AAV vectors for delivery of GLP-1 receptor agonist fusions

Abstract

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

Description

[0001] References to Electronic Sequence Listings

[0002] 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 Art

[0003] Glucagon-like peptide 1 (GLP-1) is an endogenous peptide hormone that plays an important role in glucose homeostasis. GLP-1 is a peptide hormone produced by proteolytic cleavage of glucagon precursor protein in the gastrointestinal (GI) tract. GLP-1 and other GLP-1 receptor agonists have the ability to control hyperglycemia by enhancing insulin release, increasing insulin sensitivity, preventing β cell loss and delaying gastric emptying. However, GLP-1 has a short half-life, which hinders its use as a drug. Other GLP-1 receptor agonists are currently used to treat diabetes in humans. GLP-1 receptor agonists that are engineered to overcome the short half-life of natural hormones by fusing agonists to proteins with longer half-lives have become important therapeutic agents for the treatment of type 2 diabetes (T2DM). Summary of the invention

[0004] Provided herein are viral vectors encoding glucagon-like peptide 1 (GLP-1) receptor agonist fusion protein constructs. In some embodiments, these viral vectors can achieve sustained expression and / or increased circulation half-life of the GLP-1 receptor agonist in a subject compared to vector-mediated delivery of a GLP-1 receptor agonist without a fusion partner. Further provided are methods for preparing and using such viral vectors.

[0005] On the one hand, a viral vector is provided, the viral vector comprising a nucleic acid comprising a polynucleotide sequence encoding a fusion protein. The fusion protein comprises (a) a leader sequence comprising a secretion 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.

[0006] 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.

[0007] On the other hand, the viral vector comprises an AAV capsid and a vector genome packaged in the AAV capsid, wherein the vector genome includes an AAV inverted terminal repeat (ITR), the polynucleotide sequence encoding the fusion protein, and a regulatory sequence directing the expression of the fusion protein.

[0008] In another aspect, a pharmaceutical composition suitable for 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.

[0009] In yet another aspect, there is provided use of a viral vector as described herein for the manufacture of a medicament for treating a subject suffering from a metabolic disease, optionally diabetes.

[0010] In another aspect, a method for treating a subject suffering from a metabolic disease is provided, wherein the method comprises administering to the subject an effective amount of a viral vector or composition as described herein.

[0011] Other aspects and advantages of the present invention will become apparent from the following detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is a schematic diagram of Dulaglutide.

[0013] Figure 2 In vitro inducible hGLP-1-Fc and CB7.GLP-1-Fc are shown. GLP1-Fc fusions were measured in culture supernatants of HEK293 cells transfected with plasmids of inducible human GLP-1-Fc with human thrombin signal sequence (TF.GT2A.GLP-1-Fc) and CB7.feGLP-1-Fc with cat thrombin signal sequence. Supernatants were collected 48 hours after treatment with 0nM, 4nM and 40nM rapamycin (Rapa) or 48 hours after transfection with CB7.feGLP-1-Fc. GLP1-Fc was quantified by active form GLP1 ELISA together with the STD of the kit.

[0014] Figure 3 GLP-1 is shown to be expressed in Rag1KO (RAG1 - / - ) mice (n=5 / vector). Rag1KO female mice were given 1 x 10 11GC / mice were injected with 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 the active form of GLP-1 was performed. AAV vectors were injected on day 0, and rapamycin was administered by oral gavage approximately day 14 and day 15 after AAV injection.

[0015] Figure 4 It is a schematic diagram of the plasmid map of pAAV.CMV.TF.GT2A.hGLP-1-Fc.3w.rBG.

[0016] Figure 5 AAV-mediated expression of engineered GLP-1 constructs in mice is shown.

[0017] Fig. 6A A schematic diagram of an exemplary expression cassette including an inducible construct for use in a two-vector system is shown.

[0018] Figure 6B A schematic diagram of an expression cassette comprising an inducible construct for use in a 1-vector system is shown, the expression cassette comprising an IRES linker.

[0019] Fig. 7A A schematic diagram of an expression cassette comprising an inducible construct including a F2A cleavage sequence linker and human GLP1-Fc with a secretion signal for use in a 1-vector system is shown.

[0020] Figure 7B A further detailed view of the GT2A cleavage sequence is shown, wherein GT2A_V1 includes the amino acid sequence of SEQ ID NO:21, and GT2A_V2 includes the amino acid sequence of SEQ ID NO:22.

[0021] Figure 8 Shown is expression of a rhesus macaque exemplary therapeutic transgene (rhTT) in HEK293 cell supernatants, as measured following transfection with various constructs and treatment with 0 nM, 4 nM, and 40 nM of rapamycin, and plotted as IU / mL rhTT.

[0022] Fig. 9Inducible human (h) and rhesus (rh) GLP-1 expression in vitro is shown. GLP1-Fc fusions were measured in culture supernatants of HEK293 cells transfected with inducible hGLP-1-Fc (including thrombin signal sequence), 2-vector system including rhGLP-1-Fc, and plasmids of CB7.rhGLP-1-Fc. Cells were plated on day 0, transfected on day 1, treated with 0 nM, 4 nM, and 40 nM rapamycin on day 2, and supernatants from cells were collected on day 4 or 48 hours after transfection with CB7.rhGLP-1-Fc. GLP1-Fc was quantified by active form GLP1 ELISA along with the STD of the kit.

[0023] Figures 10A to 10C Shown are analyses of rhGLP1-Fc expression and anti-rhGLP1-Fc ADA (anti-drug antibody) detection assays against NHP1(18-128). Fig. 10A Shown are rhGLP1-Fc expression levels in serum plotted in nM, as measured from day 0 to 200. Fig. 10B Shown are rapamycin levels in serum plotted in μg / L, as measured from day 0 to 200. Fig. 10C Shown are the results of the ADA detection assay plotted as OD450nm, as measured from day 0 to 200.

[0024] Figures 11A to 11C Analysis of rhGLP1-Fc expression and anti-rhGLP1-Fc ADA assay against NHP1(18-072) is shown. Fig.11A Shown are rhGLP1-Fc expression levels in serum plotted in nM, as measured from day 0 to 200. Fig. 11B Shown are rapamycin levels in serum plotted in μg / L, as measured from day 0 to 200. Fig. 11C Shown are the results of the ADA detection assay plotted as OD450nm, as measured from day 0 to 200.

[0025] Figures 12A to 12E Analysis of rhGLP1-Fc expression and anti-rhGLP1-Fc ADA assay against NHP1(18-013) is shown. Fig. 12A Shown are rhGLP1-Fc expression levels in serum plotted in nM, as measured from day 0 to 200. Fig. 12B Shown are rapamycin levels in serum plotted in μg / L, as measured from day 0 to 200. Fig. 12C Shown are the results of the ADA detection assay plotted as OD450nm, as measured from day 0 to 200. Fig.12DLong-term rhGLP-1 expression in NHP1(18-013) is shown. Arrows indicate rapamycin administration. Fig.12E Blood rapamycin levels (ng / mL) are shown.

[0026] Fig.13 The experimental design for the experiments described in Example 5 is shown.

[0027] Fig.14A and 14B Shown are analysis of rhGLP1-Fc expression and anti-rhGLP1-Fc ADA assay in animals treated with a constitutive promoter. Fig.14A Shown are rhGLP1-Fc expression levels in serum plotted in nM, as measured from day 0 to 300. Fig. 14B Shown are the results of the ADA detection assay plotted as OD450nm, as measured from day 0 to 230.

[0028] Fig.15A and 15B Shown are analyses of rhGLP1-Fc expression and anti-rhGLP1-Fc ADA assay in animals treated IM using a 2-vector inducible promoter system. Fig.15A Shown are rhGLP1-Fc expression levels in serum plotted in nM, as measured from day 0 to about 120. Fig. 15B Shown are the results of the ADA detection assay plotted as OD450nm, as measured from day 0 to 230. Arrows indicate dosing with rapamycin.

[0029] Fig.16A and 16B Shown are analyses of rhGLP1-Fc expression and anti-rhGLP1-Fc ADA assays from animals treated using the 1-vector inducible promoter system. Fig.16A Shown are rhGLP1-Fc expression levels in serum plotted in nM, as measured from day 0 to about 120. Fig. 16B Shown are the results of the ADA detection assay plotted as OD450nm, as measured from day 0 to 230.

[0030] Fig.17 Results of potency assays of GLP-1-Fc transgene products are shown. Purified human and rhesus GLP-1-Fc were compared to pharmacy-derived dulaglutide (Trulicity), with the human constructs described herein showing comparable or better potency than Trulicity.

[0031] Fig.18A Shown are hGLP1-Fc expression levels in serum plotted in nM, as measured from day 0 to 60. Fig.18B The potency of hGLP-1-Fc in NHP plasma sampled from day 0 to day 60 is shown. Fig. 18C Shown are rhGLP1-Fc expression levels in serum plotted in nM, as measured from day 0 to 80. Fig.18D Shown are the body weights of the 2 NHPs in the study. Fig.18E Blood glucose levels (mg / dL) are shown. The blood glucose reference level for rhesus monkeys is 63-130 mg / dL. Animal 18-007 appeared asymptomatic, and both animals had relatively low baseline BG on day 0. As of day 60, no ADA was detected. DETAILED DESCRIPTION

[0032] Long-acting GLP-1 receptor agonist fusion protein expression constructs have been developed for use in subjects (including humans) in need thereof. A leader sequence is provided, comprising a secretory signal peptide and a fusion domain intended to extend the circulation time of the resulting fusion protein.

[0033] Described are the delivery of these constructs to subjects in need thereof by a variety of routes, and in particular by in vivo expression mediated by a recombinant vector such as an rAAV vector. Also provided are methods of using these constructs in regimens for treating diabetes or metabolic syndrome in subjects in need thereof and increasing the half-life of GLP-1 in the subject. Additionally, methods for enhancing the activity of GLP-1 in a subject are provided. Also provided are methods for inducing weight loss in a subject in need thereof.

[0034] GLP-1 fusion protein

[0035] 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 pre-propolypeptide that undergoes proteolytic processing to form two forms of glucagon, GLP-1 and GLP-2. Initial sequencing studies indicated that GLP-1 has 37 amino acid residues. However, subsequent information has shown that this peptide is a propeptide and undergoes additional processing to remove 6 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 converted to an amide in vivo to form GLP-1 (7-36) amide. GLP-1 (7-37) and GLP-1 (7-36) amide are insulinotropic hormones with equal potency. Therefore, as used herein, the biologically "active" forms of GLP-1 useful herein are: GLP-1-(7-37) and GLP-1-(7-36) NH2.

[0036] GLP-1 receptor agonists are a class of antidiabetic agents that mimic the effects 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, thereby helping T2DM patients achieve glycemic control. As used herein, the term "GLP-1 receptor agonist" refers to at least GLP-1 or its functional fragments, amino acid sequence variants of GLP-1 or its functional fragments, and other polypeptide agonists of the GLP-1 receptor (e.g., exedin-4 and its variants). The present disclosure provides fusion proteins including one or more copies of a GLP-1 receptor agonist, as well as polynucleotides and vectors encoding such fusion proteins. In some embodiments, the fusion protein includes a polynucleotide sequence encoding the fusion protein, the fusion protein including (a) a leader sequence including a secretion 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 IgG Fc or a functional variant thereof.

[0037] In some embodiments, the GLP-1 receptor agonist comprises a variant, which may comprise up to about 10% variation from a GLP-1 nucleic acid or amino acid sequence described herein or known in the art, and the variant retains the function of the wild-type sequence. As used herein, "retaining function" means that the nucleic acid or amino acid acts in the same manner as the wild-type sequence, although not necessarily at the same expression or activity level. 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 expression or activity of the functional variant has an increase or decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the wild-type sequence.

[0038] Several human drugs that fuse a GLP-1 receptor agonist to a stable fusion domain are known in the art. These human drugs include albiglutide, liraglutide, dulaglutide, and lixisenatide (also known by its chemical name des-38-proline-exendin-4 (Heloderma suspectum)-(1-39)-peptidylpenta-L-lysyl-L-lysinamide). Dulaglutide is a disulfide-linked homodimeric fusion peptide in which each monomer consists of a GLP-1 analog portion and an IgG4 Fc region. Yu M et al. (2018) Battle of GLP-1 delivery technologies. Adv. Drug Deliv. Rev. Figure 1 A schematic diagram of dulaglutide is shown in . See WO 2005 / 000892A2, which is incorporated herein by reference.

[0039] In one embodiment, the fusion comprises a combination of a GLP-1 analog and a heterologous sequence. A GLP-1 analog means 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 analog has a maximum of 1, 2 or 3 amino acid substitutions compared to the native sequence. Native human GLP-1 (1-37) has a sequence of HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 1), wherein GLP-1 (7-37) has a sequence of HAEGTFTSDVSSYLEGQAAKEFIA WLVKGRG (SEQ ID NO: 2). In some embodiments, it is desirable to alter the native GLP-1 sequence to optimize one or more of its characteristics. For example, in one embodiment, the GLP-1 analog contains one, two or three amino acid substitutions selected from A8G, G22E and R36G compared to the native sequence. These substitutions have shown efficacy in improving the clinical characteristics of GLP-1, including protection from DPP-4 inactivation (A8G), increased solubility (G22E), and reduced immunogenicity by replacing the arginine at position 36 with a glycine residue (R36G) to remove potential T cell epitopes. 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: HGEGTFTSDVSSYLEEQAAKE FIAWLVKGGG. The GLP-1 analog albiglutide has a sequence as shown in SEQ ID NO:4. The GLP-1 analog exendin-4 has a sequence as shown in SEQ ID NO:5. The GLP-1 analog des-38-proline-exenatide-4 (Blunttail Gila)-(1-39)-peptidylpenta-L-lysyl-L-lysinamide has a sequence as shown in SEQ ID NO:6. In one 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 a DPP-IV resistant variant thereof.

[0040] The 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.

[0041] The leader sequence can be derived from the same species that is ultimately intended to be used, for example, people. As used herein, the term "origin" or "derived from" refers to that a sequence or protein is derived from a specific subject species or shares the same sequence with a protein or sequence derived from a specific subject species. For example, a leader sequence "derived from" people shares the same sequence (or its variant, as defined herein) with the same leader sequence as expressed in people. However, the specified nucleic acid or amino acid does not actually need to be derived from people. Various techniques known in the art that can produce a desired sequence include mutagenesis of similar proteins (for example, homologues) or artificial generation of nucleic acid or amino acid sequences. "Derived" nucleic acid or amino acid retains the function of the same nucleic acid or amino acid in its "derived" species, regardless of the actual source of the derived sequence.

[0042] The term "amino acid substitution" and its synonyms are intended to cover the modification of an amino acid sequence by replacing one amino acid with another substituted amino acid. The substitution may be a conservative substitution. The substitution may also be a non-conservative substitution. With respect to two amino acids, the term conservative is intended to mean that the amino acids share common properties recognized by those skilled 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 properties 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 amino acids and non-natural naturally occurring amino acids are known in the art and may be used as substituted amino acids in various embodiments. Methods for replacing amino acids are well known to those skilled in the art and include, but are not limited to, mutations in the nucleotide sequence encoding the amino acid sequence. Reference herein to "one or more" is intended to encompass, for example, 1, 2, 3, 4, 5, 6 or more separate embodiments.

[0043] In one embodiment, the leader sequence is a human thrombin (factor II) sequence. In one embodiment, the thrombin leader sequence has a sequence shown in SEQ ID NO:7:MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRR or a functional variant thereof with up to 1, 2 or 3 amino acid substitutions. In some embodiments, the leader sequence includes a signal peptide and a propeptide. In one embodiment, the secretory signal peptide of the leader sequence includes a human thrombin signal peptide. In one embodiment, the signal peptide is MAHVRGLQLPGCLALAALCSLVHS (SEQ ID NO: 8) or a functional variant thereof with up to 1, 2 or 3 amino acid substitutions. In another embodiment, the leader sequence includes a human thrombin propeptide. In one embodiment, the propeptide has a sequence of QHVFLAPQQARSLLQRVRR (SEQ ID NO: 9) or a functional variant thereof with up to 1, 2 or 3 amino acid substitutions.

[0044] In one embodiment, functional variants of a desired leader sequence comprise variants that may contain up to about 10% variation from a leader nucleic acid or amino acid sequence described herein or known in the art, which variants retain the function of the wild-type sequence.

[0045] In some embodiments, the coding regions for both the propeptide and the GLP-1 peptide are incorporated into a single nucleic acid sequence without a linker between the coding sequence for the propeptide and the coding sequence for GLP-1.

[0046] The fusion protein further comprises a fusion domain. In one embodiment, the fusion domain is a human IgG Fc fragment or a functional variant thereof. Immunoglobulins generally have a longer in vivo circulation half-life. By fusing a GLP-1 receptor agonist (and a leader sequence) to IgG Fc, the circulation time of the fusion protein is extended, and the function of GLP-1 is retained. In another embodiment, the fusion domain is a rhesus monkey IgG Fc fragment or a functional variant thereof.

[0047] As used herein, the Fc portion of an immunoglobulin has the meaning generally given to the term in the field of immunology. Specifically, this term refers to an antibody fragment that does not contain two antigen-binding regions (Fab fragments) from an antibody. The Fc portion is composed of the constant region of an antibody from two heavy chains, which are associated by non-covalent interactions and disulfide bonds. The Fc portion may include a hinge region and extend to the c-terminus of the antibody through CH2 and CH3 domains. The Fc portion may further include one or more glycosylation sites. In one embodiment, the fusion domain is human IgG Fc. The highly conserved four subclasses IgG1, IgG2, IgG3 and IgG4 differ in their constant regions, particularly in their hinges and upper CH2 domains. See, Vidarsson et al., IgG subclasses and isotypes: From structure to effector functions, Front Immunol., October 2014; 5:520, the document 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 IgG4 Fc. In one embodiment, the human IgG Fc is SEQ ID NO: 11:

[0048] 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 with SEQ ID NO:11.

[0049] In another embodiment, the fusion domain is 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, rhesus IgG Fc is IgG4 Fc. In one embodiment, rhesus IgG Fc is SEQ ID NO: 17:

[0050] PPCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVEVHNAQTKPRERQFNSTYRVV SVLTVTHQDW LNGKEYTCKV SNKGLPAPIE KTISKAKGQRPREPQVYILPPPQEELTKNQV SLTCLVTGFY PSDIAVEWES NGQPENTYKTTPPVLDSDGS YLLYSKLTVN KSRWQPGNIFTCSVMHEALH NHYTQKSLSV 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 with SEQ ID NO: 17. In one embodiment, the rhesus IgG further comprises a hinge sequence.

[0051] The in vivo function and stability of the fusion proteins disclosed herein can be optimized by adding small peptide linkers, for example, to prevent potentially unwanted domain interactions or for other reasons. In addition, the glycine-rich linker can provide some structural flexibility so that the GLP-1 analog portion can effectively interact with the GLP-1 receptor on target cells such as pancreatic beta cells. Therefore, in one embodiment, the C-terminus of the GLP-1 analog and the N-terminus of the fusion domain of the fusion protein are fused through a linker. 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).

[0052] In one embodiment, the fusion protein includes (a) a human thrombin leader sequence; (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 that is at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

[0053] SEQ ID NO:14

[0054] MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0055] 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.

[0056] SEQ ID NO:15:

[0057] atggctcacgttcgaggactgcagctgcctggatgtctggctcttgccgctctgtgtagcctggtgcacagccagcacgtgtttctggctcctcagcaagccagatcactgctgcagagagttagaaggcacggcgagggcacctttacctccgacgtgtctagctacctggaagaacaggccgccaaagagtttatcgcctggctggtcaaaggtggcggcggaggcggaggaagcggtggcggaggttcaggtggtggtggatctgccgagtctaagtacggccctccttgtcctccctgtcctgctcccgaagctgctggcggcccatccgtgtttctgttccctccaaagcctaaggacaccctgatgatcagcagaacccctgaagtgacctgcgtggtggtcgacgtgtcccaagaggatcctgaggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacagttcaacagcacctacagagtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgcctagctccatcgagaaaaccatcagcaaggccaagggccagccaagagaaccccaggtgtacacactgcctccaagccaagaggaaatgaccaagaaccaggtgtccctgacctgcctcgtgaagggcttctacccttccgatatcgccgtggaatgggagagcaatggccagcctgagaacaactacaagaccacacctcctgtgctggacagcgacggctcattcttcctgtacagcagactgaccgtggacaagagcagatggcaagagggcaacgtgttcagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagtctctgagcctgagcctgggc

[0058] In one embodiment, the fusion protein comprises (a) a human thrombin leader sequence; (b) a DPP-IV resistant variant of GLP-1 (7-37); a linker; and (c) rhesus monkey IgG Fc. In one embodiment, the fusion protein comprises (a) a rhesus monkey thrombin leader sequence; (b) a DPP-IV resistant variant of GLP-1 (7-37); a linker; and (c) rhesus monkey IgG Fc.

[0059] In one embodiment, the fusion protein has the sequence of SEQ ID NO: 37, or a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0060] SEQ ID NO:37

[0061] MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQALSLLQRVRRHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAEFTPPCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAQ TKPRERQFNSTYRVVSVLTVTHQDWLNGKEYTCKVSNKGLPAPIEKTISKAKGQPREPQVYILPPPQEELTKNQVSLTCLVTGFYPSDIAVEWESNGQPENTYKTTPPVLDSDGSYLLYSKLTVNKSRWQPGNIFTCSVMHEALHNHYTQKSLSVSPG

[0062] 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.

[0063] SEQ ID NO:36

[0064] atggctcacgttcgaggactgcagctgcctggatgtctggctcttgccgctctgtgtagcctggtgcacagccagcatgtgtttctggctcctcaacaagccctgagcctgctgcaaagagttagaaggcacggcgagggcaccttcacctccgacgtgtccagctacctggaagaacaggccgccaaagagtttatcgcctggctggtcaaaggcggtggtggtggcggaggatctggcggaggtggaagcggcggaggcggatctgctgagtttacacctccttgtcctccctgtcctgctcccgagctgctcggaggcccttccgtgtttctgttccctccaaagcctaaggacaccctgatgatcagcagaacccctgaagtgacctgcgtggtcgtggacgtgtcccaagaggatcctgaggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgcccagacaaagcccagagagcggcagttcaacagcacctacagagtggtgtccgtgctgaccgtgacacaccaggattggctgaacggcaaagagtacacctgtaaagtctccaacaagggcctgcctgctcctatcgagaaaaccatcagcaaggccaagggccagcctagagaaccccaggtgtacatcctgcctccacctcaagaggaactgaccaagaaccaggtgtccctgacctgtctggtcaccggcttctacccttccgatatcgccgtggaatgggagagcaacggacagcccgagaacacctacaagaccacacctccagtgctggacagcgacggcagctatctgctgtactccaagctgacagtgaacaagagccggtggcagcccggcaacatcttcacctgttctgtgatgcacgaggccctgcacaaccactacacccagaagtctctgagcgtcagccctggc

[0065] When a variant or fragment of a leader sequence, a GLP-1 receptor agonist or a fusion domain is desired, site-directed mutagenesis of a wild-type nucleic acid sequence can be used to generate the coding sequence of these peptides. Alternatively or in addition, web-based or commercially available computer programs and service-based companies can be used to reverse 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 and / or cDNA coding sequence is designed to be optimally expressed in the subject species (e.g., humans) that is ultimately intended to be administered.

[0066] Codon optimization can be used to design the coding sequence for optimal expression. The codon optimized coding region can be designed by various methods. This optimization can be performed using a method available online, a disclosed method, or a company providing codon optimization services. For example, a codon optimization method is described in International Patent Application Publication No. WO 2015 / 012924, which is incorporated herein by reference. In short, the nucleic acid sequence of the encoded product is modified with a synonymous codon sequence. Suitably, the entire length of the open reading frame (ORF) of the product is modified. However, in some embodiments, only the fragment of the ORF can be changed. By using one of these methods, frequency can be applied to any given polypeptide sequence, and a nucleic acid fragment of a codon optimized coding region encoding a polypeptide is produced.

[0067] In addition to the leader sequences, GLP-1 receptor agonists, fusion domains, and fusion proteins provided herein, nucleic acid sequences encoding these polypeptides are also provided. In one embodiment, a nucleic acid sequence encoding the GLP-1 fusion protein described herein is provided. In another embodiment, the nucleic acid sequence comprises any nucleic acid sequence encoding the GLP-1 fusion protein of SEQ ID NO: 14.

[0068] Expression cassette

[0069] On the other hand, an expression cassette is provided herein, comprising a nucleic acid encoding a GLP-1 fusion protein as described herein. As used herein, an "expression cassette" refers to a nucleic acid molecule comprising a biologically useful nucleic acid sequence (e.g., a gene cDNA, mRNA, etc. encoding a protein, enzyme, or other useful gene product) and a regulatory sequence operably linked thereto, the regulatory sequence directing or regulating the transcription, translation, and / or expression of the nucleic acid sequence and its gene product. As used herein, an "operably linked" sequence includes both regulatory sequences (also referred to as elements) adjacent or non-adjacent to the nucleic acid sequence and regulatory sequences acting in trans or cis nucleic acid sequences. Such regulatory sequences typically include, for example, one or more of the following: a promoter, an enhancer, a transcription factor, a transcription terminator, an intron, a sequence that enhances translation efficiency (i.e., a Kozak consensus sequence), an efficient RNA processing signal (e.g., a slicing and polyadenylation sequence), a sequence that stabilizes cytoplasmic mRNA (e.g., a woodchuck hepatitis virus (WHP) post-translational regulatory element (WPRE)), and a TATA signal. Expression cassette can contain the regulatory sequence of gene sequence upstream (5 ' place), for example, one or more of promoter, enhancer, intron etc. and one or more of enhancer, or the regulatory sequence of gene sequence downstream (3 ' place), for example, 3 ' untranslated region (3 ' UTR) including polyadenylation site, and other elements.In certain embodiments, regulatory sequence is operably connected with the nucleotide sequence of gene product, and wherein regulatory sequence is separated from the nucleotide sequence of gene product by the inserted nucleotide sequence, i.e. 5 '-untranslated region (5 ' UTR).In certain embodiments, expression cassette comprises the nucleotide sequence of one or more gene products.In certain embodiments, expression cassette can be monocistronic expression cassette or bicistronic expression cassette.In other embodiments, term " transgenic " refers to one or more DNA sequences inserted into target cell from exogenous.

[0070] In one embodiment, an expression cassette refers to a nucleic acid molecule comprising a GLP-1 construct coding sequence (e.g., a coding sequence for a GLP-1 fusion protein), a promoter, and may contain other regulatory sequences thereof, which can be engineered into genetic elements and / or packaged into the capsid of a viral vector (e.g., a viral particle). Typically, such an expression cassette for generating a viral vector contains a GLP-1 construct sequence described herein and other expression control sequences (such as the expression control sequences described herein), wherein the GLP-1 construct sequence is flanked by packaging signals of a viral genome (and referred to as a "vector genome"). Any expression control sequence can be optimized for a particular species using techniques known in the art, including, for example, codon optimization, as described herein.

[0071] In certain embodiments, the expression cassette comprises a constitutive promoter. In another embodiment, the CB7 promoter is used. CB7 is a chicken beta-actin promoter with a cytomegalovirus enhancer element. In some embodiments, the CB7 promoter has a nucleic acid sequence of SEQ ID NO: 33. In one embodiment, the promoter is a CMV promoter. In some embodiments, the CMV promoter is a nucleic acid sequence of SEQ ID NO: 27.

[0072] In one embodiment, the promoter is included in an inducible gene expression system. The inducible gene regulation / expression system contains at least the following components: a promoter operably linked to a transgene encoding a GLP-1 fusion protein described herein (also referred to as a regulatable promoter), an activation domain, a DNA binding domain, and a zinc finger homeodomain binding site. In other embodiments, additional components may be included in the expression system, as further described herein. Figure 4 A plasmid showing an exemplary inducible expression system design is shown in .

[0073] 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, such as the promoter shown in SEQ ID NO: 27. In another embodiment, the promoter is the ubiquitous inducible promoter Z12I, which includes 12 repeated copies of the binding sites of ZFHD1 and IL2 minimal promoters. See, for example, Chen et al., Hum Gene Ther Methods. 2013 August; 24(4): 270–278, which is incorporated herein.

[0074] 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 carboxyl terminus of the p65 subunit from NF-κB and the FKBP12-rapamycin binding (FRB) domain of the FKBP12-rapamycin associated protein (FRAP). In one embodiment, the activation domain is a FKBP12-rapamycin binding (FRB) domain of the human FKBP12-rapamycin associated protein (FRAP) fused to the carboxyl terminus of the p65 subunit from human NF-κB. In one embodiment, the FRB domain has the amino acid sequence shown in SEQ ID NO:24. In one embodiment, the FRB domain has the amino acid sequence shown 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 shown in SEQ ID NO:26. In one embodiment, the p65 subunit has the sequence shown in SEQ ID NO:26 encoded by the nucleic acid sequence of SEQ ID NO:25.

[0075] The inducible system can be included in a single vector including the coding sequence of the fusion protein, or in a two-vector system. Described herein are two-vectors ( Fig. 6A ) and 1-vector ( Figure 6B and Fig. 7A ) system instance.

[0076] 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 IRES or 2A peptides. In one embodiment, the linker is a cleavable 2A peptide. In one embodiment, the linker includes a GT2A_V1 peptide, which includes the amino acid sequence of SEQ ID NO:21. In one embodiment, the linker includes a GT2A_V2 peptide, which includes the amino acid sequence of SEQ ID NO:22. In one embodiment, the 2A peptide is selected to increase packaging restrictions, thereby allowing a single carrier system.

[0077] The DNA binding domain consists of a DNA binding fusion of zinc finger homology domain 1 (ZFHD1) linked to up to three copies of FK506 binding protein (FKBP). In the presence of an inducer (e.g., a rapamycin analog, 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 contained in a 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.

[0078] 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 have different nucleic acids from each other to minimize recombination. For example, SEQ ID NO:30 provides 3 "wobbled" coding sequences of FKBP, each of which encodes the sequence of SEQ ID NO:31: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIR GWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE.

[0079] The expression system further comprises a zinc finger homeodomain binding site. The nucleic acid molecule contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 ZFHD binding sites. In one embodiment, the expression system contains 8 (eight) zinc finger homeodomain binding sites (binding partners) (8XZFHD). However, the present invention encompasses expression systems having two to about twelve copies of zinc finger binding sites. An example of a single copy of a ZFHD binding site is: aatgatgggcgctcgagt (SEQ ID NO: 32).

[0080] In some embodiments, a minimal IL2 promoter is present downstream of the zinc finger homeodomain binding site. An exemplary IL2 promoter is shown in SEQ ID NO: 10.

[0081] Such inducible systems are known in the art and include, for example, the rapamycin inducible system described by, e.g., Rivera et al., A humanized system for pharmacologic control of gene expression, Nature Medicine, Vol. 2, pp. 1028-1032 (September 1996), and Rivera et al., Long-term pharmacologically regulated expression of erythropoietin in primates following AAV-mediated gene transfer, Blood, Feb. 15, 2005, Vol. 105, No. 4, both of which are incorporated herein by reference. In one embodiment, the inducible gene expression system includes a CMV promoter, an activation domain that is the FKBP12-rapamycin binding (FRB) domain of the human FKBP12-rapamycin associated protein (FRAP) fused to the carboxyl terminus of the p65 subunit of human NF-κB, a GT2A peptide, a ZFHD1 DNA binding domain, three FKBP subunits, hGH poly A, 8XZFHD, and a minimal sIL2 promoter. These sequences are in addition to the coding sequence of the GLP-1 fusion protein and optionally other regulatory sequences.

[0082] In addition to the promoter, the expression cassette and / or vector may also contain other appropriate transcription initiation, termination, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (i.e., Kozak consensus sequences), sequences that enhance protein stability, and sequences that enhance secretion of the encoded product when desired. Examples of suitable polyA sequences include, for example, SV40, bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit β-globin (also known as rabbit globin polyA; RGB), modified RGB (mRGB), and TK polyA. Examples of suitable enhancers include, for example, α-fetoprotein enhancer, TTR minimal promoter / enhancer, LSP (TH binding globulin promoter / α1 microglobulin / double Kunitz inhibitor enhancer), etc. In one embodiment, polyA is rabbit globin polyA.

[0083] 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 to the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

[0084] In one embodiment, rAAV is provided, comprising 5'ITR, CB7 promoter, chicken β-actin intron, coding sequence of fusion protein of SEQ ID NO: 14, rabbit globin poly A and 3'ITR. In another embodiment, rAAV comprises polynucleotide comprising CMV promoter, activation domain is FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin associated protein (FRAP) fused to carboxyl terminus of p65 subunit of NF-κB from human, GT2A peptide, ZFHD1 DNA binding domain, three FKBP subunits, hGH poly A, 8XZFHD, minimal sIL2 promoter, coding sequence of GLP-1 fusion protein of SEQ ID NO: 14 and rabbit β-globin poly A.

[0085] In one embodiment, an expression cassette is provided, comprising a polynucleotide comprising a CB7 promoter, a chicken β-actin intron, a coding sequence for a fusion protein of SEQ ID NO: 14, and rabbit globin poly A. In one embodiment, the expression cassette is the expression cassette found in SEQ ID NO: 34, 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, wherein SEQ ID NO: 34, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity thereto, is flanked by 5' and 3' AAV ITRs.

[0086] In another embodiment, an expression cassette is provided, comprising a polynucleotide comprising a CB7 promoter, a chicken β-actin intron, a coding sequence for a fusion protein of SEQ ID NO: 37, and rabbit globin poly A. In one embodiment, the expression cassette is the expression cassette found in SEQ ID NO: 35, 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, wherein SEQ ID NO: 35, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity thereto, is flanked by 5' and 3' AAV ITRs.

[0087] In another embodiment, an expression cassette is provided, the expression cassette comprising a polynucleotide comprising a CMV promoter, a FKBP12-rapamycin binding (FRB) domain of a human FKBP12-rapamycin associated protein (FRAP) fused to the carboxyl terminus of the p65 subunit of NF-κB from humans, a GT2A peptide, a ZFHD1 DNA binding domain, three FKBP subunits, 8XZFHD, a minimal IL2 promoter, a coding sequence of a GLP-1 fusion protein of SEQ ID NO: 14, and rabbit beta globin poly A. In one embodiment, the expression cassette is an expression cassette 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 wherein SEQ ID NO:38, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity thereto, is flanked by 5' and 3' AAV ITRs.

[0088] In another embodiment, an expression cassette is provided, the expression cassette comprising a polynucleotide comprising a CMV promoter, a FKBP12-rapamycin binding (FRB) domain of a human or rhesus FKBP12-rapamycin associated protein (FRAP) fused to the carboxyl terminus of the p65 subunit of NF-κB from a human or rhesus monkey, a GT2A peptide, a ZFHD1 DNA binding domain, three FKBP subunits, 8XZFHD, a minimal IL2 promoter, a coding sequence of a GLP-1 fusion protein of SEQ ID NO: 37, and rabbit beta globin poly A. In one embodiment, the expression cassette is an expression cassette 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 wherein SEQ ID NO:39, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity thereto, is flanked by 5' and 3' AAV ITRs.

[0089] In another embodiment, an expression cassette is provided, comprising a polynucleotide comprising a Z12I promoter (comprising 12 ZFHD1 sites and a minimal IL2 promoter), a coding sequence for a GLP-1 fusion protein of SEQ ID NO: 37, and rabbit beta globin poly A. In one embodiment, the expression cassette is the expression cassette 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, wherein SEQ ID NO: 40, 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. Provided is a second expression cassette comprising a polynucleotide comprising a CMV promoter, a chimeric intron, a FKBP12-rapamycin binding (FRB) domain of a human or rhesus FKBP12-rapamycin associated protein (FRAP) fused to a p65 subunit of NF-κB from a human or rhesus (or a portion thereof), an IRES or 2A peptide, a ZFHD1 DNA binding domain, three FKBP subunits, 8XZFHD, and a polyA sequence. In one embodiment, the expression cassette is an expression cassette found in SEQ ID NO: 41, 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 wherein SEQ ID NO:41, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity thereto, is flanked by 5' and 3' AAV ITRs.

[0090] Viral vectors

[0091] On the other hand, a viral vector comprising an expression cassette as described herein is provided. In certain embodiments of the viral vectors described herein, the viral vector is an adeno-associated virus (AAV) viral vector or a recombinant AAV (rAAV). As used herein, the term "recombinant AAV" or "rAAV" refers to naturally occurring adeno-associated viruses, adeno-associated viruses available to those skilled in the art and / or obtainable in view of the compositions and methods described herein, and artificial AAV. Adeno-associated virus (AAV) viral vectors are AAV DNA enzyme-resistant particles with AAV protein capsids, wherein the expression cassette for delivery to target cells is packaged with AAV reverse terminal repeats (ITRs) (together referred to as "vector genomes") flanked by AAV. The AAV capsid is composed of 60 capsid (cap) protein subunits VP1, VP2, and VP3, which are arranged in an icosahedral symmetric manner, with a ratio of about 1:1:10 to 1:1:20, depending on the selected AAV. Various AAVs can be selected as the source of the capsid of the AAV viral vector as identified above. In one embodiment, the AAV capsid is an AAVrh91 capsid or a variant thereof. In certain embodiments, the capsid protein is designated by a number or a combination of numbers and letters following the term "AAV" in the rAAV vector name. Unless otherwise indicated, the AAV capsids, ITRs, and other selected AAV components described herein can be readily selected from any AAV, including but not limited to AAVs identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVhu37, AAVrh32.33, AAV Anc80, AAV10, AAV11, AAV12, AAVrh8, AAVrh74, AAV-DJ8, AAV-DJ, AAVhu.37, AAVrh.64R1, and AAVhu68. See, e.g., U.S. Published Patent Application No. 2007-0036760-A1; U.S. Published Patent Application No. 2009-0197338-A1; EP 1310571. See also WO 2003 / 042397 (AAV7 and other simian AAVs), U.S. Pat. No. 7790449 and U.S. Pat. No. 7282199 (AAV8), WO 2005 / 033321 and US 7,906,111 (AAV9) and WO 2006 / 110689 and WO2003 / 042397 (rh.10), WO 2005 / 033321, WO 2018 / 160582 (AAVhu68), which are incorporated herein by reference.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, now WO 2020 / 223231 published November 5, 2020], AAVrh92, AAVrh93, AAVrh91.93 [PCT / US20 / 30281 filed April 28, 2020], all of 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 on October 21, 2019 and U.S. Provisional Patent Application No. 63 / 025,753 filed on May 15, 2020, which describe 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.AR2.09, 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 .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 or AAV3B.AR2.17, all of which are incorporated herein by reference. See also International Patent Application No. PCT / US21 / 45945 filed on August 13, 2021, U.S. Provisional Patent Application No. 63 / 065,616 filed on August 14, 2020, and U.S. Provisional Patent Application No. 63 / 109,734 filed on November 4, 2020, all of which are incorporated herein by reference in their entirety. These documents also describe other AAV capsids that can be selected for the production of rAAV and are incorporated by reference. Among the AAVs isolated or engineered from humans or non-human primates (NHPs) and well characterized, human AAV2 was the first AAV to be developed as a gene transfer vector; it has been widely used in efficient gene transfer experiments in different target tissues and animal models.

[0092] As used herein, with respect to AAV, the term "variant" means any AAV sequence derived from a known AAV sequence, including an AAV sequence with a conservative amino acid substitution and an AAV sequence sharing at least 90%, at least 95%, at least 97%, at least 99% or higher sequence identity with an amino acid or nucleic acid sequence. In another embodiment, the AAV capsid comprises a variant, which may include up to about 10% change with 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 the AAV capsid provided herein and / or known in the art. In one embodiment, the AAV capsid shares at least 95% identity with the AAV capsid. When determining the identity percentage of the AAV capsid, any variable protein (e.g., vp1, vp2 or vp3) can be compared.

[0093] In one embodiment, the viral vector is an rAAV having a capsid of AAV8 or a functional variant thereof. In one embodiment, the viral vector is an rAAV having a capsid of AAVrh91 or a functional variant thereof. In one embodiment, the viral vector is an rAAV having a capsid of AAV3.AR.2.12 or a functional variant thereof. In one embodiment, the viral vector is an rAAV having a capsid selected from AAV9, AAVrh64R1, AAVhu37 or AAVrh10.

[0094] In certain embodiments, the AAV is rh91. A nucleic acid sequence encoding an AAVrh91 capsid is provided in SEQ ID NO: 18, and an encoded amino acid sequence is provided in SEQ ID NO: 20. A rAAV is provided herein, 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 an AAVrh91 amino acid sequence is provided in SEQ ID NO: 19, and an 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 N-terminal and / or C-terminal truncations (e.g., truncations of about 1 to about 10 amino acids).

[0095] In certain embodiments, the AAVrh91 capsid is characterized by one or more of the following: (1) AAVrh91 capsid proteins comprising: a heterogeneous population of AAVrh91 vp1 proteins selected from the group consisting of vp1 proteins produced by expression of a nucleic acid sequence encoding a predicted amino acid sequence of 1 to 736 of SEQ ID NO:20, vp1 proteins produced by expression of SEQ ID NO:18, or vp1 proteins produced by a nucleic acid sequence at least 70% identical to SEQ ID NO:18 encoding a predicted amino acid sequence of 1 to 736 of SEQ ID NO:20, a heterogeneous population of AAVrh91 vp2 proteins selected from the group consisting of vp2 proteins produced by expression of a nucleic acid sequence encoding at least about amino acids 138 to 736 of SEQ ID NO:20, vp2 proteins produced by a sequence comprising at least nucleotides 412 to 2208 of SEQ ID NO:18, or vp2 proteins produced by a nucleic acid sequence at least 70% identical to SEQ ID NO:18 encoding a predicted amino acid sequence of SEQ ID NO:20. a heterogeneous population of AAVrh91 vp3 proteins selected from the group consisting of: vp3 proteins produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:20, vp3 proteins produced from a sequence comprising at least nucleotides 607 to 2208 of SEQ ID NO:18, or vp3 proteins produced from a nucleic acid sequence at least 70% identical to at least nucleotides 607 to 2208 of SEQ ID NO:18 encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:20; and / or (2) a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:20; a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:20; and / or (3) a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:20; NO:20; and a heterogeneous population of vp3 proteins, the vp3 proteins being the product of a nucleic acid sequence encoding at least about amino acids 138 to 736 of SEQ ID NO:20, wherein: the vp1, vp2 and vp3 proteins contain a subpopulation having amino acid modifications, the amino acid modifications comprising at least two highly deamidated asparagines (N) in the asparagine-glycine pairs in SEQ ID NO:20; 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 sequence and a non-AAV nucleic acid sequence encoding a product operably linked to a sequence directing expression of the product in a host cell.;

[0096] In certain embodiments, the AAVrh91 capsid is characterized by one or more of the following: (1) AAVrh91 capsid proteins comprising: a heterogeneous population of AAVrh91 vp1 proteins selected from the group consisting of vp1 proteins produced by expression of a nucleic acid sequence encoding a predicted amino acid sequence of 1 to 736 of SEQ ID NO:20, vp1 proteins produced by SEQ ID NO:19, or vp1 proteins produced by a nucleic acid sequence at least 70% identical to SEQ ID NO:19 encoding a predicted amino acid sequence of 1 to 736 of SEQ ID NO:20, a heterogeneous population of AAVrh91 vp2 proteins selected from the group consisting of vp2 proteins produced by expression of a nucleic acid sequence encoding at least about amino acids 138 to 736 of SEQ ID NO:20, vp2 proteins produced by a sequence comprising at least nucleotides 412 to 2208 of SEQ ID NO:19, or vp2 proteins produced by a nucleic acid sequence at least 70% identical to SEQ ID NO:19 encoding a predicted amino acid sequence of SEQ ID NO:20. a heterogeneous population of AAVrh91 vp3 proteins selected from the group consisting of: vp3 proteins produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:20, vp3 proteins produced from a sequence comprising at least nucleotides 607 to 2208 of SEQ ID NO:19, or vp3 proteins produced from a nucleic acid sequence at least 70% identical to at least nucleotides 607 to 2208 of SEQ ID NO:19 encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:20; and / or (2) a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:20; a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:20; and / or (3) a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:20; NO:20; and a heterogeneous population of vp3 proteins, the vp3 proteins being the product of a nucleic acid sequence encoding at least about amino acids 138 to 736 of SEQ ID NO:20, wherein: the vp1, vp2 and vp3 proteins contain a subpopulation having amino acid modifications, the amino acid modifications comprising at least two highly deamidated asparagines (N) in the asparagine-glycine pairs in SEQ ID NO:20; 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 sequence and a non-AAV nucleic acid sequence encoding a product operably linked to a sequence directing expression of the product in a host cell.;

[0097] In certain embodiments, the AAVrh91 vp1, vp2, and vp3 proteins contain a subpopulation with amino acid modifications that include at least two highly deamidated asparagines (N) in the asparagine-glycine pairs in SEQ ID NO: 20, and optionally further include a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change. A high level of deamidation is observed at NG pairs N57, N383, and / or N512 relative to numbering of SEQ ID NO: 20. Deamidation has been observed at other residues. In certain embodiments, AAVrh91 can have other deamidated residues, e.g., typically less than 10%, and / or can have other modifications comprising phosphorylation (e.g., where present, ranging from about 2 to about 30%, or about 2 to about 20%, or about 2 to about 10%) (e.g., at S149) or oxidation (e.g., at one or more of -W22, -M211, W247, M403, M435, M471, W478, W503, -M537, -M541, -M559, -M599, M635, and / or W695). Optionally, W can be oxidized to kynurenine.

[0098] Table A - AAVrh91 deamidation

[0099] AAVrh91 deamidation based on VP1 numbering Deamidation% N57+ deamidation 65-90, 70-95, 80-95, 75-100, 80-100 or 90-100 N94+ deamidation 2-15 or 2-5 N303+ deamidation 2-15 or 5-10 N383+deamidation 65-90, 70-95, 80-95, 75-100, 80-100 or 90-100 N497+deamidation 2-15 or 5-10 N512+ deamidation 65-90, 70-95, 80-95, 75-100, 80-100 or 90-100 ~N691+deamidation 2-15, 2-10 or 5-10

[0100] In certain embodiments, the AAVrh91 capsid is modified in one or more of the positions identified in the above table within the range provided as determined using mass spectrometry using trypsin. In certain embodiments, one or more of the positions are modified as described herein or a glycine after the N. The residue numbers are based on the AAVrh91 sequence provided herein. See, SEQ ID NO: 20.

[0101] In certain embodiments, the AAVrh91 capsid comprises: a heterogeneous population of vp1 proteins, wherein the vp1 proteins are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:20; a heterogeneous population of vp2 proteins, wherein the vp2 proteins are the product of a nucleic acid sequence encoding the amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:20; and a heterogeneous population of vp3 proteins, wherein the vp3 proteins are the product of a nucleic acid sequence encoding at least amino acids 203 to 736 of SEQ ID NO:20.

[0102] In certain embodiments, the modified AAVrh91 nucleic acid sequence is used to generate a mutant rAAV having a capsid that is less deamidated than the native AAVrh91 capsid. Such mutant rAAV may have reduced immunogenicity and / or increased stability during storage, particularly when stored in suspension form.

[0103] In one aspect, a recombinant AAV (rAAV) is provided. The rAAV comprises an AAV capsid from adeno-associated virus rh91 and a vector genome packaged in the AAV capsid, wherein the vector genome comprises an AAV inverted terminal repeat sequence (ITR), a coding sequence of a GLP-1 receptor agonist of SEQ ID NO: 14, and a regulatory sequence for directing the expression of the GLP-1 receptor agonist.

[0104] In certain embodiments, the AAV68 capsid is further characterized by one or more of the following. The AAV hu68 capsid protein comprises: an AAVhu68 vp1 protein produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 736 of SEQ ID NO:42, a vp1 protein produced by SEQ ID NO:43 or 44, or a vp1 protein produced by a nucleic acid sequence at least 70% identical to SEQ ID NO:43 or 44, the nucleic acid sequence encoding the predicted amino acid sequence of 1 to 736 of SEQ ID NO:42; an AAVhu68 vp2 protein produced by expression of a nucleic acid sequence encoding at least about amino acids 138 to 736 of SEQ ID NO:42, a vp2 protein produced by a sequence including at least nucleotides 412 to 2211 of SEQ ID NO:43 or 44, or a vp2 protein produced by a nucleic acid sequence at least 70% identical to at least nucleotides 412 to 2211 of SEQ ID NO:43 or 44, the nucleic acid sequence encoding NO:42; and / or an AAVhu68 vp3 protein produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:42, a vp3 protein produced from a sequence including at least nucleotides 607 to 2211 of SEQ ID NO:43 or 44, or a vp3 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 607 to 2211 of SEQ ID NO:43 or 44, wherein the nucleic acid sequence encodes a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:42.

[0105] Additionally or alternatively, an AAV capsid is provided, comprising a heterogeneous population of vp1 proteins that optionally include valine at position 157, a heterogeneous population of vp2 proteins that optionally include valine at position 157, and a heterogeneous population of vp3 proteins, wherein based on the numbering of the vp1 capsid of SEQ ID NO:42, at least one subpopulation of vp1 and vp2 proteins includes valine at position 157 and optionally further includes glutamic acid at position 67. Additionally or alternatively, an AAVhu68 capsid is provided, comprising: a heterogeneous population of vp1 proteins, wherein the vp1 proteins are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:42; a heterogeneous population of vp2 proteins, wherein the vp2 proteins are the product of a nucleic acid sequence encoding the amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:42; and a heterogeneous population of vp3 proteins, wherein the vp3 proteins are the product of a nucleic acid sequence encoding at least amino acids 203 to 736 of SEQ ID NO:42, wherein: the vp1, vp2 and vp3 proteins contain subpopulations having amino acid modifications.

[0106] AAVhu68 vp1, vp2 and vp3 proteins are generally expressed as alternative splice variants encoded by the same nucleic acid sequence encoding the full-length vp1 amino acid sequence (amino acids 1 to 736) of SEQ ID NO: 42. Optionally, the vp1 coding sequence alone is used to express the vp1, vp2 and vp3 proteins. Alternatively, this sequence can be co-expressed with one or more of the following: a nucleic acid sequence encoding the AAVhu68 vp3 amino acid sequence of SEQ ID NO:42 (about aa 203 to 736), wherein the AAVhu68 vp3 amino acid sequence is without 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 the complementary strands thereto, i.e., the corresponding mRNA or tRNA (about nt 607 to about nt 2211 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, wherein SEQ ID NO:43 or 44 encodes aa 203 to 736 of SEQ ID NO:42. Additionally or alternatively, the vp1-encoding and / or vp2-encoding sequences can be co-expressed with one or more of the following: a nucleic acid sequence encoding the AAVhu68 vp2 amino acid sequence of SEQ ID NO:42 (approximately aa 138 to 736) without the vp1 unique region (approximately aa 1 to approximately 137) or the complementary strand thereto, i.e., the corresponding mRNA or tRNA (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 approximately aa 138 to 736 of SEQ ID NO:42.

[0107] 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 an additional nucleic acid sequence encoding, for example, a vp3 protein that does not contain the unique regions of vp1 and / or vp2. rAAVhu68 produced from production using a single nucleic acid sequence vp1 produces a heterogeneous population of vp1 proteins, vp2 proteins, and vp3 proteins. More specifically, AAVhu68 capsids contain subpopulations within the vp1 protein, within the vp2 protein, and within the vp3 protein that have modifications of the predicted amino acid residues from SEQ ID NO:42. These subpopulations contain at least deamidated asparagine (N or Asn) residues. For example, the asparagine in the asparagine-glycine pair is highly deamidated.

[0108] In one embodiment, the AAVhu68 vp1 nucleic acid sequence has the sequence of SEQ ID NO:43 or 44 or a complementary strand thereto, such as a corresponding mRNA or tRNA. In certain embodiments, the vp2 and / or vp3 proteins can be expressed additionally or alternatively from a nucleic acid sequence different from vp1, such as to change 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 to 736) is also provided, wherein the AAVhu68 vp3 amino acid sequence lacks 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, i.e., 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 to 736) is also provided, wherein the AAVhu68 vp2 amino acid sequence does not have the vp1 unique region (about aa 1 to about aa137) or the complementary strand thereto, i.e., the corresponding mRNA or tRNA (nt 412 to 2211 of SEQ ID NO:43 or 44).

[0109] However, other nucleic acid sequences encoding the amino acid sequence of SEQ ID NO: 42 may be selected for use in generating rAAVhu68 capsids. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 43 or 44, or a sequence at least 70% to 99% identical, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to SEQ ID NO: 43 or 44 encoding SEQ ID NO: 42. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence 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%, at least 99% identical to about nt 412 to about nt 2211 of SEQ ID NO: 43 or 44 encoding the vp2 capsid protein (about aa 138 to 736) of SEQ ID NO: 42. In certain embodiments, the nucleic acid sequence has a nucleic acid sequence of about nt 607 to about nt 2211 of SEQ ID NO:43 or 44, or a sequence that is 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 nt SEQ ID NO:43 or 44 encoding the vp3 capsid protein of SEQ ID NO:42 (about aa 203 to 736).

[0110]

[0111]

[0112] In certain embodiments, the AAVhu68 capsid is characterized as 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 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) are deamidated. In these and other embodiments, the AAVhu68 capsid is further characterized as having a population of proteins in which about 1% to about 20% of the N residues are deamidated at one or more of the following 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 includes at least a subset of vp1, vp2, and / or vp3 proteins that are deamidated at one or more of the following 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, based on numbering of the amino acid sequence of SEQ ID NO: 42, or a combination thereof. In certain embodiments, the capsid protein may have one or more amidated amino acids.

[0113] In another embodiment, a recombinant adeno-associated virus (rAAV) is provided having an AAVhu68 capsid and a vector genome, wherein (a) the AAV hu68 capsid comprises a heterogeneous population of AAVhu68 vp1 proteins, a heterogeneous population of AAVhu68 vp2 proteins, and a heterogeneous population of AAVhu68 vp3 proteins, wherein the heterogeneous AAVhu68 vp1, AAVhu68 vp2, and AAVhu68 vp3 proteins contain a subpopulation having an amino acid modification, wherein the amino acid modification comprises a SEQ ID NO: 1 as determined using mass spectrometry. NO:42 wherein at least two asparagines (N) in an asparagine-glycine pair in two or more of N57, N329, N452, N512 are deamidated to 50% to 100%, and optionally further comprises a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change wherein the deamidated asparagine is deamidated to aspartic acid, isoaspartic acid, an interconverted aspartic acid / isoaspartic acid pair, or a combination thereof, wherein the AAVhu68 capsid further comprises a subpopulation having one or more of:

[0114] 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;

[0115] The vp1, v2 and vp3 proteins have at least 75% deamidated N in the asparagine-glycine pair in position N329 based on the residue numbering of the amino acid sequence of SEQ ID NO:42;

[0116] The vp1, v2 and vp3 proteins have at least 50% of the N in the asparagine-glycine pair in position N452, based on the residue numbering of the amino acid sequence of SEQ ID NO: 42, deamidated; and / or

[0117] at least 75% of the N in the asparagine-glycine pair in position N512 of the vp1, v2 and vp3 proteins based on the residue numbering of the amino acid sequence of SEQ ID NO: 42 is deamidated; and

[0118] The vector genome in the AAVhu68 capsid comprises a nucleic acid molecule comprising an AAV inverted terminal repeat sequence and a non-AAV nucleic acid sequence encoding a GLP-1 fusion as described herein operably linked to a sequence that directs expression of the GLP-1 fusion in a target cell.

[0119] In one embodiment, rAAV is scAAV. The abbreviation "sc" refers to self-complementary. "Self-complementary AAV" refers to a plasmid or vector having an expression cassette in which the coding region carried by the recombinant AAV nucleic acid sequence has been designed to form an intramolecular double-stranded DNA template. After infection, the second strand of cell-mediated synthesis is not waited for, but two complementary half scAAVs will associate to form a double-stranded DNA (dsDNA) that is easy to replicate and transcribe immediately. See, for example, DM McCarty et al., "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis" "Gene Therapy, (August 2001), Vol. 8, No. 16, pp. 1248-1254. Self-complementary AAVs are described, for example, in U.S. Pat. Nos. 6,596,535, 7,125,717, and 7,456,683, which are incorporated herein by reference in their entireties.

[0120] 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, bacteriophage, transposon, cosmid, RNA molecule (e.g., mRNA), episome, etc., which transfers the GLP-1 sequence carried thereon to a host cell, e.g., for use in producing nanoparticles carrying DNA or RNA in packaging host cells, viral vectors, and / or host cells for delivery to a subject. In one embodiment, the genetic element is a plasmid. The selected genetic element can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion technology, high-speed DNA-coated pellets, viral infection, and protoplast fusion. Methods for preparing 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 Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0121] As used herein, the term "host cell" may refer to a packaging cell line, wherein a vector (e.g., recombinant AAV or rAAV) is produced by a production plasmid. In an alternative, the term "host cell" may refer to any target cell that is desired to express a gene product as described herein. Therefore, "host cell" refers to a prokaryotic or eukaryotic cell (e.g., bacterial cell, human cell, or insect cell), which contains exogenous or heterologous DNA introduced into the cell by any means (e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion technology, high-speed DNA-coated pellets, viral infection, and protoplast fusion). In certain embodiments herein, the term "host cell" refers to a culture of cells of various mammalian species for in vitro evaluation of compositions described herein. In other embodiments herein, the term "host cell" refers to a cell for producing and packaging a viral vector or a recombinant virus. In a further embodiment, the term "host cell" is an intestinal cell, a small intestinal cell, a pancreatic cell, a liver cell.

[0122] As used herein, the term "target cell" refers to any target cell in which it is desired to express a heterologous nucleic acid sequence or protein. In certain embodiments, the target cell is a liver cell. In other embodiments, the target cell is a muscle cell.

[0123] In one embodiment, rAAV is provided, which includes a vector genome comprising an expression cassette, wherein the expression cassette includes a CMV promoter, an activation domain that is the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin associated protein (FRAP) fused to the carboxyl terminus of the p65 subunit of NF-κB from human, a GT2A_V1 peptide, a ZFHD1 DNA binding domain, three FKBP subunits, hGH poly A, 8XZFHD, a minimal sIL2 promoter, a coding sequence of the GLP-1 fusion protein of SEQ ID NO: 14, and rabbit β-globin polyA. In another embodiment, rAAV is provided, comprising a vector genome comprising an expression cassette, wherein the expression cassette comprises a CMV promoter, an activation domain which is the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin associated protein (FRAP) fused to the carboxyl terminus of the p65 subunit of NF-κB from human, a GT2A_V2 peptide, a ZFHD1 DNA binding domain, three FKBP subunits, hGH poly A, 8XZFHD, a minimal sIL2 promoter, a coding sequence of a GLP-1 fusion protein of SEQ ID NO: 14, and rabbit β-globin polyA.

[0124] The minimum sequence required to package the expression cassette into the AAV virus particle is AAV 5' and 3' ITRs with the same AAV origin as the capsid or with a different AAV origin (to produce an AAV pseudotype). In one embodiment, the ITR sequence from AAV2 or a deleted version thereof (ΔITR) is adopted for convenience and to speed up regulatory approval. However, ITRs from other AAV sources can be selected. Preferably, the source of ITR is the same as the source of Rep protein, and the Rep protein is provided in trans for production. Typically, the expression cassette of the AAV vector includes AAV 5'ITR, GLP-1 fusion protein coding sequence and any regulatory sequence and AAV 3'ITR. However, other configurations of these elements may be suitable. A shortened version of the 5'ITR referred to as ΔITR has been described, in which the D sequence and the terminal resolution site (trs) are deleted. In other embodiments, full-length AAV5' and 3'ITR are used.

[0125] In order to package the expression cassette into the virion, ITR is the only AAV component required in cis in the construct identical to the gene. In one embodiment, the coding sequence for replication (rep) and / or capsid (cap) is removed from the AAV genome and supplied in trans or supplied by a packaging cell line to produce an AAV vector. For example, as described above, the pseudotype AAV can contain ITRs from a source different from the source of the AAV capsid. In one embodiment, a chimeric AAV capsid can be utilized. Still other AAV components can be selected. The sources of such AAV sequences are described herein and can also be isolated or obtained from academic, commercial or public sources (e.g., the American Type Culture Collection, Manassas, VA, in Manassas, Virginia). AAV sequences can be synthesized or otherwise suitable by reference to disclosed sequences (e.g., in the literature or in, for example, obtained from publicly available sequences in databases such as those provided by

[0126] Methods for producing and isolating AAV viral vectors suitable for delivery to a subject are known in the art. [See, e.g., U.S. Pat. No. 7,790,449; U.S. Pat. No. 7,282,199; WO 2003 / 042397; WO 2005 / 033321; WO2006 / 110689; and US 7,588,772 B2]. In one system, a production cell line is transiently transfected with a construct encoding a transgene flanked by ITRs and a construct encoding rep and cap. In a second system, a packaging cell line that stably supplies rep and cap is transiently transfected with a construct encoding a transgene flanked by ITRs. In each of these systems, AAV virions are produced in response to infection with a helper adenovirus or herpes virus, thereby requiring separation of rAAV from contaminating viruses. Recently, systems have been developed that do not require infection with a helper virus to recover AAV - systems by which the desired helper functions are also provided in trans (i.e., adenovirus E1, E2a, VA and E4 or herpesvirus UL5, UL8, UL52 and UL29, and herpesvirus polymerase). In these newer systems, helper functions can be provided by transiently transfecting cells with constructs encoding the desired 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, transgenes flanked by ITRs and rep / cap genes are introduced into insect cells by infection with baculovirus-based vectors. For a review of these production systems, see generally, 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 entirety. Methods for 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, e.g., Grieger and 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 references cited below, each of which is incorporated herein by reference in its entirety. Methods for constructing any embodiment of the present invention 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 et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York City (2012). Similarly, methods for producing rAAV virions are well known, and the selection of an appropriate method is not a limitation of the present invention. See, for example, K. Fisher et al., (1993) J. Virol., 70:520-532 and U.S. Pat. No. 5,478,745.

[0127] The rAAV described herein includes a selected capsid with a vector genome packaged inside. The vector genome (or rAAV genome) includes 5' and 3' AAV inverted terminal repeats (ITRs), a polynucleotide sequence encoding a fusion protein, and a regulatory sequence that directs the polynucleotide sequence encoding the fusion protein to be inserted into the genome of the host cell. In one embodiment, the vector genome is a sequence shown in SEQ ID NO: 16 or a sequence that shares at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity thereto.

[0128] 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 nucleic acid sequences contain AAV reverse terminal repeats (ITRs). In the examples herein, the vector genome contains at least AAV 5'ITR, coding sequence (i.e., transgene) and AAV 3'ITR from 5' to 3'. It is possible to select ITRs from AAV2 (an AAV different from the capsid source) or in addition to non-full-length ITRs. In certain embodiments, ITRs are from AAVs that provide the same AAV source as the rep function during production or trans-supplementation of AAV. In addition, other ITRs can be used, for example, self-complementary (scAAV) ITRs. Both single-stranded AAV and self-complementary (sc) AAV are covered in rAAV. Transgenics is a nucleic acid coding sequence heterologous to the vector sequence, and the nucleic acid coding sequence encodes a polypeptide, a protein, a functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene products of interest. The nucleic acid coding sequence is operably linked to the regulatory components in a manner that allows the transgene to be transcribed, translated and / or expressed in the cells of the target tissue. Suitable components of the vector genome are discussed in more detail herein. In one example, a "vector genome" contains at least a vector-specific sequence from 5' to 3', a nucleic acid sequence encoding a GLP-1 construct, which is operably linked to a regulatory control sequence (which directs its expression in the target cell), wherein the vector-specific sequence can be a terminal repeat sequence that specifically packages the vector genome into a viral vector capsid or envelope protein. For example, the AAV inverted terminal repeat sequence is used for packaging into AAV and certain other parvovirus capsids.

[0129] The AAV sequence of the vector generally includes cis-acting 5' and 3' inverted terminal repeat sequences (see, e.g., BJ Carter, Handbook of Parvoviruses, edited by P. Tijsser, CRC Press, pp. 155-168 (1990)). The length of the ITR sequence is about 145 bp. Preferably, substantially the entire sequence encoding the ITR is used in the molecule, but minor modifications to these sequences are allowed to some extent. The ability to modify these ITR sequences is within the technical scope of the art. (See, e.g., texts such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., Journal of Virology, 70:520-532 (1996)). An example of such a molecule employed in the present invention is a "cis-acting" plasmid containing a transgene, in which the selected transgene sequence and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences. In one embodiment, ITR is from AAV different from the AAV supplying capsid. In one embodiment, the ITR sequence from AAV2. However, ITR from other AAV sources can be selected. A shortened version of the 5'ITR referred to as ΔITR has been described, in which the D sequence and the terminal resolution site (trs) are deleted. In certain embodiments, the vector genome comprises a shortened AAV2 ITR of 130 base pairs, in which the external A element is missing. It is not desired to be bound by theory, but it is believed that during the amplification of the vector DNA using the internal (A') element as a template, the shortened ITR returns to the wild-type length of 145 base pairs. In other embodiments, full-length AAV 5' and 3'ITR are used. In the case where the source of ITR is from AAV2 and the AAV capsid is from another AAV source, the resulting vector can be referred to as pseudotyped. However, other configurations of these elements may be suitable.

[0130] Optionally, the GLP-1 constructs described herein may be delivered by viral vectors other than rAAV. Such other viral vectors may include any virus suitable for gene therapy, including any virus suitable for gene therapy, including but not limited to adenovirus; herpes virus; lentivirus; retrovirus, etc. Suitably, when one of these other vectors is produced, it is produced as a replication-defective viral vector.

[0131] "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, wherein any viral genomic sequence also packaged in the viral capsid or envelope is replication-defective; that is, the synthetic or artificial viral particle cannot produce progeny virions but retains 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, which is flanked by the signals required for amplification and packaging of the artificial genome), but these genes can be supplied during production. Therefore, this is considered safe for use in gene therapy because replication and infection by progeny virions will not occur unless the viral enzymes required for replication are present.

[0132] Compositions comprising viral vector constructs as described herein are also provided. Pharmaceutical compositions as described herein are designed to be delivered to a subject in need by any suitable route or combination of different routes. Direct delivery to the liver (optionally intravenously, by the hepatic artery or by transplantation), oral administration routes, inhalation administration routes, intranasal administration routes, intratracheal administration routes, intraarterial administration routes, intraocular administration routes, intravenous administration routes, intramuscular administration routes, subcutaneous administration routes, intradermal administration routes and other maternal administration routes. Viral vectors as described herein can be delivered in the form of a single composition or multiple compositions. Optionally, two or more different AAVs can be delivered, or multiple viruses [see, for example, WO 2011 / 126808 and WO 2013 / 049493]. In another embodiment, multiple viruses can contain different replication defective viruses (e.g., AAV and adenovirus). In one embodiment, administration is performed intramuscularly. In another embodiment, administration is performed intravenously.

[0133] Replication-defective viruses can be formulated with physiologically acceptable carriers for 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 dosage contained in the formulation. The number of genome copies (GC) of the replication-defective virus composition of the present invention can be determined using any method known in the art. A method for titrating the number of AAV GC is as follows: a purified AAV vector sample is first treated with DNA enzyme to eliminate unencapsidated AAV genomic 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 targeting a specific region of the viral genome (usually a polyA signal). Another suitable method for determining genome copies is quantitative PCR (qPCR), in particular optimized qPCR or digital droplet PCR [LockMartin et al., Human Gene Therapy Methods. 2014 Apr, 25(2): 115-125. doi: 10.1089 / hgtb.2013.131, published online ahead of edit on Dec. 13, 2013].

[0134] Furthermore, the replication-defective virus composition can be formulated in a dosage unit to contain about 1.0 x 10 9 GC to about 1.0x 10 15 In another embodiment, this amount of viral genome can be delivered in divided doses. In one embodiment, for an average human subject of about 70 kg, the dose is about 1.0 x 10 10 GC to about 3.0x 10 14 GC. In another embodiment, the dose is about 1 x 10 9 GC. For example, the dose of AAV virus can be about 1 x 10 10 GC, 1x 10 11 Gc, about 5X 10 11 GC, about 1X 10 12 GC, about 5X10 12 GC or about 1X 10 13 In another embodiment, for human subjects, the dose is about 1.0 x 10 9 GC / kg to about 3.0x 10 14 GC / kg. In another embodiment, the dose is about 1 x 10 9 GC / kg. For example, the dose of AAV virus can be about 1 x 10 10GC / kg, 1x 10 11 GC / kg, about 5X10 11 GC / kg, about 1X 10 12 GC / kg, about 5X 10 12 GC / kg or about 1X 10 13 GC / kg. In one embodiment, the construct can be delivered in a volume of 1 μL to about 100 mL. As used herein, the term "dose" or "amount" can refer to the total dose or amount delivered to a subject during treatment or the dose or amount delivered by administering a single unit (or multiple units or divided doses).

[0135] The recombinant vector described above can be delivered to a host cell according to the disclosed method. The rAAV preferably suspended in a physiologically compatible carrier can be administered to a desired subject including a human. In view of the indications for which the transfer virus is directed, a person skilled in the art can easily select a suitable carrier. For example, a suitable carrier comprises 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.

[0136] In another embodiment, the composition comprises a carrier, a diluent, an excipient and / or an 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 pharmaceutically 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 to 9, or pH 6.0 to 7.5, or pH 6.2 to 7.7, or pH 6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8, or about 7.0. In certain embodiments, the pH of the formulation is adjusted to 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, the pH that may be desired is 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. In certain embodiments, for intravenous delivery, the pH that may be desired is about 6.8 to about 7.2. However, other pHs within the broadest range and these subranges may be selected for other delivery routes.

[0137] Optionally, in addition to rAAV and / or variants and carriers, the compositions of the invention may also contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, methyl paraben, ethyl vanillin, glycerol, phenol and p-chlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0138] 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 pharmaceutically active substances is well known in the art. Supplementary active ingredients may also be incorporated into the composition. The phrase "pharmaceutically 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, etc. can be used to introduce the composition of the present invention into suitable host cells. Specifically, the transgene delivered by the rAAV vector can be formulated for delivery or encapsulation in lipid particles, liposomes, vesicles, nanospheres or nanoparticles, etc.

[0139] In one embodiment, the composition comprises a final formulation suitable for delivery to a subject, such as 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 transported in the form of a concentrate that is diluted to be administered to a subject. In other embodiments, the composition can be lyophilized and reconstituted at the time of administration.

[0140] A suitable surfactant or combination of surfactants can be selected from non-toxic nonionic surfactants. In one embodiment, a difunctional block copolymer surfactant terminated with a primary hydroxyl group is selected, such as F68 [BASF], also known as Poloxamer 188, has a neutral pH and an average molecular weight of 8400. Other surfactants and other poloxamers may be selected, i.e., nonionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS15 (polyethylene glycol-15 hydroxystearate), LABRASOL (polyoxycaprylyl glyceride), polyoxyl 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol, and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are usually named with the letter "P" (for poloxamer), followed by three numbers: the first two digits x100 give the approximate molecular mass of the polyoxypropylene core, and the last digit x10 gives the percentage of the 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.

[0141] The dosage of the vector depends primarily on factors such as the condition being treated, the patient's age, weight, and health, and therefore may vary between patients. For example, a therapeutically effective human dose of a viral vector is typically in the range of about 25 microliters to about 1000 microliters to about 100 mL of a solution containing a concentration of about 1 x 10 9 Up to 1x 10 16 The amount of viral vector containing 10 genomes (to treat a subject with an average body weight of 70 kg) is preferably 1.0 x 10 12 GC to 1.0x 10 13GC. The compositions of the present invention can be delivered in a volume of about 0.1 μL to about 10 mL, including all numbers 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. 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.

[0142] In some embodiments, the concentration of the recombinant adeno-associated virus carrying the nucleic acid sequence encoding the desired transgene under the control of the regulatory sequence is desirably about 10 7 and 10 14 The concentration of vector genomes / mL (vg / mL) (also called genome copies / mL (GC / mL)) is in the range of 1.5 vector genomes / mL (vg / mL).

[0143] In one embodiment, the dose of rAAV in the composition is about 1.0 x 10 9 GC / kg body weight is about 1.5x10 13 GC / kg. In one embodiment, the dosage is about 1.0 x 10 10 GC / kg. In one embodiment, the dosage is about 1.0x10 11GC / kg. In one embodiment, the dosage is about 1.0 x 10 12 GC / kg. In one embodiment, the dosage is about 5.0 x 10 12 GC / kg. In one embodiment, the dosage is about 1.0 x 10 13 GC / kg. All ranges described herein are inclusive of the endpoints.

[0144] In one embodiment, the effective dose (total genome copies delivered) is about 10 7 Up to 10 13 In one embodiment, the total dose is about 10 8 In one embodiment, the total dose is about 10 9 In one embodiment, the total dose is about 10 10 In one embodiment, the total dose is about 10 11 In one embodiment, the total dose is about 10 12 In one embodiment, the total dose is about 10 13 In one embodiment, the total dose is about 10 14 In one embodiment, the total dose is about 10 15 genome copies.

[0145] It is desirable to utilize the lowest effective concentration of virus to reduce the risk of undesirable effects such as toxicity. Still other dosages and administration volumes within these ranges may 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 the extent to which the disorder (if progressive) has developed.

[0146] In certain embodiments, the composition includes rAAV, and the rAAV includes an inducible GLP-1 agonist construct. In certain embodiments, the inducer or molecule is rapamycin or a rapamycin analog. In certain embodiments, the inducer is rapamycin, and at least one or more, at least two or more times, at least three or more times are applied after the composition including rAAV. In certain embodiments, rapamycin is applied at a dosage of at least about 4nM to at least about 40nM. In certain embodiments, the inducer (i.e., rapamycin) is applied at a dosage of at least about 0.1mg / kg to at least about 3.0mg / kg. In certain embodiments, the inducer (i.e., rapamycin) is applied at a dosage of at least about 0.5mg / kg to at least about 2.0mg / kg.

[0147] The viral vectors and other constructs described herein can be used to prepare a medicament for delivering a GLP-1 fusion protein construct to a subject in need, supplying a GLP-1 with an increased half-life to a subject, and / or for treating type I diabetes, type II diabetes, or metabolic syndrome in a subject. Thus, in another aspect, a method for treating diabetes is provided. The method comprises administering a composition as described herein to a subject in need. In one embodiment, the composition comprises a viral vector comprising a GLP-1 fusion protein expression cassette, as described herein.

[0148] As used herein, the term "treatment" or "treating" is defined to encompass the administration of one or more compounds or compositions described herein to a subject for the purpose of improving one or more symptoms of type I diabetes, type II diabetes, or metabolic syndrome. Thus, "treatment" may include reducing the progression of type I diabetes, type II diabetes, or metabolic syndrome in a given subject, reducing the severity of a given subject's symptoms, eliminating a given subject's disease symptoms, delaying the progression of a given subject's disease, or increasing a given subject's therapy efficacy.

[0149] As used herein, the term "remission" refers to the ability to discontinue insulin therapy when a subject no longer exhibits clinical signs of diabetes and has normal blood glucose levels.

[0150] 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.

[0151] On the other hand, a method for 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 containing 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.

[0152] In another aspect, a method of reducing the weight of 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.

[0153] The course of treatment may optionally involve 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 regimens involving other diabetes drugs or protein-based therapies, including, for example, GLP-1 analogs, insulin, oral antihyperglycemic drugs (sulfonylureas, biguanides, thiazolidinediones, and alpha-glucosidase inhibitors). Optionally, the compositions described herein may be combined in regimens involving lifestyle changes, including diet and exercise regimens. . In certain embodiments, the AAV vector and the combination therapy are administered substantially simultaneously. In other embodiments, the AAV vector is administered first. In other embodiments, the combination therapy is administered first.

[0154] 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 Porcine Insulin Zinc Suspension Insulin glargine Insulin Lispro (Humalog), Insulin Aspart (Novolog), Insulin Glulisine (Apidra), Novolin, and Velosulin.

[0155] In some embodiments, the combination of rAAV and insulin described herein reduces the insulin dosage requirement of the subject compared to before treatment with the 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 dose of insulin required for the subject. For example, insulin or other therapies can be used to treat the subject, and the treating physician can continue treatment after administering the AAV vector. Such insulin or other synergistic therapies can then be continued, reduced or interrupted as needed.

[0156] In one embodiment, a composition comprising an expression cassette, a vector genome, rAAV, or other compositions for gene therapy described herein is delivered in a single dose / patient. In one embodiment, a therapeutically effective amount of a composition described herein is delivered to a subject. As used herein, a "therapeutically effective amount" refers to an amount in which an expression cassette or vector or a combination thereof delivers and expresses an amount sufficient to achieve the therapeutic target GLP1-Fc in a target cell. The therapeutically effective amount may be selected by a treating physician, or based on previously determined guidelines. For example, dulaglutide may be injected subcutaneously once a week at an initial dose of 0.75 mg. The dose may be increased in 1.5 mg increments to achieve additional glycemic control. Before increasing the dose to 3 mg once a week, the patient should remain at a dose of 1.5 mg once a week for at least 4 weeks. Before increasing the dose to 4.5 mg once a week, the patient should remain at a dose of 3 mg once a week for at least 4 weeks. The maintenance dose of dulaglutide may be 0.75 mg to 4.5 mg subcutaneously injected once a week, with a maximum dose of 4.5 mg per week. rAAV can be delivered to a subject and then supplemented with oral or subcutaneous dulaglutide, insulin, or other drugs as needed to achieve the equivalent of a desired dose of 0.75 mg to 4.5 mg per week.

[0157] In certain embodiments, the therapeutic goal is to improve or treat one or more of the symptoms of type I diabetes, type II diabetes or metabolic syndrome. The therapeutically effective amount can be determined based on an animal model rather than a human patient. In another embodiment, the therapeutic goal is to alleviate a metabolic disease in a subject. As used herein, when used to refer to a vp capsid protein, the term "heterogeneous" or any grammatical variant thereof refers to a population consisting of different elements, such as vp1, vp2 or vp3 monomers (proteins) with different modified amino acid sequences. SEQ ID NO: 20 provides the encoded amino acid sequence of AAVrh91 vp1 protein. The term "heterogeneous" used in conjunction with vp1, vp2 and vp3 proteins (alternatively referred to as isoforms) refers to the differences in the amino acid sequences of vp1, vp2 and vp3 proteins in the capsid. The AAV capsid contains subpopulations within the vp1 protein, within the vp2 protein and within the vp3 protein with modifications of the predicted amino acid residues. These subpopulations contain at least some deamidated asparagine (N or Asn) residues. For example, certain subpopulations include at least one, two, three, or four highly deamidated asparagine (N) positions in asparagine-glycine pairs, and optionally further include other deamidated amino acids, wherein the deamidation results in an amino acid change and other optional modifications.

[0158] As used herein, unless otherwise specified, a "subpopulation" of vp proteins refers to a group of vp proteins that have at least one defined common property and consist of at least one group member to less than all members of a reference group. For example, unless otherwise specified, a "subpopulation" of vp1 proteins is at least one (1) vp1 protein and less than all vp1 proteins in an assembled AAV capsid. Unless otherwise specified, a "subpopulation" of vp3 proteins can be one (1) vp3 protein that is less than all vp3 proteins in an assembled AAV capsid. For example, vp1 proteins can be a subpopulation of vp proteins; vp2 proteins can be a separate subpopulation of vp proteins, and vp3 is still another subpopulation of vp proteins in an assembled AAV capsid. In another example, vp1, vp2, and vp3 proteins can contain subpopulations with different modifications, for example, at least one, two, three, or four highly deamidated asparagines, for example at asparagine-glycine pairs.

[0159] As used herein, a "stock" of rAAV refers to a population of rAAVs. Although its capsid protein is heterogeneous due to deamidation, rAAVs are expected to share the same vector genome with 5 in the stock. The stock can contain rAAVs with capsids having, for example, a heterogeneous deamidation pattern unique to the selected AAV capsid protein and the selected production system. The stock can be produced from a single production system or pooled from multiple runs of a production system. Various production systems can be selected, including but not limited to the production systems described herein. As used herein, the terms "GLP-1 construct", "GLP-1 expression construct" and synonyms include a combination of a GLP-1 sequence as described herein with a leader sequence and a fusion domain. The terms "GLP-1 construct", "GLP-1 expression construct" and synonyms can be used to refer to a nucleic acid sequence encoding a GLP-1 fusion protein or its expression product.

[0160] In the context of nucleic acid sequences, the terms "percent identity (%)", "sequence identity", "percent sequence identity" or "identical percentage" refer to the bases in two sequences that are identical when aligned to obtain correspondence. The length of the sequence identity comparison can exceed the full length of the genome, the full length of the gene coding sequence, or a fragment of at least about 100 to 150 nucleotides, or as required. However, it is also possible to expect identity between smaller fragments, such as at least about nine nucleotides, typically at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides. Multiple sequence alignment programs can also be used for nucleic acid sequences. Examples of such programs include "Clustal W", "CAP sequence assembly", "BLAST", "MAP" and "MEME", which can be accessed through Web servers on the Internet. Other sources of such programs are known to those skilled in the art. Alternatively, the vector NTI utility program is also used. Many algorithms known in the art can be used to measure nucleotide sequence identity, including the algorithms contained in the above-mentioned programs. As another example, the GCG 6.1 version of the program Fasta TM Compare polynucleotide sequences. Fasta TM The alignment and percent sequence identity of the best overlap region between the query sequence and the search sequence are provided. For example, the percent sequence identity between nucleic acid sequences can be calculated using Fasta as provided in GCG version 6.1 with its default parameters (font size 6 and NOPAM coefficients for the scoring matrix). TM Where determined, such procedures are incorporated herein by reference.

[0161] The term "highly conserved" means at least 80% identity, preferably at least 90% identity, and more preferably more than 97% identity. Identity can be readily determined by those skilled in the art using algorithms and computer programs known to those skilled in the art.

[0162] Unless otherwise specified by a higher range, it should be understood that the percentage of identity is the lowest level of identity and encompasses all higher levels of identity, up to 100% identity with a reference sequence. Unless otherwise specified, it should be understood that the percentage of identity is the lowest level of identity and encompasses all higher levels of identity, up to 100% identity with a reference sequence. For example, "95% identity" and "at least 95% identity" can be used interchangeably and include 95%, 96%, 97%, 98%, 99% and up to 100% identity with a reference sequence, and all fractions therebetween.

[0163] In the context of amino acid sequences, the terms "percent identity (%)", "sequence identity", "percent sequence identity" or "identical percent" refer to the residues in two sequences that are identical when aligned for correspondence. The percent identity of an amino acid sequence over the full length of a protein, a polypeptide, about 70 amino acids to about 100 amino acids or a peptide fragment thereof, or a corresponding nucleic acid sequence encoding sequence can be readily determined. Suitable amino acid fragments may be at least about 8 amino acids in length, and may be up to about 150 amino acids. Typically, when referring to "identity", "homology" or "similarity" between two different sequences, the "identity", "homology" or "similarity" is determined with reference to an "aligned" sequence. An "aligned" sequence or "alignment" refers to a plurality of nucleic acid sequences or protein (amino acid) sequences that typically contain corrections for missing or additional bases or amino acids compared to a reference sequence. Alignment is performed using any of a variety of publicly available or commercially available multiple sequence alignment programs. Sequence alignment programs are available for amino acid sequences, for example, "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME" and "Match-Box" programs. Typically, any of these programs is used with the default settings, although those skilled in the art can change these settings as needed. Alternatively, those skilled in the art can utilize another algorithm or computer program that provides at least the same level of identity or alignment as that provided by the reference algorithm and program. See, for example, J.D. T. Homson et al., Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690 (1999).

[0164] It should be noted that the term "a" or "an" refers to one or more. Thus, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

[0165] The words "comprise", "comprises" and "comprising" are to be interpreted as inclusive rather than exclusive. The words "consist / consisting" and variations thereof are to be interpreted as exclusive rather than inclusive. Although various embodiments in the specification are presented using "comprising" language, in other cases, it is also intended to use the language of "consisting of" or "consisting essentially of" to explain and describe the relevant embodiments.

[0166] As used herein, "patient" or "subject" refers to a mammal, including humans, veterinary or farm animals, livestock or pets, and animals commonly 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.

[0167] As used herein, unless otherwise indicated, the term "about" means a variability of 10% (±10%, e.g., ±1, ±2, ±3, ±4, ±5, ±6, ±7, ±8, ±9, ±10, or values ​​therebetween) relative to a given reference.

[0168] In some cases, the term "E+#" or the term "e+#" is used to refer to an exponent. For example, "5E10" or "5e10" is 5x10 10 . These terms can be used interchangeably.

[0169] As used herein, the term "modulate" or variations thereof refers to the ability of a composition to inhibit one or more components of a biological pathway.

[0170] As used herein, "disease," "disorder," and "condition" are used interchangeably to refer to an abnormal state of a subject.

[0171] Unless otherwise defined in this specification, technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art and with reference to the public texts, which provide those skilled in the art with a general guide to many of the terms used in this application.

[0172] Reference to "one embodiment" or "another embodiment" when describing an embodiment does not mean that the referenced embodiment is mutually exclusive of another embodiment (eg, an embodiment described before the referenced embodiment) unless explicitly stated otherwise.

[0173] Examples

[0174] The following examples are provided to illustrate various embodiments of the present invention. The examples are not intended to limit the present invention in any way.

[0175] Glucagon-like peptide 1 (GLP-1) is a hormone produced by proteolytic cleavage of the glucagon precursor protein in the gastrointestinal (GI) tract. GLP-1 widely regulates glucose homeostasis by enhancing insulin release from β cells, increasing insulin sensitivity of some tissues, slowing gastric emptying (without causing hypoglycemia), and increasing satiety. GLP-1 cannot be effectively used as a drug due to its extremely short half-life, but long-acting analogs of GLP-1 have become widely used drugs for the treatment of type 2 diabetes. GLP-1 agonists have an excellent safety profile and require repeated, usually lifelong parenteral administration, making the agonists good candidates for AAV-mediated gene transfer, which can achieve long-term expression after a single administration. GLP-1 and GLP-1 agonists are difficult to express from AAV vectors because the protein cannot be expressed in its native context (glucagon protein), which requires processing by proteases specific for L cells of the small intestine. Attempts to express GLP-1 using heterologous signal peptides have failed to achieve high levels of expression. It is proposed that signal peptides may not achieve reliable expression because they do not result in proper processing of the GLP-1 N-terminus involved in receptor binding. Instead, GLP-1 is expressed using a propeptide that is cleaved to produce free GLP-1 protein. Propeptides from coagulation factors such as thrombin and factor IX are selected for GLP-1 expression because these propeptides can be cleaved by ubiquitous proteases (e.g., furin) and are endogenous peptides that will not be immunogenic. Thrombin propeptides increase the expression of human GLP-1 analogs by at least 100-fold relative to the signal peptide alone. Using this technology, two long-acting GLP-1 analogs that can be expressed from AAV vectors have been developed, one long-acting GLP-1 analog includes an IgG4 Fc fusion and one long-acting GLP-1 analog includes an albumin fusion, both carrying human propeptides. Expression cassettes have been developed for constitutively or in a controlled manner expressing these proteins by administering small molecule drugs that activate transcription of the GLP-1 agonist sequence. The target product features are designed for a single intramuscular injection. In one embodiment, a single injection includes an inducible version, such as a single pill once every 2-4 weeks, which is designed to maintain therapeutic GLP-1 agonist levels. As another embodiment, a single injection includes a constitutive version, which is designed to continue lifelong expression at therapeutic levels after a single dose. The designed product is tested in preclinical models to examine pharmacology and safety in non-human primates. Assays for GLP-1 agonist expression and activity have been developed. Safety and pharmacokinetics have been examined to analyze the ability to achieve known therapeutic concentrations.

[0176] This innovation allows for a one-time, potentially lifelong treatment for type 2 diabetes, particularly in patients who have not achieved glycated hemoglobin (also known as glycosylated hemoglobin, hemoglobin A1c, HbA1c, or A1c) goals after 3 months with metformin or other oral agents alone. The current standard of care includes long-acting subcutaneous GLP-1 agonists such as liraglutide (daily administration), dulaglutide (weekly administration), DPP (e.g., dipeptidyl peptidase-4) IV inhibitors (oral), and oral semaglutide (daily administration). Previous attempts to achieve AAV-mediated GLP-1 expression have produced significantly lower expression or required the use of xenogeneic leader sequences that are immunogenic and unsuitable for clinical use.

[0177] Example 1 - Construction of GLP-1 vector

[0178] GLP-1 agonists are challenging 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 traditional heterologous signal peptides produce low expression. Expression systems using heterologous propeptides with universal protease cleavage sites produce foreign protein sequences that can serve as targets for T cells. A system has been developed that increases GLP-1 expression in liver or muscle cells by about 300-fold without introducing foreign protein sequences. Figure 5 AAV-mediated expression of engineered GLP-1 constructs in mice is shown. Mice received intramuscular injections of AAV vectors expressing GLP-1 agonists with either a standard IL-2 signal peptide or endogenous precursor (constructs) that had been developed. Serum GLP-1 concentrations were measured by ELISA 3 weeks after injection.

[0179] More specifically, a vector was constructed in which a leader sequence was placed upstream of one of several GLP-1 receptor agonist amino acid sequences, followed by a fusion domain. See, e.g., Figure 4 . The resulting protein sequence was back translated and the kozak consensus sequence, stop codon and cloning site were subsequently added. 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 was 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; the vector genome is shown in SEQ ID NO: 16.

[0180] Currently available inducible constructs include 2-vector and 1-vector inducible systems. See, e.g., Fig. 6A and Figure 6B . Fig. 6A A schematic diagram of an exemplary expression cassette including an inducible construct for use in a two-vector system is shown. Figure 6B A schematic diagram of an expression cassette comprising an inducible construct for use in a 1-vector system is shown, the expression cassette comprising an IRES linker.

[0181] In addition, the GT2A peptide was introduced into the expression vector containing the GLP1-Fc transgene. The human GLP1-Fc with secretion signal is 954 bp. For the hGLP-1-Fc construct (described above) Figure 6B Expression in the expression vector shown, replacing the IRES linker with the GT2A cleavage sequence, which allows it to be adapted to packaging restriction ( Fig. 7A ; Single inducible cassette of 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. Fig. 7A A schematic diagram of an expression cassette comprising an inducible construct for use in a 1-vector system is shown, the cassette including a F2A cleavage sequence linker and human GLP1-Fc containing a secretion signal.

[0182] Example 2 – In vitro expression

[0183] GLP1-Fc fusions were measured in culture supernatants of HEK293 cells transfected with plasmids of inducible human dulaglutide (TF.GT2A.hGLP-1-Fc) and CB7.feline dulaglutide (feGLP-1-Fc) with a human thrombin signal sequence. Feline dulaglutide refers to a construct in which the IgG Fc portion of dulaglutide is replaced by a feline IgG sequence, optionally in combination with a feline thrombin leader sequence (feTrb). Supernatants were collected 48 hours after treatment with 0nM, 4nM, and 40nM of rapamycin (Rapa) or 48 hours after transfection with CB7.feGLP-1-Fc. GLP1-Fc was quantified by active form GLP1 ELISA together with the STD of the kit. Figure 2 The expression of the three constructs is shown in . Increasing doses of rapamycin resulted in increased expression of GLP-1.

[0184] In addition, expression of an exemplary therapeutic transgene (rhTT) in rhesus macaques in designed constructs including either GT2A_V1 or GT2A_V2 peptides was evaluated ( Figure 6B , 7A and 7B). Figure 8Shown is the expression of rhesus therapeutic transgene (rhTT) in HEK293 cell supernatant, as measured after transfection with various constructs including GT2A peptide and treatment with 0nM, 4nM and 40nM rapamycin, and plotted as IU / mL rhTT. Next, the expression of human and rhesus GLP-1Fc expression was examined in vitro using a designed single inducible cassette including GT2A_V1 and GT2A_V2 peptides. Fig. 9 Inducible human (h) and rhesus (rh) GLP-1 expression in vitro is shown. GLP1-Fc fusions were measured in culture supernatants of HEK293 cells transfected with plasmids of inducible hGLP-1-Fc, 2-vector systems including rhGLP-1-Fc, and CB7.rhGLP-1-Fc. Cells were plated on day 0, transfected on day 1, treated with 0 nM, 4 nM, and 40 nM rapamycin on day 2, and supernatants from cells were collected on day 4 or 48 hours after transfection with CB7.rhGLP-1-Fc (including rhTrb). GLP1-Fc was quantified by active form GLP1 ELISA along with the STD of the kit.

[0185] Example 3 - Pilot expression in Rag1KO mice

[0186] The following constructs were packaged into the AAVrh91 vector by triple transfection and iodixanol gradient purification as described previously.

[0187] AAVrh91.TF.hGLP-1-Fc.3w.rBG, with human thrombin signal

[0188] AAVrh91.TF.rhGLP-1-Fc.3w.rBG, with rhesus monkey thrombin signal

[0189] By IM administration, the vehicle (1 x 10 11 Rag1KO female mice (n=5 / vehicle) were treated with GC / mouse. Serum was collected continuously by separating whole blood in serum separation tubes containing 5 μl of DPP-IV inhibitor (Millipore), and active GLP-1 expression and activity were determined as described above. Vehicle was injected on day 0, and rapamycin was administered on approximately day 14 and day 15. Figure 3 Serum active GLP-1 concentrations are shown in . Serum levels reach a maximum approximately 1 week after rapamycin administration.

[0190] Example 4 - Long-term expression studies in NHPs

[0191] In this study, the expression of rhesus GLP-1 (rhGLP-1-Fc) in non-human primates (NHPs; i.e., rhesus macaques) was examined. Tables 1A and 1B show an overview of the study including AAV administration and rapamycin administration (i.e., induction). Briefly, according to the table below, NHPs were administered AAVrh91 designated vectors by intramuscular injection (IM). For NHP2, rapamycin was administered at a dose of 0.5 mg / kg on day 21, 0.5 mg / kg on day 56, and 2.0 mg / kg on day 126. For NHP3, rapamycin was administered at a dose of 0.5 mg / kg on day 21, 0.5 mg / kg on day 78, and 2.0 mg / kg on day 148.

[0192] Table 1A.

[0193]

[0194] Table 1B.

[0195] Induction NHP2(18-072) NHP3(18-013) First dose: rapamycin intravenous, 0.5 mg / kg Day 21 Day 21 Second dose: rapamycin intravenous, 0.5 mg / kg Day 56 Day 78 Third time: oral rapamycin, 2.0 mg / kg Day 126 Day 148

[0196] Figures 10A to 10C Shown are analyses of rhGLP1-Fc expression and anti-rhGLP1-Fc ADA (anti-drug antibody) detection assays against NHP1(18-128). Fig. 10A Shown are rhGLP1-Fc expression levels in serum plotted in nM, as measured from day 0 to 200. Fig. 10B Shown are rapamycin levels in serum plotted in μg / L, as measured from day 0 to 200. Fig. 10C Shown are the results of the ADA detection assay plotted as OD450nm, as measured from day 0 to 200.

[0197] Figures 11A to 11C Analysis of rhGLP1-Fc expression and anti-rhGLP1-Fc ADA assay against NHP1(18-072) is shown. Fig.11A Shown are rhGLP1-Fc expression levels in serum plotted in nM, as measured from day 0 to 200. Fig. 11B Shown are rapamycin levels in serum plotted in μg / L, as measured from day 0 to 200. Fig. 11C Shown are the results of the ADA detection assay plotted as OD450nm, as measured from day 0 to 200.

[0198] Figures 12A to 12DAnalysis of rhGLP1-Fc expression and anti-rhGLP1-Fc ADA assay against NHP1(18-013) is shown. Fig. 12A Shown are rhGLP1-Fc expression levels in serum plotted in nM, as measured from day 0 to 200. Fig. 12B Shown are rapamycin levels in serum plotted in μg / L, as measured from day 0 to 200. Fig. 12C Shown are the results of the ADA detection assay plotted as OD450nm, as measured from day 0 to 200. Fig.12D Long-term expression data for NHP1 (18-013) treated with the 2-vector system are shown. Arrows indicate administration of rapamycin.

[0199] In summary, a 1-vector inducible system for expressing human GLP1-Fc fusions has been developed. In addition, induction of human GLP1-Fc by rapamycin in Rag1KO mice was demonstrated. In NHPs, 1-vector and 2-vector inducible vectors expressing monkey GLP1-Fc were observed to respond to rapamycin and cause transient increases in serum GLP1-Fc at greater than 1 nM for a duration of more than 20 days. Low dose constitutive expression vectors were observed to provide high and sustained expression of serum GLP1-Fc in NHPs.

[0200] Example 5 - Long-term expression studies in NHPs

[0201] In this study, the expression of rhesus GLP-1 (rhGLP-1-Fc) in non-human primates (NHPs; ie, rhesus macaques) was examined. Fig.13 An overview of a study involving AAV administration and rapamycin administration (ie, induction) involving NHPs tested in Example 4 (samples utilized in this study) is shown.

[0202] Fig.14A and 14B Shown are analysis of rhGLP1-Fc expression and anti-rhGLP1-Fc ADA assay in animals treated with a constitutive promoter. Fig.14A Shown are rhGLP1-Fc expression levels in serum plotted in nM, as measured from day 0 to 300. Fig. 14B Shown are the results of the ADA detection assay plotted as OD450nm, as measured from day 0 to 230.

[0203] Fig.15A and 15B Shown are analyses of rhGLP1-Fc expression and anti-rhGLP1-Fc ADA assays of animals treated using the 2-vector inducible promoter system. Fig.15AShown are rhGLP1-Fc expression levels in serum plotted in nM, as measured from day 0 to about 120. Fig. 15B Shown are the results of the ADA detection assay plotted as OD450nm, as measured from day 0 to 230.

[0204] Fig.16A and 16B Shown are analyses of rhGLP1-Fc expression and anti-rhGLP1-Fc ADA assays from animals treated using the 1-vector inducible promoter system. Fig.16A Shown are rhGLP1-Fc expression levels in serum plotted in nM, as measured from day 0 to about 450. Fig. 16B Shown are the results of the ADA detection assay plotted as OD450nm, as measured from day 0 to 230.

[0205] Example 6 - Potency Assay

[0206] Use a stable Expression of human glucagon-like peptide 1 receptor (GLP1R) in CRE-bla CHO-K1 cell line GLP1R-CRE-bla CHO-K1 cells (ThemoFisher) compared the potency of purified human and rhesus GLP-1-Fc in plasmids as described herein. CRE-bla CHO-K1 cells contain a β-lactamase reporter gene controlled by a CRE response element.

[0207] Fig.17 Results of potency assays of GLP-1-Fc transgene products are shown. Purified human and rhesus GLP-1-Fc were compared to dulaglutide (Dulatide) from a pharmacy, with the human constructs described herein showing comparable or better potency than Dulatide.

[0208] Example 7 - Expression of constitutive GLP-1-Fc in NHPs

[0209] In this study, the expression of human GLP-1 (hGLP-1-Fc) in non-human primates (NHPs; ie, rhesus monkeys) was examined. Table 2 shows an overview of the study involving AAV administration and rapamycin administration (ie, induction).

[0210] Table 2

[0211] Animal ID Carrier dose way 18-007 AAVrh91.CB7.hGLP-1-Fc.rBG 1e12 GC / kg IM 18-122 AAVrh91.CB7.hGLP-1-Fc.rBG 1e10 GC / kg IM

[0212] Fig.18AShown are hGLP1-Fc expression levels in serum plotted in nM, as measured from day 0 to 150. Fig.18B The efficacy of hGLP-1-Fc in NHP plasma sampled from day 0 to day 60 is shown. As a comparison, Fig. 18C The rhGLP-1-Fc expression level in serum is shown.

[0213] Fig.18D Shown are the body weights of the 2 NHPs in the study. Fig.18E Blood glucose levels (mg / dL) are shown. The blood glucose reference level for rhesus monkeys is 63-130 mg / dL. Animal 18-007 appeared asymptomatic, and both animals had relatively low baseline BG on day 0. As of day 60, no ADA was detected.

[0214] In conclusion, the hGLP-1-Fc transgene with a thrombin leader sequence produced a transgene product as potent as dulaglutide. At low vector doses, the recombinant expression vector produced 100-1000-fold therapeutic levels of GLP-1-Fc in NHPs, demonstrating the potency of the vector and the safety of the transgene product. The rapamycin-inducible vector showed long-term, repeated induction of GLP-1-Fc in NHPs by intravenous or oral rapamycin.

[0215] All documents cited in this specification are incorporated herein by reference. The sequence table marked as "22-10015.PCT_Seq-Listing" submitted herewith and the sequences and text therein are incorporated by reference. U.S. Provisional Patent Application Nos. 63 / 316,220 and 63 / 384,196 are incorporated herein by reference in their entirety. Although the present invention has been described with reference to specific embodiments, it should be understood that modifications can be made without departing from the spirit of the present invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

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

3. A composition comprising a viral vector comprising: (a) Adeno-associated virus (AAV) capsid; as well as (b) a vector genome packaged in 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 IgG4 Fc, wherein the fusion protein has a sequence of SEQ ID NO: 14 or a sequence at least 99% identical thereto, and a regulatory sequence for directing 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. The composition according to one of claims 1 to 4, wherein the fusion protein is under the control of an inducible gene expression system.

6. The composition of claim 5, wherein the inducible gene expression system comprises a regulatable promoter, an activation domain, and a DNA binding domain.

7. The composition of any one of claims 3 to 6, wherein the AAV inverted terminal repeats (ITRs) are AAV2 5' ITR and AAV2 3' ITR flanking the fusion protein coding sequence and the regulatory sequence.

8. The composition of any one of claims 3 to 7, wherein the vector genome comprises a CB7 promoter and rabbit globin poly A.

9. The composition according to any one of claims 5 to 8, wherein 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 homology domain (ZFHD) and one, two or three FK506 binding protein domain (FKBP) subunit genes; and (c) at least one copy of a ZFHD binding site followed by a minimal promoter, and (d) Regulatable promoter.

10. The composition of claim 9, wherein the inducible gene expression system is included in a vector.

11. The composition of claim 9, wherein the inducible gene expression system is included in two vectors.

12. The composition of any one of claims 9 to 11, wherein the transactivation domain comprises a portion of NF-κB p65.

13. The composition of any one of claims 9 to 12, wherein the regulatable promoter is a constitutive promoter.

14. The composition of claim 12, wherein the regulatable promoter is a CMV promoter.

15. The composition of any one of claims 9 to 14, further comprising an IRES or 2A.

16. The composition of any one of claims 9 to 15, comprising at least 8 copies of the ZFHD binding site.

17. A composition according to any one of claims 5 to 16, 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 homology domain (ZFHD) and three FK506 binding protein binding domain (FKBP) subunit genes; 12 copies of the ZFHD binding site; and a sequence encoding a fusion protein comprising a GLP-1 analog and human IgG4 Fc.

18. A pharmaceutical composition suitable for treating a metabolic disease in a subject, the pharmaceutical composition comprising an aqueous liquid and the composition according to any one of claims 1 to 17.

19. The composition of any one of claims 1 to 18, for use in a method for treating a subject suffering from a metabolic disease.

20. Use of a composition according to any one of claims 1 to 19 for the preparation of a medicament for treating a subject suffering from a metabolic disease.

21. The composition or use according to any one of claims 1 to 20, wherein the composition is formulated for use at 1 x 10 9 GC / kg to 5x 10 13 The rAAV was administered at a dose of GC / kg.

22. The composition or use according to any one of claims 1 to 20, wherein the patient is a human and 1 x 10 10 GC to 1.5x 10 15 The rAAV dose for GC.

23. The composition or use of any one of claims 1 to 22, wherein the rAAV is delivered intramuscularly or intravenously.

24. A method for treating a subject with a metabolic disease, the method comprising delivering to the subject a recombinant adeno-associated virus (rAAV), the rAAV having an AAV capsid from adeno-associated virus rh91 or hu68 and a vector genome packaged in the AAV capsid, the vector genome comprising 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.

25. The method of claim 24, wherein the composition of any one of claims 1 to 18 is administered to the patient.

26. The method of claim 24 or 25, wherein 1 x 10 9 GC / kg to 5x 10 13 The rAAV dose was GC / kg body weight.

27. The method of any one of claims 24 to 26, wherein the rAAV is delivered intramuscularly or intravenously.

28. A composition according to any one of claims 1 to 18 for use in the treatment of diabetes in a human.

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