Viral vectors encoding GLP-2 receptor agonist fusions and their use in treatment of short bowel syndrome

Abstract

Compositions and methods for treating short bowel syndrome in a subject are provided. A viral vector is provided comprising a nucleic acid molecule comprising a sequence encoding a GLP-2 receptor agonist fusion protein and a regulatory sequence that directs expression thereof.

Description

[0001] References to Electronic Sequence Listings

[0002] The contents of the electronic sequence listing (22-10018PCT_Seq-Listing.xml; size: 161 kb; and creation date: March 3, 2023) are incorporated herein by reference in their entirety. Background Art

[0003] Short bowel syndrome (SBS) is a rare form of organ failure resulting from surgical removal of the intestine for either congenital or acquired reasons. SBS is a leading cause of intestinal failure (IF), in which intestinal function persistently declines to below the minimum level required to absorb macronutrients and / or water and electrolytes. Standard treatment for SBS / IF involves lifelong daily parenteral nutrition (PN), which is the intravenous infusion of a special form of food to support the patient's daily nutritional needs. In addition to the burden and risks associated with daily PN, PN can cause complications, including infection, intestinal dysgenesis, liver disease, renal dysfunction, and bone disease, which have a significant negative impact on the quality of life of SBS patients. Prior to the development and FDA and EMA approval of the glucagon-like peptide-2 (GLP-2) agonist Teduglutide, medications for SBS have been drugs that only treat the symptoms associated with SBS / IF.

[0004] GLP-2 is a 33-amino acid intestinal peptide hormone with a potent intestinal growth-promoting effect. GLP-2 does increase the length of the small intestine and the length of the villi of the intestinal epithelium, leading to efficient absorption of nutrients (even though the residual intestine is shorter in SBS patients), and reduces PN volume and number. However, due to cleavage by DPP IV, the serum half-life of native GLP-2 is very short and therefore cannot be used as an effective injection. DPP IV-resistant long-acting GLP-2 agonists, comprising fusions with the IgG Fc domain and serum albumin, have been developed as disease-modifying agents for SBS. Teduglutide is a GLP-2-like peptide with an A2G mutation that targets DPP IV resistance and extends the serum half-life from 5 minutes to 1.5 hours. Daily subcutaneous injection of teduglutide at 0.05 mg / kg results in a sustained and continuous reduction in PN volume throughout the 2-year treatment course. With an excellent safety profile, 90% of patients achieved a 20% or greater reduction in weekly PN volume from baseline with this treatment, and 70% of patients achieved greater than 1 additional day of PN discontinuation per week from baseline (Schwartz et al., 2016). In order to maintain the reduced PN volume and number, lifelong daily injections of teduglutide are required (Compher et al., 2011).

[0005] Improved treatments for SBS are needed. Summary of the invention

[0006] Provided herein are viral vectors encoding glucagon-like peptide 2 (GLP-2) receptor agonist fusion protein constructs. In some embodiments, these viral vectors can achieve sustained expression and / or increased circulation half-life of GLP-2 receptor agonists in subjects compared to vector-mediated delivery of GLP-2 receptor agonists without fusion partners. Further provided are methods for preparing and using such viral vectors.

[0007] On the one hand, a viral vector is provided, the viral vector comprising a nucleic acid comprising a polynucleotide sequence encoding a fusion protein. The fusion protein comprises (a) a leader sequence comprising a secretion signal peptide, (b) a glucagon-like peptide-1 (GLP-2) receptor agonist, and (c) a fusion domain comprising (i) IgG Fc or a functional variant thereof, (ii) albumin or a functional variant thereof, or (iii) an XTEN polypeptide (Podust et al., 240:52-66 (October 2016)). In one embodiment, the vector is an adeno-associated virus vector.

[0008] In one embodiment, (i) the secretory signal peptide of the leader sequence comprises a thrombin signal peptide; (ii) the leader sequence comprises a thrombin propeptide; and / or (iii) the leader sequence comprises a thrombin leader sequence. In another embodiment, the leader sequence comprises an IL-2 leader sequence. In one embodiment, the GLP fusion is selected from SEQ ID NO: 13, 15, 17, 19, 21 or 23 and functional variants thereof.

[0009] In one embodiment, the fusion domain is human IgG4 Fc having the sequence of SEQ ID NO: 8 or a sequence sharing at least 90% identity therewith or a functional variant thereof. In another embodiment, the fusion domain is human albumin having the sequence of SEQ ID NO: 11 or a sequence sharing at least 90% identity therewith or a functional variant thereof. In one embodiment, the fusion domain is rhesus monkey IgG4 Fc having the sequence of SEQ ID NO: 9 or a sequence sharing at least 90% identity therewith or a functional variant thereof.

[0010] On the other hand, the viral vector comprises an AAV capsid and a vector genome packaged in the AAV capsid, wherein the vector genome includes an AAV inverted terminal repeat (ITR), the polynucleotide sequence encoding the fusion protein, and a regulatory sequence directing the expression of the fusion protein.

[0011] In another aspect, a pharmaceutical composition suitable for treating short bowel syndrome in a subject is provided. The composition comprises an aqueous liquid and a viral vector as described herein. In one embodiment, the subject is a human.

[0012] In yet another aspect, there is provided use of a viral vector as described herein for the manufacture of a medicament for treating a subject suffering from short bowel syndrome, optionally diabetes.

[0013] In another aspect, a method of treating a subject suffering from short bowel syndrome is provided, wherein the method comprises administering to the subject an effective amount of a viral vector or composition as described herein.

[0014] Other aspects and advantages of the present invention will become apparent from the following detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the in vivo processing of proglucagon. The sequences of GLP-1 (SEQ ID NO: 4) and GLP-2 (SEQ ID NO: 1) are shown.

[0016] Figure 2 is a table showing the sequences of GLP-2 and analogs.

[0017] Figure 3A is a schematic diagram of the GLP2.G2.Fc construct comprising an A2G substitution in the GLP2 amino acid sequence, a thrombin leader sequence, and an IgG4 Fc fusion.

[0018] Figure 3B is a schematic diagram of the GLP2.G2.SA construct comprising an A2G substitution in the GLP2 amino acid sequence, a thrombin leader sequence, and a serum albumin fusion.

[0019] Figure 3C is an alignment of human (SEQ ID NO: 1), cynomolgus monkey (SEQ ID NO: 5) and mouse (SEQ ID NO: 6) GLP2 sequences.

[0020] Figure 4A The elution profile of hGLP2-SA protein purified using an albupure column is shown.

[0021] Figure 4B Shows that there is Figure 4A Figure 2. Gel stained with hGLP2-SA protein.

[0022] Figure 5AShown are hGLP2-SA levels in RagKO mice dosed intramuscularly with AAVrh91.CI.hGLP2.G2.SA.rBG at 1e11 gc / mouse.

[0023] Figure 5B are two graphs showing Figure 5A Small intestine lengths and weights of vehicle- and vehicle-treated mice as described.

[0024] Figure 5C It shows that Figure 5A Intestinal histology of vehicle and vehicle treated mice as described. Vehicle treated mouse intestines showed healthier villi compared to vehicle treated mice.

[0025] Fig. 6A Shown are hGLP2-SA and hGLP2-Fc levels of mice treated with AAVrh91.CI.hGLP2.G2.SA.rBG or AAVrh91.CI.hGLP2.G2.Fc.rBG at a dose of 1e11 gc / mouse. GLP-2 levels of the Fc construct were higher after approximately day 7 of the study.

[0026] Figure 6B Potency assays for hGLP2 and hGLP2.Fc are shown. The EC50 for hGLP2 was determined to be 0.4 nM, while the fusion protein had an EC = 13.6 nM.

[0027] Figure 7 The study design for the experiment as described in Example 4 is shown.

[0028] Figures 8A-8C The results of an experiment described in Example 4 are shown. 13 (1e13)GC / kg(E185NG) and 5x 10 10 AAVrh91.CB7.CI.hGLP-2-Fc.rBG was administered to 2 NHPs at a dose of (1e10) GC / kg(BM239H). Fig. 8A Plasma levels of GLP-2-Fc fusion protein are shown. Figure 8B Serum citrulline, a biomarker of intestinal surface area, is shown. Figure 8C Detection of anti-GLP-2-Fc antibodies in NHP sera diluted 1:100 is shown.

[0029] Figures 9A-9F The results of an experiment described in Example 5 are shown. Rag1KO female mice were given 1 x 10 10 GC / mouse, 3x 10 10GC / mouse or 1x 10 11 GC / mouse were treated by injection of vector AAVrh91.CB7.CI.hGLP-2-Fc.rBG. The study design is shown in Fig. 9A middle. Fig. 9B Serum GLP2 levels are shown. Fig. 9C Body weight over time is shown. Fig.9D Body weights on day 56 are shown. Fig.9E Small intestine (SI) length is shown, while Fig.9F SI weights are shown. DETAILED DESCRIPTION

[0030] Adeno-associated virus (AAV) vectors expressing GLP-2 agonists are described herein to treat SBS / IF patients via a single intramuscular vector administration. Transgenic GLP-2 agonists contain A2G mutations for DPP IV resistance, as well as fusions with human IgG Fc domains or serum albumin for further extension of serum half-life. In combination with these half-life extension techniques, the addition of thrombin propeptides enables expression of GLP-2 agonists above therapeutic levels, with significantly reduced vector doses (i.e., 1e10 to 1e12 GC / kg). Expression cassettes for constitutively or in a controlled manner expressing these proteins by administering small molecule drugs that activate transcription of GLP-2 agonist sequences are described herein.

[0031] Described are the delivery of these constructs to subjects in need thereof by a variety of routes, and in particular by in vivo expression mediated by recombinant vectors such as rAAV vectors. Also provided are methods of using these constructs in regimens for treating short bowel syndrome in subjects in need thereof and increasing the half-life of GLP-2 in subjects. Additionally, methods for enhancing the activity of GLP-2 in subjects are provided.

[0032] GLP-2 fusion protein

[0033] Post-translational processing of proglucagon, together with GLP-1, produces glucagon-like peptide-2 (GLP-2), a 33-amino acid intestinal growth-promoting peptide hormone. GLP-2 acts to slow gastric emptying, reduce gastric secretions, and increase intestinal blood flow. GLP-2 also stimulates the growth of the large and small intestines at least by enhancing crypt cell proliferation and villus length, thereby increasing the surface area of ​​the mucosal epithelium. These effects suggest that GLP-2 can be used to treat various gastrointestinal conditions. However, the administration of GLP-2 alone to human patients has not shown promise. The short half-life of GLP-2 limits its use as a therapeutic agent because dipeptidyl peptidase IV (DPP-IV) rapidly cleaves GLP-2 in vivo to produce essentially inactive peptides. The amino acid sequence of human GLP-2 is HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ ID NO: 1).

[0034] As mentioned above, a GLP-2 analogue named Teduglutide has been developed in which amino acid residue 2 (alanine) has been replaced with glycine. The sequence of this GLP-2 analogue is shown in SEQ ID NO: 2HGDGSFSDEMNTILDNLAARDFINWLIQTKITD.

[0035] The present disclosure provides fusion proteins including one or more copies of a GLP-2 receptor agonist, as well as polynucleotides and vectors encoding such fusion proteins. In some embodiments, the fusion protein includes a polynucleotide sequence encoding a fusion protein, the fusion protein including (a) a leader sequence including a secretion signal peptide; (b) a glucagon-like peptide-2 (GLP-2) receptor agonist; and (c) a fusion domain. In one embodiment, the GLP-2 receptor agonist includes a thrombin leader sequence, a GLP-2 receptor agonist, and IgG Fc or a functional variant thereof. In another embodiment, the fusion protein includes a thrombin leader sequence, a GLP-2 receptor agonist, and albumin or a functional variant thereof. In another embodiment, the fusion protein includes a thrombin leader sequence, two copies of a GLP-2 receptor agonist, and albumin or a functional variant thereof.

[0036] In some embodiments, the GLP-2 receptor agonist comprises a variant that may comprise up to about 10% variation from a GLP-2 nucleic acid or amino acid sequence described herein or known in the art, and the variant retains the function of the wild-type sequence. As used herein, "retaining function" means that the nucleic acid or amino acid acts in the same manner as the wild-type sequence, although not necessarily at the same expression or activity level. For example, in one embodiment, the functional variant has increased expression or activity compared to the wild-type sequence. In another embodiment, the functional variant has decreased expression or activity compared to the wild-type sequence. In one embodiment, the expression or activity of the functional variant has an increase or decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the wild-type sequence.

[0037] Other GLP-2 analogs have been developed and include glepaglutide (SEQ ID NO: 3), apraglutide, and other analogs, as described below and Figure 2 shown.

[0038]

[0039] In one embodiment, the fusion comprises a combination of a GLP-2 analog and a heterologous sequence. A GLP-2 analog means a polypeptide that shares at least 90%, 95%, 97%, 98%, 99% or 100% identity with native human GLP-2 (SEQ ID NO: 1) or GLP-2-A2G (SEQ ID NO: 2). In one embodiment, the GLP-2 analog has a maximum of 1, 2 or 3 amino acid substitutions compared to the native sequence. In another embodiment, the GLP-2 sequence is derived from a species other than humans. For example, GLP-2 can be from a non-human primate, dog, cat, mouse, rat, sheep, cow, horse, etc. In some embodiments, it is desirable to alter the native GLP-2 sequence to optimize one or more of its characteristics. In certain embodiments, the GLP-2 has a sequence in which amino acid residue 2 (alanine) has been substituted with glycine, such as SEQ ID NO: 2. For example, in other embodiments, the GLP-2 analog contains one, two or three, 4, 5, 6, 7, 8 or up to 9 amino acid substitutions selected from A2G, D3E, S5T, D8S, M10L, N11A, N16A, N24A, Q28A compared to the native sequence. These substitutions have been shown to improve the efficacy of the GLP-2 clinical profile, including preventing DPP-4 inactivation (A2G). In one embodiment, the GLP-2 analog is a DPP-IV resistant variant of GLP-2. In one embodiment, the GLP-2 analog has a sequence comprising or consisting of SEQ ID NO: 2. In one embodiment, the variant shares at least 90% identity, 95% identity, 97% identity, 98% identity, 99% identity or 100% identity with SEQ ID NO: 2.

[0040] The fusion protein may include a leader sequence, which may include a secretory signal peptide. As used herein, the term "leader sequence" refers to any N-terminal sequence of a polypeptide.

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

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

[0043] In one embodiment, the leader sequence is a human thrombin (factor II) sequence. In one embodiment, the thrombin leader sequence has the sequence shown in SEQ ID NO:7: MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRR or a functional variant thereof with up to 1, 2 or 3 amino acid substitutions.

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

[0045] In some embodiments, the coding regions for both the propeptide and the GLP-2 peptide are incorporated into a single nucleic acid sequence without a linker between the coding sequence for the propeptide and the coding sequence for GLP-2.

[0046] The fusion protein further comprises a fusion domain. In one embodiment, the fusion domain is a human IgG Fc fragment or a functional variant thereof. Immunoglobulins generally have a longer in vivo circulation half-life. By fusing a GLP-2 receptor agonist (and a leader sequence) to IgG Fc, the circulation time of the fusion protein is extended, and the function of GLP-2 is retained. In another embodiment, the fusion domain is a rhesus monkey IgG Fc fragment or a functional variant thereof.

[0047] As used herein, the Fc portion of an immunoglobulin has the meaning generally given to the term in the field of immunology. Specifically, this term refers to an antibody fragment that does not contain two antigen-binding regions (Fab fragments) from an antibody. The Fc portion is composed of the constant region of an antibody from two heavy chains, which are associated by non-covalent interactions and disulfide bonds. The Fc portion may include a hinge region and extend to the c-terminus of the antibody through CH2 and CH3 domains. The Fc portion may further include one or more glycosylation sites. In one embodiment, the fusion domain is human IgG Fc. The highly conserved four subclasses IgG1, IgG2, IgG3 and IgG4 differ in their constant regions, particularly in their hinges and upper CH2 domains. See, Vidarsson et al., IgG subclasses and isotypes: From structure to effector functions, Front Immunol., October 2014; 5:520, the document is incorporated herein by reference. The Fc domain can be derived from any human IgG, including human IgG1, human IgG2, human IgG3 or human IgG4. In one embodiment, the human IgG Fc is IgG4 Fc. In one embodiment, the human IgG Fc is SEQ ID NO: 8:

[0048] AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG. In another embodiment, the human IgG Fc shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity with SEQ ID NO:8.

[0049] In another embodiment, the fusion domain is rhesus IgG Fc. The Fc domain can be derived from any rhesus IgG, including rhesus IgG1, rhesus IgG2, rhesus IgG3 or rhesus IgG4. In one embodiment, the rhesus IgG Fc is IgG4 Fc. In one embodiment, the rhesus IgG Fc is SEQ ID NO:9:

[0050] PPCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVEVHNAQTKPRERQFNSTYRVV SVLTVTHQDW LNGKEYTCKV SNKGLPAPIE KTISKAKGQPREPQVYILPPPQEELTKNQV SLTCLVTGFY PSDIAVEWES NGQPENTYKTTPPVLDSDGS YLLYSKLTVN KSRWQPGNIFTCSVMHEALH NHYTQKSLSV SPGK. In another embodiment, the rhesus IgG Fc shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity with SEQ ID NO: 9. In one embodiment, the rhesus IgG further comprises a hinge sequence.

[0051] In another embodiment, the fusion domain is human albumin or a functional variant thereof. In one embodiment, human albumin is

[0052] SEQ ID NO:10:

[0053] In another embodiment, the human albumin shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity with SEQ ID NO:10.

[0054] In another embodiment, the fusion domain is rhesus albumin or a functional variant thereof. In one embodiment, the rhesus albumin is

[0055] SEQ ID NO:11:

[0056] In another embodiment, the rhesus monkey albumin shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity with SEQ ID NO:11.

[0057] The in vivo function and stability of the fusion proteins disclosed herein can be optimized by adding small peptide linkers, for example, to prevent potentially unwanted domain interactions or for other reasons. In addition, the glycine-rich linker can provide some structural flexibility so that the GLP-2 analog portion can effectively interact with the GLP-2 receptor on target cells such as pancreatic beta cells. Therefore, in one embodiment, the C-terminus of the GLP-2 analog and the N-terminus of the fusion domain of the fusion protein are fused through a linker. In one embodiment, the linker comprises 1, 1.5 or 2 repeats of a G-rich peptide linker having the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 12).

[0058] In one embodiment, the fusion protein includes (a) a human thrombin leader sequence; (b) a DPP-IV resistant variant of GLP-2 (A2G); a linker; and (c) a human IgG Fc. In one embodiment, the fusion protein has a sequence of SEQ ID NO: 13 or a sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

[0059] SEQ ID NO:13

[0060] MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRHGDGSFSDEMNTILDNLAARDFINWLIQTKITDGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*

[0061] In one embodiment, the sequence encoding the fusion protein is SEQ ID NO: 14, or a sequence at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0062] SEQ ID NO:14:

[0063] ATGGCTCACGTTCGGGGACTGCAGCTGCCTGGATGTCTGGCTCTTGCCGCTCTGTGTAGCCTGGTGCACAGCCAGCACGTGTTTCTGGCTCCTCAGCAAGCCAGATCACTGCTGCAGAGAGTTAGAAGGCACGGCGACGGCAGCTTCAGCGACGAGATGAACACCATCCTGGACAACCTGGCCGCCAGAGACTTCATCAACTGGCTGATCCAGACCAAGATCACCGACGGTGGCGGAGGCGGAGGATCTGGTGGTGGTGGATCTGGCGGCGGAGGTTCTGCCGAGTCTAAGTACGGACCTCCTTGTCCTCCCTGTCCTGCTCCAGAAGCTGCTGGCGGCCCATCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTCGACGTGTCCCAAGAGGATCCTGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCAAGAGAACCCCAGGTGTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGATGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCAGGTGGCAAGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAAAGCCTGAGCCTGTCTCTGGGCTAA

[0064] In one embodiment, the fusion protein comprises (a) a human thrombin leader sequence; (b) a DPP-IV resistant variant of GLP-2 (A2G); a linker; and (c) human serum albumin. In one embodiment, the fusion protein has the sequence of SEQ ID NO: 15 or a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. SEQ ID NO: 15:

[0065] *

[0066] In one embodiment, the sequence encoding the fusion protein is SEQ ID NO: 16, or a sequence at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0067] SEQ ID NO:16:

[0068]

[0069] In one embodiment, the fusion protein includes (a) a human thrombin leader sequence; (b) a DPP-IV resistant variant of GLP-2 (A2G); a linker; and (c) an XTEN polypeptide. In one embodiment, the fusion protein has a sequence of SEQ ID NO: 17 or a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0070] SEQ ID NO:17:

[0071] *

[0072] In one embodiment, the sequence encoding the fusion protein is SEQ ID NO: 18, or a sequence at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0073] SEQ ID NO:18:

[0074]

[0075] In one embodiment, the fusion protein includes (a) a rhesus monkey thrombin leader sequence; (b) a DPP-IV resistant variant of GLP-2 (A2G); a linker; and (c) a rhesus monkey IgG Fc. In one embodiment, the fusion protein has a sequence of SEQ ID NO: 19 or a sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

[0076] SEQ ID NO:19

[0077] MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQALSLLQRVRRHGDGSFSDEMNTVLVDNLATRDFINWLIQTKITDGGGGGGGSGGGGSGGGGSAEFTPPCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAQTKPRERQFNSTYRVVSVLTVTHQDWLNGKEYTCKVSNKGLPAPIEKTISKAKGQPREPQVYILPPPQEELTKNQVSLTCLVTGFYPSDIAVEWESNGQPENTYKTTPPVLDSDGSYLLYSKLTVNKSRWQPGNIFTCSVMHEALHNHYTQKSLSVSPG*

[0078] In one embodiment, the sequence encoding the fusion protein is SEQ ID NO: 20, or a sequence at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0079] SEQ ID NO:20:

[0080] ATGGCTCACGTTCGGGGACTGCAGCTGCCTGGATGTCTGGCTCTTGCCGCTCTGTGTAGCCTGGTGCACAGCCAGCATGTGTTTCTGGCTCCTCAGCAGGCCCTGAGCCTGCTGCAAAGAGTTAGAAGGCACGGCGACGGCAGCTTCAGCGACGAGATGAATACCGTGCTGGTGGACAACCTGGCCACCAGAGACTTCATCAACTGGCTGATCCAGACCAAGATCACCGACGGTGGTGGCGGAGGCGGAGGATCTGGTGGCGGTGGTTCTGGCGGTGGCGGATCTGCTGAGTTTACCCCTCCTTGTCCTCCCTGTCCTGCTCCAGAACTGCTCGGCGGACCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCAAGAGGATCCTGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCCAGACAAAGCCCAGAGAGCGGCAGTTCAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGACACACCAGGATTGGCTGAACGGCAAAGAGTACACCTGTAAAGTCTCCAACAAGGGCCTGCCTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAACCCCAGGTGTACATCCTGCCTCCACCTCAAGAGGAACTGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCACCGGCTTCTACCCTTCCGATATCGCCGTGGAGTGGGAGAGCAACGGACAGCCCGAGAACACCTACAAGACCACACCTCCAGTGCTGGACAGCGACGGCTCTTACCTGCTGTACTCCAAGCTGACAGTGAACAAGAGCCGGTGGCAGCCCGGCAACATCTTCACCTGTTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAAAGCCTGAGCGTGTCCCCTGGATAA

[0081] In one embodiment, the fusion protein includes (a) a rhesus monkey thrombin leader sequence; (b) a DPP-IV resistant variant of GLP-2 (A2G); a linker; and (c) rhesus monkey serum albumin. In one embodiment, the fusion protein has a sequence of SEQ ID NO: 21 or a sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

[0082] SEQ ID NO:21

[0083] MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQALSLLQRVRRHGDGSFSDEMNTVLVDNLATRDFINWLIQTKITGGGGGGGSGGGGSGGGGSDTHKSEVAHRFKDLGEEHFKGLVLVAFSQYLQQCPFEEHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLC TVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPPLVRPEVDVMCTAFHDNEATFLKKYLYEVARRHPYFYAPELLFFAARYKAAFAECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGDRAFKAWAVARLSQKFPKAEFAEVSKLVTDLTKVHTEC CHGDLLECADDRADLAKYMCENQDSISSKLKECCDKPLLEKSHCLAEVENDEMPADLPSLAADYVESKDVCKNYAEAKDVFLGMFLYEYARRHHPDYSVMLLLRLAKAYEATLEKCCAAADPHECYAKVFDEFQPLVEEPQNLVKQNCELFEQLGEYKFQNALLVRYTKK VPQVSTPTLVEVSRNLGKVGAKCCKLPEAKRMPCAEDYLSVVLNRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALELDEAYVPKAFNAETFTFHADMCTLSEKEKQVKKQTALVELVKHKPKATKEQLKGVMDNFAAFVEKCCKADDKEACFAEEGPKFVAASQAALA

[0084] In one embodiment, the sequence encoding the fusion protein is SEQ ID NO: or a sequence at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0085] SEQ ID NO:22:

[0086]

[0087] In one embodiment, the fusion protein includes (a) a rhesus monkey thrombin leader sequence; (b) a DPP-IV resistant variant of GLP-2 (A2G); a linker; and (c) an XTEN polypeptide. In one embodiment, the fusion protein has a sequence of SEQ ID NO: 23 or a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0088] SEQ ID NO:23

[0089] MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQALSLLQRVRRHGDGSFSDEMNTVLVDNLATRDFINWLIQTKITDGGGGGGSGGGSGGGSSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG STEPSEGSAPGTSEPSEGPGSPAGSPTSTEEGTSEPSEGPGSAPGTSESATPESGPGSESAPATSGSETPGTSESATPESGPGSESAPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSAP

[0090] In one embodiment, the sequence encoding the fusion protein is SEQ ID NO: 24, or a sequence at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0091] SEQ ID NO:24:

[0092]

[0093] When a variant or fragment of a leader sequence, GLP-2 receptor agonist, or fusion domain is desired, site-directed mutagenesis of a wild-type nucleic acid sequence can be used to generate coding sequences for these peptides. Alternatively or in addition, web-based or commercially available computer programs and service-based companies can be used to reverse translate amino acid sequences into nucleic acid coding sequences, including both RNA and / or cDNA. See, for example, backtranseq by EMBOSS, ebi.ac.uk / Tools / st / ; Gene Infinity (geneinfinity.org / sms- / sms_backtranslation.html); ExPasy (expasy.org / tools / ). In one embodiment, the RNA and / or cDNA coding sequence is designed to be optimally expressed in the subject species (e.g., humans) to which it is ultimately intended to be administered.

[0094] Codon optimization can be used to design the coding sequence for optimal expression. The codon optimized coding region can be designed by various methods. This optimization can be performed using a method available online, a disclosed method, or a company providing codon optimization services. For example, a codon optimization method is described in International Patent Application Publication No. WO 2015 / 012924, which is incorporated herein by reference. In short, the nucleic acid sequence of the encoded product is modified with a synonymous codon sequence. Suitably, the entire length of the open reading frame (ORF) of the product is modified. However, in some embodiments, only the fragment of the ORF can be changed. By using one of these methods, frequency can be applied to any given polypeptide sequence, and a nucleic acid fragment of a codon optimized coding region encoding a polypeptide is produced.

[0095] In addition to the leader sequences, GLP-2 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-2 peptide described herein is provided. In some embodiments, the nucleic acid sequence may include any nucleic acid sequence encoding the GLP-2 sequence of SEQ ID NO: 1. In another embodiment, the nucleic acid sequence includes any nucleic acid, and any nucleic acid includes the GLP-2 sequence of SEQ ID NO: 2.

[0096] In one embodiment, a nucleic acid sequence encoding the GLP-2 fusion protein described herein is provided. In another embodiment, the nucleic acid sequence comprises any nucleic acid sequence encoding the GLP-2 fusion protein of SEQ ID NO: 13, 15, 17, 19, 21 or 23.

[0097] Expression cassette

[0098] In another aspect, an expression cassette is provided herein, comprising a nucleic acid encoding a GLP-2 fusion protein as described herein. As used herein, an "expression cassette" refers to a nucleic acid molecule comprising a biologically useful nucleic acid sequence (e.g., a gene cDNA, mRNA, etc. encoding a protein, enzyme, or other useful gene product) and a regulatory sequence operably linked thereto, the regulatory sequence directing or regulating the transcription, translation, and / or expression of the nucleic acid sequence and its gene product. As used herein, an "operably linked" sequence includes both regulatory sequences (also referred to as elements) that are adjacent or non-adjacent to the nucleic acid sequence and regulatory sequences that function in a trans or cis nucleic acid sequence. Such regulatory sequences typically include, for example, one or more of the following: a promoter, an enhancer, a transcription factor, a transcription terminator, an intron, a sequence that enhances translation efficiency (i.e., a Kozak consensus sequence), an efficient RNA processing signal (e.g., a slicing and polyadenylation sequence), a sequence that stabilizes cytoplasmic mRNA (e.g., a woodchuck hepatitis virus (WHP) post-translational regulatory element (WPRE)), and a TATA signal. Expression cassette can contain the regulatory sequence of gene sequence upstream (5 ' place), for example, one or more of promoter, enhancer, intron etc. and one or more of enhancer, or the regulatory sequence of gene sequence downstream (3 ' place), for example, 3 ' untranslated region (3 ' UTR) including polyadenylation site, and other elements.In certain embodiments, regulatory sequence is operably connected with the nucleotide sequence of gene product, and wherein regulatory sequence is separated from the nucleotide sequence of gene product by the nucleotide sequence in between, i.e. 5 ' untranslated region (5 ' UTR).In certain embodiments, expression cassette comprises the nucleotide sequence of one or more gene products.In certain embodiments, expression cassette can be monocistronic expression cassette or bicistronic expression cassette.In other embodiments, term " transgenic " refers to one or more DNA sequences inserted into target cell from external source.

[0099] In one embodiment, an expression cassette refers to a nucleic acid molecule comprising a GLP-2 construct coding sequence (e.g., a coding sequence for a GLP-2 fusion protein), a promoter, and may contain other regulatory sequences thereof, which can be engineered into genetic elements and / or packaged into the capsid of a viral vector (e.g., a viral particle). Typically, such an expression cassette for generating a viral vector contains a GLP-2 construct sequence described herein and other expression control sequences (such as the expression control sequences described herein), wherein the GLP-2 construct sequence is flanked by packaging signals of a viral genome (and referred to as a "vector genome"). Any expression control sequence can be optimized for a particular species using techniques known in the art, including, for example, codon optimization, as described herein.

[0100] In certain embodiments, the expression cassette comprises a constitutive promoter. In another embodiment, the CB7 promoter is used. CB7 is a chicken beta-actin promoter with a cytomegalovirus enhancer element. In some embodiments, the CB7 promoter has a nucleic acid sequence of SEQ ID NO: 25. In one embodiment, the promoter is a CMV promoter. In some embodiments, the CMV promoter is a nucleic acid sequence of SEQ ID NO: 26.

[0101] In another embodiment, a tissue-specific promoter is used. Alternatively, other liver-specific promoters may be used, such as those listed in the Liver Specific Gene Promoter Database, Cold Spring Harbor (rulai.schl.edu / LSPD), and include, but are not limited to, α1 antitrypsin (A1AT); human albumin (Miyatake et al., Journal of Virology (J. Virol.), 71:5124 32 (1997)), humAlb; hepatitis B virus core promoter (Sandig et al., Gene Ther., 3:1002 9 (1996)); TTR minimal enhancer / promoter, α-antitrypsin promoter, liver-specific promoter (LSP) (Wu et al., Mol Ther. 16:280-289 (2008)), TBG liver-specific promoter. Other promoters such as viral promoters, constitutive promoters, regulatable promoters (see, for example, WO 2011 / 126808 and WO 2013 / 04943), or promoters that are responsive to physiological cues can be used in the vectors described herein.

[0102] In one embodiment, the promoter is included in an inducible gene expression system. The inducible gene regulation / expression system contains at least the following components: a promoter operably linked to a transgene encoding a GLP-2 fusion protein described herein (also referred to as a regulatable promoter), an activation domain, a DNA binding domain, and a zinc finger homeodomain binding site. In other embodiments, additional components may be included in the expression system, as further described herein.

[0103] The system includes a promoter upstream of the coding sequence of the GLP-2 fusion protein. Promoters described herein, such as CMV and CB7 promoters, can be used. In one embodiment, the promoter is a CMV promoter, such as the promoter shown in SEQ ID NO: 26. In another embodiment, the promoter is the ubiquitous inducible promoter Z12I, which includes 12 repeated copies of the binding sites of ZFHD1 and IL2 minimal promoters. See, for example, Chen et al., Hum Gene Ther Methods. 2013 August; 24(4): 270–278, which is incorporated herein.

[0104] The expression system includes an activation domain, which is preferably located upstream of the DNA binding domain. In one embodiment, the activation domain is a fusion of the carboxyl terminus of the p65 subunit of NF-κB and the FKBP12-rapamycin binding (FRB) domain of the FKBP12-rapamycin associated protein (FRAP) from the carboxyl terminus of the p65 subunit of NF-κB. In one embodiment, the activation domain is a FKBP12-rapamycin binding (FRB) domain of the human FKBP12-rapamycin associated protein (FRAP) fused to the carboxyl terminus of the p65 subunit of NF-κB from humans.

[0105] In one embodiment, there is a linker between the transactivation domain and the DNA binding domain, and the linker can be F2A or IRES. In one embodiment, the linker is selected from IRES or 2A peptide.

[0106] The DNA binding domain consists of a DNA binding fusion of zinc finger homology domain 1 (ZFHD1) linked to up to three copies of FK506 binding protein (FKBP). In the presence of an inducer (e.g., a rapamycin analog, such as rapamycin), the DNA binding domain and the activation domain dimerize through the interaction of their FKBP and FRB domains, resulting in transcriptional activation of the transgene. In some embodiments, ZFHD1 is contained in a framework with GT2A or IRES.

[0107] The expression system is designed to have one, two or three copies of the FKBP sequence. These are referred to herein as FKBP subunits. In one embodiment, the subunits are designed to express the same protein but have different nucleic acids from each other to minimize recombination. For example, SEQ ID NO:27 provides three "wobbled" coding sequences of FKBP, each of which encodes the sequence shown in SEQ ID NO:28: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIR GWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE.

[0108] The expression system further comprises a zinc finger homeodomain binding site. The nucleic acid molecule contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 ZFHD binding sites. In one embodiment, the expression system contains 8 (eight) zinc finger homeodomain binding sites (binding partners) (8XZFHD). However, the present invention encompasses expression systems having two to about twelve copies of zinc finger binding sites. An example of a single copy of a ZFHD binding site is: aatgatgggcgctcgagt (SEQ ID NO: 29).

[0109] In some embodiments, a minimal IL2 promoter is present downstream of the zinc finger homeodomain binding site. An exemplary IL2 promoter is shown in SEQ ID NO: 30.

[0110] Such inducible systems are known in the art and include, for example, the rapamycin inducible system described by, e.g., Rivera et al., A humanized system for pharmacologic control of gene expression, Nature Medicine, Vol. 2, pp. 1028-1032 (September 1996), and Rivera et al., Long-term pharmacologically regulated expression of erythropoietin in primates following AAV-mediated gene transfer, Blood, Feb. 15, 2005, Vol. 105, No. 4, both of which are incorporated herein by reference. In one embodiment, the inducible gene expression system includes a CMV promoter, an activation domain that is the FKBP12-rapamycin binding (FRB) domain of the human FKBP12-rapamycin associated protein (FRAP) fused to the carboxyl terminus of the p65 subunit of human NF-κB, a GT2A peptide, a ZFHD1 DNA binding domain, three FKBP subunits, hGH poly A, 8XZFHD, and a minimal sIL2 promoter. These sequences are in addition to the coding sequence of the GLP-2 fusion protein and optionally other regulatory sequences.

[0111] In addition to the promoter, the expression cassette and / or vector may also contain other appropriate transcription initiation, termination, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (i.e., Kozak consensus sequences), sequences that enhance protein stability, and sequences that enhance secretion of the encoded product when desired. Examples of suitable polyA sequences include, for example, SV40, bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit β-globin (also known as rabbit globin polyA; RGB), modified RGB (mRGB), and TK polyA. Examples of suitable enhancers include, for example, α-fetoprotein enhancer, TTR minimal promoter / enhancer, LSP (TH binding globulin promoter / α1 microglobulin / double Kunitz inhibitor enhancer), etc. In one embodiment, polyA is rabbit globin polyA.

[0112] These control sequences are "operably linked" to the GLP-2 construct sequence. As used herein, the term "operably linked" refers to both expression control sequences that are contiguous to the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

[0113] In one embodiment, rAAV is provided, comprising a 5'ITR, a CB7 promoter, a chicken β-actin intron, a coding sequence of a fusion protein of SEQ ID NO: 13, 15, 17, 19, 21 or 23, rabbit globin poly A and a 3'ITR. In another embodiment, rAAV comprises a polynucleotide comprising a CMV promoter, an activation domain that is a FKBP12-rapamycin binding (FRB) domain of a human FKBP12-rapamycin associated protein (FRAP) fused to the carboxyl terminus of a p65 subunit of NF-κB from humans, an IRES, a ZFHD1 DNA binding domain, three FKBP subunits, hGH poly A, 8XZFHD, a minimal sIL2 promoter, a coding sequence of a GLP-2 fusion protein of SEQ ID NO: 13, 15, 17, 19, 21 or 23, and rabbit β-globin poly A.

[0114] In another embodiment, a two-vector inducible system is provided. The first rAAV includes 12XZFHD, a minimal IL2 promoter, a coding sequence of a GLP-2 fusion protein of SEQ ID NO: 13, 15, 17, 19, 21 or 23, and rabbit beta globin polyA. A stuffer sequence may be included to increase the packaging size of the vector. The second rAAV includes a polynucleotide comprising a CMV promoter, an intron, and the activation domain is a FKBP12-rapamycin binding (FRB) domain of a human FKBP12-rapamycin associated protein (FRAP) fused to the carboxyl terminus of a p65 subunit of NF-κB from humans, an IRES, a ZFHD1 DNA binding domain, and polyA.

[0115] In one embodiment, an expression cassette is provided, the expression cassette comprising a polynucleotide comprising a CB7 promoter, a chicken β-actin intron, a coding sequence for a fusion protein of SEQ ID NO: 13, 15, 17, 19, 21 or 23, and rabbit globin poly A. In one embodiment, the expression cassette is an expression cassette found in SEQ ID NO: X, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity thereto. In another embodiment, a vector genome is provided, wherein SEQ ID NO: XX, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity thereto, is flanked by 5' and 3' AAV ITRs.

[0116] In another embodiment, an expression cassette is provided, comprising a polynucleotide comprising a CB7 promoter, a chicken β-actin intron, a coding sequence for a fusion protein of SEQ ID NO: 13, 15, 17, 19, 21 or 23, and rabbit globin poly A.

[0117] Viral vectors

[0118] On the other hand, a viral vector comprising an expression cassette as described herein is provided. In certain embodiments of the viral vectors described herein, the viral vector is an adeno-associated virus (AAV) viral vector or a recombinant AAV (rAAV). As used herein, the term "recombinant AAV" or "rAAV" refers to naturally occurring adeno-associated viruses, adeno-associated viruses available to those skilled in the art and / or obtainable in view of the compositions and methods described herein, and artificial AAV. Adeno-associated virus (AAV) viral vectors are AAV DNA enzyme-resistant particles with AAV protein capsids, wherein the expression cassette for delivery to target cells is packaged with AAV reverse terminal repeats (ITRs) (together referred to as "vector genomes") flanked by AAV. The AAV capsid is composed of 60 capsid (cap) protein subunits VP1, VP2, and VP3, which are arranged in an icosahedral symmetric manner, with a ratio of about 1:1:10 to 1:1:20, depending on the selected AAV. Various AAVs can be selected as the source of the capsid of the AAV viral vector as identified above. In one embodiment, the AAV capsid is an AAVrh91 capsid or a variant thereof. In certain embodiments, the capsid protein is designated by a number or a combination of numbers and letters following the term "AAV" in the rAAV vector name. Unless otherwise indicated, the AAV capsids, ITRs, and other selected AAV components described herein can be readily selected from any AAV, including but not limited to AAVs identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVhu37, AAVrh32.33, AAV Anc80, AAV10, AAV11, AAV12, AAVrh8, AAVrh74, AAV-DJ8, AAV-DJ, AAVhu.37, AAVrh.64R1, and AAVhu68. See, e.g., U.S. Published Patent Application No. 2007-0036760-A1; U.S. Published Patent Application No. 2009-0197338-A1; EP 1310571. See also WO 2003 / 042397 (AAV7 and other simian AAVs), U.S. Pat. No. 7790449 and U.S. Pat. No. 7282199 (AAV8), WO 2005 / 033321 and US 7,906,111 (AAV9) and WO 2006 / 110689 and WO2003 / 042397 (rh.10), WO 2005 / 033321, WO 2018 / 160582 (AAVhu68), which are incorporated herein by reference.Other suitable AAVs may include, but are not limited to, AAVrh90 [PCT / US20 / 30273 filed April 28, 2020], AAVrh91 [PCT / US20 / 030266 filed April 28, 2020, now published WO 2020 / 223231 on November 5, 2020], AAVrh92, AAVrh93, AAVrh91.93 [PCT / US20 / 30281 filed April 28, 2020], all of 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 October 21, 2019 and U.S. Provisional Patent Application No. 63 / 025,753 filed on May 15, 2020, which describe AAV3B.AR2.01, AAV3B.AR2.02, AAV3B.AR2.03, AAV3B.AR2.04, AAV3B.AR2.05, AAV3B.AR2.06, AAV3B.AR2.07, AAV3B.AR2.08, AAV3B.AR2.09, AAV3B.AR2.10, AAV3B.AR2.11, AAV3B.AR2.12, AAV3B.AR2.13, AAV3B.AR2.14, AAV3B.AR2.15, AAV3B.AR2.16, AAV3B.AR2.17, AAV3B.AR2.18, AAV3B.AR2.19, AAV3B.AR2 .05, AAV3B.AR2.06, AAV3B.AR2.07, AAV3B.AR2.08, AAV3B.AR2.10, AAV3B.AR2.11, AAV3B.AR2.12, AAV3B.AR2.13, AAV3B.AR2.14, AAV3B.AR2.15, AAV3B.AR2.16 or AAV3B.AR2.17, all of which are incorporated herein by reference. See also International Patent Application No. PCT / US21 / 45945 filed on August 13, 2021, U.S. Provisional Patent Application No. 63 / 065,616 filed on August 14, 2020, and U.S. Provisional Patent Application No. 63 / 109,734 filed on November 4, 2020, all of which are incorporated herein by reference in their entirety. These documents also describe other AAV capsids that can be selected for the production of rAAV and are incorporated by reference. Among the AAVs isolated or engineered from humans or non-human primates (NHPs) and well characterized, human AAV2 was the first AAV to be developed as a gene transfer vector; it has been widely used in efficient gene transfer experiments in different target tissues and animal models.

[0119] As used herein, with respect to AAV, the term "variant" means any AAV sequence derived from a known AAV sequence, including an AAV sequence with a conservative amino acid substitution and an AAV sequence sharing at least 90%, at least 95%, at least 97%, at least 99% or higher sequence identity with an amino acid or nucleic acid sequence. In another embodiment, the AAV capsid comprises a variant, which may include up to about 10% change with any described or known AAV capsid sequence. That is, the AAV capsid shares about 90% identity to about 99.9% identity, about 95% to about 99% identity or about 97% to about 98% identity with the AAV capsid provided herein and / or known in the art. In one embodiment, the AAV capsid shares at least 95% identity with the AAV capsid. When determining the identity percentage of the AAV capsid, any variable protein (e.g., vp1, vp2 or vp3) can be compared.

[0120] In one embodiment, the viral vector is an rAAV having a capsid of AAV8 or a functional variant thereof. In one embodiment, the viral vector is an rAAV having a capsid of AAVrh91 or a functional variant thereof. In one embodiment, the viral vector is an rAAV having a capsid of AAV3.AR.2.12 or a functional variant thereof. In one embodiment, the viral vector is an rAAV having a capsid selected from AAV9, AAVrh64R1, AAVhu37 or AAVrh10.

[0121] In certain embodiments, a novel isolated AAVrh91 capsid is provided. A nucleic acid sequence encoding the AAVrh91 capsid is provided in SEQ ID NO:31, and an encoded amino acid sequence is provided in SEQ ID NO:32. A rAAV is provided herein, comprising at least one of vp1, vp2, and vp3 of AAVrh91 (SEQ ID NO:32). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVrh91 (SEQ ID NO:31). In yet another embodiment, a nucleic acid sequence encoding the AAVrh91 amino acid sequence is provided in SEQ ID NO:19, and an encoded amino acid sequence is provided in SEQ ID NO:32. Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVrh91eng (SEQ ID NO:19). In certain embodiments, vp1, vp2 and / or vp3 are full-length capsid proteins of AAVrh91 (SEQ ID NO: 32). In other embodiments, vp1, vp2 and / or vp3 have N-terminal and / or C-terminal truncations (e.g., truncations of about 1 to about 10 amino acids).

[0122] In certain embodiments, the AAVrh91 capsid is characterized by one or more of the following: (1) AAVrh91 capsid proteins comprising: a heterogeneous population of AAVrh91 vp1 proteins selected from the group consisting of vp1 proteins produced by expression of a nucleic acid sequence encoding a predicted amino acid sequence of 1 to 736 of SEQ ID NO:32, vp1 proteins produced by expression of SEQ ID NO:31, or vp1 proteins produced by a nucleic acid sequence at least 70% identical to SEQ ID NO:31 encoding a predicted amino acid sequence of 1 to 736 of SEQ ID NO:32, a heterogeneous population of AAVrh91 vp2 proteins selected from the group consisting of vp2 proteins produced by expression of a nucleic acid sequence encoding at least about amino acids 138 to 736 of SEQ ID NO:32, vp2 proteins produced by a sequence comprising at least nucleotides 412 to 2208 of SEQ ID NO:31, or vp2 proteins produced by a nucleic acid sequence at least 70% identical to SEQ ID NO:31 encoding a predicted amino acid sequence of 1 to 736 of SEQ ID NO:32. NO:32, a heterogeneous population of AAVrh91 vp3 proteins selected from the group consisting of: vp3 proteins produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:32, vp3 proteins produced from a sequence comprising at least nucleotides 607 to 2208 of SEQ ID NO:31, or vp3 proteins produced from a nucleic acid sequence at least 70% identical to at least nucleotides 607 to 2208 of SEQ ID NO:31 encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:32; and / or (2) a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:32, a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:32, NO:32, and a heterogeneous population of vp3 proteins, the product of a nucleic acid sequence encoding at least about amino acids 138 to 736 of SEQ ID NO:32, and the product of a nucleic acid sequence encoding at least amino acids 203 to 736 of SEQ ID NO:32, wherein: the vp1, vp2 and vp3 proteins contain a subpopulation having amino acid modifications, the amino acid modifications comprising at least two highly deamidated asparagines (N) in the asparagine-glycine pairs in SEQ ID NO:32, and optionally further comprising a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change;and (B) a vector genome in an AAVrh91 capsid, the vector genome comprising a nucleic acid molecule comprising an AAV inverted terminal repeat sequence and a non-AAV nucleic acid sequence encoding a product operably linked to a sequence directing expression of the product in a host cell.;

[0123] In certain embodiments, the AAVrh91 capsid is characterized by one or more of the following: (1) AAVrh91 capsid proteins comprising: a heterogeneous population of AAVrh91 vp1 proteins selected from the group consisting of vp1 proteins produced by expression of a nucleic acid sequence encoding a predicted amino acid sequence of 1 to 736 of SEQ ID NO:32, vp1 proteins produced by expression of SEQ ID NO:19, or vp1 proteins produced by a nucleic acid sequence at least 70% identical to SEQ ID NO:19 encoding a predicted amino acid sequence of 1 to 736 of SEQ ID NO:32, a heterogeneous population of AAVrh91 vp2 proteins selected from the group consisting of vp2 proteins produced by expression of a nucleic acid sequence encoding at least about amino acids 138 to 736 of SEQ ID NO:32, vp2 proteins produced by a sequence comprising at least nucleotides 412 to 2208 of SEQ ID NO:19, or vp2 proteins produced by a nucleic acid sequence at least 70% identical to SEQ ID NO:19 encoding a predicted amino acid sequence of SEQ ID NO:32. NO:32, a heterogeneous population of AAVrh91 vp3 proteins selected from the group consisting of: vp3 proteins produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:32, vp3 proteins produced from a sequence comprising at least nucleotides 607 to 2208 of SEQ ID NO:19, or vp3 proteins produced from a nucleic acid sequence at least 70% identical to at least nucleotides 607 to 2208 of SEQ ID NO:19 encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:32; and / or (2) a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:32, a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:32, NO:32, and a heterogeneous population of vp3 proteins, the product of a nucleic acid sequence encoding at least about amino acids 138 to 736 of SEQ ID NO:32, and the product of a nucleic acid sequence encoding at least amino acids 203 to 736 of SEQ ID NO:32, wherein: the vp1, vp2 and vp3 proteins contain a subpopulation having amino acid modifications, the amino acid modifications comprising at least two highly deamidated asparagines (N) in the asparagine-glycine pairs in SEQ ID NO:32, and optionally further comprising a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change;and (B) a vector genome in an AAVrh91 capsid, the vector genome comprising a nucleic acid molecule comprising an AAV inverted terminal repeat sequence and a non-AAV nucleic acid sequence encoding a product operably linked to a sequence directing expression of the product in a host cell.;

[0124] In certain embodiments, the AAVrh91 vp1, vp2, and vp3 proteins contain a subpopulation with amino acid modifications that include at least two highly deamidated asparagines (N) in the asparagine-glycine pairs in SEQ ID NO: 32, and optionally further include a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change. Relative to the numbering of SEQ ID NO: 32, high levels of deamidation are observed at NG pairs N57, N383, and / or N512. Deamidation has been observed at other residues. In certain embodiments, AAVrh91 can have other deamidated residues, e.g., typically less than 10%, and / or can have other modifications comprising phosphorylation (e.g., where present, in the range of about 2% to about 30%, or about 2% to about 20%, or about 2% to about 10%) (e.g., at S149) or oxidation (e.g., at one or more of -W22, -M211, W247, M403, M435, M471, W478, W503, -M537, -M541, -M559, -M599, M635, and / or W695). Optionally, W can be oxidized to kynurenine.

[0125] Table A - AAVrh91 deamidation

[0126] AAVrh91 deamidation based on VP1 numbering Deamidation% N57+ deamidation 65-90, 70-95, 80-95, 75-100, 80-100 or 90-100 N94+ deamidation 2-15 or 2-5 N303+ deamidation 2-15 or 5-10 N383+deamidation 65-90, 70-95, 80-95, 75-100, 80-100 or 90-100 N497+deamidation 2-15 or 5-10 N512+ deamidation 65-90, 70-95, 80-95, 75-100, 80-100 or 90-100 ~N691+deamidation 2-15, 2-10 or 5-10

[0127] In certain embodiments, the AAVrh91 capsid is modified in one or more of the positions identified in the above table within the range provided as determined using mass spectrometry using trypsin. In certain embodiments, one or more of the positions modified as described herein or a glycine after the N. The residue numbers are based on the AAVrh91 sequence provided herein. See SEQ ID NO: 32.

[0128] In certain embodiments, the AAVrh91 capsid comprises: a heterogeneous population of vp1 proteins, wherein the vp1 proteins are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:32; a heterogeneous population of vp2 proteins, wherein the vp2 proteins are the product of a nucleic acid sequence encoding the amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:32; and a heterogeneous population of vp3 proteins, wherein the vp3 proteins are the product of a nucleic acid sequence encoding at least amino acids 203 to 736 of SEQ ID NO:32.

[0129] In certain embodiments, the modified AAVrh91 nucleic acid sequence is used to generate a mutant rAAV having a capsid that is less deamidated than the native AAVrh91 capsid. Such mutant rAAV may have reduced immunogenicity and / or increased stability during storage, particularly when stored in suspension form.

[0130] In one aspect, a recombinant AAV (rAAV) is provided. The rAAV comprises an AAV capsid from adeno-associated virus rh91 and a vector genome packaged in the AAV capsid, wherein the vector genome comprises an AAV inverted terminal repeat sequence (ITR), a coding sequence of a GLP-2 receptor agonist of SEQ ID NO: 14, and a regulatory sequence for directing the expression of the GLP-2 receptor agonist.

[0131] In certain embodiments, the AAV68 capsid is further characterized by one or more of the following. The AAV hu68 capsid proteins include: an AAVhu68 vp1 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 736 of SEQ ID NO:55, a vp1 protein produced from SEQ ID NO:53 or 54, or a vp1 protein produced from a nucleic acid sequence that is at least 70% identical to SEQ ID NO:53 or 54 encoding the predicted amino acid sequence of 1 to 736 of SEQ ID NO:55; an AAVhu68 vp2 protein produced by expression from a nucleic acid sequence encoding at least about amino acids 138 to 736 of SEQ ID NO:55, a vp2 protein produced from a sequence including at least nucleotides 412 to 2211 of SEQ ID NO:53 or 54, or a vp2 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 412 to 2211 of SEQ ID NO:53 or 54 encoding the predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:55; and / or an AAVhu68 vp3 protein produced by expression from a nucleic acid sequence encoding at least about amino acids 138 to 736 of SEQ ID NO:55, a vp3 protein produced from a sequence including at least nucleotides 412 to 2211 of SEQ ID NO:53 or 54, or a vp3 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 412 to 2211 of SEQ ID NO:53 or 54 encoding the predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:55. AAVhu68 vp3 protein produced by expressing a nucleic acid sequence that encodes the predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:55, a vp3 protein produced by a sequence that includes at least nucleotides 607 to 2211 of SEQ ID NO:53 or 54, or a vp3 protein produced by a nucleic acid sequence that is at least 70% identical to at least nucleotides 607 to 2211 of SEQ ID NO:53 or 54 that encodes the predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:55.

[0132] Additionally or alternatively, an AAV capsid is provided, comprising a heterogeneous population of vp1 proteins that optionally include valine at position 157, a heterogeneous population of vp2 proteins that optionally include valine at position 157, and a heterogeneous population of vp3 proteins, wherein based on the numbering of the vp1 capsid of SEQ ID NO:55, at least one subpopulation of vp1 and vp2 proteins includes valine at position 157 and optionally further includes glutamic acid at position 67. Additionally or alternatively, an AAVhu68 capsid is provided, comprising: a heterogeneous population of vp1 proteins, wherein the vp1 proteins are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:55; a heterogeneous population of vp2 proteins, wherein the vp2 proteins are the product of a nucleic acid sequence encoding the amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:55; and a heterogeneous population of vp3 proteins, wherein the vp3 proteins are the product of a nucleic acid sequence encoding at least amino acids 203 to 736 of SEQ ID NO:55, wherein: the vp1, vp2 and vp3 proteins contain subpopulations having amino acid modifications.

[0133] AAVhu68 vp1, vp2 and vp3 proteins are generally expressed as alternative splice variants encoded by the same nucleic acid sequence encoding the full-length vp1 amino acid sequence (amino acids 1 to 736) of SEQ ID NO: 55. Optionally, the vp1 coding sequence alone is used to express the vp1, vp2 and vp3 proteins. Alternatively, this sequence can be co-expressed with one or more of the following: a nucleic acid sequence encoding the AAVhu68 vp3 amino acid sequence of SEQ ID NO:55 (about aa 203 to 736), wherein the AAVhu68 vp3 amino acid sequence is without the vp1 unique region (about aa 1 to about aa 137) and / or the vp2 unique region (about aa 1 to about aa 202) or the complementary strands thereto, i.e., the corresponding mRNA or tRNA (about nt 607 to about nt 2211 of SEQ ID NO:53 or 54) or a sequence that is at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99%) identical to SEQ ID NO:53 or 54, wherein SEQ ID NO:53 or 54 encodes aa 203 to 736 of SEQ ID NO:55. Additionally or alternatively, the vp1 encoding and / or vp2 encoding sequences can be co-expressed with one or more of the following: a nucleic acid sequence encoding the AAVhu68 vp2 amino acid sequence of SEQ ID NO:55 (approximately aa 138 to 736) without the vp1 unique region (approximately aa 1 to approximately 137) or the complementary strand thereto, i.e., the corresponding mRNA or tRNA (nt 412 to 2212 of SEQ ID NO:53 or 54) or a sequence that is at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:53 or 54, which encodes approximately aa 138 to 736 of SEQ ID NO:55.

[0134] As described herein, rAAVhu68 has rAAVhu68 capsids produced in a production system that expresses capsids from an AAVhu68 nucleic acid encoding the vp1 amino acid sequence of SEQ ID NO:55, and optionally an additional nucleic acid sequence encoding a vp3 protein that does not contain the unique regions of vp1 and / or vp2. rAAVhu68 produced from production using a single nucleic acid sequence vp1 produces a heterogeneous population of vp1 proteins, vp2 proteins, and vp3 proteins. More specifically, AAVhu68 capsids contain subpopulations within the vp1 protein, within the vp2 protein, and within the vp3 protein that have modifications of the predicted amino acid residues from SEQ ID NO:55. These subpopulations contain at least deamidated asparagine (N or Asn) residues. For example, the asparagine in the asparagine-glycine pair is highly deamidated.

[0135] In one embodiment, the AAVhu68 vp1 nucleic acid sequence has the sequence of SEQ ID NO:53 or 54 or a complementary strand thereto, such as a corresponding mRNA or tRNA. In certain embodiments, the vp2 and / or vp3 proteins can be expressed additionally or alternatively from a nucleic acid sequence different from vp1, such as to change the ratio of vp proteins in a selected expression system. In certain embodiments, a nucleic acid sequence encoding the AAVhu68 vp3 amino acid sequence of SEQ ID NO:55 (about aa 203 to 736) is also provided, wherein the AAVhu68 vp3 amino acid sequence lacks the vp1 unique region (about aa 1 to about aa 137) and / or the vp2 unique region (about aa 1 to about aa 202) or a complementary strand thereto, i.e., the corresponding mRNA or tRNA (about nt 607 to about nt 2211 of SEQ ID NO:53 or 54). In certain embodiments, nucleic acid sequences encoding the AAVhu68 vp2 amino acid sequence of SEQ ID NO:55 (about aa 138 to 736) are also provided, wherein the AAVhu68 vp2 amino acid sequence does not have the vp1 unique region (about aa 1 to about 137) or the complementary strand thereto, i.e., the corresponding mRNA or tRNA (nt 412 to 2211 of SEQ ID NO:53 or 54).

[0136] However, other nucleic acid sequences encoding the amino acid sequence of SEQ ID NO:55 can be selected for use in generating rAAVhu68 capsids. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO:53 or 54, or a sequence that is at least 70% to 99% identical, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to SEQ ID NO:53 or 54 encoding SEQ ID NO:55. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO:53 or 54, or a sequence that is at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to about nt 412 to about nt 2211 of SEQ ID NO:53 or 54 encoding the vp2 capsid protein (about aa 138 to 736) of SEQ ID NO:55. In certain embodiments, the nucleic acid sequence has a nucleic acid sequence of about nt 607 to about nt 2211 of SEQ ID NO:53 or 54, or a sequence that is at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to nt SEQ ID NO:53 or 54 encoding the vp3 capsid protein of SEQ ID NO:55 (about aa 203 to 736).

[0137]

[0138]

[0139] In certain embodiments, the AAVhu68 capsid is characterized as having a capsid protein in which at least 45% of the N residues are deamidated at at least one of positions N57, N329, N452, and / or N512, based on the numbering of the amino acid sequence of SEQ ID NO: 55. In certain embodiments, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the N residues at one or more of these NG positions (i.e., N57, N329, N452, and / or N512, based on the numbering of the amino acid sequence of SEQ ID NO: 55) are deamidated. In these and other embodiments, the AAVhu68 capsid is further characterized as having a population of proteins in which about 1% to about 20% of the N residues are deamidated at one or more of the following positions: N94, N253, N270, N304, N409, N477, and / or Q599, based on the numbering of the amino acid sequence of SEQ ID NO: 55. In certain embodiments, AAVhu68 includes at least a subset of vp1, vp2, and / or vp3 proteins that are deamidated at one or more of the following positions: N35, N57, N66, N94, N113, N252, N253, Q259, N270, N303, N304, N305, N319, N328, N329, N336, N409, N410, N452, N477, N515, N598, Q599, N628, N651, N663, N709, N735, based on numbering of the amino acid sequence of SEQ ID NO: 55, or a combination thereof. In certain embodiments, the capsid protein may have one or more amidated amino acids.

[0140] In another embodiment, a recombinant adeno-associated virus (rAAV) is provided having an AAVhu68 capsid and a vector genome, wherein (a) the AAV hu68 capsid comprises a heterogeneous population of AAVhu68 vp1 proteins, a heterogeneous population of AAVhu68 vp2 proteins; and a heterogeneous population of AAVhu68 vp3 proteins, wherein the heterogeneous AAVhu68 vp1, AAVhu68 vp2, and AAVhu68 vp3 proteins contain a subpopulation having an amino acid modification, the amino acid modification comprising SEQ ID NO: 1 as determined using mass spectrometry. NO:55 wherein at least two asparagines (N) in an asparagine-glycine pair in two or more of N57, N329, N452, N512 are deamidated to 50% to 100%, and optionally further comprises a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change wherein the deamidated asparagine is deamidated to aspartic acid, isoaspartic acid, an interconverted aspartic acid / isoaspartic acid pair, or a combination thereof, wherein the AAVhu68 capsid further comprises a subpopulation having one or more of:

[0141] at least 65% of the asparagine (N) in the asparagine-glycine pair located at position N57 of the vp1 protein based on the numbering of SEQ ID NO: 55 is deamidated;

[0142] The vp1, v2 and vp3 proteins have at least 75% deamidated N in the asparagine-glycine pair in position N329 based on the residue numbering of the amino acid sequence of SEQ ID NO:55;

[0143] The vp1, v2 and vp3 proteins have at least 50% of the N in the asparagine-glycine pair in position N452, based on the residue numbering of the amino acid sequence of SEQ ID NO: 55, deamidated; and / or

[0144] at least 75% of the N in the asparagine-glycine pair in position N512 of the vp1, v2 and vp3 proteins based on residue numbering of the amino acid sequence of SEQ ID NO: 55 is deamidated; and

[0145] The vector genome in the AAVhu68 capsid comprises a nucleic acid molecule comprising an AAV inverted terminal repeat sequence and a non-AAV nucleic acid sequence, wherein the non-AAV nucleic acid sequence encodes a GLP-2 fusion operably linked to a sequence as described herein, wherein the sequence directs expression of the GLP-2 fusion in a target cell.

[0146] In one embodiment, rAAV is scAAV. The abbreviation "sc" refers to self-complementary. "Self-complementary AAV" refers to a plasmid or vector having an expression cassette in which the coding region carried by the recombinant AAV nucleic acid sequence has been designed to form an intramolecular double-stranded DNA template. After infection, the second strand of cell-mediated synthesis is not waited for, but two complementary half scAAVs will associate to form a double-stranded DNA (dsDNA) that is easy to replicate and transcribe immediately. See, for example, DM McCarty et al., "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis" "Gene Therapy, (August 2001), Vol. 8, No. 16, pp. 1248-1254. Self-complementary AAVs are described, for example, in U.S. Pat. Nos. 6,596,535, 7,125,717, and 7,456,683, which are incorporated herein by reference in their entireties.

[0147] In one embodiment, the nucleic acid sequence encoding the GLP-2 construct described herein is engineered into any suitable genetic element, e.g., naked DNA, bacteriophage, transposon, cosmid, RNA molecule (e.g., mRNA), episome, etc., which transfers the GLP-2 sequence carried thereon to a host cell, e.g., for use in producing nanoparticles carrying DNA or RNA in packaging host cells, viral vectors, and / or host cells for delivery to a subject. In one embodiment, the genetic element is a plasmid. The selected genetic element can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion technology, high-speed DNA-coated pellets, viral infection, and protoplast fusion. Methods for preparing such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0148] As used herein, the term "host cell" may refer to a packaging cell line, wherein a vector (e.g., recombinant AAV or rAAV) is produced by a production plasmid. In an alternative, the term "host cell" may refer to any target cell that is desired to express a gene product as described herein. Therefore, "host cell" refers to a prokaryotic or eukaryotic cell (e.g., bacterial cell, human cell, or insect cell), which contains exogenous or heterologous DNA introduced into the cell by any means (e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion technology, high-speed DNA-coated pellets, viral infection, and protoplast fusion). In certain embodiments herein, the term "host cell" refers to a culture of cells of various mammalian species for in vitro evaluation of compositions described herein. In other embodiments herein, the term "host cell" refers to a cell for producing and packaging a viral vector or a recombinant virus. In a further embodiment, the term "host cell" is an intestinal cell, a small intestinal cell, a pancreatic cell, a liver cell.

[0149] As used herein, the term "target cell" refers to any target cell in which it is desired to express a heterologous nucleic acid sequence or protein. In certain embodiments, the target cell is a liver cell. In other embodiments, the target cell is a muscle cell.

[0150] In one embodiment, rAAV is provided, which includes a vector genome comprising an expression cassette, wherein the expression cassette includes a CMV promoter, an activation domain that is the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin associated protein (FRAP) fused to the carboxyl terminus of the p65 subunit of NF-κB from human, a GT2A_V1 peptide, a ZFHD1 DNA binding domain, three FKBP subunits, hGH poly A, 12XZFHD, a minimal sIL2 promoter, a coding sequence of a GLP-2 fusion protein of SEQ ID NO: 13, 15, 17, 19, 21 or 23, and rabbit β-globin polyA. In another embodiment, rAAV is provided, comprising a vector genome comprising an expression cassette, wherein the expression cassette comprises a CMV promoter, an activation domain which is the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin associated protein (FRAP) fused to the carboxyl terminus of the p65 subunit of NF-κB from human, a GT2A_V2 peptide, a ZFHD1 DNA binding domain, three FKBP subunits, hGH poly A, 12XZFHD, a minimal sIL2 promoter, a coding sequence of a GLP-2 fusion protein of SEQ ID NO: 13, 15, 17, 19, 21 or 23, and rabbit β-globin polyA.

[0151] In one embodiment, rAAV is provided, comprising a vector genome comprising an expression cassette, wherein the expression cassette comprises a CB7 promoter, a chicken β-actin intron, a coding sequence of a fusion protein of SEQ ID NO: 13, 15, 17, 19, 21 or 23, rabbit globin poly A and 3'ITR. In another embodiment, rAAV is provided, comprising a vector genome comprising an expression cassette, wherein the expression cassette comprises a CMV promoter, an activation domain is a FKBP12-rapamycin binding (FRB) domain of a human FKBP12-rapamycin associated protein (FRAP) fused to the carboxyl terminus of a p65 subunit of NF-κB from humans, an IRES, a ZFHD1 DNA binding domain, three FKBP subunits, hGH poly A, 8XZFHD, a minimal sIL2 promoter, a coding sequence of a GLP-2 fusion protein of SEQ ID NO: 13, 15, 17, 19, 21 or 23, and rabbit β-globin poly A.

[0152] In another embodiment, a two-vector inducible system is provided. In one embodiment, a first rAAV is provided, which includes a vector genome comprising an expression cassette, wherein the expression cassette includes 12XZFHD, a minimal IL2 promoter, a coding sequence of a GLP-2 fusion protein of SEQ ID NO: 13, 15, 17, 19, 21 or 23, and rabbit beta globin polyA. A stuffer sequence may be included to increase the packaging size of the vector. The second rAAV includes a vector genome comprising an expression cassette, wherein the expression cassette includes a polynucleotide, the polynucleotide includes a CMV promoter, an intron, and the activation domain is a FKBP12-rapamycin binding (FRB) domain of a human FKBP12-rapamycin associated protein (FRAP) fused to the carboxyl terminus of a p65 subunit of NF-κB from humans, an IRES, a ZFHD1 DNA binding domain, and polyA.

[0153] In one embodiment, the rAAV comprises a vector genome comprising an expression cassette comprising a polynucleotide comprising a CB7 promoter, a chicken β-actin intron, a coding sequence for a fusion protein of SEQ ID NO: 13, 15, 17, 19, 21 or 23, and rabbit globin poly A.

[0154] The minimum sequence required to package the expression cassette into the AAV virus particle is an AAV 5' and 3' ITR with the same AAV origin as the capsid or with a different AAV origin (to produce an AAV pseudotype). In one embodiment, the ITR sequence from AAV2 or a deleted version thereof (ΔITR) is adopted for convenience and to speed up regulatory approval. However, ITRs from other AAV sources can be selected. Preferably, the source of the ITR is the same as the source of the Rep protein, and the Rep protein is provided in trans for production. Typically, the expression cassette of the AAV vector includes an AAV 5'ITR, a GLP-2 fusion protein coding sequence and any regulatory sequences and an AAV 3'ITR. However, other configurations of these elements may be suitable. A shortened version of the 5'ITR referred to as ΔITR has been described, in which the D sequence and the terminal resolution site (trs) are deleted. In other embodiments, full-length AAV5' and 3'ITR are used.

[0155] In order to package the expression cassette into the virion, ITR is the only AAV component required in cis in the construct identical to the gene. In one embodiment, the coding sequence for replication (rep) and / or capsid (cap) is removed from the AAV genome and supplied in trans or supplied by a packaging cell line to produce an AAV vector. For example, as described above, the pseudotype AAV can contain ITRs from a source different from the source of the AAV capsid. In one embodiment, a chimeric AAV capsid can be utilized. Still other AAV components can be selected. The sources of such AAV sequences are described herein and can also be isolated or obtained from academic, commercial or public sources (e.g., the American Type Culture Collection, Manassas, VA, in Manassas, Virginia). AAV sequences can be synthesized or otherwise suitable by reference to disclosed sequences (e.g., in the literature or in, for example, obtained from publicly available sequences in databases such as those provided by

[0156] Methods for producing and isolating AAV viral vectors suitable for delivery to a subject are known in the art. [See, e.g., U.S. Pat. No. 7,790,449; U.S. Pat. No. 7,282,199; WO 2003 / 042397; WO 2005 / 033321; WO2006 / 110689; and US 7,588,772 B2]. In one system, a production cell line is transiently transfected with a construct encoding a transgene flanked by ITRs and a construct encoding rep and cap. In a second system, a packaging cell line that stably supplies rep and cap is transiently transfected with a construct encoding a transgene flanked by ITRs. In each of these systems, AAV virions are produced in response to infection with a helper adenovirus or herpes virus, thereby requiring separation of rAAV from contaminating viruses. Recently, systems have been developed that do not require infection with a helper virus to recover AAV - systems by which the desired helper functions are also provided in trans (i.e., adenovirus E1, E2a, VA and E4 or herpesvirus UL5, UL8, UL52 and UL29, and herpesvirus polymerase). In these newer systems, helper functions can be provided by transiently transfecting cells with constructs encoding the desired helper functions, or the cells can be engineered to stably contain genes encoding helper functions, the expression of which can be controlled at the transcriptional or post-transcriptional level. In yet another system, transgenes flanked by ITRs and rep / cap genes are introduced into insect cells by infection with baculovirus-based vectors. For a review of these production systems, see generally, e.g., Zhang et al., 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, the contents of each of which are incorporated herein by reference in their entirety. Methods for making and using these and other AAV production systems are also described in the following U.S. Patents, the contents of each of which are incorporated herein by reference in their entirety: 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823 and 7,439,065.See generally, e.g., Grieger and Samulski, 2005, "Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications," Adv. Biochem. Engin / Biotechnol. 99: 119-145; Buning et al., 2008, "Recent developments in adeno-associated virus vector technology," J. Gene Med. 10: 717-733; and references cited below, each of which is incorporated herein by reference in its entirety. Methods for constructing any embodiment of the present invention are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York City (2012). Similarly, methods for producing rAAV virions are well known, and the selection of an appropriate method is not a limitation of the present invention. See, for example, K. Fisher et al., (1993) Journal of Virology, 70:520-532 and U.S. Pat. No. 5,478,745.

[0157] The rAAV described herein includes a selected capsid with a vector genome packaged inside. The vector genome (or rAAV genome) includes 5' and 3' AAV inverted terminal repeats (ITRs), a polynucleotide sequence encoding a fusion protein, and a regulatory sequence that directs the polynucleotide sequence encoding the fusion protein to be inserted into the genome of the host cell. In one embodiment, the vector genome is a sequence shown in SEQ ID NO: 16 or a sequence that shares at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity thereto.

[0158] As used herein, "vector genome" refers to a nucleic acid sequence packaged inside a parvovirus (e.g., rAAV) capsid that forms a viral particle. Such nucleic acid sequences contain AAV reverse terminal repeats (ITRs). In the examples herein, the vector genome contains at least AAV 5'ITRs, coding sequences (i.e., transgenes) and AAV 3'ITRs from 5' to 3'. It is possible to select ITRs from AAV2 (an AAV different from the capsid source) or in addition to non-full-length ITRs. In certain embodiments, ITRs are from the same AAV source as the AAV that provides the rep function during production or trans-supplementation of AAV. In addition, other ITRs can be used, for example, self-complementary (scAAV) ITRs. Both single-stranded AAV and self-complementary (sc) AAV are covered in rAAV. Transgenics are nucleic acid coding sequences heterologous to the vector sequence, and the nucleic acid coding sequences encode polypeptides, proteins, functional RNA molecules (e.g., miRNA, miRNA inhibitors) or other gene products of interest. The nucleic acid coding sequence is operably linked to the regulatory components in a manner that allows the transgene to be transcribed, translated and / or expressed in the cells of the target tissue. Suitable components of the vector genome are discussed in more detail herein. In one example, a "vector genome" contains at least a vector-specific sequence from 5' to 3', a nucleic acid sequence encoding a GLP-2 construct, which is operably linked to a regulatory control sequence (which directs its expression in the target cell), wherein the vector-specific sequence can be a terminal repeat sequence that specifically packages the vector genome into a viral vector capsid or envelope protein. For example, the AAV inverted terminal repeat sequence is used for packaging into AAV and certain other parvovirus capsids.

[0159] The AAV sequence of the vector generally includes cis-acting 5' and 3' inverted terminal repeats (see, e.g., BJ Carter, Handbook of Parvoviruses, edited by P. Tijsser, CRC Press, pp. 155-168 (1990)). The length of the ITR sequence is about 145 bp. Preferably, substantially the entire sequence encoding the ITR is used in the molecule, but minor modifications to these sequences are allowed to some extent. The ability to modify these ITR sequences is within the technical scope of the art. (See, e.g., texts such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., Journal of Virology, 70:520-532 (1996)). An example of such a molecule employed in the present invention is a "cis-acting" plasmid containing a transgene, in which the selected transgene sequence and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences. In one embodiment, ITR is from AAV different from the AAV supplying capsid. In one embodiment, the ITR sequence from AAV2. However, ITR from other AAV sources can be selected. A shortened version of the 5'ITR referred to as ΔITR has been described, in which the D sequence and the terminal resolution site (trs) are deleted. In certain embodiments, the vector genome comprises a shortened AAV2 ITR of 130 base pairs, in which the external A element is missing. It is not desired to be bound by theory, but it is believed that during the amplification of the vector DNA using the internal (A') element as a template, the shortened ITR returns to the wild-type length of 145 base pairs. In other embodiments, full-length AAV 5' and 3'ITR are used. In the case where the source of ITR is from AAV2 and the AAV capsid is from another AAV source, the resulting vector can be referred to as pseudotyped. However, other configurations of these elements may be suitable.

[0160] Optionally, the GLP-2 constructs described herein may be delivered by viral vectors other than rAAV. Such other viral vectors may comprise any virus suitable for gene therapy, including but not limited to adenovirus; herpes virus; lentivirus; retrovirus, etc. Suitably, when one of these other vectors is produced, it is produced as a replication-defective viral vector.

[0161] "Replication-defective virus" or "viral vector" refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, wherein any viral genomic sequence also packaged in the viral capsid or envelope is replication-defective; that is, the synthetic or artificial viral particle cannot produce progeny virions but retains the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gutless"-containing only the transgene of interest, which is flanked by the signals required for amplification and packaging of the artificial genome), but these genes can be supplied during production. Therefore, this is considered safe for use in gene therapy because replication and infection by progeny virions will not occur unless the viral enzymes required for replication are present.

[0162] Compositions comprising viral vector constructs as described herein are also provided. Pharmaceutical compositions as described herein are designed to be delivered to a subject in need by any suitable route or combination of different routes. Direct delivery to the liver (optionally intravenously, by the hepatic artery or by transplantation), oral administration routes, inhalation administration routes, intranasal administration routes, intratracheal administration routes, intraarterial administration routes, intraocular administration routes, intravenous administration routes, intramuscular administration routes, subcutaneous administration routes, intradermal administration routes and other maternal administration routes. Viral vectors as described herein can be delivered in the form of a single composition or multiple compositions. Optionally, two or more different AAVs can be delivered, or multiple viruses [see, for example, WO 2011 / 126808 and WO 2013 / 049493]. In another embodiment, multiple viruses can contain different replication defective viruses (e.g., AAV and adenovirus). In one embodiment, administration is performed intramuscularly. In another embodiment, administration is performed intravenously.

[0163] Replication-defective viruses can be formulated with physiologically acceptable carriers for gene transfer and gene therapy applications. In the case of AAV viral vectors, quantification of genome copies ("GC") can be used as a measure of the dosage contained in the formulation. The number of genome copies (GC) of the replication-defective virus composition of the present invention can be determined using any method known in the art. A method for titrating the number of AAV GC is as follows: a purified AAV vector sample is first treated with DNA enzyme to eliminate unencapsidated AAV genomic DNA or contaminating plasmid DNA from the production process. The nuclease-resistant particles are then subjected to heat treatment to release the genome from the capsid. The released genome is then quantified by real-time PCR using a primer / probe set targeting a specific region of the viral genome (usually a polyA signal). Another suitable method for determining genome copies is quantitative PCR (qPCR), in particular optimized qPCR or digital droplet PCR [LockMartin et al., Human Gene Therapy Methods. 2014 Apr, 25(2): 115-125. doi: 10.1089 / hgtb.2013.131, published online ahead of edit on Dec. 13, 2013].

[0164] Furthermore, the replication-defective virus composition can be formulated in a dosage unit to contain about 1.0 x 10 9 GC to about 1.0x 10 15 In another embodiment, this amount of viral genome can be delivered in divided doses. In one embodiment, for an average human subject of about 70 kg, the dose is about 1.0 x 10 10 GC to about 3.0x 10 14 GC. In another embodiment, the dose is about 1 x 10 9 GC. For example, the dose of AAV virus can be about 1 x 10 10 GC, 1x 10 11 GC, about 5X 10 11 GC, about 1X 10 12 GC, about 5X10 12 GC or about 1X 10 13 In another embodiment, for human subjects, the dose is about 1.0 x 10 9 GC / kg to about 3.0x 10 14 GC / kg. In another embodiment, the dose is about 1 x 10 9 GC / kg. For example, the dose of AAV virus can be about 1 x 10 10GC / kg, 1x 10 11 GC / kg, about 5X10 11 GC / kg, about 1X 10 12 GC / kg, about 5X 10 12 GC / kg or about 1X 10 13 GC / kg. In one embodiment, the construct can be delivered in a volume of 1 μL to about 100 mL. As used herein, the term "dose" or "amount" can refer to the total dose or amount delivered to a subject during treatment or the dose or amount delivered by administering a single unit (or multiple units or divided doses).

[0165] The recombinant vector described above can be delivered to a host cell according to the disclosed method. The rAAV preferably suspended in a physiologically compatible carrier can be administered to a desired subject including a human. In view of the indications for which the transfer virus is directed, a person skilled in the art can easily select a suitable carrier. For example, a suitable carrier comprises saline, which can be formulated with various buffer solutions (e.g., phosphate-buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil and water. The selection of the carrier is not a limitation of the present invention.

[0166] In another embodiment, the composition comprises a carrier, a diluent, an excipient and / or an adjuvant. In certain embodiments, for administration to a human patient, the rAAV is suitably suspended in an aqueous solution containing saline, a surfactant, and a pharmaceutically and / or physiologically compatible salt or mixture of salts. Suitably, the formulation is adjusted to a physiologically acceptable pH, for example, in the range of pH 6 to 9, or pH 6.0 to 7.5, or pH 6.2 to 7.7, or pH 6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8, or about 7.0. In certain embodiments, the pH of the formulation is adjusted to about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, or about 7.8. In certain embodiments, the pH that may be desired is about 7.28 to about 7.32, about 6.0 to about 7.5, about 6.2 to about 7.7, about 7.5 to about 7.8, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, or about 7.8. In certain embodiments, for intravenous delivery, the pH that may be desired is about 6.8 to about 7.2. However, other pHs within the broadest range and these subranges may be selected for other delivery routes.

[0167] Optionally, in addition to rAAV and / or variants and carriers, the compositions of the invention may also contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, methyl paraben, ethyl vanillin, glycerol, phenol and p-chlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0168] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients may also be incorporated into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a host. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc. can be used to introduce the composition of the present invention into suitable host cells. Specifically, the transgene delivered by the rAAV vector can be formulated for delivery or encapsulation in lipid particles, liposomes, vesicles, nanospheres or nanoparticles, etc.

[0169] In one embodiment, the composition comprises a final formulation suitable for delivery to a subject, such as an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration. Optionally, one or more surfactants are present in the formulation. In another embodiment, the composition can be transported in the form of a concentrate that is diluted to be administered to a subject. In other embodiments, the composition can be lyophilized and reconstituted at the time of administration.

[0170] A suitable surfactant or combination of surfactants can be selected from non-toxic nonionic surfactants. In one embodiment, a difunctional block copolymer surfactant terminated with a primary hydroxyl group is selected, such as F68 [BASF], also known as Poloxamer 188, has a neutral pH and an average molecular weight of 8400. Other surfactants and other poloxamers may be selected, i.e., nonionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS15 (polyethylene glycol-15 hydroxystearate), LABRASOL (polyoxycaprylyl glyceride), polyoxyl 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol, and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are usually named with the letter "P" (for poloxamer), followed by three numbers: the first two digits x100 give the approximate molecular mass of the polyoxypropylene core, and the last digit x10 gives the percentage of the polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005% to about 0.001% of the suspension.

[0171] The dosage of the vector depends primarily on factors such as the condition being treated, the patient's age, weight, and health, and therefore may vary between patients. For example, a therapeutically effective human dose of a viral vector is typically in the range of about 25 microliters to about 1000 microliters to about 100 mL of a solution containing a concentration of about 1 x 10 9 Up to 1x 10 16 The amount of viral vector containing 10 genomes (to treat a subject with an average body weight of 70 kg) is preferably 1.0 x 10 12 GC to 1.0x 10 13GC. The compositions of the present invention can be delivered in a volume of about 0.1 μL to about 10 mL, including all numbers within the range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method. In one embodiment, the volume is about 50 μL. In another embodiment, the volume is about 70 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 125 μL. In another embodiment, the volume is about 150 μL. In another embodiment, the volume is about 175 μL. In yet another embodiment, the volume is about 200 μL. In another embodiment, the volume is about 250 μL. In another embodiment, the volume is about 300 μL. In another embodiment, the volume is about 450 μL. In another embodiment, the volume is about 500 μL. In another embodiment, the volume is about 600 μL. In another embodiment, the volume is about 750 μL. In another embodiment, the volume is about 850 μL. In another embodiment, the volume is about 1000 μL. In another embodiment, the volume is about 1.5 mL. In another embodiment, the volume is about 2 mL. In another embodiment, the volume is about 2.5 mL. In another embodiment, the volume is about 3 mL. In another embodiment, the volume is about 3.5 mL. In another embodiment, the volume is about 4 mL. In another embodiment, the volume is about 5 mL. In another embodiment, the volume is about 5.5 mL. In another embodiment, the volume is about 6 mL. In another embodiment, the volume is about 6.5 mL. In another embodiment, the volume is about 7 mL. In another embodiment, the volume is about 8 mL. In another embodiment, the volume is about 8.5 mL. In another embodiment, the volume is about 9 mL. In another embodiment, the volume is about 9.5 mL. In another embodiment, the volume is about 10 mL.

[0172] In some embodiments, the concentration of the recombinant adeno-associated virus carrying the nucleic acid sequence encoding the desired transgene under the control of the regulatory sequence is desirably about 10 7 and 10 14 The range of GC / mL was 1.

[0173] In one embodiment, the dose of rAAV in the composition is about 1.0 x 10 9 GC / kg body weight is about 1.5x10 13 GC / kg. In one embodiment, the dosage is about 1.0 x 10 10 GC / kg. In one embodiment, the dosage is about 1.0x10 11 GC / kg. In one embodiment, the dosage is about 1.0 x 10 12GC / kg. In one embodiment, the dosage is about 5.0 x 10 12 GC / kg. In one embodiment, the dosage is about 1.0 x 10 13 GC / kg. All ranges described herein are inclusive of the endpoints.

[0174] In one embodiment, the effective dose (total genome copies delivered) is about 10 7 Up to 10 13 In one embodiment, the total dose is about 10 8 In one embodiment, the total dose is about 10 9 In one embodiment, the total dose is about 10 10 In one embodiment, the total dose is about 10 11 In one embodiment, the total dose is about 10 12 In one embodiment, the total dose is about 10 13 In one embodiment, the total dose is about 10 14 In one embodiment, the total dose is about 10 15 genome copies.

[0175] It is desirable to utilize the lowest effective concentration of virus to reduce the risk of undesirable effects such as toxicity. Still other dosages and administration volumes within these ranges may be selected by the attending physician taking into account the physical condition of the subject (preferably a human) being treated, the age of the subject, the particular disorder, and the extent to which the disorder (if progressive) has developed.

[0176] In certain embodiments, the composition includes rAAV, and the rAAV includes an inducible GLP-2 agonist construct. In certain embodiments, the inducer or molecule is rapamycin or a rapamycin analog. In certain embodiments, the inducer is rapamycin, and at least one or more, at least two or more times, at least three or more times are applied after the composition including rAAV. In certain embodiments, rapamycin is applied at a dosage of at least about 4nM to at least about 40nM. In certain embodiments, the inducer (i.e., rapamycin) is applied at a dosage of at least about 0.1mg / kg to at least about 3.0mg / kg. In certain embodiments, the inducer (i.e., rapamycin) is applied at a dosage of at least about 0.5mg / kg to at least about 2.0mg / kg.

[0177] The viral vectors and other constructs described herein can be used to prepare a medicament for delivering a GLP-2 fusion protein construct to a subject in need thereof, supplying a GLP-2 with an increased half-life to a subject, and / or for treating SBS in a subject. Thus, in another aspect, a method for treating SBS is provided. The method comprises administering a composition as described herein to a subject in need thereof. In one embodiment, the composition comprises a viral vector comprising a GLP-2 fusion protein expression cassette, as described herein.

[0178] As used herein, the term "treatment" or "treating" is defined to encompass the administration of one or more compounds or compositions described herein to a subject for the purpose of improving one or more symptoms of short bowel syndrome. Thus, "treatment" may include one or more of reducing the progression of SBS in a given subject, reducing the severity of symptoms in a given subject, eliminating symptoms of the disease in a given subject, delaying the progression of the disease in a given subject, or increasing the efficacy of a therapy in a given subject.

[0179] In another embodiment, a method for treating SBS in a subject is provided. The method comprises administering a viral vector comprising a nucleic acid molecule comprising a sequence encoding a fusion protein as described herein. In one embodiment, the subject is a human.

[0180] The course of treatment may optionally involve repeated administration of the same viral vector (e.g., AAVrh91 vector) or different viral vectors (e.g., AAVrh91 and AAV3B.AR2.12). Still other combinations may be selected using the viral vectors described herein. Optionally, the compositions described herein may be combined in a regimen involving nutritional therapy (enteral or parenteral nutrition), medications (such as those used to control gastric acid, reduce diarrhea, or improve intestinal absorption), or GLP-2 analogs or surgery. In certain embodiments, the AAV vector and the combination therapy are administered substantially simultaneously. In other embodiments, the AAV vector is administered first. In other embodiments, the combination therapy is administered first.

[0181] In one embodiment, the composition is administered with an effective amount of a GLP-2 analog. Various commercially available GLP-2 products are known in the art, including but not limited to teduglutide, glipaglutide, and apraglutide.

[0182] In some embodiments, the combination of rAAV and GLP-2 analogs described herein reduces the GLP-2 analog dosage requirements of the subject compared to before treatment with the viral vector. Such dosage requirements can be reduced by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. The treating physician can determine the correct dose of the GLP-2 analog required by the subject. For example, the subject can be treated with a GLP-2 analog or other therapy, and the treating physician can continue the treatment after administering the AAV vector. Such GLP-2 analogs or other synergistic therapies can then be continued, reduced or interrupted as needed.

[0183] In one embodiment, a composition including an expression cassette, a vector genome, rAAV, or other compositions for gene therapy described herein is delivered in a single dose / patient. In one embodiment, a therapeutically effective amount of a composition described herein is delivered to a subject. As used herein, a "therapeutically effective amount" refers to the amount of an expression cassette or vector or a combination thereof that delivers and expresses the amount of GLP-2-Fc sufficient to achieve a therapeutic goal in a target cell. The therapeutically effective amount can be selected by a treating physician, or based on previously determined guidelines. For example, teduglutide can be injected subcutaneously daily at an initial dose of 0.05 mg / kg. For subjects with moderate to severe renal damage, the dose can be increased in increments of 0.025 mg / kg. rAAV can be delivered to the subject, and then supplemented with oral or subcutaneous teduglutide, or other drugs as needed, to achieve the equivalent of a desired dose of 0.05 mg / kg per day.

[0184] In certain embodiments, the goal of treatment is to improve or treat one or more of the symptoms of SBS.Therapeutically effective amounts can be determined based on animal models rather than human patients.

[0185] As used herein, the term "heterogeneous" or any grammatical variation thereof, when used to refer to vp capsid proteins, refers to a population consisting of non-identical elements, e.g., having vp1, vp2, or vp3 monomers (proteins) with different modified amino acid sequences. SEQ ID NO:32 provides the encoded amino acid sequence of the AAVrh91 vp1 protein. The term "heterogeneous" used in conjunction with the vp1, vp2, and vp3 proteins (alternatively referred to as isoforms) refers to differences in the amino acid sequences of the vp1, vp2, and vp3 proteins within the capsid. The AAV capsid contains subpopulations within the vp1 protein, within the vp2 protein, and within the vp3 protein that have modifications from predicted amino acid residues. These subpopulations contain at least some deamidated asparagine (N or Asn) residues. For example, certain subpopulations include at least one, two, three, or four highly deamidated asparagine (N) positions in asparagine-glycine pairs, and optionally further include other deamidated amino acids, wherein the deamidation results in an amino acid change and other optional modifications.

[0186] As used herein, unless otherwise specified, a "subpopulation" of vp proteins refers to a group of vp proteins that have at least one defined common property and consist of at least one group member to less than all members of a reference group. For example, unless otherwise specified, a "subpopulation" of vp1 proteins is at least one (1) vp1 protein and less than all vp1 proteins in an assembled AAV capsid. Unless otherwise specified, a "subpopulation" of vp3 proteins can be one (1) vp3 protein that is less than all vp3 proteins in an assembled AAV capsid. For example, vp1 proteins can be a subpopulation of vp proteins; vp2 proteins can be a separate subpopulation of vp proteins, and vp3 is still another subpopulation of vp proteins in an assembled AAV capsid. In another example, vp1, vp2, and vp3 proteins can contain subpopulations with different modifications, for example, at least one, two, three, or four highly deamidated asparagines, for example at asparagine-glycine pairs.

[0187] As used herein, a "stock" of rAAV refers to a population of rAAVs. Although its capsid protein is heterogeneous due to deamidation, rAAVs are expected to share the same vector genome with 5 in the stock. The stock can contain rAAVs with capsids having, for example, a heterogeneous deamidation pattern unique to the selected AAV capsid protein and the selected production system. The stock can be produced from a single production system or pooled from multiple runs of a production system. Various production systems can be selected, including but not limited to the production systems described herein. As used herein, the terms "GLP-2 construct", "GLP-2 expression construct" and synonyms include a combination of a GLP-2 sequence as described herein with a leader sequence and a fusion domain. The terms "GLP-2 construct", "GLP-2 expression construct" and synonyms can be used to refer to a nucleic acid sequence encoding a GLP-2 fusion protein or its expression product.

[0188] In the context of nucleic acid sequences, the terms "percent identity (%)", "sequence identity", "percent sequence identity" or "identical percentage" refer to the bases in two sequences that are identical when aligned to obtain correspondence. The length of the sequence identity comparison can exceed the full length of the genome, the full length of the gene coding sequence, or a fragment of at least about 100 to 150 nucleotides, or as required. However, it is also possible to expect identity between smaller fragments, such as at least about nine nucleotides, typically at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides. Multiple sequence alignment programs can also be used for nucleic acid sequences. Examples of such programs include "Clustal W", "CAP sequence assembly", "BLAST", "MAP" and "MEME", which can be accessed through Web servers on the Internet. Other sources of such programs are known to those skilled in the art. Alternatively, the vector NTI utility program is also used. Many algorithms known in the art can be used to measure nucleotide sequence identity, including those contained in the above-mentioned programs. As another example, the GCG 6.1 version of the program Fasta TM Compare polynucleotide sequences. Fasta TM The alignment and percent sequence identity of the best overlap region between the query sequence and the search sequence are provided. For example, the percent sequence identity between nucleic acid sequences can be calculated using Fasta as provided in GCG version 6.1 with its default parameters (font size 6 and NOPAM coefficients for the scoring matrix). TM Where determined, such procedures are incorporated herein by reference.

[0189] The term "highly conserved" means at least 80% identity, preferably at least 90% identity, and more preferably more than 97% identity. Identity can be readily determined by those skilled in the art using algorithms and computer programs known to those skilled in the art.

[0190] Unless otherwise specified by a higher range, it should be understood that the percentage of identity is the lowest level of identity and encompasses all higher levels of identity, up to 100% identity with a reference sequence. Unless otherwise specified, it should be understood that the percentage of identity is the lowest level of identity and encompasses all higher levels of identity, up to 100% identity with a reference sequence. For example, "95% identity" and "at least 95% identity" can be used interchangeably and include 95%, 96%, 97%, 98%, 99% and up to 100% identity with a reference sequence, and all fractions therebetween.

[0191] In the context of amino acid sequences, the terms "percent identity (%)", "sequence identity", "percent sequence identity" or "identical percent" refer to the residues in two sequences that are identical when aligned for correspondence. The percent identity of an amino acid sequence over the full length of a protein, a polypeptide, about 70 amino acids to about 100 amino acids or a peptide fragment thereof, or a corresponding nucleic acid sequence encoding sequence can be readily determined. Suitable amino acid fragments may be at least about 8 amino acids in length, and may be up to about 150 amino acids. Typically, when referring to "identity", "homology" or "similarity" between two different sequences, the "identity", "homology" or "similarity" is determined with reference to an "aligned" sequence. An "aligned" sequence or "alignment" refers to a plurality of nucleic acid sequences or protein (amino acid) sequences that typically contain corrections for missing or additional bases or amino acids compared to a reference sequence. Alignment is performed using any of a variety of publicly available or commercially available multiple sequence alignment programs. Sequence alignment programs are available for amino acid sequences, for example, "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME" and "Match-Box" programs. Typically, any of these programs is used with the default settings, although those skilled in the art can change these settings as needed. Alternatively, those skilled in the art can utilize another algorithm or computer program that provides at least the same level of identity or alignment as that provided by the reference algorithm and program. See, for example, J.D. Thomson et al., Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690 (1999).

[0192] It should be noted that the term "a" or "an" refers to one or more. Thus, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

[0193] The words "comprise", "comprises" and "comprising" are to be interpreted as inclusive rather than exclusive. The words "consist / consisting" and variations thereof are to be interpreted as exclusive rather than inclusive. Although various embodiments in the specification are presented using "comprising" language, in other cases, it is also intended to use the language of "consisting of" or "consisting essentially of" to explain and describe the relevant embodiments.

[0194] As used herein, "patient" or "subject" refers to a mammal, including humans, veterinary or farm animals, livestock or pets, and animals commonly used in clinical research. In one embodiment, the subject of these methods and compositions is a human. In another embodiment, the subject is not a feline.

[0195] As used herein, unless otherwise indicated, the term "about" means a variability of 10% (±10%, e.g., ±1, ±2, ±3, ±4, ±5, ±6, ±7, ±8, ±9, ±10, or values ​​therebetween) relative to a given reference.

[0196] In some cases, the term "E+#" or the term "e+#" is used to refer to an exponent. For example, "5E10" or "5e10" is 5x10 10 . These terms can be used interchangeably.

[0197] As used herein, the term "modulate" or variations thereof refers to the ability of a composition to inhibit one or more components of a biological pathway.

[0198] As used herein, "disease," "disorder," and "condition" are used interchangeably to refer to an abnormal state of a subject.

[0199] Unless otherwise defined in this specification, technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art and with reference to the public texts, which provide those skilled in the art with a general guide to many of the terms used in this application.

[0200] Reference to "one embodiment" or "another embodiment" when describing an embodiment does not mean that the referenced embodiment is mutually exclusive of another embodiment (eg, an embodiment described before the referenced embodiment) unless explicitly stated otherwise.

[0201] Examples

[0202] The following examples are provided to illustrate various embodiments of the present invention. The examples are not intended to limit the present invention in any way.

[0203] Example 1 - Construction of GLP-2 vector

[0204] Expression of GLP-2 agonists by adeno-associated virus (AAV) is challenging. GLP-2 is usually expressed from the glucagon precursor protein, which requires tissue-specific proteases and produces unwanted proteins. Expression systems using traditional heterologous signal peptides produce low expression. Expression systems using heterologous propeptides with universal protease cleavage sites produce foreign protein sequences that can serve as targets for T cells.

[0205] More specifically, a vector was constructed in which a leader sequence was placed upstream of one of several GLP-2 receptor agonist amino acid sequences, followed by a fusion domain. See, e.g., FIG. 3 . The resulting protein sequence was back-translated, followed by the addition of a kozak consensus sequence, a stop codon, and a cloning site. The sequence was generated and cloned into an expression vector containing a CMV promoter under the control of an inducible expression system. The expression construct was flanked by AAV2 ITRs. The resulting plasmid was called pAAV.Z12I.hGLP2.G2.Fc.rBG.

[0206] In a second construct, a sequence was generated and cloned into an expression vector containing a CB7 constitutive promoter.

[0207] Example 2 – In vitro expression

[0208] The following constructs were packaged into AAVrh91 vector by triple transfection as described previously.

[0209] AAVrh91.CB7.CI.hGLP-2-Fc.rBG

[0210] AAVrh91.CB7.CI.hGLP-2-SA.rBG

[0211] GLP-2-SA fusions were measured in the culture supernatant of HEK293 cells transfected with a vector for inducible human GLP-2-SA with a human thrombin signal sequence. GLP2-SA was identified by gel electrophoresis using spyroRuby staining. Figure 4A and Figure 4B .

[0212] Example 3 - Pilot expression in Rag1KO mice

[0213] Rag1KO female mice were injected with the vector AAVrh91.CB7.CI.hGLP-2-SA.rBG (1x10 11 GC / mouse) or AAVrh91.CB7.CI.hGLP-2-Fc.rBG (1x 10 11 GC / mouse). Serum was collected continuously by separating whole blood in serum separator tubes containing 5 μl of DPP-IV inhibitor (Millipore), and active GLP-2 expression and activity were determined as described above. Vehicle was injected on day 0, and mice were necropsied on day 56. Serum GLP-2 concentration (nM) was as follows Figure 5A Shown are estimates based on Fc fusion standards. Serum expression levels reached an increase on day 14 post-dosing.

[0214] The small intestine was weighed and measured at necropsy. The length and weight of the small intestine were significantly increased compared to control animals ( Figure 5B In addition, vehicle-treated intestines showed healthy intestinal epithelial cell growth compared to vehicle-treated animals. Figure 5C Serum citrulline levels (uM), a biomarker of intestinal surface area, were measured. Figure 5D ).

[0215] Expression of GLP-2 was compared to the albumin fusion construct. The Fc fusion construct showed greater GLP-2 expression ( Fig. 6A ). GLP.G2.Fc fusion showed significantly lower potency (EC50 = 13.6 nM) compared to hGLP (EC50 = 0.4 nM) ( Figure 6B ). These data suggest that AAV-mediated expression of a GLP-2 agonist shows a significant and sustained increase in intestinal surface area, which should have therapeutic effects in short bowel syndrome.

[0216] Example 4 - Dosage Study in NHPs

[0217] In this study, the expression of human GLP-2 in non-human primates (NHPs; ie, rhesus monkeys) was examined. Figure 7 An overview of the study is shown. Briefly, 1 x 10 13 (1e13)GC / kg(E185NG) and 5x 10 10 AAVrh91.CB7.CI.hGLP-2-Fc.rBG was administered to 2 NHPs at a dose of (1e10) GC / kg(BM239H).

[0218] GLP-2 expression and efficacy, liver enzymes, and citrulline levels were measured. Necropsy was performed on day 60. Fig. 8A Plasma levels of GLP-2-Fc fusion protein are shown. Figure 8B Serum citrulline, a biomarker of intestinal surface area, is shown. Figure 8C Detection of anti-GLP-2-Fc antibodies in NHP serum diluted 1: 100 is shown. AAV-mediated expression of GLP-2-Fc fusions showed a significant increase in intestinal surface area at high doses until anti-GLP-2-Fc antibodies reduced its expression, demonstrating its therapeutic efficacy for short bowel syndrome.

[0219] Example 5 - Long-term efficacy and safety study in Rag1KO mice

[0220] Rag1KO female mice were given 1x 10 10 GC / mouse, 3x 1010 GC / mouse or 1x 10 11 GC / mouse were treated by injection of vector AAVrh91.CB7.CI.hGLP-2-Fc.rBG. The study design is shown in Fig. 9A hGLP2-Fc levels and body weight were measured throughout the study. The highest vector dose showed the highest serum GLP2 levels ( Fig. 9B ), while the body weights were relatively consistent among all groups, with the vehicle group showing the lowest trend ( Fig. 9C , D). At necropsy on day 28, small intestine length and weight were measured. Although SI length showed a modest increase with increasing dose compared with vehicle ( Fig.9E ), but SI weight increased significantly in the two highest dose groups ( Fig.9F ).

[0221] All documents cited in this specification are incorporated herein by reference. U.S. Provisional Patent Application No. 63 / 316,219, filed on March 3, 2022, is incorporated herein by reference. Although the present invention has been described with reference to specific embodiments, it should be understood that modifications may be made without departing from the spirit of the present invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. A composition comprising a nucleic acid comprising a sequence encoding a fusion protein comprising a GLP-2 protein and an IgG4 Fc, an albumin, or an XTEN polypeptide, wherein the fusion protein has a sequence of SEQ ID NO: 13, 15, 17, 19, 21, or 23, or a sequence at least 99% identical thereto.

2. The composition of any one of claims 1 to 7, wherein the sequence encoding the fusion protein is SEQ ID NO: 14, 16, 18, 20, 22 or 24 or shares a sequence at least 75% identical thereto.

3. The composition, comprising a viral vector, wherein the viral vector comprises: (a) Adeno-associated virus (AAV) capsid; as well as (b) a vector genome packaged in the AAV capsid, the vector genome comprising AAV inverted terminal repeats (ITRs), comprising coding sequences for a GLP-2 protein and an IgG4 Fc, an albumin, or an XTEN polypeptide, wherein the fusion protein has a sequence of SEQ ID NO: 13, 15, 17, 19, 21, or 23, or a sequence at least 99% identical thereto, and a regulatory sequence directing expression of the fusion protein.

4. The composition of any one of claims 1 to 3, wherein the viral vector is rAAV having the AAV capsid of AAVrh91 or AAVhu68.

5. The composition according to one of claims 1 to 4, wherein the fusion protein is under the control of an inducible gene expression system.

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

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

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

9. according to the composition described in any one of claims 5 to 8, wherein the inducible gene expression system comprises (a) an activation domain comprising a transactivation domain and a FKBP12-rapamycin binding (FRB) domain of FKBP12-rapamycin associated protein (FRAP); (b) a DNA binding domain comprising a zinc finger homology domain (ZFHD) and one, two or three FK506 binding protein domain (FKBP) subunit genes; and (c) at least one copy of a ZFHD binding site followed by a minimal promoter, and (d) Regulatable promoter.

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

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

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

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

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

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

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

17. A composition according to any one of claims 5 to 16, comprising a regulatable promoter; an activation domain comprising a p65 transactivation domain and the FKBP12-rapamycin binding (FRB) domain of FKBP12-rapamycin associated protein (FRAP); a DNA binding domain comprising a zinc finger homology domain (ZFHD) and three FK506 binding protein binding domain (FKBP) subunit genes; 12 copies of the ZFHD binding site; and a sequence encoding a fusion protein comprising a GLP-2 analog and human IgG4 Fc.

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

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

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

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

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

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

24. A method for treating a subject with a metabolic disease, the method comprising delivering a recombinant adeno-associated virus (rAAV) to the subject, the rAAV having an AAV capsid from adeno-associated virus rh91 or hu68 and a vector genome packaged in the AAV capsid, the vector genome comprising AAV inverted terminal repeats (ITRs), a sequence encoding a fusion protein comprising a GLP-2 analog and human IgG4 Fc, and a regulatory sequence directing expression of the fusion protein.

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

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

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

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

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