Viral vector encoding a GLP-1 receptor agonist fusion and its use in the treatment of metabolic diseases in cats

Abstract

This invention provides compositions and methods for treating type II diabetes in cats. [Solution] A viral vector is provided, comprising a nucleic acid comprising 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 containing either (i) feline IgG Fc or a functional variant thereof, or (ii) feline albumin or a functional variant thereof.

Description

[Background technology]

[0001] In the United States, one in 400 cats and one in 500 dogs suffers from symptoms similar to human diabetes. The current standard treatment involves frequent visits to the veterinary hospital for diagnosis, along with twice-daily insulin injections by the owner, which are expensive, time-consuming, and inconvenient for owners of these animals.

[0002] Type 2 diabetes mellitus (T2DM) is the most common form of diabetes mellitus in cats, accounting for approximately 90% of cases. Risk factors include age, sex (male), obesity, indoor living, lack of exercise, breed, and administration of long-acting or repeated steroids or megestrol acetate. These factors lead to decreased insulin sensitivity and increased demand on β-cells for insulin production. Gottleib and Rand, Managing feline diabetes: current perspectives, Veterinary Medicine: Research and Reports, June 2018:9 33-42.

[0003] Glucagon-like peptide 1 (GLP-1) is an endogenous peptide hormone that plays a central role in glucose homeostasis. GLP-1 receptor agonists are currently used in humans for the treatment of diabetes. 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. 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 T2DM. [Overview of the project]

[0004] Viral vectors encoding glucagon-like peptide 1 (GLP-1) receptor agonist fusion proteins adapted for use in cats are provided herein. In some embodiments, these viral vectors achieve sustained expression of the GLP-1 receptor agonist in cats and / or an increase in half-life, compared to vector-mediated delivery of a GLP-1 receptor agonist without a fusion partner or compared to a fusion partner not adapted for use in cats. Methods for producing and using such viral vectors are further provided.

[0005] In one aspect, a viral vector is provided that includes a nucleic acid comprising a polynucleotide sequence encoding a fusion protein. The fusion protein includes (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) feline IgG Fc or a functional variant thereof, or (ii) feline albumin or a functional variant thereof. In one embodiment, the vector is an adeno-associated viral vector.

[0006] In one embodiment, (i) the secretion signal peptide of the leader sequence comprises a feline thrombin signal peptide, (ii) the leader sequence comprises a feline thrombin propeptide, and / or (iii) the leader sequence comprises a feline thrombin leader sequence. In another embodiment, the leader sequence comprises a feline 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 functional variants thereof.

[0007] In one embodiment, the fusion domain is feline IgG Fc having the sequence of SEQ ID NO: 11, or a sequence sharing at least 90% identity therewith, or a functional variant thereof. In another embodiment, the fusion domain is feline albumin having the sequence of SEQ ID NO: 12, or a sequence sharing at least 90% identity therewith, or a functional variant thereof.

[0008] In another embodiment, the viral vector comprises an AAV capsid and a vector genome packaged within the AAV capsid, the vector genome comprising AAV inverted terminal repeats (ITRs), a polynucleotide sequence encoding a fusion protein, and a regulatory sequence directing expression of the fusion protein.

[0009] In another aspect, there is provided a pharmaceutical composition suitable for use in treating a metabolic disorder in a cat. The composition comprises an aqueous liquid and a viral vector as described herein.

[0010] In yet another aspect, use of a viral vector as described herein is provided for the manufacture of a medicament for treating a feline subject having a metabolic disorder, optionally diabetes.

[0011] In another aspect, there is provided a method of treating a feline subject having a metabolic disorder. The method comprises administering to the feline subject an effective amount of a viral vector or composition as described herein. Other aspects and advantages of the invention will be readily apparent from the following detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] [Figure 1A] Schematic of dulaglutide. [Figure 1B] Schematic of albiglutide. [Figure 2A-2B]This bar graph shows GLP-1 expression in HEK293 cells. HEK293 cells were transfected with a plasmid expressing a feline GLP1R agonist, and active GLP-1 expression was measured 48 hours after transfection by an ELISA specific for active GLP-1 (7-36) (Figure 2A). Figure 1B shows the response ratio measured relative to the control. GLP-1 activity in the culture supernatant was measured by a cell-based GLP-1 activity assay (GeneBLAzer GLP1R-CRE-bla CHO-K1 cell-based assay). The fusion protein construct using feTrb.feGLP1-IgG Fc(CB7.feline durlaglutide (feTrb).rBG) showed the highest expression / activity among the constructs tested. [Figure 3A-3B] This report demonstrates pilot expression of GLP-1 in Rag1KO mice. Female Rag1KO mice were administered 1 × 10¹¹ GC / mouse via an IM of the indicated vector. Blood samples were collected weekly. GLP-1 ELISA and GLP-1 activity assays were performed. Figure 3A shows the results of GLP-1 ELISA performed from weekly blood samples of treated mice. Figure 3B shows serum GLP-1 activity 21 days after injection. [Figure 4A-4E]The results of cat studies conducted using AAVrh91.CB7.CI. feline durlaglutide (feTrbss) and pAAV.CB7.CI. feGLP1-SA (feTrb).RBG are shown. Cats were treated with various doses of the vector via IM injection, and transgene expression and body weight were recorded for at least 28 days post-injection. Figure 4A shows the weekly body weight of individual cats treated with 5 × 10¹¹ GC / kg of AAVrh91.CB7.CI. feline durlaglutide (feTrbss) up to 17 weeks. Figure 4B shows the corresponding serum levels of fe-GLP-1-Fc for cats from Figure 4A (shown as mean + / - SD). Figure 4C shows the relationship between AAV dose and serum feline GLP-1-Fc levels 28 days after IM administration of AAVrh91.CB7.CI. feline durlaglutide (feTrbss) in either 5 × 10¹¹ GC / kg, 1 × 10¹¹ GC / cat, 1 × 10¹⁰ GC / cat, or 1 × 10⁹ GC / cat. (Data shown are mean + / - SD, n + 4 / group) Figure 4D shows the relative expression of fe-GLP-1-SA over 28 days in cats administered 1 × 10¹¹ GC / cat pAAV.CB7.CI.feGLP1-SA (feTrb).RBG (mean + / - SD, n=4). Figure 4E compares the activity of fe-GLP-1 protein in the serum of cats at day 28 from the above cohort. Figure 4F shows weight loss[]. Figure 4G shows feline GLP1Fc expression in animals. [Figure 5] This is the plasmid map of pAAV.CB7.CI.fe-durlaglutide (feTrbss).RBG. [Figure 6] This is the plasmid map of pAAV.CB7.CI.feline albiglutide (feTrb).RBG. [Figure 7] This is the plasmid map for pAAV.CB7.CI.feGLP1-SA(feTrb).RBG. [Figure 8A] The amino acid sequence of feline dulaglutide containing the feline thrombin signaling sequence (SEQ ID NO: 14) is shown. [Figure 8B] The amino acid sequence of feline albiglutide containing the feline thrombin signaling sequence (SEQ ID NO: 18) is shown. [Figure 8C] The amino acid sequence of feline GLP1-SA containing the feline thrombin signaling sequence (SEQ ID NO: 16) is shown. [Figure 9] This graph shows the serum concentrations of fGLP-1-SA at the test day, 0 to 182 days after intramuscular injection of AAV feline GLP-1-SA. The dotted line indicates the target therapy threshold. [Figure 10] This graph shows the serum concentration of feline GLP-1-Fc over 330 days after intramuscular injection of AAV feline GLP-1-Fc. [Figure 11] This graph shows the response to anti-transgene product antibodies in AAV fGLP-1-Fc treated cats. [Figure 12] This graph shows the expression of fGLP-1-SA over 336 days after intramuscular injection of AAV feline GLP-1-SA (n=4 per group). [Modes for carrying out the invention]

[0013] Long-acting GLP-1 receptor agonist fusion protein expression constructs are being developed for use in felines. 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.

[0014] The compositions and methods described herein are intended for use in felines. The term felines (Felidae) refers to any of the 37 species of cats, including, in particular, cheetahs, pumas, jaguars, leopards, lions, lynxes, tigers, and domestic cats. In one preferred embodiment, the subject is a domestic cat.

[0015] 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 for veterinary subjects requiring treatment of T2DM 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 veterinary subjects requiring it are also provided.

[0016] 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 acids It was shown to have amino acid residues. However, subsequent information showed that this peptide is a propeptide and was further processed to the active form of GLP-1, GLP-1(7-37), by removing six amino acids from the amino terminus. The glycine at position 37 was also transformed in vivo into an amide to form GLP-1(7-36)amide. GLP-1(7-37) and GLP-1(7-36)amide 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 GLP-1-(7-36)NH2.

[0017] 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 GLP-1 or its functional fragments, amino acid sequence variants of GLP-1 or its functional fragments, and other polypeptide agonists of the GLP-1 receptor (e.g., exezin-4 and its variants). This disclosure provides one or more copies of GLP-1 receptor agonists, as well as fusion proteins comprising polynucleotides and vectors encoding such fusion proteins. 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 containing either (i) feline IgG Fc or a functional variant thereof, or (ii) feline albumin or a functional variant thereof. In one embodiment, the fusion protein comprises a feline thrombin leader sequence, a GLP-1 receptor agonist, and feline IgG Fc or a functional variant thereof. In another embodiment, the fusion protein comprises a feline thrombin leader, a GLP-1 receptor agonist, and feline albumin or a functional variant thereof. In yet another embodiment, the fusion protein comprises a feline thrombin leader, two copies of the GLP-1 receptor agonist, and feline albumin or a functional variant thereof. In yet another embodiment, the fusion protein comprises a feline thrombin leader, two copies of the GLP-1 receptor agonist, and feline IgG Includes Fc or its functional variant.

[0018] In some embodiments, the GLP-1 receptor agonist includes variants that retain the function of the wild-type sequence, and may include variations of up to about 10% 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 manner 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.

[0019] Several human drugs that fuse a GLP-1 receptor agonist to a stabilizing fusion domain are known in the art. These include albiglutide, liraglutide, dulaglutide, and lixisenatide (also known chemically as des-38-proline-exendine-4 (Heroderma suspectum))-(1-39)-peptidylpenta-L-lysyl-L-lysinamide). In this disclosure, terms for human drugs preceded by the prefix "fe" are used in which the human fusion domain is replaced with a feline homolog of that fusion domain, and the GLP-1 receptor agonist is fused to a feline homolog. This refers to a variant of a human drug that is a fragment or variant of a protein, in which the GLP-1 receptor agonist is replaced with a feline homolog of that fragment or variant.

[0020] Dulaglutide is a disulfide-linked homodimer fusion peptide in which each monomer consists of one GLP-1 analog moiety and one IgG4Fc region. A schematic diagram of dulaglutide is shown in Figure 1A. See WO2005 / 000892A2 (incorporated herein by reference).

[0021] 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.

[0022] In one embodiment, the fusion includes a GLP-1 analog combined with a different feline sequence. A GLP-1 analog means a polypeptide that shares at least 90%, 95%, 97%, 98%, 99%, or 100% identity with natural feline 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 feline 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 contains one, two, or three amino acid substitutions selected from A8G, G22E, and R36G compared to the native sequence (using the full-length native GLP-1 numbering as a reference). For clarity, with respect to GLP-1(7-37), these amino acid substitutions are A2G, G16E, and R30G. 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 feline 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 comprising or consisting of SEQ ID NO: 4: HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG. In another embodiment, the GLP-1 receptor agonist has a sequence comprising or consisting of SEQ ID NO: 5: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (Exezin-4), 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: 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 resistant variant.

[0023] 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.

[0024] The leader sequence may originate from the same species to which the administration is ultimately intended, i.e., from felines. As used herein, the terms “originate” or “derived from” mean that the 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 leader sequence “originate” from cats shares the same sequence (or a variant thereof, as defined herein) as the leader sequence expressed in cats. However, the specified nucleic acid or amino acid does not actually need to be supplied from a cat. 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 acid or amino acid sequences. A “derived” nucleic acid or amino acid retains the same function as the nucleic acid or amino acid in the species from which it is “derived,” regardless of the actual source of the derived sequence.

[0025] 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 may be a conservative substitution; it may also be a non-conservative substitution. The term "conservative," when referring to two amino acids, is intended to mean that they share a common characteristic recognized by those skilled in the art. For example, amino acids with hydrophobic, non-acidic side chains; amino acids with hydrophobic, acidic side chains; amino acids with hydrophilic, non-acidic side chains; amino acids with hydrophilic, acidic side chains; and amino acids with hydrophilic, basic side chains. The common characteristic may also be an amino acid with a hydrophobic side chain, an amino acid with aliphatic, hydrophobic side chain, an amino acid with aromatic, hydrophobic side chain, an amino acid with a polar, neutral side chain, an amino acid with a charged side chain, an amino acid with a charged, acidic side chain, and an amino acid with a charged, basic side chain. Both naturally occurring and non-naturally occurring amino acids are known in the art and, in embodiments, can be used as substitute amino acids. 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, one, two, three, four, five, six, or more individual embodiments.

[0026] In one embodiment, the leader is a feline thrombin (factor II) sequence. In one embodiment, the thrombin leader has the sequence represented by SEQ ID NO: 7: MAHIRGLWLPGCLALAALCSLVHSQHVFLAPQQALSLLQRVRR, or a functional variant thereof having up to one, two, or three amino acid substitutions. In some embodiments, the leader comprises a signal peptide and a propeptide. In one embodiment, the secretory signal peptide of the leader sequence comprises a feline thrombin signal peptide. In one embodiment, the signal peptide is MAHIRGLWLPGCLALAALCSLVHS (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 comprises a feline thrombin propeptide. In one embodiment, the propeptide has the sequence QHVFLAPQQALSLLQRVRR (SEQ ID NO: 9), or a functional variant thereof having up to one, two, or three amino acid substitutions.

[0027] In one embodiment, the leader is a feline IL-2 sequence. In one embodiment, the IL-2 leader is the sequence shown in SEQ ID NO: 10:MYKIQLLSCIALTLILVTNS, or a functional variant thereof having up to one, two, or three amino acid substitutions.

[0028] 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.

[0029] 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.

[0030] The fusion protein further comprises a fusion domain. In one embodiment, the fusion domain is a feline IgG Fc fragment (e.g., IgG1a, IgG1b, or IgG2) 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 maintaining the function of GLP-1.

[0031] Two subclasses of the feline IgG constant domain, IgG1 and IgG2, have been described, with IgG1 being the dominant subclass (approximately 98%). The two alleles of the feline IGGH1 heavy chain gene (Cγ1a and Cγ1b) encode IgG heavy chain 1a and 1b proteins, respectively, with usage frequencies of approximately 62% and 36%, respectively. (Lu et al., Sequence analysis of feline immunoglobulin mRNAs and the development of a felinized monoclonal antibody specific) to feline panleukopenia virus, Sci Rep. Oct. 2017;7:12713, these are incorporated herein by reference.

[0032] As used herein, the Fc portion of an 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 constant regions of the antibody from both heavy chains, associated by non-covalent interactions and disulfide bonds. The Fc portion includes a hinge region and may extend to the c-terminus of the antibody through the CH2 and CH3 domains. The Fc portion may further include one or more glycosylation sites. In one embodiment, the fusion domain is feline IgG Fc. The Fc domain can be derived from any feline IgG, including feline IgG1a, feline IgG1b, or feline IgG2. In one embodiment, the feline IgG Fc is Sequence ID No. 11: In another embodiment, feline IgG Fc shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity with respect to sequence number 11.

[0033] In another embodiment, the fusion domain is feline albumin or a functional variant thereof. In one embodiment, feline albumin is Sequence ID No. 12: EAHQSEIAHRFNDLGEEHFRGLVLVAFSQYLQQCPFEDHVKLVNEVTEFAKGCVADQSAANCEKSLHELLGDKLCTVASLRDKYGEMADCCEKKEPERNECFLQHKDDNPGFGQLVTPEADAMCTAFHENEQRFLGKYLYEIARRHPYFYAPELLYYAEEYKGVFTECCEAADKAACLTPKVDALREKVLASSAKERLKC ASLQKFGERAFKAWSVARLSQKFPKAEFAEISKLVTDLAKIHKECCHGDLLECADDRADLAKYICENQDSISTKLKECCGKPVLEKSHCISEVERDELPADLPPLAVDFVEDKEVCKN YQEAKDVFLGTFLYEYSRRHPEYSVSLLLRLAKEYEATLEKCCATDDPPACYAHVFDEFKPLVEEPHNLVKTNCELFEKLGEYGFQNALLVRYTKKVPQVSTPTLVEVSRSLGKVGSK CCTHPEAERLSCAEDYLSVVLNRLCVLHEKTPVSERVTKCCTESLVNRRPCFSALQVDETYVPKEFSAETFTFHADLCTLPEAEKQIKKQSALVELLKHKPKATEEQLKTVMGDFGSFVDKCCAAEDKEACFAEEGPKLVAAAQAALA.In another embodiment, feline albumin shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity with respect to sequence number 12.

[0034] 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).

[0035] In one embodiment, the fusion protein comprises (a) a feline thrombin reader, (b) a DPP-IV resistant variant of GLP-1(7-37), a linker, and (c) feline IgG Fc. In one embodiment, the fusion protein has the sequence of SEQ ID NO: 14, or a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. Sequence ID 14 MAHIRGLWLPGCLALAALCSLVHSQHVFLAPQQALSLLQRVRRHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSARKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVY TAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG

[0036] In one embodiment, the sequence encoding the fusion protein is either SEQ ID NO: 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 atggctcacatcagaggactttggctgcctggctgtctggctctggctgctctgtgttctctggtgcacagccagcacgtgtttctggcccctcagcaggctctgtccctgctgcaaagagttagaaggcacggcgagg caccttcacctccgacgtgtctagctacctggaagaacaggccgccaaagagtttatcgcctggctggtcaaaggtggcggcggaggcggaggaagcggtggcggaggttcaggtggtggtggatctgccagaaagaccg accatcctcctggaccctaagccttgcgactgccctaagtgtcctccacctgagatgctcggcggacccagcatcttcatcttcccacctaagccaaaggacaccctgagcatcagcagaacccctgaagtgacctgcctggtcgttgatctgggccccgacgatagcgacgtgca gatcacttggtttgtggacaacaccaggtgtacacagccaagacaagccccagagaggaacagttcaacagcacctacagagtggtgtccgtgctgcccatcctgcaccaggattggctgaagggcaaagaattcaagtgcaaagtgaacagcaagagcctgccttctccaatc gagcggaccatcagcaaggccaagggacagcctcacgagcctcaggtgtacgtcctgcctcctgctcaagaggaactgagccggaacaaagtgtccgtgacctgtctgatcaagagctttcacccacctgatatcgccgtggaatgggagatcacaggccagcctgagcctgaga acaactaccggactacccctccacagctggactccgatggcacctacttcgtgtacagcaagctgagcgtggacagaagccactggcagcggggcaatacctacacctgttccgtgtctcacgaggccctgcacagccaccacaacagaagtctctctgacacagagccccggctga

[0037] In one embodiment, the fusion protein comprises (a) a feline thrombin reader, (b) a DPP-IV resistant variant of GLP-1(7-37), a linker, and (c) feline albumin. In one embodiment, the fusion protein has the sequence of SEQ ID NO: 16, or a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. Sequence ID 16 .

[0038] In one embodiment, the sequence encoding the fusion protein is either SEQ ID NO: 17 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 17 atggctcacatcagaggactttggctgcctggctgtctggctctggctgctctgtgttctctggtgcacagccagcacgtgtttctggcccctcagcaggctctgtccctgctgcaaaga

[0039] In another embodiment, the fusion protein is (a) feline thrombin leader, (b) feline G The fusion protein comprises two tandem copies of LP-1(7-37) or its DPP-IV resistance variant, a linker, and (c) feline albumin. In one embodiment, the fusion protein has the sequence of SEQ ID NO: 18, or a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. Sequence ID 18 MAHIRGLWLPGCLALAALCSLVHSQHVFLAPQQALSLLQRVRRHGEGTFTSDVSSYLEGQAAKEFIAWLVKGRHGEGTFTSDVSSYLEGQAAKEFIAWLVKGREAHQSEIAHRFNDLGEEHFRGLVLVAFSQYLQQCPFEDHVKLVNEVTEFAKGCVADQSAANCEKSLHE LLGDKLCTVASLRDKYGEMADCCEKKEPERNECFLQHKDDNPGFGQLVTPEADAMCTAFHENEQRFLGKYLYEIARRHPYFYAPELLYYAEEYKGVFTECCEAADKAACLTPKVDALREKVLASSAKERLKCASLQKFGERAFKAWSVARLSQKFPKAEFAEISKLVTDLAK IHKECCHGDLLECADDRADLAKYICENQDSISTKLKECCGKPVLEKSHCISEVERDELPADLPPLAVDFVEDKEVCKNYQEAKDVFLGTFLYEYSRRHPEYSVSLLLRLAKEYEATLEKCCATDDPPACYAHVFDEFKPLVEEPHNLVKTNCELFEKLGEYGFQNALLVRYT KKVPQVSTPTLVEVSRSLGKVGSKCCTHPEAERLSCAEDYLSVVLNRLCVLHEKTPVSERVTKCCTESLVNRRPCFSALQVDETYVPKEFSAETFTFHADLCTLPEAEKQIKKQSALVELLKHKPKATEEQLKTVMGDFGSFVDKCCAAEDKEACFAEEGPKLVAAAQAALA

[0040] In one embodiment, the fusion protein has the sequence of SEQ ID NO: 20, or a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. Sequence ID 20 MAHIRGLWLPGCLALAALCSLVHSQHVFLAPQQALSLLQRVRRHGEGTFTSDVSSYLEGQAAKEFIAWLVKGREAHQSEIAHRFNDLGEEHFRGLVLVAFSQYLQQCPFEDHVKLVNEVTEFAKGCVADQSAANCEKSLHELLGDKLCTVASLRDKYGEMADCC EKKEPERNECFLQHKDDNPGFGQLVTPEADAMCTAFHENEQRFLGKYLYEIARRPYFYAPELLYYAEEYKGVFTECCEAADKAACLTPKVDALREKVLASSAKERLKCASLQKFGERAFKAWSVARLSQKFPKAEFAEISKLVTDLAKIHKECCHGDLLECAD DRADLAKYICENQDSISTKLKECCGKPVLEKSHCISEVERDELPADLPPLAVDFVEDKEVCKNYQEAKDVFLGTFLYEYSRRHPEYSVSLLLRLAKEYEATLEKCCATDDPPACYAHVFDEFKPLVEEPHNLVKTNCELFEKLGEYGFQNALLVRYTKKVPQVS TPTLVEVSRSLGKVGSKCCTHPEAERLSCAEDYLSVVLNRLCVLHEKTPVSERVTKCCTESLVNRRPCFSALQVDETYVPKEFSAETFTFHADLCTLPEAEKQIKKQSALVELLKHKPKATEEQLKTVMGDFGSFVDKCCAAEDKEACFAEEGPKLVAAAQAALA

[0041] In one embodiment, the sequence encoding the fusion protein is either SEQ ID NO: 19 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 19 agaggcctgcttcgctgaagagggccctaagttggttgccgctgctcaagctgccctggcctgataa

[0042] If a leader sequence, GLP-1 receptor agonist, or variant or fragment of a fusion domain is desired, the coding sequences for these peptides can be generated using site-directed mutagenesis of wild-type nucleic acid sequences. Alternatively or additionally, the amino acid sequence can 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, namely cats.

[0043] 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.

[0044] 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.

[0045] 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. In yet another embodiment, this includes any nucleic acid sequence encoding the GLP-1 fusion protein of SEQ ID NO: 16. In yet another embodiment, this includes any nucleic acid sequence encoding the GLP-1 fusion protein of SEQ ID NO: 18. In yet another embodiment, this includes any nucleic acid sequence encoding the GLP-1 fusion protein of SEQ ID NO: 20.

[0046] In certain embodiments of the viral vectors 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 the view 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, in which an expression cassette packaged therein is delivered to target cells. The AAV capsid is adjacent to the inverted terminal repeat (ITR) of the AAV (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. As identified above, various AAVs may be selected as the source of the capsid for the AAV viral vector. 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. See also WO2003 / 042397 (AAV7 and other monkey AAVs), U.S. Patent Nos. 7,790449 and 7,282199 (AAV8), WO2005 / 033321 and U.S. 7,906,111 (AAV9), and WO2006 / 110689, as well as WO2003 / 042397 (rh10), 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, publication date November 5, 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 August 13, 2021, U.S. Provisional Patent Application 63 / 065,616 filed August 14, 2020, and U.S. Provisional Patent Application 63 / 109,734 filed November 4, 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.

[0047] 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 is derived from any described or known AAV capsid sequence up to about 1 This includes variants that may include 0% variation. 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 an AAV capsid, the comparison may be made across any of the variable proteins (e.g., vp1, vp2, or vp3).

[0048] 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.

[0049] In certain embodiments, a novel isolated AAVrh91 capsid is provided. The nucleic acid sequence encoding the AAVrh91 capsid is provided in SEQ ID NO: 24, and the encoded amino acid sequence is provided in SEQ ID NO: 26. An rAAV comprising at least one of vp1, vp2, and vp3 of AAVrh91 (SEQ ID NO: 26) is provided herein. An rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVrh91 (SEQ ID NO: 24) is also provided herein. The nucleic acid sequence encoding the amino acid sequence of AAVrh91 is provided in SEQ ID NO: 24, and the encoded amino acid sequence is provided in SEQ ID NO: 26. An rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVrh91eng (SEQ ID NO: 25) is also provided herein. In certain embodiments, vp1, vp2, and / or vp3 are the full-length capsid protein of AAVrh91 (SEQ ID NO: 26). 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).

[0050] In certain embodiments, the AAVrh91 capsid is a heterogeneous aggregate 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: 26 from 1 to 736, a vp1 protein produced from SEQ ID NO: 24, or a vp1 protein produced from a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 24 encoding the predicted amino acid sequence of SEQ ID NO: 26 from 1 to 736, a vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about 138 to 736 amino acids of SEQ ID NO: 26, a vp2 protein produced from a sequence containing at least nucleotides 412 to 2208 of SEQ ID NO: 24, 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: 24 encoding the predicted amino acid sequence of at least about 138 to 736 amino acids of SEQ ID NO: 26 AAVrh91 capsid protein comprising a heterogeneous assembly of AAVrh91 vp3 protein selected from a heterogeneous assembly of vp2 protein, a vp3 protein produced from expression of a nucleic acid sequence encoding the predicted amino acid sequence of at least approximately 203-736 amino acids of SEQ ID NO: 26, a vp3 protein produced from a sequence containing at least nucleotides 607-2208 of SEQ ID NO: 24, or a vp3 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 607-2208 of SEQ ID NO: 24 encoding the predicted amino acid sequence of at least approximately 203-736 amino acids of SEQ ID NO: 26, and / or (2) a heterogeneous assembly of vp1 protein which is the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 26, a heterogeneous assembly of vp2 protein which is the product of a nucleic acid sequence encoding the amino acid sequence of at least approximately 138-736 amino acids of SEQ ID NO: 26, and a few of SEQ ID NO: 26 (B) A heterogeneous assembly of vp3 protein, which is the product of a nucleic acid sequence encoding at least approximately 203 to 736 amino acids, wherein the vp1, vp2 and vp3 proteins contain a subassembly having amino acid modifications including at least two highly deamidated asparagine (N) in the asparagine-glycine pair of SEQ ID NO: 26, and optionally further containing a subassembly containing other deamidated amino acids, wherein deamidation results in a change in amino acids; and (B) a vector genome in an AAVrh91 capsid, wherein the vector genome contains a nucleic acid molecule containing an AAV inverted terminal repeat sequence, and a non-AAV nucleic acid sequence encoding a product that is operably linked to a sequence that directs the expression of the product in a host cell.

[0051] In certain embodiments, the AAVrh91 capsid is a heterogeneous aggregate 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: 26 from 1 to 736, a vp1 protein produced from SEQ ID NO: 25, or a vp1 protein produced from a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 25 encoding the predicted amino acid sequence of SEQ ID NO: 26 from 1 to 736, a vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about 138 to 736 amino acids of SEQ ID NO: 26, a vp2 protein produced from a sequence containing at least nucleotides 412 to 2208 of SEQ ID NO: 25, 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: 25 encoding the predicted amino acid sequence of at least about 138 to 736 amino acids of SEQ ID NO: 26 AAVrh91 is selected from a heterogeneous assembly of vp2 protein, vp3 protein produced from expression of a nucleic acid sequence encoding the predicted amino acid sequence of at least approximately 203-736 amino acids of SEQ ID NO: 26, vp3 protein produced from a sequence containing at least nucleotides 607-2208 of SEQ ID NO: 25, 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: 25, which encodes the predicted amino acid sequence of at least approximately 203-736 amino acids of SEQ ID NO: 26.AAVrh91 capsid protein containing a heterogeneous aggregate of vp3 protein, and / or (2) a heterogeneous aggregate of vp1 protein which is the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 26, a heterogeneous aggregate of vp2 protein which is the product of a nucleic acid sequence encoding the amino acid sequence of at least about 138 to 736 amino acids of SEQ ID NO: 26, and a heterogeneous aggregate of vp3 protein which is the product of a nucleic acid sequence encoding at least about 203 to 736 amino acids of SEQ ID NO: 26, wherein vp1, vp2 and vp3 proteins are asparagine- (B) A vector genome in an AAVrh91 capsid, characterized by one or more of the following: (B) a vector genome comprising 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.

[0052] 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: 26, 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: 26, high levels of deamidation are observed at N57, N383, and / or N512 of 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 phosphorylated (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 oxidized (e.g., For example, it may have other modifications including one or more of approximately W22, M211, W247, M403, M435, M471, W478, W503, approximately M537, approximately M541, approximately M559, approximately M599, M635, and / or W695. Optionally, W may be oxidized to kynurenine. [Table 1]

[0053] 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 SEQ ID NO: 26.

[0054] In certain embodiments, the AAVrh91 capsid comprises a heterogeneous population of vp1 proteins, which are products of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 26; a heterogeneous population of vp2 proteins, which are products of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 26 at least from amino acids 138 to 736; and a heterogeneous population of vp3 proteins, which are products of a nucleic acid sequence encoding at least from amino acids 203 to 736 of SEQ ID NO: 26.

[0055] 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.

[0056] 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 in the AAV capsid, the vector genome comprising an AAV inverted terminal repeat (ITR), the coding sequence for the feline GLP-1 receptor agonist of sequence number 14, and a regulatory sequence that directs the expression of the feline GLP-1 receptor agonist.

[0057] 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 trans See "Duction independently of DNA synthesis," Gene Therapy, (August 2001), Vol. 8, Number 16, Pages 1248–1254. Self-complementary AAVs are described, for example, in U.S. Patents 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated herein by reference in whole.

[0058] 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. 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).

[0059] 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.

[0060] 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.

[0061] As used herein, “expression cassette” refers to a nucleic acid molecule comprising a biologically useful nucleic acid sequence and regulatory sequences operably ligated to it, which direct or regulate the transcription, translation, and / or expression of the nucleic acid sequence (e.g., a gene cDNA encoding a protein, enzyme, or other useful gene product, such as mRNA) and the gene product. As used herein, “operably ligated” 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, 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, and sequences that stabilize cytoplasmic mRNA, such as woodchuck hepatitis virus The expression cassette includes one or more of the following: a WHP (Whole Wave Programme) post-translational regulatory element (WPRE) and a TATA signal. Among other elements, the expression cassette may include one or more upstream (5'~) regulatory sequences of the gene sequence, such as promoters, enhancers, and introns, and one or more enhancers or downstream (3'~) regulatory sequences of the 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 the target cell. Typically, such expression cassettes can be used to generate a viral vector and include a coding sequence for a gene product described herein, adjacent to the packaging signal of the viral genome, and other expression regulatory sequences, such as those described herein. In certain embodiments, the vector genome may include two or more expression cassettes.

[0062] 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 the 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 contains the GLP-1 construct sequence described herein, adjacent to the packaging signal of the viral 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.

[0063] Expression cassettes typically contain a promoter sequence as part of the expression regulatory sequence. In one embodiment, a liver-specific promoter thyroxine-binding globulin (TBG) is used. Plasmids and vectors described herein use the CB7 promoter. CB7 is a chicken β-actin promoter having a cytomegalovirus enhancer element. 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, but not limited to, 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)), or TTR minimal enhancer / promoter, α-antitrypsin promoter, or liver-specific promoter (LSP) (Wu et al. Mol Ther. 16:280-289 (2008)). Other promoters may be used, such as viral promoters, constitutive promoters, modulo promoters [see, for example, WO2011 / 126808 and WO2013 / 04943], or promoters that respond to physiological cues, and may be utilized in the vectors described herein.

[0064] In addition to promoters, expression cassettes and / or vectors may include 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 encoded products. Examples of suitable poly-A sequences include, for example, SV40, or bovine growth hormone (bGH), etc. Examples of suitable enhancers include growth hormone (hGH), SV40, rabbit β-globin (rabbit globin polyA; also known as RGB), modified RGB (mRGB), and TK polyA. Examples of suitable enhancers include, for example, α-fetoprotein enhancers, TTR minimal promoter / enhancer, and LSP (TH-binding globulin promoter / α1-microglobulin / bicin enhancer). In one embodiment, polyA is rabbit globin polyA.

[0065] 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.

[0066] In one embodiment, an rAAV is provided comprising a 5'ITR, a CB7 promoter, a chicken β-actin intron, the coding sequence of the fusion protein of SEQ ID NO: 14, rabbit globin polyA, and a 3'ITR. In another embodiment, an rAAV is provided comprising a 5'ITR, a CB7 promoter, a chicken β-actin intron, the coding sequence of the fusion protein of SEQ ID NO: 16, rabbit globin polyA, and a 3'ITR. In yet another embodiment, an rAAV is provided comprising a 5'ITR, a CB7 promoter, a chicken β-actin intron, the coding sequence of the fusion protein of SEQ ID NO: 18, rabbit globin polyA, and a 3'ITR. In yet another embodiment, an rAAV is provided comprising a 5'ITR, a CB7 promoter, a chicken β-actin intron, the coding sequence of the fusion protein of SEQ ID NO: 20, rabbit globin polyA, and a 3'ITR.

[0067] 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 degradation sites (trs), is described. In other embodiments, full-length AAV 5' and 3' ITRs are used.

[0068] 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.

[0069] A method for generating and isolating an AAV virus vector suitable for delivery to a target is: This is 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 a baculovirus-derived vector. For a review of these production systems, see, for example, Zhang et al., 2009, “Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production,” Human Gene Therapy 20:922–929 (the contents of each 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. See, for example, Grieger & Samulski, 2005, “Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications,” Adv. Biochem.Engin / Biotechnol. 99:119-145, Buning et al., 2008, “Recent developments in adeno-associated virus vector technology,” J. Gene Med. 10:717-733, and the references cited below, each of which is incorporated herein by reference in its entirety. The methods used to construct any embodiment of the present invention are known to technicians in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. For example, Green and Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold. See Spring Harbor, NY (2012). Similarly, methods for producing rAAV virions are well known, and the selection of a preferred 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.

[0070] The rAAV described herein comprises a selected capsid having an internally packaged vector genome. The vector genome (or rAAV genome) directs the insertion of 5' and 3' AAV inverted terminal repeats (ITRs), polynucleotide sequences encoding the fusion protein, and polynucleotide sequences encoding the fusion protein into the host cell genome. Includes a regulatory sequence. In one embodiment, the vector genome is the sequence shown in SEQ ID NO: 21, or a sequence that shares at least 90%, at least 95%, or at least 99% identity with respect to it. In one embodiment, the vector genome is the sequence shown in SEQ ID NO: 22, or a sequence that shares at least 90%, at least 95%, or at least 99% identity with respect to it. In one embodiment, the vector genome is the sequence shown in SEQ ID NO: 23, or a sequence that shares at least 90%, at least 95%, or at least 99% identity with respect to it. In one embodiment, an expression cassette is provided having the sequence nt199-3125 of SEQ ID NO: 21, or a sequence that shares at least 90%, at least 95%, or at least 99% identity with respect to it. In one embodiment, an expression cassette is provided having the sequence nt199-4194 of SEQ ID NO: 22, or a sequence that shares at least 90%, at least 95%, or at least 99% identity with respect to it. In one embodiment, an expression cassette is provided which has the sequence nt199~4143 of sequence number 23, or a sequence that shares at least 90%, at least 95%, or at least 99% identity with respect to it.

[0071] 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 from AAV2, AAV from a different source than the capsid, or other than a full-length ITR 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. The nucleic acid coding sequence is operably ligated to a regulatory element in a manner that enables transcription, translation, and / or expression of the transgene in cells of the target tissue. Preferred components of the vector genome are discussed in more detail herein. In one embodiment, the “vector genome” comprises, at a minimum, a vector-specific sequence and a nucleic acid encoding a GLP-1 construct operably ligated to a regulatory element (which directs its expression in the target sequence) from at least 5' to 3', wherein 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 to AAV and certain other parvovirus capsids.

[0072] The vector's AAV sequence 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 long. Preferably, the entire sequence substantially encoding the ITR is used intramolecularly, although some minor modifications to these sequences are permissible. The ability to modify these ITR sequences is within the scope of 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 a molecule used in the present invention is a “cis-acting” plasmid containing a transgene, wherein the selected transgene sequence and associated regulatory elements are adjacent to the 5' and 3' AAV ITR sequences. In one embodiment, the ITR is 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 derived from other AAV sources may be selected. A shortened version of the 5' ITR, called a ΔITR, is described, which has a deleted D sequence and terminal degradation sites (trs). In certain embodiments, the vector genome contains a 130-base pair shortened AAV2 ITR with a deleted outer A element. While we do not want 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. If the ITR source is AAV2 and the AAV capsid is from a different AAV source, the resulting vector may be called a pseudotype. However, other configurations of these elements may also be preferred.

[0073] 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 and be used any virus suitable for gene therapy. Preferably, if one of these other vectors is produced, it is produced as a replication-deficient viral vector.

[0074] 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 but retains the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be “gutless” containing only the 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 cannot occur without the presence of the viral enzymes required for replication.

[0075] Compositions comprising the viral vector constructs described herein are also provided. The pharmaceutical compositions described herein are designed to be delivered to feline 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.

[0076] 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. Next, a primer / proxy targeting a specific region of the viral genome (usually the poly-A signaling pathway) is used. Quantify the released genome by real-time PCR using a bus set. Another suitable 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]. 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 9 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 dose is about 1.0×10 10 GC to about 3.0×10 13 GC for an average feline subject of about 5 - 10 kg. In another embodiment, the dose is about 1×10 9 GC. For example, the dose of AAV virus can be about 1×10 10 GC, 1×10 11 GC, about 5×10 11 GC, about 1×10 12 GC, about 5×10 12 GC, or about 1×10 13 GC. In another embodiment, the dose is about 1.0×10 9 GC / kg to about 3.0×10 13 GC / kg for a feline subject. In another embodiment, the dose is about 1×10 9 GC / kg. For example, the dose of AAV virus can be about 1×10 10 GC / kg, 1×10 11 GC / kg, about 5×10 11 GC / kg, about 1×10 12 GC / kg, about 5×10 12 GC / kg, or about 1×10 13It may be GC / kg. In one embodiment, the construct may be delivered to a veterinary subject in a volume of 1 μL to approximately 100 mL. For a discussion of good practices regarding the administration of substances to various veterinary animals, see, for example, Diehl et al, J. Applied. See Toxicology, 21:15-23 (2001). This document is incorporated herein by reference. Where used herein, the terms “dosage” or “volume” may refer to the total dose or total amount delivered to a subject in the course of treatment, or the amount delivered in a single unit (or multiple units or divided doses).

[0077] 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 the desired subject, including cats. Suitable carriers 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 with various buffer solutions (e.g., phosphate-buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The choice of carrier is not limiting to the present invention.

[0078] In another embodiment, the composition comprises a carrier, a diluent, an excipient and / or an adjuvant.

[0079] In certain embodiments, for administration to human patients, rAAV is preferably suspended in an aqueous solution containing saline, a surfactant, and a pharmaceutically and / or physiologically compatible salt, or a 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 about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, or about 7.8. In certain embodiments, for 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 intravenous delivery, pH values ​​of approximately 6.8–7.2 may be desirable. However, other pH values, and these, are available over a wider range. A subrange of this may be selected for other delivery routes.

[0080] 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.

[0081] 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, and vesicles may be used to introduce the compositions of the present invention into suitable host cells or target cells. In particular, rAAV vector delivery transgenes may be formulated for delivery encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles, etc.

[0082] 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 diluted for administration to a subject. In yet another embodiment, the composition may be lyophilized and reconstituted at the time of administration.

[0083] Suitable surfactants, or combinations 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), polyoxy-10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol may be selected. In one embodiment, the formulation contains a poloxamer. These copolymers are generally named with the letter "P" (in the case of poloxamers) 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.

[0084] The dose of the vector depends primarily on factors such as the condition being treated, the age, weight, and health status of the feline patient, and therefore varies among patients. For example, the therapeutically effective human dose of viral vector is generally in the range of about 25 to about 1000 microliters to about 100 mL, (to treat an average 4.5 kg cat subject) about 1 × 10⁶ 9 ~1 × 10 16 The concentration of the genomic viral vector (including all integers or fractional quantities within that range). In a particular embodiment, the cat patient has a concentration of approximately 1 × 10⁻¹⁶, including all integers or fractional quantities within that range. 9 GC / Cat ~ approx. 1 x 10 12 GC / Cat, or approximately 1 x 10 10GC / Cat ~ approx. 1 x 10 11 It is administered to cats (GC).

[0085] The compositions of the present invention can be delivered in volumes ranging 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 about 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.

[0086] 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 one vector genome (vg / mL) (also called genome copies / mL (GC / mL)).

[0087] In one embodiment, the dosage of rAAV in the composition is approximately 1.0 × 10⁶ of body weight. 9 GC / kg ~ approx. 3.0×10 13 The value is GC / kg. In one embodiment, the dose is approximately 1 × 10⁻⁶. 11 The value is GC / kg. In one embodiment, the dose is approximately 1.0 × 10⁻⁶. 13 The value is GC / kg. In one embodiment, the dose is approximately 1.0 × 10⁻⁶. 12 The value is GC / kg. In one embodiment, the dose is approximately 5.0 × 10⁻⁶. 12 The value is GC / kg. All scopes described herein include endpoints.

[0088] 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 one embodiment, 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.

[0089] 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.

[0090] The viral vectors and other constructs described herein are for delivering GLP-1 fusion protein constructs to subjects requiring them, for supplying GLP-1 with an extended half-life to subjects, and / or for treating type 1 diabetes, type 2 diabetes, or metabolic syndrome in subjects. It may be used to prepare a pharmacopoeia for the treatment of diabetes. In another embodiment, a method for treating diabetes is provided. This method comprises administering the composition described herein to a feline subject in need. In one embodiment, the composition comprises a viral vector containing the GLP-1 fusion protein expression cassette described herein.

[0091] 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 (T2DM), 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.

[0092] As used herein, the term “remission” refers to the ability of a cat to discontinue insulin treatment when it no longer shows clinical signs of diabetes and has normal blood glucose levels.

[0093] In another embodiment, a method for treating T2DM in cats is provided. The method comprises administering a viral vector comprising a nucleic acid molecule containing a sequence encoding a fusion protein described herein.

[0094] In another embodiment, a method for treating metabolic disorders in cats is provided. The method comprises administering a composition described herein to a cat 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 syndrome.

[0095] In another embodiment, a method for reducing body weight in a feline 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.

[0096] 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 comprising other antidiabetic drugs or protein-based therapies (e.g., GLP-1 analogs, insulin, oral antihyperglycemic agents (sulfonylurea, biguanides, thiazolidinediones, and alpha-glucoididase 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 other embodiments, the combination therapy is administered first.

[0097] In one embodiment, the composition is administered in combination with an effective amount of insulin. Various commercially available insulin products are known in the art, including, but not limited to, recombinant protamine zinc human insulin (ProZinc®), porcine insulin zinc suspension (Vetsulin®), and insulin glargine (Lantus®). In some embodiments, the combination of rAAV and insulin described herein is used. The combination therapy 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 amount of insulin required by the subject. For example, the subject may be treated with insulin or other therapies, which the treating physician may continue during the administration of the AAV vector. Such insulin or other combination therapies may then be continued, reduced, or discontinued as needed.

[0098] In one embodiment, the expression cassette, vector genome, composition comprising rAAV, 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 goal. 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 dose” may be determined based on an animal model rather than a feline patient. In another embodiment, the therapeutic goal is remission of the metabolic disease of the subject.

[0099] In certain embodiments, an effective dose and / or method induces the expression of the fusion protein in the serum of the subject for at least 3 months, at least 6 months, or at least 12 months. In certain embodiments, an effective dose and / or method induces the expression of the fusion protein in the subject at serum concentrations of at least 3,000 picomoles (pM), at least 5,000 pM, at least 10,000 pM, at least 25,000 pM, or at least 50,000 pM for at least 3 months, at least 6 months, or at least 12 months. In other embodiments, the effective dose and / or method induces fusion protein expression in a subject at serum concentrations of 3,000 picomoles (pM) to 200,000 pM, 5,000 picomoles (pM) to 200,000 pM, 10,000 picomoles (pM) to 200,000 pM, 25,000 picomoles (pM) to 200,000 pM, or 50,000 picomoles (pM) to 200,000 pM for 3 to 12 months, 6 to 12 months, or 12 months. In specific embodiments, the effective dose and / or method induces fusion protein expression in a subject at a therapeutically effective concentration for at least 3 months, at least 6 months, or at least 12 months.

[0100] In other embodiments, the therapeutic goal is the reduction of serum fructosamine. In certain embodiments, the effective dose and / or method is effective in reducing the serum fructosamine of the target by about 6%. In another embodiment, the effective dose and / or method is effective in reducing the serum fructosamine of the target by 5% to 10%. In yet another embodiment, the effective dose and / or method is effective in reducing the serum fructosamine of the target by about 10%. In yet another embodiment, the effective dose and / or method is effective in reducing the serum fructosamine of the target by 10% to 20%. Other ranges and integers within the enumerated range are intended. As used herein, the term “heterogeneous” or any grammatical variation thereof, when used to refer to vp capsid proteins, refers to a group of non-identical elements having vp1, vp2, or vp3 monomers (proteins) having different modified amino acid sequences, for example. Sequence ID No. 20 provides the encoded amino acid sequence of the AAVrh91 vp1 protein. The term "heterogeneous" as used in relation to the 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 that have the predicted amino acid residue modifications. These subpopulations contain at least certain deamidated asparagine (N or Asn) residues. For example, certain subpopulations contain at least one or two asparagine-glycine pairs. The molecule contains three or four highly deamidated asparagine (N) positions, and optionally further contains other deamidated amino acids, where deamidation results in amino acid changes and other optional modifications.

[0101] 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 (1)vp1 protein in an assembled AAV capsid and fewer than all vp1 proteins. 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.

[0102] As used herein, a “stock” of rAAVs refers to a population of rAAVs. Despite the 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.

[0103] 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.

[0104] 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 is desired. However, identity between smaller fragments of, for example, at least about 9 nucleotides, usually at least about 20–24 nucleotides, at least about 28–32 nucleotides, or at least about 36 or more 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 is also 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 in GCG version 6.1. Fasta® provides best-in-class alignment of overlapping regions and sequence identity percentage between query and search sequences. For example, percentage sequence identity between nucleic acid sequences can be calculated using Fasta® with its default parameters (word size 6 and NOPAM factor for scoring matrix) provided in GCG Version 6.1, which are incorporated herein for reference. It is possible to make a decision.

[0105] 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.

[0106] 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.

[0107] 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 modify 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. See, for example, JDThomson et al, Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments," 27(13):2682-2690 (1999).

[0108] 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.

[0109] 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 the specification are presented using the language “comprising,” in other circumstances, the relevant embodiments are also intended to be interpreted and described using the language “consisting of” or “essentially consisting of.”

[0110] 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.

[0111] 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.

[0112] 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.

[0113] As used herein, “disease,” “disorder,” and “condition” are used interchangeably to describe an abnormal condition in the subject.

[0114] 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.

[0115] 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. [Examples]

[0116] The following examples are for illustrative purposes only and are not intended to limit the present invention.

[0117] Example 1 - Construction of GLP-1 vector 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. 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 a chicken β-actin promoter with a CMV enhancer. The expression construct was adjacent to the AAV2 ITR. The feline thrombin-durlaglutide amino acid sequence is shown in SEQ ID NO: 14. The feline thrombin-albiglutide amino acid sequence is shown in SEQ ID NO: 18. The feline GLP-1-SA amino acid sequence is shown in SEQ ID NO: 16.

[0118] Example 2 - In vitro expression Purified plasmids for the constructs were transfected into triple wells of a 6-well plate of 90% confluent HEK293 cells using lipofectamine 2000, according to the manufacturer's instructions. Supernatants were collected 48 hours after transfection, and active GLP-1 was measured using an ELISA specific for active GLP-1 (7-36). Expression of the three constructs is shown in Figure 2A. GLP-1 activity in the culture supernatant was measured using a cell-based GLP-1 activity assay (GeneBLAzer GLP1R-CRE-bla). The results were measured by a CHO-K1 cell-based assay (Figure 2B). The feline dulaglutide construct showed the best performance in both expression and activity assays.

[0119] 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.

[0120] AAVrh91.CB7.Nekodurlaglutide(hIL2).rBG with human IL2 signaling AAVrh91.CB7.Necodurlaglutide(feIL2).rBG with feline IL2 signaling AAVrh91.CB7.Nekodurlaglutide(feTrb).rBG AAVrh91.CB7.NekoGLP1-SA(feTrb).rBG AAVrh91.CB7.Feline Albiglutide (feTrb).rBG Rag1KO female mice were injected with a vector (1×10) via IM injection. 11The mice were treated with intravenous injection of GC (Glycine-Clamped / Glycine) cells. Serum was continuously collected by separating whole blood in a serum isolation tube containing 5 microliters of the DPP-IV inhibitor (Millipore), and assayed for the active GLP-1 expression and activity described above. Serum active GLP-1 concentrations are shown in Figure 3A, and activity is shown in Figure 3B. These data indicate that high levels of GLP-1 agonists can be expressed in vivo using AAV delivery technology. In addition, the use of a thrombin prepropeptide reader appears to provide a significant advantage for the in vivo expression of feline GLP-1-Fc.

[0121] Example 4 - Expression and immunogenicity study of AAV feline GLP-1-Fc and feline GLP-1-SA in cats Cats (n=4 / cohort), 5 × 10 11 GC / kg, 1 × 10 11 GC / Cat, 1x10 10 GC / Cat, or 1x10 9 Cats were treated with either GC / cat AAV-feline GLP-1-Fc (AAVrh91, CB7, CI, or feline durlaglutide (feTrbss)) and delivered intramuscularly (IM or IM). Body weight (Figure 4A) and GLP-1 expression (Figures 4B and C) were recorded. GLP-1 expression was evaluated using an ELISA specific to the active form of GLP-1(7-36).

[0122] Weight loss (average of about 10%) was observed during the first four weeks. Although the animals' weight began to increase during the study period, by week 18 they were on average 5% lighter than at the start of the study.

[0123] Long-term expression of feline GLP-1-Fc was suppressed by high dose (5 × 10⁻¹⁰ 11 The GC / kg group was evaluated over 16 weeks (Figure 4B). This data is approximately 2.6 × 10⁻⁶. 5 At average pM concentrations, the study showed sustained expression of feline GLP-1-Fc, which had stalled around 6 weeks.

[0124] By day 28 (D28), the mean expression level in each cohort was 1.8 × 10⁻⁶. 5 pM(5×1011 GC / kg), 6.5 × 10 4 pM(1×10 11 GC / Cat), and 3.1×10 3 pM(1×10 10 It was GC / Cat). 1 x 10 9 Feline GLP-1-Fc levels in the GC / feline cohort were below the assay's sensitivity level. Mean feline GLP-Fc levels in the top three dose cohorts were above the expected therapeutic dose.

[0125] In a separate cohort (n=4) of cats, 1 × 10 11 GC / feline feline GLP-1-SA [AAVrh91.CB7.CI.feline durlaglutide-SA(feTrb).rBG(p5432)] was administered (Figure 4D). Good protein expression was indicated by mean serum levels of 2.5 × 10⁶. 3 It was observed on day 28, when the temperature exceeded pM.

[0126] The activity of GLP-1 agonists in animal serum was evaluated using a cell-based GLP-1 activity assay (GeneBLAzer GLP-1R-CRE-bla CHO-K1 cell-based assay) (Figure 4E). These data demonstrate a clear dose-response of GLP-1 activity in the AAV-feline GLP-Fc cohort, and similar activity was observed at an equivalent dose of AAV-feline GLP-1-SA.

[0127] Example 5 - Tolerance development and physiological benefits after administration of AAV feline GLP-1-SA in cats with or without insulin. research design This study evaluated the efficacy and safety of AAV feline GLP-1-SA (a recombinant adeno-associated virus vector serotype rh91 containing a DNA transgene expressing feline-specific GLP-1 serum albumin fusion protein) administered as a single intramuscular (IM) injection, regardless of whether initial insulin therapy was administered, for the management of diabetes mellitus (DM) in cats.

[0128] Ten cats were randomly assigned to either arm 1 or arm 2. On day 0, the cats in both arms received 1 × 10⁶ AAV cat GLP-1-SA. 11 The animals received gene copies / intramuscular injections. Cats in Arm 2 also received daily insulin injections starting from day 0. Cats in Arm 1 were permitted to start insulin if necessary for adequate diabetes control. Researchers were permitted to use preferred brands of insulin [ProZinc (n=2), Vetsulin (n=2), or glargine human insulin (n=6)].

[0129] Registered cats had diabetes mellitus, but were either treatment-naïve or previously treated, and were not currently taking insulin or other antidiabetic medications. Inclusion criteria included a) at least one clinical sign consistent with DM [polyuria (PU), polystoliasis (PD), or unintentional weight loss despite good appetite], b) fasting blood glucose >270 mg / dL, c) glucosuria, and d) serum fructosamine >400 μmol / L.

[0130] Sustained expression of feline GLP-1-SA Prior to the clinical trial, the serum concentration of feline GLP-1-SA required for therapeutic benefit in cats was estimated based on the known human value of recombinant GLP-1-Fc fusion protein, dulaglutide (Trulicity®), which was 800 pM. A 20% increase in the target concentration was applied to account for the decreased potency of GLP-1-SA in comparative trials of GLP-1-SA and GLP-1-Fc (data not shown). The resulting value of 1000 pM was multiplied by 3 to account for the possibility that cats would be less sensitive to GLP-1 than humans. Therefore, the selected target of 3000 pM represents a conservative estimate of the minimum therapeutically effective concentration of feline GLP-1-Fc in the serum of the target cats.

[0131] As shown in Figure 9, all treated cats expressed feline GLP-1-SA protein above the targeted therapeutic threshold of 3,000 picomoles (pM) throughout the 182-day study period.

[0132] Considering that the expression level remained nearly constant, continuous expression can be expected. Approximately half of all cats expressed feline GLP1-SA at sustained levels of approximately 50,000 pM or higher.

[0133] The data demonstrate that all treated cats had expression levels above the therapeutic threshold for at least 180 days, and eight cats expressed feline GLP-1-SA at levels at least five times above the threshold.

[0134] Decreased fructosamine levels in AAV cats with or without insulin-induced GLP-1-SA Fructosamine is a glycated serum protein used by veterinarians to assess long-term diabetes control in cats, similar to the use of HbA1c as a marker in humans. As shown in Table 1, the cats in both test arms were AAV feline GLP-1- Fructosamine levels decreased in response to SA treatment. Notably, on day 14 (D14), the mean fructosamine level in arm 1 decreased without the administration of insulin. This decrease of at least approximately 9% in fructosamine levels on day 14 was attributed to AAV feline GLP-1-SA alone. In both study groups, a sustained decrease in fructosamine concentrations was observed until day 70 (D70). [Table 2]

[0135] To compare the efficacy of AAV feline GLP-1-SA in combination with insulin versus insulin monotherapy, the results were compared with publicly available data for two brands of insulin products, ProZinc® and Vetsulin® (available at animaldrugsatfda.fda.gov). Data for study subjects are reported as days since the start of insulin administration (day 0 in Arm 2, various days in Arm 1). As shown in Table 2, at 14 days, subjects previously injected with AAV feline GLP-1-SA had lower fructosamine levels than cats treated with insulin monotherapy (based on historical data for two veterinary-approved insulins, ProZinc® and Vetsulin®). In summary, the fructosamine data demonstrate that AAV feline GLP-1-SA is not only effective on its own but also improves overall diabetes control with insulin compared to insulin monotherapy. [Table 3]

[0136] Blood glucose reduction in AAV cats with or without insulin using GLP-1-SA. Blood glucose levels were direct measurements of diabetic controls and were measured at each visit. On visits on days 42 and 84, insulin was withheld 12 hours prior to the first measurement of the complete 9-hour blood glucose curve. On all other days, if the cat was administered insulin... These were single measurements taken within one hour after morning insulin administration. Table 3 shows the mean blood glucose change from D0 for each animal. AAV cat GLP-1-SA causes a decrease in blood glucose levels, either without insulin (arm 1, D14, and D28) or with insulin (other values). [Table 4]

[0137] Reduced insulin dose requirements due to AAV feline GLP-1-SA Remission is defined as the ability to discontinue insulin treatment when a cat no longer shows clinical signs of diabetes and has normal blood glucose levels. One of the subjects in Arm 1 entered remission on day 70 and remained in remission until the end of the study. Another subject was able to remove insulin from day 54 to day 84 and was therefore in remission for one month. Two subjects in Arm 2 completed the study with very low doses of insulin, 1 IU twice daily, which suggested that control is possible at doses far lower than typical doses, although close to remission. The mean insulin doses at actual study days 30, 42, and 60 are listed in Table 4 below and compared simultaneously with the historical data for Vetsulin® and ProZinc® in Table 5.

[0138] Table 5, comparing study data with historical data, shows that treatment with AAV feline GLP-1-SA can reduce the insulin dose to below the average dose in cats treated with insulin alone without AAV feline GLP-1-SA. [Table 5] [Table 6]

[0139] Example 7 - Development of tolerance after administration of AAV feline GLP-1-Fc in cats We conducted a study to evaluate the efficacy and safety of AAV feline GLP-1-Fc (a recombinant adeno-associated virus vector serotype rh91 containing a DNA transgene expressing a feline-specific GLP-1Fc receptor domain fusion protein) administered as a single intramuscular (IM) injection.

[0140] Each of the four cats received a 5x10 AAV cat GLP-1-Fc. 11 The animals received a gene copy via intramuscular injection. Transgene expression was measured in plasma every 14 days.

[0141] Prior to clinical trials, the inventors estimated the serum concentration of feline GLP-1-Fc required for therapeutic benefit in cats based on known human values ​​of 800 pM of the recombinant GLP-1-Fc fusion protein dulaglutide (trademark name Trulicity®). This value of 800 pM was multiplied by 3 to account for the possibility that cats may be less sensitive to GLP-1 than humans. Therefore, the inventors' selected target of 2400 pM represents a conservative estimate of the minimum therapeutically effective concentration of feline GLP-1-Fc in the serum of the target cats.

[0142] As shown in Figure 10, three of the four animals expressed high levels of feline GLP-1-Fc for more than 330 days, far exceeding the therapeutic efficacy threshold of 2,400 pM.

[0143] One animal acquired antibodies against the transgenic protein, as shown in Figure 11, which is consistent with the low levels of protein expression observed in that animal.

[0144] These data demonstrate that a single injection of AAV feline GLP-1-Fc induces sustained expression of the transgenic protein at high levels.

[0145] Example 8 - Development of tolerance to AAV feline GLP-1-SA after administration in healthy cats We conducted a study to evaluate the efficacy and safety of AAV feline GLP-1-SA (a recombinant adeno-associated virus vector serotype rh91 containing a DNA transgene expressing feline-specific GLP-1 serum albumin fusion protein) administered as a single intramuscular (IM) injection.

[0146] Each of the 16 cats received intramuscular injections of AAV feline GLP-1-SA at one of three dose levels: 1e10, 1e11, or 1e12 gene copies / animal. Transgene expression was measured in plasma every 14 days. As shown in Figure 12, all 16 animals expressed high levels of GLP-1-Fc well above the therapeutic efficacy threshold of 3,000 pM for over 330 days. No animals acquired antibodies against the transgene protein.

[0147] These data demonstrate that a single injection of AAV feline GLP-1-SA induces sustained expression of the transgenic protein at high levels.

[0148] Specific Embodiments 1. A viral vector comprising a nucleic acid comprising 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 containing either (i) feline IgG Fc or a functional variant thereof, or (ii) feline albumin or a functional variant thereof. 2. The viral vector according to Embodiment 1, wherein the vector is an adeno-associated virus vector. 3. The viral vector according to Embodiment 1 or 2, wherein (i) the secreted signal peptide of the leader sequence comprises a feline thrombin signal peptide, (ii) the leader sequence comprises a feline thrombin propeptide, and / or (iii) the leader sequence comprises a feline thrombin leader sequence. 4. The viral vector according to Embodiment 3, wherein (i) the signal peptide of the leader sequence comprises the polypeptide sequence MAHIRGLWLPGCLALAALCSLVHS (SEQ ID NO: 8) or a functional variant thereof having up to 1, 2, or 3 amino acid substitutions, (ii) the leader sequence comprises the polypeptide sequence QHVFLAPQQALSLLQRVRR (SEQ ID NO: 9) or a functional variant thereof having up to 1, 2, or 3 amino acid substitutions, and / or (iii) the leader sequence comprises the polypeptide sequence MAHIRGLWLPGCLALAALCSLVHSQHVFLAPQQALSLLQRVRR (SEQ ID NO: 7) or a functional variant thereof having up to 1, 2, or 3 amino acid substitutions. 5. The viral vector according to Embodiment 1 or 2, wherein the leader sequence comprises a feline IL-2 leader sequence. 6. The viral vector according to Embodiment 5, wherein the feline IL-2 reader sequence comprises the sequence of MYKIQLLSCIALTLILVTNS (SEQ ID NO: 10), or a functional variant thereof having up to one, two, or three amino acid substitutions. 7. A viral vector according to any one of Embodiments 1 to 6, wherein the GLP-1 receptor agonist is feline GLP-1(7-37) or a functional variant thereof. 8. The viral vector according to Embodiment 7, wherein the GLP-1 receptor agonist is a DPP-IV resistant variant of feline GLP-1(7-37). 9. The viral vector according to Embodiment 7 or 8, wherein the GLP-1 receptor agonist is HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG (SEQ ID NO: 3), or a functional variant thereof having up to one, two, or three amino acid substitutions. 10. The viral vector according to Embodiment 9, wherein the GLP-1 receptor agonist comprises glycine (G) at position 8 relative to feline GLP-1(1-37) and / or glutamine (E) at position 22 relative to GLP-1(1-37), and optionally the GLP-1 receptor agonist is HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG (SEQ ID NO: 3). 11. GLP-1 receptor agonists include HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 2), HGEGTFTSDVSSYLEGQAAKEF The viral vector according to Embodiment 7 or 8, which is IAWLVKGRG (SEQ ID NO: 4), or a functional variant thereof having up to one, two, or three amino acid substitutions. 12. A viral vector according to any one of Embodiments 1 to 6, wherein the GLP-1 receptor agonist is GLP-1(1-37) or a functional variant thereof. 13. A viral vector according to any one of Embodiments 1 to 6, wherein the GLP-1 receptor agonist is HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 4), or optionally a functional variant thereof that shares at least 90% or 100% identity with SEQ ID NO: 4. 14. A viral vector according to any one of Embodiments 1 to 13, wherein the fusion protein comprises a second GLP-1 receptor agonist. 15. The viral vector according to Embodiment 14, wherein the GLP-1 receptor agonist is two tandem copies of GLP-1(7-37) or its DPP-IV resistant variant. 16. The viral vector according to Embodiment 15, wherein the DPP-IV resistant variant is HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 4). 17. A viral vector according to any one of Embodiments 1 to 16, wherein the fusion protein further comprises a linker between the GLP-1 receptor agonist and the fusion domain. 18. A viral vector according to any one of Embodiments 1 to 17, wherein the fusion domain is feline IgG Fc or a functional variant thereof. 19. The viral vector according to Embodiment 18, wherein the fusion domain is feline IgG Fc. 20. The viral vector according to Embodiment 18 or 19, wherein feline IgG Fc is feline IgG2 Fc. 21. The viral vector according to Embodiment 18 or 19, wherein the feline IgG Fc is feline IgG1a Fc. 22. The viral vector according to Embodiment 18 or 19, wherein the feline IgG Fc is feline IgG1b Fc. 23. The viral vector according to Embodiment 18 or 19, wherein feline IgG Fc shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity with respect to SEQ ID NO: 11. 24. A viral vector according to any one of Embodiments 1 to 17, wherein the fusion domain is feline albumin or a functional variant thereof. 25. The viral vector according to Embodiment 18, wherein the fusion domain is feline albumin. 26. The viral vector according to Embodiment 24 or 25, wherein feline albumin shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity with respect to SEQ ID NO: 12. 27. The viral vector according to Embodiment 1, wherein the fusion protein comprises (a) a feline thrombin reader, (b) a DPP-IV resistant variant of GLP-1(7-37), a linker, and (c) feline IgG Fc. 28. The viral vector according to Embodiment 1 or 26, wherein the fusion protein has a sequence identical to, or at least 90%, at least 95%, or at least 98% of, the sequence of Sequence ID No. 14. 29. The viral vector according to Embodiment 1, 26, or 27, wherein the sequence encoding the fusion protein is at least 90%, at least 95%, or at least 98% identical to Sequence ID No. 15. 30. The viral vector according to Embodiment 1, wherein the fusion protein comprises (a) a feline thrombin reader, (b) a DPP-IV resistant variant of GLP-1(7-37), a linker, and (c) feline albumin. 31. The fusion protein has the sequence of SEQ ID NO: 16, or at least 90% of it. A viral vector according to Embodiment 1 or 30, having at least 95% or at least 98% identical sequences. 32. The viral vector according to Embodiment 1, 30, or 31, wherein the sequence encoding the fusion protein is at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 17. 33. The viral vector according to Embodiment 1, wherein the fusion protein comprises (a) a feline thrombin reader, (b) two tandem copies of feline GLP-1(7-37) or its DPP-IV resistant variant, a linker, and (c) feline albumin. 34. The viral vector according to Embodiment 1 or 33, wherein the fusion protein has a sequence identical to, or at least 90%, at least 95%, or at least 98% of, the sequence of SEQ ID NO: 18 or SEQ ID NO: 20. 35. The viral vector according to Embodiment 1, 33, or 34, wherein the sequence encoding the fusion protein is at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 19. 36. (a) AAV capsid and, (b) A viral vector according to any one of Embodiments 1 to 35, comprising a vector genome packaged in an AAV capsid, wherein the vector genome comprises an AAV inverted terminal repeat (ITR), a polynucleotide sequence encoding a fusion protein, and a regulatory sequence directing the expression of the fusion protein. 37. (a) AAV capsid and, (b) A viral vector according to any one of Embodiments 1 to 35, comprising a vector genome packaged in an AAV capsid, wherein the vector genome comprises an AAV inverted terminal repeat (ITR), a polynucleotide sequence encoding a fusion protein, and a regulatory sequence that directs the insertion of the polynucleotide sequence encoding the fusion protein into the genome of a host cell. 38. The viral vector according to any one of Embodiments 1 to 37, wherein the viral vector is a recombinant adeno-associated virus (rAAV) having the capsid of AAV8 or a functional variant thereof. 39. The viral vector according to any one of Embodiments 1 to 37, wherein the viral vector is an rAAV having a capsid of AAVrh91 or a functional variant thereof. 40. The viral vector according to any one of Embodiments 1 to 37, wherein the viral vector is an rAAV having the capsid of AAV3B.AR2.12 or a functional variant thereof. 41. The viral vector according to any one of Embodiments 1 to 37, wherein the viral vector is an rAAV having a capsid selected from AAV9, AAVrh64R1, AAVhu37, or AAVrh10. 42. A pharmaceutical composition suitable for use in the treatment of metabolic diseases in cats, comprising an aqueous liquid and a viral vector as described in any one of Embodiments 1 to 41. 43. A viral vector according to any one of Embodiments 1 to 41, or a pharmaceutical composition according to Embodiment 42, for use in a method for treating a cat subject to metabolic disease, or optionally having diabetes. 44. Use of a viral vector according to any one of Embodiments 1 to 41 or a pharmaceutical composition according to Embodiment 42 in the manufacture of a pharmaceutical for the treatment of a cat subject to metabolic disease, or optionally having diabetes. 45. The composition is 1 × 10 9 GC / kg~3×10 13 The viral vector or use according to Embodiment 43 or 44, formulated to be administered to a feline subject at a dose of GC / kg of rAAV and / or delivered intramuscularly or intravenously. 46. ​​A method for treating a cat with a metabolic disease, wherein an effective amount of [a substance] is administered to the cat. A method comprising administering a viral vector described in any one of the application forms 1 to 41 or a pharmaceutical composition described in embodiment 42. 47. The method according to embodiment 46, wherein the metabolic disease is diabetes mellitus. 48. The method according to embodiment 47, wherein the diabetes is type 1 diabetes. 49. The method according to embodiment 47, wherein the diabetes is type 2 diabetes. 50. The method according to any one of embodiments 45 to 49, wherein an effective dose is administered intravenously. 51. The method according to any one of embodiments 45 to 49, wherein an effective dose is administered intramuscularly. 52. The effective amount is 1 × 10 9 GC / kg~3×10 13 The method according to any one of embodiments 45 to 51, wherein the rAAV is GC / kg body weight. 53. The effective dose is 1 × 10⁻⁶ 10 GC / kg~3×10 13 The method according to any one of embodiments 45 to 51, wherein the rAAV is the GC body weight. 54. The method according to any one of Embodiments 54 to 51, wherein the method results in the expression of the fusion protein in the serum of the subject for a period of at least 3 months, at least 6 months, or at least 12 months. 55. The method according to any one of Embodiments 54 to 51, wherein the method brings about the expression of a fusion protein in a subject at serum concentrations of at least 3,000 picomoles (pM), at least 5,000 pM, at least 10,000 pM, at least 25,000 pM, or at least 50,000 pM for at least 3 months, at least 6 months, or at least 12 months. 56. The method according to any one of Embodiments 54 to 51, wherein the method brings about the expression of a fusion protein in a subject at serum concentrations of 3,000 picomoles (pM) to 200,000 pM, 5,000 picomoles (pM) to 200,000 pM, 10,000 picomoles (pM) to 200,000 pM, 25,000 picomoles (pM) to 200,000 pM, or 50,000 picomoles (pM) to 200,000 pM for a period of 3 to 12 months, 6 to 12 months, or 12 months. 57. The method according to any one of Embodiments 54 to 51, wherein the method brings about the expression of a fusion protein in a subject at a therapeutically effective concentration for at least 3 months, at least 6 months, or at least 12 months. 58. The method according to any one of embodiments 54 to 57, wherein the method reduces the target serum fructosamine by approximately 6%. 59. The method according to any one of embodiments 54 to 57, wherein the method reduces the target serum fructosamine by 5% to 10%. 60. The method according to any one of embodiments 54 to 59, wherein the method reduces the target serum glucose by approximately 10%. 61. The method according to any one of embodiments 54 to 60, wherein the method reduces the target serum glucose by 10% to 20%. 62. The method according to any one of Embodiments 54 to 61, wherein the method comprises administering an effective amount of insulin to the target. 63. The method according to Embodiment 61, wherein the insulin is recombinant human insulin with zinc protamine (ProZinc®), porcine insulin zinc suspension (Vetsulin®), and / or insulin glargine (Lantus®). 64. The method according to claim 61 of claim 63, wherein the method reduces the insulin dose requirement compared to the insulin dose requirement for a subject before treatment with a viral vector. 65. The method according to any one of claims 54 to 64, wherein the method causes remission of a metabolic disease in a subject.

[0149] (Sequence Listing Free Text) The following information is a numerical identifier <223> Provides an array containing free text below. [Table 7]

[0150] All documents referenced herein are incorporated herein by reference. U.S. Provisional Patent Application No. 63 / 069,492, filed on 24 August 2020, along with its sequence listing, is incorporated herein by reference in its entirety. The sequence listing filed with it, labeled “20-9292PCT_Seq-Listing_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. A viral vector comprising a nucleic acid comprising 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 containing either (i) feline IgG Fc or a functional variant thereof, or (ii) feline albumin or a functional variant thereof.

2. The viral vector according to claim 1, wherein the vector is an adeno-associated virus vector.

3. (i) The secretion signal peptide of the leader sequence comprises a feline thrombin signal peptide, (ii) the leader sequence comprises a feline thrombin propeptide, and / or (iii) the leader sequence comprises a feline thrombin leader sequence, according to claim 1 or 2.

4. (i) The signal peptide of the leader sequence comprises the polypeptide sequence of SEQ ID NO: 8 or a functional variant thereof having up to one, two, or three amino acid substitutions; (ii) The leader sequence comprises the polypeptide sequence of SEQ ID NO: 9 or a functional variant thereof having up to one, two, or three amino acid substitutions; and / or (iii) The leader sequence comprises the polypeptide sequence of SEQ ID NO: 7 or a functional variant thereof having up to one, two, or three amino acid substitutions; the viral vector according to claim 3.

5. The viral vector according to claim 1 or 2, wherein the leader sequence includes a feline IL-2 leader sequence.

6. The viral vector according to claim 5, wherein the feline IL-2 reader sequence comprises the sequence of sequence number 10, or a functional variant thereof having up to one, two, or three amino acid substitutions.

7. The viral vector according to any one of claims 1 to 6, wherein the GLP-1 receptor agonist is feline GLP-1 (7-37) or a functional variant thereof.

8. The viral vector according to claim 7, wherein the GLP-1 receptor agonist is a DPP-IV resistant variant of feline GLP-1 (7-37).

9. The viral vector according to claim 7 or 8, wherein the GLP-1 receptor agonist is SEQ ID NO: 3, or a functional variant thereof having up to one, two, or three amino acid substitutions.

10. The viral vector according to claim 9, wherein the GLP-1 receptor agonist comprises glycine (G) at position 8 relative to feline GLP-1 (1-37) and / or glutamine (E) at position 22 relative to GLP-1 (1-37), and optionally the GLP-1 receptor agonist is Sequence ID No.

3.

11. The viral vector according to claim 7 or 8, wherein the GLP-1 receptor agonist is SEQ ID NO: 2, SEQ ID NO: 4, or a functional variant thereof of SEQ ID NO: 2 or SEQ ID NO: 4 having up to one, two, or three amino acid substitutions.

12. The GLP-1 receptor agonist is GLP-1 (1-37) or its functional varian The viral vector according to any one of claims 1 to 6.

13. The viral vector according to any one of claims 1 to 6, wherein the GLP-1 receptor agonist is SEQ ID NO: 4, or optionally a functional variant thereof that shares at least 90% or 100% identity with SEQ ID NO:

4.

14. The viral vector according to any one of claims 1 to 13, wherein the fusion protein comprises a second GLP-1 receptor agonist.

15. The viral vector according to claim 14, wherein the GLP-1 receptor agonist is two tandem copies of GLP-1 (7-37) or its DPP-IV resistant variant.

16. The viral vector according to claim 15, wherein the DPP-IV resistant variant is Sequence ID No.

4.

17. The viral vector according to any one of claims 1 to 16, wherein the fusion protein further comprises a linker between the GLP-1 receptor agonist and the fusion domain.

18. The viral vector according to any one of claims 1 to 17, wherein the fusion domain is feline IgG Fc or a functional variant thereof.

19. The viral vector according to claim 18, wherein the fusion domain is feline IgG Fc.

20. The viral vector according to claim 18 or 19, wherein the feline IgG Fc is feline IgG2 Fc.

21. The viral vector according to claim 18 or 19, wherein the feline IgG Fc is feline IgG1a Fc.

22. The viral vector according to claim 18 or 19, wherein the feline IgG Fc is feline IgG1b Fc.

23. The viral vector according to claim 18 or 19, wherein the feline IgG Fc shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity with respect to sequence number 11.

24. The viral vector according to any one of claims 1 to 17, wherein the fusion domain is feline albumin or a functional variant thereof.

25. The viral vector according to claim 18, wherein the fusion domain is feline albumin.

26. The viral vector according to claim 24 or 25, wherein the feline albumin shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity with respect to sequence number 12.

27. The viral vector according to claim 1, wherein the fusion protein comprises (a) a feline thrombin reader, (b) a DPP-IV resistant variant of GLP-1 (7-37), a linker, and (c) feline IgG Fc.

28. The viral vector according to claim 1 or 26, wherein the fusion protein has the sequence of SEQ ID NO: 14, or a sequence that is at least 90%, at least 95%, or at least 98% identical thereto.

29. The viral vector according to claim 1, 26, or 27, wherein the sequence encoding the fusion protein is at least 90%, at least 95%, or at least 98% identical to sequence number 15.

30. The viral vector according to claim 1, wherein the fusion protein comprises (a) a feline thrombin reader, (b) a DPP-IV resistant variant of GLP-1 (7-37), a linker, and (c) feline albumin.

31. The viral vector according to claim 1 or 30, wherein the fusion protein has the sequence of SEQ ID NO: 16, or a sequence that is at least 90%, at least 95%, or at least 98% identical thereto.

32. The viral vector according to claim 1, 30, or 31, wherein the sequence encoding the fusion protein is at least 90%, at least 95%, or at least 98% identical to sequence number 17.

33. The viral vector according to claim 1, wherein the fusion protein comprises (a) a feline thrombin reader, (b) two tandem copies and linkers of feline GLP-1 (7-37) or its DPP-IV resistant variant, and (c) feline albumin.

34. The viral vector according to claim 1 or 33, wherein the fusion protein has the sequence of SEQ ID NO: 18 or SEQ ID NO: 20, or a sequence that is at least 90%, at least 95%, or at least 98% identical thereto.

35. The viral vector according to claim 1, 33, or 34, wherein the sequence encoding the fusion protein is at least 90%, at least 95%, or at least 98% identical to sequence number 19.

36. (a) AAV capsid and, (b) A viral vector according to any one of claims 1 to 35, comprising a vector genome packaged in the AAV capsid, wherein the vector genome comprises an AAV inverted terminal repeat (ITR), the polynucleotide sequence encoding the fusion protein, and a regulatory sequence that directs the expression of the fusion protein.

37. (a) AAV capsid and, (b) A viral vector according to any one of claims 1 to 35, comprising a vector genome packaged in the AAV capsid, wherein the vector genome comprises an AAV inverted terminal repeat (ITR), the polynucleotide sequence encoding the fusion protein, and a regulatory sequence that directs the insertion of the polynucleotide sequence encoding the fusion protein into the genome of a host cell.

38. The viral vector according to any one of claims 1 to 37, wherein the viral vector is a recombinant adeno-associated virus (rAAV) having the capsid of AAV8 or a functional variant thereof.

39. The aforementioned viral vector has a capsid of AAVrh91 or a functional variant thereof. A viral vector according to any one of claims 1 to 37, wherein the viral vector is rAAV.

40. The viral vector according to any one of claims 1 to 37, wherein the viral vector is an rAAV having a capsid of AAV3B.AR2.12 or a functional variant thereof.

41. The viral vector according to any one of claims 1 to 37, wherein the viral vector is an rAAV having a capsid selected from AAV9, AAVrh64R1, AAVhu37, or AAVrh10.

42. A pharmaceutical composition suitable for use in the treatment of metabolic diseases in cats, comprising an aqueous liquid and a viral vector according to any one of claims 1 to 41.

43. A viral vector according to any one of claims 1 to 41, or a pharmaceutical composition according to claim 42, for use in a method for treating a cat with metabolic disease, or selectively having diabetes.

44. Use of a viral vector according to any one of claims 1 to 41 or a pharmaceutical composition according to claim 42 in the manufacture of a pharmaceutical for treating a cat with metabolic disease, or selectively having diabetes.

45. The above composition is 1 × 10 ９ GC / kg ~ 3 x 10 １３ The viral vector or use according to claim 43 or 44, formulated to be administered to the feline subject in a dose of GC / kg and / or delivered intramuscularly or intravenously.

46. A method for treating a feline subject having a metabolic disorder, comprising administering to the feline subject an effective amount of a viral vector according to any one of claims 1 to 41 or a pharmaceutical composition according to claim 42.

47. The method according to claim 46, wherein the metabolic disease is diabetes mellitus.

48. The method according to claim 47, wherein the diabetes is type 1 diabetes.

49. The method according to claim 47, wherein the diabetes is type II diabetes.

50. The method according to any one of claims 45 to 49, wherein the effective amount is administered intravenously.

51. The method according to any one of claims 45 to 49, wherein the effective amount is administered intramuscularly.

52. The effective amount is 1 × 10 ９ GC / kg ~ 3 x 10 １３ The method according to any one of claims 45 to 51, wherein the rAAV is GC / kg body weight.

53. The effective amount is 1 × 10 １０ GC / kg ~ 3 x 10 １３ The method according to any one of claims 45 to 51, wherein the rAAV is the GC body weight.

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