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

Abstract

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

Description

[Technical field]

[0001] Electronic Sequence Listing Reference The contents of the electronic sequence listing (22-10018PCT_Seq-Listing.xml; size: 161 kb, and creation date: March 3, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0002] Short bowel syndrome (SBS) is a rare organ failure caused by surgical resection of the intestine for congenital or acquired reasons. SBS is the leading cause of intestinal failure (IF), a persistent reduction in intestinal function below the minimum required for absorption of macronutrients and / or water and electrolytes. Standard treatment for SBS / IF includes lifelong daily parenteral nutrition (PN), in which special forms of food are infused intravenously to replenish the patient's daily nutritional needs. In addition to the burden and risks associated with daily PN, PN can cause complications including infections, intestinal hypoplasia, liver disease, renal dysfunction, and bone disease, severely negatively impacting the quality of life of SBS patients. Medications used for SBS were intended to treat only symptoms associated with SBS / IF until the development and approval by the FDA and EMA of teduglutide, a glucagon-like peptide 2 (GLP-2) agonist.

[0003] GLP-2 is a 33-amino acid intestinal peptide hormone with a potent enterotrophic effect. GLP-2 indeed increases the length of the small intestine and the length of the intestinal epithelium villi, allowing effective absorption of nutrients even in SBS patients with a short remaining intestine, and reducing the volume and number of PN. However, native GLP-2 cannot be used as an effective injectable agent with its very short serum half-life due to cleavage by DPP IV. DPP IV-resistant long-acting GLP-2 agonists, including 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 for DPP IV resistance, which extends the serum half-life from 5 minutes to 1.5 hours. Subcutaneous teduglutide injections of 0.05 mg / kg per day result in a sustained and continuous reduction in PN volume throughout 2 years of treatment. 90% of patients achieved a >20% reduction in PN volume per week from baseline, and 70% gained >1 additional day of PN off per week from baseline with this treatment, with an excellent safety profile (Schwartz et al., 2016). Daily teduglutide injections are required for life to maintain the reduction in PN volume and number (Compher et al., 2011).

[0004] What is needed are improved treatments for SBS. Summary of the Invention

[0005] Provided herein are viral vectors encoding glucagon-like peptide 2 (GLP-2) receptor agonist fusion protein constructs. These viral vectors, in some embodiments, can achieve sustained expression and / or increased circulating half-life of GLP-2 receptor agonist in a subject compared to vector-mediated delivery of GLP-2 receptor agonist without a fusion partner. Methods of making and using such viral vectors are also provided.

[0006] In one aspect, a viral vector is provided that comprises a nucleic acid comprising a polynucleotide sequence encoding a fusion protein. The fusion protein comprises (a) a linker comprising a secretory signal peptide. (b) a glucagon-like peptide-1 (GLP-2) receptor agonist; and (c) a fusion domain comprising (i) an 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 (Oct 2016). In one embodiment, the vector is an adeno-associated virus vector.

[0007] 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 NOs: 13, 15, 17, 19, 21, or 23, and functional variants thereof.

[0008] In one embodiment, the fusion domain is a 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 a 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 a rhesus IgG4 Fc having the sequence of SEQ ID NO: 9, or a sequence sharing at least 90% identity therewith, or a functional variant thereof.

[0009] In another embodiment, the viral vector comprises an AAV capsid and a vector genome packaged within the AAV capsid, the vector genome comprising AAV inverted terminal repeats (ITRs), a polynucleotide sequence encoding a fusion protein, and regulatory sequences that direct expression of the fusion protein.

[0010] In another aspect, a pharmaceutical composition suitable for use in 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.

[0011] In yet another aspect, a use of the viral vectors described herein is provided for the manufacture of a medicament for treating a subject with short bowel syndrome, optionally, diabetes.

[0012] In another aspect, a method of treating a subject having short bowel syndrome is provided, the method comprising administering to the subject an effective amount of a viral vector or composition described herein.

[0013] Other aspects and advantages of the present invention will become readily apparent from the following detailed description of the invention. [Brief description of the drawings]

[0014] [Figure 1] 1 is a schematic diagram of proglucagon processing in vivo, showing the sequences of GLP-1 (SEQ ID NO: 4) and GLP-2 (SEQ ID NO: 1). [Diagram 2] 1 is a table showing the sequences of GLP-2 and analogs. [Figure 3A] Schematic diagram of the GLP2.G2.Fc construct, comprising an A2G substitution in the GLP2 amino acid sequence, a thrombin leader, and an IgG4 Fc fusion. [Figure 3B] Schematic diagram of the GLP2.G2.SA construct, which contains an A2G substitution in the GLP2 amino acid sequence, a thrombin leader, and a serum albumin fusion. [Figure 3C] Alignment of human (SEQ ID NO:1), cynomolgus monkey (SEQ ID NO:5), and mouse (SEQ ID NO:6) GLP2 sequences. [Figure 4A] 1 shows the elution profile of hGLP2-SA protein using albupure column purification. [Figure 4B]4B shows the gel with stained hGLP2-SA protein from FIG. 4A. [Figure 5A] Shows hGLP2-SA levels in RagKO mice administered AAVrh91.CI.hGLP2.G2.SA.rBG intramuscularly at 1e11 gc / mouse. [Figure 5B] 5B shows two graphs showing the small intestinal length and weight of mice treated with the vehicle and vector described in FIG. 5A. [Figure 5C] Intestinal histology of vehicle and vector treated mice is shown in Figure 5 A. The intestines of vector treated mice show healthier villi compared to vehicle treated mice. [Figure 6A] Figure 2 shows hGLP2-SA and hGLP2-Fc levels in 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 were higher with the Fc construct approximately 7 days into the study. [Figure 6B] Potency assays for hGLP2 and hGLP2.Fc are shown. The EC50 was determined to be 0.4 nM for hGLP2, while the EC for the fusion protein was determined to be 13.6 nM. [Figure 7] 1 shows the study design of the experiment described in Example 4. [Figure 8A] 1 shows the results of the experiment described in Example 4. Two NHPs received AAVrh91.CB7.CI.hGLP-2-Fc.rBG by intramuscular injection (IM) at a dose of 1×10 (1e13) GC / kg (E185NG) and 5×10 (1e10) GC / kg (BM239H). Plasma levels of GLP-2-Fc fusion protein are shown. [Figure 8B]1 shows the results of the experiment described in Example 4. Two NHPs received AAVrh91.CB7.CI.hGLP-2-Fc.rBG by intramuscular injection (IM) at a dose of 1×10 13 (1e13) GC / kg (E185NG) and 5×10 10 (1e10) GC / kg (BM239H). Serum citrulline, a biomarker of intestinal surface area, is shown. [Figure 8C] 1 shows the results of the experiment described in Example 4. Two NHPs received AAVrh91.CB7.CI.hGLP-2-Fc.rBG by intramuscular injection (IM) at a dose of 1×10 (1e13) GC / kg (E185NG) and 5×10 (1e10) GC / kg (BM239H). Detection of anti-GLP-2-Fc antibodies in NHP serum at a dilution of 1:100 is shown. [Figure 9A] 9A-9C show the results of the experiment described in Example 5. Rag1KO female mice were treated with injections of the vector AAVrh91.CB7.CI.hGLP-2-Fc.rBG at doses of 1×10 GC / mouse, 3×10 GC / mouse, or 1×10 GC / mouse via the IM route of administration. The study design is shown in FIG. 9A. [Figure 9B] 1 shows the results of the experiment described in Example 5. Rag1KO female mice were treated with injections of the vector AAVrh91.CB7.CI.hGLP-2-Fc.rBG via the IM route of administration at doses of 1×10 GC / mouse, 3×10 GC / mouse, or 1×10 GC / mouse. Serum GLP2 levels are shown. [Figure 9C] 1 shows the results of the experiment described in Example 5. Rag1KO female mice were treated with injections of the vector AAVrh91.CB7.CI.hGLP-2-Fc.rBG at doses of 1×10 10 GC / mouse, 3×10 10 GC / mouse, or 1×10 11 GC / mouse via the IM route of administration. Body weight over time is shown. [Figure 9D]1 shows the results of the experiment described in Example 5. Rag1KO female mice were treated with injections of the vector AAVrh91.CB7.CI.hGLP-2-Fc.rBG via the IM route of administration at doses of 1×10 GC / mouse, 3×10 GC / mouse, or 1×10 GC / mouse. Body weights on day 56 are shown. [Figure 9E] 1 shows the results of the experiment described in Example 5. Rag1KO female mice were treated with injections of the vector AAVrh91.CB7.CI.hGLP-2-Fc.rBG via the IM route of administration at doses of 1×10 GC / mouse, 3×10 GC / mouse, or 1×10 GC / mouse. The lengths of the small intestine (SI) are shown. [Figure 9F] Figure 1 shows the results of the experiment described in Example 5. Rag1KO female mice were treated with injections of the vector AAVrh91.CB7.CI.hGLP-2-Fc.rBG via the IM route of administration at doses of 1x1010GC / mouse, 3x1010GC / mouse, or 1x1011GC / mouse. SI weights are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Described herein are adeno-associated virus (AAV) vectors expressing GLP-2 agonists for treating SBS / IF patients with a single intramuscular vector administration. The transgene GLP-2 agonist includes an A2G mutation for DPP IV resistance, and a fusion with human IgG Fc domain or serum albumin for further serum half-life extension. In combination with these half-life extension technologies, the addition of thrombin propeptide allows for supratherapeutic expression of GLP-2 agonists at significantly lower vector doses (i.e., 1e10-1e12 GC / kg). Described herein are expression cassettes that express these proteins constitutively or in a controlled manner via administration of small molecule drugs that activate transcription of the GLP-2 agonist sequences.

[0016] These constructs are described for delivery to the subject in need of delivery via multiple routes, particularly by in vivo expression mediated by recombinant vectors such as rAAV vectors.Also provided is a method for using these constructs in a regimen in a subject in need of treating short bowel syndrome, and increasing the half-life of GLP-2 in the subject.In addition, a method for enhancing the activity of GLP-2 in a subject is provided.

[0017] GLP-2 fusion protein Post-translational processing of proglucagon, together with GLP-1, produces the 33 amino acid intestinal trophic peptide hormone glucagon-like peptide-2 (GLP-2). 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 intestine, at least by enhancing crypt cell proliferation and villi length, so as to increase the surface area of ​​the mucosal epithelium. These effects suggest that GLP-2 may be used to treat a wide variety of gastrointestinal conditions. However, administering GLP-2 alone to human patients has not been promising. GLP-2 has a short half-life that limits its use as a therapeutic agent, as rapid in vivo cleavage of GLP-2 by dipeptidyl peptidase IV (DPP-IV) results in an essentially inactive peptide. The amino acid sequence of human GLP-2 is HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ ID NO: 1).

[0018] As mentioned above, a GLP-2 analogue named teduglutide has been developed in which amino acid residue 2 (alanine) is replaced with glycine, the sequence of which is shown in SEQ ID NO: 2 HGDGSFSDEMNTILDNLAARDFINWLIQTKITD.

[0019] The present disclosure provides fusion proteins comprising 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 comprises a polynucleotide sequence encoding a fusion protein comprising (a) a leader sequence comprising a secretory 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 comprises a thrombin leader sequence, a GLP-2 receptor agonist, and a polynucleotide sequence encoding a fusion protein comprising a fusion domain. In another embodiment, the fusion protein comprises a thrombin leader, a GLP-2 receptor agonist, and an IgG Fc or functional variant thereof. In another embodiment, the fusion protein comprises a thrombin leader, a GLP-2 receptor agonist, and albumin or a functional variant thereof. In another embodiment, the fusion protein comprises a thrombin leader, two copies of a GLP-2 receptor agonist, and albumin or a functional variant thereof.

[0020] In some embodiments, the GLP-2 receptor agonist includes variants that retain the function of the wild-type sequence and may include up to about 10% variation from the GLP-2 nucleic acid or amino acid sequences described herein or known in the art. As used herein, "retaining function" means that the nucleic acid or amino acid functions in the same manner as the wild-type sequence, although not necessarily at the same level of expression or activity. For example, in one embodiment, the functional variant has increased expression or activity compared to the wild-type sequence. In another embodiment, the functional variant has decreased expression or activity compared to the wild-type sequence. In one embodiment, the functional variant has increased or decreased expression or activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the wild-type sequence.

[0021] Other GLP-2 analogs have been developed, including grepaglutide (SEQ ID NO: 3), apraglutide, and others shown below, and those shown in FIG. [Table 1]

[0022] In one embodiment, the fusion comprises a GLP-2 analog in combination with a heterologous sequence. A GLP-2 analog refers to 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 up to 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 human. For example, GLP-2 can be derived from non-human primates, dogs, cats, mice, rats, sheep, cows, horses, etc. In some embodiments, it is desirable to modify 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) is replaced with glycine, e.g., SEQ ID NO: 2. For example, in other embodiments, the GLP-2 analog comprises 1, 2, 3, 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 clinical profile of GLP-2 (A2G), including protection from DPP-4 inactivation. 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 that includes or consists 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.

[0023] A fusion protein may include a leader sequence, which may include a secretory signal peptide. As used herein, the term "leader sequence" refers to any N-terminal sequence of a polypeptide.

[0024] The leader sequence may be derived from the same species as that for which administration is ultimately intended, e.g., human. As used herein, the term "derived" or "derived from" means that the sequence or protein is sourced from or shares the same sequence as a protein or sequence sourced from a particular subject species. For example, a leader sequence "derived" from a human shares the same sequence (or a variant thereof as defined herein) as the same leader sequence expressed in a human. However, the specified nucleic acid or amino acid need not actually be sourced from a human. A variety of techniques are known in the art that can produce a desired sequence, including mutagenesis of similar proteins (e.g., homologs) or artificial production of a nucleic acid or amino acid sequence. A "derived" nucleic acid or amino acid retains the function of the same nucleic acid or amino acid in the species from which it is "derived", regardless of the actual source of the derived sequence.

[0025] The term "amino acid substitution" and its synonyms are intended to encompass the modification of an amino acid sequence by replacing an amino acid with another alternative amino acid. The substitution may be a conservative substitution. It may also be a non-conservative substitution. The term conservative, when referring to two amino acids, is intended to mean that the amino acids share a common property recognized by those of skill in the art. For example, amino acids with hydrophobic non-acidic side chains, amino acids with hydrophobic acidic side chains, amino acids with hydrophilic non-acidic side chains, amino acids with hydrophilic acidic side chains, and amino acids with hydrophilic basic side chains. The common property may also be amino acids with hydrophobic side chains, amino acids with aliphatic hydrophobic side chains, amino acids with aromatic hydrophobic side chains, amino acids with polar neutral side chains, amino acids with charged side chains, amino acids with charged acidic side chains, and amino acids with charged basic side chains. Both naturally occurring and non-naturally occurring amino acids are known in the art and may be used as alternative amino acids in embodiments. Methods for substituting amino acids are well known to those of skill in the art and include, but are not limited to, mutations in the nucleotide sequence encoding the amino acid sequence. References herein to "one or more" are intended to encompass, for example, one, two, three, four, five, six or more individual embodiments.

[0026] In one embodiment, the leader is a human thrombin (Factor II) sequence. In one embodiment, the thrombin leader has the sequence shown in SEQ ID NO: 7: MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRR, or a functional variant thereof having up to 1, 2, or 3 amino acid substitutions.

[0027] In one embodiment, functional variants of desired leaders include variants that retain the function of the wild-type sequence and may contain up to about 10% variation from the leader nucleic acid or amino acid sequences described herein or known in the art.

[0028] In some embodiments, the coding regions for both the propeptide and the GLP-2 peptide are incorporated into a single nucleic acid sequence with no linker between the coding sequences for the propeptide and the GLP-2 peptide.

[0029] The fusion protein further comprises a fusion domain, which in one embodiment is a human IgG Fc fragment or a functional variant thereof. Immunoglobulins typically have a long circulating half-life in vivo. By fusing the GLP-2 receptor agonist (and leader) to IgG Fc, the circulation time of the fusion protein is extended while maintaining the function of GLP-2. In another embodiment, the fusion domain is a rhesus I It is a gG Fc fragment or a functional variant thereof.

[0030] As used herein, the Fc portion of an immunoglobulin has the meaning commonly given to the term in the field of immunology. Specifically, the term refers to an antibody fragment that does not contain the two antigen-binding regions (Fab fragments) from the antibody. The Fc portion consists of the constant regions of the antibody from both heavy chains, which are associated by non-covalent interactions and disulfide bonds. The Fc portion includes the hinge region and may extend through the CH2 and CH3 domains to the c-terminus of the antibody. The Fc portion may further include one or more glycosylation sites. In one embodiment, the fusion domain is human IgG Fc. The four highly conserved subclasses, IgG1, IgG2, IgG3, and IgG4, differ in their constant regions, especially their hinge and upper CH2 domains. See Vidarsson et al, IgG Subclasses and Allotypes: From Structure to Effector Functions, Front Immunol. Oct. 2014; 5:520, which is incorporated herein by reference. The Fc domain can be derived from any human IgG, including human IgG1, human IgG2, human IgG3, or human IgG4. In one embodiment, the human IgG Fc is an IgG4 Fc. In one embodiment, the human IgG Fc is SEQ ID NO:8: AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG. In another embodiment, the human IgG Fc shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity to SEQ ID NO:8.

[0031] In another embodiment, the fusion domain is a Rhesus IgG Fc. The Fc domain can be derived from any Rhesus IgG, including Rhesus IgG1, Rhesus IgG2, Rhesus IgG3, or Rhesus IgG4. In one embodiment, the Rhesus IgG Fc is an IgG4 Fc. In one embodiment, the Rhesus IgG Fc is SEQ ID NO: 9: PPCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAQTKPRE RQFNSTYRVV SVLTVTHQDW LNGKEYTCKV SNKGLPAPIE KTISKAKGQP REPQVYILPP PQEELTKNQV SLTCLVTGFY PSDIAVEWES NGQPENTYKT TPPVLDSDGS YLLYSKLTVN KSRWQPGNIF TCSVMHEALH NHYTQKSLSV SPGK. In another embodiment, the rhesus IgG Fc shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity to SEQ ID NO: 9. In one embodiment, the rhesus IgG further comprises a hinge sequence.

[0032] In another embodiment, the fusion domain is human albumin or a functional variant thereof. In one embodiment, the human albumin is SEQ ID NO:10: DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVF DEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL. In another embodiment, the human albumin shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity to SEQ ID NO:10.

[0033] In another embodiment, the fusion domain is rhesus albumin or a functional variant thereof. In one embodiment, rhesus albumin is SEQ ID NO:11: 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 to SEQ ID NO:11.

[0034] The in vivo function and stability of the fusion protein of the present disclosure may be optimized by adding a small amount of peptide linker, for example, to prevent potentially undesirable domain interactions or for other reasons. Furthermore, the glycine-rich linker may provide some structural flexibility so that the GLP-2 analog moiety can productively interact with the GLP-2 receptor on target cells, such as pancreatic beta cells. Thus, the C-terminus of the GLP-2 analog and the N-terminus of the fusion domain of the fusion protein are fused via a linker in one embodiment. In one embodiment, the linker comprises 1, 1.5, or 2 repeats of a G-rich peptide linker having the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 12).

[0035] In one embodiment, the fusion protein comprises (a) a human thrombin leader, (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 at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. SEQ ID NO:13 MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRHGDGSDFSDEMNTILDNLAARDFINWLIQTKITDGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*

[0036] 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. SEQ ID NO:14: ATGGCTCACGTTCGGGGACTGCAGCTGCCTGGATGTCTGGCTCTTGCCGCTCTGTGTAGCCTGGTGCACAGCCAGCACGTGTTTCTGGCTCCTCAGCAAGCCAGATCACTGCTGCAGAGAGTTAGAAGGCACGGCGACGGCAGCTTCAGCGACGAGATGAACACCATCCTGGACAACCTGGCCGCCAGAGACTTCATCAACTGGCTGATCCAGACCAAGATCACCGACGGTGGCGGAGGCGGAGGATCTGGTGGTGGTGGATCTGGCGGCGGAGGTTCTGCCGAGTCTAAGTACGGACCTCCTTGTCCTCCCTGTCCTGCTCCAGAAGCTGCTGGCGGCCCATCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTCGACGTGTCCCAAGAGGATCCTGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCAAGAGAACCCCAGGTGTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGATGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCAGGTGGCAAGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAAAGCCTGAGCCTGTCTCTGGGCTAA

[0037] In one embodiment, the fusion protein comprises (a) a human thrombin leader, (b) a DPP-IV resistant variant of GLP-2 (A2G), a linker, and (c) human serum albumin. In one embodiment, the fusion protein has a sequence of SEQ ID NO: 15, or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. SEQ ID NO:15: MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRHGDGSDFSDEMNTILDNLAARDFINWLIQTKITDGGGGGGSGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAF *

[0038] 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. SEQ ID NO:16: AGGCCAAGGACGTGTTCCTGGGCATGTTTCTGTACGAGTACGCCCGCAGACACCCCGACTACTCTGTTGTGCTGCTGCTGAGACTGGCCAAAACCTACGAGACAACCCTGGAAAAGTGCTGTGCCGCCGCTGATCCTCACGAGTGTTACGCCAAGGTGTTCGACGAGTTCAAGCCACTGGTGGAAGAACCCCAGAACCTGATCAAGCAGAACTGCGAGCTGTTCGAGCAGCTGGGCGAGTACAAGTTCCAGAACGCCCTGCTCGTGCGGTACACCAAGAAGGTGCCCCAGGTTTCCACACCTACACTGGTTGAGGTGTCCCGGAACCTGGGCAAAGTGGGCAGCAAGTGTTGCAAGCACCCTGAGGCCAAGAGAATGCCCTGCGCCGAGGATTACCTGAGCGTGGTGCTGAATCAGCTGTGCGTGCTGCACGAGAAAACCCCTGTGTCCGACAGAGTGACCAAGTGCTGTACCGAGAGCCTGGTCAACAGACGGCCTTGCTTTAGCGCCCTCGAGGTGGACGAGACATACGTGCCCAAAGAGTTCAACGCCGAGACATTCACCTTCCACGCCGACATCTGTACCCTGAGCGAGAAAGAGCGGCAGATCAAGAAACAGACTGCCCTGGTGGAACTGGTCAAGCACAAGCCCAAGGCCACCAAAGAACAGCTGAAGGCCGTGATGGACGACTTCGCCGCCTTCGTGGAAAAGTGCTGCAAGGCCGACGACAAAGAGACCTGCTTCGCCGAAGAGGGCAAGAAACTGGTGGCCGCTTCTCAGGCTGCTCTGGGATAA

[0039] In one embodiment, the fusion protein comprises (a) a human thrombin leader, (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 at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. SEQ ID NO:17: MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRHGDGSFSDEMNTILDNLAARDFINWLIQTKITDGGGGGGSGGGSGGGGSSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATSPTSTEEGTSESATPESGPGSEPATS GSAPGTSTEPSEGSAPGGTSTEPSEGSAPGGTSTEPSEGSAPGGSPTSTEEGTSEGSAPGGTSESATPESGPGSEPATSGSETPPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSE GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG*

[0040] 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. SEQ ID NO:18:

[0041] In one embodiment, the fusion protein comprises (a) a rhesus thrombin leader, (b) a DPP-IV resistant variant of GLP-2 (A2G), a linker, and (c) a rhesus IgG Fc. In one embodiment, the fusion protein has a sequence of SEQ ID NO: 19, or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. SEQ ID NO:19 MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQALSLLQRVRRHGDGSFSDEMNTVLVDNLATRDFINWLIQTKITDGGGGGGGSGGGGSGGGGSAEFTPPCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAQTKPRERQFNSTYRVVSVLTVTHQDWLNGKEYTCKVSNKGLPAPIEKTISKAKGQPREPQVYILPPPQEELTKNQVSLTCLVTGFYPSDIAVEWESNGQPENTYKTTPPVLDSDGSYLLYSKLTVNKSRWQPGNIFTCSVMHEALHNHYTQKSLSVSPG*

[0042] 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. SEQ ID NO:20: ATGGCTCACGTTCGGGGACTGCAGCTGCCTGGATGTCTGG CTCTTGCCGCTCTGTGTAGCCTGGTGCACAGCCAGCATGTGTTTCTGGCTCCTCAGCAGGCCCTGAGCCTGCTGCAAAGAGTTAGAAGGCACGGCGACGGCAGCTTCAGCGACGAGATGAATACCGTGCTGGTGGACAACCTGGCCACCAGAGACTTCATCAACTGGCTGATCCAGACCAAGATCACCGACGGTGGTGGCGGAGGCGGAGGATCTGGTGGCGGTGGTTCTGGCGGTGGCGGATCTGCTGAGTTTACCCCTCCTTGTCCTCCCTGTCCTGCTCCAGAACTGCTCGGCGGACCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCAAGAGGATCCTGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCCAGACAAAGCCCAGAGAGCGGCAGTTCAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGACACACCAGGATTGGCTGAACGGCAAAGAGTACACCTGTAAAGTCTCCAACAAGGGCCTGCCTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAACCCCAGGTGTACATCCTGCCTCCACCTCAAGAGGAACTGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCACCGGCTTCTACCCTTCCGATATCGCCGTGGAGTGGGAGAGCAACGGACAGCCCGAGAACACCTACAAGACCACACCTCCAGTGCTGGACAGCGACGGCTCTTACCTGCTGTACTCCAAGCTGACAGTGAACAAGAGCCGGTGGCAGCCCGGCAACATCTTCACCTGTTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAAAGCCTGAGCGTGTCCCCTGGATAA

[0043] In one embodiment, the fusion protein comprises (a) a rhesus thrombin leader, (b) a DPP-IV resistant variant of GLP-2 (A2G), a linker, and (c) rhesus serum albumin. In one embodiment, the fusion protein has a sequence of SEQ ID NO: 21, or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. SEQ ID NO:21 MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQALSLLQRVRRHGDGSFSDEMNTVLVDNLATRDFINWLIQTKITGGGGGGGSGGGGSGGGGSDTHKSEVAHRFKDLGEEHFKGLVLVAFSQYLQQCPFEEHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLC TVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPPLVRPEVDVMCTAFHDNEATFLKKYLYEVARRHPYFYAPELLFFAARYKAAFAECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGDRAFKAWAVARLSQKFPKAEFAEVSKLVTDLTKVHTEC CHGDLLECADDRADLAKYMCENQDSISSKLKECCDKPLLEKSHCLAEVENDEMPADLPSLAADYVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVMLLLRLAKAYEATLEKCCAAADPHECYAKVFDEFQPLVEEPQNLVKQNCELFEQLGEYKFQNALLVRYTKK VPQVSTPTLVEVSRNLGKVGAKCCKLPEAKRMPCAEDYLSVVLNRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALELDEAYVPKAFNAETFTFHADMCTLSEKEKQVKKQTALVELVKHKPKATKEQLKGVMDNFAAFVEKCCKADDKEACFAEEGPKFVAASQAALA

[0044] In one embodiment, the sequence encoding the fusion protein is SEQ ID NO: The sequences are at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical. SEQ ID NO:22: TAAGCACAAGCCTAAGGCCACCAAAGAACAACTGAAGGGCGTGATGGACAACTTCGCCGCCTTTGTGGAAAAATGCTGCAAGGCCGACGACAAAGAGGCCTGCTTCGCAGAAGAGGGCCCTAAGTTTGTGCCGCCTCTCAAGCTGCTCTGGCTTAA

[0045] In one embodiment, the fusion protein comprises (a) a rhesus thrombin leader, (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 at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. SEQ ID NO:23 MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQALSLLQRVRRHGDGSFSDEMNTVLVDNLATRDFINWLIQTKITDGGGGGGSGGGSGGGSSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG STEPSEGSAPGTSEPSEGPGSPAGSPTSTEEGTSEPSEGPGSAPGTSESATPESGPGSESAPATSGSETPGTSESATPESGPGSESAPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSAP

[0046] 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. SEQ ID NO:24: ATGGCTCACGTTCGGGGACTGCAGCTGCCTGGATGTCTGGCTCTTGCCGCTCTGTGTAGCCTGGTGCACAGCCAGCATGTGTTTCTGGCTCCTCAGCAAGCCCTGAGCCTGCTGCAAAGAGTTAGAAGGCACGGCGACGGCAGCTTCAGCGACGAGATGAATACCGTGCTGGTGGACAACCTGGCCACCAGAGACTTCAT CAACTGGCTGATCCAGACCAAGATCACCGACGGTGGCGGAGGCGGAGGATCTGGTGGTGGTGGATCTGGCGGCGGAGGAAGTTCTCCTGCTGGCAGCCCTACAAGCACCGAGGAAGGCACAAGCGAGTCTGCCACACCTGAGTCTGGCCCTGGCACATCTACAGAGCCTAGCGAAGGATCTGCCCCAGGATCTCCTGCCG GAAGGCAGCCCTGCTGGCTCCCCAACGTCAACAGAAGAAGGTACTAGCACAGAGCCCAGCGAGGGTTCCGCTCCGGGAACTTCTGAATCTGCTACACCCGAGTCAGGTCCTGGTACAAGCGAGTCAGCTACGCCCGAAAGTGGACCTGGCACCTCAGAGTCTGCAACTCCTGAGAGCGGTCCAGGATCAGAACCAGCCACCTCTGGCTCTGAGACACCAGGTTCTGAGCCTGCAACGT CCGGAAGCGAAACACCAGGCAGTCCTGCCGGAAGTCCTACTTCAACCGAAGAGGGGACCTCTACAGAGCCATCAGAGGGCTCTGCACCGGGCACCTCAACAGAACCATCTGAAGGATCCGCACCGGGCTCTGAGCCTGCTACTAGTGGAAGCGAAACTCCTGGCACCAGTGAATCGCTACTCCCGAGTCTGGCCCGGGAACGTCTACTGAACCATCTGAGGGAAGTGCCCCAGGCTAA

[0047] If variants or fragments of the leader sequence, GLP-2 receptor agonist, or fusion domain are desired, the coding sequences of these peptides can be generated using site-directed mutagenesis of the wild-type nucleic acid sequence. Alternatively or additionally, web-based or commercially available computer programs and service-based companies can be used to back-translate the amino acid sequence into a nucleic acid coding sequence, including both RNA and / or cDNA. See, for example, backtranseq by EMBOSS, ebi.ac.uk / Tools / st / ; Gene Infinity (geneinfinity.org / sms- / sms_backtranslation.html); ExPasy (expasy.org / tools / ). In one embodiment, the RNA coding sequence and / or the cDNA coding sequence are designed for optimal expression in the subject species to which administration is ultimately intended, e.g., humans.

[0048] Coding sequences can be designed for optimal expression using codon optimization. Codon-optimized coding regions can be designed by a variety of different methods. This optimization can be done using methods available online, published methods, or companies that provide codon optimization services. One codon optimization method is described, for example, in International Patent Application Publication No. 2015 / 012924, which is incorporated herein by reference. Briefly, a nucleic acid sequence that encodes a product is modified with synonymous codon sequences. Preferably, the entire length of the product's open reading frame (ORF) is modified. However, in some embodiments, only a fragment of the ORF may be modified. By using one of these methods, a frequency can be applied to any given polypeptide sequence to produce a nucleic acid fragment of a codon-optimized coding region that encodes a polypeptide.

[0049] In addition to the leader sequences, GLP-2 receptor agonists, fusion domains, and fusion proteins provided herein, nucleic acid sequences encoding these polypeptides are provided. In one embodiment, a nucleic acid sequence encoding a GLP-2 peptide as described herein is provided. In some embodiments, this may include any nucleic acid sequence that encodes the GLP-2 sequence of SEQ ID NO:1. In another embodiment, this includes any nucleic acid that includes the GLP-2 sequence of SEQ ID NO:2.

[0050] In one embodiment, a nucleic acid sequence is provided that encodes a GLP-2 fusion protein as described herein, which in another embodiment includes any nucleic acid sequence encoding a GLP-2 fusion protein of SEQ ID NO: 13, 15, 17, 19, 21, or 23.

[0051] Expression cassette In another aspect, provided herein is an expression cassette comprising a nucleic acid encoding a GLP-2 fusion protein as described herein. "A nucleic acid molecule" refers to a nucleic acid molecule that includes a biologically useful nucleic acid sequence and a regulatory sequence operably linked thereto that directs or regulates the transcription, translation, and / or expression of the nucleic acid sequence (e.g., a gene cDNA encoding a protein, enzyme, or other useful gene product, mRNA, etc.) and its gene product. As used herein, "operably linked" sequences include both regulatory sequences (also referred to as elements) that are contiguous or non-contiguous with the nucleic acid sequence and regulatory sequences that act in trans or cis with the nucleic acid sequence. Such regulatory sequences typically include one or more of, for example, promoters, enhancers, transcription factors, transcription terminators, introns, sequences that increase translation efficiency (i.e., Kozak consensus sequences), efficient RNA processing signals such as slicing and polyadenylation sequences, sequences that stabilize cytoplasmic mRNA, e.g., Woodchuck Hepatitis Virus (WHP) post-translational regulatory element (WPRE), and TATA signal. An expression cassette may include, among other elements, regulatory sequences upstream (5') of a gene sequence, such as one or more of a promoter, enhancer, intron, etc., and enhancers, or regulatory sequences downstream (3') of the gene sequence, such as one or more of a 3' untranslated region (3'UTR) including a polyadenylation site. In certain embodiments, the regulatory sequences are operably linked to the nucleic acid sequence of a gene product, and the regulatory sequences are separated from the nucleic acid sequence of the gene product by an intervening nucleic acid sequence, i.e., a 5' untranslated region (5'UTR). In certain embodiments, an expression cassette includes nucleic acid sequences of one or more gene products. In some embodiments, the expression cassette may be a monocistronic or bicistronic expression cassette. In other embodiments, the term "transgene" refers to one or more DNA sequences from an exogenous source that are inserted into a target cell.

[0052] In one embodiment, an expression cassette refers to a nucleic acid molecule that includes a GLP-2 construct coding sequence (e.g., a coding sequence for a GLP-2 fusion protein), a promoter, and may include other regulatory sequences therefor, which may be engineered into a genetic element and / or packaged into a viral vector capsid (e.g., a viral particle). Typically, such an expression cassette for generating a viral vector includes a GLP-2 construct sequence as described herein adjacent to a packaging signal in the viral genome (and referred to as the "vector genome"), and other expression control sequences, such as those described herein. Any of the expression control sequences may be optimized for a particular species using techniques known in the art, including codon optimization, for example, as described herein.

[0053] In certain embodiments, the expression cassette comprises a constitutive promoter. In another embodiment, CB7 is used. CB7 is a chicken beta actin promoter with a cytomegalovirus enhancer element. In some embodiments, the CB7 promoter has the nucleic acid sequence of SEQ ID NO: 25. In one embodiment, the promoter is a CMV promoter. In some embodiments, the CMV promoter is the nucleic acid sequence of SEQ ID NO: 26.

[0054] In another embodiment, tissue-specific promoter is used.Alternatively, other liver-specific promoters can be used, for example, those listed in the Liver Specific Gene Promoter Database, Cold Spring Harbor, (rulai.schl.edu / LSPD), including but not limited to alpha 1 antitrypsin (A1AT), human albumin (Miyatake et al., J.Virol., 71:5124 32(1997)), humAlb; Hepatitis B virus core promoter (Sandig et al., Gene Ther., 3:1002 9(1996)), TTR minimal enhancer / promoter, alpha-antitrypsin promoter, liver-specific promoter (LSP) (Wu et al.Mol Ther.16:280-289(2008)), TBG liver-specific promoter. Viral promoters, constitutive promoters, regulatable promoters (see, e.g., WO2011 / 126808 and WO2013 / 04943), or Other promoters, such as promoters that respond to physiological cues, may be used and utilized in the vectors described herein.

[0055] In one embodiment, the promoter is included in an inducible gene expression system. The inducible gene regulation / expression system includes at least the following components: a promoter (also referred to as a regulatable promoter) operably linked to a transgene encoding a GLP-2 fusion protein as described herein, an activation domain, a DNA binding domain, and a zinc finger homeodomain binding site(s). In other embodiments, additional components may be included in the expression system, as further described herein.

[0056] 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 as shown in SEQ ID NO: 26. In one embodiment, the promoter is a ubiquitous inducible promoter Z12I, which contains 12 repeated copies of binding sites for ZFHD1 and an IL2 minimal promoter. See, for example, Chen et al, Hum Gene Ther Methods. 2013 Aug;24(4):270-278 (incorporated herein).

[0057] The expression system comprises an activation domain, which is preferably located upstream of the DNA binding domain. In one embodiment, the activation domain is a fusion of the carboxy terminus from the p65 subunit of NF-kappa B with the FKBP12-rapamycin binding (FRB) domain of FKBP12-rapamycin related protein (FRAP). In one embodiment, the activation domain is the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin related protein (FRAP) fused to the carboxy terminus from the p65 subunit of NF-kappa B from human origin.

[0058] In one embodiment, there is a linker between the transactivation domain and the DNA binding domain, and the linker may be an F2A or an IRES. In one embodiment, the linker is selected from an IRES or a 2A peptide.

[0059] The DNA-binding domain consists of a DNA-binding fusion of zinc finger homeodomain 1 (ZFHD1) joined to up to three copies of FK506-binding protein (FKBP). In the presence of an inducer, e.g., a rapalog such as rapamycin, the DNA-binding and activation domains dimerize through the interaction of their FKBP and FRB domains, resulting in transcriptional activation of the transgene. In some embodiments, ZFHD1 is included in frame with GT2A or an IRES.

[0060] The expression system is designed to have one, two, or three copies of the FKBP sequence. These are referred to herein as FKBP subunits. In one embodiment, the subunits are designed to express the same protein, but with different nucleic acids from each other to minimize recombination. For example, SEQ ID NO:27 provides three "wobbled" coding sequences for FKBP, each of which encodes the sequence shown in SEQ ID NO:28: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE.

[0061] The expression system further comprises a zinc finger homeodomain binding site. The nucleic acid molecule comprises binding sites for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 ZFHDs. In one embodiment, the expression system comprises eight zinc finger homeodomain binding sites (binding partners) (8XZFHD). However, the present invention does not include any zinc finger homeodomain binding sites. The present invention encompasses expression systems having from 2 to about 12 copies of a ZFHD binding site. An example of a single copy ZFHD binding site is aatgatgggcgctcgagt (SEQ ID NO: 29).

[0062] In some embodiments, there is a minimal IL2 promoter downstream of the zinc finger homeodomain binding site. An exemplary IL2 promoter is shown in SEQ ID NO:30.

[0063] Such induction systems are known in the art and are described, for example, in Rivera et al, A humanized system for pharmacologic control of gene expression, Nature Medicine volume 2, pages 1028-1032 (September 1996) and Rivera et al, Long-term pharmacologically regulated expression of erythropoietin in primates following AAV-mediated gene transfer, Blood, 15 February 2005, volume 1 105, number 4, both of which are incorporated herein by reference. In one embodiment, the inducible gene expression system comprises a CMV promoter and the activation domain is the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin related protein (FRAP) fused to the carboxy terminus from the p65 subunit of NF-kappa B of human origin, a GT2A peptide, a ZFHD1 DNA binding domain, three FKBP subunits, hGH polyA, 8XZFHD, and a minimal sIL2 promoter. These sequences are added to the coding sequence for the GLP-2 fusion protein, and optionally other regulatory sequences.

[0064] In addition to the promoter, the expression cassette and / or vector may include suitable transcription initiation, termination, and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals, sequences that stabilize cytoplasmic mRNA, sequences that increase translation efficiency (i.e., Kozak consensus sequences), sequences that enhance protein stability, and, if necessary, sequences that enhance secretion of the encoded product. Examples of suitable polyA sequences include, for example, SV40, bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit β-globin (also called rabbit globin polyA; RGB), modified RGB (mRGB), and TK polyA. Examples of suitable enhancers include, for example, alpha fetoprotein enhancer, TTR minimal promoter / enhancer, LSP (TH binding globulin promoter / alpha 1-microglobulin / bikunin enhancer), among others. In one embodiment, the polyA is rabbit globin polyA.

[0065] 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 with a gene of interest, and expression control sequences that act in trans or at a distance to control the gene of interest.

[0066] In one embodiment, a rAAV is provided that includes a 5' ITR, a CB7 promoter, a chicken beta-actin intron, a coding sequence for a fusion protein of SEQ ID NO: 13, 15, 17, 19, 21, or 23, a rabbit globin polyA, and a 3' ITR. In another embodiment, the rAAV includes a polynucleotide that includes a CMV promoter, and an activation domain that includes the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin related protein (FRAP) fused to the carboxy terminus from the p65 subunit of NF-kappa B from human, an IRES, a ZFHD1 DNA binding domain, three FKBP subunits, hGH polyA, 8XZFHD, a minimal sIL2 promoter, the sequence The coding sequence for the GLP-2 fusion protein of numbers 13, 15, 17, 19, 21, or 23, and rabbit beta globin polyA.

[0067] In another embodiment, a two vector induction system is provided. The first rAAV comprises 12XZFHD, a minimal IL2 promoter, a coding sequence for 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 package size of the vector. The second rAAV comprises a polynucleotide comprising a CMV promoter, an intron, and the activation domain is the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin related protein (FRAP) fused to the carboxy terminus from the p65 subunit of NF-kappa B from human, an IRES, a ZFHD1 DNA binding domain, and polyA.

[0068] In one embodiment, an expression cassette is provided that comprises a polynucleotide comprising a CB7 promoter, a chicken beta-actin intron, a coding sequence for a fusion protein of SEQ ID NO: 13, 15, 17, 19, 21, or 23, and a rabbit globin polyA. In one embodiment, the expression cassette is that found in SEQ ID NO:X, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity therewith. In another embodiment, a vector genome is provided that is flanked by 5' and 3' AAV ITRs with SEQ ID NO:XX, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity therewith.

[0069] In another embodiment, an expression cassette is provided comprising a polynucleotide comprising a CB7 promoter, a chicken beta-actin intron, a coding sequence for a fusion protein of SEQ ID NO: 13, 15, 17, 19, 21, or 23, and a rabbit globin polyA.

[0070] Viral Vectors In another aspect, a viral vector is provided that comprises the expression cassette described herein. In certain embodiments of the viral vector described herein, the viral vector is an adeno-associated virus (AAV) viral vector or a recombinant AAV (rAAV). The term "recombinant AAV" or "rAAV" as used herein refers to naturally occurring adeno-associated viruses, adeno-associated viruses available to those skilled in the art and / or available in the context of the composition(s) and method(s) described herein, as well as artificial AAV. An adeno-associated virus (AAV) viral vector is an AAV DNase-resistant particle that has an AAV protein capsid, in which the expression cassette packaged is flanked by AAV inverted terminal repeats (ITRs) for delivery to target cells (collectively referred to as "vector genome"). The AAV capsid is composed of 60 capsid protein subunits, VP1, VP2, and VP3, arranged in icosahedral symmetry in a ratio of approximately 1:1:10 to 1:1:20, depending on the AAV selected. Various AAVs can be selected as the source of capsids for the AAV viral vectors identified above. In one embodiment, the AAV capsid is the AAVrh91 capsid or a variant thereof. In certain embodiments, the capsid proteins are designated by a number or a combination of numbers and letters following the term "AAV" in the name of the rAAV vector. Unless otherwise indicated, the AAV capsids, ITRs, and other selected AAV components described herein may be readily selected from among any AAV, including, but not limited to, the AAVs identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAVhu37, AAVrh32.33, AAVAnc80, AAV10, AAV11, AAV12, AAVrh8, AAVrh74, AAV-DJ8, AAV-DJ, AAVhu.37, AAVrh.64R1, and AAVhu68. See, e.g., U.S. Patent Application Publication No. 2007-0036760-A1; ... See US Patent Application Publication No. 2009-0197338-A1, EP 1310571. See also WO2003 / 042397 (AAV7 and other simian AAVs), U.S. Patent No. 7,790,449 and U.S. Patent No. 7,282,199 (AAV8), WO2005 / 033321 and US 7,906,111 (AAV9), and WO2006 / 110689, and WO2003 / 042397 (rh.10), WO2005 / 033321, WO2018 / 160582 (AAVhu68), which are incorporated herein by reference. Other suitable AAVs may include, but are not limited to, AAVrh90 [filed April 28, 2020, PCT / US20 / 30273], AAVrh91 [filed April 28, 2020, PCT / US20 / 030266, current publication WO2020 / 223231 published November 5, 2020], AAVrh92, AAVrh93, AAVrh91.93 [filed April 28, 2020, PCT / US20 / 30281], which are incorporated herein by reference. Other suitable AAVs include the AAV3B variants described in U.S. Provisional Patent Application No. 62 / 924,112, filed October 21, 2019, and U.S. Provisional Patent Application No. 63 / 025,753, filed May 15, 2020, including AAV3B.AR2.01, AAV3B.AR2.02, AAV3B.AR2.03, AAV3B.AR2.04, AAV3B.AR2.05, AAV3B.AR2.06, AAV3B.AR2.07, AAV3B.AR2.09, 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.AR2.20, AAV3B.AR2.21, AAV3B.AR2.22, AAV3B.AR2.23, AAV3B.AR2.24, AAV3B.AR2.25, AAV3B.AR2.26, AAV3B.AR2.27, AAV3B.AR2.28, AAV3B.AR2.29, AAV3B.AR2.30, AAV3B.AR2.31, AAV3B.AR2.32, AAV3B.AR2.33, AAV3B.AR2.34, AAV3B.AR2.35, AAV3B.AR2.36, AAV3B.AR2.37, AAV3B.AR2.38, AAV3B.AR2.39, AAV3B.AR , AAV3B.AR2.06, AAV3B.AR2.07, AAV3B.AR2.08, AAV3B.AR2.10, AAV3B.AR2.11, AAV3B.AR2.12, AAV3B.AR2.13, AAV3B.AR2.14, AAV3B.AR2.15, AAV3B.AR2.16, or AAV3B.AR2.17, which are incorporated herein by reference. See also International Patent Application No. PCT / US21 / 45945, filed August 13, 2021, U.S. Provisional Patent Application No. 63 / 065,616, filed August 14, 2020, and U.S. Provisional Patent Application No. 63 / 109,734, filed November 4, 2020, all of which are incorporated herein by reference in their entireties.These documents also describe other AAV capsids that can be selected to generate rAAV and are incorporated by reference. Of the well-characterized AAVs isolated or engineered from humans or non-human primates (NHPs), human AAV2 was the first AAV developed as a gene transfer vector and has been widely used for efficient gene transfer experiments in various target tissues and animal models.

[0071] As used herein, with respect to AAV, the term "variant" refers to any AAV sequence derived from a known AAV sequence, including AAV sequences with conservative amino acid substitutions and AAV sequences that share at least 90%, at least 95%, at least 97%, at least 99% or more sequence identity across the amino acid or nucleic acid sequence. In another embodiment, the AAV capsid includes a variant that may include up to about 10% variation from any described or known AAV capsid sequence. That is, the AAV capsid shares about 90% identity to about 99.9% identity, about 95% to about 99% identity, or about 97% to about 98% identity with an AAV capsid provided herein and / or known in the art. In one embodiment, the AAV capsid shares at least 95% identity with an AAV capsid. When determining the percent identity of an AAV capsid, the comparison can be made across any of the variable proteins (e.g., vp1, vp2, or vp3).

[0072] In one embodiment, the viral vector is a rAAV having a capsid of AAV8 or a functional variant thereof. In one embodiment, the viral vector is a rAAV having a capsid of AAVrh91 or a functional variant thereof. In one embodiment, the viral vector is a rAAV having a capsid of AAV3.AR.2.12 or a functional variant thereof. In one embodiment, the viral vector is a rAAV having a capsid selected from AAV9, AAVrh64R1, AAVhu37, or AAVrh10.

[0073] 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 the encoded amino acid sequence is provided in SEQ ID NO: 32. Provided herein is an rAAV 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 the 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 an N-terminal and / or C-terminal truncation (e.g., a truncation of about 1 to about 10 amino acids).

[0074] In certain embodiments, the AAVrh91 capsid is characterized by one or more of the following: (1) a heterologous collection of AAVrh91 vp1 proteins selected from a vp1 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of 1-736 of SEQ ID NO:32, a vp1 protein produced from SEQ ID NO:31, or a vp1 protein produced from a nucleic acid sequence that encodes the predicted amino acid sequence of 1-736 of SEQ ID NO:32; a vp2 protein produced by expression from a nucleic acid sequence encoding at least about 138-736 amino acids of SEQ ID NO:32, a vp2 protein produced from a sequence including at least nucleotides 412-2208 of SEQ ID NO:31, or a vp2 protein produced from a nucleic acid sequence that encodes the predicted amino acid sequence of at least about 138-736 amino acids of SEQ ID NO:32; a heterologous collection of vp2 proteins, vp3 proteins produced from expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:32, vp3 proteins produced from a sequence comprising at least nucleotides 607-2208 of SEQ ID NO:31, or vp3 proteins produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 607-2208 of SEQ ID NO:31 encoding a predicted amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:32.and / or (2) a heterogeneous collection of vp1 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of at least about 138-736 amino acids of SEQ ID NO:32, a heterogeneous collection of vp2 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of at least about 203-736 amino acids of SEQ ID NO:32, wherein the vp1 proteins, vp2 proteins, and vp3 proteins are the product of a nucleic acid sequence encoding an amino acid sequence of at least about 203-736 amino acids of SEQ ID NO:32, a heterogeneous collection of vp1 proteins, a heterogeneous collection of vp2 proteins, a heterogeneous collection of vp3 proteins, each of which comprises a subpopulation having an amino acid modification comprising at least two highly deamidated asparagines (N) in lysine pairs, and optionally further comprising a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change; (B) a vector genome in an AAVrh91 capsid, the vector genome comprising a nucleic acid molecule comprising an AAV inverted terminal repeat and a non-AAV nucleic acid sequence encoding a product operably linked to a sequence that directs expression of the product in a host cell.

[0075] In certain embodiments, the AAVrh91 capsid is characterized by one or more of the following: (1) an AAVrh91 capsid protein, the vp1 protein, produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of 1-736 of SEQ ID NO:32; a heterologous collection of AAVrh91 vp1 proteins selected from: vp1 proteins produced from SEQ ID NO:19, or vp1 proteins produced from a nucleic acid sequence that is at least 70% identical to SEQ ID NO:19, which encodes the predicted amino acid sequence of at least about 138-736 amino acids of SEQ ID NO:32; a vp2 protein produced by expression from a nucleic acid sequence that encodes the predicted amino acid sequence of at least about 138-736 amino acids of SEQ ID NO:32; a vp2 protein produced from a sequence that includes at least nucleotides 412-2208 of SEQ ID NO:19, or vp2 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 412-2208 of SEQ ID NO:19, which encodes the predicted amino acid sequence of at least about 138-736 amino acids of SEQ ID NO:32. a heterologous collection of vp2 proteins, a vp3 protein produced from expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:32, a vp3 protein produced from a sequence comprising at least nucleotides 607-2208 of SEQ ID NO:19, or a vp3 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 607-2208 of SEQ ID NO:19 encoding a predicted amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:32.and / or (2) a heterogeneous collection of vp1 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of at least about 138-736 amino acids of SEQ ID NO:32, a heterogeneous collection of vp2 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of at least about 203-736 amino acids of SEQ ID NO:32, wherein the vp1 proteins, vp2 proteins, and vp3 proteins are the product of a nucleic acid sequence encoding an amino acid sequence of at least about 203-736 amino acids of SEQ ID NO:32, a heterogeneous collection of vp1 proteins, a heterogeneous collection of vp2 proteins, a heterogeneous collection of vp3 proteins, each of which comprises a subpopulation having an amino acid modification comprising at least two highly deamidated asparagines (N) in lysine pairs, and optionally further comprising a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change; and (B) a vector genome in an AAVrh91 capsid, the vector genome comprising a nucleic acid molecule comprising an AAV inverted terminal repeat and a non-AAV nucleic acid sequence encoding a product operably linked to a sequence that directs expression of the product in a host cell.

[0076] In certain embodiments, the vp1, vp2, and vp3 proteins of AAVrh91 comprise subpopulations having amino acid modifications that include at least two highly deamidated asparagines (N) in the asparagine-glycine pair of SEQ ID NO: 32, and optionally further comprise subpopulations that include other deamidated amino acids, where deamidation results in an amino acid change. High levels of deamidation are observed at NG pairs N57, N383, and / or N512, relative to the numbers in SEQ ID NO: 32. Deamidation has been observed at other residues. In certain embodiments, AAVrh91 may have other residues that are deamidated, e.g., typically less than 10%, and / or may have other modifications including phosphorylation (e.g., in a range of about 2 to about 30%, or about 2 to about 20%, or about 2 to about 10%, if present) (e.g., at S149), or oxidation (e.g., at one or more of about W22, about M211, W247, M403, M435, M471, W478, W503, about M537, about M541, about M559, about M599, M635, and / or W695). Optionally, W may be oxidized to kynurenine. [Table 2]

[0077] In certain embodiments, the AAVrh91 capsid is modified at one or more of the positions identified in the table above, as determined by mass spectrometry using trypsin enzyme, to the ranges provided below. In certain embodiments, one or more positions, or the glycine following the N, is modified as described herein. Residue numbers are based on the AAVrh91 sequence provided herein. See SEQ ID NO: 32.

[0078] In certain embodiments, the AAVrh91 capsid comprises a heterogeneous collection of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:32, a heterogeneous collection of vp2 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence at least about amino acids 138-736 of SEQ ID NO:32, and a heterogeneous collection of vp3 proteins that are the product of a nucleic acid sequence encoding at least amino acids 203-736 of SEQ ID NO:32.

[0079] In certain embodiments, modified AAVrh91 nucleic acid sequences can be used to generate mutant rAAVs having capsids that contain less deamidation than the native AAVrh91 capsid. Such mutant rAAVs can have reduced immunogenicity and / or increased stability upon storage, particularly in suspension form.

[0080] In one aspect, a recombinant AAV (rAAV) is provided that includes an AAV capsid derived from the adeno-associated virus rh91 and a vector genome packaged within the AAV capsid, the vector genome including AAV inverted terminal repeats (ITRs), a coding sequence for a GLP-2 receptor agonist of SEQ ID NO: 14, and regulatory sequences that direct expression of the GLP-2 receptor agonist.

[0081] In certain embodiments, the AAV68 capsid is further characterized by one or more of the following: The AAV hu68 capsid protein is an AAVhu68 vp1 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of 1-736 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 encodes the predicted amino acid sequence of 1-736 of SEQ ID NO:55 that is at least 70% identical to SEQ ID NO:53 or 54; an AAVhu68 vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 138-736 of SEQ ID NO:55, a vp2 protein produced from a sequence that includes at least nucleotides 412-2211 of SEQ ID NO:53 or 54, or a vp2 protein produced from a nucleic acid sequence that encodes the predicted amino acid sequence of 1-736 of SEQ ID NO:55 that is at least 70% identical to SEQ ID NO:53 or 54 ... and / or AAVhu68 vp3 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:55; vp3 protein produced from a sequence that includes at least nucleotides 607-2211 of SEQ ID NO:53 or 54; or vp3 protein produced from a nucleic acid sequence that encodes the predicted amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:55.

[0082] Additionally or alternatively, optionally, an AAV capsid is provided that comprises a heterogeneous population of vp1 proteins that comprise a valine at position 157, optionally a heterogeneous population of vp2 proteins that comprise a valine at position 157, and a heterogeneous population of vp3 proteins, wherein at least a subpopulation of vp1 and vp2 proteins comprise a valine at position 157 and optionally further comprise a glutamic acid at position 67, based on the numbering of the vp1 capsid in SEQ ID NO:55. Additionally or alternatively, there is provided an AAVhu68 capsid comprising a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:55, a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of at least about amino acids 138-736 of SEQ ID NO:55, and a heterogeneous population of vp3 proteins that are the product of a nucleic acid sequence encoding at least amino acids 203-736 of SEQ ID NO:55, wherein the vp1 proteins, vp2 proteins, and vp3 proteins include subpopulations having amino acid modifications.

[0083] The AAVhu68 vp1, vp2, and vp3 proteins are typically expressed as alternative splice variants encoded by the same nucleic acid sequence that encodes the full-length vp1 amino acid sequence (amino acids 1-736) of SEQ ID NO: 55. Optionally, the vp1 coding sequence is used alone to express the vp1, vp2, and vp3 proteins. Alternatively, this sequence may be co-expressed with one or more of a nucleic acid sequence encoding the AAVhu68 vp3 amino acid sequence of SEQ ID NO:55 (about aa203-736) without the vp1 unique region (about aa1 to about aa137) and / or the vp2 unique region (about aa1 to about aa202), or a complementary strand thereto, the corresponding mRNA or tRNA (about nt 607 to about nt 2211 of SEQ ID NO:53 or 54), or a sequence at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:53 or 54 encoding aa203-736 of SEQ ID NO:55. Additionally or alternatively, the vp1 and / or vp2 coding sequences can be co-expressed with a nucleic acid sequence encoding the AAVhu68 vp2 amino acid sequence of SEQ ID NO:55 (about aa 1 to about 137) without the vp1 unique region (about aa 1 to about 137), or a complementary strand thereto, the corresponding mRNA or tRNA (e.g., nt 412 to 2212 of SEQ ID NO: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 about aa 138 to 736 of SEQ ID NO:55.

[0084] 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, additional nucleic acid sequences, such as sequences encoding a vp3 protein that does not include the vp1 unique region and / or the vp2 unique region. rAAVhu68 resulting from production using a single nucleic acid sequence vp1 produces a heterogeneous population of vp1, vp2, and vp3 proteins. More specifically, the AAVhu68 capsids are expressed in a vp1, vp2, and vp3 protein. These subpopulations include subpopulations in the 5'-amino acid sequence of SEQ ID NO: 55, which have modifications from the predicted amino acid residue of SEQ ID NO: 55. These subpopulations contain, at a minimum, deamidated asparagine (N or Asn) residues. For example, asparagine in an asparagine-glycine pair is highly deamidated.

[0085] In one embodiment, the AAVhu68 vp1 nucleic acid sequence has the sequence of SEQ ID NO: 53 or 54, or a complementary strand thereto, e.g., the corresponding mRNA or tRNA. In certain embodiments, the vp2 and / or vp3 proteins may additionally or alternatively be expressed from a nucleic acid sequence different from vp1, e.g., to alter the ratio of vp proteins in a selected expression system. In certain embodiments, a nucleic acid sequence encoding the AAVhu68 vp3 amino acid sequence of SEQ ID NO: 55 (about aa 203-736) is also provided that does not include the vp1 unique region (about aa 1 to about aa 137) and / or the vp2 unique region (about aa 1 to about aa 202), or a complementary strand thereto, the corresponding mRNA or tRNA (about nt 607 to about nt 2211 of SEQ ID NO: 53 or 54). In certain embodiments, a nucleic acid sequence encoding the AAVhu68 vp2 amino acid sequence of SEQ ID NO:55 (about aa 138-736), without the vp1 unique region (about aa 1 to about 137), or the complementary strand thereto, the corresponding mRNA or tRNA (nt 412-2211 of SEQ ID NO:53 or 54), is also provided.

[0086] However, other nucleic acid sequences encoding the amino acid sequence of SEQ ID NO:55 may be selected for use in producing rAAVhu68 capsids. In certain embodiments, the nucleic acid sequence has a sequence at least 70% to 99% identical, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to the nucleic acid sequence of SEQ ID NO:53 or 54, or SEQ ID NO:53 or 54 encoding SEQ ID NO:55. In certain embodiments, the nucleic acid sequence has a sequence at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to the nucleic acid sequence of SEQ ID NO:53 or 54, or from about nt 412 to about nt 2211 of SEQ ID NO:53 or 54, and encodes the vp2 capsid protein of SEQ ID NO:55 (about aa 138 to 736). In certain embodiments, the nucleic acid sequence has a nucleic acid sequence from about nt 607 to about nt 2211 of SEQ ID NO: 53 or 54, or a sequence at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to nt of SEQ ID NO: 53 or 54, and encodes the vp3 capsid protein (about aa 203 to 736) of SEQ ID NO: 55. [Table 3]

[0087] In certain embodiments, the AAVhu68 capsid is characterized in having a capsid protein in which at least 45% of the N residues are deamidated at at least one of positions N57, N329, N452, and / or N512, based on the numbering of the amino acid sequence of SEQ ID NO: 55. In certain embodiments, these NG positions (i.e. At least about 60%, at least about 70%, at least about 80%, or at least 90% of the N residues are deamidated at one or more of 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 these and other embodiments, the AAVhu68 capsid is further characterized by having a population of proteins having about 1% to about 20% of the N residues deamidated at one or more of positions N94, N253, N270, N304, N409, N477, and / or Q599, based on the numbering of the amino acid sequence of SEQ ID NO: 55. In certain embodiments, AAVhu68 comprises at least a subpopulation of vp1, vp2, and / or vp3 proteins that are deamidated at one or more of positions N35, N57, N66, N94, N113, N252, N253, Q259, N270, N303, N304, N305, N319, N328, N329, N336, N409, N410, N452, N477, N515, N598, Q599, N628, N651, N663, N709, N735, or combinations thereof, based on the numbering of the amino acid sequence of SEQ ID NO: 55. In certain embodiments, the capsid protein may have one or more amidated amino acids.

[0088] In another embodiment, a recombinant adeno-associated virus (rAAV) having an AAVhu68 capsid and a vector genome is provided, 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, and wherein the heterogeneous AAVhu68 vp1, AAVhu68 vp2, and AAVhu68 the vp3 protein comprises a subpopulation having amino acid modifications comprising 50% to 100% deamidation of at least two asparagines (N) in asparagine-glycine pairs at two or more of N57, N329, N452, N512 of SEQ ID NO:55, as determined using mass spectrometry, and optionally further comprising a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change where the deamidated asparagine is deamidated to aspartic acid, isoaspartic acid, an interconverting aspartic acid / isoaspartic acid pair, or a combination thereof; and the AAVhu68 capsid comprises at least 65% of the asparagines (N) in the asparagine-glycine pair located at position N57 of the vp1 protein, based on the numbering of SEQ ID NO:55, are deamidated; at least 75% of the N in the asparagine-glycine pair at position N329 of the vp1 protein, v2 protein, and vp3 protein are deamidated, based on the residue numbering of the amino acid sequence of SEQ ID NO:55; At least 50% of the N in the asparagine-glycine pair at position N452 of the vp1 protein, v2 protein, and vp3 protein are deamidated, based on the residue numbering of the amino acid sequence of SEQ ID NO: 55; and / or at least 75% of the N in the asparagine-glycine pair at position N512 of the vp1 protein, v2 protein, and vp3 protein are deamidated, based on the residue numbering of the amino acid sequence of SEQ ID NO:55; Further included are subpopulations having one or more of the vector genomes in an AAVhu68 capsid, the vector genome comprising an AAV inverted terminal repeat sequence and a non-AAV nucleic acid sequence encoding a GLP-2 fusion described herein operably linked to a sequence that directs expression of the GLP-2 fusion in a target cell.

[0089] In one embodiment, the rAAV is a scAAV. The abbreviation "sc" refers to self-complementary. "Self-complementary AAV" refers to a plasmid or vector that has an expression cassette in which the coding region carried by the recombinant AAV nucleic acid sequence is designed to form an intramolecular double-stranded DNA template. Upon infection, the two complementary halves of the scAAV associate and immediately synthesize the second strand, rather than waiting for cell-mediated synthesis of the second strand. This will form a single double-stranded DNA (dsDNA) unit ready for replication and transcription at the time of transcription. For example, DM McCarty et al., "Self-complementary recombinant adeno-associated virus See, "Self-complementary AAV (scAAV) vectors promote efficient transduction independently of DNA synthesis", Gene Therapy, (August 2001), Vol 8, Number 16, Pages 1248-1254. Self-complementary AAVs are described, for example, in U.S. Pat. Nos. 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated by reference in its entirety.

[0090] In one embodiment, the nucleic acid sequence encoding the GLP-2 construct described herein is engineered into any suitable genetic element, such as naked DNA, phage, transposon, cosmid, RNA molecule (e.g., mRNA), episome, etc., which introduces the GLP-2 sequence carried thereon into a host cell, for example, to generate nanoparticles carrying DNA or RNA, viral vectors in a packaging host cell, and / or for delivery to a host cell in a subject. In one embodiment, the genetic element is a plasmid. The selected genetic element can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. Methods used to generate such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0091] As used herein, the term "host cell" may refer to a packaging cell line in which a vector (e.g., recombinant AAV or rAAV) is produced from a production plasmid. Alternatively, the term "host cell" may refer to any target cell in which expression of a gene product described herein is desired. Thus, "host cell" refers to a prokaryotic or eukaryotic cell (e.g., bacterial, human, or insect cell) that contains exogenous or heterologous DNA introduced into the cell by any means, such as electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. In certain embodiments herein, the term "host cell" refers to cultures of cells of various mammalian species for in vitro evaluation of the compositions described herein. In other embodiments herein, the term "host cell" refers to cells used to generate and package viral vectors or recombinant viruses. In further embodiments, the term "host cell" is an intestinal cell, a small intestinal cell, a pancreatic cell, or a liver cell.

[0092] As used herein, the term "target cell" refers to any target cell in which expression of a heterologous nucleic acid sequence or protein is desired. In certain embodiments, the target cell is a liver cell. In other embodiments, the target cell is a muscle cell.

[0093] In one embodiment, an rAAV is provided that includes a vector genome that includes an expression cassette, the expression cassette including a CMV promoter and an activation domain including the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin related protein (FRAP) fused to the carboxy terminus from the p65 subunit of NF-kappa B from human, a GT2A_V1 peptide, a ZFHD1 DNA binding domain, three FKBP subunits, hGH polyA, 12XZFHD, a minimal sIL2 promoter, a coding sequence for a GLP-2 fusion protein of SEQ ID NO: 13, 15, 17, 19, 21, or 23, and a coding sequence for a rabbit vegetative cell line comprising the CMV promoter and a ZFHD1 DNA binding domain ... In another embodiment, a rAAV is provided that includes a vector genome that includes an expression cassette, the expression cassette includes a CMV promoter and an activation domain is the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin related protein (FRAP) fused to the carboxy terminus from the p65 subunit of NF-kappa B from human, a GT2A_V2 peptide, a ZFHD1 DNA binding domain, three FKBP subunits, hGH poly A, 12XZFHD, a minimal sIL2 promoter, a coding sequence for a GLP-2 fusion protein of SEQ ID NO: 13, 15, 17, 19, 21, or 23, and a rabbit beta globin poly A.

[0094] In one embodiment, a rAAV is provided that includes a vector genome that includes an expression cassette, the expression cassette includes a CB7 promoter, a chicken beta-actin intron, a coding sequence for a fusion protein of SEQ ID NO: 13, 15, 17, 19, 21, or 23, a rabbit globin polyA, and a 3' ITR. In another embodiment, a rAAV is provided that includes a vector genome that includes an expression cassette, the expression cassette includes a CMV promoter and an activation domain is an FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin related protein (FRAP) fused to the carboxy terminus from the p65 subunit of NF-kappa B from human, an IRES, a ZFHD1 DNA binding domain, three FKBP subunits, hGH polyA, 8XZFHD, a minimal sIL2 promoter, a coding sequence for a GLP-2 fusion protein of SEQ ID NO: 13, 15, 17, 19, 21, or 23, and a rabbit beta globin polyA.

[0095] In another embodiment, a two vector induction system is provided. In one embodiment, a first rAAV is provided that includes a vector genome that includes an expression cassette, the expression cassette includes 12XZFHD, a minimal IL2 promoter, a coding sequence for a GLP-2 fusion protein of SEQ ID NO: 13, 15, 17, 19, 21, or 23, and rabbit beta globin polyA. A stuffer sequence can be included to increase the package size of the vector. A second rAAV includes a vector genome that includes an expression cassette, the expression cassette includes a polynucleotide that includes a CMV promoter, an intron, and an activation domain is the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin related protein (FRAP) fused to the carboxy terminus from the p65 subunit of NF-kappa B from human, an IRES, a ZFHD1 DNA binding domain, and polyA.

[0096] In one embodiment, the rAAV comprises a vector genome comprising an expression cassette comprising a polynucleotide comprising a CB7 promoter, a chicken beta-actin intron, a coding sequence for a fusion protein of SEQ ID NO: 13, 15, 17, 19, 21, or 23, and a polynucleotide comprising a rabbit globin polyA.

[0097] The minimal sequences required to package an expression cassette into an AAV viral particle are the AAV 5' and 3' ITRs, which may be of the same AAV origin as the capsid or of a different AAV origin (to generate AAV pseudotypes). In one embodiment, the ITR sequences from AAV2, or a deleted version thereof (ΔITR), are used for convenience and to speed regulatory approval. However, ITRs from other AAV sources may be selected. Preferably, the source of the ITRs is the same as the source of the Rep protein provided in trans for production. Typically, an expression cassette for an AAV vector includes the AAV 5' ITR, the GLP-2 fusion protein coding sequence, and any regulatory sequences, as well as the AAV 3' ITR. However, other configurations of these elements may be suitable. A shortened version of the 5' ITR, in which the D sequence and the terminal separation site (trs) are deleted (designated ΔITR), has been described. In other embodiments, the full-length AAV 5' and 3' ITRs are used.

[0098] The ITRs are the only AAV components required in cis in the same construct as the genes to package the expression cassette into virions. In one embodiment, the replication (rep) and / or capsid (cap) coding sequences are removed from the AAV genome and supplied in trans or by a packaging cell line to generate the AAV vector. For example, as described above, pseudotyped AAV may contain ITRs from a source different from the source of the AAV capsid. In one embodiment, a chimeric AAV capsid may be utilized. Still other AAV components may be selected. Sources of such AAV sequences are described herein and may be obtained from academic, commercial, or public sources (e.g., American Type AAV sequences may be isolated or obtained from the National AAV Culture Collection, Manassas, VA. AAV sequences may be obtained synthetically or through other suitable means by reference to published sequences such as those available in the literature or databases (e.g., GenBank®, PubMed®, etc.).

[0099] Methods for generating and isolating AAV viral vectors suitable for delivery to subjects are known in the art.See, for example, US Patent No. 7,790,449, US Patent No. 7,282,199, WO2003 / 042397, WO2005 / 033321, WO2006 / 110689, and US7,588,772 B2. In one system, a producer cell line is transiently transfected with a construct encoding a transgene flanked by ITRs and a construct(s) encoding rep and cap. In the 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 herpesvirus, requiring the separation of rAAV from contaminating viruses. More recently, systems have been developed that do not require infection with a helper virus to restore AAV, and the necessary helper functions (i.e., adenovirus E1, E2a, VA, and E4, or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase) are also supplied by the system in trans. In these newer systems, the helper functions can be supplied by transient transfection of cells with constructs encoding the required helper functions, or the cells can be engineered to stably contain genes encoding helper functions, the expression of which can be controlled at the transcriptional or post-transcriptional level. In yet another system, the transgene flanked by ITRs and the rep / cap genes are introduced into insect cells by infection with a baculovirus-derived vector. For a review of these production systems generally, see, e.g., Zhang et al., 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, the contents of each of which are incorporated herein by reference in their entireties.Methods of making and using these and other AAV production systems are also described in the following U.S. patents, the contents of each of which are incorporated herein by reference in their entirety: 5,139,941, 5,741,683, 6,057,152, 6,204,059, 6,268,213, 6,491,907, 6,660,514, 6,951,753, 7,094,604, 7,172,893, 7,201,898, 7,229,823, and 7,439,065. See generally, for example, Grieger & Samulski, 2005, "Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications," Adv. Biochem. Engin / Biotechnol. 99:119-145, Buning. et al., 2008, “Recent developments in adeno-associated virus vector technology,” J. Gene Med. 10:717-733, and the references cited below (these (each of which is incorporated herein by reference in its entirety). The methods used to construct any embodiment of the present invention are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Similarly, methods for producing rAAV virions are well known and the selection of a suitable method is not a limitation of the present invention. See, e.g., K. Fisher et al, (1993) J. Virol., 70:520-532 and U.S. Patent No. 5,478,745.

[0100] The rAAV described herein comprises a selected capsid having a vector genome packaged inside. The vector genome (or rAAV genome) comprises 5' and 3' AAV inverted terminal repeats (ITRs), a polynucleotide sequence encoding a fusion protein, and a regulatory sequence that directs the insertion of the polynucleotide sequence encoding the fusion protein into the genome of a host cell. In one embodiment, the vector genome is a sequence set forth in SEQ ID NO: 16, or a sequence sharing at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.

[0101] As used herein, "vector genome" refers to a nucleic acid sequence packaged inside a parvovirus (e.g., rAAV) capsid that forms a viral particle. Such a nucleic acid sequence contains AAV inverted terminal repeats (ITRs). In the examples herein, the vector genome includes at least, from 5' to 3', an AAV 5'ITR, a coding sequence(s) (i.e., transgene(s)), and an AAV 3'ITR. ITRs from AAV2, a source AAV different from the capsid, or other than full-length ITRs can be selected. In certain embodiments, the ITRs are from the same AAV source as the AAV that provides the rep function or trans-complementing AAV during production. Additionally, other ITRs can be used, such as self-complementary (scAAV) ITRs. Both single-stranded and self-complementary (sc) AAVs are included in rAAV. A transgene is a nucleic acid coding sequence heterologous to the vector sequence that encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor), or other gene product of interest. The nucleic acid coding sequence is operably linked to regulatory elements in a manner that allows transcription, translation, and / or expression of the transgene in cells of the target tissue. Suitable components of a vector genome are discussed in more detail herein. In one example, a "vector genome" includes at least, from 5' to 3', a vector-specific sequence and a nucleic acid sequence encoding a GLP-2 construct operably linked to a regulatory control sequence (that directs its expression in the target sequence), where 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, AAV inverted terminal repeats are utilized for packaging into AAV and certain other parvovirus capsids.

[0102] The AAV sequences of the vector typically contain cis-acting 5' and 3' inverted terminal repeat sequences (see, e.g., BJ Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155 168 (1990)). The ITR sequences are approximately 145 bp in length. Preferably, substantially complete sequences encoding the ITRs are used in the molecule, although some minimal modification of these sequences is tolerated. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., Sambrook et al, "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J. Virol., 70:520 532 (1996) (see ). One example of such a molecule used in the present invention is a "cis-acting" plasmid containing a transgene, where the selected transgene sequence and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences. In one embodiment, the ITRs are from a different AAV than the one supplying the capsid. In one embodiment, the ITR sequences are from AAV2. However, ITRs from other AAV sources can be selected. A shortened version of the 5'ITR (designated ΔITR) has been described, in which the D sequence and terminal separation sites (trs) are deleted. In one particular embodiment, the vector genome comprises a 130 base pair truncated AAV2 ITR, in which the external A elements are deleted. Without wishing to be bound by theory, it is believed that the shortened ITRs are restored to the wild-type length of 145 base pairs during vector DNA amplification, using the internal (A') elements as templates. In other embodiments, full-length AAV 5' and 3' ITRs are used. When the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be referred to as pseudotyped, however, other configurations of these elements may also be suitable.

[0103] Optionally, the GLP-2 construct described herein can be delivered via a viral vector other than rAAV. Such other viral vectors can include any virus suitable for gene therapy, including but not limited to adenovirus, herpesvirus, lentivirus, retrovirus, etc. Preferably, when one of these other vectors is produced, it is produced as a replication-defective viral vector.

[0104] "Replication-defective virus" or "viral vector" refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, and any viral genomic sequences also packaged in the viral capsid or envelope are replication-defective, i.e., they are unable to generate progeny virions, but retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gutless", containing only the transgene of interest flanked by signals required for amplification and packaging of the artificial genome), although these genes can be supplied during production. It is therefore considered safe for use in gene therapy, since replication and infection by progeny virions cannot occur except in the presence of viral enzymes required for replication.

[0105] Compositions comprising the viral vector constructs described herein are also provided. The pharmaceutical compositions described herein are designed to be delivered to a subject in need thereof by any suitable route or combination of different routes. Direct delivery to the liver (optionally intravenously, via the hepatic artery, or by transplantation), oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes of administration. The viral vectors described herein may be delivered in a single composition or multiple compositions. Optionally, two or more different AAVs, or multiple viruses, may be delivered [see, for example, WO2011 / 126808 and WO2013 / 049493]. In another embodiment, the multiple viruses may include different replication-deficient viruses (e.g., AAV and adenovirus). In one embodiment, administration is intramuscular. In another embodiment, administration is intravenous.

[0106] Replication-defective viruses can be formulated with physiologically acceptable carriers for use in gene transfer and gene therapy applications. In the case of AAV viral vectors, quantification of genome copies ("GC") can be used as a measure of the dose contained in the formulation. Any method known in the art can be used to determine the genome copy (GC) number of the replication-defective virus composition of the present invention. One method for performing GC number titration of AAV is as follows. The purified AAV vector sample is first treated with DNase to eliminate non-encapsidated AAV genomic DNA or contaminating plasmid DNA from the production process. Next, Then, the nuclease-resistant particles are subjected to heat treatment to release the genome from the capsid. The released genome is then quantified by real-time PCR using a primer / probe set that targets a specific region of the viral genome (usually the polyA signal). Another suitable method for determining genome copies is quantitative PCR (qPCR), especially optimized qPCR or digital droplet PCR [Lock Martin, et al, Human Gene Therapy Methods. April 2014, 25(2):115-125. doi:10.1089 / hgtb.2013.131, published online before editing on December 13, 2013].

[0107] In addition, the replication-deficient virus composition contains approximately 1.0×10 9 GC~approx. 1.0×10 15 The dosage unit can be formulated to contain an amount of replication-deficient virus in the range of GC. In another embodiment, this amount of viral genome can be delivered in divided doses. In one embodiment, the dose is about 1.0×10 for an average human subject of about 70 kg. 10 GC~approx.3.0×10 14 In another embodiment, the dose is about 1×10 9 For example, the dose of AAV virus is about 1×10 10 GC, 1×10 11 GC, approx. 5×10 11 GC, approx. 1×10 12 GC, approx. 5×10 12 GC, or approximately 1 × 10 13 In another embodiment, the dosage is about 1.0×10 9 GC / kg ~ approx. 3.0×10 14 In another embodiment, the dose is about 1×10 9 For example, the dose of AAV virus is about 1×10 10 GC / kg, 1×10 11 GC / kg, approx. 5×10 11 GC / kg, approx. 1×10 12 GC / kg, approx. 5×10 12GC / kg, or approximately 1 x 10 13 GC / kg. In one embodiment, the construct may be delivered in a volume of 1 μL to about 100 mL. As used herein, the term "dose" or "amount" may refer to the total dose or amount delivered to a subject over the course of treatment, or the dose or amount delivered in a single unit (or multiple units or split doses).

[0108] The above-mentioned recombinant vector can be delivered to a host cell according to published methods. Preferably, rAAV suspended in a physiologically compatible carrier can be administered to a desired subject, including humans. A suitable carrier can be easily selected by those skilled in the art in view of the indication for which the introduced virus is intended. For example, one suitable carrier includes saline, which can be formulated with various buffer solutions (e.g., phosphate-buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present invention.

[0109] In another embodiment, the composition comprises a carrier, diluent, excipient and / or adjuvant. In certain embodiments, for administration to a human patient, the rAAV is suitably suspended in an aqueous solution containing saline, a surfactant, and a pharma- ceutically and / or physiologically compatible salt or mixture of salts. Suitably, the formulation is adjusted to a physiologically acceptable pH, for example, in the range of pH 6-9, or pH 6.0-7.5, or pH 6.2-7.7, or pH 6.5-7.5, pH 7.0-7.7, or pH 7.2-7.8, or about pH 7.0. In certain embodiments, the formulation is adjusted to a pH of about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, or about 7.8. In certain embodiments, a pH of about 7.28 to about 7.32, about 6.0 to about 7.5, about 6.2 to about 7.7, about 7.5 to about 7.8, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, or about 7.8 may be desirable. In certain embodiments, for intravenous delivery, a pH of about 6.8 to about 7.2 may be desirable. However, other pHs within the broadest range, and subranges thereof, may be selected for other delivery routes.

[0110] Optionally, the compositions of the invention may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to the rAAV and / or variant and carrier(s). Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0111] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharma- ceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutical acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a host. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like can be used to introduce the compositions of the present invention into suitable host cells. In particular, rAAV vector-delivered transgenes can be formulated for delivery either encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles, and the like.

[0112] In one embodiment, the composition comprises a final formulation suitable for delivery to a subject, for example, an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration.Optionally, one or more surfactants are present in the formulation.In another embodiment, the composition can be delivered as a concentrate that is diluted for administration to a subject.In other embodiments, the composition can be lyophilized and reconstituted at the time of administration.

[0113] Suitable surfactants or combinations of surfactants may be selected from non-toxic non-ionic surfactants. In one embodiment, a primary hydroxyl terminated bifunctional block copolymer surfactant is selected, such as Pluronic® F68 [BASF], also known as Poloxamer 188, with neutral pH and average molecular weight of 8400. Other surfactants and other poloxamers may be selected, i.e., non-ionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (macrogol-15 hydroxystearate), LABRASOL (polyoxycaprylic acid glyceride), polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are generally named using the letter "P" (for poloxamer) followed by three digits, the first two digits x 100 giving the approximate molecular mass of the polyoxypropylene core and the last digit x 10 giving the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005% to about 0.001% of the suspension.

[0114] The dosage of a viral vector depends primarily on factors such as the condition being treated, the age, weight, and health of the patient, and may therefore vary between patients. For example, a therapeutically effective human dosage of a viral vector is generally about 1×10 (to treat an average subject weighing 70 kg) in the range of about 25 to about 1000 microliters to about 100 mL. 9 ~1×10 16 The concentration of the genomic viral vector (including all integers or fractions therein, preferably 1.0×10 for human patients) 12 GC~1.0×10 13The compositions of the present invention are administered in doses ranging from about 0.1 μL to about 10 μL, including all values ​​within the range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method. It may be delivered in a volume of 10 mL. In one embodiment, the volume is about 50 μL. In another embodiment, the volume is about 70 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 125 μL. In another embodiment, the volume is about 150 μL. In another embodiment, the volume is about 175 μL. In yet another embodiment, the volume is about 200 μL. In another embodiment, the volume is about 250 μL. In another embodiment, the volume is about 300 μL. In another embodiment, the volume is about 450 μL. In another embodiment, the volume is about 500 μL. In another embodiment, the volume is about 600 μL. In another embodiment, the volume is about 750 μL. In another embodiment, the volume is about 850 μL. In another embodiment, the volume is about 1000 μL. In another embodiment, the volume is about 1.5 mL. In another embodiment, the volume is about 2 mL. In another embodiment, the volume is about 2.5 mL. In another embodiment, the volume is about 3 mL. In another embodiment, the volume is about 3.5 mL. In another embodiment, the volume is about 4 mL. In another embodiment, the volume is about 5 mL. In another embodiment, the volume is about 5.5 mL. In another embodiment, the volume is about 6 mL. In another embodiment, the volume is about 6.5 mL. In another embodiment, the volume is about 7 mL. In another embodiment, the volume is about 8 mL. In another embodiment, the volume is about 8.5 mL. In another embodiment, the volume is about 9 mL. In another embodiment, the volume is about 9.5 mL. In another embodiment, the volume is about 10 mL.

[0115] In some embodiments, the concentration of the recombinant adeno-associated virus having a nucleic acid sequence encoding a desired transgene under the control of a regulatory sequence in the composition is desirably about 10 per milliliter. 7 ~10 14 The range is in genome copies (GC / mL).

[0116] In one embodiment, the dosage of rAAV in the composition is about 1.0×10 9 GC / kg ~ approx. 1.5×10 13 In one embodiment, the dosage is about 1.0×10 10 In one embodiment, the dosage is about 1.0×10 11 In one embodiment, the dosage is about 1.0×10 12 In one embodiment, the dosage is about 5.0×10 12 In one embodiment, the dosage is about 1.0×10 13 GC / kg. All ranges stated herein are inclusive of the endpoints.

[0117] In one embodiment, the effective dose (total genome copies delivered) is about 10 7 ~10 13 In one embodiment, the total administered dose is about 10 8 In one embodiment, the total administered dose is about 10 9 In one embodiment, the total administered dose is about 10 10 In one embodiment, the total administered dose is about 10 11 In one embodiment, the total administered dose is about 10 12 In one embodiment, the total administered dose is about 10 13 In one embodiment, the total administered dose is about 10 14 In one embodiment, the total administered dose is about 10 15 It is a genome copy.

[0118] It is desirable to utilize the lowest effective concentration of the virus to reduce the risk of undesirable effects such as toxicity, etc. Moreover, other dosage amounts and dosage 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, if progressive, the degree of progression of the disorder.

[0119] In certain embodiments, the composition comprises a rAAV comprising an inducible GLP-2 agonist construct. In certain embodiments, the inducing agent or molecule is rapamycin or a rapalog. In certain embodiments, the inducing agent is rapamycin and is administered at least once or more, at least twice or more, or at least three or more times after the composition comprising the rAAV. In some embodiments, the rapamycin is administered at a dose of at least about 4 to at least about 40 nM. In certain embodiments, the inducing agent (i.e., rapamycin) is administered at a dose of at least about 0.1 mg / kg to at least about 3.0 mg / kg. In certain embodiments, the inducing agent is administered at a dose of at least about 0.1 mg / kg to at least about 3.0 mg / kg. In embodiments, the inducing agent (ie, rapamycin) is administered at a dose of at least about 0.5 mg / kg to at least about 2.0 mg / kg.

[0120] The viral vectors and other constructs described herein can be used to deliver a GLP-2 fusion protein construct to a subject in need thereof, to provide a subject with an increased half-life of GLP-2, and / or to prepare a medicament for treating SBS in a subject. Thus, in another aspect, a method of treating SBS is provided. The method comprises administering a composition described herein to a subject in need thereof. In one embodiment, the composition comprises a viral vector comprising a GLP-2 fusion protein expression cassette described herein.

[0121] As used herein, the term "treatment" or "treating" is defined to include administering to a subject one or more compounds or compositions described herein for the purpose of alleviating one or more symptoms of short bowel syndrome. Thus, "treatment" can include one or more of reducing the progression of SBS, reducing the severity of symptoms, eliminating symptoms of the disease, slowing the progression of the disease, or increasing the effectiveness of a therapy in a given subject.

[0122] 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.

[0123] A course of treatment may optionally include repeated administration of the same viral vector (e.g., AAVrh91 vector) or different viral vectors (e.g., AAVrh91 and AAV3B.AR2.12). Still other combinations may be selected using the viral vectors described herein. Optionally, the compositions described herein may be combined in a regimen involving nutritional therapy (enteral or parenteral nutrition), drugs, such as drugs used to control gastric acid, drugs used to reduce diarrhea or improve intestinal absorption, or GLP-2 analogs, or surgery. In certain embodiments, the AAV vector and the combination therapy are administered essentially simultaneously. In other embodiments, the AAV vector is administered first. In other embodiments, the combination therapy is administered first.

[0124] In one embodiment, the composition is administered in combination with an effective amount of a GLP-2 analogue. Various commercially available GLP-2 products are known in the art, including, but not limited to, teduglutide, grepaglutide, and apraglutide.

[0125] In some embodiments, the combination of rAAV and a GLP-2 analog described herein reduces the dose requirement of the GLP-2 analog in the subject compared to before treatment with the viral vector. Such dose requirement may be reduced by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. The treating physician can determine the correct dosage of the GLP-2 analog that the subject requires. For example, the subject may be treated with a GLP-2 analog or other therapy that the treating physician may continue upon administration of the AAV vector. Such GLP-2 analog or other combination therapy may then be continued, reduced, or discontinued as necessary.

[0126] In one embodiment, the expression cassette, vector genome, rAAV-containing composition, or other composition described herein for gene therapy is delivered as a single dose per patient. In one embodiment, the subject is delivered a therapeutically effective amount of the composition described herein. As used herein, a "therapeutically effective amount" refers to an amount of an expression cassette or vector, or combination thereof, that delivers and expresses an amount of GLP-2-Fc in a target cell sufficient to achieve a therapeutic goal. The therapeutically effective amount refers to the amount of teduglutide administered subcutaneously. The therapeutically effective amount may be selected by the treating physician or may be guided based on previously determined guidelines. For example, teduglutide may be provided subcutaneously at an initial dose of 0.05 mg / kg per day. The dose may be increased in increments of 0.025 mg / kg for subjects with moderate to severe renal impairment. The rAAV may be delivered to the subject and then supplemented with oral or subcutaneous teduglutide, or other drugs as needed, to reach the equivalent of the desired dose of 0.05 mg / kg per day.

[0127] In certain embodiments, the therapeutic goal is to alleviate or treat one or more of the symptoms of SBS. Therapeutically effective amounts may be determined based on animal models rather than human patients.

[0128] As used herein, the term "heterologous" or any grammatical variation thereof when used to refer to vp capsid proteins refers to a population of non-identical members, 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 "heterologous" as used in reference to vp1, vp2, and vp3 proteins (alternatively referred to as isoforms) refers to differences in the amino acid sequences of the vp1, vp2, and vp3 proteins within the capsid. AAV capsids contain subpopulations within the vp1, vp2, and vp3 proteins with predicted amino acid residue modifications. These subpopulations contain, at a minimum, specific deamidated asparagine (N or Asn) residues. For example, certain subpopulations contain at least one, two, three, or four highly deamidated asparagine (N) positions in asparagine-glycine pairs, and optionally further contain other deamidated amino acids, where deamidation results in an amino acid change and other optional modifications.

[0129] As used herein, a "subpopulation" of vp proteins refers to a group of vp proteins that have at least one defined common feature and that consists of at least one group member and fewer than all members of the reference group, unless otherwise specified. For example, a "subpopulation" of vp1 proteins is at least one vp1 protein and fewer than all vp1 proteins in an assembled AAV capsid, unless otherwise specified. A "subpopulation" of vp3 can be one vp3 protein and fewer than all vp3 proteins in an assembled AAV capsid, unless otherwise specified. For example, in an assembled AAV capsid, vp1 protein can be a subpopulation of vp proteins, vp2 protein can be another subpopulation of vp proteins, and vp3 is yet another subpopulation of vp proteins. In another example, the vp1, vp2, and vp3 proteins can include subpopulations having, e.g., at least one, two, three, or four highly deamidated asparagines, e.g., different modifications at asparagine-glycine pairs.

[0130] As used herein, a "stock" of rAAV refers to a population of rAAV. Despite heterogeneity of capsid proteins due to deamidation, rAAV within a stock are expected to share five identical vector genomes. A stock may contain, for example, rAAV with capsids having selected AAV capsid proteins and heterogeneous deamidation patterns characteristic of a selected production system. A stock may be produced from a single production system or may be pooled from multiple runs of a production system. A variety of production systems may be selected, including, but not limited to, those described herein. As used herein, the terms "GLP-2 construct," "GLP-2 expression construct," and synonyms include the GLP-2 sequences described herein in combination with leader and fusion domains. The terms "GLP-2 construct," "GLP-2 expression construct," and synonyms include the GLP-2 fusion domain, as described herein. It may be used to refer to a nucleic acid sequence encoding a protein or its expression product.

[0131] The terms "percent identity (%)", "sequence identity", "percent sequence identity", or "percent identical" in the context of nucleic acid sequences refer to bases in two sequences that are the same when aligned to correspond. The length of sequence identity comparison can be over the entire length of a genome, the entire length of a gene coding sequence, or a fragment of at least about 100-150 nucleotides, or as desired. However, identity between smaller fragments, for example, of at least about 9 nucleotides, usually at least about 20-24 nucleotides, at least about 28-32 nucleotides, at least about 36 nucleotides or more, may also be desired. Multiple sequence alignment programs are also available for nucleic acid sequences. Examples of such programs include "Clustal W", "CAP Sequence Assembly", "BLAST", "MAP", and "MEME", which are accessible through web servers on the Internet. Other sources of such programs are known to those of skill in the art. Alternatively, the Vector NTI utility can also be used. There are also several algorithms known in the art that can be used to measure nucleotide sequence identity, including those included in the programs described above. As another example, polynucleotide sequences can be compared using Fasta™, a program in GCG version 6.1. Fasta™ provides alignment and percent sequence identity of the best overlapping regions between the query and search sequences. For example, percent sequence identity between nucleic acid sequences can be determined using Fasta™ using its default parameters (word size 6 and NOPAM factor for scoring matrix) provided in GCG version 6.1, which is incorporated herein by reference.

[0132] The term "highly conserved" means at least 80% identity, preferably at least 90% identity, more preferably more than 97% identity. Identity is easily determined by those skilled in the art by resorting to algorithms and computer programs known to those skilled in the art.

[0133] Unless otherwise stated in the upper range, the percentage of identity will be understood to be a minimum level of identity and includes all higher levels of identity up to 100% identity to the reference sequence. Unless otherwise stated, the percentage of identity will be understood to be a minimum level of identity and includes all higher levels of identity up to 100% identity to the reference sequence. For example, "95% identity" and "at least 95% identity" can be used interchangeably, including 95%, 96%, 97%, 98%, 99%, up to 100% identity to the reference sequence, and all fractions therebetween.

[0134] The terms "percent identity (%)", "sequence identity", "percent sequence identity", or "percent identical" in the context of amino acid sequences refer to residues in two sequences that are the same when aligned so that they correspond. Percent identity can be readily determined for the corresponding nucleic acid sequence encoding a full-length polypeptide, polypeptide, about 70 amino acids to about 100 amino acids, or peptide fragments thereof, or sequencers of a protein. Suitable amino acid fragments can be at least about 8 amino acids in length and can be up to about 150 amino acids in length. In general, when referring to "identity", "homology", or "similarity" between two different sequences, the "identity", "homology", or "similarity" is determined with reference to the "aligned" sequences. An "aligned" sequence or "alignment" refers to multiple nucleic acid or protein (amino acid) sequences, often including corrections for missing or additional bases or amino acids, as compared to a reference sequence. Alignments can be performed using a variety of publicly or commercially available sequences. The alignment is performed using any of the multiple sequence alignment programs available for amino acid sequences, including, for example, the "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME" and "Match-Box" programs. Generally, one of these programs is used with default settings, but one of skill in the art may change these settings as necessary. Alternatively, one of skill in the art may utilize another algorithm or computer program that provides at least the same level of identity or alignment as that provided by the referenced algorithm and program. See, for example, JD Thomson et al. al, Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690 (1999).

[0135] It should be noted that the terms "a" or "an" refer to one or more. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0136] The terms "comprise", "comprises", and "comprising" should be interpreted inclusively rather than exclusively. The terms "consist", "consisting", and variations thereof should be interpreted exclusively rather than inclusively. Although various embodiments herein are set forth using the word "comprising", it is also intended that in other circumstances the relevant embodiment should be construed and described using the word "consisting of" or "consisting essentially of".

[0137] As used herein, "patient" or "subject" refers to a mammal, meaning humans, veterinary or agricultural animals, domestic or pet animals, and animals typically used in clinical research. In one embodiment, the subject of these methods and compositions is a human. In another embodiment, the subject is not a feline.

[0138] As used herein, the term "about" means a variability of 10% (±10%, e.g., ±1, ±2, ±3, ±4, ±5, ±6, ±7, ±8, ±9, ±10, or any value therebetween) from a given reference, unless otherwise specified.

[0139] In certain cases, the term "E+#" or "e+#" is used to refer to the exponent. For example, "5E10" or "5e10" refers to 5 × 10 10 These terms may be used interchangeably.

[0140] The term "modulate" or variations thereof as used herein refers to the ability of a composition to inhibit one or more components of a biological pathway.

[0141] As used herein, "disease," "disorder," and "condition" are used interchangeably to refer to an abnormal condition in a subject.

[0142] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and by reference to published documents which provide general guidance to those of ordinary skill in the art for many of the terms used herein.

[0143] When describing an embodiment, references to "one embodiment" or "another embodiment" will be used unless expressly stated otherwise. Unless expressly specified, a referenced embodiment is not meant to be mutually exclusive with another embodiment (e.g., an embodiment described before the referenced embodiment). EXAMPLES

[0144] The following examples are provided to illustrate various embodiments of the invention and are not intended to limit the invention in any manner.

[0145] Example 1 - Construction of GLP-2 Vector GLP-2 agonists are difficult to express via adeno-associated virus (AAV). GLP-2 is usually expressed from the glucagon precursor protein, which requires tissue-specific proteases and produces unwanted proteins. Expression systems using conventional heterologous signal peptides result in low expression. Expression systems using heterologous propeptides with universal protease cleavage sites result in foreign protein sequences that can be targeted by T cells.

[0146] More specifically, a vector was constructed with a leader sequence placed upstream of one of several GLP-2 receptor agonist amino acid sequences, followed by a fusion domain. See, for example, FIG. 3. The resulting protein sequence was reverse translated, followed by the addition of a Kozak consensus sequence, a stop codon, and a cloning site. The sequence was generated and cloned into an expression vector containing a CMV promoter under the control of an inducible expression system. The expression construct was flanked by AAV2 ITRs. The resulting plasmid was called pAAV.Z12I.hGLP2.G2.Fc.rBG.

[0147] In the second construct, sequences were generated and cloned into an expression vector containing the CB7 constitutive promoter.

[0148] Example 2 - In vitro expression The following constructs were packaged into the AAVrh91 vector by triple transfection as previously described. AAVrh91.CB7.CI.hGLP-2-Fc.rBG AAVrh91.CB7.CI.hGLP-2-SA.rBG GLP-2-SA fusion was measured in the culture supernatant of HEK293 cells transfected with the vector for inducible human GLP-2-SA with a human thrombin signal sequence. GLP2-SA was identified by gel electrophoresis using spyroRuby staining (Figures 4A and 4B).

[0149] Example 3 - Pilot expression in Rag1KO mice Rag1KO female mice were administered the vector AAVrh91.CB7.CI.hGLP-2-SA.rBG (1 × 10 11 GC / mouse) or AAVrh91.CB7.CI.hGLP-2-Fc.rBG (1 × 10 11 Mice were treated with injections of 1000 mg / mL GLP-2 (GC / mouse). Serum was collected serially by separating whole blood in serum separator tubes containing 5 microliters of DPP-IV inhibitor (Millipore) and assayed for active GLP-2 expression and activity as described above. Vector was injected on day 0 and mice were necropsied on day 56. Serum GLP-2 concentrations (nM) are shown in Figure 5A and are estimates based on Fc fusion standards. Serum expression levels increased up to day 14 post-dose.

[0150] Small intestines were weighed and measured at necropsy. Small intestinal length and weight were significantly increased compared to control animals (Figure 5B). In addition, vector-treated intestines show healthy intestinal cell growth compared to vehicle-treated animals (Figure 5C). Serum citrulline levels (uM), a biomarker of intestinal surface area, were measured (Figure 5D).

[0151] Expression of GLP-2 was compared to albumin fusion constructs. The Fc fusion constructs showed greater GLP-2 expression (Figure 6A). The GLP.G2.Fc fusion shows significantly lower potency (EC50=13.6 nM) compared to hGLP (EC50=0.4 nM) (Figure 6B). These data indicate that AAV-mediated expression of GLP-2 agonists demonstrates a substantial and sustained increase in intestinal surface area that should be therapeutic in short bowel syndrome.

[0152] Example 4 - Dosage Study in NHPs In this study, we investigate the expression of human GLP-2 in non-human primates (NHPs; i.e., rhesus macaques). Figure 7 shows an overview of the study. Briefly, two NHPs were cultured at 1 × 10 13 Dose of (1e13)GC / kg(E185NG) and 5×10 10 AAVrh91.CB7.CI.hGLP-2-Fc.rBG was administered by intramuscular injection (IM) at a dose of (1e10) GC / kg (BM239H).

[0153] GLP-2 expression and potency, liver enzymes, and citrulline levels were measured. Necropsy is performed on day 60. Figure 8A shows plasma levels of GLP-2-Fc fusion protein. Figure 8B shows serum citrulline, a biomarker of intestinal surface area. Figure 8C shows detection of anti-GLP-2-Fc antibodies in NHP serum at a dilution of 1:100. AAV-mediated expression of GLP-2-Fc fusion demonstrates a substantial increase in intestinal surface area at high doses until anti-GLP-2-Fc antibodies reduce its expression, demonstrating its therapeutic efficacy against short bowel syndrome.

[0154] Example 5 - Long-term efficacy and safety studies in Rag1KO mice Rag1KO female mice were administered 1 × 10 10 GC / mouse, 3 × 10 10 GC / mouse or 1×10 11 Injections of the vector AAVrh91.CB7.CI.hGLP-2-Fc.rBG were administered at a dose of 100 mg / mouse per day. The study design is shown in Figure 9A. hGLP2-Fc levels and body weight were measured throughout the study. The highest vector doses showed the highest serum GLP2 levels (Figure 9B), while body weights were relatively consistent among all groups, with vehicle tending to be the lowest (Figure 9C,D). At necropsy on day 28, small intestinal length and weight were measured. SI length increased slightly with increasing dose compared to vehicle (Figure 9E), while SI weight was significantly increased in the two highest dose groups (Figure 9F).

[0155] All documents cited herein are incorporated herein by reference. U.S. Provisional Patent Application No. 63 / 316,219, filed March 3, 2022, is incorporated herein by reference. Although the present invention has been described with reference to certain embodiments, it will be understood that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. 1. A composition comprising a nucleic acid comprising a sequence encoding a fusion protein comprising a GLP-2 protein and an IgG4 Fc, albumin, or an XTEN polypeptide, wherein the fusion protein has the 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 a sequence sharing at least 75% identity thereto.

3. A composition comprising a viral vector, (a) an adeno-associated virus (AAV) capsid; (b) a vector genome packaged into said AAV capsid, said vector genome comprising AAV inverted terminal repeats (ITRs), said coding sequence comprising a GLP-2 protein and an IgG4 Fc, albumin or XTEN polypeptide, and said fusion protein having a sequence of SEQ ID NO: 13, 15, 17, 19, 21, or 23, or a sequence at least 99% identical thereto, and regulatory sequences that direct expression of the fusion protein.

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

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

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 the AAV2 5'ITR and the AAV2 3'ITR, which flank the fusion protein coding and regulatory sequences.

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

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

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

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

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

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

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

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

16. The composition of any one of claims 9 to 15, comprising at least eight copies of a binding site for said ZFHD.

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

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

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

20. 20. Use of a composition according to any one of claims 1 to 19 in the manufacture of a medicament for treating a subject with a metabolic disease.

21. The composition is 1×10 ９ GC / kg ~ 5 x 10 １３ 21. The composition or use of any one of claims 1 to 20, formulated to administer a dose of GC / kg of the rAAV.

22. The patient is a human and １０ ~1.5 x 10 １５ GC dose of said rAAV.

21. The composition or use according to any one of 1 to 20.

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

24. 1. A method of treating a subject having a metabolic disease, comprising delivering to the subject a recombinant adeno-associated virus (rAAV) having an AAV capsid derived 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 that directs expression of the fusion protein.

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

26. The patient is ９ GC / kg ~ 5 x 10 １３ The method of claim 24 or 25, wherein the rAAV is administered at a dose of 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 treating diabetes in humans.

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