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  • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
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  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2217/00—Genetically modified animals
  • A01K2217/07—Animals genetically altered by homologous recombination
  • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2227/00—Animals characterised by species
  • A01K2227/10—Mammal
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  • C07—ORGANIC CHEMISTRY
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  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
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  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
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  • C12N2820/00—Vectors comprising a special origin of replication system
  • C12N2820/002—Vectors comprising a special origin of replication system inducible or controllable
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
  • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
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  • C12N2840/00—Vectors comprising a special translation-regulating system
  • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
  • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

• SBS is a major cause of intestinal failure (IF), the persistent reduction of gut function below the minimum necessary for the absorption of macro nutrients and/or water and electrolytes.
• Standard treatment of SBS/IF includes lifelong daily parenteral nutrition (PN), an intravenous infusion of special form of foods, to support daily required nutrient for patients.
• PN causes complications including infection, gut hypoplasia, liver diseases, renal dysfunction, and bone diseases leading sever negative impact of the quality of life of SBS patients.
• Medications used for SBS had been those to treat only symptoms associated with SBS/IF until Teduglutide, a Glucagon-like peptide-2 (GLP-2) agonist, was developed and approved by FDA and EMA.
• GLP-2 Glucagon-like peptide-2
• GLP-2 is a 33 amino acid intestine peptide hormone which has a potent intestinotrophic effect. GLP-2 indeed increases the length of small intestine and villi length of the intestinal epithelium leading to effective absorption of nutrient even with shorter remnant bowel of SBS patients and reduction of PN volume and numbers.
• native GLP-2 cannot be used as an effective injectable with its very short serum half-life due to the cleavage by DPP IV.
• DPP IV-resistant long acting GLP-2 agonists, including fusions with IgG Fc domain and serum albumin, have been developed as disease modifying agents for SBS.
• Teduglutide is a GLP-2 like peptide with the A2G mutation for DPP IV resistance, that extends serum half-life from 5 min to 1.5 hours.
• Daily 0.05 mg/kg subcutaneous Teduglutide injections results in sustained and continuous reduction of PN volume throughout 2 years of treatment.90% of patients achieved >20% reduction of PN volume per week from the baseline, and 70% obtained 1 additional days PN off per week from the baseline with this treatment with a great safety profile (Schwartz et al., 2016).
• Daily Teduglutide injections are required for lifelong to maintain reduced PN volume and numbers (Compher et al., 2011). What is needed are improved treatments for SBS.
• Viral vectors encoding glucagon-like peptide 2 (GLP-2) receptor agonist fusion protein constructs are provided herein. These viral vectors may achieve, in some embodiments, sustained expression of the GLP-2 receptor agonist in subjects and/or increased circulating half-life, as compared to vector-mediated delivery of a GLP-2 receptor agonist without a fusion partner. Further provided are methods of making and using such viral vectors.
• a viral vector is provided which includes a nucleic acid comprising a polynucleotide sequence encoding a fusion protein.
• the fusion protein includes (a) a leader sequence comprising a secretion signal peptide, (b) a glucagon-like peptide-1 (GLP-2) receptor agonist, and (c) a fusion domain comprising (i) an IgG Fc or a functional variant thereof, (ii) an albumin or a functional variant thereof, or (iii) an XTEN polypeptide (Podust et al, 240:52-66 (Oct 2016).
• the vector is an adeno-associated viral vector.
• the secretion 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.
• the leader sequence comprises an IL-2 leader sequence.
• the GLP-fusion is selected from SEQ ID NO: 13, 15, 17, 19, 21, or 23, and functional variants thereof.
• 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.
• 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.
• 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.
• the viral vector includes an AAV capsid, and a vector genome packaged in the AAV capsid, said vector genome comprising AAV inverted terminal repeats (ITRs), the polynucleotide sequence encoding the fusion protein, and regulatory sequences which direct expression of the fusion protein.
• a pharmaceutical composition suitable for use in treating short bowel syndrome in a subject includes an aqueous liquid and the viral vector as described herein.
• the subject is a human.
• use of a viral vector as described herein is provided for the manufacture of a medicament for treating a subject having short bowel syndrome, optionally diabetes.
• a method of treating a subject having short bowel syndrome is provided. The method includes administering to the subject an effective amount of a viral vector or composition as described herein, Other aspects and advantages of the invention will be readily apparent from the following detailed description of the invention.
• FIG.1 is a schematic of the processing of proglucagon in vivo.
• FIG.2 is a table showing sequences of GLP-2 and analogs.
• FIG.3A is a schematic of the GLP2.G2.Fc construct, which includes an A2G substitution in the GLP2 amino acid sequence, a thrombin leader, and an IgG4 Fc fusion.
• FIG.3B is a schematic of the GLP2.G2.SA construct, which includes an A2G substitution in the GLP2 amino acid sequence, a thrombin leader, and a serum albumim fusion.
• FIG.3C is an alignment of the human (SEQ ID NO: 1), cyno (SEQ ID NO: 5), and mouse (SEQ ID NO: 6) GLP2 sequences.
• FIG.4A shows an elution profile of hGLP2-SA protein using albupure column purification.
• FIG.4B shows a gel with stained hGLP2 SA protein from FIG.4A.
• FIG.5A shows hGLP2-SA levels in RagKO mice dosed intramuscularly with AAVrh91.CI.hGLP2.G2.SA.rBG at 1e11 gc/mouse.
• FIG.5B are two graphs showing small intestine length and weight for vehicle and vector treated mice as described in FIG.5A.
• FIG.5C shows intestine histology for vehicle and vector treated mice as described in FIG.5A.
• Vector treated mice intestine show healthier villi as compared to vehicle treated mice.
• FIG.6A 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 dosage of 1e11 gc/mouse. GLP-2 levels for the Fc construct were higher after about study day 7.
• FIG.6B shows a potency assay for hGLP2 and hGLP2.Fc.
• FIG.7 shows the study design for an experiment as described in Example 4.
• FIGs.8A-8C show the results of the experiment as described in Example 4.2 NHPs were administered AAVrh91.CB7.CI.hGLP-2-Fc.rBG via intramuscular injection (IM) at a dose of 1 x 10 13 (1e13) GC/kg (E185NG) and a of dose 5 x 10 10 (1e10) GC/kg (BM239H).
• FIG.8A shows plasma level of GLP-2-Fc fusion protein.
• FIG.8B shows serum citrulline, a biomarker of gut surface area.
• FIG.8C shows detection of anti-GLP-2-Fc antibody in NHP serum at 1:100 dilution.
• FIGs.9A-9F show the results of the experiment as described in Example 5.
• Rag1KO female mice were treated with an injection of the vector AAVrh91.CB7.CI.hGLP-2-Fc.rBG at a dosage of 1 x 10 10 GC/ mouse, 3 x 10 10 GC/ mouse, or 1 x 10 11 GC/ mouse via IM route of administration.
• the study design is shown in FIG.9A.
• FIG.9B shows serum GLP2 levels
• FIG.9C shows body weights over time.
• FIG.9D shows body weights at day 56.
• FIG.9E shows small intestine (SI) length
• FIG.9F shows SI weight.
• adeno-associated viral (AAV) vectors expressing GLP-2 agonists to treat SBS/IF patients with a single intramuscular vector administration.
• Transgene GLP-2 agonists include the A2G mutation for DPP IV resistance and fusions with human IgG Fc domain or serum albumin for further extended serum half-life.
• a thrombin propeptide enables expression of GLP-2 agonists above the therapeutic level with remarkably lower vector doses (i.e., 1e10 to 1e12 GC/kg).
• Described herein are expression cassettes to express these proteins constitutively or in a controlled manner via administration of a small molecule drug that activates transcription of the GLP-2 agonist sequence. Delivery of these constructs to subjects in need thereof via a number of routes, and particularly by expression in vivo mediated by a recombinant vector such as a rAAV vector, is described. Also provided are methods of using these constructs in regimens for treating short bowel syndrome in a subject in need thereof and increasing the half-life of GLP-2 in a subject. In addition, methods are provided for enhancing the activity of GLP-2 in a subject.
• GLP-2 Fusion Proteins Post-translational processing of proglucagon generates glucagon-like peptide-2 (GLP- 2), a 33-amino acid intestinotrophic peptide hormone, together with GLP-1.
• GLP-2 acts to slow gastric emptying, reduce gastric secretions and increase intestinal blood flow.
• GLP-2 also stimulates growth of the large and small intestine at least by enhancing crypt cell proliferation and villus length so as to increase the surface area of the mucosal epithelium.
• GLP-2 has a short half-life that limits its use as a therapeutic because rapid in vivo cleavage of GLP-2 by dipeptidyl peptidase IV (DPP-IV) yields an essentially inactive peptide.
• the amino acid sequence of human GLP-2 is HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ ID NO: 1).
• a GLP-2 analog named teduglutide has been developed, in which amino acid residue 2 (alanine) has been substituted with glycine.
• the sequence of this GLP-2 analog is shown in SEQ ID NO: 2 HGDGSFSDEMNTILDNLAARDFINWLIQTKITD.
• the 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.
• the fusion protein comprises a polynucleotide sequence encoding a fusion protein comprising (a) a leader sequence comprising a secretion signal peptide, (b) a glucagon like peptide 2 (GLP 2) receptor agonist, and (c) a fusion domain.
• the GLP-2 receptor agonist comprises a thrombin leader sequence, a GLP-2 receptor agonist, and an IgG Fc or functional variant thereof.
• the fusion protein comprises a thrombin leader, a GLP-2 receptor agonist, and an albumin or functional variant thereof.
• the fusion protein comprises a thrombin leader, two copies of a GLP-2 receptor agonist, and an albumin or functional variant thereof.
• GLP-2 receptor agonists include variants which may include up to about 10% variation from a GLP-2 nucleic acid or amino acid sequence described herein or known in the art, which retain the function of the wild-type sequence.
• by “retain function” it is meant that the nucleic acid or amino acid functions in the same way as the wild-type sequence, although not necessarily at the same level of expression or activity.
• a functional variant has increased expression or activity as compared to the wild-type sequence.
• the functional variant has decreased expression or activity as compared to the wild-type sequence.
• the functional variant has 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater increase or decrease in expression or activity as compared to the wild-type sequence.
• Other GLP-2 analogs have been developed and include glepaglutide (SEQ ID NO: 3), apraglutide, and others shown below, and in FIG.2.
• the fusion comprises, in one embodiment, a GLP-2 analog in combination with heterologous sequences.
• GLP-2 analog is meant a polypeptide sharing 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).
• the GLP-2 analog has at most 1, 2, or 3 amino acid substitutions as compared to the native sequence.
• the GLP-2 sequence is derived from a species other than human.
• the GLP-2 may be from a non-human primate, dog, cat, mouse, rat, sheep, cow, horse, etc.
• the GLP-2 has a sequence which amino acid residue 2 (alanine) has been substituted with glycine, e.g., SEQ ID NO: 2.
• the GLP-2 analog contains one, two, three, 4, 5, 6, 7, 8 or up to 9 amino acid substitutions selected from A2G, D3E, S5T, D8S, M10L, N11A, N16A, N24A, Q28A as compared to the native sequence. These substitutions have been shown to improve efficacy of the clinical profile of GLP-2, including protection from DPP ⁇ 4 inactivation (A2G).
• the GLP-2 analog is a DPP-IV resistant variant of GLP-2.
• the GLP-2 analog has a sequence comprising, or consisting of, SEQ ID NO: 2.
• the variant shares at least 90% identity, 95% identity, 97% identity, 98% identity, 99% identity or 100% identity with SEQ ID NO: 2.
• the fusion protein may comprise a leader sequence, which may comprise a secretion signal peptide.
• leader sequence refers to any N-terminal sequence of a polypeptide.
• the leader sequence may be derived from the same species for which administration is ultimately intended, e.g., a human.
• the terms “derived” or “derived from” mean the sequence or protein is sourced from a specific subject species or shares the same sequence as a protein or sequence sourced from a specific subject species.
• a leader sequence which is “derived from” a human shares the same sequence (or a variant thereof, as defined herein) as the same leader sequence as expressed in a human.
• the specified nucleic acid or amino acid need not actually be sourced from a human.
• Various techniques are known in the art which are able to produce a desired sequence, including mutagenesis of a similar protein (e.g., a homolog) or artificial production of a nucleic acid or amino acid sequence.
• the “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 actual source of the derived sequence.
• amino acid substitution and its synonyms are intended to encompass modification of an amino acid sequence by replacement of an amino acid with another, substituting, amino acid.
• the substitution may be a conservative substitution. It may also be a non-conservative substitution.
• conservative in referring to two amino acids, is intended to mean that the amino acids share a common property recognized by one of skill in the art. For example, amino acids having hydrophobic nonacidic side chains, amino acids having hydrophobic acidic side chains, amino acids having hydrophilic nonacidic side chains, amino acids having hydrophilic acidic side chains, and amino acids having hydrophilic basic side chains.
• Common properties may also be amino acids having hydrophobic side chains, amino acids having aliphatic hydrophobic side chains, amino acids having aromatic hydrophobic side chains, amino acids with polar neutral side chains, amino acids with electrically charged side chains, amino acids with electrically charged acidic side chains, and amino acids with electrically charged basic side chains.
• Both naturally occurring and non- naturally occurring amino acids are known in the art and may be used as substituting amino acids in embodiments.
• Methods for replacing an amino acid are well known to the skilled in the art and include, but are not limited to, mutations of the nucleotide sequence encoding the amino acid sequence. Reference to “one or more” herein is intended to encompass the individual embodiments of, for example, 1, 2, 3, 4, 5, 6, or more.
• the leader is a human thrombin (Factor II) sequence.
• the thrombin leader has the sequence shown in SEQ ID NO: 7: MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRR, or a functional variant thereof having at most 1, 2, or 3 amino acid substitutions.
• functional variants of the desired leader include variants which may include up to about 10% variation from a leader nucleic acid or amino acid sequence described herein or known in the art, which retain the function of the wild type sequence.
• the coding regions for both the propeptide and GLP-2 peptide are incorporated into a single nucleic acid sequence without a linker between the coding sequences of the propeptide and GLP-2.
• the fusion protein further includes a fusion domain.
• the fusion domain in one embodiment, is a human IgG Fc fragment or a functional variant thereof. Immunoglobulins typically have long circulating half-lives in vivo. By fusing the GLP-2 receptor agonist (and leader) to an IgG Fc, the circulation time of the fusion protein is prolonged, while the function of the GLP-2 is preserved.
• the fusion domain is a rhesus IgG Fc fragment or functional variant thereof.
• the Fc portion of an immunoglobulin has the meaning commonly given to the term in the field of immunology. Specifically, this term refers to an antibody fragment which does not contain the two antigen binding regions (the Fab fragments) from the antibody.
• the Fc portion consists of the constant region of an antibody from both heavy chains, which associate through non-covalent interactions and disulfide bonds.
• the Fc portion can include the hinge regions and extend through the CH2 and CH3 domains to the c- terminus of the antibody.
• the Fc portion can further include one or more glycosylation sites.
• the fusion domain is a human IgG Fc.
• the Fc domain can be derived from any human IgG, including human IgG1, human IgG2, human IgG3, or human IgG4.
• the human IgG Fc is an IgG4 Fc.
• the human IgG Fc is SEQ ID NO: 8: AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLG.
• 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.
• 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.
• the rhesus IgG Fc is an IgG4 Fc.
• 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.
• 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.
• the rhesus IgG further comprises a hinge sequence.
• the fusion domain is a human albumin or a functional variant thereof.
• the human albumin is SEQ ID NO: 10: DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNL PRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECC QAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFP KAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEK PLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARR HPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCEL FEQLGEYKFQNALLVRYTKKVPQVS
• 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.
• the fusion domain is a rhesus albumin or a functional variant thereof.
• the rhesus albumin is SEQ ID NO: 11: DTHKSEVAHRFKDLGEEHFKGLVLVAFSQYLQQCPFEEHVKLVNEVTEFAKTCVAD ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNL PPLVRPEVDVMCTAFHDNEATFLKKYLYEVARRHPYFYAPELLFFAARYKAAFAEC CQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGDRAFKAWAVARLSQKF PKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYMCENQDSISSKLKECC DKPLLEKSHCLAEVENDEMPADL
• the rhesus albumin shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity to SEQ ID NO: 11.
• the in vivo function and stability of the fusion proteins of the present disclosure may be optimized by adding small peptide linkers, e.g., to prevent potentially unwanted domain interactions or for other reasons.
• a glycine-rich linker may provide some structural flexibility such that the GLP-2 analog portion can interact productively with the GLP-2 receptor on target cells such as the beta cells of the pancreas.
• the C- terminus of the GLP-2 analog and the N- terminus of the fusion domain of the fusion protein are, in one embodiment, fused via a linker.
• the linker includes 1, 1.5 or 2 repeats of a G-rich peptide linker having the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 12).
• the fusion protein comprises (a) human thrombin leader, (b) a DPP-IV resistant variant of GLP-2(A2G), a linker, and (c) a human IgG Fc.
• the fusion protein has the sequence of SEQ ID NO: 13, or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.
• 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.
• the fusion protein has the 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 MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRHGDGSFSDEMNTI LDNLAARDFINWLIQTKITDGGGGGGSGGGGSGGGGSDAHKSEVAHRFKDLGEENF KALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCT VATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNE ETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDE GKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTE CCHGDLLECADDRADLAKYICENQ
• the fusion protein has the 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 MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRHGDGSFSDEMNTI LDNLAARDFINWLIQTKITDGGGGGGSGGGGSGGGGSSPAGSPTSTEEGTSESATPES GPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPES GPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS APGTSTEPSEGSAPGTSESATPES GPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPES GPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS APGTSESATPESGPGTSESATPESGPGSPAGSPT
• the fusion protein has the 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 MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQALSLLQRVRRHGDGSFSDEMNTV LVDNLATRDFINWLIQTKITDGGGGGGGSGGGGSGGGGSAEFTPPCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAQTKPRER QFNSTYRVVSVLTVTHQDWLNGKEYTCKVSNKGLPAPIEKTISKAKGQPREPQVYIL PPPQEELTKNQVSLTCLVTGFYPSDIAVEWESNGQPENTYKTTPPVLDSDGSYLLYSK LTVNKSRWQPGNIFTCSVMHEALHNHYTQKSLSVSPG*
• the sequence encoding the fusion protein is
• the fusion protein has the 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 MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQALSLLQRVRRHGDGSFSDEMNTV LVDNLATRDFINWLIQTKITGGGGGGGSGGGGSGGGGSDTHKSEVAHRFKDLGEEH FKGLVLVAFSQYLQQCPFEEHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKL CTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPPLVRPEVDVMCTAFHDN EATFLKKYLYEVARRHPYFYAPELLFFAARYKAAFAECCQAADKAACLLPKLDELR DEGKASSAKQRLKCASLQKFGDRAFKAWAVARLSQKFPKAEFAEVSKLVTDLTKV HTECCHGDLLECADDRADLAKYMC
• the fusion protein has the 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 MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQALSLLQRVRRHGDGSFSDEMNTV LVDNLATRDFINWLIQTKITDGGGGGGSGGGGSGGGGSSPAGSPTSTEEGTSESATPE SGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPES GPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS APGTSTEPSEGSAPGTSESATPES GPGTSTEPSEGSAPGTSESATPES GPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTE
• RNA and/or cDNA coding sequences are designed for optimal expression in the subject species for which administration is ultimately intended, e.g., a human.
• the coding sequences may be designed for optimal expression using codon optimization.
• Codon-optimized coding regions can be designed by various different methods. This optimization may be performed using methods which are available on-line, published methods, or a company which provides codon optimizing services.
• One codon optimizing method is described, e.g., in International Patent Application Pub. No. WO 2015/012924, which is incorporated by reference herein. Briefly, the nucleic acid sequence encoding the product is modified with synonymous codon sequences. Suitably, the entire length of the open reading frame (ORF) for the product is modified. However, in some embodiments, only a fragment of the ORF may be altered.
• nucleic acid sequences encoding these polypeptides are provided.
• a nucleic acid sequence is provided which encodes for the GLP-2 peptides described herein. In some embodiments, this may include any nucleic acid sequence which encodes the GLP-2 sequence of SEQ ID NO: 1. In another embodiment, this includes any nucleic acid which includes the GLP-2 sequence of SEQ ID NO: 2.
• a nucleic acid sequence is provided which encodes for the GLP-2 fusion protein described herein. In another embodiment, this includes any nucleic acid sequence which encodes the GLP-2 fusion protein of SEQ ID NO: 13, 15, 17, 19, 21 or 23.
• Expression Cassettes Provided herein, in another aspect, is an expression cassette comprising a nucleic acid encoding a GLP-2 fusion protein as described herein.
• an “expression cassette” refers to a nucleic acid molecule which comprises a biologically useful nucleic acid sequence (e.g., a gene cDNA encoding a protein, enzyme or other useful gene product, mRNA, etc.) and regulatory sequences operably linked thereto which direct or modulate transcription, translation, and/or expression of the nucleic acid sequence and its gene product.
• a biologically useful nucleic acid sequence e.g., a gene cDNA encoding a protein, enzyme or other useful gene product, mRNA, etc.
• regulatory sequences operably linked thereto which direct or modulate transcription, translation, and/or expression of the nucleic acid sequence and its gene product.
• “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 nucleic acid sequence.
• Such regulatory sequences typically include, e.g., one or more of a promoter, an enhancer, a transcription factor, transcription terminator, an intron, sequences that enhance translation efficiency (i.e., a Kozak consensus sequence), efficient RNA processing signals such as slicing and a polyadenylation sequence, sequences that stabilize cytoplasmic mRNA, for example Woodchuck Hepatitis Virus (WHP) posttranslational Regulatory Element (WPRE), and a TATA signal.
• a promoter e.g., one or more of a promoter, an enhancer, a transcription factor, transcription terminator, an intron, sequences that enhance translation efficiency (i.e., a Kozak consensus sequence)
• efficient RNA processing signals such as slicing and a polyadenylation sequence
• sequences that stabilize cytoplasmic mRNA for example Woodchuck Hepatitis Virus (WHP) posttranslational Regulatory Element (WPRE)
• WPRE Woodchuck He
• the expression cassette may contain regulatory sequences upstream (5’ to) of the gene sequence, e.g., one or more of a promoter, an enhancer, an intron, etc., and one or more of an enhancer, or regulatory sequences downstream (3 to) a gene sequence, e.g., 3 untranslated region (3’ UTR) comprising a polyadenylation site, among other elements.
• the regulatory sequences are operably linked to the nucleic acid sequence of a gene product, wherein the regulatory sequences are separated from nucleic acid sequence of a gene product by intervening nucleic acid sequences, i.e., 5’-untranslated regions (5’UTR).
• the expression cassette comprises nucleic acid sequence of one or more of gene products.
• the expression cassette can be a monocistronic or a bicistronic expression cassette.
• the term “transgene” refers to one or more DNA sequences from an exogenous source which are inserted into a target cell.
• the expression cassette refers to a nucleic acid molecule which comprises the GLP-2 construct coding sequences (e.g., coding sequences for the GLP-2 fusion protein), promoter, and may include other regulatory sequences therefor, which cassette may be engineered into a genetic element and/or packaged into the capsid of a viral vector (e.g., a viral particle).
• such an expression cassette for generating a viral vector contains the GLP-2 construct sequences described herein flanked by packaging signals of the viral genome (and is termed a “vector genome”) and other expression control sequences such as those described herein. Any of the expression control sequences can be optimized for a specific species using techniques known in the art including, e.g., codon optimization, as described herein.
• the expression cassette includes a constitutive promoter.
• a CB7 promoter is used.
• CB7 is a chicken ⁇ -actin promoter with cytomegalovirus enhancer elements.
• the CB7 promoter has the nucleic acid sequence of SEQ ID NO: 25.
• the promoter is a CMV promoter.
• the CMV promoter is a nucleic acid sequence of SEQ ID NO: 26.
• a tissue specific promoter is used.
• other liver-specific promoters may be used such as those listed in the Liver Specific Gene Promoter Database, Cold Spring Harbor, (rulai.schl.edu/LSPD), and including, but not limited to, alpha 1 anti-trypsin (A1AT); human albumin (Miyatake et al., J.
• humAlb hepatitis B virus core promoter
• hepatitis B virus core promoter Sandig et al., Gene Ther., 3:10029 (1996)
• TTR minimal enhancer/promoter alpha-antitrypsin promoter
• LSP liver-specific promoter
• TBG liver specific promoter Other promoters, such as viral promoters, constitutive promoters, regulatable promoters (see, e.g., WO 2011/126808 and WO 2013/04943), or a promoter responsive to physiologic cues may be used may be utilized in the vectors described herein.
• the promoter is comprised in an inducible gene expression system.
• the inducible gene regulation/expression system contains at least the following components: a promoter operably linked to transgene encoding the GLP-2 fusion protein described herein (also referred to as the regulatable promoter), an activation domain, DNA binding domain, and zinc finger homeodomain binding site(s).
• additional components may be included in the expression system, as further described herein.
• the system comprises the promoter upstream of the coding sequence for the GLP-2 fusion protein. Promoters described herein, such as CMV and CB7 promoters may be used.
• the promoter is a CMV promoter, such as that shown in SEQ ID NO: 26.
• the promoter is the ubiquitous, inducible promoter Z12I which comprises 12 repeated copies of the binding site for ZFHD1 and the IL2 minimal promoter. See, e.g., Chen et al, Hum Gene Ther Methods.2013 Aug; 24(4): 270–278, which is incorporated herein.
• the expression system comprises an activation domain, which is preferably located upstream of the DNA binding domain.
• the activation domain is a fusion of the carboxy terminus from the p65 subunit of NF-kappa B and FKBP12-rapamycin binding (FRB) domain of FKBP12-rapamycin-associated protein (FRAP).
• the activation domain is a FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin-associated protein (FRAP) fused to a carboxy terminus from the p65 subunit of NF-kappa B from a human.
• FKBP12-rapamycin binding domain FKBP12-rapamycin binding domain of human FKBP12-rapamycin-associated protein (FRAP) fused to a carboxy terminus from the p65 subunit of NF-kappa B from a human.
• linker may be an F2A or an IRES.
• the linker is selected from an IRES or a 2A peptide.
• the DNA binding domain is composed of a DNA-binding fusion of zinc finger homeodomain 1 (ZFHD1) joined to up to three copies of FK506 binding protein (FKBP).
• the DNA binding domain and activation domain are dimerized through interaction of their FKBP and FRB domains, leading to transcription activation of the transgene.
• the ZFHD1 is included in frame with the GT2A or IRES.
• the expression system is designed to have one, two or three copies of the FKBP sequence. These are termed herein FKBP subunits.
• the subunits are designed to express the same protein, but to have nucleic acids which are divergent from one another in order to minimize recombination.
• SEQ ID NO: 27 provides 3 “wobbled” coding sequences for FKBP, each of which encode the sequence shown in SEQ ID NO: 28: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVI RGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE
• the expression system further comprises zinc finger homeodomain binding sites.
• the nucleic acid molecule contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 binding sites for ZFHD.
• the expression system contains 8 (eight) zinc finger homeodomains binding site (binding partners) (8XZFHD).
• the invention encompasses expression systems having from two to about twelve copies of the zinc finger binding site.
• An example of a single copy of a ZFHD binding site is: aatgatgggcgctcgagt (SEQ ID NO: 29)
• An exemplary IL2 promoter is shown in SEQ ID NO: 30.
• Such inducible systems are known in the art, and include, e.g., the rapamycin- inducible system described by e.g., 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 105, number 4, both of which are incorporated herein by reference.
• rapamycin- inducible system described by e.g., 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 105, number 4, both of which are incorporated herein by reference.
• the inducible gene expression system comprises a CMV promoter
• the activation domain is a FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin-associated protein (FRAP) fused to a carboxy terminus from the p65 subunit of NF-kappa B from a human, GT2A peptide, ZFHD1 DNA binding domain, three FKBP subunits, an hGH poly A, 8XZFHD, and a minimal sIL2 promoter.
• FBB FKBP12-rapamycin binding
• FRAP human FKBP12-rapamycin-associated protein
• an expression cassette and/or a vector may contain other appropriate transcription initiation, termination, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
• suitable polyA sequences include, e.g., SV40, bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit ⁇ -globin (also referred to as rabbit globin polyA; RGB), modified RGB (mRGB) and TK polyA.
• control sequences are “operably linked” to the GLP-2 construct sequences.
• operably linked refers to both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
• a rAAV which includes a 5’ ITR, CB7 promoter, chicken beta-actin intron, coding sequence for the fusion protein of SEQ ID NO: 13, 15, 17, 19, 21, or 23, a rabbit globin poly A, and a 3’ ITR.
• the rAAV comprises a polynucleotide comprising a CMV promoter
• the activation domain is a FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin-associated protein (FRAP) fused to a carboxy terminus from the p65 subunit of NF-kappa B from a human, IRES, ZFHD1 DNA binding domain, three FKBP subunits, an hGH poly A, 8XZFHD, a minimal sIL2 promoter, coding sequence for the GLP-2 fusion protein of SEQ ID NO: 13, 15, 17, 19, 21, or 23, and rabbit beta globin polyA.
• a two vector inducible system is provided.
• the first rAAV comprises 12XZFHD, a minimal IL2 promoter, coding sequence for the GLP-2 fusion protein of SEQ ID NO: 13, 15, 17, 19, 21, or 23, and rabbit beta globin polyA.
• a stuffer sequence may be included to increase the packaging size of the vector.
• the second rAAV comprises a polynucleotide comprising a CMV promoter, an intron, the activation domain is a FKBP12- rapamycin binding (FRB) domain of human FKBP12-rapamycin-associated protein (FRAP) fused to a carboxy terminus from the p65 subunit of NF kappa B from a human, IRES, ZFHD1 DNA binding domain, and a polyA.
• FB FKBP12- rapamycin binding
• FRAP human FKBP12-rapamycin-associated protein
• an expression cassette in one embodiment, includes a polynucleotide comprising a CB7 promoter, chicken beta-actin intron, coding sequence for the fusion protein of SEQ ID NO: 13, 15, 17, 19, 21, or 23, and a rabbit globin poly A.
• 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.
• a vector genome is provided wherein SEQ ID NO: X-X, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity therewith is flanked by 5’ and 3’ AAV ITRs.
• an expression cassette in another embodiment, includes a polynucleotide comprising a CB7 promoter, chicken beta-actin intron, coding sequence for the fusion protein of SEQ ID NO: 13, 15, 17, 19, 21, or 23, and a rabbit globin poly A.
• Viral Vectors in another aspect, viral vectors that include the expression cassettes described herein are provided. In certain embodiments of the viral vectors described herein, the viral vector is an adeno-associated virus (AAV) viral vector or recombinant AAV (rAAV).
• AAV adeno-associated virus
• rAAV recombinant AAV
• AAV adeno-associated virus
• An adeno-associated virus (AAV) viral vector is an AAV DNase-resistant particle having an AAV protein capsid into which is packaged an expression cassette flanked by AAV inverted terminal repeat sequences (ITRs) (together referred to as the “vector genome”) for delivery to target cells.
• ITRs AAV inverted terminal repeat sequences
• An AAV capsid is composed of 60 capsid (cap) protein subunits, VP1, VP2, and VP3, that are arranged in an icosahedral symmetry in a ratio of approximately 1:1:10 to 1:1:20, depending upon the selected AAV.
• Various AAVs may be selected as sources for capsids of AAV viral vectors as identified above.
• the AAV capsid is an AAVrh91 capsid or variant thereof.
• the capsid protein is designated by a number or a combination of numbers and letters following the term “AAV” in the name of the rAAV vector.
• the AAV capsid, ITRs, and other selected AAV components described herein may be readily selected from among any AAV, including, without limitation, the AAVs identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVhu37, AAVrh32.33, AAVAnc80, AAV10, AAV11, AAV12, AAVrh8, AAVrh74, AAV-DJ8, AAV-DJ, AAVhu.37, AAVrh.64R1, and AAVhu68.
• AAVrh90 [PCT/US20/30273, filed April 28, 2020]
• AAVrh91 [PCT/US20/030266, filed April 28, 2020, now a publication WO 2020/223231, published November 5, 2020]
• AAVrh92, AAVrh93, AAVrh91.93 [PCT/US20/30281, filed April 28, 2020]
• AAV3B variants which are described in US Provisional Patent Application No.62/924,112, filed October 21, 2019, and US Provisional Patent Application No.63/025,753, filed May 15, 2020, describing AAV3B.AR2.01, AAV3B.AR2.02, AAV3B.AR2.03, AAV3B.AR2.04, AAV3B.AR2.05, AAV3B.AR2.06, AAV3B.AR2.07, AAV3B.AR2.08, AAV3B.AR2.10, 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.
• the term “variant” means any AAV sequence which is derived from a known AAV sequence, including those with a conservative amino acid replacement, and those sharing at least 90%, at least 95%, at least 97%, at least 99% or greater sequence identity over the amino acid or nucleic acid sequence.
• the AAV capsid includes variants which 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 to an AAV capsid provided herein and/or known in the art.
• the AAV capsid shares at least 95% identity with an AAV capsid.
• the comparison may be made over any of the variable proteins (e.g., vp1, vp2, or vp3).
• the viral vector is an rAAV having the capsid of AAV8 or a functional variant thereof.
• the viral vector is an rAAV having the capsid of AAVrh91 or a functional variant thereof.
• the viral vector is an rAAV having the capsid of AAV3.AR.2.12 or a functional variant thereof.
• the viral vector is an rAAV having a capsid selected from AAV9, AAVrh64R1, AAVhu37, or AAVrh10.
• 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.
• an rAAV comprising at least one of the vp1, vp2 and the vp3 of AAVrh91 (SEQ ID NO: 32).
• rAAV comprising an AAV capsid encoded by at least one of the vp1, vp2 and the vp3 of AAVrh91 (SEQ ID NO: 31).
• 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.
• rAAV comprising an AAV capsid encoded by at least one of the vp1, vp2 and the vp3 of AAVrh91eng (SEQ ID NO: 19).
• the vp1, vp2 and/or vp3 is the full-length capsid protein of AAVrh91 (SEQ ID NO: 32). In other embodiments, the vp1, vp2 and/or vp3 has an N- terminal and/or a C-terminal truncation (e.g., truncation(s) of about 1 to about 10 amino acids).
• an AAVrh91 capsid is characterized by one or more of the following: (1) AAVrh91 capsid proteins comprising: a heterogeneous population of AAVrh91 vp1 proteins selected from: vp1 proteins produced by expression from a nucleic acid sequence which encodes the predicted amino acid sequence of 1 to 736 of SEQ ID NO: 32, vp1 proteins produced from SEQ ID NO: 31, or vp1 proteins produced from a nucleic acid sequence at least 70% identical to SEQ ID NO: 31 which encodes the predicted amino acid sequence of 1 to 736 of SEQ ID NO: 32, a heterogeneous population of AAVrh91 vp2 proteins selected from: vp2 proteins produced by expression from a nucleic acid sequence which encodes the predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO: 32, vp2 proteins produced from a sequence comprising at least nucleotides 412 to 2208 of SEQ ID NO: 31, or vp
• an AAVrh91 capsid is characterized by one or more of the following: (1) AAVrh91 capsid proteins comprising: a heterogeneous population of AAVrh91 vp1 proteins selected from: vp1 proteins produced by expression from a nucleic acid sequence which encodes the predicted amino acid sequence of 1 to 736 of SEQ ID NO: 32, vp1 proteins produced from SEQ ID NO: 19, or vp1 proteins produced from a nucleic acid sequence at least 70% identical to SEQ ID NO: 19 which encodes the predicted amino acid sequence of 1 to 736 of SEQ ID NO: 32, a heterogeneous population of AAVrh91 vp2 proteins selected from: vp2 proteins produced by expression from a nucleic acid sequence which encodes the predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO: 32, vp2 proteins produced from a sequence comprising at least nucleotides 412 to 2208 of SEQ ID NO: 19, or vp
• the AAVrh91 vp1, vp2 and vp3 proteins contain subpopulations with amino acid modifications comprising at least two highly deamidated asparagines (N) in asparagine – glycine pairs in SEQ ID NO: 32 and optionally further comprising subpopulations comprising other deamidated amino acids, wherein the deamidation results in an amino acid change.
• N highly deamidated asparagines
• subpopulations comprising other deamidated amino acids
• AAVrh91 may have other residues deamidated, e.g., typically at less than 10% and/or may have other modifications, including phosphorylation (e.g., where present, in the range of about 2 to about 30%, or about 2 to about 20%, or about 2 to about 10%) (e.g., at S149), or oxidation (e.g., at one or more of ⁇ W22, ⁇ M211, W247, M403, M435, M471, W478, W503, ⁇ M537, ⁇ M541, ⁇ M559, ⁇ M599, M635, and/or, W695).
• the W may oxidize to kynurenine.
• an AAVrh91 capsid is modified in one or more of the positions identified in the preceding table, in the ranges provided, as determined using mass spectrometry with a trypsin enzyme.
• one or more of the 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.
• an AAVrh91 capsid comprises: a heterogeneous population of vp1 proteins which are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 32, a heterogeneous population of vp2 proteins which are the product of a nucleic acid sequence encoding the amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO: 32, and a heterogeneous population of vp3 proteins which are the product of a nucleic acid sequence encoding at least amino acids 203 to 736 of SEQ ID NO: 32.
• the modified AAVrh91 nucleic acid sequences is be used to generate a mutant rAAV having a capsid with lower deamidation than the native AAVrh91 capsid.
• Such mutant rAAV may have reduced immunogenicity and/or increase stability on storage, particularly storage in suspension form.
• a recombinant AAV rAAV is provided.
• the rAAV includes an AAV capsid from adeno-associated virus rh91, and a vector genome packaged in the AAV capsid, said vector genome comprising AAV inverted terminal repeats (ITRs), a coding sequence for the GLP-2 receptor agonist of SEQ ID NO: 14, and regulatory sequences which direct expression of the GLP-2 receptor agonist.
• ITRs AAV inverted terminal repeats
• GLP-2 receptor agonist a coding sequence for the GLP-2 receptor agonist of SEQ ID NO: 14
• regulatory sequences which direct expression of the GLP-2 receptor agonist regulatory sequences which direct expression of the GLP-2 receptor agonist.
• an AAV68 capsid is further characterized by one or more of the following.
• AAV hu68 capsid proteins comprise: AAVhu68 vp1 proteins produced by expression from a nucleic acid sequence which encodes the predicted amino acid sequence of 1 to 736 of SEQ ID NO: 55, vp1 proteins produced from SEQ ID NO: 53 or 54, or vp1 proteins produced from a nucleic acid sequence at least 70% identical to SEQ ID NO: 53 or 54 which encodes the predicted amino acid sequence of 1 to 736 of SEQ ID NO: 55; AAVhu68 vp2 proteins produced by expression from a nucleic acid sequence which encodes the predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO: 55, vp2 proteins produced from a sequence comprising at least nucleotides 412 to 2211 of SEQ ID NO: 53 or 54, or vp2 proteins produced from a nucleic acid sequence at least 70% identical to at least nucleotides 412 to 2211 of SEQ ID NO: 53 or 54 which encodes the predicted amino acid sequence of at least about
• an AAV capsid which comprise a heterogenous population of vp1 proteins optionally comprising a valine at position 157, a heterogenous population of vp2 proteins optionally comprising a valine at position 157, and a heterogenous population of vp3 proteins, wherein at least a subpopulation of the vp1 and vp2 proteins comprise a valine at position 157 and optionally further comprising a glutamic acid at position 67 based on the numbering of the vp1 capsid of SEQ ID NO: 55.
• an AAVhu68 capsid which comprises a heterogenous population of vp1 proteins which are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 55, a heterogenous population of vp2 proteins which are the product of a nucleic acid sequence encoding the amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO: 55, and a heterogenous population of vp3 proteins which are the product of a nucleic acid sequence encoding at least amino acids 203 to 736 of SEQ ID NO: 55, wherein: the vp1, vp2 and vp3 proteins contain subpopulations with amino acid modifications
• the AAVhu68 vp1, vp2 and vp3 proteins are typically expressed as alternative splice variants encoded by the same nucleic acid sequence which encodes the full-length vp1 amino acid sequence of SEQ ID NO: 55 (amino acid 1 to 736).
• the vp1-encoding sequence is used alone to express the vp1, vp2 and vp3 proteins.
• this sequence may be co-expressed with one or more of a nucleic acid sequence which encodes the AAVhu68 vp3 amino acid sequence of SEQ ID NO: 55 (about aa 203 to 736) without the vp1-unique region (about aa 1 to about aa 137) and/or vp2-unique regions (about aa 1 to about aa 202), or a strand complementary 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 which encodes aa 203 to 736 of SEQ ID NO: 55
• the vp1-encoding and/or the vp2-encoding sequence may be co expressed with the nucleic acid sequence which encodes the AAVhu68 vp2 amino acid sequence of SEQ ID NO: 55 (about aa 138 to 736) without the vp1-unique region (about aa 1 to about 137), or a strand complementary thereto, the corresponding mRNA or tRNA (nt 412 to 2212 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 which encodes about aa 138 to 736 of SEQ ID NO: 55.
• a rAAVhu68 has a rAAVhu68 capsid produced in a production system expressing capsids from an AAVhu68 nucleic acid which encodes the vp1 amino acid sequence of SEQ ID NO: 55, and optionally additional nucleic acid sequences, e.g., encoding a vp3 protein free of the vp1 and/or vp2-unique regions.
• the rAAVhu68 resulting from production using a single nucleic acid sequence vp1 produces the heterogenous populations of vp1 proteins, vp2 proteins and vp3 proteins.
• the AAVhu68 capsid contains subpopulations within the vp1 proteins, within the vp2 proteins and within the vp3 proteins which have modifications from the predicted amino acid residues in SEQ ID NO: 55.
• These subpopulations include, at a minimum, deamidated asparagine (N or Asn) residues.
• asparagines in asparagine - glycine pairs are highly deamidated.
• the AAVhu68 vp1 nucleic acid sequence has the sequence of SEQ ID NO: 53 or 54, or a strand complementary thereto, e.g., the corresponding mRNA or tRNA.
• the vp2 and/or vp3 proteins may be expressed additionally or alternatively from different nucleic acid sequences than the vp1, e.g., to alter the ratio of the vp proteins in a selected expression system.
• a nucleic acid sequence which encodes the AAVhu68 vp3 amino acid sequence of SEQ ID NO: 55 (about aa 203 to 736) without the vp1-unique region (about aa 1 to about aa 137) and/or vp2-unique regions (about aa 1 to about aa 202), or a strand complementary thereto, the corresponding mRNA or tRNA (about nt 607 to about nt 2211 of SEQ ID NO: 53 or 54).
• nucleic acid sequence which encodes the AAVhu68 vp2 amino acid sequence of SEQ ID NO: 55 (about aa 138 to 736) without the vp1-unique region (about aa 1 to about 137), or a strand complementary thereto, the corresponding mRNA or tRNA (nt 412 to 2211 of SEQ ID NO: 53 or 54).
• nucleic acid sequences which encode the amino acid sequence of SEQ ID NO: 55 may be selected for use in producing rAAVhu68 capsids.
• the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 53 or 54 or a sequence at least 70% to 99% identical, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, identical to SEQ ID NO: 53 or 54 which encodes SEQ ID NO: 55.
• the nucleic acid sequence has the nucleic acid sequence 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 about nt 412 to about nt 2211 of SEQ ID NO: 53 or 54 which encodes the vp2 capsid protein (about aa 138 to 736) of SEQ ID NO: 55.
• the nucleic acid sequence has the nucleic acid sequence of 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 SEQ ID NO: 53 or 54 which encodes the vp3 capsid protein (about aa 203 to 736) of SEQ ID NO: 55.
• the AAVhu68 capsid is characterized, by having, capsid proteins in which at least 45% of N residues are deamidated at least one of positions N57, N329, N452, and/or N512 based on the numbering of amino acid sequence of SEQ ID NO: 55. In certain embodiments, at least about 60%, at least about 70%, at least about 80%, or at least 90% of the N residues at one or more of these N-G positions (i.e., N57, N329, N452, and/or N512, based on the numbering of amino acid sequence of SEQ ID NO: 55) are deamidated.
• an AAVhu68 capsid is further characterized by having a population of proteins in which about 1% to about 20% of the N residues have deamidations at one or more of positions: N94, N253, N270, N304, N409, N477, and/or Q599, based on the numbering of amino acid sequence of SEQ ID NO: 55.
• the AAVhu68 comprises at least a subpopulation of vp1, vp2 and/or vp3 proteins which 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, based on the numbering of amino acid sequence of SEQ ID NO: 55, or combinations thereof.
• the capsid proteins may have one or more amidated amino acids.
• a recombinant adeno-associated virus having an AAVhu68 capsid and a vector genome, wherein (a) the AAV hu68 capsid comprises a heterogenous population of AAVhu68 vp1 proteins, a heterogenous population of AAVhu68 vp2 proteins; and a heterogenous population of AAVhu68 vp3 proteins, wherein the heterogenous AAVhu68 vp1, AAVhu68 vp2 and AAVhu68 vp3 proteins contain subpopulations with amino acid modifications comprising 50% to 100% deamidation in at least two asparagines (N) in asparagine - glycine pairs in two or more of N57, N329, N452, N512 of SEQ ID NO: 55 as determined using mass spectrometry and optionally further comprising subpopulations comprising other deamidated amino acids, wherein the deamidation results in an amino acid change, wherein
• the rAAV is an scAAV.
• sc refers to self- complementary.
• Self-complementary AAV refers a plasmid or vector having an expression cassette in which a coding region carried by a recombinant AAV nucleic acid sequence has been designed to form an intra-molecular double-stranded DNA template.
• dsDNA double stranded DNA
• the nucleic acid sequences encoding the GLP-2 constructs described herein are engineered into any suitable genetic element, e.g., naked DNA, phage, transposon, cosmid, RNA molecule (e.g., mRNA), episome, etc., which transfers the GLP-2 sequences carried thereon to a host cell, e.g., for generating nanoparticles carrying DNA or RNA, viral vectors in a packaging host cell and/or for delivery to a host cell in a subject.
• the genetic element is a plasmid.
• the selected genetic element may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion.
• the methods used to make such constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).
• the term “host cell” may refer to the packaging cell line in which a vector (e.g., a recombinant AAV or rAAV) is produced from a production plasmid.
• a host cell may refer to any target cell in which expression of a gene product described herein is desired.
• a host cell refers to a prokaryotic or eukaryotic cell (e.g., bacterial cell, human cell or insect cell) that contains exogenous or heterologous DNA that has been introduced into the cell by any means, e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion.
• the term “host cell” refers to cultures of cells of various mammalian species for in vitro assessment of the compositions described herein.
• the term “host cell” refers to the cells employed to generate and package the viral vector or recombinant virus.
• the term “host cell” is an intestine cell, a small intestine cell, a pancreatic cell, a liver cell.
• the term “target cell” refers to any target cell in which expression of a heterologous nucleic acid sequence or protein is desired.
• the target cell is a liver cell.
• the target cell is a muscle cell.
• the rAAV comprises a vector genome comprising an expression cassette, wherein the expression cassette comprises a CMV promoter, the activation domain is a FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin-associated protein (FRAP) fused to a carboxy terminus from the p65 subunit of NF-kappa B from a human, GT2A_V1 peptide, ZFHD1 DNA binding domain, three FKBP subunits, an hGH poly A, 12XZFHD, a minimal sIL2 promoter, coding sequence for the GLP-2 fusion protein of SEQ ID NO: 13, 15, 17, 19, 21, or 23, and rabbit beta globin polyA.
• FVB FKBP12-rapamycin binding
• FRAP human FKBP12-rapamycin-associated protein
• the rAAV is provide which comprises a vector genome comprising an expression cassette, wherein the expression cassette comprises a CMV promoter, the activation domain is a FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin-associated protein (FRAP) fused to a carboxy terminus from the p65 subunit of NF-kappa B from a human, GT2A_V2 peptide, ZFHD1 DNA binding domain, three FKBP subunits, an hGH poly A, 12XZFHD, a minimal sIL2 promoter, coding sequence for the GLP-2 fusion protein of SEQ ID NO: 13, 15, 17, 19, 21, or 23, and rabbit beta globin polyA.
• FVB FKBP12-rapamycin binding
• FRAP human FKBP12-rapamycin-associated protein
• the rAAV comprises a vector genome comprising an expression cassette, wherein the expression cassette comprises a CB7 promoter, chicken beta-actin intron, coding sequence for the fusion protein of SEQ ID NO: 13, 15, 17, 19, 21, or 23, a rabbit globin poly A, and a 3 ITR.
• the rAAV comprises a vector genome comprising an expression cassette, wherein the expression cassette comprises a CMV promoter, the activation domain is a FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin-associated protein (FRAP) fused to a carboxy terminus from the p65 subunit of NF-kappa B from a human, IRES, ZFHD1 DNA binding domain, three FKBP subunits, an hGH poly A, 8XZFHD, a minimal sIL2 promoter, coding sequence for the GLP-2 fusion protein of SEQ ID NO: 13, 15, 17, 19, 21, or 23and rabbit beta globin polyA.
• FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin-associated protein (FRAP) fused to a carboxy terminus from the p65 subunit of NF-kappa B from a human, IRES, ZFHD1 DNA binding domain, three FKBP subunits, an
• a two vector inducible system comprising a vector genome comprising an expression cassette, wherein the expression cassette comprises a 12XZFHD, a minimal IL2 promoter, coding sequence for the GLP-2 fusion protein of SEQ ID NO: 13, 15, 17, 19, 21, or 23, and rabbit beta globin polyA.
• a stuffer sequence may be included to increase the packaging size of the vector.
• the second rAAV comprises a vector genome comprising an expression cassette, wherein the expression cassette comprises a polynucleotide comprising a CMV promoter, an intron, the activation domain is a FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin-associated protein (FRAP) fused to a carboxy terminus from the p65 subunit of NF-kappa B from a human, IRES, ZFHD1 DNA binding domain, and a polyA.
• FBB FKBP12-rapamycin binding
• FRAP human FKBP12-rapamycin-associated protein
• an rAAV includes a vector genome that includes an expression cassette that includes a polynucleotide comprising a CB7 promoter, chicken beta-actin intron, coding sequence for the fusion protein of SEQ ID NO: 13, 15, 17, 19, 21, or 23, and a rabbit globin poly A.
• the minimal sequences required to package the 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 produce an AAV pseudotype).
• the ITR sequences from AAV2, or the deleted version thereof ( ⁇ ITR) are used for convenience and to accelerate regulatory approval. However, ITRs from other AAV sources may be selected.
• the source of the ITRs is the same as the source of the Rep protein, which is provided in trans for production.
• an expression cassette for an AAV vector comprises an AAV 5’ ITR, the GLP-2 fusion protein coding sequences and any regulatory sequences, and an AAV 3 ITR.
• a shortened version of the 5’ ITR termed ⁇ ITR, has been described in which the D- sequence and terminal resolution site (trs) are deleted.
• trs terminal resolution site
• the full-length AAV 5’ and 3’ ITRs are used.
• the ITRs are the only AAV components required in cis in the same construct as the gene.
• the coding sequences for the replication (rep) and/or capsid (cap) are removed from the AAV genome and supplied in trans or by a packaging cell line in order to generate the AAV vector.
• a pseudotyped AAV may contain ITRs from a source which differs from the source of the AAV capsid.
• a chimeric AAV capsid may be utilized. Still other AAV components may be selected. Sources of such AAV sequences are described herein and may also be isolated or obtained from academic, commercial, or public sources (e.g., the American Type Culture Collection, Manassas, VA).
• the AAV sequences may be obtained through synthetic or other suitable means by reference to published sequences such as are available in the literature or in databases such as, e.g., GenBank®, PubMed®, or the like.
• Methods for generating and isolating AAV viral vectors suitable for delivery to a subject are known in the art. See, e.g., US Patent 7790449; US Patent 7282199; WO 2003/042397; WO 2005/033321, WO 2006/110689; and US 7588772 B2].
• a producer cell line is transiently transfected with a construct that encodes the transgene flanked by ITRs and a construct(s) that encodes rep and cap.
• a packaging cell line that stably supplies rep and cap is transiently transfected with a construct encoding the transgene flanked by ITRs.
• AAV virions are produced in response to infection with helper adenovirus or herpesvirus, requiring the separation of the rAAVs from contaminating virus.
• helper adenovirus or herpesvirus More recently, systems have been developed that do not require infection with helper virus to recover the AAV - the required helper functions (i.e., adenovirus E1, E2a, VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase) are also supplied, in trans, by the system.
• helper functions can be supplied by transient transfection of the cells with constructs that encode the required helper functions, or the cells can be engineered to stably contain genes encoding the helper functions, the expression of which can be controlled at the transcriptional or posttranscriptional level.
• the transgene flanked by ITRs and rep/cap genes are introduced into insect cells by infection with baculovirus-based vectors.
• the rAAV described herein comprise a selected capsid with a vector genome packaged inside.
• the vector genome (or rAAV genome) comprises 5’ and 3’ AAV inverted terminal repeats (ITRs), the polynucleotide sequence encoding the fusion protein, and regulatory sequences which direct insertion of the polynucleotide sequence encoding the fusion protein to the genome of a host cell.
• the vector genome is the sequence shown 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 therewith.
• a “vector genome” refers to the nucleic acid sequence packaged inside a parvovirus (e.g., rAAV) capsid which forms a viral particle.
• a nucleic acid sequence contains AAV inverted terminal repeat sequences (ITRs).
• ITRs AAV inverted terminal repeat sequences
• a vector genome contains, at a minimum, from 5 to 3 , an AAV 5 ITR, coding sequence(s) (i.e., transgene(s)), and an AAV 3’ ITR. ITRs from AAV2, a different source AAV than the capsid, or other than full-length ITRs may be selected.
• the ITRs are from the same AAV source as the AAV which provides the rep function during production or a transcomplementing AAV.
• ITRs e.g., self-complementary (scAAV) ITRs
• scAAV self-complementary
• Both single-stranded AAV and self-complementary (sc) AAV are encompassed with the rAAV.
• the transgene is a nucleic acid coding sequence, heterologous to the vector sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product, of interest.
• a “vector genome” contains, at a minimum, from 5’ to 3’, a vector-specific sequence, a nucleic acid sequence encoding GLP-2 constructs operably linked to regulatory control sequences (which direct their expression in a target cell), where the vector-specific sequence may be a terminal repeat sequence which specifically packages the vector genome into a viral vector capsid or envelope protein.
• AAV inverted terminal repeats are utilized for packaging into AAV and certain other parvovirus capsids.
• the AAV sequences of the vector typically comprise the cis-acting 5′ and 3′ inverted terminal repeat sequences (See, e.g., B. J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp.155168 (1990)).
• the ITR sequences are about 145 bp in length.
• substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible.
• the ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al, “Molecular Cloning.
• An example of such a molecule employed in the present invention is a “cis-acting” plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5′ and 3′ AAV ITR sequences.
• the ITRs are from an AAV different than that supplying a capsid.
• the ITR sequences from AAV2. However, ITRs from other AAV sources may be selected.
• the vector genome includes a shortened AAV2 ITR of 130 base pairs, wherein the external A elements is deleted. Without wishing to be bound by theory, it is believed that the shortened ITR reverts back to the wild-type length of 145 base pairs during vector DNA amplification using the internal (A’) element as a template. In other embodiments, full-length AAV 5’ and 3’ ITRs are used. Where the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be termed pseudotyped.
• the GLP-2 constructs described herein may be delivered via viral vectors other than rAAV.
• viral vectors may include any virus suitable for gene therapy, including but not limited to adenovirus; herpes virus; lentivirus; retrovirus; etc.
• virus suitable for gene therapy including but not limited to adenovirus; herpes virus; lentivirus; retrovirus; etc.
• one of these other vectors is generated, it is produced as a replication-defective viral vector.
• a “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, where any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient; i.e., they cannot generate progeny virions but retain the ability to infect target cells.
• the genome of the viral vector does not include genes encoding the enzymes required to replicate (the genome can be engineered to be “gutless”- containing only the transgene of interest flanked by the signals required for amplification and packaging of the artificial genome), but these genes may be supplied during production.
• compositions which include the viral vector constructs described herein are designed for delivery to subjects in need thereof by any suitable route or a combination of different routes.
• Direct delivery to the liver optionally via intravenous, via the hepatic artery, or by transplant
• the viral vectors described herein may be delivered in a single composition or multiple compositions.
• two or more different AAV may be delivered, or multiple viruses [see, e.g., WO 2011/126808 and WO 2013/049493].
• multiple viruses may contain different replication-defective viruses (e.g., AAV and adenovirus).
• administration is intramuscular.
• administration is intravenous.
• the replication-defective viruses can be formulated with a physiologically acceptable carrier for use in gene transfer and gene therapy applications.
• quantification of the genome copies (“GC”) may be used as the 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 compositions of the invention.
• AAV GC number titration One method for performing AAV GC number titration is as follows: Purified AAV vector samples are first treated with DNase to eliminate un-encapsidated AAV genome DNA or contaminating plasmid DNA from the production process. The nuclease resistant particles are then subjected to heat treatment to release the genome from the capsid. The released genomes are then quantitated by real-time PCR using primer/probe sets targeting specific region of the viral genome (usually poly A signal).
• Another suitable method for determining genome copies is quantitative- PCR (qPCR), particularly the optimized qPCR or digital droplet PCR [Lock Martin, et al, Human Gene Therapy Methods. April 2014, 25(2): 115-125.
• the replication-defective virus compositions can be formulated in dosage units to contain an amount of replication-defective virus that is in the range of about 1.0 x 10 9 GC to about 1.0 x 10 15 GC. In another embodiment, this amount of viral genome may be delivered in split doses. In one embodiment, the dose is about 1.0 x 10 10 GC to about 3.0 x 10 14 GC for an average human subject of about 70 kg. In another embodiment, the dose about 1 x 10 9 GC.
• the dose of AAV virus may be about 1 x 10 10 GC, 1 x 10 11 GC, about 5 X 10 11 GC, about 1 X 10 12 GC, about 5 X 10 12 GC, or about 1 X 10 13 GC.
• the dosage is about 1.0 x 10 9 GC/kg to about 3.0 x 10 14 GC/kg for a human subject.
• the dose about 1 x 10 9 GC/kg.
• the dose of AAV virus may be about 1 x 10 10 GC/kg, 1 x 10 11 GC/kg, about 5 X 10 11 GC/kg, about 1 X 10 12 GC/kg, about 5 X 10 12 GC/kg, or about 1 X 10 13 GC/kg.
• the constructs may be delivered in volumes from 1 ⁇ L to about 100 mL.
• the term “dosage” or “amount” can refer to the total dosage or amount delivered to the subject in the course of treatment, or the dosage or amount delivered in a single unit (or multiple unit or split dosage) administration.
• the above-described recombinant vectors may be delivered to host cells according to published methods.
• the rAAV preferably suspended in a physiologically compatible carrier, may be administered to a desired subject including a human.
• Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the transfer virus is directed.
• one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline).
• Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present invention.
• the composition includes a carrier, diluent, excipient and/or adjuvant.
• the rAAV for administration to a human patient, is suitably suspended in an aqueous solution containing saline, a surfactant, and a pharmaceutically and/or physiologically compatible salt or mixture of salts.
• the formulation is adjusted to a physiologically acceptable pH, e.g., in the range of pH 6 to 9, or pH 6.0 to 7.5, or pH 6.2 to 7.7, or pH 6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8, or about 7.0.
• 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.
• 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 desired.
• a pH of about 6.8 to about 7.2 may be desired for intravenous delivery.
• other pHs within the broadest ranges and these subranges may be selected for other route of delivery.
• compositions of the invention may contain, in addition to the rAAV and/or variants and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers.
• suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.
• Suitable chemical stabilizers include gelatin and albumin.
• 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.
• 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.
• Supplementary active ingredients can also be incorporated into the compositions.
• pharmaceutically-acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.
• a composition includes a final formulation suitable for delivery to a subject, e.g., is an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration.
• one or more surfactants are present in the formulation.
• the composition may be transported as a concentrate which is diluted for administration to a subject.
• the composition may be lyophilized and reconstituted at the time of administration.
• a suitable surfactant, or combination of surfactants may be selected from among non-ionic surfactants that are nontoxic.
• a difunctional block copolymer surfactant terminating in primary hydroxyl groups is selected, e.g., such as Pluronic® F68 [BASF], also known as Poloxamer 188, which has a neutral pH, has an average molecular weight of 8400.
• Poloxamers may be selected, i.e., nonionic triblock copolymers composed 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 (Polyoxy capryllic glyceride), polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol.
• the formulation contains a poloxamer.
• copolymers are commonly named with the letter “P” (for poloxamer) followed by three digits: the first two digits x 100 give the approximate molecular mass of the polyoxypropylene core, and the last digit x 10 gives the percentage polyoxyethylene content.
• Poloxamer 188 is selected.
• the surfactant may be present in an amount up to about 0.0005 % to about 0.001% of the suspension. Dosages of the vector depends primarily on factors such as the condition being treated, the age, weight and health of the patient, and may thus vary among patients.
• a therapeutically effective human dosage of viral vector is generally in the range of from about 25 to about 1000 microliters to about 100 mL of solution containing concentrations of from about 1 x 10 9 to 1 x 10 16 genomes virus vector (to treat an average subject of 70 kg in body weight) including all integers or fractional amounts within the range, and preferably 1.0 x 10 12 GC to 1.0 x 10 13 GC for a human patient.
• the composition of the invention may be delivered in a volume of from about 0.1 ⁇ L to about 10 mL, including all numbers within the range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method. In one embodiment, the volume is about 50 ⁇ L.
• the volume is about 70 ⁇ L. In another embodiment, the volume is about 100 ⁇ L. In another embodiment, the volume is about 125 ⁇ L. In another embodiment, the volume is about 150 ⁇ L. In another embodiment, the volume is about 175 ⁇ L. In yet another embodiment, the volume is about 200 ⁇ L. In another embodiment, the volume is about 250 ⁇ L. In another embodiment, the volume is about 300 ⁇ L. In another embodiment, the volume is about 450 ⁇ L. In another embodiment, the volume is about 500 ⁇ L. In another embodiment, the volume is about 600 ⁇ L. In another embodiment, the volume is about 750 ⁇ L. In another embodiment, the volume is about 850 ⁇ L. In another embodiment, the volume is about 1000 ⁇ L.
• 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.
• the volume is about 9.5 mL. In another embodiment, the volume is about 10 mL.
• a concentration of a recombinant adeno-associated virus carrying a nucleic acid sequence encoding the desired transgene under the control of the regulatory sequences desirably ranges from about 10 7 and 10 14 genome copies per milliliter (GC/mL) in a composition.
• the dosage of rAAV in a composition is from about 1.0 x 10 9 GC/kg of body weight to about 1.5 x 10 13 GC/kg. In one embodiment, the dosage is about 1.0 x 10 10 GC/kg. In one embodiment, the dosage is about 1.0 x 10 11 GC/kg.
• the dosage is about 1.0 x 10 12 GC/kg. In one embodiment, the dosage is about 5.0 x 10 12 GC/kg. In one embodiment, the dosage is about 1.0 x 10 13 GC/kg. All ranges described herein are inclusive of the endpoints.
• the effective dosage (total genome copies delivered) is from about 10 7 to 10 13 genome copies. In one embodiment, the total dosage is about 10 8 genome copies. In one embodiment, the total dosage is about 10 9 genome copies. In one embodiment, the total dosage is about 10 10 genome copies. In one embodiment, the total dosage is about 10 11 genome copies. In one embodiment, the total dosage is about 10 12 genome copies. In one embodiment, the total dosage is about 10 13 genome copies.
• the total dosage is about 10 14 genome copies. In one embodiment, the total dosage is about 10 15 genome copies. It is desirable that the lowest effective concentration of virus be utilized in order to reduce the risk of undesirable effects, such as toxicity. Still other dosages and administration volumes in these ranges may be selected by the attending physician, taking into account the physical state of the subject, preferably human, being treated, the age of the subject, the particular disorder and the degree to which the disorder, if progressive, has developed.
• the composition comprises an rAAV comprising an inducible GLP-2 agonist construct. In certain embodiments, the inducing agent or molecule is a rapamycin or a rapalog.
• the inducing agent is rapamycin, and is administered at least one or more, at least two or more, at least three or more times following rAAV-comprising composition. In some embodiments the rapamycin is administered at dose at least about 4 to at least about 40 nM. In certain embodiments, the inducing agent (i.e., rapamycin) is administered at a dose at least about 0.1 mg/kg to at least about 3.0 mg/kg. In certain embodiments, the inducing agent (i.e., rapamycin) is administered at a dose at least about 0.5 mg/kg to at least about 2.0 mg/kg.
• the viral vectors and other constructs described herein may be used in preparing a medicament for delivering a GLP-2 fusion protein construct to a subject in need thereof, supplying GLP-2 having an increased half-life to a subject, and/or for treating SBS in a subject.
• a method of treating SBS includes administering a composition as described herein to a subject in need thereof.
• the composition includes a viral vector containing a GLP-2 fusion protein expression cassette, as described herein.
• treatment or “treating” is defined encompassing administering to a subject one or more compounds or compositions described herein for the purposes of amelioration of one or more symptoms of short bowel syndrome.
• Treatment can thus include one or more of reducing progression of SBS, reducing the severity of the symptoms, removing the disease symptoms, delaying progression of disease, or increasing efficacy of therapy in a given subject.
• a method for treating SBS in a subject includes administering a viral vector comprising a nucleic acid molecule comprising a sequence encoding a fusion protein as described herein.
• the subject is a human.
• a course of treatment may optionally involve repeat administration of the same viral vector (e.g., an AAVrh91 vector) or a different viral vector (e.g., an AAVrh91 and an AAV3B.AR2.12). Still other combinations may be selected using the viral vectors described herein.
• the composition described herein may be combined in a regimen involving nutritional therapy (enteral or parenteral nutrition), medications, such as those used to control stomach acid, reduce diarrhea, or improve intestinal absorption, or a GLP-2 analog, or surgery.
• the AAV vector and the combination therapy are administered essentially simultaneously.
• the AAV vector is administered first.
• the combination therapy is delivered first.
• the composition is administered in combination with an effective amount of a GLP-2 analog.
• GLP-2 analog Various commercially available GLP-2 products are known in the art, including, without limitation, teduglutide, glepaglutide, and apraglutide.
• combination of the rAAV described herein with GLP-2 analog decreases GLP-2 analog dose requirements in the subject, as compared to prior to treatment with the viral vector. Such dose requirements 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 may determine the correct dosage of GLP-2 analog needed by the subject. For example, the subject may be being treated using GLP-2 analog or other therapy, which the treating physician may continue upon administration of the AAV vector. Such GLP-2 analog or other co-therapy may be continued, reduced, or discontinued as needed subsequently.
• composition comprising the expression cassette, vector genome, rAAV, or other composition described herein for gene therapy is delivered as a single dose per patient.
• the subject is delivered a therapeutically effective amount of a composition described herein.
• a “therapeutically effective amount” refers to the amount of the expression cassette or vector, or a combination thereof that delivers and expresses in the target cells an amount of GLP-2-Fc sufficient to reach therapeutic goal.
• the therapeutically effective amount may be selected by the treating physician, or guided based on previously determined guidelines. For example, teduglutide may be provided at an initial dose of 0.05 mg/kg subcutaneously daily. The dose may be increased in 0.025 mg/kg increments 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 medication as needed to reach the equivalent of the desired dosage of 0.05 mg/kg daily.
• the therapeutic goal is to ameliorate or treat one or more of the symptoms of SBS.
• a therapeutically effective amount may be determined based on an animal model, rather than a human patient.
• the term “heterogenous” or any grammatical variation thereof refers to a population consisting of elements that are not the same, for example, 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.
• heterogenous as used in connection with vp1, vp2 and vp3 proteins refers to differences in the amino acid sequence of the vp1, vp2 and vp3 proteins within a capsid.
• the AAV capsid contains subpopulations within the vp1 proteins, within the vp2 proteins and within the vp3 proteins which have modifications from the predicted amino acid residues. These subpopulations include, at a minimum, certain deamidated asparagine (N or Asn) residues.
• certain subpopulations comprise at least one, two, three or four highly deamidated asparagines (N) positions in asparagine - glycine pairs and optionally further comprising other deamidated amino acids, wherein the deamidation results in an amino acid change and other optional modifications.
• a “subpopulation” of vp proteins refers to a group of vp proteins which has at least one defined characteristic in common and which consists of at least one group member to less than all members of the reference group, unless otherwise specified.
• a “subpopulation” of vp1 proteins is at least one (1) vp1 protein and less than all vp1 proteins in an assembled AAV capsid, unless otherwise specified.
• a “subpopulation” of vp3 proteins may be one (1) vp3 protein to less than all vp3 proteins in an assembled AAV capsid, unless otherwise specified.
• vp1 proteins may be a subpopulation of vp proteins;
• vp2 proteins may be a separate subpopulation of vp proteins, and
• vp3 are yet a further subpopulation of vp proteins in an assembled AAV capsid.
• vp1, vp2 and vp3 proteins may contain subpopulations having different modifications, e.g., at least one, two, three or four highly deamidated asparagines, e.g., at asparagine - glycine pairs.
• a “stock” of rAAV refers to a population of rAAV. Despite heterogeneity in their capsid proteins due to deamidation, rAAV in a stock are expected to 5 share an identical vector genome.
• a stock can include rAAV having capsids with, for example, heterogeneous deamidation patterns characteristic of the selected AAV capsid proteins and a selected production system. The stock may be produced from a single production system or pooled from multiple runs of the production system. A variety of production systems, including but not limited to those described herein, may be selected.
• GLP-2 construct As used herein the terms “GLP-2 construct”, “GLP-2 expression construct” and synonyms include the GLP-2 sequence as described herein in combination with a leader and fusion domain.
• the terms “GLP-2 construct”, “GLP-2 expression construct” and synonyms can be used to refer to the nucleic acid sequences encoding the GLP 2 fusion protein or the expression products thereof.
• the terms “percent (%) identity”, “sequence identity”, “percent sequence identity”, or “percent identical” in the context of nucleic acid sequences refers to the bases in the two sequences which are the same when aligned for correspondence.
• the length of sequence identity comparison may be over the full-length of the genome, the full-length of a gene coding sequence, or a fragment of at least about 100 to 150 nucleotides, or as desired. However, identity among smaller fragments, e.g., of at least about nine nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides, may also be desired.
• Multiple sequence alignment programs are also available for nucleic acid sequences. Examples of such programs include, “Clustal W”, “CAP Sequence Assembly”, “BLAST”, “MAP”, and “MEME”, which are accessible through Web Servers on the internet. Other sources for such programs are known to those of skill in the art.
• Vector NTI utilities are also used.
• algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above.
• polynucleotide sequences can be compared using FastaTM, a program in GCG Version 6.1.
• FastaTM provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences.
• percent sequence identity between nucleic acid sequences can be determined using FastaTM with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 6.1, herein incorporated by reference.
• highly conserved is meant at least 80% identity, preferably at least 90% identity, and more preferably, over 97% identity.
• Identity is readily determined by one of skill in the art by resort to algorithms and computer programs known by those of skill in the art. Unless otherwise specified by an upper range, it will be understood that a percentage of identity is a minimum level of identity and encompasses all higher levels of identity up to 100% identity to the reference sequence. Unless otherwise specified, it will be understood that a percentage of identity is a minimum level of identity and encompasses all higher levels of identity up to 100% identity to the reference sequence.
• 95% identity and at least 95% identity may be used interchangeably and include 95%, 96%, 97%, 98%, 99%, and up to 100% identity to the referenced sequence, and all fractions therebetween.
• percent (%) identity refers to the residues in the two sequences which are the same when aligned for correspondence. Percent identity may be readily determined for amino acid sequences over the full-length of a protein, polypeptide, about 70 amino acids to about 100 amino acids, or a peptide fragment thereof or the corresponding nucleic acid sequence coding sequencers.
• a suitable amino acid fragment may be at least about 8 amino acids in length, and may be up to about 150 amino acids.
• identity when referring to “identity”, “homology”, or “similarity” between two different sequences, “identity”, “homology” or “similarity” is determined in reference to “aligned” sequences. “Aligned” sequences or “alignments” refer to multiple nucleic acid sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence. Alignments are performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs.
• Sequence alignment programs are available for amino acid sequences, e.g., the “Clustal X”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs. Generally, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. See, e.g., J. D. Thomson et al, Nucl. Acids. Res., “A comprehensive comparison of multiple sequence alignments”, 27(13):2682-2690 (1999).
• the term “a” or “an” refers to one or more.
• the terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein.
• the words “comprise”, “comprises”, and “comprising” are to be interpreted inclusively rather than exclusively.
• the words “consist”, “consisting”, and its variants, are to be interpreted exclusively, rather than inclusively. While various embodiments in the specification are presented using “comprising” language, under other circumstances, a related embodiment is also intended to be interpreted and described using “consisting of” or “consisting essentially of” language.
• Patient or subject as used herein means a mammalian animal, including a human, a veterinary or farm animal, a domestic animal or pet, and animals normally used for clinical research.
• the subject of these methods and compositions is a human.
• the subject is not a feline.
• the term “about” means a variability of 10% ( ⁇ 10%, e.g., ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 5, ⁇ 6, ⁇ 7, ⁇ 8, ⁇ 9, ⁇ 10, or values therebetween) from the reference given, unless otherwise specified.
• the term “E+#” or the term “e+#” is used to reference an exponent.
• 5E10 or “5e10” is 5 x 10 10 .
• regulation or variations thereof as used herein refers to the ability of a composition to inhibit one or more components of a biological pathway.
• disease As used herein, “disease”, “disorder” and “condition” are used interchangeably, to indicate an abnormal state in a subject.
• 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 texts, which provide one skilled in the art with a general guide to many of the terms used in the present application.
• Example 1 Construction of GLP-2 vectors GLP-2 agonists are challenging to express via adeno-associated virus (AAV). GLP-2 is normally expressed from the glucagon precursor protein, which requires tissue specific proteases and produces unwanted proteins. Expression systems using traditional heterologous signal peptides yield low expression.
• AAV adeno-associated virus
• Expression systems using heterologous propeptides with universal protease cleavage sites yield foreign protein sequences that could be targets for T cells. More specifically, vectors were constructed in which a leader sequence was placed upstream of one of several GLP-2 receptor agonist amino acid sequences followed by a fusion domain. See, e.g., FIG.3. The resulting protein sequence was back-translated, followed by addition of a kozak consensus sequence, stop codon, and cloning sites. The sequences were produced, 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 is called pAAV.Z12I.hGLP2.G2.Fc.rBG.
• the sequences were produced, and cloned into an expression vector containing a CB7 constitutive promoter.
• GLP2-SA was identified by gel electrophoresis using spyroRuby stain.
• Example 3 – Pilot expression in Rag1KO mice Rag1KO female mice were treated with an injection of the vector AAVrh91.CB7.CI.hGLP-2-SA.rBG (1 x 10 11 GC/ mouse) or AAVrh91.CB7.CI.hGLP-2- Fc.rBG (1 x 10 11 GC/ mouse) via IM route of administration. Serum was serially collected by separating whole blood in serum separator tubes containing 5 microliters DPP-IV inhibitor (Millipore) and assayed for active GLP-2 expression and activity as above.
• DPP-IV inhibitor Microliter
• FIG.5A Serum GLP-2 concentrations (nM) are shown in FIG.5A and are an estimation based on Fc fusion standard. Serum expression levels reached increased through day 14 post dosing. Small intestines were weighed and measured at necropsy. The length and weight of small intestine increased significantly as compared to control animals (FIG.5B). In addition, vector treated intestine show healthy enterocyte growth as compared to vehicle treated animals. (FIG.5C). Serum citrulline levels (uM), a biomarker of gut surface area, were measured. (FIG.5D). Expression of GLP-2 was compared with albumin fusion construct.
• NHPs were administered AAVrh91.CB7.CI.hGLP-2-Fc.rBG via intramuscular injection (IM) at a dose of 1 x 10 13 (1e13) GC/kg (E185NG) and a of dose 5 x 10 10 (1e10) GC/kg (BM239H).
• IM intramuscular injection
• FIG.8A shows plasma level of GLP-2-Fc fusion protein.
• FIG.8B shows serum citrulline, a biomarker of gut surface area.
• FIG.8C shows detection of anti-GLP-2-Fc antibody in NHP serum at 1:100 dilution.
• AAV-mediated expression of GLP- 2-Fc fusion demonstrates substantial increase in gut surface area at the high dose until anti- GLP-2-Fc antibodies reduced its expression demonstrating its therapeutic efficacy on short bowel syndromes.
• Example 5 Long term efficacy and safety study in Rag1KO mice Rag1KO female mice were treated with an injection of the vector AAVrh91.CB7.CI.hGLP-2-Fc.rBG at a dosage of 1 x 10 10 GC/ mouse, 3 x 10 10 GC/ mouse, or 1 x 10 11 GC/ mouse via IM route of administration. The study design is shown in FIG.9A. hGLP2-Fc levels and body weight were measured throughout the study.

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