AAV vectors for delivery of GLP-1 receptor agonist fusions

Abstract

Compositions and methods for treating metabolic disorders in a subject are provided. A viral vector is provided that includes a nucleic acid molecule that includes a sequence encoding a GLP-1 receptor agonist fusion protein and a regulatory sequence that directs its expression.

Description

[Technical field]

[0001] Electronic Sequence Listing Reference The contents of the electronic sequence listing (22-10015.PCT_Seq-Listing.xml; size: 91.5 kb, and creation date: March 3, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0002] Glucagon-like peptide 1 (GLP-1) is an endogenous peptide hormone that plays a central role in glucose homeostasis. GLP-1 is a peptide hormone produced in the gastrointestinal (GI) tract from the proteolytic cleavage of the glucagon preprotein. GLP-1 and other GLP-1 receptor agonists have the ability to control hyperglycemia by enhancing insulin release, increasing insulin sensitivity, preventing beta cell loss, and delaying gastric emptying. However, GLP-1 has a short half-life, which has hindered its use as a drug. Other GLP-1 receptor agonists are currently used in humans for the treatment of diabetes. GLP-1 receptor agonists engineered to overcome the short half-life of the native hormone by fusing the agonist to a protein with a longer half-life have emerged as important therapeutics for the treatment of type 2 diabetes mellitus (T2DM). Summary of the Invention

[0003] Provided herein are viral vectors encoding glucagon-like peptide 1 (GLP-1) receptor agonist fusion protein constructs.These viral vectors, in some embodiments, can achieve sustained expression and / or increased circulating half-life of GLP-1 receptor agonist in a subject compared to vector-mediated delivery of GLP-1 receptor agonist without fusion partner.Methods of making and using such viral vectors are also provided.

[0004] In one aspect, a viral vector is provided that comprises a nucleic acid comprising a polynucleotide sequence encoding a fusion protein. The fusion protein comprises (a) a leader sequence comprising a secretory signal peptide, (b) a glucagon-like peptide-1 (GLP-1) receptor agonist, and (c) a fusion domain comprising an IgG Fc or a functional variant thereof. In one embodiment, the vector is an adeno-associated viral vector.

[0005] In one embodiment, (i) the secretory signal peptide of the leader sequence comprises a thrombin signal peptide, (ii) the leader sequence comprises a thrombin propeptide, and / or (iii) the leader sequence comprises a thrombin leader sequence.

[0006] In another embodiment, the viral vector comprises an AAV capsid and a vector genome packaged within the AAV capsid, the vector genome comprising AAV inverted terminal repeats (ITRs), a polynucleotide sequence encoding a fusion protein, and regulatory sequences that direct expression of the fusion protein.

[0007] In another aspect, a pharmaceutical composition suitable for use in treating a metabolic disease in a subject is provided. The composition comprises an aqueous liquid and a viral vector as described herein. In one embodiment, the subject is a human.

[0008] In yet another aspect, the use of the viral vectors described herein is directed to the treatment of metabolic diseases, any Optionally, the method is provided for the manufacture of a medicament for treating a subject with diabetes.

[0009] In another aspect, a method of treating a subject having a metabolic disease is provided, the method comprising administering to the subject an effective amount of a viral vector or composition described herein.

[0010] Other aspects and advantages of the present invention will become readily apparent from the following detailed description of the invention. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram of dulaglutide. [Diagram 2] Inducible hGLP-1-Fc vs. CB7. GLP-1-Fc in vitro. GLP1-Fc fusions were measured in culture supernatants of plasmid-transfected HEK293 cells for inducible human GLP-1-Fc with human thrombin signal sequence (TF.GT2A.GLP-1-Fc) and CB7 feline dulaglutide with feline thrombin signal sequence (CB7.feGLP-1-Fc). Supernatants were collected 48 hours after treatment with rapamycin (Rapa) at 0, 4, and 40 nM, or 48 hours after transfection with CB7.feGLP-1-Fc. GLP1-Fc was quantified by active GLP1 ELISA with the STD of the kit. [Diagram 3] Figure 1 shows inducible expression of GLP-1 in Rag1KO (RAG1- / -) mice (n=5 / vector). Rag1KO female mice were administered 1x1011GC / mouse via intramuscular (IM or IM) delivery of the indicated vectors (i.e., AAVrh91.TF.hGLP-1-Fc.3w.rBG and AAVrh91.TF.rhGLP-1-Fc.3w.rBG). Blood was collected weekly. A GLP1 ELISA specific for active GLP-1 was performed. AAV vectors were injected on day 0 and rapamycin was administered by oral gavage around days 14 and 15 after AAV injection. [Figure 4] Schematic representation of the plasmid map of pAAV.CMV.TF.GT2A.hGLP-1-Fc.3w.rBG. [Diagram 5] 1 shows AAV-mediated expression of engineered GLP-1 constructs in mice. [Figure 6A] FIG. 1 shows a schematic diagram of an exemplary expression cassette containing an inducible construct for use in a two-vector system. [Figure 6B] FIG. 1 shows a schematic diagram of an expression cassette containing inducible construct for use in a one-vector system containing an IRES linker. [Figure 7A]FIG. 1 shows a schematic diagram of an expression cassette containing an inducible construct for use in the one-vector system, comprising human GLP1-Fc with an F2A cleavage sequence linker and secretion signal. [Figure 7B] Further details of the GT2A truncation sequences are shown, where GT2A_V1 comprises the amino acid sequence of SEQ ID NO:21 and GT2A_V2 comprises the amino acid sequence of SEQ ID NO:22. [Figure 8] FIG. 1 shows expression of an exemplary therapeutic transgene (rhTT) in rhesus monkeys in HEK293 cell supernatants measured after transfection with various constructs and treatment with rapamycin at 0 nM, 4 nM, and 40 nM, and plotted as IU of rhTT / mL. [Figure 9] Figure 1 shows the expression of inducible human (h) and rhesus (rh) GLP-1 in vitro. GLP1-Fc fusions were measured in culture supernatants of HEK293 cells transfected with plasmids for inducible hGLP-1-Fc (containing a thrombin signal sequence), rhGLP-1-Fc containing the two-vector system, and CB7.rhGLP-1-Fc. Cells were seeded on day 0, transfected on day 1, and treated with rapamycin at 0 nM, 4 nM, and 40 nM on day 2, and supernatants from cells were collected on day 4 or 48 hours after transfection with CB7.rhGLP-1-Fc, and GLP1-Fc was quantified by active GLP1 ELISA with the STD from the kit. [Figure 10A] Figure 1 shows expression and analysis of rhGLP1-Fc in anti-rhGLP1-Fc ADA (anti-drug antibody) detection assay in NHP1(18-128). Plotted serum rhGLP1-Fc expression levels are shown in nM, measured from days 0 to 200. [Figure 10B] Figure 1 shows expression and analysis of rhGLP1-Fc in an anti-rhGLP1-Fc ADA (anti-drug antibody) detection assay in NHP1(18-128). Plotted serum rapamycin levels are shown as μg / L, measured from days 0 to 200. [Figure 10C]Figure 1 shows expression and analysis of rhGLP1-Fc in anti-rhGLP1-Fc ADA (anti-drug antibody) detection assay of NHP1(18-128). Plotted ADA detection assay results measured from days 0 to 200 are shown as OD 450 nm. [Figure 11A] Figure 1 shows expression and analysis of rhGLP1-Fc in anti-rhGLP1-Fc ADA assay of NHP1(18-072). Plotted serum rhGLP1-Fc expression levels are shown in nM, measured from days 0-200. [Figure 11B] Figure 1 shows expression and analysis of rhGLP1-Fc in anti-rhGLP1-Fc ADA assay of NHP1(18-072). Plotted serum rapamycin levels are shown as μg / L measured from days 0 to 200. [Figure 11C] Figure 1 shows expression and analysis of rhGLP1-Fc in anti-rhGLP1-Fc ADA assay of NHP1(18-072). Plotted ADA detection assay results measured from days 0 to 200 are shown as OD 450 nm. [Figure 12A] Figure 1 shows expression and analysis of anti-rhGLP1-Fc ADA assay of NHP1 (18-013). Plotted serum rhGLP1-Fc expression levels are shown in nM, measured from days 0-200. [Figure 12B] Figure 1 shows expression and analysis of anti-rhGLP1-Fc ADA assay of NHP1(18-013). Plotted serum rapamycin levels are shown as μg / L measured from days 0 to 200. [Figure 12C] Figure 1 shows expression and analysis of anti-rhGLP1-Fc ADA assay of NHP1 (18-013). Plotted ADA detection assay results measured from days 0 to 200 are shown as OD 450 nm. [Figure 12D] Figure 1 shows expression and analysis of anti-rhGLP1-Fc ADA assay in NHP1(18-013). Figure 2 shows long-term rhGLP-1 expression in NHP1(18-013). Arrow indicates administration of rapamycin. [Figure 12E] Figure 1 shows expression and analysis of anti-rhGLP1-Fc ADA assay of NHP1 (18-013). Blood rapamycin levels (ng / mL) are shown. [Figure 13] 1 shows the experimental design of the experiment described in Example 5. [Figure 14] Figure 1 shows rhGLP1-Fc expression and analysis of anti-rhGLP1-Fc ADA assays in animals treated using the constitutive promoter. A shows plotted rhGLP1-Fc expression levels in serum as nM measured from days 0 to 300. B shows plotted ADA detection assay results as OD 450 nm measured from days 0 to 230. [Figure 15] Figure 1 shows expression of rhGLP1-Fc and analysis of anti-rhGLP1-Fc ADA assays in animals treated with IM using a two-vector inducible promoter system. A shows plotted rhGLP1-Fc expression levels in serum as nM measured from 0 to approximately 120 days. B shows plotted ADA detection assay results as OD 450 nm measured from 0 to 230 days. Arrows indicate administration of rapamycin. [Figure 16] Figure 1 shows rhGLP1-Fc expression and analysis of anti-rhGLP1-Fc ADA assays from animals treated using a one-vector inducible promoter system. A shows plotted rhGLP1-Fc expression levels in serum as nM measured from 0 to about 120 days. B shows plotted ADA detection assay results as OD 450 nm measured from 0 to 230 days. [Figure 17]

[0036] Figure 1 shows the results of a potency assay of the GLP-1-Fc transgene product. Purified human and rhesus GLP-1-Fc were compared to pharmacy-derived dulaglutide (Trulicity), with the human constructs described herein demonstrating comparable or better efficacy than Trulicity. [Figure 18A] Plotted serum hGLP1-Fc expression levels measured from days 0 to 60 are shown in nM. [Figure 18B]The potency of hGLP-1-Fc from NHP plasma sampled on days 0-60 is shown. [Figure 18C] Plotted serum rhGLP1-Fc expression levels measured from days 0 to 80 are shown in nM. [Figure 18D] Body weights of the two NHPs in the study are shown. [Figure 18E] Blood glucose levels (mg / dL) are shown. Reference blood glucose levels for rhesus monkeys are 63-130 mg / dL. Animal 18-007 was asymptomatic and both animals had relatively low baseline BG on day 0. At day 60, no ADA was detected. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Long-acting GLP-1 receptor agonist fusion protein expression constructs have been developed for use in subjects in need thereof, including humans. Leader sequences containing secretory signal peptides are provided, as well as fusion domains intended to extend the circulation time of the resulting fusion protein.

[0013] The delivery of these constructs to a subject in need thereof is described through a number of routes, particularly by in vivo expression mediated by a recombinant vector, such as an rAAV vector. Methods of using these constructs in a regimen in a subject in need of treating diabetes or metabolic syndrome to increase the half-life of GLP-1 in the subject are also provided. Additionally, methods are provided for enhancing the activity of GLP-1 in a subject. Methods are also provided for inducing weight loss in a subject in need thereof.

[0014] GLP-1 fusion protein Glucagon-like peptide 1 or GLP-1 is an incretin derived from the transcription product of the proglucagon gene. In vivo, the glucagon gene expresses a 180 amino acid prepropolypeptide that is proteolytically processed to form glucagon, which is in two forms: GLP-1 and GLP-2. The original sequencing studies showed that GLP-1 has 37 amino acid residues. However, subsequent information showed that this peptide is a propeptide and is further processed by removing six amino acids from the amino terminus to form GLP-1(7-37), the active form of GLP-1. The glycine at position 37 is also transformed to an amide in vivo to form GLP-1(7-36)amide. GLP-1(7-37) and GLP-1(7-36)amide are insulin-stimulating hormones with comparable potency. Thus, as used herein, the biologically "active" forms of GLP-1 that are useful herein are GLP-1-(7-37) and GLP-1-(7-36)NH 2 It is.

[0015] GLP-1 receptor agonists are a type of anti-diabetic drug that mimics the action of glucagon-like peptides. GLP-1 is one of several naturally occurring incretin compounds that affect the body after being released from the intestine during digestion. By binding to and activating the GLP-1 receptor, GLP-1 receptor agonists can lower blood glucose levels, which helps T2DM patients achieve glycemic control. As used herein, the term "GLP-1 receptor agonist" refers to at least GLP-1 or a functional fragment thereof, amino acid sequence variants of GLP-1 or functional fragments thereof, and other polypeptide agonists of the GLP-1 receptor (e.g., exedin-4 and variants thereof). The present disclosure relates to one or more copies of GLP-1 receptor agonists, as well as such fusions. A fusion protein is provided comprising a polynucleotide encoding a protein and a vector. In some embodiments, the fusion protein comprises a polynucleotide sequence encoding a fusion protein comprising (a) a leader sequence comprising a secretory signal peptide, (b) a glucagon-like peptide-1 (GLP-1) receptor agonist, and (c) a fusion domain. In one embodiment, the GLP-1 receptor agonist comprises a thrombin leader sequence, a GLP-1 receptor agonist, and an IgG Fc or a functional variant thereof.

[0016] In some embodiments, the GLP-1 receptor agonist includes variants that retain the function of the wild-type sequence and may include up to about 10% variation from the GLP-1 nucleic acid or amino acid sequence described herein or known in the art. As used herein, "retaining function" means that the nucleic acid or amino acid functions in the same manner as the wild-type sequence, although not necessarily at the same level of expression or activity. For example, in one embodiment, the functional variant has increased expression or activity compared to the wild-type sequence. In another embodiment, the functional variant has decreased expression or activity compared to the wild-type sequence. In one embodiment, the functional variant has increased or decreased expression or activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the wild-type sequence.

[0017] Several human drugs are known in the art that fuse a GLP-1 receptor agonist to a stabilized fusion domain. These include albiglutide, liraglutide, dulaglutide, and lixisenatide (also known by the chemical name des-38-proline-exendin-4(Heloderma suspectum)-(1-39)-peptidylpenta-L-lysyl-L-lysinamide). Dulaglutide is a disulfide-linked homodimeric fusion peptide, with each monomer consisting of one GLP-1 analog moiety and one IgG4 Fc region. Yu M, et al.(2018)Battle of GLP-1 delivery technologies, Adv. Drug Deliv. Rev. A schematic diagram of dulaglutide is shown in Figure 1. See WO2005 / 000892A2, incorporated herein by reference.

[0018] In one embodiment, the fusion comprises a GLP-1 analogue in combination with a heterologous sequence. GLP-1 analogue refers to a polypeptide that shares at least 90%, 95%, 97%, 98%, 99%, or 100% identity with native human GLP-1(7-37). In one embodiment, the GLP-1 analogue has up to 1, 2, or 3 amino acid substitutions compared to the native sequence. Native human GLP-1(1-37) has the sequence HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 1), and GLP-1(7-37) has the sequence HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 2). In some embodiments, it is desirable to modify the native GLP-1 sequence to optimize one or more of its characteristics. For example, in one embodiment, the GLP-1 analogue comprises 1, 2, or 3 amino acid substitutions selected from A8G, G22E, and R36G compared to the native sequence. These substitutions have been shown to improve the efficacy of the clinical profile of GLP-1 (A8G), including protection from DPP-4 inactivation, increased solubility (G22E), and reduced immunogenicity by replacing the arginine at position 36 (R36G) with a glycine residue to remove a potential T cell epitope. In one embodiment, the GLP-1 analog is a DPP-IV resistant variant of GLP-1. In one embodiment, the GLP-1 analog has a sequence comprising or consisting of SEQ ID NO: 3: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG. The GLP-1 analog albirglutide has a sequence as set forth in SEQ ID NO: 4. The GLP-1 analog exendin-4 has a sequence as set forth in SEQ ID NO: 5. The GLP-1 analog des-38-proline-exendin-4 (Heloderma suspectum)-(1-39)-peptidyl penta-L-lysyl-L-lysine amide has a sequence as set forth in SEQ ID NO: 6. In an embodiment, more than one copy of the GLP-1 analog is present in the fusion protein. In another embodiment, the GLP-1 receptor agonist is two tandem copies of GLP-1(7-37) or its DPP-IV resistant variant.

[0019] A fusion protein may include a leader sequence, which may include a secretory signal peptide. As used herein, the term "leader sequence" refers to any N-terminal sequence of a polypeptide.

[0020] The leader sequence may be derived from the same species as that for which administration is ultimately intended, e.g., human. As used herein, the term "derived" or "derived from" means that the sequence or protein is sourced from or shares the same sequence as a protein or sequence sourced from a particular subject species. For example, a leader sequence "derived" from a human shares the same sequence (or a variant thereof as defined herein) as the same leader sequence expressed in a human. However, the specified nucleic acid or amino acid need not actually be sourced from a human. A variety of techniques are known in the art that can produce a desired sequence, including mutagenesis of a similar protein (e.g., a homologue), or artificial production of a nucleic acid or amino acid sequence. A "derived" nucleic acid or amino acid retains the function of the same nucleic acid or amino acid in the species from which it is "derived", regardless of the actual source of the derived sequence.

[0021] The term "amino acid substitution" and its synonyms are intended to encompass the modification of an amino acid sequence by replacing an amino acid with another alternative amino acid. The substitution may be a conservative substitution. It may also be a non-conservative substitution. The term conservative, when referring to two amino acids, is intended to mean that the amino acids share a common property recognized by those of skill in the art. For example, amino acids with hydrophobic non-acidic side chains, amino acids with hydrophobic acidic side chains, amino acids with hydrophilic non-acidic side chains, amino acids with hydrophilic acidic side chains, and amino acids with hydrophilic basic side chains. The common property may also be amino acids with hydrophobic side chains, amino acids with aliphatic hydrophobic side chains, amino acids with aromatic hydrophobic side chains, amino acids with polar neutral side chains, amino acids with charged side chains, amino acids with charged acidic side chains, and amino acids with charged basic side chains. Both naturally occurring and non-naturally occurring amino acids are known in the art and may be used as alternative amino acids in embodiments. Methods for substituting amino acids are well known to those of skill in the art and include, but are not limited to, mutations in the nucleotide sequence encoding the amino acid sequence. References herein to "one or more" are intended to encompass, for example, 1, 2, 3, 4, 5, 6 or more individual embodiments.

[0022] In one embodiment, the leader is a human thrombin (Factor II) sequence. In one embodiment, the thrombin leader has the sequence shown in SEQ ID NO: 7: MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRR, or a functional variant thereof having up to 1, 2, or 3 amino acid substitutions. In some embodiments, the leader comprises a signal peptide and a propeptide. In one embodiment, the secretory signal peptide of the leader sequence comprises a human thrombin signal peptide. In one embodiment, the signal peptide is MAHVRGLQLPGCLALAALCSLVHS (SEQ ID NO: 8), or a functional variant thereof having up to 1, 2, or 3 amino acid substitutions. In another embodiment, the leader sequence comprises a human thrombin propeptide. In one embodiment, the propeptide has the sequence QHVFLAPQQARSLLQRVRR (SEQ ID NO: 9), or a functional variant thereof having up to 1, 2, or 3 amino acid substitutions.

[0023] In one embodiment, a functional variant of the desired leader retains the function of the wild-type sequence. , including variants that may contain up to about 10% variation from the leader nucleic acid or amino acid sequences described herein or known in the art.

[0024] In some embodiments, the coding regions for both the propeptide and the GLP-1 peptide are incorporated into a single nucleic acid sequence with no linker between the coding sequences for the propeptide and the GLP-1 peptide.

[0025] The fusion protein further comprises a fusion domain, which in one embodiment is a human IgG Fc fragment or a functional variant thereof. Immunoglobulins typically have a long circulating half-life in vivo. By fusing the GLP-1 receptor agonist (and leader) to the IgG Fc, the circulation time of the fusion protein is extended while maintaining the function of GLP-1. In another embodiment, the fusion domain is a rhesus IgG Fc fragment or a functional variant thereof.

[0026] As used herein, the Fc portion of an immunoglobulin has the meaning commonly given to the term in the field of immunology. Specifically, the term refers to an antibody fragment that does not contain the two antigen-binding regions (Fab fragments) from the antibody. The Fc portion consists of the constant regions of the antibody from both heavy chains, which are associated by non-covalent interactions and disulfide bonds. The Fc portion includes the hinge region and may extend through the CH2 and CH3 domains to the c-terminus of the antibody. The Fc portion may further include one or more glycosylation sites. In one embodiment, the fusion domain is human IgG Fc. The four highly conserved subclasses, IgG1, IgG2, IgG3, and IgG4, differ in their constant regions, especially their hinge and upper CH2 domains. See Vidarsson et al, IgG Subclasses and Allotypes: From Structure to Effector Functions, Front Immunol. Oct. 2014; 5:520, which is incorporated herein by reference. The Fc domain can be derived from any human IgG, including human IgG1, human IgG2, human IgG3, or human IgG4. In one embodiment, the human IgG Fc is an IgG4 Fc. In one embodiment, the human IgG Fc is represented by SEQ ID NO: 11: AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG. In another embodiment, the human IgG Fc shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity to SEQ ID NO:11.

[0027] In another embodiment, the fusion domain is a Rhesus IgG Fc. The Fc domain can be derived from any Rhesus IgG, including Rhesus IgG1, Rhesus IgG2, Rhesus IgG3, or Rhesus IgG4. In one embodiment, the Rhesus IgG Fc is an IgG4 Fc. In one embodiment, the Rhesus IgG Fc is SEQ ID NO: 17: PPCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAQTKPRE RQFNSTYRVV SVLTVTHQDW LNGKEYTCKV SNKGLPAPIE KTISKAKGQP REPQVYILPP PQEELTKNQV SLTCLVTGFY PSDIAVEWES NGQPENTYKT TPPVLDSDGS YLLYSKLTVN KSRWQPGNIF TCSVMHEALH NHYTQKSL SV SPGK. In another embodiment, the Rhesus IgG Fc shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity to SEQ ID NO: 17. In one embodiment, the Rhesus IgG further comprises a hinge sequence.

[0028] The in vivo function and stability of the fusion protein of the present disclosure may be optimized by adding a small amount of peptide linker, for example, to prevent potentially undesirable domain interactions or for other reasons. Furthermore, the glycine-rich linker may provide some structural flexibility so that the GLP-1 analog moiety can productively interact with the GLP-1 receptor on target cells, such as pancreatic beta cells. Thus, the C-terminus of the GLP-1 analog and the N-terminus of the fusion domain of the fusion protein are fused via a linker in one embodiment. In one embodiment, the linker comprises 1, 1.5, or 2 repeats of a G-rich peptide linker having the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 13).

[0029] In one embodiment, the fusion protein comprises (a) a human thrombin leader, (b) a DPP-IV resistant variant of GLP-1(7-37), a linker, and (c) a human IgG Fc. In one embodiment, the fusion protein has a sequence of SEQ ID NO: 14, or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. SEQ ID NO:14: MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0030] In one embodiment, the sequence encoding the fusion protein is SEQ ID NO: 15, or a sequence at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. SEQ ID NO:15: atggctcacgttcgaggactgcagctgcctggatgtctggctcttgccgctctgtgtagcctggtgcacagccagcacgtgtttctggctcctcagcaagccagatcactgctgcagagagttagaaggcacggcgagggcacctttacctccgacgtgtctagctacctggaagaacaggccgccaaagagtttatcgcctggctggtcaaaggtggcggcggaggcggaggaagcggtggcggaggttcaggtggtggtggatctgccgagtctaagtacggccctccttgtcctccctgtcctgctcccgaagctgctggcggcccatccgtgtttctgttccctccaaagcctaaggacaccctgatgatcagcagaacccctgaagtgacctgcgtggtggtcgacgtgtcccaagaggatcctgaggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacagttcaacagcacctacagagtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgcctagctccatcgagaaaaccatcagcaaggccaagggcc agccaagagaaccccaggtgtacacactgcctccaagccaagaggaaatgaccaagaaccaggtgtccctgacctgcctcgtgaagggcttctacccttccgatatcgccgtggaatgggagagcaatggccagcctgagaacaactacaagaccacacctcctgtgctggacagcgacggctcattcttcctgtacagcagactgaccgtggacaagagcagatggcaagagggcaacgtgttcagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagtctctgagcctgagcctgggc

[0031] In one embodiment, the fusion protein comprises (a) a human thrombin leader, (b) a DPP-IV resistant variant of GLP-1(7-37), a linker, and (c) a rhesus IgG In one embodiment, the fusion protein comprises (a) a human thrombin leader, (b) a DPP-IV-resistant variant of GLP-1(7-37), a linker, and (c) a rhesus IgG Fc.

[0032] In one embodiment, the fusion protein has the sequence of SEQ ID NO: 37, or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. SEQ ID NO:37 MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQALSLLQRVRRHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSAEFTPPCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAQ TKPRERQFNSTYRVVSVLTVTHQDWLNGKEYTCKVSNKGLPAPIEKTISKAKGQPREPQVYILPPPQEELTKNQVSLTCLVTGFYPSDIAVEWESNGQPENTYKTTPPVLDSDGSYLLYSKLTVNKSRWQPGNIFTCSVMHEALHNHYTQKSLSVSPG

[0033] In one embodiment, the sequence encoding the fusion protein is SEQ ID NO: 36, or a sequence at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. SEQ ID NO:36 atggctcacgttcgaggactgcagctgcctggatgtctggctcttgccgctctgtgtagcctggtgcacagccagcatgtgtttctggctcctcaacaagccctgagcctgctgcaaagagttagaaggcacggcgagggcaccttcacctccgacgtgtccagctacctggaagaacaggccgccaaagagtttatcgcctggctggtcaaaggcggtggtggtggcggaggatctggcggaggtggaagcggcggaggcggatctgctgagtttacacctccttgtcctccctgtcctgctcccgagctgctcggaggcccttccgtgtttctgttccctccaaagcctaaggacaccctgatgatcagcagaacccctgaagtgacctgcgtggtcgtggacgtgtcccaagaggatcctgaggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgcccagacaaagcccagagagcggcagttcaacagcacctacagagtggtgtccgtgctgaccgtgacacaccaggattggctgaacggcaaagagtacacctgtaaagtctccaacaagggcctgcctgctcctatcgagaaaaccatcagcaaggccaagggccagcctagagaaccccaggtgtacatcctgcctccacctcaagaggaactgaccaagaaccaggtgtccctgacctgtctggtcacc ggcttctacccttccgatatcgccgtggaatgggagagcaacggacagcccgagaacacctacaagaccacacctccagtgctggacagcgacggcagctatctgctgtactccaagctgacagtgaacaagagccggtggcagcccggcaacatcttcacctgttctgtgatgcacgaggccctgcacaaccactacacccagaagtctctgagcgtcagccctggc

[0034] If variants or fragments of the leader sequence, GLP-1 receptor agonist, or fusion domain are desired, the coding sequences of these peptides can be generated using site-directed mutagenesis of the wild-type nucleic acid sequence. Alternatively or additionally, web-based or commercially available computer programs and service-based companies can be used to back-translate the amino acid sequence into a nucleic acid coding sequence, including both RNA and / or cDNA. See, for example, backtranseq by EMBOSS, ebi.ac.uk / Tools / st / ; Gene Infinity (geneinfinity.org / sms- / sms_backtranslation.html); ExPasy (expasy.org / tools / ). In one embodiment, the RNA coding sequence and / or the cDNA coding sequence are designed for optimal expression in the subject species, e.g., human, to which administration is ultimately intended.

[0035] Coding sequences can be designed for optimal expression using codon optimization. Codon-optimized coding regions can be designed by a variety of different methods. This optimization can be done using methods available online, published methods, or companies that provide codon optimization services. One codon optimization method is described, for example, in International Patent Application Publication No. 2015 / 012924, which is incorporated herein by reference. Briefly, a nucleic acid sequence that encodes a product is modified with synonymous codon sequences. Preferably, the entire length of the product's open reading frame (ORF) is modified. However, in some embodiments, only a fragment of the ORF may be modified. By using one of these methods, a frequency can be applied to any given polypeptide sequence to produce a nucleic acid fragment of a codon-optimized coding region that encodes a polypeptide.

[0036] In addition to the leader sequences, GLP-1 receptor agonists, fusion domains, and fusion proteins provided herein, nucleic acid sequences encoding these polypeptides are provided. In one embodiment, a nucleic acid sequence encoding a GLP-1 fusion protein as described herein is provided. In another embodiment, this includes any nucleic acid sequence encoding the GLP-1 fusion protein of SEQ ID NO: 14.

[0037] Expression cassette In another aspect, an expression cassette is provided herein that includes a nucleic acid encoding a GLP-1 fusion protein as described herein. As used herein, "expression cassette" refers to a nucleic acid molecule that includes a biologically useful nucleic acid sequence and a regulatory sequence operably linked thereto that directs or regulates the transcription, translation, and / or expression of the nucleic acid sequence (e.g., a gene cDNA encoding a protein, enzyme, or other useful gene product, mRNA, etc.) and its gene product. As used herein, "operably linked" sequence includes both regulatory sequences (also referred to as elements) that are contiguous or non-contiguous with the nucleic acid sequence and regulatory sequences that act in trans or cis with the nucleic acid sequence. Such regulatory sequences typically include one or more of, for example, promoters, enhancers, transcription factors, transcription terminators, introns, sequences that increase translation efficiency (i.e., Kozak consensus sequences), efficient RNA processing signals such as slicing and polyadenylation sequences, sequences that stabilize cytoplasmic mRNA, such as the Woodchuck Hepatitis Virus (WHP) post-translational regulatory element (WPRE), and TATA signal. The expression cassette contains, among other elements, a gene sequence upstream ( The expression cassette may include one or more 5'-regulatory sequences, such as one or more of a promoter, enhancer, intron, etc., and enhancers, or downstream (3'-) regulatory sequences of the gene sequence, such as one or more 3'-untranslated regions (3'UTRs) including polyadenylation sites. In certain embodiments, the regulatory sequences are operably linked to the nucleic acid sequence of the gene product, and the regulatory sequences are separated from the nucleic acid sequence of the gene product by an intervening nucleic acid sequence, i.e., a 5'-untranslated region (5'UTR). In certain embodiments, the expression cassette includes one or more nucleic acid sequences of the gene product. In some embodiments, the expression cassette may be a monocistronic or bicistronic expression cassette. In other embodiments, the term "transgene" refers to one or more DNA sequences from an exogenous source that are inserted into a target cell.

[0038] In one embodiment, an expression cassette refers to a nucleic acid molecule that includes a GLP-1 construct coding sequence (e.g., a coding sequence for a GLP-1 fusion protein), a promoter, and may include other regulatory sequences therefor, which may be engineered into a genetic element and / or packaged into a viral vector capsid (e.g., a viral particle). Typically, such an expression cassette for producing a viral vector includes a GLP-1 construct sequence as described herein adjacent to a packaging signal of the viral genome (and referred to as the "vector genome"), and other expression control sequences, such as those described herein. Any of the expression control sequences may be optimized for a particular species using techniques known in the art, including, for example, codon optimization, as described herein.

[0039] In certain embodiments, the expression cassette comprises a constitutive promoter. In another embodiment, CB7 is used. CB7 is a chicken beta actin promoter with a cytomegalovirus enhancer element. In some embodiments, the CB7 promoter has the nucleic acid sequence of SEQ ID NO: 33. In one embodiment, the promoter is a CMV promoter. In some embodiments, the CMV promoter is the nucleic acid sequence of SEQ ID NO: 27.

[0040] In one embodiment, the promoter is included in an inducible gene expression system. The inducible gene regulation / expression system includes at least the following components: a promoter (also referred to as a regulatable promoter) operably linked to a transgene encoding a GLP-1 fusion protein as described herein, an activation domain, a DNA binding domain, and a zinc finger homeodomain binding site(s). In other embodiments, additional components may be included in the expression system, as further described herein. A plasmid showing the design of an exemplary inducible expression system is shown in FIG. 4.

[0041] The system includes a promoter upstream of the coding sequence of the GLP-1 fusion protein. Promoters described herein, such as CMV and CB7 promoters, can be used. In one embodiment, the promoter is a CMV promoter as shown in SEQ ID NO: 27. In one embodiment, the promoter is a ubiquitous inducible promoter Z12I, which contains 12 repeated copies of binding sites for ZFHD1 and an IL2 minimal promoter. See, for example, Chen et al, Hum Gene Ther Methods. 2013 Aug; 24(4): 270-278 (incorporated herein).

[0042] The expression system includes an activation domain, which is preferably located upstream of the DNA binding domain. In one embodiment, the activation domain is a fusion of the carboxy terminus from the p65 subunit of NF-kappa B with the FKBP12-rapamycin binding (FRB) domain of FKBP12-rapamycin related protein (FRAP). In one embodiment, the activation domain is the FKBP12 of human FKBP12-rapamycin related protein (FRAP) fused to the carboxy terminus from the p65 subunit of NF-kappa B from human. -rapamycin binding (FRB) domain. In one embodiment, the FRB domain has the amino acid sequence set forth in SEQ ID NO: 24. In one embodiment, the FRB domain has the amino acid sequence set forth in SEQ ID NO: 24 encoded by the nucleic acid sequence of SEQ ID NO: 23. In one embodiment, the p65 subunit has the sequence set forth in SEQ ID NO: 26. In one embodiment, the p65 subunit has the sequence set forth in SEQ ID NO: 26 encoded by the nucleic acid sequence of SEQ ID NO: 25.

[0043] The inducible system can be contained in a single vector containing the coding sequence for the fusion protein, or in a two-vector system. Examples of two-vector (FIG. 6A) and one-vector (FIGS. 6B and 7A) systems incorporating GLP1 fusion proteins are described herein.

[0044] In one embodiment, there is a linker between the transactivation domain and the DNA binding domain, and the linker can be F2A or IRES. In one embodiment, the linker is selected from an IRES or a 2A peptide. In one embodiment, the linker is a cleavable 2A peptide. In one embodiment, the linker comprises a GT2A_V1 peptide comprising the amino acid sequence of SEQ ID NO: 21. In one embodiment, the linker comprises a GT2A_V2 peptide comprising the amino acid sequence of SEQ ID NO: 22. In one embodiment, the 2A peptide is selected to increase the packaging limit to allow for a single vector system.

[0045] The DNA-binding domain consists of a DNA-binding fusion of zinc finger homeodomain 1 (ZFHD1) joined to up to three copies of FK506-binding protein (FKBP). In the presence of an inducer, e.g., a rapalog such as rapamycin, the DNA-binding domain and the activation domain dimerize through the interaction of their FKBP and FRB domains, resulting in transcriptional activation of the transgene. In some embodiments, ZFHD1 is included in frame with GT2A or IRES. In one embodiment, ZFHD1 has the sequence shown in SEQ ID NO:29. In one embodiment, ZFHD1 has the sequence of SEQ ID NO:28, encoded by the nucleic acid sequence of SEQ ID NO:28.

[0046] The expression system is designed to have one, two, or three copies of the FKBP sequence. These are referred to herein as FKBP subunits. In one embodiment, the subunits are designed to express the same protein, but with different nucleic acids from each other, to minimize recombination. For example, SEQ ID NO:30 provides three "wobbled" coding sequences for FKBP, each of which is represented by SEQ ID NO:31: It encodes the sequence shown in GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE.

[0047] The expression system further comprises a zinc finger homeodomain binding site. The nucleic acid molecule comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 binding sites for a ZFHD. In one embodiment, the expression system comprises eight zinc finger homeodomain binding sites (binding partners) (8XZFHD). However, the invention encompasses expression systems having from 2 to about 12 copies of a zinc finger binding site. An example of a single copy ZFHD binding site is aatgatgggcgctcgagt (SEQ ID NO: 32).

[0048] In some embodiments, there is a minimal IL2 promoter downstream of the zinc finger homeodomain binding site. An exemplary IL2 promoter is shown in SEQ ID NO:10.

[0049] Such inducible systems are known in the art and are described, for example, in Rivera et al., humanized system for pharmacological control of gene expression, Nature Medicine volume 2, pages 1028-1032 (September 1996) and Rivera et al, Long-term pharmacologically regulated expression of erythropoietin in primates following AAV-mediated gene transfer, Blood, 15 February 2005, volume 105, number 4, both of which are incorporated herein by reference. In one embodiment, the inducible gene expression system comprises a CMV promoter and the activation domain is the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin related protein (FRAP) fused to the carboxy terminus from the p65 subunit of NF-kappa B of human origin, the GT2A peptide, the ZFHD1 DNA binding domain, three FKBP subunits, hGH polyA, 8XZFHD, and a minimal sIL2 promoter. These sequences are added to the coding sequence for the GLP-1 fusion protein, and optionally other regulatory sequences.

[0050] In addition to the promoter, the expression cassette and / or vector may include suitable transcription initiation, termination, and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals, sequences that stabilize cytoplasmic mRNA, sequences that increase translation efficiency (i.e., Kozak consensus sequences), sequences that enhance protein stability, and, if necessary, sequences that enhance secretion of the encoded product. Examples of suitable polyA sequences include, for example, SV40, bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit β-globin (also called rabbit globin polyA; RGB), modified RGB (mRGB), and TK polyA. Examples of suitable enhancers include, for example, alpha fetoprotein enhancer, TTR minimal promoter / enhancer, LSP (TH binding globulin promoter / alpha 1-microglobulin / bikunin enhancer), among others. In one embodiment, the polyA is rabbit globin polyA.

[0051] These control sequences are "operably linked" to the GLP-1 construct sequence. As used herein, the term "operably linked" refers to both expression control sequences that are contiguous with a gene of interest, and expression control sequences that act in trans or at a distance to control the gene of interest.

[0052] In one embodiment, the 5' ITR, CB7 promoter, chicken beta-actin intron, coding sequence for the fusion protein of SEQ ID NO: 14, rabbit globin polyA, and 3' A rAAV comprising ITRs is provided. In another embodiment, the rAAV comprises a polynucleotide comprising a CMV promoter, an activation domain is the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin related protein (FRAP) fused to the carboxy terminus from the p65 subunit of NF-kappa B from human, a GT2A peptide, a ZFHD1 DNA binding domain, three FKBP subunits, hGH polyA, 8XZFHD, a minimal sIL2 promoter, a coding sequence for a GLP-1 fusion protein of SEQ ID NO: 14, and a rabbit beta globin polyA.

[0053] In one embodiment, an expression cassette is provided that comprises a polynucleotide comprising a CB7 promoter, a chicken beta-actin intron, a coding sequence for a fusion protein of SEQ ID NO: 14, and a rabbit globin polyA. In one embodiment, the expression cassette is that found in SEQ ID NO: 34, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity therewith. In another embodiment, the expression cassette is that found in SEQ ID NO: 34, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity therewith. Vector genomes are provided that are flanked by 5' and 3' AAV ITRs with sequences that share 9% or 100% identity.

[0054] In another embodiment, an expression cassette is provided that comprises a polynucleotide comprising a CB7 promoter, a chicken beta-actin intron, a coding sequence for a fusion protein of SEQ ID NO: 37, and a rabbit globin polyA. In one embodiment, the expression cassette is that found in SEQ ID NO: 35, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity therewith. In another embodiment, a vector genome is provided in which SEQ ID NO: 35, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity therewith, is flanked by 5' and 3' AAV ITRs.

[0055] In another embodiment, an expression cassette is provided that comprises a polynucleotide comprising a CMV promoter, the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin related protein (FRAP) fused to the carboxy terminus from the p65 subunit of NF-kappa B from human, a GT2A peptide, a ZFHD1 DNA binding domain, three FKBP subunits, 8XZFHD, a minimal IL2 promoter, a coding sequence for a GLP-1 fusion protein of SEQ ID NO: 14, and a rabbit beta globin poly A. In one embodiment, the expression cassette is that found in SEQ ID NO: 38 or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity thereto. In another embodiment, a vector genome is provided in which SEQ ID NO:38, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity thereto, is flanked by the 5' and 3' AAV ITRs.

[0056] In another embodiment, an expression cassette is provided that comprises a polynucleotide comprising a CMV promoter, the FKBP12-rapamycin binding (FRB) domain of human or rhesus FKBP12-rapamycin related protein (FRAP) fused to the carboxy terminus from the p65 subunit of NF-kappa B from human or rhesus, a GT2A peptide, a ZFHD1 DNA binding domain, three FKBP subunits, 8XZFHD, a minimal IL2 promoter, a coding sequence for a GLP-1 fusion protein of SEQ ID NO:37, and a rabbit beta globin poly A. In one embodiment, the expression cassette is that found in SEQ ID NO:39 or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity thereto. In another embodiment, a vector genome is provided in which SEQ ID NO:39, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity thereto, is flanked by the 5' and 3' AAV ITRs.

[0057] In another embodiment, an expression cassette is provided that includes a Z12I promoter (containing 12 ZFHD1 sites and a minimal IL2 promoter), a coding sequence for a GLP-1 fusion protein of SEQ ID NO: 37, and a rabbit beta globin polyA. In one embodiment, the expression cassette is that found in SEQ ID NO: 40, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity therewith. In another embodiment, a vector genome is provided in which SEQ ID NO: 40, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity therewith, is flanked by the 5' and 3' AAV ITRs. CMV promoter, a chimeric intron, the FKBP12-rapamycin binding (FRB) domain of human or rhesus FKBP12-rapamycin-related protein (FRAP) fused to the p65 subunit of NF-kappa B from human or rhesus (or a portion thereof), an IRES or 2A peptide, a ZFHD1 DNA binding domain, three FKBP subunits, 8X A second expression cassette is provided that comprises a polynucleotide comprising a ZFHD and a polyA sequence. In one embodiment, the expression cassette is that found in SEQ ID NO: 41, or a sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity therewith. In another embodiment, a vector genome is provided in which SEQ ID NO: 41, or a sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity therewith, is flanked by the 5' and 3' AAV ITRs.

[0058] Viral Vectors In another aspect, a viral vector is provided that comprises the expression cassette described herein. In certain embodiments of the viral vector described herein, the viral vector is an adeno-associated virus (AAV) viral vector or a recombinant AAV (rAAV). The term "recombinant AAV" or "rAAV" as used herein refers to naturally occurring adeno-associated viruses, adeno-associated viruses available to those skilled in the art and / or available in the context of the composition(s) and method(s) described herein, as well as artificial AAV. An adeno-associated virus (AAV) viral vector is an AAV DNase-resistant particle that has an AAV protein capsid, in which the expression cassette packaged is flanked by AAV inverted terminal repeats (ITRs) for delivery to target cells (collectively referred to as "vector genome"). The AAV capsid is composed of 60 capsid protein subunits, VP1, VP2, and VP3, arranged in icosahedral symmetry in a ratio of approximately 1:1:10 to 1:1:20, depending on the AAV selected. Various AAVs can be selected as the source of capsids for the AAV viral vectors identified above. In one embodiment, the AAV capsid is the AAVrh91 capsid or a variant thereof. In certain embodiments, the capsid proteins are designated by a number or a combination of numbers and letters following the term "AAV" in the name of the rAAV vector. Unless otherwise indicated, the AAV capsids, ITRs, and other selected AAV components described herein may be readily selected from among any AAV, including, but not limited to, the AAVs identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAVhu37, AAVrh32.33, AAVAnc80, AAV10, AAV11, AAV12, AAVrh8, AAVrh74, AAV-DJ8, AAV-DJ, AAVhu.37, AAVrh.64R1, and AAVhu68. See, e.g., U.S. Patent Application Publication No. 2007-0036760-A1, U.S. Patent Application Publication No. 2009-0197338-A1, and EP 1310571.See also WO2003 / 042397 (AAV7 and other simian AAVs), U.S. Pat. No. 7,790,449 and U.S. Pat. No. 7,282,199 (AAV8), WO2005 / 033321 and US Pat. No. 7,906,111 (AAV9), as well as WO2006 / 110689, and WO2003 / 042397 (rh.10), WO2005 / 033321, WO2018 / 160582 (AAVhu68), which are incorporated by reference herein. Other suitable AAVs may include, but are not limited to, AAVrh90 [PCT / US20 / 30273, filed April 28, 2020], AAVrh91 [PCT / US20 / 030266, filed April 28, 2020, current publication WO2020 / 223231, published November 5, 2020] AAVrh92, AAVrh93, AAVrh91.93 [PCT / US20 / 30281, filed April 28, 2020], which are incorporated herein by reference. In one embodiment, the AAV is rh91. Other suitable AAVs include the AAV3B variants described in U.S. Provisional Patent Application No. 62 / 924,112, filed October 21, 2019, and U.S. Provisional Patent Application No. 63 / 025,753, filed May 15, 2020, including AAV3B.AR2.01, AAV3B.AR2.02, AAV3B.AR2.03, AAV3B.AR2.04, AAV3B.AR2.05, AAV3B.AR2.06, AAV3B.AR2.07, AAV3B.AR2.08, AAV3B.AR2.10, AAV3B.AR2.11, AAV3B.AR2.12, AAV3B.AR2.13, AAV3B.AR2.14, AAV3B.AR2.15, AAV3B.AR2.16, AAV3B.AR2.17, AAV3B.AR2.18, AAV3B.AR2.19, AAV3B.AR2.20, AAV3B.AR2.21, AAV3B.AR2.22, AAV3B.AR2.23, AAV3B.AR2.24, AAV3B.AR2.25, AAV3B.AR2.26, AAV3B.AR2.27, AAV3B.AR2.28, AAV3B.AR2.29, AAV3B.AR2.30, AAV3B.AR2.31, AAV3B.AR2.32, AAV3B.AR2.33, AAV3B.AR2.34, AAV3B.AR2.35, AAV3B.AR2.36, AAV3B.AR2.37, AAV3B.AR2.38, AAV3B.AR2.39, AAV3B.AR AV3B.AR2.11, AAV3B.AR2.12, AAV3B.AR2.13, AAV3B.AR2.14, AAV3B.AR2.15, AAV3B.AR2.16, or AAV3B.AR2.17 are described and are incorporated herein by reference. See also International Patent Application No. PCT / US21 / 45945, filed August 13, 2021, U.S. Provisional Patent Application No. 63 / 065,616, filed August 14, 2020, and U.S. Provisional Patent Application No. 63 / 109,734, filed November 4, 2020, all of which are incorporated herein by reference in their entireties. These documents also describe other AAV capsids that may be selected to generate rAAV, and are incorporated by reference. Among the well-characterized AAVs isolated or engineered from humans or non-human primates (NHPs), human AAV2 was the first AAV developed as a gene transfer vector and has been widely used for efficient gene transfer experiments in various target tissues and animal models.

[0059] As used herein, with respect to AAV, the term "variant" refers to any AAV sequence derived from a known AAV sequence, including AAV sequences with conservative amino acid substitutions and AAV sequences that share at least 90%, at least 95%, at least 97%, at least 99% or more sequence identity across the amino acid or nucleic acid sequence. In another embodiment, the AAV capsid includes a variant that may include up to about 10% variation from any described or known AAV capsid sequence. That is, the AAV capsid shares about 90% identity to about 99.9% identity, about 95% to about 99% identity, or about 97% to about 98% identity with an AAV capsid provided herein and / or known in the art. In one embodiment, the AAV capsid shares at least 95% identity with an AAV capsid. When determining the percent identity of an AAV capsid, the comparison can be made across any of the variable proteins (e.g., vp1, vp2, or vp3).

[0060] In one embodiment, the viral vector is a rAAV having a capsid of AAV8 or a functional variant thereof. In one embodiment, the viral vector is a rAAV having a capsid of AAVrh91 or a functional variant thereof. In one embodiment, the viral vector is a rAAV having a capsid of AAV3.AR.2.12 or a functional variant thereof. In one embodiment, the viral vector is a rAAV having a capsid selected from AAV9, AAVrh64R1, AAVhu37, or AAVrh10.

[0061] In certain embodiments, the AAV is rh91. A nucleic acid sequence encoding the AAVrh91 capsid is provided in SEQ ID NO: 18, and the encoded amino acid sequence is provided in SEQ ID NO: 20. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVrh91 (SEQ ID NO: 20). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVrh91 (SEQ ID NO: 18). In yet another embodiment, a nucleic acid sequence encoding the AAVrh91 amino acid sequence is provided in SEQ ID NO: 19, and the encoded amino acid sequence is provided in SEQ ID NO: 20. Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVrh91 (SEQ ID NO: 19). In certain embodiments, vp1, vp2, and / or vp3 are full-length capsid proteins of AAVrh91 (SEQ ID NO: 20). In other embodiments, vp1, vp2, and / or vp3 have an N-terminal and / or C-terminal truncation (e.g., a truncation of about 1 to about 10 amino acids).

[0062] In certain embodiments, the AAVrh91 capsid is characterized by one or more of the following: (1) an AAVrh91 capsid protein comprising one of sequences 1-736 of SEQ ID NO: 20; A heterologous collection of AAVrh91 vp1 proteins selected from vp1 proteins produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence, vp1 proteins produced from SEQ ID NO:18, or vp1 proteins produced from a nucleic acid sequence that encodes the predicted amino acid sequence of at least about 138-736 amino acids of SEQ ID NO:20; vp2 proteins produced from a sequence comprising at least nucleotides 412-2208 of SEQ ID NO:18, or vp2 proteins produced from a nucleic acid sequence that encodes the predicted amino acid sequence of at least about 138-736 amino acids of SEQ ID NO:20; a heterologous collection of vp2 proteins, vp3 proteins produced from expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:20, vp3 proteins produced from a sequence comprising at least nucleotides 607-2208 of SEQ ID NO:18, or vp3 proteins produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 607-2208 of SEQ ID NO:18 encoding a predicted amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:20.and / or (2) a heterogeneous collection of vp1 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of at least about amino acids 138-736 of SEQ ID NO:20, a heterogeneous collection of vp2 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:20, wherein the vp1 proteins, vp2 proteins, and vp3 proteins are the product of a nucleic acid sequence encoding an amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:20, a heterogeneous collection of vp1 proteins, a heterogeneous collection of vp2 proteins, a heterogeneous collection of vp3 proteins, each of which comprises a subpopulation having an amino acid modification comprising at least two highly deamidated asparagines (N) in a syn pair, and optionally further comprising a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change, and (B) a vector genome in an AAVrh91 capsid, the vector genome comprising a nucleic acid molecule comprising an AAV inverted terminal repeat and a non-AAV nucleic acid sequence encoding a product operably linked to a sequence that directs expression of the product in a host cell.

[0063] In certain embodiments, the AAVrh91 capsid is characterized by one or more of the following: (1) a heterologous collection of AAVrh91 vp1 proteins selected from a vp1 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of 1-736 of SEQ ID NO:20, a vp1 protein produced from SEQ ID NO:19, or a vp1 protein produced from a nucleic acid sequence that encodes the predicted amino acid sequence of 1-736 of SEQ ID NO:20; a vp2 protein produced by expression from a nucleic acid sequence encoding at least about 138-736 amino acids of SEQ ID NO:20, a vp2 protein produced from a sequence including at least nucleotides 412-2208 of SEQ ID NO:19, or a vp2 protein produced from a nucleic acid sequence that encodes the predicted amino acid sequence of at least about 138-736 amino acids of SEQ ID NO:20; a heterogeneous collection of AAVrh91 vp3 proteins selected from a heterogeneous collection of vp2 proteins, vp3 proteins produced from expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:20, vp3 proteins produced from a sequence including at least nucleotides 607-2208 of SEQ ID NO:19, or vp3 proteins produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 607-2208 of SEQ ID NO:19, which encodes a predicted amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:20, and / or (2) a vp1 protein that is the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:20. a heterogeneous collection of vp1 proteins, vp2 proteins, and vp3 proteins, each of which is a product of a nucleic acid sequence encoding an amino acid sequence of at least about 138 to 736 amino acids of SEQ ID NO:20; and a heterogeneous collection of vp2 proteins, each of which is a product of a nucleic acid sequence encoding at least about 203 to 736 amino acids of SEQ ID NO:20, wherein the vp1 proteins, vp2 proteins, and vp3 proteins comprise a subpopulation having an amino acid modification comprising at least two highly deamidated asparagines (N) in the asparagine-glycine pair of SEQ ID NO:20; and (B) an AAVrh91 capsid comprising one or more of a heterogeneous collection of vp1 proteins, a heterogeneous collection of vp2 proteins, or a heterogeneous collection of vp3 proteins, wherein the deamidation results in an amino acid change, and further comprising a subpopulation that comprises other deamidated amino acids, and (C) a vector genome in the AAVrh91 capsid, the vector genome comprising an AAV inverted terminal repeat and a nucleic acid molecule that comprises a non-AAV nucleic acid sequence encoding a product operably linked to a sequence that directs expression of the product in a host cell.

[0064] In certain embodiments, the vp1, vp2, and vp3 proteins of AAVrh91 comprise subpopulations having amino acid modifications that include at least two highly deamidated asparagines (N) in the asparagine-glycine pair of SEQ ID NO:20, and optionally further comprise subpopulations that include other deamidated amino acids, where the deamidation results in an amino acid change. High levels of deamidation are observed at NG pairs N57, N383, and / or N512, relative to the numbers in SEQ ID NO:20. Deamidation has been observed at other residues. In certain embodiments, AAVrh91 may have other residues that are deamidated, e.g., typically less than 10%, and / or may have other modifications including phosphorylation (e.g., in a range of about 2 to about 30%, or about 2 to about 20%, or about 2 to about 10%, if present) (e.g., at S149), or oxidation (e.g., at one or more of about W22, about M211, W247, M403, M435, M471, W478, W503, about M537, about M541, about M559, about M599, M635, and / or W695). Optionally, W may be oxidized to kynurenine. [Table 1]

[0065] In certain embodiments, the AAVrh91 capsid is modified at one or more of the positions identified in the table above, within the ranges provided below, as determined using mass spectrometry with trypsin enzyme. In certain embodiments, one or more positions, or the glycine following the N, is modified as described herein. Residue numbers are based on the AAVrh91 sequence provided herein. See SEQ ID NO:20.

[0066] In certain embodiments, the AAVrh91 capsid comprises a heterogeneous collection of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:20, a heterogeneous collection of vp2 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence at least about amino acids 138-736 of SEQ ID NO:20, and a heterogeneous collection of vp3 proteins that are the product of a nucleic acid sequence encoding at least amino acids 203-736 of SEQ ID NO:20.

[0067] In certain embodiments, modified AAVrh91 nucleic acid sequences can be used to generate mutant rAAVs having capsids that contain less deamidation than the native AAVrh91 capsid. Such mutant rAAVs can have reduced immunogenicity and / or increased stability upon storage, particularly in suspension form.

[0068] In one aspect, a recombinant AAV (rAAV) is provided that comprises an AAV capsid derived from the adeno-associated virus rh91 and a vector genome packaged within the AAV capsid, the vector genome comprising AAV inverted terminal repeats (ITRs), a coding sequence for a GLP-1 receptor agonist of SEQ ID NO: 14, and regulatory sequences that direct expression of the GLP-1 receptor agonist.

[0069] In certain embodiments, the AAV68 capsid is further characterized by one or more of the following: The AAVhu68 capsid protein may be an AAVhu68 vp1 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 736 of SEQ ID NO:42, a vp1 protein produced from SEQ ID NO:43 or 44, or a vp1 protein produced from a nucleic acid sequence that encodes the predicted amino acid sequence of 1 to 736 of SEQ ID NO:42 that is at least 70% identical to SEQ ID NO:43 or 44 that encodes the predicted amino acid sequence of 1 to 736 of SEQ ID NO:42; an AAVhu68 vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:42, a vp2 protein produced from a sequence that includes at least nucleotides 412 to 2211 of SEQ ID NO:43 or 44, or a vp2 protein produced from a nucleic acid sequence that encodes the predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:42 that is at least 70% identical to at least nucleotides 412 to 2211 of SEQ ID NO:43 or 44 that encodes the predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:42, and / or an AAVhu68 vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:42. The present invention also includes vp3 proteins, vp3 proteins produced from a sequence including at least nucleotides 607-2211 of SEQ ID NO:43 or 44, or vp3 proteins produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 607-2211 of SEQ ID NO:43 or 44 that encodes the predicted amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:42.

[0070] Additionally or alternatively, optionally, an AAV capsid is provided that comprises a heterogeneous population of vp1 proteins that comprise a valine at position 157, optionally a heterogeneous population of vp2 proteins that comprise a valine at position 157, and a heterogeneous population of vp3 proteins, wherein at least a subpopulation of vp1 and vp2 proteins comprise a valine at position 157 and optionally further comprise a glutamic acid at position 67, based on the numbering of the vp1 capsid in SEQ ID NO:42. Additionally, or alternatively, there is provided an AAVhu68 capsid comprising a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:42, a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of at least about amino acids 138-736 of SEQ ID NO:42, and a heterogeneous population of vp3 proteins that are the product of a nucleic acid sequence encoding at least amino acids 203-736 of SEQ ID NO:42, wherein the vp1 proteins, vp2 proteins, and vp3 proteins include subpopulations having amino acid modifications.

[0071] The vp1, vp2, and vp3 proteins of AAVhu68 typically encode the full-length vp1 amino acid sequence (amino acids 1 to 736) of SEQ ID NO:42. and are expressed as alternative splice variants encoded by the same nucleic acid sequence. Optionally, the vp1 coding sequence is used alone to express the vp1, vp2, and vp3 proteins. Alternatively, this sequence may be co-expressed with one or more of a nucleic acid sequence encoding the AAVhu68 vp3 amino acid sequence (about aa203-736) of SEQ ID NO: 42 without the vp1 unique region (about aa1 to about aa137) and / or the vp2 unique region (about aa1 to about aa202), or a complementary strand thereto, the corresponding mRNA or tRNA (about nt607 to about nt2211 of SEQ ID NO: 43 or 44), or a sequence at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 43 or 44 encoding aa203-736 of SEQ ID NO: 42. Additionally or alternatively, the vp1 and / or vp2 coding sequences can be co-expressed with a nucleic acid sequence encoding the AAVhu68 vp2 amino acid sequence of SEQ ID NO:42 (about aa 1 to about 137) without the vp1 unique region (about aa 1 to about 137), or a complementary strand thereto, the corresponding mRNA or tRNA (e.g., nt 412 to 2212 of SEQ ID NO:43 or 44), or a sequence that is at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:43 or 44, which encodes about aa 138 to 736 of SEQ ID NO:42.

[0072] As described herein, rAAVhu68 has rAAVhu68 capsids produced in a production system that expresses capsids from an AAVhu68 nucleic acid encoding the vp1 amino acid sequence of SEQ ID NO:42, and, optionally, additional nucleic acid sequences, such as sequences encoding a vp3 protein that does not include the vp1 unique region and / or the vp2 unique region. rAAVhu68 resulting from production using a single nucleic acid sequence vp1 produces a heterogeneous population of vp1, vp2, and vp3 proteins. More specifically, AAVhu68 capsids contain subpopulations within the vp1, vp2, and vp3 proteins that have modifications from the predicted amino acid residues of SEQ ID NO:42. These subpopulations contain, at a minimum, deamidated asparagine (N or Asn) residues. For example, the asparagine in an asparagine-glycine pair is highly deamidated.

[0073] In one embodiment, the AAVhu68 vp1 nucleic acid sequence has the sequence of SEQ ID NO: 43 or 44, or a complementary strand thereto, e.g., the corresponding mRNA or tRNA. In certain embodiments, the vp2 and / or vp3 proteins may additionally or alternatively be expressed from a nucleic acid sequence different from vp1, e.g., to alter the ratio of vp proteins in a selected expression system. In certain embodiments, a nucleic acid sequence encoding the AAVhu68 vp3 amino acid sequence of SEQ ID NO: 42 (about aa 203-736) is also provided that does not include the vp1 unique region (about aa 1 to about aa 137) and / or the vp2 unique region (about aa 1 to about aa 202), or a complementary strand thereto, the corresponding mRNA or tRNA (about nt 607 to about nt 2211 of SEQ ID NO: 43 or 44). In certain embodiments, a nucleic acid sequence encoding the AAVhu68 vp2 amino acid sequence of SEQ ID NO: 42 (about aa 138-736), without the vp1 unique region (about aa 1 to about 137), or the complementary strand thereto, the corresponding mRNA or tRNA (nt 412-2211 of SEQ ID NO: 43 or 44), is also provided.

[0074] However, other nucleic acid sequences encoding the amino acid sequence of SEQ ID NO:42 may be selected for use in producing rAAVhu68 capsids. In certain embodiments, the nucleic acid sequence has at least 70% to 99% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity to the nucleic acid sequence of SEQ ID NO:43 or 44, or a sequence encoding SEQ ID NO:42 ... or 44, or a sequence at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the nucleic acid sequence of about nt 412 to about nt 2211 of SEQ ID NO: 43 or 44, and encodes the vp2 capsid protein (about aa 138 to 736) of SEQ ID NO: 42. In certain embodiments, the nucleic acid sequence has a sequence at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the nucleic acid sequence of about nt 607 to about nt 2211 of SEQ ID NO: 43 or 44, or a sequence at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the nucleic acid sequence of about nt 607 to about nt 2211 of SEQ ID NO: 43 or 44, and encodes the vp3 capsid protein (about aa 203 to 736) of SEQ ID NO: 42. [Table 2-1] [Table 2-2]

[0075] In certain embodiments, an AAVhu68 capsid is characterized in having a capsid protein in which at least 45% of the N residues are deamidated at at least one of positions N57, N329, N452, and / or N512, based on the numbering of the amino acid sequence of SEQ ID NO: 42. In certain embodiments, at least about 60%, at least about 70%, at least about 80%, or at least 90% of the N residues are deamidated at one or more of these NG positions (i.e., N57, N329, N452, and / or N512, based on the numbering of the amino acid sequence of SEQ ID NO: 42). In these and other embodiments, the AAVhu68 capsid is further characterized in that it has a population of proteins in which about 1% to about 20% of N residues have deamidation at one or more of positions N94, N253, N270, N304, N409, N477, and / or Q599, based on the numbering of the amino acid sequence of SEQ ID NO:42. In certain embodiments, AAVhu68 comprises at least a subpopulation of vp1, vp2, and / or vp3 proteins that are deamidated at one or more of positions N35, N57, N66, N94, N113, N252, N253, Q259, N270, N303, N304, N305, N319, N328, N329, N336, N409, N410, N452, N477, N515, N598, Q599, N628, N651, N663, N709, N735, or combinations thereof, based on the numbering of the amino acid sequence of SEQ ID NO: 42. In certain embodiments, the capsid protein may have one or more amidated amino acids.

[0076] In another embodiment, a recombinant adeno-associated virus (rAAV) having an AAVhu68 capsid and a vector genome is provided, wherein (a) the AAVhu68 capsid comprises a heterogeneous population of AAVhu68 vp1 proteins, a heterogeneous population of AAVhu68 vp2 proteins, and a heterogeneous population of AAVhu68 vp3 proteins, and the heterogeneous AAVhu68 vp1, AAVhu68 vp2, and AAVhu68 vp3 proteins are identified using mass spectrometry. and wherein the AAVhu68 capsid comprises a subpopulation having an amino acid modification comprising 50% to 100% deamidation of at least two asparagines (N) in an asparagine-glycine pair at two or more of N57, N329, N452, N512 of SEQ ID NO: 42, and optionally further comprising a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change such that the deamidated asparagine is deamidated to aspartic acid, isoaspartic acid, an interconverting aspartic acid / isoaspartic acid pair, or a combination thereof, and the AAVhu68 capsid comprises at least 65% of the asparagine (N) in the asparagine-glycine pair located at position N57 of the vp1 protein, based on the numbering of SEQ ID NO: 42, is deamidated; at least 75% of the N in the asparagine-glycine pair at position N329 of the vp1 protein, v2 protein, and vp3 protein are deamidated, based on the residue numbering of the amino acid sequence of SEQ ID NO:42; At least 50% of the N in the asparagine-glycine pair at position N452 of the vp1, v2 and vp3 proteins are deamidated, based on the residue numbering of the amino acid sequence of SEQ ID NO: 42; and / or at least 75% of the N in the asparagine-glycine pair at position N512 of the vp1 protein, v2 protein, and vp3 protein are deamidated, based on the residue numbering of the amino acid sequence of SEQ ID NO:42; Further included are subpopulations having one or more of the vector genomes in an AAVhu68 capsid, the vector genome comprising an AAV inverted terminal repeat sequence and a non-AAV nucleic acid sequence encoding a GLP-1 fusion described herein operably linked to a sequence that directs expression of the GLP-1 fusion in a target cell.

[0077] In one embodiment, the rAAV is a scAAV. The abbreviation "sc" refers to self-complementary. "Self-complementary AAV" refers to a plasmid or vector with an expression cassette in which the coding region carried by the recombinant AAV nucleic acid sequence is designed to form an intramolecular double-stranded DNA template. Upon infection, rather than waiting for cell-mediated synthesis of the second strand, the two complementary halves of the scAAV will associate to form one double-stranded DNA (dsDNA) unit ready for immediate replication and transcription. See, e.g., DM McCarty et al, "Self-complementary recombinant adeno-associated virus See, "Self-complementary AAV (scAAV) vectors promote efficient transduction independently of DNA synthesis", Gene Therapy, (August 2001), Vol 8, Number 16, Pages 1248-1254. Self-complementary AAVs are described, for example, in U.S. Pat. Nos. 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated by reference in its entirety.

[0078] In one embodiment, the nucleic acid sequence encoding the GLP-1 construct described herein is engineered into any suitable genetic element, e.g., naked DNA, phage, transposon, cosmid, RNA molecule (e.g., mRNA), episome, etc., which introduces the GLP-1 sequence carried thereon into a host cell, e.g., for generating nanoparticles carrying DNA or RNA, viral vectors in a packaging host cell, and / or for delivery to a host cell in a subject. In one embodiment, the genetic element is a plasmid. The selected genetic element may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. Methods used to generate such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular See Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0079] As used herein, the term "host cell" may refer to a packaging cell line in which a vector (e.g., recombinant AAV or rAAV) is produced from a production plasmid. Alternatively, the term "host cell" may refer to any target cell in which expression of a gene product described herein is desired. Thus, "host cell" refers to a prokaryotic or eukaryotic cell (e.g., bacterial, human, or insect cell) that contains exogenous or heterologous DNA introduced into the cell by any means, such as electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. In certain embodiments herein, the term "host cell" refers to cultures of cells of various mammalian species for in vitro evaluation of the compositions described herein. In other embodiments herein, the term "host cell" refers to cells used to produce and package viral vectors or recombinant viruses. In further embodiments, the term "host cell" is an intestinal cell, a small intestinal cell, a pancreatic cell, or a liver cell.

[0080] As used herein, the term "target cell" refers to any target cell in which expression of a heterologous nucleic acid sequence or protein is desired. In certain embodiments, the target cell is a liver cell. In other embodiments, the target cell is a muscle cell.

[0081] In one embodiment, an rAAV is provided that includes a vector genome comprising an expression cassette, the expression cassette comprising a CMV promoter and an activation domain comprising the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin related protein (FRAP) fused to the carboxy terminus from the p65 subunit of NF-kappa B of human origin, a GT2A_V1 peptide, a ZFHD1 DNA binding domain, three FKBP subunits, hGH polyA, 8XZFHD, a minimal sIL2 promoter, a coding sequence for a GLP-1 fusion protein of SEQ ID NO:14, and a rabbit beta globin polyA. In another embodiment, an rAAV is provided that includes a vector genome that includes an expression cassette, the expression cassette including a CMV promoter and an activation domain that is the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin related protein (FRAP) fused to the carboxy terminus from the p65 subunit of NF-kappa B of human origin, a GT2A_V2 peptide, a ZFHD1 DNA binding domain, three FKBP subunits, hGH polyA, 8XZFHD, a minimal sIL2 promoter, a coding sequence for a GLP-1 fusion protein of SEQ ID NO:14, and a rabbit beta globin polyA.

[0082] The minimal sequences required to package an expression cassette into an AAV viral particle are the AAV 5' and 3' ITRs, which may be of the same AAV origin as the capsid or of a different AAV origin (to generate AAV pseudotypes). In one embodiment, the ITR sequences from AAV2, or a deleted version thereof (ΔITR), are used for convenience and to speed regulatory approval. However, ITRs from other AAV sources may be selected. Preferably, the source of the ITRs is the same as the source of the Rep protein provided in trans for production. Typically, an expression cassette for an AAV vector includes the AAV 5' ITR, the GLP-1 fusion protein coding sequence, and any regulatory sequences, as well as the AAV 3' ITR. However, other configurations of these elements may be suitable. A shortened version of the 5' ITR, in which the D sequence and the terminal separation site (trs) are deleted (designated ΔITR), has been described. In other embodiments, the full-length AAV 5' and 3' ITRs are used.

[0083] The ITRs are the only AAV components required in cis in the same construct as the genes to package the expression cassette into virions. In one embodiment, the replication (rep) and / or capsid (cap) coding sequences are removed from the AAV genome and supplied in trans or by a packaging cell line to generate the AAV vector. For example, as described above, pseudotyped AAV may contain ITRs from a source different from the source of the AAV capsid. In one embodiment, a chimeric AAV capsid may be utilized. Still other AAV components may be selected. Sources of such AAV sequences are described herein and may be obtained from academic, commercial, or public sources (e.g., American Type AAV sequences may be isolated or obtained from the National AAV Culture Collection, Manassas, VA. AAV sequences may be obtained synthetically or through other suitable means by reference to published sequences such as those available in the literature or databases (e.g., GenBank®, PubMed®, etc.).

[0084] Methods for generating and isolating AAV viral vectors suitable for delivery to subjects are known in the art.See, for example, US Patent No. 7,790,449, US Patent No. 7,282,199, WO2003 / 042397, WO2005 / 033321, WO2006 / 110689, and US7,588,772 B2. In one system, a producer cell line is transiently transfected with a construct encoding a transgene flanked by ITRs and a construct encoding rep and cap. In the second system, a packaging cell line that stably supplies rep and cap is transiently transfected with a construct(s) encoding a transgene flanked by ITRs. In each of these systems, AAV virions are produced in response to infection with a helper adenovirus or herpesvirus, requiring the separation of rAAV from contaminating viruses. More recently, systems have been developed that do not require infection with a helper virus to restore AAV, and the necessary helper functions (i.e., adenovirus E1, E2a, VA, and E4, or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase) are also supplied by the system in trans. In these newer systems, the helper functions can be supplied by transient transfection of cells with constructs encoding the required helper functions, or the cells can be engineered to stably contain genes encoding helper functions, the expression of which can be controlled at the transcriptional or post-transcriptional level. In yet another system, the transgene flanked by ITRs and the rep / cap genes are introduced into insect cells by infection with a baculovirus-derived vector. For a review of these production systems generally, see, e.g., Zhang et al., 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, the contents of each of which are incorporated herein by reference in their entireties.Methods of making and using these and other AAV production systems are also described in the following U.S. patents, the contents of each of which are incorporated herein by reference in their entirety: 5,139,941, 5,741,683, 6,057,152, 6,204,059, 6,268,213, 6,491,907, 6,660,514, 6,951,753, 7,094,604, 7,172,893, 7,201,898, 7,229,823, and 7,439,065. See generally, for example, Grieger & Samulski, 2005, "Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications," Adv. Biochem. Engin / Biotechnol. 99:119-145, Buning. et al., 2008, “Recent developments in adeno-associated virus vector technology,” J. Gene Med. 10:717-733, and the references cited below (these (each of which is incorporated herein by reference in its entirety). The methods used to construct any embodiment of the present invention are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Similarly, methods for producing rAAV virions are well known and the selection of a suitable method is not a limitation of the present invention. See, e.g., K. Fisher et al, (1993) J. Virol., 70:520-532 and U.S. Patent No. 5,478,745.

[0085] The rAAV described herein comprises a selected capsid having a vector genome packaged inside. The vector genome (or rAAV genome) comprises 5' and 3' AAV inverted terminal repeats (ITRs), a polynucleotide sequence encoding a fusion protein, and a regulatory sequence that directs the insertion of the polynucleotide sequence encoding the fusion protein into the genome of a host cell. In one embodiment, the vector genome is a sequence set forth in SEQ ID NO: 16, or a sequence sharing at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.

[0086] As used herein, "vector genome" refers to a nucleic acid sequence packaged inside a parvovirus (e.g., rAAV) capsid that forms a viral particle. Such a nucleic acid sequence includes AAV inverted terminal repeats (ITRs). In the example herein, the vector genome includes at least, from 5' to 3', an AAV 5'ITR, a coding sequence(s) (i.e., transgene(s)), and an AAV 3'ITR. ITRs from AAV2, a source AAV different from the capsid, or other than full-length ITRs can be selected. In certain embodiments, the ITRs are from the same AAV source as the AAV that provides the rep function or trans-complementing AAV during production. Additionally, other ITRs can be used, such as self-complementary (scAAV) ITRs. Both single-stranded and self-complementary (sc) AAVs are included in rAAV. A transgene is a nucleic acid coding sequence heterologous to the vector sequence that encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor), or other gene product of interest. The nucleic acid coding sequence is operably linked to regulatory elements in a manner that allows transcription, translation, and / or expression of the transgene in cells of the target tissue. Suitable components of a vector genome are discussed in more detail herein. In one example, a "vector genome" includes at least, from 5' to 3', a vector-specific sequence and a nucleic acid sequence encoding a GLP-1 construct operably linked to a regulatory control sequence (that directs its expression in the target sequence), and the vector-specific sequence may be a terminal repeat sequence that specifically packages the vector genome into a viral vector capsid or envelope protein. For example, AAV inverted terminal repeats are utilized for packaging into AAV and certain other parvovirus capsids.

[0087] The AAV sequences of the vector typically contain cis-acting 5' and 3' inverted terminal repeat sequences (see, e.g., BJ Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155 168 (1990)). The ITR sequences are approximately 145 bp in length. Preferably, substantially complete sequences encoding the ITRs are used in the molecule, although some minimal modification of these sequences is tolerated. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., Sambrook et al, "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York (1989) and K. Fisher et al., J. Virol., 70:520 532 (1996) (see ). One example of such a molecule used in the present invention is a "cis-acting" plasmid containing a transgene, where the selected transgene sequence and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences. In one embodiment, the ITRs are from a different AAV than the one supplying the capsid. In one embodiment, the ITR sequences are from AAV2. However, ITRs from other AAV sources can be selected. A shortened version of the 5'ITR (designated ΔITR) has been described, in which the D sequence and terminal separation sites (trs) are deleted. In one particular embodiment, the vector genome comprises a 130 base pair truncated AAV2 ITR, in which the external A elements are deleted. Without wishing to be bound by theory, it is believed that the shortened ITRs are restored to the wild-type length of 145 base pairs during vector DNA amplification, using the internal (A') elements as templates. In other embodiments, full-length AAV 5' and 3' ITRs are used. When the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be referred to as pseudotyped, however, other configurations of these elements may also be suitable.

[0088] Optionally, the GLP-1 construct described herein can be delivered via a viral vector other than rAAV. Such other viral vectors can include any virus suitable for gene therapy, including but not limited to adenovirus, herpesvirus, lentivirus, retrovirus, etc. Preferably, when one of these other vectors is produced, it is produced as a replication-defective viral vector.

[0089] "Replication-defective virus" or "viral vector" refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, and any viral genomic sequences also packaged in the viral capsid or envelope are replication-defective, i.e., they are unable to generate progeny virions, but retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gutless", containing only the transgene of interest flanked by signals required for amplification and packaging of the artificial genome), although these genes can be supplied during production. It is therefore considered safe for use in gene therapy, since replication and infection by progeny virions cannot occur except in the presence of viral enzymes required for replication.

[0090] Compositions comprising the viral vector constructs described herein are also provided. The pharmaceutical compositions described herein are designed to be delivered to a subject in need thereof by any suitable route or combination of different routes. Direct delivery to the liver (optionally intravenously, via the hepatic artery, or by transplantation), oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes of administration. The viral vectors described herein may be delivered in a single composition or multiple compositions. Optionally, two or more different AAVs, or multiple viruses, may be delivered [see, for example, WO2011 / 126808 and WO2013 / 049493]. In another embodiment, the multiple viruses may include different replication-deficient viruses (e.g., AAV and adenovirus). In one embodiment, administration is intramuscular. In another embodiment, administration is intravenous.

[0091] Replication-defective viruses can be formulated with physiologically acceptable carriers for use in gene transfer and gene therapy applications. In the case of AAV viral vectors, quantification of genome copies ("GC") can be used as a measure of the dose contained in the formulation. Any method known in the art can be used to determine the genome copy (GC) number of the replication-defective virus composition of the present invention. One method for performing GC number titration of AAV is as follows. Purified AAV vector samples are first treated with DNase to eliminate non-encapsidated AAV genome DNA or contaminating plasmid DNA from the production process. The nuclease-resistant particles are then subjected to heat treatment to release the genome from the capsid. The released genome is then quantified by real-time PCR using a primer / probe set that targets a specific region of the viral genome (usually the polyA signal). Another suitable method for determining genome copies is quantitative PCR (qPCR), especially optimized qPCR or digital droplet PCR [Lock Martin, et al, Human Gene Therapy Methods. April 2014, 25(2):115-125. doi:10.1089 / hgtb.2013.131, published online before editing on December 13, 2013].

[0092] In addition, the replication-deficient virus composition contains approximately 1.0×10 9 GC~approx. 1.0×10 15 The dosage unit can be formulated to contain an amount of replication-deficient virus in the range of GC. In another embodiment, this amount of viral genome can be delivered in divided doses. In one embodiment, the dose is about 1.0×10 for an average human subject of about 70 kg. 10 GC~approx.3.0×10 14 In another embodiment, the dose is about 1×10 9 For example, the dose of AAV virus is about 1×10 10 GC, 1×10 11 GC, approx. 5×10 11 GC, approx. 1×10 12 GC, approx. 5×10 12 GC, or approximately 1 × 10 13 In another embodiment, the dosage is about 1.0×10 9 GC / kg ~ approx. 3.0×10 14 In another embodiment, the dose is about 1×10 9 For example, the dose of AAV virus is about 1×10 10 GC / kg, 1×10 11 GC / kg, approx. 5×10 11 GC / kg, approx. 1×10 12 GC / kg, approx. 5×10 12GC / kg, or approximately 1 x 10 13 GC / kg. In one embodiment, the construct may be delivered in a volume of 1 μL to about 100 mL. As used herein, the term "dose" or "amount" may refer to the total dose or amount delivered to a subject over the course of treatment, or the dose or amount delivered in a single unit (or multiple units or split doses).

[0093] The above-mentioned recombinant vector can be delivered to a host cell according to published methods. Preferably, rAAV suspended in a physiologically compatible carrier can be administered to a desired subject, including humans. A suitable carrier can be easily selected by those skilled in the art in view of the indication for which the introduced virus is intended. For example, one suitable carrier includes saline, which can be formulated with various buffer solutions (e.g., phosphate-buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present invention.

[0094] In another embodiment, the composition comprises a carrier, diluent, excipient and / or adjuvant. In certain embodiments, for administration to a human patient, the rAAV is suitably suspended in an aqueous solution containing saline, a surfactant, and a pharma- ceutically and / or physiologically compatible salt or mixture of salts. Suitably, the formulation is adjusted to a physiologically acceptable pH, for example, in the range of pH 6-9, or pH 6.0-7.5, or pH 6.2-7.7, or pH 6.5-7.5, pH 7.0-7.7, or pH 7.2-7.8, or about pH 7.0. In certain embodiments, the formulation is adjusted to a pH of about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, or about 7.8. In certain embodiments, a pH of about 7.28 to about 7.32, about 6.0 to about 7.5, about 6.2 to about 7.7, about 7.5 to about 7.8, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, or about 7.8 may be desirable. In certain embodiments, for intravenous delivery, a pH of about 6.8 to about 7.2 may be desirable. However, other pHs in the broader range, and subranges thereof, may be selected for other delivery routes.

[0095] Optionally, the compositions of the invention may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to the rAAV and / or variant 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.

[0096] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharma- ceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutical acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a host. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like can be used to introduce the compositions of the present invention into suitable host cells. In particular, rAAV vector-delivered transgenes can be formulated for delivery either encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles, and the like.

[0097] In one embodiment, the composition comprises a final formulation suitable for delivery to a subject, for example, an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration.Optionally, one or more surfactants are present in the formulation.In another embodiment, the composition can be delivered as a concentrate that is diluted for administration to a subject.In other embodiments, the composition can be lyophilized and reconstituted at the time of administration.

[0098] Suitable surfactants or combinations of surfactants may be selected from non-toxic non-ionic surfactants. In one embodiment, a primary hydroxyl terminated bifunctional block copolymer surfactant is selected, such as Pluronic® F68 [BASF], also known as Poloxamer 188, with neutral pH and average molecular weight of 8400. Other surfactants and other poloxamers may be selected, i.e., non-ionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (macrogol-15 hydroxystearate), LABRASOL (polyoxycaprylic acid glyceride), polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are generally named with the letter "P" (for poloxamer) followed by three digits, the first two digits x 100 giving the approximate molecular mass of the polyoxypropylene core and the last digit x 10 giving the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005% to about 0.001% of the suspension.

[0099] The dosage of a viral vector depends primarily on factors such as the condition being treated, the age, weight, and health of the patient, and may therefore vary between patients. For example, a therapeutically effective human dosage of a viral vector is generally about 1×10 (to treat an average subject weighing 70 kg) in the range of about 25 to about 1000 microliters to about 100 mL. 9 ~1×10 16 The concentration of the genomic viral vector (including all integers or fractions therein, preferably 1.0×10 for human patients) 12 GC~1.0×10 13The compositions of the present invention are administered in doses ranging from about 0.1 μL to about 10 μL, including all values ​​within the range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method. It may be delivered in a volume of 10 mL. In one embodiment, the volume is about 50 μL. In another embodiment, the volume is about 70 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 125 μL. In another embodiment, the volume is about 150 μL. In another embodiment, the volume is about 175 μL. In yet another embodiment, the volume is about 200 μL. In another embodiment, the volume is about 250 μL. In another embodiment, the volume is about 300 μL. In another embodiment, the volume is about 450 μL. In another embodiment, the volume is about 500 μL. In another embodiment, the volume is about 600 μL. In another embodiment, the volume is about 750 μL. In another embodiment, the volume is about 850 μL. In another embodiment, the volume is about 1000 μL. In another embodiment, the volume is about 1.5 mL. In another embodiment, the volume is about 2 mL. In another embodiment, the volume is about 2.5 mL. In another embodiment, the volume is about 3 mL. In another embodiment, the volume is about 3.5 mL. In another embodiment, the volume is about 4 mL. In another embodiment, the volume is about 5 mL. In another embodiment, the volume is about 5.5 mL. In another embodiment, the volume is about 6 mL. In another embodiment, the volume is about 6.5 mL. In another embodiment, the volume is about 7 mL. In another embodiment, the volume is about 8 mL. In another embodiment, the volume is about 8.5 mL. In another embodiment, the volume is about 9 mL. In another embodiment, the volume is about 9.5 mL. In another embodiment, the volume is about 10 mL.

[0100] In some embodiments, the concentration of the recombinant adeno-associated virus having a nucleic acid sequence encoding a desired transgene under the control of a regulatory sequence in the composition is desirably about 10 per milliliter. 7 ~10 14The range is 10 vector genomes (vg / mL) (also referred to as genome copies (GC / mL)).

[0101] In one embodiment, the dosage of rAAV in the composition is about 1.0×10 9 GC / kg ~ approx. 1.5×10 13 In one embodiment, the dosage is about 1.0×10 10 In one embodiment, the dosage is about 1.0×10 11 In one embodiment, the dosage is about 1.0×10 12 In one embodiment, the dosage is about 5.0×10 12 In one embodiment, the dosage is about 1.0×10 13 GC / kg. All ranges stated herein are inclusive of the endpoints.

[0102] In one embodiment, the effective dose (total genome copies delivered) is about 10 7 ~10 13 In one embodiment, the total administered dose is about 10 8 In one embodiment, the total administered dose is about 10 9 In one embodiment, the total administered dose is about 10 10 In one embodiment, the total administered dose is about 10 11 In one embodiment, the total administered dose is about 10 12 In one embodiment, the total administered dose is about 10 13 In one embodiment, the total administered dose is about 10 14 In one embodiment, the total administered dose is about 10 15 It is a genome copy.

[0103] It is desirable to utilize the lowest effective concentration of the virus to reduce the risk of undesirable effects such as toxicity. Further dosages and dosage volumes within these ranges can be selected by the attending physician, taking into account the physical condition of the subject, preferably a human, being treated, the age of the subject, the particular disorder, and, if progressive, the degree of progression of the disorder.

[0104] In certain embodiments, the composition comprises a rAAV comprising an inducible GLP-1 agonist construct. In certain embodiments, the inducing agent or molecule is rapamycin or a rapalog. In certain embodiments, the inducing agent is rapamycin and is administered at least once or more, at least twice or more, or at least three or more times after the composition comprising the rAAV. In some embodiments, the rapamycin is administered at a dose of at least about 4 to at least about 40 nM. In certain embodiments, the inducing agent (i.e., rapamycin) is administered at least It is administered at a dose of about 0.1 mg / kg to at least about 3.0 mg / kg. In certain embodiments, the inducer (i.e., rapamycin) is administered at a dose of at least about 0.5 mg / kg to at least about 2.0 mg / kg.

[0105] The viral vectors and other constructs described herein can be used to deliver GLP-1 fusion protein constructs to a subject in need thereof, to provide a subject with increased half-life GLP-1, and / or to prepare a medicament for treating type I diabetes, type II diabetes, or metabolic syndrome in a subject. Thus, in another aspect, a method of treating diabetes is provided. The method comprises administering a composition described herein to a subject in need thereof. In one embodiment, the composition comprises a viral vector comprising a GLP-1 fusion protein expression cassette described herein.

[0106] As used herein, the term "treatment" or "treating" is defined to include administering to a subject one or more compounds or compositions described herein for the purpose of alleviating one or more symptoms of type I diabetes, type II diabetes, or metabolic syndrome. Thus, "treatment" can include one or more of reducing the progression of type I diabetes, type II diabetes, or metabolic syndrome, reducing the severity of a condition, eliminating a symptom of the disease, slowing the progression of the disease, or increasing the effectiveness of a therapy in a given subject.

[0107] As used herein, the term "remission" refers to the ability to discontinue insulin treatment when a subject no longer exhibits clinical signs of diabetes and has normal blood glucose levels.

[0108] In another embodiment, a method for treating T2DM in a subject is provided. The method comprises administering a viral vector comprising a nucleic acid molecule comprising a sequence encoding a fusion protein as described herein. In one embodiment, the subject is a human.

[0109] In another aspect, a method of treating a metabolic disease in a subject is provided. The method comprises administering a composition as described herein to a subject in need thereof. In one embodiment, the composition comprises a viral vector comprising a GLP-1 fusion protein expression cassette as described herein. In one embodiment, the metabolic disease is type I diabetes. In one embodiment, the metabolic disease is type II diabetes. In one embodiment, the metabolic disease is metabolic syndrome. In one embodiment, the subject is a human.

[0110] In another aspect, a method for reducing weight in a subject is provided. The method comprises administering to a subject in need thereof a composition as described herein. In one embodiment, the composition comprises a viral vector comprising a GLP-1 fusion protein expression cassette as described herein.

[0111] The course of treatment may optionally include repeated administration of the same viral vector (e.g., AAVrh91 vector) or different viral vectors (e.g., AAVrh91 and AAV3B.AR2.12). Still other combinations may be selected using the viral vectors described herein. Optionally, the compositions described herein may be combined in a regimen that includes other diabetes drugs or protein-based therapies (e.g., including GLP-1 analogs, insulin, oral antihyperglycemic drugs (sulfonylureas, biguanides, thiazolidinediones, and alpha glucodase inhibitors)). Optionally, the compositions described herein may be combined in a regimen that involves lifestyle changes, including diet and exercise. In certain embodiments, the AAV vector and the combination therapy are administered essentially simultaneously. In other embodiments, the AAV vector is administered first. In other embodiments, the combination therapy is administered first.

[0112] In one embodiment, the composition is administered in combination with an effective amount of insulin. Various commercially available insulin products are known in the art, including, but not limited to, protamine zinc recombinant human insulin (ProZinc®), porcine insulin zinc suspension (Vetsulin®), insulin glargine (Lantus®), lispro (Humalog), aspart (Novolog), glulisine (Apidra), Novolin, and Verosulin.

[0113] In some embodiments, the combination of rAAV and insulin described herein reduces insulin dosage requirements in a subject compared to before treatment with a viral vector. Such dosage requirements can be reduced by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. The treating physician can determine the correct dosage of insulin required by the subject. For example, the subject can be treated with insulin or other therapy, which the treating physician can continue at the time of administration of the AAV vector. Such insulin or other combination therapy can then be continued, reduced, or discontinued, as necessary.

[0114] In one embodiment, the expression cassette, vector genome, rAAV-containing composition, or other composition described herein for gene therapy is delivered as a single dose per patient. In one embodiment, the subject is delivered a therapeutically effective amount of the composition described herein. As used herein, "therapeutically effective amount" refers to the amount of expression cassette or vector, or combination thereof, that delivers and expresses an amount of GLP1-Fc in a target cell sufficient to achieve a therapeutic goal. The therapeutically effective amount can be selected by the treating physician or can be guided based on previously determined guidelines. For example, dulaglutide can be provided subcutaneously at an initial dose of 0.75 mg once per week. The dose can be increased in 1.5 mg increments for additional glycemic control. The patient should remain on a dose of 1.5 mg once per week for at least 4 weeks before increasing the dose to 3 mg once per week. The patient should remain on a dose of 3 mg once per week for at least 4 weeks before increasing the dose to 4.5 mg once per week. The maintenance dose of dulaglutide can be 0.75-4.5 mg subcutaneously once weekly, with a maximum dose of 4.5 mg weekly. The rAAV can be delivered to a subject and then supplemented with oral or subcutaneous dulaglutide, insulin, or other medications as needed to reach the equivalent of the desired dose of 0.75-4.5 mg once weekly.

[0115] In certain embodiments, the therapeutic goal is to alleviate or treat one or more of the symptoms of type I diabetes, type II diabetes, or metabolic syndrome. The therapeutically effective amount may be determined based on an animal model rather than a human patient. In another embodiment, the therapeutic goal is the amelioration of a metabolic disease in a subject. As used herein, when used to refer to a vp capsid protein, the term "heterologous" or any grammatical variation thereof refers to a population of non-identical members, for example, having vp1, vp2, or vp3 monomers (proteins) with different modified amino acid sequences. SEQ ID NO: 20 provides the encoded amino acid sequence of the AAVrh91 vp1 protein. The term "heterologous" used in reference to vp1, vp2, and vp3 proteins (alternatively referred to as isoforms) refers to differences in the amino acid sequences of the vp1, vp2, and vp3 proteins within the capsid. AAV capsids contain subpopulations within the vp1, vp2, and vp3 proteins that have predicted amino acid residue modifications. These subpopulations contain, at a minimum, specific deamidated asparagine (N or Asn) residues. For example, certain subpopulations contain at least one, two, three, or four highly deamidated asparagine (N) positions in asparagine-glycine pairs, and optionally further contain other deamidated amino acids, where deamidation results in amino acid changes and other optional modifications. I agree.

[0116] As used herein, a "subpopulation" of vp proteins refers to a group of vp proteins that have at least one defined common feature and that consists of at least one group member and fewer than all members of the reference group, unless otherwise specified. For example, a "subpopulation" of vp1 proteins is at least one vp1 protein and fewer than all vp1 proteins in an assembled AAV capsid, unless otherwise specified. A "subpopulation" of vp3 can be one vp3 protein and fewer than all vp3 proteins in an assembled AAV capsid, unless otherwise specified. For example, in an assembled AAV capsid, vp1 protein can be a subpopulation of vp proteins, vp2 protein can be another subpopulation of vp proteins, and vp3 is yet another subpopulation of vp proteins. In another example, the vp1, vp2, and vp3 proteins can include subpopulations having, e.g., at least one, two, three, or four highly deamidated asparagines, e.g., different modifications at asparagine-glycine pairs.

[0117] As used herein, a "stock" of rAAV refers to a population of rAAV. Despite the heterogeneity of capsid proteins due to deamidation, rAAV within a stock are expected to share five identical vector genomes. A stock may contain, for example, rAAV with capsids having selected AAV capsid proteins and heterogeneous deamidation patterns characteristic of a selected production system. A stock may be produced from a single production system or may be pooled from multiple runs of a production system. A variety of production systems may be selected, including but not limited to those described herein. As used herein, the terms "GLP-1 construct", "GLP-1 expression construct" and synonyms include the GLP-1 sequences described herein in combination with leader and fusion domains. The terms "GLP-1 construct", "GLP-1 expression construct" and synonyms may be used to refer to a nucleic acid sequence encoding a GLP-1 fusion protein or its expression product.

[0118] The terms "percent identity (%)", "sequence identity", "percent sequence identity", or "percent identical" in the context of nucleic acid sequences refer to bases in two sequences that are the same when aligned to correspond. The length of sequence identity comparison can be over the entire length of a genome, the entire length of a gene coding sequence, or a fragment of at least about 100-150 nucleotides, or as desired. However, identity between smaller fragments, for example, of at least about 9 nucleotides, usually at least about 20-24 nucleotides, at least about 28-32 nucleotides, at least about 36 nucleotides or more, may also be desired. Multiple sequence alignment programs are also available for nucleic acid sequences. Examples of such programs include "Clustal W", "CAP Sequence Assembly", "BLAST", "MAP", and "MEME", which are accessible through web servers on the Internet. Other sources of such programs are known to those of skill in the art. Alternatively, the Vector NTI utility can also be used. There are also several algorithms known in the art that can be used to measure nucleotide sequence identity, including those included in the programs described above. As another example, polynucleotide sequences can be compared using Fasta™, a program in GCG version 6.1. Fasta™ provides alignment and percent sequence identity of the best overlapping regions between the query and search sequences. For example, percent sequence identity between nucleic acid sequences can be determined using Fasta™ using its default parameters (word size 6 and NOPAM factor for scoring matrix) provided in GCG version 6.1, which is incorporated herein by reference.

[0119] The term "highly conserved" means at least 80% identity, preferably at least 90% identity, more preferably more than 97% identity. Identity is easily determined by those skilled in the art by resorting to algorithms and computer programs known to those skilled in the art.

[0120] Unless otherwise stated in the upper range, the percentage of identity will be understood to be a minimum level of identity and includes all higher levels of identity up to 100% identity to the reference sequence. Unless otherwise stated, the percentage of identity will be understood to be a minimum level of identity and includes all higher levels of identity up to 100% identity to the reference sequence. For example, "95% identity" and "at least 95% identity" can be used interchangeably, including 95%, 96%, 97%, 98%, 99%, up to 100% identity to the reference sequence, and all fractions therebetween.

[0121] The terms "percent identity (%)", "sequence identity", "percent sequence identity", or "percent identity" in the context of amino acid sequences refer to residues in two sequences that are the same when aligned so that they correspond. Percent identity can be readily determined for the corresponding nucleic acid sequence encoding a full-length polypeptide, polypeptide, about 70 amino acids to about 100 amino acids, or peptide fragments thereof, or sequencers of a protein. Suitable amino acid fragments can be at least about 8 amino acids in length and can be up to about 150 amino acids in length. In general, when referring to "identity", "homology", or "similarity" between two different sequences, the "identity", "homology", or "similarity" is determined with reference to the "aligned" sequences. An "aligned" sequence or "alignment" refers to multiple nucleic acid or protein (amino acid) sequences, often including corrections for missing or additional bases or amino acids, as compared to a reference sequence. Alignment is performed using any of a variety of publicly or commercially available multiple sequence alignment programs. Sequence alignment programs are available for amino acid sequences, including, for example, the "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME", and "Match-Box" programs. Typically, one of these programs is used with default settings, but one of skill in the art may modify these settings as necessary. Alternatively, one of skill in the art may utilize another algorithm or computer program that provides at least the same level of identity or alignment as that provided by the referenced algorithms and programs. See, for example, J.D. Thomson et al. al, Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690 (1999).

[0122] It should be noted that the terms "a" or "an" refer to one or more. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0123] The terms "comprise", "comprises", and "comprising" should be interpreted inclusively rather than exclusively. The terms "consist", "consisting", and variations thereof should be interpreted exclusively rather than inclusively. Although various embodiments herein are set forth using the word "comprising", it is also intended that in other circumstances the relevant embodiment should be construed and described using the word "consisting of" or "consisting essentially of".

[0124] As used herein, "patient" or "subject" refers to a mammal, meaning humans, veterinary or agricultural animals, domestic or pet animals, and animals typically used in clinical research. In one embodiment, the subject of these methods and compositions is a human. In another embodiment, the subject is not a feline.

[0125] As used herein, the term "about" means a variability of 10% (±10%, e.g., ±1, ±2, ±3, ±4, ±5, ±6, ±7, ±8, ±9, ±10, or any value therebetween) from a given reference, unless otherwise specified.

[0126] In certain cases, the term "E+#" or "e+#" is used to refer to the exponent. For example, "5E10" or "5e10" refers to 5 × 10 10 These terms may be used interchangeably.

[0127] The term "modulate" or variations thereof as used herein refers to the ability of a composition to inhibit one or more components of a biological pathway.

[0128] As used herein, "disease," "disorder," and "condition" are used interchangeably to refer to an abnormal condition in a subject.

[0129] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and by reference to published documents which provide general guidance to those of ordinary skill in the art for many of the terms used herein.

[0130] References to "one embodiment" or "another embodiment" when describing an embodiment do not imply that the referenced embodiment is mutually exclusive with another embodiment (e.g., an embodiment described before the referenced embodiment), unless expressly specified otherwise. EXAMPLES

[0131] The following examples are provided to illustrate various embodiments of the invention and are not intended to limit the invention in any manner.

[0132] Glucagon-like peptide 1 (GLP-1) is a hormone produced in the gastrointestinal (GI) tract from the proteolytic cleavage of the glucagon preprotein. GLP-1 broadly regulates glucose homeostasis by enhancing insulin release from beta cells, increasing insulin sensitivity in several tissues, slowing gastric emptying (without causing hypoglycemia), and enhancing satiety. GLP-1 has an extremely short half-life, which has prevented it from being effectively used as a drug, but long-acting analogs of GLP-1 have become widely used agents for the treatment of type 2 diabetes. GLP-1 agonists have an excellent safety profile, require repeated, often lifelong parenteral administration, and make them good candidates for AAV-mediated gene transfer, where long-term expression can be achieved after a single dose. GLP-1 and GLP-1 agonists are difficult to express from AAV vectors because the protein cannot be expressed in its native content (glucagon protein), which requires processing by proteases specific to the L-cells of the small intestine. Attempts to express GLP-1 using a heterologous signal peptide have failed to achieve high levels of expression. It was proposed that reliable expression cannot be achieved because the signal peptide does not result in proper processing of the N-terminus of GLP-1, which is involved in receptor binding. Instead, GLP-1 was expressed using a propeptide that is cleaved to produce free GLP-1 protein. These can be cleaved by ubiquitous proteases (e.g., furin) and are immunogenic. We selected propeptides from clotting factors such as thrombin and factor IX for GLP-1 expression because these are endogenous peptides that should not be administered intramuscularly. The thrombin propeptide increased expression of the human GLP-1 analogue by at least 100-fold compared to the signal peptide alone. Using this technology, we developed two long-acting GLP-1 analogues that can be expressed from AAV vectors, one containing an IgG4 Fc fusion and the other containing an albumin fusion, both carrying the human propeptide. We developed expression cassettes that express these proteins constitutively or in a controlled manner via administration of a small molecule drug that activates transcription of the GLP-1 agonist sequence. The target product profile is designed as a single intramuscular injection. In one embodiment, the single injection contains an inducible version as a single tablet every 2-4 weeks, designed to maintain therapeutic GLP-1 agonist levels. In another embodiment, the single injection contains a constitutive version designed for continuous lifelong expression at therapeutic levels after a single dose. The designed products have been tested in preclinical models to investigate pharmacology and safety in non-human primates. Assays have been developed for GLP-1 agonist expression and activity. Safety and pharmacokinetics have been investigated to analyze the ability to achieve known therapeutic concentrations.

[0133] This innovation allows for a one-off, potentially lifelong treatment of type 2 diabetes, especially in patients who do not achieve glycated hemoglobin (also called glycated hemoglobin, hemoglobin A1c, HbA1c, or A1c) goals with metformin alone or other oral agents after 3 months. Standard treatments currently include long-acting subcutaneous GLP-1 agonists such as liraglutide (daily), dulaglutide (weekly), DPP (e.g., dipeptidyl peptidase-4) IV inhibitors (PO), and semaglutide PO (daily). Previous attempts to achieve AAV-mediated GLP-1 expression either resulted in dramatically lower expression or required the use of heterologous leader sequences that were immunogenic and not suitable for clinical applications.

[0134] Example 1 - Construction of GLP-1 Vector GLP-1 agonists are difficult to express via adeno-associated virus (AAV). GLP-1 is usually expressed from the glucagon precursor protein, which requires tissue-specific proteases and produces unwanted proteins. Expression systems using conventional heterologous signal peptides result in low expression. Expression systems using heterologous propeptides with universal protease cleavage sites result in foreign protein sequences that can be targeted by T cells. We developed a system that increases GLP-1 expression from liver or muscle cells by approximately 300-fold without introducing foreign protein sequences. Figure 5 shows AAV-mediated expression of engineered GLP-1 constructs in mice. Mice received intramuscular injections of AAV vectors expressing GLP-1 agonists with the developed (constructs) standard IL-2 signal peptide or endogenous precursor. Serum GLP-1 concentrations were measured by ELISA 3 weeks after injection.

[0135] More specifically, a vector was constructed with a leader sequence placed upstream of one of several GLP-1 receptor agonist amino acid sequences, followed by a fusion domain. See, for example, FIG. 4. The resulting protein sequence was reverse translated, followed by the addition of a Kozak consensus sequence, a stop codon, and a cloning site. The sequence was generated and cloned into an expression vector containing a CMV promoter under the control of an inducible expression system. The expression construct was flanked by AAV2 ITRs. The resulting plasmid is called pAAV.TF.GT2A.hGLP-1-Fc.3w.rBG. The human thrombin-GLP-1-Fc amino acid sequence is shown in SEQ ID NO: 14, the coding sequence is shown in SEQ ID NO: 15, and the vector genome is shown in SEQ ID NO: 16.

[0136] Currently available inducible constructs include two-vector inducible systems and one-vector inducible systems. See, for example, Figures 6A and 6B. Figure 6A shows a schematic diagram of an exemplary expression cassette containing an inducible construct for use in a two-vector system. Figure 6B shows a schematic diagram of an expression cassette containing an inducible construct for use in a one-vector system that includes an IRES linker.

[0137] Furthermore, the GT2A peptide was introduced into the expression vector containing the GLP1-Fc transgene. Human GLP1-Fc with secretion signal is 954bp. For the expression of the hGLP-1-Fc construct, the IRES linker is replaced with the GT2A cleavage sequence in the expression vector as shown in FIG. 6B, as described above, which allows the IRES linker to fit the packaging limit (FIG. 7A; single inducible cassette for GLP-1 Fc). The GT2A peptide is selected from the GT2A_V1 peptide comprising the amino acid sequence of SEQ ID NO: 21, or the GT2A_V2 peptide comprising the amino acid sequence of SEQ ID NO: 22. A schematic diagram of an expression cassette containing an inducible construct for use in a one-vector system, comprising human GLP1-Fc with F2A cleavage sequence linker and secretion signal, is shown.

[0138] Example 2 - In vitro expression GLP1-Fc fusions were measured in culture supernatants of HEK293 cells transfected with the plasmids for inducible human dulaglutide with human thrombin signal sequence (TF.GT2A.hGLP-1-Fc) and CB7 feline dulaglutide (feGLP-1-Fc). By feline dulaglutide we mean a construct in which the IgG Fc portion of dulaglutide is replaced with a feline IgG sequence, optionally combined with a feline thrombin leader (feTrb). Supernatants were collected 48 hours after treatment with rapamycin (Rapa) at 0, 4, and 40 nM, or 48 hours after transfection with CB7.feGLP-1-Fc). GLP1-Fc was quantified by active GLP1 ELISA with the STD of the kit. Expression of the three constructs is shown in Figure 2. Increasing doses of rapamycin led to increased expression of GLP-1.

[0139] Furthermore, the expression of an exemplary therapeutic transgene (rhTT) in rhesus monkeys was evaluated in the designed constructs containing GT2A_V1 or GT2A_V2 peptides (Figures 6B, 7A, and 7B). Figure 8 shows the expression of an exemplary therapeutic transgene (rhTT) in rhesus monkeys in HEK293 cell supernatants after transfection with various constructs containing GT2A peptides and treatment with rapamycin at 0 nM, 4 nM, and 40 nM, plotted as IU / mL of rhTT. Next, the expression of human and rhesus GLP-1 Fc expression in vitro was investigated using the designed single inducible cassettes containing GT2A_V1 and GT2A_V2 peptides. Figure 9 shows the expression of inducible human (h) and rhesus (rh) GLP-1 in vitro. GLP1-Fc fusions were measured in culture supernatants of plasmid-transfected HEK293 cells for inducible hGLP-1-Fc, rhGLP-1-Fc containing the two-vector system, and CB7.rhGLP-1-Fc. Cells were seeded on day 0, transfected on day 1, and treated with rapamycin at 0, 4, and 40 nM on day 2, and supernatants from cells were collected on day 4 or 48 hours after transfection of CB7.rhGLP-1-Fc (containing rhTrb). GLP1-Fc was quantified by active GLP1 ELISA with the STD of the kit.

[0140] Example 3 - Pilot expression in Rag1KO mice The following constructs were packaged into the AAVrh91 vector by triple transfection and iodixanol gradient purification as previously described. AAVrh91.TF.hGLP-1-Fc.3w.rBG with human thrombin signal AAVrh91.TF.rhGLP-1-Fc.3w.rBG with rhesus thrombin signal

[0141] Rag1KO female mice (n = 5 / vector) were administered vector (1 × 10 11Mice were treated with injections of GLP-1 (GC / mouse). Serum was collected serially by separating whole blood in serum separator tubes containing 5 microliters of DPP-IV inhibitor (Millipore) and assayed for active GLP-1 expression and activity as described above. Vector was injected on day 0 and rapamycin was administered around days 14 and 15. Serum active GLP-1 concentrations are shown in Figure 3. Serum levels reached a maximum approximately 1 week after rapamycin administration.

[0142] Example 4 - Long-term expression studies in NHPs In this study, expression of rhesus GLP-1 (rhGLP-1-Fc) in non-human primates (NHPs; i.e., rhesus macaques) was investigated. Tables 1A and 1B provide an overview of the study including AAV administration and rapamycin administration (i.e., induction). Briefly, NHPs were administered vectors designated AAVrh91 via intramuscular injection (IM) according to the table below. For NHP2, rapamycin was administered at a dose of 0.5 mg / kg on day 21, at a dose of 0.5 mg / kg on day 56, and at a dose of 2.0 mg / kg on day 126. For NHP3, rapamycin was administered at a dose of 0.5 mg / kg on day 21, at a dose of 0.5 mg / kg on day 78, and at a dose of 2.0 mg / kg on day 148. [Table 3] [Table 4]

[0143] Figures 10A-C show rhGLP1-Fc expression and analysis of anti-rhGLP1-Fc ADA (anti-drug antibody) detection assay of NHP1(18-128). Figure 10A shows plotted serum rhGLP1-Fc expression levels as nM measured from days 0-200. Figure 10B shows plotted serum rapamycin levels as μg / L measured from days 0-200. Figure 10C shows plotted ADA detection assay results as OD 450 nm measured from days 0-200.

[0144] Figures 11A-11C show expression and analysis of rhGLP1-Fc in anti-rhGLP1-Fc ADA assays of NHP1(18-072). Figure 11A shows the results of measurements taken on days 0-200. Figure 11B shows plotted serum rhGLP1-Fc expression levels as nM measured from day 0 to 200. Figure 11C shows plotted serum rapamycin levels as μg / L measured from day 0 to 200. Figure 11C shows plotted ADA detection assay results as OD 450 nm measured from day 0 to 200.

[0145] Figures 12A-D show rhGLP1-Fc expression and analysis of anti-rhGLP1-Fc ADA assay of NHP1(18-013). Figure 12A shows plotted serum rhGLP1-Fc expression levels as nM measured from days 0-200. Figure 12B shows plotted serum rapamycin levels as μg / L measured from days 0-200. Figure 12C shows plotted ADA detection assay results as OD450 nm measured from days 0-200. Figure 12D shows long-term expression data of NHP1(18-013) treated using the two-vector system. Arrows indicate administration of rapamycin.

[0146] In summary, we developed a one-vector inducible system for the expression of human GLP1-Fc fusions. In addition, we confirmed the induction of human GLP1-Fc upon rapamycin in Rag1KO mice. We observed that one-vector and two-vector inducible vectors expressing monkey GLP1-Fc responded to rapamycin in NHPs, resulting in a transient increase in serum GLP1-Fc of more than 1 nM with a duration of more than 20 days. We observed that low doses of constitutively expressing vectors provided high and sustained expression of serum GLP1-Fc in NHPs.

[0147] Example 5 - Long-term expression studies in NHPs This study investigated the expression of rhesus GLP-1 (rhGLP-1-Fc) in non-human primates (NHPs; i.e., rhesus macaques). Figure 13 shows an overview of the study involving AAV administration and rapamycin administration (i.e., induction) involving the NHPs tested in Example 4 (samples utilized in this study).

[0148] Figures 14A and 14B show rhGLP1-Fc expression and analysis of anti-rhGLP1-Fc ADA assays from animals treated using the constitutive promoter. Figure 14A shows plotted serum rhGLP1-Fc expression levels as nM measured from days 0 to 300. Figure 14B shows plotted ADA detection assay results as OD 450 nm measured from days 0 to 230.

[0149] Figures 15A and 15B show rhGLP1-Fc expression and analysis of anti-rhGLP1-Fc ADA assays from animals treated using the two-vector inducible promoter system. Figure 15A shows plotted serum rhGLP1-Fc expression levels as nM measured from day 0 to about 120. Figure 15B shows plotted ADA detection assay results as OD 450 nm measured from day 0 to 230.

[0150] Figures 16A and 16B show rhGLP1-Fc expression and analysis of anti-rhGLP1-Fc ADA assays from animals treated using the one vector inducible promoter system. Figure 16A shows plotted serum rhGLP1-Fc expression levels as nM measured from day 0 to about 450. Figure 16B shows plotted ADA detection assay results as OD 450 nm measured from day 0 to 230.

[0151] Example 6 - Potency Assay Human and rhesus GLP-1-Fc purified from the plasmids described herein were analyzed using GeneBLAzer® GLP1R-CRE-bla CHO-K1 cells (ThemoFisher) containing the human glucagon-like peptide 1 receptor (GLP1R) stably integrated into the CellSensor® CRE-bla CHO-K1 cell line. ) were used to compare efficacy. CellSensor® CRE-bla CHO-K1 cells contain a beta-lactamase reporter gene under the control of a CRE response element.

[0152] Figure 17 shows the results of a potency assay of the GLP-1-Fc transgene product. Purified human and rhesus GLP-1-Fc were compared to drug-derived dulaglutide (Trulicity), with the human constructs described herein demonstrating comparable or better efficacy than Trulicity.

[0153] Example 7 - Expression of constitutive GLP-1-Fc in NHPs In this study, we investigated the expression of human GLP-1 (hGLP-1-Fc) in non-human primates (NHPs; i.e., rhesus macaques). Table 2 shows an overview of the study that included AAV administration and rapamycin administration (i.e., induction). [Table 5]

[0154] Figure 18A shows plotted serum hGLP1-Fc expression levels in nM measured from days 0 to 150. Figure 18B shows the potency of hGLP-1-Fc from NHP plasma sampled from days 0 to 60. In comparison, Figure 18C shows serum rhGLP-1-Fc expression levels.

[0155] Figure 18D shows the body weights of the two NHPs in the study. Figure 18E shows the blood glucose levels (mg / dL). Reference blood glucose levels for rhesus monkeys are 63-130 mg / dL. Animal 18-007 was asymptomatic and both animals had relatively low baseline BG on day 0. At day 60, no ADA was detected.

[0156] In summary, the hGLP-1-Fc transgene with a thrombin leader results in a transgene product as potent as dulaglutide. The constitutively expressing vector results in 100-1000-fold greater therapeutic levels of GLP-1-Fc at low vector doses in NHPs, demonstrating vector efficacy and safety of the transgene product. The rapamycin-inducible vector shows long-term repeated induction of GLP-1-Fc in NHPs with intravenous or oral rapamycin.

[0157] All documents cited herein are incorporated by reference. Similarly, the sequence listing labeled "22-10015.PCT_Seq-Listing" and submitted herewith, and the sequences and text therein, are incorporated by reference. U.S. Provisional Patent Applications Nos. 63 / 316,220 and 63 / 384,196 are incorporated by reference in their entireties. While the invention has been described with reference to specific embodiments, it will be understood that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. 1. A composition comprising a nucleic acid comprising a sequence encoding a fusion protein comprising a GLP-1 analog and an IgG4 Fc, wherein the fusion protein has the sequence of SEQ ID NO: 14, or a sequence at least 99% identical thereto.

2. 2. The composition of claim 1, wherein the sequence encoding the fusion protein is SEQ ID NO: 15, or a sequence sharing at least 75% identity thereto.

3. A composition comprising a viral vector, (a) an adeno-associated virus (AAV) capsid; (b) a vector genome packaged within the AAV capsid, the vector genome comprising AAV inverted terminal repeats (ITRs), a coding sequence for a fusion protein comprising a GLP-1 analog and an IgG4 Fc, the fusion protein having the sequence of SEQ ID NO: 14, or a sequence at least 99% identical thereto, and regulatory sequences that direct expression of the fusion protein.

4. The composition of claim 3, wherein the viral vector is a recombinant AAV (rAAV) having the AAV capsid of AAVrh91 or AAVhu68.

5. 5. The composition of any one of claims 1 to 4, wherein the fusion protein is under the control of an inducible gene expression system, optionally wherein the inducible gene expression system comprises a regulatable promoter, an activation domain, and a DNA binding domain.

6. 4. The composition of claim 3, wherein the AAV inverted terminal repeats (ITRs) are AAV2 5' ITR and AAV2 3' ITR, flanking the coding and regulatory sequences of the fusion protein, and optionally, the vector genome comprises a CB7 promoter and rabbit globin polyA.

7. The inducible gene expression system comprises: (a) an activation domain comprising a transactivation domain and the FKBP12-rapamycin binding (FRB) domain of FKBP12-rapamycin-associated protein (FRAP); (b) a DNA-binding domain comprising a zinc finger homeodomain (ZFHD) and one, two, or three FK506 binding protein domain (FKBP) subunit genes; (c) at least one copy of a binding site for a ZFHD followed by a minimal promoter; (d) a regulatable promoter, optionally wherein the inducible gene expression system is contained in one vector or wherein the inducible gene expression system is contained in two vectors.

8. The composition of claim 7, wherein the transactivation domain comprises a portion of NF-κB p65 and / or the regulatable promoter is a constitutive promoter or a CMV promoter.

9. The composition of claim 7, further comprising an IRES or 2A and / or at least eight copies of a binding site for a ZFHD.

10. 6. The composition of claim 5, comprising: a regulatable promoter; an activation domain comprising a p65 transactivation domain and the FKBP12-rapamycin binding (FRB) domain of FKBP12-rapamycin-related protein (FRAP); a DNA binding domain comprising a zinc finger homeodomain (ZFHD) and three FK506 binding protein domain (FKBP) subunit genes; 12 copies of binding sites for the ZFHD; and a sequence encoding a fusion protein comprising a GLP-1 analog and human IgG4 Fc.

11. A pharmaceutical composition suitable for use in treating a metabolic disorder in a subject, comprising an aqueous liquid and a composition according to any one of claims 1 to 4.

12. 5. The composition of any one of claims 1 to 4 for use in a method for treating a subject with a metabolic disease.

13. The composition is 1×10 9 GC / kg ~ 5 x 10 13 5. The composition of claim 1, wherein the patient is a human and the rAAV is administered at a dose of 1x10 10 to 1.5x10 15 GC / kg, or wherein the patient is a human and the rAAV is administered at a dose of 1x10 10 to 1.5x10 15 GC, and optionally the rAAV is delivered intramuscularly or intravenously.

14. A recombinant adeno-associated virus (rAAV) for use in a method of treating a subject having a metabolic disease, comprising an AAV capsid derived from adeno-associated virus rh91 or hu68 and a vector genome packaged in the AAV capsid, wherein the vector genome comprises AAV inverted terminal repeats (ITRs), a sequence encoding a fusion protein comprising a GLP-1 analog and human IgG4 Fc, and a regulatory sequence that directs expression of the fusion protein, and the method comprises delivering the rAAV to the subject.

15. 15. The rAAV for use according to claim 14, wherein the patient is administered the composition of any one of claims 1 to 4, optionally wherein the patient is administered the rAAV at a dose of 1 x 10 9 GC / kg to 5 x 10 13 GC / kg body weight, and optionally wherein the rAAV is delivered intramuscularly or intravenously.

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