Viral vectors encoding parathyroid hormone fusions and their use in treatment of parathyroid hypofunction

Abstract

Compositions and methods for treating parathyroid hypofunction in a subject are provided. A viral vector is provided comprising a nucleic acid molecule comprising a sequence encoding a PTH fusion protein and a regulatory sequence that directs the expression thereof.

Description

[0001] References to Electronic Sequence Listings

[0002] The contents of the electronic sequence listing (22-10017.PCT_Seq-Listing.xml; size: 304kb; and creation date: March 1, 2023) are incorporated herein by reference in their entirety. Background Art

[0003] Parathyroid hormone (PTH) is an 84-amino acid hormone produced by the parathyroid glands. PTH regulates serum calcium concentrations through its effects on bones, kidneys, and intestines. Hypoparathyroidism (HP) is a rare condition in which the parathyroid glands are unable to secrete PTH. HP causes hypocalcemia, which leads to additional symptoms including weakness, muscle cramps, headaches, and / or uncontrollable convulsions and muscle spasms. Recombinant human PTH (rhPTH (1-84)) has been approved by the FDA for the treatment of chronic HP in adults. rhPTH (1-84) requires daily subcutaneous injections because PTH has a short serum half-life. Although rhPTH (1-84) has shown a good safety profile in clinical studies, exogenous PTH increases the risk of osteosarcoma in rats, suggesting that long-term continuous use of rhPTH (1-84) has safety issues.

[0004] There is a need for improved treatments for hypoparathyroidism. Summary of the invention

[0005] Provided herein are viral vectors encoding parathyroid hormone (PTH) fusion protein constructs. In some embodiments, these viral vectors can achieve sustained expression and / or increased circulation half-life of PTH fusions in subjects compared to vector-mediated delivery of PTH fusions without a fusion partner. Further provided are methods for preparing and using such viral vectors.

[0006] On the one hand, a viral vector is provided, the viral vector comprising a nucleic acid comprising a polynucleotide sequence encoding a fusion protein. The fusion protein comprises (a) a leader sequence comprising a secretion signal peptide and a propeptide, (b) a parathyroid hormone (PTH) fusion, and (c) a fusion domain comprising (i) IgG Fc or a functional variant thereof or (ii) albumin or a functional variant thereof. In one embodiment, the vector is an adeno-associated viral vector.

[0007] In one embodiment, (i) the secretory signal peptide of the leader sequence comprises a thrombin signal peptide; (ii) the leader sequence comprises a thrombin propeptide; and / or (iii) the leader sequence comprises a thrombin leader sequence. In another embodiment, the leader sequence comprises an IL-2 leader sequence combined with a PTH propeptide or a thrombin propeptide. In one embodiment, the PTH fusion is selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48 and functional variants thereof.

[0008] In one embodiment, the fusion domain is human IgG4 Fc, the human IgG4 Fc having the sequence of SEQ ID NO: 6 or a sequence sharing at least 90% identity therewith or a functional variant thereof. In another embodiment, the fusion domain is human albumin, the human albumin having the sequence of SEQ ID NO: 9 or a sequence sharing at least 90% identity therewith or a functional variant thereof. In one embodiment, the fusion domain is rhesus monkey IgG4 Fc, the rhesus monkey IgG4 Fc having the sequence of SEQ ID NO: 8 or a sequence sharing at least 90% identity therewith or a functional variant thereof. In one embodiment, the fusion domain is human IgG1 Fc, the human IgG1 Fc having the sequence of SEQ ID NO: 7 or a sequence sharing at least 90% identity therewith or a functional variant thereof.

[0009] On the other hand, the viral vector comprises an AAV capsid and a vector genome packaged in the AAV capsid, wherein the vector genome includes an AAV inverted terminal repeat (ITR), the polynucleotide sequence encoding the fusion protein, and a regulatory sequence directing the expression of the fusion protein.

[0010] In another aspect, a pharmaceutical composition suitable for treating hypoparathyroidism in a subject is provided. The composition comprises an aqueous liquid and a viral vector as described herein. In one embodiment, the subject is a human.

[0011] In yet another aspect, there is provided use of a viral vector as described herein for the manufacture of a medicament for treating a subject suffering from hypoparathyroidism, optionally diabetes.

[0012] In another aspect, a method of treating a subject suffering from hypoparathyroidism is provided, wherein the method comprises administering to the subject an effective amount of a viral vector or composition as described herein.

[0013] Other aspects and advantages of the present invention will become apparent from the following detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1A is a schematic diagram showing an overview of the PTH constructs described herein that utilize a heterologous leader sequence (signal peptide and propeptide) in combination with mature PTH.

[0015] Figure 1B A table showing an experimental overview of the experiments described in Example 3 is shown.

[0016] Figure 1C Shows Figure 1B Briefly, three groups of RagKO mice were treated with either a vector or vehicle utilizing either the native leader sequence or the IL2 signal (combined with the PTH propeptide). Plasma PTH levels (day 14) and serum calcium levels (day 28) were comparable for both vectors.

[0017] Figure 2A and Figure 2B Shown is a schematic diagram of the PTH constructs described herein that utilize a combination of a fusion partner and a truncated PTH.

[0018] Figure 2C Shows Figure 2B In vitro expression of the constructs described in in HEK293 cells.

[0019] Figure 3A is a gel showing stained PTH Fc fusion protein produced from the construct and purified using Protein A column purification. Fractions 3 and 4 produced under reducing (R) and non-reducing (NR) conditions are shown with a ladder.

[0020] Figure 3B It is shown Figure 3A Graph showing the results of a potency assay of purified proteins of, fractions 3 and 4 of purified PTH73 Fc fusions showed similar potency to PTH34 (the active portion of the protein).

[0021] Figure 3C is a graph showing the levels of hPTH73Fc fusion produced in vitro from inducible constructs when induced using 40 nM rapamycin.

[0022] Figure 4A A table showing an experimental overview of the experiments described in Example 3 is shown.

[0023] Figure 4B Shows Figure 4A Briefly, three groups of RagKO mice were treated with vehicle, vehicle with native leader sequence (hPTH(N)), or vehicle with native leader sequence with Fc fusion (hPTH73.P.Fc). Plasma hPTH potency was determined based on bleeding on days 14 and 28.

[0024] Figure 5A A table showing an experimental overview of the experiment described in Example 3 is shown. Briefly, three groups of RagKO mice were treated with vehicle, an inducible vector utilizing a native leader sequence with an Fc fusion. Groups 2 and 3 were administered rapamycin on day 12, and group 3 was administered again on day 38.

[0025] Figure 5B Plasma hPTH potency determined from day 14 and day 38 bleeds is shown.

[0026] Figure 5C Serum calcium levels are shown.

[0027] Figure 6 The full length sequence of human PTH as provided in SEQ ID NO: 1 is shown in FIG.

[0028] Fig. 7A The study design for the experiment described in Example 4 is shown. Figure 7B Preliminary data are shown, including NAB titers of 5 different AAV capsids.

[0029] Figures 8A-8C The results of the experiment described in Example 5 are shown. Fig. 8A Body weights of animals treated with the indicated vehicle and dose are shown. Figure 8B Serum calcium of animals treated with the vehicle and doses indicated is shown. Figure 8C Plasma hPTH levels of animals treated with the vehicle and doses indicated are shown.

[0030] Figures 9A-8B The results of the experiment described in Example 6 are shown. Fig. 9A Plasma hPTH levels of animals treated with the vehicle and doses indicated are shown. Fig. 9B Serum calcium of animals treated with the vehicle and doses indicated is shown. DETAILED DESCRIPTION

[0031] Adeno-associated virus (AAV) vectors expressing PTH fusion proteins are described herein to treat hypoparathyroidism in patients by single intramuscular vector administration. Described are delivery of these constructs to subjects in need thereof by multiple pathways, and particularly by in vivo expression mediated by recombinant vectors such as rAAV vectors. Methods of using these constructs in regimens for treating HP in subjects in need thereof and increasing the half-life of PTH in subjects are also provided. In addition, methods for enhancing the activity of PTH in subjects are provided.

[0032] PTH fusion protein

[0033] Hypoparathyroidism (HP) is characterized by low serum calcium levels caused by insufficient secretion of parathyroid hormone (PTH). Although serum calcium levels are normalized by treatment with activated vitamin D protein and calcium supplementation, patients' quality of life is impaired and their risk of developing multiple complications is increased. In many recent studies, replacement therapy with recombinant human PTH (rhPTH (1-84)) and therapy with an N-terminal PTH fragment (rhPTH (1-34)) have been investigated. Both drugs have been shown to normalize serum calcium while reducing the need for activated vitamin D and calcium supplements. However, once-daily injections result in large fluctuations in serum calcium. Twice-daily injections can reduce fluctuations but do not restore normal physiology of calcium homeostasis.

[0034] Parathyroid hormone (PTH) is a single-chain polypeptide consisting of 115 amino acids with the following sequence:

[0035]

[0036] The full-length sequence includes a signal peptide (amino acids 1-25) and a propeptide (amino acids 26-31). The mature protein is represented by SEQ ID NO: 1-

[0037] SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ (SEQ ID NO: 2).

[0038] The present disclosure provides fusion proteins including one or more copies of PTH, as well as polynucleotides and vectors encoding such fusion proteins. As used herein, "PTH" or "PTH protein" refers to full-length preparathyroid hormone (including signal sequence and propeptide), sequences covering propeptide and mature peptide (e.g., amino acid about 26-115), mature peptides (e.g., amino acid about 32-115), peptides with C-terminal or N-terminal truncations. Specifically, peptides comprising amino acids 1-33, 1-34, 1-72, 1-73, 1-74, 1-75 of the mature peptide sequence (SEQ ID NO:2) are useful herein. These truncated peptides can be combined with natural PTH propeptide and signal peptide or heterologous propeptide and / or signal peptide. Many constructs embodying various combinations of these sequences are described herein. As used herein, reference to PTH and the numbers thereafter (e.g., PTHX, PTH34, PTH73) refers to shortened PTH peptides, which include peptides of the first X number starting from the N-terminal of SEQ ID NO:2. For example, PTH34 comprises approximately the first 34 amino acids of the mature peptide, while PTH73 comprises approximately the first 73 amino acids of the mature peptide.

[0039] As used herein, the term "leader sequence" refers to any N-terminal sequence of a PTH polypeptide. In some embodiments, the leader sequence comprises a secretory signal peptide, a propeptide, or both.

[0040] In some embodiments, the fusion protein comprises a polynucleotide sequence encoding a fusion protein, the fusion protein comprising (a) a leader sequence, the leader sequence comprising a secretory signal peptide, and optionally a propeptide; (b) PTH; and optionally (c) a fusion domain. In one embodiment, the PTH fusion comprises a thrombin leader sequence, a PTH protein, and an IgG Fc or a functional variant thereof. In another embodiment, the fusion protein comprises a thrombin leader sequence, a PTH protein, and an albumin or a functional variant thereof. In another embodiment, the PTH fusion comprises a PTH leader sequence, a PTH protein, and an IgG Fc or a functional variant thereof. In another embodiment, the fusion protein comprises a PTH leader sequence, a PTH protein, and an albumin or a functional variant thereof. In another embodiment, the PTH fusion comprises an IL2 signal sequence, a PTH propeptide, a PTH protein, and an IgG Fc or a functional variant thereof. In another embodiment, the fusion protein comprises an IL2 signal sequence, a PTH propeptide, a PTH protein, and an albumin or a functional variant thereof.

[0041] In some embodiments, the PTH fusion comprises a variant, which may comprise a PTH nucleic acid or amino acid sequence as described herein or known in the art, with a change of up to about 10%, and the variant retains the function of the wild-type sequence. As used herein, "retaining function" means that the nucleic acid or amino acid acts in the same manner as the wild-type sequence, although not necessarily at the same expression or activity level. For example, in one embodiment, compared with the wild-type sequence, the functional variant has increased expression or activity. In another embodiment, compared with the wild-type sequence, the functional variant has reduced expression or activity. In one embodiment, compared with the wild-type sequence, the expression or activity of the functional variant has an increase or decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some embodiments, compared with the wild-type sequence, the functional variant has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes.

[0042] In one embodiment, the fusion comprises a combination of a PTH protein and a heterologous sequence. The PTH protein shares at least 90%, 95%, 97%, 98%, 99% or 100% identity with native human PTH (SEQ ID NO: 1 or 2). In one embodiment, the PTH protein has at most 1, 2 or 3 amino acid substitutions compared to the native sequence. In another embodiment, the PTH sequence is derived from a species other than humans. For example, PTH can be from non-human primates, dogs, cats, mice, rats, sheep, cattle, horses, etc. In some embodiments, it is desirable to change the native PTH sequence to optimize one or more characteristics thereof. In one embodiment, the PTH protein has a sequence comprising or consisting of SEQ ID NO: 1. In one embodiment, the variant shares at least 90% identity, 95% identity, 97% identity, 98% identity, 99% identity or 100% identity with SEQ ID NO: 1. In one embodiment, the PTH protein has a sequence comprising or consisting of SEQ ID NO: 2. In one embodiment, the variant shares at least 90% identity, 95% identity, 97% identity, 98% identity, 99% identity, or 100% identity with SEQ ID NO:2.

[0043] The leader sequence can be derived from the same species that is ultimately intended to be used, for example, people. As used herein, the term "origin" or "derived from" refers to that a sequence or protein is derived from a specific subject species or shares the same sequence with a protein or sequence derived from a specific subject species. For example, a leader sequence "derived from" people shares the same sequence (or its variant, as defined herein) with the same leader sequence as expressed in people. However, the specified nucleic acid or amino acid does not actually need to be derived from people. Various techniques known in the art that can produce a desired sequence include mutagenesis of similar proteins (for example, homologues) or artificial generation of nucleic acid or amino acid sequences. "Derived" nucleic acid or amino acid retains the function of the same nucleic acid or amino acid in its "derived" species, regardless of the actual source of the derived sequence.

[0044] The term "amino acid substitution" and its synonyms are intended to cover the modification of an amino acid sequence by replacing one amino acid with another substituted amino acid. The substitution may be a conservative substitution. The substitution may also be a non-conservative substitution. With respect to two amino acids, the term conservative is intended to mean that the amino acids share common properties recognized by those skilled in the art. For example, amino acids with hydrophobic non-acidic side chains, amino acids with hydrophobic acidic side chains, amino acids with hydrophilic non-acidic side chains, amino acids with hydrophilic acidic side chains, and amino acids with hydrophilic basic side chains. The common properties may also be amino acids with hydrophobic side chains, amino acids with aliphatic hydrophobic side chains, amino acids with aromatic hydrophobic side chains, amino acids with polar neutral side chains, amino acids with charged side chains, amino acids with charged acidic side chains, and amino acids with charged basic side chains. Both naturally occurring amino acids and non-natural naturally occurring amino acids are known in the art and may be used as substituted amino acids in various embodiments. Methods for replacing amino acids are well known to those skilled in the art and include, but are not limited to, mutations in the nucleotide sequence encoding the amino acid sequence. Reference herein to "one or more" is intended to encompass, for example, 1, 2, 3, 4, 5, 6 or more separate embodiments.

[0045] In one embodiment, the leader sequence is a human thrombin (factor II) sequence. In one embodiment, the thrombin leader sequence has the sequence shown in SEQ ID NO:3: MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRR or a functional variant thereof with up to 1, 2 or 3 amino acid substitutions.

[0046] In another embodiment, the leader sequence is a human IL2 sequence. In one embodiment, the IL2 leader sequence has a sequence as shown in SEQ ID NO:4:MYKMQLLSCIALTLVLVANS or a functional variant thereof with up to 1, 2 or 3 amino acid substitutions. In certain embodiments, in addition to the IL2 signal peptide, the leader sequence comprises a propeptide (e.g., from human PTH, human thrombin or other sequences).

[0047] In another embodiment, the leader sequence is a human PTH sequence. In one embodiment, the PTH leader sequence has SEQ ID NO: 5 with up to 1, 2 or 3 amino acid substitutions:

[0048] The sequence shown in MIPAKDMAKVMIVMLAICFLTKSDGKSVKKR or a functional variant thereof.

[0049] In one embodiment, functional variants of a desired leader sequence comprise variants that may contain up to about 10% variation from a leader nucleic acid or amino acid sequence described herein or known in the art, which variants retain the function of the wild-type sequence.

[0050] In some embodiments, the coding regions for both the leader sequence and the PTH peptide are incorporated into a single nucleic acid sequence without a linker between the coding sequences for the leader sequence and the coding sequences for PTH.

[0051] In certain embodiments, the fusion protein further comprises a fusion domain. In one embodiment, the fusion domain is a human IgG Fc fragment or a functional variant thereof. Immunoglobulin generally has a longer in vivo circulation half-life. By merging PTH protein (and leader sequence) with IgG Fc, the circulation time of the fusion protein is extended, and the function of PTH is retained simultaneously. In another embodiment, the fusion domain is a rhesus monkey IgG Fc fragment or a functional variant thereof.

[0052] As used herein, the Fc portion of an immunoglobulin has the meaning generally given to the term in the field of immunology. Specifically, this term refers to an antibody fragment that does not contain two antigen-binding regions (Fab fragments) from an antibody. The Fc portion is composed of the constant region of an antibody from two heavy chains, which are associated by non-covalent interactions and disulfide bonds. The Fc portion may include a hinge region and extend to the c-terminus of the antibody through CH2 and CH3 domains. The Fc portion may further include one or more glycosylation sites. In one embodiment, the fusion domain is human IgG Fc. The highly conserved four subclasses IgG1, IgG2, IgG3 and IgG4 differ in their constant regions, particularly in their hinges and upper CH2 domains. See, Vidarsson et al., IgG subclasses and isotypes: From structure to effector functions, Front Immunol., October 2014; 5:520, the document is incorporated herein by reference. The Fc domain can be derived from any human IgG, including human IgG1, human IgG2, human IgG3 or human IgG4. In one embodiment, the human IgG Fc is IgG4 Fc. In one embodiment, the human IgG Fc is SEQ ID NO:6:

[0053]

[0054] In another embodiment, the human IgG Fc shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity with SEQ ID NO:6.

[0055] In one embodiment, the human IgG Fc is an IgG1 Fc. In one embodiment, the human IgG Fc is SEQ ID NO:7: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG. In another embodiment, the human IgG Fc shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity with SEQ ID NO:7.

[0056] In another embodiment, the fusion domain is rhesus IgG Fc. The Fc domain can be derived from any rhesus IgG, including rhesus IgG1, rhesus IgG2, rhesus IgG3 or rhesus IgG4. In one embodiment, rhesus IgG Fc is IgG4 Fc. In one embodiment, rhesus IgG Fc is SEQ ID NO: 8:

[0057] PPCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEVQFNWYVDGVEVHNAQTKPRERQFNSTYRVV SVLTVTHQDW LNGKEYTCKVSNKGLPAPIE KTISKAKGQRPREPQVYILPPPQEELTKNQV SLTCLVTGFYPSDIAVEWES NGQPENTYKTTPPVLDSDGS YLLYSKLTVNKSRWQPGNIFTCSVMHEALH NHYTQKSLSV SPGK. In another embodiment, the rhesus IgG Fc shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity with SEQ ID NO: 8. In one embodiment, the rhesus IgG further comprises a hinge sequence.

[0058] In another embodiment, the fusion domain is human albumin or a functional variant thereof. In one embodiment, human albumin is

[0059] SEQ ID NO:9:

[0060] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADES

[0061] AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRL

[0062] VRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAAD

[0063] KAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEV

[0064] SKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCI

[0065] AEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLL

[0066] RLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQN

[0067] ALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCV

[0068] LHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKER

[0069] QIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALG. In another embodiment, the human albumin shares at least 90% identity, at least 95% identity, at least 99% identity or at least 100% identity with SEQ ID NO:9.

[0070] The in vivo function and stability of the fusion proteins disclosed herein can be optimized by adding small peptide linkers, for example, to prevent potentially unwanted domain interactions or for other reasons. In addition, glycine-rich linkers can provide some structural flexibility so that the PTH protein portion can effectively interact with PTH receptors on target cells such as pancreatic beta cells. Therefore, in one embodiment, the C-terminus of the PTH protein and the N-terminus of the fusion domain of the fusion protein are fused via a linker. In one embodiment, the linker comprises 1, 1.5 or 2 repeats of a G-rich peptide linker having the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 10) or a similar sequence.

[0071] In one embodiment, the fusion protein includes (a) a human thrombin leader sequence; (b) PTH (1-34); a linker; and (c) human IgG Fc. In one embodiment, the fusion protein has a sequence of SEQ ID NO: X or a sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

[0072] SEQ ID NO:11

[0073]

[0074]

[0075] In one embodiment, the sequence encoding the fusion protein is SEQ ID NO: 12, or a sequence at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0076] SEQ ID NO:12:

[0077]

[0078] In one embodiment, the fusion protein includes (a) a human thrombin leader sequence; (b) PTH (1-73); a linker; and (c) human IgG Fc. In one embodiment, the fusion protein has a sequence of SEQ ID NO: 13 or a sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

[0079] SEQ ID NO:13

[0080]

[0081] In one embodiment, the sequence encoding the fusion protein is SEQ ID NO: 14, or a sequence at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0082] SEQ ID NO:14:

[0083]

[0084] In one embodiment, the fusion protein comprises (a) a human thrombin leader sequence; (b) PTH (1-74); a linker; and (c) human IgG Fc. In one embodiment, the fusion protein comprises (a) a human thrombin leader sequence; (b) PTH (1-75); a linker; and (c) human IgG Fc. In one embodiment, the fusion protein comprises (a) a human thrombin leader sequence; (b) PTH (1-84); a linker; and (c) human IgG Fc. In one embodiment, the fusion protein has a sequence of SEQ ID NO: 15 or a sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

[0085] SEQ ID NO:15

[0086]

[0087]

[0088] In one embodiment, the sequence encoding the fusion protein is SEQ ID NO: 16, or a sequence at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0089] SEQ ID NO:16:

[0090]

[0091] In one embodiment, the fusion protein includes (a) a human PTH leader sequence; (b) PTH (1-34); a linker; and (c) a human IgG Fc. In one embodiment, the fusion protein has a sequence of SEQ ID NO: 17 or a sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

[0092] SEQ ID NO:17

[0093]

[0094]

[0095] In one embodiment, the sequence encoding the fusion protein is SEQ ID NO: 18, or a sequence at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0096] SEQ ID NO:18:

[0097]

[0098] In one embodiment, the fusion protein includes (a) a human PTH leader sequence; (b) PTH (1-73); a linker; and (c) a human IgG Fc. In one embodiment, the fusion protein has a sequence of SEQ ID NO: 19 or a sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

[0099] SEQ ID NO:19

[0100]

[0101] In one embodiment, the sequence encoding the fusion protein is SEQ ID NO: 20, or a sequence at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0102] SEQ ID NO:20:

[0103]

[0104] In one embodiment, the fusion protein includes (a) a human PTH leader sequence; (b) PTH (1-74); a linker; and (c) a human IgG Fc. In one embodiment, the fusion protein has a sequence of SEQ ID NO: 21 or a sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

[0105] SEQ ID NO:21

[0106]

[0107] In one embodiment, the sequence encoding the fusion protein is SEQ ID NO: 22, or a sequence at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0108] SEQ ID NO:22:

[0109]

[0110]

[0111] In one embodiment, the fusion protein includes (a) human PTH leader sequence (including propeptide); (b) PTH (1-75); linker; and (c) human IgG Fc. In one embodiment, 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.

[0112] SEQ ID NO:23

[0113]

[0114] In one embodiment, the sequence encoding the fusion protein is SEQ ID NO:24, or a sequence at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0115] SEQ ID NO:24:

[0116]

[0117]

[0118] In one embodiment, the fusion protein includes (a) a human PTH leader sequence; (b) PTH (1-84); a linker; and (c) human IgG Fc.

[0119] In one embodiment, the fusion protein comprises (a) a human IL2 leader sequence (comprising PTH or thrombin propeptide); (b) PTH (1-34); a linker; and (c) human IgG Fc. In one embodiment, the fusion protein comprises (a) a human IL2 leader sequence (comprising PTH or thrombin propeptide); (b) PTH (1-73); a linker; and (c) human IgG Fc. In one embodiment, the fusion protein comprises (a) a human IL2 leader sequence (comprising PTH or thrombin propeptide); (b) PTH (1-74); a linker; and (c) human IgG Fc. In one embodiment, the fusion protein comprises (a) a human IL2 leader sequence (comprising PTH or thrombin propeptide); (b) PTH (1-75); a linker; and (c) human IgG Fc. In one embodiment, the fusion protein includes (a) a human IL2 leader sequence (including PTH or propeptide of thrombin); (b) PTH (1-84); a linker; and (c) human IgG Fc.

[0120] In one embodiment, the fusion protein comprises (a) a human thrombin leader sequence; (b) PTH (1-34); a linker; and (c) human albumin. In one embodiment, the fusion protein has a sequence of SEQ ID NO: 25 or a sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

[0121] SEQ ID NO:25

[0122]

[0123]

[0124] In one embodiment, the sequence encoding the fusion protein is SEQ ID NO:26, or a sequence at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0125] SEQ ID NO:26:

[0126]

[0127]

[0128] In one embodiment, the fusion protein comprises (a) a human thrombin leader sequence; (b) PTH (1-73); a linker; and (c) human albumin. In one embodiment, the fusion protein has a sequence of SEQ ID NO: 27 or a sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

[0129] SEQ ID NO:27

[0130]

[0131] In one embodiment, the sequence encoding the fusion protein is SEQ ID NO:28, or a sequence at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0132] SEQ ID NO:28:

[0133]

[0134]

[0135] In one embodiment, the fusion protein comprises (a) a human thrombin leader sequence; (b) PTH (1-74); a linker; and (c) human albumin. In one embodiment, the fusion protein comprises (a) a human thrombin leader sequence; (b) PTH (1-75); a linker; and (c) human albumin. In one embodiment, the fusion protein comprises (a) a human thrombin leader sequence; (b) PTH (1-84); a linker; and (c) human albumin. In one embodiment, the fusion protein has a sequence of SEQ ID NO: 29 or a sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

[0136] SEQ ID NO:29

[0137]

[0138] In one embodiment, the sequence encoding the fusion protein is SEQ ID NO:30, or a sequence at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0139] SEQ ID NO:30:

[0140]

[0141]

[0142] In one embodiment, the fusion protein comprises (a) a human PTH leader sequence (including a propeptide); (b) PTH (1-34); a linker; and (c) human albumin. In one embodiment, the fusion protein comprises (a) a human PTH leader sequence (including a propeptide); (b) PTH (1-73); a linker; and (c) human albumin. In one embodiment, the fusion protein comprises (a) a human PTH leader sequence (including a propeptide); (b) PTH (1-74); a linker; and (c) human albumin. In one embodiment, the fusion protein comprises (a) a human PTH leader sequence (including a propeptide); (b) PTH (1-75); a linker; and (c) human albumin. In one embodiment, the fusion protein comprises (a) a human PTH leader sequence (including a propeptide); (b) PTH (1-84); a linker; and (c) human albumin.

[0143] In one embodiment, the fusion protein comprises (a) a human IL2 leader sequence (comprising PTH or thrombin propeptide); (b) PTH (1-34); a linker; and (c) human albumin. In one embodiment, the fusion protein comprises (a) a human IL2 leader sequence (comprising PTH or thrombin propeptide); (b) PTH (1-73); a linker; and (c) human albumin. In one embodiment, the fusion protein comprises (a) a human IL2 leader sequence (comprising PTH or thrombin propeptide); (b) PTH (1-74); a linker; and (c) human albumin. In one embodiment, the fusion protein comprises (a) a human IL2 leader sequence (comprising PTH or thrombin propeptide); (b) PTH (1-75); a linker; and (c) human albumin. In one embodiment, the fusion protein comprises (a) a human IL2 leader sequence (comprising PTH or thrombin propeptide); (b) PTH (1-84); a linker; and (c) human albumin.

[0144] When a variant or fragment of a leader sequence, PTH protein or fusion domain is desired, site-directed mutagenesis of a wild-type nucleic acid sequence can be used to generate the coding sequence of these peptides. Alternatively or in addition, a web-based or commercially available computer program and a service-based company can be used to reversely translate the amino acid sequence into a nucleic acid coding sequence, including both RNA and / or cDNA. See, for example, by EMBOSS, ebi.ac.uk / Tools / st / ; Gene Infinity (geneinfinity.org / sms- / sms_backtranslation.html); ExPasy (expasy.org / tools / ) backtranseq. In one embodiment, RNA and / or cDNA coding sequences are designed to be optimally expressed in the subject species (e.g., humans) that is ultimately intended to be administered.

[0145] Codon optimization can be used to design the coding sequence for optimal expression. The codon optimized coding region can be designed by various methods. This optimization can be performed using a method available online, a disclosed method, or a company providing codon optimization services. For example, a codon optimization method is described in International Patent Application Publication No. WO 2015 / 012924, which is incorporated herein by reference. In short, the nucleic acid sequence of the encoded product is modified with a synonymous codon sequence. Suitably, the entire length of the open reading frame (ORF) of the product is modified. However, in some embodiments, only the fragment of the ORF can be changed. By using one of these methods, frequency can be applied to any given polypeptide sequence, and a nucleic acid fragment of a codon optimized coding region encoding a polypeptide is produced.

[0146] In addition to the leader sequences, PTH proteins, fusion domains, and fusion proteins provided herein, nucleic acid sequences encoding these polypeptides are also provided. In one embodiment, a nucleic acid sequence encoding the PTH peptide described herein is provided. In some embodiments, the nucleic acid sequence may include any nucleic acid sequence encoding the PTH sequence of SEQ ID NO: 1. In another embodiment, the nucleic acid sequence includes any nucleic acid, and any nucleic acid includes the PTH sequence of SEQ ID NO: 2.

[0147] In one embodiment, a nucleic acid sequence encoding the PTH fusion protein described herein is provided. In another embodiment, the nucleic acid sequence comprises any nucleic acid sequence encoding the PTH fusion protein of SEQ ID NO: 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 39, 42, 44, 46 or 48.

[0148] Expression cassette

[0149] On the other hand, an expression cassette is provided herein, comprising a nucleic acid encoding a PTH fusion protein as described herein. As used herein, an "expression cassette" refers to a nucleic acid molecule comprising a biologically useful nucleic acid sequence (e.g., a gene cDNA, mRNA, etc. encoding a protein, enzyme, or other useful gene product) and a regulatory sequence operably connected thereto, the regulatory sequence directing or regulating the transcription, translation, and / or expression of the nucleic acid sequence and its gene product. As used herein, an "operably connected" sequence comprises both a regulatory sequence (also referred to as an element) adjacent to or non-adjacent to the nucleic acid sequence and a regulatory sequence acting in a trans or cis nucleic acid sequence. Such regulatory sequences typically include, for example, one or more of the following: a promoter, an enhancer, a transcription factor, a transcription terminator, an intron, a sequence that enhances translation efficiency (i.e., a Kozak consensus sequence), an effective RNA processing signal (e.g., a slicing and polyadenylation sequence), a sequence that stabilizes cytoplasmic mRNA (e.g., a woodchuck hepatitis virus (WHP) post-translational regulatory element (WPRE)) and a TATA signal. Expression cassette can contain the regulatory sequence of gene sequence upstream (5 ' place), for example, one or more of promoter, enhancer, intron etc. and one or more of enhancer, or the regulatory sequence of gene sequence downstream (3 ' place), for example, 3 ' untranslated region (3 ' UTR) including polyadenylation site, and other elements.In certain embodiments, regulatory sequence is operably connected with the nucleotide sequence of gene product, and wherein regulatory sequence is separated from the nucleotide sequence of gene product by the nucleotide sequence in between, i.e. 5 '-untranslated region (5 ' UTR).In certain embodiments, expression cassette comprises the nucleotide sequence of one or more gene products.In certain embodiments, expression cassette can be monocistronic expression cassette or bicistronic expression cassette.In other embodiments, term " transgenic " refers to one or more DNA sequences inserted into target cell from external source.

[0150] In one embodiment, an expression cassette refers to a nucleic acid molecule comprising a PTH construct coding sequence (e.g., a coding sequence for a PTH fusion protein), a promoter, and may contain other regulatory sequences thereof, which may be engineered into genetic elements and / or packaged into the capsid of a viral vector (e.g., a viral particle). Typically, such an expression cassette for producing a viral vector contains a PTH construct sequence described herein and other expression control sequences (such as those described herein), the PTH construct sequence being flanked by packaging signals of a viral genome (and referred to as a "vector genome"). Any expression control sequence may be optimized for a particular species using techniques known in the art, including, for example, codon optimization, as described herein.

[0151] In certain embodiments, the expression cassette comprises a constitutive promoter. In another embodiment, the CB7 promoter is used. CB7 is a chicken beta-actin promoter with a cytomegalovirus enhancer element. In some embodiments, the CB7 promoter has SEQ ID NO:31:

[0152] ctagtcgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatggagttccgcgttacataact

[0153] Nucleic acid sequence

[0154] In one embodiment, the promoter is a CMV promoter. In some embodiments, the CMV promoter has SEQ ID NO: 32:

[0155] The nucleic acid sequence of cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatc

[0156] In another embodiment, a tissue-specific promoter is used. Alternatively, other liver-specific promoters may be used, such as those listed in the Liver Specific Gene Promoter Database, Cold Spring Harbor (rulai.schl.edu / LSPD) of Cold Spring Harbor, and including, but not limited to, α1 antitrypsin (A1AT); human albumin (Miyatake et al., Journal of Virology (J. Virol.), 71:5124 32 (1997)), humAlb; hepatitis B virus core promoter (Sandig et al., Gene Ther., 3:1002 9 (1996)); TTR minimal enhancer / promoter, α-antitrypsin promoter, liver-specific promoter (LSP) (Wu et al., Mol Ther. 16:280-289 (2008)), TBG liver-specific promoter. Other promoters such as viral promoters, constitutive promoters, regulatable promoters (see, for example, WO 2011 / 126808 and WO 2013 / 04943), or promoters that are responsive to physiological cues can be used in the vectors described herein.

[0157] In one embodiment, the promoter is included in an inducible gene expression system. The inducible gene regulation / expression system contains at least the following components: a promoter operably linked to a transgene encoding a PTH fusion protein described herein (also referred to as a regulatable promoter), an activation domain, a DNA binding domain, and a zinc finger homeodomain binding site. In other embodiments, additional components may be included in the expression system, as further described herein.

[0158] The system includes a promoter upstream of the coding sequence of the PTH fusion protein. Promoters described herein, such as CMV and CB7 promoters, can be used. In one embodiment, the promoter is a CMV promoter, such as the promoter shown in SEQ ID NO: 32. In another embodiment, the promoter is the ubiquitous inducible promoter Z12I, which includes 12 repeated copies of the binding sites of ZFHD1 and IL2 minimal promoters. See, for example, Chen et al., "Human Gene Ther Methods." August 2013; 24 (4): 270–278, which is incorporated herein.

[0159] The expression system includes an activation domain, which is preferably located upstream of the DNA binding domain. In one embodiment, the activation domain is a fusion of the carboxyl terminus of the p65 subunit of NF-κB and the FKBP12-rapamycin binding (FRB) domain of the FKBP12-rapamycin associated protein (FRAP) from the carboxyl terminus of the p65 subunit of NF-κB. In one embodiment, the activation domain is a FKBP12-rapamycin binding (FRB) domain of the human FKBP12-rapamycin associated protein (FRAP) fused to the carboxyl terminus of the p65 subunit of NF-κB from humans.

[0160] In one embodiment, there is a linker between the transactivation domain and the DNA binding domain, which linker can be F2A or IRES. In one embodiment, the linker is selected from IRES or 2A peptide. In one embodiment, the linker is a cleavable 2A peptide. In one embodiment, the 2A peptide is selected to increase packaging constraints, thereby allowing a single vector system.

[0161] The DNA binding domain consists of a DNA binding fusion of zinc finger homology domain 1 (ZFHD1) linked to up to three copies of FK506 binding protein (FKBP). In the presence of an inducer (e.g., a rapamycin analog, such as rapamycin), the DNA binding domain and the activation domain dimerize through the interaction of their FKBP and FRB domains, resulting in transcriptional activation of the transgene. In some embodiments, ZFHD1 is contained in a framework with GT2A or IRES.

[0162] The expression system is designed to have one, two or three copies of the FKBP sequence. These are referred to herein as FKBP subunits. In one embodiment, the subunits are designed to express the same protein but have different nucleic acids from each other to minimize recombination. For example, SEQ ID NO:33 provides three "wobbled" coding sequences for FKBP, each of which encodes SEQ ID NO:34:

[0163] Sequence shown in GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIR GWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE

[0164] The expression system further comprises a zinc finger homeodomain binding site. The nucleic acid molecule contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 ZFHD binding sites. In one embodiment, the expression system contains 12 (twelve) zinc finger homeodomain binding sites (binding partners) (12XZFHD). In another embodiment, the expression system contains 8 (eight) zinc finger homeodomain binding sites (binding partners) (12XZFHD). However, the present invention encompasses expression systems having two to about twelve copies of zinc finger binding sites. An example of a single copy of a ZFHD binding site is: AATGATGGGCGCTCGAGT (SEQ ID NO: 35)

[0165] In some embodiments, a minimal IL2 promoter is present downstream of the zinc finger homeodomain binding site. An exemplary IL2 promoter is shown in SEQ ID NO: 36.

[0166] Such inducible systems are known in the art and include, for example, the rapamycin inducible system described by, e.g., Rivera et al., A humanized system for pharmacologic control of gene expression, Nature Medicine, Vol. 2, pp. 1028-1032 (September 1996), and Rivera et al., Long-term pharmacologically regulated expression of erythropoietin in primates following AAV-mediated gene transfer, Blood, Feb. 15, 2005, Vol. 105, No. 4, both of which are incorporated herein by reference. In one embodiment, the inducible gene expression system includes a CMV promoter, an activation domain that is a FKBP12-rapamycin binding (FRB) domain of a human FKBP12-rapamycin associated protein (FRAP) fused to the carboxyl terminus of the p65 subunit of human NF-κB, a GT2A peptide, a ZFHD1 DNA binding domain, three FKBP subunits, hGH poly A, 12XZFHD, and a minimal sIL2 promoter. These sequences are in addition to the coding sequence of the PTH fusion protein and, optionally, other regulatory sequences.

[0167] In addition to the promoter, the expression cassette and / or vector may also contain other appropriate transcription initiation, termination, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (i.e., Kozak consensus sequences), sequences that enhance protein stability, and sequences that enhance secretion of the encoded product when desired. Examples of suitable polyA sequences include, for example, SV40, bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit β-globin (also known as rabbit globin polyA; RGB), modified RGB (mRGB), and TK polyA. Examples of suitable enhancers include, for example, α-fetoprotein enhancer, TTR minimal promoter / enhancer, LSP (TH binding globulin promoter / α1 microglobulin / double Kunitz inhibitor enhancer), etc. In one embodiment, polyA is rabbit globin polyA.

[0168] These control sequences are "operably linked" to the PTH construct sequence. As used herein, the term "operably linked" refers to both expression control sequences that are contiguous to the gene of interest and expression control sequences that function in trans or at a distance to control the gene of interest.

[0169] In one embodiment, rAAV is provided, comprising a 5'ITR, a CB7 promoter, a chicken β-actin intron, a coding sequence for a fusion protein of SEQ ID NO: 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 39, 42, 44, 46 or 48, rabbit globin poly A and a 3'ITR. In another embodiment, the rAAV includes a polynucleotide comprising a CMV promoter, an activation domain that is the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin associated protein (FRAP) fused to the carboxyl terminus of the p65 subunit of NF-κB from human, an IRES, a ZFHD1 DNA binding domain, three FKBP subunits, hGH poly A, 8XZFHD, a minimal sIL2 promoter, a coding sequence for a PTH fusion protein of SEQ ID NO: 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 39, 42, 44, 46 or 48, and rabbit beta globin poly A.

[0170] In another embodiment, a two-vector inducible system is provided. The first rAAV includes 12XZFHD, a minimal IL2 promoter, a coding sequence of a PTH fusion protein of SEQ ID NO: 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 39, 42, 44, 46 or 48, and rabbit beta globin polyA. A stuffer sequence may be included to increase the packaging size of the vector. The second rAAV includes a polynucleotide comprising a CMV promoter, an intron, and the activation domain is a FKBP12-rapamycin binding (FRB) domain of a human FKBP12-rapamycin associated protein (FRAP) fused to the carboxyl terminus of a p65 subunit of NF-κB from humans, an IRES, a ZFHD1 DNA binding domain, and polyA.

[0171] In one embodiment, an expression cassette is provided, comprising a polynucleotide comprising a CB7 promoter, a chicken β-actin intron, a coding sequence for a fusion protein of SEQ ID NO: 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 39, 42, 44, 46 or 48, and rabbit globin poly A.

[0172] In another embodiment, an expression cassette is provided, comprising a polynucleotide comprising a CB7 promoter, a chicken β-actin intron, a coding sequence for a fusion protein of SEQ ID NO: 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 39, 42, 44, 46 or 48, and rabbit globin poly A.

[0173] Viral vectors

[0174] On the other hand, a viral vector comprising an expression cassette as described herein is provided. In certain embodiments of the viral vectors described herein, the viral vector is an adeno-associated virus (AAV) viral vector or a recombinant AAV (rAAV). As used herein, the term "recombinant AAV" or "rAAV" refers to naturally occurring adeno-associated viruses, adeno-associated viruses available to those skilled in the art and / or obtainable in view of the compositions and methods described herein, and artificial AAV. Adeno-associated virus (AAV) viral vectors are AAV DNA enzyme-resistant particles with AAV protein capsids, wherein the expression cassette for delivery to target cells is packaged with AAV reverse terminal repeats (ITRs) (together referred to as "vector genomes") flanked by AAV. The AAV capsid is composed of 60 capsid (cap) protein subunits VP1, VP2, and VP3, which are arranged in an icosahedral symmetric manner, with a ratio of about 1:1:10 to 1:1:20, depending on the selected AAV. Various AAVs can be selected as the source of the capsid of the AAV viral vector as identified above. In one embodiment, the AAV capsid is an AAVrh91 capsid or a variant thereof. In certain embodiments, the capsid protein is designated by a number or a combination of numbers and letters following the term "AAV" in the rAAV vector name. Unless otherwise indicated, the AAV capsids, ITRs, and other selected AAV components described herein can be readily selected from any AAV, including but not limited to AAVs identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVhu37, AAVrh32.33, AAV Anc80, AAV10, AAV11, AAV12, AAVrh8, AAVrh74, AAV-DJ8, AAV-DJ, AAVhu.37, AAVrh.64R1, and AAVhu68. See, e.g., U.S. Published Patent Application No. 2007-0036760-A1; U.S. Published Patent Application No. 2009-0197338-A1; EP 1310571. See also WO 2003 / 042397 (AAV7 and other simian AAVs), U.S. Pat. No. 7790449 and U.S. Pat. No. 7282199 (AAV8), WO 2005 / 033321 and US 7,906,111 (AAV9) and WO 2006 / 110689 and WO2003 / 042397 (rh.10), WO 2005 / 033321, WO 2018 / 160582 (AAVhu68), which are incorporated herein by reference.Other suitable AAVs may include, but are not limited to, AAVrh90 [PCT / US20 / 30273 filed April 28, 2020], AAVrh91 [PCT / US20 / 030266 filed April 28, 2020, now published WO 2020 / 223231 on November 5, 2020], AAVrh92, AAVrh93, AAVrh91.93 [PCT / US20 / 30281 filed April 28, 2020], all of which are incorporated herein by reference. Other suitable AAVs include the AAV3B variants described in U.S. Provisional Patent Application No. 62 / 924,112 filed on October 21, 2019 and U.S. Provisional Patent Application No. 63 / 025,753 filed on May 15, 2020, which describe AAV3B.AR2.01, AAV3B.AR2.02, AAV3B.AR2.03, AAV3B.AR2.04, AAV3B.AR2.05, AAV3B.AR2.06, AAV3B.AR2.07, AAV3B.AR2.08, AAV3B.AR2.09, AAV3B.AR2.10, AAV3B.AR2.11, AAV3B.AR2.12, AAV3B.AR2.13, AAV3B.AR2.14, AAV3B.AR2.15, AAV3B.AR2.16, AAV3B.AR2.17, AAV3B.AR2.18, AAV3B.AR2.19, AAV3B.AR2 .05, AAV3B.AR2.06, AAV3B.AR2.07, AAV3B.AR2.08, AAV3B.AR2.10, AAV3B.AR2.11, AAV3B.AR2.12, AAV3B.AR2.13, AAV3B.AR2.14, AAV3B.AR2.15, AAV3B.AR2.16 or AAV3B.AR2.17, all of which are incorporated herein by reference. See also International Patent Application No. PCT / US21 / 45945 filed on August 13, 2021, U.S. Provisional Patent Application No. 63 / 065,616 filed on August 14, 2020, and U.S. Provisional Patent Application No. 63 / 109,734 filed on November 4, 2020, all of which are incorporated herein by reference in their entirety. These documents also describe other AAV capsids that can be selected for the production of rAAV and are incorporated by reference. Among the AAVs isolated or engineered from humans or non-human primates (NHPs) and well characterized, human AAV2 was the first AAV to be developed as a gene transfer vector; it has been widely used in efficient gene transfer experiments in different target tissues and animal models.

[0175] As used herein, with respect to AAV, the term "variant" means any AAV sequence derived from a known AAV sequence, including an AAV sequence with a conservative amino acid substitution and an AAV sequence sharing at least 90%, at least 95%, at least 97%, at least 99% or higher sequence identity with an amino acid or nucleic acid sequence. In another embodiment, the AAV capsid comprises a variant, which may include up to about 10% change with any described or known AAV capsid sequence. That is, the AAV capsid shares about 90% identity to about 99.9% identity, about 95% to about 99% identity or about 97% to about 98% identity with the AAV capsid provided herein and / or known in the art. In one embodiment, the AAV capsid shares at least 95% identity with the AAV capsid. When determining the identity percentage of the AAV capsid, any variable protein (e.g., vp1, vp2 or vp3) can be compared.

[0176] In one embodiment, the viral vector is an rAAV having a capsid of AAV8 or a functional variant thereof. In one embodiment, the viral vector is an rAAV having a capsid of AAVrh91 or a functional variant thereof. In one embodiment, the viral vector is an rAAV having a capsid of AAV3.AR.2.12 or a functional variant thereof. In one embodiment, the viral vector is an rAAV having a capsid selected from AAV9, AAVrh64R1, AAVhu37 or AAVrh10. In one embodiment, the viral vector is an rAAV having a capsid of AAVhu68 or a functional variant thereof.

[0177] In certain embodiments, the viral vector has an AAVrh91 capsid. A nucleic acid sequence encoding an AAVrh91 capsid is provided in SEQ ID NO:37, and an encoded amino acid sequence is provided in SEQ ID NO:38. A rAAV is provided herein, comprising at least one of vp1, vp2, and vp3 of AAVrh91 (SEQ ID NO:38). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVrh91 (SEQ ID NO:37). In yet another embodiment, a nucleic acid sequence encoding an AAVrh91 amino acid sequence is provided in SEQ ID NO:19, and an encoded amino acid sequence is provided in SEQ ID NO:38. Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVrh91eng (SEQ ID NO:19). In certain embodiments, vp1, vp2 and / or vp3 are full-length capsid proteins of AAVrh91 (SEQ ID NO: 38). In other embodiments, vp1, vp2 and / or vp3 have N-terminal and / or C-terminal truncations (e.g., truncations of about 1 to about 10 amino acids).

[0178] In certain embodiments, the AAVrh91 capsid is characterized by one or more of the following: (1) AAVrh91 capsid proteins comprising: a heterogeneous population of AAVrh91 vp1 proteins selected from the group consisting of vp1 proteins produced by expression of a nucleic acid sequence encoding a predicted amino acid sequence of 1 to 736 of SEQ ID NO:38, vp1 proteins produced by expression of SEQ ID NO:37, or vp1 proteins produced by a nucleic acid sequence at least 70% identical to SEQ ID NO:37 encoding a predicted amino acid sequence of 1 to 736 of SEQ ID NO:38, a heterogeneous population of AAVrh91 vp2 proteins selected from the group consisting of vp2 proteins produced by expression of a nucleic acid sequence encoding at least about amino acids 138 to 736 of SEQ ID NO:38, vp2 proteins produced by a sequence comprising at least nucleotides 412 to 2208 of SEQ ID NO:37, or vp2 proteins produced by a nucleic acid sequence at least 70% identical to SEQ ID NO:37 encoding a predicted amino acid sequence of SEQ ID NO:38. A heterogeneous population of AAVrh91 vp3 proteins selected from the group consisting of: (1) a vp3 protein produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:38, (2) a vp3 protein produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:38, (3) a vp3 protein produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:38, (4) a vp3 protein produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:38, (5) a vp3 protein produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:38, (6) a vp3 protein produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:38, (7) a vp3 protein produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:38, (8) a vp3 protein produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:38, (9) a vp3 protein produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:38, (10) a vp3 protein produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:38, (11) a vp3 protein produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about NO:38; and a heterogeneous population of vp3 proteins, the vp1, vp2 and vp3 proteins being the product of a nucleic acid sequence encoding at least about amino acids 138 to 736 of SEQ ID NO:38, wherein: the vp1, vp2 and vp3 proteins contain a subpopulation having amino acid modifications, the amino acid modifications comprising at least two highly deamidated asparagines (N) in the asparagine-glycine pairs in SEQ ID NO:38, and optionally further comprising a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change;and (B) a vector genome in an AAVrh91 capsid, the vector genome comprising a nucleic acid molecule comprising an AAV inverted terminal repeat sequence and a non-AAV nucleic acid sequence encoding a product operably linked to a sequence directing expression of the product in a host cell.;

[0179] In certain embodiments, the AAVrh91 capsid is characterized by one or more of the following: (1) AAVrh91 capsid proteins comprising: a heterogeneous population of AAVrh91 vp1 proteins selected from the group consisting of vp1 proteins produced by expression of a nucleic acid sequence encoding a predicted amino acid sequence of 1 to 736 of SEQ ID NO:38, vp1 proteins produced by expression of SEQ ID NO:19, or vp1 proteins produced by a nucleic acid sequence at least 70% identical to SEQ ID NO:19 encoding a predicted amino acid sequence of 1 to 736 of SEQ ID NO:38, a heterogeneous population of AAVrh91 vp2 proteins selected from the group consisting of vp2 proteins produced by expression of a nucleic acid sequence encoding at least about amino acids 138 to 736 of SEQ ID NO:38, vp2 proteins produced by a sequence comprising at least nucleotides 412 to 2208 of SEQ ID NO:19, or vp2 proteins produced by a nucleic acid sequence at least 70% identical to SEQ ID NO:19 encoding a predicted amino acid sequence of SEQ ID NO:38. A heterogeneous population of AAVrh91 vp3 proteins selected from the group consisting of: (1) a vp3 protein produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:38, (2) a vp3 protein produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:38, (3) a vp3 protein produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:38, (4) a vp3 protein produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:38, (5) a vp3 protein produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:38, (6) a vp3 protein produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:38, (7) a vp3 protein produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:38, (8) a vp3 protein produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:38, (9) a vp3 protein produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:38, (10) a vp3 protein produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:38, or (11) a vp3 protein produced by expression from a nucleic acid sequence encoding a predicted amino acid sequence of at least NO:38; and a heterogeneous population of vp3 proteins, the vp1, vp2 and vp3 proteins being the product of a nucleic acid sequence encoding at least about amino acids 138 to 736 of SEQ ID NO:38, wherein: the vp1, vp2 and vp3 proteins contain a subpopulation having amino acid modifications, the amino acid modifications comprising at least two highly deamidated asparagines (N) in the asparagine-glycine pairs in SEQ ID NO:38, and optionally further comprising a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change;and (B) a vector genome in an AAVrh91 capsid, the vector genome comprising a nucleic acid molecule comprising an AAV inverted terminal repeat sequence and a non-AAV nucleic acid sequence encoding a product operably linked to a sequence directing expression of the product in a host cell.;

[0180] In certain embodiments, the AAVrh91 vp1, vp2, and vp3 proteins contain a subpopulation with amino acid modifications that include at least two highly deamidated asparagines (N) in the asparagine-glycine pairs in SEQ ID NO: 38, and optionally further include a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change. Relative to the numbering of SEQ ID NO: 38, high levels of deamidation are observed at NG pairs N57, N383, and / or N512. Deamidation has been observed at other residues. In certain embodiments, AAVrh91 can have other deamidated residues, e.g., typically less than 10%, and / or can have other modifications comprising phosphorylation (e.g., where present, ranging from about 2 to about 30%, or about 2 to about 20%, or about 2 to about 10%) (e.g., at S149) or oxidation (e.g., at one or more of -W22, -M211, W247, M403, M435, M471, W478, W503, -M537, -M541, -M559, -M599, M635, and / or W695). Optionally, W can be oxidized to kynurenine.

[0181] Table A - AAVrh91 deamidation

[0182] AAVrh91 deamidation based on VP1 numbering Deamidation% N57+ deamidation 65-90, 70-95, 80-95, 75-100, 80-100 or 90-100 N94+ deamidation 2-15 or 2-5 N303+ deamidation 2-15 or 5-10 N383+deamidation 65-90, 70-95, 80-95, 75-100, 80-100 or 90-100 N497+deamidation 2-15 or 5-10 N512+ deamidation 65-90, 70-95, 80-95, 75-100, 80-100 or 90-100 ~N691+deamidation 2-15, 2-10 or 5-10

[0183] In certain embodiments, the AAVrh91 capsid is modified in one or more of the positions identified in the above table within the range provided as determined using mass spectrometry using trypsin. In certain embodiments, one or more of the positions modified as described herein or a glycine after the N. The residue numbers are based on the AAVrh91 sequence provided herein. See, SEQ ID NO: 38.

[0184] In certain embodiments, the AAVrh91 capsid comprises: a heterogeneous population of vp1 proteins, wherein the vp1 proteins are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:38; a heterogeneous population of vp2 proteins, wherein the vp2 proteins are the product of a nucleic acid sequence encoding the amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:38; and a heterogeneous population of vp3 proteins, wherein the vp3 proteins are the product of a nucleic acid sequence encoding at least amino acids 203 to 736 of SEQ ID NO:38.

[0185] In certain embodiments, the modified AAVrh91 nucleic acid sequence is used to generate a mutant rAAV having a capsid that is less deamidated than the native AAVrh91 capsid. Such mutant rAAV may have reduced immunogenicity and / or increased stability during storage, particularly when stored in suspension form.

[0186] In one aspect, a recombinant AAV (rAAV) is provided. The rAAV comprises an AAV capsid from adeno-associated virus rh91 and a vector genome packaged in the AAV capsid, wherein the vector genome comprises an AAV inverted terminal repeat (ITR), a coding sequence of a PTH fusion of SEQ ID NO: 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 39, 42, 44, 46 or 48, and a regulatory sequence directing the expression of the PTH fusion.

[0187] In certain embodiments, the AAV68 capsid is further characterized by one or more of the following. AAV hu68 capsid proteins include: an AAVhu68 vp1 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 736 of SEQ ID NO: 100, a vp1 protein produced from SEQ ID NO: 98 or 99, or a vp1 protein produced from a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 98 encoding the predicted amino acid sequence of 1 to 736 of SEQ ID NO: 100; an AAVhu68 vp2 protein produced by expression from a nucleic acid sequence encoding at least about amino acids 138 to 736 of SEQ ID NO: 100, a vp2 protein produced from a sequence that includes at least nucleotides 412 to 2211 of SEQ ID NO: 98 or 99, or a vp2 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 412 to 2211 of SEQ ID NO: 98 or 99 encoding the predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO: 100; and / or an AAVhu68 vp3 protein produced by expression from a nucleic acid sequence encoding at least about amino acids 138 to 736 of SEQ ID NO: 100, a vp3 protein produced from a sequence that includes at least nucleotides 412 to 2211 of SEQ ID NO: 98 or 99, or a vp3 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 412 to 2211 of SEQ ID NO: 98 or 99 encoding the predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO: 100. AAVhu68 vp3 protein produced by expressing a nucleic acid sequence encoding at least about amino acids 203 to 736 of SEQ ID NO:100, a vp3 protein produced by a sequence including at least nucleotides 607 to 2211 of SEQ ID NO:98, or a vp3 protein produced by a nucleic acid sequence that is at least 70% identical to at least nucleotides 607 to 2211 of SEQ ID NO:98 or 99 that encodes the predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:100.

[0188] Additionally or alternatively, an AAV capsid is provided, comprising a heterogeneous population of vp1 proteins that optionally include valine at position 157, a heterogeneous population of vp2 proteins that optionally include valine at position 157, and a heterogeneous population of vp3 proteins, wherein based on the numbering of the vp1 capsid of SEQ ID NO:100, at least one subpopulation of vp1 and vp2 proteins includes valine at position 157 and optionally further includes glutamic acid at position 67. Additionally or alternatively, an AAVhu68 capsid is provided, comprising: a heterogeneous population of vp1 proteins, wherein the vp1 proteins are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:100; a heterogeneous population of vp2 proteins, wherein the vp2 proteins are the product of a nucleic acid sequence encoding the amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:100; and a heterogeneous population of vp3 proteins, wherein the vp3 proteins are the product of a nucleic acid sequence encoding at least amino acids 203 to 736 of SEQ ID NO:100, wherein: the vp1, vp2 and vp3 proteins contain subpopulations having amino acid modifications.

[0189] AAVhu68 vp1, vp2 and vp3 proteins are generally expressed as alternative splice variants encoded by the same nucleic acid sequence encoding the full-length vp1 amino acid sequence (amino acids 1 to 736) of SEQ ID NO: 100. Optionally, the vp1 coding sequence alone is used to express the vp1, vp2 and vp3 proteins. Alternatively, this sequence can be co-expressed with one or more of the following: a nucleic acid sequence encoding the AAVhu68 vp3 amino acid sequence of SEQ ID NO: 100 (about aa 203 to 736), wherein the AAVhu68 vp3 amino acid sequence is without the vp1 unique region (about aa 1 to about aa 137) and / or the vp2 unique region (about aa 1 to about aa 202) or the complementary strands thereto, i.e., the corresponding mRNA or tRNA (about nt 607 to about nt 2211 of SEQ ID NO: 98 or 99) or a sequence that is at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99%) identical to SEQ ID NO: 98 or 99, wherein SEQ ID NO: 98 or 99 encodes aa 203 to 736 of SEQ ID NO: 100. Additionally or alternatively, the vp1 encoding and / or vp2 encoding sequences can be co-expressed with one or more of the following: a nucleic acid sequence encoding the AAVhu68 vp2 amino acid sequence of SEQ ID NO:100 (approximately aa 138 to 736) without the vp1 unique region (approximately aa 1 to approximately 137) or the complementary strand thereto, i.e., the corresponding mRNA or tRNA (nt 412 to 22121 of SEQ ID NO:98) or a sequence that is at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:98 or 99, which encodes approximately aa 138 to 736 of SEQ ID NO:100.

[0190] As described herein, rAAVhu68 has rAAVhu68 capsids produced in a production system that expresses capsids from an AAVhu68 nucleic acid encoding the vp1 amino acid sequence of SEQ ID NO: 100, and optionally an additional nucleic acid sequence, e.g., encoding a vp3 protein that does not contain the unique regions of vp1 and / or vp2. rAAVhu68 produced from production using a single nucleic acid sequence vp1 produces a heterogeneous population of vp1 proteins, vp2 proteins, and vp3 proteins. More specifically, AAVhu68 capsids contain subpopulations within the vp1 protein, within the vp2 protein, and within the vp3 protein that have modifications of the predicted amino acid residues from SEQ ID NO: 100. These subpopulations contain at least deamidated asparagine (N or Asn) residues. For example, the asparagine in the asparagine-glycine pair is highly deamidated.

[0191] In one embodiment, the AAVhu68 vp1 nucleic acid sequence has the sequence of SEQ ID NO:98 or 99 or a complementary strand thereto, such as a corresponding mRNA or tRNA. In certain embodiments, the vp2 and / or vp3 proteins can be expressed additionally or alternatively from a nucleic acid sequence different from vp1, such as to change the ratio of vp proteins in a selected expression system. In certain embodiments, a nucleic acid sequence encoding the AAVhu68 vp3 amino acid sequence of SEQ ID NO:100 (about aa 203 to 736) is also provided, wherein the AAVhu68 vp3 amino acid sequence lacks the vp1 unique region (about aa 1 to about aa 137) and / or the vp2 unique region (about aa 1 to about aa 202) or a complementary strand thereto, i.e., the corresponding mRNA or tRNA (about nt 607 to about nt 2211 of SEQ ID NO:98 or 99). In certain embodiments, nucleic acid sequences encoding the AAVhu68 vp2 amino acid sequence of SEQ ID NO: 100 (about aa 138 to 736) are also provided, wherein the AAVhu68 vp2 amino acid sequence does not have the vp1 unique region (about aa 1 to about 137) or the complementary strand thereto, i.e., the corresponding mRNA or tRNA (nt 412 to 2211 of SEQ ID NO: 98 or 99).

[0192] However, other nucleic acid sequences encoding the amino acid sequence of SEQ ID NO: 100 may be selected for use in generating rAAVhu68 capsids. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 98 or 99, 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: 98 or 99 encoding SEQ ID NO: 100. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 98 or 99, 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: 98 or 99 encoding the vp2 capsid protein (about aa 138 to 736) of SEQ ID NO: 100. In certain embodiments, the nucleic acid sequence has a nucleic acid sequence of about nt 607 to about nt 2211 of SEQ ID NO:98 or 99, or a sequence that is at least 70% to 99.%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to nt SEQ ID NO:98 or 99 encoding the vp3 capsid protein of SEQ ID NO:100 (about aa 203 to 736).

[0193]

[0194]

[0195] In certain embodiments, the AAVhu68 capsid is characterized as having a capsid protein in which at least 45% of the N residues are deamidated at at least one of positions N57, N329, N452, and / or N512, based on the numbering of the amino acid sequence of SEQ ID NO: 100. 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 NG positions (i.e., N57, N329, N452, and / or N512, based on the numbering of the amino acid sequence of SEQ ID NO: 100) are deamidated. In these and other embodiments, the AAVhu68 capsid is further characterized as having a population of proteins in which about 1% to about 20% of the N residues are deamidated at one or more of the following positions: N94, N253, N270, N304, N409, N477, and / or Q599, based on the numbering of the amino acid sequence of SEQ ID NO: 100. In certain embodiments, AAVhu68 includes at least a subset of vp1, vp2, and / or vp3 proteins that are deamidated at one or more of the following positions: N35, N57, N66, N94, N113, N252, N253, Q259, N270, N303, N304, N305, N319, N328, N329, N336, N409, N410, N452, N477, N515, N598, Q599, N628, N651, N663, N709, N735, based on numbering of the amino acid sequence of SEQ ID NO: 100, or a combination thereof. In certain embodiments, the capsid protein may have one or more amidated amino acids.

[0196] In one aspect, a recombinant AAV (rAAV) is provided. The rAAV comprises an AAV capsid from adeno-associated virus hu68 and a vector genome packaged in the AAV capsid, wherein the vector genome comprises an AAV inverted terminal repeat (ITR), a coding sequence of a PTH fusion of SEQ ID NO: 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 39, 42, 44, 46 or 48, and a regulatory sequence directing the expression of the PTH fusion.

[0197] In another embodiment, a recombinant adeno-associated virus (rAAV) is provided having an AAVhu68 capsid and a vector genome, wherein (a) the AAV hu68 capsid comprises a heterogeneous population of AAVhu68 vp1 proteins, a heterogeneous population of AAVhu68 vp2 proteins, and a heterogeneous population of AAVhu68 vp3 proteins, wherein the heterogeneous AAVhu68 vp1, AAVhu68 vp2, and AAVhu68 vp3 proteins contain a subpopulation having an amino acid modification, wherein the amino acid modification comprises a SEQ ID NO: 1 as determined using mass spectrometry. 50% to 100% deamidation of at least two asparagines (N) in asparagine-glycine pairs in two or more of N57, N329, N452, N512 of NO:100, and optionally further comprising a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change in which the deamidated asparagine is deamidated to aspartic acid, isoaspartic acid, an interconverted aspartic acid / isoaspartic acid pair, or a combination thereof, wherein the AAVhu68 capsid further comprises a subpopulation having one or more of the following:

[0198] (a) at least 65% of the asparagine (N) in the asparagine-glycine pair located at position N57 of the vp1 protein based on the numbering of SEQ ID NO: 100 is deamidated,

[0199] (b) at least 75% of the N in the asparagine-glycine pair at position N329 of the vp1, v2 and vp3 proteins, based on the residue numbering of the amino acid sequence of SEQ ID NO: 100, is deamidated,

[0200] (c) at least 50% of the N in the asparagine-glycine pair in position N452 of the vp1, v2 and vp3 proteins based on the residue numbering of the amino acid sequence of SEQ ID NO: 100 is deamidated; and / or

[0201] (d) at least 75% of the N in the asparagine-glycine pair in position N512 of the vp1, v2 and vp3 proteins based on the residue numbering of the amino acid sequence of SEQ ID NO: 100 is deamidated, and

[0202] The vector genome in the AAVhu68 capsid comprises a nucleic acid molecule comprising an AAV inverted terminal repeat sequence and a non-AAV nucleic acid sequence encoding a PTH fusion as described herein operably linked to a sequence that directs expression of the PTH fusion in a target cell.

[0203] Still other modifications are observed, most of which do not convert an amino acid into a different amino acid residue. Optionally, at least one Lys in vp1, vp2 and vp3 of the capsid is acetylated. Optionally, at least one Asp in vp1, vp2 and / or vp3 of the capsid is isomerized to D-Asp. Optionally, at least one S (Ser, serine) in vp1, vp2 and / or vp3 of the capsid is phosphorylated. Optionally, at least one T (Thr, threonine) in vp1, vp2 and / or vp3 of the capsid is phosphorylated. Optionally, at least one W (trp, tryptophan) in vp1, vp2 and / or vp3 of the capsid is oxidized. Optionally, at least one M (Met, methionine) in vp1, vp2 and / or vp3 of the capsid is oxidized. In certain embodiments, the capsid protein has one or more phosphorylations. For example, certain vp1 capsid proteins can be phosphorylated at position 149.

[0204] In one embodiment, rAAV is scAAV. The abbreviation "sc" refers to self-complementary. "Self-complementary AAV" refers to a plasmid or vector having an expression cassette in which the coding region carried by the recombinant AAV nucleic acid sequence has been designed to form an intramolecular double-stranded DNA template. After infection, instead of waiting for the cell-mediated second chain synthesis, two complementary half scAAVs will associate to form a double-stranded DNA (dsDNA) that is easy to replicate and transcribe immediately. See, for example, D M McCarty et al., "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis" Gene Therapy, (August 2001), Vol. 8, No. 16, pp. 1248-1254. Self-complementary AAVs are described, for example, in U.S. Pat. Nos. 6,596,535, 7,125,717, and 7,456,683, which are incorporated herein by reference in their entireties.

[0205] In one embodiment, the nucleic acid sequence encoding the PTH construct described herein is engineered into any suitable genetic element, e.g., naked DNA, bacteriophage, transposon, cosmid, RNA molecule (e.g., mRNA), episome, etc., which transfers the PTH sequence carried thereon to a host cell, e.g., for producing nanoparticles, viral vectors, and / or for delivery to a subject's host cell in a packaging host cell carrying DNA or RNA. In one embodiment, the genetic element is a plasmid. The selected genetic element can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion technology, high-speed DNA-coated pellets, viral infection, and protoplast fusion. Methods for preparing such constructs are known to nucleic acid manipulation technicians 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).

[0206] As used herein, the term "host cell" may refer to a packaging cell line, wherein a vector (e.g., recombinant AAV or rAAV) is produced by a production plasmid. In an alternative, the term "host cell" may refer to any target cell that is desired to express a gene product as described herein. Therefore, "host cell" refers to a prokaryotic or eukaryotic cell (e.g., bacterial cell, human cell, or insect cell), which contains exogenous or heterologous DNA introduced into the cell by any means (e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion technology, high-speed DNA-coated pellets, viral infection, and protoplast fusion). In certain embodiments herein, the term "host cell" refers to a culture of cells of various mammalian species for in vitro evaluation of compositions described herein. In other embodiments herein, the term "host cell" refers to a cell for producing and packaging a viral vector or a recombinant virus. In a further embodiment, the term "host cell" is an intestinal cell, a small intestinal cell, a pancreatic cell, a liver cell.

[0207] As used herein, the term "target cell" refers to any target cell in which it is desired to express a heterologous nucleic acid sequence or protein. In certain embodiments, the target cell is a liver cell. In other embodiments, the target cell is a muscle cell.

[0208] In one embodiment, rAAV is provided, which includes a vector genome comprising an expression cassette, wherein the expression cassette includes a CMV promoter, an activation domain that is the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin associated protein (FRAP) fused to the carboxyl terminus of the p65 subunit of NF-κB from human, a GT2A_V1 peptide, a ZFHD1 DNA binding domain, three FKBP subunits, hGH poly A, 12XZFHD, a minimal sIL2 promoter, a coding sequence for the PTH fusion protein of SEQ ID NO: 14, and rabbit β-globin polyA. In another embodiment, rAAV is provided, comprising a vector genome comprising an expression cassette, wherein the expression cassette comprises a CMV promoter, an activation domain which is the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin associated protein (FRAP) fused to the carboxyl terminus of the p65 subunit of NF-κB from human, a GT2A_V2 peptide, a ZFHD1 DNA binding domain, three FKBP subunits, hGH poly A, 12XZFHD, a minimal sIL2 promoter, a coding sequence of a PTH fusion protein of SEQ ID NO: 14, and rabbit β-globin polyA.

[0209] In one embodiment, rAAV is provided, comprising a vector genome comprising an expression cassette, wherein the expression cassette comprises a CB7 promoter, a chicken β-actin intron, a coding sequence for a fusion protein of SEQ ID NO: 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 39, 42, 44, 46 or 48, rabbit globin poly A and 3' ITR. In another embodiment, an rAAV is provided, comprising a vector genome comprising an expression cassette, wherein the expression cassette comprises a CMV promoter, an activation domain which is the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin associated protein (FRAP) fused to the carboxyl terminus of the p65 subunit of NF-κB from human, an IRES, a ZFHD1 DNA binding domain, three FKBP subunits, hGH poly A, 8XZFHD, a minimal sIL2 promoter, a coding sequence for a PTH fusion protein of SEQ ID NO: 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 39, 42, 44, 46 or 48, and rabbit beta-globin polyA.

[0210] In another embodiment, a two-vector inducible system is provided. In one embodiment, a first rAAV is provided, which includes a vector genome comprising an expression cassette, wherein the expression cassette includes 12XZFHD, a minimal IL2 promoter, a coding sequence of a PTH fusion protein of SEQ ID NO: 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 39, 42, 44, 46 or 48, and rabbit beta globin polyA. A stuffer sequence may be included to increase the packaging size of the vector. The second rAAV includes a vector genome comprising an expression cassette, wherein the expression cassette includes a polynucleotide, the polynucleotide includes a CMV promoter, an intron, and the activation domain is a FKBP12-rapamycin binding (FRB) domain of a human FKBP12-rapamycin associated protein (FRAP) fused to the carboxyl terminus of a p65 subunit of NF-κB from a human, an IRES, a ZFHD1 DNA binding domain, and polyA.

[0211] In one embodiment, the rAAV comprises a vector genome comprising an expression cassette comprising a polynucleotide comprising a CB7 promoter, a chicken β-actin intron, a coding sequence for a fusion protein of SEQ ID NO: 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 39, 42, 44, 46 or 48, and rabbit globin poly A.

[0212] The minimum sequence required for packaging the expression cassette into the AAV virus particle is AAV 5' and 3' ITR with the same AAV origin as the capsid or with a different AAV origin (to produce an AAV pseudotype). In one embodiment, the ITR sequence from AAV2 or its deleted version (ΔITR) is adopted for convenience, and in order to speed up regulatory approval. However, ITRs from other AAV sources can be selected. Preferably, the source of ITR is the same as the source of Rep protein, and the Rep protein is provided in trans for production. Typically, the expression cassette of the AAV vector includes AAV 5'ITR, PTH fusion protein coding sequence and any regulatory sequence and AAV 3'ITR. However, other configurations of these elements may be suitable. A shortened version of the 5'ITR referred to as ΔITR has been described, in which the D sequence and the terminal resolution site (trs) are deleted. In other embodiments, full-length AAV 5' and 3'ITR are used.

[0213] In order to package the expression cassette into the virion, ITR is the only AAV component required in cis in the construct identical to the gene. In one embodiment, the coding sequence for replication (rep) and / or capsid (cap) is removed from the AAV genome and supplied in trans or supplied by a packaging cell line to produce an AAV vector. For example, as described above, the pseudotype AAV may contain ITRs from a source different from the source of the AAV capsid. In one embodiment, a chimeric AAV capsid may be utilized. Still other AAV components may be selected. The sources of such AAV sequences are described herein and may also be isolated or obtained from academic, commercial or public sources (e.g., American Type Culture Collection, Manassas, VA, in Manassas, Virginia). AAV sequences may be synthesized or otherwise suitable by reference to disclosed sequences (e.g., in the literature or in, for example, obtained from publicly available sequences in databases such as those provided by

[0214] Methods for producing and isolating AAV viral vectors suitable for delivery to a subject are known in the art. [See, e.g., U.S. Pat. No. 7,790,449; U.S. Pat. No. 7,282,199; WO 2003 / 042397; WO 2005 / 033321; WO2006 / 110689; and US 7,588,772 B2]. In one system, a production cell line is transiently transfected with a construct encoding a transgene flanked by ITRs and a construct encoding rep and cap. In a second system, a packaging cell line that stably supplies rep and cap is transiently transfected with a construct encoding a transgene flanked by ITRs. In each of these systems, AAV virions are produced in response to infection with a helper adenovirus or herpes virus, thereby requiring separation of rAAV from contaminating viruses. Recently, systems have been developed that do not require infection with a helper virus to recover AAV - systems by which the desired helper functions are also provided in trans (i.e., adenovirus E1, E2a, VA and E4 or herpesvirus UL5, UL8, UL52 and UL29, and herpesvirus polymerase). In these newer systems, helper functions can be provided by transiently transfecting cells with constructs encoding the desired helper functions, or the cells can be engineered to stably contain genes encoding helper functions, the expression of which can be controlled at the transcriptional or post-transcriptional level. In yet another system, transgenes flanked by ITRs and rep / cap genes are introduced into insect cells by infection with baculovirus-based vectors. For a review of these production systems, see generally, e.g., Zhang et al., 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, the contents of each of which are incorporated herein by reference in their entirety. Methods for making and using these and other AAV production systems are also described in the following U.S. Patents, the contents of each of which are incorporated herein by reference in their entirety: 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823 and 7,439,065.See generally, e.g., Grieger and Samulski, 2005, "Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications," Adv. Biochem. Engin / Biotechnol. 99: 119-145; Buning et al., 2008, "Recent developments in adeno-associated virus vector technology," J. Gene Med. 10: 717-733; and references cited below, each of which is incorporated herein by reference in its entirety. Methods for constructing any embodiment of the present invention are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York City (2012). Similarly, methods for producing rAAV virions are well known, and the selection of an appropriate method is not a limitation of the present invention. See, for example, K. Fisher et al., (1993) Journal of Virology, 70:520-532 and U.S. Pat. No. 5,478,745.

[0215] The rAAV described herein comprises a selected capsid having a vector genome packaged inside. The vector genome (or rAAV genome) comprises 5' and 3' AAV inverted terminal repeats (ITRs), a polynucleotide sequence encoding a fusion protein, and a regulatory sequence that directs the polynucleotide sequence encoding the fusion protein to be inserted into the genome of a host cell.

[0216] As used herein, "vector genome" refers to a nucleic acid sequence packaged inside a parvovirus (e.g., rAAV) capsid that forms a viral particle. Such nucleic acid sequences contain AAV reverse terminal repeats (ITRs). In the examples herein, the vector genome contains at least AAV 5'ITR, coding sequence (i.e., transgene) and AAV 3'ITR from 5' to 3'. It is possible to select ITRs from AAV2 (an AAV different from the capsid source) or in addition to non-full-length ITRs. In certain embodiments, ITRs are from AAVs that provide the same AAV source as the rep function during production or trans-supplementation of AAV. In addition, other ITRs can be used, for example, self-complementary (scAAV) ITRs. Both single-stranded AAV and self-complementary (sc) AAV are covered in rAAV. Transgenics is a nucleic acid coding sequence heterologous to the vector sequence, and the nucleic acid coding sequence encodes a polypeptide, a protein, a functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene products of interest. The nucleic acid coding sequence is operably linked to the regulatory components in a manner that allows the transgene to be transcribed, translated and / or expressed in the cells of the target tissue. Suitable components of the vector genome are discussed in more detail herein. In one example, a "vector genome" contains at least a vector-specific sequence from 5' to 3', a nucleic acid sequence encoding a PTH construct, which is operably linked to a regulatory control sequence (the regulatory control sequence directs its expression in the target cell), wherein the vector-specific sequence can be a terminal repeat sequence that specifically packages the vector genome into a viral vector capsid or envelope protein. For example, the AAV inverted terminal repeat sequence is used for packaging into AAV and certain other parvovirus capsids.

[0217] The AAV sequence of the vector generally includes cis-acting 5' and 3' inverted terminal repeat sequences (see, e.g., BJ Carter, Handbook of Parvoviruses, edited by P. Tijsser, CRC Press, pp. 155-168 (1990)). The length of the ITR sequence is about 145 bp. Preferably, substantially the entire sequence encoding the ITR is used in the molecule, but minor modifications to these sequences are allowed to some extent. The ability to modify these ITR sequences is within the technical scope of the art. (See, e.g., texts such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., Journal of Virology, 70:520-532 (1996)). An example of such a molecule employed in the present invention is a "cis-acting" plasmid containing a transgene, in which the selected transgene sequence and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences. In one embodiment, ITR is from AAV different from the AAV supplying capsid. In one embodiment, the ITR sequence from AAV2. However, ITR from other AAV sources can be selected. A shortened version of the 5'ITR referred to as ΔITR has been described, in which the D sequence and the terminal resolution site (trs) are deleted. In certain embodiments, the vector genome comprises a shortened AAV2 ITR of 130 base pairs, in which the external A element is missing. It is not desired to be bound by theory, but it is believed that during the amplification of the vector DNA using the internal (A') element as a template, the shortened ITR returns to the wild-type length of 145 base pairs. In other embodiments, full-length AAV 5' and 3'ITR are used. In the case where the source of ITR is from AAV2 and the AAV capsid is from another AAV source, the resulting vector can be referred to as pseudotyped. However, other configurations of these elements may be suitable.

[0218] Optionally, the PTH constructs described herein can be delivered by viral vectors other than rAAV. Such other viral vectors can include any virus suitable for gene therapy, including any virus suitable for gene therapy, including but not limited to adenovirus; herpes virus; lentivirus; retrovirus, etc. Suitably, when one of these other vectors is produced, it is produced as a replication-defective viral vector.

[0219] "Replication-defective virus" or "viral vector" refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, wherein any viral genomic sequence also packaged in the viral capsid or envelope is replication-defective; that is, the synthetic or artificial viral particle cannot produce progeny virions but retains the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gutless"-containing only the transgene of interest, which is flanked by the signals required for amplification and packaging of the artificial genome), but these genes can be supplied during production. Therefore, this is considered safe for use in gene therapy because replication and infection by progeny virions will not occur unless the viral enzymes required for replication are present.

[0220] Compositions comprising viral vector constructs as described herein are also provided. Pharmaceutical compositions as described herein are designed to be delivered to a subject in need by any suitable route or combination of different routes. Direct delivery to the liver (optionally intravenously, by the hepatic artery or by transplantation), oral administration routes, inhalation administration routes, intranasal administration routes, intratracheal administration routes, intraarterial administration routes, intraocular administration routes, intravenous administration routes, intramuscular administration routes, subcutaneous administration routes, intradermal administration routes and other maternal administration routes. Viral vectors as described herein can be delivered in the form of a single composition or multiple compositions. Optionally, two or more different AAVs can be delivered, or multiple viruses [see, for example, WO 2011 / 126808 and WO 2013 / 049493]. In another embodiment, multiple viruses can contain different replication defective viruses (e.g., AAV and adenovirus). In one embodiment, administration is performed intramuscularly. In another embodiment, administration is performed intravenously.

[0221] Replication-defective viruses can be formulated with physiologically acceptable carriers for gene transfer and gene therapy applications. In the case of AAV viral vectors, quantification of genome copies ("GC") can be used as a measure of the dosage contained in the formulation. The number of genome copies (GC) of the replication-defective virus composition of the present invention can be determined using any method known in the art. A method for titrating the number of AAV GCs is as follows: a purified AAV vector sample is first treated with DNA enzyme to eliminate unencapsidated AAV genomic DNA or contaminating plasmid DNA from the production process. The nuclease-resistant particles are then subjected to heat treatment to release the genome from the capsid. The released genome is then quantified by real-time PCR using a primer / probe set targeting a specific region of the viral genome (usually a poly A signal). Another suitable method for determining genome copies is quantitative PCR (qPCR), in particular optimized qPCR or digital droplet PCR [LockMartin et al., Human Gene Therapy Methods. 2014 Apr, 25(2): 115-125. doi: 10.1089 / hgtb.2013.131, published online ahead of edit on Dec. 13, 2013].

[0222] Furthermore, the replication-defective virus composition can be formulated in a dosage unit to contain about 1.0 x 10 9 GC to about 1.0x 10 15 In another embodiment, this amount of viral genome can be delivered in divided doses. In one embodiment, for an average human subject of about 70 kg, the dose is about 1.0 x 10 10 GC to about 3.0x 10 14 GC. In another embodiment, the dose is about 1 x 10 9 GC. For example, the dose of AAV virus can be about 1 x 10 10 GC, 1x 10 11 Gc, about 5X 10 11 GC, about 1X 10 12 GC, about 5X10 12 GC or about 1X 10 13 In another embodiment, for human subjects, the dose is about 1.0 x 10 9 GC / kg to about 3.0x 10 14 GC / kg. In another embodiment, the dose is about 1 x 10 9 GC / kg. For example, the dose of AAV virus can be about 1 x 10 10GC / kg, 1x 10 11 GC / kg, about 5X10 11 GC / kg, about 1X 10 12 GC / kg, about 5X 10 12 GC / kg or about 1X 10 13 GC / kg. In one embodiment, the construct can be delivered in a volume of 1 μL to about 100 mL. As used herein, the term "dose" or "amount" can refer to the total dose or amount delivered to a subject during treatment or the dose or amount delivered by administering a single unit (or multiple units or divided doses).

[0223] The recombinant vector described above can be delivered to a host cell according to the disclosed method. The rAAV preferably suspended in a physiologically compatible carrier can be administered to a desired subject including a human. In view of the indications for which the transfer virus is directed, a person skilled in the art can easily select a suitable carrier. For example, a suitable carrier comprises saline, which can be formulated with various buffer solutions (e.g., phosphate-buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil and water. The selection of the carrier is not a limitation of the present invention.

[0224] In another embodiment, the composition comprises a carrier, a diluent, an excipient and / or an adjuvant. In certain embodiments, for administration to a human patient, the rAAV is suitably suspended in an aqueous solution containing saline, a surfactant, and a pharmaceutically and / or physiologically compatible salt or mixture of salts. Suitably, the formulation is adjusted to a physiologically acceptable pH, for example, in the range of pH 6 to 9, or pH 6.0 to 7.5, or pH 6.2 to 7.7, or pH 6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8, or about 7.0. In certain embodiments, the pH of the formulation is adjusted to about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, or about 7.8. In certain embodiments, the pH that may be desired is about 7.28 to about 7.32, about 6.0 to about 7.5, about 6.2 to about 7.7, about 7.5 to about 7.8, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, or about 7.8. In certain embodiments, for intravenous delivery, the pH that may be desired is about 6.8 to about 7.2. However, other pHs within the broadest range and these subranges may be selected for other delivery routes.

[0225] Optionally, in addition to rAAV and / or variants and carriers, the compositions of the invention may also contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, methyl paraben, ethyl vanillin, glycerol, phenol and p-chlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0226] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients may also be incorporated into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a host. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc. can be used to introduce the composition of the present invention into suitable host cells. Specifically, the transgene delivered by the rAAV vector can be formulated for delivery or encapsulation in lipid particles, liposomes, vesicles, nanospheres or nanoparticles, etc.

[0227] In one embodiment, the composition comprises a final formulation suitable for delivery to a subject, such as an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration. Optionally, one or more surfactants are present in the formulation. In another embodiment, the composition can be transported in the form of a concentrate that is diluted to be administered to a subject. In other embodiments, the composition can be lyophilized and reconstituted at the time of administration.

[0228] A suitable surfactant or combination of surfactants can be selected from non-toxic nonionic surfactants. In one embodiment, a difunctional block copolymer surfactant terminated with a primary hydroxyl group is selected, such as F68 [BASF], also known as Poloxamer 188, has a neutral pH and an average molecular weight of 8400. Other surfactants and other poloxamers may be selected, i.e., nonionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS15 (polyethylene glycol-15 hydroxystearate), LABRASOL (polyoxycaprylyl glyceride), polyoxyl 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol, and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are usually named with the letter "P" (for poloxamer), followed by three numbers: the first two digits x100 give the approximate molecular mass of the polyoxypropylene core, and the last digit x10 gives the percentage of the polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005% to about 0.001% of the suspension.

[0229] The dosage of the vector depends primarily on factors such as the condition being treated, the patient's age, weight, and health, and therefore may vary between patients. For example, a therapeutically effective human dose of a viral vector is typically in the range of about 25 microliters to about 1000 microliters to about 100 mL of a solution containing a concentration of about 1 x 10 9 Up to 1x 10 16 The amount of viral vector containing 10 genomes (to treat a subject with an average body weight of 70 kg) is preferably 1.0 x 10 12 GC to 1.0x 10 13GC. The compositions of the present invention can be delivered in a volume of about 0.1 μL to about 10 mL, including all numbers within the range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method. In one embodiment, the volume is about 50 μL. In another embodiment, the volume is about 70 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 125 μL. In another embodiment, the volume is about 150 μL. In another embodiment, the volume is about 175 μL. In yet another embodiment, the volume is about 200 μL. In another embodiment, the volume is about 250 μL. In another embodiment, the volume is about 300 μL. In another embodiment, the volume is about 450 μL. In another embodiment, the volume is about 500 μL. In another embodiment, the volume is about 600 μL. In another embodiment, the volume is about 750 μL. In another embodiment, the volume is about 850 μL. In another embodiment, the volume is about 1000 μL. In another embodiment, the volume is about 1.5 mL. In another embodiment, the volume is about 2 mL. In another embodiment, the volume is about 2.5 mL. In another embodiment, the volume is about 3 mL. In another embodiment, the volume is about 3.5 mL. In another embodiment, the volume is about 4 mL. In another embodiment, the volume is about 5 mL. In another embodiment, the volume is about 5.5 mL. In another embodiment, the volume is about 6 mL. In another embodiment, the volume is about 6.5 mL. In another embodiment, the volume is about 7 mL. In another embodiment, the volume is about 8 mL. In another embodiment, the volume is about 8.5 mL. In another embodiment, the volume is about 9 mL. In another embodiment, the volume is about 9.5 mL. In another embodiment, the volume is about 10 mL.

[0230] In some embodiments, the concentration of the recombinant adeno-associated virus carrying the nucleic acid sequence encoding the desired transgene under the control of the regulatory sequence is desirably about 10 7 and 10 14 The range of GC / mL was 1.

[0231] In one embodiment, the dose of rAAV in the composition is about 1.0 x 10 9 GC / kg body weight is about 1.5x10 13 GC / kg. In one embodiment, the dosage is about 1.0 x 10 10 GC / kg. In one embodiment, the dosage is about 1.0x10 11 GC / kg. In one embodiment, the dosage is about 1.0 x 10 12GC / kg. In one embodiment, the dosage is about 5.0 x 10 12 GC / kg. In one embodiment, the dosage is about 1.0 x 10 13 GC / kg. All ranges described herein are inclusive of the endpoints.

[0232] In one embodiment, the effective dose (total genome copies delivered) is about 10 7 Up to 10 13 In one embodiment, the total dose is about 10 8 In one embodiment, the total dose is about 10 9 In one embodiment, the total dose is about 10 10 In one embodiment, the total dose is about 10 11 In one embodiment, the total dose is about 10 12 In one embodiment, the total dose is about 10 13 In one embodiment, the total dose is about 10 14 In one embodiment, the total dose is about 10 15 genome copies.

[0233] It is desirable to utilize the lowest effective concentration of virus to reduce the risk of undesirable effects such as toxicity. Still other dosages and administration volumes within these ranges may be selected by the attending physician taking into account the physical condition of the subject (preferably a human) being treated, the age of the subject, the particular disorder, and the extent to which the disorder (if progressive) has developed.

[0234] In certain embodiments, the composition includes rAAV, and the rAAV includes an inducible PTH agonist construct. In certain embodiments, the inducer or molecule is rapamycin or a rapamycin analog. In certain embodiments, the inducer is rapamycin, and at least one or more, at least two or more times, at least three or more times are applied after the composition including rAAV. In certain embodiments, rapamycin is applied with a dosage of at least about 4nM to at least about 40nM. In certain embodiments, the inducer (i.e., rapamycin) is applied with a dosage of at least about 0.1mg / kg to at least about 3.0mg / kg. In certain embodiments, the inducer (i.e., rapamycin) is applied with a dosage of at least about 0.5mg / kg to at least about 2.0mg / kg.

[0235] The viral vectors and other constructs described herein can be used to prepare a medicament for delivering a PTH fusion protein construct to a subject in need, supplying PTH with an increased half-life to a subject, and / or for treating HP in a subject. Therefore, in another aspect, a method for treating HP is provided. The method comprises administering a composition as described herein to a subject in need. In one embodiment, the composition comprises a viral vector containing a PTH fusion protein expression cassette, as described herein.

[0236] As used herein, the term "treatment" or "treating" is defined to encompass the administration of one or more compounds or compositions described herein to a subject for the purpose of improving one or more symptoms of short bowel syndrome. Thus, "treatment" may include reducing the progression of HP in a given subject, reducing the severity of symptoms in a given subject, eliminating symptoms of the disease in a given subject, delaying the progression of the disease in a given subject, or increasing the efficacy of a therapy in a given subject.

[0237] In another embodiment, a method for treating HP in a subject is provided. The method comprises administering a viral vector comprising a nucleic acid molecule comprising a sequence encoding a fusion protein as described herein. In one embodiment, the subject is a human.

[0238] The course of treatment may optionally involve repeated administration of the same viral vector (e.g., AAVrh91 vector) or different viral vectors (e.g., AAVrh91 and AAV3B.AR2.12). Still other combinations may be selected using the viral vectors described herein. Optionally, the compositions described herein may be combined in a regimen involving nutritional therapy (enteral or parenteral nutrition), a drug (such as a drug for controlling gastric acid, reducing diarrhea, or improving intestinal absorption), or a PTH protein or surgery. In certain embodiments, the AAV vector and the combination therapy are administered substantially simultaneously. In other embodiments, the AAV vector is administered first. In other embodiments, the combination therapy is administered first.

[0239] In one embodiment, the composition is administered with an effective amount of PTH protein. Various commercially available PTH products are known in the art, including but not limited to teriparatide (PTH1-34), abaloparatide (abaloparatide) and recombinant PTH (rhPTH1-84). In another embodiment, the composition is administered in combination with an effective amount of calcium carbonate or vitamin D.

[0240] In some embodiments, the combination of rAAV and PTH protein described herein reduces the PTH protein dosage requirement of the subject compared to before treatment with the viral vector. Such dosage requirements can be reduced by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. The treating physician can determine the correct dosage of the PTH protein required by the subject. For example, the PTH protein or other therapies can be used to treat the subject, and the treating physician can continue the treatment after the AAV vector is administered. Such PTH protein or other synergistic therapies can then be continued, reduced or interrupted as needed.

[0241] In one embodiment, a composition including an expression cassette, a vector genome, rAAV or other compositions for gene therapy described herein is delivered in a single dose / patient. In one embodiment, a therapeutically effective amount of a composition described herein is delivered to a subject. As used herein, a "therapeutically effective amount" refers to the amount of an expression cassette or vector or a combination thereof that delivers and expresses a PTH fusion protein sufficient to achieve a therapeutic goal in a target cell. The therapeutically effective amount can be selected by a treating physician, or based on previously determined guidelines. For example, teriparatide can be injected subcutaneously twice a day at an initial dose of 20 μg. rAAV can be delivered to a subject, and then supplemented with oral or subcutaneous teriparatide, or other drugs as needed, to achieve the equivalent of a desired dose of 20 μg injected subcutaneously twice a day.

[0242] In certain embodiments, the goal of treatment is to ameliorate or treat one or more of the symptoms of HP.Therapeutically effective amounts can be determined based on animal models rather than human patients.

[0243] As used herein, the term "heterogeneous" or any grammatical variation thereof, when used to refer to vp capsid proteins, refers to a population consisting of non-identical elements, e.g., having vp1, vp2, or vp3 monomers (proteins) with different modified amino acid sequences. SEQ ID NO:38 provides the encoded amino acid sequence of the AAVrh91 vp1 protein. The term "heterogeneous" used in conjunction with the vp1, vp2, and vp3 proteins (alternatively referred to as isoforms) refers to differences in the amino acid sequences of the vp1, vp2, and vp3 proteins within the capsid. The AAV capsid contains subpopulations within the vp1 protein, within the vp2 protein, and within the vp3 protein that have modifications from predicted amino acid residues. These subpopulations contain at least some deamidated asparagine (N or Asn) residues. For example, certain subpopulations include at least one, two, three, or four highly deamidated asparagine (N) positions in asparagine-glycine pairs, and optionally further include other deamidated amino acids, wherein the deamidation results in an amino acid change and other optional modifications.

[0244] As used herein, unless otherwise specified, a "subpopulation" of vp proteins refers to a group of vp proteins that have at least one defined common property and consist of at least one group member to less than all members of a reference group. For example, unless otherwise specified, a "subpopulation" of vp1 proteins is at least one (1) vp1 protein and less than all vp1 proteins in an assembled AAV capsid. Unless otherwise specified, a "subpopulation" of vp3 proteins can be one (1) vp3 protein that is less than all vp3 proteins in an assembled AAV capsid. For example, vp1 proteins can be a subpopulation of vp proteins; vp2 proteins can be a separate subpopulation of vp proteins, and vp3 is still another subpopulation of vp proteins in an assembled AAV capsid. In another example, vp1, vp2, and vp3 proteins can contain subpopulations with different modifications, for example, at least one, two, three, or four highly deamidated asparagines, for example at asparagine-glycine pairs.

[0245] As used herein, the "stock" of rAAV refers to a population of rAAV. Although its capsid protein has heterogeneity due to deamidation, rAAV is expected to share the same vector genome with 5 in the stock. The stock can contain rAAV with a capsid, and the capsid has, for example, a unique heterogeneous deamidation pattern of the selected AAV capsid protein and the selected production system. The stock can be produced from a single production system, or pooled from multiple runs of the production system. Various production systems can be selected, including but not limited to the production systems described herein. As used herein, the terms "PTH construct", "PTH expression construct" and synonyms include a combination of a PTH sequence as described herein with a leader sequence and a fusion domain. The terms "PTH construct", "PTH expression construct" and synonyms can be used to refer to a nucleic acid sequence encoding a PTH fusion protein or its expression product.

[0246] In the context of nucleic acid sequences, the terms "percent identity (%)", "sequence identity", "percent sequence identity" or "identical percentage" refer to the bases in two sequences that are identical when aligned to obtain correspondence. The length of the sequence identity comparison can exceed the full length of the genome, the full length of the gene coding sequence, or a fragment of at least about 100 to 150 nucleotides, or as required. However, it is also possible to expect identity between smaller fragments, such as at least about nine nucleotides, typically at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides. Multiple sequence alignment programs can also be used for nucleic acid sequences. Examples of such programs include "Clustal W", "CAP sequence assembly", "BLAST", "MAP" and "MEME", which can be accessed through Web servers on the Internet. Other sources of such programs are known to those skilled in the art. Alternatively, the vector NTI utility program is also used. Many algorithms known in the art can be used to measure nucleotide sequence identity, including the algorithms contained in the above-mentioned programs. As another example, the GCG 6.1 version of the program Fasta TM Compare polynucleotide sequences. Fasta TM The alignment and percent sequence identity of the best overlap region between the query sequence and the search sequence are provided. For example, the percent sequence identity between nucleic acid sequences can be calculated using Fasta as provided in GCG version 6.1 with its default parameters (font size 6 and NOPAM coefficients for the scoring matrix). TM Where determined, such procedures are incorporated herein by reference.

[0247] The term "highly conserved" means at least 80% identity, preferably at least 90% identity, and more preferably more than 97% identity. Identity can be readily determined by those skilled in the art using algorithms and computer programs known to those skilled in the art.

[0248] Unless otherwise specified by a higher range, it should be understood that the percentage of identity is the lowest level of identity and encompasses all higher levels of identity, up to 100% identity with a reference sequence. Unless otherwise specified, it should be understood that the percentage of identity is the lowest level of identity and encompasses all higher levels of identity, up to 100% identity with a reference sequence. For example, "95% identity" and "at least 95% identity" can be used interchangeably and include 95%, 96%, 97%, 98%, 99% and up to 100% identity with a reference sequence, and all fractions therebetween.

[0249] In the context of amino acid sequences, the terms "percent identity (%)", "sequence identity", "percent sequence identity" or "identical percent" refer to the residues in two sequences that are identical when aligned for correspondence. The percent identity of an amino acid sequence over the full length of a protein, a polypeptide, about 70 amino acids to about 100 amino acids or a peptide fragment thereof, or a corresponding nucleic acid sequence encoding sequence can be readily determined. Suitable amino acid fragments may be at least about 8 amino acids in length, and may be up to about 150 amino acids. Typically, when referring to "identity", "homology" or "similarity" between two different sequences, the "identity", "homology" or "similarity" is determined with reference to an "aligned" sequence. An "aligned" sequence or "alignment" refers to a plurality of nucleic acid sequences or protein (amino acid) sequences that typically contain corrections for missing or additional bases or amino acids compared to a reference sequence. Alignment is performed using any of a variety of publicly available or commercially available multiple sequence alignment programs. Sequence alignment programs are available for amino acid sequences, for example, "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME" and "Match-Box" programs. Typically, any of these programs is used with the default settings, although those skilled in the art can change these settings as needed. Alternatively, those skilled in the art can utilize another algorithm or computer program that provides at least the same level of identity or alignment as that provided by the reference algorithm and program. See, for example, J.D. T. Homson et al., Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690 (1999).

[0250] It should be noted that the term "a" or "an" refers to one or more. Thus, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

[0251] The words "comprise", "comprises" and "comprising" are to be interpreted as inclusive rather than exclusive. The words "consist / consisting" and variations thereof are to be interpreted as exclusive rather than inclusive. Although various embodiments in the specification are presented using "comprising" language, in other cases, it is also intended to use the language of "consisting of" or "consisting essentially of" to explain and describe the relevant embodiments.

[0252] As used herein, "patient" or "subject" refers to a mammal, including humans, veterinary or farm animals, livestock or pets, and animals commonly used in clinical research. In one embodiment, the subject of these methods and compositions is a human. In another embodiment, the subject is not a feline.

[0253] As used herein, unless otherwise indicated, the term "about" means a variability of 10% (±10%, e.g., ±1, ±2, ±3, ±4, ±5, ±6, ±7, ±8, ±9, ±10, or values ​​therebetween) relative to a given reference.

[0254] In some cases, the term "E+#" or the term "e+#" is used to refer to an exponent. For example, "5E10" or "5e10" is 5x10 10 . These terms can be used interchangeably.

[0255] As used herein, the term "modulate" or variations thereof refers to the ability of a composition to inhibit one or more components of a biological pathway.

[0256] As used herein, "disease," "disorder," and "condition" are used interchangeably to refer to an abnormal state of a subject.

[0257] Unless otherwise defined in this specification, technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art and with reference to the public texts, which provide those skilled in the art with a general guide to many of the terms used in this application.

[0258] Reference to "one embodiment" or "another embodiment" when describing an embodiment does not mean that the referenced embodiment is mutually exclusive of another embodiment (eg, an embodiment described before the referenced embodiment) unless explicitly stated otherwise.

[0259] Examples

[0260] The following examples are provided to illustrate various embodiments of the present invention. The examples are not intended to limit the present invention in any way.

[0261] Example 1 - Construction of PTH vector

[0262] A pharmacologically regulated gene expression system is reported by which transgene expression can be controlled by orally available drugs such as rapamycin and its proteins (rapamycin analogs (Rapalogs)). This gene circuit can be delivered to skeletal muscle via an AAV vector and provides sustained regulated transgene expression for more than 5 years in non-human primates (Rivera et al., 2005). Applying this technology to PTH could provide a solution for HP patients to avoid daily injections of PTH protein and be safe in cases where patients require treatment. Multiple versions of the PTH transgene were developed to obtain the preferred kinetics of PTH expression by rapamycin, and to focus on the level and duration of induced expression to prepare a potent AAV vector that can provide therapeutic levels of PTH expression with less vector doses and less frequent oral inducers. Figure 2B .

[0263] All transgenes were codon-optimized for normal human codon usage. Various secretion sequences were also introduced to allow for better expression in skeletal muscle cells. Figure 1A For longer serum half-life, several N-terminally truncated versions of PTH were fused to the Fc domain of human IgG1 and IgG4 or serum albumin. Figure 2A .

[0264] More specifically, vectors were constructed in which a leader sequence was placed upstream of one of several PTH amino acid sequences, followed by a fusion domain. Figure 2B Sequences were generated and cloned into expression vectors containing a CMV promoter under the control of an inducible expression system. The expression constructs were flanked by AAV2 ITRs. In other constructs, sequences were generated and cloned into expression vectors containing a CB7 constitutive promoter.

[0265] The following constructs were packaged into the AAVrh91 vector by triple transfection and iodixanol gradient purification as described previously.

[0266] Example 2 – In vitro expression

[0267] PTH fusion was measured in the culture supernatant of HEK293 cells transfected with a plasmid for inducible human PTH-Fc. PTH-Fc was expressed as Figure 3A The staining shown was quantified. Fractions 3 and 4 under reducing (R) and non-reducing (NR) conditions are shown. PTH cannot form dimers under reducing conditions. The potency of these purified proteins was tested. Both tested fusions were found to have similar potency to PTH (1-34). Figure 3B

[0268] The inducible construct was tested with 0 nM or 40 nM rapamycin. The inducible construct showed slightly lower expression compared to the constitutive construct. Figure 3C ).

[0269] Example 3 - Pilot expression in Rag1KO mice

[0270] like Figure 1B As shown, Rag1KO female mice were injected with the vector AAVrh91.CB7.CI.hPTH(N).rBG (1x 10 11 GC / mouse) (containing the native leader sequence) or AAVrh91.CB7.CI.hPTH(IL2).rBG (1x10 11 GC / mice) (containing the IL2 leader sequence). Plasma on day 14 showed similar PTH levels, with the IL2 leader sequence providing slightly higher secreted PTH. Figure 1C Serum calcium on day 28 showed similar results. Figure 1C .

[0271] Rag1KO female mice were injected with the vector AAVrh91.CB7.CI.hPTH(N).rBG (1x10 11 GC / mouse) or AAVrh91.CB7.CI.hPTH73.P.Fc.rBG (1x 10 11 GC / mouse) for treatment. Figure 4A Plasma was isolated on days 14 and 28 and tested for hPTH potency. CB7.hPTH73.P.Fc showed superior potency compared to the construct lacking the fusion. Figure 4B .

[0272] like Figure 5A As shown, Rag1KO female mice were injected with the vector AAVrh91.CMV.TF1Nc.3.hGH (1 x 10 11 GC / mouse) and AAVrh91.CB7.CI.hPTH73.P.Fc.rBG (1x10 11 GC / mouse). Rapamycin was administered at 10 mg / kg on day 12 (Groups 2-3) and 3 mg / kg on day 38 (Group 3). Groups 1 and 2 were bled and autopsied on day 14, while Group 3 was bled on days 14, 21, 28, and 40 (autopsy). Plasma was isolated and tested for efficacy ( Figure 5B ). Groups 2 and 3 showed comparable efficacy on day 14, with the second rapamycin induction increasing the efficacy of Group 3. Serum calcium is shown in Figure 5C middle.

[0273] Example 4 - Constitutive PTH-Fc Vector in AFG Monkeys

[0274] As shown in FIG7 , the vector AAVrh91.CB7.CI.hPTH73.P.Fc.rBG (1× 10 12 GC / kg) were used to treat female African green monkeys. The NAB titers of these monkeys were as follows Figure 7B shown.

[0275] Example 5-TPTX model in Rag2KO rats

[0276] Rag2KO rats were subjected to parathyroidectomy (TPTX) to validate the TPTX model. Parathyroid surgery was performed on 8-week-old Sprague Dawley rats / Rag2 KO rats using 5-aminolevulinic acid fluorescence identification technology. Intraperitoneal administration of 5-ALA (500 mg / kg) before surgery resulted in phototoxicity. Therefore, rats were placed under soft light for 2 hours before surgery. A midline vertical incision was made in the neck, and red fluorescent PTH glands were observed under white-blue light and UV filters. The glands were removed with a cold knife. Postoperative complications were low calcium levels, leading to uncontrolled muscle spasms and contractions. Death occurred within 4 days without calcium supplementation, which was provided by water or a customized diet (AIN-93G 0.5% Ca).

[0277] TPTX rats were injected IM with vehicle, inducible .hPTH73.P.Fc (1x 10 12 GC / kg or 1x 10 13 The first dose of rapamycin (10 mg / kg) was given intraperitoneally on day 14, and the second dose (intraperitoneal, 3 mg / kg) of rapamycin was given on day 40. Clinical signs are shown in the table below.

[0278]

[0279]

[0280] The results are shown in Figures 8A-8C Incomplete parathyroid removal occurred in two rats injected with vehicle. For unknown reasons, the condition of the rats was severely exacerbated.

[0281] Example 6 - Constitutive PTH-Fc Vector in Rag2KO Rats

[0282] Rag2KO rats (3 rats / group) were injected IM with AAVrh91.CB7.CI.hPTH73.P.Fc.rBG (1x 10 13 GC / kg or 3x 10 13 GC / kg). From receiving 3x 10 13 One rat in the GC / kg group developed hind limb paralysis and had to be euthanized probably due to pathological spinal cord fracture. Plasma PTH ( Fig. 9A ) and serum calcium ( Fig. 9B ).

[0283] All documents cited in this specification are incorporated herein by reference. U.S. Provisional Patent Application No. 63 / 316,215, filed on March 3, 2022, is incorporated herein by reference. Although the present invention has been described with reference to specific embodiments, it should be understood that modifications may be made without departing from the spirit of the present invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. A composition comprising a nucleic acid comprising a sequence encoding a fusion protein comprising a parathyroid hormone (PTH) protein and an IgG4 Fc, wherein the fusion protein has a sequence of SEQ ID NO: 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 39, 42, 44, 46 or 48, or a sequence at least 99% identical thereto.

2. The composition of claim 1, wherein the sequence encoding the fusion protein is SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 or shares a sequence at least 75% identical thereto.

3. The composition, comprising a viral vector, wherein the viral vector comprises: (a) adeno-associated virus (AAV) capsid, and (b) a vector genome packaged in the AAV capsid, wherein the vector genome comprises an AAV inverted terminal repeat (ITR), a coding sequence for the fusion protein, and a regulatory sequence for directing the expression of the fusion protein.

4. The composition of claim 3, wherein the viral vector is a recombinant AAV (rAAV) having the AAV capsid of AAVrh91 or AAVhu68.

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

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

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

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

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

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

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

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

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

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

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

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

17. A composition according to any one of claims 5 to 16, comprising a regulatable promoter; an activation domain comprising a p65 transactivation domain and a FKBP12-rapamycin binding (FRB) domain of a FKBP12-rapamycin associated protein (FRAP); a DNA binding domain comprising a zinc finger homology domain (ZFHD) and three FK506 binding protein binding domain (FKBP) subunit genes; 12 copies of the ZFHD binding site; and a sequence encoding a fusion protein of a PTH protein comprising any one of SEQ ID NO: 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 39, 42, 44, 46 or 48, and a regulatory sequence that directs the expression of the fusion protein.

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

19. The composition of any one of claims 1 to 18 for use in a method for treating a subject suffering from hypoparathyroidism.

20. Use of a composition according to any one of claims 1 to 18 for the preparation of a medicament for treating a subject suffering from hypoparathyroidism.

21. The composition according to any one of claims 1 to 18, wherein the composition is formulated for use in a 1×10 9 GC / kg to 5x 10 13 GC / kg of the rAAV was administered.

22. The composition according to any one of claims 1 to 18, wherein the patient is a human and 1 x 10 10 GC to 1.5x 10 15 The rAAV dose for GC.

23. The composition of any one of claims 1 to 18, wherein the rAAV is delivered intramuscularly or intravenously.

24. A method for treating a subject with hypoparathyroidism, the method comprising delivering to the subject a rAAV having an AAV capsid from AAVrh91 or AAVhu68 and a vector genome packaged in the AAV capsid, the vector genome comprising AAV inverted terminal repeats (ITRs), a sequence encoding a fusion protein of a PTH protein comprising any one of SEQ ID NO: 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 39, 42, 44, 46 or 48, and a regulatory sequence that directs expression of the fusion protein.

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

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

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

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

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