a polyethylene glycol precipitation step and two consecutive 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 titer of the viral genome 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 the 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 0.8 μg of DNA per well was transfected using Lipofectamine 2000 according to the manufacturer's instructions for use (Thermo Fisher Scientific). Untransfected HEK293 cells and HEK293 cells transfected with an AAV plasmid without the transgene (pAAV-null) were used 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 due to improved 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. HEK293 cells transfected with the pAAV-null plasmid were used as a control. As previously observed, cells transfected with pAAV-miniCMV-Ins3 showed superior performance compared to cells 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 evaluate their biological activity, we generated AAV1-Ins1, AAV1-Ins3, AAV1-Ins4, AAV1-Ins5, or AAV1-Ins6 (see Table 13) from AAV1 vectors encoding preproinsulin variants, and evaluated their efficacy in improving glucose processing in vivo in healthy mice. For this purpose, we used 3 × 10⁶ CD1 mice. 11Mice were treated with AAV-ins or AAV1-null vectors containing the viral genome (vg). Mice were anesthetized by intraperitoneal injection of ketamine (100 mg / kg) and xylazine (10 mg / kg). The hind limbs were shaved, and the vector was administered by intramuscular injection of a total volume of 180 μl divided into six injection sites distributed in the quadriceps, gastrocnemius, and craniotibial regions of each hind limb.

[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 administered glucose (2 g / kg body weight) intraperitoneally. Blood glucose levels in tail vein blood samples were measured at the indicated time points.

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

[0302] (Example 9) In vitro evaluation of TLR9 stimulation with AAV-Ins or AAV-Gck Differences in TLR9 stimulation were detected in HEK-Dual® hTLR9 cells transduced with AAV1-ratGck or AAV1-hInsB10D_2 (Figure 18A). TLR9 stimulation was reduced in cells transduced with modified AAV1-GCK constructs (1) containing the CMV promoter and modified hGck8 coding sequence (AAV1-hGck8) and (2) containing the CMV promoter and modified hGck12 coding sequence (AAV1-hGck12) compared to the wild-type AAV1-GCK control construct (AAV1-hGckWT) containing the CMV promoter and hGckWT coding sequence (Figure 18B). TLR9 stimulation was similar between control AAV1-CMV-hInsB10D cells and cells transduced with 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 slope of TLR9 stimulation is shown in Table 15. The results indicate reduced TLR9 stimulation by constructs containing modified GcK coding sequences, suggesting reduced immune activation by 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 induction model of diabetic C57BL / 6J mice after six intramuscular injections. Two cohorts of mice (8-9 weeks old at the start of treatment) were obtained. Diabetes was induced in one cohort by administering five daily intraperitoneal (ip) doses of streptozotocin (50 mg / kg; STZ). The second cohort served as a non-diabetic control by administering five daily intraperitoneal doses of sodium citrate buffer.

[0304] After induction, the animals were continuously fed and given local municipal tap water, which had been chlorinated through reverse osmosis to maintain a chlorine concentration of 1–6 ppm, via an automated water supply system. The animals were acclimated to the animal facility for four days prior to determining baseline body weight, non-fasting blood glucose, and circulating mouse insulin levels. The animals were then divided into blocks and assigned to treatment groups so that there were no significant intra-group or inter-group differences in any of these parameters.

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

[0306] Body weight and non-fasting blood glucose were determined weekly. To maintain consistency, all measurements were taken 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, circulating insulin in mice and humans, HbA1c, and other metabolic parameters. Tissue samples from muscle, liver, and pancreas were collected, weighed, and stored for assessment 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 product was effective in significantly lowering blood glucose levels to those of non-diabetic controls by day 33 (Figure 19). Once euglycemia was achieved, this effect was maintained throughout the study. The treatment appeared to prevent weight loss often associated with untreated type 1 diabetes and restored several metabolic parameters to levels associated with euglycemia. Optimal profiles of kinetics and glucose-lowering effects were shown with the B10H construct containing the natural insulin signaling peptide (high dose) and the B10H construct containing the IL-6 signaling peptide (low dose).

[0308] These data further demonstrated that insulin and glucokinase can be expressed in skeletal muscle using the AAV1 vector. Treatment with these vectors in a mouse model of type 1 diabetes rapidly restored euglycemic levels under both fed and fasting conditions, and maintained them for 8 weeks.

[0309] 10.1 Circulating insulin levels in diabetic mice after administration of AAV-Ins and AAV-Gck vectors STZ administration eliminated all detectable circulating mouse insulin. To evaluate the efficacy of AAV1 vector constructs containing either the native sequence or the IL-6 signaling sequence of hInsB10D or hInsB10H, circulating human insulin was measured in STZ mice 4 weeks after intramuscular (im) injection of the AAV1 vector construct and after 6 hours of fasting. All samples were analyzed by a validated plate-based ELISA method.

[0310] In the literature, fasting circulating insulin levels in C57Bl / 6 mice are typically in the range of 0.75–1.0 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). Ultimately, both groups were euthanized due to hypoglycemia.

[0311] As shown in Figure 19, animals administered with 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) both reduced blood glucose levels, maintaining them at or near the levels of non-diabetic control mice throughout 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 demonstrate that co-administration of hINS and hGCK to the skeletal muscle of STZ-treated mice can effectively control blood glucose when circulating insulin levels are within the range of normal fasting (i.e., basal) levels.

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

[0313] Fasting blood glucose (T=0) levels in non-diabetic control mice were significantly lower than those in STZ-treated animals (Figure 21). Treatment with the AAV1 vector construct significantly reduced fasting glucose levels compared to STZ control mice and non-diabetic controls. After forced oral administration of glucose (2 g / kg), blood glucose levels in non-STZ controls increased by 189 ± 17 mg / dL, peaking at 15 minutes and returning to near-control levels at 90 minutes. In contrast, STZ-treated mice increased by 264 ± 37 mg / dL, peaking at nearly 30 minutes and not returning to control levels even at 120 minutes. This data indicates that STZ-treated mice were unable to properly regulate glucose processing after the postprandial fluctuation range. Similar to non-diabetic controls, intramuscular injections 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 fluctuation ranges of 154±16 and 218±22 mg / dL, respectively, 15 minutes after loading. Both treatments returned blood glucose to T=0 levels within 60 minutes. Results from ANCOVA analysis using AUC supported these interpretations (Figure 22). Overall, these data suggest that intramuscular injections of these constructs can not only control fasting glucose levels but also smooth the large postprandial glucose fluctuation ranges typically observed in diabetic patients.

[0314] The goal for type 1 diabetes is to normalize blood glucose control without changing body weight, while preventing diabetic ketoacidosis and hypoglycemia as a medical consequence. Because patients with type 1 diabetes have little to no circulating insulin, they must take insulin daily to survive. Furthermore, hyperglycemia caused by streptozotocin (STZ)-induced diabetes has been reported to progressively lead to insulin resistance in peripheral tissues (Ordonez P, Moreno M, Alonso A, Fernandez R, Diaz F and Gonzalez C. Insulin sensitivity in streptozotocin-induced diabetic rats treated with different doses of 17beta-oestradiol or progesterone. Exp Physiol 92:241-9, 2007. doi: 10.1113 / expphysiol.2006.035006. Epub 2006 Oct 26.). The results presented here support the idea that improving insulin sensitivity in peripheral tissues, in addition to providing an alternative insulin supply to muscles, can provide multiple means of restoring blood glucose control.

[0315] 10.3 HbA1c levels in diabetic mice after administration of AAV-Ins and AAV-Gck vectors Blood samples were obtained during the last non-fasting blood collection 8 weeks after injection. Serum glycated hemoglobin (HbA1c) was determined from the second drop (5–10 μL) of blood taken from tail amputation using a handheld HbA1c analyzer. This method has been validated by comparative studies against methods certified by the National Glycohemoglobin Standardization Program (NGSP) as an absolute standard. 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 non-diabetic mice is 4%–5.6%, and diabetes is defined as HbA1c > 6.5%. Data from this study are shown in Figure 23. HbA1c levels in non-STZ control mice were 4.3±0.05%. STZ administration resulted in a significant increase in HbA1c (9.48±0.64%; p<0.001) compared to non-diabetic controls. Treatment with high doses (4.89±0.15%) of the vector construct hInsB10H+AAV1-GCK(containing the coding sequence of SEQ ID NO: 111)(AAV926) and low doses (5.03±0.27%) of hInsB10H+IL6(containing the coding sequence of SEQ ID NO: 120)+AAV1-GCK(AAV926) significantly reduced blood glucose and HbA1c to levels of non-diabetic controls.

[0317] HbA1c is a comprehensive signal that reflects the average blood glucose level over a certain period. In mice, the t of red blood cells 1 / 2 The duration is approximately 14 days. Clinically, this trial is a primary tool for evaluating glycemic control and has strong predictive value regarding diabetes and its comorbidities. The goal of diabetes therapy is to maintain HbA1c within the normal range (<6.5%), and most commercially available products reduce 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 non-diabetic controls within 8 weeks. Furthermore, these vectors resulted in a >4% reduction in HbA1c compared to STZ controls. HbA1c decreased in all groups, but only the 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) groups survived until the 8-week blood draw. This means that the HbA1c levels are the average over 8 weeks.

[0318] HbA1c, along with weekly temporal measurements of blood glucose, provides a quantitative measure of both improved postprandial glucose exposure over a period of time and reduced degree of blood glucose variability, suggesting that both factors can be normalized with this treatment. These results support the possibility that intramuscularly co-administered hIns and GcK AAV constructs in patients with type 1 diabetes may 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 In a mouse model of STZ-induced diabetes, both serum triglyceride and ketone levels were elevated compared to non-diabetic controls, and energy was primarily shifting to lipid-based sources. Intramuscular injection of high-dose hInsB10H (containing the coding sequence of SEQ ID NO: 111) + AAV1-GCK (AAV926) and low-dose 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 non-diabetic controls. This data showed that multiple metabolic endpoints normalized after administration of AAV-hInsB10H (containing the native sequence or the IL-6 signaling sequence) and AAV-Gck vectors.

[0320] 10.5 Liver 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. Hepatic hINS mRNA levels were assessed to determine whether the AAV-1 vector bypassed muscle, entered circulation, transduced to the liver, and was subsequently transcribed to detectable levels. Samples from non-diabetic control or STZ control mice were not measured as they were not injected with the vector. Animals that died unexpectedly or exhibited distress were not included in the analysis. Results are shown in Table 18 and Figure 25. [Table 18]

[0321] The results of this assay show a ΔCt of more than 5 cycles, which is the same as the three tests performed. The levels of hINS mRNA in the liver of mice administered with the constructs (high dose of AAV1-mWTIns + AAV1-rGck (AAV926); low dose of AAV1-mWTIns (Ins17) + AAV1-rGck (AAV926); and high dose of AAV1-mWTIns (Ins17) + AAV1-rGck (AAV926)) were extremely low or absent, suggesting that the intramuscularly injected AAV largely remained in the target muscle. These results indicate that intramuscular delivery of the AAV1-Ins construct led to normalization of blood glucose, HbA1c, ketones, and triglycerides, resulting from the transduction of AAV-Ins and AAV-Gck vectors and subsequent protein expression within the muscle. In certain embodiments, for example, the following are provided: (Item 1) A polynucleotide encoding human insulin (Ins) protein, comprising: (i) a nucleotide sequence encoding a signal peptide, wherein the signal peptide is optionally not a wild-type preproinsulin signal sequence; and (ii) 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 of these relative to the corresponding amino acid positions in wild-type proinsulin. combination A polynucleotide comprising a nucleotide sequence encoding a proinsulin polypeptide, comprising an amino acid modification at a position selected from, wherein the polynucleotide optionally further comprises a furin cleavage site. (Item 2) A polynucleotide comprising nucleic acids encoding human insulin (Ins) protein, wherein the nucleic acids are (a) nucleic acids 73-330 of any of sequence numbers 43-57, 110-116, 150-151, 154-155 or 157-159, nucleic acids 88-345 of any of sequence numbers 117-122, 152 or 156, or nucleic acids 79-336 of sequence number 153, or (b) at least 85%, 90%, 95%, or 99% of any one of sequence numbers 43-57, 110-122 or 150-159. Also This is a polynucleotide containing open reading frames (ORFs) that contain 100% identical nucleotide sequences. (Item 3) The polynucleotide described in item 2, wherein the encoded human Ins protein contains any of the amino acid sequences of SEQ ID NO: 41 (amino acids 25-110), SEQ ID NO: 144 (amino acids 25-110), SEQ ID NO: 145 (amino acids 25-110), SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO: 145. (Item 4) The human Ins protein is a polynucleotide as described in item 2 or 3, comprising a signal peptide. (Item 5) The polynucleotide according to item 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) The polynucleotide described in item 5, wherein the signal peptide comprises 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. (Item 7) The human Ins protein further comprises a polynucleotide according to any one of items 1 to 6. (Item 8) The nucleic acid further comprises a 5'UTR containing nucleotide sequences that are at least 85%, 90%, 95%, 99%, or 100% identical to nucleic acid 5-329 of SEQ ID NO: 42, item 1 or A polynucleotide as described in any of the seven items. (Item 9) The polynucleotide according to any one of items 1 to 8, wherein the nucleic acid further comprises a 5'UTR having a nucleotide sequence that is 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) The polynucleotide according to any one of items 1 to 9, wherein the nucleic acid further comprises a 3'UTR having a nucleotide sequence that is 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) The polynucleotide according to any one 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) A polynucleotide as described in any one of items 1 through 11, comprising at least two open reading frames (ORFs) encoding a human Ins protein. (Item 13) The polynucleotide according to item 12, wherein at least two of the aforementioned ORFs are operably linked by an IRES sequence. (Item 14) The polynucleotide according to item 13, wherein the IRES sequence contains a nucleotide sequence that is at least 85%, 90%, 95%, 99%, or 100% identical to sequence number 142 or sequence number 143. (Item 15) The nucleic acid is (a) Any of sequence numbers 1 to 16 Kaka Nucleotide sequences that are at least 85%, 90%, 95%, 99%, or 100% identical to the selected sequences. or any combination thereof A polynucleotide, including any of the items listed in items 1 through 10. (Item 16) The nucleic acid is (a) Any of sequence numbers 84-88 Kaka Nucleotide sequences that are at least 85%, 90%, 95%, 99%, or 100% identical to the selected sequences. or any combination thereof A polynucleotide, including any one of items 1 through 11. (Item 17) The nucleic acid is (a) Any of sequence numbers 124-126, 130-132, and 139-141 Kaka Nucleotide sequences that are at least 85%, 90%, 95%, 99%, or 100% identical to the selected sequences. or any combination thereof A polynucleotide, including any of the items listed in items 12 to 14. (Item 18) The polynucleotide according to any one of items 1 to 17, wherein the nucleic acid is operably linked to a promoter. (Item 19) An expression cassette comprising a polynucleotide described in any one of items 1 to 18 and a heterologous expression control sequence operably linked to the sequence of the nucleic acid. (Item 20) The expression cassette described in item 19, wherein the heterologous expression control sequence is the promoter. (Item 21) The polynucleotide or expression cassette described in item 20, wherein the promoter is a eukaryotic promoter. (Item 22) The polynucleotide or expression cassette described in item 21, wherein the promoter is the CMV promoter. (Item 23) The polynucleotide or expression cassette according to any of the preceding items, wherein the nucleic acid is operably linked to a polyadenylated (poly-A) element. (Item 24) A vector containing a polynucleotide or expression cassette as described in any of the preceding items. (Item 25) The vector described in item 24, wherein the vector is a viral vector, a nonviral vector, a plasmid, a lipid, or a lysosome. (Item 26) The vector described in item 24, wherein the vector is an adeno-associated virus (AAV) vector or a lentiviral vector. (Item 27) Recombinant AAV (rAAV) particles comprising an AAV capsid and a vector genome containing a polynucleotide or expression cassette as described in any of items 1 to 23. (Item 28) The vector or rAAV particle described in item 26 or 27, wherein the serotype of the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRH8, AAVrh9, AAV9, AAVrh10, AAV10, AAVH10, 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 containing a polynucleotide, expression cassette, vector, or rAAV particle as described in any of the preceding items. (Item 31) A mammalian host cell, as described in item 30. (Item 32) A method for producing human Ins protein in cells, comprising contacting the cells with a polynucleotide, expression cassette, vector, or rAAV particle described in any of items 1 to 29, and / or transforming the cells with a polynucleotide, expression cassette, vector, or rAAV particle described in any of items 1 to 29, thereby producing the human Ins protein in the cells. (Item 33) A method for causing a subject to produce a human Ins protein, comprising administering a polynucleotide, expression cassette, vector, or rAAV particle described in any of items 1 to 29 to the subject, thereby causing the subject to produce the human Ins protein. (Item 34) A method for treating or improving diabetes-related symptoms in a subject that requires treatment or improvement of diabetes-related symptoms, comprising delivering a therapeutically effective amount of a polynucleotide, expression cassette, vector, or rAAV particle described in any of items 1 to 29 to the subject, thereby treating the diabetes of the subject. (Item 35) The method according to 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 the human glucokinase (Gck) protein, wherein the nucleic acid is (a) a nucleotide sequence, wherein (a) nucleic acids 1 to 1398 of any of sequence numbers 61 to 80 or 162, or (b) sequence numbers 61 to 80 and 16 2 A nucleotide sequence that is at least 85%, 90%, 95%, 99%, or 100% identical to a sequence selected from any of the above. , or any combination thereof Polynucleotides containing ORFs. (Item 37) The encoded human Gck protein is a polynucleotide as described in item 36, comprising the amino acid sequence of sequence number 82. (Item 38) The polynucleotide according to item 36 or item 37, wherein the nucleic acid further comprises a 5'UTR having nucleotide sequences that are at least 85%, 90%, 95%, 99%, or 100% identical to nucleic acids 5-329 of SEQ ID NO: 42. (Item 39) The polynucleotide according to any one of items 36 to 38, wherein the nucleic acid further comprises a 5'UTR containing a nucleotide sequence that is 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 40) The polynucleotide according to any one of items 36 to 39, wherein the nucleic acid further comprises a 3'UTR having a nucleotide sequence that is 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) The polynucleotide according to any one 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 is (a) Any of sequence numbers 20-39 Kaka Nucleotide sequences that are at least 85%, 90%, 95%, 99%, or 100% identical to the selected sequences. or any combination thereof Polynucleotides, including those listed in any of items 36 to 40. (Item 43) The nucleic acid is (a) Any of sequence numbers 89-96 and 163-164 Kaka Nucleotide sequences that are at least 85%, 90%, 95%, 99%, or 100% identical to the selected sequences. or any combination thereof Nucleotides, including any one of items 36 to 40. (Item 44) The polynucleotide according to any one of items 36 to 43, wherein the nucleic acid is operably linked to a promoter. (Item 45) An expression cassette comprising a polynucleotide as described in any one of items 36 to 44 and a heterologous expression control sequence operably linked to the sequence of the nucleic acid. (Item 46) The expression cassette described in item 45, wherein the heterologous expression control sequence is the promoter. (Item 47) The polynucleotide or expression cassette described in item 46, wherein the promoter is a eukaryotic promoter. (Item 48) The polynucleotide or expression cassette described in item 46, wherein the promoter is the CMV promoter. (Item 49) A polynucleotide or expression cassette according to any one of items 36 to 48, wherein the nucleic acid is operably linked to a polyadenylated (poly-A) element. (Item 50) A vector containing a polynucleotide or expression cassette as described in any of items 36 to 49. (Item 51) A vector as described in item 50, which is a viral vector, a nonviral vector, a plasmid, a lipid, or a lysosome. (Item 52) The vectors described in item 51, which are adeno-associated virus (AAV) vectors or lentiviral vectors. (Item 53) Recombinant AAV (rAAV) particles comprising an AAV capsid and a vector genome containing a polynucleotide or expression cassette as described in any of items 36 to 49. (Item 54) The vector or rAAV particle described in item 52 or 53, wherein the serotype of the AAV 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) A host cell containing a polynucleotide, expression cassette, vector, or rAAV particle as described in any of items 36 to 54. (Item 56) A mammalian host cell, as described in item 55. (Item 57) A method for producing human Gck protein in cells, comprising contacting the cells with a polynucleotide, expression cassette, vector, or rAAV particle described in any of items 36 to 54, and / or transforming the cells with a polynucleotide, expression cassette, vector, or rAAV particle described in any of items 36 to 54, thereby producing the human Gck protein in the cells. (Item 58) A method for producing human Gck protein in a subject, comprising administering a polynucleotide, expression cassette, vector, or rAAV particle described in any of items 36 to 54 to the subject, thereby causing the subject to produce the human Gck protein. (Item 59) A method for treating or improving diabetes-related symptoms in a subject that requires treatment or improvement of diabetes-related symptoms, comprising delivering a therapeutically effective amount of a polynucleotide, expression cassette, vector, or rAAV particle described in any of items 36 to 54 to the subject, thereby treating the diabetes of the subject. (Item 60) The method according to item 59, wherein the diabetes is type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM). (Item 61) A method for treating or improving diabetes-related symptoms in a subject that requires treatment or improvement of diabetes-related symptoms, comprising (i) delivering a therapeutically effective amount of a polynucleotide, expression cassette, vector, or rAAV particle described in any one of items 1 to 29, and (ii) delivering a therapeutically effective amount of a polynucleotide, expression cassette, vector, or rAAV particle described in any one of items 36 to 54 to the subject, thereby treating diabetes in the subject. (Item 62) A method for treating or improving symptoms associated with diabetes in a subject that requires the production of human Ins protein and human Gck protein, and / or for treating or improving symptoms associated with diabetes, 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, a plurality of expression cassettes, a plurality of vectors, or a plurality of rAAV particles as described in any one of items 1 to 29, and (ii) a polynucleotide, a plurality of expression cassettes, a plurality of vectors, or a plurality of rAAV particles as described in any one of items 36 to 54, thereby causing the subject to produce human Ins protein and human Gck protein and / or treating diabetes. (Item 63) The method according to item 61 or 62, wherein the diabetes is type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM). (Item 64) (i) a polynucleotide, expression cassette, vector, or rAAV particle as described in any one of items 1 to 29, and (ii) a polynucleotide, expression cassette, vector, or rAAV particle as described in any one of items 36 to 54, administered together or sequentially, according to any one of items 61 to 63. (Item 65) The method according to any one of items 32 to 35, 57 to 60, and 61 to 64, wherein the delivery and / or administration is intramuscular. (Item 66) (i) the blood glycated hemoglobin (HbA1c) level decreases and / or is regulated in the subject, (ii) circulating ketones are reduced in the subject, (iii) triglycerides are reduced in the subject, or (iv) any of the above combination The method described in any of items 32 to 35, 57 to 60, and 61 to 65.

Claims

1. (i) a polynucleotide, expression cassette, vector, or recombinant adeno-associated virus (AAV) particle comprising a nucleotide sequence encoding a human insulin (Ins) protein, wherein the nucleotide sequence encoding the human Ins protein comprises (a) 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 (a) nucleic acids 73-330 of any one sequence number 43-57, 110-116, 150-151, 154-155 or 157-159, nucleic acids 88-345 of any one sequence number 117-122, 152 or 156, or nucleic acids 79-336 of sequence number 153, and the human Ins protein further comprises an IL-6 signal sequence, (ii) A polynucleotide, expression cassette, vector, or rAAV particle comprising a nucleotide sequence encoding a human glucokinase (Gck) protein, wherein the nucleotide sequence encoding the human Gck protein is (a) 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 nucleic acids 1 to 1398 of any one of sequence numbers 61 to 80 or 162, or (b) a polynucleotide, expression cassette, vector, or rAAV particle comprising any one of sequence numbers 61 to 80 or 162. A composition containing the following:

2. In the manufacture of pharmaceuticals for treating or improving symptoms related to diabetes, (i) a polynucleotide, expression cassette, vector, or rAAV particle comprising a nucleotide sequence encoding human insulin (Ins) protein, wherein the nucleotide sequence encoding the human Ins protein comprises (a) 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 (a) nucleic acids 73-330 of any one sequence number 43-57, 110-116, 150-151, 154-155 or 157-159, nucleic acids 88-345 of any one sequence number 117-122, 152 or 156, or nucleic acids 79-336 of sequence number 153, and the human Ins protein further comprises an IL-6 signal sequence, and (ii) Polynucleotides, expression cassettes, vectors, or rAAV particles comprising a nucleotide sequence encoding a human glucokinase (Gck) protein, wherein the nucleotide sequence encoding the human Gck protein is (a) 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 nucleic acids 1 to 1398 of any one of SEQ ID NOs. 61 to 80 or 162, or (b) a polynucleotide, expression cassette, vector, or rAAV particle comprising any one of SEQ ID NOs. 61 to 80 or 162. Use of compositions containing the above.

3. It is a combination, (a) A composition comprising a polynucleotide comprising a nucleic acid encoding a human insulin (Ins) protein, wherein the nucleic acid encoding the human Ins protein comprises a nucleotide 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 nucleic acids 73-330 of any one of SEQ ID NOs: 43-57, 110-116, 150-151, 154-155, or 157-159, nucleic acids 88-345 of any one of SEQ ID NOs: 117-122, 152, or 156, or nucleic acids 79-336 of SEQ ID NO: 153, and the human Ins protein further comprises an IL-6 signal sequence, (b) A composition comprising a polynucleotide comprising a nucleic acid encoding a human glucokinase (Gck) protein, wherein the nucleic acid encoding the human Gck protein comprises a nucleotide sequence which is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to one nucleic acid 1 to 1398 of SEQ ID NOs. 61 to 80 or 162, or comprises a nucleotide sequence of SEQ ID NOs. 61 to 80 or 162. A combination that includes [something].

4. A polynucleotide, expression cassette, vector, or rAAV particle comprising a nucleic acid encoding a human insulin (Ins) protein, wherein the nucleic acid encoding the human Ins protein comprises nucleotide sequences that are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to nucleic acids 73-330 of any one of SEQ ID NOs: 43-57, 110-116, 150-151, 154-155, or 157-159, nucleic acids 88-345 of any one of SEQ ID NOs: 117-122, 152, or 156, or nucleic acids 79-336 of SEQ ID NO: 153, and the human Ins protein further comprises an IL-6 signal sequence.

5. A composition comprising the polynucleotide, expression cassette, vector, or rAAV particles described in Claim 4, wherein the composition is A second composition comprising a polynucleotide, expression cassette, vector, or rAAV particles containing a nucleic acid encoding a human glucokinase (Gck) protein, wherein the nucleic acid encoding the human Gck protein comprises a nucleotide 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 a sequence selected from any one nucleic acid 1 to 1398 of SEQ ID NOs. 61 to 80 or 162, or the nucleotide sequence of any one of SEQ ID NOs. 61 to 80 or 162. A composition characterized by being administered in combination with [another substance].

6. A composition, combination, polynucleotide, expression cassette, vector, or rAAV particle according to any one of claims 1 and 3 to 5, for use in producing human Ins protein and human Gck protein in subjects where it is necessary to produce human Ins protein and human Gck protein, and / or in treating or improving diabetes-related symptoms in subjects where it is necessary to treat or improve diabetes-related symptoms.

7. The polynucleotide, expression cassette, vector, rAAV particle, composition, use, or combination according to any one of claims 1 to 6, wherein the encoded human Ins protein comprises one amino acid sequence from 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.

8. The polynucleotide, expression cassette, vector, rAAV particle, composition, use, or combination according to any one of claims 1 to 7, further comprising a promoter operably linked to the nucleotide sequence encoding the human Ins protein.

9. The polynucleotide, expression cassette, vector, rAAV particle, composition, use, or combination according to claim 8, wherein the promoter is a constitutive promoter.

10. The polynucleotide, expression cassette, vector, rAAV particle, composition, use, or combination according to claim 9, wherein the promoter is a CMV promoter.

11. The vector, composition, use, or combination according to any one of claims 1 to 10, wherein the vector comprises recombinant adeno-associated virus (rAAV) particles having the nucleotide sequence encoding the human Ins protein.

12. The polynucleotide, expression cassette, vector, rAAV particle, composition, use, or combination according to any one of claims 1 to 11, wherein the encoded human Gck protein comprises the amino acid sequence of SEQ ID NO:

82.

13. The polynucleotide, expression cassette, vector, rAAV particles, composition, use, or combination according to any one of claims 1 to 12, wherein the polynucleotide, expression cassette, vector, or rAAV particles comprising the nucleotide sequence encoding the human Gck comprises a sequence that is at least 90%, 95%, 99%, or 100% identical to any of sequence numbers 20-39, 89-96, and 163-164, or any combination thereof.

14. The polynucleotide, expression cassette, vector, rAAV particle, composition, use, or combination according to any one of claims 1 to 13, further comprising a promoter operably linked to the nucleotide sequence encoding the human Gck protein.

15. The polynucleotide, expression cassette, vector, rAAV particle, composition, use, or combination according to claim 14, wherein the promoter is a constitutive promoter.

16. The polynucleotide, expression cassette, vector, rAAV particle, composition, use, or combination according to claim 15, wherein the promoter is a CMV promoter.

17. (i) A nucleotide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to Sequence ID No. 121, and (ii) A polynucleotide, expression cassette, vector, rAAV particle, composition, use, or combination according to any one of claims 1 to 16, comprising a nucleotide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to Sequence ID No.

68.

18. The vector, rAAV particles, composition, use, or combination according to any one of claims 1 to 17, wherein the serotype of the AAV is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRH8, AAVrh9, AAV9, AAVrh10, AAV10, AAVRH10, AAV11, or AAV12.

19. The vector, rAAV particles, composition, use, or combination according to any one of claims 1 to 18, wherein the serotype of the AAV in the AAV particles is AAV1.

20. The use according to any one of claims 2 or 6 to 19, wherein the diabetes is type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM).

21. The combination according to any one of claims 3 or 6 to 19, wherein the combination is administered together or sequentially.

22. A polynucleotide, expression cassette, vector, rAAV particle, composition, use, or combination according to any one of claims 1 to 21, formulated for intramuscular delivery and / or intramuscular administration.

23. A polynucleotide, expression cassette, vector, rAAV particle, composition, use, or combination according to any one of claims 1 to 22, characterized by (i) a decrease and / or regulation of glycated hemoglobin (HbA1c) levels in a subject after administration, (ii) a reduction in circulating ketones in a subject after administration, (iii) a reduction in triglycerides in a subject after administration, or (iv) any combination thereof.

24. A polynucleotide encoding human insulin (Ins) protein, comprising: (i) a nucleotide sequence encoding an IL-6 signal peptide; and (ii) a nucleotide sequence encoding a proinsulin polypeptide, which includes amino acid modifications at positions 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 positions in wild-type proinsulin.

25. A polynucleotide comprising nucleic acid encoding human insulin (Ins) protein, wherein the nucleic acid encoding the human Ins protein comprises a nucleotide 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 the nucleic acids 73-330 of SEQ ID NOs. 43-57, 110-116, 150-151, 154-155, or 157-159, any one of the nucleic acids 88-345 of SEQ ID NOs. 117-122, 152, or 156, or any of the nucleic acids 79-336 of SEQ ID NO. 153, and the human Ins protein further comprises an IL-6 signal sequence.

26. The polynucleotide according to claim 25, wherein the encoded human Ins protein includes 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.

27. The polynucleotide, expression cassette, vector, rAAV particle, composition, use, or combination according to any one of claims 1 to 26, wherein the human Ins protein further comprises a cleavage site.

28. The polynucleotide, expression cassette, vector, rAAV particle, composition, use, or combination according to any one of claims 1 to 27, wherein the nucleic acid encoding the human Ins protein further comprises a 5'UTR having at least 90%, at least 95%, at least 99%, or 100% identical nucleotide sequences to nucleic acids 5 to 329 of SEQ ID NO:

42.

29. A polynucleotide, expression cassette, vector, rAAV particle, composition, use, or combination according to any one of claims 1 to 27, further comprising a 5'UTR having a nucleotide sequence at least 90%, at least 95%, at least 99%, or 100% identical with SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO:

148.

30. A polynucleotide, expression cassette, vector, rAAV particle, composition, use, or combination according to any one of claims 1 to 29, wherein the nucleic acid encoding the human Ins protein further comprises a 3'UTR having a nucleotide sequence that is at least 90%, at least 95%, at least 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.

31. The polynucleotide, expression cassette, vector, rAAV particle, composition, use, or combination according to claim 30, wherein the 3'UTR further comprises a restricting site selected from the group consisting of BamHI, EcoRI, NdeI, EcoRV, SpeI, XbaI, NheI, VspI, NsiI, ScaI, KpnI, SspI, and PacI.

32. A polynucleotide, expression cassette, vector, rAAV particle, composition, use, or combination according to any one of claims 1 to 31, comprising at least two open reading frames (ORFs) encoding human Ins proteins.

33. The polynucleotide, expression cassette, vector, rAAV particle, composition, use, or combination according to claim 32, wherein at least two ORFs are operably linked by an IRES sequence.

34. The polynucleotide, expression cassette, vector, rAAV particle, composition, use, or combination according to claim 33, wherein the IRES sequence comprises a nucleotide sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 142 or SEQ ID NO:

143.

35. The nucleic acid encoding the human Ins protein, Nucleotide sequences or any combination thereof that are at least 90%, at least 95%, at least 99%, or 100% identical to any of sequence numbers 1-16. A polynucleotide, expression cassette, vector, rAAV particle, composition, use, or combination according to any one of claims 1 to 34, comprising:

36. The nucleic acid encoding the human Ins protein is Nucleotide sequences or any combination thereof that are at least 90%, at least 95%, at least 99%, or 100% identical to any of sequence numbers 84-88. A polynucleotide, expression cassette, vector, rAAV particle, composition, use, or combination according to any one of claims 1 to 34, comprising:

37. The nucleic acid encoding the human Ins protein, Nucleotide sequences or any combination thereof that are at least 90%, at least 95%, at least 99%, or 100% identical to any of sequence numbers 123-126, 128-141, and 160-161. A polynucleotide, expression cassette, vector, rAAV particle, composition, use, or combination according to any one of claims 1 to 34, comprising:

38. An expression cassette comprising a polynucleotide according to any one of claims 4 and 6 to 37 and a promoter operably ligated to the sequence of the nucleic acid.

39. The expression cassette according to claim 38, wherein the promoter is a constitutive promoter.

40. The expression cassette according to claim 39, wherein the promoter is a CMV promoter.

41. The polynucleotide or expression cassette according to any one of claims 4 and 6 to 40, wherein the nucleic acid is operably linked to a polyadenylated (poly-A) element.

42. A vector comprising a polynucleotide or expression cassette according to any one of claims 4 and 6 to 41.

43. The vector according to claim 42, wherein the vector is a viral vector, a non-viral vector, a plasmid, a lipid, or a lysosome.

44. The vector according to claim 43, wherein the vector is an adeno-associated virus (AAV) vector or a lentiviral vector.

45. Recombinant AAV (rAAV) particles comprising an AAV capsid and a vector genome comprising a polynucleotide or expression cassette according to any one of claims 4 and 6 to 41.

46. The vector or rAAV particle according to claim 44 or 45, wherein the serotype of the AAV is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRH8, AAVrh9, AAV9, AAVrh10, AAV10, AAVRH10, AAV11, or AAV12.

47. A host cell comprising a polynucleotide, expression cassette, vector, or rAAV particle according to any one of claims 4 and 6 to 46.

48. The host cell according to claim 47, which is a mammalian cell.

49. A composition for use in a method for producing human Ins protein in cells, wherein the composition comprises a polynucleotide, expression cassette, vector, or rAAV particle according to any one of claims 4 and 6 to 46, the method comprising contacting the polynucleotide, expression cassette, vector, or rAAV particle with the cells and / or transforming the cells with the polynucleotide, expression cassette, vector, or rAAV particle to produce the human Ins protein in the cells.

50. A composition for producing human Ins protein in a subject, wherein the composition comprises a polynucleotide, an expression cassette, a vector, or rAAV particles as described in any one of claims 4 and 6 to 46.

51. A composition for treating or improving diabetes-related symptoms in a subject requiring treatment or improvement of diabetes-related symptoms, wherein the composition comprises a polynucleotide, expression cassette, vector, or rAAV particle according to any one of claims 4 and 6 to 46.

52. The composition according to claim 51, wherein the diabetes is type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM).

53. The composition according to any one of claims 49 to 52, characterized in that the composition is formulated for intramuscular administration or intramuscular delivery.

54. (i) a decrease and / or regulation of glycated hemoglobin (HbA1c) levels in the subject after administration; (ii) a reduction in circulating ketones in the subject after administration; (iii) a reduction in triglycerides in the subject after administration; or (iv) any combination thereof, according to any one of claims 49 to 53.

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