Viral vectors encoding GLP-1 receptor agonist fusions and their use in the treatment of metabolic diseases

Abstract

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

Description

[Background technology]

[0001] Glucagon-like peptide 1 (GLP-1) is an endogenous peptide hormone that plays a central role in glucose homeostasis. GLP-1 is a peptide hormone produced in the gastrointestinal (GI) tract from the proteolytic cleavage of glucagon preprotein. 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 has hindered its use as a drug. Other GLP-1 receptor agonists are currently used in humans for the treatment of diabetes. GLP-1 receptor agonists designed to overcome the short half-life of the natural hormone by fusing the agonist to a protein with a longer half-life are emerging as important therapeutic agents for the treatment of type 2 diabetes (T2DM). [Overview of the project]

[0002] Viral vectors encoding glucagon-like peptide 1 (GLP-1) receptor agonist fusion protein constructs are provided herein. In some embodiments, these viral vectors can achieve sustained expression and / or increased circulating half-life of GLP-1 receptor agonists in a subject compared to vector-mediated delivery of GLP-1 receptor agonists without a fusion partner. Methods for producing and using such viral vectors are further provided.

[0003] In one embodiment, a viral vector is provided that includes 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 either (i) IgG Fc or a functional variant thereof, or (ii) albumin or a functional variant thereof. In one embodiment, the vector is an adeno-associated virus vector.

[0004] 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. In another embodiment, the leader sequence comprises an IL-2 leader sequence. In one embodiment, the GLP-1 receptor agonist is selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and their functional variants.

[0005] In one embodiment, the fusion domain is human IgG4 having the sequence of SEQ ID NO: 11, or a sequence sharing at least 90% identity with it, or a functional variant thereof. In another embodiment, the fusion domain is human albumin having the sequence of SEQ ID NO: 12, or a sequence sharing at least 90% identity with it, or a functional variant thereof. In one embodiment, the fusion domain is rhesus monkey IgG4 Fc having the sequence of SEQ ID NO: 17, or a sequence sharing at least 90% identity with it, or a functional variant thereof.

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

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

[0008] In yet another embodiment, the use of the viral vector described herein is provided for the manufacture of a pharmaceutical product for the treatment of a subject having a metabolic disorder, optionally, diabetes.

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

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

Brief Description of the Drawings

[0011] [Figure 1A] It is a schematic diagram of dulaglutide. [Figure 1B] It is a schematic diagram of albiglutide. [Figure 2] Inducible hDulaglutide (Trb) versus CB7.feDulaglutide (feTrb) in vitro is shown. The GLP1-Fc fusion was measured in the culture supernatant of HEK293 cells transfected with plasmid for inducible human dulaglutide (TF.GT2A.dulaglutide (Trb)) and CB7.cat dulaglutide (feTrb) having a human thrombin signal sequence. The supernatant was collected at 0, 4, and 40 nM 48 hours after treatment with rapamycin (Rapa), or 48 hours after transfection of CB7.fe dulaglutide (feTrb). GLP1-Fc was quantified by active GLP1 ELISA together with the STD of the kit. [Figure 3] Inducible expression of GLP-1 in Rag1KO (RAG1- / -) mice (n = 5 / mouse) is shown. Rag1KO female mice were administered 1×1011 GC / mouse via intramuscular (I.M. or IM) delivery of the indicated vectors (i.e., AAVrh91.TF.h dulaglutide (Trb)3w.rBG and AAVrh91.TF.rh dulaglutide (rhTrb).3w.rBG). Blood was collected weekly. GLP1 ELISA specific for active GLP-1 was performed. The AAV vector was injected on day 0, and rapamycin was administered by oral gavage around days  14 and 15 after AAV injection. [Figure 4]This is a schematic diagram of the plasmid map for pAAV.CMV.TF.GT2A.dulaglutide(Trb).3w.rBG. [Figure 5] This shows AAV-mediated expression of an engineered GLP-1 construct in mice. [Figure 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 containing an inducible construct for use in a single-vector system including an IRES linker is shown. [Figure 7A] A schematic diagram of an expression cassette containing an inducible construct for use in a single vector system, including an F2A cleavage sequence linker with a secretion signal and human GLP1-Fc(h dulaglutide), is shown. [Figure 7B] Further detailed diagrams of the GT2A cleavage sequences are shown, where GT2A_V1 contains the amino acid sequence of SEQ ID NO: 21, and GT2A_V2 contains the amino acid sequence of SEQ ID NO: 22. [Figure 8] This shows the expression of exemplary therapeutic transgene (rhTT) in rhesus monkeys in HEK293 cell supernatant, measured after transfection with various constructs and treatment with rapamycin at 0 nM, 4 nM, and 40 nM, and plotted as IU / mL rhTT. [Figure 9] This study demonstrates in vitro expression of inducible human (h) and rhesus monkey (rh) GLP-1. Inducible h dulaglutide containing a thrombin signaling sequence, rh dulaglutide containing two vector systems, and CB7.rh dulaglutide were measured in the culture supernatant of HEK293 cells transfected with plasmids using GLP1-Fc fusions. Cells were seeded on day 0, transfected on day 1, treated with rapamycin at 0 nM, 4 nM, and 40 nM on day 2, and supernatant was collected from cells 4 days or 48 hours after transfection with CB7.rh dulaglutide (rhTrb). GLP1-Fc was quantified by active GLP1 ELISA with the kit's STD. [Figure 10A-10C]This figure shows the rhGLP1-Fc expression and analysis of the NHP1(18-128) anti-rhGLP1-Fc ADA (anti-drug antibody) detection assay. Figure 10A shows serum rhGLP1-Fc expression levels, plotted as nM, measured from day 0 to 200. Figure 10B shows serum rapamycin levels, plotted as μg / L, measured from day 0 to 200. Figure 10C shows the results of the ADA detection assay, plotted as an outer diameter of 450 nm, measured from day 0 to 200. [Figure 11A-11C] This figure shows the rhGLP1-Fc expression and analysis of the anti-rhGLP1-Fc ADA assay using NHP1(18-072). Figure 11A shows serum rhGLP1-Fc expression levels, plotted as nM, measured from day 0 to 200. Figure 11B shows serum rapamycin levels, plotted as μg / L, measured from day 0 to 200. Figure 11C shows the results of the ADA detection assay, plotted as an outer diameter of 450 nm, measured from day 0 to 200. [Figure 12A-12C] This figure shows the rhGLP1-Fc expression and analysis of the anti-rhGLP1-Fc ADA assay using NHP1(18-013). Figure 12A shows serum rhGLP1-Fc expression levels, plotted as nM, measured from day 0 to 200. Figure 12B shows serum rapamycin levels, plotted as μg / L, measured from day 0 to 200. Figure 12C shows the results of the ADA detection assay, plotted as an outer diameter of 450 nm, measured from day 0 to 200. [Modes for carrying out the invention]

[0012] Long-acting GLP-1 receptor agonist fusion protein expression constructs are being developed for use in subjects requiring them, including humans. A leader sequence containing a secretory signaling peptide, as well as a fusion domain intended to extend the circulation time of the resulting fusion protein, are provided.

[0013] It is described that these constructs can be delivered to subjects in need via numerous pathways, particularly by in vivo expression mediated by recombinant vectors such as rAAV vectors. Methods are also provided for using these constructs in regimens in subjects requiring treatment for diabetes or metabolic syndrome to increase the half-life of GLP-1 in the subjects. In addition, methods for enhancing GLP-1 activity in subjects are provided. Methods for inducing weight loss in subjects requiring it are also provided.

[0014] GLP-1 fusion protein Glucagon-like peptide 1, or GLP-1, is an incretin derived from the transcript of the proglucagon gene. In vivo, the glucagon gene expresses a 180-amino acid prepropeptide that is proteolytically processed to form glucagon, which has two forms: GLP-1 and GLP-2. Original sequencing studies showed that GLP-1 has 37 amino acid residues. However, subsequent information showed that this peptide is a propeptide and was further processed into the active form of GLP-1, GLP-1(7-37), by removing six amino acids from the amino terminus. The glycine at position 37 is also transformed into an amide in vivo to form GLP-1(7-36) amide. GLP-1(7-37) and GLP-1(7-36) amides are insulin-stimulating hormones with equivalent potency. Therefore, when used herein, the biologically "active" forms of GLP-1 that are useful herein are GLP-1-(7-37) and GL It is P-1-(7-36)NH2.

[0015] GLP-1 receptor agonists are a class of antidiabetic drugs that mimic the action of glucagon-like peptides. GLP-1 is one of several naturally occurring incretin compounds that, after being released from the intestines during digestion, exert their effects on the body. By binding to and activating the GLP-1 receptor, GLP-1 receptor agonists can lower blood glucose levels, which helps patients with T2DM achieve glycemic control. As used herein, the term “GLP-1 receptor agonist” refers to at least GLP-1 or a functional fragment thereof, an amino acid sequence variant of GLP-1 or a functional fragment thereof, and other polypeptide agonists of the GLP-1 receptor (e.g., exezin-4 and its variants). This disclosure provides one or more copies of a GLP-1 receptor agonist, as well as a fusion protein comprising polynucleotides and vectors encoding such a fusion protein. In some embodiments, the fusion protein comprises a polynucleotide sequence encoding a fusion protein comprising (a) a leader sequence containing 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. In another embodiment, the fusion protein comprises a thrombin leader, a GLP-1 receptor agonist, and albumin or a functional variant thereof. In yet another embodiment, the fusion protein comprises a thrombin leader, two copies of the GLP-1 receptor agonist, and albumin or a functional variant thereof.

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

[0017] Several human drugs that fuse GLP-1 receptor agonists to a stabilizing fusion domain are known in the art. These include albiglutide, liraglutide, dulaglutide, and lixisenatide (also known chemically as des-38-proline-exendin-4(Heloderma suspectum)-(1-39)-peptidylpenta-L-lysyl-L-lysineamide). Dulaglutide is a disulfide-linked homodimer fusion peptide in which each monomer consists of one GLP-1 analog moiety and one IgG4Fc domain. A schematic diagram of dulaglutide is shown in Figure 1A. See WO2005 / 000892A2, which is incorporated herein by reference.

[0018] Albiglutide is a recombinant protein composed of two copies of a GLP-1 analog fused to human albumin. This molecule has a Gly8 substitution for Ala in both copies of the GLP-1 analog to improve resistance to DPP-4 degradation. A schematic diagram of albiglutide is shown in Figure 1B.

[0019] In one embodiment, the fusion includes a GLP-1 analog combined with a heterogeneous sequence. A -1 analog refers to a polypeptide that shares at least 90%, 95%, 97%, 98%, 99%, or 100% identity with natural human GLP-1(7-37). In one embodiment, the GLP-1 analog has up to one, two, or three amino acid substitutions compared to the natural sequence. Natural human GLP-1(1-37) has the sequence HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 1), and GLP-1(7-37) has the sequence HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 2). In some embodiments, it is desirable to modify the natural GLP-1 sequence to optimize one or more of its features. For example, in one embodiment, the GLP-1 analog includes one, two, or three amino acid substitutions selected from A8G, G22E, and R36G compared to the natural sequence. These substitutions have been shown to improve the efficacy of the clinical profile of GLP-1, including protection from DPP-4 inactivation (A8G), increased solubility (G22E), and reduced immunogenicity by substituting the arginine at position 36 (R36G) with a glycine residue to remove a potential T cell epitope. In one embodiment, the GLP-1 analog is a DPP-IV resistant variant of GLP-1. In one embodiment, the GLP-1 analog has a sequence containing or consisting of SEQ ID NO: 3: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG. In another embodiment, the GLP-1 analog has a sequence containing or consisting of SEQ ID NO: 4: HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG. In another embodiment, the GLP-1 receptor agonist has a sequence containing or consisting of SEQ ID NO: 5: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS, or a functional variant thereof. In one embodiment, the variant shares at least 90%, 95%, 97%, 98%, 99%, or 100% identity with sequence number 5.In another embodiment, the GLP-1 receptor agonist has a sequence containing or consisting of SEQ ID NO: 6:HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK, or a functional variant thereof. In one embodiment, the variant shares at least 90%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 6. In one embodiment, two or more copies of a GLP-1 analog are present in the fusion protein. In another embodiment, the GLP-1 receptor agonist is two tandem copies of GLP-1(7-37) or its DPP-IV resistance variant.

[0020] The fusion protein may contain a leader sequence that may contain a secretion signal peptide. As used herein, the term “leader sequence” refers to any N-terminal sequence of a polypeptide.

[0021] The leader sequence may originate from the same species to which the administration is ultimately intended, e.g., humans. As used herein, the terms “originating” or “derived from” mean that a sequence or protein originates from a particular species of subject, or shares the same sequence as a protein or sequence derived from a particular species of subject. For example, a human “originating” leader sequence shares the same sequence (or a variant thereof, as defined herein) as the same leader sequence expressed in humans. However, the specified nucleic acid or amino acid does not actually need to be supplied from humans. Various techniques are known in the art that can produce a desired sequence, including mutagenesis of similar proteins (e.g., homologs) or artificial production of nucleic acids or amino acid sequences. A “derived” nucleic acid or amino acid retains the function of the same nucleic acid or amino acid in the “originating” species, regardless of the actual source of the originating sequence.

[0022] The term "amino acid substitution" and its synonyms are intended to encompass the modification of an amino acid sequence by replacing one amino acid with another substitute amino acid. A substitution can be a conserved substitution. It can also be a non-conservative substitution. The term "conservative" refers to the substitution of two amino acids. When referring to acids, it is intended that those amino acids share common properties recognized by those skilled in the art. For example, amino acids having hydrophobic non-acidic side chains, amino acids having hydrophobic acidic side chains, amino acids having hydrophilic non-acidic side chains, amino acids having hydrophilic acidic side chains, and amino acids having hydrophilic basic side chains. Common properties may also include amino acids having hydrophobic side chains, amino acids having aliphatic hydrophobic side chains, amino acids having aromatic hydrophobic side chains, amino acids having polar neutral side chains, amino acids having charged side chains, amino acids having charged acidic side chains, and amino acids having charged basic side chains. Both naturally occurring and non-naturally occurring amino acids are known in the art and may be used as substitute amino acids in embodiments. Methods for substituting 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. References to “one or more” in this specification are intended to encompass, for example, 1, 2, 3, 4, 5, 6, or more individual embodiments.

[0023] In one embodiment, the leader is a human thrombin (factor II) sequence. In one embodiment, the thrombin leader has the sequence represented by SEQ ID NO: 7: MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRR, or a functional variant thereof having up to one, two, or three amino acid substitutions. In some embodiments, the leader 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 having up to one, two, or three amino acid substitutions. In another embodiment, the leader sequence includes a human thrombin propeptide. In one embodiment, the propeptide has the sequence QHVFLAPQQARSLLQRVRR (SEQ ID NO: 9), or a functional variant thereof having up to one, two, or three amino acid substitutions.

[0024] In one embodiment, the leader is a human IL-2 sequence. In one embodiment, the IL-2 leader is the sequence represented by SEQ ID NO: 10:MYRMQLLSCIALSLALVTNS, or a functional variant thereof having up to one, two, or three amino acid substitutions.

[0025] In one embodiment, a functional variant of a desired leader may include a variant that retains the function of the wild-type sequence and may include up to about 10% variation from a leader nucleic acid or amino acid sequence described herein or known in the art.

[0026] 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 sequences of the propeptide and GLP-1.

[0027] 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 typically have a long circulating half-life in vivo. By fusing a GLP-1 receptor agonist (and leader) to IgG Fc, the circulating time of the fusion protein is extended while the function of GLP-1 is maintained. In one embodiment, the fusion domain is a rhesus monkey IgG Fc fragment or a functional variant thereof.

[0028] As used herein, the Fc portion of immunoglobulin has the meaning generally attributed to the term in the field of immunology. Specifically, this term refers to an antibody fragment that does not contain the two antigen-binding regions (Fab fragments) from the antibody. The Fc portion consists of the constant regions of the antibody from both heavy chains, associated by non-covalent interactions and disulfide bonds. The portion may include a hinge region and extend through the CH2 and CH3 domains to the c-terminus of the antibody. The Fc portion may further include one or more glycosylation sites. In one embodiment, the fusion domain is human IgG Fc. The four highly conserved subclasses, IgG1, IgG2, IgG3, and IgG4, differ in their constant regions, particularly their hinge and upper CH2 domain. See Vidarsson et al, IgG Subclasses and Allotypes: From Structure to Effector Functions, Front Immunol. Oct. 2014;5:520, which is incorporated herein by reference. The Fc domain may be derived from any human IgG, including human IgG1, human IgG2, human IgG3, or human IgG4. In one embodiment, human IgG Fc is IgG4 Fc. In one embodiment, human IgG Fc is Sequence ID No. 11: In another embodiment, 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.

[0029] In one embodiment, the fusion domain is rhesus macaque IgG Fc. The Fc domain may be derived from any rhesus macaque IgG, including rhesus macaque IgG1, rhesus macaque IgG2, rhesus macaque IgG3, or rhesus macaque IgG4. In one embodiment, rhesus macaque IgG Fc is IgG4 Fc. In one embodiment, rhesus macaque IgG Fc is Sequence ID No. 17: PPCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAQTKPRE RQFNSTYRVV SVLTVTHQDW LNGKEYTCKV SNKGLPAPIE KTISKAKGQP REPQVYILPP PQEELTKNQV SLTCLVTGFY PSDIAVEWES NGQPENTYKT TPPVLDSDGS YLLYSKLTVN KSRWQPGNIF TCSVMHEALH NHYTQKSLSV SPGK. In another embodiment, rhesus macaque 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, rhesus macaque IgG further comprises a hinge sequence.

[0030] In another embodiment, the fusion domain is human albumin or a functional variant thereof. In one embodiment, human albumin is Sequence ID 12: DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNP NLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLS QRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMF LYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKH In another embodiment, human albumin shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity with SEQ ID NO: 12.

[0031] The in vivo function and stability of the fusion proteins of this disclosure may be optimized by adding a small peptide linker, for example, to prevent potentially undesirable domain interactions or for other reasons. Furthermore, the glycine-rich linker may provide some structural flexibility so that the GLP-1 analog moiety can productively interact with the GLP-1 receptor on target cells such as pancreatic β-cells. Thus, the C-terminus of the GLP-1 analog and the N-terminus of the fusion domain of the fusion protein are fused via a linker in one embodiment. In one embodiment, the linker comprises one, 1.5, or two repeats of a G-rich peptide linker having the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 13).

[0032] In one embodiment, the fusion protein comprises (a) a human thrombin reader, (b) a DPP-IV resistant variant of GLP-1(7-37), a linker, and (c) human IgG Fc. In one embodiment, the fusion protein has a sequence identical to SEQ ID NO: 14, or at least 90%, at least 95%, at least 98%, or at least 99% thereto. Sequence ID 14 MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0033] In one embodiment, the sequence encoding the fusion protein is sequence number 15, or a sequence that is at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. Sequence ID 15: atggctcacgttcgaggactgcagctgcctggatgtctggctcttgccgctctgtgtagcctggtgcacagccagcacgtgtttctggctcctcagcaagccagatcactgctgcagagagttagaaggcacggcgagg cacctttacctccgacgtgtctagctacctggaagaacaggccgccaaagagtttatcgcctggctggtcaaaggtggcggcggaggcggaggaagcggtggcggaggttcaggtggtggtggatctgccgagtctaagt acggccctccttgtcctccctgtcctgctcccgaagctgctggcggcccatccgtgtttctgttccctccaaagcctaaggacaccctgatgatcagcagaacccctgaagtgacctgcgtggtggtcgacgtgtcccaa gaggatcctgaggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacagttcaacagcacctacagagtggtgtccgtgctgaccgtgctgcaccaggattggctgaa cggcaaagagtacaagtgcaaggtgtccaacaagggcctgcctagctccatcgagaaaaccatcagcaaggccaagggccagccaagagaaccccagg tgtacacactgcctccaagccaagaggaaatgaccaagaaccaggtgtccctgacctgcctcgtgaagggcttctacccttccgatatcgccgtggaat gggagagcaatggccagcctgagaacaactacaagaccacacctcctgtgctggacagcgacggctcattcttcctgtacagcagactgaccgtggac aagagcagatggcaagagggcaacgtgttcagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagtctctgagcctgagcctgggc

[0034] In one embodiment, the fusion protein comprises (a) a human thrombin reader, (b) a DPP-IV resistant variant of GLP-1(7-37), a linker, and (c) rhesus monkey IgG. It contains Fc. In one embodiment, the fusion protein comprises (a) a rhesus monkey thrombin leader, (b) a DPP-IV resistant variant of GLP-1(7-37), a linker, and (c) rhesus monkey IgG Fc.

[0035] In one embodiment, the fusion protein has a sequence identical to SEQ ID NO: 37, or at least 90%, at least 95%, at least 98%, or at least 99% thereto. Sequence ID 37 MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQALSLLQRVRRHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGGSGGGGSAEFTPPCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAQ TKPRERQFNSTYRVVSVLTVTHQDWLNGKEYTCKVSNKGLPAPIEKTISKAKGQPREPQVYILPPPQEELTKNQVSLTCLVTGFYPSDIAVEWESNGQPENTYKTTPPVLDSDGSYLLYSKLTVNKSRWQPGNIFTCSVMHEALHNHYTQKSLSVSPG

[0036] In one embodiment, the sequence encoding the fusion protein is sequence number 36, or a sequence that is at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. Sequence ID 36 atggctcacgttcgaggactgcagctgcctggatgtctggctcttgccgctctgtgtagcctggtgcacagccagcatgtgtttctggctcctcaacaagccctgagcctgctgcaaagagttagaaggcacggcgagggcaccttcacctccgacgtgtccagctacctggaagaacaggccgccaaagagtttatcgcctggctggtcaaaggcggtggtggtggcggaggatctggcggaggtggaagcggcggaggcggatctgctgagtttacacctccttgtcctccctgtcctgctcccgagctgctcggaggcccttccgtgtttctgttccctccaaagcctaaggacaccctgatgatcagcagaacccctgaagtgacctgcgtggtcgtggacgtgtcccaagaggatcctgaggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgcccagacaaagcccagagagcggcagttcaacagcacctacagagtggtgtccgtgctgaccgtgacacaccaggattggctgaacggcaaagagtacacctgtaaagtctccaacaagggcctgcctgctcctatcgagaaaaccatcagcaaggccaagggccagcctagagaaccccaggtgtacatcctgcctccacctcaagagga actgaccaagaaccaggtgtccctgacctgtctggtcaccggcttctacccttccgatatcgccgtggaatgggagagcaacggacagcccgagaacacctacaagaccacacctccagtgctggacagcgacggcagctatctgctgtactccaagctgacagtgaacaagagccggtggcagcccggcaacatcttcacctgttctgtgatgcacgaggccctgcacaaccactacacccagaagtctctgagcgtcagccctggc

[0037] In one embodiment, the fusion protein comprises (a) a human thrombin reader, (b) a DPP-IV resistant variant of GLP-1(7-37), a linker, and (c) human albumin. In another embodiment, the fusion protein comprises (a) a human thrombin reader, (b) two tandem copies of human GLP-1(7-37) or its DPP-IV resistant variant, a linker, and (c) human albumin.

[0038] If a leader sequence, GLP-1 receptor agonist, or variant or fragment of a fusion domain is desired, the coding sequences for these peptides may be generated using site-directed mutagenesis of wild-type nucleic acid sequences. Alternatively or additionally, the amino acid sequence may be backtranslated into a nucleic acid coding sequence containing both RNA and / or cDNA using web-based or commercially available computer programs, as well as service-based companies. See, for example, backtranseq by EMBOSS (ebi.ac.uk / Tools / st / ), Gene Infinity (geneinfinity.org / sms- / sms_backtranslation.html), and ExPasy (expasy.org / tools / ). In one embodiment, the RNA and / or cDNA coding sequence is designed for optimal expression in the target species to which administration is ultimately intended, e.g., humans.

[0039] The coding sequence can be designed for optimal expression using codon optimization. The codon-optimized coding region can be designed by a variety of different methods. This optimization can be performed using methods available online, published methods, or by using a company that provides codon optimization services. One codon optimization method is described, for example, in International Patent Application Publication 2015 / 012924, which is incorporated herein by reference. Briefly, the nucleic acid sequence encoding the product is modified with synonymous codon sequences. Preferably, the entire length of the open reading frame (ORF) of the product is modified. However, in some embodiments, only fragments of the ORF may be modified. By using one of these methods, frequencies can be applied to any given polypeptide sequence to produce nucleic acid fragments of a codon-optimized coding region encoding the polypeptide.

[0040] 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 peptide described herein is provided. In some embodiments, this may include any nucleic acid sequence encoding the GLP-1 sequence of SEQ ID NO: 1. In another embodiment, this may include any nucleic acid containing the GLP-1 sequence of SEQ ID NO: 2. In another embodiment, this may include any nucleic acid containing the GLP-1 sequence of SEQ ID NO: 3. In another embodiment, this may include any nucleic acid containing the GLP-1 sequence of SEQ ID NO: 4. In another embodiment, this may include any nucleic acid containing the GLP-1 sequence of SEQ ID NO: 5. In another embodiment, this may include any nucleic acid containing the GLP-1 sequence of SEQ ID NO: 6.

[0041] In one embodiment, a nucleic acid sequence encoding the GLP-1 fusion protein described herein is provided. In another embodiment, this includes any nucleic acid sequence encoding the GLP-1 fusion protein of SEQ ID NO: 14.

[0042] Expression Cassette In another embodiment, an expression cassette comprising a nucleic acid encoding a GLP-1 fusion protein described herein is provided herein. As used herein, “expression cassette” means a nucleic acid molecule comprising a biologically useful nucleic acid sequence and regulatory sequences operably linked to the nucleic acid sequence (e.g., a gene cDNA encoding a protein, enzyme, or other useful gene product, such as mRNA) and the transcription, translation, and / or expression of the gene product. As used herein, “operably linked” sequences include both regulatory sequences (also referred to as elements) that are continuous or discontinuous with the nucleic acid sequence and regulatory sequences that act in trans or cis nucleic acid sequences. Such regulatory sequences typically include, for example, one or more of the following: promoters, enhancers, transcription factors, transcription termination factors, introns, sequences that improve translation efficiency (i.e., Kozak consensus sequences), efficient RNA processing signals such as slicing and polyadenylation sequences, sequences that stabilize cytoplasmic mRNA, such as woodchuck hepatitis virus (WHP) post-translational regulatory elements (WPREs), and TATA signals. An expression cassette may include, among other elements, one or more upstream (5') regulatory sequences of a gene sequence, such as promoters, enhancers, and introns, and one or more enhancers or downstream (3') regulatory sequences of a gene sequence, such as a 3' untranslated region (3'UTR) containing a polyadenylation site. In certain embodiments, the regulatory sequences are operably linked to the nucleic acid sequence of the gene product, and the regulatory sequences are separated from the nucleic acid sequence of the gene product by an intervening nucleic acid sequence, i.e., a 5' untranslated region (5'UTR). In certain embodiments, the expression cassette includes the nucleic acid sequences of one or more gene products. In some embodiments, the expression cassette may be a monocistronic or bicistronic expression cassette. In other embodiments, the term “transgene” refers to one or more DNA sequences from an exogenous source that are inserted into a target cell.

[0043] 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 other regulatory sequences for them, and this cassette may be manipulated into a genetic element and / or packaged into a capsid of a viral vector (e.g., a viral particle). Typically, such an expression cassette for producing a viral vector comprises the GLP-1 construct sequence described herein, adjacent to a packaging signal of the viral genome (also referred to as the “vector genome”), and other expression regulatory sequences, such as those described herein. As described herein, any of the expression regulatory sequences can be optimized for a particular species using techniques known in the art, including, for example, codon optimization.

[0044] In certain embodiments, the expression cassette includes a constitutive promoter. In another embodiment, a CB7 promoter is used. CB7 is a chicken β-actin promoter having a cytomegalovirus enhancer element. In some embodiments, the CB7 promoter has the nucleic acid sequence of SEQ ID NO: 33. In one embodiment, the promoter is a CMV promoter. In some embodiments, the CMV promoter has the nucleic acid sequence of SEQ ID NO: 27.

[0045] In another embodiment, a tissue-specific promoter may be used. Alternatively, other liver-specific promoters may be used, such as those listed in the Liver Specific Gene Promoter Database, Cold Spring Harbor, (rulai.schl.edu / LSPD), including α1 antitrypsin (A1AT), human albumin (Miyatake et al., J. Virol., 71:5124 32 (1997)), humAlb, hepatitis B virus core promoter (Sandig et al., Gene Ther., 3:1002 9 (1996)), TTR minimal enhancer / promoter, and alpha-antitrypsin promoter. Other promoters may be used in the vectors described herein, including, but not limited to, liver-specific promoters (LSPs) (Wu et al. Mol Ther. 16:280-289 (2008)), TBG liver-specific promoters, viral promoters, constitutive promoters, moduloable promoters (see, e.g., WO2011 / 126808 and WO2013 / 04943), or promoters that respond to physiological cues.

[0046] In one embodiment, the promoter is included in the inducible gene expression system. The inducible gene regulation / expression system includes at least the following components: a promoter (also referred to as a regulatory promoter) operably ligated to a transgene encoding the GLP-1 fusion protein described herein, 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. A plasmid illustrating an exemplary inducible expression system design is shown in Figure 4.

[0047] The system includes a promoter upstream of the coding sequence of the GLP-1 fusion protein. Promoters described herein, such as the CMV and CB7 promoters, may be used. In one embodiment, the promoter is the CMV promoter, such as the one shown in SEQ ID NO: 27. In one embodiment, the promoter is the ubiquitous inducible promoter Z12I, which contains 12 repeat copies of the binding sites of the ZFHD1 and IL2 minimal promoters. See, for example, Chen et al, Hum Gene Ther Methods. 2013. See Aug;24(4):270-278 (which is incorporated herein).

[0048] 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 from the p65 subunit of NF-kappa B and the FKBP12-rapamycin-binding (FRB) domain of FKBP12-rapamycin-related protein (FRAP). In one embodiment, the activation domain is the FKBP12-rapamycin-binding (FRB) domain of human FKBP12-rapamycin-related protein (FRAP) fused to the carboxyl terminus from the p65 subunit of human-derived NF-kappa B. 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, which is 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, which is encoded by the nucleic acid sequence of SEQ ID NO: 25.

[0049] The induction system may consist of a single vector containing the coding sequence of the fusion protein, or a two-vector system. Examples of two-vector (Figure 6A) and one-vector (Figure 6B and Figure 7A) systems incorporating the GLP1 fusion protein are described herein.

[0050] In one embodiment, there is a linker between the transactivation domain and the DNA binding domain, and the linker may 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 comprises the GT2A_V1 peptide containing the amino acid sequence of SEQ ID NO: 21. In one embodiment, the linker comprises the GT2A_V2 peptide containing the amino acid sequence of SEQ ID NO: 22. In one embodiment, the 2A peptide is selected to increase the packaging limit in order to enable a single vector system.

[0051] The DNA-binding domain consists of DNA-binding fusions of zinc finger homeodomain 1 (ZFHD1) conjugated to up to three copies of FK506-binding protein (FKBP). In the presence of an inducer, such as a rapamycin or other rapagnolog, the DNA-binding domain and active The sexualization domains are dimerized via the interaction of their FKBP and FRB domains, resulting in transcriptional activation of the transgene. In some embodiments, ZFHD1 is contained within a frame together 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, which is encoded by the nucleic acid sequence of SEQ ID NO: 28.

[0052] 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 to minimize recombination. For example, Sequence ID No. 30 provides three "variant" coding sequences of FKBP, each of which codes for the sequence shown in Sequence ID No. 31 below: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE

[0053] The expression system further comprises zinc finger homeodomain binding sites. The nucleic acid molecule contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 binding sites for ZFHD. In one embodiment, the expression system comprises 8 zinc finger homeodomain binding sites (binding partners) (8XZFHD). However, the present invention encompasses expression systems having 2 to about 12 copies of the zinc finger binding site. An example of a single copy of the ZFHD binding site is aatgatgggcgctcgagt (SEQ ID NO: 32).

[0054] In some embodiments, a minimal IL2 promoter is located downstream of the zinc finger homeodomain binding site. An exemplary IL2 promoter is shown in SEQ ID NO: 10.

[0055] Such induction systems are known in the field, for example, Rivera et al, A humanized system for pharmacologic control of gene expression, Nature Medicine. The following are examples of rapamycin-inducible systems, as described in Volume 2, pages 1028-1032 (September 1996) and Rivera et al, Long-term pharmacologically regulated expression of erythropoietin in primates following AAV-mediated gene transfer, Blood, 15 February 2005, Volume 105, number 4, both of which are incorporated herein by reference. In one embodiment, the inducible gene expression system comprises a CMV promoter, the activating domain being the FKBP12-rapamycin-binding (FRB) domain of human FKBP12-rapamycin-related protein (FRAP) fused to the carboxy terminus from the p65 subunit of human-derived NF-kappa B, the GT2A peptide, the ZFHD1 DNA-binding domain, three FKBP subunits, hGH poly(A), 8XZFHD, and a minimal sIL2 promoter. These sequences are added to the coding sequence of the GLP-1 fusion protein, and optionally to other regulatory sequences.

[0056] In addition to the promoter, the expression cassette and / or vector may include other suitable transcription start sequences, termination sequences, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (poly-A) signals, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (i.e., Kozak consensus sequences), sequences that enhance protein stability, and, if necessary, sequences that enhance the secretion of the encoded product. Examples of suitable poly-A sequences include, for example, SV40, or bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit β-globin (rabbit globin poly-A Examples include RGB (also known as RGB), modified RGB (mRGB), and TK poly-A. Examples of suitable enhancers include, for example, α-fetoprotein enhancers, TTR minimal promoter / enhancer, and LSP (TH-binding globulin promoter / α1-microglobulin / bicinin enhancer). In one embodiment, poly-A is rabbit globin poly-A.

[0057] These regulatory sequences are “operatably ligated” to the GLP-1 construct sequence. As used herein, the term “operatably ligated” refers to both regulatory sequences that are contiguous to the gene of interest and regulatory sequences that act trans or detached to regulate the gene of interest.

[0058] In one embodiment, an rAAV is provided comprising a 5'ITR, a CB7 promoter, a chicken β-actin intron, the coding sequence for the fusion protein of SEQ ID NO: 14, rabbit globin polyA, and a 3'ITR. In another embodiment, the rAAV comprises a polynucleotide comprising a CMV promoter, the activation domain being the FKBP12-rapamycin-binding (FRB) domain of human FKBP12-rapamycin-associated protein (FRAP) fused to the carboxy terminus from the p65 subunit of human NF-kappa B, a GT2A peptide, a ZFHD1 DNA-binding domain, three FKBP subunits, hGH polyA, 8XZFHD, a minimal sIL2 promoter, the coding sequence for the GLP-1 fusion protein of SEQ ID NO: 14, and rabbit beta-globin polyA.

[0059] In one embodiment, an expression cassette is provided comprising a CB7 promoter, a chicken beta-actin intron, the coding sequence for the fusion protein of SEQ ID NO: 14, and a polynucleotide including rabbit globin polyA. In one embodiment, the expression cassette is the sequence found in SEQ ID NO: 34, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with it. In another embodiment, a vector genome is provided, in which the sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with it is adjacent to the 5' and 3' AAV ITRs.

[0060] In another embodiment, an expression cassette is provided comprising a CB7 promoter, a chicken beta-actin intron, the coding sequence for the fusion protein of SEQ ID NO: 37, and a polynucleotide including rabbit globin polyA. In one embodiment, the expression cassette is the sequence found in SEQ ID NO: 35, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with it. In another embodiment, a vector genome is provided, in which the sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with it is adjacent to the 5' and 3' AAV ITRs.

[0061] In another embodiment, an expression cassette is provided comprising a CMV promoter, the FKBP12-rapamycin-binding (FRB) domain of human FKBP12-rapamycin-associated protein (FRAP) fused to the carboxy terminus of the p65 subunit of human-derived NF-kappa B, the GT2A peptide, the ZFHD1 DNA-binding domain, three FKBP subunits, 8XZFHD, a minimal IL2 promoter, the coding sequence of the GLP-1 fusion protein of SEQ ID NO: 14, and a polynucleotide including rabbit betaglobin polyA. In one embodiment, the expression cassette is the sequence found in SEQ ID NO: 38, or a sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with it. In another embodiment, a vector genome is provided, and sequence SEQ ID NO: 38, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with it, is adjacent to the 5' and 3' AAV ITR. ru.

[0062] In another embodiment, an expression cassette is provided comprising a CMV promoter, an FKBP12-rapamycin-binding (FRB) domain of human or rhesus monkey FKBP12-rapamycin-related protein (FRAP) fused to the carboxy terminus of the p65 subunit of human or rhesus monkey NF-kappa B, a GT2A peptide, a ZFHD1 DNA-binding domain, three FKBP subunits, 8XZFHD, a minimal IL2 promoter, the coding sequence of the GLP-1 fusion protein of SEQ ID NO: 37, and a polynucleotide including rabbit betaglobin polyA. In one embodiment, the expression cassette is the sequence found in SEQ ID NO: 39, or a sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with it. In another embodiment, a vector genome is provided in which sequence SEQ ID NO: 39, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with it, is adjacent to the 5' and 3' AAV ITRs.

[0063] In another embodiment, an expression cassette is provided comprising a Z12I promoter (containing 12 ZFHD1 sites and a minimal IL2 promoter), the coding sequence for the GLP-1 fusion protein of SEQ ID NO: 37, and a polynucleotide containing rabbit betaglobin polyA. In one embodiment, the expression cassette is the sequence found in SEQ ID NO: 40, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with it. In another embodiment, a vector genome is provided, in which the sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with it is adjacent to the 5' and 3' AAV ITRs. A second expression cassette is provided comprising a CMV promoter, a chimeric intron, the FKBP12-rapamycin-binding (FRB) domain of human or rhesus monkey FKBP12-rapamycin-related protein (FRAP) fused to the p65 subunit of human or rhesus monkey NF-kappa B, an IRES or 2A peptide, a ZFHD1 DNA-binding domain, three FKBP subunits, 8XZFHD, and a polynucleotide including a poly-A sequence. In one embodiment, the expression cassette is the sequence found in SEQ ID NO: 41, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with it. In another embodiment, a vector genome is provided, where SEQ ID NO: 41, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with it, is adjacent to the 5' and 3' AAV ITRs.

[0064] Viral vector In another embodiment, a viral vector comprising the expression cassette described herein is provided. In certain embodiments of the viral vector described herein, the viral vector is an adeno-associated virus (AAV) viral vector or recombinant AAV (rAAV). As used herein, the terms “recombinant AAV” or “rAAV” refer to naturally occurring adeno-associated viruses, adeno-associated viruses available to those skilled in the art, and / or available in terms of the compositions and methods described herein, as well as artificial AAVs. An adeno-associated virus (AAV) viral vector is an AAV DNase-resistant particle having an AAV protein capsid, within which an expression cassette adjacent to the inverted terminal repeat (ITR) of AAV for delivery to target cells is packaged (collectively referred to as the “vector genome”). The AAV capsid consists of 60 capsid (cap) protein subunits, VP1, VP2, and VP3, which are arranged icosahedral symmetrically in a ratio of approximately 1:1:10 to 1:1:20, depending on the selected AAV. Various AAVs may be selected as the source of the capsid for the AAV viral vector described above. In one embodiment, the AAV capsid is the AAVrh91 capsid or a variant thereof. In certain embodiments, the capsid protein is designated by a number or a combination of a number and letters following the term "AAV" in the name of the rAAV vector. Unless otherwise specified, the AAV capsids, ITRs, and other selected AAV components described herein may be readily selected from any AAV, including, but not limited to, those identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVhu37, AAVrh32,33, AAVanc80, AAV10, AAV11, AAV12, AAVrh8, AAVrh74, AAV-DJ8, AAV-DJ, AAVhu.37, AAVrh.64R1, and AAVhu68. See, for example, U.S. Patent Publication No. 2007 / 0036760-A1, U.S. Patent Publication No. 2009 / 0197338-A1, and EP1310571. Also see WO2003 / 042397 (AAV7 and other monkey AAVs), U.S. Patent Nos. 7,790449 and 7,282199 (AAV8), WO2005 / 033321 and U.S. Patent Nos. 7,906,111 (AAV9), as well as WO2006 / 110689, and WO2003 / 042397 (rh.10), WO2005 / 033321, and WO2018 / 160582 (AAVhu68), which are incorporated herein by reference. Other suitable AAVs may include, but are not limited to, AAVrh90 [filed on 28 April 2020, PCT / US20 / 30273], AAVrh91 [filed on 28 April 2020, PCT / US20 / 030266, current publication WO2020 / 223231, published on 5 November 2020], AAVrh92, AAVrh93, and AAVrh91.93 [filed on 28 April 2020, PCT / US20 / 30281], which are incorporated herein by reference.Other suitable AAVs include the AAV3B variants described in U.S. Provisional Patent Application No. 62 / 924,112 filed on 21 October 2019 and U.S. Provisional Patent Application No. 63 / 025,753 filed on 15 May 2020, which include AAV3B.AR2.01, AAV3B.AR2.02, AAV3B.AR2.03, AAV3B.AR2.04, and AAV3B.AR2.05. See also 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, which are incorporated herein by reference. See also International Patent Application PCT / US21 / 45945 filed on 13 August 2021, U.S. Provisional Patent Application 63 / 065,616 filed on 14 August 2020, and U.S. Provisional Patent Application 63 / 109,734 filed on 4 November 2020, all of which are incorporated herein by reference in their entirety. These documents also describe other AAV capsids that may be selected to generate rAAV, and are incorporated by reference. Of the AAVs isolated or engineered from humans or non-human primates (NHPs) and well-characterized, human AAV2 was the first AAV developed as a gene transfer vector and is widely used in efficient gene transfer experiments in various target tissues and animal models.

[0065] As used herein, with respect to AAV, the term “variant” means any AAV sequence derived from a known AAV sequence, including AAV sequences having conserved amino acid substitutions, and AAV sequences sharing at least 90%, at least 95%, at least 97%, or at least 99% sequence identity across amino acid sequences or nucleic acid sequences. In another embodiment, an AAV capsid includes a variant that may contain up to about 10% variation from any described or known AAV capsid sequence. That is, an AAV capsid shares about 90% to about 99.9% identity, about 95% to about 99% identity, or about 97% to about 98% identity with an AAV capsid provided herein and / or known in the art. In one embodiment, an AAV capsid shares at least 95% identity with an AAV capsid. When determining the identity percentage of AAV capsids, the comparison is performed across any of the variable proteins (e.g., vp1, vp2, or vp3). obtain.

[0066] In one embodiment, the viral vector is an rAAV having the capsid of AAV8 or a functional variant thereof. In one embodiment, the viral vector is an rAAV having the capsid of AAVrh91 or a functional variant thereof. In one embodiment, the viral vector is an rAAV having the 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.

[0067] In certain embodiments, a novel isolated AAVrh91 capsid is provided. The nucleic acid sequence encoding the AAVrh91 capsid is provided in SEQ ID NO: 18, and the encoded amino acid sequence is provided in SEQ ID NO: 20. An rAAV comprising at least one of vp1, vp2, and vp3 of AAVrh91 (SEQ ID NO: 20) is provided herein. An rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVrh91 (SEQ ID NO: 18) is also provided herein. In another embodiment, the nucleic acid sequence encoding the AAVrh91 amino acid sequence is provided in SEQ ID NO: 19, and the encoded amino acid sequence is provided in SEQ ID NO: 20. An rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVrh91eng (SEQ ID NO: 19) is also provided herein. In certain embodiments, vp1, vp2, and / or vp3 are the full-length capsid protein of AAVrh91 (SEQ ID NO: 20). In other embodiments, vp1, vp2, and / or vp3 have N-terminal and / or C-terminal cleavage (e.g., cleavage of about 1 to about 10 amino acids).

[0068] In certain embodiments, the AAVrh91 capsid is a heterogeneous population of AAVrh91 vp1 proteins selected from (1) a vp1 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 20 from 1 to 736, a vp1 protein produced from SEQ ID NO: 18, or a vp1 protein produced from a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 18 encoding the predicted amino acid sequence of SEQ ID NO: 20 from 1 to 736, a vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 20 from at least approximately 138 to 736, a vp2 protein produced from a sequence containing at least nucleotides 412 to 2208 of SEQ ID NO: 18, or a vp2 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 412 to 2208 of SEQ ID NO: 20 encoding the predicted amino acid sequence of SEQ ID NO: 20 from at least approximately 138 to 736 AAVrh91 is selected from a heterogeneous population of vp2 protein, vp3 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least approximately 203-736 amino acids of SEQ ID NO: 20, vp3 protein produced from a sequence containing at least nucleotides 607-2208 of SEQ ID NO: 18, or vp3 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 607-2208 of SEQ ID NO: 18, which encodes the predicted amino acid sequence of at least approximately 203-736 amino acids of SEQ ID NO: 20.A heterogeneous population of vp3 protein, and / or (2) a heterogeneous population of vp1 protein which is the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 20, a heterogeneous population of vp2 protein which is the product of a nucleic acid sequence encoding at least approximately amino acids 138-736 of SEQ ID NO: 20, and a heterogeneous population of vp3 protein which is the product of a nucleic acid sequence encoding at least amino acids 203-736 of SEQ ID NO: 20, wherein the vp1, vp2, and vp3 proteins include a subpopulation having amino acid modifications containing at least two highly deamidated asparagine (N) in the asparagine-glycine pair of SEQ ID NO: 20, and optionally further including a subpopulation containing other deamidated amino acids, wherein deamidation results in an amino acid change, and the AAVrh91 capsid protein includes one or more AAVrh91 capsid proteins. (B) A vector genome in an AAVrh91 capsid, wherein the vector genome comprises a nucleic acid molecule containing an AAV inverted terminal repeat sequence, and a non-AAV nucleic acid sequence encoding a product operably linked to a sequence that directs the expression of the product in a host cell.

[0069] In certain embodiments, the AAVrh91 capsid is a heterogeneous population of AAVrh91 vp1 proteins selected from (1) a vp1 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 20 from 1 to 736, a vp1 protein produced from SEQ ID NO: 19, or a vp1 protein produced from a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 19 encoding the predicted amino acid sequence of SEQ ID NO: 20 from 1 to 736, a vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 20 from at least approximately 138 to 736, a vp2 protein produced from a sequence containing at least nucleotides 412 to 2208 of SEQ ID NO: 19, or a vp2 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 412 to 2208 of SEQ ID NO: 20 encoding the predicted amino acid sequence of SEQ ID NO: 20 from at least approximately 138 to 736 AAVrh91 is selected from a heterogeneous population of vp2 proteins, vp3 proteins produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least approximately 203-736 amino acids of SEQ ID NO: 20, vp3 proteins produced from a sequence containing at least nucleotides 607-2208 of SEQ ID NO: 19, or vp3 proteins produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 607-2208 of SEQ ID NO: 19, which encodes the predicted amino acid sequence of at least approximately 203-736 amino acids of SEQ ID NO: 20.(2) a heterogeneous population of vp3 protein, and / or (2) a heterogeneous population of vp1 protein which is the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 20, a heterogeneous population of vp2 protein which is the product of a nucleic acid sequence encoding at least approximately amino acids 138 to 736 of SEQ ID NO: 20, and a heterogeneous population of vp3 protein which is the product of a nucleic acid sequence encoding at least amino acids 203 to 736 of SEQ ID NO: 20, wherein the vp1, vp2, and vp3 proteins contain at least two highly deamidated asparagine pairs in the asparagine-glycine pair of SEQ ID NO: 20. (B) AAVrh91 capsid comprising one or more AAVrh91 capsid proteins having an amino acid modification including (N), and optionally further comprising a subpopulation having other deamidated amino acids, wherein deamidation results in an amino acid change; and (B) a vector genome in an AAVrh91 capsid comprising a non-AAV nucleic acid sequence encoding a product operably linked to a nucleic acid molecule having an AAV inverted terminal repeat sequence and a sequence that directs the expression of the product in a host cell, characterized by one or more of the following:

[0070] In certain embodiments, the vp1, vp2, and vp3 proteins of AAVrh91 comprise a subpopulation having amino acid modifications containing at least two highly deamidated asparagine (N) in the asparagine-glycine pair of SEQ ID NO: 20, and optionally further comprising subpopulations containing other deamidated amino acids, where deamidation results in amino acid changes. Compared to the number in SEQ ID NO: 20, high levels of deamidation are observed at N57, N383, and / or N512 in the NG pair. Deamidation is observed at other residues. In certain embodiments, AAVrh91 may have other deamidated residues (e.g., typically less than 10%) and / or other modifications including phosphorylation (e.g., in the range of 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 may be oxidized to kynurenine. [Table 1]

[0071] In certain embodiments, the AAVrh91 capsid is modified at one or more positions, within the range provided, as identified in the table above, and determined by mass spectrometry using trypsinase. In certain embodiments, one or more positions, or glycine following N, are modified as described herein. Residue numbers are based on the AAVrh91 sequence provided herein. See Sequence ID No. 20.

[0072] In certain embodiments, the AAVrh91 capsid comprises a heterogeneous population of vp1 proteins, which are products of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 20; a heterogeneous population of vp2 proteins, which are products of the nucleic acid sequence encoding at least approximately amino acids 138 to 736 of SEQ ID NO: 20; and a heterogeneous population of vp3 proteins, which are products of the nucleic acid sequence encoding at least amino acids 203 to 736 of SEQ ID NO: 20.

[0073] In certain embodiments, the nucleic acid sequence of modified AAVrh91 may be used to generate mutant rAAV having a capsid with lower deamidation than the natural AAVrh91 capsid. Such mutant rAAV may have reduced immunogenicity and / or increased stability during storage, particularly in suspension form.

[0074] In one embodiment, recombinant AAV (rAAV) is provided. The rAAV comprises an AAV capsid derived from adeno-associated virus rh91 and a vector genome packaged within the AAV capsid, the vector genome comprising an AAV inverted terminal repeat (ITR), the coding sequence for the GLP-1 receptor agonist of sequence number 14, and a regulatory sequence that directs the expression of the GLP-1 receptor agonist.

[0075] In one embodiment, rAAV is scAAV. The abbreviation "sc" stands for self-complementary. "Self-complementary AAV" refers to a plasmid or vector having an expression cassette in which the coding region supported by the recombinant AAV nucleic acid sequence is designed to form an intramolecular double-stranded DNA template. Upon infection, rather than waiting for cell-mediated synthesis of the second strand, the two complementary halves of scAAV will associate to form a single double-stranded DNA (dsDNA) unit ready for immediate replication and transcription. 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,Number 1 See pages 6, 1248–1254. Self-complementary AAVs are described, for example, in U.S. Patents 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated herein by reference in whole.

[0076] In one embodiment, the nucleic acid sequence encoding the GLP-1 construct described herein is manipulated onto any suitable gene element, such as naked DNA, phage, transposon, cosmid, RNA molecule (e.g., mRNA), episome, etc., which then introduce the GLP-1 sequence supported thereon into host cells, for example, to generate nanoparticles that carry DNA or RNA viral vectors in packaging host cells and / or for delivery to target host cells. In one embodiment, the gene element is a plasmid. The selected gene element may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA coated pellets, viral infection, and protoplast fusion. The methods used to construct such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. For example, Green and Sambrook, Molecular Cloning:A Laboratory Manual,Cold Spring See Harbor Press, Cold Spring Harbor, NY (2012).

[0077] As used herein, the term “host cell” may refer to a packaging cell line from which a vector (e.g., recombinant AAV or rAAV) is produced from a production plasmid. Alternatively, the term “host cell” may refer to any target cell in which the expression of the gene product described herein is desired. Thus, “host cell” refers to a prokaryotic or eukaryotic cell (e.g., a bacterial cell, human cell, or insect cell) containing 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 techniques, fast 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 the compositions described herein. In other embodiments herein, the term “host cell” refers to a cell used to generate and package a viral vector or recombinant virus. In a further embodiment, the term “host cell” is an intestinal cell, small intestinal cell, pancreatic cell, or hepatic cell.

[0078] As used herein, the term “target cell” refers to any target cell on which the expression of a heterologous nucleic acid sequence or protein is desired. In one embodiment, the target cell is a liver cell. In one embodiment, the target cell is a muscle cell.

[0079] In one embodiment, an rAAV is provided comprising a vector genome containing an expression cassette, the expression cassette comprising a CMV promoter, the activation domain being the FKBP12-rapamycin-binding (FRB) domain of human FKBP12-rapamycin-related protein (FRAP) fused to the carboxy terminus from the p65 subunit of human-derived NF-kappa B, the GT2A_V1 peptide, the ZFHD1 DNA-binding domain, three FKBP subunits, hGH polyA, 8XZFHD, minimal sIL2 promoter, the coding sequence of the GLP-1 fusion protein of SEQ ID NO: 14, and rabbit betaglobin polyA. In another embodiment, an rAAV is provided comprising a vector genome containing an expression cassette, the expression cassette comprising a CMV promoter, the activation domain comprising the FKBP12-rapamycin-binding (FRB) domain of human FKBP12-rapamycin-related protein (FRAP) fused to the carboxyterminus of the p65 subunit of human-derived NF-kappa B, the GT2A_V2 peptide, the ZFHD1 DNA-binding domain, and three FKBP subunits The components are hGH polyA, 8XZFHD, minimal sIL2 promoter, the coding sequence for the GLP-1 fusion protein of sequence number 14, and rabbit beta-globin polyA.

[0080] The minimum sequences required to package the expression cassette onto AAV virus particles are the AAV 5' and 3' ITRs, which may be of the same AAV origin as the capsid, or of a different AAV origin (to create an AAV pseudotype). In one embodiment, an ITR sequence from AAV2, or a deleted version thereof (ΔITR), is used for convenience and to expedite regulatory approval. However, ITRs from other AAV sources may be selected. Preferably, the source of the ITRs is the same as the source of the Rep protein provided trans for production. Typically, an expression cassette for an AAV vector includes the AAV 5' ITR, a GLP-1 fusion protein coding sequence, an optional regulatory sequence, and the AAV 3' ITR. However, other configurations of these elements may also be preferred. A shortened version of the 5' ITR, referred to as ΔITR, which has a deleted D sequence and terminal isolation sites (trs), is described. In other embodiments, full-length AAV 5' and 3' ITRs are used.

[0081] To package the expression cassette into a virion, the ITR is the only AAV component required in cis form within the same construct as the gene. In one embodiment, the coding sequences for replication (rep) and / or capsid (cap) are removed from the AAV genome and supplied trans or by a packaging cell line to generate the AAV vector. For example, as described above, a pseudotype AAV may contain ITR from a different source than the source of the AAV capsid. In one embodiment, a chimeric AAV capsid may be used. Further other AAV components may be selected. Sources of such AAV sequences are described herein and can be isolated or obtained from academic, commercial, or public resources (e.g., American Type Culture Collection, Manassas, VA). AAV sequences can be obtained by synthesis or other preferred means by referring to publicly available sequences, such as those available in literature or databases, e.g., GenBank®, PubMed®, etc.

[0082] Methods for generating and isolating AAV viral vectors suitable for delivery to a target are known in the art. See, for example, U.S. Patent Nos. 7790449, 7282199, WO2003 / 042397, WO2005 / 033321, WO2006 / 110689, and U.S. 7588772B2. In one system, a producer cell line is transiently transfected with a construct encoding a transgene adjacent to the ITR, as well as constructs 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 adjacent to the ITR. In each of these systems, AAV virions are produced in response to infection with a helper adenovirus or herpesvirus, requiring the isolation of rAAV from the contaminating virus. More recently, systems have been developed that do not require infection with helper viruses to restore AAV, and the necessary helper functions (i.e., adenoviruses E1, E2a, VA, and E4, or herpesviruses UL5, UL8, UL52, and UL29, as well as herpesvirus polymerases) are also supplied by the system in trans. In these newer systems, helper functions can be supplied by transient transfection of cells with constructs encoding the required helper functions, or cells can be engineered to stably contain genes encoding helper functions, and their expression can be controlled at the transcriptional or post-transcriptional level. In yet another system, the transgenes and rep / cap genes adjacent to the ITR are introduced into insect cells by infection with baculovirus-derived vectors. For a review of these production systems, see, for example, Zhang et al., 2009, “Adenovirus-adeno-associated virus hybrid for See, “Large-scale recombinant adeno-associated virus production,” Human Gene Therapy 20:922–929, the contents of which are incorporated herein by reference in their entirety. Methods for constructing and using these and other AAV production systems are also described in the following U.S. patents, the contents 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. Generally, see, for example, Grieger & Samulski, 2005, “Adeno-associated virus as a gene therapy.” See, “vector: Vector development, production and clinical applications,” Adv. Biochem.Engin / Biotechnol.99:119-145, Buning et al., 2008, “Recent developments in adeno-associated virus vector technology,” J.Gene Med.10:717-733, and the references cited below, each of which is incorporated herein by reference in whole. The methods used to construct any embodiment of the present invention are known to technicians in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. For example, Green and Sambrook See et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Similarly, methods for producing rAAV virions are well known, and the selection of an appropriate method does not limit the present invention. See, for example, K. Fisher et al, (1993) J. Virol., 70:520-532 and U.S. Patent No. 5,478,745.

[0083] The rAAV described herein comprises a selected capsid having a vector genome packaged inside. The vector genome (or rAAV genome) comprises 5' and 3' AAV inverted terminal repeats (ITRs), a polynucleotide sequence encoding a fusion protein, and a regulatory sequence that directs the insertion of the polynucleotide sequence encoding the fusion protein into the host cell genome. In one embodiment, the vector genome is the 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 therewith.

[0084] As used herein, “vector genome” refers to a nucleic acid sequence packaged inside a parvovirus (e.g., rAAV) capsid that forms a viral particle. Such a nucleic acid sequence contains an AAV inverted terminal repeat (ITR). In the examples herein, the vector genome includes, at least from 5' to 3', an AAV 5'ITR, a coding sequence (i.e., a transgene), and an AAV 3'ITR. ITRs other than those from AAV2, AAV from a different source than the capsid, or full-length ITRs may be selected. In certain embodiments, the ITR is from the same AAV source as the AAV that provides the rep function or trans-complementary AAV during production. Furthermore, other ITRs, such as self-complementary (scAAV) ITRs, may be used. Both single-stranded AAV and self-complementary (sc)AAV are included in rAAV. The transgene is a nucleic acid coding sequence heterogeneous to the vector sequence that codes for the polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor), or other gene product of interest. Nucleic acid coding sequences are operably linked to regulatory elements in a manner that enables transcription, translation, and / or expression of the transgene in cells of the target tissue. Preferred components of a vector genome are discussed in more detail herein. In one example, the “vector genome” has, at a minimum, a vector-specific sequence and a regulatory element (which directs its expression in the target sequence) at least from 5' to 3'. The vector comprises a nucleic acid encoding a GLP-1 construct operably ligated to a section control sequence, and the vector-specific sequence may be a terminal repeat sequence that specifically packages the vector genome to a viral vector capsid or envelope protein. For example, the AAV inverted terminal repeat is used to package AAV and certain other parvovirus capsids.

[0085] The AAV sequence of the vector typically contains cis-acting 5' and 3' inverted terminal repeats (see, e.g., B.J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155-168 (1990)). The ITR sequence is approximately 145 bp in length. Preferably, a substantially complete sequence encoding the ITR is used in the molecule, although some minimal modification of these sequences is permissible. The ability to modify these ITR sequences is within the scope of those skilled in the art (see, e.g., Sambrook et al., “Molecular Cloning. A Laboratory Manual”, 2nd ed., Cold Spring Harbor Laboratory, New York (1989), and K. Fisher et al., J. Virol., 70:520-532 (1996)). An example of such molecules used in the present invention is a “cis-acting” plasmid containing a transgene, where the selected transgene sequence and associated regulatory elements are adjacent to 5' and 3' AAV ITR sequences. In one embodiment, the ITR is derived from a different AAV than the one supplying the capsid. In one embodiment, it is an ITR sequence derived from AAV2. However, ITRs from other AAV sources may be selected. A shortened version of the 5' ITR, referred to as a ΔITR, is described, with a deletion of the D sequence and terminal segregation sites (trs). In certain embodiments, the vector genome contains a 130-base pair shortened AAV2 ITR with a deletion of the outer A element. While we do not wish to be constrained by theory, it is thought that the shortened ITR is reverted to the 145-base pair wild-type length during vector DNA amplification using the inner (A') element as a template. In other embodiments, full-length AAV 5' and 3' ITRs are used. When the ITR source is AAV2 and the AAV capsid is from another AAV source, the resulting vector may be referred to as a pseudotype. However, other configurations of these elements may also be preferable.

[0086] Optionally, the GLP-1 constructs described herein may be delivered via viral vectors other than rAAV. Such other viral vectors include, but are not limited to, adenoviruses, herpesviruses, lentiviruses, and retroviruses, and may include any virus suitable for gene therapy. Preferably, if one of these other vectors is produced, it is produced as a replication-deficient viral vector.

[0087] A “replication-deficient virus” or “viral vector” refers to a synthetic or artificial viral particle in which an expression cassette containing the gene of interest is packaged within a viral capsid or envelope, and any viral genome sequence packaged within the viral capsid or envelope is replication-deficient, i.e., it cannot produce progeny virions but can retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be “gutless” containing only the target transgene adjacent to the signals required for amplification and packaging of the artificial genome), but these genes can be supplied during production. Therefore, it is considered safe for use in gene therapy because replication and infection by progeny virions cannot occur without the presence of the viral enzymes required for replication.

[0088] Compositions comprising the viral vector constructs described herein are also provided. The pharmaceutical compositions are designed to be delivered to subjects requiring them by any preferred route or combination of different routes. Direct delivery to the liver (optionally, intravenously, via hepatic artery, or by transplantation), oral, inhalation, intranasal, intratracheal, intra-arterial, intraocular, intravenous, intramuscular, subcutaneous, intracutaneous, and other parenteral administration routes. The viral vectors described herein may be delivered in a single composition or in multiple compositions. Optionally, two or more different AAVs, or multiple viruses, may be delivered [see, for example, WO2011 / 126808 and WO2013 / 049493]. In another embodiment, the multiple viruses may contain different replication-deficient viruses (e.g., AAV and adenovirus). In one embodiment, administration is intramuscular. In another embodiment, administration is intravenous.

[0089] Defective replication viruses can be formulated with physiologically acceptable carriers for use in gene transfer and gene therapy applications. In the case of AAV viral vectors, the quantification of genome copies ("GC") can be used as a measure of the dose contained in the formulation. The number of genome copies (GC) of the defective replication virus composition of the present invention can be determined using any method known in the art. One method for titrating the GC number of AAV is as follows: A purified AAV vector sample is first treated with DNase to remove uncapsidized AAV genomic DNA or contaminating plasmid DNA from the production process. 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 the poly-A signal). Another preferred method for determining genome copies is quantitative PCR (qPCR), particularly optimized qPCR or digital droplet PCR [Lock Martin, et al, Human Gene Therapy Methods. April 2014, 25(2):115-125. doi:10.1089 / hgtb.2013.131, published online prior to editing, December 13, 2013].

[0090] In addition, the replication-deficient virus composition can be formulated in a dosage unit containing an amount of replication-deficient virus in the range of about 1.0×10 10 GC to about 1.0×10 15 GC. In another embodiment, this amount of viral genome can be delivered in divided doses. In one embodiment, the dosage is about 1.0×10 10 GC to about 3.0×10 14 GC for an average human subject of about 70 kg. In another embodiment, the dosage is about 1×10 9 GC. For example, the dosage of AAV virus can be about 1×10 10 GC, 1×10<00​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​The recombinant vector described above can be delivered to host cells according to the published method. Preferably, rAAV suspended on a physiologically compatible carrier can be administered to a desired subject, including humans. A suitable carrier can be readily selected by those skilled in the art in terms of the indications targeted by the introduced virus. For example, one suitable carrier comprises saline and can be formulated using various buffering 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 choice of carrier is not limiting to the present invention.

[0092] In another embodiment, the composition comprises a carrier, a diluent, an excipient and / or an adjuvant. In a particular embodiment, for administration to a human patient, rAAV is preferably suspended in an aqueous solution containing saline, a surfactant, and a pharmaceutically and / or physiologically compatible salt or mixture of salts. Preferably, the formulation is adjusted to a physiologically acceptable pH range, for example, 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 pH 7.0. In certain embodiments, the formulation is adjusted to a pH of approximately 6.0, approximately 6.1, approximately 6.2, approximately 6.3, approximately 6.4, approximately 6.5, approximately 6.6, approximately 6.7, approximately 6.8, approximately 6.9, approximately 7.0, approximately 7.1, approximately 7.2, approximately 7.3, approximately 7.4, approximately 7.5, approximately 7.6, approximately 7.7, or approximately 7.8. In certain embodiments, for intrathecal delivery, pH values ​​of approximately 7.28–7.32, 6.0–7.5, 6.2–7.7, 7.5–7.8, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8 may be desirable. In certain embodiments, for venous delivery, pH values ​​of approximately 6.8–7.2 may be desirable. However, a wider range of other pH values, and sub-ranges thereof, may be selected for other delivery routes.

[0093] Optionally, the compositions of the present invention may include other conventional pharmaceutical components, such as preservatives or chemical stabilizers, in addition to rAAV and / or variants and carriers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0094] As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antimicrobial and antifungal agents, isotonic and absorption retardants, buffers, carrier solutions, suspensions, colloids, etc. 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., may be used to introduce the compositions of the present invention into suitable host cells. In particular, rAAV vector delivery transgenes may be formulated for delivery encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles, etc.

[0095] In one embodiment, the composition comprises a final formulation suitable for delivery to a subject, for example, an aqueous liquid suspension buffered to a physiologically suitable pH and salt concentration. Optionally, one or more surfactants are present in the formulation. In another embodiment, the composition may be transported as a concentrate that is diluted for administration to a subject. In yet another embodiment, the composition may be lyophilized and reconstituted at the time of administration.

[0096] A suitable surfactant, or combination of surfactants, may be selected from non-toxic nonionic surfactants. In one embodiment, for example, a primary hydroxyl-terminated bifunctional block copolymer surfactant such as Pluronic® F68 [BASF], also known as poloxamer 188, which has a neutral pH and an average molecular weight of 8400, is selected. Other surfactants and other poloxamers, namely nonionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) adjacent to two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (macrogol-15 hydroxystearate), LABRASOL (polyoxycaprylic acid glyceride), polyoxy10 oleyl Possible surfactants include tetrapropylmethylnitrate, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol. In one embodiment, the formulation contains poloxamer. These copolymers are generally named with the letter "P" (in the case of poloxamer) followed by a three-digit number, where the first two digits × 100 give the approximate molecular mass of the polyoxypropylene core, and the last digit × 10 gives the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount of up to about 0.0005% to about 0.001% of the suspension.

[0097] The dose of the vector depends primarily on factors such as the condition being treated, the patient's age, weight, and health status, and therefore varies among patients. For example, the therapeutically effective human dose of a viral vector generally ranges from about 25 to about 1000 microliters to about 100 mL, approximately 1 × 10¹⁶ (to treat an average subject weighing 70 kg). 9 ~1 × 10 16 The concentration of the genomic viral vector (including all integers or fractional quantities within that range, preferably 1.0 × 10 for a human patient) 12 GC~1.0×10 13The solution contains GC. The composition of the present invention can be delivered in volumes from about 0.1 μL to about 10 mL, including all values ​​within the range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method. 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 approximately 850 μL. In another embodiment, the volume is approximately 1000 μL. In another embodiment, the volume is approximately 1.5 mL. In another embodiment, the volume is approximately 2 mL. In another embodiment, the volume is approximately 2.5 mL. In another embodiment, the volume is approximately 3 mL. In another embodiment, the volume is approximately 3.5 mL. In another embodiment, the volume is approximately 4 mL. In another embodiment, the volume is approximately 5 mL. In another embodiment, the volume is approximately 5.5 mL. In another embodiment, the volume is approximately 6 mL. In another embodiment, the volume is approximately 6.5 mL. In another embodiment, the volume is approximately 7 mL. In another embodiment, the volume is approximately 8 mL. In another embodiment, the volume is approximately 8.5 mL. In another embodiment, the volume is approximately 9 mL. In another embodiment, the volume is approximately 9.5 mL. In another embodiment, the volume is approximately 10 mL.

[0098] In some embodiments, the concentration of recombinant adeno-associated virus having a nucleic acid sequence encoding a desired transgene under the control of a regulatory sequence is preferably about 10 per milliliter in the composition. 7 ~10 14 This is the range of a vector genome (vg / mL) (also called genome copies / mL (GC / mL)).

[0099] In one embodiment, the dosage of rAAV in the composition is approximately 1.0 × 10⁶ of body weight. 9 GC / kg ~ approx. 1.5×10 13 The concentration is GC / kg. In one embodiment, the dosage is approximately 1.0 × 10⁻⁶. 10 The concentration is GC / kg. In one embodiment, the dosage is approximately 1.0 × 10⁻⁶. 11 The concentration is GC / kg. In one embodiment, the dosage is approximately 1.0 × 10⁻⁶. 12 The concentration is GC / kg. In one embodiment, the dosage is approximately 5.0 × 10⁻⁶. 12 The concentration is GC / kg. In one embodiment, the dosage is approximately 1.0 × 10⁻⁶. 13 The value is GC / kg. All scopes described herein include endpoints.

[0100] In one embodiment, the effective dose (total genome copies delivered) is approximately 10 7 ~10 13 It is a vector genome. In one embodiment, the total dose is approximately 10 8 It is a genome copy. In terms of administration method, the total dose is approximately 10 9 It is a genome copy. In one embodiment, the total dose is approximately 10 10 It is a genome copy. In one embodiment, the total dose is approximately 10 11 It is a genome copy. In one embodiment, the total dose is approximately 10 12 It is a genome copy. In one embodiment, the total dose is approximately 10 13 It is a genome copy. In one embodiment, the total dose is approximately 10 14 It is a genome copy. In one embodiment, the total dose is approximately 10 15 It is a genome copy.

[0101] To reduce the risk of undesirable effects such as toxicity, it is desirable to use the lowest effective concentration of the virus. Furthermore, other doses and dosages within these ranges may be selected by the attending physician, taking into account the physical condition of the subject being treated (preferably a human), the subject's age, the specific disorder, and, in progressive cases, the degree of the disorder that has developed.

[0102] In certain embodiments, the composition comprises an rAAV containing an inducible GLP-1 agonist construct. In certain embodiments, the inducer or molecule is rapamycin or rapalog. In certain embodiments, the inducer is rapamycin and is administered at least once, at least twice, or at least three times after the composition containing the rAAV. In some embodiments, rapamycin is administered in doses of at least about 4 to at least about 40 nM. In certain embodiments, the inducer (i.e., rapamycin) is administered in doses of at least about 0.1 mg / kg to at least about 3.0 mg / kg. In certain embodiments, the inducer (i.e., rapamycin) is administered in doses of at least about 0.5 mg / kg to at least about 2.0 mg / kg.

[0103] The viral vectors and other constructs described herein may be used to deliver GLP-1 fusion protein constructs to subjects requiring them, to supply subjects with increased half-life GLP-1, and / or to prepare pharmaceuticals for the treatment of type 1 diabetes, type 2 diabetes, or metabolic syndrome in subjects. Thus, in another embodiment, a method for treating diabetes is provided. This method comprises administering a composition described herein to a subject requiring it. In one embodiment, the composition comprises a viral vector containing a GLP-1 fusion protein expression cassette described herein.

[0104] As used herein, the terms “treatment” or “to treat” are defined to include administering one or more of the compounds or compositions described herein to a subject for the purpose of alleviating one or more symptoms of type 1 diabetes, type 2 diabetes, or metabolic syndrome. Accordingly, “treatment” may include, in a given subject, one or more of the following: reducing the progression of type 1 diabetes, type 2 diabetes, or metabolic syndrome; reducing the severity of symptoms; delaying the progression of the disease; or increasing the effectiveness of the therapy.

[0105] As used herein, the term “remission” refers to the ability to discontinue insulin therapy when the subject no longer exhibits clinical signs of diabetes and has normal blood glucose levels.

[0106] 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 containing a sequence encoding a fusion protein described herein. In one embodiment, the subject is a human.

[0107] In another embodiment, a method for treating metabolic disorders in a subject is provided. The method comprises administering a composition described herein to a subject in need thereof. In one embodiment, the composition comprises a viral vector containing a GLP-1 fusion protein expression cassette described herein. In one embodiment, the metabolic disorder is type 1 diabetes. In one embodiment, the metabolic disorder is type 2 diabetes. In one embodiment, the metabolic disorder is metabolic This is a dyslexia syndrome. In one embodiment, the subject is a human.

[0108] In another embodiment, a method for reducing body weight in a subject is provided. This method involves administering a composition 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 described herein.

[0109] 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). Further combinations may be selected using the viral vectors described herein. Optionally, the compositions described herein may be combined with regimens including other antidiabetic drugs or protein-based therapies (e.g., GLP-1 analogs, insulin, oral antihyperglycemic agents (sulfonylurea, biguanides, thiazolidinediones, and alpha-glucoidase inhibitors)). Optionally, the compositions described herein may be combined with regimens involving lifestyle changes, including diet and exercise therapy. In certain embodiments, the AAV vector and the combination therapy are administered essentially simultaneously. In other embodiments, the AAV vector is administered first. In certain embodiments, the combination therapy is administered first.

[0110] 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 are not limited to, recombinant protamine zinc human insulin (ProZinc®), porcine insulin zinc suspension (Vetsulin®), insulin glargine (Lantus®), lispro (Humalog), aspart (Novolog), glulisine (Apidra), Novolin, and veloslin.

[0111] In some embodiments, the rAAV-insulin combination described herein reduces the insulin dose requirements in the subject compared to before treatment with the viral vector. Such dose requirements may be reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more. The treating physician can determine the correct dosage of insulin required by the subject. For example, the subject may be treated with insulin or other therapies, which the treating physician may continue at the time of AAV vector administration. Such insulin or other combination therapy may then be continued, reduced, or discontinued as needed.

[0112] In one embodiment, the expression cassette, vector genome, rAAV-containing composition, or other composition described herein for gene therapy is delivered as a single dose per patient. In one embodiment, the subject is delivered a therapeutically effective dose of the composition described herein. As used herein, “therapeutic dose” refers to the amount of expression cassette or vector, or combination thereof, that delivers and expresses a sufficient amount of GLP1-Fc to target cells to achieve a therapeutic objective. The therapeutic dose may be selected by the treating physician or guided based on previously determined guidelines. For example, dulaglutide may be delivered subcutaneously once a week in an initial dose of 0.75 mg. The dose may be increased in 1.5 mg increments for additional glycemic control. The patient should remain on a dose of 1.5 mg once a week for at least four weeks before increasing the dose to 3 mg once a week. The patient should remain on a dose of 3 mg once a week for at least four weeks before increasing the dose to 4.5 mg once a week. The maintenance dose of dulaglutide is 0.75–4.5 mg subcutaneously once weekly, with a maximum dose of 4.5 mg per week. rAAV is delivered to the subject, and then, if necessary, oral or subcutaneous dulaglutide, insulin, or other medications may be supplemented to reach the desired dose equivalent of 0.75–4.5 mg per week.

[0113] In certain embodiments, the therapeutic goal is to alleviate or treat one or more symptoms of type 1 diabetes, type 2 diabetes, or metabolic syndrome. The “therapeutic effective dose” may be determined based on an animal model rather than a human patient. In another embodiment, the therapeutic goal is remission of the metabolic disease in question. As used herein, when used to refer to vp capsid proteins, the term “heterogeneous” or any grammatical variation thereof refers to a population of non-identical elements having, for example, vp1, vp2, or vp3 monomers (proteins) having different modified amino acid sequences. Sequence ID No. 20 provides the encoded amino acid sequence of the AAVrh91 vp1 protein. The term “heterogeneous” as used in relation to vp1, vp2, and vp3 proteins (alternatively referred to as isoforms) refers to the differences in the amino acid sequences of the vp1, vp2, and vp3 proteins within the capsid. The AAV capsid contains subpopulations within the vp1, vp2, and vp3 proteins having the predicted amino acid residue modifications. These subpopulations contain, at a minimum, specific deamidated asparagine (N or Asn) residues. For example, a particular subpopulation contains at least one, two, three, or four highly deamidated asparagine (N) positions in an asparagine-glycine pair, and optionally further contains other deamidated amino acids, where deamidation results in amino acid changes and other optional modifications.

[0114] As used herein, a “subpopulation” of vp proteins means, unless otherwise specified, a group of vp proteins that share at least one defined common feature and consist of at least one group member and fewer members than all members of the reference group. For example, a “subpopulation” of vp1 proteins is, unless otherwise specified, at least one vp1 protein and fewer than all vp1 proteins in an assembled AAV capsid. A “subpopulation” of vp3 proteins may be, unless otherwise specified, one vp3 protein and fewer than all vp3 proteins in an assembled AAV capsid. For example, in an assembled AAV capsid, vp1 proteins may be a subpopulation of vp proteins, vp2 proteins may be another subpopulation of vp proteins, and vp3 may be yet another subpopulation of vp proteins. In another example, the vp1, vp2, and vp3 proteins may comprise subpopulations having different modifications, for example, at least one, two, three, or four highly deamidated asparagines, such as asparagine-glycine pairs.

[0115] As used herein, a “stock” of rAAV refers to a population of rAAVs. Despite heterogeneity of capsid proteins resulting from deamidation, rAAVs within a stock are expected to share five identical vector genomes. A stock may include, for example, rAAVs having a selected AAV capsid protein and capsids with heterogeneous deamidation patterns characteristic of a selected production system. A stock may be produced from a single production system or pooled from multiple runs of a production system. A variety of production systems may be selected, including but not limited to those described herein. As used herein, “GLP-1 construct,” “GLP-1 expression construct,” and synonyms include the GLP-1 sequences described herein in combination with the leader and fusion domain. The terms “GLP-1 construct,” “GLP-1 expression construct,” and synonyms may be used to refer to nucleic acid sequences encoding a GLP-1 fusion protein or their expression products.

[0116] In the context of nucleic acid sequences, the terms “identity percentage (%)”, “sequence identity”, “sequence identity percentage”, or “identity percentage” refer to bases in two sequences that are identical when aligned for correspondence. The length of the sequence identity comparison may be the full length of the genome, the full length of the gene coding sequence, or a fragment of at least about 100–150 nucleotides, or this may be desired. However, for example, at least about 9 nucleotides, usually at least about 20–24 nucleotides, or at least about 28–32 nucleotides Identity between smaller fragments of nucleotides, at least about 36 nucleotides, may also be desired. Multiple sequence alignment programs are also available for nucleic acid sequences. Examples of such programs include "Clustal W," "CAP Sequence Assembly," "BLAST," "MAP," and "MEME," which are accessible through web servers on the Internet. Other sources of such programs are known to those skilled in the art. Alternatively, the Vector NTI utility can also be used. In addition, several algorithms known in the art exist, including those included in the programs described above, and can be used to measure nucleotide sequence identity. As another example, polynucleotide sequences can be compared using Fasta®, a program from GCG version 6.1. Fasta® provides the best overlap region alignment and sequence identity percentage between query and search sequences. For example, the percentage sequence identity between nucleic acid sequences can be determined using Fasta® with its default parameters (word size 6 and NOPAM factor for the scoring matrix) provided in GCG Version 6.1, which is incorporated herein for reference.

[0117] The term "highly preserved" 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.

[0118] Unless otherwise specified in the upper limit, the percentage of identity is understood to be the minimum level of identity and encompasses all higher levels up to 100% identity with respect to the reference sequence. Unless otherwise specified, the percentage of identity is understood to be the minimum level of identity and encompasses all higher levels up to 100% identity with respect to the reference sequence. For example, "95% identity" and "at least 95% identity" can be used interchangeably and include 95%, 96%, 97%, 98%, 99%, up to 100% identity with respect to the reference sequence, and all fractions in between.

[0119] In the context of amino acid sequences, the terms “identity percentage (%),” “sequence identity,” “sequence identity percentage,” or “identical percentage” refer to residues in two sequences that are identical when aligned to correspond. The identity percentage can be readily determined for a full-length polypeptide of a protein, a polypeptide, an amino acid sequence spanning approximately 70 amino acids, approximately 100 amino acids, or a peptide fragment thereof, or for a corresponding nucleic acid sequence encoding a sequence. A suitable amino acid fragment may be at least approximately 8 amino acids long and up to approximately 150. Generally, when referring to “identity,” “homology,” or “similarity” between two different sequences, “identity,” “homology,” or “similarity” is determined by referring to an “aligned” sequence. An “aligned” sequence or “alignment” refers to multiple nucleic acid sequences or protein (amino acid) sequences that, compared to a reference sequence, often include corrections for missing or additional bases or amino acids. Alignment is performed using one of the various publicly or commercially available multiple sequence alignment programs. Sequence alignment programs are available for amino acid sequences, including, for example, the "Clustal X," "MAP," "PIMA," "MSA," "BLOCKMAKER," "MEME," and "Match-Box" programs. Generally, one of these programs is used with its default settings, but those skilled in the art may change these settings as needed. Alternatively, those skilled in the art may use other algorithms or computer programs that provide at least the same level of identity or alignment as those provided by the reference algorithms and programs. For example, JDThomson et al, Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments," 27( See 13):2682-2690(1999).

[0120] Please note that the terms "a" or "an" refer to one or more. Therefore, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably in this specification.

[0121] The terms “comprise,” “comprises,” and “comprising” should be interpreted comprehensively, not exclusively. The terms “consist,” “consisting,” and their variations should be interpreted exclusively, not comprehensively. While various embodiments in this specification are presented using the language “comprising,” in other circumstances, relevant embodiments are also intended to be interpreted and described using the language “consisting of” or “essentially consisting of.”

[0122] As used herein, “patient” or “subject” means mammal, and includes humans, veterinary or agricultural animals, domestic or companion animals, and animals typically used in clinical research. In one embodiment, the subject of these methods and compositions is human. In another embodiment, the subject is not cat.

[0123] Where used herein, the term “about” means a variability of 10% (±10%, e.g., ±1, ±2, ±3, ±4, ±5, ±6, ±7, ±8, ±9, ±10, or values ​​in between) from the given reference, unless otherwise specified.

[0124] In certain cases, the term "E+#" or "e+#" is used to refer to an exponent. For example, "5E10" or "5e10" means 5 × 10⁻¹⁰ 10 These terms can be used synonymously.

[0125] As used herein, the terms “modulation” or its variants refer to the ability of a composition to inhibit one or more components of a biological pathway.

[0126] As used herein, “disease,” “disorder,” and “condition” are used interchangeably to describe an abnormal condition in the subject.

[0127] Unless otherwise defined herein, the technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art and by referring to published literature that provides a general guide to those skilled in the art for the many terms used herein.

[0128] When describing one embodiment, references to "one embodiment" or "another embodiment" do not imply that the referenced embodiment is mutually exclusive with another embodiment (for example, an embodiment described before the referenced embodiment) unless otherwise explicitly specified.

[0129] Specific Embodiments 1. A viral vector containing nucleic acids that include a sequence encoding a fusion protein containing a GLP-1 analog and IgG4 Fc. 2. The vector according to Embodiment 1, wherein the vector is an adeno-associated virus vector. 3. The viral vector according to Embodiment 1 or Embodiment 2, wherein the fusion protein further comprises a thrombin reader sequence. 4. The viral vector according to Embodiment 3, wherein the thrombin reader sequence comprises the sequence of SEQ ID NO: 7, or a functional variant thereof having at most 1, 2, or 3 amino acid substitutions. Tar. 5. A viral vector according to any one of Embodiments 1 to 4, wherein the fusion protein further comprises a spacer. 6. A viral vector according to any one of Embodiments 1 to 5, wherein the fusion protein comprises a human thrombin reader, a GLP-1 analog, a spacer, and human IgG4 Fc. 7. The viral vector according to Embodiments 1 to 6, wherein the fusion protein has the sequence of SEQ ID NO: 14, or a sequence that is at least 99% identical thereto. 8. A viral vector according to any one of Embodiments 1 to 7, wherein the sequence encoding the fusion protein is Sequence ID No. 15. 9. (a) AAV capsid and, (b) A viral vector according to any one of Embodiments 1 to 8, comprising a vector genome packaged in an AAV capsid, wherein the vector genome includes an AAV inverted terminal repeat (ITR), a coding sequence for a fusion protein, and a regulatory sequence that directs the expression of the fusion protein. 10. The viral vector according to any one of Embodiments 1 to 9, wherein the viral vector is recombinant adeno-associated virus (rAAV) having the AAV capsid of AAV8 or a functional variant thereof. 11. The viral vector according to any one of Embodiments 1 to 9, wherein the viral vector is an AAV capsid of AAVrh91 or an rAAV having a functional variant thereof. 12. The viral vector according to any one of Embodiments 1 to 9, wherein the viral vector is an AAV capsid of AAV3B.AR2.12 or an rAAV having a functional variant thereof. 13. The viral vector according to any one of Embodiments 1 to 9, wherein the viral vector is an rAAV having an AAV capsid or a functional variant thereof selected from AAV9, AAVrh64R1, AAVhu37, or AAVrh10. 14. A viral vector according to any one of Embodiments 1 to 13, comprising a vector genome including an inducible gene expression system, a controllable promoter, a sequence encoding a fusion protein, and a polyadenylation signal. 15. A viral vector according to any one of Embodiments 9 to 14, wherein the AAV inverted terminal repeats (ITRs) are AAV2 5' ITR and AAV2 3' ITR adjacent to the fusion protein coding sequence and regulatory sequence. 16. A viral vector according to any one of Embodiments 9 to 15, wherein the vector genome comprises a human cytomegalovirus promoter and rabbit globin polyA. 17. A viral vector according to any one of Embodiments 1 to 16, comprising an inducible gene expression system. 18. Inducible gene expression systems, (a) an activation domain including a transactivation domain and the FKBP12-rapamycin-binding (FRB) domain of FKBP12-rapamycin-related protein (FRAP), (b) A DNA-binding domain comprising a zinc finger homeodomain (ZFHD) and one, two, or three FK506-binding protein domain (FKBP) subunit genes, (c) ZFHD, followed by at least one copy of the binding site to the minimal IL2 promoter, (d) an adjustable promoter, The viral vector according to Embodiment 17, wherein the presence of an effective amount of rapamycin or rapalog induces the expression of a transgene in host cells. 19. The viral vector according to Embodiment 18, wherein the FKBP subunit gene sequences share less than approximately 85% identity with one another. 20. The viral vector according to Embodiment 18 or 19, wherein one of the FKBP subunit gene sequences is a natural FKBP gene sequence. 21. A viral vector according to any one of Embodiments 18 to 20, wherein the transactivation domain comprises a portion of NF-κB p65. 22. The viral vector according to any one of embodiments 18 to 21, wherein the adjustable promoter is a constitutive promoter. 23. The viral vector according to any one of embodiments 18 to 21, wherein the adjustable promoter is a tissue-specific promoter. 24. The viral vector according to any one of embodiments 18 to 22, wherein the adjustable promoter is a CMV promoter. 25. A viral vector according to any one of embodiments 18 to 24, further comprising IRES or 2A. 26. A viral vector according to any one of embodiments 18 to 25, further comprising a 2A linker selected from GT2A_V1 (SEQ ID NO: 21) or GT2A_V2 (SEQ ID NO: 22). 27. A viral vector according to any one of embodiments 18 to 26, comprising at least eight copies of the binding site to ZFHD. 28. The viral vector according to any one of embodiments 18 to 27, wherein the vector genome contains the sequence of SEQ ID NO: 16, or a sequence that is at least 95% to 99.9% identical thereto. 29. A viral vector comprising a nucleic acid molecule comprising a controllable promoter, an activation domain containing a p65 transactivation domain and an FKBP12-rapamycin-binding (FRB) domain of FKBP12-rapamycin-related protein (FRAP), a DNA-binding domain containing a zinc finger homeodomain (ZFHD) and three FK506-binding protein domain (FKBP) subunit genes, eight copies of the binding site for ZFHD, and a sequence encoding a fusion protein containing a GLP-1 analog and human IgG4 Fc. 30. A pharmaceutical composition suitable for use in the treatment of metabolic disorders in a subject, comprising an aqueous liquid and a viral vector as described in any one of Embodiments 1 to 20. 31. The pharmaceutical composition according to Embodiment 30, wherein the fusion protein comprises a human thrombin reader, a GLP-1 analog, a spacer, and human IgG4 Fc. 32. A viral vector according to any one of Embodiments 1 to 29, or a pharmaceutical composition according to Embodiment 30 or 31, for use in a method of treating a subject having a metabolic disease. 33. Use of a viral vector according to any one of Embodiments 1 to 29, or a pharmaceutical composition according to any one of Embodiments 29 to 31, in the manufacture of a pharmaceutical for treating a subject having a metabolic disease. 34. The composition is 1 × 10 9 GC / kg~5×10 13 The viral vector or use according to Embodiment 32 or 33, which is formulated to administer rAAV in a dose of GC / kg. 35. The patient is a human being, 1 x 10 10 ~1.5×10 15 The viral vector or use according to Embodiment 32 or 33, wherein rAAV is administered in a GC dose. 36. A viral vector or use according to any one of embodiments 32 to 35, wherein rAAV is delivered intramuscularly or intravenously. 37. A method for treating a subject with a metabolic disorder, comprising delivering to the subject a recombinant adeno-associated virus (rAAV) having an AAV capsid derived from adeno-associated virus rh91, and a vector genome packaged within the AAV capsid, wherein the vector genome comprises an AAV inverted terminal repeat (ITR), a sequence encoding a fusion protein containing a GLP-1 analog and human IgG4 Fc, and a regulatory sequence that directs the expression of the fusion protein. 38. The patient has a viral vector or embodiment described in any one of Embodiments 1 to 29. The method according to Embodiment 37, wherein the pharmaceutical composition described in Embodiment 30 or 31 is administered. 39. The patient, 1 x 10 9 GC / kg~5×10 13 The method according to embodiment 37 or 38, wherein rAAV is administered in a dose of GC / kg body weight. 40. The method according to any one of embodiments 37 to 39, wherein rAAV is delivered intramuscularly or intravenously. 41. A viral vector according to any one of Embodiments 1-29, 32, or 34-36, a composition according to any one of Embodiments 30-32, a use according to any one of Embodiments 33-36, or a method according to any one of Embodiments 37-40, for the treatment of diabetes in humans. [Examples]

[0130] The following embodiments are provided to illustrate various embodiments of the present invention. These embodiments are not intended to limit the present invention in any way.

[0131] Glucagon-like peptide 1 (GLP-1) is a hormone produced from the proteolytic cleavage of glucagon preprotein in the gastrointestinal (GI) tract. GLP-1 broadly regulates glucose homeostasis by enhancing insulin release from beta cells, increasing insulin sensitivity in several tissues, slowing gastric emptying (without causing hypoglycemia), and increasing satiety. Although GLP-1 has not been effectively used as a drug due to its extremely short half-life, long-acting analogs of GLP-1 are widely used drugs for the treatment of type 2 diabetes. GLP-1 agonists have an excellent safety profile, require repeated, often lifelong, parenteral administration, and can achieve long-term expression after a single dose, making them good candidates for AAV-mediated gene transfer. GLP-1 and GLP-1 agonists are difficult to express from AAV vectors because the protein cannot be expressed in its native contents (glucagon protein), which requires processing by proteases specific to L cells in the small intestine. Attempts to express GLP-1 using heterologous signal peptides have failed to achieve high levels of expression. It was suggested that reliable expression may not be achieved because signal peptides do not properly process the N-terminus of GLP-1 involved in receptor binding. Instead, GLP-1 was expressed using propeptides that are cleaved to produce free GLP-1 protein. These propeptides were selected from coagulation factors such as thrombin and factor IX for GLP-1 expression because they are endogenous peptides that can be cleaved by ubiquitous proteases (e.g., furin) and are not immunogenic. Thrombin propeptides increased the expression of human GLP-1 analogs by at least 100-fold compared to signal peptides alone. Using this technique, two long-acting GLP-1 analogs were developed that can be expressed from AAV vectors: one containing an IgG4 Fc fusion and the other containing an albumin fusion, both carrying human propeptides. We developed expression cassettes that constitutively or regulatedly express GLP-1 agonist sequences by administering small molecule drugs that activate their transcription. Target product profiles are designed for single intramuscular injection.In one embodiment, the single injection includes an inducible version as a single tablet administered every 2–4 weeks, designed to maintain therapeutic GLP-1 agonist levels. In another embodiment, the single injection includes a constitutive version designed for continuous lifetime expression at therapeutic levels after a single dose. The designed product was the testis in a preclinical model to investigate pharmacology and safety in non-human primates. Assays were developed for the expression and activity of the GLP-1 agonist. Safety and pharmacokinetics have been investigated to analyze the ability to achieve known therapeutic concentrations.

[0132] This innovation is particularly relevant in patients with type 2 diabetes who have not achieved their target glycated hemoglobin (also known as glycated hemoglobin, hemoglobin A1c, HbA1c, or A1c) after 3 months with metformin alone or other oral medications, potentially leading to a single, potentially lifetime, outcome. This enables treatment. The standard of care currently includes long-acting subcutaneous GLP-1 agonists such as liraglutide (daily), dulaglutide (weekly), DPP (e.g., dipeptidyl peptidase-4) IV inhibitors (PO), and semaglutide PO (daily). Previous attempts to achieve AAV-mediated GLP-1 expression either resulted in dramatically lower expression or required the use of heterologous leader sequences that were immunogenic and unsuitable for clinical application.

[0133] Example 1 - Construction of GLP-1 vector GLP-1 agonists are difficult to express via adeno-associated virus (AAV). GLP-1 is typically expressed from a glucagon precursor protein, which requires a tissue-specific protease and produces unwanted proteins. Conventional expression systems using heterologous signal peptides result in low expression. Expression systems using heterologous propeptides with universal protease cleavage sites introduce exogenous protein sequences that can be targeted by T cells. We developed a system that increases GLP-1 expression by approximately 300-fold from liver or muscle cells without introducing exogenous protein sequences. Figure 5 shows AAV-mediated expression of the engineered GLP-1 construct in mice. Mice received intramuscular injections of AAV vectors expressing GLP-1 agonists using the developed standard IL-2 signal peptide or endogenous precursor. Serum GLP-1 concentrations were measured by ELISA three weeks after injection.

[0134] More specifically, a vector was constructed by placing a leader sequence upstream of one of several GLP-1 receptor agonist amino acid sequences, followed by a fusion domain. See, for example, Figure 4. The resulting protein sequence was back-translated, and then a Kozak consensus sequence, stop codon, and cloning site were added. The sequence was constructed and cloned into an expression vector containing the CMV promoter under the control of an inducible expression system. The expression construct was adjacent to the AAV2 ITR. The resulting plasmid is named pAAV.TF.GT2A.dulaglutide(trb).3w.rBG. The human thrombin-dulaglutide amino acid sequence is shown in SEQ ID NO: 14, the coding sequence in SEQ ID NO: 15, and the vector genome in SEQ ID NO: 16.

[0135] Currently available inducible constructs include two-vector and one-vector inducible systems. See, for example, Figures 6A and 6B. Figure 6A shows a schematic diagram of an exemplary expression cassette containing an inducible construct for use in a two-vector system. Figure 6B shows a schematic diagram of an expression cassette containing an inducible construct for use in a one-vector system including an IRES linker.

[0136] Furthermore, the GT2A peptide was introduced into the expression vector containing the GLP1-Fc transgene. The human GLP1-Fc (h-dulaglutide) with a secretory signal is 954 bp. As described above, for the expression of the h-dulaglutide construct, the IRES linker is replaced with the GT2A cleavage sequence in the expression vector shown in Figure 6B, thereby enabling the IRES linker to conform to the packaging limit (Figure 7A; single inducible cassette for GLP-1 Fc). The GT2A peptide is selected from the GT2A_V1 peptide containing the amino acid sequence of SEQ ID NO: 21, or the GT2A_V2 peptide containing the amino acid sequence of SEQ ID NO: 22. Figure 7A shows a schematic diagram of an expression cassette containing an inducible construct for use in a single vector system, including the F2A cleavage sequence linker with a secretory signal and human GLP1-Fc (h-dulaglutide).

[0137] Example 2 - In vitro expression In the culture supernatant of HEK293 cells transfected with a plasmid containing a GLP1-Fc fusion, inducible human dulaglutide (TF.GT2A.dulaglutide (Trb)) and CB7.fenea dulaglutide (feTrb) containing a human thrombin signaling sequence were detected. Measurements were taken. Feline dulaglutide refers to a construct in which the IgG Fc portion of dulaglutide is replaced with a feline IgG sequence and optionally combined with a feline thrombin reader (feTrb). The supernatant was collected at 0, 4, and 40 nM 48 hours after treatment with rapamycin (Rapa), or 48 hours after transfection with CB7.fe dulaglutide (feTrb). GLP1-Fc was quantified by active GLP1 ELISA with the kit's STD. The expression of the three constructs is shown in Figure 2. Increasing the dose of rapamycin resulted in increased GLP-1 expression.

[0138] Furthermore, we evaluated the expression of exemplary therapeutic transgene (rhTT) in rhesus monkeys in designed constructs containing GT2A_V1 or GT2A_V2 peptides (Figures 6B, 7A, and 7B). Figure 8 shows the expression of therapeutic transgene (rhTT) in rhesus monkeys in HEK293 cell supernatant, measured after transfection with various constructs containing GT2A peptides at 0 nM, 4 nM, and 40 nM, and plotted as IU / mL rhTT after treatment with rapamycin. Next, we investigated the expression of human and rhesus monkey GLP-1 Fc in vitro using designed single inducible cassettes containing GT2A_V1 and GT2A_V2 peptides. Figure 9 shows the expression of inducible human (h) and rhesus monkey (rh) GLP-1 in vitro. Inducible h-dulaglutide containing a thrombin signaling sequence, rh-dulaglutide containing a two-vector system, and CB7.rh-dulaglutide were measured in the culture supernatant of HEK293 cells transfected with a plasmid containing a GLP1-Fc fusion. Cells were seeded on day 0, transfected on day 1, treated with rapamycin at 0 nM, 4 nM, and 40 nM on day 2, and the supernatant was collected from the cells on day 4 or 48 hours after transfection with CB7.rh-dulaglutide (rhTrb). GLP1-Fc was quantified by active GLP1 ELISA with the kit's STD.

[0139] Example 3 - Pilot expression in Rag1KO mice The following constructs were packaged into the AAVrh91 vector by triple transfection and iodine xanol gradient purification, as described above. AAVrh91.TF.h dulaglutide (Trb).3w.rBG with human thrombin signaling AAVrh91.TF.rh dulaglutide (rhTrb).3w.rBG with rhesal thrombin signaling Rag1KO female mice (n=5 / vector) were administered via the IM route using a vector (1×10 11The mice were treated with GC / mouse injection. Serum was continuously collected by separating whole blood in a serum isolation tube containing 5 microliters of DPP-IV inhibitor (Millipore), and the expression and activity of active GLP-1 described above were assayed. The vector was injected on day 0, and rapamycin was administered around days 14 and 15. Serum active GLP-1 concentrations are shown in Figure 3. Serum levels reached their maximum approximately one week after rapamycin administration.

[0140] Example 4 - Long-term expression study in NHP This study investigated the expression of rhesus macaque GLP-1 (rh dulaglutide) in non-human primates (NHPs; i.e., rhesus macaques). Tables 1A and 1B summarize the study, including AAV administration and rapamycin administration (i.e., induction). Briefly, NHP1-3 were administered the AAVrh91-designated vector via intramuscular injection (IM) - NHP1: 1 × 10⁻¹⁶ 12 (1e12)GC / kg doses: AAVrh91.CB7.rh dulaglutide.rBG;NHP2: 5 × 10 12 AAVrh91.CMV.TFNc.3 AAVrh91.Z12I.rh dulaglutide.rBG and AAVrh91.Z12I.rh dulaglutide.rBG at a dose of (5e12)GC / kg; and NHP3: 1×10 13 (1e13)GC / kg. For NHP2, rapamycin was administered at a dose of 0.5 mg / kg on day 21, 0.5 mg / kg on day 56, and 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. [Table 2] [Table 3]

[0141] Figures 10A-10C show rhGLP1-Fc expression and analysis in the NHP1(18-128) anti-rhGLP1-Fc ADA (anti-drug antibody) detection assay. Figure 10A shows serum rhGLP1-Fc expression levels, plotted as nM, measured from day 0 to 200. Figure 10B shows serum rapamycin levels, plotted as μg / L, measured from day 0 to 200. Figure 10C shows the results of the ADA detection assay, plotted as an outer diameter of 450 nm, measured from day 0 to 200.

[0142] Figures 11A-11C show rhGLP1-Fc expression and analysis of the anti-rhGLP1-Fc ADA assay for NHP1(18-072). Figure 11A shows serum rhGLP1-Fc expression levels, plotted as nM, measured from day 0 to 200. Figure 11B shows serum rapamycin levels, plotted as μg / L, measured from day 0 to 200. Figure 11C shows the results of the ADA detection assay, plotted as an outer diameter of 450 nm, measured from day 0 to 200.

[0143] Figures 12A-12C show rhGLP1-Fc expression and analysis of the anti-rhGLP1-Fc ADA assay for NHP1(18-013). Figure 12A shows serum rhGLP1-Fc expression levels, plotted as nM, measured from day 0 to 200. Figure 12B shows serum rapamycin levels, plotted as μg / L, measured from day 0 to 200. Figure 12C shows the results of the ADA detection assay, plotted as an outer diameter of 450 nm, measured from day 0 to 200.

[0144] In summary, we developed a one-vector induction system for the expression of human GLP1-Fc fusion. In addition, we confirmed the induction of human GLP1-Fc in Rag1KO mice upon rapamycin. In NHP, we observed that one-vector and two-vector inducible vectors expressing monkey GLP1-Fc responded to rapamycin, resulting in a transient increase in serum GLP1-Fc exceeding 1 nM for a duration of more than 20 days. We observed that low doses constitutively expressing the vector provided high and sustained serum GLP1-Fc expression in NHP. (Sequence listing-free testing)

[0145] The following information is a numerical identifier <223> Provides an array containing free text below. [Table 4-1] [Table 4-2]

[0146] All documents referenced herein are incorporated herein by reference. U.S. Provisional Patent Application No. 63 / 069,500, filed on 24 August 2020, is incorporated herein by reference in its entirety, along with its sequence listing. The sequence listing filed with this specification, labeled “20-9429PCT_Seq_List_ST25,” and the sequences and text therein, are incorporated herein by reference. While the present invention is described with reference to specific embodiments, it will be understood that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. AAVrh91 capsid, A nucleic acid comprising a nucleotide sequence encoding a fusion protein containing a GLP-1 analog and IgG4Fc, wherein the fusion protein has the sequence of SEQ ID NO: 14, or an amino sequence that is at least 99% identical thereto, and furthermore, the nucleotide sequence encoding the fusion protein is SEQ ID NO: 15, A vector genome packaged in an AAV capsid includes an AAV inverted terminal repeat (ITR), the coding sequence for the fusion protein, and a regulatory sequence that directs the expression of the fusion protein. Adeno-associated virus (AAV) vectors containing this virus.

2. The AAV vector according to claim 1, comprising an inducible gene expression system, a controllable promoter, the nucleotide sequence encoding the fusion protein, and a vector genome comprising a polyadenylation signal.

3. The AAV vector according to claim 1 or 2, wherein the AAV inverted terminal repeat (ITR) is the 5'ITR of AAA2 and the 3'ITR of AAV2 adjacent to the nucleotide sequences encoding the fusion protein coding sequence and the regulatory sequence.

4. The AAV vector according to any one of claims 2 to 3, wherein the vector genome comprises a CB7 promoter and rabbit globin polyA.

5. The inducible gene expression system described above is (a) an activation domain comprising a transactivation domain and the FKBP12-rapamycin-binding (FRB) domain of FKBP12-rapamycin-related protein (FRAP), (b) A DNA-binding domain comprising a zinc finger homeodomain (ZFHD) and one, two, or three FK506-binding protein domain (FKBP) subunit genes, (c) ZFHD, followed by at least one copy of the binding site to the minimal IL2 promoter, (d) A controllable promoter, comprising The AAV vector according to claim 2, wherein the presence of an effective amount of rapamycin or rapalog induces the expression of the transgene in host cells.

6. The AAV vector according to claim 5, wherein the FKBP subunit gene sequences share approximately 70% to approximately 85% identity with one another, and furthermore, one of the FKBP subunit gene sequences is a natural FKBP gene sequence.

7. The AAV vector according to any one of claims 5 to 6, wherein the transactivation domain comprises a portion of NF-κB p65.

8. The AAV vector according to any one of claims 5 to 7, wherein the adjustable promoter is a constitutive promoter or a CMV promoter.

9. An AAV vector according to any one of claims 5 to 8, further comprising IRES or 2A.

10. The AAV vector according to any one of claims 5 to 9, further comprising a 2A linker selected from GT2A_V1 consisting of the amino acid sequence of SEQ ID NO: 21 or GT2A_V2 consisting of the amino acid sequence of SEQ ID NO:

22.

11. The AAV vector according to any one of claims 5 to 10, comprising at least eight copies of the binding site to the ZFHD.

12. The AAV vector according to any one of claims 5 to 11, wherein the vector genome comprises the nucleotide sequence of sequence number 16.

Images