Aav-mediated insulin and glucokinase gene therapy for the treatment of diabetes

Abstract

This disclosure relates to a combination therapy comprising a first AAV vector genome containing an insulin expression cassette and a second AAV vector genome containing a glucokinase expression cassette, wherein the first AAV vector genome and the second AAV vector genome are administered intramuscularly (e.g., to at least two skeletal muscle groups), and optionally wherein the combination therapy is accompanied by an immunosuppressive regimen; and a method of treating diabetes using the combination therapy.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 595,296, filed November 1, 2023, and U.S. Provisional Application No. 63 / 615,452, filed December 28, 2023, each of which is hereby incorporated in its entirety.

[0003] References to sequence lists submitted electronically

[0004] The contents of the .XML file format sequence list (4525_129PC02_SequenceListing_ST26.xml; size: 302,607 bytes; and creation date: October 24, 2024), which was submitted electronically with this application, are incorporated herein by reference in their entirety. Technical Field

[0005] This disclosure relates to the medical field, including AAV gene therapy compositions comprising nucleic acids encoding insulin and glucokinase, and immunosuppressive regimens used together with them for the treatment of diabetes. Background Technology

[0006] The two main forms of diabetes are type 1 (T1DM) and type 2 (T2DM) (Diabetescare, 1997, 20-1183-1197).

[0007] Type 1 diabetes mellitus (T1DM) is characterized by a severe deficiency of insulin production due to specific destruction of pancreatic β-cells. β-cell loss in T1DM results from an autoimmune-mediated process in which chronic inflammation known as islet inflammation causes 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 disorders in childhood, and its incidence is rapidly increasing, especially in young children. T1DM is diagnosed when autoimmune-mediated β-cell destruction is nearly complete, leading to a need for insulin replacement therapy for survival. In adults, T1DM may present similarly to T2DM, with a slow deterioration in metabolic control, subsequently progressing to insulin dependence. This form is known as latent autoimmune diabetes mellitus in adults (LADA) (Diabetes Atlas 4th Edition, 2009, International Diabetes Federation).

[0008] Type 2 diabetes mellitus (T2DM) is the most common form of diabetes and has been attributed to the interaction of genetic, environmental, and behavioral risk factors. T2DM is characterized by insulin insensitivity, decreased insulin production, and eventual pancreatic β-cell failure (Olokoba, A. et al., 2012, Oman Med. J. 27(4):269-273).

[0009] Lowering hyperglycemia and maintaining normal blood glucose levels are the goals of any treatment for type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). Currently, therapies for all patients with T1DM and a large proportion of those with T2DM are based on regular subcutaneous injections of both short-acting and long-acting insulin preparations.

[0010] Lifelong insulin therapy is typically the first-line treatment for both type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). While lifelong exogenous insulin therapy has been very successful in managing diabetes, complications can still occur due to the difficulty in maintaining strict glycemic control. Prolonged hyperglycemia can lead to serious microvascular or macrovascular complications, most commonly retinopathy, neuropathy, nephropathy, stroke, or myocardial infarction. These devastating complications can be prevented by improving glycemic control. Notably, brittle diabetes is a particularly unstable form that can be difficult to manage even with lifelong exogenous insulin therapy.

[0011] AAV gene therapy offers a novel treatment paradigm for diabetes. A challenge of AAV gene therapy generally includes the risk of a host immune response to the AAV capsid or the protein encoded by the transgene, which has been observed in various clinical and preclinical studies. This immune response can adversely affect patient health, inhibit viral transduction, prevent repeated dosing strategies, eliminate transduced cells, and pose a significant obstacle to the potential efficacy of AAV gene therapy. This challenge is particularly significant in the diabetic population, as these patients are more sensitive to immunosuppressive regimens (such as steroids) that are typically used in conjunction with gene therapy. Summary of the Invention

[0012] Certain aspects of this disclosure relate to a method for treating or improving diabetes-related symptoms in a subject of need, said method comprising administering a therapy (e.g., combination gene therapy) to said subject, said therapy comprising: one or more adeno-associated virus (AAV) vectors comprising (a) a first promoter operably linked to a polynucleotide encoding a human insulin (hIns) protein; and (b) a second promoter operably linked to a polynucleotide encoding a human glucokinase (hGck) protein; wherein said therapy is administered intramuscularly (IM) to at least two different skeletal muscle groups.

[0013] In some respects, therapies (such as combination gene therapy) include:

[0014] (a) A first AAV vector genome containing an insulin expression cassette, the insulin expression cassette containing a first promoter operatively linked to a multinucleotide encoding the human insulin (hIns) protein, wherein the insulin expression cassette is side-mounted with an inverted terminal repeat (ITR); and

[0015] (b) A second AAV vector genome containing a glucokinase expression cassette, the glucokinase expression cassette containing a second promoter operatively linked to a multinucleotide encoding a human glucokinase (hGck) protein, wherein the glucokinase expression cassette is side-mounted with an inverted terminal repeat (ITR).

[0016] The therapy described herein is administered intramuscularly (IM) to at least two different skeletal muscle groups.

[0017] In some respects, the first AAV vector genome and the second AAV vector genome are administered in the same composition (e.g., via the same IM injection).

[0018] In some respects, the first AAV vector genome and the second AAV vector genome are administered in the same pharmaceutical composition (e.g., via the same IM injection).

[0019] In some aspects, one or more AAV vectors are AAV vector genomes containing a single expression cassette, the single expression cassette containing a promoter (e.g., first and second promoters), a polynucleotide encoding the hIns protein, and a polynucleotide encoding the hGck protein, wherein the single expression cassette is side-mounted with an inverted terminal repeat (ITR).

[0020] In some aspects, the combination therapy intramuscularly (IM) is applied to at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten different skeletal muscle groups. In some aspects, the combination therapy intramuscularly (IM) is applied to 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, or 8-10 different skeletal muscle groups. In some aspects, the combination therapy intramuscularly (IM) is applied to two, three, four, five, six, seven, eight, nine, or ten different skeletal muscle groups.

[0021] In some respects, combination therapy is administered intramuscularly (IM) to (i) 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, or 9-10 different skeletal muscle groups; (ii) 2-8, 3-8, 4-8, 5-8, 6-8, or 7-8 different skeletal muscle groups; (iii) 2-6, 3-6, 4-6, or 5-6 different skeletal muscle groups; (iv) 2-5, 3-5, or 4-5 different skeletal muscle groups; or (iv) 2-4 or 3-4 different skeletal muscle groups.

[0022] In some aspects, the combination therapy comprises: (i) a first recombinant AAV (rAAV) particle comprising a first AAV vector genome containing an insulin expression cassette, the insulin expression cassette containing a promoter operatively linked to a multinucleotide encoding a human insulin (hIns) protein, wherein the insulin expression cassette is side-mounted with an inverted terminal repeat (ITR); and (ii) a second recombinant AAV (rAAV) particle comprising a second AAV vector genome containing a glucokinase expression cassette, the glucokinase expression cassette containing a promoter operatively linked to a multinucleotide encoding a human glucokinase (hGck) protein, wherein the glucokinase expression cassette is side-mounted with an inverted terminal repeat (ITR).

[0023] In some respects, different skeletal muscle groups may include the quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscles), gluteus maximus, hamstrings, deltoids, trapezius, pectoralis major (e.g., pectoral muscles), and latissimus dorsi (lat). In some respects, the skeletal muscle groups are selected from the quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscles), gluteus maximus, hamstrings, deltoids, trapezius, pectoralis major (e.g., pectoral muscles), and latissimus dorsi (lat), optionally two or more of these different skeletal muscle groups include any combination thereof. In some respects, the two or more different skeletal muscle groups are any combination of two or more of the following: quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscles), gluteus maximus, hamstrings, deltoids, trapezius, pectoralis major (e.g., pectoral muscles), or latissimus dorsi (lat).

[0024] In some respects, the skeletal muscle group is selected from the quadriceps, biceps, gastrocnemius (e.g., calf muscles), gluteus maximus, and hamstrings, optionally two or more of these different skeletal muscle groups comprising any combination thereof. In some respects, the two or more different skeletal muscle groups are any combination of two or more of the following: quadriceps, biceps, gastrocnemius (e.g., calf muscles), gluteus maximus, or hamstrings.

[0025] In some respects, the skeletal muscle groups are selected from i) the left and right quadriceps, ii) the left and right biceps, iii) the left and right triceps, iv) the left and right gastrocnemius, v) the left and right gluteus maximus, vi) the left and right hamstrings, vii) the left and right deltoids, viiii) the left and right trapezius, ix) the left and right pectoralis major, and x) the left and right latissimus dorsi.

[0026] In some respects, the skeletal muscle group is selected from the quadriceps, biceps, triceps, gastrocnemius, gluteus maximus, and hamstrings, and optionally two or more of these different skeletal muscle groups include any combination thereof.

[0027] In some aspects, the administration involves bilateral injection into one or more skeletal muscle groups (e.g., IM injection into the right and left quadriceps, right and left biceps, right and left gastrocnemius, right and left gluteus maximus, and / or right and left hamstrings). In some aspects, the administration involves bilateral injection into one or more skeletal muscle groups (e.g., IM injection into the right and left quadriceps, right and left biceps, right and left gastrocnemius, right and left gluteus maximus, right and left triceps, right and left hamstrings, and any combination thereof).

[0028] In some respects, bilateral injections are equally distributed among the various skeletal muscle groups (e.g., the right quadriceps and left quadriceps receive approximately the same volume and / or dose).

[0029] In some aspects, the administration of an IM comprising a total of 20-150 injections of the therapy (e.g., combination gene therapy) to at least two different skeletal muscle groups. In some aspects, the administration of an IM comprising a total of 20-120, 20-110, 20-100, 20-80, 20-60, 25-150, 25-120, 25-110, or 25-100 injections to at least two different skeletal muscle groups.

[0030] In some respects, the administration includes 1-80 (e.g., 2-80, 4-80, 6-80, 8-80, 10-80, 20-80, 30-80, 40-80, 50-80, or 60-80) IM injections to the first skeletal muscle group and 1-80 (e.g., 2-80, 4-80, 6-80, 8-80, 10-80, 20-80, 30-80, 40-80, 50-80, or 60-80) IM injections to the second skeletal muscle group. In some aspects, the administration includes 1-50 (e.g., 2-50, 4-50, 6-50, 8-50, 10-50, 20-50, 30-50, or 40-50) IM injections to the first skeletal muscle group, 1-50 (e.g., 2-50, 4-50, 6-50, 8-50, 10-50, 20-50, 30-50, or 40-50) IM injections to the second skeletal muscle group, and 1-50 (e.g., 2-50, 4-50, 6-50, 8-50, 10-50, 20-50, 30-50, or 40-50) IM injections to the third skeletal muscle group.

[0031] In some respects, the administration includes (i) 1-80 IM injections to a first skeletal muscle group and 1-80 IM injections to a second skeletal muscle group; (ii) 1-60 IM injections to a first skeletal muscle group, 1-60 IM injections to a second skeletal muscle group, and 1-60 IM injections to a third skeletal muscle group; (iii) 1-40 IM injections to a first skeletal muscle group, 1-40 IM injections to a second skeletal muscle group, 1-40 IM injections to a third skeletal muscle group, and 1-40 IM injections to a fourth skeletal muscle group; (iv) administration to a first skeletal muscle group... Administer 1-30 IM injections to the second skeletal muscle group, the third skeletal muscle group, the fourth skeletal muscle group, and the fifth skeletal muscle group; and (v) administer 1-25 IM injections to the first skeletal muscle group, the second skeletal muscle group, the third skeletal muscle group, the fourth skeletal muscle group, the fifth skeletal muscle group, and the sixth skeletal muscle group.

[0032] In some respects, the administration includes: (i) 2-80, 4-80, 6-80, 8-80, 10-80, 20-80, 30-80, 40-80, 50-80, or 60-80 IM injections to the first skeletal muscle group, and 2-80, 4-80, 6-80, 8-80, 10-80, 20-80, 30-80, 40-80, 50-80, or 60-80 IM injections to the second skeletal muscle group; (ii) 2-60, 4-60, 6-60, 8-60, 10-60, 20-60, or 60-80 IM injections to the first skeletal muscle group. (iii) 30-60, 40-60, or 50-60 IM injections; 2-60, 4-60, 6-60, 8-60, 10-60, 20-60, 30-60, 40-60, or 50-60 IM injections for the second skeletal muscle group; and 2-60, 4-60, 6-60, 8-60, 10-60, 20-60, 30-60, 40-60, or 50-60 IM injections for the third skeletal muscle group; (iii) 2-40, 4-40, 6-40, 8-40, 10-40, 20-40, or 30-60 IM injections for the first skeletal muscle group. -40 IM injections, with 2-40, 4-40, 6-40, 8-40, 10-40, 20-40, or 30-40 IM injections for the second skeletal muscle group, 2-40, 4-40, 6-40, 8-40, 10-40, 20-40, or 30-40 IM injections for the third skeletal muscle group, and 2-40, 4-40, 6-40, 8-40, 10-40, 20-40, or 30-40 IM injections for the fourth skeletal muscle group; (iv) 2-30, 4-30, 6-30, 8-30, 1 0-30 or 20-30 IM injections; 2-30, 4-30, 6-30, 8-30, 10-30 or 20-30 IM injections for the second skeletal muscle group; 2-30, 4-30, 6-30, 8-30, 10-30 or 20-30 IM injections for the third skeletal muscle group; 2-30, 4-30, 6-30, 8-30, 10-30 or 20-30 IM injections for the fourth skeletal muscle group; and 2-30, 4-30, 6-30, 8-30, 10-30 or 20-30 IM injections for the fifth skeletal muscle group.Or (v) administer 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 IM injections to the first skeletal muscle group, 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 IM injections to the second skeletal muscle group, and 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 IM injections to the third skeletal muscle group. The injection regimen is as follows: 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 intramural injections for the fourth skeletal muscle group; 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 intramural injections for the fifth skeletal muscle group; and 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 intramural injections for the sixth skeletal muscle group.

[0033] In some respects, the administration includes 8-50 intramural injections to the quadriceps (e.g., 4-25 bilateral intramural injections to each quadriceps), 8-50 intramural injections to the biceps (e.g., 4-25 bilateral intramural injections to each biceps), and 8-50 intramural injections to the hamstrings (e.g., 4-25 bilateral intramural injections to each hamstring).

[0034] In some respects, each IM injection is administered in a volume of about 100 μL to about 1.5 mL (e.g., about 100 μL to about 1.4 mL, about 100 μL to about 1.35 mL, about 100 μL to about 1.2 mL, about 100 μL to about 1.0 mL, about 100 μL to about 800 μL, about 200 μL to about 800 μL, about 200 μL to about 600 μL, or about 400 μL to about 800 μL).

[0035] In some respects, each IM injection is administered in the following volumes: about 100 μL to about 2 mL, 100 μL to about 1.9 mL, 100 μL to about 1.8 mL, 100 μL to about 1.7 mL, 100 μL to about 1.6 mL, 100 μL to about 1.5 mL, 100 μL to about 1.4 mL, about 100 μL to about 1.35 mL, about 100 μL to about 1.2 mL, about 100 μL to about 1.0 mL, about 100 μL to about 800 μL, about 200 μL to about 800 μL, about 200 μL to about 600 μL, about 400 μL to about 800 μL, about 500 μL to about 1.5 mL, about 600 μL to about 1.4 mL, about 700 μL to about 1.3 mL, about 800 μL to about 1.2 mL, or about 900 μL to about 1.1 mL.

[0036] In some respects, each IM injection is administered in a volume of about 0.5 mL to about 1 mL.

[0037] In some respects, the total cumulative volume administered by all IM injections is about 20 mL to about 70 mL, about 20 mL to about 50 mL, or about 30 mL to about 40 mL.

[0038] In some respects, each IM injection contains approximately 2 × 10 13 Up to 5×10 13 vg / mL.

[0039] In some respects, each IM injection contains approximately 8 × 10 10 Up to 5×10 13 vg / mL. In some respects, each IM injection contains approximately 2 × 10 13 Approximately 4×10 13 vg / mL.

[0040] In some respects, IM injections are administered with intervals of at least 1-5 cm. In some respects, IM injections are administered with intervals of at least 1-3 cm. In some respects, the intervals between IM injection sites on the subject are at least 5 cm. In some respects, the intervals between IM injection sites on the subject are at least 4 cm. In some respects, the intervals between IM injection sites on the subject are at least 3 cm. In some respects, the intervals between IM injection sites on the subject are at least 2 cm. In some respects, the intervals between IM injection sites on the subject are at least 1 cm.

[0041] In some aspects, the application involves IM injections at depths of at least 0.5 cm, at least 0.6 cm, at least 0.7 cm, at least 0.8 cm, at least 0.9 cm, at least 1.0 cm, at least 1.1 cm, at least 1.2 cm, at least 1.3 cm, at least 1.4 cm, at least 1.5 cm, at least 2.0 cm, at least 2.5 cm, at least 3.0 cm, at least 3.5 cm, at least 4.0 cm, at least 4.5 cm, or at least 5.0 cm. In some aspects, each IM injection is approximately 0.5 cm to 2 cm deep. In some aspects, the depth of each IM injection is between 1 and 2 cm.

[0042] In some respects, regardless of the thickness of subcutaneous fat, each IM injection should be at least 0.5 cm below the surface of the injected muscle.

[0043] In some aspects, the first AAV vector genome and the second AAV vector genome are administered simultaneously or sequentially, optionally wherein the first AAV vector genome and the second AAV vector genome are administered in the same pharmaceutical composition. In some aspects, AAV particles containing the first AAV vector genome and the second AAV vector genome are mixed prior to administration.

[0044] In some respects, the method further includes administering an immunosuppressive regimen to the subject.

[0045] Certain aspects of this disclosure relate to a method of treating or improving diabetes-related symptoms in a subject of need, comprising administering to the subject (i) an immunosuppressive regimen and (ii) a therapy comprising one or more adeno-associated virus (AAV) vectors (e.g., combination gene therapy), said one or more AAV vectors comprising (a) a promoter (e.g., first and second promoters), (b) a polynucleotide encoding the human insulin (hIns) protein, and (c) a polynucleotide encoding the human glucokinase (hGck) protein. In some aspects, the combination therapy is administered intramuscularly (IM) to at least two different skeletal muscle groups.

[0046] In some aspects, the method further comprises administering an immunosuppressive regimen containing an immunosuppressant to the subject.

[0047] Certain aspects of this disclosure relate to a method of treating or improving diabetes-related symptoms in a subject of need, comprising administering to the subject (i) an immunosuppressive regimen comprising an immunosuppressant, and (ii) a therapy comprising one or more adeno-associated virus (AAV) vectors (e.g., combination gene therapy), said one or more AAV vectors comprising (a) a promoter (e.g., first and second promoters), (b) a polynucleotide encoding a human insulin (hIns) protein, and (c) a polynucleotide encoding a human glucokinase (hGck) protein. In some aspects, the combination therapy is administered intramuscularly (IM) to at least two different skeletal muscle groups.

[0048] In some aspects, the therapy (e.g., combination gene therapy) comprises (a) a first AAV vector genome containing an insulin expression cassette, the insulin expression cassette containing a first promoter operatively linked to a multinucleotide encoding a human insulin (hIns) protein, wherein the insulin expression cassette is side-mounted with an inverted terminal repeat (ITR); and (b) a second AAV vector genome containing a glucokinase expression cassette, the glucokinase expression cassette containing a second promoter operatively linked to a multinucleotide encoding a human glucokinase (hGck) protein, wherein the glucokinase expression cassette is side-mounted with an inverted terminal repeat (ITR).

[0049] In some respects, the first AAV vector genome and the second AAV vector genome are administered in the same composition (e.g., via the same IM injection).

[0050] In some respects, the first AAV vector genome and the second AAV vector genome are administered in the same pharmaceutical composition (e.g., via the same IM injection).

[0051] In some aspects, one or more AAV vectors are AAV vector genomes containing a single expression cassette comprising a first promoter operatively linked to a multinucleotide encoding an hIns protein and a second promoter operatively linked to a multinucleotide encoding an hGck protein, wherein the single expression cassette is side-mounted with an inverted terminal repeat (ITR).

[0052] Some aspects of this disclosure relate to a dosing regimen comprising:

[0053] (a) Primary immunosuppressant;

[0054] (b) A therapy administered intramuscularly following (a) (e.g., combination gene therapy), said therapy comprising one or more adeno-associated virus (AAV) vectors, said one or more AAV vectors comprising (a) a promoter (e.g., first and second promoters), (b) a polynucleotide encoding human insulin (hIns) protein, and (c) a polynucleotide encoding human glucokinase (hGck) protein; and

[0055] (c) A second immunosuppressant administered after (b).

[0056] In some aspects, the therapy comprises (i) a first recombinant AAV (rAAV) particle comprising a first AAV vector genome containing an insulin expression cassette, the insulin expression cassette containing a first promoter operatively linked to a multinucleotide encoding a human insulin (hIns) protein, wherein the insulin expression cassette is side-mounted with an inverted terminal repeat (ITR); and (ii) a second recombinant AAV (rAAV) particle comprising a second AAV vector genome containing a glucokinase expression cassette, the glucokinase expression cassette containing a second promoter operatively linked to a multinucleotide encoding a human glucokinase (hGck) protein, wherein the glucokinase expression cassette is side-mounted with an inverted terminal repeat (ITR).

[0057] In some aspects, the intramuscular (IM) administration of the dosing regimen is applied to at least two different muscle groups. In some aspects, the combined intramuscular (IM) administration is applied to at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten different skeletal muscle groups. In some aspects, the combined intramuscular (IM) administration is applied to 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, or 8-10 different skeletal muscle groups. In some aspects, the combined intramuscular (IM) administration is applied to two, three, four, five, six, seven, eight, nine, or ten different skeletal muscle groups.

[0058] In some respects, the first AAV vector genome and the second AAV vector genome are administered in the same pharmaceutical composition.

[0059] In some respects, immunosuppressive regimens involve administering one or more immunosuppressants before (before AAV), concurrently with, and / or after (after AAV) therapies.

[0060] In some respects, immunosuppressive regimens include administering an immunosuppressant (pre-AAV) prior to the administration of combination therapy.

[0061] In some respects, immunosuppressive regimens include the administration of immunosuppressants (after AAV) following the administration of combination therapy.

[0062] In some respects, pre-AAV immunosuppressants are administered 1 to 14 days prior to the administration of combination therapy.

[0063] In some cases, pre-AAV immunosuppressants are administered 1 to 7 days prior to the administration of combination therapy.

[0064] In some cases, pre-AAV immunosuppressants are administered 1 to 2 days prior to the administration of combination therapy.

[0065] In some cases, post-AAV immunosuppressants are initially administered 1, 2, 3, or 4 weeks after the administration of combination therapy.

[0066] In some cases, immunosuppressants are initially administered approximately 3 weeks after the administration of combination therapy.

[0067] In some cases, after initial administration, AAV post-immunosuppressants are administered twice daily, once daily, or every other day.

[0068] In some respects, after initial administration, AAV post-immunosuppressant administration lasts approximately 6 to 40, approximately 8 to 40, approximately 10 to 40, approximately 12 to 40, approximately 6 to 30, approximately 8 to 30, approximately 10 to 30, approximately 12 to 30, approximately 6 to 20, approximately 8 to 20, approximately 10 to 20, or approximately 12 to 20 weeks.

[0069] In some respects, immunosuppressive regimens involve administering immunosuppressants prior to the administration of combination therapy.

[0070] In some respects, immunosuppressive regimens include the administration of immunosuppressants following the administration of combination therapy.

[0071] In some cases, immunosuppressants are administered 1 to 14 days prior to the application of combination therapy.

[0072] In some cases, immunosuppressants are administered 1 to 7 days prior to the application of combination therapy.

[0073] In some cases, immunosuppressants are administered 1 to 3 days prior to the application of combination therapy.

[0074] In some cases, immunosuppressants are administered 3 to 7 days prior to the application of combination therapy.

[0075] In some cases, immunosuppressants are further administered 1, 2, 3, or 4 weeks after the administration of combination therapy.

[0076] In some cases, immunosuppressants are administered approximately every 1–3 weeks after combination therapy, and optionally approximately every 1–2 weeks.

[0077] In some cases, immunosuppressants are administered for approximately 1–6 months after combination therapy, optionally for approximately 1–3 months.

[0078] In some respects, immunosuppressive regimens include a second immunosuppressant.

[0079] In some respects, immunosuppressive regimens include administering a second immunosuppressant prior to the combination therapy.

[0080] In some respects, immunosuppressive regimens include the administration of a second immunosuppressant following the administration of combination therapy.

[0081] In some cases, the second immunosuppressant is administered 1 to 14 days prior to the administration of combination therapy.

[0082] In some cases, the second immunosuppressant is administered 1 to 7 days prior to the combination therapy.

[0083] In some cases, the second immunosuppressant is administered 1 to 3 days prior to the administration of combination therapy.

[0084] In some cases, the second immunosuppressant is administered 3 to 7 days prior to the administration of combination therapy.

[0085] In some cases, a second immunosuppressant is administered 1, 2, 3, or 4 weeks after the combination therapy.

[0086] In some cases, the second immunosuppressant is administered twice daily, once daily, or every other day.

[0087] In some cases, the second immunosuppressant is administered for approximately 1–6 months after combination therapy, optionally for approximately 1–3 months.

[0088] In some respects, immunosuppressive regimens include IL-6 antagonists, IL-1 antagonists, TNFα antagonists, mTOR pathway inhibitors, mycophenolate mofetil (MMF), tyk2 inhibitors, methotrexate, rapamycin, abatacept, antithymocyte globulin (ATG), B-cell depletion agents, T-cell co-stimulation antagonists, T-cell depletion agents, corticosteroids, and / or calcineurin inhibitors.

[0089] In some respects, immunosuppressive regimens include the administration of immunosuppressants selected from the group consisting of: IL-6 antagonists, IL-1 antagonists, TNFα antagonists, mTOR pathway inhibitors, mycophenolate mofetil (MMF), tyk2 inhibitors, methotrexate, abatacept, antithymocyte globulin (ATG), B-cell depletion agents, T-cell co-stimulation antagonists, T-cell depletion agents, corticosteroids, calcineurin inhibitors, or any combination thereof.

[0090] In some respects, pre-AAV immunosuppressants comprise IL-6 antagonists, IL-1 antagonists, mycophenolate mofetil (MMF), tyk2 inhibitors, rapamycin, and / or abatacept.

[0091] In some respects, post-AAV immunosuppressants include calcineurin inhibitors, MMF, methotrexate, tyk2 inhibitors, rapamycin, and / or abatacept.

[0092] In some respects, IL-6 antagonists are selected from the group consisting of: tocilizumab, sarilumab, siltuximab, olokizumab, elsilimomab, clazakizumab, sirukumab, levilimab, or any combination thereof. In some respects, the IL-6 antagonist is tocilizumab.

[0093] In some respects, calcineurin inhibitors are selected from the group consisting of cyclosporine, tacrolimus, voclosporin, and any combination thereof. In some respects, the calcineurin inhibitor is tacrolimus.

[0094] In some respects, IL-6 antagonists are selected from the group consisting of: tocilizumab, sarlizumab, saturizumab, staxicizumab, olozazumab, esimomomab, clazazumab, cilukumab, levelizumab, or any combination thereof.

[0095] In some respects, the IL-6 antagonist is tocilizumab.

[0096] In some respects, the second immunosuppressant includes calcineurin inhibitors, MMF, methotrexate, tyk2 inhibitors, rapamycin, abatacept, anti-thymocyte globulin, etanercept, basiliximab, sirolimus, or any combination thereof.

[0097] In some respects, immunosuppressive regimens involve administering tocilizumab, optionally subcutaneously, on approximately day -3 and approximately weekly or every other week, relative to combination therapy, up to approximately 12 weeks.

[0098] In some respects, immunosuppressive regimens involve the administration of tacrolimus, optionally orally, on approximately day -3 and every 1-3 days, relative to combination therapy, for up to approximately 3-12 weeks.

[0099] In some respects, the total administration dose of the first AAV vector genome containing the insulin expression cassette comprises approximately 7 × 10⁻⁶. 10 Approximately 5×10 13 Vector genome (vg) / kg.

[0100] In some respects, the total dose of the second AAV vector genome containing the glucokinase expression cassette comprises approximately 5 × 10⁻⁶ units. 11 Approximately 5×10 13 Vector genome (vg) / kg.

[0101] In some respects, the ratio of a first AAV vector genome containing an insulin expression cassette to a second AAV vector genome containing a glucokinase expression cassette is approximately 1:0.25–1.25, 1:1–20, 1:2–20, 1:3–20, 1:4–20, 1:5–20, 1:1–18, 1:2–18, 1:3–18, 1:4–18, 1:5–18, 1:1–16, 1:2–16, and 1:3–16. 1:4-16, 1:5-16, 1:1-15, 1:2-15, 1:3-15, 1:4-15, 1:5-15, 1:1-14, 1:2-14, 1:3-14, 1:4-14, 1:5-14, 1:1-12, 1:2-12, 1:3-12, 1:4-12, 1:5-12, 1:1-10, 1:2-10, 1:3-10, 1:4-10 or 1:5-10.

[0102] In some respects, the ratio of a first AAV vector genome containing an insulin expression cassette to a second AAV vector genome containing a glucokinase expression cassette is approximately 1:1-15, 1:2-15, 1:3-15, 1:4-15, 1:5-15, 1:1-14, 1:2-14, 1:3-14, 1:4-14, 1:5-14, 1:1-12, 1:2-12, 1:3-12, 1:4-12, 1:5-12, 1:1-10, 1:2-10, 1:3-10, 1:4-10, or 1:5-10.

[0103] In some respects, the ratio of a first AAV vector genome containing an insulin expression cassette to a second AAV vector genome containing a glucokinase expression cassette is approximately 1:1-10, 1:2-10, 1:3-10, 1:4-10, or 1:5-10.

[0104] In some respects, the ratio of a first AAV vector genome containing an insulin expression cassette to a second AAV vector genome containing a glucokinase expression cassette is approximately 1:1, approximately 1:2, approximately 1:3, approximately 1:4, approximately 1:5, approximately 1:6, approximately 1:8, approximately 1:9, approximately 1:10, approximately 1:11, approximately 1:12, approximately 1:13, approximately 1:14, or approximately 1:15.

[0105] In some respects, the ratio of a first AAV vector genome containing an insulin expression cassette to a second AAV vector genome containing a glucokinase expression cassette is approximately 1:2, approximately 1:3, approximately 1:4, approximately 1:5, approximately 1:6, approximately 1:8, approximately 1:9, or approximately 1:10.

[0106] In some respects, the first AAV vector genome containing the insulin expression cassette is approximately 5 × 10⁻⁶. 11 With approximately 1×10 13Dosage between vg / kg.

[0107] In some respects, the first AAV vector genome containing the insulin expression cassette is approximately 2 × 10⁻⁶. 12 With approximately 1×10 13 Dosage between vg / kg.

[0108] In some respects, the first AAV vector genome containing the insulin expression cassette is approximately 4 × 10⁻⁶. 12 To approximately 1×10 13 Administer at a dose of vg / kg.

[0109] In some respects, the first AAV vector genome containing the insulin expression cassette is approximately 7 × 10⁻⁶. 10 Approximately 7×10 12 Administer at a dose of vg / kg.

[0110] In some respects, the first AAV vector genome containing the insulin expression cassette is approximately 5 × 10⁻⁶. 11 Approximately 5×10 12 Administer at a dose of vg / kg.

[0111] In some respects, the first AAV vector genome containing the insulin expression cassette is approximately 1 × 10⁻⁶. 11 To approximately 1×10 12 Administer at a dose of vg / kg.

[0112] In some respects, the second AAV vector genome containing a glucokinase expression cassette is approximately 1 × 10 12 With approximately 5×10 12 Dosage between vg / kg.

[0113] In some respects, the second AAV vector genome containing a glucokinase expression cassette is approximately 2 × 10 12 With approximately 5×10 12 Dosage between vg / kg.

[0114] In some respects, the second AAV vector genome containing a glucokinase expression cassette is approximately 2 × 10 12 Approximately 3.5 × 10 12 Administer at a dose of vg / kg.

[0115] In some respects, the second AAV vector genome containing a glucokinase expression cassette is approximately 3.5 × 10⁻⁶. 12 Approximately 5×10 12 Administer at a dose of vg / kg.

[0116] In some respects, within 15 days to 6 months, 30 to 120 days, or 30 to 60 days after administration of combination therapy: (i) a decrease and / or regulation of glycated hemoglobin (HbA1c) levels in the subject, (ii) a decrease in circulating ketones in the subject, (iii) a decrease in triglycerides in the subject, (iv) a decrease in exogenous insulin in the subject, (v) an increase in circulating C-peptide levels in the subject, or (vi) any combination thereof.

[0117] In some respects, when measured 15-6 months, 30-120 days, 30-60 days, or 60-90 days after administration of combination therapy, the circulating insulin level in the subject's blood was approximately 1-40 μU / mL, optionally approximately 5-40 μU / mL, approximately 5-25 μU / mL, approximately 5-10 μU / mL, approximately 10-20 μU / mL, approximately 10-15 μU / mL, approximately 15-20 μU / mL, or approximately 15-25 μU / mL.

[0118] In some respects, within 15-6 months, 30-120 days, 30-60 days, or 60-90 days after administration of combination therapy, subjects’ HbA1C levels decreased by approximately 0.5 to 1.5% compared to pre-treatment levels.

[0119] In some respects, within 15 days to 6 months, 30 to 120 days, 30 to 60 days, or 60 to 90 days after administration of combination therapy, subjects’ HbA1C levels were less than about 7.0%, optionally less than about 6.5%, about 6.0%, about 5.7%, and optionally about 5.0% to 6.5%.

[0120] In some respects, within 15 days to 6 months, 30 to 120 days, 30 to 60 days, or 60 to 90 days after administration of combination therapy, the subjects’ circulating glucose in the blood was at least about 70 to 140 mg / dL.

[0121] In some respects, the subjects did not exhibit insulin resistance.

[0122] In some respects, the subjects exhibited insulin resistance.

[0123] Certain aspects of this disclosure relate to a dosing regimen comprising: (a) a first immunosuppressant; (b) a combination therapy administered intramuscularly (IM) after (a), the combination therapy comprising (i) a first recombinant AAV (rAAV) particle comprising a first AAV vector genome comprising an insulin expression cassette comprising a first promoter operatively linked to a polynucleotide encoding human insulin (hIns) protein, wherein the insulin expression cassette is side-mounted with an inverted terminal repeat (ITR); and (ii) a second recombinant AAV (rAAV) particle comprising a second AAV vector genome comprising a glucokinase expression cassette comprising a second promoter operatively linked to a polynucleotide encoding human glucokinase (hGck) protein, wherein the glucokinase expression cassette is side-mounted with an inverted terminal repeat (ITR); and (c) a second immunosuppressant.

[0124] In some respects, the first immunosuppressant comprises an IL-6 antagonist selected from the group consisting of: tocilizumab, sarrizumab, staxiciumab, olozazumab, esimozumab, clazazumab, cilukumab, levelizumab, and any combination thereof.

[0125] In some respects, the second immunosuppressant is a calcineurin inhibitor selected from the group consisting of cyclosporine, tacrolimus, vorticol, and any combination thereof.

[0126] In some cases, the primary immunosuppressant includes tocilizumab, and the secondary immunosuppressant includes tacrolimus.

[0127] In some respects, the dosing regimen includes tocilizumab administered on approximately day -3 and approximately weekly or every other week, alternatively subcutaneously, for up to approximately 12 weeks, in contrast to combination therapy.

[0128] In some respects, immunosuppressive regimens involve the administration of tacrolimus, optionally orally, on approximately day -3 and every 1-3 days, relative to combination therapy, for up to approximately 3-12 weeks.

[0129] In some respects, the first AAV vector genome and the second AAV vector genome each have a density of approximately 5 × 10⁻⁶. 11 Approximately 5×10 13 Dosage administration was between vector genome (vg) / kg. In some respects, the first AAV vector genome and the second AAV vector genome were administered at a rate of approximately 1 × 10⁻⁶. 12 To approximately 1×10 13 Dosage between vg / kg.

[0130] In some respects, the first AAV vector genome containing the insulin expression cassette is approximately 2 × 10⁻⁶. 12 To approximately 1×10 13 Dosage administration between vg / kg. In some respects, the first AAV vector genome containing the insulin expression cassette is administered at approximately 4 × 10⁻⁶. 12 To approximately 1×10 13 Administered at doses between vg / kg. In some respects, the first AAV vector genome containing the insulin expression cassette is approximately 4.5 × 10⁻⁶. 12 Approximately 5×10 12 Administered at a dose of vg / kg. In some respects, the first AAV vector genome containing the insulin expression cassette is delivered at approximately 5 × 10⁻⁶. 12 Approximately 9×10 12 Administer at a dose of vg / kg.

[0131] In some respects, the second AAV vector genome containing a glucokinase expression cassette is approximately 1 × 10 12 Approximately 5×10 12 Administered at doses between vg / kg. In some respects, the second AAV vector genome containing the glucokinase expression cassette is administered at approximately 2 × 10⁻⁶. 12 Approximately 5×10 12 Administered at doses between vg / kg. In some respects, the second AAV vector genome containing the glucokinase expression cassette is administered at approximately 2 × 10⁻⁶. 12 Approximately 2.8 × 10 12 Administered at a dose of vg / kg. In some respects, the second AAV vector genome containing the glucokinase expression cassette is delivered at approximately 4 × 10⁻⁶. 12 vg / kg to approximately 5×10 12 Administer at a dose of vg / kg.

[0132] In some respects, the first AAV vector genome containing the insulin expression cassette is approximately 7 × 10⁻⁶. 10 vg / kg to approximately 1.8×10 12 Administer at a dose of vg / kg.

[0133] In some respects, the first AAV vector genome containing the insulin expression cassette is approximately 7 × 10⁻⁶. 11 From approximately 1.8 × 10 12 Administer at a dose of vg / kg.

[0134] In some respects, the first AAV vector genome containing the insulin expression cassette is approximately 5 × 10⁻⁶. 11 Approximately 5×10 12 Administer at a dose of vg / kg.

[0135] In some respects, the first AAV vector genome containing the insulin expression cassette is approximately 1 × 10⁻⁶. 11 To approximately 1×10 12 Administer at a dose of vg / kg.

[0136] In some respects, the second AAV vector genome containing a glucokinase expression cassette is approximately 3.5 × 10⁻⁶. 12 Administer at a dose of vg / kg.

[0137] In some respects, the first AAV vector genome (containing an expression cassette encoding hIns) and the second AAV vector genome (containing an expression cassette encoding hGck) are administered at a selected ratio of 1:0.25–1.25. In some respects, the ratio is approximately 1:1. In some respects, the ratio is 1:0.35–0.75. In some respects, the ratio is 1:0.4–0.6. In some respects, the ratio is 1:0.45–0.55. In some respects, the ratio is approximately 1:0.5.

[0138] In some respects, the ratio is approximately 1:1-15. In some respects, the ratio is approximately 1:3-5. In some respects, the ratio is approximately 1:2. In some respects, the ratio is approximately 1:3. In some respects, the ratio is approximately 1:4. In some respects, the ratio is approximately 1:5.

[0139] In some aspects, the polynucleotide encoding the hIns protein comprises an open reading frame (ORF) containing a nucleotide sequence that is at least 85%, 90%, 95%, 99%, or 100% identical to any of the following: (i) nucleic acids 73-330 of any of SEQ ID NO: 43-57 or 110-116, nucleic acids 88-345 of any of SEQ ID NO: 117-122, 152, or 156, or nucleic acids 79-336 of SEQ ID NO: 153; or (ii) SEQ ID NO: 43-57 or SEQ ID NO: 110-122; and / or the polynucleotide encoding the human glucokinase hGck protein comprises an ORF containing (i) a nucleotide sequence that is at least 85%, 90%, 95%, 99%, or 100% identical to any of the following: (a) SEQ ID NO: Nucleic acid 1-1398 of any one of 61-80 or 162; or (ii) SEQ ID NO: 61-80 and 162.

[0140] In some respects, the polynucleotide encoding the hIns protein contains an open reading frame (ORF) containing at least 85%, 90%, 95%, 99%, or 100% of the same nucleotide sequence as SEQ ID NO: 121; and / or the polynucleotide encoding the human glucokinase hGck protein contains an ORF containing at least 85%, 90%, 95%, 99%, or 100% of the same nucleotide sequence as SEQ ID NO: 68.

[0141] In some respects, the hIns protein contains any of the following amino acid sequences: amino acids 25-110 of SEQ ID NO: 41, amino acids 25-110 of SEQ ID NO: 144, amino acids 25-110 of SEQ ID NO: 145, SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO: 145.

[0142] In some aspects, the hIns protein contains a signal peptide. In some aspects, the signal peptide is a wild-type proinsulin signal sequence, an IL-6 signal sequence, a fibronectin signal sequence, or a non-wild-type proinsulin signal sequence. In some aspects, the signal peptide contains amino acids 1-24 of SEQ ID NO: 41, amino acids 1-24 of SEQ ID NO: 144, or amino acids 1-24 of SEQ ID NO: 145. In some aspects, the hIns protein further contains a cleavage site.

[0143] In some respects, the polynucleotide encoding the hIns protein further comprises a 5' UTR, said 5' UTR comprising at least 85%, 90%, 95%, 99%, or 100% identical nucleotide sequences to nucleic acids 5-329, SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148.

[0144] In some respects, the polynucleotide encoding the hIns protein further comprises a 3' UTR, said 3' UTR comprising at least 85%, 90%, 95%, 99%, or 100% identical nucleotide sequences to those of 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, SEQ ID NO: 149, or SEQ ID NO: 171.

[0145] In some respects, the encoded hGck protein contains the amino acid sequence of SEQ ID NO: 82.

[0146] In some respects, the polynucleotide encoding the hGck protein further comprises a 5' UTR, said 5' UTR comprising at least 85%, 90%, 95%, 99%, or 100% identical nucleotide sequences to nucleic acids 5-329, SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148.

[0147] In some aspects, the polynucleotide encoding the hGck protein further comprises a 3' UTR, said 3' UTR comprising at least 85%, 90%, 95%, 99%, or 100% identical nucleotide sequences to those 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, SEQ ID NO: 149, SEQ ID NO: 169, or SEQ ID NO: 171.

[0148] In some respects, the first promoter is a eukaryotic promoter. In some respects, the first promoter is a constitutive promoter. In some respects, the first promoter is a ubiquitous promoter. In some respects, the first promoter is a muscle-specific promoter. In some respects, the first promoter is a CMV promoter. In some respects, the second promoter is a eukaryotic promoter. In some respects, the first promoter is a constitutive promoter. In some respects, the first promoter is a ubiquitous promoter. In some respects, the first promoter is a muscle-specific promoter. In some respects, the second promoter is a CMV promoter.

[0149] In some respects, the insulin expression cassette promoter and / or glucokinase expression cassette promoter are eukaryotic promoters. In some respects, the insulin expression cassette promoter and / or glucokinase expression cassette promoter are constitutive promoters. In some respects, the insulin expression cassette promoter and / or glucokinase expression cassette promoter are ubiquitous promoters. In some respects, the insulin expression cassette promoter and / or glucokinase expression cassette promoter are CMV promoters. In some respects, the insulin expression cassette promoter and / or glucokinase expression cassette promoter are muscle-specific promoters.

[0150] In some respects, the insulin expression cassette and / or glucokinase expression cassette further include polyadenylation (polyA) elements.

[0151] In some aspects, the first recombinant AAV (rAAV) particle contains a first AAV vector genome. In some aspects, the second recombinant AAV (rAAV) particle contains a second vector genome. In some aspects, the AAV serotype of the first rAAV particle and / or the second rAAV particle is selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh9, AAV9, AAVrh10, AAV10, AAV11, AAV12, and AAVrh74. In some aspects, the AAV serotype of the first rAAV particle and / or the second rAAV particle is AAV1.

[0152] In some respects, the first rAAV particles and the second rAAV particles are formulated in a single composition.

[0153] In some respects, the first rAAV particle and the second rAAV particle are applied simultaneously or sequentially.

[0154] In some respects, diabetes is classified as type 1 diabetes (T1DM) or type 2 diabetes (T2DM).

[0155] In some respects, within 15 days and 6 months, 30–120 days or 30–60 days after administration of combination therapy: (i) a decrease and / or regulation of glycated hemoglobin (HbA1c) levels in the subject, (ii) a decrease in circulating ketones in the subject, (iii) a decrease in triglycerides in the subject, (iv) a decrease in exogenous insulin in the subject, (v) an increase in circulating C-peptide levels in the subject, or (vi) any combination thereof.

[0156] Some aspects of this disclosure relate to a method of immunosuppression in a subject receiving AAV gene therapy for the treatment of diabetes, wherein the subject is given an immunosuppression regimen comprising (a) administering an immunosuppressant before AAV gene therapy (pre-AAV); and (b) administering an immunosuppressant after AAV gene therapy (post-AAV).

[0157] In some respects, pre-AAV immunosuppressants are administered 1 to 14 days prior to AAV gene therapy.

[0158] In some respects, pre-AAV immunosuppressants are administered 1 to 7 days prior to AAV gene therapy.

[0159] In some cases, pre-AAV immunosuppressants are administered 1 to 2 days before AAV gene therapy.

[0160] In some cases, pre-AAV immunosuppressants are administered 1 to 3 days prior to AAV gene therapy.

[0161] In some cases, pre-AAV immunosuppressants are administered 3 to 7 days prior to AAV gene therapy.

[0162] In some cases, post-AAV immunosuppressants are administered 1, 2, 3, or 4 weeks after AAV gene therapy.

[0163] In some respects, post-AAV immunosuppressants are administered approximately every 1–3 weeks after the administration of combination therapy, and optionally approximately every 1–2 weeks.

[0164] In some respects, post-AAV immunosuppressants are administered for approximately 1–6 months, optionally approximately 1–3 months, following AAV gene therapy.

[0165] In some cases, immunosuppressants are initially administered approximately 3 weeks after AAV gene therapy.

[0166] In some cases, after initial administration, AAV post-immunosuppressants are administered twice daily, once daily, or every other day.

[0167] In some respects, after initial administration, AAV post-immunosuppressant administration lasts approximately 6 to 40, approximately 8 to 40, approximately 10 to 40, approximately 12 to 40, approximately 6 to 30, approximately 8 to 30, approximately 10 to 30, approximately 12 to 30, approximately 6 to 20, approximately 8 to 20, approximately 10 to 20, or approximately 12 to 20 weeks.

[0168] Some aspects of this disclosure relate to a method of immunosuppression in a subject receiving AAV gene therapy for the treatment of diabetes, wherein the subject is given an immunosuppression regimen comprising (a) administering a first immunosuppressant prior to the administration of AAV gene therapy; and optionally, (b) administering a second immunosuppressant after the administration of AAV gene therapy.

[0169] In some respects, the first immunosuppressant is administered 1 to 14 days prior to the administration of AAV gene therapy.

[0170] In some cases, the first immunosuppressant is administered 1 to 7 days prior to the administration of AAV gene therapy.

[0171] In some cases, the first immunosuppressant is administered 1 to 3 days before AAV gene therapy.

[0172] In some respects, the second immunosuppressant is administered 1 to 14 days prior to the administration of AAV gene therapy.

[0173] In some cases, the second immunosuppressant is administered 1 to 7 days prior to the administration of AAV gene therapy.

[0174] In some cases, the second immunosuppressant is administered 3 to 7 days prior to the administration of AAV gene therapy.

[0175] In some cases, after initial administration, the second immunosuppressant is administered twice daily, once daily, or every other day.

[0176] In some cases, the primary and / or secondary immunosuppressants are administered for approximately 2, 3, 4, 5, or 6 months. In some cases, the immunosuppressive regimen includes IL-6 antagonists, IL-1 antagonists, TNFα antagonists, mTOR pathway inhibitors, mycophenolate mofetil (MMF), tyk2 inhibitors, methotrexate, rapamycin, abatacept, anti-thymocyte globulin (ATG), B-cell depletion agents, T-cell co-stimulation antagonists, T-cell depletion agents, corticosteroids, and / or calcineurin inhibitors.

[0177] In some respects, AAV pre-immunosuppressants include IL-6 antagonists, IL-1 antagonists, MMF, tyk2 inhibitors, rapamycin, or abatacept.

[0178] In some respects, post-AAV immunosuppressants include calcineurin inhibitors, MMF, methotrexate, tyk2 inhibitors, rapamycin, and / or abatacept.

[0179] In some respects, IL-6 antagonists are selected from the group consisting of: tocilizumab, sarrizumab, staxiciumab, olozazumab, esimomomab, clazazumab, cilukumab, levelizumab, or any combination thereof. In some respects, the IL-6 antagonist is tocilizumab.

[0180] In some respects, calcineurin inhibitors are selected from the group consisting of cyclosporine, tacrolimus, vorticol, and any combination thereof. In some respects, the calcineurin inhibitor is tacrolimus.

[0181] In some cases, pre-AAV immunosuppressants and / or post-AAV immunosuppressants are administered for approximately 2 months, 3 months, 4 months, 5 months, or 6 months.

[0182] In some respects, pre-AAV immunosuppressants and / or post-AAV immunosuppressants include IL-6 antagonists, IL-1 antagonists, TNFα antagonists, mTOR pathway inhibitors, mycophenolate mofetil (MMF), tyk2 inhibitors, methotrexate, rapamycin, abatacept, anti-thymocyte globulin (ATG), B-cell depletion agents, T-cell co-stimulation antagonists, T-cell depletion agents, corticosteroids, calcineurin inhibitors, or any combination thereof.

[0183] In some respects, pre-AAV immunosuppressants and / or post-AAV immunosuppressants include IL-6 antagonists, IL-1 antagonists, MMF, tyk2 inhibitors, rapamycin, abatacept, or any combination thereof.

[0184] In some respects, pre-AAV immunosuppressants and / or post-AAV immunosuppressants include calcineurin inhibitors, MMF, methotrexate, tyk2 inhibitors, rapamycin, abatacept, or any combination thereof.

[0185] In some aspects, the method comprises administering tocilizumab, optionally subcutaneously, on approximately day -3 and approximately weekly or every other week, relative to AAV gene therapy, up to approximately 12 weeks.

[0186] In some aspects, the method comprises administering tacrolimus, optionally orally, on approximately day -3 and every 1-3 days relative to AAV gene therapy, for up to approximately 3-12 weeks. Attached Figure Description

[0187] Figure 1A-1J The levels of the mentioned biomarkers are shown in a non-human primate (NHP) model of type 1 diabetes induced by STZ, in first-order cynomolgus monkeys (NHP1) administered AAV1-hINS+AAV1-hGCK after streptozotocin (STZ) treatment. Figure 1A The figures show daily pre- and postprandial blood glucose levels (mg / dL) before and after administration of AAV1-hINS+AAV1-hGCK (day 0), as well as the amount (U / kg / day) of exogenous long-acting insulin (insulin glargine, "Lantus") and short-acting insulin (insulin lispro, "Humalog") administered, with time points up to day 180 after AAV administration. The percentage reduction in exogenous insulin administration compared to the 7-day mean (baseline) of exogenous insulin administered before AAV administration (after STZ treatment, before day 0) is shown at the top of the figure. Blood samples were drawn throughout the in-life phase, and C-peptide levels were analyzed from day 0 to day 182 after AAV1-hINS+AAV1-hGCK administration. Figure 1B; ng / mL), HbA1C level ( Figure 1C ;%) and creatine kinase (CK) levels ( Figure 1D (U / L). Figure 1E The weight (kg) is shown before and after AAV administration and up to day 182. Figure 1F This shows (i) blood glucose levels (mg / dL) during an intravenous glucose tolerance test (IVGTT) of NHP1 before STZ administration (before STZ), (ii) after STZ and before AAV1-hIns+AAV1-hGck administration (after STZ), and (iii) after STZ and at 56 days (D+56), 92 days (D+92), or 176 days (D+176) after AAV1-hIns+AAV1-hGck administration. Figure 1G This shows the C-peptide levels (ng / mL) of NHP1 during IVGTT at (i) before STZ administration, (ii) after STZ and before AAV1-hIns+AAV1-hGck administration (post-STZ), and (iii) after STZ and at 56 days (D+56), 92 days (D+92), or 176 days (D+176) after AAV1-hIns+AAV1-hGck administration. Figure 1H This shows the interferon-γ response (spots / million cells) in peripheral blood mononuclear cells against AAV1, glucokinase (Gck), insulin, and negative controls, measured from 14 days (-14) before (AAV1-hINS+AAV1-hGCK) administration to 161 days after AAV administration. Positive responses are indicated by boxes. Figure 1I Anti-insulin IgG (dilution factor) was measured 14 days (-14) before AAV1-hINS+AAV1-hGCK administration until 182 days after administration. Figure 1J Anti-glucokinase IgG (ECL signal) was measured 14 days (-14) before AAV1-hINS+AAV1-hGCK administration until 161 days after administration.

[0188] Figure 2A-2I The levels of the mentioned biomarkers are shown in a non-human primate (NHP) model of type 1 diabetes induced by STZ, in second cynomolgus macaques (NHP2) administered AAV1-hINS+AAV1-hGCK after streptozotocin (STZ) treatment. Figure 2AThe graph shows daily pre- and post-meal blood glucose levels (mg / dL) before and after administration of AAV1-hINS+AAV1-hGCK (day 0), as well as the amount (U / kg / day) of exogenous long-acting insulin (insulin glargine, "Lantus") and short-acting insulin (insulin lispro, "Humalog") administered, up to day 132 after AAV administration. The percentage reduction in exogenous insulin administration compared to the 7-day mean of exogenous insulin administered before AAV administration (baseline) (before day 0) is shown at the top of the graph. Blood samples were drawn weekly from day 0 until day 132 after AAV1-hINS+AAV1-hGCK administration, and pre-meal, fasting, or stimulated C-peptide levels were analyzed. Figure 2B ; ng / mL), Hb1A1C level ( Figure 2C ; %) level and creatine kinase (CK) level ( Figure 2D (U / L). Figure 2E The weight (kg) is shown before and after AAV administration and up to day 132. Figure 2F This shows (i) blood glucose levels (mg / dL) during an intravenous glucose tolerance test (IVGTT) of NHP2 before STZ administration (before STZ), after STZ and before AAV1-hIns+AAV1-hGck administration (after STZ), and (iii) after STZ administration of AAV1-hINS+AAV1-hGCK for 58 days (D+58) or 91 days (D+91) after STZ administration of AAV1-hINS+AAV1-hGCK. Figure 2G This shows the C-peptide levels (ng / mL) of NHP2 during IVGTT at (i) before STZ administration, (ii) after STZ and before AAV1-hIns+AAV1-hGck administration (post-STZ), and (iii) after STZ and at 58 days (D+58) or 91 days (D+91) after AAV1-hIns+AAV1-hGck administration. Figure 2H This displays the interferon-γ responses (spots / million cells) in peripheral blood mononuclear cells against AAV1, glucokinase (Gck), insulin, positive controls, and negative controls, measured from 14 days (-14) before AAV administration to 105 days after AAV administration. Positive test responses are indicated by boxes. Figure 2I The anti-insulin IgG (anti-insulin dilution titer) and anti-glucokinase IgG (anti-glucokinase ECL signal) were measured from 14 days before AAV administration until 132 days after administration.

[0189] Figure 3A-3IThe levels of the mentioned biomarkers are shown in a non-human primate (NHP) model of STZ-induced type 1 diabetes, in third-order cynomolgus macaques (NHP3) administered AAV1-hINS+AAV1-hGCK after streptozotocin (STZ) treatment. Figure 3A The graph shows daily pre- and post-meal blood glucose levels (mg / dL) before and after administration of AAV1-hINS+AAV1-hGCK (day 0), as well as the amount (U / kg / day) of exogenous long-acting insulin (insulin glargine, "Lantus") and short-acting insulin (insulin lispro, "Humalog") administered, up to day 90 after AAV administration. The percentage reduction in exogenous insulin administration compared to the 7-day mean of exogenous insulin administered before AAV administration (baseline) (before day 0) is shown at the top of the graph. Blood samples were drawn weekly from day 0 until day 90 after AAV1-hINS+AAV1-hGCK administration, and pre-meal, fasting, or stimulated C-peptide levels were analyzed. Figure 3B ; ng / mL), Hb1A1C level ( Figure 3C ; %) level and creatine kinase (CK) level ( Figure 3D (U / L). Figure 3E The weight (kg) is shown before and after AAV administration up to day 90. Figure 3F Show (i) blood glucose levels (mg / dL) during an intravenous glucose tolerance test (IVGTT) of NHP3 before STZ administration (before STZ), after STZ administration and before administration of AAV1-hIns+AAV1-hGck (after STZ), and (iii) after STZ administration and 56 days (D+56) or 84 days (D+84) after administration of AAV1-hIns+AAV1-hGck. Figure 3G This shows the C-peptide levels (ng / mL) of NHP3 during IVGTT at (i) before STZ administration, (ii) after STZ administration and before AAV1-hIns+AAV1-hGck administration (post-STZ), and (iii) after STZ administration and at 56 days (D+56) or 84 days (D+84) after AAV1-hIns+AAV1-hGck administration. Figure 3H This displays the interferon-γ response (spots / million cells) in peripheral blood mononuclear cells against AAV1, glucokinase (Gck), insulin, positive control, and negative control, measured from 14 days (-14) before AAV administration to 91 days after AAV administration. Positive test responses are indicated by boxes. Figure 3IThe values ​​of anti-insulin IgG (anti-insulin dilution titer) and anti-glucokinase IgG (anti-glucokinase ECL signal) were measured from 14 days before AAV administration until 91 days after administration.

[0190] Figure 4A-4I The levels of the mentioned biomarkers are shown in a non-human primate (NHP) model of STZ-induced type 1 diabetes, in cynomolgus fourth macaques (NHP4) administered AAV1-hINS+AAV1-hGCK after streptozotocin (STZ) treatment. Figure 4A The graph shows daily pre- and postprandial blood glucose levels (mg / dL) before and after administration of AAV1-hINS+AAV1-hGCK (day 0), as well as the amount (U / kg / day) of exogenous long-acting insulin (insulin glargine, "Lantus") and short-acting insulin (insulin lispro, "Humalog") administered, up to day 90 after AAV administration. The percentage reduction in exogenous insulin administration compared to the 7-day mean of exogenous insulin administered before AAV administration (baseline) (before day 0) is shown at the top of the graph. Blood samples were drawn weekly from day 0 until day 90 after AAV1-hINS+AAV1-hGCK administration, and C-peptide levels were analyzed randomly (samples obtained without regard to fasting or eating status), fasted, or stimulated (samples obtained as part of IVGTT). Figure 4B ; ng / mL), Hb1A1C level ( Figure 4C ; %) level and creatine kinase (CK) level ( Figure 4D (U / L). Figure 4E The weight (kg) is shown before and after AAV administration and up to day 90. Figure 4F This shows (i) blood glucose levels (mg / dL) during intravenous glucose tolerance tests (IVGTT) of NHP4 before STZ administration (before STZ), (ii) after STZ administration and before administration of AAV1-hIns+AAV1-hGck (after STZ), and (iii) after STZ administration and at 56 days (D+56) and 84 days (D+84) after administration of AAV1-hIns+AAV1-hGck. Figure 4G This shows the C-peptide levels (ng / mL) of NHP4 during IVGTT at (i) before STZ administration, (ii) after STZ administration and before AAV1-hIns+AAV1-hGck administration (post-STZ), and (iii) 56 days after STZ administration and 56 days after AAV1-hIns+AAV1-hGck administration (D+56). Figure 4HThis displays the interferon-γ response (spots / million cells) in peripheral blood mononuclear cells against AAV1, glucokinase (Gck), insulin, positive control, and negative control, measured from 14 days (-14) before AAV administration to 91 days after AAV administration. Positive test responses are indicated by boxes. Figure 4I The values ​​of anti-insulin IgG (anti-insulin dilution titer) and anti-glucokinase IgG (anti-glucokinase ECL signal) were measured from 14 days before AAV administration until 80 days after administration.

[0191] Figure 5A-5K The levels of the mentioned biomarkers are shown in a non-human primate (NHP) model of STZ-induced type 1 diabetes, in fifth-order cynomolgus macaques (NHP5) treated with AAV1-hINS+AAV1-hGCK after streptozotocin (STZ) treatment. Figure 5A This displays daily pre- and post-meal blood glucose levels (mg / dL) from 30 days prior to (-30 days) of AAV1-hINS+AAV1-hGCK administration until 180 days after AAV1-hINS+AAV1-hGCK administration. Blood samples were drawn weekly from day 0 until day 180 after AAV1-hINS+AAV1-hGCK administration, and C-peptide levels were analyzed. Figure 5B ; ng / mL) and Hb1A1C levels ( Figure 5C ;%). From day 0 until day 180 after AAV1-hINS+AAV1-hGCK administration, blood samples were drawn weekly and creatine kinase (CK) levels were analyzed. Figure 5D (U / L). Figure 5E The body weight (kg) was shown before and after administration of AAV1-hINS+AAV1-hGCK and up to day 182. Figure 5F The data show the blood glucose levels (mg / dL) of NHP5 during intravenous glucose tolerance tests (IVGTT) before STZ administration (before STZ), (ii) after STZ administration and before administration of AAV1-hIns+AAV1-hGck (after STZ), and (iii) after STZ administration and at 56 days (D+56), 91 days (D+91), and 175 days (D+175) after administration of AAV1-hIns+AAV1-hGck. Figure 5G This shows the C-peptide levels (ng / mL) of NHP5 during IVGTT at (i) before STZ administration, (ii) after STZ administration and before AAV1-hIns+AAV1-hGck administration (post-STZ), and (iii) after STZ administration and at 56 days (D+56), 91 days (D+91), and 175 days (D+175) after AAV1-hIns+AAV1-hGck administration. Figure 5H This shows the amount (U / kg / day) of exogenous long-acting insulin (glargine insulin) and short-acting insulin (lispro insulin) at time points from the administration of AAV1-hINS+AAV1-hGCK (day 0) up to day 180 after administration of AAV1-hINS+AAV1-hGCK. Figure 5I This shows the percentage reduction (change relative to baseline) of exogenous insulin administration compared to the 30-day average (baseline) of exogenous insulin administration prior to AAV1-hINS+AAV1-hGCK administration. The 30-day average reduction percentage is shown at the top of the graph. Figure 5J This shows insulin (µU / mL) measured after fasting or at a randomized time from the administration of AAV1-hINS+AAV1-hGCK (day 0) to day 182. Figure 5K This displays the interferon-γ responses (spots / million cells) in peripheral blood mononuclear cells against AAV1, glucokinase (Gck), insulin, positive controls, and negative controls, measured from 14 days (-14) before (AAV1-hINS+AAV1-hGCK) administration to 182 days after AAV1-hINS+AAV1-hGCK administration. Positive test responses are indicated by boxes.

[0192] Figure 6A-6L The levels of the mentioned biomarkers are shown in a non-human primate (NHP) model of STZ-induced type 1 diabetes, in sixth cynomolgus macaques (NHP6) administered AAV1-hINS+AAV1-hGCK after streptozotocin (STZ) treatment. Figure 6A This displays daily pre- and post-meal blood glucose levels (mg / dL) from 30 days prior to (-30 days) of AAV1-hINS+AAV1-hGCK administration until 182 days after AAV1-hINS+AAV1-hGCK administration. Blood samples were drawn weekly from day 0 until day 182 after AAV1-hINS+AAV1-hGCK administration, and C-peptide levels were analyzed. Figure 6B ; ng / mL) and Hb1A1C levels ( Figure 6C ;%). From day 0 until day 182 after AAV1-hINS+AAV1-hGCK administration, blood samples were drawn weekly and creatine kinase (CK) levels were analyzed. Figure 6D (U / L). Figure 6E The body weight (kg) was shown before and after administration of AAV1-hINS+AAV1-hGCK and up to day 182. Figure 6F The cyclic tacrolimus levels (µg / L) are shown from day 21 to day 182 following administration of AAV1-hINS+AAV1-hGCK. Figure 6GThis shows (i) blood glucose levels (mg / dL) during intravenous glucose tolerance tests (IVGTT) of NHP6 before STZ administration (before STZ), (ii) after STZ administration and before administration of AAV1-hIns+AAV1-hGck (after STZ), and (iii) after STZ administration and at 56 days (D+56), 91 days (D+91), and 175 days (D+175) after administration of AAV1-hIns+AAV1-hGck. Figure 6H This shows the C-peptide levels (ng / mL) of NHP6 during IVGTT at (i) before STZ administration, (ii) after STZ administration and before AAV1-hIns+AAV1-hGck administration (post-STZ), and (iii) after STZ administration and at 56 days (D+56), 91 days (D+91), and 175 days (D+175) after AAV1-hIns+AAV1-hGck administration. Figure 6I This shows the amount (U / kg / day) of exogenous long-acting insulin (glargine insulin) and short-acting insulin (lispro insulin) at time points from the administration of AAV1-hINS+AAV1-hGCK (day 0) up to day 182 after administration of AAV1-hINS+AAV1-hGCK. Figure 6J This shows the percentage reduction (change relative to baseline) of exogenous insulin administration compared to the 30-day average (baseline) of exogenous insulin administration prior to AAV1-hINS+AAV1-hGCK administration. The 30-day average reduction percentage is shown at the top of the graph. Figure 6K This shows insulin (µU / mL) measured after fasting or at a randomized time from the administration of AAV1-hINS+AAV1-hGCK (day 0) to day 180. Figure 6L This displays the interferon-γ responses (spots / million cells) in peripheral blood mononuclear cells against AAV1, glucokinase (Gck), insulin, positive controls, and negative controls, measured from 14 days (-14) before (AAV1-hINS+AAV1-hGCK) administration to 182 days after AAV1-hINS+AAV1-hGCK administration. Positive test responses are indicated by boxes.

[0193] Figure 7A-7L The levels of the mentioned biomarkers are shown in a non-human primate (NHP) model of STZ-induced type 1 diabetes, in cynomolgus seventh macaques (NHP7) administered AAV1-hINS+AAV1-hGCK after streptozotocin (STZ) treatment. Figure 7AThis shows daily pre- and post-meal blood glucose levels (mg / dL) from 30 days (-30) before injection of AAV1-hINS+AAV1-hGCK until 122 days after AAV1-hINS+AAV1-hGCK administration. Figure 7B This shows the amount (U / kg / day) of exogenous long-acting insulin (glargine insulin) and short-acting insulin (lispro insulin) at time points from the administration of AAV1-hINS+AAV1-hGCK (day 0) up to day 122 after administration of AAV1-hINS+AAV1-hGCK. Figure 7C This shows the percentage reduction (change relative to baseline) of exogenous insulin administration compared to the 30-day average (baseline) of exogenous insulin administration prior to AAV1-hINS+AAV1-hGCK administration. The 30-day average reduction percentage is shown at the top of the graph. Figure 7D This shows the Hb1A1C level (%) from day 0 until day 122 after AAV1-hINS+AAV1-hGCK administration. Figure 7E This shows (i) blood glucose levels (mg / dL) during intravenous glucose tolerance tests (IVGTT) of NHP7 patients before STZ administration (before STZ), (ii) after STZ administration and before administration of AAV1-hIns+AAV1-hGck (after STZ), and (iii) after STZ administration and at 64 days (D+64) and 91 days (D+91) after administration of AAV1-hIns+AAV1-hGck. Figure 7F This shows the C-peptide levels (ng / mL) of NHP7 during IVGTT at (i) before STZ administration, (ii) after STZ administration and before AAV1-hIns+AAV1-hGck administration (post-STZ), and (iii) after STZ administration and at 64 days (D+64) and 91 days (D+91) after AAV1-hIns+AAV1-hGck administration. Figure 7G This shows the creatine kinase (CK) levels (U / L) from day 0 until day 122 after AAV1-hINS+AAV1-hGCK administration. Figure 7H The cyclic tacrolimus levels (µg / L) are shown from day 21 to day 90 following AAV1-hINS+AAV1-hGCK administration. Figure 7I This shows human insulin levels (µU / mL) measured from the administration of AAV1-hIns+AAV1-hGck (day 0) up to day 122. Figure 7J This shows the C-peptide levels (ng / mL) from day 0 to day 122 after administration of AAV1-hINS+AAV1-hGCK. Figure 7KThis displays the interferon-γ responses (spots / million cells) in peripheral blood mononuclear cells against AAV1, glucokinase (Gck), insulin, positive controls, and negative controls, measured from 14 days (-14) before (AAV1-hINS+AAV1-hGCK) administration to 122 days after AAV1-hINS+AAV1-hGCK administration. Positive test responses are indicated by boxes. Figure 7L The body weight (kg) was shown before and after administration of AAV1-hINS+AAV1-hGCK and up to day 122.

[0194] Figure 8A-8L The levels of the mentioned biomarkers are shown in a non-human primate (NHP) model of STZ-induced type 1 diabetes, in cynomolgus macaques (NHP8) treated with streptozotocin (STZ) and administered AAV1-hINS+AAV1-hGCK. Figure 8A This shows daily pre- and post-meal blood glucose levels (mg / dL) from 30 days (-30) before injection of AAV1-hINS+AAV1-hGCK until 122 days after AAV1-hINS+AAV1-hGCK administration. Figure 8B This shows the amount (U / kg / day) of exogenous long-acting insulin (glargine insulin) and short-acting insulin (lispro insulin) at time points from the administration of AAV1-hINS+AAV1-hGCK (day 0) up to day 122 after administration of AAV1-hINS+AAV1-hGCK. Figure 8C This shows the percentage reduction (change relative to baseline) of exogenous insulin administration compared to the 30-day average (baseline) of exogenous insulin administration prior to AAV1-hINS+AAV1-hGCK administration. The 30-day average reduction percentage is shown at the top of the graph. Figure 8D This shows the Hb1A1C level (%) from day 0 until day 122 after AAV1-hINS+AAV1-hGCK administration. Figure 8E This shows (i) blood glucose levels (mg / dL) during intravenous glucose tolerance tests (IVGTT) at NHP8 before STZ administration (before STZ), (ii) after STZ administration and before administration of AAV1-hIns+AAV1-hGck (after STZ), and (iii) after STZ administration and at 56 days (D+56) and 91 days (D+91) after administration of AAV1-hIns+AAV1-hGck. Figure 8FThis shows the C-peptide levels (ng / mL) of NHP8 during IVGTT at (i) before STZ administration, (ii) after STZ administration and before AAV1-hIns+AAV1-hGck administration (post-STZ), and (iii) after STZ administration and at 56 days (D+64) and 91 days (D+91) after AAV1-hIns+AAV1-hGck administration. Figure 8G This shows the creatine kinase (CK) levels (U / L) from day 0 until day 122 after AAV1-hINS+AAV1-hGCK administration. Figure 8H The cyclic tacrolimus level (µg / L) is shown from day 21 after administration of AAV1-hINS+AAV1-hGCK until termination. Figure 8I This shows the human insulin level (µU / mL) measured from the administration of AAV1-hIns+AAV1-hGck (day 0) up to day 122. Figure 8J This shows the C-peptide levels (ng / mL) from day 0 to day 122 after administration of AAV1-hINS+AAV1-hGCK. Figure 8K This displays the interferon-γ responses (spots / million cells) in peripheral blood mononuclear cells against AAV1, glucokinase (Gck), insulin, positive controls, and negative controls, measured from 14 days (-14) before (AAV1-hINS+AAV1-hGCK) administration to 122 days after AAV1-hINS+AAV1-hGCK administration. Positive test responses are indicated by boxes. Figure 8L The body weight (kg) was shown before and after administration of AAV1-hINS+AAV1-hGCK and up to day 122.

[0195] Figure 9A This study summarizes the anti-insulin IgG (dilution factor) of NHP1-NHP4 measured 14 days (-14) prior to administration of AAV1-hINS+AAV1-hGCK. Figure 9B Anti-glucokinase IgG (ECL signal) of NHP1-NHP6 was measured 14 days (-14) prior to AAV administration. Figure 9C This displays the levels of anti-drug antibody (ADA) in NHP1 and NHP2 insulin (screening signal) measured from day -14 to day 182.

[0196] Figure 10A-10I This indicates the amount of human glucokinase protein (ng / mg) in the muscle at or near the injection site of AAV1-hINS+AAV1-hGCK from NHP1-NHP8. Figure 10AThis displays glucokinase protein levels (ng / mg) from various muscle samples obtained at or 1 cm away from the injection site of AAV1-hINS+AAV1-hGCK. The lower limit of quantification (LLOQ) is shown as a dashed line. NHP1-NHP8 are also shown. Figure 10B Specifically, this displays the glucokinase level (ng / mg) in muscle samples or samples taken 1 cm away from the injection site of AAV1-hINS+AAV1-hGCK. The lower limit of quantitation (LLOQ) is shown as a dashed line. Figure 10C Specifically, this displays the glucokinase level (ng / mg) in muscle samples or samples taken 1 cm away from the injection site of AAV1-hINS+AAV1-hGCK containing NHP2. The lower limit of quantitation (LLOQ) is shown as a dashed line. Figure 10D Specifically, this displays the glucokinase level (ng / mg) in muscle samples or samples taken 1 cm away from the injection site of AAV1-hINS+AAV1-hGCK containing NHP3. The lower limit of quantitation (LLOQ) is shown as a dashed line. Figure 10E Specifically, this displays the glucokinase level (ng / mg) in muscle samples of NHP4 obtained from the injection site of AAV1-hINS+AAV1-hGCK, or samples taken 1 cm away. The lower limit of quantitation (LLOQ) is shown as a dashed line. Figure 10F Specifically, this displays the glucokinase level (ng / mg) in muscle samples of NHP5 obtained from the injection site of AAV1-hINS+AAV1-hGCK, or samples taken 1 cm away. The lower limit of quantitation (LLOQ) is shown as a dashed line. Figure 10G Specifically, this displays the glucokinase level (ng / mg) in muscle samples of NHP6 obtained from the injection site of AAV1-hINS+AAV1-hGCK, or samples taken 1 cm away. The lower limit of quantitation (LLOQ) is shown as a dashed line. Figure 10H Specifically, this displays the glucokinase level (ng / mg) in muscle samples of NHP7 obtained from the injection site of AAV1-hINS+AAV1-hGCK, or samples taken 1 cm away. The lower limit of quantitation (LLOQ) is shown as a dashed line. Figure 10I Specifically, this displays the glucokinase level (ng / mg) in muscle samples of NHP8 obtained from the injection site of AAV1-hINS+AAV1-hGCK, or samples taken 1 cm away. The lower limit of quantitation (LLOQ) is shown as a dashed line.

[0197] Figure 11AThis shows the vector genome level (Vg; copies / µg gDNA) and insulin mRNA level (copy / µg RNA) obtained from muscle samples obtained at or near the injection site (IS) of AAV1-hINS+AAV1-hGCK from NHP1 and NHP2. Figure 11B This shows the vector genome level (Vg; copies / µg gDNA) and glucokinase mRNA level (copy / µg RNA) obtained from muscle samples at or near the injection site (IS) of AAV1-hINS+AAV1-hGCK from NHP1 and NHP2. Figure 11C This shows the vector genome level (Vg; copies / µg gDNA) and insulin mRNA level (copy / µg RNA) obtained from muscle samples at or near the injection site (IS) of AAV1-hINS+AAV1-hGCK from NHP3 and NHP4. Figure 11D This shows the vector genome level (Vg; copies / µg gDNA) and glucokinase mRNA level (copy / µg RNA) obtained from muscle samples at or near the injection site (IS) of AAV1-hINS+AAV1-hGCK from NHP3 and NHP4. Figure 11E This shows the vector genome level (Vg; copies / µg gDNA) and insulin mRNA level (copy / µg RNA) obtained from muscle samples at or near the injection site (IS) of AAV1-hINS+AAV1-hGCK from NHP5 and NHP6. Figure 11F This shows the vector genome level (Vg; copies / µg DNA) and glucokinase mRNA level (copies / µg RNA) obtained from muscle samples at or near the injection site (IS) of AAV1-hINS+AAV1-hGCK from NHP5 and NHP6. Figure 11G This shows the vector genome level (Vg; copies / µg gDNA) and insulin mRNA level (copy / µg RNA) obtained from muscle samples at or near the injection site (IS) of AAV1-hINS+AAV1-hGCK from NHP7 and NHP8. Figure 11H This shows the vector genome level (Vg; copies / µg gDNA) and glucokinase mRNA level (copy / µg RNA) obtained from muscle samples at or near the injection site (IS) of AAV1-hINS+AAV1-hGCK from NHP7 and NHP8.

[0198] Figure 12A-12GMetabolic data from an ongoing study of a group of cynomolgus macaques (NHP9-NHP11) in a non-human primate (NHP) model of STZ-induced type 1 diabetes, administered AAV1-hINS+AAV1-hGCK after streptozotocin (STZ) treatment. Figure 12A Displays lactate levels (mol / L) measured from NHP9 to NHP11, starting from the day of AAV1-hINS+AAV1-hGCK injection (day 0). Figure 12B This displays the HbA1C levels (%) of NHP9-NHP11 measured from the day of AAV1-hINS+AAV1-hGCK injection (day 0). Figure 12C This displays the C-peptide levels (ng / mL) of NHP9 and NHP10 measured from the day of AAV1-hINS+AAV1-hGCK injection (day 0). The limit of quantitation (LLOQ) is shown as a dashed line. Figure 12D This displays human insulin levels (µU / mL) of NHP9 and NHP10 measured from the day of AAV1-hINS+AAV1-hGCK injection (day 0). Figure 12E This shows the glucagon levels (pmol / L) of NHP9 and NHP10 measured from the day of AAV1-hINS+AAV1-hGCK injection (day 0). Figure 12F This displays the triglyceride levels (mg / dL) of NHP9-NHP11 measured from the day of AAV1-hINS+AAV1-hGCK injection (day 0). Figure 12G The data shows HbA1c levels for NHP 9-14 on days 0 and 28.

[0199] Figures 13A-13D The levels of the mentioned biomarkers are shown in a non-human primate (NHP) model of STZ-induced type 1 diabetes, in cynomolgus monkey 9 (NHP9) administered AAV1-hINS+AAV1-hGCK after streptozotocin (STZ) treatment. Figure 13A This displays daily pre- and post-meal blood glucose levels (mg / dL) from 30 days (-30) before injection of AAV1-hINS+AAV1-hGCK until 184 days after AAV1-hINS+AAV1-hGCK administration. Arrows indicate days when the tacrolimus dose was reduced. Figure 13BThis chart shows the dosage (U / kg / day) of exogenous long-acting insulin (glargine insulin) and short-acting insulin (lispro insulin) administered from 30 days (-30) prior to AAV1-hINS+AAV1-hGCK administration up to day 184 following AAV1-hINS+AAV1-hGCK administration. For each 30-day window starting from day 0, the percentage change in glargine and lispro insulin compared to the 30-day window prior to AAV is shown at the top of the graph. Arrows indicate days when the tacrolimus dose was reduced due to an anemia event unrelated to the test substance observed on day 120. Figure 13C The data show the blood glucose levels (mg / dL) of NHP9 during an intravenous glucose tolerance test (IVGTT) before STZ administration (before STZ), (ii) after STZ administration and before administration of AAV1-hIns+AAV1-hGck (after STZ), and (iii) after STZ administration and at 56 days (D+56) and 91 days (D+91) after administration of AAV1-hIns+AAV1-hGck. Figure 13D Displayed as AUC (Area Under the Curve) Figure 13C Quantitatively.

[0200] Figures 14A-14D The levels of the mentioned biomarkers are shown in a non-human primate (NHP) model of STZ-induced type 1 diabetes, in tenth cynomolgus macaques (NHP10) administered AAV1-hINS+AAV1-hGCK after streptozotocin (STZ) treatment. Figure 14A This shows daily pre- and post-meal blood glucose levels (mg / dL) from 30 days (-30) before injection of AAV1-hINS+AAV1-hGCK until 184 days after AAV1-hINS+AAV1-hGCK administration. Figure 14B The figure shows the amount (U / kg / day) of exogenous long-acting insulin (glargine insulin) and short-acting insulin (lispro insulin) administered from 30 days (-30) before AAV1-hINS+AAV1-hGCK administration up to 184 days after AAV1-hINS+AAV1-hGCK administration. For each 30-day window starting from day 0, the percentage change in glargine insulin and lispro insulin compared to the 30-day window before AAV administration is shown at the top of the graph. Figure 14C The data show the blood glucose levels (mg / dL) during the intravenous glucose tolerance test (IVGTT) of NHP10 before STZ administration (before STZ), (ii) after STZ administration and before administration of AAV1-hIns+AAV1-hGck (after STZ), and (iii) after STZ administration and at 56 days (D+56) and 91 days (D+91) after administration of AAV1-hIns+AAV1-hGck. Figure 14DDisplayed as AUC (Area Under the Curve) Figure 14C Quantitatively.

[0201] Figures 15A-15B The levels of the mentioned biomarkers are shown in an STZ-induced type 1 diabetes non-human primate (NHP) model, in eleventh-year cynomolgus monkeys (NHP11) administered AAV1-hINS+AAV1-hGCK after streptozotocin (STZ) treatment. Figure 15A This shows daily pre- and post-meal blood glucose levels (mg / dL) from 30 days (-30) before injection of AAV1-hINS+AAV1-hGCK until 43 days after AAV1-hINS+AAV1-hGCK administration. Figure 15B This chart shows the dosage (U / kg / day) of exogenous long-acting insulin (glargine insulin) and short-acting insulin (lispro insulin) administered from 30 days (-30) before AAV1-hINS+AAV1-hGCK administration up to 43 days after AAV1-hINS+AAV1-hGCK administration. For each window starting from day 0, the percentage change in glargine insulin and lispro insulin compared to the 30-day window before AAV administration is shown at the top of the graph.

[0202] Figures 16A-16B The levels of the mentioned biomarkers are shown in a non-human primate (NHP) model of STZ-induced type 1 diabetes, in 12th cynomolgus monkeys (NHP12) administered AAV1-hINS+AAV1-hGCK after streptozotocin (STZ) treatment. Figure 16A This shows daily pre- and post-meal blood glucose levels (mg / dL) from 30 days (-30) before injection of AAV1-hINS+AAV1-hGCK until 43 days after AAV1-hINS+AAV1-hGCK administration. Figure 16B This chart shows the dosage (U / kg / day) of exogenous long-acting insulin (glargine insulin) and short-acting insulin (lispro insulin) administered from 30 days (-30) before AAV1-hINS+AAV1-hGCK administration up to 43 days after AAV1-hINS+AAV1-hGCK administration. For each window starting from day 0, the percentage change in glargine insulin and lispro insulin compared to the 30-day window before AAV administration is shown at the top of the graph.

[0203] Figures 17A-17B The levels of the mentioned biomarkers are shown in a non-human primate (NHP) model of STZ-induced type 1 diabetes, in 13th cynomolgus monkeys (NHP13) administered AAV1-hINS+AAV1-hGCK after streptozotocin (STZ) treatment. Figure 17AThis shows daily pre- and post-meal blood glucose levels (mg / dL) from 30 days (-30) before injection of AAV1-hINS+AAV1-hGCK until 43 days after AAV1-hINS+AAV1-hGCK administration. Figure 17B This chart shows the dosage (U / kg / day) of exogenous long-acting insulin (glargine insulin) and short-acting insulin (lispro insulin) administered from 30 days (-30) before AAV1-hINS+AAV1-hGCK administration up to 43 days after AAV1-hINS+AAV1-hGCK administration. For each window starting from day 0, the percentage change in glargine insulin and lispro insulin compared to the 30-day window before AAV administration is shown at the top of the graph.

[0204] Figures 18A-18B The levels of the mentioned biomarkers are shown in a non-human primate (NHP) model of STZ-induced type 1 diabetes, in fourteenth cynomolgus macaques (NHP14) administered AAV1-hINS+AAV1-hGCK after streptozotocin (STZ) treatment. Figure 18A This shows daily pre- and post-meal blood glucose levels (mg / dL) from 30 days (-30) before injection of AAV1-hINS+AAV1-hGCK until 43 days after AAV1-hINS+AAV1-hGCK administration. Figure 18B This chart shows the dosage (U / kg / day) of exogenous long-acting insulin (glargine insulin) and short-acting insulin (lispro insulin) administered from 30 days (-30) before AAV1-hINS+AAV1-hGCK administration up to 43 days after AAV1-hINS+AAV1-hGCK administration. For each window starting from day 0, the percentage change in glargine insulin and lispro insulin compared to the previous 30-day window is shown at the top of the graph.

[0205] Figures 19A-19B Display from the front view ( Figure 19A ) and rear view ( Figure 19B (See the diagram of the muscles and tendons in the leg.) Detailed Implementation

[0206] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In case of any conflict, this application (including the definitions) shall prevail. Unless the context otherwise requires, singular terms shall include plural terms and plural terms shall include singular terms.

[0207] Throughout this disclosure, the term "a" or "an" refers to one or more of the entities described; for example, "a polynucleotide" should be understood to mean one or more polynucleotides. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0208] Furthermore, the term “and / or” as used herein should be understood to explicitly disclose each of the two specified features or components, with or without the other. Therefore, 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 herein in phrases such as “A, B, and / or C” is intended to cover all of the following: 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).

[0209] The term “about” is used herein to mean approximately, roughly, around, or within its range. When the term “about” is used in conjunction with a numerical range, the term varies the range by extending the boundaries above and below the stated numerical value. Generally, unless otherwise indicated, the term “about” is used herein to mean values ​​above and below a 10% difference in terms of upward and downward (higher or lower) variation of the stated value.

[0210] The term "at least" preceding a number or series of numbers is understood to include the number adjacent to the term "at least," as well as all subsequent numbers or integers that can logically be included, as is clearly apparent from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For instance, "at least 18 nucleotides in a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated property. When "at least" precedes a series of numbers or a range, it should be understood that "at least" can modify individual numbers within the series or range. "At least" is also not limited to integers (e.g., without considering the number of significant figures, "at least 5%" includes 5.0%, 5.1%, and 5.18%).

[0211] Unless otherwise specifically indicated, nucleotide sequences are presented in this document as single strands from left to right in a 5' to 3' orientation. Nucleotides and amino acids are represented in this document in the manner recommended by the IUPAC-IUB Committee on Biochemistry Nomenclature, or (for amino acids) by single-letter or three-letter codes, both in accordance with 37 CFR §1.822 and established usage.

[0212] As used herein, "polynucleotide" or "nucleic acid" refers to a sequence of nucleotides linked by phosphodiester bonds. Polynucleotides are presented herein in a 5' to 3' orientation. The polynucleotides disclosed herein may be deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules. Nucleotide bases are indicated herein by single-letter codes: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I), and uracil (U).

[0213] As used herein, unless otherwise indicated, the term "polypeptide" encompasses both peptides and proteins.

[0214] The terms “coding sequence” or “sequence encoding…” are used herein to refer to a DNA or RNA region (transcriptional region) that “encodes” a specific protein (e.g., insulin or glucokinase). When placed under the control of appropriate regulatory regions (e.g., promoters), coding sequences are transcribed (DNA) and translated (RNA) into polypeptides in vitro or in vivo. The boundaries of a coding sequence are defined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. Coding sequences may include, but are not limited to, cDNA from prokaryotes or eukaryotes, genomic DNA from prokaryotes or eukaryotes, and synthetic DNA sequences. The transcription stop sequence may be located at the 3' end of the coding sequence.

[0215] In some respects, the expression cassette may contain several operatively linked fragments, such as one or more of the following: a promoter, a 5' untranslated sequence, a leader sequence, an intron, a coding sequence, and a 3' untranslated sequence (e.g., containing a polyadenylation site or a signal sequence).

[0216] As used in this article, “gene expression” refers to the process in which genes are transcribed into RNA and / or translated into active proteins.

[0217] As used herein, an open reading frame (ORF) is the translatable portion of a reading frame. An ORF is a continuous codon extension that begins with a start codon and ends with a stop codon. In some respects, an ORF sequence may be displayed or referenced with or without a start codon sequence and / or a stop codon sequence.

[0218] A Kozak consensus sequence (or Kozak consensus) is a sequence present on eukaryotic mRNA that shares the common (gcc) sequence (gccRccAUGG), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another "G". In some respects, the polynucleotide contains a nucleic acid sequence having at least 95%, at least 99%, or higher sequence identity with the Kozak consensus sequence.

[0219] The term "sequence identity" is used herein to mean a relationship between two or more amino acid (peptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparison of said sequences. In some respects, sequence identity is calculated based on the full length of two given SEQ ID NOs or based on a portion thereof. A portion may mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or any other specified percentage, of the two SEQ ID NOs. The term "identity" may also mean the degree of sequence correlation between two or more amino acid or nucleic acid sequences, as determined on a case-by-case basis, such as by matching such sequence strings.

[0220] In some respects, methods for determining identity are designed to produce the maximum match between the tested sequences. The methods for determining identity and similarity are encoded in publicly available computer programs.

[0221] When referring to nucleic acids or fragments thereof, “gross homology” or “gross similarity” indicates that when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, there is nucleotide sequence identity in at least about 95% to 99% of the sequence.

[0222] As used herein, and unless otherwise indicated, the term "complementarity" when used to describe a first nucleic acid sequence relative to a second nucleic acid sequence refers to the ability of an oligonucleotide or polynucleotide containing the first nucleic acid sequence to hybridize with an oligonucleotide or polynucleotide containing the second nucleic acid sequence under certain conditions and form a double helix structure, as understood by those skilled in the art. 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 for 12–16 hours, followed by washing (see, for example, "Molecular Cloning: A Laboratory Manual," Sambrook et al., (1989) Cold Spring Harbor Laboratory Press). Other conditions may be used, such as physiologically relevant conditions that may be encountered in vivo. Those skilled in the art will be able to determine the set of conditions most suitable for the complementarity test of the two sequences based on the final application of the hybridized nucleotides.

[0223] The term "promoter" is used herein to mean a nucleic acid sequence or segment upstream of the transcription start site of one or more genes (or coding sequences) for controlling transcription of one or more genes (or coding sequences). In some respects, promoters are structurally identified by the presence of a DNA-dependent RNA polymerase binding site, a transcription start site, and optionally any other DNA sequence (including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those skilled in the art to directly or indirectly regulate the amount of transcription by 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 depending on physiological or developmental conditions, or in some respects, an inducible promoter can be induced by exogenous molecules (e.g., chemicals or drugs) or other exogenous stimuli (e.g., light or radiation). A "tissue-specific" promoter is preferentially active in a specific type of differentiated cell / tissue.

[0224] As used herein, the term "enhancer" is a cis-acting element that stimulates or inhibits the transcription of adjacent genes. Enhancers that inhibit transcription are also called "silencers." Enhancers can act in either orientation at a distance of several kilobase pairs (kb) from the coding sequence and downstream of the transcribed region (e.g., they can bind to the coding sequence).

[0225] The terms "operably linked," "operably inserted," "operably located," "under control," or "under transcriptional control" mean that the promoter is in the correct position and orientation relative to the nucleic acid to control RNA polymerase initiation and gene (or coding sequence) expression. DNA sequences and regulatory sequences are considered operably linked if they are linked in a way that allows gene expression when an appropriate molecule (e.g., a transcription activating protein) binds to the regulatory sequence. DNA is operably inserted if the DNA of interest is located near a DNA sequence that guides the transcription and translation of the DNA of interest in the cell (i.e., promotes, for example, the production of a polypeptide encoded by the DNA of interest).

[0226] The term "transgenic" is used herein to mean a gene or nucleic acid molecule introduced into a cell. Examples of transgenics are nucleic acids encoding therapeutic polypeptides (e.g., genes encoding insulin and / or genes encoding glucokinase). In some embodiments, the gene may be present, but in some cases it is not typically expressed in the cell or is expressed at an insufficient level. In this case, "insufficient" means that although the gene (e.g., insulin and / or glucokinase) is typically expressed in the cell, it is still possible to develop symptoms and / or diseases (e.g., diabetes) as disclosed herein. In some aspects, transgenics allow for increased or overexpression of genes (e.g., insulin and / or glucokinase). Transgenics may contain sequences native to the cell, sequences not naturally present in the cell, or combinations of both. In some aspects, transgenics may contain modified sequences encoding insulin, glucokinase, both insulin and glucokinase, and / or other proteins, said modified sequences being operatively linked to appropriate regulatory sequences for expression in the cell of sequences encoding insulin, glucokinase, or both insulin and glucokinase. In some aspects, transgenics do not integrate into the genome of the host cell.

[0227] The terms “modified gene” and “modified nucleic acid” are used interchangeably herein to refer to the introduction of one or more modifications or changes relative to the natural sequence of a gene or nucleic acid sequence. 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 or a fragment thereof.

[0228] As used herein, the term "derived from" means a component isolated from or prepared using a specified molecule or organism, or information (e.g., amino acid or nucleic acid sequences) derived from a specified molecule or organism. For example, a nucleic acid sequence derived from a second nucleic acid sequence (e.g., the wild-type human insulin gene) may include a nucleotide sequence that is identical or substantially similar to the nucleotide sequence of the second nucleic acid sequence, or a portion thereof. In some respects, mutants, analogs, or derivatives may be derived from wild-type sequences.

[0229] In the case of polynucleotides, derived species can be obtained, for example, through naturally occurring mutagenesis, artificial directed mutagenesis, or artificial random mutagenesis. Mutagenesis used to derive polynucleotides can be intentionally directed, intentionally random, or a mixture of both.

[0230] As used in this article, "vector" usually refers to a recombinant plasmid or virus that contains polynucleotides delivered to host cells in vitro or in vivo.

[0231] As used herein, the term "delivery vector" or "vector" includes any genetic element capable of replicating when bound to a suitable control element and capable of transferring gene or nucleic acid sequences between cells, such as plasmids, bacteriophages, transposons, kinases, chromosomes, artificial chromosomes, viruses, viral particles, etc. Therefore, the term includes cloning and expression vectors, as well as viral vectors. In some aspects, useful vectors are anticipated to be those in which the nucleic acid segment to be transcribed is located under the transcriptional control of a promoter. In some aspects, delivery vectors are selected from the group consisting of viral vectors, plasmids, lipids, and lysosomes.

[0232] The terms “expression vector,” “expression construct,” or “expression cassette” refer to any type of genetic construct containing nucleic acids, in which some or all of the nucleic acid coding sequences can be transcribed.

[0233] In some respects, biological vectors include viruses, particularly attenuated and / or replication-defective viruses. In some embodiments, chemical vectors include lipid complexes and naked DNA constructs.

[0234] As used herein, the terms “naked DNA” or “naked nucleic acid” refer to nucleic acid molecules that are not encapsulated or capsidated in viral particles, bacterial cells, or in the cytoplasm that facilitates the delivery of nucleic acids to target cells. Naked nucleic acids may be combined with means for facilitating the delivery of nucleic acids to target cell sites (e.g., to facilitate the entry of nucleic acids into target cells through the digestive tract, to protect nucleic acids from gastric acid, and / or to penetrate intestinal mucus) and / or to deliver nucleic acids to the surface of target epithelial cells.

[0235] "Viral genome" or "viral vector" can refer to a viral sequence containing one or more polynucleotide regions encoding or containing molecules of interest (e.g., proteins, peptides, and polynucleotides, or more thereof). Viral vectors can be used to deliver genetic material into cells. Viral vectors can be modified for specific applications. In some aspects, the delivery vectors disclosed herein are viral vectors selected from the group consisting of adeno-associated virus (AAV) vectors, adenovirus vectors, lentiviral vectors, or retroviral vectors.

[0236] As used herein, the term "adeno-associated virus vector" or "AAV vector" refers to any vector containing or derived from adeno-associated virus components suitable for infecting mammalian cells (preferably human cells). The term AAV vector may refer to AAV-type viral particles or viral particles containing a payload. AAV vectors may be derived from various serotypes, including combinations of serotypes (i.e., "pseudotyped" AAV), or from various genomes (e.g., single-stranded or self-complementary). Additionally, AAV vectors may be replication-defective and / or targeted. As used herein, the term “adeno-associated virus” (AAV) includes, but is not limited to, AAV types 1, 2, 3 (including 3A and 3B), 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, AAVrh8, AAVrh10, AAVrh.74, snake AAV, avian AAV, bovine AAV, canine AAV, horse AAV, sheep AAV, goat AAV, shrimp AAV, those AAV serotypes and clades disclosed by Gao et al. (J. Virol. 78:6381 (2004)) and Moris et al. (Virol. 33:375 (2004)), and any other AAV. See, for example, FIELDS et al., Virology, Volume 2, Chapter 69 (4th edition, Lippincott-Raven Publishers). In some respects, “AAV vector” includes derivatives of known AAV vectors. In some respects, “AAV vector” includes modified or artificial AAV vectors. In some respects, AAV vectors are modified or mutated relative to the wild-type AAV serotype sequence. In some respects, the terms “AAV genome” and “AAV vector” are used interchangeably.

[0237] As used herein, a “recombinant AAV particle” or “rAAV particle” is an AAV virus comprising a capsid protein and an AAV vector having at least one payload region (e.g., an expression cassette comprising a polynucleotide encoding insulin and / or Gck) and at least one inverted terminal repeat (ITR) region. In some aspects, the term “AAV vector of this disclosure” or “AAV vector” refers, for example, an AAV vector encapsulated in an AAV capsid containing a polynucleotide encoding insulin, GcK, or a combination thereof.

[0238] Viral “transduction” of cells refers to the transfer of nucleic acids from viral particles to cells. In some respects, transduction refers to the delivery of one or more nucleic acids encoding insulin and / or glucokinase to a recipient host cell via a viral vector. For example, transduction of target cells via the rAAV vector of this disclosure results in the transfer of the rAAV genome contained in the vector (e.g., containing polynucleotides of this disclosure) into the transduced cells.

[0239] "Transfection" of cells refers to the introduction of genetic material into cells for the purpose of gene modification. Transfection can be accomplished by a variety of methods known in the art, such as transduction or electroporation.

[0240] The terms "host cell" or "target cell" are used herein to refer to cells in which polynucleotide delivery occurs in vitro or in vivo. In some respects, AAV vectors are capable of transducing both dividing and non-dividing cells.

[0241] "Recombination" means something different from those usually found in nature.

[0242] The “serotype” of a vector or viral capsid is defined by different immune profiles based on capsid protein sequences and capsid structure.

[0243] “AAV Cap” refers to AAV Cap proteins: VP1, VP2, and VP3, and their analogues.

[0244] “AAV Rep” refers to the AAV Rep protein and its analogues.

[0245] Regarding sequences flanking other elements, "flanking" indicates the presence of one or more flanking elements upstream and / or downstream (i.e., 5' and / or 3') relative to the sequence. The term "flanking" is not intended to indicate that the sequence must be continuous. For example, intercalation sequences may exist between the nucleic acid encoding a transgene and the flanking element. A sequence flanking two other elements (e.g., an ITR) (e.g., a transgene) indicates that one element is located at the 5' of the sequence and the other at the 3' of the sequence; however, intercalation sequences may exist between them.

[0246] As used herein, the terms “effective amount,” “therapeutic effective amount,” and “sufficient amount” in gene therapy compositions comprising the rAAV particles and / or polynucleotides disclosed herein refer to an amount sufficient to achieve a beneficial or desired outcome (including clinical outcomes) when administered to a subject (including humans), and therefore “effective amount” or its synonyms depend on the context of its application. In some aspects, the amount of a given therapeutic agent or composition may vary depending on a variety of factors, such as the given agent, the drug formulation, the route of administration, the type of disease or condition, or the identity of the subject or host being treated (e.g., age, sex, and / or weight).

[0247] As used herein, the term "administration" means delivering the molecules or compositions of this disclosure (e.g., polynucleotides, expression cassettes, delivery vectors, expression constructs, rAAVs, combination gene therapies, immunosuppressants, etc.) to a subject or system. In some aspects, administration may include one or more doses delivered via an appropriate route.

[0248] As used herein, the term "skeletal muscle group" refers to a muscle that can be identified using nomenclature based on its anatomical location or its relationship to a specific skeleton within a vertebrate. Typically, for each skeletal muscle group, the subject has both right and left (bilateral) muscles. In some respects, application may be performed on a single (right or left) muscle of a skeletal muscle group. In other respects, application may be performed on both the right and left muscles of a skeletal muscle group, i.e., bilateral application.

[0249] As used herein, in the context of intramuscular application, the terms "bilateral" or "bilaterally" refer to application to both sides of the same muscle or muscle group in the subject (e.g., application to both the right and left quadriceps, both the right and left biceps, and / or both the right and left hamstrings). In contrast, "unilateral" refers to application to only one side of the subject (e.g., the right or left side).

[0250] As used herein, the terms “immunosuppression” or “immunosuppressive” refer to a temporary or permanent reduction or limitation of the immune response in a subject. “Immunosuppressant” or “immunomodulator” is a substance capable of inducing a temporary or permanent reduction or limitation of the immune response in a subject, or a change in the type of immune response. For example, an immunomodulator can redirect T cell fate from TH1 pro-inflammatory T cells to TH2 or Treg wound-healing or regulatory T cells. In some respects, immunosuppression can reduce or limit immune-mediated responses to viral vectors (e.g., rAAV). In some respects, immune-mediated responses can be innate and / or adaptive immune responses.

[0251] The term "ratio" refers to a comparison of two or more numbers that indicates their quantitative relationship to each other. In some respects, ratios can be used to compare two parts within a whole or a total.

[0252] As used herein, the term "vector ratio" refers to the comparison of the amount of one AAV vector in a vector genome (vg) to the amount of another AAV vector in the vector genome. In some respects, the vector ratio refers to the amount of AAV1-hINS vg:AAV1-hGCK vg. For example, equal amounts of AAV1-hINS vg and AAV1-hGCK vg can also be understood as a one-to-one vector ratio or a 1:1 vector ratio.

[0253] As used herein, the term "gene therapy" refers to the insertion of a nucleic acid sequence (e.g., a nucleic acid containing a promoter operatively linked to a polynucleotide encoding a therapeutic molecule) into the cells and / or tissues of an individual to treat a disease or condition. Gene therapy may also include the insertion of transgenes that are inherently repressive (i.e., suppress, reduce, or decrease the expression, activity, or function of endogenous genes or proteins (e.g., unwanted or abnormal (e.g., pathogenic) genes or proteins)). Such transgenes may be exogenous. Exogenous molecules or sequences should be understood as molecules or sequences that are not normally present in the cells, tissues, and / or individual to be treated. Both acquired and congenital diseases are suitable candidates for gene therapy.

[0254] In some aspects, this disclosure provides polynucleotides encoding wild-type or mutant insulin and / or wild-type glucokinase or functional fragments thereof. This disclosure also provides nucleic acid constructs comprising, as part of their sequence, polynucleotides encoding wild-type or mutant insulin and / or wild-type glucokinase or functional fragments thereof. For example, this disclosure includes expression cassettes, plasmids, and / or other vectors comprising polynucleotides and other elements (e.g., regulatory elements). In some aspects, this disclosure provides a packaged gene delivery medium, such as a viral capsid (e.g., an AAV capsid), comprising polynucleotides encoding wild-type or mutant insulin and / or wild-type glucokinase or functional fragments thereof.

[0255] This disclosure also includes methods for expressing wild-type or mutant insulin and / or wild-type glucokinase or functional fragments thereof by delivering a polynucleotide, along with elements required to promote expression in the cell, into the cell. This disclosure also provides gene therapy methods in which a polynucleotide encoding wild-type or mutant insulin and / or wild-type glucokinase or functional fragments thereof is administered to a subject, for example as a component of one or more carriers and / or as a component packaged as one or more viral gene delivery mediators (e.g., AAV particles).

[0256] This disclosure also provides gene therapy methods in which a polynucleotide encoding wild-type or mutant insulin and / or wild-type glucokinase or a functional fragment thereof is administered intramuscularly to a subject, for example as a component of one or more carriers and / or a component packaged as one or more viral gene delivery mediators (e.g., AAV particles), particularly wherein intramuscular administration is performed on at least two different skeletal muscle groups (e.g., quadriceps, biceps, and hamstrings), preferably as bilateral administration.

[0257] This disclosure also provides an immunosuppressive regimen accompanying a gene therapy approach (e.g., administration of an IL-6 antagonist (e.g., tocilizumab) for a period prior to gene therapy administration and / or administration of a calcineurin inhibitor (e.g., tacrolimus) for a period after gene therapy administration), wherein a polynucleotide encoding wild-type or mutant insulin and / or wild-type glucokinase or a functional fragment thereof is administered intramuscularly to the subject, for example as a component of one or more carriers and / or as a component packaged as one or more viral gene delivery mediators (e.g., AAV particles), particularly wherein intramuscular administration is performed on at least two different skeletal muscle groups (e.g., quadriceps, biceps, and hamstrings), preferably as bilateral administration. The treatment can, for example, be used to treat or alleviate various symptoms of diabetes in a subject in need.

[0258] Furthermore, certain aspects of this disclosure, such as the disclosed intramuscular administration (e.g., to two or more skeletal muscle groups, preferably bilaterally) and / or the use of immunosuppressive regimens (e.g., administering an IL-6 antagonist (e.g., tocilizumab) for a period of time prior to gene therapy administration and / or administering a calcineurin inhibitor (e.g., tacrolimus) for a period of time after gene therapy administration), may provide improvements in the safety and efficacy of the gene therapy treatments disclosed herein.

[0259] Other aspects of this disclosure relate to a method of immunosuppression in a subject receiving AAV gene therapy for the treatment of diabetes, wherein the subject is given an immunosuppression regimen comprising (a) administering an immunosuppressant before AAV gene therapy (pre-AAV); and (b) administering an immunosuppressant after AAV gene therapy (post-AAV).

[0260] In some respects, the methods disclosed herein result in (i) a decrease in glycated hemoglobin (HbA1c) levels in treated subjects, (ii) a decrease in circulating ketones in treated subjects, (iii) a decrease in triglycerides in treated subjects, (iv) a decrease in exogenous insulin in treated subjects, (v) an increase in circulating C-peptide levels in treated subjects, (vi) a decrease in the immune response to exogenous AAV, insulin protein, and / or glucokinase protein, or (vii) any combination thereof.

[0261] This document further elaborates on various aspects of these aspects of the disclosure.

[0262] Insulin and glucokinase gene therapy combination and administration method

[0263] Certain aspects of this disclosure relate to a therapy, such as an AAV combination gene therapy, comprising intramuscular (IM) delivery of a polynucleotide encoding an insulin (Ins) protein and a polynucleotide encoding a glucokinase (Gck) protein. In several preferred aspects, the combination gene therapy is administered to at least two different skeletal muscle groups. In some aspects, the combination therapy comprises IM administration of the polynucleotide encoding insulin and glucokinase in a single pharmaceutical composition, for example (i) two separate rAAV particles, each containing a polynucleotide encoding insulin and glucokinase, in the same pharmaceutical composition, or (ii) rAAV particles in a pharmaceutical composition containing a polynucleotide encoding both insulin and glucokinase. In some aspects, the combination therapy of this disclosure is administered intramuscularly (IM) to 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, or 8-10 different skeletal muscle groups.

[0264] In some respects, combination therapy is administered intramuscularly (IM) to (i) 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, or 9-10 different skeletal muscle groups; (ii) 2-8, 3-8, 4-8, 5-8, 6-8, or 7-8 different skeletal muscle groups; (iii) 2-6, 3-6, 4-6, or 5-6 different skeletal muscle groups; (iv) 2-5, 3-5, or 4-5 different skeletal muscle groups; or (iv) 2-4 or 3-4 different skeletal muscle groups.

[0265] Some aspects involve applying the combination therapy of this disclosure intramuscularly (IM) to at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten different skeletal muscle groups. In some aspects, the combination therapy of this disclosure intramuscularly (IM) is applied to 3-10, 4-10, 5-10, 6-10, 7-10, or 8-10 different skeletal muscle groups. In some aspects, the combination therapy intramuscularly (IM) is applied to two, three, four, five, six, seven, eight, nine, or ten different skeletal muscle groups.

[0266] There are three (3) types of muscles: skeletal muscle, cardiac muscle, and smooth muscle. Each type of muscle has a different structure and function. Within each skeletal muscle, muscle fibers are organized into bundles surrounded by a layer of connective tissue, called fascicles. In vertebrates, skeletal muscles are typically attached to bones and responsible for skeletal movement. Skeletal muscles also play a role in maintaining body posture and position, maintaining body temperature, storing nutrients, and stabilizing joints. The skeletal muscle groups of the upper body include, but are not limited to, the biceps, triceps, abdominal muscles, pectoral muscles, deltoid muscles, trapezius muscles, latissimus dorsi, and erector spinae muscles. The skeletal muscle groups of the lower body include, but are not limited to, the quadriceps, hamstrings, gastrocnemius (e.g., calf muscles), soleus muscles, and gluteal muscles (e.g., gluteus maximus).

[0267] In some respects, different skeletal muscle groups may include the quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscles), gluteus maximus, hamstrings, deltoids, trapezius, pectoralis major (e.g., pectoral muscles), and latissimus dorsi (lat). In some respects, skeletal muscle groups are selected from the quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscles), gluteus maximus, hamstrings, deltoids, trapezius, pectoralis major (e.g., pectoral muscles), and latissimus dorsi (lat), or any combination thereof. In some respects, skeletal muscle groups are selected from the quadriceps, biceps, gastrocnemius (e.g., calf muscles), gluteus maximus, and hamstrings, or any combination thereof. In some respects, skeletal muscle groups are selected from the quadriceps, biceps, and hamstrings, or any combination thereof.

[0268] In some respects, different skeletal muscle groups may include the quadriceps, biceps (e.g., biceps brachii or biceps femoris), triceps, gastrocnemius (e.g., calf muscles), gluteus maximus, hamstrings (e.g., semitendinosus, semimembranosus or biceps femoris), deltoid, trapezius, pectoral muscles (e.g., pectoralis major), thigh muscles (e.g., vastus intermedius, vastus lateralis or vastus medialis), hip muscles (gluteus medius), and latissimus dorsi.

[0269] In some respects, different skeletal muscle groups may include the iliopsoas, tensor fasciae latae, vastus lateralis, iliotibial band, pectineus, adductor longus, gracilis, rectus femoris, vastus medialis, gluteus maximus, adductor magnus, semitendinosus, gracilis, semimembranosus, sartorius, gluteus medius, biceps femoris, plantar muscles, or gastrocnemius. Figures 19A-19B Any muscles publicly disclosed.

[0270] In some respects, administration involves bilateral injection into one or more skeletal muscle groups (e.g., IM injection into the right and left quadriceps, right and left biceps, right and left gastrocnemius, right and left gluteus maximus, right and left triceps and / or right and left hamstrings).

[0271] In some respects, administration involves bilateral injection into one or more skeletal muscle groups (e.g., IM injection into the right and left quadriceps, right and left biceps, right and left gastrocnemius, right and left gluteus maximus and / or right and left hamstrings).

[0272] In some respects, bilateral injections are equally distributed among the various skeletal muscle groups (e.g., the right quadriceps and left quadriceps receive approximately the same volume and / or dose).

[0273] In some respects, different skeletal muscle groups may include the iliopsoas, tensor fasciae latae, vastus lateralis, iliotibial band, pectineus, adductor longus, gracilis, rectus femoris, vastus medialis, gluteus maximus, adductor magnus, semitendinosus, gracilis, semimembranosus, sartorius, gluteus medius, biceps femoris, plantar muscles, or gastrocnemius. Figures 19A-19B Any muscles publicly disclosed.

[0274] In some respects, the skeletal muscle groups are selected from the quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscles), gluteus maximus, hamstrings, deltoids, trapezius, pectoralis major (e.g., pectoral muscles), and latissimus dorsi (lat). In other respects, two or more distinct skeletal muscle groups are any combination of two or more of the following: quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscles), gluteus maximus, hamstrings, deltoids, trapezius, pectoralis major (e.g., pectoral muscles), or latissimus dorsi (lat).

[0275] In some respects, the skeletal muscle group is selected from the quadriceps, biceps, gastrocnemius (e.g., calf muscles), gluteus maximus, and hamstrings. In other respects, two or more different skeletal muscle groups comprise any combination of two or more of the following: quadriceps, biceps, gastrocnemius (e.g., calf muscles), gluteus maximus, or hamstrings.

[0276] In some aspects, administration involves a total of 20-150 injections of the therapy (e.g., combination gene therapy) targeting at least two different skeletal muscle groups. In other aspects, administration involves a total of 20-120 IM injections, 20-110 IM injections, 20-100 IM injections, 20-80 IM injections, 20-60 IM injections, 25-150 IM injections, 25-120 IM injections, 25-110 IM injections, or 25-100 IM injections.

[0277] In some respects, the administration includes 1-80 (e.g., 2-80, 4-80, 6-80, 8-80, 10-80, 20-80, 30-80, 40-80, 50-80, or 60-80) IM injections to the first skeletal muscle group and 1-80 (e.g., 2-80, 4-80, 6-80, 8-80, 10-80, 20-80, 30-80, 40-80, 50-80, or 60-80) IM injections to the second skeletal muscle group. In some aspects, the administration includes 1-50 (e.g., 2-50, 4-50, 6-50, 8-50, 10-50, 20-50, 30-50, or 40-50) IM injections to the first skeletal muscle group, 1-50 (e.g., 2-50, 4-50, 6-50, 8-50, 10-50, 20-50, 30-50, or 40-50) IM injections to the second skeletal muscle group, and 1-50 (e.g., 2-50, 4-50, 6-50, 8-50, 10-50, 20-50, 30-50, or 40-50) IM injections to the third skeletal muscle group.

[0278] In some respects, the administration includes 8-50 intramural injections to the quadriceps (e.g., 4-25 bilateral intramural injections to each quadriceps), 8-50 intramural injections to the biceps (e.g., 4-25 bilateral intramural injections to each biceps), and 8-50 intramural injections to the hamstrings (e.g., 4-25 bilateral intramural injections to each hamstring).

[0279] In some respects, the administration includes (i) 1-80 IM injections to a first skeletal muscle group and 1-80 IM injections to a second skeletal muscle group; (ii) 1-60 IM injections to a first skeletal muscle group, 1-60 IM injections to a second skeletal muscle group, and 1-60 IM injections to a third skeletal muscle group; (iii) 1-40 IM injections to a first skeletal muscle group, 1-40 IM injections to a second skeletal muscle group, 1-40 IM injections to a third skeletal muscle group, and 1-40 IM injections to a fourth skeletal muscle group; (iv) administration to a first skeletal muscle group... Administer 1-30 IM injections to the second skeletal muscle group, the third skeletal muscle group, the fourth skeletal muscle group, and the fifth skeletal muscle group; and (v) administer 1-25 IM injections to the first skeletal muscle group, the second skeletal muscle group, the third skeletal muscle group, the fourth skeletal muscle group, the fifth skeletal muscle group, and the sixth skeletal muscle group.

[0280] In some respects, the administration includes: (i) 2-80, 4-80, 6-80, 8-80, 10-80, 20-80, 30-80, 40-80, 50-80, or 60-80 IM injections to the first skeletal muscle group, and 2-80, 4-80, 6-80, 8-80, 10-80, 20-80, 30-80, 40-80, 50-80, or 60-80 IM injections to the second skeletal muscle group; (ii) 2-60, 4-60, 6-60, 8-60, 10-60, 20-60, or 60-80 IM injections to the first skeletal muscle group. (iii) 30-60, 40-60, or 50-60 IM injections; 2-60, 4-60, 6-60, 8-60, 10-60, 20-60, 30-60, 40-60, or 50-60 IM injections for the second skeletal muscle group; and 2-60, 4-60, 6-60, 8-60, 10-60, 20-60, 30-60, 40-60, or 50-60 IM injections for the third skeletal muscle group; (iii) 2-40, 4-40, 6-40, 8-40, 10-40, 20-40, or 30-60 IM injections for the first skeletal muscle group. -40 IM injections, with 2-40, 4-40, 6-40, 8-40, 10-40, 20-40, or 30-40 IM injections for the second skeletal muscle group, 2-40, 4-40, 6-40, 8-40, 10-40, 20-40, or 30-40 IM injections for the third skeletal muscle group, and 2-40, 4-40, 6-40, 8-40, 10-40, 20-40, or 30-40 IM injections for the fourth skeletal muscle group; (iv) 2-30, 4-30, 6-30, 8-30, 1 0-30 or 20-30 IM injections; 2-30, 4-30, 6-30, 8-30, 10-30 or 20-30 IM injections for the second skeletal muscle group; 2-30, 4-30, 6-30, 8-30, 10-30 or 20-30 IM injections for the third skeletal muscle group; 2-30, 4-30, 6-30, 8-30, 10-30 or 20-30 IM injections for the fourth skeletal muscle group; and 2-30, 4-30, 6-30, 8-30, 10-30 or 20-30 IM injections for the fifth skeletal muscle group.Or (v) administer 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 IM injections to the first skeletal muscle group, 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 IM injections to the second skeletal muscle group, and 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 IM injections to the third skeletal muscle group. The injection regimen is as follows: 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 intramural injections for the fourth skeletal muscle group; 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 intramural injections for the fifth skeletal muscle group; and 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 intramural injections for the sixth skeletal muscle group.

[0281] In some respects, the administration of IM injections comprises a total of 20-70 (e.g., 20-70, 25-70, 30-70, 35-70, 40-70, 45-70, 50-70, 50-55, 55-60, 60-65, 65-70, 50-70, 50-60, 60-70, 55-65, or 55-70) doses of therapy (e.g., combination gene therapy) to at least two different skeletal muscle groups. In some respects, the administration includes 10-35 (e.g., 10-15, 15-20, 20-25, 25-30, 10-20, 20-30, 15-25, or 30-35) IM injections to the first skeletal muscle group and 10-35 (e.g., 10-15, 15-20, 20-25, 25-30, 10-20, 20-30, 15-25, or 30-35) IM injections to the second skeletal muscle group. In some respects, the administration includes 10-35 (e.g., 10-15, 15-20, 20-25, 25-30, 10-20, 20-30, 15-25, or 30-35) IM injections to the first skeletal muscle group, 10-35 (e.g., 10-15, 15-20, 20-25, 25-30, 10-20, 20-30, 15-25, or 30-35) IM injections to the second skeletal muscle group, and 10-35 (e.g., 10-15, 15-20, 20-25, 25-30, 10-20, 20-30, 15-25, or 30-35) IM injections to the third skeletal muscle group.

[0282] In some respects, each IM injection is administered in the following volumes: about 100 μL to about 2 mL, 100 μL to about 1.9 mL, 100 μL to about 1.8 mL, 100 μL to about 1.7 mL, 100 μL to about 1.6 mL, 100 μL to about 1.5 mL, 100 μL to about 1.4 mL, about 100 μL to about 1.35 mL, about 100 μL to about 1.2 mL, about 100 μL to about 1.0 mL, about 100 μL to about 800 μL, about 200 μL to about 800 μL, about 200 μL to about 600 μL, about 400 μL to about 800 μL, about 500 μL to about 1.5 mL, about 600 μL to about 1.4 mL, about 700 μL to about 1.3 mL, about 800 μL to about 1.2 mL, or about 900 μL to about 1.1 mL.

[0283] In some respects, each IM injection is administered in a volume of about 100 μL to about 1.5 mL (e.g., about 100 μL to about 1.4 mL, about 100 μL to about 1.35 mL, about 100 μL to about 1.2 mL, about 100 μL to about 1.0 mL, about 100 μL to about 800 μL, about 200 μL to about 800 μL, about 200 μL to about 600 μL, or about 400 μL to about 800 μL).

[0284] In some respects, each IM injection is administered in a volume of approximately 500 µL to approximately 1 mL.

[0285] In some respects, the total volume of the administered therapy is about 25 mL, about 30 mL, about 35 mL, about 40 mL, about 45 mL, about 50 mL, about 55 mL, about 60 mL, about 65 mL, or about 70 mL. In other respects, the total volume of the administered therapy is about 20 mL to about 70 mL, about 25 mL to about 50 mL, about 25 mL to about 35 mL, about 35 mL to about 45 mL, about 30 mL to about 35 mL, or about 35 mL to about 40 mL.

[0286] In some respects, each IM injection contains approximately 2 × 10 13 Approximately 5×10 13 vg / mL.

[0287] In some respects, each IM injection contains approximately 8 × 10 10 Approximately 5×10 13vg / mL. In some respects, each IM injection contains approximately 8 × 10 vg / mL. 10 Up to 2×10 13 vg / mL. In some respects, each IM injection contains approximately 2 × 10 13 Approximately 4×10 13 vg / mL.

[0288] In some respects, each IM injection contains approximately 8 × 10 10 Approximately 8×10 12 vg / mL, approximately 8×10 10 Approximately 8×10 11 vg / mL, approximately 8×10 10 Approximately 2×10 12 vg / mL, approximately 8×10 10 Approximately 4×10 12 vg / mL, approximately 2×10 12 Approximately 8×10 12 vg / mL or approximately 8×10 11 Approximately 4×10 12 vg / mL.

[0289] In some respects, each IM injection contains approximately 1×10 11 To approximately 1×10 13 vg / mL, approximately 1×10 11 To approximately 1×10 12 vg / mL, approximately 1×10 11 To approximately 2.6 × 10 12 vg / mL, approximately 1×10 11 Approximately 5×10 12 vg / mL, approximately 2.6×10 12 To approximately 1×10 13 vg / mL or approximately 1×10 12 Approximately 5×10 12 vg / mL.

[0290] In some respects, each IM injection contains approximately 1.2 × 10⁻⁶. 11 From approximately 1.2 × 10 13 vg / mL, approximately 1.2×10 11 From approximately 1.2 × 10 12 vg / mL, approximately 1.2×10 11 Approximately 3.1 × 10 12 vg / mL, approximately 1.2×10 11 Approximately 6×10 12 vg / mL, approximately 3.1×10 12 From approximately 1.2 × 10 13vg / mL or approximately 1.2 × 10 12 Approximately 6×10 12 vg / mL.

[0291] In some respects, each IM injection contains approximately 1.4 × 10⁻⁶. 11 From approximately 1.4 × 10 13 vg / mL, approximately 1.4 × 10 11 From approximately 1.4 × 10 12 vg / mL, approximately 1.4 × 10 11 Approximately 3.6 × 10 12 vg / mL, approximately 1.4 × 10 11 Approximately 7×10 12 vg / mL, approximately 3.6×10 12 From approximately 1.4 × 10 13 vg / mL or approximately 1.4 × 10 12 Approximately 7×10 12 vg / mL.

[0292] In some respects, each IM injection contains approximately 1.6 × 10 11 From approximately 1.6 × 10 13 vg / mL, approximately 1.6 × 10 11 From approximately 1.6 × 10 12 vg / mL, approximately 1.6 × 10 11 Approximately 4.1 × 10 12 vg / mL, approximately 1.6 × 10 11 Approximately 8×10 12 vg / mL, approximately 4.1×10 12 From approximately 1.6 × 10 13 vg / mL or approximately 1.6 × 10 12 Approximately 8×10 12 vg / mL.

[0293] In some respects, each IM injection contains approximately 1.8 × 10 11 From approximately 1.8 × 10 13 vg / mL, approximately 1.8×10 11 From approximately 1.8 × 10 12 vg / mL, approximately 1.8×10 11 Approximately 4.6 × 10 12 vg / mL, approximately 1.8×10 11 Approximately 9×10 12 vg / mL, approximately 4.6×10 12 From approximately 1.8 × 10 13 vg / mL or approximately 1.8 × 10 12Approximately 9×10 12 vg / mL.

[0294] In some respects, each IM injection contains approximately 2 × 10 11 Approximately 2×10 13 vg / mL, approximately 2×10 11 Approximately 2×10 12 vg / mL, approximately 2×10 11 To approximately 5.1 × 10 12 vg / mL, approximately 2×10 11 To approximately 1×10 13 vg / mL, approximately 5.1×10 12 Approximately 2×10 13 vg / mL or approximately 2×10 12 To approximately 1×10 13 vg / mL.

[0295] In some respects, each IM injection contains approximately 8 × 10 10 Approximately 2×10 11 vg / mL, approximately 8×10 10 To approximately 1×10 11 vg / mL, approximately 8×10 10 From approximately 1.2 × 10 11 vg / mL, approximately 8×10 10 From approximately 1.4 × 10 11 vg / mL, approximately 8×10 10 From approximately 1.6 × 10 11 vg / mL, approximately 8×10 10 From approximately 1.8 × 10 11 vg / mL, approximately 1.4 × 10 11 Approximately 2×10 11 vg / mL, approximately 1×10 11 From approximately 1.8 × 10 11 vg / mL or approximately 1.2 × 10 11 From approximately 1.6 × 10 11 vg / mL.

[0296] In some respects, each IM injection contains approximately 8 × 10 11 Approximately 2×10 12 vg / mL, approximately 8×10 11 To approximately 1×10 12 vg / mL, approximately 8×10 11 From approximately 1.2 × 10 12 vg / mL, approximately 8×10 11 From approximately 1.4 × 10 12vg / mL, approximately 8×10 11 From approximately 1.6 × 10 12 vg / mL, approximately 8×10 11 From approximately 1.8 × 10 12 vg / mL, approximately 1.4 × 10 12 Approximately 2×10 12 vg / mL, approximately 1×10 12 From approximately 1.8 × 10 12 vg / mL or approximately 1.2 × 10 12 From approximately 1.6 × 10 12 vg / mL.

[0297] In some respects, each IM injection contains approximately 2 × 10 12 To approximately 5.1 × 10 12 vg / mL, approximately 2×10 12 To approximately 2.6 × 10 12 vg / mL, approximately 2×10 12 Approximately 3.1 × 10 12 vg / mL, approximately 2×10 12 Approximately 3.6 × 10 12 vg / mL, approximately 2×10 12 Approximately 4.1 × 10 12 vg / mL, approximately 2×10 12 Approximately 4.6 × 10 12 vg / mL, approximately 3.6×10 12 To approximately 5.1 × 10 12 vg / mL, approximately 2.6×10 12 Approximately 4.6 × 10 12 vg / mL or approximately 3.1×10 12 Approximately 4.1 × 10 12 vg / mL.

[0298] In some respects, each IM injection contains approximately 4 × 10 12 To approximately 1×10 13 vg / mL, approximately 4×10 12 Approximately 5×10 12 vg / mL, approximately 4×10 12 Approximately 6×10 12 vg / mL, approximately 4×10 12 Approximately 7×10 12 vg / mL, approximately 4×10 12 Approximately 8×10 12 vg / mL, approximately 4×10 12 Approximately 9×10 12vg / mL, approximately 7×10 12 To approximately 1×10 13 vg / mL, approximately 5×10 12 Approximately 9×10 12 vg / mL or approximately 6×10 12 Approximately 8×10 12 vg / mL.

[0299] In some respects, each IM injection contains approximately 8 × 10 12 Approximately 2×10 13 vg / mL, approximately 8×10 12 To approximately 1×10 13 vg / mL, approximately 8×10 12 From approximately 1.2 × 10 13 vg / mL, approximately 8×10 12 From approximately 1.4 × 10 13 vg / mL, approximately 8×10 12 From approximately 1.6 × 10 13 vg / mL, approximately 8×10 12 From approximately 1.8 × 10 13 vg / mL, approximately 1.4 × 10 13 Approximately 2×10 13 vg / mL, approximately 1×10 13 From approximately 1.8 × 10 13 vg / mL or approximately 1.2 × 10 13 From approximately 1.6 × 10 13 vg / mL.

[0300] In some respects, IM injections are administered with intervals of at least 1-7 cm. In some respects, IM injections are administered with intervals of at least 1-3 cm. In some respects, the intervals between IM injection sites on the subject are at least 5 cm. In some respects, the intervals between IM injection sites on the subject are at least 3 cm. In some respects, the intervals between IM injection sites on the subject are at least 2 cm. In some respects, the intervals between IM injection sites on the subject are at least 1 cm.

[0301] In some cases, IM injections are administered with intervals of at least 1-7 cm. In some cases, the intervals between IM injection sites on the subject are at least 7 cm. In some cases, the intervals between IM injection sites on the subject are at least 6 cm. In some cases, the intervals between IM injection sites on the subject are at least 5 cm. In some cases, the intervals between IM injection sites on the subject are at least 4 cm. In some cases, the intervals between IM injection sites on the subject are at least 3 cm. In some cases, the intervals between IM injection sites on the subject are at least 2 cm. In some cases, the intervals between IM injection sites on the subject are at least 1 cm.

[0302] In some cases, the interval between IM injection sites on the subject was approximately 7 cm. In some cases, the interval between IM injection sites on the subject was approximately 6 cm. In some cases, the interval between IM injection sites on the subject was approximately 5 cm. In some cases, the interval between IM injection sites on the subject was approximately 4 cm. In some cases, the interval between IM injection sites on the subject was approximately 3 cm. In some cases, the interval between IM injection sites on the subject was approximately 2 cm. In some cases, the interval between IM injection sites on the subject was approximately 1 cm.

[0303] In some aspects, the application involves IM injections, said IM injections being at least 0.5 cm deep, at least 0.6 cm deep, at least 0.7 cm deep, at least 0.8 cm deep, at least 0.9 cm deep, at least 1.0 cm deep, at least 1.1 cm deep, at least 1.2 cm deep, at least 1.3 cm deep, at least 1.4 cm deep, at least 1.5 cm deep, or at least 2.0 cm deep. In some aspects, the depth of each IM injection is between 1 and 2 cm.

[0304] In some respects, regardless of the thickness of subcutaneous fat, each IM injection should be at least 0.5 cm below the surface of the injected muscle.

[0305] In some aspects, the application includes an IM injection, said IM injection being at least 0.5 cm deep, at least 0.6 cm deep, at least 0.7 cm deep, at least 0.8 cm deep, at least 0.9 cm deep, at least 1.0 cm deep, at least 1.1 cm deep, at least 1.2 cm deep, at least 1.3 cm deep, at least 1.4 cm deep, at least 1.5 cm deep, at least 2.0 cm deep, at least 2.5 cm deep, at least 3.0 cm deep, at least 3.5 cm deep, at least 4.0 cm deep, at least 4.5 cm deep, or at least 5.0 cm deep.

[0306] In some aspects, the application includes IM injection, the IM injection being about 0.5 cm deep, about 0.6 cm deep, about 0.7 cm deep, about 0.8 cm deep, about 0.9 cm deep, about 1.0 cm deep, about 1.1 cm deep, about 1.2 cm deep, about 1.3 cm deep, about 1.4 cm deep, about 1.5 cm deep, about 2.0 cm deep, about 2.5 cm deep, about 3.0 cm deep, about 3.5 cm deep, about 4.0 cm deep, about 4.5 cm deep, or about 5.0 cm deep.

[0307] In some respects, the depth of each IM injection is between approximately 3.5 and approximately 5 cm.

[0308] In some respects, combination therapy (e.g., a single formulation or separate formulations) is administered intramuscularly (IM) to at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten different skeletal muscle groups. In some respects, combination therapy intramuscularly (IM) is administered to two, three, four, five, six, seven, eight, nine, or ten different skeletal muscle groups. In some respects, the skeletal muscle groups are selected from the quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscles), gluteus maximus, hamstrings, deltoids, trapezius, pectoralis major (e.g., pectoralis major), and latissimus dorsi. In some respects, the skeletal muscle groups are selected from the quadriceps, biceps, gastrocnemius (e.g., calf muscles), gluteus maximus, and hamstrings. In some respects, the skeletal muscle groups are selected from the quadriceps, biceps, and hamstrings. In some respects, the skeletal muscle group is selected from the quadriceps, biceps, triceps, gastrocnemius, gluteus maximus, and hamstrings, and optionally two or more of these different skeletal muscle groups include any combination thereof.

[0309] In some respects, the skeletal muscle groups are selected from the quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscles), gluteus maximus, hamstrings, deltoids, trapezius, pectoralis major (e.g., pectoral muscles), and latissimus dorsi (lat). In other respects, two or more distinct skeletal muscle groups are any combination of two or more of the following: quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscles), gluteus maximus, hamstrings, deltoids, trapezius, pectoralis major (e.g., pectoral muscles), or latissimus dorsi (lat).

[0310] In some respects, the skeletal muscle group is selected from the quadriceps, biceps, gastrocnemius (e.g., calf muscles), gluteus maximus, and hamstrings. In other respects, two or more distinct skeletal muscle groups are any combination of two or more of the following: quadriceps, biceps, gastrocnemius (e.g., calf muscles), gluteus maximus, or hamstrings.

[0311] In some respects, administration involves bilateral injection into one or more skeletal muscle groups (e.g., IM injection into the right and left quadriceps, right and left biceps, right and left gastrocnemius, right and left gluteus maximus and / or right and left hamstrings).

[0312] In some aspects, this disclosure relates to a combination therapy formulated for IM administration, comprising: a) an insulin expression cassette containing a first promoter operatively linked to a multinucleotide encoding a human insulin (hIns) protein, wherein the insulin expression cassette is side-mounted with an inverted terminal repeat (ITR); and b) a glucokinase expression cassette containing a second promoter operatively linked to a multinucleotide encoding a human glucokinase (hGck) protein, wherein the glucokinase expression cassette is side-mounted with an ITR, wherein the combination therapy is administered to at least two different skeletal muscle groups.

[0313] In some aspects, a first AAV vector genome containing an insulin expression cassette and a second AAV vector genome containing a glucokinase expression cassette are administered. In other aspects, an AAV vector genome containing both an insulin expression cassette and a glucokinase expression cassette is administered.

[0314] In some aspects, this disclosure relates to a combination therapy formulated for IM administration, comprising: a) a first AAV vector genome comprising an insulin expression cassette, the insulin expression cassette comprising a first promoter operatively linked to a multinucleotide encoding a human insulin (hIns) protein, wherein the insulin expression cassette is side-mounted with an inverted terminal repeat (ITR); and b) a second AAV vector genome comprising a glucokinase expression cassette, the glucokinase expression cassette comprising a second promoter operatively linked to a multinucleotide encoding a human glucokinase (hGck) protein, wherein the glucokinase expression cassette is side-mounted with an ITR, wherein the vector ratio of the first AAV vector genome and the second AAV vector genome is selected from the group consisting of: 1:0.25–0.75, 1:0.75–1.25, 1:1.75–2.25, and 1:3.75–1:4.25 (e.g., about 1:0.5, about 1:1, about 1:2, or about 1:4).

[0315] In some aspects, the carrier ratio is approximately 1:0.25-0.75, 1:0.25-0.7, 1:0.25-0.65, 1:0.25-0.60, 1:0.25-0.55, or 1:0.25-0.5. In other aspects, the carrier ratio is approximately 1:0.25-0.75, 1:0.3-0.75, 1:0.35-0.75, 1:0.40-0.75, 1:0.45-0.75, 1:0.5-0.75, 1:0.3-0.7, 1:0.3-0.65, 1:0.3-0.6, 1:0.3-0.55, 1:0.3-0.5, 1:0.35-0.7, or 1:0.3. 5:0.65, 1:0.35-0.6, 1:0.35-0.55, 1:0.35-0.5, 1:0.4-0.7, 1:0.4-0.65, 1:0.4-0.6, 1:0.4-0.55, 1:0.4-0.5, 1:0.45-0.7, 1:0.45-0.65, 1:0.45-0.6, 1:0.45-0.55, or 1:0.45-0.5. In some aspects, the carrier ratio is about 1:0.25-0.75, 1:0.3-0.70, 1:0.35-0.65, 1:0.35-0.75, 1:0.4-0.60, or 1:0.45-0.55.

[0316] In some aspects, the carrier ratio is selected from the following groups: 1:0.75-1.25, 1:0.8-1.25, 1:0.85-1.25, 1:0.9-1.25, 1:0.95-1.25, and 1:1.0-1.25. In some aspects, the carrier ratio is selected from the following groups: 1:0.75-1.25, 1:0.75-1.20, 1:0.75-1.15, 1:0.75-1.10, 1:0.75-1.05, and 1:0.75-1.00. In some aspects, the carrier ratio is selected from the following groups: 1:0.75-1:25, 1:0.80-1.20, 1:0.85-1.15, 1:0.9-1.1, and 1:0.95-1.05.

[0317] In some respects, the carrier ratio is approximately 1:0.25-0.75.

[0318] In some respects, the carrier ratio is approximately 1:0.4-0.6.

[0319] In some respects, the carrier ratio is approximately 1:0.5.

[0320] In some aspects, the carrier ratio is approximately 1:0.9-1.1.

[0321] In some respects, the carrier ratio is approximately 1:1.

[0322] In some respects, the carrier ratio is approximately 1:2.

[0323] In some respects, the carrier ratio is approximately 1:3.

[0324] In some respects, the carrier ratio is approximately 1:4.

[0325] In some respects, the carrier ratio is approximately 1:5.

[0326] In some respects, the carrier ratio is approximately 1:10.

[0327] In some respects, the carrier ratio is approximately 1:20.

[0328] In some respects, the carrier ratio is between approximately 1:3 and approximately 1:5.

[0329] In some respects, the carrier ratio is between: about 1:1 to about 1:15, about 1:1 to about 1:10, about 1:1 to about 1:5, about 1:1 to about 1:4, about 1:1 to about 1:3, about 1:3 to about 1:5, about 1:3 to about 1:10, about 1:3 to about 1:15, about 1:5 to about 1:10, about 1:5 to about 1:15, or about 1:10 to about 1:15.

[0330] In some aspects, the first AAV vector genome containing an insulin expression cassette has the following dosages: producing approximately 1 µU / mL to approximately 3 µU / mL, approximately 1 µU / mL to approximately 5 µU / mL, approximately 1 µU / mL to approximately 10 µU / mL, approximately 3 µU / mL to approximately 10 µU / mL, approximately 4 µU / mL to approximately 10 µU / mL, approximately 5 µU / mL to approximately 10 µU / mL, approximately 6 µU / mL to approximately 10 µU / mL, approximately 7 µU / mL to approximately 10 µU / mL, approximately 8 µU / mL to approximately 10 µU / mL, approximately 9 µU / mL to approximately 10 µU / mL, approximately 3 µU / mL to approximately 9 µU / mL, approximately 3 µU / mL to approximately 8 µU / mL, approximately 3 µU / mL to approximately 7 µU / mL, approximately 3 µU / mL to approximately 6 µU / mL, approximately 3 µU / mL to approximately 5 µU / mL, approximately 5 µU / mL to approximately 9 µU / mL, approximately 5 µU / mL to approximately 9 µU / mL, approximately 5 µU / mL to approximately 9 µU / mL, approximately 5 µU / mL to approximately 9 µU / mL, approximately 5 µU / mL to approximately 10 ... About 8 µU / mL to about 8 µU / mL, about 5 µU / mL to about 7 µU / mL, about 5 µU / mL to about 6 µU / mL, about 6 µU / mL to about 9 µU / mL, about 6 µU / mL to about 8 µU / mL or about 6 µU / mL to about 7 µU / mL, about 1 µU / mL to about 40 µU / mL, about 3 µU / mL to about 40 µU / mL, about 5 µU / mL to about 40 µU / mL, about 10 µU / mL to about 40 µU / mL, about 15 µU / mL to about 40 µU / mL, about 20 µU / mL to about 40 µU / mL, about 25 µU / mL to about 40 µU / mL, about 30 µU / mL to about 40 µU / mL, about 35 µU / mL to about 40 µU / mL, about 20 µU / mL to about 30 µU / mL, about 10 µU / mL to about 30 µU / mL or about 10 µU / mL Insulin levels between µU / mL and approximately 20 µU / mL.

[0331] In some aspects, the combination therapy comprises a first AAV vector genome containing an insulin expression cassette and a second AAV vector genome containing a glucokinase expression cassette in a vector ratio of about 1:0.4-0.6 (e.g., about 1:0.5), optionally wherein the first and second vector genomes are combined in a single intramuscular formulation, said single intramuscular formulation being applied to at least two different skeletal muscle groups. In some aspects, the skeletal muscle groups include the quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscles), gluteus maximus, hamstrings, deltoids, trapezius, pectoralis major (e.g., pectoralis major), and latissimus dorsi. In some aspects, the at least two different skeletal muscle groups are selected from two or more of the following: quadriceps, biceps, hamstrings, or any combination thereof. In some aspects, the at least two different skeletal muscle groups include the quadriceps, biceps, hamstrings, or any combination thereof.

[0332] In some aspects, the combination therapy comprises a first AAV vector genome containing an insulin expression cassette and a second AAV vector genome containing a glucokinase expression cassette in a vector ratio of about 1:1-15 (e.g., about 1:5), optionally wherein the first and second vector genomes are combined in a single intramuscular formulation, said intramuscular formulation being applied to at least two different skeletal muscle groups. In some aspects, the skeletal muscle groups include the quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscles), gluteus maximus, hamstrings, deltoids, trapezius, pectoralis major (e.g., pectoralis major), and latissimus dorsi.

[0333] In some respects, the skeletal muscle groups are selected from the quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscles), gluteus maximus, hamstrings, deltoids, trapezius, pectoralis major (e.g., pectoral muscles), and latissimus dorsi (lat). In other respects, two or more distinct skeletal muscle groups are any combination of two or more of the following: quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscles), gluteus maximus, hamstrings, deltoids, trapezius, pectoralis major (e.g., pectoral muscles), or latissimus dorsi (lat).

[0334] In some respects, the skeletal muscle group is selected from the quadriceps, biceps, gastrocnemius (e.g., calf muscles), gluteus maximus, and hamstrings. In other respects, two or more distinct skeletal muscle groups are any combination of two or more of the following: quadriceps, biceps, gastrocnemius (e.g., calf muscles), gluteus maximus, or hamstrings.

[0335] In some respects, administration involves bilateral injection into one or more skeletal muscle groups (e.g., IM injection into the right and left quadriceps, right and left biceps, right and left gastrocnemius, right and left gluteus maximus and / or right and left hamstrings).

[0336] In some aspects, the combination therapy comprises a first AAV vector genome containing an insulin expression cassette and a second AAV vector genome containing a glucokinase expression cassette in a vector ratio of about 1:0.4-0.6 (e.g., about 1:0.5), optionally wherein the first and second vector genomes are combined in a single intramuscular formulation, said intramuscular formulation being administered to at least three different skeletal muscle groups. In some aspects, the different skeletal muscle groups include three or more of the following: quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscle), gluteus maximus, hamstrings, deltoid, trapezius, pectoralis major (e.g., pectoralis major), or latissimus dorsi.

[0337] In some aspects, the combination therapy comprises a first AAV vector genome containing an insulin expression cassette and a second AAV vector genome containing a glucokinase expression cassette in a vector ratio of about 1:1-15 (e.g., about 1:5), optionally wherein the first and second vector genomes are combined in a single intramuscular modulator, said intramuscular modulator being applied to at least three different skeletal muscle groups.

[0338] In some aspects, the combination therapy comprises: (i) a first recombinant AAV (rAAV) particle comprising a first AAV vector genome containing an insulin expression cassette, the insulin expression cassette containing a promoter operatively linked to a multinucleotide encoding a human insulin (hIns) protein, wherein the insulin expression cassette is side-mounted with an inverted terminal repeat (ITR); and (ii) a second recombinant AAV (rAAV) particle comprising a second AAV vector genome containing a glucokinase expression cassette, the glucokinase expression cassette containing a promoter operatively linked to a multinucleotide encoding a human glucokinase (hGck) protein, wherein the glucokinase expression cassette is side-mounted with an inverted terminal repeat (ITR).

[0339] In some respects, at least three distinct skeletal muscle groups are selected from the quadriceps, biceps, and hamstrings, or any combination thereof. In some respects, at least three distinct skeletal muscle groups include the quadriceps, biceps, and / or hamstrings.

[0340] In some respects, the administration involves bilateral injection into one or more of at least three different skeletal muscle groups. In some respects, the administration involves bilateral injection into one or more skeletal muscle groups (e.g., IM injection into the right and left quadriceps, right and left biceps, right and left gastrocnemius, right and left gluteus maximus and / or right and left hamstrings).

[0341] In some aspects, the combination therapy comprises a first AAV vector genome containing an insulin expression cassette and a second AAV vector genome containing a glucokinase expression cassette in a vector ratio of about 1:0.9-1.1 (e.g., about 1:1.0), optionally wherein the first and second vector genomes are combined in a single intramuscular modulator, said intramuscular modulator being administered to at least two different skeletal muscle groups. In some aspects, the at least two different skeletal muscle groups comprise at least two of the following: quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscle), gluteus maximus, hamstrings, deltoid, trapezius, pectoralis major (e.g., pectoralis major), or latissimus dorsi. In some aspects, the at least two different skeletal muscle groups comprise at least two of the following: quadriceps, biceps, and hamstrings. In some aspects, the combination therapy comprises a first AAV vector genome containing an insulin expression cassette and a second AAV vector genome containing a glucokinase expression cassette in a vector ratio of about 1:1-15 (e.g., about 1:5), optionally wherein the first and second vector genomes are combined in a single intramuscular modulator, said intramuscular modulator being applied to at least two different skeletal muscle groups.

[0342] In some respects, administration involves bilateral injection into one or more skeletal muscle groups (e.g., IM injection into the right and left quadriceps, right and left biceps, right and left gastrocnemius, right and left gluteus maximus and / or right and left hamstrings).

[0343] In some aspects, the combination therapy comprises a first AAV vector genome containing an insulin expression cassette and a second AAV vector genome containing a glucokinase expression cassette in a vector ratio of about 1:0.9-1.1 (e.g., about 1:1), optionally wherein the first and second vector genomes are combined in a single intramuscular modulator administered to at least three distinct skeletal muscle groups including: quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscles), gluteus maximus, hamstrings, deltoids, trapezius, pectoralis major (e.g., pectoralis major), and latissimus dorsi. In some aspects, at least two distinct skeletal muscle groups include at least two of the following: quadriceps, biceps, and / or hamstrings.

[0344] In some aspects, the combination therapy comprises a first AAV vector genome containing an insulin expression cassette and a second AAV vector genome containing a glucokinase expression cassette in a vector ratio of about 1:1 to 15 (e.g., about 1:5), optionally wherein the first and second vector genomes are combined in a single intramuscular modulator, said intramuscular modulator being applied to at least three distinct skeletal muscle groups including: quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscle), gluteus maximus, hamstrings, deltoid, trapezius, pectoralis major (e.g., pectoralis major), and latissimus dorsi.

[0345] In some respects, the administration involves bilateral injection into one or more of at least three different skeletal muscle groups. In some respects, the administration involves bilateral injection into one or more skeletal muscle groups (e.g., IM injection into the right and left quadriceps, right and left biceps, right and left gastrocnemius, right and left gluteus maximus and / or right and left hamstrings).

[0346] In some respects, each IM injection is administered in a volume of about 100 μL to about 1.5 mL (e.g., about 100 μL to about 1.4 mL, about 100 μL to about 1.35 mL, about 100 μL to about 1.2 mL, about 100 μL to about 1.0 mL, about 100 μL to about 800 μL, about 200 μL to about 800 μL, about 200 μL to about 600 μL, or about 400 μL to about 800 μL).

[0347] In some respects, each IM injection contains approximately 2 × 10 13 Up to 5×10 13 vg / mL.

[0348] In some respects, each IM injection contains approximately 7 × 10 10 Up to 7×10 12 The first AAV vector genome (e.g., hIns) is administered at a dose of vg / kg. In some respects, each IM injection contains approximately 7 × 10⁻⁶ units. 10 Up to 7×10 11 The first AAV vector genome (e.g., hIns) is administered at a dose of vg / kg. In some respects, each IM injection contains approximately 7 × 10⁻⁶ units. 10 Up to 1.8×10 12 The first AAV vector genome (e.g., hIns) is administered at a dose of vg / kg. In some respects, each IM injection contains approximately 7 × 10⁻⁶ units. 10 Up to 3.5×10 12 The first AAV vector genome (e.g., hIns) is vg / kg. In some respects, each IM injection contains approximately 1.8 × 10⁻⁶ units. 12 Up to 7×10 12 The first AAV vector genome (e.g., hIns) is vg / kg. In some respects, each IM injection contains approximately 1.8 × 10⁻⁶ units. 12 Up to 3.5×10 12 The first AAV vector genome (e.g., hIns) is administered at a dose of vg / kg. In some respects, each IM injection contains approximately 3.5 × 10⁻⁶ units. 12 Up to 7×10 12 The first AAV vector genome (e.g., hIns) is vg / kg.

[0349] In some respects, each IM injection contains a dose of approximately 3.2 × 10⁻⁶. 12 Up to 3.5×10 12 A second AAV vector genome (e.g., hGck) is administered at a dose of vg / kg. In some respects, each IM injection contains approximately 2 × 10⁻⁶ units. 12 Up to 5×10 12 A second AAV vector genome (e.g., hGck) is administered at a dose of vg / kg. In some respects, each IM injection contains approximately 2 × 10⁻⁶ units. 12 Up to 3.5×10 12 The second AAV vector genome (e.g., hGck) is administered at a dose of vg / kg. In some respects, each IM injection contains approximately 3.5 × 10⁻⁶ units. 12 Up to 5×10 12 The second AAV vector genome (e.g., hGck) is vg / kg.

[0350] In some respects, IM injections are administered with intervals of at least 1-3 cm. In some respects, the intervals between IM injection sites on the subject are at least 3 cm. In some respects, the intervals between IM injection sites on the subject are at least 2 cm. In some respects, the intervals between IM injection sites on the subject are at least 1 cm.

[0351] In some cases, IM injections are administered with intervals of at least 1-7 cm. In some cases, the intervals between IM injection sites on the subject are at least 5 cm. In some cases, the intervals between IM injection sites on the subject are at least 4 cm. In some cases, the intervals between IM injection sites on the subject are at least 3 cm. In some cases, the intervals between IM injection sites on the subject are at least 2 cm. In some cases, the intervals between IM injection sites on the subject are at least 1 cm.

[0352] In some cases, the interval between IM injection sites on the subject was approximately 7 cm. In some cases, the interval between IM injection sites on the subject was approximately 6 cm. In some cases, the interval between IM injection sites on the subject was approximately 5 cm. In some cases, the interval between IM injection sites on the subject was approximately 4 cm. In some cases, the interval between IM injection sites on the subject was approximately 3 cm. In some cases, the interval between IM injection sites on the subject was approximately 2 cm. In some cases, the interval between IM injection sites on the subject was approximately 1 cm.

[0353] In some aspects, the application involves IM injections, said IM injections being at least 0.5 cm deep, at least 0.6 cm deep, at least 0.7 cm deep, at least 0.8 cm deep, at least 0.9 cm deep, at least 1.0 cm deep, at least 1.1 cm deep, at least 1.2 cm deep, at least 1.3 cm deep, at least 1.4 cm deep, at least 1.5 cm deep, or at least 2.0 cm deep. In some aspects, the depth of each IM injection is between 1 and 2 cm.

[0354] In some respects, the first AAV vector genome, the second AAV vector genome, or both the first AAV vector genome and the second AAV vector genome are approximately 5 × 10 11 To approximately 1×10 14 The vector genome was administered intramuscularly at a dose of vg / kg. In some respects, the first and second AAV vector genomes were each administered at approximately 5 × 10⁻⁶. 11 Approximately 5×10 13 Dosage administration between vector genome (vg) / kg. In some respects, the first AAV vector genome, the second AAV vector genome, or both the first AAV vector genome and the second AAV vector genome are administered at a dose of approximately 1 × 10⁻⁶. 12 To approximately 1×10 13 Administer intramuscularly at a dose of vg / kg.

[0355] In some respects, the first AAV vector genome, the second AAV vector genome, or both the first AAV vector genome and the second AAV vector genome are administered intramuscularly at the following dose: approximately 1 × 10⁻⁶. 12 vg / kg to approximately 1×10 14 vg / kg, approximately 2×10 12 vg / kg to approximately 1×10 14 vg / kg, approximately 3×10 12 vg / kg to approximately 1×10 14 vg / kg, approximately 4×10 12 vg / kg to approximately 1×10 14 vg / kg, approximately 5×10 12 vg / kg to approximately 1×10 14 vg / kg, approximately 6×10 12 vg / kg to approximately 1×10 14 vg / kg, approximately 7×10 12 vg / kg to approximately 1×10 14 vg / kg, approximately 8×10 12 vg / kg to approximately 1×10 14 vg / kg, or approximately 9×10 12vg / kg to approximately 1×10 14 vg / kg.

[0356] In some respects, the first AAV vector genome, the second AAV vector genome, or both the first AAV vector genome and the second AAV vector genome are administered intramuscularly at the following dosage: approximately 7 × 10⁻⁶. 10 vg / kg to approximately 1×10 14 vg / kg, approximately 8×10 10 vg / kg to approximately 1×10 14 vg / kg, approximately 9×10 10 vg / kg to approximately 1×10 14 vg / kg, approximately 1×10 11 vg / kg to approximately 1×10 14 vg / kg, approximately 2×10 11 vg / kg to approximately 1×10 14 vg / kg, approximately 3×10 11 vg / kg to approximately 1×10 14 vg / kg, approximately 4×10 11 vg / kg to approximately 1×10 14 vg / kg, approximately 7×10 10 vg / kg to approximately 1×10 13 vg / kg, approximately 7×10 10 vg / kg to approximately 1×10 12 vg / kg, approximately 7×10 10 vg / kg to approximately 1×10 11 vg / kg, approximately 7×10 10 vg / kg to approximately 7×10 12 vg / kg, approximately 7×10 10 vg / kg to approximately 7×10 11 vg / kg, approximately 7×10 10 vg / kg to approximately 1.8×10 12 vg / kg, approximately 7×10 10 vg / kg to approximately 3.5×10 12 vg / kg, approximately 1.8×10 12 vg / kg to approximately 7×10 12 vg / kg, approximately 1.8×10 12 vg / kg to approximately 3.5×10 12 vg / kg, approximately 3.5×10 12 vg / kg to approximately 7×10 12 vg / kg, approximately 2×10 12 vg / kg to approximately 5×1012 vg / kg, approximately 2×10 12 vg / kg to approximately 3.5×10 12 vg / kg, approximately 3.5×10 12 vg / kg to approximately 5×10 12 vg / kg, approximately 5×10 11 vg / kg to approximately 1×10 14 vg / kg, approximately 6×10 11 vg / kg to approximately 1×10 14 vg / kg, approximately 7×10 11 vg / kg to approximately 1×10 14 vg / kg, approximately 8×10 11 vg / kg to approximately 1×10 14 vg / kg, or approximately 9×10 11 vg / kg to approximately 1×10 14 vg / kg.

[0357] In some respects, the first AAV vector genome, the second AAV vector genome, or both the first AAV vector genome and the second AAV vector genome are administered intramuscularly at the following dosage: approximately 2 × 10⁻⁶. 12 vg / kg to approximately 1×10 14 vg / kg, approximately 3×10 12 vg / kg to approximately 1×10 14 vg / kg, approximately 4×10 12 vg / kg to approximately 1×10 14 vg / kg, approximately 5×10 12 vg / kg to approximately 1×10 14 vg / kg, approximately 6×10 12 vg / kg to approximately 1×10 14 vg / kg, approximately 7×10 12 vg / kg to approximately 1×10 14 vg / kg, or approximately 8×10 12 vg / kg to approximately 1×10 14 vg / kg.

[0358] In some respects, the first AAV vector genome, the second AAV vector genome, or both the first AAV vector genome and the second AAV vector genome are administered intramuscularly at the following dosage: approximately 5 × 10⁻⁶. 11 vg / kg to approximately 1×10 14 vg / kg, approximately 6×10 11 vg / kg to approximately 1×10 14 vg / kg, approximately 7×1011 vg / kg to approximately 1×10 14 vg / kg, approximately 8×10 11 vg / kg to approximately 1×10 14 vg / kg, or approximately 9×10 11 vg / kg to approximately 1×10 14 vg / kg.

[0359] In some respects, the first AAV vector genome, the second AAV vector genome, or both the first AAV vector genome and the second AAV vector genome are administered intramuscularly at the following dose: approximately 1 × 10⁻⁶. 13 vg / kg to approximately 1×10 14 vg / kg, approximately 2×10 13 vg / kg to approximately 1×10 14 vg / kg, approximately 3×10 13 vg / kg to approximately 1×10 14 vg / kg, approximately 4×10 13 vg / kg to approximately 1×10 14 vg / kg, approximately 5×10 13 vg / kg to approximately 1×10 14 vg / kg, approximately 6×10 13 vg / kg to approximately 1×10 14 vg / kg, approximately 7×10 13 vg / kg to approximately 1×10 14 vg / kg, approximately 8×10 13 vg / kg to approximately 1×10 14 vg / kg, or approximately 8×10 13 vg / kg to approximately 1×10 14 vg / kg.

[0360] In some respects, the first AAV vector genome, the second AAV vector genome, or both the first AAV vector genome and the second AAV vector genome are administered intramuscularly at the following dosage: approximately 2 × 10⁻⁶. 13 vg / kg to approximately 1×10 14 vg / kg, approximately 3×10 13 vg / kg to approximately 1×10 14 vg / kg, approximately 4×10 13 vg / kg to approximately 1×10 14 vg / kg, approximately 5×10 13 vg / kg to approximately 1×10 14 vg / kg, approximately 6×10 13 vg / kg to approximately 1×1014 vg / kg, approximately 7×10 13 vg / kg to approximately 1×10 14 vg / kg, or approximately 8×10 13 vg / kg to approximately 1×10 14 vg / kg.

[0361] In some respects, the first AAV vector genome has a density of approximately 7 × 10⁻⁶. 10 The second AAV vector genome was administered at a dose of approximately 3.5 × 10⁻⁶ vg / kg. 12 The dose was administered at a rate of vg / kg. In some respects, the first AAV vector genome was delivered at approximately 8 × 10⁻⁶. 10 The second AAV vector genome was administered at a dose of approximately 3.5 × 10⁻⁶ vg / kg. 12 The dose was administered at a rate of vg / kg. In some respects, the first AAV vector genome was delivered at approximately 9 × 10⁻⁶. 10 The second AAV vector genome was administered at a dose of approximately 3.5 × 10⁻⁶ vg / kg. 12 The dose was administered at a rate of vg / kg. In some respects, the first AAV vector genome was administered at approximately 1 × 10⁻⁶. 10 The second AAV vector genome was administered at a dose of approximately 3.5 × 10⁻⁶ vg / kg. 12 The dose was administered at a rate of vg / kg. In some respects, the first AAV vector genome was administered at approximately 2 × 10⁻⁶. 11 The second AAV vector genome was administered at a dose of approximately 3.5 × 10⁻⁶ vg / kg. 12 The dose was administered at a rate of vg / kg. In some respects, the first AAV vector genome was delivered at approximately 3 × 10⁻⁶. 11 The second AAV vector genome was administered at a dose of approximately 3.5 × 10⁻⁶ vg / kg. 12 The dose was administered at a rate of vg / kg. In some respects, the first AAV vector genome was delivered at approximately 4 × 10⁻⁶. 11 The second AAV vector genome was administered at a dose of approximately 3.5 × 10⁻⁶ vg / kg. 12 The dose was administered at a rate of vg / kg. In some respects, the first AAV vector genome was delivered at approximately 5 × 10⁻⁶. 11 The second AAV vector genome was administered at a dose of approximately 3.5 × 10⁻⁶ vg / kg. 12 The dose was administered at a rate of vg / kg. In some respects, the first AAV vector genome was delivered at approximately 6 × 10⁻⁶. 11 The second AAV vector genome was administered at a dose of approximately 3.5 × 10⁻⁶ vg / kg. 12 The dose was administered at a rate of vg / kg. In some respects, the first AAV vector genome was delivered at approximately 7 × 10⁻⁶. 11The second AAV vector genome was administered at a dose of approximately 3.5 × 10⁻⁶ vg / kg. 12 The dose was administered at a rate of vg / kg. In some respects, the first AAV vector genome was delivered at approximately 8 × 10⁻⁶. 11 The second AAV vector genome was administered at a dose of approximately 3.5 × 10⁻⁶ vg / kg. 12 The dose was administered at a rate of vg / kg. In some respects, the first AAV vector genome was delivered at approximately 9 × 10⁻⁶. 11 The second AAV vector genome was administered at a dose of approximately 3.5 × 10⁻⁶ vg / kg. 12 The dose was administered at a rate of vg / kg. In some respects, the first AAV vector genome was administered at approximately 1 × 10⁻⁶. 12 The second AAV vector genome was administered at a dose of approximately 3.5 × 10⁻⁶ vg / kg. 12 The dose was administered at a rate of vg / kg. In some respects, the first AAV vector genome was delivered at approximately 1.8 × 10⁻⁶. 12 The second AAV vector genome was administered at a dose of approximately 3.5 × 10⁻⁶ vg / kg. 12 The dose was administered at a rate of vg / kg. In some respects, the first AAV vector genome was administered at approximately 2 × 10⁻⁶. 12 The second AAV vector genome was administered at a dose of approximately 3.5 × 10⁻⁶ vg / kg. 12 Administer at a dose of vg / kg.

[0362] In some respects, the first AAV vector genome is approximately 3 × 10⁻⁶. 13 The second AAV vector genome was administered at a dose of approximately 1.5 × 10⁻⁶ vg / kg. 13 The dose was administered at a rate of vg / kg. In some respects, the first AAV vector genome was delivered at approximately 4 × 10⁻⁶. 13 The second AAV vector genome was administered at a dose of approximately 2 × 10 vg / kg. 13 The dose was administered at a rate of vg / kg. In some respects, the first AAV vector genome was delivered at approximately 5 × 10⁻⁶. 13 The second AAV vector genome was administered at a dose of approximately 2.5 × 10⁻⁶ vg / kg. 13 The dose was administered at a rate of vg / kg. In some respects, the first AAV vector genome was delivered at approximately 6 × 10⁻⁶. 13 The second AAV vector genome was administered at a dose of approximately 3 × 10 vg / kg. 13 The dose was administered at a rate of vg / kg. In some respects, the first AAV vector genome was delivered at approximately 7 × 10⁻⁶. 13 The second AAV vector genome was administered at a dose of approximately 3.5 × 10⁻⁶ vg / kg. 13 The dose was administered at a rate of vg / kg. In some respects, the first AAV vector genome was delivered at approximately 8 × 10⁻⁶. 13The second AAV vector genome was administered at a dose of approximately 4 × 10 vg / kg. 13 The dose was administered at a rate of vg / kg. In some respects, the first AAV vector genome was delivered at approximately 9 × 10⁻⁶. 13 The dose was administered at a dose of vg / kg, and the second AAV vector genome was administered at approximately 4.5 × 10⁻⁶. 13 The dose was administered at a rate of vg / kg. In some respects, the first AAV vector genome was administered at approximately 1 × 10⁻⁶. 14 The second AAV vector genome was administered at a dose of approximately 5 × 10 vg / kg. 13 Administer at a dose of vg / kg.

[0363] In some respects, the first AAV vector genome containing the insulin expression cassette is approximately 2 × 10⁻⁶. 12 To approximately 1×10 13 Dosage administration between vg / kg. In some respects, the first AAV vector genome containing the insulin expression cassette is administered at approximately 4 × 10⁻⁶. 12 To approximately 1×10 13 Administered at doses between vg / kg. In some respects, the first AAV vector genome containing the insulin expression cassette is approximately 4.5 × 10⁻⁶. 12 Approximately 5×10 12 Administered at a dose of vg / kg. In some respects, the first AAV vector genome containing the insulin expression cassette is delivered at approximately 5 × 10⁻⁶. 12 Approximately 9×10 12 Administer at a dose of vg / kg.

[0364] In some respects, the second AAV vector genome containing a glucokinase expression cassette is approximately 1 × 10 12 Approximately 5×10 12 Between vg / kg (e.g., 3.5×10) 12 Administered at a dose of (vg / kg). In some respects, the second AAV vector genome containing the glucokinase expression cassette is administered at approximately 2 × 10⁻⁶. 12 Approximately 5×10 12 Administered at doses between vg / kg. In some respects, the second AAV vector genome containing the glucokinase expression cassette is administered at approximately 2 × 10⁻⁶. 12 Approximately 2.8 × 10 12 Administered at a dose of vg / kg. In some respects, the second AAV vector genome containing the glucokinase expression cassette is delivered at approximately 4 × 10⁻⁶. 12 vg / kg to approximately 5×10 12 Dosage of vg / kg

[0365] In some respects, the combination therapy comprises a first AAV vector genome containing an insulin expression cassette and a second AAV vector genome containing a glucokinase expression cassette, with a vector ratio of approximately 1:0.4-0.6 (e.g., approximately 1:0.5), wherein the first AAV vector genome, the second AAV vector genome, or both the first AAV vector genome and the second AAV vector genome are in a ratio of approximately 1 × 10⁻⁶. 12 To approximately 1×10 14 The administration is performed intramuscularly (IM) at a dose of vg / kg, wherein the IM administration is directed at at least two distinct skeletal muscle groups. In some aspects, the distinct skeletal muscle groups include the quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscles), gluteus maximus, hamstrings, deltoids, trapezius, pectoralis major (e.g., pectoralis major), and / or latissimus dorsi. In some aspects, the at least two distinct skeletal muscle groups include at least two of the following: quadriceps, biceps, and / or hamstrings. In some aspects, the administration comprises bilateral injection into one or more of the at least two distinct skeletal muscle groups. In some aspects, the administration comprises bilateral injection into one or more skeletal muscle groups (e.g., IM injection into the right and left quadriceps, right and left biceps, right and left gastrocnemius, right and left gluteus maximus, and / or right and left hamstrings).

[0366] In some aspects, IM administration targets at least three distinct skeletal muscle groups selected from the following: quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscles), gluteus maximus, hamstrings, deltoids, trapezius, pectoralis major (e.g., pectoralis major), and / or latissimus dorsi. In some aspects, the at least three distinct skeletal muscle groups include the quadriceps, biceps, and / or hamstrings. In some aspects, the administration involves bilateral injection into one or more of the at least three distinct skeletal muscle groups. In some aspects, the administration involves bilateral injection into one or more skeletal muscle groups (e.g., IM injection into the right and left quadriceps, right and left biceps, right and left gastrocnemius, right and left gluteus maximus, and / or right and left hamstrings). In some aspects, IM administration involves multiple injections, with individual injection volumes between 100 µL and 1.5 mL. In some aspects, the individual injection volume is from about 100 μL to about 1.5 mL (e.g., from about 100 μL to about 1.4 mL, from about 100 μL to about 1.35 mL, from about 100 μL to about 1.2 mL, from about 100 μL to about 1.0 mL, from about 100 μL to about 800 μL, from about 200 μL to about 800 μL, from about 200 μL to about 600 μL, or from about 400 μL to about 800 μL). In some aspects, the individual injection volume is between 200 µL and 1.0 mL. In some aspects, the individual injection volume is about 500 µL. In some aspects, the individual injection volume is between 1 µL and 500 µL. In some aspects, the individual injection volume is between 10 µL and 300 µL. In some aspects, the individual injection volume is between 50 µL and 250 µL. In some aspects, the individual injection volume is about 100 µL.

[0367] In some respects, IM administration involves multiple injections, with individual injection doses ranging from 1×10⁻⁶. 11 vg / injection site and 5×10 13 Between vg and injection site. In some cases, individual injection doses contain approximately 2 × 10 13 Up to 5×10 13 vg / injection site. In some cases, the individual injection dose is approximately 1 × 10⁻⁶. 11 Vg / injection site approximately 1×10 13 vg / injection site. In some cases, the individual injection dose is approximately 1.25 × 10⁻⁶. 11 VG / injection site approximately 1.75 × 10 12 vg / injection site. In some cases, the individual injection dose is approximately 1.5 × 10⁻⁶. 11 VG / injection site approximately 1.5 × 10 12 vg / injection site. In some cases, a single injection site is approximately 1.75 × 10⁻⁶. 11VG / injection site approximately 1.25 × 10 12 vg / injection site. In some cases, the individual injection dose is approximately 1 × 10⁻⁶. 12 vg / injection site.

[0368] In some aspects, IM application involves multiple injections with a minimum distance between injection sites. In some aspects, the distance between injection sites is at least 0.5 cm, at least 0.6 cm, at least 0.7 cm, at least 0.8 cm, at least 0.9 cm, at least 1.0 cm, at least 1.1 cm, at least 1.2 cm, at least 1.3 cm, at least 1.4 cm, at least 1.5 cm, at least 2.0 cm, or at least 3.0 cm. In some aspects, the distance between injection sites is between 0.5 cm and 3 cm, between 0.75 cm and 2.5 cm, between 1 cm and 2 cm, between 0.75 cm and 1.25 cm, or approximately 1 cm.

[0369] In some respects, IM administration involves injections spaced at least 1-3 cm apart. In some respects, the intervals between IM injection sites on the subject are at least 3 cm apart. In some respects, the intervals between IM injection sites on the subject are at least 2 cm apart. In some respects, the intervals between IM injection sites on the subject are at least 1 cm apart.

[0370] In some aspects, IM application includes injections at a depth of at least 0.5 cm, at least 0.6 cm, at least 0.7 cm, at least 0.8 cm, at least 0.9 cm, at least 1.0 cm, at least 1.1 cm, at least 1.2 cm, at least 1.3 cm, at least 1.4 cm, at least 1.5 cm, or at least 2.0 cm. In some aspects, the depth of each IM injection is between 1 and 2 cm.

[0371] In some respects, combination therapy is accompanied by an immunosuppressive regimen. In some respects, combination therapy is accompanied by an immunosuppressive regimen containing immunosuppressants. In some respects, the immunosuppressive regimen involves administering one or more immunosuppressants before, concurrently with, and / or after the combination therapy. In some respects, the immunosuppressants include IL-6 antagonists, IL-1 antagonists, TNFα antagonists, mTOR pathway inhibitors, mycophenolate mofetil (MMF), tyk2 inhibitors, methotrexate, rapamycin, abatacept, anti-thymocyte globulin (ATG), B-cell depletion agents, T-cell co-stimulation antagonists, T-cell depletion agents, corticosteroids, and / or calcineurin inhibitors. In some respects, the immunosuppressants include IL-6 antagonists and / or calcineurin inhibitors.

[0372] In some respects, immunosuppressive regimens include administering an immunosuppressant (e.g., an IL-6 antagonist) before administering combination therapy. In some respects, immunosuppressive regimens include administering an immunosuppressant (e.g., a calcineurin inhibitor) after administering combination therapy. In some respects, immunosuppressive regimens include administering an immunosuppressant (e.g., an IL-6 antagonist) before administering combination therapy and administering an immunosuppressant (e.g., a calcineurin inhibitor) after administering combination therapy.

[0373] In some cases, an immunosuppressant (e.g., an IL-6 antagonist) is administered 1 to 14 days (e.g., 1 to 3 days) prior to the administration of combination therapy. In other cases, an immunosuppressant (e.g., an IL-6 antagonist) is administered 1 to 7 days (e.g., 1 to 3 days) prior to the administration of combination therapy. In still other cases, an immunosuppressant is administered 3 to 7 days prior to the administration of combination therapy.

[0374] In some cases, immunosuppressants (such as calcineurin inhibitors) are started 1, 2, 3, or 4 weeks (e.g., 3 weeks) after the administration of combination therapy.

[0375] In some cases, a first immunosuppressant (e.g., an IL-6 antagonist) is administered 1 to 7 days (e.g., 1 to 2 days) before the administration of combination therapy, and a second immunosuppressant (e.g., a calcineurin inhibitor) is initiated 1, 2, 3, or 4 weeks (e.g., 3 weeks) after the administration of combination therapy.

[0376] In some cases, a first immunosuppressant (e.g., an IL-6 antagonist) is administered 1 to 7 days (e.g., 1 to 3 days) prior to the administration of combination therapy, and a second immunosuppressant (e.g., a calcineurin inhibitor) is administered 1 to 14 days (e.g., 1 to 3 days) prior to the administration of combination therapy.

[0377] In some cases, the first immunosuppressant is administered once every week, two weeks, three weeks, or four weeks.

[0378] In some cases, the first immunosuppressant is administered for about 3 months, 4 months, 5 months, or 6 months.

[0379] In some cases, immunosuppressants are administered approximately every 1–3 weeks after combination therapy, and optionally approximately every 1–2 weeks.

[0380] In some cases, immunosuppressants are administered for approximately 1–6 months after combination therapy, optionally for approximately 1–3 months.

[0381] In some respects, immunosuppressive regimens include a second immunosuppressant.

[0382] In some respects, immunosuppressive regimens include administering a second immunosuppressant prior to the combination therapy.

[0383] In some respects, immunosuppressive regimens include the administration of a second immunosuppressant following the administration of combination therapy.

[0384] In some cases, the second immunosuppressant is administered 1 to 14 days prior to the administration of combination therapy.

[0385] In some cases, the second immunosuppressant is administered 1 to 7 days prior to the combination therapy.

[0386] In some cases, the second immunosuppressant is administered 1 to 3 days prior to the administration of combination therapy.

[0387] In some cases, the second immunosuppressant is administered 3 to 7 days prior to the administration of combination therapy.

[0388] In some cases, a second immunosuppressant is administered 1, 2, 3, or 4 weeks after the combination therapy.

[0389] In some cases, the second immunosuppressant is administered twice daily, once daily, or every other day.

[0390] In some cases, the second immunosuppressant is administered for approximately 1–6 months after combination therapy, optionally for approximately 1–3 months.

[0391] In some cases, the second immunosuppressant is administered once or twice daily for 4 to 52 weeks.

[0392] In some cases, the second immunosuppressant is administered once daily for 4 to 24 weeks, 4 to 16 weeks, or 4 to 8 weeks.

[0393] In some cases, the second immunosuppressant is administered twice daily for approximately 1 month, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, or approximately 6 months.

[0394] In some respects, the IL-6 antagonist is selected from the group consisting of: tocilizumab, sarrizumab, secuximab, olozazumab, esimomomab, clazazumab, cilukumab, and lenvelimab. In some respects, the IL-6 antagonist is tocilizumab. In some respects, the IL-6 antagonist is selected from the group consisting of: tocilizumab, sarrizumab, secuximab, olozazumab, esimomomab, clazazumab, cilukumab, lenvelimab, or any combination thereof. In some respects, the IL-6 antagonist is tocilizumab.

[0395] In some cases, calcineurin inhibitors are selected from the group consisting of cyclosporine, tacrolimus, and vorticol. In other cases, tacrolimus is selected as the calcineurin inhibitor.

[0396] In some respects, the second immunosuppressant is selected from the group consisting of: anti-thymocyte globulin, etanercept, balithimab, tacrolimus, sirolimus, and mycophenolate mofetil.

[0397] In some aspects, the combination therapy is accompanied by an immunosuppressive regimen (e.g., administration of an IL-6 antagonist (e.g., tocilizumab) for a period prior to gene therapy administration, and / or administration of a calcineurin inhibitor (e.g., tacrolimus) for a period after gene therapy administration) to accompany a gene therapy approach comprising intramuscular administration of the combination therapy, the combination therapy comprising a first AAV vector genome containing an insulin expression cassette and a second AAV vector genome containing a glucokinase expression cassette in a vector ratio of about 1:0.9-1.1 (e.g., about 1:1), optionally wherein the first and second vector genomes are combined in a single intramuscular formulation administered to at least two distinct skeletal muscle groups. In some aspects, the distinct skeletal muscle groups may include the quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscles), gluteus maximus, hamstrings, deltoids, trapezius, pectoralis major (e.g., pectoralis major), and / or latissimus dorsi. In some respects, different skeletal muscle groups are selected from the quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscles), gluteus maximus, hamstrings, deltoids, trapezius, pectoral muscles (e.g., pectoralis major), and / or latissimus dorsi. In some respects, at least two different skeletal muscle groups are selected from the quadriceps, biceps, hamstrings, or any combination thereof.

[0398] In some respects, the combination therapy comprises a first AAV vector genome containing an insulin expression cassette and a second AAV vector genome containing a glucokinase expression cassette, with a vector ratio of approximately 1:0.9–1.1 (e.g., approximately 1:1.1), wherein the first AAV vector genome, the second AAV vector genome, or both the first AAV vector genome and the second AAV vector genome are in a ratio of approximately 1 × 10⁻⁶. 12 To approximately 1×10 14 vg / kg (e.g., approximately 1×10) 13 To approximately 1×10 14 Intramuscular (IM) administration at a dose of (vg / kg). In some aspects, a single intramuscular formulation is administered to at least three distinct skeletal muscle groups selected from: quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscles), gluteus maximus, hamstrings, deltoids, trapezius, pectoralis major, and / or latissimus dorsi. In some aspects, at least three distinct skeletal muscle groups include the quadriceps, biceps, and / or hamstrings.

[0399] In some aspects, the combination therapy is accompanied by an immunosuppressive regimen (e.g., administration of an IL-6 antagonist (e.g., tocilizumab) for a period prior to gene therapy administration, and / or administration of a calcineurin inhibitor (e.g., tacrolimus) for a period after gene therapy administration) to accompany a gene therapy approach comprising intramuscular administration of the combination therapy, the combination therapy comprising a first AAV vector genome containing an insulin expression cassette and a second AAV vector genome containing a glucokinase expression cassette in a vector ratio of about 1:0.4-0.6 (e.g., about 1:0.5), optionally wherein the first and second vector genomes are combined in a single intramuscular formulation administered to at least two different skeletal muscle groups selected from: quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscles), gluteus maximus, hamstrings, deltoids, trapezius, pectoralis major (e.g., pectoralis major), and latissimus dorsi. In some aspects, the at least two different skeletal muscle groups are selected from the quadriceps, biceps, and / or hamstrings.

[0400] In some respects, the combination therapy comprises a first AAV vector genome containing an insulin expression cassette and a second AAV vector genome containing a glucokinase expression cassette, with a vector ratio of approximately 1:0.4-0.6 (e.g., approximately 1:0.5), wherein the first AAV vector genome, the second AAV vector genome, or both the first AAV vector genome and the second AAV vector genome are in a ratio of approximately 1 × 10⁻⁶. 12 To approximately 1×10 14 vg / kg (e.g., approximately 1×10) 13 To approximately 1×10 14 Intramuscular (IM) administration at a dose of (vg / kg). In some aspects, a single intramuscular formulation is administered to at least three different skeletal muscle groups selected from three or more of the following: quadriceps, biceps, triceps, gastrocnemius (e.g., calf muscles), gluteus maximus, hamstrings, deltoids, trapezius, pectoralis major (e.g., pectoralis major), and latissimus dorsi. In some aspects, at least three different skeletal muscle groups include the quadriceps, biceps, and hamstrings, or any combination thereof.

[0401] In some aspects of this disclosure, methods comprising intramuscular administration (e.g., to the quadriceps, biceps, and hamstrings, preferably bilaterally) and the use of immunosuppressive regimens (e.g., administration of an IL-6 antagonist (e.g., tocilizumab) for a period prior to gene therapy administration, and / or administration of a calcineurin inhibitor (e.g., tacrolimus) for a period after gene therapy administration) may provide improvements in the safety, efficacy, and durability of the combination gene therapies disclosed herein. In some aspects, said methods result in (i) a decrease in glycated hemoglobin (HbA1c) levels in treated subjects, (ii) a decrease in circulating ketones in treated subjects, (iii) a decrease in triglycerides in treated subjects, (iv) a decrease in exogenous insulin in treated subjects, (v) an increase in circulating C-peptide levels in treated subjects, (vi) a decrease in the immune response to exogenous AAV, insulin protein, and / or glucokinase protein, and / or (vii) any combination thereof.

[0402] In some aspects, the polynucleotide encoding hIns is a modified polynucleotide (e.g., proinsulin or proinsulin, its mutants, analogs, or variants). In some aspects, the polynucleotide encoding hGck is a modified polynucleotide (e.g., Gck, its mutants, analogs, or variants). In some aspects, the modification of the coding sequence preserves the wild-type or mutant amino acid sequence of insulin and / or glucokinase. In some aspects, the encoded human Ins protein comprises a signal sequence and a proinsulin polypeptide. In some aspects, the encoded human Ins protein comprises the amino acid sequence of any one of the following: amino acids 25-110 of SEQ ID NO: 41, amino acids 25-110 of SEQ ID NO: 144, or amino acids 25-110 of SEQ ID NO: 145. In some aspects, the modified nucleic acid sequence encodes human proinsulin (e.g., SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO: 145). In some aspects, the modified nucleic acid sequence encodes human Gck (e.g., SEQ ID NO: 82).

[0403] In some respects, the modified nucleic acid is codon-optimized. In some respects, codon optimization involves modifying codons in the open reading frame of the nucleic acid encoding insulin or glucokinase. In some respects, the modified nucleic acid contains a reduced CpG content compared to the corresponding wild-type sequence and / or unmodified sequence.

[0404] In some respects, modified nucleic acids exhibit reduced innate immunogenicity compared to their corresponding wild-type sequences and / or unmodified sequences. In some respects, modified nucleic acids exhibit increased expression compared to their corresponding wild-type sequences and / or unmodified sequences. In some respects, modified nucleic acids exhibit reduced expression compared to their corresponding wild-type sequences and / or unmodified sequences. In some respects, through… computer The method involves subsequent manual sequence examination to develop modified sequences. The nucleic acids disclosed herein can be generated using molecular biology techniques; for example, modified cDNA encoding insulin or glucokinase can be obtained via PCR amplification or cDNA cloning techniques.

[0405] In some respects, nucleic acid sequences are modified to reduce CpG content, for example, to minimize inflammatory responses via TLR9 dimerization and related pathways. In some respects, certain CpGs have inhibitory or neutralizing effects on their inflammatory effects. In some embodiments, one or more of these motifs may be retained. In some respects, such CpG motifs may be introduced into nucleic acid sequences to suppress downstream effects of TLR9 dimerization.

[0406] In some respects, codon modification can reduce the immunogenicity of polynucleotides encoding insulin and / or glucokinase compared to their wild-type counterparts and / or unmodified polynucleotides. In some respects, codon modification can improve the expression of polynucleotides encoding insulin or glucokinase compared to their wild-type counterparts and / or unmodified Gck polynucleotides. In some respects, codon modification can reduce the immunogenicity of polynucleotides encoding glucokinase compared to their wild-type counterparts and / or unmodified Gck polynucleotides.

[0407] The polynucleotides and modified nucleic acids disclosed herein may be present in whole cells, in cell lysates, or in partially purified or substantially pure forms. The polynucleotides and modified nucleic acids may be isolated.

[0408] As used herein, polynucleotides or nucleic acids are “isolated” or “substantially pure” when purified from other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) using 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., eds. (1987), Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York.

[0409] In some aspects, the polynucleotides or modified nucleic acids disclosed herein may be, for example, DNA or RNA, and may or may not contain intron sequences. In some aspects, the nucleic acid may be a cDNA molecule.

[0410] Insulin nucleic acid

[0411] In some aspects, the polynucleotide encoding human insulin comprises a sequence encoding wild-type human insulin (SEQ ID NO: 147) and / or a human Ins mutant or analogue (e.g., SEQ ID NO: 110 or SEQ ID NO: 111). In some aspects, the polynucleotide encoding human insulin is modified relative to wild-type (SEQ ID NO: 147) and / or unmodified human insulin (Ins) or a human Ins mutant or analogue (e.g., SEQ ID NO: 110 or SEQ ID NO: 111). In some aspects, the polynucleotide encoding human insulin comprises, for example, a sequence corresponding to SEQ ID NO: 1, SEQ ID NO: 127, SEQ ID NO: 138, or SEQ ID NO: 170, said sequence comprising a 5' UTR, an ORF, and / or a 3' UTR. In some aspects, the polynucleotide encoding human insulin comprises sequences including a 5' UTR (e.g., nucleic acid 5-329 of SEQ ID NO: 42), an ORF (e.g., SEQ ID NO: 121), and / or a 3' UTR (e.g., SEQ ID NO: 171), such as the polynucleotide comprising SEQ ID NO: 170. In some aspects, the polynucleotide encoding human insulin is modified relative to the sequence corresponding to SEQ ID NO: 1, SEQ ID NO: 127, SEQ ID NO: 138, or SEQ ID NO: 170. In some aspects, the polynucleotide encoding human insulin is modified relative to the sequence corresponding to SEQ ID NO: 170. In some aspects, the polynucleotide encoding human insulin 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 a functional fragment thereof.

[0412] Insulin consists of two polypeptide chains linked together by disulfide bonds: the A chain and the B chain. It is first synthesized into a single polypeptide called proinsulin. Proinsulin is the primary translation product of the insulin gene. It is a peptide of 110 amino acids in length. Proinsulin comprises the proinsulin molecule with a signal peptide linked to its N-terminus. The N-terminal portion containing the proinsulin signal peptide is cleaved, leaving the remaining amino acids as proinsulin. Amino acids 1-30 of the resulting cleaved sequence form the B chain, and here "B10" corresponds to position 34 in proinsulin. Therefore, for example, the "B10" proinsulin mutation corresponds to the H34 mutation in proinsulin. In some respects, as mentioned herein, "B10H" refers to the wild-type histidine amino acid at position B10 (also called H34 in the wild-type proinsulin sequence). Both proinsulin and proinsulin also include a C-peptide between the A and B chains. In mature insulin protein, the C-peptide is cleaved by proteolysis, and the A and B chains are linked by disulfide bonds.

[0413] In some aspects, the polynucleotide encoding the human insulin disclosed herein encodes a proinsulin mutant, said proinsulin mutant comprising one or more mutations at positions H34, P52, K53, R55, and / or L86 corresponding to the positions in wild-type proinsulin (SEQ ID NO: 41). In some aspects, the polynucleotide encoding the human insulin encodes a proinsulin mutant, said proinsulin mutant comprising one or more of the following mutations corresponding to the positions in SEQ ID NO: 41: H34D, H34I, H34V, P52D, K53R, R55K, and / or L86R. In some aspects, the polynucleotide encoding the human insulin encodes a proinsulin mutant, said proinsulin mutant comprising mutations H34D, H34I, H34V, P52D, K53R, R55K, and / or L86R corresponding to the positions in SEQ ID NO: 41. In some aspects, the polynucleotide encoding human insulin encodes a proinsulin mutant, said proinsulin mutant comprising the mutations P52D, K53R, R55K, and / or L86R at the corresponding positions in SEQ ID NO: 41. In some aspects, the polynucleotide encoding human insulin 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 aspects, the polynucleotide encoding human insulin 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, said amino acid sequence comprising one or more of the following mutations at the corresponding positions in SEQ ID NO: 41: H34D, H34I, H34V, P52D, K53R, R55K, and / or L86R. In some respects, the polynucleotide encoding human insulin does not include the amino acid sequence corresponding to the H34 mutation in SEQ ID NO: 41.

[0414] In some respects, the polynucleotides encoding human insulin contain cleavage sites, such as the furin endopeptide cleavage site.

[0415] In some aspects, the polynucleotide encoding human insulin comprises a nucleic acid encoding a signal peptide (e.g., a wild-type proinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence). In some aspects, proinsulin comprises a wild-type insulin signal sequence (e.g., amino acids 1-24 of MALWMRLLPLLALLALWGPDPAAA (SEQ ID NO: 165) or SEQ ID NO: 41). In some aspects, the signal sequence of wild-type proinsulin is replaced with a non-insulin-secreting peptide, such as an IL-6 signal sequence (e.g., MNSFSTSAFGPVAFSLGLLLVLPAAFPAP (SEQ ID NO: 166)) or a fibronectin signal sequence (e.g., MLRGPGPGLLLLAVQCLGTAVPSTGA (SEQ ID NO: 167)).

[0416] In some aspects, the polynucleotide encoding human insulin encodes human insulin containing an amino acid modification selected from H34D, H34I, or H34V corresponding to the amino acid position of wild-type proinsulin (or a mutation of histidine (H) at position B10 of the proinsulin B chain to aspartic acid (D), isoleucine (I), or valine (V)). In some respects, the polynucleotide encoding human insulin encodes human insulin containing an amino acid modification selected from H34D, H34I, or H34V corresponding to the amino acid position of wild-type proinsulin (or the histidine (H) at position B10 of proinsulin B chain is mutated to aspartic acid (D), isoleucine (I), or valine (V)), wherein the human insulin optionally contains a cleavage site (e.g., a furin cleavage site) and a signal peptide (e.g., a wild-type proinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence).

[0417] In some aspects, the polynucleotide encoding human insulin encodes human insulin containing amino acid modifications K53R, R55K, and L86R corresponding to the amino acid positions of wild-type proinsulin (or corresponding to the following modifications: lysine (K) at position B29 of proinsulin mutated to arginine (R), arginine (R) at position C1 mutated to lysine (K), and leucine (L) at position C32 mutated to arginine (R)). In some aspects, the polynucleotide encoding human insulin encodes human insulin containing amino acid modifications K53R, R55K, and L86R corresponding to the amino acid positions of wild-type proinsulin (or corresponding to the following modifications: lysine (K) at position B29 of proinsulin mutated to arginine (R), arginine (R) at position C1 mutated to lysine (K), and leucine (L) at position C32 mutated to arginine (R)), wherein human insulin optionally includes a cleavage site (e.g., a furin cleavage site) and a signal peptide (e.g., a wild-type proinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence).

[0418] In some respects, the polynucleotide encoding human insulin encodes human insulin containing amino acid modifications H34D, K53R, R55K, and L86R corresponding to the amino acid positions of wild-type proinsulin (or corresponding to the following modifications: histidine (H) at position B10 of proinsulin is mutated to aspartic acid (D), lysine (K) at position B29 is mutated to arginine (R), arginine (R) at position C1 is mutated to lysine (K), and leucine (L) at position C32 is mutated to arginine (R)). In some respects, the modified nucleic acid encodes human insulin containing amino acid modifications H34D, K53R, R55K, and L86R corresponding to the amino acid positions of wild-type proinsulin (or corresponding to the following modifications: histidine (H) at position B10 of proinsulin is mutated to aspartic acid (D), lysine (K) at position B29 is mutated to arginine (R), arginine (R) at position C1 is mutated to lysine (K), and leucine (L) at position C32 is mutated to arginine (R)), wherein the human insulin optionally contains a cleavage site (e.g., a furin cleavage site) and a signal peptide (e.g., a wild-type proinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence).

[0419] In some respects, the polynucleotide encoding human insulin encodes human insulin containing amino acid modifications H34I, K53R, R55K, and L86R corresponding to the amino acid positions of wild-type proinsulin (or corresponding to the following modifications: histidine (H) at position B10 of proinsulin is mutated to isoleucine (I), lysine (K) at position B29 is mutated to arginine (R), arginine (R) at position C1 is mutated to lysine (K), and leucine (L) at position C32 is mutated to arginine (R)). In some respects, the polynucleotide encoding human insulin encodes human insulin containing amino acid modifications H34I, K53R, R55K, and L86R corresponding to the amino acid positions of wild-type proinsulin (or corresponding to the following modifications: histidine (H) at position B10 of proinsulin is mutated to isoleucine (I), lysine (K) at position B29 is mutated to arginine (R), arginine (R) at position C1 is mutated to lysine (K), and leucine (L) at position C32 is mutated to arginine (R)), wherein human insulin optionally contains a cleavage site (e.g., a furin cleavage site) and a signal peptide (e.g., a wild-type proinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence).

[0420] In some respects, the polynucleotide encoding human insulin encodes human insulin containing amino acid modifications H34V, K53R, R55K, and L86R corresponding to the amino acid positions of wild-type proinsulin (or corresponding to the following modifications: histidine (H) at position B10 of proinsulin is mutated to valine (V), lysine (K) at position B29 is mutated to arginine (R), arginine (R) at position C1 is mutated to lysine (K), and leucine (L) at position C32 is mutated to arginine (R)). In some respects, the polynucleotide encoding human insulin encodes human insulin containing amino acid modifications H34V, K53R, R55K, and L86R corresponding to the amino acid positions of wild-type proinsulin (or corresponding to the following modifications: histidine (H) at position B10 of proinsulin is mutated to valine (V), lysine (K) at position B29 is mutated to arginine (R), arginine (R) at position C1 is mutated to lysine (K), and leucine (L) at position C32 is mutated to arginine (R)), wherein human insulin optionally contains a cleavage site (e.g., a furin cleavage site) and a signal peptide (e.g., a wild-type proinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence).

[0421] In some aspects, the polynucleotide encoding human insulin encodes human insulin containing amino acid modifications P49D, K53R, R55K, and L86R corresponding to the amino acid positions of wild-type proinsulin (or corresponding to the following modifications: proline (P) at position B28 of proinsulin mutated to aspartic acid (D), lysine (K) at position B29 mutated to arginine (R), arginine (R) at position C1 mutated to lysine (K), and leucine (L) at position C32 mutated to arginine (R)). In some aspects, the polynucleotide encoding human insulin encodes human insulin containing amino acid modifications P49D, K53R, R55K, and L86R (corresponding to the amino acid positions of wild-type proinsulin), wherein human insulin optionally contains a cleavage site (e.g., a furin cleavage site) and a signal peptide (e.g., a wild-type proinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence).

[0422] In some aspects, the polynucleotide encoding human insulin encodes a human insulin (Ins) protein (e.g., proinsulin or a variant thereof), wherein the nucleic acid comprises: (i) a nucleotide sequence encoding a signal peptide (e.g., a wild-type proinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence); and (ii) a nucleotide sequence encoding a proinsulin polypeptide containing an amino acid modification at a position selected from the following positions relative to the corresponding amino acid in wild-type proinsulin: 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 (or an amino acid modification at a position selected from the following positions relative to the corresponding amino acid in wild-type proinsulin: amino acids H34, P52, K53, R55, L86, or any combination thereof). In some aspects, the signal peptide is not a wild-type proinsulin signal sequence (e.g., the wild-type proinsulin sequence is replaced by an IL-6 signal sequence or a fibronectin signal sequence). In some aspects, the polynucleotide encoding human insulin further comprises a cleavage site (e.g., a furin cleavage site). In some respects, the encoded human Ins protein (e.g., proinsulin or a variant thereof) contains an amino acid modification selected from: (i) H34D, H34I or H34V (or a mutation of histidine (H) at position B10 of the proinsulin B chain to aspartic acid (D), isoleucine (I) or valine (V)), and / or (ii) one or more amino acid modifications relative to the wild-type proinsulin sequence 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). In some respects, 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 including a mutation of proline (P) at position B28 of the proinsulin B chain to aspartic acid (D), a mutation of lysine (K) at position B29 of the proinsulin B chain to arginine (R), a mutation of arginine (R) at position C1 of the proinsulin C chain to lysine (K), a mutation of leucine (L) at position C32 of the proinsulin C chain to arginine (R) or any combination thereof).

[0423] In some aspects, the polynucleotide encoding human insulin encodes a variant or mutant human insulin protein or a functional fragment thereof. In some aspects, 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 aspects, 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 SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO: 145. In some aspects, the human insulin protein comprises insertions, deletions, substitutions, or combinations thereof relative to wild-type human insulin. In some aspects, the human insulin protein comprises at least one substitution. In some aspects, at least one substitution is a conserved substitution. In some aspects, at least one substitution is a non-conserved substitution.

[0424] In some aspects, the polynucleotide discloses an open reading frame (ORF) encoding human insulin, said ORF comprising a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to nucleic acids 73-330 of any one of SEQ ID NO: 43-57, 110-116, or nucleic acids 88-345 of any one of SEQ ID NO: 117-122. In some aspects, the polynucleotide discloses an open reading frame (ORF) encoding human insulin, said ORF comprising a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to SEQ ID NO: 121. In some respects, the ORF further contains a nucleic acid sequence encoding a signal peptide.

[0425] In some aspects, the polynucleotides of this disclosure comprise an open reading frame (ORF) encoding human insulin, said 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 the following: 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 or SEQ ID NO: 122, wherein the polynucleotide encodes human insulin protein (e.g., SEQ ID NO: 41, SEQ ID NO: 144 or SEQ ID NO: 145) or a functional fragment thereof.

[0426] In some aspects, the polynucleotides of this disclosure include an open reading frame (ORF) comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 121. In some aspects, the polynucleotides of this disclosure include an open reading frame (ORF) comprising a nucleic acid sequence having the sequence of SEQ ID NO: 121.

[0427] In some aspects, the polynucleotide encoding human insulin disclosed herein comprises an open reading frame (ORF) containing a nucleic acid having a sequence having the following sequences: 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, or SEQ ID NO: 122. In some aspects, the polynucleotide encoding human insulin of this disclosure comprises an open reading frame containing a nucleic acid having the sequence of SEQ ID NO: 121. In some aspects, the polynucleotide encoding human insulin comprises the ORF sequence presented or mentioned in Table 8.

[0428] In some aspects, the polynucleotides of this disclosure comprise two or more ORFs. In some aspects, the two or more ORFs are operatively linked. In some aspects, the ORFs are operatively linked via IRES.

[0429] In some aspects, the polynucleotide comprising an ORF encoding human insulin further comprises a 5' UTR nucleic acid sequence. In some aspects, the polynucleotide comprising an ORF encoding human insulin further comprises a modified 5' UTR nucleic acid sequence. In some aspects, the polynucleotide comprising an ORF encoding human insulin further comprises a 5' UTR, said 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 acid 5-329 of SEQ ID NO: 42, or SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some respects, the 5' UTR 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 nucleic acid 5-329 of SEQ ID NO: 42.

[0430] In some aspects, the polynucleotide comprising the ORF encoding human insulin contains the Kozak consensus sequence (or Kozak sequence). In some aspects, the 5' UTR contains a nucleic acid having the sequence of SEQ ID NO: 42, SEQ ID NO: 42, nucleic acid 5-329, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some aspects, the polynucleotide comprising the ORF encoding human insulin contains the 5' UTR nucleic acid sequence presented or mentioned in Table 8.

[0431] In some aspects, the polynucleotide comprising an ORF encoding human insulin further comprises a modified 3' UTR nucleic acid sequence. In some aspects, the polynucleotide comprising an ORF encoding human insulin further comprises a 3' UTR, said 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 the following sequences: 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: 149, or SEQ ID NO: 171. In some aspects, the 3' UTR comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 171. In some aspects, the 3' UTR comprises a nucleic acid having a 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: 149, or SEQ ID NO: 171. In some aspects, the 3' UTR comprises a restriction site selected from the group consisting of: Bam HI、 Eco RI Nde I, Eco RV, Spe I, Xba I, Nhe I, Vsp I, Nsi I, Sca I, Kpn I, Ssp I and Pac I, and any combination thereof. In some respects, the polynucleotides containing the ORF encoding human insulin contain the 3' UTR nucleic acid sequence presented or mentioned in Table 8.

[0432] In some aspects, the polynucleotide comprising an ORF encoding human insulin disclosed herein comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence selected from the following: 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: 89, SEQ ID NO: 80, SEQ ID NO: 89, SEQ ID NO: 80, SEQ ID NO: 89, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 80 ... SEQ ID NO: 88, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, 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: 160, SEQ ID NO: 161 or SEQ ID NO: 170, wherein the 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 aspects, the polynucleotide comprising an ORF encoding human insulin of this disclosure comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 138. In some aspects, the polynucleotide comprising an ORF encoding human insulin of this disclosure comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 170.

[0433] In some aspects, the polynucleotide comprising an ORF encoding human insulin disclosed herein comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with nucleic acid sequences selected from the following: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In some aspects, the polynucleotide encoding human insulin of this disclosure comprises nucleic acids having the sequences of the following: 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: 127, SEQ ID NO: 128, SEQ ID NO: 129, 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: 160, SEQ ID NO: 161, or SEQ ID NO: 170. In some aspects, the polynucleotide encoding human insulin of this disclosure comprises a nucleic acid having the sequence of SEQ ID NO: 138. In some aspects, the polynucleotide encoding human insulin of this disclosure comprises a nucleic acid having the sequence of SEQ ID NO: 170.In some aspects, the polynucleotide encoding human insulin of this disclosure comprises nucleic acids 5-957 of the following: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In some aspects, the polynucleotide comprising the ORF encoding human insulin comprises nucleic acids containing the 5' UTR, ORF, and 3' UTR presented in Table 8.

[0434] In some aspects, the polynucleotides of this disclosure encode human insulin containing a wild-type proinsulin secretion signal peptide. In some aspects, the polynucleotides of this disclosure encoding human insulin do not encode a wild-type proinsulin secretion signal peptide. In some aspects, wild-type proinsulin is replaced by a non-insulin secretion signal. In some aspects, the polynucleotides of this disclosure encoding human insulin encode human proinsulin containing an interleukin-6 (IL-6) secretion signal peptide. In some aspects, the polynucleotides of this disclosure encoding human insulin encode human proinsulin containing a fibronectin secretion signal peptide.

[0435] Glucokinase Nucleic Acid

[0436] In some aspects, the polynucleotide encoding human glucokinase comprises a sequence encoding wild-type human glucokinase (SEQ ID NO: 82) or a functional fragment thereof. In some aspects, the polynucleotide encoding human glucokinase comprises a modified nucleic acid sequence encoding wild-type human glucokinase (SEQ ID NO: 82) or a functional fragment thereof. In some aspects, the polynucleotides disclosed herein comprising sequences encoding human glucokinase are modified relative to wild-type and / or unmodified nucleic acid sequences, for example, comprising 5' UTR (e.g., SEQ ID NO: 42), ORF (e.g., SEQ ID NO: 68), and / or 3' UTR (e.g., SEQ ID NO: 60 or 171). In some aspects, the polynucleotide encoding human Gck comprises 5' UTR (e.g., SEQ ID NO: 42), ORF (e.g., SEQ ID NO: 68), and / or 3' UTR (e.g., SEQ ID NO: 60 or 171), for example, comprising polynucleotides of SEQ ID NO: 19, SEQ ID NO: 27, or SEQ ID NO: 172.

[0437] In some aspects, the polynucleotides of this disclosure comprise an ORF encoding human glucokinase, said 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 the following: 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 human glucokinase protein (SEQ ID NO: 82) or a functional fragment thereof.

[0438] In some aspects, the polynucleotides of this disclosure comprise an ORF encoding human glucokinase, said 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 SEQ ID NO: 68. In some aspects, the polynucleotides of this disclosure comprise an open reading frame encoding human glucokinase, said open reading frame comprising a nucleic acid having a sequence having any of the following: SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO: 162. In some aspects, the polynucleotides of this disclosure comprise an open reading frame encoding human glucokinase, said open reading frame comprising a nucleic acid having the sequence having SEQ ID NO: 68. In some respects, the polynucleotides encoding human glucokinase contain the ORF sequences presented or mentioned in Table 9.

[0439] In some aspects, the polynucleotide comprising an ORF encoding human glucokinase of this disclosure further comprises a modified 5' UTR nucleic acid sequence. In some aspects, the polynucleotide comprising an ORF encoding human glucokinase of this disclosure further comprises a 5' UTR, said 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 nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some aspects, the polynucleotide comprising an ORF encoding human glucokinase disclosed herein further comprises a 5' UTR, said 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.

[0440] In some aspects, the polynucleotide comprising the ORF encoding human glucokinase contains a Kozak consensus sequence (or Kozak sequence). In some aspects, the 5' UTR contains a nucleic acid having the sequence of SEQ ID NO: 42, SEQ ID NO: 42, nucleic acid 5-329, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some aspects, the polynucleotide comprising the ORF encoding human glucokinase contains the 5' UTR sequence presented or mentioned in Table 9.

[0441] In some aspects, the polynucleotide comprising an ORF encoding human glucokinase of this disclosure further comprises a modified 3' UTR nucleic acid sequence. In some aspects, the polynucleotide comprising an ORF encoding human glucokinase of this disclosure further comprises a 3' UTR, said 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 the following: 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, SEQ ID NO: 149, SEQ ID NO: 169, or SEQ ID NO: 171. In some aspects, the polynucleotide comprising an ORF encoding human glucokinase disclosed herein further comprises a 3' UTR, said 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: 171. In some aspects, the 3' UTR comprises a nucleic acid having a sequence having the following: 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, SEQ ID NO: 149, SEQ ID NO: 169, or SEQ ID NO: 171. In some respects, the 3' UTR contains restriction sites selected from the following groups: Bam HI、 Eco RI Nde I, Eco RV, Spe I, Xba I, Nhe I, Vsp I, Nsi I, Sca I, Kpn I, Ssp I and PacI, and any combination thereof. In some respects, the polynucleotides containing the ORF encoding human glucokinase contain the 3' UTR sequence presented or mentioned in Table 9.

[0442] In some aspects, the polynucleotide encoding human glucokinase disclosed herein comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence selected from the following: 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: 3 ... 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, SEQ ID NO: 164, SEQ ID NO: 168, or SEQ ID NO: 172, wherein the nucleic acid sequence encodes a human glucokinase protein (e.g., SEQ ID NO: 82) or a functional fragment thereof. In some aspects, the polynucleotide encoding human glucokinase of this disclosure comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 27. In some respects, the polynucleotide encoding human glucokinase disclosed herein comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 168.In some respects, the polynucleotide encoding human glucokinase disclosed herein comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 172. In some aspects, the polynucleotide encoding human glucokinase disclosed herein comprises a 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 nucleic acid selected from the following sequences: 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 aspects, the polynucleotide encoding human glucokinase disclosed herein comprises nucleic acids having sequences having the following: 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: 16 ... SEQ ID NO: 164, SEQ ID NO: 168, or SEQ ID NO: 172. In some aspects, the polynucleotide encoding human glucokinase of this disclosure comprises a nucleic acid having the sequence of SEQ ID NO: 168. In some aspects, the polynucleotide encoding human glucokinase of this disclosure comprises nucleic acids 5-2025 of the following: 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 respects, the polynucleotides encoding human glucokinase are the nucleic acids presented or mentioned in Table 9.

[0443] Expression Box

[0444] Certain aspects of this disclosure relate to a combination gene therapy, such as a combination AAV gene therapy, for delivering an expression cassette (or expression construct) containing a polynucleotide encoding an insulin (Ins) protein and an expression cassette containing a polynucleotide encoding a glucokinase (Gck) protein. In some aspects, the combination therapy includes the separate administration of the Ins and Gck expression cassettes. In some aspects, the combination therapy includes the administration of the insulin and glucokinase expression cassettes in a single formulation, such as two separate rAAV particles in the same pharmaceutical composition.

[0445] In some aspects, the combination therapy includes an insulin expression cassette comprising a promoter operatively linked to a multinucleotide encoding a human insulin protein, wherein the insulin expression cassette is side-mounted with an inverted terminal repeat sequence. In some aspects, the combination therapy includes a glucokinase expression cassette comprising a promoter operatively linked to a multinucleotide encoding a human glucokinase protein, wherein the glucokinase expression cassette is side-mounted with an inverted terminal repeat sequence. In some aspects, the first and second promoters are the same or substantially the same promoters; or in some aspects, the first and second promoters are different promoters driving substantially equivalent levels of transgene expression.

[0446] Expression cassettes containing a eukaryotic promoter operably linked to a DNA of interest (e.g., DNA encoding insulin or DNA encoding glucokinase) may be used in this disclosure. In some aspects, the expression cassette containing a DNA sequence (or corresponding RNA sequence) that may be used according to this disclosure may be any eukaryotic expression cassette containing a DNA or RNA sequence of interest. For example, plasmids or viral constructs (e.g., AAV vectors) may be lysed to provide linear DNA with linkable ends. These ends may bind to exogenous DNA having complementarity (e.g., linkable ends) to provide a biologically functional recombinant DNA molecule with intact replicons and desired phenotypic properties. In some aspects, the expression cassette is capable of replicating in both eukaryotic and prokaryotic hosts.

[0447] In some respects, the exogenous DNA used in this disclosure is derived from suitable cells, and the constructs are prepared using techniques known in the art. Similarly, techniques for obtaining the expression of exogenous DNA or RNA sequences in genetically modified host cells are known in the art (see, for example, Kormal et al., Proceedings of the National Academy of Sciences (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, New York; methods and compositions for the expression of DNA of interest in eukaryotes, each of which is hereby incorporated by reference).

[0448] In some respects, the DNA expression construct includes a promoter to facilitate the expression of DNA of interest (e.g., a polynucleotide encoding insulin or a polynucleotide encoding glucokinase) within secretory cells. In some respects, the promoter is a strong eukaryotic promoter, such as a promoter derived from cytomegalovirus (CMV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), or adenovirus. Exemplary promoters include, but are not limited to, promoters of immediate early genes from human CMV (Boshart et al., Cell 41:521-530 (1985)) and promoters of long terminal repeat (LTR) sequences from RSV (Gorman et al., Proceedings of the National Academy of Sciences, 79:6777-6781 (1982)). In some respects, the promoter is used in conjunction with an intron sequence. In some respects, the CMV promoter is a mini-CMV promoter. Alternatively, the promoter used may be a tissue-specific promoter. In some aspects, the insulin expression cassette contains a CMV promoter. In some aspects, the glucokinase expression cassette contains a CMV promoter. In some aspects, the insulin expression cassette contains a constitutive promoter. In some aspects, the glucokinase expression cassette contains a constitutive promoter. In some aspects, the insulin expression cassette contains a ubiquitous promoter. In some aspects, the glucokinase expression cassette contains a ubiquitous promoter. In some aspects, the insulin expression cassette contains a muscle-specific promoter. In some aspects, the glucokinase expression cassette contains a muscle-specific promoter.

[0449] The expression cassette disclosed herein may also include other components, such as: markers (e.g., antibiotic resistance genes (e.g., ampicillin resistance genes) or β-galactosidase) to help select cells containing and / or expressing constructs; origin of replication (preferably, high copy number origin of replication) for stable replication of the construct in bacterial cells; nuclear localization signals; or other elements that promote the production of DNA expression constructs, proteins encoded therefrom, or both.

[0450] For eukaryotic expression, the expression cassette may contain at least a eukaryotic promoter operably linked to a DNA of interest (e.g., a polynucleotide encoding insulin or glucokinase), which is then operably linked to a polyadenylation sequence. The polyadenylation signal sequence may be selected from any of a variety of polyadenylation signal sequences known in the art. In some aspects, the polyadenylation signal sequence is the SV40 early polyadenylation signal sequence. In some aspects, the polyadenylation signal sequence is a growth hormone polyadenylation signal sequence (e.g., bovine growth hormone polyA or human growth hormone polyA). In some aspects, the glucokinase expression cassette contains the SV40 polyadenylation signal sequence. In some aspects, the insulin expression cassette contains the bovine or human growth hormone polyadenylation signal sequence.

[0451] In some respects, the expression cassette may also include one or more introns that can increase the expression level of the DNA of interest, particularly when the DNA of interest is cDNA (e.g., introns that do not contain naturally occurring sequences). Any of a variety of introns known in the art can be used (e.g., the human β-globulin intron, which is inserted at the 5' position of the DNA of interest in the construct).

[0452] DNA of interest (e.g., a polynucleotide encoding insulin or a polynucleotide encoding glucokinase) can be inserted into an expression cassette to express a therapeutic molecule (e.g., a protein) as a fusion protein (e.g., a fusion protein having a β-galactosidase or a portion thereof at the N-terminus and a therapeutic protein at the C-terminus). The generation of the fusion protein can facilitate the identification of transformed cells expressing the protein (e.g., using enzyme-linked immunosorbent assay (ELISA) with an antibody that binds to the fusion protein).

[0453] The vector used to deliver DNA of interest (e.g., polynucleotides encoding insulin, polynucleotides encoding glucokinase) can be viral or non-viral vectors, or can consist of naked DNA mixed with an adjuvant (e.g., viral particles (e.g., AAV particles) or cationic lipids or liposomes). An "adjuvant" is a substance that does not produce the desired effect on its own, but is used to enhance or otherwise improve the action of the active compound. The precise vector and vector formulation used will depend on several factors, such as the target cells and / or organs for gene transfer.

[0454] Examples of suitable promoters include: early intermediate promoters of cytomegalovirus (CMV); viral long terminal repeat (LTR) promoters, such as promoters from murine moloney leukemia virus (MMLV), Rous sarcoma virus, or HTLV-1; early promoters of simian virus 40 (SV 40); RSV promoters; and herpes simplex virus thymidine kinase promoters. In some respects, the promoter is a constitutive promoter. In some respects, the promoter is a ubiquitous promoter. In some respects, the promoter is a cell-specific and / or tissue-specific promoter. In some respects, the promoter is a muscle-specific promoter. In some respects, the promoter is used in conjunction with an intron sequence. In some respects, the promoter is tissue-specific. In some respects, the first promoter is a CMV promoter. In some respects, the second promoter is a CMV promoter. In some respects, the CMV promoter is a mini-CMV promoter. In some respects, the insulin expression cassette contains a CMV promoter. In some respects, the glucokinase expression cassette contains a CMV promoter. In some respects, the first and second promoters are the same or substantially the same promoters; or in some respects, the first and second promoters are different promoters that drive substantially equivalent levels of transgene expression.

[0455] In some respects, the insulin expression cassette contains a constitutive promoter. In some respects, the glucokinase expression cassette contains a constitutive promoter. In some respects, the insulin expression cassette contains a ubiquitous promoter. In some respects, the glucokinase expression cassette contains a ubiquitous promoter. In some respects, the insulin expression cassette contains a muscle-specific promoter. In some respects, the glucokinase expression cassette contains a muscle-specific promoter.

[0456] In some aspects, the expression cassette includes a promoter operatively linked to a polynucleotide comprising an ORF having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence selected from the following: 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 or SEQ ID NO: 122, wherein the polynucleotide 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 aspects, the expression cassette includes a promoter operatively linked to a polynucleotide encoding a human insulin protein, the polynucleotide comprising an ORF having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 121 or SEQ ID NO: 122.In some aspects, the polynucleotide encoding human insulin protein of this disclosure comprises an open reading frame (ORF) containing a nucleic acid having a sequence having the following sequences: 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, or SEQ ID NO: 122. In some aspects, the polynucleotide encoding the human insulin protein of this disclosure comprises an open reading frame containing a nucleic acid having the sequence of SEQ ID NO: 122. In some aspects, the polynucleotide encoding the human insulin protein of this disclosure comprises an open reading frame containing a nucleic acid having the sequence of SEQ ID NO: 121. In some aspects, the polynucleotide encoding the human insulin protein comprises the ORF sequence presented or mentioned in Table 8.

[0457] In some aspects, the expression cassette contains a polynucleotide encoding human insulin, which includes a wild-type proinsulin secretion signal peptide. In some aspects, the polynucleotide encoding human insulin protein of this disclosure does not encode a wild-type proinsulin secretion signal peptide. In some aspects, wild-type proinsulin is replaced by a non-insulin secretion signal. In some aspects, the expression cassette contains a polynucleotide encoding human proinsulin, which includes an interleukin-6 (IL-6) secretion signal peptide. In some aspects, the expression cassette contains a polynucleotide encoding human proinsulin, which includes a fibronectin secretion signal peptide.

[0458] In some aspects, the expression cassette comprises a polynucleotide encoding the human insulin protein, said polynucleotide further comprising a 5' UTR, said 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 acids 5-329, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some aspects, the polynucleotide encoding the human insulin protein comprises a 5' UTR sequence presented or mentioned in Table 8.

[0459] In some aspects, the expression cassette contains a polynucleotide encoding a human insulin protein, the polynucleotide further comprising a 3' UTR, the 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 any of the following: 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: 149, or SEQ ID NO: 171. In some respects, the 3' UTR comprises nucleic acids having sequences 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: 149, and SEQ ID NO: 171. In some respects, the 3' UTR comprises restriction sites selected from the group consisting of: Bam HI、 Eco RI Nde I, Eco RV, Spe I, Xba I, Nhe I, Vsp I, Nsi I, Sca I, Kpn I, Ssp I and Pac I, and any combination thereof. In some respects, the polynucleotide encoding the human insulin protein contains the 3' UTR sequence presented or mentioned in Table 8.

[0460] In some aspects, the expression cassette comprises a polynucleotide 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 following: 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: 127, SEQ ID NO: 128, SEQ ID NO: 129, 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: 160, SEQ ID NO: 161 or SEQ ID NO: 170, wherein the polynucleotide 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 aspects, the expression cassette contains a polynucleotide encoding a human insulin protein, said polynucleotide 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 or SEQ ID NO: 171.In some aspects, the expression cassette contains a polynucleotide encoding a human insulin protein, said polynucleotide 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 selected from the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In some aspects, the expression cassette comprises a polynucleotide encoding a human insulin protein, said polynucleotide having the sequences of the following: 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: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 19, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 129, 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: 160, SEQ ID NO: 161, or SEQ ID NO: 170. In some aspects, the expression cassette contains a polynucleotide encoding a human insulin protein, said polynucleotide having the sequence of SEQ ID NO: 138 or SEQ ID NO: 170.In some aspects, the expression cassette contains a polynucleotide encoding a human insulin protein, said polynucleotide 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 selected from the following sequences: 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 respects, the expression cassette contains a polynucleotide encoding the human insulin protein, the polynucleotide comprising the 5'UTR, ORF, and 3'UTR presented or mentioned in Table 8.

[0461] In some aspects, the expression cassette includes a promoter operatively linked to a polynucleotide encoding a human glucokinase protein sequence, said polynucleotide comprising an ORF having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence selected from the following: SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO: 162. In some aspects, the expression cassette comprises a promoter operatively linked to a polynucleotide encoding a human glucokinase protein sequence, said polynucleotide comprising an ORF having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 68. In some aspects, the expression cassette includes a promoter operatively linked to a polynucleotide encoding a human glucokinase protein, said polynucleotide having the sequence of any of the following: SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO: 162.In some aspects, the expression cassette includes a promoter operatively linked to a polynucleotide encoding a human glucokinase protein, said polynucleotide having the sequence of SEQ ID NO: 68. In some aspects, the polynucleotide encoding the human glucokinase protein includes the ORF sequence presented or mentioned in Table 9.

[0462] In some aspects, the expression cassette comprises a polynucleotide encoding a human glucokinase protein, said polynucleotide further comprising a 5' UTR, said 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 nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some aspects, the polynucleotide encoding a human glucokinase protein comprises a 5' UTR sequence presented or mentioned in Table 9.

[0463] In some aspects, the expression cassette comprises a polynucleotide encoding a human glucokinase protein, said polynucleotide further comprising a 3' UTR, said 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 any of the following: 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, SEQ ID NO: 149, or SEQ ID NO: 169. In some aspects, the 3' UTR contains nucleic acids having sequences of the following: 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, SEQ ID NO: 149, SEQ ID NO: 169, or SEQ ID NO: 171. In some aspects, the 3' UTR contains restriction sites selected from the group consisting of: Bam HI、 Eco RI Nde I, Eco RV, Spe I, Xba I, Nhe I, Vsp I, Nsi I, Sca I, Kpn I, Ssp I and Pac I, or any combination thereof. In some respects, the polynucleotide encoding the human glucokinase protein contains the 3' UTR sequence presented or mentioned in Table 9.

[0464] In some aspects, the expression cassette comprises a polynucleotide 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 following: 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, SEQ ID NO: 164, SEQ ID NO: 168, or SEQ ID NO: 172, wherein the nucleic acid sequence encodes a human glucokinase protein (e.g., SEQ ID NO: 82) or a functional fragment thereof. In some aspects, the expression cassette comprises a polynucleotide 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: 168, wherein the nucleic acid sequence encodes a human glucokinase protein (e.g., SEQ ID NO: 82) or a functional fragment thereof. In some aspects, the expression cassette comprises a polynucleotide 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: 172, wherein the nucleic acid sequence encodes a human glucokinase protein (e.g., SEQ ID NO: 82) or a functional fragment thereof.In some aspects, the expression cassette comprises a polynucleotide encoding a human glucokinase protein, said polynucleotide 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 selected from the following sequences: 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 aspects, the expression cassette comprises a polynucleotide encoding a human glucokinase protein, said polynucleotide having the sequences of the following: 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: 97, SEQ ID NO: 98, SEQ ID NO: 9 ...9, 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: 99, SEQ ID NO: 99, SEQ ID NO: 99, SEQ ID NO: 99, SEQ ID NO: 90, NO:163, SEQ ID NO:164, or SEQ ID NO:168.In some aspects, the expression cassette comprises a polynucleotide encoding a human glucokinase protein, said polynucleotide comprising nucleic acids 5-2025 of the following: 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 aspects, the expression cassette comprises a polynucleotide encoding a human glucokinase protein, said polynucleotide comprising the 5' UTR, ORF, and 3' UTR presented or mentioned in Table 9.

[0465] In some aspects, the combination therapy comprises: administration of an insulin expression cassette, the insulin expression cassette comprising a polynucleotide encoding human insulin, the polynucleotide comprising an ORF having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the following: 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 or SEQ ID NO: 122;And administer a glucokinase expression cassette, the glucokinase expression cassette comprising a polynucleotide encoding human glucokinase, the polynucleotide comprising an ORF having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or 100% sequence identity with the following: SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO: 162. In some aspects, the combination therapy comprises: an insulin expression cassette comprising a polynucleotide encoding human insulin, said polynucleotide comprising an ORF having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 121; and a glucokinase expression cassette comprising a polynucleotide encoding human glucokinase, said polynucleotide comprising an ORF having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity with SEQ ID NO: 68.

[0466] In some aspects, the combination therapy's insulin expression cassette and glucokinase expression cassette are administered at the following ratios: approximately 1:0.25-0.75, 1:0.25-0.7, 1:0.25-0.65, 1:0.25-0.60, 1:0.25-0.55, 1:0.25-0.5, 1:0.3-0.7, 1:0.3-0.65, 1:0.3-0.6, 1:0.3-0.55, 1:0.3-0.5, 1:0. 35-0.7, 1:0.35-0.65, 1:0.35-0.6, 1:0.35-0.55, 1:0.35-0.5, 1:0.4-0.7, 1:0.4-0.65, 1:0.4-0.6, 1:0.4-0.55, 1:0.4-0.5, 1:0.45-0.7, 1:0.45-0.65, 1:0.45-0.6, 1:0.45-0.55 or 1:0.45-0.5. In some aspects, the combination therapy's insulin expression cassette and glucokinase expression cassette are administered at ratios of approximately 1:0.25-0.75, 1:0.3-0.75, 1:0.35-0.75, 1:0.40-0.75, 1:0.45-0.75, or 1:0.5-0.75. In some aspects, the combination therapy's insulin expression cassette and glucokinase expression cassette are administered at ratios of approximately 1:0.25-0.75, 1:0.3-0.70, 1:0.35-0.65, 1:0.35-0.75, 1:0.4-0.60, or 1:0.45-0.55. In some aspects, the combination therapy's insulin expression cassette and glucokinase expression cassette are administered at ratios of approximately 1:0.5, approximately 1:1, approximately 1:2, or approximately 1:4. In some respects, the vector ratio of the first AAV vector genome to the second AAV vector genome is selected from the following groups: 1:0.25–0.75, 1:0.75–1.25, 1:1.75–2.25, and 1:3.75–1:4.25 (e.g., approximately 1:0.5, approximately 1:1, approximately 1:2, and approximately 1:4). In some respects, the vector ratio (i.e., the first AAV vector containing the hIns expression cassette: the second AAV vector containing the hGck expression cassette) is 1:0.4–0.6. In some respects, the vector ratio (i.e., the first AAV vector containing the hIns expression cassette: the second AAV vector containing the hGck expression cassette) is 1:0.5.

[0467] In some aspects, the insulin expression cassette and glucokinase expression cassette of the combination therapy are administered at ratios of approximately 1:0.75–1.25, 1:0.8–1.25, 1:0.85–1.25, 1:0.9–1.25, 1:0.95–1.25, and 1:1.0–1.25. In some aspects, the carrier ratio is selected from groups of the following compositions: 1:0.75–1.25, 1:0.75–1.20, 1:0.75–1.15, 1:0.75–1.10, 1:0.75–1.05, and 1:0.75–1.00. In some respects, the vector ratio is selected from the following groups: 1:0.75–1:25, 1:0.80–1.20, 1:0.85–1.15, 1:0.90–1.10, and 1:0.95–1.05. In some respects, the vector ratio (i.e., the first AAV vector containing the hIns expression cassette: the second AAV vector containing the hGck expression cassette) is 1:1.

[0468] In some aspects, the vector ratio (i.e., the first AAV vector containing the hIns expression cassette: the second AAV vector containing the hGck expression cassette) is 1:2. In some aspects, the vector ratio (i.e., the first AAV vector containing the hIns expression cassette: the second AAV vector containing the hGck expression cassette) is 1:3. In some aspects, the vector ratio (i.e., the first AAV vector containing the hIns expression cassette: the second AAV vector containing the hGck expression cassette) is 1:4. In some aspects, the vector ratio (i.e., the first AAV vector containing the hIns expression cassette: the second AAV vector containing the hGck expression cassette) is 1:5. In some aspects, the vector ratio (i.e., the first AAV vector containing the hIns expression cassette: the second AAV vector containing the hGck expression cassette) is 1:10. In some aspects, the vector ratio (i.e., the first AAV vector containing the hIns expression cassette: the second AAV vector containing the hGck expression cassette) is 1:15. In some respects, the carrier ratio (i.e., the first AAV carrier containing the hIns expression cassette: the second AAV carrier containing the hGck expression cassette) is between: about 1:1 to about 1:15, about 1:1 to about 1:10, about 1:1 to about 1:5, about 1:1 to about 1:4, about 1:1 to about 1:3, about 1:3 to about 1:5, about 1:3 to about 1:10, about 1:3 to about 1:15, about 1:5 to about 1:10, about 1:5 to about 1:15, or about 1:10 to about 1:15.

[0469] In some respects, the polynucleotide encoding the human insulin protein encodes human insulin containing the wild-type proinsulin secretion signal peptide. In some respects, the polynucleotide encoding the human insulin protein does not encode the wild-type proinsulin secretion signal peptide. In some respects, the wild-type proinsulin is replaced by a non-insulin secretion signal. In some respects, the polynucleotide encoding the human insulin protein encodes human proinsulin containing the interleukin-6 (IL-6) secretion signal peptide. In some respects, the polynucleotide encoding the human insulin protein encodes human proinsulin containing the fibronectin secretion signal peptide.

[0470] In some respects, insulin expression cassettes containing polynucleotides encoding human insulin protein include the 5' UTR, ORF, and 3' UTR presented or mentioned in Table 8, and glucokinase expression cassettes containing polynucleotides encoding human glucokinase protein include the 5' UTR, ORF, and 3' UTR presented or mentioned in Table 9.

[0471] Some aspects of this disclosure relate to vectors, such as viral vectors, that contain expression constructs. In some aspects, the expression construct includes an expression cassette. In some aspects, the expression construct further includes a genome capable of being stable and remaining free in a cell. Within the context of this disclosure, in some aspects, the cell or host cell may encompass the cell used to prepare the construct or the cell to which the construct is administered. In some aspects, the construct is capable of integration into the genome of a cell, for example, through homologous recombination or otherwise. In some aspects, the expression construct is an expression construct in which nucleotide sequences encoding insulin and / or glucokinase, as disclosed herein, are operatively linked to a promoter as provided herein, wherein the promoter is capable of directing the expression of the nucleotide sequences (i.e., the coding sequences) in a cell. In some aspects, the expression cassette, as used herein, comprises or consists of nucleotide sequences encoding insulin and / or glucokinase, in each case, the nucleotide sequences being operatively linked to a promoter, wherein the promoter is capable of directing the expression of said nucleotide sequences. In some aspects, the viral expression construct is an expression construct intended for use in gene therapy. It may be designed to contain a portion of a viral genome as disclosed herein.

[0472] In some respects, the expression construct further includes one or more of the following: an ITR sequence (e.g., AAV2ITR), a polyA sequence (e.g., SV40 polyadenylation signal, bGH polyadenylation signal), and an enhancer sequence (e.g., SV40 enhancer sequence).

[0473] In some respects, recombinant techniques were used to prepare the expression constructs disclosed herein, in which nucleic acid sequences encoding insulin and / or glucokinase were expressed in suitable cells (e.g., cultured cells or cells of multicellular organisms), as described in Ausubel et al., “Current Protocols in Molecular Biology,” Green Press and Wiley International Science Press, New York (1987), and Sambrook and Russell (2001, ibid.), both of which are incorporated herein by reference in their entirety. See also Kunkel (1985), Proceedings of the National Academy of Sciences, 82:488 (describing site-directed mutagenesis), Roberts et al. (1987), Nature, 328:731-734, or Wells, JA et al. (1985), Gene, 34:315 (describing cassette mutagenesis).

[0474] Delivery carrier

[0475] This disclosure also provides vectors comprising any of the polynucleotides, expression cassettes, or constructs described herein. In some aspects, the delivery vector is a viral vector, a non-viral vector, a plasmid, a lipid, or a lysosome. In some aspects, the delivery vector is a viral vector. In some aspects, the viral vector is an adeno-associated virus (AAV) expression vector.

[0476] In some respects, polynucleotides encoding insulin and / or glucokinase are used in expression constructs or expression vectors. The phrase "expression vector" generally refers to a nucleotide sequence capable of enabling gene expression in a host compatible with such sequences. These expression vectors may include at least a suitable promoter sequence and an optional transcription termination signal. Other factors necessary or helpful for achieving expression may also be used, as disclosed herein. Polynucleotides encoding insulin and / or glucokinase may be incorporated into expression vectors capable of being introduced into and expressed in in vitro cell cultures. In some respects, expression vectors are adapted for replication in prokaryotic hosts (e.g., bacteria, such as E. coli), or for introduction into cultured mammalian, plant, insect (e.g., Sf9), yeast, fungal, or other eukaryotic cell lines. In some respects, expression constructs are adapted for expression in vivo.

[0477] In some aspects, the delivery vector comprises an insulin expression cassette containing a promoter operatively linked to a polynucleotide comprising an ORF having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence selected from the following: 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: 110, SEQ ID NO: 1 ... 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 or SEQ ID NO: 122, wherein the polynucleotide 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 aspects, the delivery vector comprises an insulin expression cassette containing a promoter operatively linked to a polynucleotide comprising an ORF having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 122. In some aspects, the delivery vector comprises an insulin expression cassette containing a promoter operatively linked to a polynucleotide comprising an ORF having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 121.In some aspects, the polynucleotide comprises an ORF having the sequences of the following: 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 or SEQ ID NO: 122. In some aspects, the polynucleotide comprises an ORF having the sequence of SEQ ID NO: 122. In some aspects, the polynucleotide comprises an ORF having the sequence of SEQ ID NO: 121. In some aspects, the polynucleotide encoding the human insulin protein comprises an ORF sequence presented or mentioned in Table 8.

[0478] In some aspects, the delivery vector comprises an insulin expression cassette containing a polynucleotide encoding human insulin protein, the polynucleotide encoding human insulin containing a wild-type proinsulin secretion signal peptide. In some aspects, the delivery vector comprises an insulin expression cassette containing a polynucleotide encoding human insulin protein, the polynucleotide not encoding a wild-type proinsulin secretion signal peptide. In some aspects, wild-type proinsulin is replaced by a non-insulin secretion signal. In some aspects, the delivery vector comprises an insulin expression cassette containing a polynucleotide encoding human insulin protein, the polynucleotide encoding human proinsulin containing an interleukin-6 (IL-6) secretion signal peptide. In some aspects, the delivery vector comprises an insulin expression cassette containing a polynucleotide encoding a sequence of human insulin protein, the polynucleotide encoding human proinsulin containing a fibronectin secretion signal peptide.

[0479] In some aspects, the delivery vector comprises an insulin expression cassette, the insulin expression cassette comprising a polynucleotide encoding a human insulin protein, the polynucleotide further comprising a 5' UTR, the 5' UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, 100% sequence identity with nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some aspects, the 5' UTR comprises a nucleic acid having a sequence of nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some respects, the polynucleotide encoding the human insulin protein contains the 5' UTR sequence presented or mentioned in Table 8.

[0480] In some aspects, the delivery vector comprises an insulin expression cassette, the insulin expression cassette comprising a polynucleotide encoding a human insulin protein, the polynucleotide further comprising a 3' UTR, the 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, SEQ ID NO: 149, or SEQ ID NO: 171. In some aspects, the 3' UTR comprises a restriction site selected from the group consisting of: Bam HI、 Eco RI Nde I, Eco RV, Spe I, Xba I, Nhe I, Vsp I, Nsi I, Sca I, Kpn I, Ssp I and PacI, and any combination thereof. In some respects, the 3' UTR comprises a nucleic acid having a 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: 149, or SEQ ID NO: 171. In some respects, the polynucleotide encoding the human insulin protein comprises a 3' UTR sequence presented or mentioned in Table 8.

[0481] In some aspects, the delivery vector comprises an insulin expression cassette containing a polynucleotide 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 following: 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: 127, SEQ ID NO: 128, SEQ ID NO: 129, 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: 160, SEQ ID NO: 161 or SEQ ID NO: 170, wherein the polynucleotide 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 aspects, the delivery vector comprises an insulin expression cassette containing a polynucleotide 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 or SEQ ID NO: 170.In some aspects, the delivery vector comprises an insulin expression cassette containing a polynucleotide encoding a human insulin protein, the polynucleotide 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 selected from the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In some aspects, the delivery vector comprises an insulin expression cassette, the insulin expression cassette comprising a polynucleotide encoding a human insulin protein, the polynucleotide having the sequences of the following: 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: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 134, SEQ ID NO: 135 ...9, SEQ ID NO: 129, SEQ ID NO: 129, SEQ ID NO: 129, SEQ ID NO: 129, SEQ ID NO: 129, SEQ ID NO: 129, SEQ ID NO: 129, SEQ ID NO: 12 SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 160, SEQ ID NO: 161, or SEQ ID NO: 170. In some aspects, the delivery vector comprises an insulin expression cassette containing a polynucleotide encoding a human insulin protein having the sequence of SEQ ID NO: 138.In some aspects, the delivery vector comprises an insulin expression cassette containing a polynucleotide encoding a human insulin protein, the polynucleotide having the sequence of SEQ ID NO: 170. In some aspects, the delivery vector comprises an insulin expression cassette containing a polynucleotide encoding a human insulin protein, the polynucleotide comprising nucleic acids 5-957 of each of the following: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In some aspects, the delivery vector comprises an insulin expression cassette containing a polynucleotide encoding a human insulin protein, the polynucleotide comprising the 5' UTR, ORF, and 3' UTR presented or mentioned in Table 8.

[0482] In some aspects, the delivery vector comprises a glucokinase expression cassette, the glucokinase expression cassette comprising a promoter operatively linked to a polynucleotide encoding a human glucokinase protein, the polynucleotide comprising an ORF sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence selected from the following: SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76. SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80 or SEQ ID NO: 162. In some aspects, the delivery vector comprises a glucokinase expression cassette containing a promoter operatively linked to a polynucleotide encoding a human glucokinase protein, the polynucleotide having a sequence of any of the following: SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO: 162. In some aspects, the polynucleotide encoding the human glucokinase protein comprises an ORF sequence presented or mentioned in Table 9.

[0483] In some aspects, the delivery vector comprises a glucokinase expression cassette, the glucokinase expression cassette comprising a polynucleotide encoding a human glucokinase protein, the polynucleotide further comprising a 5' UTR, the 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 nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some aspects, the 5' UTR comprises a nucleic acid having a sequence of nucleic acid 5-329 of SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some respects, the polynucleotide encoding the human glucokinase protein contains the 5' UTR sequence presented or mentioned in Table 9.

[0484] In some aspects, the delivery vector comprises a glucokinase expression cassette, the glucokinase expression cassette comprising a polynucleotide encoding a human glucokinase protein, the polynucleotide further comprising a 3' UTR, the 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 any of the following: 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, SEQ ID NO: 149, SEQ ID NO: 169, or SEQ ID NO: 171. In some aspects, the 3' UTR comprises a restriction site selected from the group consisting of: Bam HI、 Eco RI Nde I, Eco RV, Spe I, Xba I, Nhe I, Vsp I, Nsi I, Sca I, Kpn I, Ssp I and Pac I, and any combination thereof. In some respects, the 3' UTR comprises nucleic acids having sequences having the following: 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, SEQ ID NO: 149, SEQ ID NO: 169, SEQ ID NO: 171. In some respects, the polynucleotide encoding the human glucokinase protein comprises the 3' UTR sequence presented or mentioned in Table 9.

[0485] In some aspects, the delivery vector comprises a glucokinase expression cassette, the glucokinase expression cassette comprising a polynucleotide 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 following: 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, SEQ ID NO: 164, SEQ ID NO: 168, SEQ ID NO: 172, wherein the nucleic acid sequence encodes a human glucokinase protein (e.g., SEQ ID NO: 82) or a functional fragment thereof.In some aspects, the delivery vector comprises a glucokinase expression cassette, the glucokinase expression cassette comprising a polynucleotide encoding a human glucokinase protein, the polynucleotide 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 5-2025 selected from the following sequences: 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 aspects, the delivery vector comprises a glucokinase expression cassette, the glucokinase expression cassette comprising a polynucleotide encoding a human glucokinase protein, the polynucleotide having the sequences of the following: 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: 9 ...98, SEQ ID NO: 99, SEQ ID NO: 99, 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: 99, SEQ ID NO: 99, SEQ ID NO: NO: 96, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 168 or SEQ ID NO: 172.In some aspects, the delivery vector comprises a glucokinase expression cassette containing a polynucleotide encoding a human glucokinase protein, the polynucleotide comprising nucleic acids 5-2025 of the following: 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 aspects, the delivery vector comprises a glucokinase expression cassette containing a polynucleotide encoding a human glucokinase protein, the polynucleotide comprising the 5' UTR, ORF, and 3' UTR presented or mentioned in Table 9.

[0486] In some aspects, the delivery vector may contain a sequence encoding a protein (e.g., insulin and / or Gck) operatively linked to a control or regulatory sequence, optional marker, any fusion complex, and / or other element. In some aspects, a modified nucleic acid is placed in a functional relationship with another nucleic acid sequence. The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of a protein. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology, Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). In some aspects, the expression vector includes transcriptional and translational regulatory nucleic acids operatively linked to a nucleic acid encoding a protein and is generally adapted to a host cell for protein expression. Generally, transcriptional and translational regulatory sequences may include promoter sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or activator sequences. As also known in the art, expression vectors may contain selectable genes or markers to allow selection of transformed host cells containing the expression vector. Selectable genes are known in the art and will vary depending on the host cell used. For example, typically, selectable marker genes confer resistance to drugs (e.g., G418, hygromycin, or methotrexate) in host cells introduced with the vector. In some aspects, selectable marker genes include the dihydrofolate reductase (DHFR) gene (for DHFR host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0487] In some respects, the delivery vector is a viral vector or a gene therapy vector containing a viral expression construct. In other respects, the viral vector or gene therapy vector is a suitable vector for gene therapy.

[0488] In some aspects, the delivery vector containing the expression cassette encoding human insulin and the delivery vector containing the expression cassette encoding human glucokinase are delivered at a ratio selected from the group consisting of: about 1:0.25-0.75 and about 1:0.75-1.25. In some aspects, the delivery vector containing the expression cassette encoding human insulin and the delivery vector containing the expression cassette encoding human glucokinase are delivered at a ratio selected from the group consisting of: about 1:0.4-0.6 and about 1:0.9-1.1. In some respects, delivery vectors containing expression cassettes encoding human insulin and delivery vectors containing expression cassettes encoding human glucokinase are delivered at ratios selected from the group consisting of: approximately 1:0.25–0.75, 1:0.25–0.7, 1:0.25–0.65, 1:0.25–0.60, 1:0.25–0.55, 1:0.25–0.5, 1:0.3–0.7, 1:0.3–0.65, 1:0.3–0.6, 1:0.3–0.55, 1 :0.3-0.5, 1:0.35-0.7, 1:0.35-0.65, 1:0.35-0.6, 1:0.35-0.55, 1:0.35-0.5, 1:0.4-0.7, 1:0.4-0.65, 1:0.4-0.6, 1:0.4-0.55, 1:0.4-0.5, 1:0.45-0.7, 1:0.45-0.65, 1:0.45-0.6, 1:0.45-0.55 and 1:0.45-0.5. In some aspects, delivery vectors comprising expression cassettes encoding human insulin and delivery vectors comprising expression cassettes encoding human glucokinase are delivered at ratios selected from the group consisting of: approximately 1:0.25–0.75, 1:0.3–0.75, 1:0.35–0.75, 1:0.40–0.75, 1:0.45–0.75, and 1:0.5–0.75. In some aspects, delivery vectors comprising expression cassettes encoding human insulin and delivery vectors comprising expression cassettes encoding human glucokinase are delivered at ratios selected from the group consisting of: approximately 1:0.25–0.75, 1:0.3–0.70, 1:0.35–0.65, 1:0.4–0.60, and 1:0.45–0.55.

[0489] In some aspects, the delivery vector containing an expression cassette encoding human insulin and the delivery vector containing an expression cassette encoding human glucokinase are delivered at a ratio of 1:1 to 1:15. In some aspects, the delivery vector containing an expression cassette encoding human insulin and the delivery vector containing an expression cassette encoding human glucokinase are delivered at a ratio of 1:2. In some aspects, the delivery vector containing an expression cassette encoding human insulin and the delivery vector containing an expression cassette encoding human glucokinase are delivered at a ratio of 1:3. In some aspects, the delivery vector containing an expression cassette encoding human insulin and the delivery vector containing an expression cassette encoding human glucokinase are delivered at a ratio of 1:4. In some aspects, the delivery vector containing an expression cassette encoding human insulin and the delivery vector containing an expression cassette encoding human glucokinase are delivered at a ratio of 1:5. In some aspects, the delivery vector containing an expression cassette encoding human insulin and the delivery vector containing an expression cassette encoding human glucokinase are delivered at a ratio of 1:10. In some aspects, the delivery vector containing an expression cassette encoding human insulin and the delivery vector containing an expression cassette encoding human glucokinase are delivered at a ratio of 1:15.

[0490] In some aspects, delivery vectors containing expression cassettes encoding human insulin and delivery vectors containing expression cassettes encoding human glucokinase are delivered at ratios selected from the group consisting of approximately 1:0.25–0.75 and approximately 1:0.75–1.25 (e.g., approximately 1:0.5 and approximately 1:1). In some aspects, the vector ratio (i.e., AAV vector containing the hIns expression cassette: AAV vector containing the hGck expression cassette) is 1:0.4–0.6. In some aspects, the delivery vectors (i.e., AAV vector containing the expression cassette: AAV vector containing the hGck expression cassette) are delivered at a ratio of 1:0.5.

[0491] In some aspects, delivery vectors comprising expression cassettes encoding human insulin and delivery vectors comprising expression cassettes encoding human glucokinase are delivered at ratios selected from the group consisting of: approximately 1:0.75–1.25, 1:0.8–1.25, 1:0.85–1.25, 1:0.9–1.25, 1:0.95–1.25, and 1:1.0–1.25. In some aspects, the vector ratios are selected from the group consisting of: 1:0.75–1.25, 1:0.75–1.20, 1:0.75–1.15, 1:0.75–1.10, 1:0.75–1.05, and 1:0.75–1.00. In some aspects, the vector ratios are selected from the group consisting of: 1:0.75–1:25, 1:0.80–1.20, 1:0.85–1.15, 1:0.90–1.10, and 1:0.95–1.05. In some aspects, delivery vectors containing expression cassettes encoding human insulin and delivery vectors containing expression cassettes encoding human glucokinase are delivered at a 1:1 ratio.

[0492] In some respects, a delivery vector containing an expression cassette encoding human insulin and a delivery vector containing an expression cassette encoding human glucokinase are delivered at a 1:2 ratio.

[0493] In some respects, the delivery vector containing the expression cassette encoding human insulin and the delivery vector containing the expression cassette encoding human glucokinase are delivered in a 1:3 ratio.

[0494] In some respects, the delivery vector containing the expression cassette encoding human insulin and the delivery vector containing the expression cassette encoding human glucokinase are delivered at a 1:4 ratio.

[0495] In some respects, the delivery vector containing the expression cassette encoding human insulin and the delivery vector containing the expression cassette encoding human glucokinase are delivered at a 1:5 ratio.

[0496] In some respects, delivery vectors containing expression cassettes encoding human insulin and delivery vectors containing expression cassettes encoding human glucokinase are delivered at a 1:10 ratio.

[0497] In some respects, the delivery vector containing the expression cassette encoding human insulin...

Claims

1. A method for treating or improving diabetes-related symptoms in a subject in need, wherein the method comprises administering a combination therapy to the subject, the combination therapy comprising: one or more adeno-associated virus (AAV) vectors, the one or more AAV vectors comprising (a) A first promoter operatively linked to a multinucleotide encoding the human insulin (hIns) protein; and (b) A second promoter operatively linked to a multinucleotide encoding the human glucokinase (hGck) protein; The combined therapy is administered intramuscularly (IM) to at least two different skeletal muscle groups.

2. The method of claim 1, wherein the combination therapy comprises: (a) A first AAV vector genome containing an insulin expression cassette, the insulin expression cassette containing a first promoter operatively linked to the polynucleotide encoding the hIns protein, wherein the insulin expression cassette is side-mounted with an inverted terminal repeat (ITR); and (b) A second AAV vector genome containing a glucokinase expression cassette, the glucokinase expression cassette containing a second promoter operatively linked to the polynucleotide encoding the hGck protein, wherein the glucokinase expression cassette is side-mounted with an inverted terminal repeat (ITR).

3. The method of claim 2, wherein the first AAV vector genome and the second AAV vector genome are administered simultaneously or sequentially, optionally wherein the first AAV vector genome and the second AAV vector genome are administered in the same pharmaceutical composition.

4. The method according to any one of claims 1 to 3, wherein the polynucleotide encoding human insulin (hIns) protein and the polynucleotide encoding human glucokinase (hGck) protein are administered in the same pharmaceutical composition.

5. The method according to any one of claims 2 to 4, wherein the first AAV vector genome and the second AAV vector genome are mixed prior to IM injection.

6. The method of claim 1, wherein the one or more AAV vectors are AAV vector genomes comprising an expression cassette, the expression cassette comprising a first promoter operatively linked to the polynucleotide encoding the hIns protein and a second promoter operatively linked to the polynucleotide encoding the hGck protein, wherein the expression cassette is side-mounted with an inverted terminal repeat (ITR).

7. The method according to any one of claims 1 to 6, wherein the combination therapy comprises: (i) a first recombinant AAV (rAAV) particle comprising the genome of the first AAV vector, the first AAV vector genome comprising the insulin expression cassette, the insulin expression cassette comprising the promoter operatively linked to the polynucleotide encoding the human insulin (hIns) protein, wherein the insulin expression cassette is side-mounted with an inverted terminal repeat (ITR); and (ii) a second recombinant AAV (rAAV) particle comprising the genome of the second AAV vector, the second AAV vector genome comprising the glucokinase expression cassette, the glucokinase expression cassette comprising the promoter operatively linked to the polynucleotide encoding the human glucokinase (hGck) protein, wherein the glucokinase expression cassette is side-mounted with an inverted terminal repeat (ITR).

8. The method according to any one of claims 1 to 7, wherein the combination therapy is administered intramuscularly (IM) to at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten different skeletal muscle groups.

9. The method according to any one of claims 1 to 8, wherein the combined therapy is administered intramuscularly (IM) to (i) 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, or 9-10 different skeletal muscle groups; (ii) 2-8, 3-8, 4-8, 5-8, 6-8, or 7-8 different skeletal muscle groups; (iii) 2-6, 3-6, 4-6, or 5-6 different skeletal muscle groups; (iv) 2-5, 3-5, or 4-5 different skeletal muscle groups; or (iv) 2-4 or 3-4 different skeletal muscle groups.

10. The method according to any one of claims 1 to 9, wherein the combined therapy is administered intramuscularly (IM) to two, three, four, five, six, seven, eight, nine, or ten different skeletal muscle groups.

11. The method according to any one of claims 1 to 10, wherein the skeletal muscle group is selected from the quadriceps, biceps, triceps, gastrocnemius, gluteus maximus, hamstrings, deltoids, trapezius, pectoralis major, and latissimus dorsi (lat), optionally wherein the two or more different skeletal muscle groups comprise any combination thereof, optionally wherein the two or more different skeletal muscle groups are selected from i) left quadriceps and right quadriceps, ii) left biceps and right biceps, iii) left triceps and right triceps, iv) left gastrocnemius and right gastrocnemius, v) left gluteus maximus and right gluteus maximus, vi) left hamstrings and right hamstrings, vii) left deltoids and right deltoids, viiii) left trapezius and right trapezius, ix) left pectoralis major and right pectoralis major, and x) left latissimus dorsi and right latissimus dorsi.

12. The method according to any one of claims 1 to 11, wherein the skeletal muscle group is selected from the quadriceps, biceps, triceps, gastrocnemius, gluteus maximus and hamstrings, optionally wherein the two or more different skeletal muscle groups include any combination thereof.

13. The method according to any one of claims 1 to 12, wherein the skeletal muscle group is selected from the quadriceps, biceps, gastrocnemius, gluteus maximus and hamstrings, optionally wherein the two or more different skeletal muscle groups include any combination thereof.

14. The method according to any one of claims 1 to 13, wherein the administration comprises bilateral injection into one or more of the different skeletal muscle groups, optionally wherein the bilateral injection is equally distributed among the respective skeletal muscle groups.

15. The method of claim 14, wherein the bilateral injection comprises IM injection into the right and left quadriceps, the right and left biceps, the right and left triceps, the right and left gastrocnemius, the right and left gluteus maximus and / or the right and left hamstrings, and any combination thereof.

16. The method according to any one of claims 1 to 15, wherein the administration comprises administering a total of 20 to 150 injections of the combined therapy to the at least two different skeletal muscle groups.

17. The method of claim 16, wherein the application comprises administering a total of 20-120 IM injections, 20-110 IM injections, 20-100 IM injections, 20-80 IM injections, 20-60 IM injections, 25-150 IM injections, 25-120 IM injections, 25-110 IM injections, or 25-100 IM injections to the at least two different skeletal muscle groups.

18. The method according to any one of claims 1 to 17, wherein the application comprises: (i) Administer 1-80 IM injections to the first skeletal muscle group and 1-80 IM injections to the second skeletal muscle group; (ii) Administer 1-60 IM injections to the first skeletal muscle group, 1-60 IM injections to the second skeletal muscle group, and 1-60 IM injections to the third skeletal muscle group. (iii) Administer 1-40 IM injections to the first skeletal muscle group, 1-40 IM injections to the second skeletal muscle group, 1-40 IM injections to the third skeletal muscle group, and 1-40 IM injections to the fourth skeletal muscle group. (iv) Administer 1-30 intramural injections to the first skeletal muscle group, 1-30 intramural injections to the second skeletal muscle group, 1-30 intramural injections to the third skeletal muscle group, 1-30 intramural injections to the fourth skeletal muscle group, and 1-30 intramural injections to the fifth skeletal muscle group; and (v) Administer 1-25 IM injections to the first skeletal muscle group, 1-25 IM injections to the second skeletal muscle group, 1-25 IM injections to the third skeletal muscle group, 1-25 IM injections to the fourth skeletal muscle group, 1-25 IM injections to the fifth skeletal muscle group, and 1-25 IM injections to the sixth skeletal muscle group.

19. The method according to any one of claims 1 to 18, wherein the application comprises: (i) Administer 2-80, 4-80, 6-80, 8-80, 10-80, 20-80, 30-80, 40-80, 50-80 or 60-80 IM injections to the first skeletal muscle group and 2-80, 4-80, 6-80, 8-80, 10-80, 20-80, 30-80, 40-80, 50-80 or 60-80 IM injections to the second skeletal muscle group; (ii) Administer 2-60, 4-60, 6-60, 8-60, 10-60, 20-60, 30-60, 40-60, or 50-60 IM injections to the first skeletal muscle group; administer 2-60, 4-60, 6-60, 8-60, 10-60, 20-60, 30-60, 40-60, or 50-60 IM injections to the second skeletal muscle group; and administer 2-60, 4-60, 6-60, 8-60, 10-60, 20-60, 30-60, 40-60, or 50-60 IM injections to the third skeletal muscle group; (iii) Administer 2-40, 4-40, 6-40, 8-40, 10-40, 20-40, or 30-40 IM injections to the first skeletal muscle group; administer 2-40, 4-40, 6-40, 8-40, 10-40, 20-40, or 30-40 IM injections to the second skeletal muscle group; administer 2-40, 4-40, 6-40, 8-40, 10-40, 20-40, or 30-40 IM injections to the third skeletal muscle group; and administer 2-40, 4-40, 6-40, 8-40, 10-40, 20-40, or 30-40 IM injections to the fourth skeletal muscle group; (iv) Administer 2-30, 4-30, 6-30, 8-30, 10-30, or 20-30 IM injections to the first skeletal muscle group; 2-30, 4-30, 6-30, 8-30, 10-30, or 20-30 IM injections to the second skeletal muscle group; 2-30, 4-30, 6-30, 8-30, 10-30, or 20-30 IM injections to the third skeletal muscle group; 2-30, 4-30, 6-30, 8-30, 10-30, or 20-30 IM injections to the fourth skeletal muscle group; and 2-30, 4-30, 6-30, 8-30, 10-30, or 20-30 IM injections to the fifth skeletal muscle group; or (v) Administer 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 IM injections to the first skeletal muscle group; administer 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 IM injections to the second skeletal muscle group; administer 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 IM injections to the third skeletal muscle group; Administer 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 IM injections to the fourth skeletal muscle group; administer 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 IM injections to the fifth skeletal muscle group; and administer 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 IM injections to the sixth skeletal muscle group.

20. The method of claim 19, wherein the application comprises administering 2-50, 4-50, 6-50, 8-50, 10-50, 20-50, 30-50, or 40-50 intramolecular injections to a first skeletal muscle group, administering 2-50, 4-50, 6-50, 8-50, 10-50, 20-50, 30-50, or 40-50 intramolecular injections to a second skeletal muscle group, and administering 2-50, 4-50, 6-50, 8-50, 10-50, 20-50, 30-50, or 40-50 intramolecular injections to a third skeletal muscle group.

21. The method according to any one of claims 1 to 20, wherein the application comprises 8-50 intramimetic injections to the quadriceps, 8-50 intramimetic injections to the biceps, and 8-50 intramimetic injections to the hamstrings.

22. The method of claim 21, wherein the application comprises bilateral 4-25 IM injections to each quadriceps muscle, bilateral 4-25 IM injections to each biceps muscle, and bilateral 4-25 IM injections to each hamstring muscle.

23. The method according to any one of claims 1 to 22, wherein each IM injection is administered in a volume of about 100 μL to about 1.5 mL, optionally about 0.5 mL to about 1 mL.

24. The method of claim 23, wherein each IM injection is administered in the following volumes: about 100 μL to about 2 mL, 100 μL to about 1.9 mL, 100 μL to about 1.8 mL, 100 μL to about 1.7 mL, 100 μL to about 1.6 mL, 100 μL to about 1.5 mL, 100 μL to about 1.4 mL, about 100 μL to about 1.35 mL, about 100 μL to about 1.2 mL, about 100 μL to about 1.0 mL, about 100 μL to about 800 μL, about 200 μL to about 800 μL, about 200 μL to about 600 μL, about 400 μL to about 800 μL, about 500 μL to about 1.5 mL, about 600 μL to about 1.4 mL, about 700 μL to about 1.3 mL, about 800 μL to about 1.2 mL, or about 900 μL to about 1.1 mL.

25. The method according to any one of claims 1 to 24, wherein the total cumulative volume of all IM injections administered is about 20 mL to about 70 mL, about 20 mL to about 50 mL, or about 30 mL to about 40 mL.

26. The method according to any one of claims 1 to 25, wherein each IM injection comprises about 8 × 10 10 Approximately 5×10 13 vg / mL, optionally the IM injection contains about 2 × 10 vg / mL. 13 Approximately 4×10 13 vg / mL.

27. The method according to any one of claims 1 to 26, wherein the IM application comprises injections spaced at least about 1-5 cm, optionally at least about 1-3 cm apart.

28. The method according to any one of claims 1 to 27, wherein the interval between each IM injection is at least 1 cm, at least 2 cm, at least 3 cm, at least 4 cm, or at least 5 cm.

29. The method according to any one of claims 1 to 28, wherein the application comprises an intramural injection, the intramural injection being at least 0.5 cm deep, at least 0.6 cm deep, at least 0.7 cm deep, at least 0.8 cm deep, at least 0.9 cm deep, at least 1.0 cm deep, at least 1.1 cm deep, at least 1.2 cm deep, at least 1.3 cm deep, at least 1.4 cm deep, at least 1.5 cm deep, at least 2.0 cm deep, at least 2.5 cm deep, at least 3.0 cm deep, at least 3.5 cm deep, at least 4.0 cm deep, at least 4.5 cm deep, or at least 5.0 cm deep.

30. The method according to any one of claims 1 to 29, wherein each IM injection is to a depth of about 0.5 cm to 2.0 cm, optionally about 1-2 cm.

31. The method according to any one of claims 1 to 30, wherein each IM injection is performed at least 0.5 cm below the surface of the injected muscle, regardless of the thickness of the subcutaneous fat.

32. The method according to any one of claims 1 to 31, further comprising administering an immunosuppressive regimen comprising an immunosuppressant to the subject.

33. A method of treating or improving diabetes-related symptoms in a subject in need, comprising administering to the subject (i) an immunosuppressive regimen comprising an immunosuppressant, and (ii) a combination therapy comprising one or more adeno-associated virus (AAV) vectors, said one or more AAV vectors comprising: (a) A first promoter operatively linked to a multinucleotide encoding the human insulin (hIns) protein; and (b) A second promoter operatively linked to a multinucleotide encoding a human glucokinase (hGck) protein, wherein the combination therapy is administered intramuscularly (IM) to at least two different skeletal muscle groups.

34. The method of claim 32 or 33, wherein the immunosuppressive regimen comprises administering one or more immunosuppressants before (before AAV), simultaneously with and / or after (after AAV) administration of the combination therapy.

35. The method according to any one of claims 32 to 34, wherein the immunosuppressive regimen comprises administering the immunosuppressant prior to administering the combination therapy.

36. The method according to any one of claims 32 to 35, wherein the immunosuppressive regimen comprises administering the immunosuppressant after administering the combination therapy.

37. The method according to any one of claims 32 to 36, wherein the immunosuppressant is administered 1 to 14 days prior to the administration of the combination therapy.

38. The method according to any one of claims 34 to 37, wherein the immunosuppressant is administered 1 to 7 days prior to the administration of the combination therapy.

39. The method according to any one of claims 34 to 38, wherein the immunosuppressant is administered 1 to 3 days prior to the administration of the combination therapy.

40. The method according to any one of claims 32 to 38, wherein the immunosuppressant is administered 3 to 7 days prior to the administration of the combination therapy.

41. The method according to any one of claims 32 to 40, wherein the immunosuppressant is further administered 1, 2, 3 or 4 weeks after administration of the combination therapy.

42. The method according to any one of claims 32 to 41, wherein the immunosuppressant is administered approximately every 1 to 3 weeks after administration of the combination therapy, optionally approximately every 1 to 2 weeks.

43. The method according to any one of claims 32 to 42, wherein the immunosuppressant is administered for about 1 to 6 months, optionally about 1 to 3 months, following the administration of the combination therapy.

44. The method according to any one of claims 32 to 43, wherein the immunosuppressive regimen comprises a second immunosuppressant.

45. The method of claim 44, wherein the immunosuppressive regimen comprises administering the second immunosuppressant prior to administering the combination therapy.

46. ​​The method of claim 44 or 45, wherein the immunosuppressive regimen comprises administering the second immunosuppressant after administering the combination therapy.

47. The method according to any one of claims 44 to 46, wherein the second immunosuppressant is administered 1 to 14 days prior to administration of the combination therapy.

48. The method according to any one of claims 44 to 47, wherein the second immunosuppressant is administered 1 to 7 days prior to the administration of the combination therapy.

49. The method according to any one of claims 44 to 48, wherein the second immunosuppressant is administered 1 to 3 days prior to the administration of the combination therapy.

50. The method according to any one of claims 44 to 48, wherein the second immunosuppressant is administered 3 to 7 days prior to the administration of the combination therapy.

51. The method according to any one of claims 44 to 50, wherein the second immunosuppressant is further administered 1, 2, 3 or 4 weeks after administration of the combination therapy.

52. The method according to any one of claims 44 to 51, wherein the second immunosuppressant is administered twice daily, once daily, or every other day.

53. The method according to any one of claims 44 to 52, wherein the second immunosuppressant is administered for about 1 to 6 months, optionally about 1 to 3 months, following the administration of the combination therapy.

54. The method according to any one of claims 32 to 53, wherein the immunosuppressive regimen comprises administering an immunosuppressant selected from the group consisting of: IL-6 antagonists, IL-1 antagonists, TNFα antagonists, mTOR pathway inhibitors, mycophenolate mofetil (MMF), tyk2 inhibitors, methotrexate, abatacept, anti-thymocyte globulin (ATG), B-cell depletion agents, T-cell co-stimulation antagonists, T-cell depletion agents, corticosteroids, calcineurin inhibitors, or any combination thereof.

55. The method according to any one of claims 32 to 54, wherein the immunosuppressant comprises an IL-6 antagonist, an IL-1 antagonist, MMF, a tyk2 inhibitor, rapamycin, abatacept, or any combination thereof.

56. The method of claim 54 or claim 55, wherein the IL-6 antagonist is selected from the group consisting of: tocilizumab, sarrizumab, satelizumab, staxicizumab, olozazumab, esimomomab, clazazumab, cilukumab, levelizumab, or any combination thereof.

57. The method of claim 56, wherein the IL-6 antagonist is tocilizumab.

58. The method according to any one of claims 32 to 57, wherein the second immunosuppressant comprises a calcineurin inhibitor, MMF, methotrexate, tyk2 inhibitor, rapamycin, abatacept, anti-thymocyte globulin, etanercept, balithimab, sirolimus, or any combination thereof.

59. The method of claim 58, wherein the calcineurin inhibitor is selected from the group consisting of cyclosporine, tacrolimus, vorciclosporine, and any combination thereof.

60. The method of claim 59, wherein the calcineurin inhibitor is tacrolimus.

61. The method according to any one of claims 32 to 60, wherein the immunosuppressive regimen comprises administering tocilizumab, optionally subcutaneously, on about day -3 and about weekly or about every other week relative to the combination therapy, up to about 12 weeks.

62. The method according to any one of claims 32 to 61, wherein the immunosuppressive regimen comprises administering tacrolimus, optionally orally, on approximately day -3 and every 1 to 3 days relative to the combination therapy, for approximately 3 to 12 weeks.

63. The method according to any one of claims 2 to 62, wherein the total administration dose of the first AAV vector genome comprising the insulin expression cassette comprises about 7 × 10⁻⁶. 10 Approximately 5×10 13 Vector genome (vg) / kg.

64. The method according to any one of claims 2 to 63, wherein the total administration dose of the second AAV vector genome comprising the glucokinase expression cassette comprises about 5 × 10⁻⁶. 11 Approximately 5×10 13 Vector genome (vg) / kg.

65. The method according to any one of claims 32 to 64, wherein the ratio of the first AAV vector genome comprising the insulin expression cassette to the second AAV vector genome comprising the glucokinase expression cassette is approximately 1:0.25-1.25, 1:1-20, 1:2-20, 1:3-20, 1:4-20, 1:5-20, 1:1-18, 1:2-18, 1:3-18, 1:4-18, 1:5-18, or 1:1-1 6, 1:2-16, 1:3-16, 1:4-16, 1:5-16, 1:1-15, 1:2-15, 1:3-15, 1:4-15, 1:5-15, 1:1-14, 1:2-14, 1:3-14, 1:4-14, 1:5-14, 1:1-12, 1:2-12, 1:3-12, 1:4-12, 1:5-12, 1:1-10, 1:2-10, 1:3-10, 1:4-10 or 1:5-10.

66. The method according to any one of claims 32 to 65, wherein the ratio of the first AAV vector genome comprising the insulin expression cassette to the second AAV vector genome comprising the glucokinase expression cassette is about 1:1-15, 1:2-15, 1:3-15, 1:4-15, 1:5-15, 1:1-14, 1:2-14, 1:3-14, 1:4-14, 1:5-14, 1:1-12, 1:2-12, 1:3-12, 1:4-12, 1:5-12, 1:1-10, 1:2-10, 1:3-10, 1:4-10, or 1:5-10.

67. The method according to any one of claims 32 to 66, wherein the ratio of the first AAV vector genome comprising the insulin expression cassette to the second AAV vector genome comprising the glucokinase expression cassette is about 1:1-10, 1:2-10, 1:3-10, 1:4-10, or 1:5-10.

68. The method according to any one of claims 32 to 66, wherein the ratio of the first AAV vector genome comprising the insulin expression cassette to the second AAV vector genome comprising the glucokinase expression cassette is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, or about 1:

15.

69. The method according to any one of claims 32 to 66, wherein the ratio of the first AAV vector genome comprising the insulin expression cassette to the second AAV vector genome comprising the glucokinase expression cassette is about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:8, about 1:9, or about 1:

10.

70. The method according to any one of claims 2 to 69, wherein the first AAV vector genome comprising an insulin expression cassette is at approximately 5 × 10⁻⁶. 11 With approximately 1×10 13 Dosage between vg / kg.

71. The method according to any one of claims 2 to 70, wherein the first AAV vector genome comprising an insulin expression cassette is at approximately 2 × 10⁻⁶. 12 With approximately 1×10 13 Dosage between vg / kg.

72. The method according to any one of claims 2 to 71, wherein the first AAV vector genome comprising an insulin expression cassette is at approximately 4 × 10⁻⁶. 12 To approximately 1×10 13 Administer at a dose of vg / kg.

73. The method according to any one of claims 2 to 69, wherein the first AAV vector genome comprising the insulin expression cassette is at approximately 7 × 10⁻⁶. 10 Approximately 7×10 12 Administer at a dose of vg / kg.

74. The method according to any one of claims 2 to 69, wherein the first AAV vector genome comprising an insulin expression cassette is at approximately 5 × 10⁻⁶. 11 Approximately 5×10 12 Administer at a dose of vg / kg.

75. The method according to any one of claims 2 to 69, wherein the first AAV vector genome comprising the insulin expression cassette is expressed at approximately 1 × 10⁻⁶. 11 To approximately 1×10 12 Administer at a dose of vg / kg.

76. The method according to any one of claims 2 to 75, wherein the second AAV vector genome comprising a glucokinase expression cassette is expressed at approximately 1 × 10⁻⁶. 12 With approximately 5×10 12 Dosage between vg / kg.

77. The method according to any one of claims 2 to 76, wherein the second AAV vector genome comprising a glucokinase expression cassette is expressed at approximately 2 × 10⁻⁶. 12 With approximately 5×10 12 Dosage between vg / kg.

78. The method according to any one of claims 2 to 77, wherein the second AAV vector genome comprising a glucokinase expression cassette is expressed at approximately 2 × 10⁻⁶. 12 Approximately 3.5 × 10 12 Administer at a dose of vg / kg.

79. The method according to any one of claims 2 to 77, wherein the second AAV vector genome comprising a glucokinase expression cassette is at approximately 3.5 × 10⁻⁶. 12 Approximately 5×10 12 Administer at a dose of vg / kg.

80. The method according to any one of claims 1 to 79, wherein within 15 days to 6 months, 30 to 120 days, or 30 to 60 days after administration of the combination therapy: (i) the glycated hemoglobin (HbA1c) level in the subject is reduced and / or regulated, (ii) the circulating ketones in the subject are reduced, (iii) the triglycerides in the subject are reduced, (iv) the exogenous insulin in the subject is reduced, (v) the circulating C-peptide level in the subject is increased, or (vi) any combination thereof.

81. The method according to any one of claims 1 to 80, wherein, when measured 15 days to 6 months, 30 to 120 days, 30 to 60 days, or 60 to 90 days after administration of the combination therapy, the circulating insulin level in the subject's blood is about 1-40 μU / mL, optionally about 5-40 μU / mL, about 5-25 μU / mL, about 5-10 μU / mL, about 10-20 μU / mL, about 10-15 μU / mL, about 15-20 μU / mL, or about 15-25 μU / mL.

82. The method according to any one of claims 1 to 81, wherein within 15 days to 6 months, 30 to 120 days, 30 to 60 days, or 60 to 90 days after administration of the combination therapy, the subject’s HbA1C level decreased by about 0.5 to 1.5% compared to before treatment.

83. The method according to any one of claims 1 to 82, wherein the subject's HbA1C level is less than about 7.0%, optionally less than about 6.5%, about 6.0%, about 5.7%, or optionally about 5.0% to 6.5%, within 15 days to 6 months, 30 to 120 days, 30 to 60 days, or 60 to 90 days after administration of the combination therapy.

84. The method according to any one of claims 1 to 83, wherein the circulating glucose in the blood of the subject is at least about 70 to 140 mg / dL within 15 days to 6 months, 30 to 120 days, 30 to 60 days, or 60 to 90 days after administration of the combination therapy.

85. The method according to any one of claims 1 to 84, wherein the diabetes is type 1 diabetes mellitus (T1DM).

86. The method according to any one of claims 1 to 84, wherein the diabetes is type 2 diabetes mellitus (T2DM).

87. The method according to any one of claims 1 to 86, wherein the subject does not have insulin resistance.

88. The method according to any one of claims 1 to 86, wherein the subject has insulin resistance.

89. A dosing regimen comprising: (a) Primary immunosuppressant; (b) A combination therapy administered intramuscularly (IM) following (a), the combination therapy comprising (i) a first recombinant AAV (rAAV) particle containing a first AAV vector genome containing an insulin expression cassette, the insulin expression cassette containing a first promoter operatively linked to a polynucleotide encoding human insulin (hIns) protein, wherein the insulin expression cassette is side-mounted with an inverted terminal repeat (ITR); and (ii) a second recombinant AAV (rAAV) particle containing a second AAV vector genome containing a glucokinase expression cassette, the glucokinase expression cassette containing a second promoter operatively linked to a polynucleotide encoding human glucokinase (hGck) protein, wherein the glucokinase expression cassette is side-mounted with an inverted terminal repeat (ITR); and (c) Secondary immunosuppressant.

90. The dosing regimen of claim 89, wherein the first immunosuppressant comprises an IL-6 antagonist selected from the group consisting of tocilizumab, sarrizumab, staxiciumab, olozazumab, esimomomab, clazazumab, cilukumab, levelizumab, and any combination thereof.

91. The dosing regimen according to claim 89 or 90, wherein the second immunosuppressant is a calcineurin inhibitor selected from the group consisting of cyclosporine, tacrolimus, vorticol, and any combination thereof.

92. The dosing regimen according to any one of claims 89 to 91, wherein the first immunosuppressant comprises tocilizumab and the second immunosuppressant comprises tacrolimus.

93. The dosing regimen according to any one of claims 89 to 92, comprising administering tocilizumab, optionally subcutaneously, on about day -3 and about weekly or about every other week relative to the combination therapy, up to about 12 weeks.

94. The dosing regimen according to any one of claims 89 to 93, wherein the immunosuppressive regimen comprises administration of tacrolimus, optionally orally, on approximately day -3 and every 1 to 3 days relative to the combination therapy, for approximately 3 to 12 weeks.

95. The method or dosing regimen according to any one of claims 1 to 94, wherein: (a) The polynucleotide encoding the hIns protein comprises an open reading frame (ORF) containing at least 85%, 90%, 95%, 99%, or 100% identical nucleotide sequences to any one of the following: (i) nucleic acids 73-330 of any one of SEQ ID NO: 43-57 or 110-116, or nucleic acids 88-345 of any one of SEQ ID NO: 117-122; or (ii) SEQ ID NO: 43-57 or SEQ ID NO: 110-122; and / or (b) The polynucleotide encoding the human glucokinase hGck protein comprises an ORF, the ORF comprising (i) a nucleotide sequence that is at least 85%, 90%, 95%, 99%, or 100% identical to a sequence selected from any of the following: (a) nucleic acids 1-1398 of any of SEQ ID NO: 61-80 or 162; or (ii) SEQ ID NO: 61-80 and 162.

96. The method or dosing regimen according to any one of claims 1 to 95, wherein: (a) The polynucleotide encoding the hIns protein comprises an open reading frame (ORF) containing at least 85%, 90%, 95%, 99%, or 100% of the same nucleotide sequence as SEQ ID NO: 121; and / or (b) The polynucleotide encoding the human glucokinase hGck protein comprises an ORF containing at least 85%, 90%, 95%, 99%, or 100% of the same nucleotide sequence as SEQ ID NO:

68.

97. The method or administration regimen according to any one of claims 1 to 96, wherein the hIns protein comprises the amino acid sequence of any one of the following: amino acids 25-110 of SEQ ID NO: 41, amino acids 25-110 of SEQ ID NO: 144, amino acids 25-110 of SEQ ID NO: 145, SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO:

145.

98. The method or administration regimen according to any one of claims 1 to 97, wherein the hIns protein comprises a signal peptide.

99. The method or administration regimen of claim 98, wherein the signal peptide is a wild-type proinsulin signal sequence, an IL-6 signal sequence, a fibronectin signal sequence, or a non-wild-type proinsulin signal sequence.

100. The method or dosing regimen according to claim 98 or 99, wherein the signal peptide comprises amino acids 1-24 of SEQ ID NO: 41, amino acids 1-24 of SEQ ID NO: 144, amino acids 1-24 of SEQ ID NO: 145, or SEQ ID NO:

165.

101. The method or administration regimen according to any one of claims 1 to 100, wherein the hIns protein further comprises a cleavage site.

102. The method or administration regimen according to any one of claims 1 to 101, wherein the polynucleotide encoding the hIns protein further comprises a 5' UTR, the 5' UTR comprising at least 85%, 90%, 95%, 99%, or 100% identical nucleotide sequences to nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO:

148.

103. The method or dosing regimen according to any one of claims 1 to 102, wherein the polynucleotide encoding the hIns protein further comprises a 3' UTR, the 3' UTR comprising at least 85%, 90%, 95%, 99%, or 100% identical nucleotide sequences 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, SEQ ID NO: 149, or SEQ ID NO:

171.

104. The method or administration regimen according to any one of claims 1 to 103, wherein the encoded hGck protein comprises the amino acid sequence of SEQ ID NO:

82.

105. The method or administration regimen according to any one of claims 1 to 104, wherein the polynucleotide encoding the hGck protein further comprises a 5' UTR, the 5' UTR comprising at least 85%, 90%, 95%, 99%, or 100% identical nucleotide sequences to nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO:

148.

106. The method or dosing regimen according to any one of claims 1 to 105, wherein the polynucleotide encoding the hGck protein further comprises a 3' UTR, the 3' UTR comprising at least 85%, 90%, 95%, 99%, or 100% identical nucleotide sequences 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, SEQ ID NO: 149, SEQ ID NO: 169, or SEQ ID NO:

171.

107. The method or dosing regimen according to any one of claims 1 to 106, wherein the insulin expression cassette promoter and / or the glucokinase expression cassette promoter are eukaryotic promoters.

108. The method or dosing regimen according to any one of claims 1 to 106, wherein the insulin expression cassette promoter and / or the glucokinase expression cassette promoter are constitutive promoters.

109. The method or dosing regimen according to any one of claims 1 to 106, wherein the insulin expression cassette promoter and / or the glucokinase expression cassette promoter are ubiquitous promoters.

110. The method or dosing regimen according to any one of claims 1 to 109, wherein the insulin expression cassette promoter and / or the glucokinase expression cassette promoter is a CMV promoter.

111. The method or dosing regimen according to any one of claims 1 to 107, wherein the insulin expression cassette promoter and / or the glucokinase expression cassette promoter are muscle-specific promoters.

112. The method or administration regimen according to any one of claims 1 to 111, wherein the insulin expression cassette further comprises a polyadenylation (polyA) element.

113. The method or administration regimen according to any one of claims 1 to 112, wherein the glucokinase expression cassette further comprises a polyadenylation (polyA) element.

114. The method or dosing regimen according to any one of claims 2 to 113, wherein the first recombinant AAV (rAAV) particle contains the first AAV vector genome, and the second recombinant AAV (rAAV) particle contains the second vector genome.

115. The method or dosing regimen of claim 114, wherein the AAV serotype of the first rAAV particle and / or the second rAAV particle is selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh9, AAV9, AAVrh10, AAV10, AAV11, AAV12, and AAVrh74.

116. The method or dosing regimen according to claim 114 or 115, wherein the AAV serotype of the first rAAV particle and / or the second rAAV particle is AAV1.

117. The method or administration regimen according to any one of claims 114 to 116, wherein the first rAAV particle and the second rAAV particle are formulated in a single pharmaceutical composition.

118. The method or administration regimen according to any one of claims 114 to 116, wherein the first rAAV particle and the second rAAV particle are administered simultaneously or sequentially.

119. A method of immunosuppression in a subject receiving AAV gene therapy for the treatment of diabetes, wherein the subject is given an immunosuppression regimen comprising (a) administering an immunosuppressant before the administration of the AAV gene therapy (pre-AAV); and (b) administering an immunosuppressant after the administration of the AAV gene therapy (post-AAV).

120. The method of claim 119, wherein the pre-AAV immunosuppressant is administered 1 to 14 days, 1 to 7 days, 1 to 3 days, or 3 to 7 days prior to administration of the AAV gene therapy.

121. The method of claim 119 or 120, wherein the AAV post-immunosuppressant is further administered for 1, 2, 3 or 4 weeks after administration of the AAV gene therapy.

122. The method according to any one of claims 119 to 121, wherein the AAV post-immunosuppressant is administered approximately every 1 to 3 weeks, optionally approximately every 1 to 2 weeks, following the administration of the combination therapy.

123. The method according to any one of claims 119 to 122, wherein the AAV post-immunosuppressant is administered for about 1 to 6 months, optionally about 1 to 3 months, after administration of the AAV gene therapy.

124. The method according to any one of claims 119 to 123, wherein the AAV post-immunosuppressant administration lasts for about 6 to 40, about 8 to 40, about 10 to 40, about 12 to 40, about 6 to 30, about 8 to 30, about 10 to 30, about 12 to 30, about 6 to 20, about 8 to 20, about 10 to 20, or about 12 to 20 weeks.

125. The method according to any one of claims 119 to 124, wherein the pre-AAV immunosuppressant and / or the post-AAV immunosuppressant are administered for about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.

126. The method according to any one of claims 119 to 125, wherein the pre-AAV immunosuppressant and / or the post-AAV immunosuppressant comprises an IL-6 antagonist, an IL-1 antagonist, a TNFα antagonist, an mTOR pathway inhibitor, mycophenolate mofetil (MMF), a tyk2 inhibitor, methotrexate, rapamycin, abatacept, anti-thymocyte globulin (ATG), a B-cell depletion agent, a T-cell co-stimulation antagonist, a T-cell depletion agent, a corticosteroid, a calcineurin inhibitor, or any combination thereof.

127. The method according to any one of claims 119 to 126, wherein the pre-AAV immunosuppressant and / or post-AAV immunosuppressant comprises an IL-6 antagonist, an IL-1 antagonist, MMF, a tyk2 inhibitor, rapamycin, abatacept, or any combination thereof.

128. The method according to any one of claims 119 to 127, wherein the pre-AAV immunosuppressant and / or the post-AAV immunosuppressant comprises calcineurin inhibitor, MMF, methotrexate, tyk2 inhibitor, rapamycin, abatacept, or any combination thereof.

129. The method according to any one of claims 119 to 128, wherein the IL-6 antagonist is selected from the group consisting of tocilizumab, sarrlumab, staxicimab, olozazumab, esimomomab, clazazumab, cilukumab, levelizumab, or any combination thereof.

130. The method of claim 129, wherein the IL-6 antagonist is tocilizumab.

131. The method according to any one of claims 126 to 130, wherein the calcineurin inhibitor is selected from the group consisting of cyclosporine, tacrolimus, vorciclosporine, and any combination thereof.

132. The method of claim 131, wherein the calcineurin inhibitor is tacrolimus.

133. The method according to any one of claims 119 to 132, comprising administering tocilizumab, optionally subcutaneously, on about day -3 and about weekly or about every other week, relative to the AAV gene therapy, up to about 12 weeks.

134. The method according to any one of claims 119 to 133, comprising administering tacrolimus, optionally orally, on approximately day -3 and every 1 to 3 days relative to the AAV gene therapy, for up to approximately 3 to 12 weeks.

135. The method of claims 119 to 134, wherein the AAV gene therapy is administered to the subject via intramuscular (IM) injection.

136. The method of claim 135, wherein the IM injection is performed on at least two different skeletal muscle groups.

137. The method of claim 136, wherein the skeletal muscle group is selected from the quadriceps, biceps, triceps, gastrocnemius, gluteus maximus, hamstrings, deltoid, trapezius, pectoralis major, and latissimus dorsi.

138. The method according to claim 136 or 137, wherein the skeletal muscle group is selected from the quadriceps, biceps, triceps, gastrocnemius, gluteus maximus and hamstrings.

139. The method according to any one of claims 119 to 137, wherein the administration comprises bilateral injection into one or more of the said skeletal muscle groups.

140. The method of claim 139, wherein the bilateral injection comprises injecting IM into the right and left quadriceps, right and left biceps, right and left triceps, right and left gastrocnemius, right and left gluteus maximus and / or right and left hamstrings.

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