Aav mediated insulin and glucokinase gene therapy for treating diabetes

Abstract

The present disclosure relates to a combination therapy comprising a first AAV vector genome comprising an insulin expression cassette and a second AAV vector genome comprising a glucokinase expression cassette which 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 methods for using the same for treating diabetes.

Description

AAV MEDIATED INSULIN AND GLUCOKINASE GENE THERAPY FOR TREATING DIABETESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the priority benefit of 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 by reference in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The content of the electronically submitted sequence listing in .XML file (4525_129PC02_SequenceListing_ST26.xml; Size: 302,607 bytes; and Date of Creation: October 24, 2024) filed with the application is incorporated herein by reference in its entirety.BACKGROUND

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

[0004] T1DM is characterized by a severe lack of insulin production due to specific destruction of the pancreatic P-cells. [3-cell loss in T1DM is the result of an autoimmune mediated process, in which a chronic inflammation called insulitis causes [3-cell destruction (Eizirik D. L. et al, 2001, Diabetologia, 44:2115-2133 and Mathis D et al, 2001, Nature, 414: 792-798). T1DM is one of the most common endocrine and metabolic conditions of childhood, and incidence is rapidly increasing, especially among young children. T1DM is diagnosed when the autoimmune-mediated [3-cell destruction is almost complete, causing patients to need insulin-replacement therapy to survive. T1DM in an adult may present itself similarly to T2DM, with a slow deterioration in metabolic control, and subsequent progression to insulin dependency. This form is called latent autoimmune diabetes mellitus in adults (LADA) (Diabetes Atlas 4th edition, 2009, International Diabetes Federation).

[0005] T2DM is the most common form of diabetes mellitus and has been attributed to an interaction between genetic, environmental, and behavioral risk factors. T2DM is characterized by insulin insensitivity, declining insulin production, and eventual pancreatic [3-cell failure (Olokoba, A. et al, 2012, Oman Med. J. 27(4):269-273).

[0006] The reduction of hyperglycemia and maintenance of normoglycemia is a goal of any therapeutic approach to T1DM and T2DM. The current therapy for all T1DM and a large subset of T2DM patients is based on regular subcutaneous injections of both shortacting and long-acting insulin preparations.

[0007] Lifelong insulin treatment is often the therapy of choice for both T1DM and T2DM. While lifelong treatment with exogenous insulin has been largely successful in managing diabetes, diabetic complications can still occur due to difficulties with maintaining tight glycemic control. States of prolonged hyperglycemia can lead to severe microvascular or macrovascular complications, most commonly presenting as retinopathies, neuropathies, nephropathies, cerebrovascular accidents, or myocardial infarctions. These devastating complications can be prevented with improvements in glycemic control. Of note, brittle diabetes, which is a particularly labile form, can be very difficult to manage even with lifelong exogenous insulin.

[0008] AAV gene therapy strategies offer a new treatment paradigm for diabetes. One challenge with AAV gene therapy generally includes the risk of host immune responses to the AAV capsid or the transgene-encoded protein(s), which have been observed in various clinical and preclinical studies. Such immune responses can adversely impact patients’ health, prevent viral transduction, prevent repeated dosing strategies, eliminate transduced cells, and pose a significant barrier to the potential effectiveness of AAV gene therapy. This challenge is particularly great in the diabetes patient population because these patients are more sensitive to the immunosuppressive regimens commonly used with gene therapy, like steroids.FIELD OF DISCLOSURE

[0009] The present disclosure pertains to the medical field, including AAV gene therapy compositions comprising nucleic acids encoding insulin and glucokinase as well as immunosuppressive regimens for use with the same for treatment of diabetes.BRIEF SUMMARY

[0010] Certain aspects of the disclosure are directed to a method of treating or ameliorating the symptoms associated with diabetes in a subject in need thereof, wherein the method comprises administering a therapy (e.g., a combination gene therapy) to the subject comprising: one or more adeno-associated virus (AAV) vectors comprising (a) a first promoter operably linked to a polynucleotide encoding a human insulin (hlns) protein; and (b) a second promoter operably linked to a polynucleotide encoding a human glucokinase (hGck) protein; wherein the therapy is administered intramuscularly (IM) to at least two different skeletal muscle groups.

[0011] In some aspects, the therapy (e.g., combination gene therapy) comprises:(a) a first AAV vector genome comprising an insulin expression cassette comprising a first promoter operably linked to a polynucleotide encoding a human insulin (hlns) protein, wherein the insulin expression cassette is flanked by inverted terminal repeats (ITRs); and(b) a second AAV vector genome comprising a glucokinase expression cassette comprising a second promoter operably linked to a polynucleotide encoding a human glucokinase (hGck) protein, wherein the glucokinase expression cassette is flanked by inverted terminal repeats (ITRs); wherein the therapy is administered intramuscularly (IM) to at least two different skeletal muscle groups.

[0012] In some aspects, the first AAV vector genome and the second AAV vector genome are administered in the same composition (e.g., via the same IM injection).

[0013] In some aspects, 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).

[0014] In some aspects, the one or more AAV vectors is an AAV vector genome comprising an single expression cassette comprising a promoter (e.g., a first and a second promoter), the polynucleotide encoding the hlns protein, and the polynucleotide encoding the hGck protein, wherein the single expression cassette is flanked by inverted terminal repeats (ITRs).

[0015] In some aspects, 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 leasteight, at least nine, or at least ten different skeletal muscle groups. In some aspects, wherein the combination therapy is administered intramuscularly (IM) 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 is administered intramuscularly (IM) to two, three, four, five, six, seven, eight, nine, or ten different skeletal muscle groups.

[0016] In some aspects, the 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; (ii) 2-5, 3-5, or 4-5 different skeletal muscle groups; (iv) 2-4 or 3-4 different skeletal muscle groups.

[0017] In some aspects, the combination therapy comprises (i) a first recombinant AAV (rAAV) particle comprising the first AAV vector genome comprising the insulin expression cassette comprising the promoter operably linked to the polynucleotide encoding the human insulin (hlns) protein, wherein the insulin expression cassette is flanked by inverted terminal repeats (ITRs); and (ii) a second recombinant AAV (rAAV) particle comprising the second AAV vector genome comprising the glucokinase expression cassette comprising the promoter operably linked to the polynucleotide encoding the human glucokinase (hGck) protein, wherein the glucokinase expression cassette is flanked by inverted terminal repeats (ITRs).

[0018] In some aspects, the different skeletal muscle groups can comprise a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and a latissimus dorsi (lat). In some aspects, the skeletal muscle group is selected from a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and a latissimus dorsi (lat), optionally wherein the two or more different skeletal muscle groups comprise any combination thereof. In some aspects, the two or more different skeletal muscle groups is any combination of two or more of a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), or a latissimus dorsi (lat).

[0019] In some aspects, the skeletal muscle group is selected from a quadriceps, a bicep, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, and a hamstring, optionally wherein the two or more different skeletal muscle groups comprise any combinationthereof. In some aspects, the two or more different skeletal muscle groups is any combination of two or more of a quadriceps, a bicep, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, or a hamstring.

[0020] In some aspects, the skeletal muscle group is selected from i) a left quadriceps and a right quadriceps, ii) a left bicep and a right bicep, iii) a left triceps and a right triceps, iv) a left gastrocnemius and a right gastrocnemius, v) a left gluteus maximus and a right gluteus maximus, vi) a left hamstring and a right hamstring, vii) a left deltoid and a right deltoid, viii) a left trapezius and a right trapezius, ix) a left pectoral muscle and a right pectoral muscle, and x) a left latissimus dorsi and a right latissimus dorsi.

[0021] In some aspects, the skeletal muscle group is selected from a quadriceps, a bicep, a triceps, a gastrocnemius, a gluteus maximus, and a hamstring, optionally wherein the two or more different skeletal muscle groups comprise any combination thereof.

[0022] In some aspects, the administration comprises bilateral injections to one or more of the skeletal muscle groups (e.g., IM injections to the right and left quadriceps, the right and left biceps, the right and left gastrocnemius muscles, the right and left gluteus maximus muscles, and / or the right and left hamstrings). In some aspects, the administration comprises bilateral injections to one or more of the skeletal muscle groups (e.g., IM injections to the right and left quadriceps, the right and left biceps, the right and left gastrocnemius muscles, the right and left gluteus maximus muscles, the right and left triceps, the right and left hamstrings, and any combinations thereof).

[0023] In some aspects, the bilateral injections are divided equally for each skeletal muscle group (e.g., the right quadriceps and the left quadriceps receive approximately the same administration volume and / or dose).

[0024] In some aspects, the administration comprises a total of 20-150 IM injections of the therapy (e.g., combination gene therapy) to the at least two different skeletal muscle groups. In some aspects, the administration comprises 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.

[0025] In some aspects, the administration comprises 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 a 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 a second skeletal muscle group. In some aspects, the administrationcomprises 1-50 (e.g., 2-50, 4-50, 6-50, 8-50, 10-50, 20-50, 30-50, or 40-50) IM injections to a 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 a 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 a third skeletal muscle group.

[0026] In some aspects, the administration comprises (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) 1-30 IM injections to a first skeletal muscle group, 1-30 IM injections to a second skeletal muscle group, 1-30 IM injections to a third skeletal muscle group, 1-30 IM injections to a fourth skeletal muscle group, and 1-30 IM injections to a fifth skeletal muscle group; and (v) 1-25 IM injections to a first skeletal muscle group, 1-25 IM injections to a second skeletal muscle group, 1-25 IM injections to a third skeletal muscle group, 1-25 IM injections to a fourth skeletal muscle group, 1-25 IM injections to a fifth skeletal muscle group, and 1-25 IM injections to a sixth skeletal muscle group.

[0027] In some aspects, the administration comprises: (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 a 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 a second skeletal muscle group; (ii) 2-60, 4-60, 6-60, 8-60, 10-60, 20-60, 30-60, 40-60, or 50-60 IM injections to a first skeletal muscle group; 2-60, 4-60, 6-60, 8-60, 10-60, 20-60, 30-60, 40-60, or 50-60 IM injections to a 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 to a third skeletal muscle group; (iii) 2-40, 4-40, 6-40, 8-40, 10-40, 20-40, or 30-40 IM injections to a first skeletal muscle group; 2-40, 4-40, 6-40, 8-40, 10-40, 20-40, or 30-40 IM injections to a second skeletal muscle group; 2-40, 4-40, 6-40, 8-40, 10-40, 20-40, or 30-40 IM injections to a third skeletal muscle group; and 2-40, 4-40, 6-40, 8-40, 10-40, 20-40, or 30-40 IM injections to a fourth skeletal muscle group; (iv) 2-30, 4-30, 6-30, 8-30, 10-30, or 20-30 IM injections to a first skeletal muscle group; 2-30, 4-30, 6-30, 8-30, 10-30, or 20-30 IM injections to a second skeletal muscle group; 2-30, 4-30, 6-30, 8-30, 10-30, or 20-30 IM injections to a third skeletal muscle group; 2-30, 4-30, 6-30, 8-30, 10-30, or 20-30 IMinjections to a fourth skeletal muscle group; and 2-30, 4-30, 6-30, 8-30, 10-30, or 20-30 IM injections to a fifth skeletal muscle group; or (v) 2-25, 4-25, 6-25, 8-25, 10-25, or 20- 25 IM injections to a first skeletal muscle group; 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 IM injections to a second skeletal muscle group; 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 IM injections to a third skeletal muscle group; 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 IM injections to a fourth skeletal muscle group; 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 IM injections to a fifth skeletal muscle group; and 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 IM injections to a sixth skeletal muscle group.

[0028] In some aspects, the administration comprises 8-50 IM injections to the quadriceps (e.g., 4-25 IM injections bilaterally to each quadriceps), 8-50 IM injections to the biceps (e.g., 4-25 IM injections bilaterally to each bicep), and 8-50 IM injections to the hamstrings (e.g., 4-25 IM injections bilaterally to each hamstring).

[0029] In some aspects, each IM injection is administered in a volume of about 100 pL to about 1.5 mL (e.g., about 100 pL to about 1.4 mL, about 100 pL to about 1.35 mL, about 100 pL to about 1.2 mL, about 100 pL to about 1.0 mL, about 100 pL to about 800 pL, about 200 pL to about 800 pL, about 200 pL to about 600 pL, or about 400 pL to about 800 pL).

[0030] In some aspects, each IM injection is administered in a volume of about 100 pL to about 2 mL, 100 pL to about 1.9 mL, 100 pL to about 1.8 mL, 100 pL to about 1.7 mL, 100 pL to about 1.6 mL, 100 pL to about 1.5 mL, 100 pL to about 1.4 mL, about 100 pL to about 1.35 mL, about 100 pL to about 1.2 mL, about 100 pL to about 1.0 mL, about 100 pL to about 800 pL, about 200 pL to about 800 pL, about 200 pL to about 600 pL, about 400 pL to about 800 pL, about 500 pL to about 1.5 mL, about 600 pL to about 1.4 mL, about 700 pL to about 1.3 mL, about 800 pL to about 1.2 mL, or about 900 pL to about 1.1 mL.

[0031] In some aspects, each IM injection is administered in a volume of about 0.5 mL to about 1 mL.

[0032] In some aspects, the total cumulative volume administered for 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.

[0033] In some aspects, the dose per IM injection comprises about 2xl013to 5xl013vgs per mL.

[0034] In some aspects, each IM injection comprises about 8xlO10to 5xl013vgs per mL. In some aspects, each IM injection comprises about 2xl013to about 4xl013vgs per mL.

[0035] In some aspects, the administration comprises IM injections that are separated by a distance of at least 1-5 cm. In some aspects, the administration comprises IM injections that are separated by a distance of at least 1-3 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 5 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 4 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 3 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 2 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 1 cm.

[0036] In some aspects, the administration comprises IM injections that are 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. In some aspects, each of the IM injections is about 0.5 cm to 2 cm deep. In some aspects, each of the IM injections is between 1-2 cm deep.

[0037] In some aspects, each of the IM injections is at least 0.5 cm below the surface of the injected muscle, regardless of the thickness of the subcutaneous fat.

[0038] 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 comprising the first AAV vector genome and the second AAV vector genome are admixed prior to administration.

[0039] In some aspects, the method further comprises administering to the subject an immunosuppressive regimen.

[0040] Certain aspects of the disclosure are directed to a method of treating or ameliorating the symptoms associated with diabetes in a subject in need thereof, comprising administering to the subject (i) an immunosuppressive regimen and (ii) a therapy (e.g., combination gene therapy) comprising one or more adeno-associated virus (AAV) vectors comprising (a) a promoter (e.g., a first and a second promoter), (b) a polynucleotide encoding a human insulin (hlns) protein; and (c) a polynucleotideencoding a human glucokinase (hGck) protein. In some aspects, the combination therapy is administered intramuscularly (IM) to at least two different skeletal muscle groups.

[0041] In some aspects, the method further comprises administering to the subject an immunosuppressive regimen comprising an immunosuppressive agent.

[0042] Certain aspects of the disclosure are directed to a method of treating or ameliorating the symptoms associated with diabetes in a subject in need thereof, comprising administering to the subject (i) an immunosuppressive regimen comprising an immunosuppressive agent, and (ii) a therapy (e.g., combination gene therapy) comprising one or more adeno-associated virus (AAV) vectors comprising (a) a promoter (e.g., a first and a second promoter), (b) a polynucleotide encoding a human insulin (hlns) 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.

[0043] In some aspects the therapy (e.g., combination gene therapy) comprises (a) a first AAV vector genome comprising an insulin expression cassette comprising a first promoter operably linked to a polynucleotide encoding a human insulin (hlns) protein, wherein the insulin expression cassette is flanked by inverted terminal repeats (ITRs); and (b) a second AAV vector genome comprising a glucokinase expression cassette comprising a second promoter operably linked to a polynucleotide encoding a human glucokinase (hGck) protein, wherein the glucokinase expression cassette is flanked by inverted terminal repeats (ITRs).

[0044] In some aspects, the first AAV vector genome and the second AAV vector genome are administered in the same composition (e.g., via the same IM injection).

[0045] In some aspects, 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).

[0046] In some aspects, the one or more AAV vectors is an AAV vector genome comprising an single expression cassette comprising the first promoter operably linked to the polynucleotide encoding the hlns protein and a second promoter operably linked to the polynucleotide encoding the hGck protein, wherein the single expression cassette is flanked by inverted terminal repeats (ITRs).

[0047] Certain aspects of the disclosure are directed to a dosing regimen comprising: (a) a first immunosuppressive agent;(b) a therapy (e.g., combination gene therapy) administered intramuscularly after (a) which comprises one or more adeno-associated virus (AAV) vectors comprising (a) a promoter (e.g., a first and a second promoter), (b) a polynucleotide encoding a human insulin (hlns) protein; and (c) a polynucleotide encoding a human glucokinase (hGck) protein; and(c) a second immunosuppressive agent administered after (b).

[0048] In some aspects, the therapy comprises (i) a first recombinant AAV (rAAV) particle comprising a first AAV vector genome comprising an insulin expression cassette comprising a first promoter operably linked to a polynucleotide encoding a human insulin (hlns) protein, wherein the insulin expression cassette is flanked by inverted terminal repeats (ITRs); and (ii) a second recombinant AA V (rAAV) particle comprising a second AAV vector genome comprising a glucokinase expression cassette comprising a second promoter operably linked to a polynucleotide encoding a human glucokinase (hGck) protein, wherein the glucokinase expression cassette is flanked by inverted terminal repeats (ITRs).

[0049] In some aspects, the therapy of the dosing regimen is administered intramuscularly (IM) at least two different muscle groups. In some aspects, 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. In some aspects, the combination is administered intramuscularly (IM) 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 is administered intramuscularly (IM) to two, three, four, five, six, seven, eight, nine, or ten different skeletal muscle groups.

[0050] In some aspects, the first AAV vector genome and the second AAV vector genome are administered in the same pharmaceutical composition.

[0051] In some aspects, the immunosuppressive regimen comprises administering one or more immunosuppressive agents prior to (pre- AAV), at the same time as, and / or after (post-AAV) administering the combination therapy.

[0052] In some aspects, the immunosuppressive regimen comprises administering an immunosuppressive agent (pre- AAV) prior to administering the combination therapy.

[0053] In some aspects, the immunosuppressive regimen comprises administering an immunosuppressive agent (post-AAV) after administering the combination therapy.

[0054] In some aspects, the pre- AAV immunosuppressive agent is administered 1 to 14 days prior to administering the combination therapy.

[0055] In some aspects, the pre- AAV immunosuppressive agent is administered 1 to 7 days prior to administering the combination therapy.

[0056] In some aspects, the pre- AAV immunosuppressive agent is administered 1 to 2 days prior to administering the combination therapy.

[0057] In some aspects, the post-AAV immunosuppressive agent is initially administered 1, 2, 3, or 4 weeks after administering the combination therapy.

[0058] In some aspects, the immunosuppressive agent is initially administered about 3 weeks after administering the combination therapy.

[0059] In some aspects, after the initial administration, the post-AAV immunosuppressive agent is administered 2 times per day, daily, or every other day.

[0060] In some aspects, after the initial administration, the post-AAV immunosuppressive agent is administered 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.

[0061] In some aspects, the immunosuppressive regimen comprises administering the immunosuppressive agent prior to administering the combination therapy.

[0062] In some aspects, the immunosuppressive regimen comprises administering the immunosuppressive agent after administering the combination therapy.

[0063] In some aspects, the immunosuppressive agent is administered 1 to 14 days prior to administering the combination therapy.

[0064] In some aspects, the immunosuppressive agent is administered 1 to 7 days prior to administering the combination therapy.

[0065] In some aspects, the immunosuppressive agent is administered 1 to 3 days prior to administering the combination therapy.

[0066] In some aspects, the immunosuppressive agent is administered 3 to 7 days prior to administering the combination therapy.

[0067] In some aspects, the immunosuppressive agent is further administered 1, 2, 3, or 4 weeks after administering the combination therapy.

[0068] In some aspects, immunosuppressive agent is administered once about every 1-3 weeks, optionally once about every 1-2 weeks after administering the combination therapy.

[0069] In some aspects, the immunosuppressive agent is administered for about 1-6 months, optionally about 1-3 months after administering the combination therapy.

[0070] In some aspects, immunosuppressive regimen comprises a second immunosuppressive agent.

[0071] In some aspects, the immunosuppressive regimen comprises administering the second immunosuppressive agent prior to administering the combination therapy.

[0072] In some aspects, the immunosuppressive regimen comprises administering the second immunosuppressive agent after administering the combination therapy.

[0073] In some aspects, the second immunosuppressive agent is administered 1 to 14 days prior to administering the combination therapy.

[0074] In some aspects, the second immunosuppressive agent is administered 1 to 7 days prior to administering the combination therapy.

[0075] In some aspects, the second immunosuppressive agent is administered 1 to 3 days prior to administering the combination therapy.

[0076] In some aspects, the second immunosuppressive agent is administered 3 to 7 days prior to administering the combination therapy.

[0077] In some aspects, the second immunosuppressive agent is further administered 1, 2, 3, or 4 weeks after administering the combination therapy.

[0078] In some aspects, second immunosuppressive agent is administered 2 times per day, daily, or every other day.

[0079] In some aspects, the second immunosuppressive agent is administered for about 1- 6 months, optionally about 1-3 months after administering the combination therapy.

[0080] In some aspects, the immunosuppressive regimen comprises an IL-6 antagonist, an IL-1 antagonist, a TNFa antagonist, an mTOR pathway inhibitor, mycophenolate mofetil (MMF), a tyk2 inhibitor, methotrexate, rapamycin, abatacept, antithymocyte globulin (ATG), a B cell depletion agent, a T cell costimulation antagonist, a T cell depletion agent, a corticosteroid, and / or a calcineurin inhibitor.

[0081] In some aspects, the immunosuppressive regimen comprises administering an immunosuppressive agent selected from the group consisting of an IL-6 antagonist, an IL- 1 antagonist, a TNFa antagonist, an mTOR pathway inhibitor, mycophenolate mofetil (MMF), a tyk2 inhibitor, methotrexate, abatacept, antithymocyte globulin (ATG), a B cell depletion agent, a T cell costimulation antagonist, a T cell depletion agent, a corticosteroid, a calcineurin inhibitor, or any combination thereof.

[0082] In some aspects, the pre-AAV immunosuppressive agent comprises an IL-6 antagonist, an IL-1 antagonist, mycophenolate mofetil (MMF), a tyk2 inhibitor, rapamycin, and / or abatacept. In some aspects, the pre-AAV immunosuppressive agent comprises an IL-6 antagonist, an IL-1 antagonist, mycophenolate mofetil (MMF), a tyk2 inhibitor, rapamycin, abatacept, or any combination thereof.

[0083] In some aspects, the post-AAV immunosuppressive agent comprises a calcineurin inhibitor, MMF, methotrexate, a tyk2 inhibitor, rapamycin, and / or abatacept.

[0084] In some aspects, the IL-6 antagonist is selected from the group consisting of tocilizumab, sarilumab, siltuximab, olokizumab, elsilimomab, clazakizumab, sirukumab, levilimab, or any combination thereof. In some aspects, the IL-6 antagonist is tocilizumab.

[0085] In some aspects, the calcineurin inhibitor is selected from the group consisting of cyclosporine, tacrolimus, voclosporin, and any combination thereof. In some aspects, the calcineurin inhibitor is tacrolimus.

[0086] In some aspects, the IL-6 antagonist is selected from the group consisting of tocilizumab, sarilumab, satralizumab, siltuximab, olokizumab, elsilimomab, clazakizumab, sirukumab, levilimab, or any combination thereof.

[0087] In some aspects, the IL-6 antagonist is tocilizumab.

[0088] In some aspects, the second immunosuppressive agent comprises a calcineurin inhibitor, MMF, methotrexate, a tyk2 inhibitor, rapamycin, abatacept, thymoglobulin, etanercept, basiliximab, sirolimus, or any combination thereof.

[0089] In some aspects, the immunosuppressive regimen comprises administering tocilizumab, optionally subcutaneously, on about day -3 and about every week or about every other week through about 12 weeks relative to the combination therapy.

[0090] In some aspects, the immunosuppressive regimen comprises administering tacrolimus, optionally orally, on about day -3 and every 1-3 days through about 3-12 weeks relative to the combination therapy.

[0091] In some aspects, the total administered dose of the first AAV vector genome comprising the insulin expression cassette comprises about 7xlO10to about 5xl013vector genomes (vg) / kg.

[0092] In some aspects, the total administered dose of the second AAV vector genome comprising the glucokinase expression cassette comprises about 5xl0nto about 5xl013vector genomes (vg) / kg.

[0093] In some aspects, 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 1:0.25-1.25, 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, 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.

[0094] In some aspects, 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.

[0095] In some aspects, 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.

[0096] In some aspects, 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, abut 1:13, about 1:14, or about 1:15.

[0097] In some aspects, 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.

[0098] In some aspects, the first AAV vector genome comprising an insulin expression cassette is administered at a dose between about 5xl0nand about IxlO13vg / kg.

[0099] In some aspects, the first AAV vector genome comprising an insulin expression cassette is administered at a dose between about 2xl012and about IxlO13vg / kg.

[0100] In some aspects, the first AAV vector genome comprising an insulin expression cassette is administered at a dose of about 4xl012to about IxlO13vg / kg.

[0101] In some aspects, the first AAV vector genome comprising an insulin expression cassette is administered at a dose of about 7xlO10to about 7xl012vg / kg.

[0102] In some aspects, the first AAV vector genome comprising an insulin expression cassette is administered at a dose of about 5xl0nto about 5xl012vg / kg.

[0103] In some aspects, the first AAV vector genome comprising an insulin expression cassette is administered at a dose of about IxlO11to about IxlO12vg / kg.

[0104] In some aspects, the second AAV vector genome comprising a glucokinase expression cassette is administered at a dose between about IxlO12and about 5xl012vg / kg.

[0105] In some aspects, the second AAV vector genome comprising a glucokinase expression cassette is administered at a dose between about 2xl012and about 5xl012vg / kg.

[0106] In some aspects, the second AAV vector genome comprising a glucokinase expression cassette is administered at a dose of about 2xl012to about 3.5xl012vg / kg.

[0107] In some aspects, the second AAV vector genome comprising a glucokinase expression cassette is administered at a dose of about 3.5xl012to about 5xl012vg / kg.

[0108] In some aspects, within 15 days-6 months, 30-120 days, or 30-60 days after administration of the combination therapy: (i) glycated blood hemoglobin (HbAlc) levels are reduced and / or regulated in the subject; (ii) circulating ketones are reduced in the subject, (iii) triglycerides are reduced in the subject, (iv) exogenous insulin is reduced in the subject, (v) circulating C-peptide levels are increased in the subject, or (vi) any combination thereof.

[0109] In some aspects, within 15 days-6 months, 30-120 days, 30-60 days, or 60-90 days after administration of the combination therapy the subject’s circulating insulin level in the blood, when measured in the absence of exsanguinous insulin treatment, is about 1- 40 pU / mL, optionally about 5-40 pU / mL, about 5-25 pU / mL, about 5-10 pU / mL, about 10-20 pU / mL, about 10-15 pU / mL, about 15-20 pU / mL, or about 15-25 pU / mL.

[0110] In some aspects, within 15 days-6 months, 30-120 days, 30-60 days, or 60-90 days after administration of the combination therapy the subject’s HbAlC level is reduced by about 0.5 to 1.5% compared to pre-treatment.[OHl] In some aspects, within 15 days-6 months, 30-120 days, 30-60 days, or 60-90 days after administration of the combination therapy the subject’s HbAlC level is less than about 7.0%, optionally less than about 6.5%, about 6.0%, about 5.7%, optionally about 5.0% to 6.5%.

[0112] In some aspects, within 15 days-6 months, 30-120 days, 30-60 days, or 60-90 days after administration of the combination therapy the subject’s circulating glucose in the blood is at least about 70 to 140 mg / dL.

[0113] In some aspects, the subject is not insulin resistant.

[0114] In some aspects, the subject is insulin resistant.

[0115] Certain aspects of the disclosure are directed to a dosing regimen comprising: (a) a first immunosuppressive agent; (b) a combination therapy administered intramuscularly (IM) after (a) which comprises (i) a first recombinant AAV (rAAV) particle comprising a first AAV vector genome comprising an insulin expression cassette comprising a first promoter operably linked to a polynucleotide encoding a human insulin (hlns) protein, wherein the insulin expression cassette is flanked by inverted terminal repeats (ITRs); and (ii) a second recombinant AAV (rAAV) particle comprising a second AAV vector genome comprising a glucokinase expression cassette comprising a second promoter operably linked to a polynucleotide encoding a human glucokinase (hGck) protein, wherein the glucokinase expression cassette is flanked by inverted terminal repeats (ITRs); and (c) a second immunosuppressive agent.

[0116] In some aspects, the first immunosuppressive agent comprises an IL-6 antagonist selected from the group consisting of tocilizumab, sarilumab, siltuximab, olokizumab, elsilimomab, clazakizumab, sirukumab, levilimab, and any combination thereof.

[0117] In some aspects, the second immunosuppressive agent is a calcineurin inhibitor selected from the group consisting of cyclosporine, tacrolimus, voclosporin, and any combination thereof.

[0118] In some aspects, the first immunosuppressive agent comprises tocilizumab and the second immunosuppressive agent comprises tacrolimus.

[0119] In some aspects, the dosing regimen comprises administering tocilizumab, optionally subcutaneously, on about day -3 and about every week or about every other week through about 12 weeks relative to the combination therapy.

[0120] In some aspects, the immunosuppressive regimen comprises administering tacrolimus, optionally orally, on about day -3 and every 1-3 days through about 3-12 weeks relative to the combination therapy.

[0121] In some aspects, the first AAV vector genome and second AAV vector genome are each administered at a dose between about 5xl0nto about 5xl013vector genomes (vg) / kg. In some aspects, the first AAV vector genome and second AAV vector genome are administered at a dose between about lxl012to about 1 xlO13vg / kg.

[0122] In some aspects, the first AAV vector genome comprising an insulin expression cassette is administered at a dose between about 2xl012to about IxlO13vg / kg. In someaspects, the first AAV vector genome comprising an insulin expression cassette is administered at a dose between about 4xl012to about IxlO13vg / kg. In some aspects, the first AAV vector genome comprising an insulin expression cassette is administered at a dose of about 4.5xl012to about 5xl012vg / kg. In some aspects, the first AAV vector genome comprising an insulin expression cassette is administered at a dose of about 5xl012to about 9xl012vg / kg.

[0123] In some aspects, the second AAV vector genome comprising a glucokinase expression cassette is administered at a dose between about lxl012to about 5 xlO12vg / kg. In some aspects, the second AAV vector genome comprising a glucokinase expression cassette is administered at a dose between about 2xl012to about 5 xlO12vg / kg. In some aspects, the second AAV vector genome comprising a glucokinase expression cassette is administered at a dose of about 2xl012to about 2.8xl012vg / kg. In some aspects, the second AAV vector genome comprising a glucokinase expression cassette is administered at a dose of about 4xl012vg / kg to about 5xl012vg / kg.

[0124] In some aspects, the first AAV vector genome comprises an insulin expression cassette is administered at a dose of about 7xlO10vg / kg to about 1.8xl012vg / kg.

[0125] In some aspects, the first AAV vector genome comprises an insulin expression cassette is administered at a dose of about 7xlOnto about 1.8xl012vg / kg.

[0126] In some aspects, the first AAV vector genome comprising an insulin expression cassette is administered at a dose of about 5xl0nto about 5xl012vg / kg.

[0127] In some aspects, the first AAV vector genome comprising an insulin expression cassette is administered at a dose of about IxlO11to about IxlO12vg / kg.

[0128] In some aspects, the second AAV vector genome comprises a glucokinase expression cassette is administered at a dose of about 3.5xl012vg / kg.

[0129] In some aspects, the first AAV vector genome (comprising the expression cassette encoding hlns) and the second AAV vector genome (comprising the expression cassette encoding hGck) are administered at a ratio selected of 1 :0.25-1.25. In some aspects, the ratio is about 1 : 1. In some aspects, the ratio is 1 :0.35-0.75. In some aspects, the ratio is 1 :0.4-0.6. In some aspects, the ratio is 1 :0.45-0.55. In some aspects, the ratio is about 1 :0.5.

[0130] In some aspects, the ratio is about 1 : 1-15. In some aspects, the ratio is about 1 :3- 5. In some aspects, the ratio is about 1 :2. In some aspects, the ratio is about 1 :3. In some aspects, the ratio is about 1 :4. Is some aspects, the ratio is about 1 :5.

[0131] In some aspects, the polynucleotide encoding the hlns protein comprises an open reading frame (ORF) comprising: a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to any one of: (i) nucleic acids 73-330 of any of SEQ ID NOs: 43-57, or 110-116, nucleic acids 88-345 of any of SEQ ID NOs: 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 comprising (i) a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to a sequence selected from any of (a) nucleic acids 1-1398 of any of SEQ ID NO: 61-80 or 162; or (ii) SEQ ID NO: 61-80 and 162.

[0132] In some aspects, the polynucleotide encoding the hlns protein comprises an open reading frame (ORF) comprising a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 121; and / or the polynucleotide encoding the human glucokinase hGck protein comprises an ORF comprising a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 68.

[0133] In some aspects, the hlns protein comprises the amino acid sequence of any of amino acids 25-110 of SEQ ID NO: 41, amino acids 25-110 of SEQ ID NO: 144, amino acids 25-110 of SEQ ID NO: 145, SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO: 145.

[0134] In some aspects, the hlns protein comprises a signal peptide. In some aspects, the signal peptide is a wild-type preproinsulin signal sequence, an IL-6 signal sequence, a fibronectin signal sequence, or a non-wild-type preproinsulin signal sequence. In some aspects, the signal peptide comprises 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 hlns protein further comprises a cleavage site.

[0135] In some aspects, the polynucleotide encoding the hlns protein further comprises a 5’ UTR comprising a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148.

[0136] In some aspects, the polynucleotide encoding the hlns protein further comprises a 3’ UTR comprising a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 60, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, or SEQ ID NO: 101, SEQ ID NO: 149, or SEQ ID NO: 171.

[0137] In some aspects, the encoded hGck protein comprises the amino acid sequence of SEQ ID NO: 82.

[0138] In some aspects, the polynucleotide encoding the hGck protein further comprises a 5’ UTR comprising a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148.

[0139] In some aspects, the polynucleotide encoding the hGck protein further comprises a 3’ UTR comprising a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 60, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 149, SEQ ID NO: 169, or SEQ ID NO: 171.

[0140] In some aspects, the first promoter is a eukaryotic promoter. In some aspects, the first promoter is a constitutive promoter. In some aspects, the first promoter is a ubiquitous promoter. In some aspects, the first promoter is a muscle specific promoter. In some aspects, the first promoter is a CMV promoter. In some aspects, the second promoter is a eukaryotic promoter. In some aspects, the first promoter is a constitutive promoter. In some aspects, the first promoter is a ubiquitous promoter. In some aspects, the first promoter is a muscle specific promoter. In some aspects, the second promoter is a CMV promoter.

[0141] In some aspects, the insulin expression cassette promoter and / or the glucokinase expression cassette promoter is a eukaryotic promoter. In some aspects, the insulin expression cassette promoter and / or the glucokinase expression cassette promoter is a constitutive promoter. In some aspects, the insulin expression cassette promoter and / or the glucokinase expression cassette promoter is a ubiquitous promoter. In some aspects, the insulin expression cassette promoter and / or the glucokinase expression cassette promoter is a CMV promoter. In some aspects, the insulin expression cassette promoter and / or the glucokinase expression cassette promoter is a muscle specific promoter.

[0142] In some aspects, the insulin expression cassette and / or the glucokinase expression cassette further comprises a polyadenylation (poly A) element.

[0143] In some aspects, a first recombinant AAV (rAAV) particle comprises the first AAV vector genome. In some aspects, a second recombinant AAV (rAAV) particle comprises the 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 ofAAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh9, AAV9, AAVrhlO, AAV10, AAV11, AAV12, and AAVrh74. In some aspects, the AAV serotype of the first rAAV particle and / or the second rAAV particle is AAV1.

[0144] In some aspects, the first rAAV particle and the second rAAV particle are formulated in a single composition.

[0145] In some aspects, the first rAAV particle and the second rAAV particle are administered simultaneously or sequentially.

[0146] In some aspects, the diabetes is diabetes mellitus type 1 (T1DM) or diabetes mellitus type 2 (T2DM). In some aspects, the diabetes is diabetes mellitus type 1 (T1DM). In some aspects, the diabetes is diabetes mellitus type 2 (T2DM).

[0147] In some aspects, within 15 days and 6 months, 30-120 days, or 30-60 days after administration of the combination therapy: (i) glycated blood hemoglobin (HbAlc) levels are reduced and / or regulated in the subject; (ii) circulating ketones are reduced in the subject, (iii) triglycerides are reduced in the subject, (iv) exogenous insulin is reduced in the subject, (v) circulating C-peptide levels are increased in the subject, or (vi) any combination thereof.

[0148] Certain aspects of the disclosure are directed to a method of immunosuppression in a subject administered an AAV gene therapy for the treatment of diabetes, wherein the subject is administered an immunosuppressive regimen comprising administering (a) an immunosuppressive agent (pre- AAV) prior to administering the AAV gene therapy; and (b) an immunosuppressive agent (post-AAV) after administering the AAV gene therapy.

[0149] In some aspects, the pre- AAV immunosuppressive agent is administered 1 to 14 days prior to administering the AAV gene therapy.

[0150] In some aspects, the pre- AAV immunosuppressive agent is administered 1 to 7 days prior to administering the AAV gene therapy.

[0151] In some aspects, the pre- AAV immunosuppressive agent is administered 1 to 2 days prior to administering the AAV gene therapy.

[0152] In some aspects, the pre- AAV immunosuppressive agent is administered 1 to 3 days prior to administering the AAV gene therapy.

[0153] In some aspects, the pre- AAV immunosuppressive agent is administered 3 to 7 days prior to administering the AAV gene therapy.

[0154] In some aspects, the post-AAV immunosuppressive agent is further administered for 1, 2, 3, or 4 weeks after administering the AAV gene therapy.

[0155] In some aspects, post-AAV immunosuppressive agent is administered once about every 1-3 weeks, optionally once about every 1-2 weeks after administering the combination therapy.

[0156] In some aspects, the post-AAV immunosuppressive agent is administered for about 1-6 months, optionally about 1-3 months after administering the AAV gene therapy.

[0157] In some aspects, the immunosuppressive agent is initially administered about 3 weeks after administering the AAV gene therapy.

[0158] In some aspects, after the initial administration, the post-AAV immunosuppressive agent is administered 2 times per day, daily, or every other day.

[0159] In some aspects, after the initial administration, the post-AAV immunosuppressive agent is administered 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.

[0160] Certain aspects of the disclosure are directed to a method of immunosuppression in a subject administered an AAV gene therapy for the treatment of diabetes, wherein the subject is administered an immunosuppressive regimen comprising administering (a) a first immunosuppressive agent prior to administering the AAV gene therapy; and, optionally, (b) a second immunosuppressive agent after administering the AAV gene therapy.

[0161] In some aspects, the first immunosuppressive agent is administered 1 to 14 days prior to administering the AAV gene therapy.

[0162] In some aspects, the first immunosuppressive agent is administered 1 to 7 days prior to administering the AAV gene therapy.

[0163] In some aspects, the first immunosuppressive agent is administered 1 to 3 days prior to administering the AAV gene therapy.

[0164] In some aspects, the second immunosuppressive agent is administered 1 to 14 days prior to administering the AAV gene therapy.

[0165] In some aspects, the second immunosuppressive agent is administered 1 to 7 days prior to administering the AAV gene therapy.

[0166] In some aspects, the second immunosuppressive agent is administered 3 to 7 days prior to administering the AAV gene therapy.

[0167] In some aspects, after the initial administration, the second immunosuppressive agent is administered 2 times per day, daily, or every other day.

[0168] In some aspects, the first immunosuppressive agent and / or the second immunosuppressive agent is administered for about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months. In some aspects, the immunosuppressive regimen comprises an IL-6 antagonist, an IL-1 antagonist, a TNFa antagonist, an mTOR pathway inhibitor, mycophenolate mofetil (MMF), a tyk2 inhibitor, methotrexate, rapamycin, abatacept, antithymocyte globulin (ATG), a B cell depletion agent, a T cell costimulation antagonist, a T cell depletion agent, a corticosteroid, and / or a calcineurin inhibitor.

[0169] In some aspects, the pre-AAV immunosuppressive agent comprises an IL-6 antagonist, an IL-1 antagonist, MMF, a tyk2 inhibitor, rapamycin, or abatacept.

[0170] In some aspects, the post-AAV immunosuppressive agent comprises a calcineurin inhibitor, MMF, methotrexate, a tyk2 inhibitor, rapamycin, and / or abatacept.

[0171] In some aspects, the IL-6 antagonist is selected from the group consisting of tocilizumab, sarilumab, siltuximab, olokizumab, elsilimomab, clazakizumab, sirukumab, levilimab, or any combination thereof. In some aspects, the IL-6 antagonist is tocilizumab.

[0172] In some aspects, the calcineurin inhibitor is selected from the group consisting of cyclosporine, tacrolimus, voclosporin, and any combination thereof. In some aspects, the calcineurin inhibitor is tacrolimus.

[0173] In some aspects, the pre-AAV immunosuppressive agent and / or the post-AAV immunosuppressive agent is administered for about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.

[0174] In some aspects, the pre-AAV immunosuppressive agent and / or the post-AAV immunosuppressive agent comprises an IL-6 antagonist, an IL-1 antagonist, a TNFa antagonist, an mTOR pathway inhibitor, mycophenolate mofetil (MMF), a tyk2 inhibitor, methotrexate, rapamycin, abatacept, antithymocyte globulin (ATG), a B cell depletion agent, a T cell costimulation antagonist, a T cell depletion agent, a corticosteroid, a calcineurin inhibitor, or any combination thereof.

[0175] In some aspects, the pre- AAV immunosuppressive agent and / or post-AAV immunosuppressive agent comprises an IL-6 antagonist, an IL-1 antagonist, MMF, a tyk2 inhibitor, rapamycin, abatacept, or any combination thereof.

[0176] In some aspects, the pre- AAV immunosuppressive agent and / or the post-AAV immunosuppressive agent comprises a calcineurin inhibitor, MMF, methotrexate, a tyk2 inhibitor, rapamycin, abatacept, or any combination thereof.

[0177] In some aspects, the method comprises administering tocilizumab, optionally subcutaneously, on about day -3 and about every week or about every other week through about 12 weeks relative to the AAV gene therapy.

[0178] In some aspects, the method comprises administering tacrolimus, optionally orally, on about day -3 and every 1-3 days through about 3-12 weeks relative to the AAV gene therapy.BRIEF DESCRIPTION OF FIGURES

[0179] FIGs. 1A-1J show noted biomarker levels measured in a first cynomolgus macaque (NHP1) administered AAVl-hINS+AAVl-hGCK following streptozotocin (STZ) treatment in a STZ-induced non-human primate (NHP) model of Type 1 diabetes. FIG. 1A shows daily pre- and post-prandial blood glucose levels (mg / dL) and the amount of exogenous long-acting insulin (insulin glargine, “Lantus”) and short-acting insulin (insulin lispro, “Humalog”) administered (U / kg / day) prior to and following administration of AAVl-hINS+AAVl-hGCK (at day 0), with timepoints up to day 180 following AAV dosing. The % reduction of exogenous insulin administered compared to the 7-day average of exogenous insulin administered prior to AAV administration (baseline) (post-STZ treatment, pre-day 0) is shown at the top of the graph. Blood samples were drawn throughout the in-life phase and analyzed for C-peptide levels (FIG. IB; ng / mL), HbAlC levels (FIG. 1C; %), and creatine kinase (CK) levels (FIG. ID; U / L) from day 0 up to day 182 following AAVl-hINS+AAVl-hGCK administration. FIG. IE shows body weight (kg) before and after AAV dosing and up to day 182. FIG. IF shows blood glucose levels (mg / dL) during an intravenous glucose tolerance test (IVGTT) of NHP1 (i) prior to STZ administration (Pre STZ), (ii) after STZ and before administration of AAVl-hlns+AAVl-hGck (Post STZ), and (iii) after STZ at 56 days (D+56), 92 days (D+92), or 176 days (D+176) after AAVl-hlns+AAVl-hGck dosing.FIG. 1G shows C-peptide levels (ng / mL) during IVGTT of NHP1 (i) prior to STZ administration (Pre STZ), (ii) after STZ and before administration of AAVl-hlns+AAVl- hGck (Post STZ), and (iii) after STZ at 56 days (D+56), 92 days (D+92), or 176 days (D+176) after AAVl-hlns+AAVl-hGck dosing. FIG. 1H shows interferon-y response (spots / million cells) in peripheral blood mononuclear cells against AAV1, glucokinase (Gck), insulin, and a negative control measured from 14 days prior to AAV1- hlNS+AAVl-hGCK administration (-14) up to 161 days following AAV administration. Positive responses are indicated by boxes. FIG. II shows anti-insulin IgG (dilution factor) measured at 14 days prior to AAVl-hINS+AAVl-hGCK administration (-14) up to 182 days after. FIG 1J shows anti-glucokinase IgG (ECLsignal) measured at 14 days prior to AAVl-hINS+AAVl-hGCK administration (-14) up to 161 days after.

[0180] FIGs. 2A-2I show noted biomarker levels measured in a second cynomolgus macaque (NHP2) administered AAVl-hINS + AAVl-hGCK following streptozotocin (STZ) treatment in a STZ-induced non-human primate (NHP) model of Type 1 diabetes. FIG. 2A shows daily pre- and post-prandial blood glucose levels (mg / dL) and the amount of exogenous long-acting insulin (insulin glargine, “Lantus”) and short-acting insulin (insulin lispro, “Humalog”) administered (U / kg / day) prior to and following administration of AAVl-hINS+AAVl-hGCK (at day 0), with timepoints up to day 132 following AAV dosing. The % reduction of exogenous insulin administered compared to the 7-day average of exogenous insulin administered prior to AAV administration (baseline) (pre-day 0) is shown at the top of the graph. Blood samples were drawn weekly and analyzed for preprandial, fasted, or stimulated C-peptide levels (FIG. 2B; ng / mL), HblAlC levels (FIG. 2C; %) levels, and creatine kinase (CK) levels (FIG. 2D; U / L) from day 0 up to day 132 following AAVl-hINS+AAVl-hGCK administration. FIG. 2E shows body weight (kg) before and after AAV dosing and up to day 132. FIG. 2F shows blood glucose levels (mg / dL) during an intravenous glucose tolerance test (IVGTT) of NHP2 (i) prior to STZ administration (Pre STZ), after STZ and before administration of AAVl-hlns+AAVl-hGck (Post STZ) and (iii) after STZ at 58 days (D+58) or 91 days (D+91) after AAVl-hINS+AAVl-hGCK dosing. FIG. 2G shows C-peptide levels (ng / mL) during IVGTT of NHP2 (i) prior to STZ administration (Pre STZ), (ii) after STZ and before administration of AAVl-hlns+AAVl-hGck (Post STZ), and (iii) after STZ at either 58 days (D+58) or 91 days (D+91) after AAVl-hlns+AAVl-hGck dosing. FIG. 2H shows interferon-y response (spots / million cells) in peripheral blood mononuclearcells against AAV1, glucokinase (Gck), insulin, a positive control, and a negative control measured from 14 days prior to AAV administration (-14) up to 105 days following AAV administration. Positive test responses are indicated by boxes. FIG. 21 shows anti-insulin IgG (Anti-Insulin dilutional titer) and anti-glucokinase IgG (anti-glucokinase ECL signal) measured at -14 days prior to AAV administration up to 132 days after.

[0181] FIGs. 3A-3I show noted biomarker levels measured in a third cynomolgus macaque (NHP3) administered AAVl-hINS+AAVl-hGCK following streptozotocin (STZ) treatment in a STZ-induced non-human primate (NHP) model of Type 1 diabetes. FIG. 3A shows daily pre- and post-prandial blood glucose levels (mg / dL) and the amount of exogenous long-acting insulin (insulin glargine, “Lantus”) and short-acting insulin (insulin lispro, “Humalog”) administered (U / kg / day) prior to and following administration of AAVl-hINS+AAVl-hGCK (at day 0), with timepoints up to day 90 following AAV dosing. The % reduction of exogenous insulin administered compared to the 7-day average of exogenous insulin administered prior to AAV administration (baseline) (pre-day 0) is shown at the top of the graph. Blood samples were drawn weekly and analyzed for preprandial, fasted, or stimulated C-peptide levels (FIG. 3B; ng / mL), HblAlC levels (FIG. 3C; %) levels, and creatine kinase (CK) levels (FIG. 3D; U / L) from day 0 up to day 90 following AAVl-hINS+AAVl-hGCK administration. FIG. 3E shows body weight (kg) before and after AAV dosing up to day 90. FIG. 3F shows blood glucose levels (mg / dL) during an intravenous glucose tolerance test (IVGTT) of NHP3 (i) prior to STZ administration (Pre STZ), after STZ administration and before administration of AAVl-hlns+AAVl-hGck (Post STZ), and (iii) after STZ administration at 56 days (D+56) or 84 days (D+84) after AAVl-hlns+AAVl-hGck dosing. FIG. 3G shows C-peptide levels (ng / mL) during IVGTT of NHP3 (i) prior to STZ administration (Pre STZ), (ii) after STZ administration and before administration of AAVl-hlns+AAVl- hGck (Post STZ), and (iii) after STZ administration at 56 days (D+56) or 84 days (D+84) after AAVl-hlns+AAVl-hGck dosing. FIG. 3H shows interferon-y response (spots / million cells) in peripheral blood mononuclear cells against AAV1, glucokinase (Gck), insulin, a positive control, and a negative control measured from 14 days prior to AAV administration (-14) up to 91 days following AAV administration. Positive test responses are indicated by boxes. FIG. 31 shows anti-insulin IgG (Anti-Insulin dilutional titer) and anti-glucokinase IgG (anti-glucokinase ECL signal) measured at 14 days prior to AAV administration up to 91 days after.

[0182] FIGs. 4A-4I show noted biomarker levels measured in a fourth cynomolgus macaque (NHP4) administered AAV I -hINS+AAV I -hGCK following streptozotocin (STZ) treatment in a STZ-induced non-human primate (NHP) model of Type 1 diabetes. FIG. 4A shows daily pre- and post-prandial blood glucose levels (mg / dL) and the amount of exogenous long-acting insulin (insulin glargine, “Lantus”) and short-acting insulin (insulin lispro, “Humalog”) administered (U / kg / day) prior to and following administration of AAVI-hINS+AAVI-hGCK (at day 0), with timepoints up to day 90 following AAV dosing. The % reduction of exogenous insulin administered compared to the 7-day average of exogenous insulin administered prior to AAV administration (baseline) (pre-day 0) is shown at the top of the graph. Blood samples were drawn weekly and analyzed for random (sample taken without regard for fasted or fed status), fasted, or stimulated (sample taken as part of IVGTT) C-peptide levels (FIG. 4B; ng / mL), HblAlC levels (FIG. 4C; %) levels, and creatine kinase (CK) levels (FIG. 4D; U / L) from day 0 up to day 90 following AAVI-hINS+AAVI-hGCK administration. FIG. 4E shows body weight (kg) before and after AAV dosing and up to day 90. FIG. 4F shows blood glucose levels (mg / dL) during an intravenous glucose tolerance test (IVGTT) of NHP4 (i) prior to STZ administration (Pre STZ), (ii) after STZ administration and before administration of AAVl-hlns+AAVl-hGck (Post STZ), and (iii) after STZ administration at 56 days (D+56) and 84 days (D+84) after AAVl-hlns+AAVl-hGck dosing. FIG. 4G shows C- peptide levels (ng / mL) during IVGTT of NHP4 (i) prior to STZ administration (Pre STZ), (ii) after STZ administration and before administration of AAVl-hlns+AAVl-hGck (Post STZ), and (iii) after STZ administration at 56 days (D+56) after AAVl-hlns+AAVl- hGck dosing. FIG. 4H shows interferon-y response (spots / million cells) in peripheral blood mononuclear cells against AAV1, glucokinase (Gck), insulin, a positive control, and a negative control measured from 14 days prior to AAV administration (-14) up to 91 days following AAV administration. Positive test responses are indicated by boxes. FIG. 41 shows anti-insulin IgG (Anti-Insulin dilutional titer) and anti-glucokinase IgG (antiglucokinase ECL signal) measured at 14 days prior to AAV administration up to 80 days after.

[0183] FIGs. 5A-5K show noted biomarker levels measured in a fifth cynomolgus macaque (NHP5) administered AAVI-hINS+AAVI-hGCK following streptozotocin (STZ) treatment in a STZ-induced non-human primate (NHP) model of Type 1 diabetes. FIG. 5A shows daily pre- and post-prandial blood glucose levels (mg / dL) from 30 daysprior to injection of AAVl-hINS+AAVl-hGCK (-30) until 180 days following AAV1- hlNS+AAVl-hGCK dosing. Blood samples were drawn weekly and analyzed for C- peptide levels (FIG. 5B; ng / mL) and HblAlC levels (FIG. 5C; %) from day 0 to day 180 following AAVl-hINS+AAVl-hGCK administration. Blood samples were drawn weekly and analyzed for creatine kinase (CK) levels (FIG. 5D; U / L) from day 0 through day 180 following AAVl-hINS+AAVl-hGCK administration. FIG. 5E shows body weight (kg) before and after AAVl-hINS+AAVl-hGCK dosing and through day 182. FIG. 5F shows blood glucose levels (mg / dL) during an intravenous glucose tolerance test (IVGTT) of NHP5 prior to STZ administration (Pre STZ), (ii) after STZ administration and before administration of AAVl-hlns+AAVl-hGck (Post STZ), and (iii) after STZ administration at 56 days (D+56), 91 days (D+91), and 175 days (D+175) after AAVl-hlns+AAVl- hGck dosing. FIG. 5G shows C-peptide levels (ng / mL) during IVGTT of NHP5 (i) prior to STZ administration (Pre STZ), (ii) after STZ administration and before administration of AAVl-hlns+AAVl-hGck (Post STZ), and (iii) after STZ administration at 56 days (D+56), 91 days (D+91), and 175 days (D+175) after AAVl-hlns+AAVl-hGck dosing. FIG. 5H shows the amount of exogenous long-acting insulin (glargine) and short-acting insulin (lispro) (U / kg / day) following administration of AAVl-hINS+AAVl-hGCK (at day 0) and timepoints through day 180 following AAVl-hINS+AAVl-hGCK dosing.FIG. 51 shows the % reduction of exogenous insulin (change from baseline) administered compared to the 30-day average of exogenous insulin administered prior to AAVl- hINS+AAVl-hGCK administration (baseline). The 30 day average % reduction is shown at the top of the graph. FIG. 5J shows insulin (pU / mL) measured either after fasting or at randomly selected times, from administration of AAVl-hINS+AAVl-hGCK (at day 0) through day 182. FIG. 5K shows interferon-y response (spots / million cells) in peripheral blood mononuclear cells against AAV1, glucokinase (Gck), insulin, a positive control, and a negative control measured from 14 days prior to AAVl-hINS+AAVl-hGCK administration (-14) up to 182 days following AAVl-hINS+AAVl-hGCK administration. Positive test responses are indicated by boxes.

[0184] FIGs. 6A-6L show noted biomarker levels measured in a sixth cynomolgus macaque (NHP6) administered AAVl-hINS+AAVl-hGCK following streptozotocin (STZ) treatment in a STZ-induced non-human primate (NHP) model of Type 1 diabetes. FIG. 6A shows daily pre- and post-prandial blood glucose levels (mg / dL) from 30 days prior to injection of AAVl-hINS+AAVl-hGCK (-30) until 182 days following AAV1-hINS+AAVl-hGCK dosing. Blood samples were drawn weekly and analyzed for C- peptide levels (FIG. 6B; ng / mL) and HblAlC levels (FIG. 6C; %) from day 0 through day 182 following AAVl-hINS+AAVl-hGCK administration. Blood samples were drawn weekly and analyzed for creatine kinase (CK) levels (FIG. 6D; U7L) from day 0 through day 182 following AAVl-hINS+AAVl-hGCK administration. FIG. 6E shows body weight (kg) before and after AAVl-hINS+AAVl-hGCK dosing and through day 182. FIG. 6F shows circulating tacrolimus levels (pg / L), beginning at day 21 following AAVl-hINS+AAVl-hGCK administration through day 182. FIG. 6G shows blood glucose levels (mg / dL) during an intravenous glucose tolerance test (IVGTT) of NHP6 (i) prior to STZ administration (Pre STZ), (ii) after STZ administration and before administration of AAVl-hlns+AAVl-hGck (Post STZ), and (iii) after STZ administration at 56 days (D+56), 91 days (D+91), and 175 days (D+175) after AAVl-hlns+AAVl- hGck dosing. FIG. 6H shows C-peptide levels (ng / mL) during IVGTT of NHP6 (i) prior to STZ administration (Pre STZ), (ii) after STZ administration and before administration of AAVl-hlns+AAVl-hGck (Post STZ), and (iii) after STZ administration at 56 days (D+56), 91 days (D+91), and 175 days (D+175) after AAVl-hlns+AAVl-hGck dosing. FIG. 61 shows the amount of exogenous long-acting insulin (glargine) and short-acting insulin (lispro) (U / kg / day) following administration of AAVl-hINS+AAVl-hGCK (at day 0) and timepoints through day 182 following AAVl-hINS+AAVl-hGCK dosing. FIG. 6J shows the % reduction of exogenous insulin administered (change from baseline) compared to the 30-day average of exogenous insulin administered prior to AAVl- hINS+AAVl-hGCK administration (baseline). The 30 day average % reduction is shown at the top of the graph. FIG. 6K shows insulin (pU / mL) measured either after fasting or at randomly selected times, from administration of AAVl-hINS+AAVl-hGCK (at day 0) through day 180. FIG. 6L shows interferon-y response (spots / million cells) in peripheral blood mononuclear cells against AAV1, glucokinase (Gck), insulin, a positive control, and a negative control measured from 14 days prior to AAVl-hINS+AAVl-hGCK administration (-14) up to 182 days following AAVl-hINS+AAVl-hGCK administration. Positive test responses are indicated by boxes.

[0185] FIGs. 7A-7L show noted biomarker levels measured in a seventh cynomolgus macaque (NHP7) administered AAVl-hINS+AAVl-hGCK following streptozotocin (STZ) treatment in a STZ-induced non-human primate (NHP) model of Type 1 diabetes. FIG. 7A shows daily pre- and post-prandial blood glucose levels (mg / dL) from 30 daysprior to injection of AAVl-hINS+AAVl-hGCK (-30) until 122 days following AAV1- hlNS+AAVl-hGCK dosing. FIG. 7B shows the amount of exogenous long-acting insulin (glargine) and short-acting insulin (lispro) (U / kg / day) following administration of AAV1- hlNS+AAVl-hGCK (at day 0) and timepoints through day 122 following AAV1- hlNS+AAVl-hGCK dosing. FIG. 7C shows the % reduction of exogenous insulin administered (change from baseline) compared to the 30-day average of exogenous insulin administered prior to AAVl-hINS+AAVl-hGCK administration (baseline). The 30 day average % reduction is shown at the top of the graph. FIG. 7D shows Hbl A1C levels (%) from day 0 through day 122 following AAVl-hINS+AAVl-hGCK administration. FIG. 7E shows blood glucose levels (mg / dL) during an intravenous glucose tolerance test (IVGTT) of NHP7 (i) prior to STZ administration (Pre STZ), (ii) after STZ administration and before administration of AAVl-hlns+AAVl-hGck (Post STZ), and (iii) after STZ administration at 64 days (D+64) and 91 days (D+91) after AAVl-hlns+AAVl-hGck dosing. FIG. 7F shows C-peptide levels (ng / mL) during IVGTT ofNHP7 (i) prior to STZ administration (Pre STZ), (ii) after STZ administration and before administration of AAVl-hlns+AAVl-hGck (Post STZ), and (iii) after STZ administration at 64 days (D+64) and 91 days (D+91) after AAVl-hlns+AAVl-hGck dosing. FIG. 7G shows creatine kinase (CK) levels (U / L) from day 0 through day 122 following AAVl-hINS+AAVl-hGCK administration. FIG. 7H shows circulating tacrolimus levels (pg / L), beginning at day 21 following AAVl-hINS+AAVl-hGCK administration through day 90. FIG. 71 shows levels of human insulin (pU / mL) measured from administration of AAVl-hlns+AAVl-hGck (day 0) until day 122. FIG. 7J shows C-peptide levels (ng / mL) from day 0 to day 122 following AAVl-hINS+AAVl-hGCK administration. FIG. 7K shows interferon-y response (spots / million cells) in peripheral blood mononuclear cells against AAV1, glucokinase (Gck), insulin, a positive control, and a negative control measured from 14 days prior to AAVl-hINS+AAVl-hGCK administration (-14) up to 122 days following AAVl-hINS+AAVl-hGCK administration. Positive test responses are indicated by boxes. FIG. 7L shows body weight (kg) before and after AAVl-hINS+AAVl-hGCK dosing and through day 122.

[0186] FIGs. 8A-8L show noted biomarker levels measured in an eighth cynomolgus macaque (NHP8) administered AAVl-hINS+AAVl-hGCK following streptozotocin (STZ) treatment in a STZ-induced non-human primate (NHP) model of Type 1 diabetes. FIG. 8A shows daily pre- and post-prandial blood glucose levels (mg / dL) from 30 daysprior to injection of AAVl-hINS+AAVl-hGCK (-30) until 122 days following AAV1- hlNS+AAVl-hGCK dosing. FIG. 8B shows the amount of exogenous long-acting insulin (glargine) and short-acting insulin (lispro) (U / kg / day) following administration of AAV1- hlNS+AAVl-hGCK (at day 0) and timepoints through day 122 following AAV1- hlNS+AAVl-hGCK dosing. FIG. 8C shows the % reduction of exogenous insulin administered (change from baseline) compared to the 30-day average of exogenous insulin administered prior to AAVl-hINS+AAVl-hGCK administration (baseline). The 30 day average % reduction is shown at the top of the graph. FIG. 8D shows Hbl A1C levels (%) from day 0 through day 122 following AAVl-hINS+AAVl-hGCK administration. FIG. 8E shows blood glucose levels (mg / dL) during an intravenous glucose tolerance test (IVGTT) of NHP8 (i) prior to STZ administration (Pre STZ), (ii) after STZ administration and before administration of AAVl-hlns+AAVl-hGck (Post STZ), and (iii) after STZ administration at 56 days (D+56) and 91 days (D+91) after AAVl-hlns+AAVl-hGck dosing. FIG. 8F shows C-peptide levels (ng / mL) during IVGTT ofNHP8 (i) prior to STZ administration (Pre STZ), (ii) after STZ administration and before administration of AAVl-hlns+AAVl-hGck (Post STZ), and (iii) after STZ administration at 56 days (D+64) and 91 days (D+91) after AAVl-hlns+AAVl-hGck dosing. FIG. 8G shows creatine kinase (CK) levels (U / L) from day 0 through day 122 following AAVl-hINS+AAVl-hGCK administration. FIG. 8H shows circulating tacrolimus levels (pg / L), beginning at day 21 following AAVl-hINS+AAVl-hGCK administration through termination. FIG. 81 shows levels of human insulin (pU / mL) measured from administration of AAVl-hlns+AAVl-hGck (day 0) until 122. FIG. 8J shows C-peptide levels (ng / mL) from day 0 to day 122 following AAVl-hINS+AAVl- hGCK administration. FIG. 8K shows interferon-y response (spots / million cells) in peripheral blood mononuclear cells against AAV1, glucokinase (Gck), insulin, a positive control, and a negative control measured from 14 days prior to AAVl-hINS+AAVl- hGCK administration (-14) up to 122 days following AAVl-hINS+AAVl-hGCK administration. Positive test responses are indicated by boxes. FIG. 8L shows body weight (kg) before and after AAVl-hINS+AAVl-hGCK dosing and through day 122.

[0187] FIG. 9A summarizes anti -insulin IgG (dilution factor) for NHP1-NHP4 measured at 14 days prior to AAVl-hINS+AAVl-hGCK administration (-14). FIG. 9B shows antiglucokinase IgG (ECLsignal) for NHP1-NHP6 measured at 14 days prior to AAVadministration (-14). FIG. 9C shows levels of anti-drug antibodies (ADA) for insulin (screening signal) measured for NHPl and NHP2 starting from day -14 until day 182.

[0188] FIGs. 10A-10I show measures of human glucokinase protein (ng / mg) from muscles at or near the injection site of AAVl-hINS+AAVl-hGCK in NHP1-NHP8. FIG. 10A shows glucokinase (ng / mg) protein levels from various muscles samples taken at the injection site of AAVl-hINS+AAVl-hGCK or 1 cm away. The lower limit of quantification (LLOQ) is displayed as a dashed line. NHP1-NHP8 are shown. FIG. 10B specifically shows the glucokinase levels (ng / mg) in muscle samples of NHP1 taken from the injection sites of AAVl-hINS+AAVl-hGCK or samples 1 cm away. The lower limit of quantification (LLOQ) is displayed as a dashed line. FIG. 10C specifically shows the glucokinase levels (ng / mg) in muscle samples of NHP2 taken from the injection sites of AAVl-hINS+AAVl-hGCK or samples 1 cm away. The lower limit of quantification (LLOQ) is displayed as a dashed line. FIG. 10D specifically shows the glucokinase levels (ng / mg) in muscle samples of NHP3 taken from the injection sites of AAVl- hINS+AAVl-hGCK or samples 1 cm away. The lower limit of quantification (LLOQ) is displayed as a dashed line. FIG. 10E specifically shows the glucokinase levels (ng / mg) in muscle samples of NHP4 taken from the injection sites of AAVl-hINS+AAVl-hGCK or samples 1 cm away. The lower limit of quantification (LLOQ) is displayed as a dashed line. FIG. 10F specifically shows the glucokinase levels (ng / mg) in muscle samples of NHP5 taken from the injection sites of AAVl-hINS+AAVl-hGCK or samples 1 cm away. The lower limit of quantification (LLOQ) is displayed as a dashed line. FIG. 10G specifically shows the glucokinase levels (ng / mg) in muscle samples of NHP6 taken from the injection sites of AAVl-hINS+AAVl-hGCK or samples 1 cm away. The lower limit of quantification (LLOQ) is displayed as a dashed line. FIG. 10H specifically shows the glucokinase levels (ng / mg) in muscle samples of NHP7 taken from the injection sites of AAVl-hINS+AAVl-hGCK or samples 1 cm away. The lower limit of quantification (LLOQ) is displayed as a dashed line. FIG. 101 specifically shows the glucokinase levels (ng / mg) in muscle samples of NHP8 taken from the injection sites of AAVl- hINS+AAVl-hGCK or samples 1 cm away. The lower limit of quantification (LLOQ) is displayed as a dashed line.

[0189] FIG. 11A shows the levels of vector genome (Vg; copies / pg gDNA) and insulin mRNA (copies / pg RNA) taken from muscle samples at or near the injection site (IS) of AAVl-hINS+AAVl-hGCK for NHPl and NHP2. FIG. 11B shows the levels of vectorgenome (Vg; copies / pg gDNA) and glucokinase mRNA (copies / pg RNA) taken from muscle samples at or near the injection site (IS) of AAVl-hINS+AAVl-hGCK for NHP1 and NHP2. FIG. 11C shows the levels of vector genome (Vg; copies / pg gDNA) and insulin mRNA (copies / pg RNA) taken from muscle samples at or near the injection site (IS) of AAVl-hINS+AAVl-hGCK for NHP3 and NHP4. FIG. 11D shows the levels of vector genome (Vg; copies / pg gDNA) and glucokinase mRNA (copies / pg RNA) taken from muscle samples at or near the injection site (IS) of AAVl-hINS+AAVl-hGCK for NHP3 and NHP4. FIG. HE shows the levels of vector genome (Vg; copies / pg gDNA) and insulin mRNA (copies / pg RNA) taken from muscle samples at or near the injection site (IS) of AAVl-hINS+AAVl-hGCK for NHP5 and NHP6. FIG. HF shows the levels of vector genome (Vg; copies / pg gDNA) and glucokinase mRNA (copies / pg RNA) taken from muscle samples at or near the injection site (IS) of AAVl-hINS+AAVl-hGCK for NHP5 and NHP6. FIG. HG shows the levels of vector genome (Vg; copies / pg gDNA) and insulin mRNA (copies / pg RNA) taken from muscle samples at or near the injection site (IS) of AAVl-hINS+AAVl-hGCK for NHP7 and NHP8. FIG. HH shows the levels of vector genome (Vg; copies / pg gDNA) and glucokinase mRNA (copies / pg RNA) taken from muscle samples at or near the injection site (IS) of AAVl-hINS+AAVl-hGCK for NHP7 and NHP8.

[0190] FIGs. 12A-12G show metabolic data in an ongoing study from a group of cynomolgus macaques (NHP9-NHP11) administered AAVl-hINS+AAVl-hGCK following streptozotocin (STZ) treatment in a STZ-induced non-human primate (NHP) model of Type 1 diabetes. FIG. 12A shows lactate levels (mol / L) measured from NHP9- NHP11 beginning on the day of AAVl-hINS+AAVl-hGCK injection (day 0). FIG. 12B shows HbAlC levels (%) measured for NHP9-NHP11 beginning on the day of AAVl- hINS+AAVl-hGCK injection (day 0). FIG. 12C shows c-peptide levels (ng / mL) measured for NHP9 and NHP 10 beginning on the day of AAVl-hINS+AAVl-hGCK injection (day 0). The lower limit of quantification (LLOQ) is displayed as a dashed line. FIG. 12D shows human insulin levels (pU / mL) measured for NHP9 and NHP10 beginning on the day of AAVl-hINS+AAVl-hGCK injection (day 0). FIG. 12E shows glucagon levels (pmol / L) measured for NHP9 and NHP 10 beginning on the day of AAVl-hINS+AAVl-hGCK injection (day 0). FIG. 12F shows triglyceride levels (mg / dL) measured for NHP9-NHP11 beginning on the day of AAVl-hINS+AAVl- hGCK injection (day 0). FIG. 12G shows HbAlc at days 0 and 28 for NHPs 9-14.

[0191] FIGs. 13A-13D show noted biomarker levels measured in a ninth cynomolgus macaque (NHP9) administered AAVl-hINS+AAVl-hGCK following streptozotocin (STZ) treatment in a STZ-induced non-human primate (NHP) model of Type 1 diabetes. FIG. 13A shows daily pre- and post-prandial blood glucose levels (mg / dL) from 30 days prior to injection of AAVl-hINS+AAVl-hGCK (-30) until 184 days following AAV1- hlNS+AAVl-hGCK dosing. An arrow indicates the day that the dose of tacrolimus was reduced. FIG. 13B shows the amount of exogenous long-acting insulin (glargine) and short-acting insulin (lispro) (U / kg) administered from 30 days prior to injection of AAVl- hINS+AAVl-hGCK (-30) until 184 days following AAVl-hINS+AAVl-hGCK dosing. For each 30-day window beginning at day 0, the percent change compared to the pre- AAV 30-day window for glargine and lispro is displayed on top of the graph. An arrow indicates the day that the dose of tacrolimus was reduced due to an observed anemic event on Day 120 unrelated to the test article. FIG 13C shows blood glucose levels (mg / dL) during an intravenous glucose tolerance test (IVGTT) of NHP9 prior to STZ administration (Pre STZ), (ii) after STZ administration and before administration of AAVl-hlns+AAVl-hGck (Post STZ), and (iii) after STZ administration at 56 days (D+56) and 91 days (D+91) after AAVl-hlns+AAVl-hGck dosing. FIG 13D displays the quantification for FIG. 13C as AUC (area under the curve).

[0192] FIGs. 14A-14D show noted biomarker levels measured in a tenth cynomolgus macaque (NHP10) administered AAVl-hINS+AAVl-hGCK following streptozotocin (STZ) treatment in a STZ-induced non-human primate (NHP) model of Type 1 diabetes. FIG. 14A shows daily pre- and post-prandial blood glucose levels (mg / dL) from 30 days prior to injection of AAVl-hINS+AAVl-hGCK (-30) until 184 days following AAVl- hINS+AAVl-hGCK dosing. FIG. 14B shows the amount of exogenous long-acting insulin (glargine) and short-acting insulin (lispro) (U / kg) administered from 30 days prior to injection of AAVl-hINS+AAVl-hGCK (-30) until 184 days following AAVl- hINS+AAVl-hGCK dosing. For each 30-day window beginning at day 0, the percent change compared to the pre-AAV 30-day window for glargine and lispro is displayed on top of the graph. FIG 14C shows blood glucose levels (mg / dL) during an intravenous glucose tolerance test (IVGTT) of NHP10 prior to STZ administration (Pre STZ), (ii) after STZ administration and before administration of AAVl-hlns+AAVl-hGck (Post STZ), and (iii) after STZ administration at 56 days (D+56) and 91 days (D+91) afterAAVl-hlns+AAVl-hGck dosing. FIG 14D displays the quantification for FIG. 14C as AUC (area under the curve).

[0193] FIGs. 15A-15B show noted biomarker levels measured in an eleventh cynomolgus macaque (NHP11) administered AAVl-hINS+AAVl-hGCK following streptozotocin (STZ) treatment in a STZ-induced non-human primate (NHP) model of Type 1 diabetes. FIG. 15A shows daily pre- and post-prandial blood glucose levels (mg / dL) from 30 days prior to injection of AAVl-hINS+AAVl-hGCK (-30) until 43 days following AAVl-hINS+AAVl-hGCK dosing. FIG. 15B shows the amount of exogenous long-acting insulin (glargine) and short-acting insulin (lispro) (U / kg) administered from 30 days prior to injection of AAVl-hINS+AAVl-hGCK (-30) until 43 days following AAVl-hINS+AAVl-hGCK dosing. For each window beginning at day 0, the percent change compared to the pre- AAV 30-day window for glargine and lispro is displayed on top of the graph.

[0194] FIGs. 16A-16B show noted biomarker levels measured in a twelfth cynomolgus macaque (NHP12) administered AAVl-hINS+AAVl-hGCK following streptozotocin (STZ) treatment in a STZ-induced non-human primate (NHP) model of Type 1 diabetes. FIG. 16A shows daily pre- and post-prandial blood glucose levels (mg / dL) from 30 days prior to injection of AAVl-hINS+AAVl-hGCK (-30) until 43 days following AAVl- hINS+AAVl-hGCK dosing. FIG. 16B shows the amount of exogenous long-acting insulin (glargine) and short-acting insulin (lispro) (U / kg) administered from 30 days prior to injection of AAVl-hINS+AAVl-hGCK (-30) until 43 days following AAVl- hINS+AAVl-hGCK dosing. For each window beginning at day 0, the percent change compared to the pre- AAV 30-day window for glargine and lispro is displayed on top of the graph.

[0195] FIGs. 17A-17B show noted biomarker levels measured in a thirteenth cynomolgus macaque (NHP 13) administered AAVl-hINS+AAVl-hGCK following streptozotocin (STZ) treatment in a STZ-induced non-human primate (NHP) model of Type 1 diabetes. FIG. 17A shows daily pre- and post-prandial blood glucose levels (mg / dL) from 30 days prior to injection of AAVl-hINS+AAVl-hGCK (-30) until 43 days following AAVl-hINS+AAVl-hGCK dosing. FIG. 17B shows the amount of exogenous long-acting insulin (glargine) and short-acting insulin (lispro) (U / kg) administered from 30 days prior to injection of AAVl-hINS+AAVl-hGCK (-30) until 43 days following AAVl-hINS+AAVl-hGCK dosing. For each window beginning at day 0,the percent change compared to the pre- AAV 30-day window for glargine and lispro is displayed on top of the graph.

[0196] FIGs. 18A-18B show noted biomarker levels measured in a fourteenth cynomolgus macaque (NHP14) administered AAVl-hINS+AAVl-hGCK following streptozotocin (STZ) treatment in a STZ-induced non-human primate (NHP) model of Type 1 diabetes. FIG. 18A shows daily pre- and post-prandial blood glucose levels (mg / dL) from 30 days prior to injection of AAVl-hINS+AAVl-hGCK (-30) until 43 days following AAVl-hINS+AAVl-hGCK dosing. FIG. 18B shows the amount of exogenous long-acting insulin (glargine) and short-acting insulin (lispro) (U / kg) administered from 30 days prior to injection of AAVl-hINS+AAVl-hGCK (-30) until 43 days following AAVl-hINS+AAVl-hGCK dosing. For each window beginning at day 0, the percent change compared to the previous 30-day window for glargine and lispro is displayed on top of the graph.

[0197] FIGs. 19A-19B show schematics of muscles and tendons in a leg from an anterior view (FIG. 19A) and a posterior view (FIG. 19B).DETAILED DESCRIPTION OF THE DISCLOSURE

[0198] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present application, including the definitions, will control. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0199] Throughout this disclosure, the term “a” or “an” entity refers to one or more of that entity; for example, “a polynucleotide,” is understood to represent one or more polynucleotides. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0200] Furthermore, “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: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).

[0201] The term “about” is used herein to mean approximately, roughly, around, or in the regions of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 10 percent, up or down (higher or lower), unless indicated otherwise.

[0202] The term “at least” prior to a number or series of numbers is understood to include the number adjacent to the term “at least,” and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 18 nucleotides of a 21 -nucleotide nucleic acid molecule” means that 18, 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range. “At least” is also not limited to integers (e.g., “at least 5%” includes 5.0%, 5.1%, 5.18% without consideration of the number of significant figures).

[0203] Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three letter code, both in accordance with, 37 CFR §1.822 and established usage.

[0204] “Polynucleotide” or “nucleic acid” as used herein means a sequence of nucleotides connected by phosphodiester linkages. Polynucleotides are presented herein in the direction from the 5' to the 3' direction. A polynucleotide of the present disclosure can be a deoxyribonucleic acid (DNA) molecule or ribonucleic acid (RNA) molecule. Nucleotide bases are indicated herein by a single letter code: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I) and uracil (U).

[0205] As used herein, the term “polypeptide” encompasses both peptides and proteins, unless indicated otherwise.

[0206] The term “coding sequence” or “sequence encoding” is used herein to mean a DNA or RNA region (the transcribed region) which “encodes” a particular protein, e.g.,such as an insulin or a glucokinase. A coding sequence is transcribed (DNA) and translated (RNA) into a polypeptide, in vitro or in vivo, when placed under the control of an appropriate regulatory region, such as a promoter. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotes or eukaryotes, genomic DNA from prokaryotes or eukaryotes, and synthetic DNA sequences. A transcription termination sequence can be located 3' to the coding sequence.

[0207] In some aspects, an expression cassette can comprise several operably linked fragments, such as one or more of a promoter, a 5 ’-untranslated sequence, a leader sequence, an intron, a coding sequence and a 3 '-untranslated sequence (e.g., comprising a polyadenylation site or a signal sequence).

[0208] As used herein, “expression of a gene” refers to the process wherein a gene is transcribed into an RNA and / or translated into an active protein.

[0209] An open reading frame (ORF) as used herein is the part of a reading frame that has the ability to be translated. An ORF is a continuous stretch of codons that begins with a start codon and ends at a stop codon. In some aspects, an ORF sequence can be shown or referenced with or without the start codon sequence and / or the stop codon sequence.

[0210] A Kozak consensus sequence, Kozak consensus or Kozak sequence, is known as a sequence which occurs on eukaryotic mRNA and has the consensus (gcc)gccRccAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another “G ” In some aspects, the polynucleotide comprises a nucleic acid sequence having at least 95%, at least 99% sequence identity, or more to the Kozak consensus sequence. In some aspects, the polynucleotide comprises a Kozak consensus sequence.

[0211] The term “sequence identity” is used herein to mean a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In certain aspects, sequence identity is calculated based on the full length of two given SEQ ID NO or on a part thereof. Part thereof can mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NO, or any other specified percentage. The term “identity” can also mean the degree of sequence relatedness between two or moreamino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences.

[0212] In certain aspects, methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs.

[0213] Substantial homology” or “substantial similarity,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95% to 99% of the sequence.

[0214] As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleic acid sequence in relation to a second nucleic acid sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleic acid sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleic acid sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions can 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, e.g., “Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions as can be encountered inside an organism, can be used. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

[0215] The term “promoter” is used herein to mean a nucleic acid sequence or fragment that functions to control the transcription of one or more genes (or coding sequences), located upstream with respect to the direction of transcription of the transcription initiation site of the gene (or coding sequence). In some aspects, the promoter is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and, optionally, any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A “constitutive” promoter is a promoter that is active under most physiological and developmental conditions. An “inducible” promoter is a promoter that is regulateddepending on physiological or developmental conditions, or in some aspects an inducible promoter can be induced by an exogenous molecule (e.g., a chemical or drug) or other exogenous stimulus (e.g., light, or radiation). A “tissue specific” promoter is preferentially active in specific types of differentiated cells / tissues.

[0216] As used herein, the term “enhancer” is a cis-acting element that stimulates or inhibits transcription of adjacent genes. An enhancer that inhibits transcription is also referred to as a “silencer.” Enhancers can function (e.g., can be associated with a coding sequence) in either orientation, over distances of up to several kilobase pairs (kb) from the coding sequence and from a position downstream of a transcribed region.

[0217] The terms “operatively linked,” “operatively inserted,” “operatively positioned,” “under control” or “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene (or coding sequence). DNA sequence and a regulatory sequence(s) are considered operably linked if they are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s). DNA can be operably inserted if the DNA of interest is positioned adjacent a DNA sequence which directs transcription and translation of the DNA of interest in a cell (i.e., facilitates the production of, e.g., a polypeptide encoded by a DNA of interest).

[0218] The term “transgene” is used herein to mean a gene or a nucleic acid molecule that is introduced into a cell. An example of a transgene is a nucleic acid encoding a therapeutic polypeptide (e.g., a gene encoding an insulin and / or a gene encoding a glucokinase). In some embodiments, the gene can be present but in some cases normally not expressed or expressed at an insufficient level in the cell. In this context, “insufficient” means that although said gene, e.g., insulin and / or glucokinase, is normally expressed in a cell, a condition and / or disease as disclosed herein (e.g., diabetes) could still develop. In certain aspects, the transgene allows for the increased expression or overexpression of the gene, e.g., an insulin and / or a glucokinase. The transgene can comprise sequences that are native to the cell, comprise sequences that do not naturally occur in the cell, or it can comprise combinations of both. In certain aspects, the transgene can comprise modified sequences coding for an insulin, a glucokinase, both an insulin and a glucokinase, and / or additional protein(s) that can be operably linked to appropriate regulatory sequences for expression of the sequences coding for an insulin, a glucokinase,or both an insulin and a glucokinase in the cell. In some aspects, the transgene is not integrated into the host cell’s genome.

[0219] The terms “modified genes”, “modified nucleic acids”, and the like are used interchangeably herein to mean the introduction of one or more modifications or changes relative to the in the natural sequence of the genes or nucleic acid sequence. Such modifications may or may not result in mutations to the encoded protein sequence. In some embodiments, the modified nucleic acid encodes a wild-type or mutant protein or fragment thereof.

[0220] The term “derived from,” as used herein, refers to a component that is isolated from or made using a specified molecule or organism, or information (e.g., amino acid or nucleic acid sequence) from the specified molecule or organism. For example, a nucleic acid sequence (e.g., a modified human insulin gene) that is derived from a second nucleic acid sequence (e.g., a wild-type human insulin gene) can include a nucleotide sequence or portion thereof that is identical or substantially similar to the nucleotide sequence of the second nucleic acid sequence. In some aspects, mutants, analogs or derivatives can be derived from a wild-type sequence.

[0221] In the case of a polynucleotide, the derived species can be obtained by, for example, naturally occurring mutagenesis, artificial directed mutagenesis or artificial random mutagenesis. The mutagenesis used to derive polynucleotides can be intentionally directed or intentionally random, or a mixture of each.

[0222] “Vector” as used herein typically refers to a recombinant plasmid or virus that comprises a polynucleotide to be delivered into a host cell, either in vitro or in vivo.

[0223] As used herein, the term “delivery vector” or “vector” includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene or nucleic acid sequences between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors. In some aspects, useful vectors are contemplated to be those vectors in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter. In some aspects, the delivery vector is selected from the group consisting of a viral vector, a plasmid, lipid, and a lysosome.

[0224] The term “expression vector”, “expression construct” or “expression cassette” means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed.

[0225] In some aspects, the biological vectors include viruses, particularly attenuated and / or replication-deficient viruses. In some embodiments, chemical vectors include lipid complexes and naked DNA constructs.

[0226] As used herein, the term “naked DNA” or “naked nucleic acid” and the like refers to a nucleic acid molecule that is not contained within a viral particle, bacterial cell, or other encapsulating or encapsidating means that facilitates delivery of nucleic acid into the cytoplasm of the target cell. Naked nucleic acid can be associated with means for facilitating delivery of the nucleic acid to the site of the target cell (e.g., to facilitate travel into the target cell of the nucleic acid through the alimentary canal, protect the nucleic acid from stomach acid, and / or serve to penetrate intestinal mucus) and / or to the surface of the target epithelial cell.

[0227] A “viral genome” or “viral vector” can refer to a viral sequence that comprises one or more polynucleotide regions encoding or comprising a molecule of interest, e.g., a protein, a peptide, and a polynucleotide or a plurality thereof. Viral vectors can be used to deliver genetic materials into cells. Viral vectors can be modified for specific applications. In some aspects, the delivery vector of the disclosure is a viral vector selected from the group consisting of an adeno-associated viral (AAV) vector, an adenoviral vector, a lentiviral vector, or a retroviral vector.

[0228] The term “adeno-associated virus vector” or “AAV vector” as used herein refers to any vector which comprises or derives from components of an adeno-associated virus and is suitable to infect mammalian cells, preferably human cells. The term AAV vector can designate an AAV-type viral particle or virion comprising a payload. The AAV vector can be derived from various serotypes, including combinations of serotypes (i.e., “pseudotyped” AAV) or from various genomes (e.g., single stranded or self- complementary). In addition, the AAV vector can be replication defective and / or targeted. As used herein, the term “adeno-associated virus” (AAV), includes but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3 A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAVrh8, AAVrhlO, AAVrh.74, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, thoseAAV 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, e.g., FIELDS et al. VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). In some aspects, an “AAV vector” includes a derivative of a known AAV vector. In some aspects, an “AAV vector” includes a modified or an artificial AAV vector. In some aspects, the AAV vector is modified or mutated relative to the wild-type AAV serotype sequence. In some aspects, the terms “AAV genome” and “AAV vector” can be used interchangeably.

[0229] As used herein, a “recombinant AAV particle” or “rAAV particle” is an AAV virus that comprises a capsid protein and an AAV vector having at least one payload region (e.g., an expression cassette including a polynucleotide encoding insulin and / or Gck) and at least one inverted terminal repeat (ITR) region. In some aspects, the terms “AAV vectors of the present disclosure” or “AAV vectors” refer to AAV vectors comprising a polynucleotide encoding an insulin, a GcK, or a combination thereof, e.g., encapsulated in an AAV capsid.

[0230] Transduction” of a cell by a virus means that there is transfer of a nucleic acid from the virus particle to the cell. In some aspects, transduction refers to the delivery of a nucleic acid or nucleic acids encoding an insulin and / or a glucokinase into a recipient host cell by a viral vector. For example, transduction of a target cell by a rAAV vector of the disclosure leads to transfer of the rAAV genome (e.g., comprising a polynucleotide of the disclosure) contained in that vector into the transduced cell.

[0231] Transfection” of a cell means that genetic material is introduced into a cell for the purpose of genetically modifying the cell. Transfection can be accomplished by a variety of means known in the art, e.g., transduction or electroporation.

[0232] The term “host cell” or “target cell” is used herein to mean the cell into which the polynucleotide delivery takes place, either in vitro or in vivo. In some aspect, AAV vectors are able to transduce both dividing and non-dividing cells.

[0233] “Recombinant” means distinct from that generally found in nature.

[0234] “Serotype” with respect to vector or virus capsid is defined by a distinct immunological profile based on the capsid protein sequences and capsid structure.

[0235] “AAV Cap” means AAV Cap proteins, VP1, VP2 and VP3 and analogs thereof.

[0236] “AAV Rep” means AAV Rep proteins and analogs thereof.

[0237] “Flanked,” with respect to a sequence that is flanked by other elements, indicates the presence of one or more the flanking elements upstream and / or downstream, i.e., 5'and / or 3', relative to the sequence. The term “flanked” is not intended to indicate that the sequences are necessarily contiguous. For example, there may be intervening sequences between the nucleic acid encoding the transgene and a flanking element. A sequence (e.g., a transgene) that is “flanked” by two other elements (e.g., ITRs), indicates that one element is located 5' to the sequence and the other is located 3' to the sequence; however, there may be intervening sequences between.

[0238] As used herein, the terms “effective amount,” “therapeutically effective amount,” and a “sufficient amount” of, e.g., a gene therapy composition comprising a rAAV particle and / or a polynucleotide disclosed herein, refer to a quantity sufficient to, when administered to the subject, including a human, effect beneficial or desired results, including clinical results, and, as such, an “effective amount” or synonym thereto depends on the context in which it is being applied. In some aspects, the amount of a given therapeutic agent or composition can vary depending upon various factors, such as the given agent, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, and / or weight) or host being treated, and the like.

[0239] As used herein, the term “administration” refers to the delivery of a molecule or composition of the present disclosure (e.g., polynucleotides, expression cassettes, delivery vectors, expression constructs, rAAV, combination gene therapy, immunosuppressive agent, etc.) to a subject or system. In some aspects, the administration can include one or more doses delivered by an appropriate route.

[0240] As used herein, the term “skeletal muscle group” refers to muscles that can be identified using nomenclature based their anatomical location or relationship to a particular bone in a vertebrate body. Typically, a subject has a right and a left (bilateral) muscle for each skeletal muscle group. In some aspects, administration can be to a single (right or left) muscle of a skeletal muscle group. In some aspects, administration can be to both the right and left muscle of a skeletal muscle group, i.e., bilateral administration.

[0241] As used herein, the terms “bilateral” or “bilaterally” in the context of intramuscular administration refers to administering to the same muscle or muscle group on both sides of a subject (e.g., to both the right and left side quadriceps, both the right and left side biceps, and / or both the right and left side hamstring muscles). In contrast, “unilateral” refers to administration to a single side of a subject, e.g., the right or the left side.

[0242] As used herein, the terms “immunosuppression” or “immunosuppressive” refers to a temporary or permanent reduction in or limit on an immune response in a subject. An “immunosuppressor” or an “immunomodulator” is a substance capable of inducing a temporary or permanent reduction in or limit to an immune response in a subject, or a change in the type of immune response. For example, an immunomodulator may direct T cell fate from a TH1 proinflammatory T cell, to a TH2 or Treg wound healing or regulatory T cell. In some aspects, the immunosuppression can be to reduce or limit an immune-mediated response to a viral vector (e.g., rAAV). In some aspects, the immune- mediated response can be an innate and / or an adaptive immune response.

[0243] The term “ratio” refers to the comparison of two or more numbers that indicates their quantitative relation to each other. In some aspects, a ratios can be used to compare two portions within a whole or total amount.

[0244] As used herein, the term “vector ratio” refers to the amount, in vector genomes (vg), of one AAV vector compared to the amount, in vector genomes, of another AAV vector. In some aspects, the vector ratio refers to the amount of AAVl-hINS vg to the amount of AAVl-hGCK vg. For example, equal number of AAVl-hINS vg and AAV1- hGCK vg can also be understood as a one-to-one vector ratio or a 1 : 1 vector ratio.

[0245] As used herein, the term “gene therapy” is the insertion of nucleic acid sequences (e.g., a nucleic acid comprising a promoter operably linked to a polynucleotide encoding a therapeutic molecule) into an individual’s cells and / or tissues to treat a disease or condition. Gene therapy can also include insertion of a transgene that is inhibitory in nature, i.e., that inhibit, decrease or reduce expression, activity or function of an endogenous gene or protein, such as an undesirable or aberrant (e.g., pathogenic) gene or protein. Such transgenes can be exogenous. An exogenous molecule or sequence is understood to be molecule or sequence not normally occurring in the cell, tissue and / or individual to be treated. Both acquired and congenital diseases can be amenable to gene therapy.

[0246] In some aspects, the disclosure provides polynucleotides encoding wild-type or mutant insulin and / or wild-type glucokinase or a functional fragment thereof. The disclosure also provides nucleic acid constructs that include as part of their sequence the polynucleotides encoding wild-type or mutant insulin and / or wild-type glucokinase or a functional fragment thereof. For example, the disclosure includes expression cassettes, plasmids and / or other vectors that include the polynucleotides along with other elements,such as regulatory elements. In some aspects, the disclosure provides a packaged gene delivery vehicle, such as a viral capsid (e.g., AAV capsid), including the polynucleotides encoding wild-type or mutant insulin and / or wild-type glucokinase or a functional fragment thereof.

[0247] The disclosure also includes methods of expressing wild-type or mutant insulin and / or wild-type glucokinase or a functional fragment thereof by delivering the polynucleotides into a cell along with elements required to promote expression in the cell. The disclosure also provides gene therapy methods in which the polynucleotides encoding wild-type or mutant insulin and / or wild-type glucokinase or a functional fragment thereof is / are administered to a subject, e.g., as a component of one or more vectors and / or packaged as a component of one or more viral gene delivery vehicles (e.g., AAV particles).

[0248] The disclosure also provides gene therapy methods in which the polynucleotides encoding wild-type or mutant insulin and / or wild-type glucokinase or a functional fragment thereof is / are intramuscularly administered to a subject, e.g., as a component of one or more vectors and / or packaged as a component of one or more viral gene delivery vehicles (e.g., AAV particles), in particular, where the intramuscular administration is to at least two different skeletal muscle groups (e.g., quadriceps, biceps, and hamstrings), preferably, as bilateral administrations.

[0249] The disclosure also provides an immunosuppressive regimen (e.g., administration of an IL-6 antagonist such as tocilizumab prior to the gene therapy administration and / or administration of a calcineurin inhibitor such as tacrolimus for a time period after the gene therapy administration) to accompany the gene therapy methods in which the polynucleotides encoding wild-type or mutant insulin and / or wild-type glucokinase or a functional fragment thereof is / are intramuscularly administered to a subject, e.g., as a component of one or more vectors and / or packaged as a component of one or more viral gene delivery vehicles (e.g., AAV particles), in particular, where the intramuscular administration is to at least two different skeletal muscle groups (e.g., quadriceps, biceps, and hamstrings), preferably, as bilateral administrations. Treatment can, for example, be used to treat or reduce the symptoms of diabetes in a subject in need thereof.

[0250] Furthermore, certain aspects of the disclosure, e.g., disclosed methods of intramuscular administration (e.g., two or more skeletal muscle groups, preferably, bilaterally) and / or use of an immunosuppressive regimen (e.g., administration of an IL-6antagonist such as tocilizumab prior to the gene therapy administration and / or administration of a calcineurin inhibitor such as tacrolimus for a time period after the gene therapy administration) can provide improvements in the safety and efficacy of the gene therapy treatments disclosed herein.

[0251] Additional aspects of the disclosure are directed to a method of immunosuppression in a subject administered an AAV gene therapy for the treatment of diabetes, wherein the subject is administered an immunosuppressive regimen comprising administering (a) an immunosuppressive agent (pre- AAV) prior to administering the AAV gene therapy; and (b) an immunosuppressive agent (post-AAV) after administering the AAV gene therapy.

[0252] In some aspects, the methods disclosed herein result in (i) a reduction in glycated blood hemoglobin (HbAlc) levels in the treated subject; (ii) a reduction in circulating ketones in the treated subject, (iii) a reduction in triglycerides in the treated subject, (iv) a reduction in exogenous insulin in the treated subject, (v) an increase in circulating C- peptide levels the treated subject, (vi) reduction in immune response to exogenous AAV, insulin protein, and / or glucokinase protein, or (vii) any combination thereof.

[0253] Each of these aspects of the disclosure is discussed in further detail herein.Insulin and Glucokinase Gene Therapy Combination and Methods of Administration

[0254] Certain aspects of the disclosure are directed to a therapy, e.g., 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 preferred aspects, the combination gene therapy is administered to at least two different skeletal muscle groups. In some aspects, the combination therapy includes IM administration of the insulin and glucokinase encoding polynucleotides in a single pharmaceutical composition, e.g., (i) two separate rAAV particles comprising the insulin and glucokinase encoding polynucleotides, respectively, in the same pharmaceutical composition or (ii) a rAAV particle comprising both the insulin and glucokinase encoding polynucleotides in the pharmaceutical composition. In some aspects, the combination therapy of the 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.

[0255] In some aspects, the 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; (ii) 2-5, 3-5, or 4-5 different skeletal muscle groups; (iv) 2-4 or 3-4 different skeletal muscle groups.

[0256] Certain aspects are directed to administering a combination therapy of the 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 the disclosure is administered intramuscularly (IM) to 3-10, 4-10, 5-10, 6-10, 7-10 or 8-10 different skeletal muscle groups. In some aspects, the combination therapy is administered intramuscularly (IM) to two, three, four, five, six, seven, eight, nine or ten different skeletal muscle groups.

[0257] There are three (3) types of muscle: skeletal muscle, cardiac muscle, and smooth muscle. Each type of muscle has a different structure and function. Inside each skeletal muscle, muscle fibers are organized into bundles, called fascicles, surrounded by a middle layer of connective tissue. Skeletal muscles in vertebrates are typically attached to bones and are responsible for skeletal movements. Skeletal muscles also have a role in sustaining body posture and position, maintaining body temperature, storing nutrients, and stabilizing joints. Skeletal muscle groups of the upper body include, but are not limited to, biceps, triceps, abdominal, pectoral, deltoid, trapezius, latissimus dorsi, and erector spinae. Skeletal muscle groups of the lower body include, but are not limited to, quadriceps, hamstrings, gastrocnemius (e.g., calf), soleus, and gluteus (e.g., gluteus maximus).

[0258] In some aspects, the different skeletal muscle groups can comprise a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and a latissimus dorsi (lat). In some aspects, the skeletal muscle groups are selected from a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and a latissimus dorsi (lat), or any combination thereof. In some aspects, the skeletal muscle groups are selected from a quadriceps, a bicep, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, and a hamstring, or any combination thereof. In some aspects, the skeletal muscle groups are selected from a quadriceps, a bicep, and a hamstring, or any combination thereof.

[0259] In some aspects, the different skeletal muscle groups can comprise a quadriceps, a bicep (e.g., the biceps brachii or the biceps femoris), a triceps, a gastrocnemius (e.g., acalf muscle), a gluteus maximus, a hamstring (e.g., the semitendinosus, the semimembranosus, or the biceps femoris), a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), a thigh muscle (e.g., vastus intermedius, vastus lateralis, or vastus medialis), a hip muscle (gluteus medius), and a latissimus dorsi (lat).

[0260] In some aspects, the different skeletal muscle groups can comprise a iliopsoas muscle, a tensor fasciae latae muscle, a vastus lateralis muscle, the iliotibial tract, a pectineus muscle, a adductor longus muscle, a gracillis muscle, a rectus femoris muscle, a vastus medialis muscle, a gluteus maximus muscle, a adductor magnus muscle, a semitendinosus muscle, a gracillis muscle, a semimembranosus muscle, a sartorius muscle, a gluteus medius muscle, a biceps femoris muscle, a plantaris muscle, or a gastrocnemius muscle. In some aspects, the different skeletal muscle groups can comprise any of the muscles disclosed in FIGs. 19A-19B.

[0261] In some aspects, the administration comprises bilateral injections to one or more of the skeletal muscle groups (e.g., IM injections to the right and left quadriceps, the right and left biceps, the right and left gastrocnemius muscles, the right and left gluteus maximus muscles, the right and left triceps, and / or the right and left hamstrings).

[0262] In some aspects, the administration comprises bilateral injections to one or more of the skeletal muscle groups (e.g., IM injections to the right and left quadriceps, the right and left biceps, the right and left gastrocnemius muscles, the right and left gluteus maximus muscles, and / or the right and left hamstrings).

[0263] In some aspects, the bilateral injections are divided equally for each skeletal muscle group (e.g., the right quadriceps and the left quadriceps receive approximately the same administration volume and / or dose).

[0264] In some aspects, the different skeletal muscle groups can comprise a iliopsoas muscle, a tensor fasciae latae muscle, a vastus lateralis muscle, the iliotibial tract, a pectineus muscle, a adductor longus muscle, a gracillis muscle, a rectus femoris muscle, a vastus medialis muscle, a gluteus maximus muscle, a adductor magnus muscle, a semitendinosus muscle, a gracillis muscle, a semimembranosus muscle, a sartorius muscle, a gluteus medius muscle, a biceps femoris muscle, a plantaris muscle, or a gastrocnemius muscle. In some aspects, the different skeletal muscle groups can comprise any of the muscles disclosed in FIGs. 19A-19B.

[0265] In some aspects, the skeletal muscle group is selected from a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid,a trapezius, a pectoral muscle (e.g., pectoralis major), and a latissimus dorsi (lat). In some aspects, the two or more different skeletal muscle groups is any combination of two or more of a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), or a latissimus dorsi (lat).

[0266] In some aspects, the skeletal muscle group is selected from a quadriceps, a bicep, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, and a hamstring. In some aspects, the two or more different skeletal muscle groups comprises any combination of two or more of a quadriceps, a bicep, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, or a hamstring.

[0267] In some aspects, the administration comprises a total of 20-150 IM injections of the therapy (e.g., combination gene therapy) to the at least two different skeletal muscle groups. In some aspects, the administration comprises 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.

[0268] In some aspects, the administration comprises 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 a 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 a second skeletal muscle group. In some aspects, the administration comprises 1-50 (e.g., 2-50, 4-50, 6-50, 8-50, 10-50, 20-50, 30-50, or 40-50) IM injections to a 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 a 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 a third skeletal muscle group.

[0269] In some aspects, the administration comprises 8-50 IM injections to the quadriceps (e.g., 4-25 IM injections bilaterally to each quadriceps), 8-50 IM injections to the biceps (e.g., 4-25 IM injections bilaterally to each bicep), and 8-50 IM injections to the hamstrings (e.g., 4-25 IM injections bilaterally to each hamstring).

[0270] In some aspects, the administration comprises (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 afourth skeletal muscle group; (iv) 1-30 IM injections to a first skeletal muscle group, 1-30 IM injections to a second skeletal muscle group, 1-30 IM injections to a third skeletal muscle group, 1-30 IM injections to a fourth skeletal muscle group, and 1-30 IM injections to a fifth skeletal muscle group; and (v) 1-25 IM injections to a first skeletal muscle group, 1-25 IM injections to a second skeletal muscle group, 1-25 IM injections to a third skeletal muscle group, 1-25 IM injections to a fourth skeletal muscle group, 1-25 IM injections to a fifth skeletal muscle group, and 1-25 IM injections to a sixth skeletal muscle group.

[0271] In some aspects, the administration comprises: (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 a 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 a second skeletal muscle group; (ii) 2-60, 4-60, 6-60, 8-60, 10-60, 20-60, 30-60, 40-60, or 50-60 IM injections to a first skeletal muscle group; 2-60, 4-60, 6-60, 8-60, 10-60, 20-60, 30-60, 40-60, or 50-60 IM injections to a 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 to a third skeletal muscle group; (iii) 2-40, 4-40, 6-40, 8-40, 10-40, 20-40, or 30-40 IM injections to a first skeletal muscle group; 2-40, 4-40, 6-40, 8-40, 10-40, 20-40, or 30-40 IM injections to a second skeletal muscle group; 2-40, 4-40, 6-40, 8-40, 10-40, 20-40, or 30-40 IM injections to a third skeletal muscle group; and 2-40, 4-40, 6-40, 8-40, 10-40, 20-40, or 30-40 IM injections to a fourth skeletal muscle group; (iv) 2-30, 4-30, 6-30, 8-30, 10-30, or 20-30 IM injections to a first skeletal muscle group; 2-30, 4-30, 6-30, 8-30, 10-30, or 20-30 IM injections to a second skeletal muscle group; 2-30, 4-30, 6-30, 8-30, 10-30, or 20-30 IM injections to a third skeletal muscle group; 2-30, 4-30, 6-30, 8-30, 10-30, or 20-30 IM injections to a fourth skeletal muscle group; and 2-30, 4-30, 6-30, 8-30, 10-30, or 20-30 IM injections to a fifth skeletal muscle group; or (v) 2-25, 4-25, 6-25, 8-25, 10-25, or 20- 25 IM injections to a first skeletal muscle group; 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 IM injections to a second skeletal muscle group; 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 IM injections to a third skeletal muscle group; 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 IM injections to a fourth skeletal muscle group; 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 IM injections to a fifth skeletal muscle group; and 2-25, 4-25, 6-25, 8-25, 10-25, or 20-25 IM injections to a sixth skeletal muscle group.

[0272] In some aspects, the administration 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) IM injections of the therapy (e.g., combination gene therapy) to the at least two different skeletal muscle groups. In some aspects, the administration comprises 10-35 (e.g., 10-15, 15-20, 20-25, 25-30, 10-20, 20-30, 15-25, or 30-35) IM injections to a 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 a second skeletal muscle group. In some aspects, the administration comprises 10-35 (e.g., 10-15, 15-20, 20-25, 25-30, 10-20, 20-30, 15-25, or 30-35) IM injections to a 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 a 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 a third skeletal muscle group.

[0273] In some aspects, each IM injection is administered in a volume of about 100 pL to about 2 mL, 100 pL to about 1.9 mL, 100 pL to about 1.8 mL, 100 pL to about 1.7 mL, 100 pL to about 1.6 mL, 100 pL to about 1.5 mL, 100 pL to about 1.4 mL, about 100 pL to about 1.35 mL, about 100 pL to about 1.2 mL, about 100 pL to about 1.0 mL, about 100 pL to about 800 pL, about 200 pL to about 800 pL, about 200 pL to about 600 pL, about 400 pL to about 800 pL, about 500 pL to about 1.5 mL, about 600 pL to about 1.4 mL, about 700 pL to about 1.3 mL, about 800 pL to about 1.2 mL, or about 900 pL to about 1.1 mL.

[0274] In some aspects, each IM injection is administered in a volume of about 100 pL to about 1.5 mL (e.g., about 100 pL to about 1.4 mL, about 100 pL to about 1.35 mL, about 100 pL to about 1.2 mL, about 100 pL to about 1.0 mL, about 100 pL to about 800 pL, about 200 pL to about 800 pL, about 200 pL to about 600 pL, or about 400 pL to about 800 pL).

[0275] In some aspects, each IM injection is administered in a volume of about 500 pL to about 1 mL. In some aspects, each IM injection is administered in a volume of about 500 pL. In some aspects, each IM injection is administered in a volume of about 1 mL

[0276] In some aspects, the total volume of the therapy administered 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 some aspects, the total volume of therapy administered 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.

[0277] In some aspects, the dose per IM injection comprises about 2xl013to about 5xl013vgs per mL.

[0278] In some aspects, each IM injection comprises about 8xlO10to about 5xl013vgs per mL. In some aspects, each IM injection comprises about 8xlO10to 2xl013vgs per mL. In some aspects, each IM injection comprises about 2xl013to about 4xl013vgs per mL.

[0279] In some aspects, each IM injection comprises about 8xlO10to about 8xl012vgs per mL, about 8xlO10to about 8xl0nvgs per mL, about 8xlO10to about 2xl012vgs per mL, about 8xlO10to about 4xl012vgs per mL, about 2xl012to about 8xl012vgs per mL, or about 8xl0nto about 4xl012vgs per mL.

[0280] In some aspects, each IM injection comprises about IxlO11to about IxlO13vgs per mL, about IxlO11to about IxlO12vgs per mL, about IxlO11to about 2.6xl012vgs per mL, about IxlO11to about 5xl012vgs per mL, about 2.6xl012to about IxlO13vgs per mL, or about IxlO12to about 5xl012vgs per mL.

[0281] In some aspects, each IM injection comprises about 1.2xlOnto about 1.2xl013vgs per mL, about 1.2xlOnto about 1.2xl012vgs per mL, about 1.2xlOnto about 3. IxlO12vgs per mL, about 1.2xlOnto about 6xl012vgs per mL, about 3. IxlO12to about 1.2xl013vgs per mL, or about 1.2xl012to about 6xl012vgs per mL.

[0282] In some aspects, each IM injection comprises about 1.4xlOnto about 1.4xl013vgs per mL, about 1.4xlOnto about 1.4xl012vgs per mL, about 1.4xlOnto about 3.6xl012vgs per mL, about 1.4xlOnto about 7xl012vgs per mL, about 3.6xl012to about 1.4xl013vgs per mL, or about 1.4xl012to about 7xl012vgs per mL.

[0283] In some aspects, each IM injection comprises about 1.6xlOnto about 1.6xl013vgs per mL, about 1.6xlOnto about 1.6xl012vgs per mL, about 1.6xlOnto about 4. IxlO12vgs per mL, about 1.6xlOnto about 8xl012vgs per mL, about 4. IxlO12to about 1.6xl013vgs per mL, or about 1.6xl012to about 8xl012vgs per mL.

[0284] In some aspects, each IM injection comprises about 1.8xl0nto about 1.8xl013vgs per mL, about 1.8xl0nto about 1.8xl012vgs per mL, about 1.8xl0nto about 4.6xl012vgs per mL, about 1.8xl0nto about 9xl012vgs per mL, about 4.6xl012to about 1.8xl013vgs per mL, or about 1.8xl012to about 9xl012vgs per mL.

[0285] In some aspects, each IM injection comprises about 2xlOnto about 2xl013vgs per mL, about 2xlOnto about 2xl012vgs per mL, about 2xlOnto about 5. IxlO12vgs per mL, about 2xlOnto about IxlO13vgs per mL, about 5. IxlO12to about 2xl013vgs per mL, or about 2xl012to about IxlO13vgs per mL.

[0286] In some aspects, each IM injection comprises about 8xlO10to about 2xlOnvgs per mL, about 8xlO10to about IxlO11vgs per mL, about 8xlO10to about 1.2xlOnvgs per mL, about 8xlO10to about L4xlOnvgs per mL, about 8xlO10to about 1.6xlOnvgs per mL, about 8xlO10to about 1.8xl0nvgs per mL, about L4xlOnto about 2xlOnvgs per mL, about IxlO11to about 1.8xl0nvgs per mL, or about 1.2xlOnto about 1.6xlOnvgs per mL.

[0287] In some aspects, each IM injection comprises about 8xl0nto about 2xl012vgs per mL, about 8xl0nto about IxlO12vgs per mL, about 8xl0nto about 1.2xl012vgs per mL, about 8xl0nto about 1.4xl012vgs per mL, about 8xl0nto about 1.6xl012vgs per mL, about 8xl0nto about 1.8xl012vgs per mL, about 1.4xl012to about 2xl012vgs per mL, about IxlO12to about 1.8xl012vgs per mL, or about 1.2xl012to about 1.6xl012vgs per mL.

[0288] In some aspects, each IM injection comprises about 2xl012to about 5. IxlO12vgs per mL, about 2xl012to about 2.6xl012vgs per mL, about 2xl012to about 3. IxlO12vgs per mL, about 2xl012to about 3.6xl012vgs per mL, about 2xl012to about 4. IxlO12vgs per mL, about 2xl012to about 4.6xl012vgs per mL, about 3.6xl012to about 5. IxlO12vgs per mL, about 2.6xl012to about 4.6xl012vgs per mL, or about 3. IxlO12to about 4. IxlO12vgs per mL.

[0289] In some aspects, each IM injection comprises about 4xl012to about IxlO13vgs per mL, about 4xl012to about 5xl012vgs per mL, about 4xl012to about 6xl012vgs per mL, about 4xl012to about 7xl012vgs per mL, about 4xl012to about 8xl012vgs per mL, about 4xl012to about 9xl012vgs per mL, about 7xl012to about IxlO13vgs per mL, about 5xl012to about 9xl012vgs per mL, or about 6xl012to about 8xl012vgs per mL.

[0290] In some aspects, each IM injection comprises about 8xl012to about 2xl013vgs per mL, about 8xl012to about IxlO13vgs per mL, about 8xl012to about 1.2xl013vgs per mL, about 8xl012to about 1.4xl013vgs per mL, about 8xl012to about 1.6xl013vgs per mL, about 8xl012to about 1.8xl013vgs per mL, about 1.4xl013to about 2xl013vgs per mL, about IxlO13to about 1.8xl013vgs per mL, or about 1.2xl013to about 1.6xl013vgs per mL.

[0291] In some aspects, the administration comprises IM injections that are separated by a distance of at least 1-7 cm. In some aspects, the administration comprises IM injections that are separated by a distance of at least 1-3 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 5 cm. In some aspects,each of the IM injection sites on the subject are separated by a distance of at least 3 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 2 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 1 cm.

[0292] In some aspects, the administration comprises IM injections that are separated by a distance of at least 1-7 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 7 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 6 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 5 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 4 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 3 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 2 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 1 cm.

[0293] In some aspects, each of the IM injection sites on the subject are separated by a distance of about 7 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of about 6 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of about 5 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of about 4 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of about 3 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of about 2 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of about 1 cm.

[0294] In some aspects, the administration comprises IM injections that are 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, each of the IM injections is between 1-2 cm deep.

[0295] In some aspects, each of the IM injections is at least 0.5 cm below the surface of the injected muscle, regardless of the thickness of the subcutaneous fat.

[0296] In some aspects, the administration comprises IM injections that are 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.

[0297] In some aspects, the administration comprises IM injections that are 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, a 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.

[0298] In some aspects, each of the IM injections is between about 3.5 to about 5 cm deep.

[0299] In some aspects, the combination therapy (e.g. either a single formulation or separate formulations) is administered 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 aspects, the combination therapy is administered intramuscularly (IM) to two, three, four, five, six, seven, eight, nine, or ten different skeletal muscle groups. In some aspects, the skeletal muscle groups are selected from a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and a latissimus dorsi (lat). In some aspects, the skeletal muscle groups are selected from a quadriceps, a bicep, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, and a hamstring. In some aspects, the skeletal muscle groups are selected from a quadriceps, a bicep, and a hamstring. In some aspects, the skeletal muscle group is selected from a quadriceps, a bicep, a triceps, a gastrocnemius, a gluteus maximus, and a hamstring, optionally wherein the two or more different skeletal muscle groups comprise any combination thereof.

[0300] In some aspects, the skeletal muscle group is selected from a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and a latissimus dorsi (lat). In some aspects, the two or more different skeletal muscle groups is any combination of two or more of a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), or a latissimus dorsi (lat).

[0301] In some aspects, the skeletal muscle group is selected from a quadriceps, a bicep, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, and a hamstring. In some aspects, the two or more different skeletal muscle groups is any combination of two or more of a quadriceps, a bicep, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, or a hamstring.

[0302] In some aspects, the administration comprises bilateral injections into one or more of the skeletal muscle groups (e.g., IM injections into the right and left quadriceps, the right and left biceps, the right and left gastrocnemius muscles, the right and left gluteus maximus muscles, and / or the right and left hamstrings).

[0303] In some aspects, the present disclosure is directed to a combination therapy formulated for IM administration comprising a) an insulin expression cassette comprising a first promoter operably linked to a polynucleotide encoding a human insulin (hlns) protein, wherein the insulin expression cassette is flanked by inverted terminal repeats (ITRs), and b) a glucokinase expression cassette comprising a second promoter operably linked to a polynucleotide encoding a human glucokinase (hGck) protein, wherein the glucokinase expression cassette is flanked by ITRs, wherein the combination therapy is administered to at least two different skeletal muscle groups.

[0304] In some aspects, a first AAV vector genome comprising the insulin expression cassette and a second AAV vector genome comprising the glucokinase expression cassette are administered. In some aspects, a AAV vector genome comprising both the insulin expression cassette and the glucokinase expression cassette is administered.

[0305] In some aspects, the present disclosure is directed to a combination therapy formulated for IM administration comprising a) a first AAV vector genome comprising an insulin expression cassette comprising a first promoter operably linked to a polynucleotide encoding a human insulin (hlns) protein, wherein the insulin expression cassette is flanked by inverted terminal repeats (ITRs), and b) a second AAV vector genome comprising a glucokinase expression cassette comprising a second promoter operably linked to a polynucleotide encoding a human glucokinase (hGck) protein, wherein the glucokinase expression cassette is flanked by ITRs, wherein the first AAV vector genome and the second AAV vector genome are in a vector ratio 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).

[0306] In some aspects, the vector ratio is about 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 some aspects, the vector ratio is about 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, l:0.3-0.7, 1:0.3-0.65, 1:0.3-0.6, 1:0.3-0.55, l: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, l:0.4-0.6, 1:0.4-0.55, l: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 vector 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.

[0307] In some aspects, the vector ratio is selected from the group consisting of 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 ratio is 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 ratio is selected from the group consisting of 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.

[0308] In some aspects, the vector ratio is about 1:0.25-0.75.

[0309] In some aspects, the vector ratio is about 1:0.4-0.6.

[0310] In some aspects, the vector ratio is about 1:0.5.

[0311] In some aspects, the vector ratio is about 1:0.9-1.1

[0312] In some aspects, the vector ratio is about 1:1.

[0313] In some aspects, the vector ratio is about 1 :2.

[0314] In some aspects, the vector ratio is about 1 :3.

[0315] In some aspects, the vector ratio is about 1 :4.

[0316] In some aspects, the vector ratio is about 1:5.

[0317] In some aspects, the vector ratio is about 1:10.

[0318] In some aspects, the vector ratio is about 1 :20.

[0319] In some aspects, the vector ratio is between about 1 :3 and about 1:5.

[0320] In some aspects, the vector 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.

[0321] In some aspects, the first AAV vector genome comprising an insulin expression cassette has a dosage that produces between about 1 pU / mL to about 3 pU / mL, about 1 pU / mL to about 5 pU / mL, about 1 pU / mL to about 10 pU / mL, about 3 pU / mL to about 10 pU / mL, about 4 pU / mL to about 10 pU / mL, about 5 pU / mL to about 10 pU / mL,about 6 pU / mL to about 10 pU / mL, about 7 pU / mL to about 10 pU / mL, about 8 pU / mL to about 10 pU / mL, about 9 pU / mL to about 10 pU / mL, about 3 pU / mL to about 9 pU / mL, about 3 pU / mL to about 8 pU / mL, about 3 pU / mL to about 7 pU / mL, about 3 pU / mL to about 6 pU / mL, about 3 pU / mL to about 5 pU / mL, about 5 pU / mL to about 9 pU / mL, about 5 pU / mL to about 8 pU / mL, about 5 pU / mL to about 7 pU / mL, about 5 pU / mL to about 6 pU / mL, about 6 pU / mL to about 9 pU / mL, about 6 pU / mL to about 8 pU / mL, or about 6 pU / mL to about 7 pU / mL, about 1 pU / mL to about 40 pU / mL, about 3 pU / mL to about 40 pU / mL, about 5 pU / mL to about 40 pU / mL, about 10 pU / mL to about 40 pU / mL, about 15 pU / mL to about 40 pU / mL, about 20 pU / mL to about 40 pU / mL, about 25 pU / mL to about 40 pU / mL, about 30 pU / mL to about 40 pU / mL, about 35 pU / mL to about 40 pU / mL, about 20 pU / mL to about 30 pU / mL, about 10 pU / mL to about 30 pU / mL, or about 10 pU / mL to about 20 pU / mL of insulin in the serum of a subject.

[0322] In some aspects, the combination therapy comprise a first AAV vector genome comprising an insulin expression cassette and a second AAV vector genome comprising an 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, which is administered to at least two different skeletal muscle groups. In some aspects, the skeletal muscle groups comprise a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and a latissimus dorsi (lat). In some aspects, the at least two different skeletal muscle groups are selected from two or more of a quadriceps, a bicep, a hamstring, or any combination thereof. In some aspects, the at least two different skeletal muscle groups are comprise a quadriceps, a bicep, a hamstring, or any combination thereof.

[0323] In some aspects, the combination therapy comprise a first AAV vector genome comprising an insulin expression cassette and a second AAV vector genome comprising an 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, which is administered to at least two different skeletal muscle groups. In some aspects, the skeletal muscle groups comprise a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and a latissimus dorsi (lat).

[0324] In some aspects, the skeletal muscle group is selected from a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and a latissimus dorsi (lat). In some aspects, the two or more different skeletal muscle groups is any combination of two or more of a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), or a latissimus dorsi (lat).

[0325] In some aspects, the skeletal muscle group is selected from a quadriceps, a bicep, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, and a hamstring. In some aspects, the two or more different skeletal muscle groups is any combination of two or more of a quadriceps, a bicep, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, or a hamstring.

[0326] In some aspects, the administration comprises bilateral injections into one or more of the skeletal muscle groups (e.g., IM injections into the right and left quadriceps, the right and left biceps, the right and left gastrocnemius muscles, the right and left gluteus maximus muscles, and / or the right and left hamstrings).

[0327] In some aspects, the combination therapy comprises a first AAV vector genome comprising an insulin expression cassette and a second AAV vector genome comprising an 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, which is administered to at least three different skeletal muscle groups. In some aspects, the different skeletal muscle groups comprise three or more of a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), or a latissimus dorsi (lat).

[0328] In some aspects, the combination therapy comprises a first AAV vector genome comprising an insulin expression cassette and a second AAV vector genome comprising an 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, which is administered to at least three different skeletal muscle groups.

[0329] In some aspects, the combination therapy comprises (i) a first recombinant AAV (rAAV) particle comprising the first AAV vector genome comprising the insulinexpression cassette comprising the promoter operably linked to the polynucleotide encoding the human insulin (hlns) protein, wherein the insulin expression cassette is flanked by inverted terminal repeats (ITRs); and (ii) a second recombinant AAV (rAAV) particle comprising the second AAV vector genome comprising the glucokinase expression cassette comprising the promoter operably linked to the polynucleotide encoding the human glucokinase (hGck) protein, wherein the glucokinase expression cassette is flanked by inverted terminal repeats (ITRs).

[0330] In some aspects, the at least three different skeletal muscle groups are selected from a quadriceps, a bicep, and a hamstring, or any combination thereof. In some aspects, the at least three different skeletal muscle groups comprise a quadriceps, a bicep, and / or a hamstring.

[0331] In some aspects, the administration comprises bilateral injections into one or more of the at least three different skeletal muscle groups. In some aspects, the administration comprises bilateral injections into one or more of the skeletal muscle groups (e.g., IM injections into the right and left quadriceps, the right and left biceps, the right and left gastrocnemius muscles, the right and left gluteus maximus muscles, and / or the right and left hamstrings).

[0332] In some aspects, the combination therapy comprise a first AAV vector genome comprising an insulin expression cassette and a second AAV vector genome comprising an 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 formulation, which is 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 a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), or a latissimus dorsi (lat). In some aspects, the at least two different skeletal muscle groups comprise at least two of a quadriceps, a bicep, and a hamstring. In some aspects, the combination therapy comprise a first AAV vector genome comprising an insulin expression cassette and a second AAV vector genome comprising an 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, which is administered to at least two different skeletal muscle groups.

[0333] In some aspects, the administration comprises bilateral injections into one or more of the skeletal muscle groups (e.g., IM injections into the right and left quadriceps, the right and left biceps, the right and left gastrocnemius muscles, the right and left gluteus maximus muscles, and / or the right and left hamstrings).

[0334] In some aspects, the combination therapy comprises a first AAV vector genome comprising an insulin expression cassette and a second AAV vector genome comprising an 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, which is administered to at least three different skeletal muscle groups comprising a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and a latissimus dorsi (lat). In some aspects, the at least two different skeletal muscle groups comprise at least two of a quadriceps, a bicep, and / or a hamstring.

[0335] In some aspects, the combination therapy comprises a first AAV vector genome comprising an insulin expression cassette and a second AAV vector genome comprising an 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, which is administered to at least three different skeletal muscle groups comprising a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and a latissimus dorsi (lat).

[0336] In some aspects, the administration comprises bilateral injections into one or more of the at least three different skeletal muscle groups. In some aspects, the administration comprises bilateral injections into one or more of the skeletal muscle groups (e.g., IM injections into the right and left quadriceps, the right and left biceps, the right and left gastrocnemius muscles, the right and left gluteus maximus muscles, and / or the right and left hamstrings).

[0337] In some aspects, each IM injection is administered in a volume of about 100 pL to about 1.5 mL (e.g., about 100 pL to about 1.4 mL, about 100 pL to about 1.35 mL, about 100 pL to about 1.2 mL, about 100 pL to about 1.0 mL, about 100 pL to about 800 pL, about 200 pL to about 800 pL, about 200 pL to about 600 pL, or about 400 pL to about 800 pL).

[0338] In some aspects, the dose per IM injection comprises about 2xl013to 5xl013vg per mL.

[0339] In some aspects, the dose per IM injection comprises about 7xlO10to 7xl012vg / kg of the first AAV vector genome (e.g., hlns). In some aspects, the dose per IM injection comprises about 7xlO10to 7xlOnvg / kg of the first AAV vector genome (e.g., hlns). In some aspects, the dose per IM injection comprises about 7xlO10to 1.8xl012vg / kg of the first AAV vector genome (e.g., hlns). In some aspects, the dose per IM injection comprises about 7xlO10to 3.5xl012vg / kg of the first AAV vector genome (e.g., hlns). In some aspects, the dose per IM injection comprises about 1.8xl012to 7xl012vg / kg of the first AAV vector genome (e.g., hlns). In some aspects, the dose per IM injection comprises about 1.8xl012to 3.5xl012vg / kg of the first AAV vector genome (e.g., hlns). In some aspects, the dose per IM injection comprises about 3.5xl012to 7xl012vg / kg of the first AAV vector genome (e.g., hlns).

[0340] In some aspects, the dose per IM injection comprises about 3.2xl012to 3.5xl012vg / kg of the second AAV vector genome (e.g., hGck). In some aspects, the dose per IM injection comprises about 2xl012to 5xl012vg / kg of the second AAV vector genome (e.g., hGck). In some aspects, the dose per IM injection comprises about 2xl012to 3.5xl012vg / kg of the second AAV vector genome (e.g., hGck). In some aspects, the dose per IM injection comprises about 3.5xl012to 5xl012vg / kg of the second AAV vector genome (e.g., hGck).

[0341] In some aspects, the administration comprises IM injections that are separated by a distance of at least 1-3 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 3 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 2 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 1 cm.

[0342] In some aspects, the administration comprises IM injections that are separated by a distance of at least 1-7 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 5 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 4 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 3 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 2 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 1 cm.

[0343] In some aspects, each of the IM injection sites on the subject are separated by a distance of about 7 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of about 6 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of about 5 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of about 4 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of about 3 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of about 2 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of about 1 cm.

[0344] In some aspects, the administration comprises IM injections that are 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, each of the IM injections is between 1-2 cm deep.

[0345] In some aspects, the first AAV vector genome, the second AAV vector genome, or both the first AAV vector genome and second AAV vector genome are administered intramuscularly at a dose of about 5xl0nto about IxlO14vector genomes vg / kg. In some aspects, the first AAV vector genome and second AAV vector genome are each administered at a dose between about 5xl0nto about 5xl013vector genomes (vg) / kg. In some aspects, the first AAV vector genome, the second AAV vector genome, or both the first AAV vector genome and second AAV vector genome are administered intramuscularly at a dose of about lxl012to about IxlO13vg / kg.

[0346] In some aspects, the first AAV vector genome, the second AAV vector genome, or both the first AAV vector genome and second AAV vector genome are administered intramuscularly at a dose of about IxlO12vg / kg to about IxlO14vg / kg, about 2xl012vg / kg to about IxlO14vg / kg, about 3xl012vg / kg to about IxlO14vg / kg, about 4xl012vg / kg to about IxlO14vg / kg, about 5xl012vg / kg to about IxlO14vg / kg, about 6xl012vg / kg to about IxlO14vg / kg, about 7xl012vg / kg to about IxlO14vg / kg, about 8xl012vg / kg to about IxlO14vg / kg, or about 9xl012vg / kg to about IxlO14vg / kg.

[0347] In some aspects, the first AAV vector genome, the second AAV vector genome, or both the first AAV vector genome and second AAV vector genome are administered intramuscularly at a dose of about 7xlO10vg / kg to about IxlO14vg / kg, about 8xl010vg / kg to about IxlO14vg / kg, about 9xlO10vg / kg to about IxlO14vg / kg, about IxlO11vg / kg toabout IxlO14vg / kg, about 2xlOnvg / kg to about IxlO14vg / kg, about 3xlOnvg / kg to about IxlO14vg / kg, about 4xlOnvg / kg to about IxlO14vg / kg, about 7xlO10vg / kg to about IxlO13vg / kg, about 7xlO10vg / kg to about IxlO12vg / kg, about 7xlO10vg / kg to about IxlO11vg / kg, about 7xlO10vg / kg to about 7xl012vg / kg, about 7xlO10vg / kg to about 7xlOnvg / kg, about 7xlO10vg / kg to about 1.8xl012vg / kg, about 7xlO10vg / kg to about 3.5xl012vg / kg, about 1.8xl012vg / kg to about 7xl012vg / kg, about 1.8xl012vg / kg to about 3.5xl012vg / kg, about 3.5xl012vg / kg to about 7xl012vg / kg, about 2xl012vg / kg to about 5xl012vg / kg, about 2xl012vg / kg to about 3.5xl012vg / kg, about 3.5xl012vg / kg to about 5xl012vg / kg, about 5xlOnvg / kg to about IxlO14vg / kg, about 6xlOnvg / kg to about IxlO14vg / kg, about 7xlOnvg / kg to about IxlO14vg / kg, about 8xlOnvg / kg to about IxlO14vg / kg, or about 9xlOnvg / kg to about IxlO14vg / kg.

[0348] In some aspects, the first AAV vector genome, the second AAV vector genome, or both the first AAV vector genome and second AAV vector genome are administered intramuscularly at a dose of about 2xl012vg / kg to about IxlO14vg / kg, about 3xl012vg / kg to about IxlO14vg / kg, about 4xl012vg / kg to about IxlO14vg / kg, about 5xl012vg / kg to about IxlO14vg / kg, about 6xl012vg / kg to about IxlO14vg / kg, about 7xl012vg / kg to about IxlO14vg / kg, or about 8xl012vg / kg to about IxlO14vg / kg.

[0349] In some aspects, the first AAV vector genome, the second AAV vector genome, or both the first AAV vector genome and second AAV vector genome are administered intramuscularly at a dose of about 5xl0nvg / kg to about IxlO14vg / kg, about 6xlOnvg / kg to about IxlO14vg / kg, about 7xlOnvg / kg to about IxlO14vg / kg, about 8xl0nvg / kg to about IxlO14vg / kg, or about 9xlOnvg / kg to about IxlO14vg / kg.

[0350] In some aspects, the first AAV vector genome, the second AAV vector genome, or both the first AAV vector genome and second AAV vector genome are administered intramuscularly at a dose of about IxlO13vg / kg to about IxlO14vg / kg, about 2xl013vg / kg to about IxlO14vg / kg, about 3xl013vg / kg to about IxlO14vg / kg, about 4xl013vg / kg to about IxlO14vg / kg, about 5xl013vg / kg to about IxlO14vg / kg, about 6xl013vg / kg to about IxlO14vg / kg, about 7xl013vg / kg to about IxlO14vg / kg, about 8xl013vg / kg to about IxlO14vg / kg, or about 8xl013vg / kg to about IxlO14vg / kg.

[0351] In some aspects, the first AAV vector genome, the second AAV vector genome, or both the first AAV vector genome and second AAV vector genome are administered intramuscularly at a dose of about 2xl013vg / kg to about IxlO14vg / kg, about 3xl013vg / kg to about IxlO14vg / kg, about 4xl013vg / kg to about IxlO14vg / kg, about 5xl013vg / kg to about IxlO14vg / kg, about 6xl013vg / kg to about IxlO14vg / kg, about 7xl013vg / kg to about IxlO14vg / kg, or about 8xl013vg / kg to about IxlO14vg / kg.

[0352] In some aspects, the first AAV vector genome is administered at a dose of about 7xlO10vg / kg and the second AAV vector genome is administered at a dose of about 3.5xl012vg / kg. In some aspects, the first AAV vector genome is administered at a dose of about 8xlO10vg / kg and the second AAV vector genome is administered at a dose of about 3.5xl012vg / kg. In some aspects, the first AAV vector genome is administered at a dose of about 9xlO10vg / kg and the second AAV vector genome is administered at a dose of about 3.5xl012vg / kg. In some aspects, the first AAV vector genome is administered at a dose of about IxlO10vg / kg and the second AAV vector genome is administered at a dose of about 3.5xl012vg / kg. In some aspects, the first AAV vector genome is administered at a dose of about 2xlOnvg / kg and the second AAV vector genome is administered at a dose of about 3.5xl012vg / kg. In some aspects, the first AAV vector genome is administered at a dose of about 3xl0nvg / kg and the second AAV vector genome is administered at a dose of about 3.5xl012vg / kg. In some aspects, the first AAV vector genome is administered at a dose of about 4xlOnvg / kg and the second AAV vector genome is administered at a dose of about 3.5xl012vg / kg. In some aspects, the first AAV vector genome is administered at a dose of about 5xl0nvg / kg and the second AAV vector genome is administered at a dose of about 3.5xl012vg / kg. In some aspects, the first AAV vector genome is administered at a dose of about 6xlOnvg / kg and the second AAV vector genome is administered at a dose of about 3.5xl012vg / kg. In some aspects, the first AAV vector genome is administered at a dose of about 7xlOnvg / kg and the second AAV vector genome is administered at a dose of about 3.5xl012vg / kg. In some aspects, the first AAV vector genome is administered at a dose of about 8xl0nvg / kg and the second AAV vector genome is administered at a dose of about 3.5xl012vg / kg. In some aspects, the first AAV vector genome is administered at a dose of about 9xlOnvg / kg and the second AAV vector genome is administered at a dose of about 3.5xl012vg / kg. In some aspects, the first AAV vector genome is administered at a dose of about IxlO12vg / kg and the second AAV vector genome is administered at a dose of about 3.5xl012vg / kg. In some aspects, the first AAV vector genome is administered at a dose of about 1.8xl012vg / kg and the second AAV vector genome is administered at a dose of about 3.5xl012vg / kg. In some aspects, the first AAV vector genome is administered at a dose of about 2xl012vg / kg and the second AAV vector genome is administered at a dose of about 3.5xl012vg / kg.

[0353] In some aspects, the first AAV vector genome is administered at a dose of about 3xl013vg / kg and the second AAV vector genome is administered at a dose of about 1.5xl013vg / kg. In some aspects, the first AAV vector genome is administered at a dose of about 4xl013vg / kg and the second AAV vector genome is administered at a dose of about 2xl013vg / kg. In some aspects, the first AAV vector genome is administered at a dose of about 5xl013vg / kg and the second AAV vector genome is administered at a dose of about 2.5xl013vg / kg. In some aspects, the first AAV vector genome is administered at a dose of about 6xl013vg / kg and the second AAV vector genome is administered at a dose of about 3xl013vg / kg. In some aspects, the first AAV vector genome is administered at a dose of about 7xl013vg / kg and the second AAV vector genome is administered at a dose of about 3.5xl013vg / kg. In some aspects, the first AAV vector genome is administered at a dose of about 8xl013vg / kg and the second AAV vector genome is administered at a dose of about 4xl013vg / kg. In some aspects, the first AAV vector genome is administered at a dose of about 9xl013vg / kg and the second AAV vector genome is administered at a dose of about 4.5xl013vg / kg. In some aspects, the first AAV vector genome is administered at a dose of about IxlO14vg / kg and the second AAV vector genome is administered at a dose of about 5xl013vg / kg.

[0354] In some aspects, the first AAV vector genome comprising an insulin expression cassette is administered at a dose between about 2xl012to about IxlO13vg / kg. In some aspects, the first AAV vector genome comprising an insulin expression cassette is administered at a dose between about 4xl012to about IxlO13vg / kg. In some aspects, the first AAV vector genome comprising an insulin expression cassette is administered at a dose of about 4.5xl012to about 5xl012vg / kg. In some aspects, the first AAV vector genome comprising an insulin expression cassette is administered at a dose of about 5xl012to about 9xl012vg / kg.

[0355] In some aspects, the second AAV vector genome comprising a glucokinase expression cassette is administered at a dose between about IxlO12to about 5 xlO12vg / kg (e.g., 3.5xl012vg / kg). In some aspects, the second AAV vector genome comprising a glucokinase expression cassette is administered at a dose between about 2xl012to about 5 xlO12vg / kg. In some aspects, the second AAV vector genome comprising a glucokinase expression cassette is administered at a dose of about 2xl012to about 2.8xl012vg / kg. In some aspects, the second AAV vector genome comprising a glucokinase expression cassette is administered at a dose of about 4xl012vg / kg to about 5xl012vg / kg

[0356] In some aspects, the combination therapy comprise a first AAV vector genome comprising an insulin expression cassette and a second AAV vector genome comprising an glucokinase expression cassette in a vector ratio of about 1 :0.4-0.6 (e.g., about 1 :0.5), wherein the first AAV vector genome, the second AAV vector genome, or both the first AAV vector genome and second AAV vector genome are administered intramuscularly (IM) at a dose of about lxl012to about IxlO14vg / kg, wherein the IM administration is to at least two different skeletal muscle groups. In some aspects, the different skeletal muscle groups comprise a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and / or a latissimus dorsi (lat). In some aspects, the at least two different skeletal muscle groups comprise at least two of a quadriceps, a bicep, and / or a hamstring. In some aspects, the administration comprises bilateral injections into one or more of the at least two different skeletal muscle groups. In some aspects, the administration comprises bilateral injections into one or more of the skeletal muscle groups (e.g., IM injections into the right and left quadriceps, the right and left biceps, the right and left gastrocnemius muscles, the right and left gluteus maximus muscles, and / or the right and left hamstrings).

[0357] In some aspects, the IM administration is to at least three different skeletal muscle groups skeletal muscle groups selected from a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and / or a latissimus dorsi (lat). In some aspects, the at least three different skeletal muscle groups comprise a quadriceps, a bicep, and / or a hamstring. In some aspects, the administration comprises bilateral injections into one or more of the at least three different skeletal muscle groups. In some aspects, the administration comprises bilateral injections into one or more of the skeletal muscle groups (e.g., IM injections into the right and left quadriceps, the right and left biceps, the right and left gastrocnemius muscles, the right and left gluteus maximus muscles, and / or the right and left hamstrings). In some aspects, the IM administration comprises multiple injections with an individual injection volume of between 100 pL and 1.5 mL. In some aspects, an individual injection volume is about 100 pL to about 1.5 mL (e.g., about 100 pL to about 1.4 mL, about 100 pL to about 1.35 mL, about 100 pL to about 1.2 mL, about 100 pL to about 1.0 mL, about 100 pL to about 800 pL, about 200 pL to about 800 pL, about 200 pL to about 600 pL, or about 400 pL to about 800 pL)Insome aspects, an individual injection volume is between 200 pL and 1.0 mL. In some aspects the individual injection volume is about 500 pL. In some aspects, the individual injection volume is between 1 pL and 500 pL. In some aspects, the individual injection volume is between 10 pL and 300 pL. In some aspects, the individual injection volume is between 50 pL and 250 pL. In some aspects, the individual injection volume is about 100 pL.

[0358] In some aspects, the IM administration comprises multiple injections with an individual injection dose of between 1 xlO11vg / injection site and 5 xlO13vg / injection site. In some aspects, an individual injection dose of comprises about 2xl013to 5xl013vg / injection site. In some aspects, the individual injection dose is about IxlO11vg / injection site to about IxlO13vg / injection site. In some aspects, the individual injection dose is about 1.25xlOnvg / injection site to about 1.75xl012vg / injection site. In some aspects, the individual injection dose is about 1.5xl0nvg / injection site to about 1.5xl012vg / injection site. In some aspects, the individual injection site is about 1.75xlOnvg / injection site to about 1.25xl012vg / injection site. In some aspects, the individual injection dose is about IxlO12vg / injection site.

[0359] In some aspects, the IM administration comprises multiple injections with a minimum distance between each injection site. In some aspects the distance between each injection site 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, at least 3.0 cm. In some aspects, the distance between each injection site is between 0.5 cm and 3 cm, between 0.75 cm and 2.5 cm, between 1 cm and 2 cm, between .75 cm and 1.25 cm, or about 1 cm.

[0360] In some aspects, the IM administration comprises injections that are separated by a distance of at least 1-3 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 3 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 2 cm. In some aspects, each of the IM injection sites on the subject are separated by a distance of at least 1 cm.

[0361] In some aspects, the IM administration comprises injections that are 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, each of the IM injections is between 1-2 cm deep.

[0362] In some aspects, the combination therapy is accompanied by an immunosuppressive regimen. In some aspects, the combination therapy is accompanied by an immunosuppressive regimen comprising an immunosuppressive agent. In some aspects, the immunosuppressive regimen comprises administering one or more immunosuppressive agents prior to, at the same time as, and / or after administering the combination therapy. In some aspects, the immunosuppressive agent comprises an IL-6 antagonist, an IL-1 antagonist, a TNFa antagonist, an mTOR pathway inhibitor, mycophenolate mofetil (MMF), a tyk2 inhibitor, methotrexate, rapamycin, abatacept, antithymocyte globulin (ATG), a B cell depletion agent, a T cell costimulation antagonist, a T cell depletion agent, a corticosteroid, and / or a calcineurin inhibitor. In some aspects, the immunosuppressive agent comprises an IL-6 antagonist and / or a calcineurin inhibitor.

[0363] In some aspects, the immunosuppressive regimen comprises administering an immunosuppressive agent (e.g., an IL-6 antagonist) prior to administering the combination therapy. In some aspects, the immunosuppressive regimen comprises administering an immunosuppressive agent (e.g., a calcineurin inhibitor) after administering the combination therapy. In some aspects, the immunosuppressive regimen comprises administering an immunosuppressive agent (e.g., an IL-6 antagonist) prior to administering the combination therapy and administering an immunosuppressive agent (e.g., a calcineurin inhibitor) after administering the combination therapy.

[0364] In some aspects, an immunosuppressive agent (e.g., an IL-6 antagonist) is administered 1 to 14 days (e.g., 1 to 3 days) prior to administering the combination therapy. In some aspects, an immunosuppressive agent (e.g., an IL-6 antagonist) is administered 1 to 7 days (e.g., 1 to 3 days) prior to administering the combination therapy. In some aspects, an immunosuppressive agent is administered 3 to 7 days prior to administering the combination therapy.

[0365] In some aspects, an immunosuppressive agent (e.g., a calcineurin inhibitor) is administered starting at 1, 2, 3, or 4 weeks (e.g., 3 weeks) after administering the combination therapy.

[0366] In some aspects, a first immunosuppressive agent (e.g., an IL-6 antagonist) is administered 1 to 7 days (e.g., 1 to 2 days) prior to administering the combination therapy and a second immunosuppressive agent (e.g., a calcineurin inhibitor) is administered starting at 1, 2, 3, or 4 weeks (e.g., 3 weeks) after administering the combination therapy.

[0367] In some aspects, a first immunosuppressive agent (e.g., an IL-6 antagonist) is administered 1 to 7 days (e.g., 1 to 3 days) prior to administering the combination therapy and a second immunosuppressive agent (e.g., a calcineurin inhibitor) is administered 1 to 14 days (e.g., 1 to 3 days) prior to administering the combination therapy.

[0368] In some aspects, the first immunosuppressive agent is administered once every one week, two weeks, three weeks, or four weeks.

[0369] In some aspects, the first immunosuppressive agent is administered for about 3 months, about 4 months, about 5 months, or about 6 months.

[0370] In some aspects, immunosuppressive agent is administered once about every 1-3 weeks, optionally once about every 1-2 weeks after administering the combination therapy.

[0371] In some aspects, the immunosuppressive agent is administered for about 1-6 months, optionally about 1-3 months after administering the combination therapy.

[0372] In some aspects, immunosuppressive regimen comprises a second immunosuppressive agent.

[0373] In some aspects, the immunosuppressive regimen comprises administering the second immunosuppressive agent prior to administering the combination therapy.

[0374] In some aspects, the immunosuppressive regimen comprises administering the second immunosuppressive agent after administering the combination therapy.

[0375] In some aspects, the second immunosuppressive agent is administered 1 to 14 days prior to administering the combination therapy.

[0376] In some aspects, the second immunosuppressive agent is administered 1 to 7 days prior to administering the combination therapy.

[0377] In some aspects, the second immunosuppressive agent is administered 1 to 3 days prior to administering the combination therapy.

[0378] In some aspects, the second immunosuppressive agent is administered 3 to 7 days prior to administering the combination therapy.

[0379] In some aspects, the second immunosuppressive agent is further administered 1, 2, 3, or 4 weeks after administering the combination therapy.

[0380] In some aspects, second immunosuppressive agent is administered 2 times per day, daily, or every other day.

[0381] In some aspects, the second immunosuppressive agent is administered for about 1- 6 months, optionally about 1-3 months after administering the combination therapy.

[0382] In some aspects, the second immunosuppressive agent is administered once or twice daily for 4 to 52 weeks.

[0383] In some aspects, the second immunosuppressive agent is administered once daily for 4 to 24 weeks, 4 to 16 weeks, or 4 to 8 weeks.

[0384] In some aspects, the second immunosuppressive agent is administered twice daily for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months. In some aspects, the second immunosuppressive agent is administered twice dail for about 1 to about 6 months, about 2 months to about 6 months, about 3 months to about 6 months, about 4 months to about 6 months, about 5 months to about 6 months, about 2 months to about 4 months, or about 3 months to about 4 months.

[0385] In some aspects, the IL-6 antagonist is selected from the group consisting of tocilizumab, sarilumab, siltuximab, olokizumab, elsilimomab, clazakizumab, sirukumab, and levilimab. In some aspects, the IL-6 antagonist is tocilizumab. In some aspects, the IL-6 antagonist is selected from the group consisting of tocilizumab, sarilumab, siltuximab, olokizumab, elsilimomab, clazakizumab, sirukumab, levilimab, or any combination thereof. In some aspects, the IL-6 antagonist is tocilizumab

[0386] In some aspects, the calcineurin inhibitor is selected from the group consisting of cyclosporine, tacrolimus, and voclosporin. In some aspects, the calcineurin inhibitor is tacrolimus.

[0387] In some aspects, the second immunosuppressive agent is selected from the group consisting of thymoglobulin, etanercept, basiliximab, tacrolimus, sirolimus, and my cophenolate mofetil.

[0388] In some aspects, the combination therapy is accompanied by an immunosuppressive regimen (e.g., administration of an IL-6 antagonist such as tocilizumab prior to the gene therapy administration and / or administration of a calcineurin inhibitor such as tacrolimus for a time period after the gene therapy administration) to accompany the gene therapy methods comprising intramuscular administration of a combination therapy comprising a first AAV vector genome comprising an insulin expression cassette and a second AAV vector genome comprising an 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, which is administered to at least two different skeletal muscle groups. In some aspects, the different skeletal muscle groups can comprise a quadriceps, abicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and / or a latissimus dorsi (lat). In some aspects, the different skeletal muscle groups are selected from a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and / or a latissimus dorsi (lat). In some aspects, the at least two different skeletal muscle groups are selected from a quadriceps, a bicep, a hamstring, or any combination thereof.

[0389] In some aspects, the combination therapy comprises a first AAV vector genome comprising an insulin expression cassette and a second AAV vector genome comprising a glucokinase expression cassette in a vector ratio of about 1 :0.9-1.1 (e.g., about 1 : 1.1), wherein the first AAV vector genome, the second AAV vector genome, or both the first AAV vector genome and second AAV vector genome are administered intramuscularly (IM) at a dose of about lxl012to about IxlO14vg / kg (e.g., about IxlO13to about IxlO14vg / kg). In some aspects, the single intramuscular formulation is administered to at least three different skeletal muscle groups selected from a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and / or a latissimus dorsi (lat). In some aspects, the at least three different skeletal muscle groups comprise a quadriceps, a bicep, and / or a hamstring.

[0390] In some aspects, the combination therapy is accompanied by an immunosuppressive regimen (e.g., administration of an IL-6 antagonist such as tocilizumab prior to the gene therapy administration and / or administration of a calcineurin inhibitor such as tacrolimus for a time period after the gene therapy administration) to accompany the gene therapy methods comprising intramuscular administration of a combination therapy comprising a first AAV vector genome comprising an insulin expression cassette and a second AAV vector genome comprising an 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, which is administered to at least two different skeletal muscle groups selected from a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and a latissimus dorsi (lat). In some aspects, the at least two different skeletal muscle groups are selected from a quadriceps, a bicep, and / or a hamstring.

[0391] In some aspects, the combination therapy comprises a first AAV vector genome comprising an insulin expression cassette and a second AAV vector genome comprising a glucokinase expression cassette in a vector ratio of about 1 :0.4-0.6 (e.g., about 1 :0.5), wherein the first AAV vector genome, the second AAV vector genome, or both the first AAV vector genome and second AAV vector genome are administered intramuscularly (IM) at a dose of about lxl012to about IxlO14vg / kg (e.g., about IxlO13to about IxlO14vg / kg). In some aspects, the single intramuscular formulation is administered to at least three different skeletal muscle groups selected from three or more of a quadriceps, a bicep, a triceps, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and a latissimus dorsi (lat). In some aspects, the at least three different skeletal muscle groups comprise a quadriceps, a bicep, and a hamstring, or any combination thereof.

[0392] In some aspects of the disclosure, the methods comprising intramuscular administration (e.g., to quadriceps, biceps, and hamstrings, preferably, bilaterally) and use of an immunosuppressive regimen (e.g., administration of an IL-6 antagonist such as tocilizumab prior to the gene therapy administration and / or administration of a calcineurin inhibitor such as tacrolimus for a time period after the gene therapy administration), can provide improvements to the safety, efficacy and durability of the combination gene therapy disclosed herein. In some aspects, the methods result in (i) a reduction in glycated blood hemoglobin (HbAlc) levels in the treated subject; (ii) a reduction in circulating ketones in the treated subject, (iii) a reduction in triglycerides in the treated subject, (iv) a reduction in exogenous insulin in the treated subject, (v) an increase in circulating C-peptide levels in the treated subject, (vi) reduction in immune response to exogenous AAV, insulin protein, and / or glucokinase protein, and / or (vii) any combination thereof.

[0393] In some aspects, the polynucleotide encoding a hlns is a modified polynucleotide (e.g., preproinsulin or proinsulin, a mutant, an analogue, or a variant thereof). In some aspects, the polynucleotide encoding a hGck is a modified polynucleotide (e.g., Gck, a mutant, an analogue, or variant thereof). In some aspects, the modifications to the coding sequence preserve the wild-type or mutant amino acid sequence for 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 of amino acids 25-110 of SEQ ID NO: 41, amino acids25-110 of SEQ ID NO: 144, or amino acids 25-110 of SEQ ID NO: 145. In some aspects, the modified nucleic acid sequence encodes a human preproinsulin (e.g., SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO: 145). In some aspects, the modified nucleic acid sequence encodes a human Gck (e.g., SEQ ID NO: 82).

[0394] In some aspects, the modified nucleic acids are codon optimized. In some aspects, the codon optimization includes modifying codons in the open reading frame of the nucleic acid encoding insulin or glucokinase. In some aspects, the modified nucleic acids comprise reduced CpG content relative to the corresponding wild-type sequence and / or unmodified sequence.

[0395] In some aspects, the modified nucleic acid has reduced innate immunogenicity relative to the corresponding wild-type sequence and / or unmodified sequence. In some aspects, the modified nucleic acid has increased expression relative to the corresponding wild-type sequence and / or unmodified sequence. In some aspects, the modified nucleic acid has decreased expression relative to the corresponding wild-type sequence and / or unmodified sequence. In some aspects, the modified sequences are developed through in silico methods followed by manual sequence examination. Nucleic acids of the disclosure can be produced using molecular biology techniques, e.g., modified cDNAs encoding insulin or glucokinase can be obtained by PCR amplification or cDNA cloning techniques.

[0396] In some aspects, the nucleic acid sequences are modified to reduce CpG content, e.g., to minimize the inflammatory response through TLR9 dimerization and related pathways. In some aspects, certain CpG motifs are inhibitory or neutralizing for their inflammatory effects. In some embodiments, one or more of these motifs can be preserved. In some aspects, such CpG motifs can be introduced into a nucleic acid sequence for inhibition of the downstream effects of TLR9 dimerization.

[0397] In some aspects, the codon modifications can reduce the immunogenicity of the insulin and / or glucokinase encoding polynucleotides relative to a corresponding wild-type polynucleotide and / or unmodified polynucleotide. In some aspects, the codon modifications improve the expression of the insulin or glucokinase encoding polynucleotide relative to a corresponding wild-type and / or unmodified polynucleotide. In some aspects, the codon modifications can reduce the immunogenicity of the glucokinase encoding polynucleotides relative to a corresponding wild-type Gck polynucleotide and / or unmodified Gck polynucleotide.

[0398] The polynucleotides and modified nucleic acids of the disclosure can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. The polynucleotides and modified nucleic acids can be isolated.

[0399] As used herein, a polynucleotide or nucleic acid is “isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art, see e.g. F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York.

[0400] In some aspects, a polynucleotide or modified nucleic acid of the disclosure can be, for example, DNA or RNA and may or may not contain intron sequences. In some aspects, the nucleic acid can be a cDNA molecule.Insulin Nucleic Acids

[0401] In some aspects, the polynucleotide encoding the human insulin comprises a sequence encoding a 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 the human insulin is modified relative to a wild-type (SEQ ID NO: 147) and / or an 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 the human insulin comprises a sequence including a 5’ UTR, an ORF, and / or a 3’ UTR, e.g, 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 the human insulin comprises a sequence including a 5’ UTR (e.g., nucleic acids 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), e.g, a polynucleotide comprising SEQ ID NO: 170. In some aspects, the polynucleotide encoding the human insulin is modified relative to a 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 the human insulin is modified relative to a sequence corresponding to SEQ ID NO: 170. In some aspects, the polynucleotide encoding the human insulin encodes wild-type human insulin (SEQ ID NO: 41), variants or mutants thereof (e.g., SEQ ID NO: 144 or SEQ ID NO: 145) or a functional fragment thereof.

[0402] Insulin includes two polypeptide chains, the A- and B- chains, linked together by disulfide bonds. It is first synthesized as a single polypeptide called preproinsulin. “Preproinsulin” is the primary translational product of the insulin gene. It is a peptide that is 110 amino acids in length. Preproinsulin includes a proinsulin molecule with a signal peptide attached to its N-terminus. Part of the N-terminus including the signal peptide of the preproinsulin is cleaved off, leaving the remaining amino acids as “proinsulin”. Amino acids 1-30 of the resulting cleaved sequence is the “B chain”, and here “BIO” corresponds to position 34 of preproinsulin. Thus, for example, a “BIO” proinsulin mutation corresponds to a H34 mutation in preproinsulin. In certain aspects, as referenced herein, “B10H” refers the wild-type histidine amino acid at the BIO position (also referenced as H34 in the wild-type preproinsulin sequence). The preproinsulin and proinsulin also include a C-peptide between the A- and B- chains. In the mature insulin protein, the C-peptide is proteolytically cleaved and the A- and B- chains are linked by disulfide bonds.

[0403] In some aspects, the polynucleotide encoding the human insulin disclosed herein encodes a preproinsulin mutant comprising one or more mutations at position(s) H34, P52, K53, R55, and / or L86 relative to the corresponding position in wild-type preproinsulin (SEQ ID NO: 41). In some aspects, the polynucleotide encoding the human insulin encodes a preproinsulin mutant comprising one or more of the following mutations H34D, H34I, H34V, P52D, K53R, R55K, and / or L86R relative to the corresponding position in SEQ ID NO: 41. In some aspects, the polynucleotide encoding the human insulin encodes a preproinsulin mutant comprising mutations H34D, H34I, H34V, P52D, K53R, R55K, and / or L86R relative to the corresponding position in SEQ ID NO: 41. In some aspects, the polynucleotide encoding the human insulin encodes a preproinsulin mutant comprising mutations P52D, K53R, R55K, and / or L86R relative to the corresponding position in SEQ ID NO: 41. In some aspects, the polynucleotide encoding the human insulin encodes an amino acids sequence 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 the human insulin encodes an amino acids sequence at least 90%, 95%, 99% or 100% similar to an amino acid sequence selected from SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO: 145, wherein the amino acid sequence comprises one or more of the following mutations H34D, H34I, H34V, P52D, K53R, R55K, and / or L86R relative to the correspondingposition in SEQ ID NO: 41. In some aspects, the polynucleotide encoding the human insulin encodes an amino acids sequence that does not include a H34 mutation relative to the corresponding position in SEQ ID NO: 41.

[0404] In some aspects, the polynucleotide encoding the human insulin comprises a cleavage site, e.g., a furin endoprotease cleavage site.

[0405] In some aspects, the polynucleotide encoding the human insulin comprises a nucleic acid encoding a signal peptide (e.g., a wild-type preproinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence). In some aspects, the preproinsulin comprises a wild-type insulin signal sequence (e.g., MALWMRLLPLLALLALWGPDPAAA (SEQ ID NO: 165) or amino acids 1-24 of SEQ ID NO: 41). In some aspects, the signal sequence of wild-type preproinsulin is replaced with a non-insulin secretion peptide, e.g., an IL-6 signal sequence (e.g., MNSFSTSAFGPVAFSLGLLLVLPAAFPAP (SEQ ID NO: 166)) or a fibronectin signal sequence (e.g, MLRGPGPGLLLLAVQCLGTAVPSTGA (SEQ ID NO: 167)).

[0406] In some aspects, the polynucleotide encoding the human insulin encodes a human insulin comprising an amino acid modification selected from H34D, H34I, or H34V corresponding to wild-type preproinsulin amino acid positions (or a histidine (H) to aspartic acid (D), isoleucine (I) or valine (V) at position B10 of the proinsulin B-chain). In some aspects, the polynucleotide encoding the human insulin encodes a human insulin comprising an amino acid modification H34D corresponding to wild-type preproinsulin amino acid positions (or a histidine (H) to aspartic acid (D) at position B10 of the proinsulin B-chain). In some aspects, the polynucleotide encoding the human insulin encodes a human insulin comprising an amino acid modification selected from H34D, H34I, or H34V corresponding to wild-type preproinsulin amino acid positions (or a histidine (H) to aspartic acid (D), isoleucine (I) or valine (V) at position B10 of the proinsulin B-chain), wherein the human insulin optionally comprises a cleavage site, e.g., a furin cleavage site, and a signal peptide (e.g., a wild-type preproinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence).

[0407] In some aspects, the polynucleotide encoding the human insulin encodes a human insulin comprising amino acid modifications K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to the proinsulin lysine (K) to arginine (R) at position B29, arginine (R) to lysine (K) at position Cl, and leucine (L) to arginine (R) at position C32). In some aspects, the polynucleotideencoding the human insulin encodes a human insulin comprising amino acid modifications K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to the proinsulin lysine (K) to arginine (R) at position B29, arginine (R) to lysine (K) at position Cl, and leucine (L) to arginine (R) at position C32), wherein the human insulin optionally comprises a cleavage site, e.g., a furin cleavage site, and a signal peptide (e.g., a wild-type preproinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence).

[0408] In some aspects, the polynucleotide encoding the human insulin encodes a human insulin comprising amino acid modifications H34D, K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to the proinsulin a histidine (H) to aspartic acid (D) at position BIO, lysine(K) to arginine (R) at position B29, arginine (R) to lysine (K) at position Cl, and leucine(L) to arginine (R) at position C32). In some aspects, the modified nucleic acid encodes a human insulin comprising amino acid modifications H34D, K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to the proinsulin a histidine (H) to aspartic acid (D) at position BIO, lysine(K) to arginine (R) at position B29, arginine (R) to lysine (K) at position Cl, and leucine(L) to arginine (R) at position C32), wherein the human insulin optionally comprises a cleavage site, e.g., a furin cleavage site, and a signal peptide (e.g., a wild-type preproinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence).

[0409] In some aspects, the polynucleotide encoding the human insulin encodes a human insulin comprising amino acid modifications H34I, K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to the proinsulin a histidine (H) to isoleucine (I) at position BIO, lysine (K) to arginine (R) at position B29, arginine (R) to lysine (K) at position Cl, and leucine (L) to arginine (R) at position C32). In some aspects, the polynucleotide encoding the human insulin encodes a human insulin comprising amino acid modifications H34I, K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to the proinsulin a histidine (H) to isoleucine (I) at position BIO, lysine (K) to arginine (R) at position B29, arginine (R) to lysine (K) at position Cl, and leucine (L) to arginine (R) at position C32), wherein the human insulin optionally comprises a cleavage site, e.g., a furin cleavage site, and a signal peptide (e.g., a wild-type preproinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence).

[0410] In some aspects, the polynucleotide encoding the human insulin encodes a human insulin comprising amino acid modifications H34V, K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to the proinsulin a histidine (H) to valine (V) at position BIO, lysine (K) to arginine (R) at position B29, arginine (R) to lysine (K) at position Cl, and leucine (L) to arginine (R) at position C32). In some aspects, the polynucleotide encoding the human insulin encodes a human insulin comprising amino acid modifications H34V, K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to the proinsulin a histidine (H) to valine (V) at position BIO, lysine (K) to arginine (R) at position B29, arginine (R) to lysine (K) at position Cl, and leucine (L) to arginine (R) at position C32), wherein the human insulin optionally comprises a cleavage site, e.g., a furin cleavage site, and a signal peptide (e.g., a wildtype preproinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence).

[0411] In some aspects, the polynucleotide encoding the human insulin encodes a human insulin comprising amino acid modifications P49D, K53R, R55K, and L86R corresponding to wild-type preproinsulin amino acid positions (or modifications corresponding to the proinsulin a proline (P) to aspartic acid (D) at position B28, lysine(K) to arginine (R) at position B29, arginine (R) to lysine (K) at position Cl, and leucine(L) to arginine (R) at position C32). In some aspects, the polynucleotide encoding the human insulin encodes a human insulin comprising amino acid modifications P49D, K53R, R55K, and L86R (corresponding to wild-type preproinsulin amino acid positions), wherein the human insulin optionally comprises a cleavage site, e.g., a furin cleavage site, and a signal peptide (e.g., a wild-type preproinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence).

[0412] In some aspects, the polynucleotide encoding the human insulin encodes a human insulin (Ins) protein (e.g., a preproinsulin or variant thereof), wherein the nucleic acid comprises: (i) a nucleotide sequence encoding a signal peptide (e.g., a wild-type preproinsulin signal sequence, an IL-6 signal sequence, or a fibronectin signal sequence) and (ii) a nucleotide sequence encoding a proinsulin polypeptide comprising an amino acid modification at a position selected from amino acid BIO, B28, and / or B29 of thehuman insulin B-chain, Cl and / or C32 of the human insulin C-chain, or any combination thereof relative to the corresponding amino acid in wild-type proinsulin (or an amino acid modification at a position selected from amino acid H34, P52, K53, R55, L86, or any combination thereof relative to the corresponding amino acid in wild-type preproinsulin). In some aspects, the signal peptide is not a wild-type preproinsulin signal sequence, e.g., the wild-type preproinsulin sequence is replaced with an IL-6 signal sequence or fibronectin signal sequence). In some aspects, the polynucleotide encoding the human insulin further comprises a cleavage site (e.g., a furin cleavage site). In some aspects, the encoded human Ins protein (e.g., a preproinsulin or variant thereof) comprises an amino acid modification selected from (i) H34D, H34I, or H34V (or a histidine (H) to aspartic acid (D), isoleucine (I) or valine (V) at position BIO of the proinsulin B-chain), and / or (ii) one or more amino acid modifications at P52, K53, R55, and / or L86 relative to the wildtype preproinsulin sequence (or positions B28 and / or B29 of the proinsulin B-chain or positions Cl and / or C32 of the proinsulin C-chain). In some aspects, the one or more amino acid modifications at P52, K53, R55, and / or L86 comprise P52D, K53R, R55K, L86R, or any combination thereof (or the one or more modifications in the proinsulin B- chain or C-chain comprise a proline (P) to aspartic acid (D) at position B28 of the proinsulin B-chain, a lysine (K) to arginine (R) at position B29 of the proinsulin B-chain, arginine (R) to lysine (K) at position Cl of the proinsulin C-chain, leucine (L) to arginine (R) at position C32 of the proinsulin C-chain, or any combination thereof).

[6413] In some aspects, the polynucleotide encoding the 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 least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acids 25-110 of SEQ ID NO: 41, amino acids 25-110 of SEQ ID NO: 144, or amino acids 25-110 of SEQ ID NO: 145. In some aspects, the human insulin protein comprises an amino acid sequence having least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO: 145. In some aspects, the human insulin protein comprises an insertion, a deletion, a substitution, or combinations thereof relative to wild-type human insulin. In some aspects, the human insulin protein comprisesat least one substitution. In some aspects, the at least one substitution is a conservative substitution. In some aspects, the at least one substitution is a non-conservative substitution.

[0414] In some aspects, a polynucleotide of the disclosure comprises an open reading frame (ORF) encoding a human insulin comprising a nucleic acid sequence having a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to nucleic acids 73-330 of any of SEQ ID NOs: 43-57, 110-116, or nucleic acids 88-345 of any of SEQ ID NO s: 117-122. In some aspects, a polynucleotide of the disclosure comprises an open reading frame (ORF) encoding a human insulin comprising a nucleic acid sequence having a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 121. In some aspects, the ORF further comprises a nucleic acid sequence encoding a signal peptide.

[0415] In some aspects, a polynucleotide of the disclosure comprises an open reading frame (ORF) encoding a human insulin 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 least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ IDNO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ IDNO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ IDNO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ IDNO: 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 a human insulin protein (e.g., SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO: 145) or a functional fragment thereof.

[0416] In some aspects, a polynucleotide of the disclosure comprises an open reading frame (ORF) comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 121. In some aspects, a polynucleotide of the disclosure comprises an open reading frame (ORF) comprising a nucleic acid sequence having the sequence of SEQ ID NO: 121.

[0417] In some aspects, the polynucleotide encoding the human insulin of the disclosure comprises an open reading frame comprising a nucleic acid having the sequence of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122. In some aspects, the polynucleotide encoding the human insulin of the disclosure comprises an open reading frame comprising a nucleic acid having the sequence of SEQ ID NO: 121. In some aspects, the polynucleotide encoding the human insulin comprises an ORF sequence present or referenced in Table 8.

[0418] In some aspects, a polynucleotide of the disclosure comprises two or more ORFs. In some aspects, the two or more ORFs are operably linked. In some aspects, the ORFs are operably linked by an IRES.

[0419] In some aspects, the polynucleotide comprising an ORF encoding the human insulin of the disclosure further comprises a 5’ UTR nucleic acid sequence. In some aspects, the polynucleotide comprising an ORF encoding the human insulin of the disclosure further comprises a modified 5’ UTR nucleic acid sequence. In some aspects, the polynucleotide comprising an ORF encoding the human insulin of the disclosure further comprises a 5’ UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42, nucleic acids 5-329 of SEQ ID NO: 42, or SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some aspects, 5’ 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 to nucleic acids 5-329 of SEQ ID NO: 42.

[0420] In some aspects, the polynucleotide comprising an ORF encoding the human insulin comprises a Kozak consensus sequence (Kozak consensus or Kozak sequence). In some aspects, the 5’ UTR comprises a nucleic acid having the sequence of SEQ ID NO: 42, nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQID NO: 148. In some aspects, the polynucleotide comprising an ORF encoding the human insulin comprises a 5’ UTR nucleic acid sequence present or referenced in Table 8.

[0421] In some aspects, the polynucleotide comprising an ORF encoding the human insulin of the disclosure further comprises a modified 3’ UTR nucleic acid sequence. In some aspects, the polynucleotide comprising an ORF encoding the human insulin of the disclosure further comprises a 3’ UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 60, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 149, or SEQ ID NO: 171. In some aspects, 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 to SEQ ID NO: 171. In some aspects, the 3’ UTR comprises a nucleic acid having the sequence of SEQ ID NO: 60, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 149, or SEQ ID NO: 171. In some aspects, the 3’ UTR comprises a restriction site selected from the group consisting of amHI, EcoRI, Ndel, Eco N, Spel, Xba\. Nhd, VspI, Nsil, Seal, Kpnl, SspI, and Pad, and any combination thereof. In some aspects, the polynucleotide comprising an ORF encoding the human insulin comprises a 3’ UTR nucleic acid sequence present or referenced in Table 8.

[0422] In some aspects, the polynucleotide comprising an ORF encoding the human insulin of the disclosure comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from 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 nucleic acid sequence encodes ahuman 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 the human insulin of the disclosure comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 138. In some aspects, the polynucleotide encoding the human insulin of the disclosure comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 170.

[0423] In some aspects, the polynucleotide comprising an ORF encoding the human insulin of the disclosure comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to nucleic acids 5-957 of a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In some aspects, the polynucleotide encoding the human insulin of the disclosure comprises a nucleic acid having the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 123, SEQ ID NO: 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 the human insulin of the disclosure comprises a nucleic acid having the sequence of SEQ ID NO: 138. In some aspects, the polynucleotide encoding the human insulin of the disclosure comprises a nucleic acid having the sequence of SEQ ID NO: 170. In some aspects, the polynucleotide encoding the human insulin of the disclosure comprises nucleic acids 5- 957 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In some aspects, the polynucleotide comprising an ORF encoding the human insulin comprises a nucleic acid comprising a 5’ UTR, an ORF, and a 3’ UTR presented in Table 8.

[0424] In some aspects, the polynucleotide of the disclosure encodes a human insulin comprising a wild-type preproinsulin secretion signal peptide. In some aspects, the polynucleotide encoding the human insulin of the disclosure does not encode a wild-type preproinsulin secretion signal peptide. In some aspects, the wild-type preproinsulin is replaced by a non-insulin secretion signal. In some aspects, the polynucleotide encoding the human insulin of the disclosure encodes a human preproinsulin comprising an interleukin 6 (IL-6) secretion signal peptide. In some aspects, the polynucleotide encoding the human insulin of the disclosure encodes a human preproinsulin comprising a fibronectin secretion signal peptide.Glucokinase Nucleic Acids

[0425] In some aspects, the polynucleotide encoding a 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 the human glucokinase comprises a modified nucleic acid sequence which encodes wild-type human glucokinase (SEQ ID NO: 82) or a functional fragment thereof. In some aspects, the polynucleotide comprising a sequence encoding the human glucokinase disclosed herein is modified relative to the wild-type and / or unmodified, e.g., including a 5’ UTR (e.g., SEQ ID NO: 42), an ORF (e.g., SEQ ID NO: 68), and / or a 3’ UTR (e.g., SEQ ID NO: 60 or 171), nucleic acid sequence. In some aspects, the polynucleotide encoding a human Gck comprises a 5’ UTR (e.g., SEQ ID NO: 42), an ORF (e.g., SEQ ID NO: 68), and / or a 3’ UTR (e.g., SEQ ID NO: 60 or 171), e.g., a polynucleotide comprising SEQ ID NO: 19, SEQ ID NO: 27, or SEQ ID NO: 172.

[0426] In some aspects, the polynucleotide of the disclosure comprises an ORF encoding the human glucokinase comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 61, SEQ IDNO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ IDNO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ IDNO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ IDNO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO: 162, wherein the nucleic acid sequence encodes a human glucokinase protein (SEQ ID NO: 82) or a functional fragment thereof.

[0427] In some aspects, the polynucleotide of the disclosure comprises an ORF encoding the human glucokinase comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 68. In some aspects, the polynucleotide of the disclosure comprises an open reading frame encoding the human glucokinase comprising a nucleic acid having the sequence of SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO: 162. In some aspects, the polynucleotide of the disclosure comprises an open reading frame encoding the human glucokinase comprising a nucleic acid having the sequence of SEQ ID NO: 68. In some aspects, the polynucleotide encoding the human glucokinase comprises an ORF sequence present or referenced in Table 9.

[0428] In some aspects, the polynucleotide comprising an ORF encoding the human glucokinase of the disclosure further comprises a modified 5’ UTR nucleic acid sequence. In some aspects, the polynucleotide comprising an ORF encoding the human glucokinase of the disclosure further comprises a 5’ UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42, nucleic acids 5- 329 of SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some aspects, the polynucleotide comprising an ORF encoding the human glucokinase of the disclosure further comprises a 5’ UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42.

[0429] In some aspects, the polynucleotide comprising an ORF encoding the human glucokinase further comprises a Kozak consensus sequence (Kozak consensus or Kozak sequence). In some aspects, the 5’ UTR comprises a nucleic acid having the sequence of SEQ ID NO: 42, nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some aspects, the polynucleotide comprising an ORF encoding the human glucokinase comprises a 5’ UTR sequence present or referenced in Table 9

[0430] In some aspects, the polynucleotide comprising an ORF encoding the human glucokinase of the disclosure further comprises a modified 3’ UTR nucleic acid sequence. In some aspects, the polynucleotide comprising an ORF encoding the human glucokinase of the disclosure further comprises a 3’ UTR comprising a nucleic acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 60, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 149, SEQ ID NO: 169, or SEQ ID NO: 171. In some aspects, the polynucleotide comprising an ORF encoding the human glucokinase of the disclosure further comprises a 3’ UTR comprising a nucleic acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 171. In some aspects, the 3’ UTR comprises a nucleic acid having the sequence of SEQ ID NO: 60, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, 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 amHI, EcoRI, Ndel, EcoRV, Spel, Xbal, Nhel, VspI, Nsil, Seal, Kpnl, SspI, and Pad, and any combination thereof. In some aspects, the polynucleotide comprising an ORF encoding the human glucokinase comprises a 3’ UTR sequence present or referenced in Table 9.

[0431] In some aspects, the polynucleotide encoding the human glucokinase of the disclosure comprises a nucleic acid sequence having at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ IDNO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ IDNO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ IDNO: 38, SEQ ID NO: 39, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ IDNO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ IDNO: 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 functional fragment thereof. In some aspects, the polynucleotide encoding the human glucokinase of the disclosure comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 27. In some aspects, the polynucleotide encoding the human glucokinase of the disclosure comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 168. In some aspects, the polynucleotide encoding the human glucokinase of the disclosure comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 172. In some aspects, the polynucleotide encoding the human glucokinase of the disclosure 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 to nucleic acids 5-2025 of a sequence selected from SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25,SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30,SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36,SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39. In some aspects, the polynucleotide encoding the human glucokinase of the disclosure comprises a nucleic acid having thesequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ IDNO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ IDNO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ IDNO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ IDNO: 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. In some aspects, the polynucleotide encoding the human glucokinase of the disclosure comprises a nucleic acid having the sequence of SEQ ID NO: 168. In some aspects, the polynucleotide encoding the human glucokinase of the disclosure comprises nucleic acids 5-2025 of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ IDNO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ IDNO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ IDNO: 38, or SEQ ID NO: 39. In some aspects, the polynucleotide encoding the human glucokinase is a nucleic acid present or referenced in Table 9.Expression cassettes

[0432] Certain aspects of the disclosure are directed to a combination gene therapy, e.g., a combination AAV gene therapy for delivery of an expression cassette (or expression construct) comprising a polynucleotide encoding an insulin (Ins) protein and an expression cassette comprising a polynucleotide encoding a glucokinase (Gck) protein. In some aspects, the combination therapy includes separate administration of the Ins and Gck expression cassettes. In some aspects, the combination therapy includes administration of the insulin and glucokinase expression cassettes in a single formulation, e.g., two separate rAAV particles in the same pharmaceutical composition.

[0433] In some aspects, the combination therapy comprises an insulin expression cassette comprising a promoter operably linked to a polynucleotide encoding a human insulin protein, wherein the insulin expression cassette is flanked by inverted terminal repeats. In some aspects, the combination therapy comprises a glucokinase expression cassette comprising a promoter operably linked to a polynucleotide encoding a human glucokinase protein, wherein the glucokinase expression cassette is flanked by inverted terminal repeats. In some aspects the first and second promoter are the same orsubstantially the same promoter; or in some aspects the first and second promoters are different promoters that drive substantially equivalent levels of transgene expression.

[0434] An expression cassette comprising a eukaryotic promoter operably linked to a DNA of interest (e.g., a DNA encoding insulin or a DNA encoding glucokinase) can be used in the disclosure. In some aspects, the expression cassette containing the DNA sequence (or the corresponding RNA sequence), which can be used in accordance with the disclosure, can be any eukaryotic expression cassette containing the DNA or the RNA sequence of interest. For example, a plasmid or viral construct (e.g., an AAV vector) can be cleaved to provide linear DNA having ligatable termini. These termini can be bound to exogenous DNA having complementary, like ligatable termini to provide a biologically functional recombinant DNA molecule having an intact replicon and a desired phenotypic property. In some aspects, the expression cassette is capable of replication in both eukaryotic and prokaryotic hosts.

[0435] In some aspects, the exogenous DNA used in the disclosure is obtained from suitable cells, and the constructs prepared using techniques known in the art. Likewise, techniques for obtaining expression of exogenous DNA or RNA sequences in a genetically altered host cell are known in the art (see e.g., Kormal et al., Proc. Natl. Acad. Sci. USA, 84:2150-2154 (1987); Sambrook et al. Molecular Cloning: a Laboratory Manual, 2nd Ed., 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; each of which are hereby incorporated by reference with respect to methods and compositions for eukaryotic expression of a DNA of interest).

[0436] In some aspects, the DNA expression construct comprises a promoter to facilitate expression of the DNA of interest (e.g., polynucleotide encoding an insulin or a polynucleotide encoding a glucokinase) within a secretory cell. In some aspects, the promoter is a strong, eukaryotic promoter such as a promoter from cytomegalovirus (CMV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RS V), or adenovirus. Exemplary promoters include, but are not limited to the promoter from the immediate early gene of human CMV (Boshart et al., Cell 41 :521-530 (1985) and the promoter from the long terminal repeat (LTR) of RSV (Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777-6781 (1982)). In some aspects, the promoter is used together with an intronic sequence. In some aspects, the CMV promoter is a mini CMV promoter. Alternatively, the promoter used can be a tissue-specific promoter. In some aspects, the insulin expression cassette comprises a CMV promoter. In some aspects, the glucokinaseexpression cassette comprises a CMV promoter. In some aspects, the insulin expression cassette comprises a constitutive promoter. In some aspects, the glucokinase expression cassette comprises a constitutive promoter. In some aspects, the insulin expression cassette comprises a ubiquitous promoter. In some aspects, the glucokinase expression cassette comprises a ubiquitous promoter. In some aspects, the insulin expression cassette comprises a muscle specific promoter. In some aspects, the glucokinase expression cassette comprises a muscle specific promoter.

[0437] The expression cassettes of the disclosure can also include other components such as a marker (e.g., an antibiotic resistance gene (such as an ampicillin resistance gene) or P-galactosidase) to aid in selection of cells containing and / or expressing the construct, an origin of replication for stable replication of the construct in a bacterial cell (preferably, a high copy number origin of replication), a nuclear localization signal, or other elements which facilitate production of the DNA expression construct, the protein encoded thereby, or both.

[0438] For eukaryotic expression, the expression cassette can contain at a minimum a eukaryotic promoter operably linked to a DNA of interest (e.g., a polynucleotide encoding an insulin or a glucokinase,), which is in turn operably linked to a polyadenylation sequence. The polyadenylation signal sequence can 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., a bovine growth hormone polyA or a human growth hormone poly A). In some aspects, the glucokinase expression cassette comprises an SV40 polyadenylation signal sequence. In some aspects, the insulin expression cassette comprises a bovine or human growth hormone polyadenylation signal sequence.

[0439] In some aspects, the expression cassette can also include one or more introns, which can increase levels of expression of the DNA of interest, particularly where the DNA of interest is a cDNA (e.g., contains no introns of the naturally-occurring sequence). Any of a variety of introns known in the art can be used (e.g., the human P-globin intron, which is inserted in the construct at a position 5' to the DNA of interest).

[0440] The DNA of interest (e.g., a polynucleotide encoding an insulin or a polynucleotide encoding a glucokinase) can be inserted into an expression cassette so that the therapeutic molecule (e.g., a protein) is expressed as a fusion protein (e.g., a fusionprotein having P-galactosidase or a portion thereof at the N-terminus and the therapeutic protein at the C-terminal portion). Production of a fusion protein can facilitate identification of transformed cells expressing the protein (e.g., by enzyme-linked immunosorbent assay (ELISA) using an antibody which binds to the fusion protein).

[0441] The vectors for delivery of the DNA of interest (e.g., the polynucleotide encoding an insulin, the polynucleotide encoding a glucokinase can be either viral or non-viral, or can be composed of naked DNA admixed with an adjuvant such as viral particles (e.g., AAV particle) or cationic lipids or liposomes. An “adjuvant” is a substance that does not by itself produce the desired effect, but acts to enhance or otherwise improve the action of the active compound. The precise vector and vector formulation used will depend upon several factors such as the cell and / or organ targeted for gene transfer.

[0442] Examples of suitable promoters include cytomegalovirus (CMV) intermediate early promoter, viral long terminal repeat promoters (LTRs), such as those from murine moloney leukaemia virus (MMLV) rous sarcoma virus, or HTLV-1, the simian virus 40 (SV 40) early promoter, RSV promoter, and the herpes simplex virus thymidine kinase promoter. In some aspects, the promoter is a constitutive promoter. In some aspects, the promoter is a ubiquitous promoter. In some aspects, the promoter is a cell-specific and / or a tissue-specific promoter. In some aspects, the promoter is a muscle specific promoter. In some aspects, the promoter is used together with an intronic sequence. In some aspects, the promoter is tissue specific. In some aspects, the first promoter is a CMV promoter. In some aspects, the second promoter is a CMV promoter. In some aspects, the CMV promoter is a mini CMV promoter. In some aspects, the insulin expression cassette comprises a CMV promoter. In some aspects, the glucokinase expression cassette comprises a CMV promoter. In some aspects the first and second promoter are the same or substantially the same promoter; or in some aspects the first and second promoters are different promoters that drive substantially equivalent levels of transgene expression.

[0443] In some aspects, the insulin expression cassette comprises a constitutive promoter. In some aspects, the glucokinase expression cassette comprises a constitutive promoter. In some aspects, the insulin expression cassette comprises a ubiquitous promoter. In some aspects, the glucokinase expression cassette comprises a ubiquitous promoter. In some aspects, the insulin expression cassette comprises a muscle specific promoter. In some aspects, the glucokinase expression cassette comprises a muscle specific promoter.

[0444] In some aspects, the expression cassette comprises a promoter operably linked to 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 to a sequence selected from SEQ ID NO: 43, SEQ ID NO: 44, SEQ IDNO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ IDNO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ IDNO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 110, SEQ ID NO: 111, SEQ IDNO: 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 a human insulin protein (e.g., SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO: 145) or a functional fragment thereof. In some aspects, the expression cassette comprises a promoter operably linked to a polynucleotide encoding the human insulin protein comprises an ORF having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 121 or SEQ ID NO: 122. In some aspects, the polynucleotide encoding the human insulin protein of the disclosure comprises an open reading frame comprising a nucleic acid having the sequence of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122. In some aspects, the polynucleotide encoding the human insulin protein of the disclosure comprises an open reading frame comprising a nucleic acid having the sequence of SEQ ID NO: 122. In some aspects, the polynucleotide encoding the human insulin protein of the disclosure comprises an open reading frame comprising a nucleic acid having the sequence of SEQ ID NO: 121. In some aspects, the polynucleotide encoding the human insulin protein comprises an ORF sequence present or referenced in Table 8.

[0445] In some aspects, the expression cassette comprises a polynucleotide encoding a human insulin comprising a wild-type preproinsulin secretion signal peptide. In someaspects, the polynucleotide encoding the human insulin protein of the disclosure does not encode a wild-type preproinsulin secretion signal peptide. In some aspects, the wild-type preproinsulin is replaced by a non-insulin secretion signal. In some aspects, the expression cassette comprises a polynucleotide encoding a human preproinsulin comprising an interleukin 6 (IL-6) secretion signal peptide. In some aspects, the expression cassette comprises a polynucleotide encoding a human preproinsulin comprising a fibronectin secretion signal peptide.

[0446] In some aspects, the expression cassette comprises the polynucleotide encoding the human insulin protein further comprises a 5’ UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42, nucleic acids 5-329 of SEQ ID NO: 42, or SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some aspects, the 5’ UTR comprises a nucleic acid having the sequence of SEQ ID NO: 42, nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some aspects, the polynucleotide encoding the human insulin protein comprises a 5’ UTR sequence present or referenced in Table 8.

[0447] In some aspects, the expression cassette comprises a polynucleotide encoding a human insulin protein further comprising a 3’ UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 60, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 149, or SEQ ID NO: 171. In some aspects, the 3’ UTR comprises a nucleic acid having the sequence of SEQ ID NO: 60, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 149, SEQ ID NO: 171. In some aspects, the 3’ UTR comprises a restriction site selected from the group consisting of 7>< / 7?HI, ECORI, Nde , Eco N, Spel, Xba , Nhel, Vsp , Nsi , Seal, Kpn , Ssp , and Pad, and any combination thereof. In some aspects, the polynucleotide encoding the human insulin protein comprises a 3’ UTR sequence present or referenced in Table 8.

[0448] 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%, atleast 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87 SEQ ID NO: 88, SEQ ID NO: 123, SEQ ID NO: 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 a human insulin protein (e.g., SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO: 145) or a functional fragment thereof. In some aspects, the expression cassette comprises a polynucleotide encoding a human insulin protein having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 138 or SEQ ID NO: 171. In some aspects, the expression cassette comprises a polynucleotide encoding a human insulin protein having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to nucleic acids 5-957 of a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In some aspects, the expression cassette comprises a polynucleotide encoding a human insulin protein having the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87 SEQ ID NO: 88, SEQ ID NO: 123, SEQ ID NO: 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 expression cassette comprises a polynucleotide encoding a human insulin protein having the sequence of SEQ ID NO: 138 or SEQ ID NO: 170. In some aspects, the expression cassette comprises a polynucleotide encoding a human insulin protein having at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to nucleic acids 5-957 of a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In some aspects, the expression cassette comprises a polynucleotide encoding a human insulin protein comprising a 5’ UTR, an ORF, and a 3’ UTR present or referenced in Table 8.

[0449] In some aspects, the expression cassette comprises a promoter operably linked to a polynucleotide encoding a human glucokinase protein sequence comprising an ORF having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65,SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70,SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75,SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, orSEQ ID NO: 162. In some aspects, the expression cassette comprises a promoter operably linked to a polynucleotide encoding a human glucokinase protein sequence comprising an ORF having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 68. In some aspects, the expression cassette comprises a promoter operably linked to a polynucleotide encoding a human glucokinase protein having the sequence of SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ IDNO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ IDNO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ IDNO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, or SEQ IDNO: 162. In some aspects, the expression cassette comprises a promoter operably linked to a polynucleotide encoding a human glucokinase protein having the sequence of SEQ ID NO: 68. In some aspects, the polynucleotide encoding a human glucokinase protein comprises an ORF sequence present or referenced in Table 9.

[0450] In some aspects, the expression cassette comprises a polynucleotide encoding a human glucokinase protein further comprising a 5’ UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42, nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some aspects, the 5’ UTR comprises a nucleic acid having the sequence of SEQ ID NO: 42, nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some aspects, the polynucleotide encoding a human glucokinase protein comprises a 5’ UTR sequence present or referenced in Table 9.

[0451] In some aspects, the expression cassette comprises a polynucleotide encoding a human glucokinase protein further comprising a 3’ UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 60, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 149, or SEQ ID NO: 169. In some aspects, the 3’ UTR comprises a nucleic acid having the sequence of SEQ ID NO: 60, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, 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 WmHI, EcoRI, Wei, EcoRV, Spel, Xbal, Nhel, VspI, Nsil, Seal, Kpnl, SspI, and Pad, or any combination thereof. In some aspects, the polynucleotide encoding a human glucokinase protein comprises a 3’ UTR sequence present or referenced in Table 9.

[0452] 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 to a sequence selected from SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ IDNO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ IDNO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ IDNO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 89, SEQ IDNO: 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 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 to SEQ ID NO: 168, wherein the nucleic acid sequence encodes a human glucokinase protein (e.g., SEQ ID NO: 82) or 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 to SEQ ID NO: 172, wherein the nucleic acid sequence encodes a human glucokinase protein (e.g., SEQ ID NO: 82) or functional fragment thereof. In some aspects, the expression cassette comprises a polynucleotide encoding a human glucokinase protein having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to nucleic acids 5-2025 of a sequence selected from SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25,SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30,SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36,SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39. In some aspects, the expression cassette comprises a polynucleotide encoding a human glucokinase protein having the sequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ IDNO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ IDNO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ IDNO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ IDNO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID 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 comprising nucleic acids 5-2025 of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25,SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39. In some aspects, the expression cassette comprises polynucleotide encoding a human glucokinase protein comprising a 5’ UTR, an ORF, and a 3’ UTR present or referenced in Table 9.

[0453] In some aspects, the combination therapy comprises administering an insulin expression cassette comprising a polynucleotide encoding a human insulin comprising a ORF having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 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 administering a glucokinase expression cassette comprising a polynucleotide encoding a human glucokinase comprising a 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 to SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ IDNO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ IDNO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ IDNO: 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 a human insulin comprising a ORF having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 121 and glucokinase expression cassette comprising a polynucleotide encoding a human glucokinase comprising a 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 to SEQ ID NO: 68.

[0454] In some aspects, the insulin expression cassette and the glucokinase expression cassette of the combination therapy are administered at a ratio of about 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, l:0.3-0.7, 1:0.3-0.65, 1:0.3- 0.6, 1:0.3-0.55, l: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, l:0.4-0.6, 1:0.4-0.55, l: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 insulin expression cassette and the glucokinase expression cassette of the combination therapy are administered at a ratio of about 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 insulin expression cassette and the glucokinase expression cassette of the combination therapy are administered at a ratio of 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. In some aspects, the insulin expression cassette and the glucokinase expression cassette of the combination therapy are administered at a ratio of about 1:0.5, about 1:1, about 1:2, or about 1:4. In some aspects, the first AAV vector genome and the second AAV vector genome are in a vector ratio 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, and about 1:4). In some aspects, the vector ratio (i.e., first AAV vector comprising the hlns expression cassette to second AAV vector comprising the hGck expression cassette) is 1:0.4-0.6. In some aspects, the vector ratio (i.e., first AAV vector comprising the hlns expression cassette to second AAV vector comprising the hGck expression cassette) is 1:0.5.

[0455] In some aspects, the insulin expression cassette and the glucokinase expression cassette of the combination therapy are administered at a ratio of about 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 ratio is 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 ratio is 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, the vector ratio (i.e., first AAV vector comprising the hlns expression cassette to second AAV vector comprising the hGck expression cassette) is 1:1.

[0456] In some aspects, the vector ratio (i.e., first AAV vector comprising the hlns expression cassette to second AAV vector comprising the hGck expression cassette) is 1 :2. In some aspects, the vector ratio (i.e., first AAV vector comprising the hlns expression cassette to second AAV vector comprising the hGck expression cassette) is1 :3. In some aspects, the vector ratio (i.e., first AAV vector comprising the hlns expression cassette to second AAV vector comprising the hGck expression cassette) is 1 :4. In some aspects, the vector ratio (i.e., first AAV vector comprising the hlns expression cassette to second AAV vector comprising the hGck expression cassette) is 1 :5. In some aspects, the vector ratio (i.e., first AAV vector comprising the hlns expression cassette to second AAV vector comprising the hGck expression cassette) is 1 : 10. In some aspects, the vector ratio (i.e., first AAV vector comprising the hlns expression cassette to second AAV vector comprising the hGck expression cassette) is 1 : 15. In some aspects, the vector ratio (i.e., first AAV vector comprising the hlns expression cassette to second AAV vector comprising 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.

[0457] In some aspects, the polynucleotide encoding a human insulin protein encodes a human insulin comprising a wild-type preproinsulin secretion signal peptide. In some aspects, the polynucleotide encoding a human insulin protein does not encode a wild-type preproinsulin secretion signal peptide. In some aspects, the wild-type preproinsulin is replaced by a non-insulin secretion signal. In some aspects, the polynucleotide encoding a human insulin protein encodes a human preproinsulin comprising an interleukin 6 (IL-6) secretion signal peptide. In some aspects, the polynucleotide encoding a human insulin protein encodes a human preproinsulin comprising a fibronectin secretion signal peptide.

[0458] In some aspects, the insulin expression cassette comprising a polynucleotide encoding a human insulin protein comprises a 5’ UTR, an ORF, and a 3’ UTR present or referenced in Table 8 and the glucokinase expression cassette comprising a polynucleotide encoding a human glucokinase protein comprises a 5’ UTR, an ORF, and a 3’ UTR present or referenced in Table 9.

[0459] Certain aspects of the disclosure are directed to vector, e.g., a viral vector, comprising an expression construct. In some aspects, the expression construct comprises an expression cassette. In some aspects, the expression construct further comprises a genome that is able to stabilize and remain episomal in a cell. Within the context of the disclosure, in some aspects, a cell or host cell can encompass a cell used to make the construct or a cell to which the construct is administered. In some aspects, a construct iscapable of integrating into a cell’s genome, e.g. through homologous recombination or otherwise. In some aspects, the expression construct is one wherein a nucleotide sequence encoding an insulin and / or a glucokinase as disclosed herein, is operably linked to a promoter as provided herein wherein the promoter is capable of directing expression of the nucleotide sequence(s) (i.e. coding sequence(s)) in a cell. In some aspects, an expression cassette as used herein comprises or consists of a nucleotide sequence encoding an insulin and / or a nucleotide sequence encoding a glucokinase, in each case the nucleotide sequence is operably linked to a promoter wherein the promoter is capable of directing expression of said nucleotide sequences. In some aspects, a viral expression construct is an expression construct that is intended to be used in gene therapy. It can be designed to comprise part of a viral genome as disclosed herein.

[0460] In some aspects, the expression construct further comprises one or more of: an ITR sequence (e.g., AAV2 ITRs), a polyA sequence (e.g., a SV40 polyadenylation signal, a bGH polyadenylation signal), and an enhancer sequence (e.g., a SV40 enhancer sequence).

[0461] In some aspects, expression constructs disclosed herein are prepared using recombinant techniques in which nucleic acid sequences encoding an insulin and / or a glucokinase are expressed in a suitable cell, e.g. cultured cells or cells of a multicellular organism, such as described in Ausubel et al., “Current Protocols in Molecular Biology”, Greene Publishing and Wiley-Interscience, New York (1987) and in Sambrook and Russell (2001, supra); both of which are incorporated herein by reference in their entirety. Also see, Kunkel (1985) Proc. Natl. Acad. Sci. 82:488 (describing site directed mutagenesis) and Roberts et al. (1987) Nature 328:731-734 or Wells, J. A., et al. (1985) Gene 34: 315 (describing cassette mutagenesis).Delivery Vectors

[0462] The present 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 vectors, 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.

[0463] In some aspects, a polynucleotide encoding an insulin and / or a glucokinase are used in an expression construct or expression vector. The phrase “expression vector”generally refers to a nucleotide sequence that is capable of effecting expression of a gene in a host compatible with such sequences. These expression vectors can include at least suitable promoter sequences and optionally, transcription termination signals. An additional factor necessary or helpful in effecting expression can also be used as disclosed herein. A polynucleotide encoding an insulin and / or a glucokinase can be incorporated into an expression vector capable of introduction into and expression in an in vitro cell culture. In some aspects, the expression vector is suitable for replication in a prokaryotic host, such as bacteria, e.g., E. coli, or can be introduced into a cultured mammalian, plant, insect, (e.g., Sf9), yeast, fungi or other eukaryotic cell lines. In some aspects, the expression construct is suitable for expression in vivo.

[0464] In some aspects, the delivery vector comprises an insulin expression cassette comprising a promoter operably 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 to a sequence selected from SEQ IDNO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ IDNO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ IDNO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ IDNO: 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 a human insulin protein (e.g., SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO: 145) or a functional fragment thereof. In some aspects, the delivery vector comprises an insulin expression cassette comprising a promoter operably 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 to SEQ ID NO: 122. In some aspects, the delivery vector comprises an insulin expression cassette comprising a promoter operably 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 to SEQ ID NO: 121. In some aspects, the polynucleotide comprises an ORF having the sequence of SEQ ID NOs: 43, SEQ IDNO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ IDNO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ IDNO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 110, SEQ IDNO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQID 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 a human insulin protein comprises an ORF sequence present or referenced in Table 8.

[0465] In some aspects, delivery vector comprises an insulin expression cassette comprising a polynucleotide encoding a human insulin protein encodes a human insulin comprising a wild-type preproinsulin secretion signal peptide. In some aspects, delivery vector comprises an insulin expression cassette comprising a polynucleotide encoding a human insulin protein does not encode a wild-type preproinsulin secretion signal peptide. In some aspects, the wild-type preproinsulin is replaced by a non-insulin secretion signal. In some aspects, delivery vector comprises an insulin expression cassette comprising a polynucleotide encoding a human insulin protein encodes a human preproinsulin comprising an interleukin 6 (IL-6) secretion signal peptide. In some aspects, delivery vector comprises an insulin expression cassette comprising a polynucleotide encoding a human insulin protein sequence encodes a human preproinsulin comprising a fibronectin secretion signal peptide.

[0466] In some aspects, the delivery vector comprises an insulin expression cassette comprising a polynucleotide encoding a human insulin protein further comprising a 5’ UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, 100% sequence identity to SEQ ID NO: 42, nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some aspects, the 5’ UTR comprises a nucleic acid having the sequence of SEQ ID NO: 42, nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some aspects, the polynucleotide encoding a human insulin protein comprises a 5’ UTR sequence present or referenced in Table 8.

[0467] In some aspects, the delivery vector comprises an insulin expression cassette comprising a polynucleotide encoding a human insulin protein further comprising a 3’ UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 60, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, 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 / U / / 7 / HI, EcoRI, Nde , Eco N, Spel, Xba , Nhel, Vsp , Nsi , Seal, Kpn , Ssp , and Pac , and any combination thereof. In some aspects, the 3’ UTR comprises a nucleic acid having the sequence of SEQ ID NO: 60, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 149, or SEQ ID NO: 171. In some aspects, the polynucleotide encoding a human insulin protein comprises a 3’ UTR sequence present or referenced in Table 8.

[0468] In some aspects, the delivery vector comprises an insulin 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 to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 123, SEQ ID NO: 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 a human insulin protein (e.g., SEQ ID NO: 41, SEQ ID NO: 144, or SEQ ID NO: 145) or a functional fragment thereof. In some aspects, the delivery vector comprises an insulin 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 to SEQ ID NO: 138 or SEQ ID NO: 170. In some aspects, the delivery vector comprises an insulin expression cassette comprising a polynucleotide encoding a human insulin protein having at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to nucleic acids 5-957 of a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In some aspects, the delivery vector comprises an insulin expression cassette comprising a polynucleotide encoding a human insulin protein having the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, 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 delivery vector comprises an insulin expression cassette comprising 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 comprising a polynucleotide encoding a human insulin protein having the sequence of SEQ ID NO: 170. In some aspects, the delivery vector comprises an insulin expression cassette comprising a polynucleotide encoding a human insulin protein comprising nucleic acids 5-957 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In some aspects, the delivery vector comprises an insulin expression cassette comprising a polynucleotide encoding a human insulin protein comprising a 5’ UTR, an ORF, and a 3’ UTR present or referenced in Table 8

[0469] In some aspects, the delivery vector comprises a glucokinase expression cassette comprising a promoter operably linked to a polynucleotide encoding a human glucokinase protein comprising an ORF sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 61, SEQ ID NO: 62,SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67,SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72,SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77,SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO: 162. In some aspects, the delivery vector comprises a glucokinase expression cassette comprising a promoter operably linked to a polynucleotide encoding a human glucokinase protein having the sequence of SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ IDNO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ IDNO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ IDNO: 80, or SEQ ID NO: 162. In some aspects, the polynucleotide encoding a human glucokinase protein comprises an ORF sequence present or referenced in Table 9.

[0470] In some aspects, the delivery vector comprises a glucokinase expression cassette comprising a polynucleotide encoding a human glucokinase protein further comprising a 5’ UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42, nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some aspects, the 5’ UTR comprises a nucleic acid having the sequence of SEQ ID NO: 42, nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148. In some aspects, the polynucleotide encoding a human glucokinase protein comprises a 5’ UTR sequence present or referenced in Table 9.

[0471] In some aspects, the delivery vector comprises a glucokinase expression cassette comprising a polynucleotide encoding a human glucokinase protein further comprising a 3’ UTR comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 60, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, 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 amHI, EcoRI, Ndel, Eco N, Spel, Xbal, Nhel, VspI, Nsil, Seal, Kpnl, Ssp\, and Pad, and anycombination thereof. In some aspects, the 3’ UTR comprises a nucleic acid having the sequence of SEQ ID NO: 60, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 149, SEQ ID NO: 169, SEQ ID NO: 171. In some aspects, the polynucleotide encoding a human glucokinase protein comprises a 3’ UTR sequence present or referenced in Table 9.

[0472] In some aspects, the delivery vector comprises a 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 to a sequence selected from SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ IDNO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ IDNO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ IDNO: 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 functional fragment thereof. In some aspects, the delivery vector comprises a glucokinase expression cassette comprising a polynucleotide encoding a human glucokinase protein having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to nucleic acids 5-2025 of a sequence selected from SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39. In some aspects, the delivery vector comprises a glucokinase expression cassette comprising a polynucleotide encoding a human glucokinase protein having the sequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26,SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31,SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37,SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91,SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 168, or SEQ ID NO: 172. In some aspects, the delivery vector comprises a glucokinase expression cassette comprising a polynucleotide encoding a human glucokinase protein comprising nucleic acids 5-2025 of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24,SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35,SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39. In some aspects, the delivery vector comprises a glucokinase expression cassette comprising polynucleotide encoding a human glucokinase protein comprising a 5’ UTR, an ORF, and a 3’ UTR present or referenced in Table 9.

[0473] In some aspects, the delivery vectors can comprise sequences encoding a protein (e.g., insulin and / or Gck) operably linked with control or regulatory sequences, selectable markers, any fusion partners, and / or additional elements. In certain aspects, the modified nucleic acid is placed into 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 the 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 vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the protein, and are typically appropriate to the host cell used to express the protein. In general, the transcriptional and translational regulatory sequences may include promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. As is also known in the art, expression vectors can contain a selection gene or marker to allow the selection of transformed host cells containing the expression vector. Selection genes are known in the art and will vary with the host cell used. For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. In some aspects, selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0474] In some aspects, the delivery vector is a viral vector or a gene therapy vector comprising a viral expression construct. In certain aspects, the viral vector or a gene therapy vector is a vector that is suitable for gene therapy.

[0475] In certain aspects, the delivery vector comprising the expression cassette encoding the human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are delivered in a ratio selected from the group consisting of about 1 :0.25-0.75 and about 1 :0.75-1.25. In certain aspects, the delivery vector comprising the expression cassette encoding the human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are delivered in a ratio selected from the group consisting of about 1 :0.4-0.6 and about 1 :0.9-1.1. In some aspects, the delivery vector comprising the expression cassette encoding the human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are delivered in a ratio selected from the group consisting of about 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, l :0.3-0.7, 1 :0.3-0.65, l :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, l:0.4-0.7, 1 :0.4-0.65, 1 :0.4-0.6, 1 :0.4-0.55, l :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, the delivery vector comprising the expression cassette encoding human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are delivered in a ratio selected from the group consisting of about 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, the delivery vector comprising the expression cassette encoding the human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are delivered in a ratio selected from the group consisting of about 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.

[0476] In some aspects, the delivery vector comprising the expression cassette encoding the human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are delivered in a ratio of 1 :1 to 1 : 15. In some aspects, the delivery vector comprising the expression cassette encoding the human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are delivered in a ratio of 1 :2. In some aspects, the delivery vector comprising the expression cassette encoding the human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are delivered in a ratio of 1 :3. In some aspects, the delivery vector comprising the expression cassette encoding the human insulin and the delivery- I l l - vector comprising the expression cassette encoding human glucokinase are delivered in a ratio of 1 :4. In some aspects, the delivery vector comprising the expression cassette encoding the human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are delivered in a ratio of 1 :5. In some aspects, the delivery vector comprising the expression cassette encoding the human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are delivered in a ratio of 1 : 10. In some aspects, the delivery vector comprising the expression cassette encoding the human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are delivered in a ratio of 1 : 15.

[0477] In certain aspects, the delivery vector comprising the expression cassette encoding the human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are delivered in a ratio selected from the group consisting of about 1 :0.25-0.75 and about 1 :0.75-1.25 (e.g., about 1 :0.5 and about 1 : 1). In some aspects, the vector ratio (i.e., AAV vector comprising the hlns expression cassette to AAV vector comprising the hGck expression cassette) is 1 :0.4-0.6. In some aspects, the delivery vectors (i.e., AAV vector comprising the hlns expression cassette to AAV vector comprising the hGck expression cassette) are delivered in a ratio of 1 :0.5.

[0478] In some aspects, the delivery vector comprising the expression cassette encoding the human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are delivered in a ratio selected from the group consisting of about 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 ratio is 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 ratio is 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, the delivery vector comprising the expression cassette encoding the human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are delivered in a 1 :1 ratio.

[0479] In some aspects, the delivery vector comprising the expression cassette encoding the human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are delivered in a 1 :2 ratio.

[0480] In some aspects, the delivery vector comprising the expression cassette encoding the human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are delivered in a 1 :3 ratio.

[0481] In some aspects, the delivery vector comprising the expression cassette encoding the human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are delivered in a 1 :4 ratio.

[0482] In some aspects, the delivery vector comprising the expression cassette encoding the human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are delivered in a 1 :5 ratio.

[0483] In some aspects, the delivery vector comprising the expression cassette encoding the human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are delivered in a 1 : 10 ratio.

[0484] In some aspects, the delivery vector comprising the expression cassette encoding the human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are delivered in a 1 : 15 ratio.

[0485] In some aspects, the delivery vector comprising the expression cassette encoding human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are administered at a dose of about 5xl0nto about 5xl013vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are administered at a dose of about lxl012to about IxlO14vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are administered at a dose of about lxl013to about IxlO14vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are administered at a dose of about IxlO12, about 2xl012, about 3xl012, about 4xl012, about 5xl012, about 6xl012, about 7xl012, about 8xl012, or about 9xl012vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are administered at a dose of about 2xl012, about 3xl012, about 4xl012, about 5xl012, about 6xl012, about 7xl012, or about 8xl012vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are administered at a dose of about IxlO13, about 2xl013, about 3xl013, about 4xl013, about 5xl013, about 6xl013, about 7xl013, about 8xl013, or about 8xl013vg / kg. In some aspects, the deliveryvector comprising the expression cassette encoding human insulin and the delivery vector comprising the expression cassette encoding human glucokinase are administered at a dose of about 2xl013, about 3xl013, about 4xl013, about 5xl013, about 6xl013, about 7xl013, or about 8xl013vg / kg.

[0486] In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about 7xlO10vg / kg to about IxlO14vg / kg, about 8xlO10vg / kg to about IxlO14vg / kg, about 9xlO10vg / kg to about IxlO14vg / kg, about IxlO11vg / kg to about IxlO14vg / kg, about 2xlOnvg / kg to about IxlO14vg / kg, about 3xl0nvg / kg to about IxlO14vg / kg, about 4xlOnvg / kg to about IxlO14vg / kg, about 7xlO10vg / kg to about IxlO13vg / kg, about 7xlO10vg / kg to about IxlO12vg / kg, about 7xlO10vg / kg to about IxlO11vg / kg, about 5xl0nvg / kg to about IxlO14vg / kg, about 6xlOnvg / kg to about IxlO14vg / kg, about 7xlOnvg / kg to about IxlO14vg / kg, about 8xl0nvg / kg to about IxlO14vg / kg, about 9xlOnvg / kg to about IxlO14vg / kg, about 7xlO10vg / kg to about IxlO14vg / kg, about 8xlO10vg / kg to about IxlO14vg / kg, about 9xlO10vg / kg to about IxlO14vg / kg, about IxlO11vg / kg to about IxlO14vg / kg, about 2xlOnvg / kg to about IxlO14vg / kg, about 3xl0nvg / kg to about IxlO14vg / kg, about 4xlOnvg / kg to about IxlO14vg / kg, about 7xlO10vg / kg to about IxlO13vg / kg, about 7xlO10vg / kg to about IxlO12vg / kg, about 7xlO10vg / kg to about IxlO11vg / kg, about 7xlO10vg / kg to about 7xl012vg / kg, about 7xlO10vg / kg to about 7xlOnvg / kg, about 7xlOlovg / kg to about 1.8xl012vg / kg, about 7xlOlovg / kg to about 3.5xl012vg / kg, about 1.8xl012vg / kg to about 7xl012vg / kg, about 1.8xl012vg / kg to about 3.5xl012vg / kg, or about 3.5xl012vg / kg to about 7xl012vg / kg.

[0487] In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about 5xl0nto about 5xl012vg / kg.

[0488] In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about IxlO11to about IxlO12vg / kg.

[0489] In some aspects, the delivery vector comprising the expression cassette encoding human glucokinase is administered at a dose of about 2xl012vg / kg to about 5xl012vg / kg, about 2xl012vg / kg to about 3.5xl012vg / kg, or about 3.5xl012vg / kg to about 5xl012vg / kg,

[0490] In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about 7xlO10vg / kg and the delivery vector comprising the expression cassette encoding human glucokinase is administered at a doseof about 3.5xl012vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about 7xlOnvg / kg and the delivery vector comprising the expression cassette encoding human glucokinase is administered at a dose of about 3.5xl012vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about 8xlO10vg / kg and the delivery vector comprising the expression cassette encoding human glucokinase is administered at a dose of about 3.5xl012vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about 9xlO10vg / kg and the delivery vector comprising the expression cassette encoding human glucokinase is administered at a dose of about 3.5xl012vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about 2xlOnvg / kg and the delivery vector comprising the expression cassette encoding human glucokinase is administered at a dose of about 3.5xl012vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about 3xlOnvg / kg and the delivery vector comprising the expression cassette encoding human glucokinase is administered at a dose of about 3.5x1012vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about 4xlOnvg / kg and the delivery vector comprising the expression cassette encoding human glucokinase is administered at a dose of about 3.5xl012vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about 5xl0nvg / kg and the delivery vector comprising the expression cassette encoding human glucokinase is administered at a dose of about 3.5xl012vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about 6xlOnvg / kg and the delivery vector comprising the expression cassette encoding human glucokinase is administered at a dose of about 3.5x1012vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about 7xlOnvg / kg and the delivery vector comprising the expression cassette encoding human glucokinase is administered at a dose of about 3.5xl012vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about 8xl0nvg / kg and the delivery vector comprising the expression cassette encoding human glucokinase is administered ata dose of about 3.5xl012vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about 9xlOnvg / kg and the delivery vector comprising the expression cassette encoding human glucokinase is administered at a dose of about 3.5x1012vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about IxlO12vg / kg and the delivery vector comprising the expression cassette encoding human glucokinase is administered at a dose of about 3.5xl012vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about 1.8xl012vg / kg and the delivery vector comprising the expression cassette encoding human glucokinase is administered at a dose of about 3.5xl012vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about 2xl012vg / kg and the delivery vector comprising the expression cassette encoding human glucokinase is administered at a dose of about 3.5x1012vg / kg.

[0491] In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about 3xl013vg / kg and the delivery vector comprising the expression cassette encoding human glucokinase is administered at a dose of about 1.5xl013vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about 4xl013vg / kg and the delivery vector comprising the expression cassette encoding human glucokinase is administered at a dose of about 2xl013vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about 5xl013vg / kg and the delivery vector comprising the expression cassette encoding human glucokinase is administered at a dose of about 2.5xl013vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about 6xl013vg / kg and the delivery vector comprising the expression cassette encoding human glucokinase is administered at a dose of about 3xl013vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about 7xl013vg / kg and the delivery vector comprising the expression cassette encoding human glucokinase is administered at a dose of about 3.5xl013vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about 8xl013vg / kg and the delivery vector comprising the expression cassette encoding human glucokinase isadministered at a dose of about 4xl013vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about 9xl013vg / kg and the delivery vector comprising the expression cassette encoding human glucokinase is administered at a dose of about 4.5xl013vg / kg. In some aspects, the delivery vector comprising the expression cassette encoding human insulin is administered at a dose of about IxlO14vg / kg and the delivery vector comprising the expression ca...

Claims

WHAT IS CLAIMED IS:

1. A method of treating or ameliorating the symptoms associated with diabetes in a subject in need thereof, wherein the method comprises administering a combination therapy to the subject comprising: one or more adeno-associated virus (AAV) vectors comprising(a) a first promoter operably linked to a polynucleotide encoding a human insulin (hlns) protein; and(b) a second promoter operably linked to a polynucleotide encoding a human glucokinase (hGck) protein; wherein the combination 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 comprising an insulin expression cassette comprising the first promoter operably linked to the polynucleotide encoding a hlns protein, wherein the insulin expression cassette is flanked by inverted terminal repeats (ITRs); and(b) a second AAV vector genome comprising a glucokinase expression cassette comprising the second promoter operably linked to the polynucleotide encoding the hGck protein, wherein the glucokinase expression cassette is flanked by inverted terminal repeats (ITRs).

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 of any one of claims 1-3, wherein the polynucleotide encoding a human insulin (hlns) protein and the polynucleotide encoding a human glucokinase (hGck) protein are administered in the same pharmaceutical composition.

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

6. The method of claim 1, wherein the one or more AAV vectors is an AAV vector genome comprising an expression cassette comprising the first promoter operably linked to the polynucleotide encoding the hlns protein and the second promoter operably linked to the polynucleotide encoding the hGck protein, wherein the expression cassette is flanked by inverted terminal repeats (ITRs).

7. The method of any one of claims 1-6, wherein the combination therapy comprises (i) a first recombinant AAV (rAAV) particle comprising the first AAV vector genome comprising the insulin expression cassette comprising the promoter operably linked to the polynucleotide encoding the human insulin (hlns) protein, wherein the insulin expression cassette is flanked by inverted terminal repeats (ITRs); and (ii) a second recombinant AAV (rAAV) particle comprising the second AAV vector genome comprising the glucokinase expression cassette comprising the promoter operably linked to the polynucleotide encoding the human glucokinase (hGck) protein, wherein the glucokinase expression cassette is flanked by inverted terminal repeats (ITRs).

8. The method of any one of claims 1-7, 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 of any one of claims 1-8, wherein the 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; (ii) 2-5, 3-5, or 4-5 different skeletal muscle groups; (iv) 2-4 or 3-4 different skeletal muscle groups.

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

11. The method of any one of claims 1-10, wherein the skeletal muscle group is selected from a quadriceps, a bicep, a triceps, a gastrocnemius, a gluteus maximus, a hamstring, adeltoid, a trapezius, a pectoral muscle, and a 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 group is selected from i) a left quadriceps and a right quadriceps, ii) a left bicep and a right bicep, iii) a left triceps and a right triceps, iv) a left gastrocnemius and a right gastrocnemius, v) a left gluteus maximus and a right gluteus maximus, vi) a left hamstring and a right hamstring, vii) a left deltoid and a right deltoid, viii) a left trapezius and a right trapezius, ix) a left pectoral muscle and a right pectoral muscle, and x) a left latissimus dorsi and a right latissimus dorsi.

12. The method of any one of claims 1-11, wherein the skeletal muscle group is selected from a quadriceps, a bicep, a triceps, a gastrocnemius, a gluteus maximus, and a hamstring, optionally wherein the two or more different skeletal muscle groups comprise any combination thereof.

13. The method of any one of claims 1-12, wherein the skeletal muscle group is selected from a quadriceps, a bicep, a gastrocnemius, a gluteus maximus, and a hamstring, optionally wherein the two or more different skeletal muscle groups comprise any combination thereof.

14. The method of any one of claims 1-13, wherein the administration comprises bilateral injections into one or more of the different skeletal muscle groups, optionally wherein the bilateral injections are divided equally for each skeletal muscle group.

15. The method of claim 14, wherein the bilateral injections comprise IM injections to the right and left quadriceps, the right and left biceps, the right and left triceps, the right and left gastrocnemius muscles, the right and left gluteus maximus muscles, and / or the right and left hamstrings, and any combinations thereof.

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

17. The method of claim 16, wherein the administration comprises 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 of any one of claims 1-17, wherein the administration comprises:(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) 1-30 IM injections to a first skeletal muscle group, 1-30 IM injections to a second skeletal muscle group, 1-30 IM injections to a third skeletal muscle group, 1-30 IM injections to a fourth skeletal muscle group, and 1-30 IM injections to a fifth skeletal muscle group; and(v) 1-25 IM injections to a first skeletal muscle group, 1-25 IM injections to a second skeletal muscle group, 1-25 IM injections to a third skeletal muscle group, 1-25 IM injections to a fourth skeletal muscle group, 1-25 IM injections to a fifth skeletal muscle group, and 1-25 IM injections to a sixth skeletal muscle group.

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

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

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

22. The method of claim 21, wherein the administration comprises 4-25 IM injections bilaterally to each quadriceps, 4-25 IM injections bilaterally to each bicep, and 4-25 IM injections bilaterally to each hamstring.

23. The method of any one of claims 1-22, wherein each IM injection is administered in a volume of about 100 pL 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 a volume of about 100 pL to about 2 mL, 100 pL to about 1.9 mL, 100 pL to about 1.8 mL, 100 pL to about 1.7 mL, 100 pL to about 1.6 mL, 100 pL to about 1.5 mL, 100 pL to about 1.4 mL, about 100 pL to about 1.35 mL, about 100 pL to about 1.2 mL, about 100 pL to about 1.0 mL, about 100 pL to about 800 pL, about 200 pL to about 800 pL, about 200 pL to about 600 pL, about 400 pL to about 800 pL, about 500 pL to about 1.5 mL, about 600 pL to about 1.4 mL, about 700 pL to about 1.3 mL, about 800 pL to about 1.2 mL, or about 900 pL to about 1.1 mL.

25. The method of any one of claims 1-24, wherein the total cumulative volume administered for 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.

26. The method of any one of claims 1-25, wherein each IM injection comprises about 8xl010to about 5xl013vgs per mL, optionally wherein the IM injection comprises about 2xl013to about 4xl013vgs per mL.

27. The method of any one of claims 1-26, wherein the IM administration comprises injections that are separated by a distance of at least about 1-5 cm, optionally at least about 1-3 cm.

28. The method of any one of claims 1-27, wherein each of the IM injections are separated by a distance of at least 1 cm, at least 2 cm, at least 3 cm, at least 4 cm, or at least 5 cm.

29. The method of any one of claims 1-28, wherein the administration comprises IM injections that are 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 of any one of claims 1-29, wherein each of the IM injections is about 0.5 cm to 2.0 cm deep, optionally about 1-2 cm deep.

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

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

33. A method of treating or ameliorating the symptoms associated with diabetes in a subject in need thereof, comprising administering to the subject (i) an immunosuppressive regimen comprising an immunosuppressive agent, and (ii) a combination therapy comprising one or more adeno-associated virus (AAV) vectors comprising:(a) a first promoter operably linked to a polynucleotide encoding a human insulin (hlns) protein; and(b) a second promoter operably linked to a polynucleotide encoding a human glucokinase (hGck) protein, optionally, 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 immunosuppressive agents prior to (pre- AAV), at the same time as, and / or after (post-AAV) administering the combination therapy.

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

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

37. The method of any one of claims 32-36, wherein the immunosuppressive agent is administered 1 to 14 days prior to administering the combination therapy.

38. The method of any one of claims 34-37, wherein the immunosuppressive agent is administered 1 to 7 days prior to administering the combination therapy.

39. The method of any one of claims 34-38, wherein the immunosuppressive agent is administered 1 to 3 days prior to administering the combination therapy.

40. The method of any one of claims 32-38, wherein the immunosuppressive agent is administered 3 to 7 days prior to administering the combination therapy.

41. The method of any one of claims 32-40, wherein the immunosuppressive agent is further administered 1, 2, 3, or 4 weeks after administering the combination therapy.

42. The method of any one of claims 32-41, wherein immunosuppressive agent is administered once about every 1-3 weeks, optionally once about every 1-2 weeks after administering the combination therapy.

43. The method of any one of claims 32-42, wherein the immunosuppressive agent is administered for about 1-6 months, optionally about 1-3 months after administering the combination therapy.

44. The method of any one of claims 32-43, wherein immunosuppressive regimen comprises a second immunosuppressive agent.

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

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

47. The method of any one of claims 44-46, wherein the second immunosuppressive agent is administered 1 to 14 days prior to administering the combination therapy.

48. The method of any one of claims 44-47, wherein the second immunosuppressive agent is administered 1 to 7 days prior to administering the combination therapy.

49. The method of any one of claims 44-48, wherein the second immunosuppressive agent is administered 1 to 3 days prior to administering the combination therapy.

50. The method of any one of claims 44-48, wherein the second immunosuppressive agent is administered 3 to 7 days prior to administering the combination therapy.

51. The method of any one of claims 44-50, wherein the second immunosuppressive agent is further administered 1, 2, 3, or 4 weeks after administering the combination therapy.

52. The method of any one of claims 44-51, wherein second immunosuppressive agent is administered 2 times per day, daily, or every other day.

53. The method of any one of claims 44-52, wherein the second immunosuppressive agent is administered for about 1-6 months, optionally about 1-3 months after administering the combination therapy.

54. The method of any one of claims 32-53, wherein the immunosuppressive regimen comprises administering an immunosuppressive agent selected from the group consisting of an IL-6 antagonist, an IL-1 antagonist, a TNFa antagonist, an mTOR pathway inhibitor, mycophenolate mofetil (MMF), a tyk2 inhibitor, methotrexate, abatacept, antithymocyte globulin (ATG), a B cell depletion agent, a T cell costimulation antagonist, a T cell depletion agent, a corticosteroid, a calcineurin inhibitor, or any combination thereof.

55. The method of any one of claims 32-54, wherein the immunosuppressive agent 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, sarilumab, satralizumab, siltuximab, olokizumab, elsilimomab, clazakizumab, sirukumab, levilimab, or any combination thereof.

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

58. The method of any one of claims 32-57, wherein the second immunosuppressive agent comprises a calcineurin inhibitor, MMF, methotrexate, a tyk2 inhibitor, rapamycin, abatacept, thymoglobulin, etanercept, basiliximab, sirolimus, or any combination thereof.

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

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

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

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

63. The method of any one of claims 2-62, wherein the total administered dose of the first AAV vector genome comprising the insulin expression cassette comprises about 7xlO10to about 5xl013vector genomes (vg) / kg.

64. The method of any one of claims 2-63, wherein the total administered dose of the second AAV vector genome comprising the glucokinase expression cassette comprises about 5xl0nto about 5xl013vector genomes (vg) / kg.

65. The method of any one of claims 32-64, 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 about 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, 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 of any one of claims 32-65, 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 of any one of claims 32-66, 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 of any one of claims 32-66, 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, abut 1:13, about 1:14, or about 1:15.

69. The method of any one of claims 32-66, 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 of any one of claims 2-69, wherein the first AAV vector genome comprising an insulin expression cassette is administered at a dose between about 5xl0nand about IxlO13vg / kg.

71. The method of any one of claims 2-70, wherein the first AAV vector genome comprising an insulin expression cassette is administered at a dose between about 2xl012and about IxlO13vg / kg.

72. The method of any one of claims 2-71, wherein the first AAV vector genome comprising an insulin expression cassette is administered at a dose of about 4xl012to about IxlO13vg / kg.

73. The method of any one of claims 2-69, wherein the first AAV vector genome comprising an insulin expression cassette is administered at a dose of about 7xlO10to about 7xl012vg / kg.

74. The method of any one of claims 2-69, wherein the first AAV vector genome comprising an insulin expression cassette is administered at a dose of about 5xl0nto about 5xl012vg / kg.

75. The method of any one of claims 2-69, wherein the first AAV vector genome comprising an insulin expression cassette is administered at a dose of about IxlO11to about IxlO12vg / kg.

76. The method of any one of claims 2-75, wherein the second AAV vector genome comprising a glucokinase expression cassette is administered at a dose between about IxlO12and about 5xl012vg / kg.

77. The method of any one of claims 2-76, wherein the second AAV vector genome comprising a glucokinase expression cassette is administered at a dose between about 2xl012and about 5xl012vg / kg.

78. The method of any one of claims 2-77, wherein the second AAV vector genome comprising a glucokinase expression cassette is administered at a dose of about 2xl012to about 3.5xl012vg / kg.

79. The method of any one of claims 2-77, wherein the second AAV vector genome comprising a glucokinase expression cassette is administered at a dose of about 3.5xl012to about 5xl012vg / kg.

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

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

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

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

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

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

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

87. The method of any one of claims 1-86, wherein the subject is not insulin resistant.

88. The method of any one of claims 1-86, wherein the subject is insulin resistant.

89. A dosing regimen comprising:(a) a first immunosuppressive agent;(b) a combination therapy administered intramuscularly (IM) after (a) which comprises (i) a first recombinant AAV (rAAV) particle comprising a first AAV vector genome comprising an insulin expression cassette comprising a first promoter operably linked to a polynucleotide encoding a human insulin (hlns) protein, wherein the insulin expression cassette is flanked by inverted terminal repeats (ITRs); and (ii) a second recombinant AAV (rAAV) particle comprising a second AAV vector genome comprising a glucokinase expression cassette comprising a second promoter operably linked to a polynucleotide encoding a human glucokinase (hGck) protein, wherein the glucokinase expression cassette is flanked by inverted terminal repeats (ITRs); and(c) a second immunosuppressive agent.

90. The dosing regimen of claim 89, wherein the first immunosuppressive agent comprises an IL-6 antagonist selected from the group consisting of tocilizumab, sarilumab, siltuximab, olokizumab, elsilimomab, clazakizumab, sirukumab, levilimab, and any combination thereof.

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

92. The dosing regimen of any one of claims 89-91, wherein the first immunosuppressive agent comprises tocilizumab and the second immunosuppressive agent comprises tacrolimus.

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

94. The dosing regimen of any one of claims 89-93, wherein the immunosuppressive regimen comprises administering tacrolimus, optionally orally, on about day -3 and every 1-3 days through about 3-12 weeks relative to the combination therapy95. The method or dosing regimen of any one of claims 1-94, wherein:(a) the polynucleotide encoding the hlns protein comprises an open reading frame (ORF) comprising: a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to any one of: (i) nucleic acids 73-330 of any of SEQ ID NOs: 43-57, or 110-116, nucleic acids 88-345 of any of SEQ ID NOs: 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 comprising (i) a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to a sequence selected from any of (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 of any one of claims 1-95, wherein:(a) the polynucleotide encoding the hlns protein comprises an open reading frame (ORF) comprising a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 121; and / or(b) the polynucleotide encoding the human glucokinase hGck protein comprises an ORF comprising a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 68.

97. The method or dosing regimen of any one of claims 1-96, wherein the hlns protein comprises the amino acid sequence of any of amino acids 25-110 of SEQ ID NO: 41, amino acids 25-110 of SEQ ID NO: 144, 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 dosing regimen of any one of claims 1-97, wherein the hlns protein comprises a signal peptide.

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

100. The method or dosing regimen of 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 dosing regimen of any one of claims 1-100, wherein the hlns protein further comprises a cleavage site.

102. The method or dosing regimen of any one of claims 1-101, wherein the polynucleotide encoding the hlns protein further comprises a 5’ UTR comprising a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148.

103. The method or dosing regimen of any one of claims 1-102, wherein the polynucleotide encoding the hlns protein further comprises a 3’ UTR comprising a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 60, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, or SEQ ID NO: 101, SEQ ID NO: 149, or SEQ ID NO: 171.

104. The method or dosing regimen of any one of claims 1-103, wherein the encoded hGck protein comprises the amino acid sequence of SEQ ID NO: 82.

105. The method or dosing regimen of any one of claims 1-104, wherein the polynucleotide encoding the hGck protein further comprises a 5’ UTR comprising a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to nucleic acids 5-329 of SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: 83, SEQ ID NO: 146, or SEQ ID NO: 148.

106. The method or dosing regimen of any one of claims 1-105, wherein the polynucleotide encoding the hGck protein further comprises a 3’ UTR comprising a nucleotide sequence at least 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 60, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 149, SEQ ID NO: 169, or SEQ ID NO: 171.

107. The method or dosing regimen of any one of claims 1-106, wherein the insulin expression cassette promoter and / or the glucokinase expression cassette promoter is a eukaryotic promoter.

108. The method or dosing regimen of any one of claims 1-106, wherein the insulin expression cassette promoter and / or the glucokinase expression cassette promoter is a constitutive promoter.

109. The method or dosing regimen of any one of claims 1-106, wherein the insulin expression cassette promoter and / or the glucokinase expression cassette promoter is a ubiquitous promoter.

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

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

112. The method or dosing regimen of any one of claims 1-111, wherein the insulin expression cassette further comprises a polyadenylation (poly A) element.

113. The method or dosing regimen of any one of claims 1-112, wherein the glucokinase expression cassette further comprises a polyadenylation (poly A) element.

114. The method or dosing regimen of any one of claims 2-113, wherein a first recombinant AAV (rAAV) particle comprises the first AAV vector genome and a second recombinant AAV (rAAV) particle comprises 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, AAVrhlO, AAV10, AAV11, AAV12, and AAVrh74.

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

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

119. A method of immunosuppression in a subject administered an AAV gene therapy for the treatment of diabetes, wherein the subject is administered an immunosuppressive regimen comprising administering (a) an immunosuppressive agent (pre-AAV) prior to administering the AAV gene therapy; and (b) an immunosuppressive agent (post-AAV) after administering the AAV gene therapy.

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

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

122. The method of any one of claims 119-121, wherein post-AAV immunosuppressive agent is administered once about every 1-3 weeks, optionally once about every 1-2 weeks after administering the combination therapy.

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

124. The method of any one of claims 119-123, wherein the post-AAV immunosuppressive agent is administered 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 of any one of claims 119-124, wherein the pre- AAV immunosuppressive agent and / or the post-AAV immunosuppressive agent is administered for about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.

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

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

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

129. The method of any one of claims 119-128, wherein the IL-6 antagonist is selected from the group consisting of tocilizumab, sarilumab, siltuximab, olokizumab, elsilimomab, clazakizumab, sirukumab, levilimab, or any combination thereof.

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

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

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

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

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

135. The method of claim 119-134, wherein the subject is administered the AAV gene therapy by intramuscular (IM) injection.

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

137. The method of claim 136, wherein the skeletal muscle group is selected from a quadriceps, a bicep, a triceps, a gastrocnemius, a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle, and a latissimus dorsi (lat).

138. The method of claim 136 or 137, wherein the skeletal muscle group is selected from a quadriceps, a bicep, a triceps, a gastrocnemius, a gluteus maximus, and a hamstring.

139. The method of any one of claims 119-137, wherein the administration comprises bilateral injections into one or more of the skeletal muscle groups.

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

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