Gene Therapy for the Treatment of Diabetes

Abstract

Provided are recombinant adeno-associated virus (rAAV) vectors containing a transgene that expresses Pax4, virions containing the vectors (rAAV virions), methods for their production, and methods for their use, including methods for treating diabetes, methods for increasing insulin production, and methods for transdifferentiation of alpha cells to beta cells, as well as pharmaceutical compositions and kits containing them.

Description

[Technical field]

[0001] Sequence Listing The electronic sequence listing submitted as file 38061_0004P1.xml has a size of 64,269 bytes, was created on November 30, 2022, and is incorporated by reference in its entirety herein.

[0002] Viral constructs, particles and compositions for use in the treatment of diabetes are provided, which have a transgene encoding PAX4 for expression in pancreatic cells. [Background technology]

[0003] According to the 2020 National Diabetes Statistics Report (hereinafter the "Report") issued by the Centers for Disease Control and Prevention (CDC), the number of people with diabetes mellitus (hereinafter the "Diabetes") in the United States is estimated to reach approximately 34.2 million people of all ages, accounting for approximately 10.5% of the population. The CDC estimates that of these people, 26.9 million have been diagnosed with diabetes, and that there are 7.3 million people who have diabetes but have not yet been diagnosed.

[0004] The islets of Langerhans (sometimes called "pancreatic islets") in the pancreas contain alpha cells, which produce glucagon, and beta cells, which produce insulin, a hormone that helps cells absorb glucose from the bloodstream. In type 1 diabetes (sometimes called "T1DM"), an individual's immune system destroys the individual's beta cells, so insulin must be taken daily for the rest of their life. Type 2 diabetes (sometimes called "T2DM") occurs when an individual's body becomes resistant to the effects of insulin and the body cannot produce enough insulin to overcome that resistance. Both conditions result in too much glucose in the blood, which over time can cause damage to multiple organs.

[0005] Currently, insulin therapy is the main treatment for T1D. Despite significant advances, insulin therapy does not provide smooth and precise glycemic control that allows β cells to function in response to fluctuations in blood glucose levels. As a result, insulin therapy cannot prevent the eventual onset of diabetes-related complications that impair patients' quality of life and longevity.

[0006] According to the report, in 2017, diabetes was the leading cause of major cardiovascular disease and limb amputations in the United States, as well as the leading cause of end-stage renal disease, the leading cause of new cases of blindness in adults aged 18-64, and the seventh leading cause of death in the U.S. Therefore, the ultimate treatment for T1D requires not only the hormone insulin, but also beta cell therapy, i.e., the ability to replace lost beta cells.

[0007] In the early stages of T1DM, beta cells are reduced but alpha cells are normal, resulting in an imbalance between alpha and beta cell function, leading to excess glucagon and several clinical symptoms, such as ketosis. This imbalance increases over time, but alpha cells remain in significant excess compared to the reduced beta cell function. In type 2 diabetes, insulin deficiency also leads to relative alpha cell dysfunction with glucagon hypersecretion, but less severe than in type 1. The alpha cell dysfunction that occurs in type 2 diabetes reflects an impairment in glucose sensing that is partially ameliorated by GLP-1 (Dunning B, Diabetologia 2005, 48 (9): 1700-13). However, because the underlying abnormalities within the islets remain, the disease continues to progress, even when exogenous GLP-1 is administered.

[0008] Pancreatic β-cell embryonic development is controlled by the sequential expression of a series of transcription factors. One of the transcription factors, Pax4, has been shown to play a key role in β-cell proliferation and survival as well as β-cell differentiation. Lorenzo et al.(2017), Brun et al., Lorenzo et al.(2015), Sosa-Pineda et al. Ectopic expression of Pax4 in glucagon-producing α-cells induces their conversion to insulin-producing β-cells. Collomat et al. Adenovirus (Ad5)-mediated delivery of the Pax4 gene directly into the pancreas of T1D mouse models showed some beneficial effects but lacked efficiency. Zhang et al.(2016). Ad5-mediated delivery of the Pax4 gene into primary islet cells not only promoted β-cell survival but also induced α-cell to β-cell transdifferentiation in primary islet cells when transplanted into mice, demonstrating activity. Parajuli et al. These data suggest that Pax4 is a candidate target for T1D treatment aimed at preserving and regenerating β-cells.

[0009] Recent studies have demonstrated therapeutic benefits by inducing transdifferentiation of α-cells to β-cells. Furuyama et al., Xiao et al. Specifically, gene delivery of Pdx1 and MafA to the pancreas was shown to normalize blood glucose in both STZ-induced T1D and NOD mice, and further testing confirmed the conversion of α-cells to β-cells in treated mice. Xiao et al.

[0010] Therapies that promote the formation of beta cells for the treatment of diabetes and thereby reduce the symptoms or damage caused by such deficiencies would be desirable. The compositions and methods disclosed herein meet these and other needs. Summary of the Invention

[0011] Provided are viral constructs, viral particles, and compositions for use in treating diabetes and preventing or delaying the progression of a pre-diabetic state to diabetes, the viral constructs, viral particles, and compositions having a transgene encoding PAX4 expressed in pancreatic cells. The vector constructs are used to generate recombinant AAV virions or particles, which are administered to a subject suffering from diabetes or at high risk of developing diabetes for rAAV gene therapy to deliver nucleic acid encoding Pax4 to cells, including pancreatic cells, of the subject. In some embodiments, expression of Pax4 in pancreatic islet cells, including alpha cells, induces transdifferentiation of alpha cells to insulin-producing beta cells, thereby treating and ameliorating diabetes in the subject and / or reducing the risk of progression of a pre-diabetic state to diabetes. In some embodiments, expression of Pax4 results in the maintenance or protection of the beta cells of the subject, thereby treating and / or ameliorating and / or delaying the progression of diabetes in the subject.

[0012] Provided is an AAV vector comprising an expression cassette comprising a nucleotide sequence encoding Pax4 (including human Pax4) operably linked to one or more regulatory elements and a polyadenylation (poly(A)) tail signal that facilitate expression of the Pax4 coding sequence, a promoter regulatory element comprising a constitutive promoter comprising a cytomegalovirus (CMV) early enhancer / chicken beta actin / rabbit beta globin splice acceptor (CAG) promoter, or a pancreatic cell or alpha cell or beta cell specific promoter (such as a human or rodent insulin promoter or a human or rodent glucagon promoter), and a poly(A) tail signal. In some embodiments, the promoter is a CAG promoter (e.g., having a nucleotide sequence of SEQ ID NO: 4). The polyA signal sequence can be an SV40 polyadenylation signal sequence, e.g., having a nucleotide sequence of SEQ ID NO: 7. In some embodiments, the promoter is a mouse glucagon promoter (MGP, e.g., having a nucleotide sequence of SEQ ID NO: 5) or a rat insulin promoter (RIP, e.g., having a nucleotide sequence of SEQ ID NO: 6). The expression cassette may be flanked by inverted terminal repeat (ITR) nucleotide sequences that include AAV2 ITR sequences. The encoded human Pax4 may have the amino acid sequence of SEQ ID NO: 1 and may be encoded by a nucleic acid that includes a nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 2 encoding human Pax4, or its reverse complementary sequence. In some embodiments, the nucleotide sequence encoding Pax4 is SEQ ID NO: 2. The 5' and 3' ITR sequences may be wild-type sequences, such as the AAV2 ITRs, or may have modified ITR sequences (either the 5' ITR or the 3'' ITR) to create a double-stranded, "self-complementary" AAV vector.

[0013] Particular expression cassettes are provided, which have the nucleotide sequence of SEQ ID NO:10 (CAG-hPax4), SEQ ID NO:11 (MGP-hPax4), or SEQ ID NO:12 (RIP-hPax4) (or a reverse complementary sequence), or have at least 85% identity to the nucleotide sequence of SEQ ID NO:10 (CAG-hPax4), SEQ ID NO:11 (MGP-hPax4), or SEQ ID NO:12 (RIP-hPax4), and can encode human Pax4.

[0014] Also provided are viral vectors (including AAV genomes) that contain a Pax4 expression cassette flanked by 5' and 3' ITRs (including modified ITR sequences to generate double-stranded or self-complementary genomes), including genomes having the nucleotide sequence of SEQ ID NO: 13 (scAAV-CAG-hPax4), SEQ ID NO: 14 (scAAV-MGP-hPax4), or SEQ ID NO: 15 (scAAV-RIP-hPax4).

[0015] Also provided are plasmids containing these expression cassettes, including the plasmid pscAAV-CAG-hPax4-KanR (shown in Figure 1), in which one strand of the nucleotide sequence is SEQ ID NO: 16, the plasmid pscAAV-MGP-hPax4-KanR (shown in Figure 2), in which one strand of the nucleotide sequence is SEQ ID NO: 17, and the plasmid pscAAV-RIP-hPax4-KanR (shown in Figure 3), in which one strand of the nucleotide sequence is SEQ ID NO: 18. The nucleotide sequence shown is one strand of a double stranded plasmid, the other strand being the reverse complement of the nucleotide sequence provided.

[0016] Also provided is a recombinant AAV virion comprising a nucleic acid comprising an expression cassette described herein encoding hPax4 (e.g., an expression cassette of SEQ ID NO: 10, 11, or 12) or a viral genome described herein (SEQ ID NO: 13, 14, or 15) and an AAV capsid. The AAV capsid can be an AAV6 capsid (having a capsid protein comprising the amino acid sequence of SEQ ID NO: 19) or an AAV8 capsid (having a capsid protein comprising the amino acid sequence of SEQ ID NO: 23) or an AAV9 capsid (having a capsid protein comprising the amino acid sequence of SEQ ID NO: 21).

[0017] Methods are provided for increasing insulin production in a subject in need of increased insulin production, increasing the number of beta cells in pancreatic islets in a subject in need of increased beta cell number, and promoting transdifferentiation of alpha cells to beta cells in a subject in need of promoting transdifferentiation of alpha cells to beta cells by administering a composition comprising an effective amount of an AAV virion disclosed herein that expresses Pax4 in pancreatic cells. Also provided are methods for treating diabetes, including type 1 diabetes (T1D) or type 2 diabetes (T2D), in a subject in need of treatment, including T1D or T2D, and for preventing or reducing the risk of developing diabetes in an individual at high risk of developing diabetes (e.g., pre-diabetic state, positive autoantibodies against pancreatic islet cells) by administering a composition comprising an effective amount of an AAV virion disclosed herein that expresses Pax4 in pancreatic cells. In some embodiments, the subject is a human. Pharmaceutical compositions and methods of administration, including delivery to the pancreas, are provided, but are not limited thereto.

[0018] Host cells for and methods of producing the recombinant AAV virions described herein are also provided.

[0019] Pax4 gene therapy has several unique advantages, such as 1) the use of a single transcription factor, Pax4, which eliminates the need to consider the order or balance of gene expression of multiple proteins, thus facilitating gene delivery; and 2) Pax4 has dual functions in promoting alpha-to-beta transdifferentiation and beta cell survival, thereby acting synergistically to increase beta cell mass.

[0020] Embodiment Embodiment 1. A recombinant adeno-associated virus (AAV) vector comprising an expression cassette having a nucleotide sequence encoding hPax4 operably linked to a promoter and a polyadenylation (poly(A)) signal sequence that promotes expression of the nucleotide sequence encoding hPax4 in pancreatic islet cells, wherein the promoter comprises a CAG promoter, a rodent or human glucagon promoter, or a rodent or human insulin promoter, and the expression cassette is flanked by inverted terminal repeat (ITR) nucleotide sequences.

[0021] Embodiment 2. The recombinant AAV vector of embodiment 1, wherein the hPax4 has the amino acid sequence of SEQ ID NO:1.

[0022] Embodiment 3. The recombinant AAV vector of embodiment 1 or 2, wherein the nucleic acid encoding hPax4 comprises a nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO:2 or its reverse complementary sequence.

[0023] Embodiment 4. A recombinant AAV vector according to any one of embodiments 1 to 3, wherein the nucleic acid encoding hPax4 has or consists of the nucleotide sequence of SEQ ID NO: 2 or its reverse complementary sequence.

[0024] Embodiment 5. The recombinant AAV vector according to any one of embodiments 1 to 4, wherein the promoter has the nucleotide sequence of SEQ ID NO: 4 (CAG promoter), SEQ ID NO: 5 (mouse glucagon promoter), or SEQ ID NO: 6 (rat insulin promoter).

[0025] Embodiment 6. The recombinant AAV vector according to any one of embodiments 1 to 5, wherein the polyA signal sequence is an SV40 polyadenylation signal sequence having the nucleotide sequence of SEQ ID NO: 7 or its reverse complementary sequence.

[0026] Embodiment 7. The recombinant AAV vector of any one of embodiments 1 to 6, wherein the recombinant AAV vector is a self-complementary vector.

[0027] Embodiment 8. A recombinant AAV vector according to any one of embodiments 1 to 7, wherein the ITR nucleotide sequence comprises a 5'ITR having the nucleotide sequence of SEQ ID NO:8 and a 3'ITR having the nucleotide sequence of SEQ ID NO:25 or a modified 3'ITR having the nucleotide sequence of SEQ ID NO:9 or its reverse complementary sequence, or has a modified 5'ITR sequence of SEQ ID NO:25 and a 3'ITR sequence or its reverse complementary sequence.

[0028] Embodiment 9. The recombinant AAV vector of any one of embodiments 1 to 8, wherein the expression cassette has the nucleotide sequence of SEQ ID NO: 10 (CAT-hPax4), SEQ ID NO: 11 (MGP-hPax4), or SEQ ID NO: 12 (RIP-hPax4), or a reverse complementary sequence thereof.

[0029] Embodiment 10. A recombinant AAV vector according to any one of embodiments 1 to 9, having the nucleotide sequence of SEQ ID NO: 13 (scAAV-CAG-hPax), SEQ ID NO: 14 (scAAV-MGP-Pax4), or SEQ ID NO: 15 (scAAV-RIP-hPax), or a reverse complementary sequence thereof.

[0030] Embodiment 11. A recombinant AAV virion comprising: 1) an AAV capsid comprising an AAV capsid protein and having islet cell tropism; and 2) a recombinant AAV vector according to any one of embodiments 1 to 10.

[0031] Embodiment 12. The AAV virion of embodiment 11, wherein the AAV capsid protein has an amino acid sequence that has at least 85% identity to the AAV6 capsid protein (SEQ ID NO: 19), or the AAV8 capsid protein (SEQ ID NO: 23), or the AAV9 capsid protein (SEQ ID NO: 21).

[0032] Embodiment 13. The AAV virion of embodiment 12, wherein the AAV capsid protein has the amino acid sequence of SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID NO:23.

[0033] Embodiment 14. A composition for use in increasing insulin production in a subject in need thereof, comprising a therapeutically effective amount of an AAV virion according to any one of embodiments 11 to 13, such that a nucleotide sequence encoding hPax4 is expressed in pancreatic islet cells when administered to the subject.

[0034] Embodiment 15. A composition for use in increasing the number of beta cells in a subject in need thereof, comprising a therapeutically effective amount of an AAV virion according to any one of embodiments 11 to 13, such that a nucleotide sequence encoding hPax4 is expressed in pancreatic islet cells when administered to the subject.

[0035] Embodiment 16. A composition for use in a subject for treating, ameliorating, or alleviating a symptom of diabetes in a subject suffering from diabetes, comprising a therapeutically effective amount of an AAV virion described in any one of embodiments 11 to 13 such that a nucleotide sequence encoding hPax4 is expressed in pancreatic islet cells when administered to the subject.

[0036] Embodiment 17. The composition for use according to embodiment 16, wherein the diabetes is type 1 diabetes.

[0037] Embodiment 18. The composition for use according to embodiment 16, wherein the diabetes is type 2 diabetes.

[0038] Embodiment 19. A composition for use in reducing the risk of developing or delaying the onset of diabetes in a subject positive for islet-associated autoantibodies, comprising a therapeutically effective amount of an AAV virion described in any one of embodiments 11 to 13 such that a nucleotide sequence encoding hPax4 is expressed in pancreatic islet cells when administered to the subject.

[0039] Embodiment 20. The composition for use according to any one of embodiments 14 to 19, wherein the composition is administered by systemic administration, intravenous administration, intramuscular administration, intraperitoneal administration, or subcutaneous administration.

[0040] Embodiment 21. The composition for use according to any one of embodiments 14 to 19, wherein the composition is administered by direct administration to the pancreas, intrapancreatic administration, intrabiliary (IBD) administration, or endoscopic retrograde cholangiopancreatography (ERCP).

[0041] Embodiment 22. A composition for use according to any one of embodiments 14 to 21, wherein the AAV virion of any one of embodiments 11 to 13 is formulated in a suitable carrier.

[0042] Embodiment 23. A composition for use according to any one of embodiments 14 to 22, wherein at least one of fasting blood glucose level, GTT level, C-peptide level, or A1C level in the subject is reduced at least 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months after administration of the composition, compared to the level before administration.

[0043] Embodiment 24. A composition for use according to any one of embodiments 14 to 23, wherein at least one of fasting blood glucose level, GTT level, C-peptide level, or A1C level in the subject is in a pre-diabetic state at least 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months after administration of the composition.

[0044] Embodiment 25. A composition for use according to any one of embodiments 14 to 24, wherein the fasting blood glucose level is 100 to 125 mg / dL, or less than 100 mg / dL, the glucose tolerance test (GTT) level is 140 to 199 mg / dL, the C-peptide level is about 0.5 ng / mL to about 2.0 ng / mL, or the A1C level is 5.7 to 6.5%.

[0045] Embodiment 26. A composition for use according to any one of embodiments 14 to 25, wherein the subject is a human.

[0046] Embodiment 27. A method for increasing insulin production in a subject in need thereof, comprising administering a therapeutically effective amount of an AAV virion described in any one of embodiments 11 to 13, such that a nucleotide sequence encoding hPax4 is expressed in pancreatic islet cells.

[0047] Embodiment 28. A method for increasing the number of beta cells in a subject in need thereof, comprising the composition comprising a therapeutically effective amount of an AAV virion described in any one of embodiments 11 to 13, such that a nucleotide sequence encoding hPax4 is expressed in pancreatic islet cells.

[0048] Embodiment 29. A method for treating, ameliorating, or alleviating a symptom of diabetes in a subject suffering from diabetes, comprising administering a therapeutically effective amount of an AAV virion described in any one of embodiments 11 to 13 such that a nucleotide sequence encoding hPax4 is expressed in pancreatic islet cells.

[0049] Embodiment 30 The method of embodiment 29, wherein the diabetes is type 1 diabetes.

[0050] Embodiment 31 The method of embodiment 29, wherein the diabetes is type 2 diabetes.

[0051] Embodiment 32. A method for reducing the risk of developing diabetes or delaying the onset of diabetes in a subject positive for islet-associated autoantibodies, comprising administering a therapeutically effective amount of an AAV virion described in any one of embodiments 11 to 13 such that a nucleotide sequence encoding hPax4 is expressed in pancreatic islet cells.

[0052] Embodiment 33. The method of any one of embodiments 27-32, wherein the administration is by systemic administration, intravenous administration, intramuscular administration, intraperitoneal administration, or subcutaneous administration.

[0053] Embodiment 34. The method of any one of embodiments 27-32, wherein the administration is direct administration to the pancreas, intrapancreatic administration, intrabiliary (IBD) administration, or administration by endoscopic retrograde cholangiopancreatography (ERCP).

[0054] Embodiment 35. The method of any one of embodiments 27 to 34, wherein the AAV virion of any one of embodiments 11 to 13 is formulated in a suitable carrier.

[0055] Embodiment 36. The method of any one of embodiments 27 to 35, wherein at least one of fasting plasma glucose, GTT, C-peptide, or A1C levels in the subject is reduced at least 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months after administration of the composition, compared to the level before administration.

[0056] Embodiment 37. The method of any one of embodiments 27 to 36, wherein at least one of fasting plasma glucose, GTT, C-peptide, or A1C levels in the subject is in a prediabetic state at least 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months after administration of the composition.

[0057] Embodiment 38. The method according to any one of embodiments 27 to 37, wherein the fasting blood glucose level is 100 to 125 mg / dL, or less than 100 mg / dL, the glucose tolerance test (GTT) level is 140 to 199 mg / dL, the C-peptide level is about 0.5 ng / mL to about 2.0 ng / mL, or the A1C level is 5.7 to 6.5%.

[0058] Embodiment 39. The method of any one of embodiments 27 to 38, wherein the subject is a human.

[0059] Embodiment 40. An AAV vector plasmid comprising: 1) an origin of replication; and 2) a recombinant AAV vector according to any one of embodiments 1 to 10.

[0060] Embodiment 41. The AAV vector plasmid of embodiment 40, having the nucleotide sequence of SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.

[0061] Embodiment 42. A cell for AAV virion production, comprising an AAV vector plasmid according to embodiment 40 or 41, a second plasmid having nucleotide sequences encoding rep and cap, wherein the cap encodes VP1, VP2, and VP3, and the rep encodes rep78, rep68, rep52, and rep40, and a third plasmid comprising nucleotide sequences encoding viral helper factors required for AAV virion production.

[0062] Embodiment 43. The cell of embodiment 42, wherein the cap is an AAV6 cap or an AAV8 cap or an AAV9 cap.

[0063] Embodiment 44. A method of producing AAV virions, comprising culturing a host cell containing the AAV vector plasmid of embodiment 40 or 41, a second plasmid encoding a cap gene and a rep gene, wherein the cap encodes VP1, VP2, and VP3, and the rep encodes rep78, rep68, rep52, and rep40, and a third plasmid encoding adenovirus helper factors under conditions sufficient to produce the AAV virions.

[0064] Embodiment 45. A method for producing an AAV virion according to embodiment 44, further comprising isolating the AAV virion. [Brief description of the drawings]

[0065] [Figure 1] 1 shows a map of the plasmid pscAAV-CAG-hPax4-KanR (SEQ ID NO: 16), which contains the 5'AAV2 inverted terminal repeat (ITR) (SEQ ID NO: 8) and 3'scITR (SEQ ID NO: 9) flanking an expression cassette containing the CAG promoter (SEQ ID NO: 4), a nucleic acid encoding human Pax4 (hPax4) (SEQ ID NO: 2), and the SV40 poly(A) signal sequence (SEQ ID NO: 7). The plasmid further contains the ampicillin resistance gene (AmpR) promoter controlling expression of the kanamycin resistance gene sequence (KanR), the CMV origin of replication (ori), and the catabolite activator protein (CAP) binding site, the lactose (lac) promoter controlling the lac operator, and the M13 reverse primer region. [Diagram 2]1 shows a map of the plasmid pscAAV-MGP-hPax4-KanR (SEQ ID NO:17), which contains the 5'AAV2 inverted terminal repeat (ITR) (SEQ ID NO:8) and 3'scITR (SEQ ID NO:9) flanked by an expression cassette containing the mouse glucagon promoter (SEQ ID NO:5), a nucleic acid encoding human Pax4 (hPax4) (SEQ ID NO:2), and the SV40 poly(A) signal sequence (SEQ ID NO:7). The plasmid further contains the ampicillin resistance gene (AmpR) promoter controlling expression of the kanamycin resistance gene sequence (KanR), a CMV origin of replication (ori), and a catabolite activator protein (CAP) binding site, the lactose (lac) promoter controlling the lac operator, and an M13 reverse primer region. [Diagram 3] 1 shows a map of the plasmid pscAAV-RIP-hPax4-KanR (SEQ ID NO: 18), which contains the 5'AAV2 inverted terminal repeat (ITR) (SEQ ID NO: 8) and 3'scITR (SEQ ID NO: 9) flanking an expression cassette containing the rat insulin promoter (SEQ ID NO: 6), a nucleic acid encoding human Pax4 (hPax4) (SEQ ID NO: 2), and the SV40 poly(A) signal sequence (SEQ ID NO: 7). The plasmid further contains the ampicillin resistance gene (AmpR) promoter controlling expression of the kanamycin resistance gene sequence (KanR), a CMV origin of replication (ori), and a catabolite activator protein (CAP) binding site, the lactose (lac) promoter controlling the lac operator, and an M13 reverse primer region. [Figure 4] Graph showing body weight (grams) over several days following administration of STZ-treated mouse models and normal (wild-type) controls not treated with STZ or vector in each group of AAV vector or PBS control, as indicated in the figure legend. [Diagram 5] Non-fasting blood glucose (BG) (mg / dL) is shown. The average BG for several days following administration of vector or PBS control for all STZ-treated mice in each treatment group (treated with vector or control PBS as indicated in the figure legend) and normal controls not treated with STZ or vector. [Figure 6]As indicated in the figure legend, non-fasting BG (mg / dL) is shown only for STZ-treated mice that received vector or PBS control one week after the last STZ dose (i.e., STZ mice with delayed progression to T1D) compared to normal mice not treated with STZ or vector. The average BG of these mice over the study period is shown. Mice treated with AAV9.CAG-Pax4 and AAV9.MGP-Pax4 had significantly lower non-fasting BG than control AAV9.CAG-GFP treated mice (assessed by 2-way ANOVA with p=0.0085 and p=0.0327, respectively). AAV9.CAG-Pax4 also showed significantly lower BG than the PBS control group (p=0.0259). [Figure 7] Glucose tolerance test (GTT) (mg / dL blood glucose) of STZ-treated mice administered vector and control PBS one week after the last STZ dose compared to normal mice not treated with STZ or vector. After overnight fasting, mice were injected with 1 g / Kg body weight of glucose and BG of mice was measured before glucose injection and 15, 30, 45, 60, 90, and 120 minutes after glucose injection. (A) GTT data. (B) Area under the curve (AUC) analysis of GTT data. Data are expressed as mean ± SEM. *: p<0.05, **: p<0.01. [Figure 8] Glucose-stimulated insulin secretion (GSIS) assays of test article and control treated mice 1 week after the last STZ administration. Data are presented as mean ± SEM. Panels A and B show the same data as bar graphs or line graphs, respectively. [Figure 9] Blood hormone concentrations of all STZ-treated mice and normal controls were analyzed at the end of the animal study (3 months after vector administration): A. Insulin (ng / ml), B. Glucagon (ng / ml), CC peptide (pM). [Figure 10]Blood hormone concentrations were analyzed at the end of the animal study in STZ-treated mice that received vector or PBS control 1 week after the last STZ dose (i.e., STZ mice with slower progression to T1D) and in normal untreated controls only. A. Insulin, B. Glucagon, CC peptide. *: p<0.05, **: p<0.01. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] definition When interpreting the detailed description and the claims, "comprising," "consisting of," and "consisting essentially of" should be given their ordinary meaning. For purposes of this disclosure, the term "comprising" contemplates and supports embodiments in which the term "comprising" is replaced with the term "consisting of" and / or the term "consisting essentially of." For example, when "comprising A, B, or C" is recited, the alternatives "is A, B, or C," "consisting of A, B, and C," "consisting essentially of A, B, or C," or equivalents thereof are contemplated and support embodiments including these alternatives.

[0067] Additionally, statements of "or" contemplate and support "one or more," "one or a combination thereof," or "and / or." For example, "A, B, or C" contemplates and supports embodiments that include A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, and A, B, and C in combination. Additionally, in statements of "closed" language (e.g., consisting of) and statements of "open" language (e.g., comprising) enumerations such as "A, B, or C" also contemplate one or a combination of the enumeration, unless otherwise indicated. For example, "consisting of A, B, or C" contemplates and supports embodiments that include A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, and A, B, and C in combination. The statement "and / or" contemplates and supports all combinations of the enumeration (i.e., "A, B, and / or C" contemplates "A, B, and C") as well as "one or more" or "one or a combination thereof." For example, "A, B, and C" contemplates A alone, B alone, C alone, A in combination with B and C, A in combination with B, A in combination with C, and B in combination with C.

[0068] For example, a statement listing alternatives with "and," such as "selected from the group consisting of," contemplates and supports the listed combination unless expressly stated. For example, "selected from the group consisting of A, B, and C" contemplates and supports "selected from the group consisting of A, B, C, and combinations thereof," and is in the same scope as "at least one selected from the group consisting of A, B, and C," or the "group" is understood to include A alone, B alone, C alone, the combination of A and B, the combination of A and C, the combination of B and C, and the combination of A, B, and C.

[0069] Furthermore, the recitation of an element in an embodiment also contemplates and supports the explicit exclusion of that element from the embodiment. For example, "comprising A, B, or C" supports embodiments that include A or B, but specifically exclude C.

[0070] As used herein, the articles "a" and "an" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. As an example, "an element" means one element or more than one element. For example, "comprising an A, a B, or a C" contemplates and supports embodiments including two or more A's, two or more B's, and two or more C's.

[0071] Unless otherwise defined, all technical and scientific terms have the same meaning as commonly understood by those skilled in the art to which the embodiments belong. Although preferred materials and methods are described, it is understood that any methods and materials similar or equivalent to those described can be used in the practice of the embodiments. The terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. In describing and claiming the present invention, the following terms are used.

[0072] The term "about" as used herein when referring to a measurable value, such as an amount, temporal duration, and the like, is intended to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as such variations are appropriate to practice the embodiments.

[0073] The term "cell line" as used herein refers to a population of cells that have the ability to grow and divide continuously or over a long period of time in vitro. In many cases, a cell line is a clonal population derived from a single progenitor cell. Furthermore, karyotypic changes may occur naturally or be induced during storage or transfer of such a clonal population. Thus, cells derived from the cell line referred to may not be strictly identical to their ancestor cells or cultures, and the cell line referred to includes such variants.

[0074] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0075] The term "host cell" as used herein is any cell that carries or can carry a substance of interest. In many cases, the host cell can be a mammalian cell (e.g., a non-human primate, rodent, or human cell). In some embodiments, the host cell can be a mammalian cell, a yeast cell, a bacterial cell, an insect cell, a plant cell, or a fungal cell. The host cell can be used as a recipient for AAV helper constructs, AAV plasmids encoding recombinant AAV genomes containing transgenes, accessory function vectors, or other transfer DNA involved in the production of recombinant AAV (rAAV) virions. This term includes the progeny of the original transfected cell. Thus, "host cell" as used herein can refer to a cell transfected with an exogenous DNA sequence. It is understood that the progeny of a single parent cell may not necessarily be completely identical to the original parent in morphology or in genome or total DNA complement due to natural, accidental, or deliberate mutations.

[0076] "Identity" as used herein refers to the subunit sequence identity between two polymer molecules, particularly between two amino acid molecules, such as between two polypeptide molecules or between two nucleic acid molecules (e.g., polynucleotides). If two amino acid sequences have the same residue at the same position, e.g., if a position is occupied by arginine in each of the two polypeptide molecules, they are identical at that position. The identity or the degree to which two amino acid sequences have the same residue at the same position in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions. For example, if half of the positions in the two sequences (e.g., 5 positions in a polymer that is 10 amino acids in length) are identical, the two sequences have 50% identity. If 90% of the positions (e.g., 9 out of 10 positions) are matched or identical, the two amino acid sequences have 90% identity. In the case of an insertion or deletion, it is understood that identity is readjusted after the insertion or deletion to what was considered to be non-identical at the time of the insertion or deletion.

[0077] By "substantially identical" is meant that a polypeptide or nucleic acid molecule exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Preferably, such sequences have at least 60%, more preferably 80% or 85%, and more preferably 90%, 95%, or even 99% identity at the amino acid level or at the nucleic acid level to the sequence used as a comparison example.

[0078] The term "modified" as used herein means that the state or structure of a molecule or cell of the invention has been altered. Molecules can be modified in many ways, including chemical, structural, and functional ways. Cells can be modified by the introduction of nucleic acids.

[0079] The term "modulate" as used herein means to mediate a detectable increase or decrease in the level of a response in a subject compared to the level of the response in the subject in the absence of a therapeutic agent or compound and / or compared to the level of the response in an otherwise identical subject not receiving treatment. The term encompasses perturbing and / or affecting a natural signal or response in a subject, preferably a human, thereby mediating a beneficial therapeutic response.

[0080] "Nucleic acid" as used herein is interchangeable with "polynucleotide" or "specific sequence of nucleotides". These terms refer to a discrete sequence that performs a specific function in a cell, directly or indirectly. The function includes encoding a sequence of a gene that is transcribed into mRNA and translated into a protein, and controlling said transcription (i.e., by a promoter) and / or translation (i.e., by a microRNA). Nucleic acids essentially have a sequence. Thus, "nucleic acid comprising SEQ ID NO:X" can be used to contemplate and support "nucleic acid having the sequence of SEQ ID NO:X". In recombinant molecular biology, it is possible to combine discrete nucleic acids. In some embodiments, a nucleic acid encoding a protein can be linked to a promoter (which is a nucleic acid) and a cis-acting element of a viral vector (i.e., an inverted terminal repeat (ITR) that is also a nucleic acid). For convenience, "nucleic acid" can be used to refer to a separate element within a larger nucleic acid, which can also be referred to as a "polynucleotide", "expression region" (i.e., a polynucleotide comprising a promoter and a nucleic acid encoding a protein), or a "vector" (see definition below).

[0081] "Encoding" refers to the inherent property of a nucleic acid to serve as a template for the direct (i.e., sense strand) or indirect (i.e., antisense strand) synthesis of a peptide, polypeptide, protein, or another nucleic acid (i.e., rRNA, tRNA, microRNA). A nucleic acid can "encode" whether it is a sense strand, an antisense strand, or a double-stranded segment. The sense strand directly encodes the rRNA, tRNA, microRNA, or mRNA. The mRNA then serves as a template for translation of the peptide, polypeptide, or protein. The antisense strand is generally considered to be the reverse complementary sequence, and is sometimes referred to in the art as the "non-coding" strand (although for present purposes "non-coding" is a misnomer, since the non-coding strand still "encodes" genetic information by serving as a template for the polymerization of new sense strands, thereby perpetuating that genetic information during semi-conservative replication). For semi-conservative replication, the two single strands in a double-stranded nucleic acid are separated, and a new strand is polymerized from the information from each single-stranded nucleic acid (i.e., the single-stranded template), whether one single-stranded template is the sense strand (e.g., used to transcribe an mRNA, thereby translating or encoding a protein, or directly) or the antisense strand. By perpetuating the genetic information, the antisense strand still encodes the genetic information, for example, for a protein. Thus, a "nucleic acid encoding X" includes the sense and antisense sequences or strands, whether X is a peptide, polypeptide, or protein, or whether X is a sequence encoding rRNA, tRNA, microRNA, antisense RNA, etc.

[0082] Furthermore, since nucleic acids can be synthesized by natural or artificial processes (e.g., recombinant biology, molecular biology), such as transcription, reverse transcription, and replication, a "nucleic acid encoding X" includes RNA, DNA, and combinations thereof.

[0083] Thus, the described nucleic acid sequences contemplate and support their complementary, reverse complement, and double-stranded forms. That is, a "nucleic acid comprising SEQ ID NO:X" should be understood to contemplate and support a "nucleic acid having the reverse complement of SEQ ID NO:X" or, using nomenclature with a prime symbol such as "'", a "nucleic acid comprising SEQ ID NO:X'", unless otherwise indicated. For example, a "nucleic acid comprising SEQ ID NO:X", where SEQ ID NO:X is 5'-ATGCC-3', contemplates and supports the reverse complement of SEQ ID NO:X, specifically 5'-GGCAT-3.

[0084] As noted above, the nucleic acid sequences described contemplate and support conversion between their RNA and DNA forms. For example, SEQ ID NO:X is "5'-ATGCC-3'" and contemplates and supports 5'-AUGCC-3', as well as its reverse complement, 5'-GGCAU-3.

[0085] With respect to AAV vectors or AAV virions, the incorporation of reverse complementary sequences and double-stranded segments into the definition of "nucleic acid" above, and the use of the term "encoding" to include sense and antisense strands as above, is intended to incorporate the means by which an AAV vector can introduce an exogenous nucleic acid sequence encoding a nucleic acid or protein into a cell. In some embodiments, it is further intended to incorporate the process by which such incorporation results in expression of such a nucleic acid (i.e., miRNA or antisense RNA) or protein (i.e., Pax4).

[0086] For example, the nucleic acid coding for a protein and the AAV vector containing the nucleic acid coding for the protein.When a typical AAV vector containing one sense strand or one antisense strand of the nucleic acid coding for the protein enters a cell, the inverted terminal repeat (ITR) primes the synthesis of the sequence that is reverse-complementary to the sense strand or antisense strand of the nucleic acid coding for the protein.Therefore, regardless of whether the sense or antisense type is introduced into the cell first, polymerization forms a double-stranded DNA segment that contains the sense strand and the antisense strand.In this regard, the entire nucleic acid, including the ITR and the sense and antisense nucleic acid coding for the protein, can be a single-stranded DNA that loops around itself to form a double-stranded segment, and the base pairs of the sense and antisense nucleic acid coding for the protein are aligned.

[0087] This segment of double-stranded DNA achieves transcription of mRNA and translation of protein from the sense strand of said DNA, regardless of whether the AAV vector contained only the sense strand or only the antisense strand when it first entered the cell. In this regard, an "AAV vector comprising a nucleic acid encoding protein X" includes, contemplates, and supports embodiments in which the nucleic acid is a sense strand encoding protein X, an antisense strand encoding protein X, a double-stranded nucleic acid encoding protein X, and a single-stranded nucleic acid comprising a sense strand and an antisense strand, where the sense strand and the antisense strand form a segment of double-stranded nucleic acid.

[0088] The phrase "operably linked" refers to the functional connection between a regulatory sequence and a heterologous nucleic acid sequence, resulting in the expression of the linked target. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.

[0089] The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery necessary to initiate specific transcription of a polynucleotide sequence. In some instances, this sequence may be a core promoter, and in other instances, this sequence may also include an enhancer alone or may be an enhancer alone and / or may include other regulatory elements required for expression of the gene product.

[0090] In certain instances, a promoter may contain enhancer elements, exons, and introns from one or a variety of viruses and animals. Thus, the term "promoter" should be understood to be not limited to non-expressed sequences, does not exclude non-expressed sequences that are between expressed sequences (i.e., introns), and does not exclude only enhancers, so long as the combination of sequences used to construct the promoter is capable of initiating specific transcription of a polynucleotide sequence.

[0091] A "constitutive" promoter is a nucleotide sequence that, when operably linked with a polynucleotide encoding or specific for a gene product, allows the gene product to be produced in a cell under most or all physiological conditions of the cell, without the need to add exogenous factors or introduce a different phenotype into the cell. This constitutive promoter can be cell-specific, as long as it is produced in a specific or target cell under most or all physiological conditions of the cell. The CAG promoter is an example of a constitutive promoter in a wide range of target cell types.

[0092] As used herein, the term "recombinant cell" refers to a cell into which an exogenous DNA segment has been introduced, such as a DNA segment that results in the transcription of a biologically active polypeptide or the production of a biologically active nucleic acid, such as RNA.

[0093] "Target gene" refers to a nucleic acid that encodes a target protein that is expressed in a target cell when a vector carrying the target gene enters the cell. Target genes include naturally occurring polymorphisms (i.e., variants) and artificial modifications to wild-type genes, so long as the target protein is still expressed. Examples of such artificial modifications include codon optimization.

[0094] "Target protein" refers to an artificial or naturally occurring protein of interest that is introduced into a host cell by a vector. In some embodiments, the target protein encoded by the genome of the host cell is non-functional due to a polymorphism in the gene sequence that causes mistranscription, missense, or mistranslation of a portion of the gene, resulting in reduced or no target protein being produced, or a non-functional target protein being produced (i.e., the polymorphism causes a premature stop codon), or attenuated activity of the target protein encoded by and expressed from the genome of interest.

[0095] In some embodiments, the target protein comprises mammalian Pax4, and in one embodiment, the target protein comprises human Pax4. It is understood and contemplated that "Pax4" encompasses naturally occurring forms (i.e., human Pax4 and other mammalian Pax4, such as mouse and rat Pax4) and non-naturally occurring Pax4 (i.e., amino acid additions, deletions, or substitutions of Pax4 that increase or decrease activity compared to naturally occurring Pax4), so long as the protein referred to as Pax4 has at least the activity, when expressed in an alpha cell, to transdifferentiate that cell into a beta cell. In some embodiments, the non-native Pax4 has at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or 200% or less of the activity of the corresponding native Pax4, where "corresponding" contemplates and supports additions, deletions, or substitutions made. In some embodiments, Pax4 is human Pax4, and in some embodiments, Pax4 has the amino acid sequence of SEQ ID NO: 1. In alternative embodiments, Pax4 has an amino acid sequence having at least 99%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO: 1 and has Pax4 activity. In other embodiments, Pax4 is encoded by a nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence encoding hPAX4 having at least 99%, 95%, 90%, 85%, or 80% identity to SEQ ID NO: 2 and having the amino acid sequence of SEQ ID NO: 1, or hPAX4 having an amino acid sequence having at least 99%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO: 1 and having PAX4 activity.

[0096] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a gene-encoding or gene-specific polynucleotide, causes a gene product to be produced in a cell only if the cell is substantially a cell of the tissue type corresponding to the promoter. The mammalian insulin and glucagon promoters are examples of tissue-specific promoters for expression in pancreatic islet cells.

[0097] A "vector" is a nucleic acid capable of delivering a target gene into a cell, and includes not only an expression region (i.e., a promoter and a nucleic acid encoding a protein or even a nucleic acid), but also several cis-acting genetic elements that provide for expression within the cell, replication within the cell, or a combination thereof, for packaging into virions.

[0098] As an example, an inverted terminal repeat (ITR) from an adeno-associated virus (AAV) is bound to a nucleic acid encoding a target protein to constitute a vector, since the ITR provides the nucleic acid encoding the target protein to be packaged in the AAV virion. The ITR may also provide another cis-acting function to express the nucleic acid encoding the target protein in the host cell when the vector enters the host cell. Such cis-acting functions of the ITR include assisting in the formation of concatemers for genome insertion; initiating second strand formation in the case of single-stranded (ss)AAV (ssAAV) vectors; or initiating replication and transcription in the case of ssAAV and self-complementary (sc)AAV (scAAV) vectors. In this regard, AAV ITRs may be characterized based on the nucleic acid sequences that provide such cis-acting functions from the serotype of AAV. That is, an ITR isolated from the AAV2 serotype may be known as an AAV2 ITR, even though the ITRs do not generally contribute to the serotype of AAV.

[0099] Additionally, the scAAV ITRs (e.g., SEQ ID NO:9) are formed by mutating or altering the wild-type AAV2 ITRs (e.g., SEQ ID NO:8, the 5' flanking ITR), particularly the terminal resolution site (trs) in the D sequence that is responsible for signaling packaging (the packaging sequence), but it is understood that the scAAV ITRs provide functions that generate the AAV vector, including the expression region and its reverse complement, before it is packaged into the AAV virion to generate the scAAV, functions not provided by the wild-type AAV ITRs. In some embodiments, the scAAV vector is then packaged into a recombinant scAAV virion.

[0100] The scAAV vectors may exhibit at least 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, or 140-fold or less efficient transduction than a corresponding vector that contains wild-type AAV2 ITRs and does not contain the complement of the expressed region. Without wishing to be bound by theory, it is believed that synthesis of the double-stranded segment in the ssAAV vector by the host cell is the rate-limiting step in ssAAV vector transduction, and that by providing the expressed region and its reverse complement, the scAAV ITRs (and vectors containing the scAAV ITRs) increase the efficiency of transduction as described above.

[0101] As an example, a plasmid may contain an origin of replication (eg, an ori from cytomegalovirus) that allows replication of a target gene within a cell, and thus, such a plasmid is a vector.

[0102] "Expression vector" refers to a vector that includes an expression region. The expression region includes a recombinant polynucleotide that includes a nucleic acid that controls expression (i.e., a promoter) and a coding nucleic acid. The coding nucleic acid includes a nucleic acid that encodes a protein. Generally, the promoter is operably linked to the nucleic acid encoding the target protein in a manner that can promote expression of the protein when the vector enters a host cell. In some embodiments, the promoter can be operably linked by ensuring that there are no codon misalignments.

[0103] Ranges: Throughout this disclosure, various aspects of the invention can be presented in a range format. The description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, any description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, a description of a range such as 1-6 should be considered to include the specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numerical values ​​within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6.

[0104] The nucleotide and amino acid sequences provided herein are shown in Table 1.

[0105] AAV vectors In some embodiments, a recombinant adeno-associated virus (AAV) vector is provided that includes an expression region or expression cassette and at least two inverted terminal repeats (ITRs) flanking the expression cassette, the expression cassette includes a promoter and a nucleic acid encoding Pax4, including human Pax4, the promoter is operably linked to express the nucleic acid encoding Pax4, a polyadenylation signal is further operably linked to the Pax4 coding sequence, and the at least two ITRs flank the expression region or expression cassette. The expression cassette may further include other regulatory sequences, such as a Kozak sequence, an enhancer, an intron sequence, a WPRE sequence, etc.

[0106] The Pax4 in some embodiments encoded by the vector, and in some embodiments human Pax4, has an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or less identity to the amino acid sequence of SEQ ID NO: 1, or that contains 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within the amino acid sequence of SEQ ID NO: 1, while maintaining Pax4 activity. In some embodiments, Pax4 comprises or consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid encoding Pax4 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or less identity to SEQ ID NO:2 or its reverse complementary sequence, or includes a nucleic acid containing 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within SEQ ID NO:2 or its reverse complementary sequence, and encodes a human Pax4 having the amino acid sequence of SEQ ID NO:1 or a variant that maintains Pax4 activity. In some embodiments, the nucleic acid encoding Pax4 has or consists of SEQ ID NO:2 or its reverse complementary sequence. In some embodiments, the nucleic acid has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to SEQ ID NO: 2 and encodes a human Pax4 protein having the amino acid sequence of SEQ ID NO: 1, or a variant having human Pax4 activity. In some embodiments, a Kozak sequence is introduced 5' to the Pax4 coding sequence to facilitate translation of the Pax4 protein from the expression cassette.The Kozak sequence may be SEQ ID NO:3, 5'GCCACC3'.

[0107] In some embodiments, the ITR, or at least two ITRs, comprise AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, or AAV9 ITR, and in some embodiments may be scAAV ITR. In some embodiments, the ITR, or at least two ITRs, comprise AAV2 ITR or scAAV ITR. In some embodiments, the ITR, or at least two ITRs, comprise AAV2 ITR and scAAV ITR, and the 5'ITR is AAV2 ITR and the 3'ITR is scAAV ITR, or the 5'ITR is scAAV ITR and the 3'ITR is AAV2 ITR. In some embodiments, the scAAV ITR is an AAV2 ITR that lacks at least one functional terminal resolution site (trs) in the D sequence. In some embodiments, the scAAV ITR lacks at least one functional trs. In some embodiments, the scAAV ITRs have at least one substitution, addition, or deletion in at least one trs, and the at least one substitution, addition, or deletion confers dysfunction to at least one trs. In some embodiments, the AAV ITRs have at least one D sequence deleted. In some embodiments, the deleted D-sequence is at the 3' end of the ITR (ssD[-]). In some embodiments, the deleted D-sequence is at the 5' end of the ITR (ssD[+]). In some embodiments, the ssD[-] sequence has at least one substitution, deletion, or addition that prevents binding of 52-kDa-FK506 binding protein (FKBP52). In some embodiments, the AAV ITRs have a D-sequence substituted into a transcription factor binding site. In some embodiments, the transcription factor binding site has an S sequence. In some embodiments, the S sequence includes a Foxd3 binding site or an NF-μE1 binding site. In some embodiments, the transcription factor binding site or S sequence includes GATA-1 and GATA-2 binding sites.In some embodiments, an ITR, or at least two ITRs, have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or less identity to SEQ ID NO:8 or its reverse complementary sequence inclusive, or have a nucleic acid that contains 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or a combination thereof within SEQ ID NO:8 or its reverse complementary sequence inclusive. In some embodiments, the ITR, or at least two ITRs, have a nucleic acid that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or less identity to SEQ ID NO:8 or its reverse complementary sequence inclusive, or that contains 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within SEQ ID NO:8 or its reverse complementary sequence inclusive, SEQ ID NO:25. In some embodiments, the ITR, or at least two ITRs, have SEQ ID NO:8 (5'ITR) and / or SEQ ID NO:25 (3'ITR), or its reverse complementary sequence. In some embodiments, the ITR, or at least two ITRs, have SEQ ID NO:8 (5'ITR) and / or SEQ ID NO:9 (3'scITR), or reverse complements thereof. In the case of a self-complementary vector, one of the ITRs has a mutated sequence, e.g., the 3'ITR has the mutated sequence of SEQ ID NO:9, and the ITR at the 5' end of the expression cassette is an unmodified ITR, e.g., the AAV2 ITR, having the nucleotide sequence of SEQ ID NO:8.

[0108] In some embodiments, the sequence encoding Pax4 is operably linked to a constitutive promoter. In some embodiments, the promoter is a CAG promoter. The CAG promoter is a composite synthetic promoter that includes a CMV early enhancer element, a chicken β-actin promoter, a chicken β-actin promoter, and the first exon and first intron of the chicken β-actin gene and a splice acceptor of the rabbit β-globin gene. See, for example, Miyazaki et al, Gene 79:269-277 (1989) and Niwa et al, Gene 108:193-199 (1991). In some embodiments, the CAG "promoter" can have the nucleotide sequence of SEQ ID NO: 4 or at least a 200, 300, 400, 500, or 600 nucleotide fragment (or a reverse complement thereof, as appropriate) that exhibits promoter activity to promote expression of a target gene in an appropriate tissue. In some embodiments, the promoter has a nucleic acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or less identity to SEQ ID NO:4 or its reverse complement, or that contains 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within SEQ ID NO:4 or its reverse complement to promote Pax4 gene expression in appropriate tissues.In some alternative embodiments, the promoter comprises or consists of a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, a simian virus (SV40) promoter, a dihydrofolate reductase promoter, a β-actin promoter, a phosphoglycerol kinase (PGK) promoter, a P5 promoter, a Ubc promoter, a tetracycline response element promoter, a UAS promoter, an Ac5 promoter, a polyhedrin promoter, a calmodulin-dependent protein kinase II-α (CaMKIIα) promoter, a Pax4 promoter, an H1 promoter, a U6 promoter, or an alpha-1-antitrypsin promoter. In some embodiments, the β-actin promoter is a chicken β-actin ("CBA") promoter or a human β-actin promoter.

[0109] In some embodiments, the Pax4 coding sequence is operably linked to a pancreatic islet cell tissue-specific promoter, including an alpha cell or beta cell specific promoter. Such promoters include a human or rodent (such as mouse or rat) insulin promoter, or a human or rodent (such as mouse or rat) glucagon promoter. In some embodiments, the promoter is a mouse glucagon promoter. In one embodiment, the promoter can be at least a 200, 300, or 400 nucleotide fragment having or consisting of the nucleotide sequence of SEQ ID NO:5, or a reverse complement thereof, as appropriate, that exhibits promoter activity to promote expression of a target gene in an appropriate tissue, such as pancreatic islet cells, including alpha cells and beta cells. In some embodiments, the promoter has a nucleic acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or less identity to SEQ ID NO:5 or its reverse complement, or that contains 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within SEQ ID NO:5 or its reverse complement to promote Pax4 gene expression in appropriate tissues, such as pancreatic islet cells.In some embodiments, the promoter is the rat insulin promoter (see Ullrich et al. "Rat insulin genes: construction of plasmids containing the coding sequences," Science, 196(4296):1313-1319(1977) (database Sequence ID: J00747.1 (BLAST)) and Dandoy-Dron et al. "Tissue-specific expression of the rat insulin 1 gene in vivo requires both the enhancer and promoter regions." Differentiation. 58(4):291-5(1995)).

[0110] In one embodiment, the promoter may be at least a 200, 300, or 400 nucleotide fragment having or consisting of the nucleotide sequence of SEQ ID NO:6 and exhibiting promoter activity that promotes expression of a target gene in an appropriate tissue (such as a pancreatic islet cell or its reverse complement, as appropriate). In some embodiments, the promoter has a nucleic acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or less identity to SEQ ID NO:6 or its reverse complement, or that contains 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within SEQ ID NO:6 or its reverse complement, and promotes Pax4 gene expression in appropriate tissues, such as pancreatic islet cells (including alpha cells and / or beta cells).

[0111] In some embodiments, the expression region is in an antisense (e.g., reverse complement) orientation or a sense orientation. In some embodiments, the vector comprises two or more expression regions. In some embodiments, the two or more expression regions comprise one in an antisense orientation and another in a sense orientation (e.g., as in scAAV).

[0112] In some embodiments, the expression region further comprises a nucleic acid encoding a polyadenylation (poly(A)) signal 3' of the target gene coding sequence, such that the expressed mRNA has a poly A tail. In some embodiments, the nucleic acid encoding the poly(A) signal comprises the bovine growth hormone (bGH) poly(A) tail signal or the simian virus 40 (SV40) poly(A) tail signal. In some embodiments, the polyA signal has the nucleotide sequence of SEQ ID NO:7 (SV40 polyA signal) (or its reverse complement), or the polyA signal has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or less identity to SEQ ID NO:7 or its reverse complement, or has 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof within SEQ ID NO:7 or its reverse complement to cause polyadenylation of the transcribed sequence.

[0113] In some embodiments, the recombinant AAV vector can be packaged into recombinant AAV virions.

[0114] Thus, an expression cassette is provided that can be incorporated into a plasmid used to generate an AAV vector or rAAV particle for gene replacement expression of a target gene. The expression cassette is flanked by AAV ITRs. In certain embodiments, the expression cassette can have elements arranged as follows: 5'AAV2 ITR-CAG promoter sequence-Kozak sequence-hPax4 coding sequence-SV40 polyA signal sequence-3'scAAV2 ITR. In some embodiments, the expression cassette has the nucleotide sequence of SEQ ID NO: 10 (without flanking ITR sequences) or SEQ ID NO: 13 (with flanking ITR sequences). In other embodiments, the gene expression cassette can have elements arranged as follows: 5'AAV2 ITR-MGP promoter sequence-Kozak sequence-hPax4 coding sequence-SV40 polyA signal sequence-3'scAAV2 ITR. In some embodiments, the expression cassette has the nucleotide sequence of SEQ ID NO: 11 (without flanking ITR sequences) or SEQ ID NO: 14 (with flanking ITR sequences). In certain embodiments, the expression cassette may have elements arranged as follows: 5'AAV2 ITR-RIP promoter sequence-Kozak sequence-hPax4 coding sequence-SV40 polyA signal sequence-3'scAAV2 ITR. In some embodiments, the expression cassette has the nucleotide sequence of SEQ ID NO: 12 (without the flanking ITR sequences) or SEQ ID NO: 15 (with the flanking ITR sequences).

[0115] In some embodiments, the scAAV vector is then packaged into recombinant AAV virions.

[0116] In some embodiments, the expressed region is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.10%, 99.11%, 99.12, 99.13, 99.14, 99.15, 99.16, 99.17, 99.18, 99.19, 99.20, 99.21, 99.22, 99.23, 99.24, 99.25, 99.26, 99.27, 99.28, 99.29, 99.30, 99.31, 99.32, 99.33, 99.34, 99.35, 99.36, 99.37, 99.38, 99.39 ... %, 99.7%, 99.8%, or 99.9% or less identity to, or containing 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof, within SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 (or SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15 with adjacent ITRs), or a reverse complement thereof, and an expression cassette for expressing hPax4 in appropriate human tissues. In some embodiments, the expression region or expression cassette has SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 (or SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15 with adjacent ITRs), or a reverse complement thereof.

[0117] AAV Vector Plasmids In some embodiments, AAV vector plasmids are provided that can be used to prepare recombinant AAV virions having a recombinant genome with a nucleotide sequence encoding a target gene operably linked to a regulatory element that facilitates expression in an appropriate tissue. The plasmids provided herein generally have an origin of replication and a selectable marker, allowing for replication of the plasmid and use in a host cell to generate recombinant AAV viral particles as described herein. Exemplary plasmids are shown in Figures 1-3. The plasmids provided herein include plasmids that contain an expression cassette as described herein. In particular, the AAV vector plasmid can contain an expression cassette having a nucleotide sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some embodiments, the AAV vector plasmid comprises a nucleic acid having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or less identity to SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, or containing 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or a combination thereof within SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. Plasmids provided include pscAAV-CAG-hPax4-KanR (Figure 1, SEQ ID NO: 16), pscAAV-MGP-hPax4-KanR (Figure 2, SEQ ID NO: 17), and pscAAV-RIP-hPax4-KanR (Figure 3, SEQ ID NO: 18).

[0118] In some embodiments, the AAV vector plasmid further comprises a bacterial expression region. In some embodiments, the bacterial expression region comprises a bacterial promoter and a nucleic acid encoding a bacterial selection region. In some embodiments, the nucleic acid encoding the bacterial selection region is operably linked to the bacterial promoter. In some embodiments, the nucleic acid encoding the bacterial selection region comprises a nucleic acid encoding an antibiotic resistance gene or protein. In some embodiments, the antibiotic resistance gene or protein comprises an ampicillin resistance gene (AmpR) or a kanamycin resistance gene sequence (kanR). In some embodiments, the bacterial promoter comprises an AmpR promoter or a KanR promoter. In some embodiments, the AAV vector plasmid further comprises an origin of replication. In some embodiments, the origin of replication comprises a CMV origin of replication (ori). In some embodiments, the AAV vector plasmid further comprises a eukaryotic expression region. In some embodiments, the eukaryotic expression region comprises a eukaryotic promoter and a nucleic acid encoding a eukaryotic selection region. In some embodiments, the nucleic acid encoding the eukaryotic selection region is operably linked to a eukaryotic promoter. In some embodiments, the eukaryotic promoter comprises a nucleic acid catabolite activator protein (CAP) binding site or a lactose (lac) promoter. In some embodiments, the eukaryotic selection region comprises a lac operator. In some embodiments, the plasmid comprises an M13 reverse primer region.

[0119] AAV virion In some embodiments, a recombinant AAV virion is provided.In some embodiments, the AAV virion comprises AAV capsid protein and a recombinant AAV vector having a nucleotide sequence encoding hPax4 operably linked to a regulatory element.In some embodiments, the recombinant AAV vector is a recombinant scAAV vector.In some embodiments, the recombinant AAV is a single-stranded DNA vector.In some embodiments, the AAV capsid protein encapsulates the recombinant AAV vector.

[0120] In some embodiments, the AAV capsid protein comprises VP1, VP2, and / or VP3 capsid protein. The capsid preferably has tropism for appropriate cells and tissues, such as pancreatic islet tissue. In some embodiments, the capsid protein comprises AAV1 capsid protein, AAV2 capsid protein, AAV3 capsid protein, AAV4 capsid protein, AAV5 capsid protein, AAV6 capsid protein, AAV7 capsid protein, AAV8 capsid protein, or AAV9 capsid protein. In some embodiments, the rAAV particle has an AAV6 capsid protein, for example, having an amino acid sequence of SEQ ID NO: 19. Alternatively, the capsid protein has an amino acid sequence with 99%, 98%, 95%, 90%, or 85% identity to the AAV6 capsid, and has AAV6 capsid protein tropism and transduction activity. The AAV6 capsid protein may be encoded by the nucleotide sequence of SEQ ID NO:20 (or having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:20 and encoding the AAV6 capsid protein). In some embodiments, the rAAV particles have an AAV8 capsid protein, for example, having the amino acid sequence of SEQ ID NO:23. Alternatively, the capsid protein has an amino acid sequence that is 99%, 98%, 95%, 90%, or 85% identical to the AAV8 capsid and has AAV8 capsid protein tropism and transduction activity. The AAV8 capsid protein may be encoded by the nucleotide sequence of SEQ ID NO:24 (or having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:24 and encoding the AAV8 capsid protein). In some embodiments, the rAAV particles have an AAV9 capsid protein, for example, having the amino acid sequence of SEQ ID NO:21. Alternatively, the capsid protein has an amino acid sequence that is 99%, 98%, 95%, 90%, or 85% identical to the AAV9 capsid and has AAV9 capsid protein tropism and transduction activity.The AAV9 capsid protein can be encoded by the nucleotide sequence of SEQ ID NO:22 (or having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:22 and encoding the AAV9 capsid protein).

[0121] In some embodiments, the isolated nucleic acids and / or rAAVs described herein can be modified and / or selected to enhance targeting of the isolated rAAV to target tissues (e.g., pancreatic islet cells). Non-limiting methods of modification and / or selection include AAV capsid serotypes (e.g., AAV6, AAV8, or AAV9), tissue-specific promoters, and / or targeting peptides. In some embodiments, the isolated nucleic acids and rAAVs disclosed herein can comprise AAV capsid serotypes with enhanced targeting to pancreatic islet cell tissues. In some embodiments, the isolated nucleic acids and rAAVs described herein can comprise tissue-specific promoters. In some embodiments, the isolated nucleic acids and rAAVs described herein can comprise AAV capsid serotypes and tissue-specific promoters with enhanced targeting to pancreatic islet cell tissues.

[0122] Methods for obtaining recombinant AAV with desired capsid proteins can be obtained, for example, from US Patent Publication No. 2003 / 0138772, which is incorporated herein by reference in its entirety. Typically, the methods involve culturing a host cell that contains a nucleic acid sequence encoding an AAV capsid protein or a fragment thereof, a functional rep gene, sufficient helper functions to allow packaging of the recombinant AAV vector into the AAV capsid protein, and a recombinant AAV vector plasmid that contains the AAV vector. Typically, the capsid protein is a structural protein encoded by the cap gene of AAV. In some embodiments, when the capsid protein includes VP1, VP2, and VP3, the VP1, VP2, and VP3 are transcribed from a single cap gene via alternative splicing. In some embodiments, the molecular weights of VP1, VP2, and VP3 are about 87 kDa, about 72 kDa, and about 62 kDa, respectively. In some embodiments, upon translation, the capsid protein forms a spherical 60-mer protein shell around the viral genome. In some embodiments, the capsid protein protects the viral genome, delivers the genome, and / or interacts with the host cell. In some embodiments, the capsid protein delivers the viral genome to the host in a tissue-specific manner.

[0123] In some embodiments, the components to be cultured in a host cell to package a recombinant AAV vector into an AAV capsid may be provided in trans to the host cell. Alternatively, any one or more of the required components (e.g., recombinant AAV vector, rep sequences, cap sequences, and / or helper functions) may be provided by a stable host cell engineered to contain one or more of the required components. In certain embodiments, a host cell is provided that contains a recombinant AAV construct or plasmid with a target gene sequence, a plasmid providing the AAV rep and cap gene sequences, and optionally a construct providing adenoviral helper proteins to produce recombinant viral particles.

[0124] In some embodiments, such stable host cells may contain the required component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters provided herein are provided in the discussion of regulatory elements suitable for use with transgenes herein. In some embodiments, the selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be made that is derived from 293 cells (containing El helper functions under the control of a constitutive promoter) but contains rep and / or cap proteins under the control of an inducible promoter. Still other stable host cells may be made by one of skill in the art.

[0125] The recombinant AAV vectors, rep sequences, cap sequences, and helper functions useful for producing the rAAV described herein can be delivered to the packaging host cell using any suitable genetic element (vector, e.g., plasmid). The selected genetic element can be delivered by any suitable method, including those described herein. The methods used to construct any of the components disclosed herein are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for producing rAAV virions are known, and the present disclosure does not limit the selection of a suitable method. See, e.g., K. Fisher et al, J. Virol., 70:520-532 (1993) and U.S. Patent No. 5,478,745.

[0126] In some embodiments, recombinant AAV can be produced using a triple transfection method (described in U.S. Pat. No. 6,001,650). Typically, recombinant AAV can be produced by transfecting a host cell with a recombinant AAV vector (including a transgene) packaged in an AAV particle, an AAV helper function vector, and an auxiliary function vector. The AAV helper function vector encodes "AAV helper function" sequences (i.e., rep and cap) that function in trans for efficient AAV replication and encapsidation with the cap gene encoding the capsid protein of the desired serotype, e.g., AAV9 capsid. In some embodiments, the AAV helper function vector can support efficient AAV vector production without producing any detectable wild-type AAV virions (i.e., AAV virions containing functional rep and cap genes). Non-limiting examples of vectors suitable for use in the present disclosure include pHLP19, described in U.S. Patent No. 6,001,650, and pRep6cap6 vector, described in U.S. Patent No. 6,156,303, both of which are incorporated herein by reference in their entirety. Accessory function vectors encode nucleotide sequences for non-AAV derived viral and / or cellular functions (i.e., "accessory functions") on which AAV depends for replication. Accessory functions encompass functions required for AAV replication, including, but not limited to, portions involved in transcription of AAV genes, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and activation of AAV capsid assembly. Viral-based accessory functions can be derived from any of the well-known helper viruses, such as adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus.

[0127] Cells Transfected host cells are disclosed herein. The term transfection refers to the uptake of foreign DNA by a cell, and a cell is transfected when foreign DNA is introduced through the cell membrane. Examples of transfection methods include Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more foreign nucleic acids, such as nucleotide integration vectors and other nucleic acid molecules, into a suitable host cell.

[0128] In one aspect, a cell is provided, which may be a mammalian host cell, e.g., a HEK293 cell or other suitable cell. In some embodiments, the cell comprises a second AAV plasmid (or repcap plasmid) and an AAV vector plasmid, which in some embodiments comprises a helper or accessory plasmid encoding adenoviral helper factors useful or necessary for production of AAV particles in the cell. In some embodiments, the second AAV plasmid comprises rep and cap. In some embodiments, cap encodes VP1, VP2, and VP3. In some embodiments, rep encodes rep78, rep68, rep52, and rep40. In some embodiments, the AAV vector plasmid comprises a recombinant AAV vector or a recombinant scAAV vector. In some embodiments, the cap is an AAV6 cap, AAV8 cap, or AAV9 cap, e.g., having an AAV6 capsid protein having the amino acid sequence of SEQ ID NO: 19, or encoded by the nucleotide sequence of SEQ ID NO: 20, or an AAV8 cap, e.g., having an AAV8 capsid protein having the amino acid sequence of SEQ ID NO: 23, or encoded by the nucleotide sequence of SEQ ID NO: 24, or an AAV9 capsid protein having the amino acid sequence of SEQ ID NO: 21, or encoded by the nucleotide sequence of SEQ ID NO: 22. In some embodiments, the AAV vector plasmid comprises an expression cassette of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, or may be pscAAV-CAG-hPax4-KanR (Figure 1, SEQ ID NO: 16), pscAAV-MGP-hPax4-KanR (Figure 2, SEQ ID NO: 17), or pscAAV-RIP-hPax4-KanR (Figure 3, SEQ ID NO: 18).

[0129] In another aspect, a method of producing AAV virions is provided. In some embodiments, the method includes transfecting at least one of a second plasmid and a vector plasmid or an AAV vector construct into a cell. In some embodiments, the vector plasmid or AAV vector construct includes a recombinant AAV vector or a recombinant scAAV vector. In some embodiments, the second plasmid includes cap and rep. In some embodiments, cap encodes VP1, VP2, and VP3. In some embodiments, rep encodes rep78, rep68, rep52, and rep40. In some embodiments, the vector plasmid or AAV vector construct includes an expression cassette having the nucleotide sequence of SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12 (not flanked by ITRs), or the nucleotide sequence of SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15 (flanked by ITRs). The plasmids used include pscAAV-CAG-hPax4-KanR (Figure 1, SEQ ID NO: 16), pscAAV-MGP-hPax4-KanR (Figure 2, SEQ ID NO: 17), and pscAAV-RIP-hPax4-KanR (Figure 3, SEQ ID NO: 18).

[0130] How to use Methods are provided for delivering Pax4 (including human Pax4) to a cell by introducing a Pax4 expression vector disclosed herein into the cell, resulting in expression of the Pax4 nucleotide sequence in the cell. Embodiments include administering to a subject a recombinant AAV vector that includes an expression cassette having a nucleotide sequence encoding Pax4 operably linked to a regulatory element that promotes expression of Pax4 in a cell of the subject. In some embodiments, the cell is a pancreatic cell, which includes a pancreatic islet cell, and further includes an alpha cell and / or a beta cell. In some embodiments, the regulatory element promotes Pax4 expression specifically in an alpha cell and / or a beta cell.

[0131] Without intending to be bound by a particular mechanism of action, expression of Pax4 in pancreatic islet cells can promote beta cell function and increase the number of beta cells in the pancreas by promoting beta cell proliferation and / or survival and / or transdifferentiation of alpha cells to beta cells. The beta cells exhibit glucose-responsive insulin secretion, which results in improved blood glucose levels and glucose tolerance, reducing the risk of sequelae of diabetic disease in a subject, or reducing the risk of developing diabetes in a subject who is pre-diabetic.

[0132] Thus, provided herein are methods of increasing insulin production in cells, including pancreatic islet cells, including β cells, in a subject in need of administration of an effective amount of an AAV virion by administering an effective amount of an AAV virion disclosed herein. In some embodiments, the disclosed methods result in a 20%, 50%, 100%, or 2-fold, 5-fold, or 10-fold increase in fasting insulin levels within 1 month, 2 months, 4 months, 6 months, 8 months, or 1 year of administration of the rAAV, as compared to the subject's insulin levels prior to administration under comparable conditions. Also provided are methods of increasing the number of β cells in the pancreas and / or increasing the proportion of β cells (including the ratio of β cells to α cells) in pancreatic islet cells in a subject in need of an increased proportion of β cells. The number of β cells can be increased 2-fold, 10-fold, 50-fold, 100-fold, 1000-fold, or more. In some embodiments, the methods promote transdifferentiation of α cells to β cells in the pancreas of a subject in need of promotion of transdifferentiation of α cells to β cells.

[0133] Thus, there is provided a method of treating, ameliorating symptoms, or slowing the progression of diabetes by administering to a subject in need thereof an effective amount of an AAV virion comprising a Pax4 transgene as disclosed herein. The method results in increased blood glucose levels (either fasting or in response to a standard glucose load), glucose tolerance, and / or insulin levels (fasting and / or in response to a glucose load) in the subject in need thereof within one month, two months, four months, six months, eight months, or one year of administration. Such methods include improvements in measures of blood glucose and / or glucose tolerance, C-peptide levels, A1C levels, and the like.

[0134] In some embodiments, the improvement in blood glucose levels, glucose tolerance test, and / or insulin secretion achieved by gene therapy described herein is compared to the individual subject prior to treatment with the AAV virions of the present disclosure. In some embodiments, treatment according to the present disclosure results in an indication of a pre-diabetic state or an indication of a near-pre-diabetic state within 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months after administration. In some embodiments, treatment according to the present disclosure results in a fasting blood glucose level of 100-125 mg / dL, or less than 100 mg / dL. In some embodiments, treatment according to the present disclosure results in a glucose tolerance test (GTT) level of 140-199 mg / dL, or less than 140 mg / dL. In some embodiments, treatment according to the present disclosure results in a C-peptide level of about 0.5 ng / mL to about 2.0 ng / mL, or about 0.17 nmol / L to about 0.83 nmol / L. In some embodiments, the improvement in A1C level results in an A1C level of 5.7-6.5%.

[0135] The methods disclosed herein are useful for treating, ameliorating, and / or alleviating symptoms of diabetes, including type 1 diabetes (T1D) or type 2 diabetes (T2D). In some embodiments, the methods include preventing disease progression, including reducing or delaying progression from prediabetes to diabetes, or worsening of T1D or T2D. In other embodiments, methods are provided for preventing or delaying progression from a prediabetic state to diabetes, including type 1 diabetes, in subjects with indicators of a prediabetic state, such as a predisposition to diabetes (including when a close family member has been diagnosed with type 1 diabetes), or in subjects with the presence of autoantibodies to pancreatic islet cells, including autoantibodies to GAD65, IA-2, and / or insulin. In other embodiments, methods are provided for preventing or delaying progression from a prediabetic state to diabetes, including type 2 diabetes, in subjects characterized by a fasting blood glucose level of 100-125 mg / dL, or a glucose tolerance test blood glucose level of 140-199 mg / dL, or an A1C level of 5.7%-6.4%.

[0136] The subject may be a human or a non-human mammal, including rodents, dogs, cats, horses, livestock, etc. In the case of a non-human subject, the Pax4 may be from the species to be treated, e.g., Pax4 from a mouse is used when treating a mouse.

[0137] In some embodiments, administration of an rAAV comprising an expression cassette encoding Pax4 as described herein reduces the incidence or severity of complications or sequelae of diabetes mellitus type 1 or type 2. In some embodiments, the method comprises administering to a subject in need of treatment a therapeutically effective amount of an AAV virion, including a pharmaceutical composition comprising the AAV virion, that alleviates or reduces the severity or progression of a diabetic complication, including, but not limited to, neuropathy, hypertension, kidney disease, cardiovascular disease, skin infections, hearing loss, or retinopathy.

[0138] In some embodiments, administration or treatment includes systemic administration, including intravenous, intraarterial, intramuscular, intraarticular, intraperitoneal, intradermal, subcutaneous, transdermal, or transmucosal administration. In some embodiments, rAAV is delivered directly to the pancreas, for example, by intraductal administration into the pancreas (also called intrabiliary (IBD) administration) or endoscopic retrograde cholangiopancreatography (ERCP).

[0139] In some embodiments, an effective amount of rAAV virions can be an amount sufficient to cause a therapeutic effect, such as an increase in insulin production or an improvement in indicators associated with T1D or T2D, such as blood glucose, C-peptide, or A1C levels. The effective amount will depend primarily on factors such as the subject's species, age, weight, health, and tissue to be targeted, and therefore may vary between animals and tissues. For example, an effective amount of rAAV virions can be about 10 6 ~10 16 genome copy number (e.g., 1 × 10 6 ~1×10 16 In the methods disclosed herein, a therapeutically effective dose can range from about 1 ml to about 100 ml of a solution containing 1×10 10 gc / kg~1×10 15 gc / kg, and the effective dose is 1×10 11 gc / kg, 1×10 12 gc / kg, 1×10 13 gc / kg, 1×10 14 gc / kg, 1×10 15 gc / kg (or genome copies per volume of pancreatic cells or islet cells, or other unit of measurement appropriate for pancreatic delivery), or absolute numbers of rAAV virions of 1 × 10 6 ~1×10 16 In some embodiments, the amount is about 10 per kg (inclusive). 11 ~10 13 Alternatively, a dosage of about 10 to about 150 mg of rAAV genome copies may be appropriate. 11 ~10 14Dosages of about 10 / kg or an appropriate measured rAAV genome copy number may be appropriate. 11 ~10 15 Dosages of about 1 x 10 per kg or an appropriate measured rAAV genome copy number may be appropriate. In some embodiments, dosages of about 1 x 10 per kg 14 Dosage of vector genome (vg) copy number or an appropriate measure may be appropriate.

[0140] In some embodiments, the number of administrations or treatments may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or less. In some embodiments, the treatment consists of a single administration of a therapeutically effective amount of rAAV particles. In some embodiments, successful treatment and / or repair is determined when one or more of the following are detected: alleviation or amelioration of one or more symptoms of the disease, disorder, or condition being treated; reduction in the severity of the disease, disorder, or condition of the subject; stabilization (i.e., not worsening) of the disease, disorder, or condition; delay or slowing of the progression of the disease, disorder, or condition; improvement or alleviation of the disease, disorder, or condition. In some embodiments, successful treatment is determined by detecting the presence of a repaired target polynucleotide in one or more cells, tissues, or organs isolated from the subject. In some embodiments, successful treatment is determined by detecting the presence of a polypeptide encoded by the repaired target polynucleotide in one or more cells, tissues, or organs isolated from the subject. In some embodiments, the success of the treatment is determined by detecting a decrease in fasting blood glucose level compared to before the treatment, in some embodiments, to a level of 100-125 mg / dL. In some embodiments, the success of the treatment is determined by detecting a decrease in fasting blood glucose level compared to before the treatment, in some embodiments, to a level of 90-125 mg / dL. In some embodiments, the success of the treatment is determined by detecting a decrease in blood glucose level in a glucose tolerance test compared to before the treatment, in some embodiments, to a level of 140-199 mg / dL. In some embodiments, the success of the treatment is determined by detecting a decrease in A1C level compared to before the treatment, in some embodiments, to a level of 5.7%-6.4%.

[0141] In some embodiments, recombinant AAV (rAAV) virions can be administered in a composition that includes a suitable carrier, if necessary. A suitable carrier can be selected depending on the indication for which the rAAV is intended. For example, one suitable carrier includes saline, which can be formulated with various buffer solutions (e.g., phosphate buffered saline). Other examples of suitable carriers include, but are not limited to, sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The compositions disclosed herein can optionally include other pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to the rAAV virions and carrier(s). Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0142] In certain embodiments, the rAAV virions are administered in a pharmaceutical composition comprising phosphate buffered saline (PBS), pH 7.3 and 0.001% pharma- ceutically acceptable non-ionic surfactant, such as Pluronic F-68 (PF68), or other suitable pharma- ceutically acceptable buffer or excipient, etc. In some embodiments, the pharmaceutical composition may comprise rAAV in an aqueous buffer comprising or consisting of 10 mM Tris, 150 mM NaCl, 0.02% poloxamer 188, 1 mM MgCl2 (adjusted to pH 8.0).

[0143] In some embodiments, the compositions disclosed herein may include rAAV virions alone or in combination with one or more other viruses (e.g., a second rAAV virion encoding one or more different transgenes). In some embodiments, the compositions may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different rAAV virions, each of which has one or more different transgenes.

[0144] As used herein, "immunosuppressed" or "immunosuppression" refers to a reduction in the activation or effectiveness of the immune response in a subject. Immunosuppression can be induced in a subject using one or more (e.g., a plurality, such as 2, 3, 4, 5, or more) agents, including, but not limited to, rituximab, methylprednisolone, prednisolone, sirolimus, immunoglobulin injections, prednisone, methotrexate, interleukin-6 inhibitors, anti-interleukin-6 antibodies, interleukin-6 receptor inhibitors, anti-interleukin-6 receptor antibodies, and any combination thereof.

[0145] In some embodiments, the methods disclosed herein may further include inducing immunosuppression in the subject (e.g., administering one or more immunosuppressants) prior to administering a rAAV virion (e.g., a rAAV virion or pharmaceutical composition disclosed herein) to the subject. In some embodiments, the subject is immunosuppressed (e.g., immunosuppression is induced in the subject) for about 30 days to about 0 days (e.g., any time between 30 days prior to administration of the rAAV virion, inclusive) prior to administering the rAAV virion to the subject. In some embodiments, the subject is pretreated with an immunosuppressant (e.g., rituximab, sirolimus, and / or prednisone) for at least 7 days.

[0146] In some embodiments, the subject's immunosuppression is maintained during and / or after administration of the rAAV virion or pharmaceutical composition, hi some embodiments, the subject can be immunosuppressed (e.g., can be administered one or more immunosuppressive agents) for between one day and one year after administration of the rAAV virion or pharmaceutical composition.

[0147] When administering an injectable aqueous solution, for example, the solution can be suitably buffered as necessary, and the liquid diluent is first made isotonic with sufficient saline or glucose. These particular aqueous solutions can be suitable for intraductal (pancreatic), endoscopic retrograde cholangiopancreatography (ERCP), intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, a sterile aqueous medium can be used. For example, a single dosage can be dissolved in 1 mL of isotonic NaCl solution and added to 1000 mL of subcutaneous injection or injected at the proposed site of injection (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject. In any event, the person responsible for administration will determine the appropriate dosage for the individual subject.

[0148] Injectable sterile solution is prepared by incorporating the required amount of active rAAV virion into a suitable solvent together with various other ingredients listed herein, and then optionally sterilizing by filtration.In general, dispersion can be prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other required ingredients from among those listed above.In the case of sterile powder for preparing sterile injectable solution, the preparation method can be vacuum drying and freeze-drying technology, which can obtain a powder of active ingredient and any desired additional ingredient from a previously sterile-filtered solution.

[0149] As used herein, the term "treating" refers to the application or administration of a composition (e.g., an rAAV as described herein) to a subject having a disease or disorder associated with low levels of beta cell or insulin production, for the purpose of treating, curing, ameliorating, alleviating, altering, relieving, ameliorating, improving, or affecting the disorder, the symptoms of the disease, or the predisposition to the disease.

[0150] Alleviating a disease associated with a decrease in beta cell number and / or insulin production includes delaying the onset or progression of the disease or reducing the severity of the disease. Alleviating a disease does not necessarily require a curative outcome. As used herein, "delaying" the onset of a disease means delaying, preventing, slowing, retarding, stabilizing, and / or postponing the progression of the disease. This delay may vary in duration depending on the disease history and / or the individual being treated. A method of "delaying" or alleviating the onset of cancer, or delaying the onset of a disease, is a method that reduces the probability of developing one or more symptoms of the disease within a given time frame and / or reduces the extent of symptoms within a given time frame compared to not using the method. Such comparisons are typically based on clinical studies with a sufficient number of subjects to obtain statistically significant results.

[0151] In particular, administration of a rAAV virion described herein to a human subject suffering from beta cell deficiency and / or insulin deficiency reduces one or more biomarkers or characteristics of the disease within 5 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, 50 weeks, or one year after administration. In some embodiments, a human subject suffering from beta cell deficiency and / or insulin deficiency reduces fasting plasma glucose to a level including 100-125 mg / dL within 5 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, 50 weeks, or one year after administration. In some embodiments, a human subject suffering from beta cell deficiency and / or insulin deficiency has a blood glucose level in a glucose tolerance test reduced to a level including 140-199 mg / dL within 5 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, 50 weeks, or 1 year after administration. In some embodiments, a human subject suffering from beta cell deficiency and / or insulin deficiency has an A1C level reduced to a level including 5.7%-6.4% within 5 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, 50 weeks, or 1 year after administration.

[0152] "Onset" or "progression" of a disease refers to the initial symptoms and / or subsequent progression of a disease. Onset of a disease can be detectable and assessed using standard clinical techniques well known in the art. However, onset also refers to undetectable progression. As used herein, the terms onset or progression refer to the biological course of a condition. "Onset" includes occurrence, recurrence, and onset.

[0153] In some embodiments, the rAAV virions disclosed herein can be administered in sufficient amounts to transduce cells of the desired tissue and provide sufficient levels of gene transfer and expression without undue adverse effects. Pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to a selected organ (e.g., pancreas), intraductal (including intrabiliary) administration, or endoscopic retrograde cholangiopancreatography (ERCP) administration. Other routes of administration include intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes of administration. Routes of administration can be combined as needed.

[0154] kit Disclosed herein are kits that include any of the agents described herein. In some embodiments, any of the agents disclosed herein can be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic, or research applications. The kits can include one or more containers housing the components of the present disclosure and instructions for use. In particular, such kits can include one or more agents described herein, along with instructions describing the intended use and proper use of these agents. In some embodiments, the agents in the kits can be in pharmaceutical formulations and dosages suitable for the particular use and method of administration of the agent. For research purposes, the kits can contain components in concentrations or amounts suitable for conducting various experiments.

[0155] Also disclosed herein is a kit for producing rAAV virions.In some embodiments, the kit may comprise a container that contains an isolated nucleic acid encoding Pax4 protein or a portion thereof.In some embodiments, the kit may further comprise instructions for producing rAAV virions.In some embodiments, the kit further comprises at least one container that contains a recombinant AAV vector that contains a transgene (i.e., Pax4).

[0156] In some embodiments, the kit can include a container that contains the above-mentioned recombinant AAV virion.In some embodiments, the kit can further include a container that contains a pharmaceutically acceptable carrier.For example, the kit can include one container that contains rAAV virion and a second container that contains a suitable buffer for injecting rAAV virion into a subject.In some embodiments, the container can be a syringe.

[0157] In some embodiments, the kits can be designed to facilitate the use of the methods described herein by researchers and can take many forms. Each of the compositions of the kit may be provided in liquid form (e.g., solution) or solid form (e.g., dry powder), as applicable. In some embodiments, some compositions may be configurable or processable (e.g., into an active form), for example, by the addition of appropriate solvents or other species (such as water or cell culture medium) that may or may not be provided with the kit. As used herein, "instructions for use" defines an instructional and / or promotional component, and can typically be accompanied by written instructions on or associated with the packaging of the present disclosure. Instructions for use can also include oral or electronic instructions provided in any manner, such as audiovisual (e.g., videotape, DVD, etc.), internet, and / or web-based communications, etc., that clearly identify to the user that the instructions are to be associated with the kit. The written instructions may be in a form prescribed by a government agency regulating the manufacture, use, or sale of drugs or biological products, and these instructions may also reflect approval by the agency of the manufacture, use, or sale for animal administration.

[0158] The kits disclosed herein may contain any one or more of the components described herein in one or more containers. In some embodiments, the kits may include instructions for mixing one or more components of the kit and / or for isolating and mixing a sample and applying it to a subject. The kits may include a container that contains an agent described herein. The agent may be in liquid, gel, or solid (powder) form. The agent may be sterilely prepared and packaged in a syringe and shipped refrigerated. Alternatively, it may be contained in a vial or other container for storage. A second container may have another agent that is sterilely prepared. Alternatively, the kit may contain an active agent that is premixed and shipped in a syringe, vial, tube, or other container. The kits may have one or more or all of the components required to administer the agent to an animal, such as a syringe, topical application device, or intravenous (iv) needle tube and bag, especially in the case of kits for producing certain somatic cell animal models.

[0159] The kits may have various forms, such as blister pouches, shrink-wrap pouches, vacuum sealable pouches, sealable thermoformed trays, or similar pouch or tray forms, with loosely packed accessories within the pouch, one or more tubes, containers, boxes, or bags. The kits may be sterilized after the accessories are added, allowing the individual accessories within the container to be unpacked in other ways. The kits may be sterilized using any suitable sterilization technique, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kits may also include other components, such as containers, cell culture media, salts, buffers, reagents, syringes, needles, cloths such as gauze for applying or removing disinfectants, disposable gloves, support for medication prior to administration, etc., depending on the particular application.

[0160] The instructions included in the kit may include methods for detecting potential AAV in cells. Additionally, kits of the present disclosure may include instructions, negative and / or positive controls, containers for samples, diluents and buffers, sample preparation tubes, and printed or electronic tables of reference AAV sequences for sequence comparison. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12]

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

Table 1-28

Table 1-29

Table 1-30

Table 1-31

Table 1-32

Table 1-33

Table 1-34

Table 1-35

Table 1-36

Table 1-37

Table 1-38

Table 1-39

Table 1-40

[0161] Example 1 The aim of this study is to evaluate the therapeutic efficacy of AAV9-based Pax4 gene delivery vectors in a streptozotocin (STZ)-induced T1D mouse model. To achieve efficient in vivo Pax4 gene delivery, we generate AAV9-based Pax4 gene delivery vectors that control human Pax4 cDNA by either the chicken beta actin core promoter region containing the cytomegalovirus (CMV) early enhancer element or CAG promoter (SEQ ID NO: 4), or the rat insulin promoter (RIP) (SEQ ID NO: 6), or the mouse glucagon promoter (MGP) (SEQ ID NO: 5). The CAG promoter provides strong and ubiquitous gene expression, whereas the RIP and MGP promoters provide beta cell-specific and alpha cell-specific gene expression, respectively. The efficacy of AAV9-based Pax4 gene delivery is evaluated by the expected improvement of T1D parameters in the STZ mouse model.

[0162] Experimental Design: Six experimental groups of mice are used in this study (n>5 mice / group, female): 1. Normal mice as controls 2. T1D untreated mice as controls 3. T1D mice treated with AAV9.CAG-eGFP (control treatment) 4. T1D mice treated with AAV9.CAG-Pax4 5. T1D mice treated with AAV9.RIP-Pax4 6. T1D mice treated with AAV9.MGP-Pax4

[0163] Six to eight week old C57BL / 6 mice (Jackson Laboratories) are housed for at least 7 days and then injected intraperitoneally with 65 mg / kg body weight STZ daily for 5 consecutive days. Blood glucose is measured every other day for 2 weeks using an ALPHA TRAK2 blood glucose monitoring meter and test strips. One to two weeks after the last STZ injection, when the blood glucose of the mice exceeds 300 mg / dL, 75 μl total of rAAV vectors are introduced via intrabiliary injection at a dose of 3.0E11vg / mouse. Mice are observed three times a day for the first two days after surgery, twice a day for the next week, and daily for one week thereafter. Blood glucose monitoring is performed three times a week for the first two weeks after surgery and twice a week for the following 10 weeks. All measurements are performed at approximately the same time of day. Glucose tolerance tests (GTTs) are performed at weeks 2, 6, and 10. Glucose-stimulated insulin secretion (GSIS) assays are performed at weeks 4, 8, and 12. At week 12 after surgery, mice are sacrificed by cardiac puncture under anesthesia to collect blood and pancreas, liver, spleen, intestine, and kidney for biochemical and immunohistochemical analysis. Hormone concentrations (insulin, glucagon, C-peptide) in blood samples are measured using ELISA kits. Immunohistochemical evaluation of tissue sections (for alpha cell, beta cell examination, and Pax4 expression examination).

[0164] statistical analysis Statistical analysis is performed using GraphPad Prism 9 software to compare blood glucose, GTT, and GSIS data of different treatment groups. When analyzing blood glucose levels between multiple groups over time (two parameters such as GTT and GSIS), a two-way ANOVA is performed to determine the significance of those differences. For each data point (fixed time point), a one-way ANOVA is performed to determine the significance of differences between three or more groups. If two groups need to be compared, a Student's t-test is performed. Data are expressed as mean ± SEM unless otherwise stated. P<0.05 is considered statistically significant.

[0165] Example 2 - Preclinical pilot study of AAV9-mediated delivery of Pax4 gene to the pancreas for the treatment of type 1 diabetes AAV9-based vectors were developed to deliver the Pax4 gene in vivo. AAV9 was chosen because it has been shown to mediate significant gene delivery to the pancreas after systemic or intrabiliary (IBD) administration, and AAV9-based vectors have been used in clinical trials. In this study, three AAV9.Pax4 vectors were generated to deliver human Pax4 cDNA. The three AAV9.Pax4 vectors were i) AAV9.CAG-Pax4, in which Pax4 expression is driven by the ubiquitously active CAG promoter, ii) AAV9.MGP-Pax4, in which Pax4 is driven by the mouse glucagon promoter, and iii) AAV9.RIP-Pax4, in which Pax4 is driven by the rat insulin promoter.

[0166] Research Overview The aim of this preclinical study was to evaluate the therapeutic efficacy of these AAV9.Pax4 vectors in a streptozotocin (STZ)-induced T1D mouse model. The STZ-T1D model was established in C57BL / 6 mice by multiple low-dose injections of STZ (65 mg / kg body weight for 5 days). When non-fasting blood glucose (BG) exceeded 300 mg / dL, mice were injected with 3 × 10 AAV9.Pax4 vectors or control (AAV9.CAG-GFP) by IBD injection. 11T1D mice were treated with a single dose of 1000 mg / vg / mouse, allowing the vector to be administered directly to the pancreas through the pancreatic duct system. PBS (phosphate buffered saline) was injected into the T1D mice as a vehicle control. In addition, five mice from the same batch were maintained as normal controls (i.e., they did not receive STZ treatment or IBD injection). Non-fasting blood glucose was monitored 2-3 times a week in STZ mice and once a week in normal mice for approximately 3 months. An intraperitoneal glucose tolerance test (GTT) was performed approximately 8 weeks after vector administration. A glucose-stimulated insulin secretion (GSIS) assay was performed approximately 10 weeks after vector administration. Mice were sacrificed approximately 3 months after treatment. Mice blood was collected for hormone (insulin, glucagon, C-peptide) measurements by corresponding ELISA kits, and pancreases were removed and processed to perform immunohistochemical analysis of islet α- and β-cells, as well as Pax4 (or GFP) transgene expression.

[0167] Mice that received STZ treatment developed hyperglycemia at a significantly faster rate than expected following STZ treatment compared to their previous STZ administration experience. Therefore, STZ-treated mice that developed hyperglycemia sooner than expected were treated with rAAV vectors as early as 3 days after the last STZ administration (at least 4 days earlier than originally expected). Therefore, if treated with these vectors within 7 days after the last STZ administration, the effect may be complicated by the presence of ongoing STZ-induced β-cell death. For this reason, data from mice that developed hyperglycemia 1 week after the last STZ administration and were subsequently treated were analyzed separately from data generated from mice that developed hyperglycemia 1 week prior to the last STZ administration. The results obtained from these mice that were treated 1 week after the last STZ administration indicate that the AAV9.Pax4 vector showed signs of therapeutic efficacy compared to control treatment. Specifically, T1D mice treated with AAV9.CAG-Pax4 and AAV9.MGP-Pax4 had significantly lower nonfasting BG than control-treated T1D mice, despite small sample sizes (n=2–4) (Figure 6). For the animals evaluated, mice treated with AAV9.CAG-Pax4 showed a marked improvement in GTT (Figure 7A and B), and all T1D model mice treated with any of the three AAV9.Pax4 vectors showed a trend toward higher insulin secretion in response to glucose loading than control treatment when evaluated by GSIS assay (Figure 8A and B). Furthermore, blood insulin and C-peptide in mice treated with AAV9.Pax4 were significantly higher than mice treated with control PBS or AAV9.CAG-GFP (Figure 9A and C), suggesting that Pax4 treatment improved β-cell mass / function. On the other hand, blood glucagon did not show significant differences between treatment groups (Figure 9B), suggesting that Pax4 gene therapy did not cause glucagon deficiency.Finally, IHC staining of pancreatic sections demonstrated Pax4 and GFP expression, confirming that AAV9-mediated transgene expression was sustained over time (data not shown).

[0168] In summary, despite a suboptimal T1D model, the observation that a single administration of AAV9.Pax4 vector improves β-cell function and attenuates hyperglycemia supports the continued development of AAV9-based Pax4 gene therapy as a therapeutic avenue for T1D.

[0169] overview Three AAV9-based human Pax4 gene delivery vectors were generated for testing. i) In AAV9.CAG-Pax4, expression of Pax4 is driven by the chicken beta actin core promoter region with the cytomegalovirus (CMV) early enhancer element (CAG) promoter. Universal activation of the CAG promoter promotes expression of Pax4 in both α and β cells, induces transdifferentiation in α cells, and promotes survival of remaining and newly transdifferentiated β cells. However, strong and universal promoter activation also allows expression of Pax4 in non-islet and non-pancreatic transduced cells, resulting in off-target transgene expression. ii) In AAV9.MGP-Pax4, Pax4 is driven by the mouse glucagon promoter (MGP) to direct cell-specific transgene expression in islet α-cells, whereas minimal promoter activation is predicted in glucagon-negative islet β-cells. iii) In AAV9.RIP-Pax4, Pax4 is driven by the rat insulin promoter (RIP) to allow cell-specific transgene expression in islet β cells, whereas minimal promoter activation is predicted in insulin-negative islet α cells.

[0170] the purpose The aim of this study was to evaluate the therapeutic efficacy of AAV9-based Pax4 gene delivery vectors in a streptozotocin (STZ)-induced T1D mouse model following direct vector administration into the pancreas by intrabiliary (IBD) injection. Three vectors were tested: AAV9.CAG-Pax4, AAV9.MGP-Pax4, and AAV9.RIP-Pax4.

[0171] Materials and Methods The rAAV constructs and concentrations used in this study are shown in Tables 2 and 3. [Table 2] [Table 3]

[0172] The vehicle controls used in this study are listed in Table 4. [Table 4]

[0173] Materials / Supplies:

[0174] Animals: C57BL / 6 mice, female, approximately 11 weeks old, obtained from Charles River (stock no. 027). Weight at randomization: 19.1 g ± 0.15 g.

[0175] Glucose Measurement: AlphaTRAK2 Blood Glucose Monitoring System (Zoetis), which includes a glucose meter and blood glucose test strips.

[0176] Mouse Ultrasensitive Insulin ELISA Kit: Catalog number 80-INSMSU-E01, obtained from ALPCO.

[0177] Glucagon ELISA kit: Catalog number 48-GLUHU-E01, obtained from ALPCO.

[0178] Mouse C-peptide ELISA kit: catalog number 80-CPTMS-E01, obtained from ALPCO.

[0179] The antibodies used in the study are shown in Table 5. [Table 5]

[0180] Experimental design The study included 6 groups (n≧5 mice / group, female). 1) Normal, untreated mice (as controls) 2) PBS-treated STZ-T1D mice (as vehicle control) 3) STZ-T1D mice treated with AAV9.CAG-GFP (as vector control) 4) STZ-T1D mice treated with AAV9.CAG-Pax4 5) STZ-T1D mice treated with AAV9.MGP-Pax4 6) STZ-T1D mice treated with AAV9.RIP-Pax4

[0181] The details of the procedure are shown in Table 6. [Table 6]

[0182] Mice were administered a dose of 65 mg / kg body weight by intraperitoneal injection for 5 days. Animals were randomly assigned to treatment based on blood glucose levels. Each mouse was administered 3 × 10 11 AAV9 vectors of VG were injected into the pancreas by IBD injection. Details of each treatment group are shown in Table 7. Grey shaded areas indicate mice that developed hyperglycemia 1 week after the last STZ administration and were used for further hormone and biomarker analysis. [Table 7-1] [Table 7-2]

[0183] Body weight and clinical observations Body weight was measured every time blood glucose was measured, 2–3 times a week for approximately 3 months.

[0184] Blood glucose (nonfasting) was monitored 2–3 times per week for approximately 3 months.

[0185] A glucose tolerance test (GTT) was performed approximately 8 weeks after vector administration.

[0186] Approximately 10 weeks after vector administration, a glucose-stimulated insulin secretion (GSIS) assay was performed.

[0187] Lab Procedures T1D was induced by multiple low-dose STZ administration. T1D induction was performed as previously described (Zhang et al., (2016), Bone et al.). Specifically, approximately 11-week-old female C57Bl / 6 mice (obtained from Charles River) were fasted for 5 h and then administered 65 mg / kg body weight STZ by intraperitoneal (ip) injection for 5 consecutive days. Blood glucose was monitored every other day using an Alpha TRAK2 blood glucose monitoring system. Treatment with test article or control was initiated when blood glucose (non-fasting) exceeded 300 mg / dL.

[0188] Intrabiliary (IBD) injection. The procedure was performed as described in previous studies (Zhang et al., (2016), Bone et al.), with modifications including reducing the injection volume and clamping the duodenum. Specifically, all surgical instruments were sterilized by autoclaving before surgery. During surgery, the workstation was thoroughly cleaned with 70% ethanol. Between surgeries, instruments were sterilized in a dry bead sterilizer. The surgery was performed using a Leica M80 stereomicroscope with a Leica IC90 E camera (microinjection system). A 70% ethanol spray was used to ensure that the working area was clean and sterile. For the surgery, mice were anesthetized with ketamine 100mg / Kg + xylazine 10mg / ml via intraperitoneal injection. Before incision, abdominal hair was removed with a shaving blade. Buprenex (0.1mg / Kg body weight) was administered via subcutaneous injection before making the incision. Next, a midline incision was made to access the mouse abdominal cavity. After exposing the duodenum and visualizing the common bile duct, the needle (31G) of a syringe containing 50 μl of PBS or AAV vector was inserted into the common bile duct adjacent to the duodenum toward the papilla of Vater. The duodenal tissue surrounding the needle was then clamped with a curved microtemporary clip to fix the needle in place and prevent the injected solution from entering the duodenum. The solution in the syringe was then slowly injected into the pancreatic duct. After removing the needle, the injection site was held for 10–15 seconds with a cotton swab to prevent leakage. The abdominal organs were then carefully placed in place, and the abdominal cavity and skin were sutured with absorbable sutures. The mice were placed on a heating pad until they recovered from anesthesia, and were returned to their cages after recovering from anesthesia.

[0189] Glucose tolerance test (GTT). GTT was performed essentially as previously described by Zhang et al. (2019). Specifically, after an overnight fast, mice were injected with 1 g glucose / Kg body weight via intraperitoneal injection. Blood glucose was measured using an AlphaTRAK glucose meter before glucose injection (0 h) and at various time points after glucose injection (15, 30, 45, 60, 90, 120 min).

[0190] Glucose-responsive insulin secretion (GSIS). In vivo GSIS was performed with an identical setup to GTT, except that blood was collected for insulin measurement. Zhang et al. (2019). Specifically, after overnight fasting, mice were injected intraperitoneally with 1 g glucose / Kg body weight. Approximately 20 μl of blood was collected from the tail vein before glucose injection (0 h) and at 15 and 30 min after glucose injection. Insulin concentrations in serum were measured using an ultrasensitive mouse insulin ELISA kit (obtained from ALPCO) according to the manufacturer's instructions.

[0191] Hormone measurements. Hormones including mouse insulin, glucagon, and C-peptide were measured using the corresponding ELISA kits from ALPCO (Salem, New Hampshire) as previously described (Parajuli et al., Zhang et al. (2016), Zhang et al. (2019)). Specifically, mouse insulin was measured using Mouse Ultrasensitive Insulin Kit 80-INSMSU-E01, mouse C-peptide was measured using Kit 80-CPTMS-E01, and glucagon was measured using Kit 48-GLUHU-E01. All assays were performed according to the manufacturer's protocols. Note that the glucagon kit can be used to measure both human and mouse glucagon following the manufacturer's instructions.

[0192] Immunohistochemistry (IHC) staining of pancreatic tissue. IHC staining of pancreatic tissue was performed as previously described by Parajuli et al. Specifically, mouse pancreases were fixed in 10% buffered formalin solution overnight at room temperature and then processed for paraffin embedding and sectioning. For IHC staining, pancreatic sections were first deparaffinized and sequentially incubated in xylene (4 × 2 min), 100% ethanol (2 × 1 min), 95% ethanol (30 s), 70% ethanol (45 s), and deionized water (1 min). Antigen retrieval (optional) was then performed by boiling the pancreatic sections in citrate buffer (pH 6.0) for 20 min, followed by natural cooling to below 40 °C and washing with deionized water. (Note: antigen retrieval is required for Pax4 staining, but not for insulin, glucagon, or GFP staining). Prior to antibody incubation, tissue sections were permeabilized with 0.25% Triton X-100 in PBS for 30 min and blocked in blocking solution (2% glycine, 2% bovine serum albumin, 5% FBS, 50 mM NH4Cl in PBS) for 1 h at room temperature. Sections were then incubated overnight at 4°C with primary antibodies diluted in 3% FBS in PBS, followed by incubation with corresponding secondary antibodies conjugated to various fluorophores. For nuclear staining, after secondary antibody incubation, slides were added with Hoechst33342 (2 μg / ml in PBS) and incubated for 10 min at room temperature. Sections were then washed with PBS and water, air-dried, mounted on glass coverslips, and processed for fluorescence microscopy. Images were captured with a Photometrics IRIS15 digital camera attached to a Nikon Ti-S / L100 inverted microscope. Fluorescent images were collected using NIS-Elements imaging software.

[0193] Final blood collection by cardiac puncture: Approximately 3 months after surgery, blood (1.2 ml) was collected from mice by cardiac puncture under anesthesia, a procedure that resulted in the death of the mice.

[0194] Tissue harvest: Pancreas, liver, kidneys, intestines, and spleen were harvested and preserved in 10% formalin solution.

[0195] Immunohistochemistry: Formalin-fixed pancreatic tissue was embedded in paraffin, sectioned, and processed for IHC staining for insulin, glucagon, and Pax4 (or GFP) to assess α- and β-cell status, and transgene (Pax4 or GFP) expression.

[0196] Statistical evaluation: Statistical analysis was performed using GraphPad Prism9 software. When analyzing blood glucose levels among multiple groups over time (two parameters such as GTT and GSIS), a two-way analysis of variance was performed to determine the significance of their differences. For each data point (fixed time point), a one-way analysis of variance was performed to determine the significance of differences among three or more groups, and a Student's t-test was used to compare differences between two groups. All quantitative data were expressed as mean ± SEM. P<0.05 was considered statistically significant. Statistical significance was defined as *: p<0.05, **: p<0.01, ***: p<0.001.

[0197] result STZ-induced T1D mice (5 × 65 mg / kg body weight) developed hyperglycemia (diabetes developed over a 2-week period) at a significantly faster rate than expected compared to previous experience with STZ 5 × 50 mg / kg body weight. rAAV vectors were administered when blood glucose levels reached 300 mg / dL. In mice treated within 1 week of the last STZ dose, STZ-induced β-cell death may have still been ongoing, complicating evaluation of the therapeutic efficacy of the test substance (Goyal et al. and Szkudelski). Data obtained from mice that developed hyperglycemia after 1 week and were subsequently treated were analyzed separately (Figure 6).

[0198] Body weight. The body weight of the mice was monitored throughout the study period. There were no significant differences between the treatment groups (Figure 4). Diabetic mice (treated with test or control substances) lost more weight than normal mice due to weight loss during STZ induction and subsequent IBD surgery.

[0199] Blood glucose (non-fasting). Non-fasting blood glucose (BG) of each mouse was monitored for approximately 12 weeks after test article and control IBD injection. The average data of all mice in each group was plotted (Figure 5). Overall, no significant differences were detected between treatment groups. Data obtained from mice that developed hyperglycemia after 1 week and were subsequently treated were analyzed separately as shown in Figure 6. Within this group of mice, T1D mice treated with AAV9.CAG-Pax4 and AAV9.MGP-Pax4 showed significantly lower BG than the control-treated group, especially in the first 2-3 weeks after vector administration. While statistically significant differences were observed between AAV9.CAG-Pax4-treated mice and both control groups (PBS and AAV9.CAG-GFP), AAV9.MGP-Pax4-treated mice showed a significant difference from the AAV9.CAG-GFP group (p=0.0327) but not from the PBS control group (p=0.0973), which may be due to insufficient sample size (n=2).

[0200] Glucose Tolerance Test (GTT). On average, T1D mice in the PBS, AAV9.CAG-GFP, and AAV9.Pax4-treated groups had non-fasting BG >450 mg / dL (Figure 5), suggesting that glycemic control was very poor and glucose loads were poorly tolerated. Nevertheless, a GTT was attempted at approximately 8 weeks after vector administration. To perform the GTT, mice were fasted overnight. GTT was assessed in treated mice at least 1 week after the last STZ treatment (see Figure 7A and B). The data show that AAV9.CAG-Pax4 significantly improved glucose tolerance compared to the control PBS or GFP groups, while AAV9.MGP-Pax4 showed improvement but did not reach statistical significance, which may be due to the small sample size. AAV9.RIP-Pax4 did not show any effect, which may be because there were few β-cells remaining in the diabetic mice and therefore the vector was unable to exert a β-cell protective effect. Parajuli et al. *: p<0.05, **: p<0.01 between the indicated groups.

[0201] Glucose-responsive insulin secretion (GSIS). GSIS assay was performed on mice that received IBD injections more than 1 week after the last STZ treatment, approximately 10 weeks after vector administration. Mice were fasted overnight and then injected with 1 g / Kg body weight glucose via intraperitoneal injection. Blood was collected before glucose injection (0 h), 15 min and 30 min after glucose injection. Insulin concentrations were measured using Zhang et al. (2019) ultrasensitive mouse insulin ELISA kit. As shown in Figure 8A and B, all mice treated with Pax4-expressing AAV9 vectors tended to secrete more insulin upon glucose injection than the PBS and GFP control groups, suggesting that they had more functional β cells, although statistically significant differences are not shown due to the small sample size.

[0202] Mouse blood insulin, glucagon, and C-peptide. At the end of the animal experiment (approximately 3 months after vector administration by IBD injection), blood was collected from each mouse by cardiac puncture and hormone measurements were performed using mouse insulin, C-peptide, and glucagon ELISA kits (ALPCO). Figure 9A to C show the data for all mice, and no significant differences were detected between the different groups for any of the three hormones.

[0203] At the end of the study (3 months after vector administration), 1 week after the last STZ administration, hormone concentrations (insulin, glucagon, and C-peptide) were further analyzed from vector-treated and control mice. Several significant differences were not detected (Figure 10A to C). Specifically, mice treated with AAV9.CAG-Pax4 produced significantly more insulin than control PBS or AAV9.CAG-GFP control treatments (Figure 10A). All Pax4-containing treatment groups showed significantly more C-peptide than control treatments (Figure 10C). Because C-peptide has a long half-life in the blood, blood C-peptide reflects accumulated β-cell function, with higher blood C-peptide levels indicating more functional β-cells. Note: Normal mice are normoglycemic and do not show elevated insulin or C-peptide than diabetic groups because β-cells were not stimulated in the experimental setting. Moreover, blood glucagon levels in all groups did not show any significant differences (FIG. 10B), suggesting that the treatment did not induce glucagon deficiency.

[0204] Immunohistochemical assessment of pancreatic islet α- and β-cells and transgene (Pax4, GFP) expression. After mice were euthanized, pancreases were harvested and processed for IHC staining of insulin, glucagon, and Pax4 (or GFP). GFP staining demonstrated highly efficient gene delivery to the pancreas by the AAV9 vector. Pax4 gene expression was detectable, but no significant expression of Pax4 was observed, even in the AAV9.CAG-Pax4 treatment group. + The number of cells was GFP +The expression of Pax4 was very low compared to cells expressing high levels of Pax4 (data not shown). The sensitivity of the Pax4 antibody was very low, and only cells expressing high levels of Pax4 were detected. Nevertheless, the results show that the expression of Pax4 persisted for 3 months after a single administration of the vector by IBD injection.

[0205] Consideration This study was designed to investigate the therapeutic efficacy of AAV9-based Pax4 gene delivery in STZ-induced T1D mice after direct administration of the vector into the pancreas by IBD injection.Several results support that Pax4 gene delivery into pancreatic islets increases insulin secretion and improves glycemic control in T1D.

[0206] Induction of T1D in mice with multiple, low-dose STZ treatments (5 × 65 mg / kg body weight) resulted in the onset of severe hyperglycemia earlier than observed in our previous experience with this model (Goyal et al.). One-third of mice (15 / 45) achieved blood glucose (BG) >300 mg / dL within 5 days of the last STZ administration, and more than 50% of mice (24 / 45) achieved BG >300 mg / dL within 1 week of the last STZ administration. Vector was administered once BG reached 300 mg / dL. Mice that reached the desired BG value before 1 week of the last STZ administration may have been undergoing STZ-induced β-cell death at the time of vector administration, which could complicate evaluation of the therapeutic utility of the construct. Furthermore, acute β-cell death in the severe T1D model may have left few residual β-cells to be protected by Pax4 expression. Indeed, data analysis only analyzed mice that achieved the desired BG and hyperglycemia after one week and detected benefits of Pax4 gene therapy as assessed by GTT, GSIS, and basal blood hormone secretion (insulin, C-peptide, glucagon).

[0207] Among the vectors, AAV9.CAG-Pax4 and AAV9.MGP-Pax4 showed the most significant improvement, followed by AAV9.RIP-Pax4. Without intending to be bound by any particular theory, the MGP promoter is expected to selectively drive Pax4 gene expression in α cells but not in β cells, thus inducing β cell regeneration (by inducing α cell to β cell transdifferentiation). The RIP promoter is expected to selectively drive Pax4 gene expression in β cells, thus protecting only existing β cells. In contrast, the universally activated CAG promoter is expected to drive Pax4 gene expression in both α and β cells (as well as other cells), thus not only promoting β cell regeneration from α cells but also protecting existing β cells. The results are consistent. It should be noted that AAV9.CAG-Pax4 can mediate Pax4 expression in transduced cells other than pancreatic islets by combining the universality of the CAG promoter with the broad biodistribution of AAV9.

[0208] The dosage of AAV9 vector used in this study was 3 × 10 11 vg / mouse, which appears to be a sufficient dose for GFP gene delivery via the AAV9.CAG-GFP vector. The data also show that AAV9-mediated gene expression is sustained (lasting for 3 months with a single dose). Due to the sensitivity of Pax4 detection, we were unable to reliably assess the efficiency of Pax4 gene delivery.

[0209] conclusion A single dose of AAV9.Pax4 vectors, particularly AAV9.CAG-Pax4 and AAV9.MGP-Pax4, improved glycemic control as assessed by non-fasting glucose monitoring and glucose tolerance testing. Second, a single dose of AAV9.Pax4 vectors demonstrated sustained improvements in beta-cell function as assessed by GSIS assay and basal plasma insulin and C-peptide levels 3 months after treatment. These improvements reached statistical significance even with very small sample sizes (n=2-4). References All publications, patent applications, patents, and other references (e.g., sequence database reference numbers) mentioned herein are incorporated by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences mentioned herein are incorporated by reference. Unless otherwise indicated, sequence accession numbers identified herein, including any tables herein, refer to the current database entries as of the filing date of this application. When a gene or protein refers to multiple sequence accession numbers, all sequence variants are encompassed. 1. Parajuli KR, Zhang Y, Cao AM, Wang H, Fonseca VA, Wu H. Pax4 Gene Delivery Improves Islet Transplantation Efficacy by Promoting beta Cell Survival and alpha-to-beta Cell Transdifferentiation. Cell Transplant.2020;29:963689720958655. 2.Lorenzo PI, Juarez-Vicente F, Cobo-Vuilleumier N, Garcia-Dominguez M, Gauthier BR. The Diabetes-Linked Transcription Factor PAX4: From Gene to Functional Consequences. Genes. 2017;8(3). 3.Brun T, Gauthier BR. A focus on the role of Pax4 in mature pancreatic islet beta-cell expansion and survival in health and disease. Journal of molecular endocrinology.2008;40(2):37-45. 4.Lorenzo PI,Fuente-Martin E,Brun T,Cobo-Vuilleumier N,Jimenez-Moreno CM,I GHG,Lopez Noriega L,Mellado-Gil JM,Martin-Montalvo A,Soria B,Gauthier BR. PAX4 Defines an Expandable beta-Cell Subpopulation in the Adult Pancreatic Islet. Scientific reports. 2015;5:15672. 5.Sosa-Pineda B,Chowdhury K,Torres M,Oliver G,Gruss P.The Pax4 gene is essential for differentiation of insulin-producing beta cells in the mammalian pancreas. Nature.1997;386(6623):399-402. 6.Collombat P,Xu X,Ravassard P,Sosa-Pineda B,Dussaud S,Billestrup N,Madsen OD,Serup P,Heimberg H,Mansouri A.The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into alpha and subsequently beta cells. Cell.2009;138(3):449-462. 7.Zhang Y,Fava GE,Wang H,Mauvais-Jarvis F,Fonseca VA,Wu H.PAX4 Gene Transfer Induces alpha-to-beta Cell Phenotypic Conversion and Confers Therapeutic Benefits for Diabetes Treatment. Molecular therapy:the journal of the American Society of Gene Therapy.2016;24(2):251-260. 8.Mendell JR,Al-Zaidy SA,Rodino-Klapac LR,Goodspeed K,Gray SJ,Kay CN,Boye SL,Boye SE,George LA,Salabarria S,Corti M,Byrne BJ,Tremblay JP. Current Clinical Applications of In Vivo Gene Therapy with AAVs. Molecular therapy:the journal of the American Society of Gene Therapy.2021;29(2):464-488. 9.Inagaki K,Fuess S,Storm TA,Gibson GA,McTiernan CF,Kay MA,Nakai H.Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8. Molecular therapy:the journal of the American Society of Gene Therapy.2006;14(1):45-53. 10.Jimenez V,Ayuso E,Mallol C,Agudo J,Casellas A,Obach M,Munoz S,Salavert A,Bosch F.In vivo genetic engineering of murine pancreatic beta cells mediated by single-stranded adeno-associated viral vectors of serotypes 6, 8 and 9. Diabetologia.2011;54(5):1075-1086. 11.Bone RN,Icyuz M,Zhang Y,Zhang Y,Cui W,Wang H,Peng JB,Matthews QL,Siegal GP,Wu H.Gene transfer of active Akt1 by an infectivity-enhanced adenovirus impacts beta-cell survival and proliferation differentially in vitro and in vivo. Islets.2012;4(6):366-378. 12.Zhang Y,Parajuli KR,Fava GE,Gupta R,Xu W,Nguyen LU,Zakaria AF,Fonseca VA,Wang H,Mauvais-Jarvis F,Sloop KW,Wu H.GLP-1 Receptor in Pancreatic alpha-Cells Regulates Glucagon Secretion in a Glucose-Dependent Bidirectional Manner. Diabetes.2019;68(1):34-44. 13.Goyal SN,Reddy NM,Patil KR,Nakhate KT,Ojha S,Patil CR,Agrawal YO. Challenges and issues with streptozotocin-induced diabetes-A clinically relevant animal model to understand the diabetes pathogenesis and evaluate therapeutics. Chem Biol Interact.2016;244:49-63. 14.Szkudelski T.The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas. Physiol Res.2001;50(6):537-546. 15.Furuyama K,Chera S,van Gurp L,Oropeza D,Ghila L,Damond N,Vethe H,Paulo JA,Joosten AM,Berney T,Bosco D,Dorrell C,Grompe M,Raeder H,Roep BO,Thorel F,Herrera PL. Diabetes relief in mice by glucose-sensing insulin-secreting human alpha-cells. Nature.2019;567(7746):43-48. 16.Xiao X,Guo P,Shiota C,Zhang T,Coudriet GM,Fischbach S,Prasadan K,Fusco J,Ramachandran S,Witkowski P,Piganelli JD,Gittes GK. Endogenous Reprogramming of Alpha Cells into Beta Cells,Induced by Viral Gene Therapy,Reverses Autoimmune Diabetes. Cell stem cell.2018;22(1):78-90e74.

Claims

1. A recombinant adeno-associated virus (AAV) vector comprising an expression cassette having a nucleotide sequence encoding hPax4 operably linked to a promoter and a polyadenylation (poly(A)) signal sequence that promotes expression of the nucleotide sequence encoding hPax4 in pancreatic islet cells, wherein the promoter comprises a CAG promoter, a rodent or human glucagon promoter, or a rodent or human insulin promoter, and the expression cassette is flanked by inverted terminal repeat (ITR) nucleotide sequences.

2. The recombinant AAV vector of claim 1 , wherein the hPax4 has the amino acid sequence of SEQ ID NO:

1.

3. 2. The recombinant AAV vector of claim 1, wherein the nucleotide sequence encoding hPax4 comprises a nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 2 or its reverse complementary sequence.

4. 2. The recombinant AAV vector of claim 1, wherein the nucleotide sequence encoding hPax4 comprises or consists of the nucleotide sequence of SEQ ID NO: 2 or its reverse complement.

5. 2. The recombinant AAV vector of claim 1, wherein the promoter is a CAG promoter, a rodent glucagon promoter, a rodent insulin promoter, or a human glucagon promoter.

6. The recombinant AAV vector described in Claim 5, wherein the promoter is a human glucagon promoter.

7. The recombinant AAV vector described in claim 2, wherein the promoter is a human glucagon promoter.

8. 2. The recombinant AAV vector of claim 1, wherein the polyA signal sequence is an SV40 polyA signal sequence having the nucleotide sequence of SEQ ID NO: 7 or its reverse complementary sequence.

9. The recombinant AAV vector of claim 1, wherein the recombinant AAV vector is a self-complementary vector.

10. 2. The recombinant AAV vector of claim 1, wherein the ITR nucleotide sequence comprises: i) a 5' ITR having the nucleotide sequence of SEQ ID NO: 8, or its reverse complementary sequence, and a 3' ITR having the nucleotide sequence of SEQ ID NO: 25, or its reverse complementary sequence; or ii) a 5' ITR having the nucleotide sequence of SEQ ID NO: 8, or its reverse complementary sequence, and a modified 3' ITR having the nucleotide sequence of SEQ ID NO: 9, or its reverse complementary sequence; or iii) a modified 5' ITR sequence, or its reverse complementary sequence, and a 3' ITR sequence having the nucleotide sequence of SEQ ID NO: 25, or its reverse complementary sequence.

11. 2. The recombinant AAV vector of claim 1, wherein the expression cassette has the nucleotide sequence of SEQ ID NO: 10 (CAG-hPax4), SEQ ID NO: 11 (MGP-hPax4), SEQ ID NO: 12 (RIP-hPax4), SEQ ID NO: 13 (scAAV-CAG-hPax), SEQ ID NO: 14 (scAAV-MGP-Pax4), or SEQ ID NO: 15 (scAAV-RIP-hPax), or a reverse complementary sequence thereof.

12. A recombinant AAV virion comprising: 1) an AAV capsid containing an AAV capsid protein and having pancreatic islet cell tropism; and 2) the recombinant AAV vector according to any one of claims 1 to 11.

13. 13. The recombinant AAV virion of claim 12, wherein the AAV capsid protein has an amino acid sequence that is at least 85% identical to i) an AAV6 capsid protein (SEQ ID NO: 19), ii) an AAV8 capsid protein (SEQ ID NO: 23), or iii) an AAV9 capsid protein (SEQ ID NO: 21).

14. 14. The recombinant AAV virion of claim 13, wherein the AAV capsid protein has the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 23, or SEQ ID NO:

21.

15. A recombinant AAV virion as described in claim 14, wherein the AAV capsid protein has the amino acid sequence of SEQ ID NO:

21.

16. The recombinant AAV virion described in claim 13, wherein the recombinant AAV vector comprises an expression cassette comprising a nucleotide sequence encoding hPax4 operably linked to a human glucagon promoter, and the AAV capsid protein is an AAV6 capsid protein or an AAV9 capsid protein.

17. A pharmaceutical composition for increasing insulin production in a subject in need thereof, comprising a therapeutically effective amount of a recombinant AAV virion described in claim 12 so that, when administered to the subject, a nucleotide sequence encoding hPax4 is expressed in pancreatic islet cells.

18. A pharmaceutical composition for increasing the number of beta cells in a subject in need thereof, comprising a therapeutically effective amount of a recombinant AAV virion described in claim 12 such that, when administered to the subject, a nucleotide sequence encoding hPax4 is expressed in pancreatic islet cells.

19. A pharmaceutical composition for use in a subject to treat diabetes in a subject suffering from diabetes, the pharmaceutical composition comprising a therapeutically effective amount of a recombinant AAV virion described in claim 12 so that, when administered to the subject, a nucleotide sequence encoding hPax4 is expressed in pancreatic islet cells.

20. 20. The pharmaceutical composition of claim 19, wherein the diabetes is type 1 diabetes.

21. 20. The pharmaceutical composition of claim 19, wherein the diabetes is type 2 diabetes.

22. The pharmaceutical composition described in claim 19, wherein the recombinant AAV virion is an AAV virion comprising an expression cassette including a nucleotide sequence encoding hPax4 operably linked to a human glucagon promoter, and the AAV capsid protein is an AAV6 capsid protein or an AAV9 capsid protein.

23. A pharmaceutical composition for reducing the risk of developing diabetes in a subject positive for islet-associated autoantibodies, comprising a therapeutically effective amount of a recombinant AAV virion described in claim 12 so that, when administered to the subject, a nucleotide sequence encoding hPax4 is expressed in pancreatic islet cells.

24. 20. The pharmaceutical composition of claim 19, wherein the pharmaceutical composition is administered by systemic administration, intravenous administration, intramuscular administration, intraperitoneal administration, or subcutaneous administration.

25. 20. The pharmaceutical composition of claim 19, wherein the pharmaceutical composition is administered by direct administration to the pancreas, intrapancreatic administration, or endoscopic retrograde cholangiopancreatography (ERCP).

26. 20. The pharmaceutical composition of claim 19, wherein the pharmaceutical composition further comprises a suitable carrier.

27. 20. The pharmaceutical composition of claim 19, wherein the subject is a human.

28. An AAV vector plasmid comprising: 1) an origin of replication; and 2) the recombinant AAV vector according to any one of claims 1 to 11.

29. The pharmaceutical composition described in Claim 28, wherein the recombinant AAV vector comprises an expression cassette comprising a nucleotide sequence encoding hPax4 operably linked to a human glucagon promoter.

30. A cell comprising an AAV vector including an expression cassette, wherein the expression cassette has a nucleotide sequence including a human glucagon promoter operably linked to hPax4.

31. A method for producing recombinant AAV virions, comprising culturing a host cell containing the AAV vector plasmid of claim 28 and a second plasmid comprising nucleotide sequences encoding cap and rep, wherein the cap encodes VP1, VP2, and VP3, and the rep encodes rep78, rep68, rep52, and rep40, wherein the AAV virions are cultured under conditions sufficient to produce the AAV virions.

32. 32. The method of claim 31, further comprising isolating AAV virions produced by the host cell.

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